P63 EXPRESSION LEVELS IN SIDE POPULATION AND LOW LIGHT SCATTERING OCULAR SURFACE EPITHELIAL CELLS

PubMed Central

Epstein, Seth P; Wolosin, J. Mario; Asbell, Penny A

2005-01-01

Purpose Because stem cells exhibit high self-renewal capacity, slow cycling, and high proliferative potential, and one of many markers postulated for epithelial stem cells, p63, is challenged by widespread expression within stem cell–free regions, we examined p63 expression in these stem cell–associated cohorts compared with their controls. Methods Rabbit limbocorneal cryosections, cytospun cell-sorted (by fluorescence-activated cell sorter) side population (SP) and low side scatter (LSSC) cells, and limbal epithelial cells over feeders were stained for p63 by indirect immunofluorescence. Clones were fixed and stained daily for 7 days. Image analysis measured p63 intensity, plotting it against colony size. Results All basal limbal cells were positive for p63, yet only 5% to 7% expressed high p63 intensities, 40% intermediate, and the majority low. Side population cells were less than 1% of total cells. The average intensity of SP staining was three times that of controls. Subpopulations displaying stemlike features exhibited highest p63 expression. Replication rates of isolated cells differed. Day 5 colonies contained 256 (16 hours/cycle) to two (96 hours/cycle) cells. Whereas all cells were positive for p63, intensity in slow-cycling cells was three to four times that in rapidly proliferating congeners. Increased cell doublings did not decrease fluorescence. Conclusions Results suggest that p63 concentration is maximal in stem cells and decreases with differentiation. High p63 levels seem to correlate with cells of the SP and LSSC phenotypes, indicating high cell stemness. With identification of stem cells, further studies can elucidate their use in supporting ocular surface health. PMID:17057802

Autoradiography and the Cell Cycle.

ERIC Educational Resources Information Center

Jones, C. Weldon

1992-01-01

Outlines the stages of a cell biology "pulse-chase" experiment in which the students apply autoradiography techniques to learn about the concept of the cell cycle. Includes (1) seed germination and plant growth; (2) radioactive labeling and fixation of root tips; (3) feulgen staining of root tips; (4) preparation of autoradiograms; and…

Biomarkers of Selenium Chemoprevention of Prostate Cancer

DTIC Science & Technology

2005-01-01

than Se-Met in inhibiting Flow Kit from BD Pharmigen (San Diego, CA). Stained cells were then quantified by flow cytometry , and the data were analyzed...decrease in Quantitation of Apoptosis by Flow Cytometry . PC-3 cells were plated at cell number accumulation by MSA was related to cell cycle arrest, we... flow exposed to either 5 or 10Mm MSA for 48 or 72 h. Adherent cells harvested by mild cytometry of ethanol-permeabilized cells stained with Pl. Synchro

A fluorescence in situ staining method for investigating spores and vegetative cells of Clostridia by confocal laser scanning microscopy and structured illuminated microscopy.

PubMed

D'Incecco, P; Ong, L; Gras, S; Pellegrino, L

2018-04-18

Non-pathogenic spore-forming Clostridia are of increasing interest due to their application in biogas production and their capability to spoil different food products. The life cycle for Clostridium includes a spore stage that can assist in survival under environmentally stressful conditions, such as extremes of temperature or pH. Due to their size, spores can be investigated by a range of microscopic techniques, many of which involve sample pre-treatment. We have developed a quick, simple and non-destructive fluorescent staining procedure that allows a clear differentiation between spores and vegetative cells and effectively stains spores, allowing recovery and tracking in subsequent experiments. Hoechst 34580, Propidium iodide and wheat germ agglutinin WGA 488 were used in combination to stain four strains of Clostridia at different life cycle stages. Staining was conducted without drying the sample, preventing changes induced by dehydration and cells observed by confocal laser scanner microscopy or using a super-resolution microscope equipped with a 3D-structured illumination module. Dual staining with Hoechst/Propidium iodide differentiated spores from vegetative cells, provided information on the viability of cells and was successfully applied to follow spore production induced by heating. Super-resolution microscopy of spores probed by Hoechst 34580 also allowed chromatin to be visualised. Direct staining of a cheese specimen using Nile Red and Fast Green allowed in situ observation of spores within the cheese and their position within the cheese matrix. The proposed staining method has broad applicability and can potentially be applied to follow Clostridium spore behaviour in a range of different environments. Copyright © 2018 Elsevier Ltd. All rights reserved.

A Thiazole Coumarin (TC) Turn-On Fluorescence Probe for AT-Base Pair Detection and Multipurpose Applications in Different Biological Systems

NASA Astrophysics Data System (ADS)

Narayanaswamy, Nagarjun; Kumar, Manoj; Das, Sadhan; Sharma, Rahul; Samanta, Pralok K.; Pati, Swapan K.; Dhar, Suman K.; Kundu, Tapas K.; Govindaraju, T.

2014-09-01

Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based thiazole coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-activated cell sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology.

A Thiazole Coumarin (TC) Turn-On Fluorescence Probe for AT-Base Pair Detection and Multipurpose Applications in Different Biological Systems

PubMed Central

Narayanaswamy, Nagarjun; Kumar, Manoj; Das, Sadhan; Sharma, Rahul; Samanta, Pralok K.; Pati, Swapan K.; Dhar, Suman K.; Kundu, Tapas K.; Govindaraju, T.

2014-01-01

Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based thiazole coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-activated cell sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology. PMID:25252596

Resveratrol Improves Cell Cycle Arrest in Chronic Prostatitis Rats, by C-kit/SCF Suppression.

PubMed

He, Yi; Zeng, Huizhi; Yu, Yang; Zhang, Jiashu; Zeng, Xiaona; Gong, Fengtao; Liu, Qi; Yang, Bo

2017-08-01

Chronic prostatitis (CP) with complex pathogenesis is difficult for treatment. c-kit has been associated with the control of cell proliferation of prostate cells. This study aims to evaluate the role of resveratrol, an activator of Sirt1, in regulating the expression of c-kit in CP and investigate the consequent effects on cell cycle. Rat model of CP was established through subcutaneous injections of diphtheria-pertussis-tetanus vaccine and subsequently treated with resveratrol. Hematoxylin and eosin staining was performed to identify the histopathological changes in prostates. Western blotting and immunohistochemical staining examined the expression level of c-kit, stem cell factor (SCF), Sirt1, and cell cycle-associated proteins. The model group exhibited severe diffuse chronic inflammation, characterized by leukocyte infiltration and papillary frond protrusion into the gland cavities, and a notable increase in prostatic epithelial height. Gland lumen diameter was also significantly smaller; the activity of c-kit/SCF in the CP rats was increased significantly compared to the control group. Meanwhile, the cell cycle proteins are dysregulated significantly in CP rats. Resveratrol treatment significantly improved these factors by Sirt1 activation. Dysregulation of cell cycle was involved in the pathological processes of CP, which was improved after resveratrol treatment by the downregulation of c-kit/SCF by activating Sirt1.

Identification of Novel Targets of the Human Cell Cycle Regulatory Protein Cdc34

DTIC Science & Technology

1998-07-01

Bohman, for stains and plamids, J. La Baer for the cDNA library, C. Molina, H. P. Roest, J. Hoeijmaker, P. Sassone-Corsi for antisera, and to Shirong...mammalian CDC34 cell cycle gene. American Association of Cancer Research, 36, A191 (1995). Hershko, A., Role of ubiquitin-mediated proteolysis in cell

Anti-inflammatory and immunosuppressive effect of phloretin.

PubMed

Lu, Xiao-yu; Zeng, Yao-ying; Ye, Yan-xia; Zhou, Yu-ying; Mu, Jing-jing; Zhao, Xiao-hui

2009-05-01

This study investigated the effect of phloretin (Ph) on the proliferation, activation, and cell-cycle distribution of mouse T lymphocytes and NO production and phagocytosis of macrophages. Carboxyfluorescein diacetatesuccinimidyl ester (CFDA-SE) staining plus flow cytometry assay was employed to obtain the proliferation-related index (PI) of lymphocytes. The expression levels of CD69 and CD25 on T lymphocytes stimulated with Con A were evaluated with flow cytometry after staining with fluorescent monoclonal antibody. Cell-cycle distribution of T lymphocytes was analyzed by propidium iodide staining. Griess kit was used to evaluate the NO production and fluorescent microbeads were used to analyze the phagocytosis ability of macrophages. Our results showed that phloretin (40, 60, and 80 micromol x L(-7)) significantly inhibited the proliferation of T lymphocytes and the PI reduced from 1.41 +/- 0.13 to 1.34 +/- 0.16, 1.19 +/- 0.12 and 1.07 +/- 0.06, respectively. Phloretin significantly inhibited the expression of CD69 and CD25 (P < 0.01). The cell cycle distribution analysis showed that phloretin could induce a cell cycle arrest at G0/G1 phase. NO production of LPS +IFN-gamma group of macrophages was (26.72 +/- 3.57) micromol x L(-1), and was significantly reduced by phloretin (P < 0.01). And phagocytosis rate of macrophages was significantly reduced by phloretin (P < 0.01). The results demonstrate that phloretin might be developed into a new immuosuppressive drug.

In vitro effects of histone deacetylase inhibitors and mitomycin C on tenon capsule fibroblasts and conjunctival melanoma cells.

PubMed

Cunneen, Thomas S; Conway, R Max; Madigan, Michele C

2009-04-01

To investigate the effects of mitomycin C and the histone deacetylase inhibitors sodium butyrate and trichostatin on the viability and growth of conjunctival melanoma cell lines and Tenon capsule fibroblasts. Cells were treated with a range of concentrations of sodium butyrate, trichostatin, and mitomycin C. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assays were performed 48 hours after treatment. Treated cells were stained with acridine orange/ethidium bromide to assess for cell death. Cell-cycle changes in histone deacetylase inhibitor-treated melanoma cells were quantified using flow cytometry. All agents induced dose-dependent cell death in the melanoma cell lines; however, sodium butyrate and trichostatin were relatively nontoxic to Tenon capsule fibroblasts. Acridine orange/ethidium bromide staining indicated that sodium butyrate and trichostatin induced apoptotic cell death. At low doses, sodium butyrate and trichostatin induced a G1 cell-cycle block in the melanoma cells. Sodium butyrate and trichostatin induced cell death in melanoma cells, comparable with mitomycin C, with minimal effect on Tenon capsule fibroblasts. In addition, they induced a G1 cell-cycle block. These findings support the need for further investigation into the in vivo efficacy of these agents.

Phosphorylation of Smad2/3 at the specific linker threonine residue indicates slow-cycling esophageal stem-like cells before re-entry to the cell cycle.

PubMed

Takahashi, Y; Fukui, T; Kishimoto, M; Suzuki, R; Mitsuyama, T; Sumimoto, K; Okazaki, T; Sakao, M; Sakaguchi, Y; Yoshida, K; Uchida, K; Nishio, A; Matsuzaki, K; Okazaki, K

2016-01-01

The stem cell compartment in the esophageal epithelium is possibly located in the basal layer. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in the epithelial cells of murine stomach and intestine, and have suggested that these cells are epithelial stem cells. In this study, we explore whether pSmad2/3L-Thr could serve as a biomarker for esophageal stem cells. We examined esophageal tissues from normal C57BL/6 mice and those with esophagitis. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, CDK4, p63, or CK14 was performed. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. We used the 5-bromo-2-deoxyuridine (BrdU) labeling assay to examine label retention of pSmad2/3L-Thr immunostaining-positive cells. We collected specimens 5, 10, 15 and 20 days after repeated BrdU administrations and observed double immunofluorescent staining of pSmad2/3L-Thr with BrdU. In the esophagus, pSmad2/3L-Thr immunostaining-positive cells were detected in the basal layer. These cells were detected between Ki67 immunostaining-positive cells, but they were not co-localized with Ki67. pSmad2/3L-Thr immunostaining-positive cells showed co-localization with CDK4, p63, and CK14. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features. Until 20 days follow-up period, pSmad2/3L-Thr immunostaining-positive cells were co-localized with BrdU. pSmad2/3L-Thr immunostaining-positive cells significantly increased in the regeneration phase of esophagitis mucosae, as compared with control mice (esophagitis vs. 6.889 ± 0.676/cm vs. 4.293 ± 0.659/cm; P < 0.001). We have identified significant expression of pSmad2/3L-Thr in the specific epithelial cells of murine esophagi. We suggest that these cells are slow-cycling epithelial stem-like cells before re-entry to the cell cycle. © 2016 International Society for Diseases of the Esophagus.

Characterisation of the nucleolar organising regions during the cell cycle in two varieties of Petunia hybrida as visualised by fluorescence in situ hybridisation and silver staining.

PubMed

Montijn, M B; ten Hoopen, R; Fransz, P F; Oud, J L; Nanninga, N

1998-05-01

The cell cycle-dependent spatial position, morphology and activity of the four nucleolar organising regions (NORs) of the Petunia hybrida cultivar Mitchell and the inbred line V26 have been analysed. Application of the silver staining technique and fluorescence in situ hybridisation on fixed root-tip material revealed that these interspecific hybrids possess four NORs of which only those of chromosome 2 are active during interphase, which implies that the NOR activity is not of parental origin. However, at the end of mitosis, activity of all NOR regions could be detected, suggesting that the high demand for ribosomes at this stage of the cell cycle requires temporal activity of all NORs. Using actin DNA probes as markers in fluorescence in situ hybridisation experiments enabled the identification of the individual petunia chromosomes.

Factors influencing the abundance of the side population in a human myeloma cell line.

PubMed

Mo, Sui-Lin; Li, Jia; Loh, Yen S; Brown, Ross D; Smith, Adrian L; Chen, Yuling; Joshua, Douglas; Roufogalis, Basil D; Li, George Q; Fan, Kei; Ng, Michelle C H; Sze, Daniel Man-Yuen

2011-01-01

Side population (SP) refers to a group of cells, which is capable to efflux Hoechst 33342, a DNA-binding dye. SP cells exist both in normal and tumor tissues. Although SP abundance has been used as an indicator for disease prognostic and drug screening in many research projects, few studies have systematically examined the factors influencing SP analysis. In this study we aim to develop a more thorough understanding of the multiple factors involved in SP analysis including Hoechst 33342 staining and cell culture. RPMI-8226, a high SP percentage (SP%) human myeloma cell line was employed here. The results showed that SP% was subject to staining conditions including: viable cell proportion, dye concentration, staining cell density, incubation duration, staining volume, and mix interval. In addition, SP% was highest in day one after passage, while dropped steadily over time. This study shows that both staining conditions and culture duration can significantly affect SP%. In this case, any conclusions based on SP% should be interpreted cautiously. The relation between culture duration and SP% suggests that the incidence of SP cells may be related to cell proliferation and cell cycle phase. Maintaining these technical variables consistently is essential in SP research.

[The effect of Foxc2 overexpression on the osteogenic properties of C3H10T1/2 cells].

PubMed

Wang, Min-Jiao; Si, Jia-Wen; Li, Hong-Liang; Ouyang, Ning-Juan; Shen, Guo-Fang

2016-08-01

To investigate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation of C3H10T1/2 cells. C3H10T1/2 cells were transfected with plenti-Foxc2 and selected with puromycin for stable clones. The expression of Foxc2 was determined by real-time PCR and Western blot. Cell proliferation was detected by CCK-8 kit. Cell cycle and apoptosis were detected by flow cytometry. The level of osteogenic biomarkers Runx2, OPN, OCN and adipogenic biomarker PPARγ were quantified by real-time PCR and Western blot. Alkaline phosphatase (ALP) staining and oil red staining were conducted to evaluate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation. Statistical analysis was performed using SPSS 17.0 software package. C3H10T1/2-Foxc2 cell line was successfully constructed and verified by direct sequencing and Foxc2 overexpression in vitro. Cell proliferation was reduced and cell cycle was blocked in G1/G0 phase. Enhanced ALP staining and reduced oil red staining were observed in C3H10T1/2-Foxc2 cells as compared with the control. Foxc2 overexpression up-regulated Runx2, OPN, OCN during osteogenic differentiation and down-regulated PPARγduring adipogenic differentiation. C3H10T1/2 cell line stably expressing Foxc2 gene was successfully established, cell proliferation was reduced, osteogenesis biomarkers were up-regulated during the osteogenesis by overexpression Foxc2, PPARγwas down-regulated during adipogenesis.

A superoxide anion generator, pyrogallol, inhibits the growth of HeLa cells via cell cycle arrest and apoptosis.

PubMed

Kim, Sang Wook; Han, Yong Whan; Lee, Soo Teik; Jeong, Hey Jin; Kim, Seong Hun; Kim, In Hee; Lee, Seung Ok; Kim, Dae Ghon; Kim, Suhn Hee; Kim, Sung Zoo; Park, Woo Hyun

2008-02-01

We investigated the in vitro effects of pyrogallol on cell growth, cell cycle regulation, and apoptosis in HeLa cells. Pyrogallol inhibited the growth of HeLa cells with an IC(50) of approximately 45 microM. Pyrogallol induced arrest during all phases of the cell cycle and also very efficiently resulted in apoptosis in HeLa cells, as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay, and DAPI staining. This apoptotic process was accompanied by the loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bcl-2 decrease, caspase-3 activation, and PARP cleavage. Pan-caspase inhibitor (Z-VAD) could rescue some HeLa cells from pyrogallol-induced cell death, while caspase-8 and -9 inhibitors unexpectedly enhanced the apoptosis. When we examined the changes of the ROS, H(2)O(2) or O(2)(*-) in pyrogallol-treated cells, H(2)O(2) was slightly increased and O(2)(*-) significantly was increased. In addition, we detected a decreased GSH content in pyrogallol-treated cells. Only pan-caspase inhibitor showing recovery of GSH depletion and reduced intracellular O(2)(*-) level decreased PI staining in pyrogallol-treated HeLa cells, which indicates dead cells. In summary, we have demonstrated that pyrogallol as a generator of ROS, especially O(2) (*-), potently inhibited the growth of HeLa cells through arrests during all phases of the cell cycle and apoptosis. (c) 2007 Wiley-Liss, Inc.

Acetylcholinesterase in blood vessels of the guinea-pig ovary during different phases of the reproductive cycle.

PubMed

Silver, A

1977-05-01

The distribution of acetylcholinesterase-containing blood vessels in the ovary has been investigated histochemically during the reproductive cycle of the guinea-pig. Whole mounts as well as frozen sections have been studied. Stained vessels were found in the stroma throughout the oestrous cycle and pregnancy. In the corpus luteum the vascular reaction varied at different stages of the oestrous cycle; while never very pronounced, it was more marked in lactating than in non-lactating animals. A feregnancy advanced an increasing number of vessels were strongly stained. At the end of pregnancy, stained vessels were less prominent. Acetylcholinesterase appeared to be localized prinicipally in the vessels themselves (possibly in muscle cells) rather than in associated nerves. Experiments in which ovaries were injected, via their arterial or venous supply, with starch or coloured gelatine suggested that most stained vessels were arterioles but a reaction also occurred in some vessels which were probably arteries and others which could have been the postulated arterio-venous shunts. Capillaries were unstained; whether the veins were also totally unreactive could not be established. The significance of the changes in acetylcholinesterase staining in varying functional states remains obscure; they may or may not reflect the emergence of certain types of vessel at different stages.

DNA fragmentation and cell cycle arrest: a hallmark of apoptosis induced by crocin from kashmiri saffron in a human pancreatic cancer cell line.

PubMed

Bakshi, Hamid; Sam, Smitha; Rozati, Roya; Sultan, Phalisteen; Islam, Tajamul; Rathore, Babita; Lone, Zahoor; Sharma, Manik; Triphati, Jagrati; Saxena, Ramesh Chand

2010-01-01

Apoptosis, a widely important mechanism that contributes to cell growth reduction, is reported to be induced by Crocus sativus in different cancer types. The present study was designed to elucidate apoptosis induction by crocin, a main component of Crocus sativus in a human pancreatic cancer cell line (BxPC-3). Cell viability was measured by MTT assay, Hoechest33258 staining was used to detect the chromatin condensation characteristic of apoptosis, and DNA fragmentation was assessed by gel electrophoresis and cell cycle analysis by flow cytometry. Crocin induced apoptosis and G1-phase cell cycle arrest of BxPC-3 cells, while decreasing cell viability in a dose dependent and time dependent manner. Cells treated with 10μg/L crocin exhibited apoptotic morphology (brightly blue-fluorescent condensed nuclei on Hoechst 33258 staining) and reduction of volume. DNA analysis revealed typical ladders as early as 12 hours after treatment indicative of apoptosis. Our preclinical study demonstrated a pancreatic cancer cell line to be highly sensitive to crocin-mediated growth inhibition and apoptotic cell death. Although the molecular mechanisms of crocin action are not yet clearly understood, it appears to have potential as a therapeutic agent.

Overexpression of AQP3 Modifies the Cell Cycle and the Proliferation Rate of Mammalian Cells in Culture.

PubMed

Galán-Cobo, Ana; Ramírez-Lorca, Reposo; Serna, Ana; Echevarría, Miriam

2015-01-01

Abnormal AQP3 overexpression in tumor cells of different origins has been reported and a role for this enhanced AQP3 expression in cell proliferation and tumor processess has been indicated. To further understand the role AQP3 plays in cell proliferation we explore the effect that stable over expression of AQP3 produces over the proliferation rate and cell cycle of mammalian cells. The cell cycle was analyzed by flow cytometry with propidium iodide (PI) and the cell proliferation rate measured through cell counting and BrdU staining. Cells with overexpression of AQP3 (AQP3-o) showed higher proliferation rate and larger percentage of cells in phases S and G2/M, than wild type cells (wt). Evaluation of the cell response against arresting the cell cycle with Nocodazole showed that AQP3-o exhibited a less modified cell cycle pattern and lower Annexin V specific staining than wt, consistently with a higher resistance to apoptosis of AQP3-overexpressing cells. The cell volume and complexity were also larger in AQP3-o compared to wt cells. After transcriptomic analysis, RT-qPCR was performed to highlight key molecules implicated in cell proliferation which expression may be altered by overexpression of AQP3 and the comparative analysis between both type of cells showed significant changes in the expression of Zeb2, Jun, JunB, NF-kβ, Cxcl9, Cxcl10, TNF, and TNF receptors. We conclude that the role of AQP3 in cell proliferation seems to be connected to increments in the cell cycle turnover and changes in the expression levels of relevant genes for this process. Larger expression of AQP3 may confer to the cell a more tumor like phenotype and contributes to explain the presence of this protein in many different tumors.

Dynamics of tobacco DNA topoisomerases II in cell cycle regulation: to manage topological constrains during replication, transcription and mitotic chromosome condensation and segregation.

PubMed

Singh, Badri Nath; Achary, V Mohan Murali; Panditi, Varakumar; Sopory, Sudhir K; Reddy, Malireddy K

2017-08-01

The topoisomerase II expression varies as a function of cell proliferation. Maximal topoisomerase II expression was tightly coupled to S phase and G2/M phase via both transcriptional and post-transcriptional regulation. Investigation in meiosis using pollen mother cells also revealed that it is not the major component of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed. Synchronized tobacco BY-2 cell cultures were used to study the role of topoisomerase II in various stages of the cell cycle. Topoisomerase II transcript accumulation was observed during the S- and G2/M- phase of cell cycle. This biphasic expression pattern indicates the active requirement of topoisomerase II during these stages of the cell cycle. Through immuno-localization of topoisomerase II was observed diffusely throughout the nucleoplasm in interphase nuclei, whereas, the nucleolus region exhibited a more prominent immuno-positive staining that correlated with rRNA transcription, as shown by propidium iodide staining and BrUTP incorporation. The immuno-staining analysis also showed that topoisomerase II is the major component of mitotic chromosomes and remain attached to the chromosomes during cell division. The inhibition of topoisomerase II activity using specific inhibitors revealed quite dramatic effect on condensation of chromatin and chromosome individualization from prophase to metaphase transition. Partially condensed chromosomes were not arranged on metaphase plate and chromosomal perturbations were observed when advance to anaphase, suggesting the importance of topoisomerase II activity for proper chromosome condensation and segregation during mitosis. Contrary, topoisomerase II is not the major component of meiotic chromosomes, even though mitosis and meiosis share many processes, including the DNA replication, chromosome condensation and precisely regulated partitioning of chromosomes into daughter cells. Even if topoisomerase II is required for individualization and condensation of meiotic chromosomes, it seems to diffuse out once meiotic chromosomal condensation is completed.

Phosphorylation of Smad2/3 at specific linker threonine indicates slow-cycling intestinal stem-like cells before reentry to cell cycle.

PubMed

Kishimoto, Masanobu; Fukui, Toshiro; Suzuki, Ryo; Takahashi, Yu; Sumimoto, Kimi; Okazaki, Takashi; Sakao, Masayuki; Sakaguchi, Yutaku; Yoshida, Katsunori; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

2015-02-01

Quiescent (slow-cycling) and active (rapid-cycling) stem cells are demonstrated in small intestines. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in murine stomach, and suggested these cells are epithelial stem cells. Here, we explore whether pSmad2/3L-Thr could serve as a biomarker for small intestine and colon stem cells. We examined small intestines and colons from C57BL/6 mice and colons with dextran sulfate sodium (DSS)-induced colitis. We performed double-immunofluorescent staining of pSmad2/3L-Thr with Ki67, cytokeratin 8, chromogranin A, CDK4, DCAMKL1, and Musashi-1. Small intestines and colons from Lgr5-EGFP knock-in mice were examined by pSmad2/3L-Thr immunofluorescent staining. To examine BrdU label retention of pSmad2/3L-Thr immunostaining-positive cells, we collected specimens after BrdU administration and observed double-immunofluorescent staining of pSmad2/3L-Thr with BrdU. In small intestines and colons, pSmad2/3L-Thr immunostaining-strongly positive cells were detected around crypt bases. Immunohistochemical co-localization of pSmad2/3L-Thr with Ki67 was not observed. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with cytokeratin 8, CDK4, and Musashi-1 and different localization from chromogranin A and DCAMKL1 immunostaining-positive cells. Under a light microscope, pSmad2/3L-Thr immunostaining-strongly positive cells were morphologically undifferentiated. In Lgr5-EGFP knock-in mice, some but not all pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with Lgr5. pSmad2/3L-Thr immunostaining-strongly positive cells showed co-localization with BrdU at 5, 10, and 15 days after administration. In DSS-induced colitis, pSmad2/3L-Thr and Ki67 immunostaining-positive cells increased in the regeneration phase and decreased in the injury phase. In murine small intestines and colons, we suggest pSmad2/3L-Thr immunostaining-strongly positive cells are epithelial stem-like cells just before reentry to the cell cycle.

Electronic waste leachate-mediated DNA fragmentation and cell death by apoptosis in mouse fibroblast (NIH/3T3) cell line.

PubMed

Alabi, Okunola A; Bakare, Adekunle A; Filippin-Monteiro, Fabíola B; Sierra, Jelver A; Creczynski-Pasa, Tânia B

2013-08-01

This study investigated the apoptotic effect of electronic waste on fibroblast cell line. Cells were treated with different concentrations of the leachate for 24h. Cell viability was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, nuclear morphology of cells was explored by acridine orange (AO)/ethidium bromide (EB) double staining, mitochondrial membrane potential was evaluated using JC-1 probe while cell cycle analysis was conducted using flow cytometry. The oxidative status was detected using DCFH-DA (dichlorofluorescin diacetate) probe and the relationship between cell death and ROS (reactive oxygen species) was investigated using N-acetylcysteine. Results showed an increased cell death as detected by MTT assay and AO/EB staining. Cell cycle analysis indicated an induction of sub/G1 events while JC-1 probe showed significant disruption of mitochondrial membrane potential. There was significant induction of ROS, while N-acetylcysteine protected the cells from DNA damage. These suggest apoptotic pathway as a possible mechanism of e-waste induced cyto-genotoxicity. Copyright © 2013 Elsevier Inc. All rights reserved.

Juglans mandshurica Maxim extracts exhibit antitumor activity on HeLa cells in vitro.

PubMed

Xin, Nian; Hasan, Murtaza; Li, Wei; Li, Yan

2014-04-01

The present study examined the potential application of Juglans mandshurica Maxim extracts (HT) for cancer therapy by assessing their anti‑proliferative activity, reduction of telomerase activity, induction of apoptosis and cell cycle arrest in S phase in HeLa cells. From the perspective of using HT as a herbal medicine, photomicroscopy and florescent microscopy techniques were utilized to characterize the effect of the extracts on telomerase activity and cell morphology. Flow cytometry was employed to study apoptosis and cell cycle of HeLa cells, and DNA laddering was performed. The results showed that HT inhibited cell proliferation and telomerase activity, induced apoptosis and caused S phase arrest of HeLa cells in vitro. HT inhibited HeLa cell proliferation significantly, and the highest inhibition rate was 83.7%. A trap‑silver staining assay showed that HT was capable of markedly decreasing telomerase activity of HeLa cells and this inhibition was enhanced in a time‑ and dose‑dependent manner. Results of a Hoechst 33258 staining assay showed that HeLa cells treated by HT induced cell death. Through DNA agarose gel electrophoresis, DNA ladders of HeLa cells treated with HT were observed, indicating apoptosis. In conclusion, the present study demonstrated that HT exhibited anti‑tumor effects comprising the inhibition of growth and telomerase activity as well as apoptosis and cell cycle arrest in HeLa cells.

Combinatorial Study of a Novel Poly (ADP-ribose) Polymerase Inhibitor and an HDAC Inhibitor, SAHA, in Leukemic Cell Lines.

PubMed

Hegde, Mahesh; Mantelingu, Kempegowda; Pandey, Monica; Pavankumar, Chottanahalli S; Rangappa, Kanchugarakoppal S; Raghavan, Sathees C

2016-10-01

Cancer is a multifactorial disease, which makes it difficult to cure. Since more than one defective cellular component is often involved during oncogenesis, combination therapy is gaining prominence in the field of cancer therapeutics. The purpose of this study was to investigate the combinatorial effects of a novel PARP inhibitor, P10, and HDAC inhibitor, SAHA, in leukemic cells. Combinatorial effects of P10 and SAHA were tested using propidium iodide staining in different leukemic cells. Further, flowcytometry-based assays such as calcein-AM/ethidium homodimer staining, annexin-FITC/PI staining, and JC-1 staining were carried out to elucidate the mechanism of cell death. In addition, cell-cycle analysis, immunocytochemistry studies, and western blotting analysis were conducted to check the combinatorial effect in Nalm6 cells. Propidium iodide staining showed that P10 in combination with SAHA induced cell death in Nalm6 cells, in which PARP expression and activity is high with a combination index of <0.2. Annexin-FITC/PI staining, JC-1 staining, and other biochemical assays revealed that P10 in combination with SAHA induced apoptosis by causing a change in mitochondrial membrane potential in >65 % cells. Importantly, combinatorial treatment induced S phase arrest in 40-45 % cells due to DNA damage and plausible replicative stress. Finally, we demonstrated that treatment with P10 led to DNA strand breaks, which were further potentiated by SAHA (p < 0.01), leading to activation of apoptosis and increased cell death in PARP-positive leukemic cells. Our study reveals that coadministration of PARP inhibitor with SAHA could be used as a combination therapy against leukemic cells that possess high levels of intrinsic PARP activity.

Enzymes of the γ-Glutamyl Cycle in the Ciliary Body and Lens

PubMed Central

Ross, Leonard L.; Barber, Lee; Tate, Suresh S.; Meister, Alton

1973-01-01

The enzymes of the γ-glutamyl cycle have been found in rabbit ciliary body and, except for 5-oxoprolinase, also in the ocular lens. Histochemical studies show that γ-glutamyl transpeptidase is localized mainly in the basal portions of the epithelial cells of the ciliary body; the findings are similar to those observed in the chloroid plexuses. The histochemical staining reaction in the ciliary epithelium is more intense than in the chloroid plexus, intestine, and kidney. γ-Glutamyl transpeptidase staining activity in the epithelium of the intestinal and renal proximal convoluted tubules is confined to the microvillus border. Moderate transpeptidase activity was found in the cytoplasm of nonpigmented epithelial cells of the iris at the posterior pupillary margin. The histochemical and enzyme activity studies are consistent with the thesis that the γ-glutamyl cycle functions in transport of amino acids across the blood-aqueous humor barrier. Images PMID:4152058

Seleno-short-chain chitosan induces apoptosis in human non-small-cell lung cancer A549 cells through ROS-mediated mitochondrial pathway.

PubMed

Zhao, Yana; Zhang, Shaojing; Wang, Pengfei; Fu, Shengnan; Wu, Di; Liu, Anjun

2017-12-01

Seleno-short-chain chitosan (SSCC) is a synthesized chitosan derivative. In this study, antitumor activity and underlying mechanism of SSCC on human non-small-cell lung cancer A549 cells were investigated in vitro. The MTT assay showed that SSCC could inhibit cell viability in a dose- and time-dependent manner, and 200 μg/ml SSCC exhibited significantly toxic effects on A549 cells. The cell cycle assay showed that SSCC triggered S phase cell cycle arrest in a dose- and time-dependent manner, which was related to a downregulation of S phase associated cyclin A. The DAPI staining and Annexin V-FITC/PI double staining identified that the SSCC could induce A549 cells apoptosis. Further studies found that SSCC led to the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) by DCFH-DA and Rhodamin 123 staining, respectively. Meanwhile, free radical scavengers N-acetyl-L-cysteine (NAC) pretreatment confirmed that SSCC-induced A549 cells apoptosis was associated with ROS generation. Furthermore, real-time PCR and western blot assay showed that SSCC up-regulated Bax and down-regulated Bcl-2, subsequently incited the release of cytochrome c from mitochondria to cytoplasm, activated the increase of cleaved-caspase 3 and finally induced A549 cells apoptosis in vitro. In general, the present study demonstrated that SSCC induced A549 cells apoptosis via ROS-mediated mitochondrial apoptosis pathway.

Expression of aquaporin-7 and aquaporin-9 in tanycyte cells and choroid plexus during mouse estrus cycle.

PubMed

Yaba, A; Sozen, B; Suzen, B; Demir, N

2017-03-01

Tanycytes are special ependymal cells located in the ventrolateral wall and floor of the third ventricle having processes extending nuclei that regulate reproductive functions and around of vessels in median eminance. The aquaporins (AQPs) are a family of transmembrane proteins that transport water and glycerol. AQP-7 and -9 are permeable to other small molecules as glycerol and therefore called aquaglyceroporins. In this study, we aimed to show localization of AQP-7 and -9 in epithelial cells of choroid plexus and tanycytes during female mouse estrus cycle. AQP-7 and -9 proteins were detected in α2 and β1 tanycytes in prœstrus stage. Interestingly, there is no staining in estrus stage in any type of tanycytes. We observed weak immunoreactivity in α1, α2 and β1 tanycyte cells in metestrus stage for AQP-7 and α1 for AQP-9 protein. AQP-7 and -9 showed intense immunoreactivity in α2, β1 and β2 tanycyte cells during diestrus stage. Consequently, AQP-7 and -9 showed differential staining pattern in different stages of mouse estrus cycle. In the light of our findings and other recent publications, we suggest that AQP-7 and -9-mediated glycerol transport in tanycyte cells might be under hormonal control to use glycerol as a potential energy substrate during mouse estrus cycle. Copyright © 2016. Published by Elsevier Masson SAS.

Andrographolide potentiates the antitumor effect of topotecan in acute myeloid leukemia cells through an intrinsic apoptotic pathway.

PubMed

Hodroj, Mohammad Hassan; Jardaly, Achraf; Abi Raad, Sarah; Zouein, Annalise; Rizk, Sandra

2018-01-01

Topotecan (TP) is an anticancer drug acting as topoisomerase I inhibitor that is used in the treatment of many types of cancers including leukemia, but it has significant side effects. Andrographolide, a compound extracted from Andrographis paniculata , was recently proven to inhibit the growth of cancer cells and can induce apoptosis. The aim of this study is to investigate the possible synergism between TP and andrographolide in acute myeloid cells in vitro. U937 acute myeloid leukemic cells were cultured using Roswell Park Memorial Institute (RPMI) medium and then treated for 24 h with TP and andrographolide prepared through the dilution of dimethyl sulfoxide (DMSO) stocks with RPMI on the day of treatment. Cell proliferation was assessed using cell proliferation assay upon treatment with both compounds separately and in combination. Cell-cycle study and apoptosis detection were performed by staining the cells with propidium iodide (PI) stain and Annexin V/PI stain, respectively, followed by flow cytometry analysis. Western blotting was used to assess the expression of various proteins involved in apoptotic pathways. Both TP and andrographolide showed an antiproliferative effect in a dose-dependent manner when applied on U937 cells separately; however, pretreating the cells with andrographolide before applying TP exhibited a synergistic effect with lower inhibitory concentrations (half-maximal inhibitory concentration). Treating the cells with TP alone led to specific cell-cycle arrest at S phase that was more prominent upon pretreatment combination with andrographolide. Using Annexin V/PI staining to assess the proapoptotic effect following the pretreatment combination showed an increase in the number of apoptotic cells, which was supported by the Western blot results that manifested an upregulation of several proapoptotic proteins expression. The pretreatment of U937 with andrographolide followed by low doses of TP showed an enhancement in inducing apoptosis when compared to the application of each compound separately.

Andrographolide potentiates the antitumor effect of topotecan in acute myeloid leukemia cells through an intrinsic apoptotic pathway

PubMed Central

Hodroj, Mohammad Hassan; Jardaly, Achraf; Abi Raad, Sarah; Zouein, Annalise; Rizk, Sandra

2018-01-01

Background Topotecan (TP) is an anticancer drug acting as topoisomerase I inhibitor that is used in the treatment of many types of cancers including leukemia, but it has significant side effects. Andrographolide, a compound extracted from Andrographis paniculata, was recently proven to inhibit the growth of cancer cells and can induce apoptosis. The aim of this study is to investigate the possible synergism between TP and andrographolide in acute myeloid cells in vitro. Materials and methods U937 acute myeloid leukemic cells were cultured using Roswell Park Memorial Institute (RPMI) medium and then treated for 24 h with TP and andrographolide prepared through the dilution of dimethyl sulfoxide (DMSO) stocks with RPMI on the day of treatment. Cell proliferation was assessed using cell proliferation assay upon treatment with both compounds separately and in combination. Cell-cycle study and apoptosis detection were performed by staining the cells with propidium iodide (PI) stain and Annexin V/PI stain, respectively, followed by flow cytometry analysis. Western blotting was used to assess the expression of various proteins involved in apoptotic pathways. Results Both TP and andrographolide showed an antiproliferative effect in a dose-dependent manner when applied on U937 cells separately; however, pretreating the cells with andrographolide before applying TP exhibited a synergistic effect with lower inhibitory concentrations (half-maximal inhibitory concentration). Treating the cells with TP alone led to specific cell-cycle arrest at S phase that was more prominent upon pretreatment combination with andrographolide. Using Annexin V/PI staining to assess the proapoptotic effect following the pretreatment combination showed an increase in the number of apoptotic cells, which was supported by the Western blot results that manifested an upregulation of several proapoptotic proteins expression. Conclusion The pretreatment of U937 with andrographolide followed by low doses of TP showed an enhancement in inducing apoptosis when compared to the application of each compound separately. PMID:29785137

Flow cytomeric measurement of DNA and incorporated nucleoside analogs

DOEpatents

Dolbeare, Frank A.; Gray, Joe W.

1989-01-01

A method is provided for simultaneously measuring total cellular DNA and incorporated nucleoside analog. The method entails altering the cellular DNA of cells grown in the presence of a nucleoside analog so that single stranded and double stranded portions are present. Separate stains are used against the two portions. An immunochemical stain is used against the single stranded portion to provide a measure of incorporated nucleoside analog, and a double strand DNA-specific stain is used against the double stranded portion to simultaneously provide a measure of total cellular DNA. The method permits rapid flow cytometric analysis of cell populations, rapid identification of cycling and noncycling subpopulations, and determination of the efficacy of S phase cytotoxic anticancer agents.

13-cis Retinoic acid induces apoptosis and cell cycle arrest in human SEB-1 sebocytes.

PubMed

Nelson, Amanda M; Gilliland, Kathryn L; Cong, Zhaoyuan; Thiboutot, Diane M

2006-10-01

Isotretinoin (13-cis retinoic acid (13-cis RA)) is the most potent inhibitor of sebum production, a key component in the pathophysiology of acne, yet its mechanism of action remains largely unknown. The effects of 13-cis RA, 9-cis retinoic acid (9-cis RA), and all-trans retinoic acid (ATRA) on cell proliferation, apoptosis, and cell cycle proteins were examined in SEB-1 sebocytes and keratinocytes. 13-cis RA causes significant dose-dependent and time-dependent decreases in viable SEB-1 sebocytes. A portion of this decrease can be attributed to cell cycle arrest as evidenced by decreased DNA synthesis, increased p21 protein expression, and decreased cyclin D1. Although not previously demonstrated in sebocytes, we report that 13-cis RA induces apoptosis in SEB-1 sebocytes as shown by increased Annexin V-FITC staining, increased TUNEL staining, and increased cleaved caspase 3 protein. Furthermore, the ability of 13-cis RA to induce apoptosis cannot be recapitulated by 9-cis RA or ATRA, and it is not inhibited by the presence of a retinoid acid receptor (RAR) pan-antagonist AGN 193109. Taken together these data indicate that 13-cis RA causes cell cycle arrest and induces apoptosis in SEB-1 sebocytes by a RAR-independent mechanism, which contributes to its sebosuppressive effect and the resolution of acne.

Flow cytometric cell cycle analysis of muscle precursor cells cultured within 3D scaffolds in a perfusion bioreactor.

PubMed

Flaibani, Marina; Luni, Camilla; Sbalchiero, Elisa; Elvassore, Nicola

2009-01-01

It has been widely demonstrated that perfusion bioreactors improve in vitro three-dimensional (3D) cultures in terms of high cell density and uniformity of cell distribution; however, the studies reported in literature were primarily based on qualitative analysis (histology, immunofluorescent staining) or on quantitative data averaged on the whole population (DNA assay, PCR). Studies on the behavior, in terms of cell cycle, of a cell population growing in 3D scaffolds in static or dynamic conditions are still absent. In this work, a perfusion bioreactor suitable to culture C(2)C(12) muscle precursor cells within 3D porous collagen scaffolds was designed and developed and a method based on flowcytometric analyses for analyzing the cell cycle in the cell population was established. Cells were extracted by enzymatic digestion of the collagen scaffolds after 4, 7, and 10 days of culture, and flow cytometric live/dead and cell cycle analyses were performed with Propidium Iodide. A live/dead assay was used for validating the method for cell extraction and staining. Moreover, to investigate spatial heterogeneity of the cell population under perfusion conditions, two stacked scaffolds in the 3D domain, of which only the upstream layer was seeded, were analyzed separately. All results were compared with those obtained from static 3D cultures. The live/dead assay revealed the presence of less than 20% of dead cells, which did not affect the cell cycle analysis. Cell cycle analyses highlighted the increment of cell fractions in proliferating phases (S/G(2)/M) owing to medium perfusion in long-term cultures. After 7-10 days, the percentage of proliferating cells was 8-12% for dynamic cultures and 3-5% for the static controls. A higher fraction of proliferating cells was detected in the downstream scaffold. From a general perspective, this method provided data with a small standard deviation and detected the differences between static and dynamic cultures and between upper and lower scaffolds. Our methodology can be extended to other cell types to investigate the influence of 3D culture conditions on the expression of other relevant cell markers.

Involvement of cell proliferation in the process of follicular atresia in the guinea pig.

PubMed

Wang, Wei; Liu, Honglin; Ding, Wei; Gong, Yan; Chen, Jingwei; Hutz, Reinhold J; Mao, Dagan; Shi, Fangxiong

2010-08-01

Cell morphology and proliferation was investigated in the atretic follicles during estrous cycles in the guinea pig. Ovarian samples on days 1, 4, 8, 12 and 16 of the estrous cycle in the guinea pig were taken in the morning for histologic staining with hematoxylin and eosin (HE), and immunohistochemical staining of the protein proliferating cell nuclear antigen (PCNA). The results indicated that the granulosa cells degenerated and eliminated first in atretic follicles, while the fibroblast-like cells appeared in the innermost layer of theca interna cells. When the fibroblast-like cells migrated to the antrum, they proliferated and formed a new tissue in peripheral to the zona pellucida of the oocyte. Our results also revealed that the orientation of the theca interna cell arrangement changed twice during the process of atresia, and the loose connective tissue in the antrum was critical for follicular atresia. Therefore, follicular atresia was not a simple process of cell death and elimination, but coexisted with cell proliferation. To our knowledge, we have for the first time confirmed cell proliferation and the presence of new tissue in atretic follicles in guinea pigs. Copyright 2010 Elsevier Ltd. All rights reserved.

Geraniol and geranyl acetate induce potent anticancer effects in colon cancer Colo-205 cells by inducing apoptosis, DNA damage and cell cycle arrest.

PubMed

Qi, Fei; Yan, Qiang; Zheng, Zhaozheng; Liu, Jian; Chen, Yan; Zhang, Guiyang

2018-01-01

Colon cancer ranks second in mortality among all human malignancies, creating thus a need for exploration of novel molecules that would prove effective, cost-effective and with lower toxicity. In the recent past monoterpenes have gained tremendous attention for their anticancer activity. In the present study we evaluated the anticancer effects of two important monoterpenes, geraniol and geranyl acetate against colo-205 cancer cells. The antiproliferative activity was determined by MTT assay. Apoptosis was assessed by DAPI staining and DNA damage was checked by comet assay. The cell cycle analysis was carried out by flow cytometry and protein expression was examined by western blotting. The results showed that both geraniol and geranyl acetate exhibited significant anticancer activity against colo-205 cancer cell line with IC50 values of 20 and 30 μM respectively. To find out the underlying mechanism, DAPI staining was carried out and it was observed that both the monoterpenes, geraniol and geranyl acetate, induced apoptosis in colo-205 cells. The apoptosis was also associated with upregulation of Bax and downregulation of Bcl-2 expressions, indicative of mitochondrial apoptosis. Moreover, these two monoterpenes could trigger DNA damage and G2/M cell cycle arrest in colo-205 cells. Taken together, we propose that geraniol and geranyl acetate may prove to be important lead molecular candidates for the treatment of colon cancer. Their anticancer activity can be attributed to the ability to trigger apoptosis, DNA damage and cell cycle arrest.

Berberine displays antitumor activity in esophageal cancer cells in vitro.

PubMed

Jiang, Shu-Xian; Qi, Bo; Yao, Wen-Jian; Gu, Cheng-Wei; Wei, Xiu-Feng; Zhao, Yi; Liu, Yu-Zhen; Zhao, Bao-Sheng

2017-04-14

To investigate the effects of berberine on esophageal cancer (EC) cells and its molecular mechanisms. Human esophageal squamous cell carcinoma cell line KYSE-70 and esophageal adenocarcinoma cell line SKGT4 were used. The effects of berberine on cell proliferation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For cell cycle progression, KYSE-70 cells were stained with propidium iodide (PI) staining buffer (10 mg/mL PI and 100 mg/mL RNase A) for 30 min and cell cycle was analyzed using a BD FACSCalibur flow cytometer. For apoptosis assay, cells were stained with an Annexin V-FITC/PI apoptosis detection kit. The rate of apoptotic cells was analyzed using a dual laser flow cytometer and estimated using BD ModFit software. Levels of proteins related to cell cycle and apoptosis were examined by western blotting. Berberine treatment resulted in growth inhibition of KYSE-70 and SKGT4 cells in a dose-dependent and time-dependent manner. KYSE-70 cells were more susceptible to the inhibitory activities of berberine than SKGT4 cells were. In KYSE-70 cells treated with 50 μmol/L berberine for 48 h, the number of cells in G 2 /M phase (25.94% ± 5.01%) was significantly higher than that in the control group (9.77% ± 1.28%, P < 0.01), and berberine treatment resulted in p21 up-regulation in KYSE-70 cells. Flow cytometric analyses showed that berberine significantly augmented the KYSE-70 apoptotic population at 12 and 24 h post-treatment, when compared with control cells (0.83% vs 43.78% at 12 h, P < 0.05; 0.15% vs 81.86% at 24 h, P < 0.01), and berberine-induced apoptotic effect was stronger at 24 h compared with 12 h. Western blotting showed that berberine inhibited the phosphorylation of Akt, mammalian target of rapamycin and p70S6K, and enhanced AMP-activated protein kinase phosphorylation in a sustained manner. Berberine is an inhibitor of human EC cell growth and could be considered as a potential drug for the treatment of EC patients.

Berberine displays antitumor activity in esophageal cancer cells in vitro

PubMed Central

Jiang, Shu-Xian; Qi, Bo; Yao, Wen-Jian; Gu, Cheng-Wei; Wei, Xiu-Feng; Zhao, Yi; Liu, Yu-Zhen; Zhao, Bao-Sheng

2017-01-01

AIM To investigate the effects of berberine on esophageal cancer (EC) cells and its molecular mechanisms. METHODS Human esophageal squamous cell carcinoma cell line KYSE-70 and esophageal adenocarcinoma cell line SKGT4 were used. The effects of berberine on cell proliferation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For cell cycle progression, KYSE-70 cells were stained with propidium iodide (PI) staining buffer (10 mg/mL PI and 100 mg/mL RNase A) for 30 min and cell cycle was analyzed using a BD FACSCalibur flow cytometer. For apoptosis assay, cells were stained with an Annexin V-FITC/PI apoptosis detection kit. The rate of apoptotic cells was analyzed using a dual laser flow cytometer and estimated using BD ModFit software. Levels of proteins related to cell cycle and apoptosis were examined by western blotting. RESULTS Berberine treatment resulted in growth inhibition of KYSE-70 and SKGT4 cells in a dose-dependent and time-dependent manner. KYSE-70 cells were more susceptible to the inhibitory activities of berberine than SKGT4 cells were. In KYSE-70 cells treated with 50 μmol/L berberine for 48 h, the number of cells in G2/M phase (25.94% ± 5.01%) was significantly higher than that in the control group (9.77% ± 1.28%, P < 0.01), and berberine treatment resulted in p21 up-regulation in KYSE-70 cells. Flow cytometric analyses showed that berberine significantly augmented the KYSE-70 apoptotic population at 12 and 24 h post-treatment, when compared with control cells (0.83% vs 43.78% at 12 h, P < 0.05; 0.15% vs 81.86% at 24 h, P < 0.01), and berberine-induced apoptotic effect was stronger at 24 h compared with 12 h. Western blotting showed that berberine inhibited the phosphorylation of Akt, mammalian target of rapamycin and p70S6K, and enhanced AMP-activated protein kinase phosphorylation in a sustained manner. CONCLUSION Berberine is an inhibitor of human EC cell growth and could be considered as a potential drug for the treatment of EC patients. PMID:28465635

Anticancer activity of 7-epiclusianone, a benzophenone from Garcinia brasiliensis, in glioblastoma.

PubMed

Sales, Leilane; Pezuk, Julia Alejandra; Borges, Kleiton Silva; Brassesco, María Sol; Scrideli, Carlos Alberto; Tone, Luiz Gonzaga; dos Santos, Marcelo Henrique; Ionta, Marisa; de Oliveira, Jaqueline Carvalho

2015-10-30

Glioblastoma is the most common tumor of the central nervous system and one of the hardest tumors to treat. Consequently, the search for novel therapeutic options is imperative. 7-epiclusianone, a tetraprenylated benzophenone isolated from the epicarp of the native plant Garcinia brasiliensis, exhibits a range of biological activities but its prospect anticancer activity is underexplored. Thus, the aim of the present study was to evaluate the influence of 7-epiclusianone on proliferation, clonogenic capacity, cell cycle progression and induction of apoptosis in two glioblastoma cell lines (U251MG and U138MG). Cell viability was measured by the MTS assay; for the clonogenic assay, colonies were stained with Giemsa and counted by direct visual inspection; For cell cycle analysis, cells were stained with propidium iodide and analyzed by cytometry; Cyclin A expression was determined by immunoblotting; Apoptotic cell death was determined by annexin V fluorescein isothiocyanate labeling and Caspase-3 activity in living cells. Viability of both cell lines was drastically inhibited; moreover, the colony formation capacity was significantly reduced, demonstrating long-term effects even after removal of the drug. 7-epiclusianone treatment at low concentrations also altered cell cycle progression, decreased the S and G2/M populations and at higher concentrations increased the number of cells at sub-G1, in concordance with the increase of apoptotic cells. The present study demonstrates for the first time the anticancer potential of 7-epiclusianone against glioblastoma cells, thus meriting its further investigation as a potential therapeutic agent.

Avenanthramide-C reduces the viability of MDA-MB-231 breast cancer cells through an apoptotic mechanism.

PubMed

Hastings, Jordan; Kenealey, Jason

2017-01-01

Avenanthramides (AVN) are a relatively unstudied family of phytochemicals that could be novel chemotherapeutics. These compounds, found in oats, are non-toxic to healthy cells and have been shown to reduce viability of human colon and liver cancers in vitro. However, these studies do not elucidate a molecular mechanism for individual AVN. In this study we aim to see the effects of AVN on MDA-MB-231 breast cancer cells. An MTT assay was used to determine cell viability. Staining and analysis with a flow cytometer was used to identify cell cycle progression and apoptosis. FloJo software was used to analyze the cytometric data. In all experiments, statistical significance was determined by a two-tailed t test. This study demonstrates that AVN-A, B, and C individually reduce viability in the MDA-MB-231 breast cancer cell line. AVN-C has the most potent decrease in tumor cell viability, decreasing viable cells to below 25% at 400 µM when compared to control after 96 h. We demonstrate that treatment with AVN-C causes DNA fragmentation and accumulation of over 90% of cells into a sub G 1 cell cycle population. Further, we conclude that AVN-C treated cells activate apoptosis because 97% of treated cells stain positive for annexin V while 91% have caspase-3/7 activity, a late marker of apoptosis. Breast cancer cells treated with AVN-C have a decrease in cell viability, an increase in the sub G 1 population, and stain positive for both annexin V and caspase activity, indicating that AVN-C induces apoptosis in breast cancer cells. These compounds may be able to act as chemotherapeutics as demonstrated through future in vivo studies.

Effects of SOV-induced phosphatase inhibition and expression of protein tyrosine phosphatases in rat corneal endothelial cells.

PubMed

Chen, Wei-Li; Harris, Deshea L; Joyce, Nancy C

2005-11-01

Contact inhibition is an important mechanism for maintaining corneal endothelium in a non-replicative state. Protein tyrosine phosphatases (PTPs) play a role in regulating the integrity of cell-cell contacts, differentiation, and growth. In this study, we aimed to evaluate whether phosphatases are involved in the maintenance of contact-dependent inhibition of proliferation in corneal endothelial cells and to identify candidate PTPs that are expressed in these cells and might be involved in regulation of contact inhibition. Confluent cultures of rat corneal endothelial cells or endothelium in ex vivo corneas were treated with the general phosphatase inhibitor, sodium orthovanadate (SOV). Immunocytochemistry (ICC) evaluated the effect of SOV on cell-cell contacts by staining for ZO-1, and on cell cycle progression by staining for Ki67. Transverse sections of rat cornea and cultured rat corneal endothelial cells were used to test for expression of the candidate PTPs: PTP-mu, PTP-LAR, PTP1B, SHP-1, SHP-2, and PTEN using ICC and either Western blots or RT-PCR. ZO-1 staining demonstrated that SOV induced a time-dependent release of cell-cell contacts in confluent cultures of corneal endothelial cells and in the endothelium of ex vivo corneas. Staining for Ki67 indicated that SOV promoted limited cell cycle progression in the absence of serum. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN, but not PTP-LAR, were expressed in rat corneal endothelial cells in situ and in culture. The subcellular location of PTP-mu and PTP1B differed in subconfluent and confluent cells, while that of SHP-1, SHP-2, and PTEN was similar, regardless of confluent status. Western blots confirmed the expression of PTP1B, SHP-1, SHP-2, and PTEN. RT-PCR confirmed expression of PTP-mu mRNA. Phosphatases are involved in regulation of junctional integrity and of cell proliferation in corneal endothelial cells. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN are expressed in rat corneal endothelium and may be involved in regulation of contact inhibition in these normally non-proliferating cells.

Sensitization of breast cancer cells to doxorubicin via stable cell line generation and overexpression of DFF40.

PubMed

Bagheri, Fatemeh; Safarian, Shahrokh; Eslaminejad, Mohamadreza Baghaban; Sheibani, Nader

2015-12-01

There are a number of reports demonstrating a relationship between the alterations in DFF40 expression and development of some cancers. Here, increased DFF40 expression in T-47D cells in the presence of doxorubicin was envisaged for therapeutic usage. The T-47D cells were transfected with an eukaryotic expression vector encoding the DFF40 cDNA. Following incubation with doxorubicin, propidium iodide (PI) staining was used for cell cycle distribution analysis. The rates of apoptosis were determined by annexin V/PI staining. Apoptosis was also evaluated using the DNA laddering analysis. The viability of DFF40-transfected cells incubated with doxorubicin was significantly decreased compared with control cells. However, there were no substantial changes in the cell cycle distribution of pIRES2-DFF40 cells incubated with doxorubicin compared to control cells. The expression of DFF40, without doxorubicin incubation, had also no significant effect on the cell cycle distribution. There was no DNA laddering in cells transfected with the empty pIRES2 vector when incubated with doxorubicin. In contrast, DNA laddering was observed in DFF40 transfected cells in the presence of doxorubicin after 48 h. Also, the expression of DFF40 and DFF45 was increased in DFF40 transfected cells in the presence of doxorubicin enhancing cell death. Collectively our results indicated that co-treatment of DFF40-transfected cells with doxorubicin can enhance the killing of these tumor cells via apoptosis. Thus, modulation of DFF40 level may be a beneficial strategy for treatment of chemo-resistant cancers.

Determination of cell cycle phases in live B16 melanoma cells using IRMS.

PubMed

Bedolla, Diana E; Kenig, Saša; Mitri, Elisa; Ferraris, Paolo; Marcello, Alessandro; Grenci, Gianluca; Vaccari, Lisa

2013-07-21

The knowledge of cell cycle phase distribution is of paramount importance for understanding cellular behaviour under normal and stressed growth conditions. This task is usually assessed using Flow Cytometry (FC) or immunohistochemistry. Here we report on the use of FTIR microspectroscopy in Microfluidic Devices (MD-IRMS) as an alternative technique for studying cell cycle distribution in live cells. Asynchronous, S- and G0-synchronized B16 mouse melanoma cells were studied by running parallel experiments based on MD-IRMS and FC using Propidium Iodide (PI) staining. MD-IRMS experiments have been done using silicon-modified BaF2 devices, where the thin silicon layer prevents BaF2 dissolution without affecting the transparency of the material and therefore enabling a better assessment of the Phosphate I (PhI) and II (PhII) bands. Hierarchical Cluster Analysis (HCA) of cellular microspectra in the 1300-1000 cm(-1) region pointed out a distribution of cells among clusters, which is in good agreement with FC results among G0/G1, S and G2/M phases. The differentiation is mostly driven by the intensity of PhI and PhII bands. In particular, PhI almost doubles from the G0/G1 to G2/M phase, in agreement with the trend followed by nucleic acids during cellular progression. MD-IRMS is then proposed as a powerful method for the in situ determination of the cell cycle stage of an individual cell, without any labelling or staining, which gives the advantage of possibly monitoring specific cellular responses to several types of stimuli by clearly separating the spectral signatures related to the cellular response from those of cells that are normally progressing.

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

PubMed Central

Niikura, Yohei; Kitagawa, Katsumi

2016-01-01

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells. PMID:26967065

Molecular mechanism of G1 arrest and cellular senescence induced by LEE011, a novel CDK4/CDK6 inhibitor, in leukemia cells.

PubMed

Tao, Yan-Fang; Wang, Na-Na; Xu, Li-Xiao; Li, Zhi-Heng; Li, Xiao-Lu; Xu, Yun-Yun; Fang, Fang; Li, Mei; Qian, Guang-Hui; Li, Yan-Hong; Li, Yi-Ping; Wu, Yi; Ren, Jun-Li; Du, Wei-Wei; Lu, Jun; Feng, Xing; Wang, Jian; He, Wei-Qi; Hu, Shao-Yan; Pan, Jian

2017-01-01

Overexpression of cyclin D1 dependent kinases 4 and 6 (CDK4/6) is a common feature of many human cancers including leukemia. LEE011 is a novel inhibitor of both CDK4 and 6. To date, the molecular function of LEE011 in leukemia remains unclear. Leukemia cell growth and apoptosis following LEE011 treatment was assessed through CCK-8 and annexin V/propidium iodide staining assays. Cell senescence was assessed by β-galactosidase staining and p16 INK4a expression analysis. Gene expression profiles of LEE011 treated HL-60 cells were investigated using an Arraystar Human LncRNA array. Gene ontology and KEGG pathway analysis were then used to analyze the differentially expressed genes from the cluster analysis. Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and could induce apoptosis. Hoechst 33,342 staining analysis showed DNA fragmentation and distortion of nuclear structures following LEE011 treatment. Cell cycle analysis showed LEE011 significantly induced cell cycle G 1 arrest in seven of eight acute leukemia cells lines, the exception being THP-1 cells. β-Galactosidase staining analysis and p16 INK4a expression analysis showed that LEE011 treatment can induce cell senescence of leukemia cells. LncRNA microarray analysis showed 2083 differentially expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with controls. Molecular function analysis showed that LEE011 induced senescence in leukemia cells partially through downregulation of the transcriptional expression of MYBL2. We demonstrate for the first time that LEE011 treatment results in inhibition of cell proliferation and induction of G 1 arrest and cellular senescence in leukemia cells. LncRNA microarray analysis showed differentially expressed mRNAs and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2. These results may open new lines of investigation regarding the molecular mechanism of LEE011 induced cellular senescence.

Cell cycle accumulation of the proliferating cell nuclear antigen PCN-1 transitions from continuous in the adult germline to intermittent in the early embryo of C. elegans.

PubMed

Kocsisova, Zuzana; Kornfeld, Kerry; Schedl, Tim

2018-05-30

The proliferating cell nuclear antigen (PCNA or PCN-1 in C. elegans), an essential processivity factor for DNA polymerase δ, has been widely used as a marker of S-phase. In C. elegans early embryos, PCN-1 accumulation is cyclic, localizing to the nucleus during S-phase and the cytoplasm during the rest of the cell cycle. The C. elegans larval and adult germline is an important model systems for studying cell cycle regulation, and it was observed that the cell cycle regulator cyclin E (CYE-1 in C. elegans) displays a non-cyclic, continuous accumulation pattern in this tissue. The accumulation pattern of PCN-1 has not been well defined in the larval and adult germline, and the objective of this study was to determine if the accumulation pattern is cyclic, as in other cells and organisms, or continuous, similar to cyclin E. To study the larval and adult germline accumulation of PCN-1 expressed from its native locus, we used CRISPR/Cas9 technology to engineer a novel allele of pcn-1 that encodes an epitope-tagged protein. S-phase nuclei were labeled using EdU nucleotide incorporation, and FLAG::PCN-1 was detected by antibody staining. All progenitor zone nuclei, including those that were not in S-phase (as they were negative for EdU staining) showed PCN-1 accumulation, indicating that PCN-1 accumulated during all cell cycle phases in the germline progenitor zone. The same result was observed with a GFP::PCN-1 fusion protein expressed from a transgene. pcn-1 loss-of-function mutations were analyzed, and pcn-1 was necessary for robust fertility and embryonic development. In the C. elegans early embryo as well as other organisms, PCN-1 accumulates in nuclei only during S-phase. By contrast, in the progenitor zone of the germline of C. elegans, PCN-1 accumulated in nuclei during all cell cycle stages. This pattern is similar to accumulation pattern of cyclin E. These observations support the model that mitotic cell cycle regulation in the germline stem and progenitor cells is distinct from somatic cells, as it does not heavily rely on cyclic accumulation of classic cell cycle proteins.

Tea Polysaccharide Prevents Colitis-Associated Carcinogenesis in Mice by Inhibiting the Proliferation and Invasion of Tumor Cells

PubMed Central

Liu, Li-Qiao; Li, Hai-Shan; Shen, Ming-Yue; Hu, Jie-Lun; Xie, Ming-Yong

2018-01-01

The imbalance between cell proliferation and apoptosis can lead to tumor progression, causing oncogenic transformation, abnormal cell proliferation and cell apoptosis suppression. Tea polysaccharide (TPS) is the major bioactive component in green tea, it has showed antioxidant, antitumor and anti-inflammatory bioactivities. In this study, the chemoprophylaxis effects of TPS on colitis-associated colon carcinogenesis, especially the cell apoptosis activation and inhibition effects on cell proliferation and invasion were analyzed. The azoxymethane/dextran sulfate sodium (AOM/DSS) was used to induce the colorectal carcinogenesis in mice. Results showed that the tumor incidence was reduced in TPS-treated AOM/DSS mice compared to AOM/DSS mice. TUNEL staining and Ki-67 immunohistochemistry staining showed that the TPS treatment increased significantly the cell apoptosis and decreased cell proliferation among AOM/DSS mice. Furthermore, TPS reduced the expression levels of the cell cycle protein cyclin D1, matrix metalloproteinase (MMP)-2, and MMP-9. In addition, in vitro studies showed that TPS, suppressed the proliferation and invasion of the mouse colon cancer cells. Overall, our findings demonstrated that TPS could be a potential agent in the treatment and/or prevention of colon tumor, which promoted the apoptosis and suppressed the proliferation and invasion of the mouse colon cancer cells via arresting cell cycle progression. PMID:29419740

Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

PubMed Central

Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

2014-01-01

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies. PMID:25460012

Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection.

PubMed

Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

2014-01-01

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

Silencing of B7-H4 suppresses the tumorigenicity of the MGC-803 human gastric cancer cell line and promotes cell apoptosis via the mitochondrial signaling pathway.

PubMed

Zhou, Donghui; Zhou, Yong; Li, Chao; Yang, Lina

2018-04-01

B7-H4 is a transmembrane protein which is a member of the B7 superfamily. It is overexpressed in various types of cancer, including gastric cancer. However, the effects of B7-H4 on the tumorigenicity of gastric cancer and the underlying mechanisms have not yet been fully explored. Thus, the aim of this study was to examine the effects of B7-H4 on the tumorigenicity of gastric cancer cells and to elucidate the underlying mechanisms. For this purpose, B7-H4 expression in gastric cancer tissues was detected by immunohistochemical staining. The effects of B7-H4 on the biological behavior of the MGC-803 human gastric cancer cell line were examined by Cell Counting kit-8 (CCK-8) assay, cell cycle analysis, wound healing assay, Annexin V/propidium iodide staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Moreover, the expression levels of apoptotic markers, such as cleaved caspase‑3, cleaved caspase‑9, Bcl-2 and Bax were examined by western blot analysis. Immunohistochemical staining revealed that a high expression of B7-H4 was found in about 41.8% of tissues obtained from patients with gastric cancer. Comparative analysis revealed that B7-H4 expression significantly correlated with lymph node metastasis and the TNM stage. The results of CCK-8 assay, cell cycle analysis, wound healing assay, Annexin V/propidium iodide staining assay and TUNEL assay all demonstrated that the silencing of B7-H4 by small interfering RNA decreased cell proliferation, suppressed cell motility, and induced cell cycle arrest and the apoptosis of MGC-803 human gastric cancer cells. Furthermore, the results of western blot analysis indicated that the downregulation of B7-H4 induced the apoptosis of the MGC-803 cells via the mitochondrial signaling pathway through the activation of caspase‑3 and caspase‑9, and by altering the Bax/Bcl-2 ratio in a manner that favored apoptosis. Based on the findings on human gastric cancer cell line MGC-803, the findings of this study suggested that B7-H4 may have the potential to be a valuable prognostic marker and a target for individualized therapies for gastric cancer. However, further investigations are required in order to confirm our findings on a larger scale.

Panaxadiol, a purified ginseng component, enhances the anti-cancer effects of 5-fluorouracil in human colorectal cancer cells.

PubMed

Li, Xiao-Li; Wang, Chong-Zhi; Mehendale, Sangeeta R; Sun, Shi; Wang, Qi; Yuan, Chun-Su

2009-11-01

Colorectal cancer is a major cause of morbidity and mortality for cancer worldwide. Although 5-fluorouracil (5-FU) is one of the most widely used chemotherapeutic agents in first-line therapy for colorectal cancer, serious side effects limit its clinical usefulness. Panaxadiol (PD) is the purified sapogenin of ginseng saponins, which exhibit anti-tumor activity. In this study, we investigated the possible synergistic anti-cancer effects of PD and 5-FU on a human colorectal cancer cell line, HCT-116. Cell viability was evaluated by an MTS cell proliferation assay. Morphological observation was performed by crystal violet cell viability staining assay. Cell cycle distribution and apoptotic effects were analyzed by flow cytometry after staining with PI/RNase or Annexin V/PI. Cell growth was markedly suppressed in HCT-116 cells treated by 5-FU (20-100 microM) for 24 or 48 h with time-dependent effects. The significant suppression on HCT-116 cell proliferation was observed after treatment with PD (25 microM) for 24 and 48 h. Panaxadiol (25 microM) markedly (P < 0.05) enhanced the anti-proliferative effects of 5-FU (5, 10, 20 microM) on HCT-116 cells compared to single treatment of 5-FU for 24 and 48 h. Flow cytometric analysis on DNA indicated that PD and 5-FU selectively arrested cell cycle progression in the G1 phase and S phase (P < 0.01), respectively, compared to the control condition. Combination use of 5-FU with PD significantly (P < 0.001) increased cell cycle arrest in the S phase compared to that treated by 5-FU alone. The combination of 5-FU and PD significantly enhanced the percentage of apoptotic cells when compared with the corresponding cell groups treated by 5-FU alone (P < 0.001). Panaxadiol enhanced the anti-cancer effects of 5-FU on human colorectal cancer cells through the regulation of cell cycle transition and the induction of apoptotic cells.

Molecular hydrogen protects human lymphocyte AHH-1 cells against 12C6+ heavy ion radiation.

PubMed

Yang, Yanyong; Gao, Fu; Zhang, Hong; Hunag, Yijuan; Zhang, Pei; Liu, Cong; Li, Bailong; Cai, Jianming

2013-12-01

To investigate the potential protective role of molecular hydrogen (H(2)) against (12)C(6+) heavy ion radiation, which is a major hazard for space travel and has been also widely used in heavy ion radiotherapy. H(2) was dissolved in Roswell Park Memorial Institute (RPMI) 1640 medium under high pressure (0.4 Mpa) to a saturated level by using an apparatus produced by our department. A 2-[6-(4'-hydroxy) phenoxy-3H-xanthen-3-on-9-yl] benzoate (HPF) probe and a 2',7'-Dichlorodihydrofluorescein diacetate (H2DCFH-DA) fluorescent dye were used to measure the intracellular reactive oxygen species (ROS) level. Cell apoptosis were determined by double-staining with Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI) as well as a Hoechst 33342 staining method alternatively. Subsequently, cell cycle analysis was performed using a PI staining method and the expression of apoptotic protein was examined by Western blot. In this study, we demonstrated H(2) reduced ROS level in Human lymphocyte AHH-1 cells as well as in the radiolysis of water. Our data also showed H(2) attenuated (12)C(6+) radiation- induced cell apoptosis and also alleviated radiation-induced G2/M cell cycle arrest. Heavy ion radiation-induced Caspase 3 activation was also inhibited by H(2) treatment. In conclusion, these data showed that H(2) attenuated (12)C(6+) radiation-induced cell apoptosis through reducing the ROS level and modulating apoptotic molecules, thus indicating the potential of H(2) as a safe and effective radioprotectant.

Magnolol Inhibits the Growth of Non-Small Cell Lung Cancer via Inhibiting Microtubule Polymerization.

PubMed

Shen, Jia; Ma, Hailin; Zhang, Tiancheng; Liu, Hui; Yu, Linghua; Li, Guosheng; Li, Huishuang; Hu, Meichun

2017-01-01

The tubulin/microtubule system, which is an integral component of the cytoskeleton, plays an essential role in mitosis. Targeting mitotic progression by disturbing microtubule dynamics is a rational strategy for cancer treatment. Microtubule polymerization assay was performed to examine the effect of Magnolol (a novel natural phenolic compound isolated from Magnolia obovata) on cellular microtubule polymerization in human non-small cell lung cancer (NSCLC) cells. Cell cycle analysis, mitotic index assay, cell proliferation assay, colony formation assay, western blotting analysis of cell cycle regulators, Annexin V-FITC/PI staining, and live/dead viability staining were carried out to investigate the Magnolol's inhibitory effect on proliferation and viability of NSCLS cells in vitro. Xenograft model of human A549 NSCLC tumor was used to determine the Magnolol's efficacy in vivo. Magnolol treatment effectively inhibited cell proliferation and colony formation of NSCLC cells. Further study proved that Magnolol induced the mitotic phase arrest and inhibited G2/M progression in a dose-dependent manner, which were mechanistically associated with expression alteration of a series of cell cycle regulators. Furthermore, Magnolol treatment disrupted the cellular microtubule organization via inhibiting the polymerization of microtubule. We also found treatment with NSCLC cells with Magnolol resulted in apoptosis activation through a p53-independent pathway, and autophgy induction via down-regulation of the Akt/mTOR pathway. Finally, Magnolol treatment significantly suppressed the NSCLC tumor growth in mouse xenograft model in vivo. These findings identify Magnolol as a promising candidate with anti-microtubule polymerization activity for NSCLC treatment. © 2017 The Author(s). Published by S. Karger AG, Basel.

Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

DOEpatents

Dolbeare, F.A.; Gray, J.W.

1983-10-18

A method for the simultaneous flow cylometric measurement of total cellular DNA content and of the uptake of DNA precursors as a measure of DNA synthesis during various phases of the cell cycle in normal and malignant cells in vitro and in vivo is described. The method comprises reacting cells with labelled halodeoxyuridine (HdU), partially denaturing cellular DNA, adding to the reaction medium monoclonal antibodies (mabs) reactive with HdU, reacting the bound mabs with a second labelled antibody, incubating the mixture with a DNA stain, and measuring simultaneously the intensity of the DNA stain as a measure of the total cellular DNA and the HdU incorporated as a measure of DNA synthesis. (ACR)

The antiangiogenic activity of Kushecarpin D, a novel flavonoid isolated from Sophora flavescens Ait.

PubMed

Pu, Li-Ping; Chen, He-Ping; Cao, Mei-Ai; Zhang, Xiu-Li; Gao, Qing-Xiang; Yuan, Cheng-Shan; Wang, Chun-Ming

2013-11-13

Kushecarpin D (KD) is a novel flavonoid isolated from the traditional Chinese herbal medicine Kushen (the dried root of Sophora flavescens Ait). As part of our continuous effort to explore Chinese traditional medicinal herbs and to identify novel natural anticancer products, the antiangiogenic properties of KD were examined in vitro using a human umbilical vein endothelial cell line (ECV304). The SRB and Trypan Blue exclusion assays were used to evaluate the effect of KD on cell proliferation. The antiangiogenic activities of KD were evaluated through studies of cell migration, cell adhesion, and tube formation. DCFH-DA and DHE fluorescent assays were used to detect the reactive oxygen species (ROS) levels. Catalase activity was detected using the colorimetric ammonium molybdate method. Cell cycle and apoptosis were measured using flow cytometry and the Hoechst 33258 staining assay. The results indicated that KD showed antiangiogenic activity via inhibitory effects on cell proliferation, cell migration, cell adhesion, and tube formation. ROS levels were down-regulated and catalase activity was up-regulated after treatment with KD. The cell cycle was arrested at the G2/M phase, while no apoptosis was observed using the Hoechst 33258 staining assay or following the flow cytometric analysis of the sub-G1 proportion. The antiangiogenic properties of KD, in combination with its anti-proliferative effect and ability to induce cell cycle arrest without inducing apoptosis, make it a good candidate for development as antitumor agent. However, further studies are essential to elucidate its mechanism of action. © 2013.

A phosphorescent silver(I)-gold (I) cluster complex that specifically lights up the nucleolus of living cells with FLIM imaging.

PubMed

Chen, Min; Lei, Zhen; Feng, Wei; Li, Chunyan; Wang, Quan-Ming; Li, Fuyou

2013-06-01

The phosphorescent silver(I)-gold(I) cluster complex [CAu6Ag2(dppy)6](BF4)4 (N1) selectively stains the nucleolus, with a much lower uptake in the nucleus and cytoplasm, and exhibits excellent photostability. This Ag-Au cluster, which has a photoluminescent lifetime of microseconds, is particularly attractive as a probe in applications of time-gated microscopy. Investigation of the pathway of cellular entry indicated that N1 permeates the outer membrane and nuclear membrane of living cells through an energy-dependent and non-endocytic route within 10 min. High concentrations of N1 in the nucleolus have been quantified by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and transmission electron microscopy coupled with an energy dispersive X-ray analysis (TEM-EDXA), which also helped to elucidate the mechanism of the specific staining. Intracellular selective staining may be correlated with the microenvironment of the nucleolus, which is consistent with experiments conducted at different phases of the cell cycle. These results prove that N1 is a very attractive phosphorescent staining reagent for visualizing the nucleolus of living cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

Acute exposure to vibration is an apoptosis-inducing stimulus in the vocal fold epithelium.

PubMed

Novaleski, Carolyn K; Kimball, Emily E; Mizuta, Masanobu; Rousseau, Bernard

2016-10-01

Clinical voice disorders pose significant communication-related challenges to patients. The purpose of this study was to quantify the rate of apoptosis and tumor necrosis factor-alpha (TNF-α) signaling in vocal fold epithelial cells in response to increasing time-doses and cycle-doses of vibration. 20 New Zealand white breeder rabbits were randomized to three groups of time-doses of vibration exposure (30, 60, 120min) or a control group (120min of vocal fold adduction and abduction). Estimated cycle-doses of vocal fold vibration were extrapolated based on mean fundamental frequency. Laryngeal tissue specimens were evaluated for apoptosis and gene transcript and protein levels of TNF-α. Results revealed that terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was significantly higher after 120min of vibration compared to the control. Transmission electron microscopy (TEM) revealed no significant effect of time-dose on the mean area of epithelial cell nuclei. Extrapolated cycle-doses of vibration exposure were closely related to experimental time-dose conditions, although no significant correlations were observed with TUNEL staining or mean area of epithelial cell nuclei. TUNEL staining was positively correlated with TNF-α protein expression. Our findings suggest that apoptosis can be induced in the vocal fold epithelium after 120min of modal intensity phonation. In contrast, shorter durations of vibration exposure do not result in apoptosis signaling. However, morphological features of apoptosis are not observed using TEM. Future studies are necessary to examine the contribution of abnormal apoptosis to vocal fold diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

Acute Exposure to Vibration is an Apoptosis-Inducing Stimulus in the Vocal Fold Epithelium

PubMed Central

Novaleski, Carolyn K.; Kimball, Emily E.; Mizuta, Masanobu; Rousseau, Bernard

2016-01-01

Clinical voice disorders pose significant communication-related challenges to patients. The purpose of this study was to quantify the rate of apoptosis and tumor necrosis factor-alpha (TNF-α) signaling in vocal fold epithelial cells in response to increasing time-doses and cycle-doses of vibration. 20 New Zealand white breeder rabbits were randomized to three groups of time-doses of vibration exposure (30, 60, 120 minutes) or a control group (120 minutes of vocal fold adduction and abduction). Estimated cycle-doses of vocal fold vibration were extrapolated based on mean fundamental frequency. Laryngeal tissue specimens were evaluated for apoptosis and gene transcript and protein levels of TNF-α. Results revealed that terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was significantly higher after 120 minutes of vibration compared to the control. Transmission electron microscopy (TEM) revealed no significant effect of time-dose on the mean area of epithelial cell nuclei. Extrapolated cycle-doses of vibration exposure were closely related to experimental time-dose conditions, although no significant correlations were observed with TUNEL staining or mean area of epithelial cell nuclei. TUNEL staining was positively correlated with TNF-α protein expression. Our findings suggest that apoptosis can be induced in the vocal fold epithelium after 120 minutes of modal intensity phonation. In contrast, shorter durations of vibration exposure do not result in apoptosis signaling. However, morphological features of apoptosis are not observed using TEM. Future studies are necessary to examine the contribution of abnormal apoptosis to vocal fold diseases. PMID:27577014

Changes in the oviducal epithelium during the estrous cycle in the marsupial Monodelphis domestica

PubMed Central

Kress, Annetrudi; Morson, Gianni

2007-01-01

The Monodelphis oviduct can be divided into four anatomical segments: preampulla (comprising fimbriae and infundibulum), ampulla, isthmus with crypts and uterotubal junction. Ovaries are enclosed in a periovarial sac, the bursa, and in some specimens tubules of an epoophoron could be identified. In both structures non-ciliated cells develop small translucent vesicles, which accumulate in the cell apices and presumably produce fluid as often seen in the bursa and in the tubules of the epooophoron. These vesicles do not stain with Alcian blue or PAS. The same applies also to the non-ciliated cells of the fimbriae. The oviducal epithelium of ampulla and the surface epithelium of the isthmus consisting of ciliated and non-ciliated, secretory cells undergo considerable changes during the estrous cycle. Proestrus shows low numbers of ciliated cells, some are in the process of neo-ciliogenesis, non-ciliated cells carry solitary cilia and few remnant secretory granules from the previous cycle may be found. At estrus the amount of ciliated cells in ampulla and isthmus has increased, most non-cililated cells lost the solitary cilia, developed longer microvilli and formed numerous secretory granules in their cell apices. At postestrus secretory products, often surrounded by membranes, are extruded into the oviducal lumen and contribute towards egg coat formation. First signs of deciliation processes are apparent. Solitary cilia reappear. At metestrus only few secretory cells are left with some secretory material. The lumen is often filled with shed cilia and cell apices. Proliferation of basal bodies within non-secretory cells indicate the formation of new ciliated cells. The non-ciliated epithelial cells of the isthmic crypts form no secretory granules but accumulate a great number of translucent vesicles, which in contrast to the secretory granules do not stain with Alcian blue or PAS. PMID:17883438

Gallic acid induced apoptotic events in HCT-15 colon cancer cells.

PubMed

Subramanian, Aruna Priyadharshni; Jaganathan, Saravana Kumar; Mandal, Mahitosh; Supriyanto, Eko; Muhamad, Ida Idayu

2016-04-21

To investigate the inhibitory action of diet-derived phenolic compound gallic acid (GA) against HCT-15 colon cancer cells. The antiproliferative effect of GA against colon cancer cells was determined by performing thiazolyl blue tetrazolium bromide (MTT) assay. The colony forming ability of GA treated colon cancer cells was evaluated using the colony forming assay. The cell cycle changes induced by GA in HCT-15 cells were analyzed by propidium iodide staining. Levels of reactive oxygen species (ROS) and mitochondrial membrane potential of HCT-15 exposed to GA was assessed using 2',7'-dichlorfluorescein-diacetate and rhodamine-123 respectively, with the help of flow cytometry. Morphological changes caused by GA treatment in the colon cancer cells were identified by scanning electron microscope and photomicrograph examination. Apoptosis was confirmed using flow cytometric analysis of GA treated HCT-15 cells after staining with Yo-Pro-1. MTT assay results illustrated that GA has an inhibitory effect on HCT-15 cells with IC50 value of 740 μmol/L. A time-dependent inhibition of colony formation was evident with GA treatment. Cell cycle arrest was evident from the accumulation of GA treated HCT-15 cells at sub-G1 phase (0.98 ± 1.03 vs 58.01 ± 2.05) with increasing exposure time. Flow cytometric analysis of GA treated HCT-15 cells depicted early events associated with apoptosis like lipid layer breakage and fall in mitochondrial membrane potential apart from an increase in the generation of ROS which were in a time dependent manner. SEM and photomicrograph images of the GA-treated cells displayed membrane blebbing and cell shrinking characteristics of apoptosis. Further apoptosis confirmation by Yo-Pro-1 staining also showed the time-dependent increase of apoptotic cells after treatment. These results show that GA induced ROS dependent apoptosis and inhibited the growth of colon cancer cells.

Gallic acid induced apoptotic events in HCT-15 colon cancer cells

PubMed Central

Subramanian, Aruna Priyadharshni; Jaganathan, Saravana Kumar; Mandal, Mahitosh; Supriyanto, Eko; Muhamad, Ida Idayu

2016-01-01

AIM: To investigate the inhibitory action of diet-derived phenolic compound gallic acid (GA) against HCT-15 colon cancer cells. METHODS: The antiproliferative effect of GA against colon cancer cells was determined by performing thiazolyl blue tetrazolium bromide (MTT) assay. The colony forming ability of GA treated colon cancer cells was evaluated using the colony forming assay. The cell cycle changes induced by GA in HCT-15 cells were analyzed by propidium iodide staining. Levels of reactive oxygen species (ROS) and mitochondrial membrane potential of HCT-15 exposed to GA was assessed using 2’,7’-dichlorfluorescein-diacetate and rhodamine-123 respectively, with the help of flow cytometry. Morphological changes caused by GA treatment in the colon cancer cells were identified by scanning electron microscope and photomicrograph examination. Apoptosis was confirmed using flow cytometric analysis of GA treated HCT-15 cells after staining with Yo-Pro-1. RESULTS: MTT assay results illustrated that GA has an inhibitory effect on HCT-15 cells with IC50 value of 740 μmol/L. A time-dependent inhibition of colony formation was evident with GA treatment. Cell cycle arrest was evident from the accumulation of GA treated HCT-15 cells at sub-G1 phase (0.98 ± 1.03 vs 58.01 ± 2.05) with increasing exposure time. Flow cytometric analysis of GA treated HCT-15 cells depicted early events associated with apoptosis like lipid layer breakage and fall in mitochondrial membrane potential apart from an increase in the generation of ROS which were in a time dependent manner. SEM and photomicrograph images of the GA-treated cells displayed membrane blebbing and cell shrinking characteristics of apoptosis. Further apoptosis confirmation by Yo-Pro-1 staining also showed the time-dependent increase of apoptotic cells after treatment. CONCLUSION: These results show that GA induced ROS dependent apoptosis and inhibited the growth of colon cancer cells. PMID:27099438

Osthole induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells.

PubMed

Chao, Xu; Zhou, Xiaojun; Zheng, Gang; Dong, Changhu; Zhang, Wei; Song, Xiaomei; Jin, Tianbo

2014-05-01

Osthole [7-methoxy-8-(3-methyl-2-butenyl) coumarin] isolated from the fruit of Cnidium monnieri (L.) Cuss, one of the commonly used Chinese medicines listed in the Shennong's Classic of Materia Medica in the Han Dynasty, had remarkable antiproliferative activity against human hepatocellular carcinoma HepG2 cells in culture. This study evaluated the effects of osthole on cell growth, nuclear morphology, cell cycle distribution, and expression of apoptosis-related proteins in HepG2 cells. Cytotoxic activity of osthole was determined by the MTT assay at various concentrations ranging from 0.004 to 1.0 µmol/ml in HepG2 cells. Cell morphology was assessed by Hoechst staining and fluorescence microscopy. Apoptosis and cell-cycle distribution was determined by annexin V staining and flow cytometry. Apoptotic protein levels were assessed by Western blot. Osthole exhibited significant inhibition of the survival of HepG2 cells and the half inhibitory concentration (IC₅₀) values were 0.186, 0.158 and 0.123 µmol/ml at 24, 48 and 72 h, respectively. Cells treated with osthole at concentrations of 0, 0.004, 0.02, 0.1 and 0.5 μmol/ml showed a statistically significant increase in the G2/M fraction accompanied by a decrease in the G0/G1 fraction. The increase of apoptosis induced by osthole was correlated with down-regulation expression of anti-apoptotic Bcl-2 protein and up-regulation expression of pro-apoptotic Bax and p53 proteins. Osthole had significant growth inhibitory activity and the pro-apoptotic effect of osthole is mediated through the activation of caspases and mitochondria in HepG2 cells. Results suggest that osthole has promising therapeutic potential against hepatocellular carcinoma.

Mast cell dynamics in the house rat (Rattus rattus) ovary during estrus cycle, pregnancy and lactation.

PubMed

Batth, B K; Parshad, R K

2000-02-01

The distribution of mast cells in various ovarian compartments was studied during different stages of the reproductive cycles in Rattus rattus. Two types of mast cell populations were recognized with light microscopy i.e., light purple and deep purple, the latter also includes deeply stained cells with extruded granules. Mast cells identified by electron microscopy showed the ultrastructural features during granule formation and release of their content. Significantly higher numbers of mast cells per unit area of ovary were seen at estrus and diestrus. Numbers of mast cells also remained high during pregnancy with possible involvement of mast cell products in vascularization of corpora lutea. A positive correlation existed between mast cell counts and embryo number during pregnancy. However, numbers of mast cells declined significantly after parturition.

Nuclear RNA quantification in protoplast cell-cycle phases.

PubMed

Bergounioux, C; Perennes, C; Brown, S C; Gadal, P

1988-01-01

Using acridine orange staining and flow cytometry the DNA and RNA levels (arbitrary units) of individual cells may be established. Here, this method has been applied to nuclei isolated from plant protoplasts during culture. The specificity of the technique has been validated for such plant material; ribonuclease markedly reduced nuclear staining without modifying the DNA histogram; ribonuclease inhibitor prevented the action of released cell nucleases; and protoplasts cultivated with actinomycin D did not synthesize RNA. First RNA synthesis was evident 18 h after Petunia hybrida protoplasts had been put into culture. An increase of RNA above a critical level was required for cells to be able to initiate DNA replication from G1, termed G1B. G2 nuclei had an RNA:DNA ratio similar to that of G1 nuclei.

[Effects of sinensetin on proliferation and apoptosis of human gastric cancer AGS cells].

PubMed

Dong, Yang; Ji, Guang; Cao, Aili; Shi, Jianrong; Shi, Hailian; Xie, Jianqun; Wu, Dazheng

2011-03-01

To study the effects and mechanisms of sinensetin on proliferation and apoptosis of human AGS gastric cancer cells. MTT assay was used to detect the growth inhibition rates of human AGS gastric cancer cells treated with sinsesectin in different concentrations and times. The cell cycle distribution was measured by flow cytometry. The apoptosis was examined by Annexin-FITC/PI staining and DNA fragment analysis. The apoptosis morphology was observed by inverted fluorescence microscope after Hoechst 33342 staining. The protein expressions of p21 and p53 were detected by western blot. MTT assay showed that sinensetin inhibited the growth of AGS gastric cancer cells in a dose- and time-dependent manner. Sinensetin blocked AGS cells in G2/ M and increased the apoptosis rates of AGS cells in a dose-dependent manner. DNA ladder was observed in cells treated with 60 micromol x L(-1) sinensetin for 48 h. The typical apoptotic morphological changes including cell nucleus shrinkage, chromatin condensation and apoptotic bodies were observed when treated with different dose of sinensetin. Western blot showed that sinensetin increased expressions of p53 and p21 in a dose-dependent manner. Sinensetin could inhibit human AGS gastric cancer cells proliferation and induce cell cycle block in G2/M phase and apoptosis. The up regulation of p53 and p21 protein might be one of the mechanisms.

Curcumin inhibits growth potential by G1 cell cycle arrest and induces apoptosis in p53-mutated COLO 320DM human colon adenocarcinoma cells.

PubMed

Dasiram, Jade Dhananjay; Ganesan, Ramamoorthi; Kannan, Janani; Kotteeswaran, Venkatesan; Sivalingam, Nageswaran

2017-02-01

Curcumin, a natural polyphenolic compound and it is isolated from the rhizome of Curcuma longa, have been reported to possess anticancer effect against stage I and II colon cancer. However, the effect of curcumin on colon cancer at Dukes' type C metastatic stage III remains still unclear. In the present study, we have investigated the anticancer effects of curcumin on p53 mutated COLO 320DM human colon adenocarcinoma cells derived from Dukes' type C metastatic stage. The cellular viability and proliferation were assessed by trypan blue exclusion assay and MTT assay, respectively. The cytotoxicity effect was examined by lactate dehydrogenase (LDH) cytotoxicity assay. Apoptosis was analyzed by DNA fragmentation analysis, Hoechst and propidium iodide double fluorescent staining and confocal microscopy analysis. Cell cycle distribution was performed by flow cytometry analysis. Here we have observed that curcumin treatment significantly inhibited the cellular viability and proliferation potential of p53 mutated COLO 320DM cells in a dose- and time-dependent manner. In addition, curcumin treatment showed no cytotoxic effects to the COLO 320DM cells. DNA fragmentation analysis, Hoechst and propidium iodide double fluorescent staining and confocal microscopy analysis revealed that curcumin treatment induced apoptosis in COLO 320DM cells. Furthermore, curcumin caused cell cycle arrest at the G1 phase, decreased the cell population in the S phase and induced apoptosis in COLO 320DM colon adenocarcinoma cells. Together, these data suggest that curcumin exerts anticancer effects and induces apoptosis in p53 mutated COLO 320DM human colon adenocarcinoma cells derived from Dukes' type C metastatic stage. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Arctigenin anti-tumor activity in bladder cancer T24 cell line through induction of cell-cycle arrest and apoptosis.

PubMed

Yang, Shucai; Ma, Jing; Xiao, Jianbing; Lv, Xiaohong; Li, Xinlei; Yang, Huike; Liu, Ying; Feng, Sijia; Zhang, Yafang

2012-08-01

Bladder cancer is the most common neoplasm in the urinary system. This study assesses arctigenin anti-tumor activity in human bladder cancer T24 cells in vitro and the underlying molecular events. The flow cytometry analysis was used to detect cell-cycle distribution and apoptosis. Western blotting was used to detect changes in protein expression. The data showed that arctigenin treatment reduced viability of bladder cancer T24 cells in a dose- and time-dependent manner after treatment with arctigenin (10, 20, 40, 80, and 100 μmol/L) for 24 hr and 48 hr. Arctigenin treatment clearly arrested tumor cells in the G1 phase of the cell cycle. Apoptosis was detected by hoechst stain and flow cytometry after Annexin-V-FITC/PI double staining. Early and late apoptotic cells were accounted for 2.32-7.01% and 3.07-7.35%, respectively. At the molecular level, arctigenin treatment decreased cyclin D1 expression, whereas CDK4 and CDK6 expression levels were unaffected. Moreover, arctigenin selectively altered the phosphorylation of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 and activated phosphorylation of p38 significantly in a dose-dependent manner. These results suggest that arctigenin may inhibit cell viability and induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen-activated protein kinase pathway may play an important role in the anti-tumor effect of arctigenin. The data from the current study demonstrate the usefulness of arctigenin in bladder cancer T24 cells, which should further be evaluated in vivo before translation into clinical trials for the chemoprevention of bladder cancer. Copyright © 2012 Wiley Periodicals, Inc.

Acacia catechu ethanolic bark extract induces apoptosis in human oral squamous carcinoma cells.

PubMed

Lakshmi, Thangavelu; Ezhilarasan, Devaraj; Vijayaragavan, Rajagopal; Bhullar, Sukhwinder Kaur; Rajendran, Ramasamy

2017-01-01

Oral cancer is in approximately 30% of all cancers in India. This study was conducted to evaluate the cytotoxic activity of ethanolic extract of Acacia catechu bark (ACB) against human squamous cell carcinoma cell line-25 (SCC-25). Cytotoxic effect of ACB extract was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide assay. A. catechu extract was treated SCC-25 cells with 25 and 50 μg/mL for 24 h. Apoptosis markers such as caspases-8 and 9, bcl-2, bax, and cytochrome c (Cyt-c) were done by RT-PCR. Morphological changes of ACB treated cells were evaluated using acridine orange/ethidium bromide (AO/EB) dual staining. Nuclear morphology and DNA fragmentation were evaluated using propidium iodide (PI) staining. Further, cell cycle analysis was performed using flow cytometry. A. catechu treatment caused cytotoxicity in SCC-25 cells with an IC 50 of 52.09 μg/mL. Apoptotic marker gene expressions were significantly increased on ACB treatment. Staining with AO/EB and PI shows membrane blebbing and nuclear membrane distortion, respectively, and it confirms the apoptosis induction in SCC-25 cells. These results suggest that ACB extract can be used as a modulating agent in oral squamous cell carcinoma.

Cell cycle stage-specific differential expression of topoisomerase I in tobacco BY-2 cells and its ectopic overexpression and knockdown unravels its crucial role in plant morphogenesis and development.

PubMed

Singh, Badri Nath; Mudgil, Yashwanti; John, Riffat; Achary, V Mohan Murali; Tripathy, Manas Kumar; Sopory, Sudhir K; Reddy, Malireddy K; Kaul, Tanushri

2015-11-01

DNA topoisomerases catalyze the inter-conversion of different topological forms of DNA. Cell cycle coupled differential accumulation of topoisomerase I (Topo I) revealed biphasic expression maximum at S-phase and M/G1-phase of cultured synchronized tobacco BY-2 cells. This suggested its active role in resolving topological constrains during DNA replication (S-phase) and chromosome decondensation (M/G1 phase). Immuno-localization revealed high concentrations of Topo I in nucleolus. Propidium iodide staining and Br-UTP incorporation patterns revealed direct correlation between immunofluorescence intensity and rRNA transcription activity within nucleolus. Immuno-stained chromosomes during metaphase and anaphase suggested possible role of Topo I in resolving topological constrains during mitotic chromosome condensation. Inhibitor studies showed that in comparison to Topo I, Topo II was essential in resolving topological constrains during chromosome condensation. Probably, Topo II substituted Topo I functioning to certain extent during chromosome condensation, but not vice-versa. Transgenic Topo I tobacco lines revealed morphological abnormalities and highlighted its crucial role in plant morphogenesis and development. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Houttuynia cordata Thunb extract modulates G0/G1 arrest and Fas/CD95-mediated death receptor apoptotic cell death in human lung cancer A549 cells

PubMed Central

2013-01-01

Background Houttuynia cordata Thunb (HCT) is commonly used in Taiwan and other Asian countries as an anti-inflammatory, antibacterial and antiviral herbal medicine. In this study, we investigated the anti-human lung cancer activity and growth inhibition mechanisms of HCT in human lung cancer A549 cells. Results In order to investigate effects of HCT on A549 cells, MTT assay was used to evaluate cell viability. Flow cytometry was employed for cell cycle analysis, DAPI staining, and the Comet assay was used for DNA fragmentation and DNA condensation. Western blot analysis was used to analyze cell cycle and apoptotic related protein levels. HCT induced morphological changes including cell shrinkage and rounding. HCT increased the G0/G1 and Sub-G1 cell (apoptosis) populations and HCT increased DNA fragmentation and DNA condensation as revealed by DAPI staining and the Comet assay. HCT induced activation of caspase-8 and caspase-3. Fas/CD95 protein levels were increased in HCT-treated A549 cells. The G0/G1 phase and apoptotic related protein levels of cyclin D1, cyclin A, CDK 4 and CDK 2 were decreased, and p27, caspase-8 and caspase-3 were increased in A549 cells after HCT treatment. Conclusions The results demonstrated that HCT-induced G0/G1 phase arrest and Fas/CD95-dependent apoptotic cell death in A549 cells PMID:23506616

Cell cycle re-entry sensitizes podocytes to injury induced death.

PubMed

Hagen, Manuel; Pfister, Eva; Kosel, Andrea; Shankland, Stuart; Pippin, Jeffrey; Amann, Kerstin; Daniel, Christoph

2016-07-17

Podocytes are terminally differentiated renal cells, lacking the ability to regenerate by proliferation. However, during renal injury, podocytes re-enter into the cell cycle but fail to divide. Earlier studies suggested that re-entry into cell cycle results in loss of podocytes, but a direct evidence for this is lacking. Therefore, we established an in vitro model to test the consequences of re-entry into the cell cycle on podocyte survival. A mouse immortalized podocyte cell line was differentiated to non-permissive podocytes and stimulated with e.g. growth factors. Stimulated cells were analyzed for mRNA-expression or stained for cell cycle analysis using flow cytometry and immunocytofluorescence microscopy. After stimulation to re-entry into cell cycle, podocytes were stressed with puromycin aminonucleoside (PAN) and analyzed for survival. During permissive stage more than 40% of immortalized podocytes were in the S-phase. In contrast, S-phase in non-permissive differentiated podocytes was reduced to 5%. Treatment with b-FGF dose dependently induced re-entry into cell cycle increasing the number of podocytes in the S-phase to 10.7% at an optimal bFGF dosage of 10 ng/ml. Forty eight hours after stimulation with bFGF the number of bi-nucleated podocytes significantly increased. A secondary injury stimulus significantly reduced podocyte survival preferentially in bi-nucleated podocytes In conclusion, stimulation of podocytes using bFGF was able to induce re-entry of podocytes into the cell cycle and to sensitize the cells for cell death by secondary injuries. Therefore, this model is appropriate for testing new podocyte protective substances that can be used for therapy.

Cyclic mechanical stretch enhances BMP9-induced osteogenic differentiation of mesenchymal stem cells.

PubMed

Song, Yang; Tang, Yinhong; Song, Jinlin; Lei, Mingxing; Liang, Panpan; Fu, Tiwei; Su, Xudong; Zhou, Pengfei; Yang, Li; Huang, Enyi

2018-04-01

The purpose of this study was to investigate whether mechanical stretch can enhance the bone morphogenetic protein 9 (BMP9)-induced osteogenic differentiation in MSCs. Recombinant adenoviruses were used to overexpress the BMP9 in C3H10T1/2 MSCs. Cells were seeded onto six-well BioFlex collagen I-coated plates and subjected to cyclic mechanical stretch [6% elongation at 60 cycles/minute (1 Hz)] in a Flexercell FX-4000 strain unit for up to 12 hours. Immunostaining and confocal microscope were used to detect cytoskeleton organization. Cell cycle progression was checked by flow cytometry. Alkaline phosphatase activity was measured with a Chemiluminescence Assay Kit and was quantified with a histochemical staining assay. Matrix mineralization was examined by Alizarin Red S Staining. Mechanical stretch induces cytoskeleton reorganization and inhibits cell proliferation by preventing cells entry into S phase of the cell cycle. Although mechanical stretch alone does not induce the osteogenic differentiation of C3H10T1/2 MSCs, co-stimulation with mechanical stretch and BMP9 enhances alkaline phosphatase activity. The expression of key lineage-specific regulators (e.g., osteocalcin (OCN), SRY-related HMG-box 9, and runt-related transcription factor 2) is also increased after the co-stimulation, compared to the mechanical stretch stimulation along. Furthermore, mechanical stretch augments the BMP9-mediated bone matrix mineralization of C3H10T1/2 MSCs. Our results suggest that mechanical stretch enhances BMP9-induced osteoblastic lineage specification in C3H10T1/2 MSCs.

Events associated with apoptotic effect of p-Coumaric acid in HCT-15 colon cancer cells

PubMed Central

Jaganathan, Saravana Kumar; Supriyanto, Eko; Mandal, Mahitosh

2013-01-01

AIM: To investigate the events associated with the apoptotic effect of p-Coumaric acid, one of the phenolic components of honey, in human colorectal carcinoma (HCT-15) cells. METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tertazolium-bromide assay was performed to determine the antiproliferative effect of p-Coumaric acid against colon cancer cells. Colony forming assay was conducted to quantify the colony inhibition in HCT 15 and HT 29 colon cancer cells after p-Coumaric acid treatment. Propidium Iodide staining of the HCT 15 cells using flow cytometry was done to study the changes in the cell cycle of treated cells. Identification of apoptosis was done using scanning electron microscope and photomicrograph evaluation of HCT 15 cells after exposing to p-Coumaric acid. Levels of reactive oxygen species (ROS) of HCT 15 cells exposed to p-Coumaric acid was evaluated using 2’, 7’-dichlorfluorescein-diacetate. Mitochondrial membrane potential of HCT-15 was assessed using rhodamine-123 with the help of flow cytometry. Lipid layer breaks associated with p-Coumaric acid treatment was quantified using the dye merocyanine 540. Apoptosis was confirmed and quantified using flow cytometric analysis of HCT 15 cells subjected to p-Coumaric acid treatment after staining with YO-PRO-1. RESULTS: Antiproliferative test showed p-Coumaric acid has an inhibitory effect on HCT 15 and HT 29 cells with an IC50 (concentration for 50% inhibition) value of 1400 and 1600 μmol/L respectively. Colony forming assay revealed the time-dependent inhibition of HCT 15 and HT 29 cells subjected to p-Coumaric acid treatment. Propidium iodide staining of treated HCT 15 cells showed increasing accumulation of apoptotic cells (37.45 ± 1.98 vs 1.07 ± 1.01) at sub-G1 phase of the cell cycle after p-Coumaric acid treatment. HCT-15 cells observed with photomicrograph and scanning electron microscope showed the signs of apoptosis like blebbing and shrinkage after p-Coumaric acid exposure. Evaluation of the lipid layer showed increasing lipid layer breaks was associated with the growth inhibition of p-Coumaric acid. A fall in mitochondrial membrane potential and increasing ROS generation was observed in the p-Coumaric acid treated cells. Further apoptosis evaluated by YO-PRO-1 staining also showed the time-dependent increase of apoptotic cells after treatment. CONCLUSION: These results depicted that p-Coumaric acid inhibited the growth of colon cancer cells by inducing apoptosis through ROS-mitochondrial pathway. PMID:24282361

Influence of homeobox B2 antisense oligodeoxynucleotides on the biological characteristics of in vitro cultured primary human umbilical vein endothelial cells.

PubMed

Liu, X S; Zhang, X Q; Tian, T; Liu, L; Ming, J

2008-01-01

This study aims to explore the influence of homeobox B2 (HOXB2) antisense oligodeoxynucleotides (asodn) on the biological characteristics of in vitro cultured primary human umbilical vein endothelial cells (HUVECs). The distribution of HOXB2 asodn in the HUVECs was observed by fluorescent labelling, and the influence of different concentrations of HOXB2 asodn on the DNA synthesis of HUVECs was assessed. Flow cytometry and a reverse transcriptase-polymerase chain reaction (RT- PCR) method were employed to observe the influence of HOXB2 asodn on HOXB2 expression and the HUVEC cell cycle. After the induction of liposome, the nuclear fluorescent staining of HOXB2 asodn was weaker 15 min after transfection and the staining reached the strongest level at 4-8 h but then weakened and disappeared by 16 h after transfection. This indicated that endothelial DNA synthesis could be inhibited by HOXB2 asodn in a dose-dependent manner. Furthermore, the HUVECs could be delayed in their passage from G1 to S. Simultaneously, expression of HOXB2 mRNA had decreased significantly by 24-48 h after transfection. Clearly, HOXB2 plays important roles in the proliferation of endothelial cells and also affects the cell cycle.

Low dose radiation induced senescence of human mesenchymal stromal cells and impaired the autophagy process

PubMed Central

Alessio, Nicola; Del Gaudio, Stefania; Capasso, Stefania; Di Bernardo, Giovanni; Cappabianca, Salvatore; Cipollaro, Marilena; Peluso, Gianfranco; Galderisi, Umberto

2015-01-01

Low doses of radiation may have profound effects on cellular function. Individuals may be exposed to low doses of radiation either intentionally for medical purposes or accidentally, such as those exposed to radiological terrorism or those who live near illegal radioactive waste dumpsites. We studied the effects of low dose radiation on human bone marrow mesenchymal stromal cells (MSC), which contain a subpopulation of stem cells able to differentiate in bone, cartilage, and fat; support hematopoiesis; and contribute to body's homeostasis. The main outcome of low radiation exposure, besides reduction of cell cycling, is the triggering of senescence, while the contribution to apoptosis is minimal. We also showed that low radiation affected the autophagic flux. We hypothesize that the autophagy prevented radiation deteriorative processes, and its decline contributed to senescence. An increase in ATM staining one and six hours post-irradiation and return to basal level at 48 hours, along with persistent gamma-H2AX staining, indicated that MSC properly activated the DNA repair signaling, though some damages remained unrepaired, mainly in non-cycling cells. This suggested that the impaired DNA repair capacity of irradiated MSC seemed mainly related to the reduced activity of a non-homologous end-joining (NHEJ) system rather than HR (homologous recombination). PMID:25544750

[In vitro differentiation of synovial-derived mesenchymal stem cells infected by adenovirus vector mediated by bone morphogenetic protein 2/7 genes into fibrocartilage cells in rabbits].

PubMed

Fu, Peiliang; Zhang, Lei; Wu, Haishan; Cong, Ruijun; Chen, Song; Ding, Zheru; Hu, Kaimen

2013-03-01

To investigate the feasibility of rabbit synovial-derived mesenchymal stem cells (SMSCs) differentiating into fibrocartilage cells by the recombinant adenovirus vector mediated by bone morphogenetic protein 2/7 (BMP-2/7) genes in vitro. SMSCs were isolated and purified from 3-month-old New Zealand white rabbits [male or female, weighing (2.1 +/- 0.3) kg]; the morphology was observed; the cells were identified with immunocytological fluorescent staining, flow cytometry, and cell cycles. The adipogenic, osteogenic, and chondrogenic differentiations were detected. The recombinant plasmid of pAdTrack-BMP-2-internal ribosome entry site (IRES)-BMP-7 was constructed and then was used to infect SMSCs. The cell DNA content and the oncogenicity were tested to determine the safety. Then infected SMSCs were cultured in incomplete chondrogenic medium in vitro. Chondrogenic differentiation of infected SMSCs was detected by RT-PCR, immunofluorescent staining, and toluidine blue staining. SMSCs expressed surface markers of stem cells, and had multi-directional potential. The transfection efficiency of SMSCs infected by recombinant plasmid of pAdTrack-BMP-2-IRES-BMP-7 was about 70%. The safety results showed that infected SMSCs had normal double time, normal chromosome number, and normal DNA content and had no oncogenicity. At 21 days after cultured in incomplete chondrocyte medium, RT-PCR results showed SMSCs had increased expressions of collegan type I and collegan type II, particularly collegan type II; the expressions of RhoA and Sox-9 increased obviously. Immunofluorescent staining and toluidine blue staining showed differentiation of SMSCs into fibrocartilage cells. It is safe to use pAdTrack-BMP-2-IRES-BMP-7 for infecting SMSCs. SMSCs infected by pAdTrack-BMP-2-IRES-BMP-7 can differentiate into fibrocartilage cells spontaneously in vitro.

A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells.

PubMed

Chen, C; Yang, R L

2013-08-01

MP [4-(3',3'-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27(KIP1) protein and p21(CIP1) mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21(CIP1), p16(INK4a) and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer.

Regulation of accumulation of ammonium-inducible glutamate dehydrogenase catalytic activity and antigen during the cell cycle of fully induced, synchronous Chlorella sorokiniana cells.

PubMed

Yeung, A T; Bascomb, N F; Turner, K J; Schmidt, R R

1981-05-01

By use of a rocket immunoelectrophoresis-activity stain procedure, it was shown that catalytic activity of an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) was accompanied by a coincident increase in enzyme antigen during the cell cycle of preinduced synchronous Chlorella sorokiniana cells growing in the continuous presence of ammonia. Between the fourth and fifth hours of the G-1 phase of the cell cycle, a three- to fourfold increase in linear accumulation of enzyme antigen was observed. Pulse-chase studies with [35S]sulfate, coupled with a specific indirect immunoadsorption procedure for enzyme antigen, showed that NADP-GDH antigen undergoes continuous degradation (i.e., a half-life of 88 to 110 min) during its linear pattern of accumulation during the cell cycle. The apparent half-life of the enzyme increased by approximately 23% of the 4.5-h positive rate change in antigen accumulation during the cell cycle. This increase in half-life is insufficient in itself to account for the large change in rate of NADP-GDH antigen accumulation. The data from immunoelectrophoresis, pulse-chase, and initial 35S incorporation rate experiments taken together support the inference that changes in the rate of NADP-GDH synthesis are primarily responsible for the accumulation patterns of NADP-GDH activity during the C. sorokiniana cell cycle.

[Microscopic structure of the epithelium of the oviducts in cows during the estrus cycle].

PubMed

Uhrín, V

1983-03-01

The mucous membrane of a cow is covered with ciliary and secretory cells. The so-called basal cells occur at the basal membrane. The counts of ciliary cells vary during the sexual cycle: they reach the maximum (up to 68%) during oestrus. About 13% of cells lose cilia during metoestrus and at the beginning of dioestrus. Reciliation occurs during pro-oestrus. Light and dark ciliary cells can be discerned by the staining of cytoplasm and by the density of nuclei. A higher variability was found in the secretory cells. There are light and dark cells, cells with a wedge shape and rod-shaped cells. Their frequency and function are discussed. Mitoses of epithelium were found in rare cases. The relative volume of epithelium and the mucous membrane of connective tissues change during the sexual cycle. The volume of secretory cells increases during metoestrus and dioestrus and the volume of ciliary cells increases during pro-oestrus and heat. The volume of nuclei decreases in metoestrus and mainly in dioestrus. PAS positive granules occur in the cytoplasm of secretory cells, mainly during metoestrus, in the apical regions. Ptyalin-resistant polysaccharides, besides glycogen, were detected in the cells. The occurrence rate of lipids varies just slightly during the oestrous cycle.

Practical selection methods for rat and mouse round spermatids without DNA staining by flow cytometric cell sorting.

PubMed

Hayama, Tomonari; Yamaguchi, Tomoyuki; Kato-Itoh, Megumi; Ishii, Yumiko; Mizuno, Naoaki; Umino, Ayumi; Sato, Hideyuki; Sanbo, Makoto; Hamanaka, Sanae; Masaki, Hideki; Hirabayashi, Masumi; Nakauchi, Hiromitsu

2016-06-01

Round spermatid injection (ROSI) into unfertilized oocytes enables a male with a severe spermatogenesis disorder to have children. One limitation of the application of this technique in the clinic is the identification and isolation of round spermatids from testis tissue. Here we developed an efficient and simple method to isolate rodent haploid round spermatids using flow cytometric cell sorting, based on DNA content (stained with Hoechst 33342 or Dye Cycle Violet) or by cell diameter and granularity (forward and side scatter). ROSI was performed with round spermatids selected by flow cytometry, and we obtained healthy offspring from unstained cells. This non-invasive method could therefore be an effective option for breeding domestic animals and human male infertility treatment. Mol. Reprod. Dev. 83: 488-496, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Cell cycle re-entry sensitizes podocytes to injury induced death

PubMed Central

Hagen, Manuel; Pfister, Eva; Kosel, Andrea; Shankland, Stuart; Pippin, Jeffrey; Amann, Kerstin; Daniel, Christoph

2016-01-01

ABSTRACT Podocytes are terminally differentiated renal cells, lacking the ability to regenerate by proliferation. However, during renal injury, podocytes re-enter into the cell cycle but fail to divide. Earlier studies suggested that re-entry into cell cycle results in loss of podocytes, but a direct evidence for this is lacking. Therefore, we established an in vitro model to test the consequences of re-entry into the cell cycle on podocyte survival. A mouse immortalized podocyte cell line was differentiated to non-permissive podocytes and stimulated with e.g. growth factors. Stimulated cells were analyzed for mRNA-expression or stained for cell cycle analysis using flow cytometry and immunocytofluorescence microscopy. After stimulation to re-entry into cell cycle, podocytes were stressed with puromycin aminonucleoside (PAN) and analyzed for survival. During permissive stage more than 40% of immortalized podocytes were in the S-phase. In contrast, S-phase in non-permissive differentiated podocytes was reduced to 5%. Treatment with b-FGF dose dependently induced re-entry into cell cycle increasing the number of podocytes in the S-phase to 10.7% at an optimal bFGF dosage of 10 ng/ml. Forty eight hours after stimulation with bFGF the number of bi-nucleated podocytes significantly increased. A secondary injury stimulus significantly reduced podocyte survival preferentially in bi-nucleated podocytes In conclusion, stimulation of podocytes using bFGF was able to induce re-entry of podocytes into the cell cycle and to sensitize the cells for cell death by secondary injuries. Therefore, this model is appropriate for testing new podocyte protective substances that can be used for therapy. PMID:27232327

Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342.

PubMed

Petersen, Timothy W; Ibrahim, Sherrif F; Diercks, Alan H; van den Engh, Ger

2004-08-01

Many methods in flow cytometry rely on staining DNA with a fluorescent dye to gauge DNA content. From the relative intensity of the fluorescence signature, one can then infer position in cell cycle, amount of DNA (i.e., for sperm selection), or, as in the case of flow karyotyping, to distinguish individual chromosomes. This work examines the staining of murine thymocytes with a common DNA dye, Hoechst 33342, to investigate nonlinearities in the florescence intensity as well as chromatic shifts. Murine thymocytes were stained with Hoechst 33342 and measured in a flow cytometer at two fluorescence emission bands. In other measurements, cells were stained at different dye concentrations, and then centrifuged. The supernatant was then used for a second round of staining to test the amount of dye uptake. Finally, to test for resonant energy transfer, we measured fluorescence anisotropy at two different wavelengths. The fluorescence of cells stained with Hoechst 33342 is a nonlinear process that shows an overall decrease in intensity with increased dye uptake, and spectral shift to the red. Along with the spectral shift of the fluorescence to the longer wavelengths, we document decreases in the fluorescence anisotropy that may indicate resonant energy transfer. At low concentrations, Hoechst 33342 binds to the minor groove of DNA and shows an increase in fluorescence and a blue shift upon binding. At higher concentrations, at which the dye molecules can no longer bind without overlapping, the blue fluorescence decreases and the red fluorescence increases until there is approximately one dye molecule per DNA base pair. The ratio of the blue fluorescence to the red fluorescence is an accurate indicator of the cellular dye concentration.

Evaluation of a menstrual cup to collect shed endometrium for in vitro studies.

PubMed

Koks, C A; Dunselman, G A; de Goeij, A F; Arends, J W; Evers, J L

1997-09-01

To evaluate whether a menstrual cup is a suitable instrument to collect antegradely shed endometrium for in vitro studies. A prospective, descriptive, cell biological and immunohistochemical study. Tertiary care university medical center. Nine female volunteers with regular cycles. Menstrual effluent was collected with a menstrual cup. Experience with the menstrual cup was described. Cytospin specimens, frozen sections, and cultures were prepared from the obtained menstrual tissue. The acceptability of the menstrual cup. The presence and viability of endometrial tissue was evaluated using immunohistochemical staining and culture outcome. All women except one described the menstrual cup as acceptable. Menstrual effluent contained single cells, clumps of cells, and glandlike structures. After 5 days of culture, the endometrial tissue appeared to be viable. Immunohistochemistry showed positive staining for vimentin in most cytospin specimens, in all cryostat specimens, and in 10 of 17 cultures. Cytokeratin 18 stained most cytospin specimens, all cryostat specimens, and 10 of 17 cultures. Positive staining for BW495/36 was observed in most cytospin specimens, all cryostat specimens, and 11 of 17 cultures. A menstrual cup in an acceptable instrument to collect antegradely shed menstrual tissue. Menstruum contains viable endometrial tissue that can be used for in vitro studies of endometrium and endometriosis.

A protocol for the simultaneous identification of chitin-containing particles and their associated bacteria.

PubMed

Biancalana, Florencia; Kopprio, Germán A; Lara, Rubén J; Alonso, Cecilia

2017-07-01

Chitin is the second most abundant polymer on Earth, playing a crucial role in the biogeochemical cycles. A core issue for studying its processing in aquatic systems is the identification and enumeration of chitin-containing particles and organisms, ideally in a manner that can be directly linked to bulk chitin quantification. The aim of this study was the development of such a technique. We successfully combined the methodology of bulk chitin determination using wheat germ agglutinin (FITC-WGA) for staining chitin-containing particles and organisms along with CARD-FISH staining of either chitin-containing eukaryotic cells or bacteria associated with them. Environmental chitin staining was successfully applied to natural water samples. Fungal hyphae, diatoms, and dinoflagellates, sestonic aggregates and chitin-containing structures derived from metazoa were observed. Also, hybridized bacteria attached to chitinaceous debris were clearly visualized. Finally, as proof of principle, cultured yeast cells were simultaneously-targeted by FITC-WGA and the EUK516 probe without exhibiting any interference between both stains. The presented approach appears as a powerful tool to evaluate the contribution of different size classes and organisms to chitin production and consumption, opening the possibility for application of single-cell approaches targeting the ecophysiology of chitin transformations in aquatic systems. Copyright © 2017 Elsevier GmbH. All rights reserved.

Single-Cell in Situ RNA Analysis With Switchable Fluorescent Oligonucleotides.

PubMed

Xiao, Lu; Guo, Jia

2018-01-01

Comprehensive RNA analyses in individual cells in their native spatial contexts promise to transform our understanding of normal physiology and disease pathogenesis. Here we report a single-cell in situ RNA analysis approach using switchable fluorescent oligonucleotides (SFO). In this method, transcripts are first hybridized by pre-decoding oligonucleotides. These oligonucleotides subsequently recruit SFO to stain their corresponding RNA targets. After fluorescence imaging, all the SFO in the whole specimen are simultaneously removed by DNA strand displacement reactions. Through continuous cycles of target staining, fluorescence imaging, and SFO removal, a large number of different transcripts can be identified by unique fluorophore sequences and visualized at the optical resolution. To demonstrate the feasibility of this approach, we show that the hybridized SFO can be efficiently stripped by strand displacement reactions within 30 min. We also demonstrate that this SFO removal process maintains the integrity of the RNA targets and the pre-decoding oligonucleotides, and keeps them hybridized. Applying this approach, we show that transcripts can be restained in at least eight hybridization cycles with high analysis accuracy, which theoretically would enable the whole transcriptome to be quantified at the single molecule sensitivity in individual cells. This in situ RNA analysis technology will have wide applications in systems biology, molecular diagnosis, and targeted therapies.

MERTK Inhibition Induces Polyploidy and Promotes Cell Death and Cellular Senescence in Glioblastoma Multiforme

PubMed Central

Sufit, Alexandra; Lee-Sherick, Alisa B.; DeRyckere, Deborah; Rupji, Manali; Dwivedi, Bhakti; Varella-Garcia, Marileila; Pierce, Angela M.; Kowalski, Jeanne; Wang, Xiaodong; Frye, Stephen V.; Earp, H. Shelton

2016-01-01

Background MER receptor tyrosine kinase (MERTK) is expressed in a variety of malignancies, including glioblastoma multiforme (GBM). Our previous work demonstrated that inhibition of MERTK using RNA interference induced cell death and chemosensitivity in GBM cells, implicating MERTK as a potential therapeutic target. Here we investigate whether a novel MERTK-selective small molecule tyrosine kinase inhibitor, UNC2025, has similar anti-tumor effects in GBM cell lines. Methods Correlations between expression of GAS6, a MERTK ligand, and prognosis were determined using data from the TCGA database. GBM cell lines (A172, SF188, U251) were treated in vitro with increasing doses of UNC2025 (50-400nM). Cell count and viability were determined by trypan blue exclusion. Cell cycle profiles and induction of apoptosis were assessed by flow cytometric analysis after BrdU or Po-Pro-1/propidium iodide staining, respectively. Polyploidy was detected by propidium iodide staining and metaphase spread. Cellular senescence was determined by β-galactosidase staining and senescence-associated secretory cytokine analysis. Results Decreased overall survival significantly correlated with high levels of GAS6 expression in GBM, highlighting the importance of TAM kinase signaling in GBM tumorigenesis and/or therapy resistance and providing strong rationale for targeting these pathways in the clinic. All three GBM cell lines exhibited dose dependent reductions in cell number and colony formation (>90% at 200nM) after treatment with UNC2025. Cell cycle analysis demonstrated accumulation of cells in the G2/M phase and development of polyploidy. After extended exposure, 60–80% of cells underwent apoptosis. The majority of surviving cells (65–95%) were senescent and did not recover after drug removal. Thus, UNC2025 mediates anti-tumor activity in GBM by multiple mechanisms. Conclusions The findings described here provide further evidence of oncogenic roles for MERTK in GBM, demonstrate the importance of kinase activity for MERTK tumorigenicity and validate UNC2025, a novel MERTK inhibitor, as a potential therapeutic agent for treatment of GBM. PMID:27783662

Comprehensive Mass Cytometry Analysis of Cell Cycle, Activation, and Coinhibitory Receptors Expression in CD4 T Cells from Healthy and HIV-Infected Individuals.

PubMed

Corneau, Aurélien; Cosma, Antonio; Even, Sophie; Katlama, Christine; Le Grand, Roger; Frachet, Véronique; Blanc, Catherine; Autran, Brigitte

2017-01-01

Mass cytometry allows large multiplex analysis of cell cycle stages together with differentiation, activation, and exhaustion markers, allowing further assessment of the quiescence status of resting CD4 T cells. Peripheral blood CD4 T lymphocytes from 8 individuals, 4 healthy donors, and 4 HIV-infected on antiretroviral treatment (T) were stained with the same 26 monoclonal antibodies and dyes targeting surface and intracellular markers of differentiation, activation, exhaustion, and cell cycle stages. Samples were run on a CYTOF-2. Patterns of naïve [TN] CD4 T cells strongly differed from all other memory subsets central-memory (CM), transitional-memory (TM), effector-memory (EM), and terminally differentiated RA-expressing (TEMRA) subsets, while stem-cell memory (SCM) and T follicular-helper cells (TfH) were close to CM and TM cells with the highest percentages in cell cycle. EM and TEMRA were the most altered by HIV infection, with an increased frequency of activated and cycling cells. Activation markers and coinhibitory receptor expression differed among cell cycle stages, with HLA-DR fitting better than CD25 or CD38 with cycle, and opposite PD-1 gradients along differentiation and cell cycle. "Resting" DR-CD25- CD4+ T cells contained similar amounts of cells in G1 than the activated DR ± CD25± ones but three fold lower cells in S-G2-M. This broad multiplex mass cytometry analysis demonstrates some subsets of the so-called "resting" CD25-DR- CD4+ T cells contain noticeable amounts of cells into cycle or expressing coinhibitory receptors, opening new avenues for a redefinition of resting peripheral blood CD4 T cells harboring the HIV reservoirs. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

Cell cycle-dependent protein fingerprint from a single cancer cell: image cytometry coupled with single-cell capillary sieving electrophoresis.

PubMed

Hu, Shen; Le, Zhang; Krylov, Sergey; Dovichi, Norman J

2003-07-15

Study of cell cycle-dependent protein expression is important in oncology, stem cell research, and developmental biology. In this paper, we report the first protein fingerprint from a single cell with known phase in the cell cycle. To determine that phase, we treated HT-29 colon cancer cells with Hoescht 33342, a vital nuclear stain. A microscope was used to measure the fluorescence intensity from one treated cell; in this form of image cytometry, the fluorescence intensity is proportional to the cell's DNA content, which varies in a predictable fashion during the cell cycle. To generate the protein fingerprint, the cell was aspirated into the separation capillary and lysed. Proteins were fluorescently labeled with 3-(2-furoylquinoline-2-carboxaldehyde, separated by capillary sieving electrophoresis, and detected by laser-induced fluorescence. This form of electrophoresis is the capillary version of SDS-PAGE. The single-cell electropherogram partially resolved approximately 25 components in a 30-min separation, and the dynamic range of the detector exceeded 5000. There was a large cell-to-cell variation in protein expression, averaging 40% relative standard deviation across the electropherogram. The dominant source of variation was the phase of the cell in the cell cycle; on average, approximately 60% of the cell-to-cell variance in protein expression was associated with the cell cycle. Cells in the G1 and G2/M phases of the cell cycle had 27 and 21% relative standard deviations in protein expression, respectively. Cells in the G2/M phase generated signals that were twice the amplitude of the signals generated by G1 phase cells, as expected for cells that are soon to divide into two daughter cells. When electropherograms were normalized to total protein content, the expression of only one component was dependent on cell cycle at the 99% confidence limit. That protein is tentatively identified as cytokeratin 18 in a companion paper.

Cinematographic observations of growth cycles of Chlamydia trachomatis in primary cultures of human amniotic cells.

PubMed Central

Neeper, I D; Patton, D L; Kuo, C C

1990-01-01

Time-lapse cinematography was used to study the growth cycle of Chlamydia trachomatis in primary cell cultures of human amnion. Twelve preterm and twelve term placentas were obtained within 8 h of delivery, and epithelial cells were dissociated from the amniotic membranes by trypsinization and grown in Rose chambers. The epithelial nature of the cultured cells was documented by morphology and by immunofluorescence staining for cytoskeletal proteins, which matched the staining of intact amnion. With regular feedings, uninfected cultures remained healthy for up to 30 days. Confluent cultures (7 to 10 days) were infected with a genital strain (E/UW-5/CX) of C. trachomatis at 10(5) infectious units per chamber. Infections were done in culture medium without cycloheximide, which is often used to induce susceptibility of the cells. Between 66 and 90% of the cells were infected. Intracytoplasmic inclusions were visible by 18 h post infection (p.i.) and grew larger as the organisms inside multiplied. By 72 h p.i., the inclusions occupied the entire cytoplasm of the host cells. Further growth of the inclusions overdistended and ruptured the host cells on days 3 to 7. Cells not infected by the original inoculum became infected on day 5 or 6 p.i. by the chlamydial particles released from the ruptured cells. No amniotic cell was ever observed to survive the infection. The data presented support the hypothesis that amniotic epithelium is susceptible to infection and damage by C. trachomatis. This culture system provided detailed and dynamic observations of chlamydial infection under conditions more nearly physiologic than previously reported. Images PMID:2365450

Cnidium officinale Makino extract induces apoptosis through activation of caspase-3 and p53 in human liver cancer HepG2 cells

PubMed Central

Hong, Heeok; An, Jeong Cheol; de La Cruz, Joseph F.; Hwang, Seong-Gu

2017-01-01

A number of diverse studies have reported the anticancer properties of Cnidium officinale Makino (CO). However, the apoptotic effect of this traditional medicinal herb in human hepatocellular carcinoma cells (HepG2) remains to be elucidated. Therefore, the present study investigated the ability of CO to reduce cell viability through apoptotic pathways. Cell viability was determined using the 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide assay. CO extract-induced apoptosis in HepG2 cells was assessed by Hoechst 33258 staining. The cell cycle was monitored using fluorescence-activated cell sorting analysis with propidium iodide staining. Furthermore, the present study explored whether various signaling molecules associated with HepG2 cell death were affected by CO treatment, including caspase-3, B-cell lymphoma 2 (Bcl-2), tumor protein p53 (p53), cyclin-dependent kinase 4 (CDK4) and cyclin D. The expression levels of these genes were examined by reverse-transcription polymerase chain reaction and western blotting. The expression levels of caspase-3 and p53 were upregulated with CO extract treatment, whereas those of Bcl-2, CDK4 and cyclin D were significantly downregulated. Cleaved caspase-3 expression was upregulated following treatment with CO extract in a dose-dependent manner. Collectively, the data suggest that CO extract has the potential to induce apoptosis of HepG2 cells and may act by suppressing the cell cycle, which leads to caspase-3 cleavage and p53 signaling. PMID:28966688

Replicative stress and alterations in cell cycle checkpoint controls following acetaminophen hepatotoxicity restrict liver regeneration.

PubMed

Viswanathan, Preeti; Sharma, Yogeshwar; Gupta, Priya; Gupta, Sanjeev

2018-03-05

Acetaminophen hepatotoxicity is a leading cause of hepatic failure with impairments in liver regeneration producing significant mortality. Multiple intracellular events, including oxidative stress, mitochondrial damage, inflammation, etc., signify acetaminophen toxicity, although how these may alter cell cycle controls has been unknown and was studied for its significance in liver regeneration. Assays were performed in HuH-7 human hepatocellular carcinoma cells, primary human hepatocytes and tissue samples from people with acetaminophen-induced acute liver failure. Cellular oxidative stress, DNA damage and cell proliferation events were investigated by mitochondrial membrane potential assays, flow cytometry, fluorescence staining, comet assays and spotted arrays for protein expression after acetaminophen exposures. In experimental groups with acetaminophen toxicity, impaired mitochondrial viability and substantial DNA damage were observed with rapid loss of cells in S and G2/M and cell cycle restrictions or even exit in the remainder. This resulted from altered expression of the DNA damage regulator, ATM and downstream transducers, which imposed G1/S checkpoint arrest, delayed entry into S and restricted G2 transit. Tissues from people with acute liver failure confirmed hepatic DNA damage and cell cycle-related lesions, including restrictions of hepatocytes in aneuploid states. Remarkably, treatment of cells with a cytoprotective cytokine reversed acetaminophen-induced restrictions to restore cycling. Cell cycle lesions following mitochondrial and DNA damage led to failure of hepatic regeneration in acetaminophen toxicity but their reversibility offers molecular targets for treating acute liver failure. © 2018 John Wiley & Sons Ltd.

Induction of apoptosis in K562 cells by dicyclohexylammonium salt of hyperforin through a mitochondrial-related pathway.

PubMed

Liu, Jin-Yun; Liu, Zhong; Wang, Dong-Mei; Li, Man-Mei; Wang, Shao-Xiang; Wang, Rui; Chen, Jian-Ping; Wang, Yi-Fei; Yang, De-Po

2011-04-25

Hyperforin is an abundant phloroglucinol-type constituent isolated from the extract of the flowering upper portion of the plant Hypericum perforatum L. The dicyclohexylammonium salt of hyperforin (DCHA-HF) has exhibited antitumor and antiangiogenic activities in various cancer cells. Here, the antitumor effects of DCHA-HF on the chronic myeloid leukemia K562 cell line were investigated for the first time. DCHA-HF exhibited dose- and time-dependent inhibitory activities against K562 cells, with IC(50) values of 8.6 and 3.2 μM for 48 h and 72 h of treatment, respectively, which was more effective than that of the hyperforin. In contrast, little cytotoxic activity was observed with DCHA-HF on HUVECs. DCHA-HF treatment resulted in induction of apoptosis as evidenced from DNA fragmentation, nuclear condensation and increase of early apoptotic cells by DAPI staining analysis, TUNEL assay and Annexin V-FITC/PI double-labeled staining analysis, respectively. Moreover, DCHA-HF elicited dissipation of mitochondrial transmembrane potential that commenced with the release of cytochrome c through down-regulation of expression of anti-apoptotic proteins and up-regulation of expression of pro-apoptotic proteins. DCHA-HF treatment induced activation of the caspase 3, 8, and 9 cascade and subsequent PARP cleavage, and DCHA-HF-induced apoptosis was significantly inhibited by caspase inhibitors. Treated cells were arrested at the G1 phase of the cell cycle and the expression of p53 and p27(Kip1), two key regulators related to cell cycle and apoptosis, was up-regulated. These results suggest that DCHA-HF inhibits K562 cell growth by inducing caspase-dependent apoptosis mediated by a mitochondrial pathway and arresting the cell cycle at the G1 phase. Therefore, DCHA-HF is a potential chemotherapeutic antitumor drug for chronic myeloid leukemia therapy. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Expression of progesterone receptor protein in the ovine uterus during the estrous cycle: Effects of nutrition, arginine and FSH.

PubMed

Grazul-Bilska, Anna T; Thammasiri, Jiratti; Kraisoon, Aree; Reyaz, Arshi; Bass, Casie S; Kaminski, Samantha L; Navanukraw, Chainarong; Redmer, Dale A

2018-03-01

To evaluate expression of progesterone receptor (PGR) AB in follicle stimulating hormone (FSH)-treated or non-treated sheep administered with arginine (Arg) or saline (Sal) fed a control (C), excess (O) or restricted (U) diet, uterine tissues were collected at the early, mid and/or late luteal phases. In exp. 1, ewes from each diet were randomly assigned to one of two treatments, Arg or Sal administration three times daily from day 0 of the first estrous cycle until uterine tissue collection. In exp. 2, ewes were injected twice daily with FSH on days 13-15 of the first estrous cycle. Uterine tissues were immunostained to detect PGR followed by image analysis. PGR were detected in luminal epithelium (LE), endometrial glands (EG), endometrial stroma (ES), myometrium (Myo), and endometrial and myometrial blood vessels. The percentage of PR-positive cells and/or intensity of staining were affected by phase of the estrous cycle, plane of nutrition, and/or FSH but not by Arg. In exp. 1, percentage of PGR-positive cells in LE and EG but not in ES and Myo was greater at the early and mid than late luteal phase, was not affected by plane of nutrition, and was similar in LE and EG. Intensity of staining was affected by phase of the estrous cycle and plane of nutrition in LE, EG and Myo, and was the greatest in LE, less in EG, and least in ES and Myo. In exp. 2, percentage of PGR-positive cells in LE, EG, ES and Myo was affected by phase of the estrous cycle, but not by plane of nutrition; was greater at the early than mid luteal phase; and was greatest in LE and EG, less in luminal (superficial) ES and Myo and least in deep ES. Intensity of staining was affected by phase of the estrous cycle and plane of nutrition in all compartments but ES, and was the greatest in LE and luminal EG, less in deep EG, and least in ES and Myo. Comparison of data for FSH (superovulated) and Sal-treated (non-superovulated) ewes demonstrated that FSH affected PR expression in all evaluated uterine compartments depending on plane of nutrition and phase of the estrous cycle. Thus, PGR are differentially distributed in uterine compartments, and PGR expression is affected by nutritional plane and FSH, but not Arg depending on phase of the estrous cycle. Such changes in dynamics of PGR expression indicate that diet plays a regulatory role and that FSH-treatment may alter uterine functions. Copyright © 2017 Elsevier Inc. All rights reserved.

Breast cancer metastasizing to the stomach mimicking primary gastric cancer: A case report

PubMed Central

Yim, Kwangil; Ro, Sang Mi; Lee, Jieun

2017-01-01

Breast cancer with stomach metastasis rare with an incidence of 1% or less among metastatic breast cancer patients. We experienced a case of breast cancer metastasizing to the stomach in 65-year-old female patient. She experienced dyspepsia and poor oral intake before visiting the clinic. Diffuse infiltration with nodular mucosal thickening of the stomach wall was observed, suggesting advanced gastric cancer based on gross endoscopic finding. Spread of poorly cohesive tumor cells in the gastric mucosa observed upon hematoxylin and eosin stain resembled signet ring cell carcinoma, but diffuse positive staining for GATA3 in immunohistochemical stain allowed for a conclusive diagnosis of breast cancer metastasizing to the stomach. Based on the final diagnosis, systemic chemotherapy was administered instead of primary surgical resection. After 2 cycles of docetaxel administration, she showed a partial response based on abdominal computed tomography scan. This case is an unusual presentation of breast cancer metastasizing to the gastrointestinal tract. PMID:28405154

Breast cancer metastasizing to the stomach mimicking primary gastric cancer: A case report.

PubMed

Yim, Kwangil; Ro, Sang Mi; Lee, Jieun

2017-03-28

Breast cancer with stomach metastasis rare with an incidence of 1% or less among metastatic breast cancer patients. We experienced a case of breast cancer metastasizing to the stomach in 65-year-old female patient. She experienced dyspepsia and poor oral intake before visiting the clinic. Diffuse infiltration with nodular mucosal thickening of the stomach wall was observed, suggesting advanced gastric cancer based on gross endoscopic finding. Spread of poorly cohesive tumor cells in the gastric mucosa observed upon hematoxylin and eosin stain resembled signet ring cell carcinoma, but diffuse positive staining for GATA3 in immunohistochemical stain allowed for a conclusive diagnosis of breast cancer metastasizing to the stomach. Based on the final diagnosis, systemic chemotherapy was administered instead of primary surgical resection. After 2 cycles of docetaxel administration, she showed a partial response based on abdominal computed tomography scan. This case is an unusual presentation of breast cancer metastasizing to the gastrointestinal tract.

Tributyltin induces G2/M cell cycle arrest via NAD(+)-dependent isocitrate dehydrogenase in human embryonic carcinoma cells.

PubMed

Asanagi, Miki; Yamada, Shigeru; Hirata, Naoya; Itagaki, Hiroshi; Kotake, Yaichiro; Sekino, Yuko; Kanda, Yasunari

2016-04-01

Organotin compounds, such as tributyltin (TBT), are well-known endocrine-disrupting chemicals (EDCs). We have recently reported that TBT induces growth arrest in the human embryonic carcinoma cell line NT2/D1 at nanomolar levels by inhibiting NAD(+)-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the irreversible conversion of isocitrate to α-ketoglutarate. However, the molecular mechanisms by which NAD-IDH mediates TBT toxicity remain unclear. In the present study, we examined whether TBT at nanomolar levels affects cell cycle progression in NT2/D1 cells. Propidium iodide staining revealed that TBT reduced the ratio of cells in the G1 phase and increased the ratio of cells in the G2/M phase. TBT also reduced cell division cycle 25C (cdc25C) and cyclin B1, which are key regulators of G2/M progression. Furthermore, apigenin, an inhibitor of NAD-IDH, mimicked the effects of TBT. The G2/M arrest induced by TBT was abolished by NAD-IDHα knockdown. Treatment with a cell-permeable α-ketoglutarate analogue recovered the effect of TBT, suggesting the involvement of NAD-IDH. Taken together, our data suggest that TBT at nanomolar levels induced G2/M cell cycle arrest via NAD-IDH in NT2/D1 cells. Thus, cell cycle analysis in embryonic cells could be used to assess cytotoxicity associated with nanomolar level exposure of EDCs.

[CHROMATIN ORGANIZATION IN CELL CYCLE OF AMOEBA PROTEUS ACCORDING TO OPTICAL TOMOGRAPHY DATA].

PubMed

Demin, S Yu; Berdieva, M A; Podlipaeva, Yu I; Yudin, A L; Goodkov, A V

2015-01-01

For the first time the nuclear cycle of large freshwater amoeba Amoeba proteus was studied by the method of optical tomography. The nuclei were fixed in situ in the cells of synchronized culture, stained by DAPI and examined by confocal laser scanning microscope. 3D-images of intranuclear chromatin were studied in details at different stages of nuclear cycle. The obtained data, together with literary ones allow represent the dynamics of structural organization of the nucleus in Amoeba proteus cell cycle in a new fashion. It was concluded that in this species the two-stage interphase takes place, as well as mitosis of peculiar type which does not correspond to any known type of mitosis according to classification existing now. It is presumed that in the course of nuclear cycle the chromosomes and/or their fragments are amplified, this presumption being in a good correspondence with the data about nuclear DNA hyperreplication in the cell cycle of A. proteus. As a result of chromosomes amplification their number may vary at different stages of cell cycle, and it allows to explain the contradictory data concerning the exact number of chromosomes in this species. The elimination of extra-DNA occurs mainly at the stage between prophase and prometaphase. We presume the majority of chromosomes, or may be even all of them to be referred to cholocentric type according to their behaviour during the mitosis.

Cryptotanshinone induces cell cycle arrest and apoptosis through the JAK2/STAT3 and PI3K/Akt/NFκB pathways in cholangiocarcinoma cells.

PubMed

Ke, Fayong; Wang, Zheng; Song, Xiaoling; Ma, Qiang; Hu, Yunping; Jiang, Lin; Zhang, Yijian; Liu, Yingbin; Zhang, Yong; Gong, Wei

2017-01-01

Cholangiocarcinoma (CCA) is the most common biliary tract malignancy in the world with high resistance to current chemotherapies and extremely poor prognosis. The main objective of this study was to investigate the inhibitory effects of cryptotanshinone (CTS), a natural compound isolated from Salvia miltiorrhiza Bunge , on CCA both in vitro and in vivo and to explore the underlying mechanisms of CTS-induced apoptosis and cell cycle arrest. The anti-tumor activity of CTS on HCCC-9810 and RBE cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and colony forming assays. Cell cycle changes were detected by flow cytometric analysis. Apoptosis was detected by annexin V/propidium iodide double staining and Hoechst 33342 staining assays. The efficacy of CTS in vivo was evaluated using a HCCC-9810 xenograft model in athymic nude mice. The expression of key proteins involved in cell apoptosis and signaling pathway in vitro was analyzed by Western blot analysis. CTS induced potent growth inhibition, S-phase arrest, apoptosis, and colony-forming inhibition in HCCC-9810 and RBE cells in a dose-dependent manner. Intraperitoneal injection of CTS (0, 10, or 25 mg/kg) for 4 weeks significantly inhibited the growth of HCCC-9810 xenografts in athymic nude mice. CTS treatment induced S-phase arrest with a decrease of cyclin A1 and an increase of cyclin D1 protein level. Bcl-2 expression was downregulated remarkably, while Bax expression was increased after apoptosis occurred. Additionally, the activation of JAK2/STAT3 and PI3K/Akt/NFκB was significantly inhibited in CTS-treated CCA cells. CTS induced CCA cell apoptosis by suppressing both the JAK2/STAT3 and PI3K/Akt/NFκB signaling pathways and altering the expression of Bcl-2/Bax family, which was regulated by these two signaling pathways. CTS may serve as a potential therapeutic agent for CCA.

DNA replication stress induces deregulation of the cell cycle events in root meristems of Allium cepa

PubMed Central

Żabka, Aneta; Polit, Justyna Teresa; Maszewski, Janusz

2012-01-01

Background and Aims Prolonged treatment of Allium cepa root meristems with changing concentrations of hydroxyurea (HU) results in either premature chromosome condensation or cell nuclei with an uncommon form of biphasic chromatin organization. The aim of the current study was to assess conditions that compromise cell cycle checkpoints and convert DNA replication stress into an abnormal course of mitosis. Methods Interphase-mitotic (IM) cells showing gradual changes of chromatin condensation were obtained following continuous 72 h treatment of seedlings with 0·75 mm HU (without renewal of the medium). HU-treated root meristems were analysed using histochemical stainings (DNA-DAPI/Feulgen; starch-iodide and DAB staining for H2O2 production), Western blotting [cyclin B-like (CBL) proteins] and immunochemistry (BrdU incorporation, detection of γ-H2AX and H3S10 phosphorylation). Key Results Continuous treatment of onion seedlings with a low concentration of HU results in shorter root meristems, enhanced production of H2O2, γ-phosphorylation of H2AX histones and accumulation of CBL proteins. HU-induced replication stress gives rise to axially elongated cells with half interphase/half mitotic structures (IM-cells) having both decondensed and condensed domains of chromatin. Long-term HU treatment results in cell nuclei resuming S phase with gradients of BrdU labelling. This suggests a polarized distribution of factors needed to re-initiate stalled replication forks. Furthermore, prolonged HU treatment extends both the relative time span and the spatial scale of H3S10 phosphorylation known in plants. Conclusions The minimum cell length and a threshold level of accumulated CBL proteins are both determining factors by which the nucleus attains commitment to induce an asynchronous course of chromosome condensation. Replication stress-induced alterations in an orderly route of the cell cycle events probably reflect a considerable reprogramming of metabolic functions of chromatin combined with gradients of morphological changes spread along the nucleus. PMID:23087128

20(S)-Protopanaxadiol-Induced Apoptosis in MCF-7 Breast Cancer Cell Line through the Inhibition of PI3K/AKT/mTOR Signaling Pathway.

PubMed

Zhang, Hong; Xu, Hua-Li; Wang, Yu-Chen; Lu, Ze-Yuan; Yu, Xiao-Feng; Sui, Da-Yun

2018-04-02

20(S)-Protopanaxadiol (PPD) is one of the major active metabolites of ginseng. It has been reported that 20(S)-PPD shows a broad spectrum of antitumor effects. Our research study aims were to investigate whether apoptosis of human breast cancer MCF-7 cells could be induced by 20(S)-PPD by targeting the Phosphatidylinositol 3-kinase/Protein kinase B/Mammalian target of rapamycin (PI3K/AKT/mTOR) signal pathway in vitro and in vivo. Cell cycle analysis was performed by Propidium Iodide (PI) staining. To overexpress and knock down the expression of mTOR, pcDNA3.1-mTOR and mTOR small interfering RNA (siRNA) transient transfection assays were used, respectively. Cell viability and apoptosis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-test and Annexin V /PI double-staining after transfection. The antitumor effect in vivo was determined by the nude mice xenograft assay. After 24 h of incubation, treatment with 20(S)-PPD could upregulate phosphorylated-Phosphatase and tensin homologue deleted on chromosome 10 (p-PTEN) expression and downregulate PI3K/AKT/mTOR-pathway protein expression. Moreover, G0/G1 cell cycle arrest in MCF-7 cells could be induced by 20(S)-PPD treatment at high concentrations. Furthermore, overexpression or knockdown of mTOR could inhibit or promote the apoptotic effects of 20(S)-PPD. In addition, tumor volumes were partially reduced by 20(S)-PPD at 100 mg/kg in a MCF-7 xenograft model. Immunohistochemical staining indicated a close relationship between the inhibition of tumor growth and the PI3K/AKT/mTOR signal pathway. PI3K/AKT/mTOR pathway-mediated apoptosis may be one of the potential mechanisms of 20(S)-PPD treatment.

Antioxidants Modulate the Antiproliferative Effects of Nitric Oxide on Vascular Smooth Muscle Cells and Adventitial Fibroblasts by Regulating Oxidative Stress

PubMed Central

Gregory, Elaine K.; Vavra, Ashley K.; Moreira, Edward S.; Havelka, George E.; Jiang, Qun; Lee, Vanessa R.; Van Lith, Robert; Ameer, Guillermo A.; Kibbe, Melina R.

2011-01-01

Background S-nitrosothiols (SNO) release nitric oxide (NO) through interaction with ascorbic acid (AA). However, little is known about their combined effect in the vasculature. The aim of this study is to investigate the effect of AA on SNO-mediated NO release, proliferation, cell cycle progression, cell death and oxidative stress in vascular cells. Methods VSMC and adventitial fibroblasts (AF) harvested from the aortae of Sprague Dawley rats were treated with AA, ± S-nitrosoglutathione (GSNO), or ± diethylenetriamine NONOate (DETA/NO). NO release, proliferation, cell cycle progression, cell death, and oxidative stress were determined by the Greiss reaction, [3H]-thymidine incorporation, flow cytometry, trypan blue exclusion, and DCF staining, respectively. Results AA increased NO release from GSNO 3-fold (p<0.001). GSNO and DETA/NO significantly decreased proliferation, but AA abrogated this effect (p<0.05). Mirroring the proliferation data, changes in cell cycle progression induced by GSNO and DETA/NO were reversed by addition of AA. GSNO- and DETA/NO-mediated increases in oxidative stress were significantly decreased by addition of AA (p<0.001). Conclusion Despite causing increased NO release from GSNO, AA reduced the antiproliferative and cell cycle effects of GSNO and DETA/NO through modulation of oxidative stress. PMID:21944289

A phthalide derivative isolated from endophytic fungi Pestalotiopsis photiniae induces G1 cell cycle arrest and apoptosis in human HeLa cells

PubMed Central

Chen, C.; Yang, R.L.

2013-01-01

MP [4-(3′,3′-dimethylallyloxy)-5-methyl-6-methoxyphthalide] was obtained from liquid culture of Pestalotiopsis photiniae isolated from the Chinese Podocarpaceae plant Podocarpus macrophyllus. MP significantly inhibited the proliferation of HeLa tumor cell lines. After treatment with MP, characteristic apoptotic features such as DNA fragmentation and chromatin condensation were observed in DAPI-stained HeLa cells. Flow cytometry showed that MP induced G1 cell cycle arrest and apoptosis in a dose-dependent manner. Western blotting and real-time reverse transcription-polymerase chain reaction were used to investigate protein and mRNA expression. MP caused significant cell cycle arrest by upregulating the cyclin-dependent kinase inhibitor p27KIP1 protein and p21CIP1 mRNA levels in HeLa cells. The expression of p73 protein was increased after treatment with various MP concentrations. mRNA expression of the cell cycle-related genes, p21CIP1, p16INK4a and Gadd45α, was significantly upregulated and mRNA levels demonstrated significantly increased translation of p73, JunB, FKHR, and Bim. The results indicate that MP may be a potential treatment for cervical cancer. PMID:23903687

Where Do All the Phytoplankton Go? Challenges in Keeping Track of Viable Cells in Phytoplankton Communities Using Flow Cytometry and Cell Staining

NASA Astrophysics Data System (ADS)

Simmons, L. J.; Fobbe, D. J.; Berges, J. A.

2016-02-01

Understanding the dynamics of phytoplankton communities has traditionally focused on differences in growth and related processes among taxa. It is now appreciated that differences in mortality could be equally important in contributing to these dynamics. Studying mortality in communities is difficult, especially on relevant time scales, which could be as short as hours to days. Flow cytometry can potentially provide solutions, because it can allow discrimination of different taxa, and when combined with staining, distinguish live and dead cells. We applied flow cytometry and staining to phytoplankton communities in a model system: a small, well-studied, urban pond in southeastern Wisconsin. Using flow cytometry, it was possible to resolve up to six dominant taxa (most <37 µm) and track them through an annual cycle. However, the axes traditionally used, forward scatter (FSC, related to cell size) and red fluorescence (FL3, related to chlorophyll a content) offered poor discrimination. Addition of orange fluorescence (FL2, traditionally related to phycobilipigments) and side scatter (SSC, related to cell surface characteristics) improved separation of taxa, but reproducibility (i.e. the specific position of the taxa on axes) was also more sensitive to environmental variation in the case of the fluorescence parameters. Dead cells could be distinguished by green fluorescence (FL1, using SYTOX Green©), but the stain also affected other fluorescence channels, requiring compensation. Correlations of numbers of dead cells with environmental factors (e.g. temperature, nutrient concentrations, irradiance) were generally poor, suggesting the greater importance of biotic versus abiotic variables in community mortality dynamics. Ongoing work is focusing on the effects of viral pathogens, grazing and allelopathic interactions using experimental manipulations and individual-based modeling.

Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Kanayo; Sakaguchi, Minoru, E-mail: sakaguti@gly.oups.ac.jp; Tanaka, Satoshi

2014-01-03

Highlights: •We examined the effects of prolyl oligopeptidase (POP) inhibition on p53 null gastric cancer cell growth. •POP inhibition-induced cell growth suppression was associated with an increase in a quiescent G{sub 0} state. •POP might regulate the exit from and/or reentry into the cell cycle. -- Abstract: Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G{sub 0}/G{sub 1} cell cycle arrest and increased levels of the CDKmore » inhibitor p27{sup kip1} and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-((4-[2-(E)-styrylphenoxy]butanoyl)-L-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G{sub 0}/G{sub 1} cell cycle phase arrest and increased levels of p27{sup kip1} in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G{sub 0} state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.« less

Scorpion (Androctonus bicolor) venom exhibits cytotoxicity and induces cell cycle arrest and apoptosis in breast and colorectal cancer cell lines

PubMed Central

Al-Asmari, Abdulrahman K.; Riyasdeen, Anvarbatcha; Abbasmanthiri, Rajamohamed; Arshaduddin, Mohammed; Al-Harthi, Fahad Ali

2016-01-01

Objectives: The defective apoptosis is believed to play a major role in the survival and proliferation of neoplastic cells. Hence, the induction of apoptosis in cancer cells is one of the targets for cancer treatment. Researchers are considering scorpion venom as a potent natural source for cancer treatment because it contains many bioactive compounds. The main objective of the current study is to evaluate the anticancer property of Androctonus bicolor scorpion venom on cancer cells. Materials and Methods: Scorpions were milked by electrical stimulation of telsons and lyophilized. The breast (MDA-MB-231) and colorectal (HCT-8) cancer cells were maintained in appropriate condition. The venom cytotoxicity was assessed by 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay, and the cellular and nuclear changes were studied with propidium iodide and 4’,6-diamidino-2-phenylindole stain, respectively. The cell cycle arrest was examined using muse cell analyzer. Results: The A. bicolor venom exerted cytotoxic effects on MDA-MB-231 and HCT-8 cells in a dose- and duration-dependent manner and induced apoptotic cell death. The treatment with this venom arrests the cancer cells in G0/G1 phase of cell cycle. Conclusions: The venom selectively induces the rate of apoptosis in MDA-MB-231 and HCT-8 cells as reflected by morphological and cell cycle studies. To the best of our knowledge, this is the first scientific evidence demonstrating the induction of apoptosis and cell cycle arrest by A. bicolor scorpion venom. PMID:27721540

Scorpion (Androctonus bicolor) venom exhibits cytotoxicity and induces cell cycle arrest and apoptosis in breast and colorectal cancer cell lines.

PubMed

Al-Asmari, Abdulrahman K; Riyasdeen, Anvarbatcha; Abbasmanthiri, Rajamohamed; Arshaduddin, Mohammed; Al-Harthi, Fahad Ali

2016-01-01

The defective apoptosis is believed to play a major role in the survival and proliferation of neoplastic cells. Hence, the induction of apoptosis in cancer cells is one of the targets for cancer treatment. Researchers are considering scorpion venom as a potent natural source for cancer treatment because it contains many bioactive compounds. The main objective of the current study is to evaluate the anticancer property of Androctonus bicolor scorpion venom on cancer cells. Scorpions were milked by electrical stimulation of telsons and lyophilized. The breast (MDA-MB-231) and colorectal (HCT-8) cancer cells were maintained in appropriate condition. The venom cytotoxicity was assessed by 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay, and the cellular and nuclear changes were studied with propidium iodide and 4',6-diamidino-2-phenylindole stain, respectively. The cell cycle arrest was examined using muse cell analyzer. The A. bicolor venom exerted cytotoxic effects on MDA-MB-231 and HCT-8 cells in a dose- and duration-dependent manner and induced apoptotic cell death. The treatment with this venom arrests the cancer cells in G0/G1 phase of cell cycle. The venom selectively induces the rate of apoptosis in MDA-MB-231 and HCT-8 cells as reflected by morphological and cell cycle studies. To the best of our knowledge, this is the first scientific evidence demonstrating the induction of apoptosis and cell cycle arrest by A. bicolor scorpion venom.

Epoxy Stearic Acid, an Oxidative Product Derived from Oleic Acid, Induces Cytotoxicity, Oxidative Stress, and Apoptosis in HepG2 Cells.

PubMed

Liu, Ying; Cheng, Yajun; Li, Jinwei; Wang, Yuanpeng; Liu, Yuanfa

2018-05-23

In the present study, effects of cis-9,10-epoxy stearic acid (ESA) generated by the thermal oxidation of oleic acid on HepG2 cells, including cytotoxicity, apoptosis, and oxidative stress, were investigated. Our results revealed that ESA decreased the cell viability and induced cell death. Cell cycle analysis with propidium iodide staining showed that ESA induced cell cycle arrest at the G0/G1 phase in HepG2 cells. Cell apoptosis analysis with annexin V and propidium iodide staining demonstrated that ESA induced HepG2 cell apoptotic events in a dose- and time-dependent manner; the apoptosis of cells after treated with 500 μM ESA for 12, 24, and 48 h was 32.16, 38.70, and 65.80%, respectively. Furthermore, ESA treatment to HepG2 cells resulted in an increase in reactive oxygen species and malondialdehyde (from 0.84 ± 0.02 to 8.90 ± 0.50 nmol/mg of protein) levels and a reduction in antioxidant enzyme activity, including superoxide dismutase (from 1.34 ± 0.27 to 0.10 ± 0.007 units/mg of protein), catalase (from 100.04 ± 5.05 to 20.09 ± 3.00 units/mg of protein), and glutathione peroxidase (from 120.44 ± 7.62 to 35.84 ± 5.99 milliunits/mg of protein). These findings provide critical information on the effects of ESA on HepG2 cells, particularly cytotoxicity and oxidative stress, which is important for the evaluation of the biosafety of the oxidative product of oleic acid.

Fluorometric method of quantitative cell mutagenesis

DOEpatents

Dolbeare, Frank A.

1982-01-01

A method for assaying a cell culture for mutagenesis is described. A cell culture is stained first with a histochemical stain, and then a fluorescent stain. Normal cells in the culture are stained by both the histochemical and fluorescent stains, while abnormal cells are stained only by the fluorescent stain. The two stains are chosen so that the histochemical stain absorbs the wavelengths that the fluorescent stain emits. After the counterstained culture is subjected to exciting light, the fluorescence from the abnormal cells is detected.

Fluorometric method of quantitative cell mutagenesis

DOEpatents

Dolbeare, F.A.

1980-12-12

A method for assaying a cell culture for mutagenesis is described. A cell culture is stained first with a histochemical stain, and then a fluorescent stain. Normal cells in the culture are stained by both the histochemical and fluorescent stains, while abnormal cells are stained only by the fluorescent stain. The two stains are chosen so that the histochemical stain absorbs the wavelengths that the fluorescent stain emits. After the counterstained culture is subjected to exciting light, the fluorescence from the abnormal cells is detected.

Disabled infectious single cycle herpes simplex virus (DISC-HSV) is a candidate vector system for gene delivery/expression of GM-CSF in human prostate cancer therapy.

PubMed

Parkinson, Richard J; Mian, Shahid; Bishop, Michael C; Gray, Trevor; Li, Geng; McArdle, Stephanie E B; Ali, Selman; Rees, Robert C

2003-06-15

DISC-HSV is a replication incompetent herpes simplex virus that is a highly efficient vector for the transduction of genes in vivo and in vitro. We examine the ability of DISC-HSV to infect human prostate cancer cell-lines and xenograft tumor models, and induce expression of reporter and therapeutic cytokine genes. Infection was confirmed by cellular staining for the beta-galactosidase reporter gene product, and by EM. Human GM-CSF production following DISC-hGMCSF infection was measured using ELISA. The metabolic activity of infected cells was determined by NADP/NADPH assay. Cell death was estimated by cell-cycle analysis using flow cytometry with propidium iodide staining. Infection of DU145, PC3 and LNCaP cells with DISC-HSV was dose dependent. Cells infected with DISC-hGM-CSF released significant levels of hGM-CSF for 3 days. NADP/NADPH assay suggested that infected cells continued to be metabolically active for 3 days post-infection, which was consistent with flow cytometry findings that cell death did not occur within 7 days of infection. Tumor xenografts injected with DISC-HSV expressed beta-galactosidase, and intracellular viral particles were demonstrated using EM. We have previously reported the rejection of established tumors following intra-tumoral injection of DISC-GMCSF. This study demonstrates the ability of DISC-HSV to infect prostate cancer and express GMCSF at significant levels. We suggest that prostate cancer is a potential target for therapy using DISC-HSV containing GM-CSF. Copyright 2003 Wiley-Liss, Inc.

[Effects of HOXB2 antisense oligodeoxynucleotides on the biological properties of primary human umbilical vein endothelial cells].

PubMed

Zhang, Xiaoqi; Liu, Xusheng

2002-07-01

To explore the effects of HOXB2 antisense oligodeoxynuc leotides (Asodn) on the biological properties of primary human umbilical vein endothelial cells (ECs). Fluorescent labelled Asodn was transfected into the endothelial cells of human unbilical vein mediated liposome and its distribution within endothelia was observed. (3)H-TdR incorporation test was employed to determine its effects on the DNA synthesis. Flow cytometry was applied to determine the change of the cell cycle. In the same time, RT-PCR was adopted to study the influence of Asodn on the expression of target genes. Fifteen minutes after the transfection, weak nucleic staining was observed. The fluorescent staining was the strongest 4 approximately 8 hours after the transfection and began to weaken in 16 hours. The proportion of cells in G1/0 phase in Asodn group was 53.4 +/- 3.1, significantly higher than that in control group (35.8 +/- 7.3, P < 0.01), and the proportion of cells in S phase in Asodn group was 42.2 +/- 3.5, significantly lower than that in control group (60.8 +/- 6.2, P < 0.01). The expression of HOXB2 mRNA was remarkably decreased during 24 to 48 hours. HOXB2 Asodn exerts inhibitory effects on EC proliferation dose-dependently, delays the conversion of G1 phase to S Phase, and inhibits the expression of HOXB2 mRNA. HOXB2 gene plays an important role in proliferation of endothelial cells and the mechanism is related to cell cycle.

In Vitro Antiproliferative Effect of Arthrocnemum indicum Extracts on Caco-2 Cancer Cells through Cell Cycle Control and Related Phenol LC-TOF-MS Identification

PubMed Central

Boulaaba, Mondher; Mkadmini, Khaoula; Tsolmon, Soninkhishig; Han, Junkyu; Smaoui, Abderrazak; Kawada, Kiyokazu; Ksouri, Riadh; Isoda, Hiroko; Abdelly, Chedly

2013-01-01

This study aimed to determinate phenolic contents and antioxidant activities of the halophyte Arthrocnemum indicum shoot extracts. Moreover, the anticancer effect of this plant on human colon cancer cells and the likely underlying mechanisms were also investigated, and the major phenols were identified by LC-ESI-TOF-MS. Results showed that shoot extracts had an antiproliferative effect of about 55% as compared to the control and were characterised by substantial total polyphenol content (19 mg GAE/g DW) and high antioxidant activity (IC50 = 40 μg/mL for DPPH test). DAPI staining revealed that these extracts decrease DNA synthesis and reduce the proliferation of Caco-2 cells which were stopped at the G2/M phase. The changes in the cell-cycle-associated proteins (cyclin B1, p38, Erk1/2, Chk1, and Chk2) correlate with the changes in cell cycle distribution. Eight phenolic compounds were also identified. In conclusion, A. indicum showed interesting antioxidant capacities associated with a significant antiproliferative effect explained by a cell cycle blocking at the G2/M phase. Taken together, these data suggest that A. indicum could be a promising candidate species as a source of anticancer molecules. PMID:24348703

Mechanisms of G1 cell cycle arrest and apoptosis in myeloma cells induced by hybrid-compound histone deacetylase inhibitor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujii, Seiko; Division of Maxillofacial Surgery, Kyushu Dental University; Okinaga, Toshinori

2013-05-10

Highlights: •Novel histone deacetylase inhibitor Ky-2, remarkably inhibits myeloma cell growth. •Ky-2 demonstrates no cytotoxicity against normal lymphocytic cells. •Ky-2 induces cell cycle arrest through the cell cycle-associated proteins. •Ky-2 induces Bcl-2-inhibitable apoptosis through a caspase-dependent cascade. -- Abstract: Objectives: Histone deacetylase (HDAC) inhibitors are new therapeutic agents, used to treat various types of malignant cancers. In the present study, we investigated the effects of Ky-2, a hybrid-compound HDAC inhibitor, on the growth of mouse myeloma cells. Materials and methods: Myeloma cells, HS-72, P3U1, and mouse normal cells were used in this study. Effect of HDAC inhibitors on cell viabilitymore » was determined by WST-assay and trypan blue assay. Cell cycle was analyzed using flow cytometer. The expression of cell cycle regulatory and the apoptosis associated proteins were examined by Western blot analysis. Hoechst’s staining was used to detect apoptotic cells. Results: Our findings showed that Ky-2 decreased the levels of HDACs, while it enhanced acetylation of histone H3. Myeloma cell proliferation was inhibited by Ky-2 treatment. Interestingly, Ky-2 had no cytotoxic effects on mouse normal cells. Ky-2 treatment induced G1-phase cell cycle arrest and accumulation of a sub-G1 phase population, while Western blotting analysis revealed that expressions of the cell cycle-associated proteins were up-regulated. Also, Ky-2 enhanced the cleavage of caspase-9 and -3 in myeloma cells, followed by DNA fragmentation. In addition, Ky-2 was not found to induce apoptosis in bcl-2 overexpressing myeloma cells. Conclusion: These findings suggest that Ky-2 induces apoptosis via a caspase-dependent cascade and Bcl-2-inhibitable mechanism in myeloma cells.« less

The effect of SiO2/Au core-shell nanoparticles on breast cancer cell's radiotherapy.

PubMed

Darfarin, Ghazal; Salehi, Roya; Alizadeh, Effat; Nasiri Motlagh, Behnam; Akbarzadeh, Abolfazl; Farajollahi, Alireza

2018-05-09

Recently it has been shown that radiation dose enhancement could be achievable in radiotherapy using nanoparticles (NPs). In this study, evaluation was made to determine efficiency of gold-silica shell-core NP in megavoltage irradiation of MCF7 breath cancer cells. Gold-silicon oxide shell-core NPs were obtained by conjugation of gold NP with amine or thiol functionalized silica NPs (AuN@SiO 2 and AuS@SiO 2 ). Cellular uptake and cytotoxicity of NPs were examined by fluorescent microscopy and MTT assay, respectively. MCF-7 breast cancer cells were treated with both NPs and irradiation was made with X-ray energies of 6 and 18 MV to the absorbed dose of 2, 4 and 8 Gy using Simense linear accelerator. The efficiency of radiation therapy was then evaluated by MTT and Brdu assay, DAPI staining and cell cycle analysis. TEM images indicated that synthesized NPs had average diameter of 25 nm. Cellular uptake demonstrated that the internalization of AuS@SiO 2 and AuN@SiO 2 NPs amounted to 18% and 34%, 3 h post treatment, respectively. Nontoxicity of prepared NPs on MCF-7 cells was proved by MTT and Brdu assays as well as DAPI staining and cell cycle studies. The highest enhancement in radiation dose was observed in the cells that irradiated with radiation energy of 18 MV and absorbed of 8 Gy at NPs concentration of 200 ppm. The Brdu findings revealed that the cytotoxicity and apoptosis on MCF-7 cells are dose dependent with a significantly more death in AuN@SiO 2 (amine) exposed cells (p < .05). Analysis also revealed interruption in cell cycle by demonstrating lack of cells, in S phase in amine treated cells (AuN@SiO 2 ) at given dose of 8 Gy using 18 MV X-ray in comparison to thiol treated cells. Based on the results of the study it can be concluded that the gold-silicon oxide shell-core NPs could play an effective role in radiotherapy of MCF-7 breast cancer cells.

Intravital imaging of cardiac function at the single-cell level.

PubMed

Aguirre, Aaron D; Vinegoni, Claudio; Sebas, Matt; Weissleder, Ralph

2014-08-05

Knowledge of cardiomyocyte biology is limited by the lack of methods to interrogate single-cell physiology in vivo. Here we show that contracting myocytes can indeed be imaged with optical microscopy at high temporal and spatial resolution in the beating murine heart, allowing visualization of individual sarcomeres and measurement of the single cardiomyocyte contractile cycle. Collectively, this has been enabled by efficient tissue stabilization, a prospective real-time cardiac gating approach, an image processing algorithm for motion-artifact-free imaging throughout the cardiac cycle, and a fluorescent membrane staining protocol. Quantification of cardiomyocyte contractile function in vivo opens many possibilities for investigating myocardial disease and therapeutic intervention at the cellular level.

The new reports on life cycle of Heterosigma akashiwo (Raphidophyceae)

NASA Astrophysics Data System (ADS)

Kim, J. H.

2016-02-01

Heterosigma akashiwo (Hada) Hada (Raphidophyceae) is a noxious bloom-forming algal species that has damaged many fish farms in coastal waters during recent decades. Consequently, many studies focused on the population dynamics of H. akashiwo, while its life cycle was not well studied. In this study, we investigated veiled life cycle of H. akashiwo through culture based method. We cultured eight H. akashiwostrains originated from Korea, Japan, USA under various conditions (water temp., light intensity, salinity, pH). Morphological diversity of cells were observed via light microscopy and scanning electron microscopy. To observe nucleus of living cell, cells were stained with Hoechst and changes of cells in culture were observed through time-lapse. For observation of cysts and their germination process, cysts were isolated from sediment. Different from the previous knowledge, H. akashiwo has only vegetative cell stage and cyst stage, we discovered that H. akashiwo has extra small cell stage and large cell stage. Large cells are much bigger (20-45 µm) than vegetative cells. Large cell formation was resulted from fusion of vegetative cells. Small cells were very small (6.88 ± 0.85 µm), these cell divided from large cell or formed in germination process of cysts rarely. Small cells have lower motility than vegetative cells. These results improved the study of life stages of H. akashiwo and this fundamental investigation provide important new information and improve our understanding of the life cycle of H. akashiwo.

Effects of exogenous zinc on cell cycle, apoptosis and viability of MDAMB231, HepG2 and 293 T cells.

PubMed

Wang, Yan-hong; Li, Ke-jin; Mao, Li; Hu, Xin; Zhao, Wen-jie; Hu, An; Lian, Hong-zhen; Zheng, Wei-juan

2013-09-01

As a non-toxic metal to humans, zinc is essential for cell proliferation, differentiation, regulation of DNA synthesis, genomic stability and mitosis. Zinc homeostasis in cells, which is crucial for normal cellular functioning, is maintained by various protein families including ZnT (zinc transporter/SLC30A) and ZIP (Zrt-, Irt-like proteins/SLC39A) that decrease and increase cytosolic zinc availability, respectively. In this study, we investigated the influences of a specific concentration range of ZnSO4 on cell cycle and apoptosis by flow cytometry, and cell viability by MTT method in MDAMB231, HepG2 and 293 T cell lines. Fluorescent sensors NBD-TPEA and the counterstain for nuclei Hoechst 33342 were used to stain the treated cells for observing the localisation and amount of Zn(2+) via laser scanning confocal microscope. It was found that the influence manners of ZnSO4 on cell cycle, apoptosis and cell viability in various cell lines were different and corresponding to the changes of Zn(2+) content of the three cell lines, respectively. The significant increase on intracelluar zinc content of MDAMB231 cells resulted in cell death, G1 and G2/M cell cycle arrest and increased apoptotic fraction. Additionally, the mRNA expression levels of ZnT and ZIP families in the three cell lines, when treated with high concentration of ZnSO4, increased and decreased corresponding to their functions, respectively.

Bevacizumab inhibits proliferation of choroidal endothelial cells by regulation of the cell cycle.

PubMed

Rusovici, Raluca; Patel, Chirag J; Chalam, Kakarla V

2013-01-01

The purpose of this study was to evaluate cell cycle changes in choroidal endothelial cells treated with varying doses of bevacizumab in the presence of a range of concentrations of vascular endothelial growth factor (VEGF). Bevacizumab, a drug widely used in the treatment of neovascular age-related macular degeneration, choroidal neovascularization, and proliferative diabetic retinopathy, neutralizes all isoforms of VEGF. However, the effect of intravitreal administration of bevacizumab on the choroidal endothelial cell cycle has not been established. Monkey choroidal endothelial (RF/6A) cells were treated with VEGF 50 ng/mL and escalating doses of bevacizumab 0.1-2 mg/mL for 72 hours. Cell cycle changes in response to bevacizumab were analyzed by flow cytometry and propidium iodide staining. Cell proliferation was measured using the WST-1 assay. Morphological changes were recorded by bright field cell microscopy. Bevacizumab inhibited proliferation of choroidal endothelial cells by stabilization of the cell cycle in G0/G1 phase. Cell cycle analysis of VEGF-enriched choroidal endothelial cells revealed a predominant increase in the G2/M population (21.84%, P, 0.01) and a decrease in the G0/G1 phase population (55.08%, P, 0.01). Addition of escalating doses of bevacizumab stabilized VEGF-enriched cells in the G0/G1 phase (55.08%, 54.49%, 56.3%, and 64% [P, 0.01]) and arrested proliferation by inhibiting the G2/M phase (21.84%, 21.46%, 20.59%, 20.94%, and 16.1% [P, 0.01]). The increase in G0/G1 subpopulation in VEGF-enriched and bevacizumab-treated cells compared with VEGF-enriched cells alone was dose-dependent. Bevacizumab arrests proliferation of VEGF-enriched choroidal endothelial cells by stabilizing the cell cycle in the G0/G1 phase and inhibiting the G2/M phase in a dose-dependent fashion.

Autophagy mediates cell cycle response by regulating nucleocytoplasmic transport of PAX6 in limbal stem cells under ultraviolet-A stress

PubMed Central

Laggner, Maria; Pollreisz, Andreas; Schmidinger, Gerald; Schmidt-Erfurth, Ursula; Chen, Ying-Ting

2017-01-01

Limbal stem cells (LSC) account for homeostasis and regeneration of corneal epithelium. Solar ultraviolet A (UVA) is the major source causing oxidative damage in the ocular surface. Autophagy, a lysosomal degradation mechanism, is essential for physiologic function and stress defense of stem cells. PAX6, a master transcription factor governing corneal homeostasis by regulating cell cycle and cell fate of LSC, responds to oxidative stress by nucleocytoplasmic shuttling. Impaired autophagy and deregulated PAX6 have been reported in oxidative stress-related ocular surface disorders. We hypothesize a functional role for autophagy and PAX6 in LSC’s stress response to UVA. Therefore, human LSC colonies were irradiated with a sub-lethal dose of UVA and autophagic activity and intracellular reactive oxygen species (ROS) were measured by CYTO-ID assay and CM-H2DCFDA live staining, respectively. Following UVA irradiation, the percentage of autophagic cells significantly increased in LSC colonies while intracellular ROS levels remained unaffected. siRNA-mediated knockdown (KD) of ATG7 abolished UVA-induced autophagy and led to an excessive accumulation of ROS. Upon UVA exposure, LSCs displayed nuclear-to-cytoplasmic translocation of PAX6, while ATG7KD or antioxidant pretreatment largely attenuated the intracellular trafficking event. Immunofluorescence showing downregulation of proliferative marker PCNA and induction of cell cycle regulator p21 indicates cell cycle arrest in UVA-irradiated LSC. Abolishing autophagy, adenoviral-assisted restoration of nuclear PAX6 or antioxidant pretreatment abrogated the UVA-induced cell cycle arrest. Adenoviral expression of an ectopic PAX gene, PAX7, did not affect UVA cell cycle response. Furthermore, knocking down PAX6 attenuated the cell cycle progression of irradiated ATG7KD LSC by de-repressing p21 expression. Collectively, our data suggest a crosstalk between autophagy and PAX6 in regulating cell cycle response of ocular progenitors under UVA stress. Autophagy deficiency leads to impaired intracellular trafficking of PAX6, perturbed redox balance and uncurbed cell cycle progression in UVA-stressed LSCs. The coupling of autophagic machinery and PAX6 in cell cycle regulation represents an attractive therapeutic target for hyperproliferative ocular surface disorders associated with solar radiation. PMID:28700649

New aspects of membrane dynamics of Amoeba proteus contractile vacuole revealed by vital staining with FM 4-64.

PubMed

Nishihara, E; Shimmen, T; Sonobe, S

2007-01-01

The contractile vacuole (CV) cycle of Amoeba proteus has been studied by phase contrast and electron microscopy. However, the understanding of membrane dynamics in this cycle is still poor. In this study, we used live imaging by fluorescence microscopy to obtain new insights. We succeeded in staining the CV with a styryl dye, FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide), and obtained the following results. (1) The CV membrane was directly stained with the dye in the external medium when the CV pore opened upon contraction. This indicates that transfer of plasma membrane to the CV does not occur. (2) The membrane dynamics during the CV cycle were elucidated. In particular, the fluorescent CV membrane was maintained as an aggregate just after contraction and the vacuole re-formed from the aggregate. Staining was maintained during continued contraction cycles. We conclude that the CV membrane is maintained during the CV cycle.

MLN8054, A Small Molecule Inhibitor of Aurora Kinase A, Sensitizes Androgen-Resistant Prostate Cancer to Radiation;Aurora kinase A; MLN8054; Prostate cancer; Radiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moretti, Luigi; Department of Radiation Oncology, Institut Jules Bordet, Universite Libre de Bruxelles, Brussels; Niermann, Kenneth

2011-07-15

Purpose: To determine whether MLN8054, an Aurora kinase A (Aurora-A) inhibitor causes radiosensitization in androgen-insensitive prostate cancer cells in vitro and in vivo. Methods and Materials: In vitro studies consisted of culturing PC3 and DU145 prostate cancer cells and then immunoblotting Aurora A and phospho-Aurora A after radiation and/or nocodazole with MLN8054. Phases of the cell cycle were measured with flow cytometry. PC3 and DU145 cell lines were measured for survival after treatment with MLN8054 and radiation. Immunofluorescence measured {gamma}-H2AX in the PC3 and DU145 cells after treatment. In vivo studies looked at growth delay of PC3 tumor cells inmore » athymic nude mice. PC3 cells grew for 6 to 8 days in mice treated with radiation, MLN8054, or combined for 7 more days. Tumors were resected and fixed on paraffin and stained for von Willebrand factor, Ki67, and caspase-3. Results: In vitro inhibition of Aurora-A by MLN8054 sensitized prostate cancer cells, as determined by dose enhancement ratios in clonogenic assays. These effects were associated with sustained DNA double-strand breaks, as evidenced by increased immunofluorescence for {gamma}-H2AX and significant G2/M accumulation and polyploidy. In vivo, the addition of MLN8054 (30 mg/kg/day) to radiation in mouse prostate cancer xenografts (PC3 cells) significantly increased tumor growth delay and apoptosis (caspase-3 staining), with reduction in cell proliferation (Ki67 staining) and vascular density (von Willebrand factor staining). Conclusion: MLN8054, a novel small molecule Aurora-A inhibitor showed radiation sensitization in androgen-insensitive prostate cancer in vitro and in vivo. This warrants the clinical development of MLN8054 with radiation for prostate cancer patients.« less

RNA interference-mediated survivin gene knockdown induces growth arrest and reduced migration of vascular smooth muscle cells.

PubMed

Nabzdyk, Christoph S; Lancero, Hope; Nguyen, Khanh P; Salek, Sherveen; Conte, Michael S

2011-11-01

Survivin (SVV) is a multifunctional protein that has been implicated in the development of neointimal hyperplasia. Nuclear SVV is essential for mitosis, whereas in mitochondria SVV has a cytoprotective function. Here, we investigated the effects of RNA interference (RNAi)-mediated SVV knockdown on cell cycle kinetics, apoptosis, migration, and gene expression in primary cultured vascular smooth muscle cells (VSMCs) from the human saphenous vein. Primary Human VSMCs were obtained from saphenous veins and cultured under standard conditions. SVV knockdown was achieved by either small interfering RNA or lentiviral transduction of short hairpin RNA, reducing SVV gene expression by quantitative PCR (>75%, P < 0.01) without a loss of cell viability. Subcellular fractionation revealed that RNAi treatment effectively targeted the nuclear SVV pool, whereas the larger mitochondrial pool was much less sensitive to transient knockdown. Both p53 and p27 protein levels were notably increased. SVV RNAi treatment significantly blocked VSMC proliferation in response to serum and PDGF-AB, arresting VSMC growth. Cell cycle analysis revealed an increased G(2)/M fraction consistent with a mitotic defect; 4',6-diamidino-2-phenylindole staining confirmed an increased frequency of polyploid and abnormal nuclei. In a transwell assay, SVV knockdown reduced migration to PDGF-AB, and actin-phalloidin staining revealed disorganized actin filaments and polygonal cell shape. However, apoptosis (DNA content and annexin V flow cytometry) was not directly induced by SVV RNAi, and sensitivity to apoptotic agonists (e.g., staurosporine and cytokines) was unchanged. In conclusion, RNAi-mediated SVV knockdown in VSMCs leads to profound cell cycle arrest at G(2)/M and impaired chemotaxis without cytotoxicity. The regulation of mitosis and apoptosis in VSMC involves differentially regulated subcellular pools of SVV. Thus, treatment of VSMC with RNAi targeting SVV might limit the response to vascular injury without destabilizing the vessel wall.

RNA interference-mediated survivin gene knockdown induces growth arrest and reduced migration of vascular smooth muscle cells

PubMed Central

Nabzdyk, Christoph S.; Lancero, Hope; Nguyen, Khanh P.; Salek, Sherveen

2011-01-01

Survivin (SVV) is a multifunctional protein that has been implicated in the development of neointimal hyperplasia. Nuclear SVV is essential for mitosis, whereas in mitochondria SVV has a cytoprotective function. Here, we investigated the effects of RNA interference (RNAi)-mediated SVV knockdown on cell cycle kinetics, apoptosis, migration, and gene expression in primary cultured vascular smooth muscle cells (VSMCs) from the human saphenous vein. Primary Human VSMCs were obtained from saphenous veins and cultured under standard conditions. SVV knockdown was achieved by either small interfering RNA or lentiviral transduction of short hairpin RNA, reducing SVV gene expression by quantitative PCR (>75%, P < 0.01) without a loss of cell viability. Subcellular fractionation revealed that RNAi treatment effectively targeted the nuclear SVV pool, whereas the larger mitochondrial pool was much less sensitive to transient knockdown. Both p53 and p27 protein levels were notably increased. SVV RNAi treatment significantly blocked VSMC proliferation in response to serum and PDGF-AB, arresting VSMC growth. Cell cycle analysis revealed an increased G2/M fraction consistent with a mitotic defect; 4′,6-diamidino-2-phenylindole staining confirmed an increased frequency of polyploid and abnormal nuclei. In a transwell assay, SVV knockdown reduced migration to PDGF-AB, and actin-phalloidin staining revealed disorganized actin filaments and polygonal cell shape. However, apoptosis (DNA content and annexin V flow cytometry) was not directly induced by SVV RNAi, and sensitivity to apoptotic agonists (e.g., staurosporine and cytokines) was unchanged. In conclusion, RNAi-mediated SVV knockdown in VSMCs leads to profound cell cycle arrest at G2/M and impaired chemotaxis without cytotoxicity. The regulation of mitosis and apoptosis in VSMC involves differentially regulated subcellular pools of SVV. Thus, treatment of VSMC with RNAi targeting SVV might limit the response to vascular injury without destabilizing the vessel wall. PMID:21856925

Resveratrol Suppresses Growth and Migration of Myelodysplastic Cells by Inhibiting the Expression of Elevated Cyclin D1 (CCND1).

PubMed

Zhou, Wei; Xu, Shilin; Ying, Yi; Zhou, Ruiqing; Chen, Xiaowei

2017-11-01

Myelodysplastic syndromes (MDS) are a group of heterogeneous diseases characterized by poorly formed blood cells. We wanted to elucidate the underlying molecular mechanism to better determine pathogenesis, prognosis, diagnosis, and treatment for patients with MDS. We compared gene expression levels between normal and MDS tissue samples by immunohistochemical analysis. We studied the proliferation, survival, and migration of MDS cells using the EDU assay, colony formation, and transwell assays. We assessed the apoptotic rate and cell cycle status using flow cytometry and Hoechst staining. Finally, we evaluated RNA and protein expressions using polymerase chain reaction and Western blots, respectively. We found that resveratrol suppressed SKM-1 (an advanced MDS cell line) proliferation in a dose-dependent manner. Consistent with this finding, the EDU and colony formation assays also showed that resveratrol inhibited SKM-1 growth. Moreover, flow cytometry and Hoechst 33258 staining demonstrated that resveratrol induced apoptosis and a change in cell cycle status in SKM-1 cells, while the transwell assay showed that resveratrol reduced the migratory ability of SKM-1 cells. Resveratrol also decreased the expression of CCND1 (a gene that encodes the cyclin D1 protein) and increased expressions of KMT2A [lysine (K)-specific methyltransferase 2A] and caspase-3, suggesting that resveratrol exerts its effect by regulating CCND1 in SKM-1 cells. In addition, a combination of resveratrol and the PI3K/AKT inhibitor LY294002 exhibited a stronger inhibitory effect on the SKM-1 cells, compared with resveratrol alone. Our study proved that resveratrol suppresses SKM-1 growth and migration by inhibiting CCND1 expression. This finding provides novel insights into the pathogenesis of MDS and might help develop new diagnosis and treatment for patients with MDS.

Antioxidants modulate the antiproliferative effects of nitric oxide on vascular smooth muscle cells and adventitial fibroblasts by regulating oxidative stress.

PubMed

Gregory, Elaine K; Vavra, Ashley K; Moreira, Edward S; Havelka, George E; Jiang, Qun; Lee, Vanessa R; Van Lith, Robert; Ameer, Guillermo A; Kibbe, Melina R

2011-11-01

S-nitrosothiols (SNO) release nitric oxide (NO) through interaction with ascorbic acid (AA). However, little is known about their combined effect in the vasculature. The aim of this study was to investigate the effect of AA on SNO-mediated NO release, proliferation, cell cycle progression, cell death, and oxidative stress in vascular cells. Vascular smooth muscle cells and adventitial fibroblasts harvested from the aortae of Sprague-Dawley rats were treated with AA, ± S-nitrosoglutathione (GSNO), or ± diethylenetriamine NONOate (DETA/NO). NO release, proliferation, cell cycle progression, cell death, and oxidative stress were determined by the Griess reaction, [(3)H]-thymidine incorporation, flow cytometry, trypan blue exclusion, and 5-(and-6)chloromethyl-2',7'dichlorodihydrofluorescein staining, respectively. AA increased NO release from GSNO 3-fold (P < .001). GSNO and DETA/NO significantly decreased proliferation, but AA abrogated this effect (P < .05). Mirroring the proliferation data, changes in cell cycle progression induced by GSNO and DETA/NO were reversed by the addition of AA. GSNO- and DETA/NO-mediated increases in oxidative stress were significantly decreased by the addition of AA (P < .001). Despite causing increased NO release from GSNO, AA reduced the antiproliferative and cell cycle effects of GSNO and DETA/NO through the modulation of oxidative stress. Copyright © 2011 Elsevier Inc. All rights reserved.

Naturally Ocurring Polyphosphate-accumulating Bacteria in Benthic Biofilms

NASA Astrophysics Data System (ADS)

Locke, N. A.; Saia, S. M.; Walter, M. T.; Carrick, H. J.; Buda, A. R.; Regan, J. M.

2014-12-01

Polyphosphate accumulating organisms (PAOs), known to store excess phosphorus (P) as polyphosphate (poly-P), influence P transport in the environment. Enhanced biological phosphorus removal (EBPR) from wastewater has long served as a basis to study bacterial PAOs, yet little research has genetically identified similar organisms in natural settings. Aerobic/anaerobic cycles, used to select for PAOs in EBPR, can result from changing environmental conditions such as night/day cycles for benthic biofilms. Benthic biofilms from eight Pennsylvanian streams were studied for naturally-occurring bacterial PAOs similar to those typically found in EBPR systems. PAOs were confirmed in the benthic biofilms by a characteristic yellow fluorescent emission from DAPI staining. Cells containing yellow fluorescence were separated from the rest of the sample using a flow cytometer, resulting in a physically enriched culture of PAOs from the benthic biofilms. Amplicon-based metagenomic sequencing will reveal the phylogeny of bacteria responsible for poly-P accumulation in these benthic biofilms. Sequencing data will be used to develop fluorescent in-situ hybridization (FISH) probes, and hybridizations will be performed on DAPI-stained cells to confirm poly-P accumulation by targeted phylotypes. Identifying PAOs in natural settings is a critical step towards studying environments that support high concentrations of PAOs, serving as significant factors in the P cycle. PAOs can then be connected to P transport models to help understand and mitigate P pollution in agricultural watersheds.

Paris Saponin I Sensitizes Gastric Cancer Cell Lines to Cisplatin via Cell Cycle Arrest and Apoptosis.

PubMed

Song, Shuichuan; Du, Leiwen; Jiang, Hao; Zhu, Xinhai; Li, Jinhui; Xu, Ji

2016-10-18

BACKGROUND Dose-related toxicity is the major restriction of cisplatin and cisplatin-combination chemotherapy, and is a challenge for advanced gastric cancer treatment. We explored the possibility of using Paris saponin I as an agent to sensitize gastric cancer cells to cisplatin, and examined the underlying mechanism. MATERIAL AND METHODS Growth inhibition was detected by MTT assay. The cell cycle and apoptosis were detected using flow cytometry and Annexin V/PI staining. The P21waf1/cip1, Bcl-2, Bax, and caspase-3 protein expression were detected using Western blot analysis. RESULTS The results revealed that PSI sensitized gastric cancer cells to cisplatin, with low toxicity. The IC50 value of cisplatin in SGC-7901 cell lines was decreased when combined with PSI. PSI promoted cisplatin-induced G2/M phase arrest and apoptosis in a cisplatin concentration-dependent manner. Bcl-2 protein expression decreased, but Bax, caspase-3, and P21waf1/cip1 protein expression increased with PSI treatment. CONCLUSIONS The underlying mechanism of Paris saponin I may be related to targeting the apoptosis pathway and cell cycle blocking, which suggests that PSI is a potential therapeutic sensitizer for cisplatin in treating gastric cancer.

Function of cell-cycle regulators in predicting silent pituitary adenoma progression following surgical resection

PubMed Central

Park, Sung Hyun; Jang, Ji Hwan; Lee, Young Min; Kim, Joon Soo; Kim, Kyu Hong; Kim, Young Zoon

2017-01-01

The present study investigated the use of cell-cycle regulators for predicting the progression of silent pituitary adenoma (SPA) following surgical resection, via immunohistochemical analysis of tumor samples obtained by surgical resection. The medical records of patients diagnosed with SPA between January 2000 and December 2013 in the Samsung Changwon Hospital, Sungkyunkwan University School of Medicine (Changwon, South Korea) were reviewed. Immunohistochemical staining was performed on sections of the archived, paraffin-embedded tissues obtained by surgery, with all tissues stained for cell-cycle regulatory proteins p16, p15, p21, cyclin-dependent kinase (CDK)4, CDK6, retinoblastoma protein (pRb) and cyclin D1, as well as E3 ubiquitin-protein ligase mib1 (MIB-1) antigen and p53. The primary end-point was to investigate the expression of cell-cycle regulatory proteins in SPA. The secondary end-point was to estimate the progression-free survival of patients with SPA following surgical resection and to identify its association with the expression of cell-cycle regulatory proteins. Of the 127 SPA samples, 44 (34.6%) were from patients with progression during a mean follow-up period of 62.4 months (range, 24.2–118.9 months). Immunohistochemical overexpression was identified in 61 samples (48.0%) for p16, 38 samples (29.9%) for p15, 19 samples (15.0%) for p21, 49 samples (38.6%) for CDK4, 17 samples (13.4%) for CDK6, 57 samples (44.9%) for pRb and in 65 samples (51.2%) for cyclin D1. Multivariate analysis revealed that null cell adenoma [95% confidence interval (CI), 0.276–0.808], somatotroph SPAs (95% CI, 1.296–3.121), corticotroph SPAs (95% CI, 1.811–4.078), pluripotent SPAs (95% CI, 2.264–5.194), decreased expression of p16 (95% CI, 2.724–5.588), overexpression of pRb (95% CI, 2.557–5.333), cyclin D1 (95% CI, 1.894–4.122) and MIB-1 (95% CI, 1.561–4.133), increased mitotic index (95% CI, 1.228–4.079), increased p53 expression (95% CI, 1.307–4.065) and invasion into the cavernous sinus (95% CI, 3.842–7.502) predicted SPA progression following resection. The results of the present study suggested that specific cell-cycle regulators, including p16, cyclin D1 and pRb, were associated with SPA progression. PMID:29344143

Distribution and heterogeneity of mast cells in female reproductive tract and ovary on different days of the oestrus cycle in Angora goats.

PubMed

Karaca, T; Arikan, S; Kalender, H; Yoruk, M

2008-08-01

The physiological distribution of mast cells (MCs) in the reproductive tract and ovary of 12 Angora goats was determined using light microscopic histochemical techniques. Uterus (corpus uteri and cornu uteri), uterine cervix, uterine tubes (isthmus and ampulla) and ovary samples were obtained by laparatomy from groups of animals during metoestrus, dioestrus and proestrus (days 5, 10 and 16 of the oestrous cycle). Tissues were fixed in Mota's fixative (basic lead acetate) for 48 h and embedded in paraffin. Six-micrometre-thick sections were stained with toluidine blue in 1% aqueous solution at pH 1.0 for 5 min and alcian blue-Safranin at pH 1.0 for 30 min. MCs were generally associated with blood vessels in all reproductive organs. In the uterus, they were concentrated mainly in the close of the uterine gland and deep stroma in the endometrium. Higher MC numbers were observed by toluidine blue staining in the uterus, uterine cervix and uterine tubes on days 10 (corpus uterine: 4.7 +/- 3.8 and cornu uterine: 4.9 +/- 3.5) and 16 (corpus uterine: 5.9 +/- 4.5 and cornu uterine: 5.4 +/- 2.4) of the oestrous cycle compared with day 5 (p < 0.05). Mast cells were not observed in the follicles, the corpus luteum and the underside of the surface epithelium of the ovarian cortex, but were observed in the interstitial cortical stroma and the ovarian medulla. In the ovary, MC numbers were significantly higher on day 16 of the oestrous cycle (cortex: 3.4 +/- 2.4 and medulla: 5.7 +/- 4.5, p < 0.05). Safranin-positive connective tissue MCs were not observed in the uterine tube on any occasion. These results indicate oestrous cycle-related changes in the number and location of MCs in goat reproductive organs.

Expression of human telomerase reverse transcriptase protein in oral epithelial dysplasia and oral squamous cell carcinoma: An immunohistochemical study

PubMed Central

Raghunandan, Bangalore Nagarajachar; Sanjai, Karpagaselvi; Kumaraswamy, Jayalakshmi; Papaiah, Lokesh; Pandey, Bhavna; Jyothi, Bellur MadhavaRao

2016-01-01

Background: Telomerase is an RNA-dependent DNA polymerase that synthesizes TTAGGG telomeric DNA sequences and almost universally provides the molecular basis for unlimited proliferative potential. The telomeres become shorter with each cycle of replication and reach a critical limit; most cells die or enter stage of replicative senescence. Telomere length maintenance by telomerase is required for all the cells that exhibit limitless replicative potential. It has been postulated that reactivation of telomerase expression is necessary for the continuous proliferation of neoplastic cells to attain immortality. Use of immunohistochemistry (IHC) is a useful, reliable method of localizing the human telomerase reverse transcriptase (hTERT) protein in tissue sections which permits cellular localization. Although there exists a lot of information on telomerase in oral cancer, little is known about their expression in oral epithelial dysplasia and their progression to oral squamous cell carcinoma (OSCC) compared to normal oral mucosa. This study addresses this lacuna. Aims: To compare the expression of hTERT protein in oral epithelial dysplasia and OSCC with normal oral mucosa by Immunohistochemical method. Subjects and Methods: In this preliminary study, IHC was used to detect the expression of hTERT protein in OSCC (n = 20), oral epithelial dysplasia (n = 21) and normal oral mucosa (n = 10). The tissue localization of immunostain, cellular localization of immunostain, nature of stain, intensity of stain, percentage of cells stained with hTERT protein were studied. A total number of 100 cells were counted in each slide. Statistical Analysis: All the data were analyzed using SPSS software version 16.0. The tissue localization, cellular localization of cytoplasmic/nuclear/both of hTERT stain, staining intensity was compared across the groups using Pearson's Chi-square test. The mean percentage of cells stained for oral epithelial dysplasia, OSCC and normal oral mucosa were compared using analysis of variance (ANOVA). A P < 0.05 was considered to be statistically significant. Results: The mean hTERT positive cells in the study groups were as follows, 62.91% in normal oral mucosa samples, 77.06% in oral epithelial dysplasia cases, and 81.48% in OSCC. In 61.9% of oral epithelial dysplasia and 65% of OSCC in our study, staining was visualized within the nucleus predominantly in the dot like pattern. There was a statistically significant difference in the nature of nuclear stain between oral epithelial dysplasia and OSCC (P = 0.023). Conclusions: Our results suggests that the mean percentage of cells showing hTERT expression steadily increased from normal oral mucosa to oral epithelial dysplasia to OSCC. The steady trend of increase in the percentage of cells was evident in different grades of oral epithelial dysplasia group and OSCC. The nature of hTERT staining did show variations among the three groups and promise to be a potential surrogate marker for malignant transformation. Further studies using IHC on larger sample size and clinical follow-up of these patients will be ascertaining the full potential of hTERT as a surrogate marker of epithelial transformation. PMID:27194869

Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells

PubMed Central

Hu, Lifang; Su, Peihong; Li, Runzhi; Yan, Kun; Chen, Zhihao; Shang, Peng; Qian, Airong

2015-01-01

Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function. [BMB Reports 2015; 48(10): 583-588] PMID:26277981

Knockdown of microtubule actin crosslinking factor 1 inhibits cell proliferation in MC3T3-E1 osteoblastic cells.

PubMed

Hu, Lifang; Su, Peihong; Li, Runzhi; Yan, Kun; Chen, Zhihao; Shang, Peng; Qian, Airong

2015-10-01

Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function.

Hibiscus sabdariffa leaf polyphenolic extract induces human melanoma cell death, apoptosis, and autophagy.

PubMed

Chiu, Chun-Tang; Hsuan, Shu-Wen; Lin, Hui-Hsuan; Hsu, Cheng-Chin; Chou, Fen-Pi; Chen, Jing-Hsien

2015-03-01

Melanoma is the least common but most fatal form of skin cancer. Previous studies have indicated that an aqueous extract of Hibiscus sabdariffa leaves possess hypoglycemic, hypolipidemic, and antioxidant effects. In this study, we want to investigate the anticancer activity of Hibiscus leaf polyphenolic (HLP) extract in melanoma cells. First, HLP was exhibited to be rich in epicatechin gallate (ECG) and other polyphenols. Apoptotic and autophagic activities of HLP and ECG were further evaluated by DAPI stain, cell-cycle analysis, and acidic vascular organelle (AVO) stain. Our results revealed that both HLP and ECG induced the caspases cleavages, Bcl-2 family proteins regulation, and Fas/FasL activation in A375 cells. In addition, we also revealed that the cells presented AVO-positive after HLP treatments. HLP could increase the expressions of autophagy-related proteins autophagy-related gene 5 (ATG5), Beclin1, and light chain 3-II (LC3-II), and induce autophagic cell death in A375 cells. These data indicated that the anticancer effect of HLP, partly contributed by ECG, in A375 cells. HLP potentially could be developed as an antimelanoma agent. © 2015 Institute of Food Technologists®

The influence of the oestrous cycle on the radiation response of solid tumours

NASA Astrophysics Data System (ADS)

Swann, Patricia R.

Oestrogen increases the transcription of nitric oxide synthase, thus increasing nitric oxide production, which can result in vasodilation of blood vessels. Fluctuating levels of oestrogen throughout the menstrual cycle has the potential to affect tumour blood flow. Variations of blood supply to a solid tumour can influence tumour oxygenation and subsequently the percentage of hypoxic cells. As hypoxic cells are more resistant to radiation than well-oxygenated cells, this could potentially affect the radiation response of the tumour. This project evaluated the impact of the oestrous stage on the radiation response of BCHT, RIF-1 and SCCvii tumours in syngeneic C3H mice. The oestrous cycle consists of the following stages, pro-oestrus, oestrus, metoestrus and dioestrus and each stage can be determined by the cellular composition of vaginal smears. The peak of oestrogen occurs in the ovulatory phase and a second smaller peak occurs in dioestrus. Subcutaneous tumour were treated at a volume of 200 - 250 mm3 with local irradiation of 10 Gy ionising radiation at different stages of the oestrous cycle. Tumours were excised either immediately or 24 hours after irradiation and disaggregated into a single cell suspension. Tumour cell survival was assessed by clonogenic assay of the excised tumour relative to untreated tumours excised at the corresponding oestrous stage. Tumours irradiated in oestrus consistently produced the lowest surviving fraction after immediate and delayed excision. Tumours irradiated in pro-oestrus and excised immediately after irradiation, showed a two-fold increase in surviving fraction compared to tumours irradiated in oestrus. The surviving fractions of tumours excised 24 hours after irradiation were less than for tumours excised immediately after irradiation. Surviving fractions of irradiated, clamped KHT tumours were independent of oestrous stage. To confirm that these oestrous stage dependent changes were due to changes in tumour perfusion, the degree of transient perfusion in the tumours was assessed. This used a fluorescent double-staining technique by intravenous injection of the fluorescent dyes Hoechst 33342 and diheptyloxacarbocyanine with a 20 minute interval between dye administrations. These dyes stain functional blood vessels and can be viewed under the fluorescent microscope. Regions of vasculature stained with both dyes indicate constant perfusion throughout the experiment, whereas only one dye indicates mismatch or transient perfusion. Tumour vasculature that experiences intermittent perfusion will result in areas of acute hypoxia that can impact on the radiation response of the tumour. The results shows that in oestrus, KHT and RIF-1 tumours showed the lowest proportion of transient perfusion, where as this oestrous stage produced the most mismatch perfusion in the SCCvii tumour. The metastatic spread of KHT tumour cells was influenced by the oestrous cycle. Fractionated irradiation of a primary tumour during metoestrus and dioestrus showed less tumour control by radiation when compared to tumours irradiated in oestrus. The intravenous injection of KHT tumour cells in oestrus and dioestrus also produced a less metastatic burden to the lungs than cells injected in pro-oestrus and metoestrus. The results of this project suggest that there are oestrous stage dependent effects that could alter the radiation response of tumours.

Functions of the Type 1 BMP Receptor Acvr1 (Alk2) in Lens Development: Cell Proliferation, Terminal Differentiation, and Survival

PubMed Central

Rajagopal, Ramya; Dattilo, Lisa K.; Kaartinen, Vesa; Deng, Chu-Xia; Umans, Lieve; Zwijsen, An; Roberts, Anita B.; Bottinger, Erwin P.; Beebe, David C.

2009-01-01

Purpose Bone morphogenetic protein (BMP) signaling is essential for the induction and subsequent development of the lens. The purpose of this study was to analyze the function(s) of the type 1 BMP receptor, Acvr1, in lens development. Methods Acvr1 was deleted from the surface ectoderm of mouse embryos on embryonic day 9 using the Cre-loxP method. Cell proliferation, cell cycle exit, and apoptosis were measured in tissue sections by immunohistochemistry, immunofluorescence, and TUNEL staining. Results Lenses formed in the absence of Acvr1. However, Acvr1CKO (conditional knockout) lenses were small. Acvr1 signaling promoted proliferation at early stages of lens formation but inhibited proliferation at later stages. Inhibition of cell proliferation by Acvr1 was necessary for the proper regionalization of the lens epithelium and promoted the withdrawal of lens fiber cells from the cell cycle. In spite of the failure of all Acvr1CKO fiber cells to withdraw from the cell cycle, they expressed proteins characteristic of differentiated fiber cells. Although the stimulation of proliferation was Smad independent, the ability of Acvr1 to promote cell cycle exit later in development depended on classical R-Smad-Smad4 signaling. Loss of Acvr1 led to an increase in apoptosis of lens epithelial and fiber cells. Increased cell death, together with the initial decrease in proliferation, appeared to account for the smaller sizes of the Acvr1CKO lenses. Conclusions This study revealed a novel switch in the functions of Acvr1 in regulating lens cell proliferation. Previously unknown functions mediated by this receptor included regionalization of the lens epithelium and cell cycle exit during fiber cell differentiation. PMID:18566469

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue

PubMed Central

Gerdes, Michael J.; Sevinsky, Christopher J.; Sood, Anup; Adak, Sudeshna; Bello, Musodiq O.; Bordwell, Alexander; Can, Ali; Corwin, Alex; Dinn, Sean; Filkins, Robert J.; Hollman, Denise; Kamath, Vidya; Kaanumalle, Sireesha; Kenny, Kevin; Larsen, Melinda; Lazare, Michael; Lowes, Christina; McCulloch, Colin C.; McDonough, Elizabeth; Pang, Zhengyu; Rittscher, Jens; Santamaria-Pang, Alberto; Sarachan, Brion D.; Seel, Maximilian L.; Seppo, Antti; Shaikh, Kashan; Sui, Yunxia; Zhang, Jingyu; Ginty, Fiona

2013-01-01

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics. PMID:23818604

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue.

PubMed

Gerdes, Michael J; Sevinsky, Christopher J; Sood, Anup; Adak, Sudeshna; Bello, Musodiq O; Bordwell, Alexander; Can, Ali; Corwin, Alex; Dinn, Sean; Filkins, Robert J; Hollman, Denise; Kamath, Vidya; Kaanumalle, Sireesha; Kenny, Kevin; Larsen, Melinda; Lazare, Michael; Li, Qing; Lowes, Christina; McCulloch, Colin C; McDonough, Elizabeth; Montalto, Michael C; Pang, Zhengyu; Rittscher, Jens; Santamaria-Pang, Alberto; Sarachan, Brion D; Seel, Maximilian L; Seppo, Antti; Shaikh, Kashan; Sui, Yunxia; Zhang, Jingyu; Ginty, Fiona

2013-07-16

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.

β-sitosterol induces G1 arrest and causes depolarization of mitochondrial membrane potential in breast carcinoma MDA-MB-231 cells

PubMed Central

2013-01-01

Backgrounds It is suggested that dietary phytosterols, such as β-sitosterol (ST), have cancer chemopreventive effects; however, studies are limited to support such claims. Here, we evaluated the efficacy of ST on three different human cancer cell lines including skin epidermoid carcinoma A431 cells, lung epithelial carcinoma A549 cells and breast adenocarcinoma MDA-MB-231. Methods Cell growth assay, cell cycle analysis, FACS, JC-1 staining, annexin V staining and immunoblotting were used to study the efficacy of ST on cancer cells. Results ST (30–90 μM) treatments for 48 h and 72 h did not show any significant effect on cell growth and death in A431 cells. Whereas similar ST treatments moderately inhibited the growth of A549 cells by up to 13% (p ≤ 0.05) in 48 h and 14% (p ≤ 0.05-0.0001) in 72 h. In MDA-MB-231 cells, ST caused a significant dose-dependent cell growth inhibition by 31- 63% (p ≤ 0.0001) in 48 h and 40-50% (p ≤ 0.0001) in 72 h. While exploring the molecular changes associated with strong ST efficacy in breast cancer cells, we observed that ST induced cell cycle arrest as well as cell death. ST caused G0/G1 cell cycle arrest which was accompanied by a decrease in CDK4 and cyclin D1, and an increase in p21/Cip1and p27/Kip1 protein levels. Further, cell death effect of ST was associated with induction of apoptosis. ST also caused the depolarization of mitochondrial membrane potential and increased Bax/Bcl-2 protein ratio. Conclusions These results suggest prominent in vitro anti-proliferative and pro-apoptotic effects of ST in MDA-MB-231 cells. This study provides valuable insight into the chemopreventive efficacy and associated molecular alterations of ST in breast cancer cells whereas it had only moderate efficacy on lung cancer cells and did not show any considerable effect on skin cancer cells. These findings would form the basis for further studies to understand the mechanisms and assess the potential utility of ST as a cancer chemopreventive agent against breast cancer. PMID:24160369

Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds.

PubMed

Schuck, Desirée Cigaran; Ribeiro, Ramira Yuri; Nery, Arthur A; Ulrich, Henning; Garcia, Célia R S

2011-11-01

Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO-1 allowing precise discrimination between mono- and multinucleated forms of P. falciparum-infected red blood cell. The use of YOYO-1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont-stage parasites were about 10-fold greater than those of ring-trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N-acetyl-serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non-treated control cultures. YOYO-1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds. Copyright © 2011 International Society for Advancement of Cytometry.

Amygdala Kindling Alters Estrus Cycle and Ovarian Morphology in the Rat.

PubMed

Pan, Juan; Zhang, Lingwu; Wang, Feng; Liu, Dan; Li, P Andy; Sun, Tao

2013-11-01

The objective of this study is to explore the effects of amygdala kindling on estrus cycle and ovarian morphology. Thirty-five female rats at the age of 8 weeks were randomly designated to electrode kindled, sham-kindled, and normal controls. Kindled rats were implanted with kindling electrodes in the left basolateral amygdala and kindled by brief suprathreshold stimulations with a bipolar electrode. Estrous cycles were daily monitored through vaginal smears. Electrographic and behavioral seizures were recorded and ovarian morphology was evaluated by light and electron microscopies. Our results showed that the kindled rats lost their ovarian periodicity displayed significant ovarian enlargement. H&E staining revealed increased number of growing follicles and total follicles, as well as polycysts in the ovaries of the kindled animals compared to sham and control animals. Ultrastructural study detected numerous apoptotic granulosa cells in growing follicles and thecal cell hyperplasia with secretary granules in the thecal cells in the kindled rats. The results suggest that amygdala kindling is a risk factor for the development of polycystic ovary syndrome.

Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

DOEpatents

Dolbeare, Frank A.; Gray, Joe W.

1986-01-01

A method for the simultaneous flow cytometric measurement of the total DNA content and the level of DNA synthesis in normal and malignant cells is disclosed. The sensitivity of the method allows a study of cell cycle traverse rates for large scale cell populations as well as single cell measurements. A DNA stain such as propidium iodide is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as bromodeoxyuridine (BrdU) is used as a probe for the measurement of BrdU uptake by the cells as a measure of DNA synthesis.

Alteration of Cell Cycle Mediated by Zinc in Human Bronchial ...

EPA Pesticide Factsheets

Zinc (Zn2+), a ubiquitous ambient air contaminant, presents an oxidant challenge to the human lung and is linked to adverse human health effects. To further elucidate the adaptive and apoptotic cellular responses of human airway cells to Zn2+, we performed pilot studies to examine cell cycle perturbation upon exposure using a normal human bronchial epithelial cell culture (BEAS-2B). BEAS-2B cells were treated with low (0, 1, 2 µM) and apoptotic (3 µM) doses of Zn2+ plus 1 µM pyrithione, a Zn2+-specific ionophore facilitating cellular uptake, for up to 24 h. Fixed cells were then stained with propidium iodine (PI) and cell cycle phase was determined by fluorescent image cytometry. Initial results report the percentage of cells in the S phase after 18 h exposure to 1, 2, and 3 µM Zn2+ were similar (8%, 7%, and 12%, respectively) compared with 7% in controls. Cells exposed to 3 µM Zn2+ increased cell populations in G2/M phase (76% versus 68% in controls). Interestingly, exposure to 1 µM Zn2+ resulted in decreased (59%) cells in G2/M. While preliminary, these pilot studies suggest Zn2+ alters cell cycle in BEAS-2B cells, particularly in the G2/M phase. The G2/M checkpoint maintains DNA integrity by enabling initiation of DNA repair or apoptosis. Our findings suggest that the adaptive and apoptotic responses to Zn2+ exposure may be mediated via perturbation of the cell cycle at the G2/M checkpoint. This work was a collaborative summer student project. The st

Localization of estrogen receptor α and progesterone receptor B in bovine cervix and vagina during the follicular and luteal phases of the sexual cycle.

PubMed

Sağsöz, H; Akbalik, M E; Saruhan, B G; Ketani, M A

2011-08-01

The localization and distribution of estrogen receptors (ERα) and progesterone receptors (PR-B) in the cervix and vagina of sexually mature bovines during the follicular and luteal phases of the sexual cycle were studied using immunohistochemistry. The estrous cycle stage of 23 Holstein bovines was assessed by gross and histological appearance of ovaries and blood steroid hormone values. Tissue samples from cervix and vagina were fixed in 10% formaldehyde for routine histological processing. Nuclear staining for ERα and PR-B was observed in the epithelial cells of the surface epithelium, stromal cells and smooth muscle cells. Generally, in the cervix, ERα immunoreactivity was more intense in the epithelial and smooth muscle cells during the follicular phase and in the epithelial cells during the luteal phase (p < 0.05). PR-B immunoreactivity was more intense in the epithelial and smooth muscle cells than in the superficial and deep stromal cells during the follicular and luteal phases (p < 0.05). In the vagina, ERα and PR-B immunoreactivities were more intense in the epithelial cells than in the connective tissue cells and smooth muscle cells during the follicular and luteal phases (p < 0.05). These results indicated that the frequency and intensity of ERα and PR-B immunoreactivity in the cervix and vagina of bovines varied according to the cervical and vaginal cell types and the phases of the sexual cycle.

Assessing cell cycle progression of neural stem and progenitor cells in the mouse developing brain after genotoxic stress.

PubMed

Etienne, Olivier; Bery, Amandine; Roque, Telma; Desmaze, Chantal; Boussin, François D

2014-05-07

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage. An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

LSD1 is Required for Hair Cell Regeneration in Zebrafish.

PubMed

He, Yingzi; Tang, Dongmei; Cai, Chengfu; Chai, Renjie; Li, Huawei

2016-05-01

Lysine-specific demethylase 1 (LSD1/KDM1A) plays an important role in complex cellular processes such as differentiation, proliferation, apoptosis, and cell cycle progression. It has recently been demonstrated that during development, downregulation of LSD1 inhibits cell proliferation, modulates the expression of cell cycle regulators, and reduces hair cell formation in the zebrafish lateral line, which suggests that LSD1-mediated epigenetic regulation plays a key role in the development of hair cells. However, the role of LSD1 in hair cell regeneration after hair cell loss remains poorly understood. Here, we demonstrate the effect of LSD1 on hair cell regeneration following neomycin-induced hair cell loss. We show that the LSD1 inhibitor trans-2-phenylcyclopropylamine (2-PCPA) significantly decreases the regeneration of hair cells in zebrafish after neomycin damage. In addition, immunofluorescent staining demonstrates that 2-PCPA administration suppresses supporting cell proliferation and alters cell cycle progression. Finally, in situ hybridization shows that 2-PCPA significantly downregulates the expression of genes related to Wnt/β-catenin and Fgf activation. Altogether, our data suggest that downregulation of LSD1 significantly decreases hair cell regeneration after neomycin-induced hair cell loss through inactivation of the Wnt/β-catenin and Fgf signaling pathways. Thus, LSD1 plays a critical role in hair cell regeneration and might represent a novel biomarker and potential therapeutic approach for the treatment of hearing loss.

Anti-cancer Activity of Osmanthus matsumuranus Extract by Inducing G2/M Arrest and Apoptosis in Human Hepatocellular Carcinoma Hep G2 Cells

PubMed Central

Jin, Soojung; Park, Hyun-Jin; Oh, You Na; Kwon, Hyun Ju; Kim, Jeong-Hwan; Choi, Yung Hyun; Kim, Byung Woo

2015-01-01

Background: Osmanthus matsumuranus, a species of Oleaceae, is found in East Asia and Southeast Asia. The bioactivities of O. matsumuranus have not yet been fully understood. Here, we studied on the molecular mechanisms underlying anti-cancer effect of ethanol extract of O. matsumuranus (EEOM). Methods: Inhibitory effect of EEOM on cell growth and proliferation was determined by WST assay in various cancer cells. To investigate the mechanisms of EEOM-mediated cytotoxicity, HepG2 cells were treated with various concentration of EEOM and analyzed the cell cycle arrest and apoptosis induction by flow cytometry, Western blot analysis, 4,6-diamidino-2-phenylindole (DAPI) staining and DNA fragmentation. Results: EEOM showed the cytotoxic activities in a dose-dependent manner in various cancer cell lines but not in normal cells, and HepG2 cells were most susceptible to EEOM-induced cytotoxicity. EEOM induced G2/M arrest in HepG2 cells associated with decreased expression of cyclin-dependent kinase 1 (CDK1), cyclin A and cylcin B, and increased expression of phospho-checkpoint kinase 2, p53 and CDK inhibitor p21. Immunofluorescence staining showed that EEOM-treated HepG2 increased doublet nuclei and condensed actin, resulting in cell rounding. Furthermore, EEOM-mediated apoptosis was determined by Annexin V staining, chromatin condensation and DNA fragmentation. EEOM caused upregulation of FAS and Bax, activation of caspase-3, -8, -9, and fragmentation of poly ADP ribose polymerase. Conclusions: These results suggest that EEOM efficiently inhibits proliferation of HepG2 cells by inducing both G2/M arrest and apoptosis via intrinsic and extrinsic pathways, and EEOM may be used as a cancer chemopreventive agent in the food or nutraceutical industry. PMID:26734586

Mouse bone marrow-derived mesenchymal stem cells inhibit leukemia/lymphoma cell proliferation in vitro and in a mouse model of allogeneic bone marrow transplant

PubMed Central

SONG, NINGXIA; GAO, LEI; QIU, HUIYING; HUANG, CHONGMEI; CHENG, HUI; ZHOU, HONG; LV, SHUQING; CHEN, LI; WANG, JIANMIN

2015-01-01

The allogeneic hematopoietic stem cell (HSC) transplantation of mesenchymal stem cells (MSCs) contributes to the reconstitution of hematopoiesis by ameliorating acute graft-versus-host disease (aGVHD). However, the role of MSCs in graft-versus-leukemia remains to be determined. In the present study, we co-cultured C57BL/6 mouse bone marrow (BM)-derived MSCs with A20 murine B lymphoma, FBL3 murine erythroleukemia and P388 murine acute lymphocytic leukemia cells. Cell proliferation, apoptosis, cell cycle progression and the amount of cytokine secretion were then measured using a Cell Counting kit-8, Annexin V/propidium iodide staining, flow cytometry and ELISA, respectively. We also established a model of allogeneic bone marrow transplantation (BMT) using BALB/c mice. Following the administration of A20 cells and MSCs, we recorded the symptoms and the survival of the mice for 4 weeks, assessed the T cell subsets present in peripheral blood, and, after the mice were sacrifice, we determined the infiltration of MSCs into the organs by histological staining. Our results revealed that the MSCs inhibited the proliferation of the mouse lymphoma and leukemia cells in vitro, leading to cell cycle arrest and reducing the secretion of interleukin (IL)-10. In our model of allogeneic BMT, the intravenous injection of MSCs into the mice injected wth A20 cells decreased the incidence of lymphoma, improved survival, increased the fraction of CD3+CD8+ T cells, decreased the fraction of CD3+CD4+ T cells and CD4+CD25+ T cells in peripheral blood, and ameliorated the manifestation of aGVHD. The results from the present study indicate that MSCs may be safe and effective when used in allogeneic BMT for the treatment of hemotological malignancies. PMID:25901937

Mouse bone marrow-derived mesenchymal stem cells inhibit leukemia/lymphoma cell proliferation in vitro and in a mouse model of allogeneic bone marrow transplant.

PubMed

Song, Ningxia; Gao, Lei; Qiu, Huiying; Huang, Chongmei; Cheng, Hui; Zhou, Hong; Lv, Shuqing; Chen, Li; Wang, Jianmin

2015-07-01

The allogeneic hematopoietic stem cell (HSC) transplantation of mesenchymal stem cells (MSCs) contributes to the reconstitution of hematopoiesis by ameliorating acute graft‑versus‑host disease (aGVHD). However, the role of MSCs in graft‑versus‑leukemia remains to be determined. In the present study, we co‑cultured C57BL/6 mouse bone marrow (BM)‑derived MSCs with A20 murine B lymphoma, FBL3 murine erythroleukemia and P388 murine acute lymphocytic leukemia cells. Cell proliferation, apoptosis, cell cycle progression and the amount of cytokine secretion were then measured using a Cell Counting kit‑8, Annexin V/propidium iodide staining, flow cytometry and ELISA, respectively. We also established a model of allogeneic bone marrow transplantation (BMT) using BALB/c mice. Following the administration of A20 cells and MSCs, we recorded the symptoms and the survival of the mice for 4 weeks, assessed the T cell subsets present in peripheral blood, and, after the mice were sacrifice, we determined the infiltration of MSCs into the organs by histological staining. Our results revealed that the MSCs inhibited the proliferation of the mouse lymphoma and leukemia cells in vitro, leading to cell cycle arrest and reducing the secretion of interleukin (IL)‑10. In our model of allogeneic BMT, the intravenous injection of MSCs into the mice injected wth A20 cells decreased the incidence of lymphoma, improved survival, increased the fraction of CD3+CD8+ T cells, decreased the fraction of CD3+CD4+ T cells and CD4+CD25+ T cells in peripheral blood, and ameliorated the manifestation of aGVHD. The results from the present study indicate that MSCs may be safe and effective when used in allogeneic BMT for the treatment of hemotological malignancies.

Butyrate-induced proapoptotic and antiangiogenic pathways in EAT cells require activation of CAD and downregulation of VEGF

DOE Office of Scientific and Technical Information (OSTI.GOV)

Belakavadi, Madesh; Prabhakar, B.T.; Salimath, Bharathi P.

Butyrate, a short-chain fatty acid produced in the colon, induces cell cycle arrest, differentiation, and apoptosis in transformed cell lines. In this report, we study the effects of butyrate (BuA) on the growth of Ehrlich ascites tumor (EAT) cells in vivo. BuA, when injected intraperitoneally (i.p) into mice, inhibited proliferation of EAT cells. Further, induction of apoptosis in EAT cells was monitored by nuclear condensation, annexin-V staining, DNA fragmentation, and translocation of caspase-activated DNase into nucleus upon BuA-treatment. Ac-DEVD-CHO, a caspase-3 inhibitor, completely inhibited BuA-induced apoptosis, indicating that activation of caspase-3 mediates the apoptotic pathway in EAT cells. The proapoptoticmore » effect of BuA also reflects on the antiangiogenic pathway in EAT cells. The antiangiogenic effect of BuA in vivo was demonstrated by the downregulation of the secretion of VEGF in EAT cells. CD31 immunohistochemical staining of peritoneum sections clearly indicated a potential angioinhibitory effect of BuA in EAT cells. These results suggest that BuA, besides regulating other fundamental cellular processes, is able to modulate the expression/secretion of the key angiogenic growth factor VEGF in EAT cells.« less

Apigenin inhibits renal cell carcinoma cell proliferation.

PubMed

Meng, Shuai; Zhu, Yi; Li, Jiang-Feng; Wang, Xiao; Liang, Zhen; Li, Shi-Qi; Xu, Xin; Chen, Hong; Liu, Ben; Zheng, Xiang-Yi; Xie, Li-Ping

2017-03-21

Apigenin, a natural flavonoid found in vegetables and fruits, has antitumor activity in several cancer types. The present study evaluated the effects and mechanism of action of apigenin in renal cell carcinoma (RCC) cells. We found that apigenin suppressed ACHN, 786-0, and Caki-1 RCC cell proliferation in a dose- and time-dependent manner. A comet assay suggested that apigenin caused DNA damage in ACHN cells, especially at higher doses, and induced G2/M phase cell cycle arrest through ATM signal modulation. Small interfering RNA (siRNA)-mediated p53 knockdown showed that apigenin-induced apoptosis was likely p53 dependent. Apigenin anti-proliferative effects were confirmed in an ACHN cell xenograft mouse model. Apigenin treatment reduced tumor growth and volume in vivo, and immunohistochemical staining revealed lower Ki-67 indices in tumors derived from apigenin-treated mice. These findings suggest that apigenin exposure induces DNA damage, G2/M phase cell cycle arrest, p53 accumulation and apoptosis, which collectively suppress ACHN RCC cell proliferation in vitro and in vivo. Given its antitumor effects and low in vivo toxicity, apigenin is a highly promising agent for treatment of RCC.

Enhanced detection of fluorescence quenching in labeled cells

DOEpatents

Crissman, H.A.; Steinkamp, J.A.

1987-11-30

A method is provided for quantifying BrdU labeled DNA in cells. The BrdU is substituted onto the DNA and the DNA is stained with a first fluorochrome having a fluorescence which is quenchable by BrdU. The first fluorochrome is preferably a thymidine base halogen analogue, such as a Hoechst fluorochrome. The DNA is then stained with a second fluorochrome having a fluorescence which is substantially uneffected by BrdU. The second fluorochrome may be selected from the group consisting of mithramycin, chromomycin A3, olivomycin, propidium iodide and ethidium bromine. The fluorescence from the first and second fluorochromes is then measured to obtain first and second output signals, respectively. The first output signal is subtracted from the second output signal to obtain a difference signal which is functionally related to the quantity of BrdU incorporated into DNA. The technique is particularly useful for quantifying the synthesis of DNA during the S-phase of the cell cycle. 2 figs.

Enhanced detection of fluorescence quenching in labeled cells

DOEpatents

Crissman, Harry A.; Steinkamp, John A.

1992-01-01

A method is provided for quantifying BrdU labeled DNA in cells. The BrdU is incorporated into the DNA and the DNA is stained with a first fluorochrome having a fluorescence which is quenchable by BrdU. The first fluorochrome is preferably a thymidine base halogen analogue, such as a Hoechst fluorochrome. The DNA is then stained with a second fluorochrome having a fluorescence that is substantially uneffected by BrdU. The second fluorochrome may be selected from the group consisting of mithramycin, chromomycin A3, olivomycin, propidium iodide and ethidium bromine. The fluorescence from the first and second fluorochromes is then measured to obtain first and second output signals, respectively. The first output signal is substracted from the second output signal to obtain a difference signal which is functionally related to the quantity of BrdU incorporated into DNA. The technique is particularly useful for quantifying the synthesis of DNA during the S-phase of the cell cycle.

Cryptotanshinone induces cell cycle arrest and apoptosis through the JAK2/STAT3 and PI3K/Akt/NFκB pathways in cholangiocarcinoma cells

PubMed Central

Ke, Fayong; Wang, Zheng; Song, Xiaoling; Ma, Qiang; Hu, Yunping; Jiang, Lin; Zhang, Yijian; Liu, Yingbin; Zhang, Yong; Gong, Wei

2017-01-01

Background Cholangiocarcinoma (CCA) is the most common biliary tract malignancy in the world with high resistance to current chemotherapies and extremely poor prognosis. The main objective of this study was to investigate the inhibitory effects of cryptotanshinone (CTS), a natural compound isolated from Salvia miltiorrhiza Bunge, on CCA both in vitro and in vivo and to explore the underlying mechanisms of CTS-induced apoptosis and cell cycle arrest. Methods The anti-tumor activity of CTS on HCCC-9810 and RBE cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and colony forming assays. Cell cycle changes were detected by flow cytometric analysis. Apoptosis was detected by annexin V/propidium iodide double staining and Hoechst 33342 staining assays. The efficacy of CTS in vivo was evaluated using a HCCC-9810 xenograft model in athymic nude mice. The expression of key proteins involved in cell apoptosis and signaling pathway in vitro was analyzed by Western blot analysis. Results CTS induced potent growth inhibition, S-phase arrest, apoptosis, and colony-forming inhibition in HCCC-9810 and RBE cells in a dose-dependent manner. Intraperitoneal injection of CTS (0, 10, or 25 mg/kg) for 4 weeks significantly inhibited the growth of HCCC-9810 xenografts in athymic nude mice. CTS treatment induced S-phase arrest with a decrease of cyclin A1 and an increase of cyclin D1 protein level. Bcl-2 expression was downregulated remarkably, while Bax expression was increased after apoptosis occurred. Additionally, the activation of JAK2/STAT3 and PI3K/Akt/NFκB was significantly inhibited in CTS-treated CCA cells. Conclusion CTS induced CCA cell apoptosis by suppressing both the JAK2/STAT3 and PI3K/Akt/NFκB signaling pathways and altering the expression of Bcl-2/Bax family, which was regulated by these two signaling pathways. CTS may serve as a potential therapeutic agent for CCA. PMID:28670110

PSCA expression is associated with favorable tumor features and reduced PSA recurrence in operated prostate cancer.

PubMed

Heinrich, Marie-Christine; Göbel, Cosima; Kluth, Martina; Bernreuther, Christian; Sauer, Charlotte; Schroeder, Cornelia; Möller-Koop, Christina; Hube-Magg, Claudia; Lebok, Patrick; Burandt, Eike; Sauter, Guido; Simon, Ronald; Huland, Hartwig; Graefen, Markus; Heinzer, Hans; Schlomm, Thorsten; Heumann, Asmus

2018-05-31

Prostate Stem Cell Antigen (PSCA) is frequently expressed in prostate cancer but its exact function is unclear. To clarify contradictory findings on the prognostic role of PSCA expression, a tissue microarray containing 13,665 prostate cancers was analyzed by immunohistochemistry. PSCA staining was absent in normal epithelial and stromal cells of the prostate. Membranous and cytoplasmic PSCA staining was seen in 53.7% of 9642 interpretable tumors. Staining was weak in 22.4%, moderate in 24.5% and strong in 6.8% of tumors. PSCA expression was associated with favorable pathological and clinical tumor features: Early pathological tumor stage (p < 0.0001), low Gleason grade (p < 0.0001), absence of lymph node metastasis (p < 0.0001), low pre-operative PSA level (p = 0.0118), negative surgical margin (p < 0.0001) and reduced PSA recurrence (p < 0.0001). PSCA expression was an independent predictor of prognosis in multivariate analysis (hazard ratio 0.84, p < 0.0001). The absence of statistical relationship to TMPRSS2:ERG fusion status, chromosomal deletion or high tumor cell proliferation argues against a major role of PSCA for regulation of cell cycle or genomic integrity. PSCA expression is linked to favorable prognosis. PSCA measurement is a candidate for inclusion in multi-parametric prognostic prostate cancer tests.

Maize histone H2B-mCherry: a new fluorescent chromatin marker for somatic and meiotic chromosome research.

PubMed

Howe, Elizabeth S; Clemente, Thomas E; Bass, Hank W

2012-06-01

Cytological studies of fluorescent proteins are rapidly yielding insights into chromatin structure and dynamics. Here we describe the production and cytological characterization of new transgenic maize lines expressing a fluorescent histone fusion protein, H2B-mCherry. The transgene is expressed under the control of the maize ubiquitin1 promoter, including its first exon and intron. Polymerase chain reaction-based genotyping and root-tip microscopy showed that most of the lines carrying the transgene also expressed it, producing bright uniform staining of nuclei. Further, plants showing expression in root tips at the seedling stage also showed expression during meiosis, late in the life cycle. Detailed high-resolution three-dimensional imaging of cells and nuclei from various somatic and meiotic cell types showed that H2B-mCherry produced remarkably clear images of chromatin and chromosome fiber morphology, as seen in somatic, male meiotic prophase, and early microgametophyte cells. H2B-mCherry also yielded distinct nucleolus staining and was shown to be compatible with fluorescence in situ hybridization. We found several instances where H2B-mCherry was superior to DAPI as a generalized chromatin stain. Our study establishes these histone H2B-mCherry lines as new biological reagents for visualizing chromatin structure, chromosome morphology, and nuclear dynamics in fixed and living cells in a model plant genetic system.

The Analysis of Cell Cycle-related Proteins in Ovarian Clear Cell Carcinoma Versus High-grade Serous Carcinoma.

PubMed

Hazama, Yukiko; Moriya, Takuya; Sugihara, Mika; Sano, Rikiya; Shiota, Mitsuru; Nakamura, Takafumi; Shimoya, Koichiro

2017-10-10

In Japan, the frequency of ovarian clear cell carcinoma (CCC) is twice as high as that in the United States and Europe. Often, patient prognosis of CCC is poor because of chemoresistance. Here, we focus on the cell cycle, which is one of the mechanisms of chemoresistance. To detect the informative markers and improve the strategy of chemotherapy for CCC, we performed immunochemical staining of cell cycle-related proteins in ovarian malignant tumors. We detected that each of the 29 samples of CCC and high-grade serous carcinoma (HGSC) were necessary to reveal the significant differences in immunostaining and prognosis. We performed the immunostaining analysis using the antibodies of cell cycle-related proteins such as Ki-67, Cdt1, MCM7, and geminin. The positive rate of Cdt1 in the CCC group was significantly higher than that in the HGSC group (P<0.0001). However, the positive rate of geminin in the HGSC group was significantly higher than that in the CCC group (P<0.0001). The overall survival of CCC patients with high labeling index of Cdt1 was significantly worse than that of CCC patients with low labeling index of Cdt1 (P=0.004). The study results suggested that the cancer cells of CCC and HGSC exist in the G1 phase and S, G2, and M phases, respectively. The differences in cell cycle of CCC might be one of the reasons for chemotherapy resistance. Further investigations are necessary to reveal the usefulness of Cdt1 as a biomarker in CCC.

Preventive effects of 125I seeds on benign restenosis following esophageal stent implantation in a dog model

PubMed Central

GAN, ZHEN; JING, JIAN; ZHU, GUANGYU; QIN, YONGLIN; TENG, GAOJUN; GUO, JINHE

2015-01-01

The present study aimed to evaluate the effects of iodine-125 (125I) seeds on the proliferation of primary esophageal fibroblasts in dogs, and to assess the safety and preventive efficacy of 125I seed-pre-loaded esophageal stents in benign restenosis following implantation. Primary fibroblasts were cultured with various 125I seed activities, which were then evaluated using cell proliferation and apoptosis assays as well as cell cycle analysis using Annexin V/propidium iodide (PI) double staining and PI staining. Prior to sacrification, animals were submitted to esophageal radiography under digital subtraction angiography. Esophageal tissues were collected and examined for macroscopic, microscopic and pathological alterations. The results demonstrated a significant and dose-dependent inhibition of fibroblast proliferation and increased apoptosis following exposure to 125I seeds. G0/G1 fibroblast populations increased in a dose-dependent manner following treatment with 125I seeds, in contrast to cells in S phase. Four weeks following implantation, α-smooth muscle actin and proliferating cell nuclear antigen expression levels in the experimental group were significantly lower compared with those in the control group; in addition, eight weeks following implantation, esophageal inner diameters were increased in the experimental group. 125I seeds inhibited proliferation of dog esophageal fibroblasts via cell cycle arrest and apoptosis. In conclusion, 125I seed-pre-loaded esophageal stents inhibited benign hyperplasia in the upper edge of the stent to a certain extent, which relieved benign restenosis following implantation with a good safety profile. PMID:25543838

Therapeutic effect of a novel Wnt pathway inhibitor on cardiac regeneration after myocardial infarction.

PubMed

Yang, Dezhong; Fu, Wenbin; Li, Liangpeng; Xia, Xuewei; Liao, Qiao; Yue, Rongchuan; Chen, Hongmei; Chen, Xiongwen; An, Songzhu; Zeng, Chunyu; Wang, Wei Eric

2017-12-15

After myocardial infarction (MI), the heart is difficult to repair because of great loss of cardiomyoctyes and lack of cardiac regeneration. Novel drug candidates that aim at reducing pathological remodeling and stimulating cardiac regeneration are highly desirable. In the present study, we identified if and how a novel porcupine inhibitor CGX1321 influenced MI and cardiac regeneration. Permanent ligation of left anterior descending (LAD) coronary artery was performed in mice to induce MI injury. Cardiac function was measured by echocardiography, infarct size was examined by TTC staining. Fibrosis was evaluated with Masson's trichrome staining and vimentin staining. As a result, CGX1321 administration blocked the secretion of Wnt proteins, and inhibited both canonical and non-canonical Wnt signaling pathways. CGX1321 improved cardiac function, reduced myocardial infarct size, and fibrosis of post-MI hearts. CGX1321 significantly increased newly formed cardiomyocytes in infarct border zone of post-MI hearts, evidenced by the increased EdU + cardiomyocytes. Meanwhile, CGX1321 increased Ki67 + and phosphohistone H3 (PH3 + ) cardiomyocytes in culture, indicating enhanced cardiomyocyte proliferation. The mRNA microarray showed that CGX1321 up-regulated cell cycle regulating genes such as Ccnb1 and Ccne1 CGX1321 did not alter YAP protein phosphorylation and nuclear translocation in cardiomyocytes. In conclusion, porcupine inhibitor CGX1321 reduces MI injury by limiting fibrosis and promoting regeneration. It promotes cardiomyocyte proliferation by stimulating cell cycle regulating genes with a Hippo/YAP-independent pathway. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Multi-tasking Sulf1/Sulf2 enzymes do not only facilitate extracellular cell signalling but also participate in cell cycle related nuclear events.

PubMed

Krishnakumar, Kavithanjali; Chakravorty, Ishani; Foy, Wendy; Allen, Steve; Justo, Tiago; Mukherjee, Abir; Dhoot, Gurtej K

2018-03-01

This study demonstrates highly dynamic spatial and temporal pattern of SULF1/SULF2 expression in a number of neuronal cell types growing in normal culture medium that included their transient nuclear mobilisation. Their nuclear translocation became particularly apparent during cell proliferation as both SULF1/SULF2 demonstrated not only cell membrane associated expression, their known site of function but also transient nuclear mobilisation during nuclear cell division. Nuclear localisation was apparent not only by immunocytochemical staining but also confirmed by immunoblotting staining of isolated nuclear fractions of C6, U87 and N2A cells. Immunocytochemical analysis demonstrated rapid nuclear exit of both SULF1/SULF2 following cell division that was slightly delayed but not blocked in a fraction of the polyploid cells observed in C6 cells. The overexpression of both Sulf1 and Sulf2 genes in C6 and U87 cells markedly promoted in vitro growth of these cells accompanied by nuclear mobilisation while inhibition of both these genes inhibited cell proliferation with little or no nuclear SULF1/SULF2 mobilisation. SULF1/SULF2 activity in these cells thus demonstrated a clear co-ordination of extracellular cell signalling with nuclear events related to cell proliferation. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.

Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2-independent mechanism.

PubMed

Wang, Rong; Yu, Zhen; Sunchu, Bharath; Shoaf, James; Dang, Ivana; Zhao, Stephanie; Caples, Kelsey; Bradley, Lynda; Beaver, Laura M; Ho, Emily; Löhr, Christiane V; Perez, Viviana I

2017-06-01

Senescent cells contribute to age-related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild-type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA-β-galactosidase (β-gal) staining, senescence-associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased β-gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in β-gal staining and pro-inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Novel purification of 1'S-1'-Acetoxychavicol acetate from Alpinia galanga and its cytotoxic plus antiproliferative activity in colorectal adenocarcinoma cell line SW480.

PubMed

Baradwaj, R G; Rao, M V; Senthil Kumar, T

2017-07-01

Alpinia galanga (L.) Willd. is a valuable medicinal crop found in specific tropical regions of southeast Asia. Its crude extracts are well known for their wide medicinal properties and many compounds identified from these extracts are of great interest currently. 1'S-1'-Acetoxychavicol acetate (ACA) obtained from rhizomes of A.galanga is one such well-illustrated compound. This study strives to progress and simplifies the purification protocol for ACA from A.galanga rhizomes. It also studies the cytotoxicity and antiproliferative activity of ACA against Dukes' type B, colorectal adenocarcinoma (SW480). HPLC standardisation was carried out for purification of ACA from rhizomes of Alpinia galanga. MTT assay was executed to estimate the IC 50 value of ACA against SW480 cell line. This value was used to study the apoptosis, nuclear morphological changes and mitochondrial membrane permeability using Acridine orange/ethidium bromide, DAPI, and JC-1 staining. The DNA fragmentation assay was used to substantiate the nuclear fragmentation of DNA observed in the DAPI staining. Further, cell cycle analysis was performed using flow cytometry to study the exact stage of the cell cycle where SW480 cells are arrested due to ACA, western blot analysis of relevant genes were done to further understand at molecular level. A comprehensive 1.89g of 1'S-1'-Acetoxychavicol acetate (ACA) was recovered from 500g of A.galanga rhizomes. ACA significantly suppressed the proliferation of SW480 cells at an IC 50 of 80μM (48h). The mode of SW480 cell death due to ACA was initially identified as apoptosis and cell cycle halted at G 0 /G 1 checkpoint with considerable DNA damage and mitochondrial depolarization. The expression of p21 was increased and concomitantly Cyclin D was downregulated in ACA treated in comparison to control. This study suggests that 1'S-1'-Acetoxychavicol acetate has potent anti-colorectal adenocarcinoma activity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

PubMed

Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

2013-10-01

Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

A novel histological technique for distinguishing between epithelial cells in forensic casework.

PubMed

French, Claire E V; Jensen, Cynthia G; Vintiner, Susan K; Elliot, Douglas A; McGlashan, Susan R

2008-06-10

There are a number of forensic cases in which the identification of the epithelial cell type from which DNA originated would provide important probative evidence. This study aimed to develop a technique using histological staining of fixed cells to distinguish between skin, buccal and vaginal epithelium. First, 11 different stains were screened on formalin-fixed, wax-embedded cells from five women. Samples were analysed qualitatively by examining staining patterns (colour) and morphology (absence or presence of nuclei). Three of the staining methods--Dane's, Csaba's and Ayoub-Shklar--were successful in distinguishing skin epithelial cells from buccal and vaginal. Second, cells were smeared directly onto slides, fixed with one of five fixatives and stained with one of the three stains mentioned above. Methanol fixation, coupled with the Dane's staining method, specific to keratin, was the only technique that distinguished between all three cell types. Skin cells stained magenta, red and orange and lacked nuclei; buccal cells stained predominantly orange-pink with red nuclei; while vaginal cells stained bright orange with orange nuclei and a blue extracellular hue. This staining pattern in vaginal cells was consistent in samples collected from 50 women aged between 18 and 67. Identification of cell type from unlabelled micrographs by 10 trained observers showed a mean success rate of 95%. The results of this study demonstrate that histological staining may provide forensic scientists with a technique for distinguishing between skin, buccal and vaginal epithelial cells and thus would enable more conclusive analyses when investigating sexual assault cases.

Role of potassium channels in chlorogenic acid-induced apoptotic volume decrease and cell cycle arrest in Candida albicans.

PubMed

Yun, JiEun; Lee, Dong Gun

2017-03-01

Chlorogenic acid (CRA) is an abundant phenolic compound in the human diet. CRA has a potent antifungal effect, inducing cell death in Candida albicans. However, there are no further studies to investigate the antifungal mechanism of CRA, associated with ion channels. To evaluate the inhibitory effects on CRA-induced cell death, C. albicans cells were pretreated with potassium and chloride channel blockers, separately. Flow cytometry was carried out to detect several hallmarks of apoptosis, such as cell cycle arrest, caspase activation, and DNA fragmentation, after staining of the cells with SYTOX green, FITC-VAD-FMK, and TUNEL. CRA caused excessive potassium efflux, and an apoptotic volume decrease (AVD) was observed. This change, in turn, induced cytosolic calcium uptake and cell cycle arrest in C. albicans. Moreover, CRA induced caspase activation and DNA fragmentation, which are considered apoptotic markers. In contrast, the potassium efflux and proapoptotic changes were inhibited when potassium channels were blocked, whereas there was no inhibitory effect when chloride channels were blocked. CRA induces potassium efflux, leading to AVD and G2/M cell cycle arrest in C. albicans. Therefore, potassium efflux via potassium channels regulates the CRA-induced apoptosis, stimulating several apoptotic processes. This study improves the understanding of the antifungal mechanism of CRA and its association with ion homeostasis, thereby pointing to a role of potassium channels in CRA-induced apoptosis. Copyright © 2016. Published by Elsevier B.V.

The neem limonoids azadirachtin and nimbolide induce cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells.

PubMed

Priyadarsini, R Vidya; Murugan, R Senthil; Sripriya, P; Karunagaran, D; Nagini, S

2010-06-01

Limonoids from the neem tree (Azadirachta indica) have attracted considerable research attention in recent years owing to their potent antioxidant and anti-proliferative effects. The present study was designed to investigate the cellular and molecular mechanisms by which azadirachtin and nimbolide exert cytotoxic effects in the human cervical cancer (HeLa) cell line. Both azadirachtin and nimbolide significantly suppressed the viability of HeLa cells in a dose-dependent manner by inducing cell cycle arrest at G0/G1 phase accompanied by p53-dependent p21 accumulation and down-regulation of the cell cycle regulatory proteins cyclin B, cyclin D1 and PCNA. Characteristic changes in nuclear morphology, presence of a subdiploid peak and annexin-V staining pointed to apoptosis as the mode of cell death. Increased generation of reactive oxygen species with decline in the mitochondrial transmembrane potential and release of cytochrome c confirmed that the neem limonoids transduced the apoptotic signal via the mitochondrial pathway. Altered expression of the Bcl-2 family of proteins, inhibition of NF-kappaB activation and over-expression of caspases and survivin provide compelling evidence that azadirachtin and nimbolide induce a shift of balance toward a pro-apoptotic phenotype. Antioxidants such as azadirachtin and nimbolide that can simultaneously arrest the cell cycle and target multiple molecules involved in mitochondrial apoptosis offer immense potential as anti-cancer therapeutic drugs.

Effects of ionizing radiation on bone cell differentiation in an experimental murine bone cell model

NASA Astrophysics Data System (ADS)

Baumstark-Khan, Christa; Lau, Patrick; Hellweg, Christine; Reitz, Guenther

During long-term space travel astronauts are exposed to a complex mixture of different radiation types under conditions of dramatically reduced weight-bearing activity. It has been validated that astronauts loose a considerable amount of bone mass at a rate up to one to two percent each month in space. Therapeutic doses of ionizing radiation cause bone damage and increase fracture risks after treatment for head-and-neck cancer and in pelvic irradiation. For low radiation doses, the possibility of a disturbed healing potential of bone was described. Radiation induced damage has been discussed to inflict mainly on immature and healing bone. Little is known about radiation effects on bone remodelling and even less on the combined action of microgravity and radiation. Bone remodelling is a life-long process performed by balanced action of cells from the osteoblast and osteoclast lineages. While osteoblasts differentiate either into bone-lining cells or into osteocytes and play a crucial role in bone matrix synthesis, osteoclasts are responsible for bone resorption. We hypothesize that the balance between bone matrix assembly by osteocytes and bone degradation by osteoclasts is modulated by microgravity as well as by ionizing radiation. To address this, a cell model consisting of murine cell lines with the potential to differentiate into bone-forming osteoblasts (OCT-1, MC3T3-E1 S24, and MC3T3-E1 S4) was used for studying radiation response after exposure to simulated components of cosmic radiation. Cells were exposed to graded doses of 150 kV X-rays, α particles (0.525 MeV/u, 160 keV/µm; PTB, Braunschweig, Germany) and accelerated heavy ions (75 MeV/u carbon, 29 keV/µm; 95 MeV/u argon, 230 keV/µm; GANIL, Caen, France). Cell survival was measured as colony forming ability; cell cycle progression was analyzed via fluorescence-activated cell scanning (FACS) by measurement of the content of propidium iodide-stained DNA, DNA damage was visualized by γH2AX-immunostaining. Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (von-Kossa staining, Alizarin Red staining). During the process of osteoblastic cell differentiation, the expression of the bone specific marker genes osteocalcin (OCN) and osteopontin (OPN) were recorded by quantitative real time reverse transcription PCR (qRT-PCR). Compared with standard culture conditions, the osteogenic marker genes OCN and OPN were highly expressed during the differentiation process induced either by osteo-inductive media additives (50 µg/ml ascorbic acid, 10 mmol/l β-glycero phosphate) or by sparsely ionizing radiation (X-rays). After 21 days of postirradiation incubation sparsely ionizing radiation could be shown to induce the formation of bone-like nodules (von-Kossa staining) for OCT-1 and MC3T3-E1 S4 cells but nor for MC3T3- E1 S24 cells. Ionizing radiation leads to a cell cycle arrest which is resolved in a dose and time dependent way. This was accompanied by a dose dependent regulation of the cyclin kinase inhibitor CDKN1A (p21/WAF) and transforming growth factor beta 1 (TGF-β1). TGF-β1 is known to affect osteoblast differentiation, matrix formation and mineralization. Modulation of its expression could influence the expression of main osteogenic transcription factors. For exposure with high LET radiation a pronounced cell cycle block was evident. The expression of the osteogenic marker genes OCN and Osterix (OSX) was increased in the OCT-1 cells with differentiation potential for exposure to α particles and accelerated carbon and argon ions. The results on the expression of differentiation markers during radiation-induced premature differentiation of bone cells of the osteoblast lineage show that densely ionizing radiation results in expression of proteins essential for bone formation and consequently in an increase in bone volume. Such an effect has been observed in in-vivo carbon ion irradiated rats. As radiation dependent permanent cell cycle blocks lead to a depletion of proliferation-competent cells from the osteoblastic precursor pool in the body, a gradual decrease of bone mass in weightlessness may be attributed to synergistic effects of radiation and weightlessness.

Comparative stain removal properties of four commercially available denture cleaning products: an in vitro study.

PubMed

Alam, M; Jagger, R; Vowles, R; Moran, J

2011-02-01

Formulations of commercially available denture cleaners vary widely. Unfortunately, comparative data to suggest which products are the most effective can become invalid as newer products are introduced or formulations are changed. The aim of this in vitro study was to measure the stain removal properties of four currently available denture cleaners. Stain was deposited on multi-well polystyrene saliva coated microplates using multiple chlorhexidine and tea solutions. Following drying, each stained well was exposed to a solution of denture cleaner, dried again and the amount of stain remaining measured using a microplate reader. The cleaning procedure was repeated with further multiple exposures of the wells to solutions of the denture cleaners. All denture cleaners removed stain better than water used as a control. At five cleaning cycles only one of the cleaners (Superdrug Cleaning Powder) had removed 100% of the stain. At 30 cycles three of the cleaners had removed 100% of the stain. All the commercial denture cleaners removed stain. Superdrug Cleaning Powder, which contains sodium percarbonate and sodium lauryl sulphate, was particularly effective. © 2010 John Wiley & Sons A/S.

Effects of Bauhinia championii (Benth.) Benth. polysaccharides on the proliferation and cell cycle of chondrocytes.

PubMed

Cai, Liangliang; Ye, Hongzhi; Yu, Fangrong; Li, Huiting; Chen, Jiashou; Liu, Xianxiang

2013-05-01

It has been recently shown that polysaccharides isolated from plants exhibit a number of beneficial therapeutic properties. Bauhinia championii (Benth.) Benth. has been widely used for the clinical treatment of knee osteoarthritis (OA) in China. However, the underlying molecular mechanisms of knee OA treatment have yet to be elucidated. In the present study, we investigated the effects of Bauhinia championii (Benth.) Benth. polysaccharides (BCBPs) on the proliferation and cell cycle of chondrocytes on 4-week-old male Sprague Dawley rats. Immunohistochemical staining was used to identify chondrocytes and an MTT assay was used to evaluate cell viability. Flow cytometry was used for cell cycle analysis. The mRNA and protein expression levels of cyclin D1, CDK4 and CDK6 in chondrocytes were detected using reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, respectively. The data demonstrate that BCBP treatment increased the viability of chondrocytes. In addition, BCBP treatment reduced the cell population in the G0/G1 phase, whereas the cell population was increased in the S phase. Furthermore, BCBP treatment enhanced the expression of cyclin D1, CDK4 and CDK6. These results indicate that BCBP treatment promotes cell proliferation by accelerating the G1/S transition.

Relationships between host and symbiont cell cycles in sea anemones and their symbiotic dinoflagellates.

PubMed

Dimond, James L; Pineda, Rea R; Ramos-Ascherl, Zullaylee; Bingham, Brian L

2013-10-01

The processes by which cnidarians and their algal endosymbionts achieve balanced growth and biomass could include coordination of host and symbiont cell cycles. We evaluated this theory with natural populations of sea anemones hosting symbiotic dinoflagellates, focusing on the temperate sea anemone Anthopleura elegantissima symbiotic with Symbiodinium muscatinei in Washington State, USA, and the tropical anemone Stichodactyla helianthus associating with unknown Symbiodinium spp. in Belize. By extruding symbiont-containing gastrodermal cells from the relatively large tentacles of these species and using nuclear staining and flow cytometry, we selectively analyzed cell cycle distributions of the symbionts and the host gastrodermal cells that house them. We found no indications of diel synchrony in host and symbiont G2/M phases, and we observed evidence of diel periodicity only in Symbiodinium spp. associated with S. helianthus but not in the anemone itself. Seasonally, S. muscatinei showed considerable G2/M phase variability among samples collected quarterly over an annual period, while the G2/M phase of its host varied much less. Within samples taken at different times of the year, correlations between host and symbiont G2/M phases ranged from very weakly to very strongly positive, with significant correlations in only half of the samples (two of four A. elegantissima samples and one of two S. helianthus samples). Overall, the G2/M phase relationships across species and sampling periods were positive. Thus, while we found no evidence of close cell cycle coupling, our results suggest a loose, positive relationship between cell cycle processes of the symbiotic partners.

Leptospermum flavescens Constituent-LF1 Causes Cell Death through the Induction of Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma Cells

PubMed Central

Navanesan, Suerialoasan; Abdul Wahab, Norhanom; Manickam, Sugumaran; Sim, Kae Shin

2015-01-01

Leptospermum flavescens Sm. (Myrtaceae), locally known as ‘Senna makki’ is a smallish tree that is widespread and recorded to naturally occur in the montane regions above 900 m a.s.l from Burma to Australia. Although the species is recorded to be used traditionally to treat various ailments, there is limited data on biological and chemical investigations of L. flavescens. The aim of the present study was to investigate and understand the ability of L. flavescens in inducing cell death in lung cancer cells. The cytotoxic potentials of the extraction yields (methanol, hexane, ethyl acetate and water extracts as wells as a semi pure fraction, LF1) were evaluated against two human non-small cell lung carcinoma cell lines (A549 and NCI-H1299) using the MTT assay. LF1 showed the greatest cytotoxic effect against both cell lines with IC50 values of 7.12 ± 0.07 and 9.62 ± 0.50 μg/ml respectively. LF1 treated cells showed a sub-G1 region in the cell cycle analysis and also caused the presence of apoptotic morphologies in cells stained with acridine orange and ethidium bromide. Treatment with LF1 manifested an apoptotic population in cells that were evaluated using the Annexin V/ propidium iodide assay. Increasing dosage of LF1 caused a rise in the presence of activated caspase-3 enzymes in treated cells. Blockage of cell cycle progression was also observed in LF1-treated cells. These findings suggest that LF1 induces apoptosis and cell cycle arrest in treated lung cancer cells. Further studies are being conducted to isolate and identify the active compound as well to better understand the mechanism involved in inducing cell death. PMID:26287817

The live cell DNA stain SiR-Hoechst induces DNA damage responses and impairs cell cycle progression.

PubMed

Sen, Onur; Saurin, Adrian T; Higgins, Jonathan M G

2018-05-21

SiR-Hoechst (SiR-DNA) is a far-red fluorescent DNA probe being used widely for time-lapse imaging of living cells that is reported to be minimally toxic at concentrations as high as 10-25 µM. However, measuring nuclear import of Cyclin B1, inhibition of mitotic entry, and the induction of γH2AX foci in cultured human cells reveals that SiR-Hoechst induces DNA damage responses and G2 arrest at concentrations well below 1 µM. SiR-Hoechst is useful for live cell imaging, but it should be used with caution and at the lowest practicable concentration.

Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

DOEpatents

Dolbeare, Frank A.; Gray, Joe W.

1988-01-01

A method for the simultaneous flow cytometric measurement of the total DNA content and the level of DNA synthesis in normal and malignant cells is disclosed. The sensitivity of the method allows a study of cell cycle traverse rates for large scale cell populations as well as single cell measurements. A DNA stain such as propidium iodide or Hoechst 33258 is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as halodeoxy-uridine (HdU), more specifically bromodeoxyuridine (BrdU) is used as a probe for the measurement of HdU or BrdU uptake by the cells as a measure of DNA synthesis.

Novel Double-Hit Model of Radiation and Hyperoxia-Induced Oxidative Cell Damage Relevant to Space Travel

PubMed Central

Pietrofesa, Ralph A.; Velalopoulou, Anastasia; Lehman, Stacey L.; Arguiri, Evguenia; Solomides, Pantelis; Koch, Cameron J.; Mishra, Om P.; Koumenis, Constantinos; Goodwin, Thomas J.; Christofidou-Solomidou, Melpo

2016-01-01

Spaceflight occasionally requires multiple extravehicular activities (EVA) that potentially subject astronauts to repeated changes in ambient oxygen superimposed on those of space radiation exposure. We thus developed a novel in vitro model system to test lung cell damage following repeated exposure to radiation and hyperoxia. Non-tumorigenic murine alveolar type II epithelial cells (C10) were exposed to >95% O2 for 8 h only (O2), 0.25 Gy ionizing γ-radiation (IR) only, or a double-hit combination of both challenges (O2 + IR) followed by 16 h of normoxia (ambient air containing 21% O2 and 5% CO2) (1 cycle = 24 h, 2 cycles = 48 h). Cell survival, DNA damage, apoptosis, and indicators of oxidative stress were evaluated after 1 and 2 cycles of exposure. We observed a significant (p < 0.05) decrease in cell survival across all challenge conditions along with an increase in DNA damage, determined by Comet analysis and H2AX phosphorylation, and apoptosis, determined by Annexin-V staining, relative to cells unexposed to hyperoxia or radiation. DNA damage (GADD45α and cleaved-PARP), apoptotic (cleaved caspase-3 and BAX), and antioxidant (HO-1 and Nqo1) proteins were increased following radiation and hyperoxia exposure after 1 and 2 cycles of exposure. Importantly, exposure to combination challenge O2 + IR exacerbated cell death and DNA damage compared to individual exposures O2 or IR alone. Additionally levels of cell cycle proteins phospho-p53 and p21 were significantly increased, while levels of CDK1 and Cyclin B1 were decreased at both time points for all exposure groups. Similarly, proteins involved in cell cycle arrest was more profoundly changed with the combination challenges as compared to each stressor alone. These results correlate with a significant 4- to 6-fold increase in the ratio of cells in G2/G1 after 2 cycles of exposure to hyperoxic conditions. We have characterized a novel in vitro model of double-hit, low-level radiation and hyperoxia exposure that leads to oxidative lung cell injury, DNA damage, apoptosis, and cell cycle arrest. PMID:27322243

Novel Double-Hit Model of Radiation and Hyperoxia-Induced Oxidative Cell Damage Relevant to Space Travel.

PubMed

Pietrofesa, Ralph A; Velalopoulou, Anastasia; Lehman, Stacey L; Arguiri, Evguenia; Solomides, Pantelis; Koch, Cameron J; Mishra, Om P; Koumenis, Constantinos; Goodwin, Thomas J; Christofidou-Solomidou, Melpo

2016-06-16

Spaceflight occasionally requires multiple extravehicular activities (EVA) that potentially subject astronauts to repeated changes in ambient oxygen superimposed on those of space radiation exposure. We thus developed a novel in vitro model system to test lung cell damage following repeated exposure to radiation and hyperoxia. Non-tumorigenic murine alveolar type II epithelial cells (C10) were exposed to >95% O₂ for 8 h only (O₂), 0.25 Gy ionizing γ-radiation (IR) only, or a double-hit combination of both challenges (O₂ + IR) followed by 16 h of normoxia (ambient air containing 21% O₂ and 5% CO₂) (1 cycle = 24 h, 2 cycles = 48 h). Cell survival, DNA damage, apoptosis, and indicators of oxidative stress were evaluated after 1 and 2 cycles of exposure. We observed a significant (p < 0.05) decrease in cell survival across all challenge conditions along with an increase in DNA damage, determined by Comet analysis and H2AX phosphorylation, and apoptosis, determined by Annexin-V staining, relative to cells unexposed to hyperoxia or radiation. DNA damage (GADD45α and cleaved-PARP), apoptotic (cleaved caspase-3 and BAX), and antioxidant (HO-1 and Nqo1) proteins were increased following radiation and hyperoxia exposure after 1 and 2 cycles of exposure. Importantly, exposure to combination challenge O₂ + IR exacerbated cell death and DNA damage compared to individual exposures O₂ or IR alone. Additionally levels of cell cycle proteins phospho-p53 and p21 were significantly increased, while levels of CDK1 and Cyclin B1 were decreased at both time points for all exposure groups. Similarly, proteins involved in cell cycle arrest was more profoundly changed with the combination challenges as compared to each stressor alone. These results correlate with a significant 4- to 6-fold increase in the ratio of cells in G2/G1 after 2 cycles of exposure to hyperoxic conditions. We have characterized a novel in vitro model of double-hit, low-level radiation and hyperoxia exposure that leads to oxidative lung cell injury, DNA damage, apoptosis, and cell cycle arrest.

Double-Staining Method for Differentiation of Morphological Changes and Membrane Integrity of Campylobacter coli Cells

PubMed Central

Alonso, Jose L.; Mascellaro, Salvatore; Moreno, Yolanda; Ferrús, María A.; Hernández, Javier

2002-01-01

We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells. PMID:12324366

Cyclin d1 expression in odontogenic cysts.

PubMed

Taghavi, Nasim; Modabbernia, Shirin; Akbarzadeh, Alireza; Sajjadi, Samad

2013-01-01

In the present study expression of cyclin D1 in the epithelial lining of odontogenic keratocyst, radicular cyst, dentigerous cyst and glandular odontogenic cyst was investigated to compare proliferative activity in these lesions. Immunohistochemical staining of cyclin D1 on formalin-fixed, paraffin-embedded tissue sections of odontogenic keratocysts (n=23), dentigerous cysts (n=20), radicular cysts (n=20) and glandular odontogenic cysts (n=5) was performed by standard EnVision method. Then, slides were studied to evaluate the following parameters in epithelial lining of cysts: expression, expression pattern, staining intensity and localization of expression. The data analysis showed statistically significant difference in cyclin D1 expression in studied groups (p < 0.001). Assessment of staining intensity and staining pattern showed more strong intensity and focally pattern in odontogenic keratocysts, but difference was not statistically significant among groups respectively (p=0.204, 0.469). Considering expression localization, cyclin D1 positive cells in odontogenic keratocysts and dentigerous cysts were frequently confined in parabasal layer, different from radicular cysts and glandular odontogenic cysts. The difference was statistically significant (p < 0.01). Findings showed higher expression of cyclin D1 in parabasal layer of odontogenic keratocyst and the entire cystic epithelium of glandular odontogenic cysts comparing to dentigerous cysts and radicular cysts, implying the possible role of G1-S cell cycle phase disturbances in the aggressiveness of odontogenic keratocyst and glandular odontogenic cyst.

Low-level laser irradiation induces in vitro proliferation of stem cells from human exfoliated deciduous teeth.

PubMed

Ginani, Fernanda; Soares, Diego Moura; de Oliveira Rocha, Hugo Alexandre; de Souza, Lélia Batista; Barboza, Carlos Augusto Galvão

2018-01-01

The aim of this study was to evaluate the effect of low-level laser irradiation (LLLI) on the proliferation and viability of stem cells from human exfoliated deciduous teeth (SHED). Cells were irradiated or not (control) with an InGaAlP laser diode (660 nm, 30 mW, continuous action mode) using two different energy densities (0.5 J/cm 2 -16 s; 1.0 J/cm 2 -33 s). Irradiation was performed at 0 and 48 h, with the laser probe fixed at a distance of 0.5 cm from the cells. Cell proliferation was analyzed at 0, 24, 48, and 72 h by the Trypan blue exclusion method and MTT assay. Cell cycle and Ki67 expression were analyzed by flow cytometry. Apoptosis-related events were evaluated by expression of annexin V/PI and nuclear morphological changes by staining with DAPI. Differences between groups at each time were analyzed by the Kruskal-Wallis and Mann-Whitney tests, adopting a level of significance of 5% (p < 0.05). The results showed that an energy density of 1.0 J/cm 2 promoted an increase in cell proliferation at 48 and 72 h compared to the control and 0.5 J/cm 2 groups. Cell cycle analysis revealed a predominance of cells in the S and G2/M phases in the irradiated groups. This finding was confirmed by the increased expression of Ki67. Low positive staining for annexin V and PI was observed in all groups, and no nuclear changes were detected, indicating that cell viability was not affected by the energy densities tested. It can be concluded that the LLLI parameters used (660 nm, 30 mW, 1.0 J/cm 2 ) promote the proliferation of SHEDs and the maintenance of cell viability.

The life-cycle of Emiliania huxleyi: A brief review and a study of relative ploidy levels analysed by flow cytometry

NASA Astrophysics Data System (ADS)

Green, J. C.; Course, P. A.; Tarran, G. A.

1996-10-01

Emiliania huxleyi exists in several principal forms including the familiar coccolith-bearing C-cell, non-motile naked N-cells, and scale-bearing swarmers (S-cells), but the relationships between these cells are unclear. Flow cytometric analyses have been undertaken on whole cells using fluorochrome staining of the DNA in order to determine the relative DNA content and the relative GC content of the S- and C-cells of selected clones. Results showed that the DNA complement of the S-cells was half that of the C-cells and the two cell types are, therefore, haploid and diploid relative to each other. The S-cells may, therefore, represent a gametic stage, though processes such as sexual fusion and meiosis have not been observed.

A case of primary mucosa-associated lymphoid tissue lymphoma of the vagina.

PubMed

Yoshinaga, Kousuke; Akahira, Jun-Ichi; Niikura, Hitoshi; Ito, Kiyoshi; Moriya, Takuya; Murakami, Takashi; Kameoka, Jun-Ichi; Ichinohasama, Ryo; Okamura, Kunihiro; Yaegashi, Nobuo

2004-09-01

We report the first case of primary mucosa-associated lymphoid tissue (MALT) lymphoma of the vagina, the diagnosis of which is supported by genetic and immunophenotypic studies. A 65-year-old, para 2 woman presented to our hospital in July 1997 with a history of prolonged vaginal discharge. Although cytologic examination suggested possible malignancy, a biopsy of the vaginal wall was diagnosed as chronic inflammation. In June 2000, she underwent gynecologic examination because of anuria. Excisional biopsy revealed subepithelial infiltration of atypical lymphoid cells that stained for CD20, CD79a, and BCL-2; stained weakly for IgM; and did not stain for CD3, CD5, CD7, CD10, CD56, CD23, and IgD, suggesting marginal zone B-cell lineage. Monoclonality was detected by Southern blot analysis, and this patient was finally diagnosed as having primary MALT lymphoma of the vagina. She received 3 cycles of chemotherapy (THP-COP) and concurrent radiation to the whole pelvis. The patient is alive and well 40 months after treatment. Because the vagina is one of the mucosa-associated tissues, MALT lymphoma, though rare, must be included in the differential diagnosis of the vaginal neoplasms.

Tropomodulin3-null mice are embryonic lethal with anemia due to impaired erythroid terminal differentiation in the fetal liver

PubMed Central

Sui, Zhenhua; Nowak, Roberta B.; Bacconi, Andrea; Kim, Nancy E.; Liu, Hui; Li, Jie; Wickrema, Amittha; An, Xiu-li

2014-01-01

Tropomodulin (Tmod) is a protein that binds and caps the pointed ends of actin filaments in erythroid and nonerythoid cell types. Targeted deletion of mouse tropomodulin3 (Tmod3) leads to embryonic lethality at E14.5-E18.5, with anemia due to defects in definitive erythropoiesis in the fetal liver. Erythroid burst-forming unit and colony-forming unit numbers are greatly reduced, indicating defects in progenitor populations. Flow cytometry of fetal liver erythroblasts shows that late-stage populations are also decreased, including reduced percentages of enucleated cells. Annexin V staining indicates increased apoptosis of Tmod3−/− erythroblasts, and cell-cycle analysis reveals that there are more Ter119hi cells in S-phase in Tmod3−/− embryos. Notably, enucleating Tmod3−/− erythroblasts are still in the process of proliferation, suggesting impaired cell-cycle exit during terminal differentiation. Tmod3−/− late erythroblasts often exhibit multilobular nuclear morphologies and aberrant F-actin assembly during enucleation. Furthermore, native erythroblastic island formation was impaired in Tmod3−/− fetal livers, with Tmod3 required in both erythroblasts and macrophages. In conclusion, disruption of Tmod3 leads to impaired definitive erythropoiesis due to reduced progenitors, impaired erythroblastic island formation, and defective erythroblast cell-cycle progression and enucleation. Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion, coordination of cell cycle with differentiation, and F-actin assembly and remodeling during erythroblast enucleation. PMID:24159174

Expression of CDK6 in Oral Squamous Cell Carcinomas

PubMed

Andisheh-Tadbir, Azadeh; Ashraf, Mohammad Javad; Jeiroodi, Naghmeh

2018-04-25

Background: CDK6 is the key factor in regulation of the cell cycle and essential for passage into the G1 phase. It also plays an important role in the development of various tumors. In this cross-sectional study expression of the CDK6 protein in oral squamous cell carcinoma (OSCC) and healthy oral mucosa of controls was assessed to determine relations with malignant transformation and clinicopathologic factors. Method: A total of 60 samples, 45 from OSCCs and 15 from healthy tissue, underwent immunohistochemistry for CDK6. Nuclear and cytoplasmic staining of keratinocytes was considered as positive and the percentages of positive cells were calculated. Results: Expression of CDK6 was detected in 55.6% of OSCC samples (25 cases) and 13.3% of controls (2 cases), the difference being significant. Mean percentage of CDK6 stained cells was 24.2±29.3 in the OSCC cases and 4.33±2.1 in the control group, again statistically significant. No relationship was detected between CDK6 expression and clinicopathologic factors. Conclusion: Overexpression of CDK6 observed in OSCC points to a role for this protein in oral carcinogenesis. Creative Commons Attribution License

Distribution of calmodulin in pea seedlings: immunocytochemical localization in plumules and root apices

NASA Technical Reports Server (NTRS)

Dauwalder, M.; Roux, S. J.; Hardison, L.

1986-01-01

Immunofluorescence techniques have been used to study the distribution of calmodulin in several tissues in young etiolated pea (Pisum sativum L.) seedlings. A fairly uniform staining was seen in the nucleoplasm and background cytoplasm of most cell types. Cell walls and nucleoli were not stained. In addition, patterned staining reactions were seen in many cells. In cells of the plumule, punctate staining of the cytoplasm was common, and in part this stain appeared to be associated with the plastids. A very distinctive staining of amyloplasts was seen in the columella of the root cap. Staining associated with cytoskeletal elements could be shown in division stages. By metaphase, staining of the spindle region was quite evident. In epidermal cells of the stem and along the underside of the leaf there was an intense staining of the vacuolar contents. Guard cells lacked this vacuolar stain. Vacuolar staining was sometimes seen in cells of the stele, but the most distinctive pattern in the stele was associated with young conducting cells of the xylem. These staining patterns are consistent with the idea that the interactions of plastids and the cytoskeletal may be one of the Ca(2+)-mediated steps in the response of plants to environmental stimuli. Nuclear functions may also be controlled, at least in part, by Ca2+.

New observations on endometrial physiology after transcervical injection of methylene blue dye.

PubMed

Marconi, Guillermo; Vilela, Martín; Quintana, Ramiro; Diradourián, Marco; Young, Edgardo; Sueldo, Carlos

2004-12-01

We describe the in vivo features of endometrium stained with methylene blue dye and observed via microhysteroscopy, showing the patterns of endometrial glands and superficial cell changes during the midproliferative, periovulatory, and midluteal phases. These preliminary observations have allowed us to identify a series of changes occurring in the different phases of the ovulatory cycle of potential value in reproductive medicine for specific groups of infertile patients.

Histology of a Woolly Mammoth (Mammuthus primigenius) Preserved in Permafrost, Yamal Peninsula, Northwest Siberia.

PubMed

Papageorgopoulou, Christina; Link, Karl; Rühli, Frank J

2015-06-01

In 2007, the baby woolly mammoth (Mammuthus primigenius) named Lyuba was found frozen in the Siberian tundra permafrost along the Yuribey River. She was proclaimed the best-preserved mammoth discovery. As part of the endoscopic examination of Lyuba, tissue samples of hair, muscle, and internal organs were taken. The sectioned biopsies were stained using standard and special histological stains. In general, the microscopic preservation of the tissue was good although no clearly identifiable cell nuclei were found by standard staining methods. Only a few cell nuclei could be identified in some samples when fluorescence stained with DAPI. The best-preserved structures were collagen fibers and muscle tissue, which gave some structural resemblance to the organs. In the hairs, evidence of pigmentation, a scaly surface, diagonal intra-hair structures, and a medulla were seen. Fat droplets could be identified with Sudan Red in the subcutaneous fat sample and in several organs. Bacteria were seen on the lumen side of the small intestine and caecum, and in the liver and lung tissue. In addition, fungi and pollen were seen in the lung sample. In the wall of the caecum and small intestine, blood vessels and nerves were visualized. Iron was identified in the vivianite sample. Some biopsies compared well structurally with the African elephant tissue sections. The histological findings support the theory that Lyuba drowned in muddy water. The microscopic tissue preservation and cell nuclei destruction indicate that Lyuba's body underwent at least one freeze-thaw cycle. © 2015 Wiley Periodicals, Inc.

Ocular anatomy, ganglion cell distribution and retinal resolution of a killer whale (Orcinus orca).

PubMed

Mass, Alla M; Supin, Alexander Y; Abramov, Andrey V; Mukhametov, Lev M; Rozanova, Elena I

2013-01-01

Retinal topography, cell density and sizes of ganglion cells in the killer whale (Orcinus orca) were analyzed in retinal whole mounts stained with cresyl violet. A distinctive feature of the killer whale's retina is the large size of ganglion cells and low cell density compared to terrestrial mammals. The ganglion cell diameter ranged from 8 to 100 µm, with the majority of cells within a range of 20-40 µm. The topographic distribution of ganglion cells displayed two spots of high cell density located in the temporal and nasal quadrants, 20 mm from the optic disk. The high-density areas were connected by a horizontal belt-like area passing below the optic disk of the retina. Peak cell densities in these areas were evaluated. Mean peak cell densities were 334 and 288 cells/mm(2) in the temporal and nasal high-density areas, respectively. With a posterior nodal distance of 19.5 mm, these high-density data predict a retinal resolution of 9.6' (3.1 cycles/deg.) and 12.6' (2.4 cycles/deg.) in the temporal and nasal areas, respectively, in water. Copyright © 2012 S. Karger AG, Basel.

Brilliant Blue G double staining enhances successful internal limiting membrane peeling with minimal adverse effect by low cellular permeability into live cells.

PubMed

Hisatomi, Toshio; Notomi, Shoji; Tachibana, Takashi; Oishi, Seiichiro; Asato, Ryo; Yamashita, Takehiro; Murakami, Yusuke; Ikeda, Yasuhiro; Enaida, Hiroshi; Sakamoto, Taiji; Ishibashi, Tatsuro

2015-02-01

Brilliant Blue G is used as a surgical adjuvant for retinal surgery. Although BBG double or multiple staining was reported, the effectiveness and safety of repeated staining is still elusive. To further examine the effectiveness and safety, we examined BBG in clinical cases in vivo, primary cell culture in vitro, and surgically resected specimen ex vivo. A retrospective interventional case series with in vitro and ex vivo studies were performed. Vitrectomy was performed in 28 cases of epiretinal membrane with BBG single to multiple staining. The surgically resected membranes were stained by BBG with or without cellular fixation. Primary cell cultures were examined with BBG and live/death cell markers, such as Calcein AM and TUNEL. Single staining provided satisfactory staining in seven cases. Double or multiple staining substantially visualized internal limiting membrane (21 cases), especially the edges of remaining internal limiting membrane (11 cases). Adverse retinal staining was not noted and the final visual acuity showed no difference with multiple staining. The live cells barely stained with BBG, while some dead cells were stained. Brilliant Blue G multiple staining substantially enhanced the visualization of internal limiting membrane. The absence of abnormal staining supports the safety of repeated BBG staining.

Regulation of a Rho-associated kinase expression during the corneal epithelial cell cycle.

PubMed

Anderson, S C; SundarRaj, N

2001-04-01

It has been recognized that an increased expression of the Rho-associated kinase (ROCK-I), a downstream target of Rho (a Ras-related small guanosine triphosphatase [GTPase]), is associated with limbal-to-corneal epithelial transition. The purpose of the present study was to determine whether the expression of ROCK-I is regulated during the cell cycle of corneal epithelial cells. Rabbit corneal epithelial cells in culture were subjected to different culture conditions to enrich them in the G0, G1, and S phases of the cell cycle. Indirect immunofluorescence staining and western blot techniques were used for analyzing the changes in the relative intracellular concentrations of ROCK-I. Northern blot analysis of the isolated cellular RNA was performed to estimate the relative concentrations of ROCK-I mRNA. Serum deprivation did not cause all the corneal epithelial cells in culture to be arrested in the G0 phase of the cell cycle. However, the cells could be arrested in G0 by treating them with culture medium supplemented with transforming growth factor (TGF)-beta1. The relative concentration of ROCK-I in the G0-arrested cells was higher than in the corresponding control untreated cultures. G0-arrested cells were induced to enter G1, followed by the S phase of the cell cycle, by refeeding them with the medium devoid of TGF-beta1. The total intracellular concentration of ROCK-I significantly decreased during the G1 phase of the cell cycle and increased again during the S phase. The decrease in intracellular ROCK-I during the G1 phase was confirmed by arresting the cells in G1 with isoleucine deprivation and thymidine-mimosine treatments. ROCK-I mRNA levels were also found to be decreased during the G1 phase of the cell cycle. The levels of ROCK-I in the corneal epithelial cells were significantly lower in the G1 phase than those in the S and G0 phases of the cell cycle. Therefore, a Rho signaling pathway(s) involving ROCK-I may be regulated during the corneal epithelial cell cycle. The downregulation of ROCK-I during the G1 phase, at least in part, is due to the decreased levels of its mRNA. Based on these findings, ROCK-I may have a role in the progression of the cell cycle in the corneal epithelial cells as they migrate centripetally from the limbal to the corneal surface.

Amygdala Kindling Alters Estrus Cycle and Ovarian Morphology in the Rat

PubMed Central

Pan, Juan; Zhang, Lingwu; Wang, Feng; Liu, Dan

2014-01-01

The objective of this study is to explore the effects of amygdala kindling on estrus cycle and ovarian morphology. Thirty-five female rats at the age of 8 weeks were randomly designated to electrode kindled, sham-kindled, and normal controls. Kindled rats were implanted with kindling electrodes in the left basolateral amygdala and kindled by brief suprathreshold stimulations with a bipolar electrode. Estrous cycles were daily monitored through vaginal smears. Electrographic and behavioral seizures were recorded and ovarian morphology was evaluated by light and electron microscopies. Our results showed that the kindled rats lost their ovarian periodicity displayed significant ovarian enlargement. H&E staining revealed increased number of growing follicles and total follicles, as well as polycysts in the ovaries of the kindled animals compared to sham and control animals. Ultrastructural study detected numerous apoptotic granulosa cells in growing follicles and thecal cell hyperplasia with secretary granules in the thecal cells in the kindled rats. The results suggest that amygdala kindling is a risk factor for the development of polycystic ovary syndrome. PMID:25285307

Anti-proliferative effect of 20-hydroxyecdysone in a lepidopteran cell line.

PubMed

Auzoux-Bordenave, Stéphanie; Hatt, Philippe-Jacques; Porcheron, Patrick

2002-02-01

Ecdysteroids are steroid hormones involved in the epidermal growth of arthropods, controlling cell proliferation and further differentiation of target cells. The epidermal cell line IAL-PID2, established from imaginal discs of the Indian meal moth Plodia interpunctella kept its sensitivity to ecdysteroids in vitro, cells being able to respond to them by cytological and biochemical changes. When added to the culture medium, 20-hydroxyecdysone (20E) stopped cell proliferation and induced formation of epithelial-like aggregates. In order to better understand the cellular sequence of ecdysteroids signalling in epidermal cells we used the IAL-PID2 cell line for in vitro investigations of cytological events induced by the moulting hormone. After a 40 h serum deprivation, formazan assay (XTT) was routinely used to evaluate anti-proliferative effects of 20E during cell cycle. We established a more precise timing of the period of cell sensitivity to the hormone during the cell cycle, by the use of the mitotic index and the BrdU incorporation test. These in vitro assays were performed in parallel with the description of some hormone dependant cytological events, using immunofluorescent labelling with anti-beta tubulin/FITC antibodies and DNA staining.

Developmental competence of Dromedary camel (Camelus dromedarius) oocytes selected using brilliant cresyl blue staining.

PubMed

Fathi, Mohamed; Ashry, Mohamed; Salama, Ali; Badr, Magdy R

2017-08-01

The objectives of the present studies were to investigate the developmental capacity of dromedary camel oocytes selected by brilliant cresyl blue (BCB) staining and to investigate the expression of select transcripts in germinal vesicle (GV) stage oocytes. These transcripts included BMP15 and GDF9 as important transcripts for folliculogenesis and oocyte development, Zar1 and Mater as maternal transcripts required for embryonic development, Cyclin B1 and CDK1 as cell cycle regulators and Oct4 and STAT3 as transcription factors. Dromedary camel oocytes were retrieved from ovaries collected at a local slaughterhouse. After exposure to BCB staining, cumulus-oocyte complexes (COCs) from BCB+, BCB- and control (selected based on morphological criteria) groups were subjected to in vitro maturation, in vitro fertilization and in vitro culture. For gene expression studies, after BCB staining cumulus cells were stripped off and the completely denuded GV stage oocytes were used for RT-PCR analysis of selected transcripts. BCB+ oocytes showed higher maturation, and fertilization rates compared with BCB- and control groups. Indices of early embryonic development, namely, cleavage at 48 hours post insemination (hpi), and development to morula at day 5 and day 7 blastocyst rates were also significantly higher in the BCB+ group. RT-PCR revealed a higher expression of BMP15, GDF9, Zar1, Mater, Cyclin B1, CDK1, OCT4 and STAT3 in good quality oocytes that stained positively for BCB (BCB+). Collectively, results provide novel information about the use of BCB screening for selecting good quality oocytes to improve in vitro embryo production in the dromedary camel.

Effects of nanosecond pulsed electrical fields (nsPEFs) on the cell cycle of CHO and Jurkat cells

NASA Astrophysics Data System (ADS)

Mahlke, Megan A.; Navara, Christopher; Ibey, Bennett L.

2014-03-01

Exposure to nano-second pulsed electrical fields (nsPEFs) can cause poration of external and internal cell membranes, DNA damage, and disassociation of cytoskeletal components, all of which are capable of disrupting a cell's ability to replicate. Variations between cell lines in membrane and cytoskeletal structure as well as in survival of nsPEF exposure should correspond to unique line-dependent cell cycle effects. Additionally, phase of cell cycle during exposure may be linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. Populations of Jurkat and Chinese Hamster Ovary (CHO) cells were examined post-exposure (10 ns pulse trains at 150kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis at various time points (1, 6, and 12h post-exposure) to determine population distribution in cell cycle phases. Additionally, CHO and Jurkat cells were synchronized in G1/S and G2/M phases, pulsed, and analyzed to evaluate role of cell cycle phase in survival of nsPEFs. CHO populations recovered similarly to sham populations postnsPEF exposure and did not exhibit a phase-specific change in response. Jurkat cells exhibited considerable apoptosis/necrosis in response to nsPEF exposure and were unable to recover and proliferate in a manner similar to sham exposed cells. Additionally, Jurkat cells appear to be more sensitive to nsPEFs in G2/M phases than in G1/S phases. Recovery of CHO populations suggests that nsPEFs do not inhibit proliferation in CHO cells; however, inhibition of Jurkat cells post-nsPEF exposure coupled with preferential cell death in G2/M phases suggest that cell cycle phase during exposure may be an important factor in determining nsPEF toxicity in certain cell lines. Interestingly, CHO cells have a more robust and rigid cytoskeleton than Jurkat cells which is thought to contribute to their ability to survive nsPEFs. The ability of the CHO cytoskeleton to recover and complete mitosis after nsPEF-induced damage in G2/M phase may be integral to the cell line's higher tolerance of nsPEF exposure.

In-silico and in-vitro anti-cancer potential of a curcumin analogue (1E, 6E)-1, 7-di (1H-indol-3-yl) hepta-1, 6-diene-3, 5-dione.

PubMed

Sufi, Shamim Akhtar; Adigopula, Lakshmi Narayana; Syed, Safiulla Basha; Mukherjee, Victor; Coumar, Mohane S; Rao, H Surya Prakash; Rajagopalan, Rukkumani

2017-01-01

Previously we showed that BDMC, an analogue of curcumin suppresses growth of human breast and laryngeal cancer cell line by causing apoptosis. Here, we demonstrate the enhanced anti-cancer activity of a heterocyclic ring (indole) incorporated curcumin analogue ((1E, 6E)-1, 7-di (1H-indol-3-yl) hepta-1, 6-diene-3, 5-Dione), ICA in short, in comparison to curcumin. ICA was synthesized by a one pot condensation reaction. Anti-cancer potential of ICA was assessed in three human cancer cell lines of different origin (Lung adenocarcinoma (A549), leukemia (K562) and colon cancer (SW480)) by MTT assay. Mode of cell death was determined by acridine orange-ethidium bromide (Ao-Eb) staining. Putative cellular targets of ICA were investigated by molecular docking studies. Cell cycle analysis following curcumin or ICA treatment in SW480 cell line was carried out by flow cytometry. Expression levels of Cyclin D1 and apoptotic markers, such as Caspase 3, 8 and 9 were studied by western blot analysis in SW480 cell line treated with or without ICA and curcumin. The yield of ICA synthesis was found to be 69% with a purity of 98%. ICA demonstrated promising anti-cancer activity compared to curcumin alone, as discerned by MTT assay. ICA was non-toxic to the cell line of normal origin. We further observed that ICA is ∼2 fold more potent than curcumin in inhibiting the growth of SW480 cells. Ao-Eb staining revealed that ICA could induce apoptosis in all the cell lines tested. Molecular docking studies suggest that ICA may possibly exhibit its anticancer effect by inhibiting EGFR in A549, Bcr-Abl in K562 and GSK-3β kinase in SW480 cell line. Moreover, ICA showed strong binding avidity for Bcl-2 protein in silico, which could result in induction of apoptosis. Cell cycle analysis revealed that both curcumin and ICA induced concomitant cell cycle arrest at G0/G1 and G2/M phase. Western blot shows that ICA could effectively down regulate the expression of cell cycle protein cyclin D1, while promoting the activation of Caspase 3, 8 and 9 when compared to curcumin in human colon cancer cell line SW480. The result of this study indicates that ICA could hold promise to be a potential anti-cancer agent. Since ICA has shown encouraging results in terms of its anti-cancer activity compared to curcumin, further research is necessary to fully delineate the underlying molecular mechanism of its anticancer potential. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Live-cell imaging visualizes frequent mitotic skipping during senescence-like growth arrest in mammary carcinoma cells exposed to ionizing radiation.

PubMed

Suzuki, Masatoshi; Yamauchi, Motohiro; Oka, Yasuyoshi; Suzuki, Keiji; Yamashita, Shunichi

2012-06-01

Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% and 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO(2)-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ß-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation. Copyright © 2012 Elsevier Inc. All rights reserved.

Evaluation of Papanicolaou stain for studying micronuclei in buccal cells under field conditions.

PubMed

Ayyad, Sohair B A; Israel, Ebenezer; El-Setouhy, Maged; Nasr, Ghada Radwan; Mohamed, Mostafa K; Loffredo, Christopher A

2006-01-01

To compare Papanicolaou (Pap) and May-Grünwald Giemsa (MGG) stain as 2 techniques for staining for buccal mucosal cells to detect micronuclei (MN) infield studies. Eighty cytologic smears (2 per individual) were taken from the buccal mucosa of 40 cigarette smokers recruited at a rural village in Egypt. Forty smears were stained with Pap stain and 40 with MGG stain. All were assessed for cellularity and scored for MN. Pap stain was faster and easier to process and transport in the field study than was MGG stain. Regarding MGG smears, bacteria and cell debris masked the MN as compared to Pap smears, in which the fixative destroyed the bacteria and made the cell boundaries clearly demarcated. Using Pap stain, MN were seen easily in transparent cytoplasm. Pap stain is the preferred method infield studies for scoring and detecting MN in cells of buccal mucosa.

Bone marrow cells stained by azide-conjugated Alexa fluors in the absence of an alkyne label.

PubMed

Lin, Guiting; Ning, Hongxiu; Banie, Lia; Qiu, Xuefeng; Zhang, Haiyang; Lue, Tom F; Lin, Ching-Shwun

2012-09-01

Thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) has recently been introduced as an alternative to 5-bromo-2-deoxyuridine (BrdU) for cell labeling and tracking. Incorporation of EdU into replicating DNA can be detected by azide-conjugated fluors (eg, Alexa-azide) through a Cu(i)-catalyzed click reaction between EdU's alkyne moiety and azide. While this cell labeling method has proven to be valuable for tracking transplanted stem cells in various tissues, we have found that some bone marrow cells could be stained by Alexa-azide in the absence of EdU label. In intact rat femoral bone marrow, ~3% of nucleated cells were false-positively stained, and in isolated bone marrow cells, ~13%. In contrast to true-positive stains, which localize in the nucleus, the false-positive stains were cytoplasmic. Furthermore, while true-positive staining requires Cu(i), false-positive staining does not. Reducing the click reaction time or reducing the Alexa-azide concentration failed to improve the distinction between true- and false-positive staining. Hematopoietic and mesenchymal stem cell markers CD34 and Stro-1 did not co-localize with the false-positively stained cells, and these cells' identity remains unknown.

Apoptosis inhibitor of macrophage (AIM) reduces cell number in canine histiocytic sarcoma cell lines.

PubMed

Uchida, Mona; Saeki, Kohei; Maeda, Shingo; Tamahara, Satoshi; Yonezawa, Tomohiro; Matsuki, Naoaki

2016-10-01

Apoptosis inhibitor of macrophage (AIM) is initially reported to protect macrophages from apoptosis. In this study, we determined the effect of AIM on the macrophage-derived tumor, histiocytic sarcoma cell lines (HS) of dogs. Five HS and five other tumor cell lines were used. When recombinant canine AIM was applied to non-serum culture media, cell numbers of all the HS and two of other tumor cell lines decreased dose-dependently. The DNA fragmentation, TUNEL staining and flow cytometry tests revealed that AIM induced both of apoptosis and cell cycle arrest in the HS. Although AIM is known as an apoptosis inhibitor, these results suggest that a high dose of AIM could have an opposite function in HS and some tumor cell lines.

Apigenin inhibits renal cell carcinoma cell proliferation

PubMed Central

Meng, Shuai; Zhu, Yi; Li, Jiang-Feng; Wang, Xiao; Liang, Zhen; Li, Shi-Qi; Xu, Xin; Chen, Hong; Liu, Ben; Zheng, Xiang-Yi; Xie, Li-Ping

2017-01-01

Apigenin, a natural flavonoid found in vegetables and fruits, has antitumor activity in several cancer types. The present study evaluated the effects and mechanism of action of apigenin in renal cell carcinoma (RCC) cells. We found that apigenin suppressed ACHN, 786-0, and Caki-1 RCC cell proliferation in a dose- and time-dependent manner. A comet assay suggested that apigenin caused DNA damage in ACHN cells, especially at higher doses, and induced G2/M phase cell cycle arrest through ATM signal modulation. Small interfering RNA (siRNA)-mediated p53 knockdown showed that apigenin-induced apoptosis was likely p53 dependent. Apigenin anti-proliferative effects were confirmed in an ACHN cell xenograft mouse model. Apigenin treatment reduced tumor growth and volume in vivo, and immunohistochemical staining revealed lower Ki-67 indices in tumors derived from apigenin-treated mice. These findings suggest that apigenin exposure induces DNA damage, G2/M phase cell cycle arrest, p53 accumulation and apoptosis, which collectively suppress ACHN RCC cell proliferation in vitro and in vivo. Given its antitumor effects and low in vivo toxicity, apigenin is a highly promising agent for treatment of RCC. PMID:28423637

CIP-36, a novel topoisomerase II-targeting agent, induces the apoptosis of multidrug-resistant cancer cells in vitro.

PubMed

Cao, Bo; Chen, Hong; Gao, Ying; Niu, Cong; Zhang, Yuan; Li, Ling

2015-03-01

The need to overcome cancer multidrug resistance (MDR) has fueled considerable interest in the development of novel synthetic antitumor agents with cytotoxicity against cancer cell lines with MDR. In this study, we aimed to investigate CIP-36, a novel podophyllotoxin derivative, for its inhibitory effects on human cancer cells from multiple sources, particularly cells with MDR in vitro. The human leukemia cell line, K562, and the adriamycin-resistant subline, K562/A02, were exposed to CIP-36 or anticancer agents, and various morphological and biochemical properties were assessed by Hoechst 33342 staining under a fluorescence microscope. Subsequently, cytotoxicity, cell growth curves and the cell cycle were analyzed. Finally, the effects of CIP-36 on topoisomerase IIα (Topo IIα) activity were determined. Treatment with CIP-36 significantly inhibited the growth of the K562 and MDR K562/A02 cells. Our data demonstrated that CIP-36 induced apoptosis, inhibited cell cycle progression and inhibited Topo IIα activity. These findings suggest that CIP-36 has the potential to overcome the multidrug resistance of K562/A02 cells by mediating Topo IIα activity.

BAG3 protects bovine papillomavirus type 1-transformed equine fibroblasts against pro-death signals.

PubMed

Cotugno, Roberta; Gallotta, Dario; d'Avenia, Morena; Corteggio, Annunziata; Altamura, Gennaro; Roperto, Franco; Belisario, Maria Antonietta; Borzacchiello, Giuseppe

2013-07-22

In human cancer cells, BAG3 protein is known to sustain cell survival. Here, for the first time, we demonstrate the expression of BAG3 protein both in equine sarcoids in vivo and in EqS04b cells, a sarcoid-derived fully transformed cell line harbouring bovine papilloma virus (BPV)-1 genome. Evidence of a possible involvement of BAG3 in equine sarcoid carcinogenesis was obtained by immunohistochemistry analysis of tumour samples. We found that most tumour samples stained positive for BAG3, even though to a different grade, while normal dermal fibroblasts from healthy horses displayed very weak staining pattern for BAG3 expression. By siRNA technology, we demonstrate in EqS04b the role of BAG3 in counteracting basal as well as chemical-triggered pro-death signals. BAG3 down-modulation was indeed shown to promote cell death and cell cycle arrest in G0/G1. In addition, we found that BAG3 silencing sensitized EqS04b cells to phenethylisothiocyanate (PEITC), a promising cancer chemopreventive/chemotherapeutic agent present in edible cruciferous vegetables. Notably, such a pro-survival role of BAG3 was less marked in E. Derm cells, an equine BPV-negative fibroblast cell line taken as a normal counterpart. Altogether our findings might suggest a mutual cooperation between BAG3 and viral oncoproteins to sustain cell survival.

BAG3 protects Bovine Papillomavirus type 1-transformed equine fibroblasts against pro-death signals

PubMed Central

2013-01-01

In human cancer cells, BAG3 protein is known to sustain cell survival. Here, for the first time, we demonstrate the expression of BAG3 protein both in equine sarcoids in vivo and in EqS04b cells, a sarcoid-derived fully transformed cell line harbouring bovine papilloma virus (BPV)-1 genome. Evidence of a possible involvement of BAG3 in equine sarcoid carcinogenesis was obtained by immunohistochemistry analysis of tumour samples. We found that most tumour samples stained positive for BAG3, even though to a different grade, while normal dermal fibroblasts from healthy horses displayed very weak staining pattern for BAG3 expression. By siRNA technology, we demonstrate in EqS04b the role of BAG3 in counteracting basal as well as chemical-triggered pro-death signals. BAG3 down-modulation was indeed shown to promote cell death and cell cycle arrest in G0/G1. In addition, we found that BAG3 silencing sensitized EqS04b cells to phenethylisothiocyanate (PEITC), a promising cancer chemopreventive/chemotherapeutic agent present in edible cruciferous vegetables. Notably, such a pro-survival role of BAG3 was less marked in E. Derm cells, an equine BPV-negative fibroblast cell line taken as a normal counterpart. Altogether our findings might suggest a mutual cooperation between BAG3 and viral oncoproteins to sustain cell survival. PMID:23876161

Nanosecond pulsed electric fields and the cell cycle

NASA Astrophysics Data System (ADS)

Mahlke, Megan A.

Exposure to nanosecond pulsed electrical fields (nsPEFs) can cause poration of external and internal cell membranes, DNA damage, and disassociation of cytoskeletal components, all of which are capable of disrupting a cell's ability to replicate. The phase of the cell cycle at the time of exposure is linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. Additionally, nsPEFs are capable of activating cell cycle checkpoints, which could lead to apoptosis or slow population growth. NsPEFs are emerging as a method for treating tumors via apoptotic induction; therefore, investigating the relevance of nsPEFs and the cell cycle could translate into improved efficacy in tumor treatment. Populations of Jurkat and Chinese Hamster Ovary (CHO) cells were examined post-exposure (10 ns pulse trains at 150kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis at various time points (1, 6, and 12h post-exposure) to determine population distribution in cell cycle phases. Additionally, CHO and Jurkat cells were synchronized in G1/S and G2/M phases, pulsed, and analyzed to evaluate the role of cell cycle phase in survival of nsPEFs. CHO populations appeared similar to sham populations post-nsPEFs but exhibited arrest in the G1 phase at 6h after exposure. Jurkat cells exhibited increased cell death after nsPEFs compared to CHO cells but did not exhibit checkpoint arrest at any observed time point. The G1/S phase checkpoint is partially controlled by the action of p53; the lack of an active p53 response in Jurkat cells could contribute to their ability to pass this checkpoint and resist cell cycle arrest. Both cell lines exhibited increased sensitivity to nsPEFs in G2/M phase. Live imaging of CHO cells after nsPEF exposure supports the theory of G1/S phase arrest, as a reduced number of cells undergo mitosis within 24 h when compared to sham treated cells. CHO cells undergoing mitosis after exposure also exhibit improper separation of chromatids which could indicate loss of function of the mitotic spindle checkpoint. Activation and loss of function of checkpoints in CHO but not Jurkat cells after nsPEF exposure suggests that activation of cell cycle checkpoints could be important in defining the character of cell line specific recovery after nsPEF exposure. Moreover, the increased sensitivity in G2/M phase exhibited by both cell lines indicates that cell cycle phase is an important consideration during nsPEF exposure, particularly when aiming to induce apoptosis.

The crosstalk: Tumor-infiltrating lymphocytes rich in regulatory T cells suppressed cancer-associated fibroblasts

PubMed Central

2013-01-01

Background. The interactions between cancer-associated fibroblasts (CAFs) and cancer cells or tumor-infiltrating lymphocytes (TILs) and cancer cells play important roles in cancer progression and metastasis. However, studies related to the crosstalk between CAFs and TILs in tumor microenvironment (TME) are still lacking. In this study, we mainly investigated the interactions between CAFs and TILs. Material and methods. The distribution of TILs rich in regulatory T cells (Tregs) in breast cancer tissues was evaluated using hematoxylin-eosin staining and immunohistochemistry with anti-CD3, anti-Foxp3, and anti-α-smooth muscle actin antibodies. Homologous CAFs/normal fibroblasts (NFs) and TILs cultured in vitro were identified and detected using immunocytochemistry and flow cytometry (FCM). The direct interaction among these cell types was studied via a factorial design in a co-cultured system. Their indirect interaction was assayed using Transwell plates. The cell cycle and apoptosis of CAFs/NFs co-cultured with TILs was analyzed using propidium iodide staining. Results. Histochemistry demonstrated most of the TILs including Tregs, were distributed in the cancer stroma, adjoining to CAFs. This finding implies that both cell types interact closely in the TME. Identification of the cultured cells showed that CAFs maintained their activated phenotype within limited passages in vitro, and that the TILs population contained a high percentage of Tregs. Data analysis of the factorial design suggests significant interactions among CAFs, NFs, and TILs in both direct and indirect contact ways. The CAFs and NFs were suppressed signally by TILs, which are probably induced by the secretory cytokines derived from TILs or Tregs. Although apoptosis was not detected in CAFs/NFs, the cell cycle assay suggested that the CAFs/NFs were arrested in the G2/M phase by the TILs and their secretory cytokines. Conclusion. CAFs and NFs were dramatically suppressed by Tregs-rich TILs. This suggests the interaction between TILs and CAFs might modify the TME in an unknown manner. PMID:23336253

The amino-terminal matrix assembly domain of fibronectin stabilizes cell shape and prevents cell cycle progression.

PubMed

Christopher, R A; Judge, S R; Vincent, P A; Higgins, P J; McKeown-Longo, P J

1999-10-01

Adhesion to the extracellular matrix modulates the cellular response to growth factors and is critical for cell cycle progression. The present study was designed to address the relationship between fibronectin matrix assembly and cell shape or shape dependent cellular processes. The binding of fibronectin's amino-terminal matrix assembly domain to adherent cells represents the initial step in the assembly of exogenous fibronectin into the extracellular matrix. When added to monolayers of pulmonary artery endothelial cells, the 70 kDa fragment of fibronectin (which contains the matrix assembly domain) stabilized both the extracellular fibronectin matrix as well as the actin cytoskeleton against cytochalasin D-mediated structural reorganization. This activity appeared to require specific fibronectin sequences as fibronectin fragments containing the cell adhesion domain as well as purified vitronectin were ineffective inhibitors of cytochalasin D-induced cytoarchitectural restructuring. Such pronounced morphologic consequences associated with exposure to the 70 kDa fragment suggested that this region of the fibronectin molecule may affect specific growth traits known to be influenced by cell shape. To assess this possibility, the 70 kDa fragment was added to scrape-wounded monolayers of bovine microvessel endothelium and the effects on two shape-dependent processes (i.e. migration and proliferation) were measured as a function of time after injury and location from the wound. The addition of amino-terminal fragments of fibronectin to the monolayer significantly inhibited (by >50%) wound closure. Staining of wounded monolayers with BrdU, moreover, indicated that either the 70 kDa or 25 kDa amino-terminal fragments of fibronectin, but not the 40 kDa collagen binding fragment, also inhibited cell cycle progression. These results suggest that the binding of fibronectin's amino-terminal region to endothelial cell layers inhibits cell cycle progression by stabilizing cell shape.

Estimation of the radiation-induced DNA double-strand breaks number by considering cell cycle and absorbed dose per cell nucleus

PubMed Central

Mori, Ryosuke; Matsuya, Yusuke; Yoshii, Yuji; Date, Hiroyuki

2018-01-01

Abstract DNA double-strand breaks (DSBs) are thought to be the main cause of cell death after irradiation. In this study, we estimated the probability distribution of the number of DSBs per cell nucleus by considering the DNA amount in a cell nucleus (which depends on the cell cycle) and the statistical variation in the energy imparted to the cell nucleus by X-ray irradiation. The probability estimation of DSB induction was made following these procedures: (i) making use of the Chinese Hamster Ovary (CHO)-K1 cell line as the target example, the amounts of DNA per nucleus in the logarithmic and the plateau phases of the growth curve were measured by flow cytometry with propidium iodide (PI) dyeing; (ii) the probability distribution of the DSB number per cell nucleus for each phase after irradiation with 1.0 Gy of 200 kVp X-rays was measured by means of γ-H2AX immunofluorescent staining; (iii) the distribution of the cell-specific energy deposition via secondary electrons produced by the incident X-rays was calculated by WLTrack (in-house Monte Carlo code); (iv) according to a mathematical model for estimating the DSB number per nucleus, we deduced the induction probability density of DSBs based on the measured DNA amount (depending on the cell cycle) and the calculated dose per nucleus. The model exhibited DSB induction probabilities in good agreement with the experimental results for the two phases, suggesting that the DNA amount (depending on the cell cycle) and the statistical variation in the local energy deposition are essential for estimating the DSB induction probability after X-ray exposure. PMID:29800455

Estimation of the radiation-induced DNA double-strand breaks number by considering cell cycle and absorbed dose per cell nucleus.

PubMed

Mori, Ryosuke; Matsuya, Yusuke; Yoshii, Yuji; Date, Hiroyuki

2018-05-01

DNA double-strand breaks (DSBs) are thought to be the main cause of cell death after irradiation. In this study, we estimated the probability distribution of the number of DSBs per cell nucleus by considering the DNA amount in a cell nucleus (which depends on the cell cycle) and the statistical variation in the energy imparted to the cell nucleus by X-ray irradiation. The probability estimation of DSB induction was made following these procedures: (i) making use of the Chinese Hamster Ovary (CHO)-K1 cell line as the target example, the amounts of DNA per nucleus in the logarithmic and the plateau phases of the growth curve were measured by flow cytometry with propidium iodide (PI) dyeing; (ii) the probability distribution of the DSB number per cell nucleus for each phase after irradiation with 1.0 Gy of 200 kVp X-rays was measured by means of γ-H2AX immunofluorescent staining; (iii) the distribution of the cell-specific energy deposition via secondary electrons produced by the incident X-rays was calculated by WLTrack (in-house Monte Carlo code); (iv) according to a mathematical model for estimating the DSB number per nucleus, we deduced the induction probability density of DSBs based on the measured DNA amount (depending on the cell cycle) and the calculated dose per nucleus. The model exhibited DSB induction probabilities in good agreement with the experimental results for the two phases, suggesting that the DNA amount (depending on the cell cycle) and the statistical variation in the local energy deposition are essential for estimating the DSB induction probability after X-ray exposure.

Regulation of plant cells, cell walls and development by mechanical signals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyerowitz, Elliot M.

2016-06-14

The overall goal of the revised scope of work for the final year of funding was to characterize cell wall biosynthesis in developing cotyledons and in the shoot apical meristem of Arabidopsis thaliana, as a way of learning about developmental control of cell wall biosynthesis in plants, and interactions between cell wall biosynthesis and the microtubule cytoskeleton. The proposed work had two parts – to look at the effect of mutation in the SPIRAL2 gene on microtubule organization and reorganization, and to thoroughly characterize the glycosyltransferase genes expressed in shoot apical meristems by RNA-seq experiments, by in situ hybridization ofmore » the RNAs expressed in the meristem, and by antibody staining of the products of the glycosyltransferases in meristems. Both parts were completed; the spiral2 mutant was found to speed microtubule reorientation after ablation of adjacent cells, supporting our hypothesis that reorganization correlates with microtubule severing, the rate of which is increased by the mutation. The glycosyltransferase characterization was completed and published as Yang et al. (2016). Among the new things learned was that primary cell wall biosynthesis is strongly controlled both by cell type, and by stage of cell cycle, implying not only that different, even adjacent, cells can have different sugar linkages in their (nonshared) walls, but also that a surprisingly large proportion of glycosyltransferases is regulated in the cell cycle, and therefore that the cell cycle regulates wall maturation to a degree previously unrecognized.« less

Association of pKi-67 with satellite DNA of the human genome in early G1 cells.

PubMed

Bridger, J M; Kill, I R; Lichter, P

1998-01-01

pKi-67 is a nucleolar antigen that provides a specific marker for proliferating cells. It has been shown previously that pKi-67's distribution varies in a cell cycle-dependent manner: it coats all chromosomes during mitosis, accumulates in nuclear foci during G1 phase (type I distribution) and localizes within nucleoli in late G1 S and G2 phase (type II distribution). Although no function has as yet been ascribed to pKi-67, it has been found associated with centromeres in G1. In the present study the distribution pattern of pKi-67 during G1 in human dermal fibroblasts (HDFs) was analysed in more detail. Synchronization experiments show that in very early G1 cells pKi-67 coincides with virtually all satellite regions analysed, i.e. with centromeric (alpha-satellite), telomeric (minisatellite) and heterochromatic blocks (satellite III) on chromosomes 1 and Y (type Ia distribution). In contrast, later in the G1 phase, a smaller fraction of satellite DNA regions are found collocalized with pKi-67 foci (type Ib distribution). When all pKi-67 becomes localized within nucleoli, even fewer satellite regions remain associated with the pKi-67 staining. However, all centromeric and short arm regions of the acrocentric chromosomes, which are in very close proximity to or even contain the rRNA genes, are collocalized with anti-pKi-67 staining throughout the remaining interphase of the cell cycle. Thus, our data demonstrate that during post-mitotic reformation and nucleogenesis there is a progressive decline in the fraction of specific satellite regions of DNA that remain associated with pKi-67. This may be relevant to nucleolar reformation following mitosis.

Genistein induced anticancer effects on pancreatic cancer cell lines involves mitochondrial apoptosis, G0/G1cell cycle arrest and regulation of STAT3 signalling pathway.

PubMed

Bi, Yi-Liang; Min, Min; Shen, Wei; Liu, Yan

2018-01-15

Genistein is a natural flavonoid that has been reported to exhibit anticancer effects against different types of cancers which include, but are not limited to, breast and oral squamous cell carcinoma. The present study was designed to evaluate the anticancer effects of the natural flavonoid genistein against pancreatic cancer cell lines and to explore the underlying mechanism. Antiproliferative activity was investigated by MTT assay. Apoptosis was detected by DAPI and annexin V/PI staining. DNA damage was assessed by comet assay. Reactive oxygen species (ROS) and reduction of mitochondrial membrane potential (MMP) were determined by flow cytometry. Cell migration was examined by wound healing assay. Protien expressions were determined by western blotting. Antiproliferative assay revealed that genistein reduced the cell viability of pancreatic cancer cells in a dose dependent manner with an IC 50 of 20 and 25 µM against Mia-PaCa2 and PANC-1 cancer cell lines respectively. However, its antiproliferative effects were less pronounced against non-cancerous pancreatic ductal epithelial cell line (H6C7) as evident from the IC 50 of 120 µM. Genistein induced significant morphological changes in pancreatic cancer cells and triggered cell cycle arrest in G 0 /G 1 phase. DAPI staining and flow cytometric analysis revealed that genistein induced apoptosis in a dose dependent manner through generation of substantial amounts of ROS and reduction of MMP. However, treatment of the pancreatic cancer with genistein and ascorbic acid could abrogate the effects of genistein on cell viability. Protien expression analysis revealed that genistein upregulated cytosolic cytochrome c, Bax, cleaved Caspase-3 and cleaved caspase-9 expressions with concomitant downregulation of Bcl-2 expression. Moreover, genistein inhibited the phosphorylation of signal transducer and activator of transcription STAT3 proteins and downregulated the expression of survivin, cyclin D1 and ALDH1A1 in Mia-PaCa2 cells in a dose dependent manner. Interestingly, genistein could inhibit the cell migration potential of the Mia-PaCa2 cells which was further associated with the downregulation of metalloproteinases (MPP-2 and MPP-9). Taken together, we propose that genistein exerts anticancer activity in pancreatic cancer cells through induction of ROS mediated mitochondrial apoptosis, cell cycle arrest and regulation of STAT3 and may therefore prove beneficial in the management of pancreatic cancers cancer. Copyright © 2017 Elsevier GmbH. All rights reserved.

Alpha-santalol, a chemopreventive agent against skin cancer, causes G2/M cell cycle arrest in both p53-mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells

PubMed Central

2010-01-01

Background α-Santalol, an active component of sandalwood oil, has shown chemopreventive effects on skin cancer in different murine models. However, effects of α-santalol on cell cycle have not been studied. Thus, the objective of this study was to investigate effects of α-santalol on cell cycle progression in both p53 mutated human epidermoid carcinoma A431 cells and p53 wild-type human melanoma UACC-62 cells to elucidate the mechanism(s) of action. Methods MTT assay was used to determine cell viability in A431 cells and UACC-62; fluorescence-activated cell sorting (FACS) analysis of propidium iodide staining was used for determining cell cycle distribution in A431 cells and UACC-62 cells; immunoblotting was used for determining the expression of various proteins and protein complexes involved in the cell cycle progression; siRNA were used to knockdown of p21 or p53 in A431 and UACC-62 cells and immunofluorescence microscopy was used to investigate microtubules in UACC-62 cells. Results α-Santalol at 50-100 μM decreased cell viability from 24 h treatment and α-santalol at 50 μM-75 μM induced G2/M phase cell cycle arrest from 6 h treatment in both A431 and UACC-62 cells. α-Santalol altered expressions of cell cycle proteins such as cyclin A, cyclin B1, Cdc2, Cdc25c, p-Cdc25c and Cdk2. All of these proteins are critical for G2/M transition. α-Santalol treatment up-regulated the expression of p21 and suppressed expressions of mutated p53 in A431 cells; whereas, α-santalol treatment increased expressions of wild-type p53 in UACC-62 cells. Knockdown of p21 in A431 cells, knockdown of p21 and p53 in UACC-62 cells did not affect cell cycle arrest caused by α-santalol. Furthermore, α-santalol caused depolymerization of microtubules similar to vinblastine in UACC-62 cells. Conclusions This study for the first time identifies effects of α-santalol in G2/M phase arrest and describes detailed mechanisms of G2/M phase arrest by this agent, which might be contributing to its overall cancer preventive efficacy in various mouse skin cancer models. PMID:20682067

Effects of Methylation Status of CpG Sites within the HPV16 Long Control Region on HPV16-Positive Head and Neck Cancer Cells.

PubMed

Zhang, Chunlin; Deng, Zeyi; Pan, Xiaoli; Uehara, Takayuki; Suzuki, Mikio; Xie, Minqiang

2015-01-01

To map comprehensively the methylation status of the CpG sites within the HPV16 long control region (LCR) in HPV-positive cancer cells, and to explore further the effects of methylation status of HPV16 LCR on cell bioactivity and E6 and E7 expression. In addition, to analyze the methylation status of the LCR in HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) patients. Methylation patterns of HPV16 LCR in UM-SCC47, CaSki, and SiHa cells and HPV16-positiive OPSCC specimens were detected by bisulfite-sequencing PCR and TA cloning. For cells treated with 5-aza-2'-deoxycytidine and E6 and E7 knockdown, MTS and trypan blue staining, annexin-V and 7-AAD staining, and prodidium iodide were used to evaluate cell growth and cell proliferation, cell apoptosis, and cell cycle arrest, respectively. E6 and E7 mRNA and protein expression were analyzed by quantitative real-time PCR and immunocytochemistry, respectively. Hypermethylation status of the LCR in UM-SCC47 (79.8%) and CaSki cells (90.0%) and unmethylation status of the LCR in SiHa cells (0%) were observed. Upon demethylation, the cells with different methylation levels responded differently during growth, apoptosis, and cell cycle arrest, as well as in terms of their E6 and E7 expression. In HPV16-positive OPSCC patients, the methylation rates were 9.5% in the entire LCR region, 13.9% in the 5'-LCR, 6.0% in the E6 enhancer, and 9.5% in the p97 promoter, and hypermethylation of p97 promoter was found in a subset of cases (20.0%, 2/10). Our study revealed two different methylation levels of the LCR in HPV16-positive cancer cells and OPSCC patients, which may represent different carcinogenesis mechanisms of HPV-positive cancers cells. Demethylating the meCpGs in HPV16 LCR might be a potential target for a subgroup of HPV16-positive patients with head and neck squamous cell carcinoma.

Comparison of hepatocellular carcinoma in American and Asian patients by tissue array analysis.

PubMed

Song, Tae-Jin; Fong, Yuman; Cho, Sung-Jin; Gönen, Mithat; Hezel, Michael; Tuorto, Scott; Choi, Sang-Yong; Kim, Young-Chul; Suh, Sung-Ock; Koo, Bum-Hwan; Chae, Yang-Seok; Jarnagin, William R; Klimstra, David S

2012-07-01

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Although some epidemiologic and etiologic differences between Asian and Western HCC are known, detailed comparative studies with pathologic correlations have not been performed. Paraffin sections of resected HCC specimens from Memorial Sloan-Kettering Cancer Center and Korea University Medical Center were used to construct tissue microarrays. Immunohistochemical staining of microarray sections was performed using antibodies against markers of proliferation and regulators of cell cycle. Patient data were correlated with staining results. When comparing both cohorts, significant differences were found in expression of p53 and MDM2. In the Asian group, more frequent positive staining for p53 (24%) was observed compared with the American group (9%; P = 0.037). For MDM2, 26% of American cases stained positive compared with 2% of Asian cases (P = 0.0003). No significant differences were found in expression of Ki67, p21, p27, cyclin D1, or bcl2. Female gender, vascular invasion, and lack of viral hepatitis infection correlated with positive MDM2 staining. These data likely correlate with differences in molecular pathogenesis of HCC based on racial and regional differences. These findings may have implications in choice of molecular targeted therapies based on patient ethnicity. Copyright © 2012 Wiley Periodicals, Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Masatoshi, E-mail: msuzuki@nagasaki-u.ac.jp; Yamauchi, Motohiro; Oka, Yasuyoshi

Purpose: Senescence-like growth arrest in human solid carcinomas is now recognized as the major outcome of radiotherapy. This study was designed to analyze cell cycle during the process of senescence-like growth arrest in mammary carcinoma cells exposed to X-rays. Methods and Materials: Fluorescent ubiquitination-based cell cycle indicators were introduced into the human mammary carcinoma cell line MCF-7. Cell cycle was sequentially monitored by live-cell imaging for up to 5 days after exposure to 10 Gy of X-rays. Results: Live-cell imaging revealed that cell cycle transition from G2 to G1 phase without mitosis, so-called mitotic skipping, was observed in 17.1% andmore » 69.8% of G1- and G2-irradiated cells, respectively. Entry to G1 phase was confirmed by the nuclear accumulation of mKO{sub 2}-hCdt1 as well as cyclin E, which was inversely correlated to the accumulation of G2-specific markers such as mAG-hGeminin and CENP-F. More than 90% of cells skipping mitosis were persistently arrested in G1 phase and showed positive staining for the senescent biochemical marker, which is senescence-associated ss-galactosidase, indicating induction of senescence-like growth arrest accompanied by mitotic skipping. While G2 irradiation with higher doses of X-rays induced mitotic skipping in approximately 80% of cells, transduction of short hairpin RNA (shRNA) for p53 significantly suppressed mitotic skipping, suggesting that ionizing radiation-induced mitotic skipping is associated with p53 function. Conclusions: The present study found the pathway of senescence-like growth arrest in G1 phase without mitotic entry following G2-irradiation.« less

Fluorescein-labeled β-Glucosidase as a Bacterial Stain

PubMed Central

Pital, Abe; Janowitz, Sheldon L.; Hudak, Charles E.; Lewis, Evelyn E.

1967-01-01

Fluorescein isothiocyanate-labeled β-glucosidase was used as a simple staining reagent with selected gram-positive and gram-negative organisms. Staining in situ appeared to be dependent on the presence of accessible glycosidic-type linkages in the bacterial cell wall. Extensive wall damage or lysis did not occur when stained cells were suspended in washing and mounting solutions. The apparent specificity of labeled enzyme for wall substance was tested by blocking reactions, staining of isolated cell walls, and failure to stain substances lacking appropriate glycosidic linkages. Severe cell wall lesions were produced after prolonged contact with labeled enzyme, and this phenomenon may also be related to staining specificity. Gram-negative organisms and spores were poorly stained unless protected glycopeptide substrate was previously exposed by treatment of cells with thioglycolic acid or dilute alkaline sodium hypochlorite solution. A potential for staining tissues and cell lines may also exist. Some possible applications of labeled enzymes are briefly discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:4169543

p38 MAPK pathway is essential for self-renewal of mouse male germline stem cells (mGSCs).

PubMed

Niu, Zhiwei; Mu, Hailong; Zhu, Haijing; Wu, Jiang; Hua, Jinlian

2017-02-01

Male germline stem cells (mGSCs), also called spermatogonial stem cells (SSCs), constantly generate spermatozoa in male animals. A number of preliminary studies on mechanisms of mGSC self-renewal have previously been conducted, revealing that several factors are involved in this regulated process. The p38 MAPK pathway is widely conserved in multiple cell types in vivo, and plays an important role in cell proliferation, differentiation, inflammation and apoptosis. However, its role in self-renewal of mGSCs has not hitherto been determined. Here, the mouse mGSCs were cultured and their identity was verified by semi-RT-PCR, alkaline phosphatase (AP) staining and immunofluorescence staining. Then, the p38 MAPK pathway was blocked by p38 MAPK-specific inhibitor SB202190. mGSC self-renewal ability was then analysed by observation of morphology, cell number, cell growth analysis, TUNEL incorporation assay and cell cycle analysis. Results showed that mouse mGSC self-renewal ability was significantly inhibited by SB202190. This study showed for the first time that the p38 MAPK pathway plays a key role in maintaining self-renewal capacity of mouse mGSCs, which offers a new self-renewal pathway for these cells and contributes to overall knowledge of the mechanisms of mGSC self-renewal. © 2016 John Wiley & Sons Ltd.

Automated single-slide staining device. [in clinical bacteriology

NASA Technical Reports Server (NTRS)

Wilkins, J. R.; Mills, S. M.

1975-01-01

An automatic single-slide Gram staining device is described. A timer-actuated solenoid controls the dispensing of gentian violet, Gram iodine solution, decolorizer, and 1% aqueous safranin in proper sequence and for the time required for optimum staining. The amount of stain or reagent delivered is controlled by means of stopcocks below each solenoid. Used stains and reagents can be flushed automatically or manually. Smears Gram stained automatically are equal in quality to those prepared manually. The time to complete one Gram cycle is 4.80 min.

P53-dependent antiproliferative and pro-apoptotic effects of trichostatin A (TSA) in glioblastoma cells.

PubMed

Bajbouj, K; Mawrin, C; Hartig, R; Schulze-Luehrmann, J; Wilisch-Neumann, A; Roessner, A; Schneider-Stock, R

2012-05-01

Glioblastomas are known to be highly chemoresistant, but HDAC inhibitors (HDACi) have been shown to be of therapeutic relevance for this aggressive tumor type. We treated U87 glioblastoma cells with trichostatin A (TSA) to define potential epigenetic targets for HDACi-mediated antitumor effects. Using a cDNA array analysis covering 96 cell cycle genes, cyclin-dependent kinase inhibitor p21(WAF1) was identified as the major player in TSA-induced cell cycle arrest. TSA slightly inhibited proliferation and viability of U87 cells, cumulating in a G1/S cell cycle arrest. This effect was accompanied by a significant up-regulation of p53 and its transcriptional target p21(WAF1) and by down-regulation of key G1/S regulators, such as cdk4, cdk6, and cyclin D1. Nevertheless, TSA did not induce apoptosis in U87 cells. As expected, TSA promoted the accumulation of total acetylated histones H3 and H4 and a decrease in endogenous HDAC activity. Characterizing the chromatin modulation around the p21(WAF1) promoter after TSA treatment using chromatin immunoprecipitation, we found (1) a release of HDAC1, (2) an increase of acetylated H4 binding, and (3) enhanced recruitment of p53. p53-depleted U87 cells showed an abrogation of the G1/S arrest and re-entered the cell cycle. Immunofluorescence staining revealed that TSA induced the nuclear translocation of p21(WAF1) verifying a cell cycle arrest. On the other hand, a significant portion of p21(WAF1) was present in the cytoplasmic compartment causing apoptosis resistance. Furthermore, TSA-treated p53-mutant cell line U138 failed to show an induction in p21(WAF1), showed a deficient G2/M checkpoint, and underwent mitotic catastrophe. We suggest that HDAC inhibition in combination with other clinically used drugs may be considered an effective strategy to overcome chemoresistance in glioblastoma cells.

Sporophytic and gametophytic functions of the cell cycle-associated Mob1 gene in Arabidopsis thaliana L.

PubMed

Galla, Giulio; Zenoni, Sara; Marconi, Gianpiero; Marino, Giada; Botton, Alessandro; Pinosa, Francesco; Citterio, Sandra; Ruperti, Benedetto; Palme, Klaus; Albertini, Emidio; Pezzotti, Mario; Mau, Martin; Sharbel, Timothy F; De Storme, Nico; Geelen, Danny; Barcaccia, Gianni

2011-09-15

Mob1 genes are primarily involved in the cell cycle progression and mitosis exit in yeasts and animals. The function of a Mob1-like gene (At5g45550) from Arabidopsis thaliana was investigated using RNAi and immunological staining. AtMob1-like RNAi silenced lines showed a reduced radial expansion of the inflorescence stem and a reduced elongation zone of the primary root. Morphological features of plant organs were accompanied by a reduction in cell size. The fertility of AtMob1-like RNAi silenced lines was very low as seed production was strongly reduced. About 2% of the progeny of AtMob1-like RNAi silenced plants were tetraploid. The female and male sporogenesis was affected differentially. The ovules developed irregularly and one third of the megaspores and embryo sacs degenerated prematurely. Up to 20% of the ovules produced binucleated megaspores that failed to develop further, being their degeneration likely accompanied with a delayed programmed cell death. The anthers produced about 30% of aborted pollen grains, showing also a strong variation in their size. Together, the results show that Arabidopsis MOB1-like is required to regulate cell expansion and cell division, presumably by affecting the mitotic as well as the meiotic cell cycle. Copyright © 2011 Elsevier B.V. All rights reserved.

Remodeling of bovine oviductal epithelium by mitosis of secretory cells.

PubMed

Ito, Sayaka; Kobayashi, Yoshihiko; Yamamoto, Yuki; Kimura, Koji; Okuda, Kiyoshi

2016-11-01

Two types of oviductal epithelial cells, secretory and ciliated, play crucial roles in the first days after fertilization in mammals. Secretory cells produce various molecules promoting embryo development, while ciliated cells facilitate transport of oocytes and zygotes by ciliary beating. The proportions of the two cell types change during the estrous cycle. The proportion of ciliated cells on the oviductal luminal surface is abundant at the follicular phase, whereas the proportion of secretory cells gradually increases with the formation of the corpus luteum. In the present study, we hypothesize that the proportions of ciliated and secretory epithelial cells are regulated by mitosis. The proportion of the cells being positive for FOXJ1 (a ciliated cell marker) or Ki67 (a mitosis marker) in epithelial cells during the estrous cycle were immunohistochemically examined. Ki67 and FOXJ1 or PAX8 (a secretory cell marker), were double-stained to clarify which types of epithelial cells undergo mitosis. In the ampulla, the percentage of FOXJ1-positive cells was highest at the day of ovulation (Day 0) and decreased by about 50 % by Days 8-12, while in the isthmus it did not change during the estrous cycle. The proportion of Ki67-positive cells was highest at around the time of ovulation in both the ampulla and isthmus. All the Ki67-positive cells were PAX8-positive and FOXJ1-negative in both the ampulla and isthmus. These findings suggest that epithelial remodeling, which is regulated by differentiation and/or proliferation of secretory cells of the oviduct, provides the optimal environment for gamete transport, fertilization and embryonic development.

Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens

PubMed Central

Zupan, John R.; Cameron, Todd A.; Anderson-Furgeson, James; Zambryski, Patricia C.

2013-01-01

Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about to divide, however, both poles are equally labeled with FM 4-64, but the constriction site is not. Second, the cell-division protein FtsA alternates from unipolar foci in the shortest cells to unipolar and midcell localization in cells of intermediate length, to strictly midcell localization in the longest cells undergoing septation. Third, the cell division protein FtsZ localizes in a cell-cycle pattern similar to, but more complex than, FtsA. Finally, because PG synthesis is spatially and temporally regulated during the cell cycle, we treated cells with sublethal concentrations of carbenicillin (Cb) to assess the role of penicillin-binding proteins in growth and cell division. Cb-treated cells formed midcell circumferential bulges, suggesting that interrupted PG synthesis destabilizes the septum. Midcell bulges contained bands or foci of FtsA-GFP and FtsZ-GFP and no FM 4-64 label, as in untreated cells. There were no abnormal morphologies at the growth poles in Cb-treated cells, suggesting unipolar growth uses Cb-insensitive PG synthesis enzymes. PMID:23674672

Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis.

PubMed

Hong, Kyung Uk; Choi, Yong-Bock; Lee, Jung-Hwa; Kim, Hyun-Jun; Kwon, Hye-Rim; Seong, Yeon-Sun; Kim, Heung Tae; Park, Joobae; Bae, Chang-Dae; Hong, Kyeong-Man

2008-08-31

Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 recognizes the amino acid sequence between 569 and 625 and that phosphorylation at T596 completely abolishes the reactivity of the antibody, suggesting that the differential reactivity originates from the phosphorylation status at T596. Immunofluorescence staining showed that mAb D-12-3 fails to detect TMAP/CKAP2 in mitotic cells between prophase and metaphase, but the staining becomes evident again in anaphase, suggesting that phosphorylation at T596 occurs transiently during early phases of mitosis. These results suggest that the cellular functions of TMAP/CKAP2 might be regulated by timely phosphorylation and dephosphorylation during the course of mitosis.

Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis

PubMed Central

Hong, Kyung Uk; Choi, Yong-Bock; Lee, Jung-Hwa; Kim, Hyun-Jun; Kwon, Hye-Rim; Seong, Yeon-Sun; Kim, Heung Tae; Park, Joobae

2008-01-01

Tumor associated microtubule associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2) is a mitotic spindle-associated protein whose expression is cell cycle-regulated and also frequently deregulated in cancer cells. Two monoclonal antibodies (mAbs) against TMAP/CKAP2 were produced: B-1-13 and D-12-3. Interestingly, the reactivity of mAb D-12-3 to TMAP/CKAP2 was markedly decreased specifically in mitotic cell lysate. The epitope mapping study showed that mAb D-12-3 recognizes the amino acid sequence between 569 and 625 and that phosphorylation at T596 completely abolishes the reactivity of the antibody, suggesting that the differential reactivity originates from the phosphorylation status at T596. Immunofluorescence staining showed that mAb D-12-3 fails to detect TMAP/CKAP2 in mitotic cells between prophase and metaphase, but the staining becomes evident again in anaphase, suggesting that phosphorylation at T596 occurs transiently during early phases of mitosis. These results suggest that the cellular functions of TMAP/CKAP2 might be regulated by timely phosphorylation and dephosphorylation during the course of mitosis. PMID:18779650

Growth fraction in non-small cell lung cancer estimated by proliferating cell nuclear antigen and comparison with Ki-67 labeling and DNA flow cytometry data.

PubMed Central

Fontanini, G.; Pingitore, R.; Bigini, D.; Vignati, S.; Pepe, S.; Ruggiero, A.; Macchiarini, P.

1992-01-01

Results generated by the immunohistochemical staining with PC10, a new monoclonal antibody recognizing PCNA (a nuclear protein associated with cell proliferation) in formalin-fixed and paraffin-embedded tissue were compared with those of Ki-67 labeling and DNA flow cytometry in 47 consecutive non-small cell lung cancer (NSCLC). PCNA reactivity was observed in all samples and confined to the nuclei of cancer cells. Its frequency ranged from 0 to 80% (37.7 +/- 23.6) and larger sized, early-staged and DNA aneuploid tumors expressed a significant higher number of PCNA-reactive cells. The PCNA and Ki-67 labeling rates were closely correlated (r = 0.383, P = 0.009). By flow cytometry, we observed a good correlation among PCNA labeling and S-phase fraction (r = 0.422, P = .0093) and G1 phase (r = 0.303, P = .051) of the cell cycle. Results indicate that PCNA labeling with PC10 is a simple method for assessing the proliferative activity in formalin-fixed, paraffin-embedded tissue of NSCLC and correlates well with Ki-67 labeling and S-phase fraction of the cell cycle. Images Figure 2 PMID:1361306

Induction of apoptosis and cell cycle arrest in L-1210 murine lymphoblastic leukaemia cells by (2E)-3-(2-naphthyl)-1-(3'-methoxy-4'-hydroxy-phenyl)-2-propen-1-one.

PubMed

Pedrini, Fernanda Spezia; Chiaradia, Louise Domeneghini; Licínio, Marley Aparecida; de Moraes, Ana Carolina Rabello; Curta, Juliana Costa; Costa, Aline; Mascarello, Alessandra; Creczinsky-Pasa, Tânia Beatriz; Nunes, Ricardo José; Yunes, Rosendo Augusto; Santos-Silva, Maria Cláudia

2010-09-01

New compounds with biological targets and less cytotoxicity to normal cells are necessary for cancer therapy. In this work ten synthetic chalcones derived from 2-naphtaldehyde were evaluated for their cytotoxic effect in murine acute lymphoblastic leukemia cells L-1210. A series of ten chalcones derived from 2-naphtaldehyde and corresponding acetophenones were prepared by aldolic condensation, using methanol as solvent under basic conditions, at room temperature for 24 h. The cell viability was determined by MTT colorimeter method. The cell cycle phase analysis was carried out by flow cytometry after propidium iodide staining. The apoptosis induction was assessed by exposure to phosphatidylserine (ANNEXIN V-FITC). Cytometric analysis was performed to evaluate the expression of p53, Bcl-2 and Bax protein. The caspase-3 expression was studied by immunoblotting analysis. A preliminary screening of a series of ten chalcones derived from 2-naphtaldehyde showed that chalcone 8, (2E)-3-(2-naphtyl)-1-(3'-methoxy-4'-hydroxy-phenyl)-2-propen-1-one, had the highest cytotoxic effect (IC50 of 54 microM), but not in normal human lymphocytes. To better understand the cytotoxic mechanism of chalcone 8, its effect on cell cycle and apoptosis was assessed. Our results showed that chalcone 8 caused cell cycle arrest in the G2/M phase and a significant increase in the proportion of cells in the subG0/G1 phase. Our results also demonstrated that chalcone 8 promoted a modification in Bax:Bcl-2 ratio and increased p53 expression and caspase-3 activation. The studied chalcone 8 has cytotoxic effect against L-1210 lymphoblastic leukaemic cells, and this effect is associated with increase of p-53 and Bax expression.

Quantum Dot Platform for Single-Cell Molecular Profiling

NASA Astrophysics Data System (ADS)

Zrazhevskiy, Pavel S.

In-depth understanding of the nature of cell physiology and ability to diagnose and control the progression of pathological processes heavily rely on untangling the complexity of intracellular molecular mechanisms and pathways. Therefore, comprehensive molecular profiling of individual cells within the context of their natural tissue or cell culture microenvironment is essential. In principle, this goal can be achieved by tagging each molecular target with a unique reporter probe and detecting its localization with high sensitivity at sub-cellular resolution, primarily via microscopy-based imaging. Yet, neither widely used conventional methods nor more advanced nanoparticle-based techniques have been able to address this task up to date. High multiplexing potential of fluorescent probes is heavily restrained by the inability to uniquely match probes with corresponding molecular targets. This issue is especially relevant for quantum dot probes---while simultaneous spectral imaging of up to 10 different probes is possible, only few can be used concurrently for staining with existing methods. To fully utilize multiplexing potential of quantum dots, it is necessary to design a new staining platform featuring unique assignment of each target to a corresponding quantum dot probe. This dissertation presents two complementary versatile approaches towards achieving comprehensive single-cell molecular profiling and describes engineering of quantum dot probes specifically tailored for each staining method. Analysis of expanded molecular profiles is achieved through augmenting parallel multiplexing capacity with performing several staining cycles on the same specimen in sequential manner. In contrast to other methods utilizing quantum dots or other nanoparticles, which often involve sophisticated probe synthesis, the platform technology presented here takes advantage of simple covalent bioconjugation and non-covalent self-assembly mechanisms for straightforward probe preparation and specimen labeling, requiring no advanced technical skills and being directly applicable for a wide range of molecular profiling studies. Utilization of quantum dot platform for single-cell molecular profiling promises to greatly benefit both biomedical research and clinical diagnostics by providing a tool for addressing phenotypic heterogeneity within large cell populations, opening access to studying low-abundance events often masked or completely erased by batch processing, and elucidating biomarker signatures of diseases critical for accurate diagnostics and targeted therapy.

Minichromosome maintenance (Mcm) proteins, cyclin B1 and D1, phosphohistone H3 and in situ DNA replication for functional analysis of vulval intraepithelial neoplasia.

PubMed

Davidson, E J; Morris, L S; Scott, I S; Rushbrook, S M; Bird, K; Laskey, R A; Wilson, G E; Kitchener, H C; Coleman, N; Stern, P L

2003-01-27

Vulval intraepithelial neoplasia (VIN) is defined histopathologically by distinctive abnormalities of cellular maturation and differentiation. To investigate the functional properties of VIN, the expression of several proteins involved in the regulation of the cell cycle as well as in situ DNA replication competence was analysed by immunohistochemistry. Snap-frozen vulval biopsies were graded as normal squamous epithelium (n=6), undifferentiated HPV positive VIN 1 (n=3), VIN 2 (n=8) and VIN 3 (n=20). Immunohistochemistry was performed using the following markers: cyclin D1 (expressed in middle/late G1), cyclin B1 (expressed in G2/early M), phosphorylated histone H3 (expressed during mitosis) and minichromosome maintenance (Mcm) proteins 2 and 5 (expressed during the cell cycle, but not in differentiated or quiescent cells). In situ DNA replication competence was used to identify S-phase cells. The percentage of positively stained nuclei in three representative microscopic fields was calculated per biopsy. In normal vulva, the expression of all markers was restricted to the proliferative compartment of the basal layer of the epithelium. In contrast in high-grade VIN, the majority of epithelial cells expressed the Mcm proteins from basal to superficial layer. The detection of cyclins B1 and D1, phospho-histone H3 and in situ DNA replication was also found through the full thickness of these lesions but by a lower proportion of the cells. This is consistent with these markers providing a series of 'snapshots' of the cell cycle status of individual cells. The low-grade VIN showed reduced expression of the cell cycle markers in relation to the level of dysplasia. The combination of these analyses establishes that the majority of VIN cells remain in a functional replicative or prereplicative state of the cell cycle. Clinical application of these analyses may provide a basis for improved diagnosis of VIN.

Ki67 and proliferation in breast cancer.

PubMed

Pathmanathan, Nirmala; Balleine, Rosemary L

2013-06-01

New approaches to the prognostic assessment of breast cancer have come from molecular profiling studies. A major feature of this work has been to emphasise the importance of cancer cell proliferation as a key discriminative indicator of recurrence risk for oestrogen receptor positive breast cancer in particular. Mitotic count scoring, as a component of histopathological grade, has long formed part of a routine evaluation of breast cancer biology. However, there is an increasingly compelling case to include a specific proliferation score in breast cancer pathology reports based on expression of the cell cycle regulated protein Ki67. Immunohistochemical staining for Ki67 is a widely available and economical test with good tolerance of pre-analytical variations and staining conditions. However, there is currently no evidence based protocol established to derive a reliable and informative Ki67 score for routine clinical use. In this circumstance, pathologists must establish a standardised framework for scoring Ki67 and communicating results to a multidisciplinary team.

Knockdown of Immature Colon Carcinoma Transcript 1 Inhibits Proliferation and Promotes Apoptosis of Non–Small Cell Lung Cancer Cells

PubMed Central

He, Jiantao; Zhang, Shenghui; Yang, Qingbo; Wang, Bo; Liu, Zhiyu; Wu, Xintian

2016-01-01

Non–small cell lung cancer, as the most frequent type lung cancer, has lower survival rate of 5 years, despite improvements in surgery and chemotherapy. Previous studies showed immature colon carcinoma transcript 1 is closely related to tumorigenesis of human cancer cells. In the present study, we found immature colon carcinoma transcript 1 was overexpressed in lung cancer tissues using Oncomine database mining, and the biological effect of immature colon carcinoma transcript 1 was investigated in non–small cell lung cancer cell lines 95D and A549. Lentivirus-mediated RNA interference was used to knock down immature colon carcinoma transcript 1 expression in 95D and A549 cells in vitro, and the knockdown efficiency was determined using quantitative real-time polymerase chain reaction and Western blot assay. Knockdown of immature colon carcinoma transcript 1 significantly suppressed non–small cell lung cancer cell proliferation and colony formation ability confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assay. Flow cytometry was applied to measure cell cycle arrest, and the result showed the cell cycle arrested in G2/M phase in 95D cells and arrested in G0/G1 phase in A549 cells. Furthermore, we measured the levels of cell cycle–associated proteins by Western blot analysis and found immature colon carcinoma transcript 1–mediated cell proliferation inhibition appeared due to downregulation of cell cycle activator cyclin D1 and upregulation of cell cycle inhibitor p21. In addition, immature colon carcinoma transcript 1 silencing significantly induced non–small cell lung cancer cell apoptosis by annexin V/7-amino-actinomycin D double-staining assay. All our data suggest that immature colon carcinoma transcript 1 may play an important role for non–small cell lung cancer cell proliferation and could be a potential molecular target for diagnosing and treating human non–small cell lung cancer. PMID:27413166

Determining Proportion of Exfoliative Vaginal Cell during Various Stages of Estrus Cycle Using Vaginal Cytology Techniques in Aceh Cattle

PubMed Central

Siregar, Tongku N.; Melia, Juli; Rohaya; Thasmi, Cut Nila; Masyitha, Dian; Wahyuni, Sri; Rosa, Juliana; Nurhafni; Panjaitan, Budianto; Herrialfian

2016-01-01

The aim of this study was to investigate the period of estrus cycle in aceh cattle, Indonesia, based on vaginal cytology techniques. Four healthy females of aceh cattle with average weight of 250–300 kg, age of 5–7 years, and body condition score of 3-4 were used. All cattle were subjected to ultrasonography analysis for the occurrence of corpus luteum before being synchronized using intramuscular injections of PGF2 alpha 25 mg. A vaginal swab was collected from aceh cattle, stained with Giemsa 10%, and observed microscopically. Period of estrus cycle was predicted from day 1 to day 24 after estrus synchronization was confirmed using ultrasonography analysis at the same day. The result showed that parabasal, intermediary, and superficial epithelium were found in the vaginal swabs collected from proestrus, metestrus, and diestrus aceh cattle. Proportions of these cells in the particular period of estrus cycle were 36.22, 32.62, and 31.16 (proestrus); 21.33, 32.58, and 46.09 (estrus); 40.75, 37.58, and 21.67 (metestrus); and 41.07, 37.38, and 21.67 (diestrus), respectively. In conclusion, dominant proportion of superficial cell that occurred in estrus period might be used as the base for determining optimal time for insemination. PMID:26977335

Protein tyrosine phosphatase, PTP1B, expression and activity in rat corneal endothelial cells

PubMed Central

Harris, Deshea L.

2007-01-01

Purpose The current studies were conducted to determine whether the protein tyrosine phosphatase, PTP1B, plays a role in regulating epidermal growth factor receptor (EGFR) Tyr992 phosphorylation and cell cycle entry in rat corneal endothelial cells. Methods Corneas were obtained from male Sprague-Dawley rats. PTP1B mRNA and protein expression were compared in confluent and subconfluent cells by RT-PCR and western blots. Immunocytochemistry was used to determine the subcellular localization of both PTP1B and EGFR following epidermal growth factor (EGF) stimulation. Western blots were used to analyze the time-dependent effect of EGF on phosphorylation of EGFR Tyr992 plus or minus CinnGEL 2Me, an inhibitor of PTP1B activity. The effect of PTP1B inhibition on cell cycle entry was determined by calculating the percent of Ki67-positive cells following EGF treatment. Results PTP1B mRNA expression was similar in confluent and subconfluent cells, but PTP1B protein was expressed at 3 fold higher levels in subconfluent cells. Positive staining for PTP1B was localized in vesicular structures below the plasma membrane. EGFR staining was located at cell-cell borders in untreated endothelium, but was mainly cytoplasmic by 15 min after EGF treatment. In control cultures, phosphorylation of EGFR Tyr992 peaked by 5 min following EGF stimulation and rapidly decreased to basal levels by 30 min. In cultures pretreated with CinnGEL 2Me, Tyr992 phosphorylation peaked 2 min following EGF addition and was consistently sustained at a higher level than controls until 60 min after treatment. By 18 h following EGF treatment, cultures pretreated with CinnGEL 2Me exhibited a 1.7 fold increase in the number of Ki67-positive cells compared with control cultures. Conclusions Comparison of PTP1B mRNA and protein levels indicates that PTP1B expression is regulated mainly at the protein level and is higher in subconfluent cells. PTP1B was located in vesicles below the plasma membrane. The fact that EGFR is internalized in response to EGF stimulation suggests that it could interact with and be regulated by PTP1B. The ability of PTP1B inhibitor to sustain EGFR Tyr992 phosphorylation and increase the number of Ki67-positive cells indicates that PTP1B plays a role in the negative regulation of EGF-induced signaling and helps suppress cell cycle entry. PMID:17563729

Immunocytochemistry of cell surface heparan sulfate proteoglycan in mouse tissues. A light and electron microscopic study.

PubMed

Hayashi, K; Hayashi, M; Jalkanen, M; Firestone, J H; Trelstad, R L; Bernfield, M

1987-10-01

The core protein of the proteoglycan at the cell surface of NMuMG mouse mammary epithelial cells bears both heparan and chondroitin sulfate chains and is recognized by the monoclonal antibody 281-2. Using this antibody and the peroxidase-antiperoxidase staining technique in adult mouse tissues, we found that the antibody recognizes the antigen in a highly restricted distribution, staining a variety of epithelial cells but no cells derived from embryonic mesoderm or neural crest. The antibody fails to stain any stromal (mesenchymal) or neuronal cells, with the exception of plasma cells and Leydig cells. Squamous and transitional epithelia stain intensely over their entire surfaces, whereas cuboidal and columnar epithelia stain moderately and only at the lateral surface of the basal cells. Within squamous and transitional epithelial tissues that undergo physiological regeneration (e.g., epidermis), the most superficial and differentiated cell types fail to stain. Within glandular and branched epithelia (e.g., pancreas), the secretory alveolar cells fail to stain. When evaluated by electron microscopy, granular deposits of stain are seen on the plasma membrane, especially on lateral surfaces, but none are noted within the cells or the basement membrane. These results indicate that in adult tissues the core protein of this heparan sulfate-rich proteoglycan is expressed almost exclusively at epithelial cell surfaces. Expression appears to be lost as the cells become either mature or highly differentiated.

Estrous cycle and ovarian changes in a rat mammary carcinogenesis model after irradiation, tamoxifen chemoprevention, and aging.

PubMed

Karim, Baktiar O; Landolfi, Jennifer A; Christian, Archie; Ricart-Arbona, Rodolfo; Qiu, Weiping; McAlonis, Melissa; Eyabi, Paul O; Khan, Khalid A; Dicello, John F; Mann, Jill F; Huso, David L

2003-10-01

Variation in the effects of selective estrogen receptor modulators (SERMs) on the estrous cycle and reproductive organs during aging could play an important role in the observed heterogeneity of tamoxifen chemoprevention efficacy against breast cancer. Of the 1,022 female Sprague Dawley rats enrolled in a long-term tamoxifen chemoprevention study, 87 were randomly chosen from four groups (irradiated, irradiated and tamoxifen treated, tamoxifen treated, and control). Vaginal smears were evaluated for determination of cycle stage, and vaginal pathologic changes. Correlation with the histologic features of reproductive tissues in 43 animals was made. More tamoxifen-treated (21.9%; 7/32) rats had irregular cycling than did control (9%; 3/23) rats. Ovarian granulosa cell hyperplasia was present in 50% (3/6) of tamoxifen-treated rats, and 20% (2/10) of control rats. Endometrial-type cells (ETCs) were present only in tamoxifen-treated (tamoxifen alone 6.25% [2/32]) and tamoxifen/ radiation-treated (28.6% [4/14]) rats. The modified Papanicolaou stain used here provided excellent morphologic detail for evaluating the estrous cycle in rodents. Tamoxifen altered vaginal cytologic and ovarian histologic features during aging. Results indicated that tamoxifen had direct and indirect effects on the reproductive tract, causing disturbance of the estrous cycle, shedding of ETCs, and promoting granulosa cell hyperplasia. Understanding of the heterogeneous response to tamoxifen chemoprevention during aging in rodents may provide important insights into the basis for tamoxifen chemoprevention failures in humans.

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks.

PubMed

Poojan, Shiv; Kim, Han-Seong; Yoon, Ji-Woon; Sim, Hye Won; Hong, Kyeong-Man

2018-05-20

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological information is also necessary. The protocol of immunocytochemical staining on paraffin-embedded cell blocks, presented herein, is an excellent alternative to immunofluorescent staining on non-paraffin-embedded fixed cells. In this protocol, a paraffin cell block from HeLa cells was prepared using the thromboplastin-plasma method, and immunocytochemistry was performed for the evaluation of two proliferation markers, CKAP2 and Ki-67. The nuclei and cytoplasmic morphology of the HeLa cells were well preserved in the cell-block slides. At the same time, the CKAP2 and Ki-67 staining patterns in the immunocytochemistry were quite similar to those in immunohistochemical staining in paraffin cancer tissues. With modified cell-culture conditions, including pre-incubation of HeLa cells under serum-free conditions, the effect could be evaluated while preserving architectural information. In conclusion, immunocytochemistry on paraffin-embedded cell blocks is an excellent alternative to immunofluorescent staining.

Quercetin suppresses HeLa cells by blocking PI3K/Akt pathway.

PubMed

Xiang, Tao; Fang, Yong; Wang, Shi-Xuan

2014-10-01

To explore the effect of quercetin on the proliferation and apoptosis of HeLa cells, HeLa cells were incubated with quercetin at different concentrations. Cell viability was evaluated by MTT assay, cell apoptosis was detected by Annexin-V/PI double labeled cytometry and DNA ladder assay. Cell cycle was flow cytometrically determined and the morphological changes of the cells were observed under a fluorescence microscope after Hoechst 33258 staining and the apoptosis-related proteins in the HeLa cells were assessed by Western blotting. The results showed that quercetin significantly inhibited the growth of HeLa cells and induced obvious apoptosis in vitro in a time- and dose-dependent manner. Moreover, quercetin induced apoptosis of HeLa cells in cell cycle-dependent manner because quercetin could induce arrest of HeLa cells at G0/G1 phase. Quercetin treatment down-regulated the expression of the PI3K and p-Akt. In addition, quercetin could down-regulate expression of bcl-2, up-regulate Bax, but exerted no effect on the overall expression of Akt. We are led to conclude that quercetin induces apoptosis via PI3k/Akt pathways, and quercetin has potential to be used as an anti-tumor agent against human cervix cancer.

Lithium Chloride Dependent Glycogen Synthase Kinase 3 Inactivation Links Oxidative DNA Damage, Hypertrophy and Senescence in Human Articular Chondrocytes and Reproduces Chondrocyte Phenotype of Obese Osteoarthritis Patients.

PubMed

Guidotti, Serena; Minguzzi, Manuela; Platano, Daniela; Cattini, Luca; Trisolino, Giovanni; Mariani, Erminia; Borzì, Rosa Maria

2015-01-01

Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA), but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy. In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763) were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS) production (2',7'-dichlorofluorescin diacetate staining). Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining. In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3β, oxidative damage and expression of GADD45β and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3β inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in γH2AX, GADD45β and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10. In articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via induction of hypertrophy and senescence. Indeed, GSK3β inactivation is responsible for ROS production, triggering oxidative stress and DNA damage response.

The Citrus Flavanone Naringenin Protects Myocardial Cells against Age-Associated Damage

PubMed Central

Costa, Barbara; Cavallini, Chiara; Testai, Lara; Martelli, Alma; Calderone, Vincenzo; Martini, Claudia

2017-01-01

In recent years, the health-promoting effects of the citrus flavanone naringenin have been examined. The results have provided evidence for the modulation of some key mechanisms involved in cellular damage by this compound. In particular, naringenin has been revealed to have protective properties such as an antioxidant effect in cardiometabolic disorders. Very recently, beneficial effects of naringenin have been demonstrated in old rats. Because aging has been demonstrated to be directly related to the occurrence of cardiac disorders, in the present study, the ability of naringenin to prevent cardiac cell senescence was investigated. For this purpose, a cellular model of senescent myocardial cells was set up and evaluated using colorimetric, fluorimetric, and immunometric techniques. Relevant cellular senescence markers, such as X-gal staining, cell cycle regulator levels, and the percentage of cell cycle-arrested cells, were found to be reduced in the presence of naringenin. In addition, cardiac markers of aging-induced damage, including radical oxidative species levels, mitochondrial metabolic activity, mitochondrial calcium buffer capacity, and estrogenic signaling functions, were also modulated by the compound. These results suggested that naringenin has antiaging effects on myocardial cells. PMID:28386313

The relationship of fibroblast translocations to cell morphology and stress fibre density.

PubMed

Lewis, L; Verna, J M; Levinstone, D; Sher, S; Marek, L; Bell, E

1982-02-01

Translocation of human fibroblasts in culture was studied using techniques of time-lapse cinemicrography, indirect immunofluorescence, and computer analysis. An inverse relationship between the velocity of cells during the last hour of life and the density of stress fibers seen by immune staining was demonstrated. Translocating cells generally assumed one of two interconvertible morphologies: a triangular tailed shape or tailed fibroblast (TF), and a tailless form that resembled a half-moon, which we call a half-moon fibroblast (HMF). The tail of TFs formed only on regions of substrate that had been previously traversed by cells. The half-moon morphology developed either on previously used or on virgin substrate. Cells adopted the HMF rather than the TF morphology with a four-fold greater frequency. HMFs translocated slightly faster than TFs. The foregoing observation suggest that the fibroblast tail is not an organelle essential for translocation. Since our technique allowed us to distinguish between cells which were cycling and those which had left cycle, we compared their velocities and found them to be similar. Also the average velocities of cells of different population-doubling levels (10th, 30th, 40th) were approximately equal.

Rosiglitazone ameliorates senescence-like phenotypes in a cellular photoaging model.

PubMed

Chen, Liang; Bi, Bo; Zeng, Jiping; Zhou, Yiqun; Yang, Ping; Guo, Yu; Zhu, Jingjing; Yang, Qingjian; Zhu, Ningwen; Liu, Tianyi

2015-03-01

Rosiglitazone (RO), a second-generation thiazolidinedione used mainly in the treatment of non-insulin-dependent diabetes mellitus, has been discovered to be a high-affinity ligand for peroxisome proliferator-activated receptor-γ (PPAR-γ). Several studies have revealed that PPAR-γ is also involved in the regulation of oxidative stress and chronic inflammation associated with aging process in vivo as well as with cellular senescence in vitro. We sought to investigate whether RO pretreatment will counteract the photoaging process using a well-established cellular photoaging model. Murine dermal fibroblasts (MDFs) were cultured in the absence or presence of RO for 48h, followed by exposure to repeated UVB irradiation. The senescent phenotypes were evaluated including cell viability, senescence-associated β-galactosidase (SA-β-gal) expression, cell morphology, ROS generation, cell cycle, production and degradation of extracellular matrix (ECM), and the potential mechanisms were discussed. Pretreatment with RO (40μM) significantly decreased the staining intensity and the percentage of SA-β-gal-positive cells and reserved the elongated cell shape compared with UVB group. The cells pretreated with RO also showed decreased UVB-induced degradation of type I collagen by decreasing MMPs expressions. In addition, we observed counteraction of cell-cycle arrest and repression of UVB-induced p53 and p21 in the presence of RO. We further confirmed a significant decrease in ROS accumulation accompanied by an increase in catalase in RO group. RO, a potent PPAR-γ activator, counteracts senescence-like phenotypes, including long-term growth arrest, flattened morphology, degradation of ECM and SA-β-gal-positive staining in MDFs by inhibiting the expression of MMPs and increasing the synthesis of catalase when administered to repeated UVB irradiation. The novel application of RO may lead to innovative and effective anti-photoaging therapies. Copyright © 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells.

PubMed

Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

2015-01-01

Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays.

Study of P21 Expression in Oral Lichen Planus and Oral Squamous Cell Carcinoma by Immunohistochemical Technique.

PubMed

Baghaei, Fahimeh; Shojaei, Setareh; Afshar-Moghaddam, Noushin; Zargaran, Massoumeh; Rastin, Verisheh; Nasr, Mohsen; Moghimbeigi, Abbas

2015-09-01

Lichen planus is a mucocutaneous disease that is relatively common in middle aged individuals. Some studies have shown that oral lichen planus has a potential to progress to squamous cell carcinoma.p21 is a cyclin-dependent kinase inhibitor that regulates the cell cycle, thus it acts as an inhibitor in cell proliferation. This study was aimed to evaluate and compare the immunostaining of p21 (as a proliferation inhibitory factor) in oral lichen planus (OLP) and oral squamous cell carcinoma (OSCC). In this descriptive cross-sectional study, p21expression was investigated in 24 samples of oral lichen planus (OLP), 24 samples of oral squamous cell carcinoma (OSCC) and 24 samples of oral epithelial hyperplasia (OEH) by employing immunohistochemical staining. The mean percentage of p21-positive cells in OSCC (54.5±6.6) was significantly higher than that in OLP (32.8±6.08) and OEH (9.4±3.8). Moreover, OLP samples expressed p21 significantly higher than the OEH. Kruskal Wallis test revealed a statistically significant difference between the groups regarding the intensity of staining (p< 0.001). According to the findings of this study, the expression of p21 might be related to the potential carcinogenic transformation of lichen planus to SCC. Therefore, continuous follow-up periods for OLP are recommended for diagnosis of the malignant transformations in early stages.

Microwave-assisted one-pot synthesis of new phenanthrene fused-tetrahydrodibenzo-acridinones as potential cytotoxic and apoptosis inducing agents.

PubMed

Kumar, Niggula Praveen; Sharma, Pankaj; Reddy, T Srinivasa; Shankaraiah, Nagula; Bhargava, Suresh K; Kamal, Ahmed

2018-05-10

An expeditious microwave-assisted one-pot synthesis of new cytotoxic phenanthrene fused-tetrahydrodibenzo-acridinones has been successfully accomplished. This protocol offers wide substrate scope, catalyst-free synthesis, atom-economy, simple recrystallization, high yields, and ethanol was used as green solvent. These new compounds were tested for their in vitro cytotoxicity against cervical (HeLa), prostate (PC-3), fibrosarcoma (HT-1080), ovarian (SKOV-3) cancer cells, and were safer to normal (Hek-293T) kidney cell line. All the compounds have displayed significant cytotoxicity profile, among them 8m being the most potent compound with an IC 50 0.24 ± 0.05 μM against SKOV-3 ovarian cancer cells. Flow cytometry analysis revealed that cells were blocked at the G2/M phase of the cell cycle. The effect of 8m on F-actin polymerisation was also studied. Hoechst staining clearly showed the decreased number of viable cells and indicated apoptosis progression. Compound 8m caused the collapse of mitochondrial membrane potential as observed via JC-1 staining and also enhanced the generation of reactive oxygen species. The increase of caspase-3 activation by 3.7 folds supported the strong apoptosis induction. In addition, an in vitro 3D-spheroid progression assay was performed with 8m that significantly suppressed the tumor cells. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Overexpression of SPAG9 in human gastric cancer is correlated with poor prognosis.

PubMed

Miao, Zhi-Feng; Wang, Zhen-Ning; Zhao, Ting-Ting; Xu, Ying-Ying; Wu, Jian-Hua; Liu, Xing-Yu; Xu, Hao; You, Yi; Xu, Hui-Mian

2015-11-01

Sperm associated antigen 9 (SPAG9) protein has been found to play an important role in cancer progression but the involved mechanisms are still obscure. Its clinical significance in human gastric cancers remains unexplored. In the present study, SPAG9 expression was analyzed in 147 gastric cancer specimens. We observed weak staining in normal gastric mucosa and positive staining in 65 out of 147 (44.2 %) cancer samples. Overexpression of SPAG9 correlated with local invasion (p = 0.0101), lymph node metastasis (p = 0.0488), TNM stage (p = 0.0002), and relapse (p = 0.0018). Importantly, SPAG9 overexpression correlated with poor overall survival (p = 0.0008). Furthermore, we performed siRNA knockdown of SPAG9 in HGC-27 cells with high endogenous expression and transfected SPAG9 plasmid in SGC-7901 cell line with low endogenous level. SPAG9 overexpression promoted while its depletion inhibited cell proliferation, cell cycle transition, and invasive cell growth. SPAG9 overxpression also increased chemoresistance to 5--fluorouracil (5-FU) in SGC-7901 cells. Further analysis showed that SPAG9 knockdown downregulated and its overexpression upregulated cyclin D1, MMP9, and p-p38 expression. In conclusion, SPAG9 overexpression in gastric cancer correlates with poor prognosis and contributes to gastric cancer cell proliferation, invasion, and chemoresistance. SPAG9 promotes gastric cancer invasion, possibly through p38-MMP9 signaling pathways.

Induction of cell death by pyropheophorbide-α methyl ester-mediated photodynamic therapy in lung cancer A549 cells.

PubMed

Tu, Ping-Hua; Huang, Wen-Jun; Wu, Zhan-Ling; Peng, Qing-Zhen; Xie, Zhi-Bin; Bao, Ji; Zhong, Ming-Hua

2017-03-01

Pyropheophorbide-α methyl ester (MPPa) was a promising photosensitizer with stable chemical structure, strong absorption, higher tissue selectivity and longer activation wavelengths. The present study investigated the effect of MPPa-mediated photodynamic treatment on lung cancer A549 cells as well as the underlying mechanisms. Cell Counting Kit-8 was employed for cell viability assessment. Reactive oxygen species levels were determined by fluorescence microscopy and flow cytometry. Cell morphology was evaluated by Hoechst staining and transmission electron microscopy. Mitochondrial membrane potential, cellular apoptosis and cell cycle distribution were evaluated flow-cytometrically. The protein levels of apoptotic effectors were examined by Western blot. We found that the photocytotoxicity of MPPa showed both drug- and light- dose dependent characteristics in A549 cells. Additionally, MPPa-PDT caused cell apoptosis by reducing mitochondrial membrane potential, increasing reactive oxygen species (ROS) production, inducing caspase-9/caspase-3 signaling activation as well as cell cycle arrest at G 0 /G 1 phase. These results suggested that MPPa-PDT mainly kills cells by apoptotic mechanisms, with overt curative effects, indicating that MPPa should be considered a potent photosensitizer for lung carcinoma treatment. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Reduced Mitogenicity of Sera Following Weight Loss in Premenopausal Women

PubMed Central

Azrad, Maria; Chang, Pi-Ling; Gower, Barbara A.; Hunter, Gary R.; Nagy, Tim R.

2011-01-01

We investigated whether serum from normal weight women is less mitogenic and more apoptotic than sera from the same women in the overweight state. Sera from premenopausal women, age (mean ±SEE) 34.6±0.53 years, who were randomized to caloric restriction (CR) (n=13), CR + aerobic exercise (AE) (n=14) or CR + resistance training (RT) (n=20) were used to culture endometrial cancer cells. Phases of the cell cycle were determined, proliferating cell nuclear antigen (PCNA) positivity was used to assess proliferation and apoptosis was assessed by determining cleaved caspase-3 and poly-ADP-ribose polymerase (PARP). Analyses showed that overall, cells grown in sera from the weight-reduced state had significantly more cells in G0/G1 and significantly fewer cells in the S and G2/M phases of the cell cycle than cells grown in sera from the overweight state. PCNA staining confirmed that cells grown in sera from the weight-reduced state had fewer proliferating cells. Cleaved caspase-3 and PARP were not different in cells grown in sera from the weight-reduced state compared to the overweight state. We conclude that weight loss with or without exercise could lower the risk for cancer through changes in serum that result in reduced cellular mitogenicity. PMID:21774593

The intermediate filament network protein, vimentin, is required for parvoviral infection.

PubMed

Fay, Nikta; Panté, Nelly

2013-09-01

Intermediate filaments (IFs) have recently been shown to serve novel roles during infection by many viruses. Here we have begun to study the role of IFs during the early steps of infection by the parvovirus minute virus of mice (MVM). We found that during early infection with MVM, after endosomal escape, the vimentin IF network was considerably altered, yielding collapsed immunofluorescence staining near the nuclear periphery. Furthermore, we found that vimentin plays an important role in the life cycle of MVM. The number of cells, which successfully replicated MVM, was reduced in infected cells in which the vimentin network was genetically or pharmacologically modified; viral endocytosis, however, remained unaltered. Perinuclear accumulation of MVM-containing vesicles was reduced in cells lacking vimentin. Our data suggests that vimentin is required for the MVM life cycle, presenting possibly a dual role: (1) following MVM escape from endosomes and (2) during endosomal trafficking of MVM. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Comparison of staining of mitotic figures by haematoxylin and eosin-and crystal violet stains, in oral epithelial dysplasia and squamous cell carcinoma.

PubMed

Ankle, Madhuri R; Kale, Alka D; Charantimath, Seema

2007-01-01

Mitosis of cells gives rise to tissue integrity. Defects during mitosis bring about abnormalities. Excessive proliferation of cells due to increased mitosis is one such outcome, which is the hallmark in precancer and cancer. The localization of proliferating cells or their precursors may not be obvious and easy. Establishing an easy way to distinguish these mitotic cells will help in grading and understanding their biological potential. Although immunohistochemistry is an advanced method in use, the cost and time factor makes it less feasible for many laboratories. Selective histochemical stains like toluidine blue, giemsa and crystal violet have been used in tissues including the developing brain, neural tissue and skin. 1) To compare the staining of mitotic cells in haematoxylin and eosin with that in crystal violet. 2) To compare the number of mitotic figures present in normal oral mucosa, epithelial dysplasia and oral squamous cell carcinoma in crystal violet-stained sections with that in H and E-stained sections. Ten tissues of normal oral mucosa and 15 tissues each of oral epithelial dysplasia seen in tobacco-associated leukoplakia and squamous cell carcinoma were studied to evaluate the selectivity of 1% crystal violet for mitotic figures. The staining was compared with standard H and E staining. Statistical analysis was done using Mann-Whitney U test. A statistically significant increase in the mean mitotic count was observed in crystal violet-stained sections of epithelial dysplasia as compared to the H and E-stained sections (p=0.0327). A similar increase in the mitotic counts was noted in crystal violet-stained sections of oral squamous cell carcinoma as compared to the H and E-stained sections.(p=0.0443). No significant difference was found in the mitotic counts determined in dysplasia or carcinoma by either the crystal violet (p=0.4429) or the H and E-staining techniques (p=0.2717). One per cent crystal violet provides a definite advantage over the H and E-stained sections in selectively staining the mitotic figures.

Effects of Long-Term 50Hz Power-Line Frequency Electromagnetic Field on Cell Behavior in Balb/c 3T3 Cells

PubMed Central

An, Guang-Zhou; Xu, Hui; Zhou, Yan; Du, Le; Miao, Xia; Jiang, Da-Peng; Li, Kang-Chu; Guo, Guo-Zhen; Zhang, Chen; Ding, Gui-Rong

2015-01-01

Power-line frequency electromagnetic field (PF-EMF) was reported as a human carcinogen by some epidemiological research, but the conclusion is lack of robust experiment evidence. To identify the effects of long-term PF-EMF exposure on cell behavior, Balb/c 3T3 cells in exponential growth phase were exposed or sham-exposed to 50 Hertz (Hz) PF-EMF at 2.3 mT for 2 hours (h) one day, 5 days every week. After 11 weeks exposure, cells were collected instantly. Cell morphology was observed under invert microscope and Giemsa staining, cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell cycle and apoptosis was examined by flow cytometry, the protein level of Proliferating Cell Nuclear Antigen (PCNA) and CyclinD1 was detected by western blot, cell transformation was examined by soft agar clone assay and plate clone forming test, and cell migration ability was observed by scratch adhesion test. It was found that after PF-EMF exposure, cell morphology, apoptosis, cell migration ability and cell transformation didn’t change. However, compared with sham group, cell viability obviously decreased and cell cycle distribution also changed after 11 weeks PF-EMF exposure. Meanwhile, the protein level of PCNA and CyclinD1 significantly decreased after PF-EMF exposure. These data suggested that although long-term 50Hz PF-EMF exposure under this experimental condition had no effects on apoptosis, cell migration ability and cell transformation, it could affect cell proliferation and cell cycle by down-regulation the expression of PCNA and CyclinD1 protein. PMID:25695503

Effects of long-term 50Hz power-line frequency electromagnetic field on cell behavior in Balb/c 3T3 cells.

PubMed

An, Guang-Zhou; Xu, Hui; Zhou, Yan; Du, Le; Miao, Xia; Jiang, Da-Peng; Li, Kang-Chu; Guo, Guo-Zhen; Zhang, Chen; Ding, Gui-Rong

2015-01-01

Power-line frequency electromagnetic field (PF-EMF) was reported as a human carcinogen by some epidemiological research, but the conclusion is lack of robust experiment evidence. To identify the effects of long-term PF-EMF exposure on cell behavior, Balb/c 3T3 cells in exponential growth phase were exposed or sham-exposed to 50 Hertz (Hz) PF-EMF at 2.3 mT for 2 hours (h) one day, 5 days every week. After 11 weeks exposure, cells were collected instantly. Cell morphology was observed under invert microscope and Giemsa staining, cell viability was detected by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell cycle and apoptosis was examined by flow cytometry, the protein level of Proliferating Cell Nuclear Antigen (PCNA) and CyclinD1 was detected by western blot, cell transformation was examined by soft agar clone assay and plate clone forming test, and cell migration ability was observed by scratch adhesion test. It was found that after PF-EMF exposure, cell morphology, apoptosis, cell migration ability and cell transformation didn't change. However, compared with sham group, cell viability obviously decreased and cell cycle distribution also changed after 11 weeks PF-EMF exposure. Meanwhile, the protein level of PCNA and CyclinD1 significantly decreased after PF-EMF exposure. These data suggested that although long-term 50Hz PF-EMF exposure under this experimental condition had no effects on apoptosis, cell migration ability and cell transformation, it could affect cell proliferation and cell cycle by down-regulation the expression of PCNA and CyclinD1 protein.

Green synthesis of platinum nanoparticles that induce cell death and G2/M-phase cell cycle arrest in human cervical cancer cells.

PubMed

Alshatwi, Ali A; Athinarayanan, Jegan; Vaiyapuri Subbarayan, Periasamy

2015-01-01

Platinum-based chemotherapeutic drugs, including cisplatin, carboplatin, and oxaliplatin, have been used to manage cancer in spite of dose-dependent side effects, including nephrotoxicity, neurotoxicity and ototoxicity. These disadvantages have prompted the development of new strategies for cancer therapy that utilize functionalized nanoparticles as nanomedicines. In the present investigation, we have synthesized platinum nanoparticles using tea polyphenol (TPP) as both a reducing and surface modifying agent. The crystalline nature and morphology of the prepared TPP-functionalized platinum nanoparticles (TPP@Pt) were analyzed using X-ray diffraction (XRD) and transmission electron microscopy (TEM). The XRD results revealed that the TPP@Pt had a crystalline nature with a face-centered cubic structure. TEM imaging suggested that the TTP@Pt are flower shaped with a well-dispersed 30-60 nm-sized TPP@Pt formation. Cervical cancer cells (SiHa) were then treated with different concentrations of TPP@Pt. The effects of TPP@Pt on cell viability, nuclear morphology and cell cycle distribution were investigated. A cell viability assay revealed that the proliferation of SiHa cells was inhibited by TPP@Pt. Propidium iodide nuclear staining indicated that TPP@Pt induced nuclear fragmentation and chromatin condensation. Treatment with TPP@Pt significantly increased the percentage of cells in the G2/M phase, which indicates induced cell cycle arrest in the G2/M phase and an increased number of cells in the subG0 cell death phase. These findings highlight a potential use of TPP@Pt in cervical cancer treatment.

7 Methyl indole ethyl isothiocyanate causes ROS mediated apoptosis and cell cycle arrest in endometrial cancer cells.

PubMed

Kristjansdottir, Katrin; Kim, Kyukwang; Choi, Joong Sub; Horan, Timothy C; Brard, Laurent; Moore, Richard G; Singh, Rakesh K

2012-08-01

Chemotherapy options for advanced endometrial cancer are limited and newer therapeutic agents are urgently needed. This study describes the therapeutic potential of 7 Methyl-indole ethyl isothiocyanate (7Me-IEITC) in endometrial cancer cell lines. 7Me-IEITC was synthesized in our laboratory. The cell viability of 7Me-IEITC treated ECC-1 and KLE endometrial cancer cell was determined by MTS assay. Morphology and apoptosis were further confirmed by DAPI-staining and TUNEL assay. The measurement of reactive oxygen species (ROS), mitochondrial transmembrane depolarization potential (ΔΨm) and cell cycle phase was determined by FACS analysis. Expression of proteins involved in apoptosis, survival and cell-cycle progression was analyzed by Western blotting. 7Me-IEITC reduced the viability of the ECC-1 and KLE cancer cell-lines (IC(50)~2.5-10 μM) in a dose dependent fashion. 7Me-IEITC treatment caused mitochondrial transmembrane potential reduction, elevated the production of ROS, leading to activation of apoptosis in endometrial cancer KLE and ECC-1 cells. 7Me-IEITC treatment activated Bad, suppressed Bcl2 phosphorylation followed by PARP-1 deactivation and caspase 3 and 7 activation. 7Me-IEITC treatment arrested the progression of KLE cells in S-phase and caused CDC25 and cyclin-D1 downregulation. Pre-treatment with ascorbic acid abrogated 7Me-IEITC induced apoptosis in ECC-1 and KLE cells, suggesting that 7Me-IEITC mediated cytotoxicity is primarily through ROS production. 7Me-IEITC demonstrated promising cytotoxic effects in endometrial cancer cell line model. Copyright © 2012 Elsevier Inc. All rights reserved.

Lamprey Prohibitin2 Arrest G2/M Phase Transition of HeLa Cells through Down-regulating Expression and Phosphorylation Level of Cell Cycle Proteins.

PubMed

Shi, Ying; Guo, Sicheng; Wang, Ying; Liu, Xin; Li, Qingwei; Li, Tiesong

2018-03-02

Prohibitin 2(PHB2) is a member of the SFPH trans-membrane family proteins. It is a highly conserved and functionally diverse protein that plays an important role in preserving the structure and function of the mitochondria. In this study, the lamprey PHB2 gene was expressed in HeLa cells to investigate its effect on cell proliferation. The effect of Lm-PHB2 on the proliferation of HeLa cells was determined by treating the cells with pure Lm-PHB2 protein followed by MTT assay. Using the synchronization method with APC-BrdU and PI double staining revealed rLm-PHB2 treatment induced the decrease of both S phase and G0/G1 phase and then increase of G2/M phase. Similarly, cells transfected with pEGFP-N1-Lm-PHB2 also exhibited remarkable reduction in proliferation. Western blot and quantitative real-time PCR(qRT-PCR) assays suggested that Lm-PHB2 caused cell cycle arrest in HeLa cells through inhibition of CDC25C and CCNB1 expression. According to our western blot analysis, Lm-PHB2 was also found to reduce the expression level of Wee1 and PLK1 and the phosphorylation level of CCNB1, CDC25C and CDK1 in HeLa cells. Lamprey prohibitin 2 could arrest G2/M phase transition of HeLa cells through down-regulating expression and phosphorylation level of cell cycle proteins.

Neuronal Cell Death Induced by Mechanical Percussion Trauma in Cultured Neurons is not Preceded by Alterations in Glucose, Lactate and Glutamine Metabolism.

PubMed

Jayakumar, A R; Bak, L K; Rama Rao, K V; Waagepetersen, H S; Schousboe, A; Norenberg, M D

2016-02-01

Traumatic brain injury (TBI) is a devastating neurological disorder that usually presents in acute and chronic forms. Brain edema and associated increased intracranial pressure in the early phase following TBI are major consequences of acute trauma. On the other hand, neuronal injury, leading to neurobehavioral and cognitive impairments, that usually develop months to years after single or repetitive episodes of head trauma, are major consequences of chronic TBI. The molecular mechanisms responsible for TBI-induced injury, however, are unclear. Recent studies have suggested that early mitochondrial dysfunction and subsequent energy failure play a role in the pathogenesis of TBI. We therefore examined whether oxidative metabolism of (13)C-labeled glucose, lactate or glutamine is altered early following in vitro mechanical percussion-induced trauma (5 atm) to neurons (4-24 h), and whether such events contribute to the development of neuronal injury. Cell viability was assayed using the release of the cytoplasmic enzyme lactate dehydrogenase (LDH), together with fluorescence-based cell staining (calcein and ethidium homodimer-1 for live and dead cells, respectively). Trauma had no effect on the LDH release in neurons from 1 to 18 h. However, a significant increase in LDH release was detected at 24 h after trauma. Similar findings were identified when traumatized neurons were stained with fluorescent markers. Additionally (13)C-labeling of glutamate showed a small, but statistically significant decrease at 14 h after trauma. However, trauma had no effect on the cycling ratio of the TCA cycle at any time-period examined. These findings indicate that trauma does not cause a disturbance in oxidative metabolism of any of the substrates used for neurons. Accordingly, such metabolic disturbance does not appear to contribute to the neuronal death in the early stages following trauma.

Steroids Regulate CXCL4 in the Human Endometrium During Menstruation to Enable Efficient Endometrial Repair

PubMed Central

Maybin, Jacqueline A.; Thiruchelvam, Uma; Madhra, Mayank; Saunders, Philippa T.K.

2017-01-01

Context: Repair of the endometrial surface at menstruation must be efficient to minimize blood loss and optimize reproductive function. The mechanism and regulation of endometrial repair remain undefined. Objective: To determine the presence/regulation of CXCL4 in the human endometrium as a putative repair factor at menses. Patients/Setting: Endometrial tissue was collected throughout the menstrual cycle from healthy women attending the gynecology department. Menstrual blood loss was objectively measured in a subset, and heavy menstrual bleeding (HMB) was defined as >80 mL per cycle. Monocytes were isolated from peripheral blood. Design: CXCL4 messenger RNA (mRNA) and protein were identified by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The function/regulation of endometrial CXCL4 was explored by in vitro cell culture. Results: CXCL4 mRNA concentrations were significantly increased during menstruation. Intense staining for CXCL4 was detected in late secretory and menstrual tissue, localized to stromal, epithelial and endothelial cells. Colocalization identified positive staining in CD68+ macrophages. Treatment of human endometrial stromal and endothelial cells (hESCs and HEECs, respectively) with steroids revealed differential regulation of CXCL4. Progesterone withdrawal resulted in significant increases in CXCL4 mRNA and protein in hESCs, whereas cortisol significantly increased CXCL4 in HEECs. In women with HMB, CXCL4 was reduced in endothelial cells during the menstrual phase compared with women with normal menstrual bleeding. Cortisol-exposed macrophages displayed increased chemotaxis toward CXCL4 compared with macrophages incubated with estrogen or progesterone. Conclusions: These data implicate CXCL4 in endometrial repair after menses. Reduced cortisol at the time of menses may contribute to delayed endometrial repair and HMB, in part by mechanisms involving aberrant expression of CXCL4. PMID:28323919

Steroids Regulate CXCL4 in the Human Endometrium During Menstruation to Enable Efficient Endometrial Repair.

PubMed

Maybin, Jacqueline A; Thiruchelvam, Uma; Madhra, Mayank; Saunders, Philippa T K; Critchley, Hilary O D

2017-06-01

Repair of the endometrial surface at menstruation must be efficient to minimize blood loss and optimize reproductive function. The mechanism and regulation of endometrial repair remain undefined. To determine the presence/regulation of CXCL4 in the human endometrium as a putative repair factor at menses. Endometrial tissue was collected throughout the menstrual cycle from healthy women attending the gynecology department. Menstrual blood loss was objectively measured in a subset, and heavy menstrual bleeding (HMB) was defined as >80 mL per cycle. Monocytes were isolated from peripheral blood. CXCL4 messenger RNA (mRNA) and protein were identified by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. The function/regulation of endometrial CXCL4 was explored by in vitro cell culture. CXCL4 mRNA concentrations were significantly increased during menstruation. Intense staining for CXCL4 was detected in late secretory and menstrual tissue, localized to stromal, epithelial and endothelial cells. Colocalization identified positive staining in CD68+ macrophages. Treatment of human endometrial stromal and endothelial cells (hESCs and HEECs, respectively) with steroids revealed differential regulation of CXCL4. Progesterone withdrawal resulted in significant increases in CXCL4 mRNA and protein in hESCs, whereas cortisol significantly increased CXCL4 in HEECs. In women with HMB, CXCL4 was reduced in endothelial cells during the menstrual phase compared with women with normal menstrual bleeding. Cortisol-exposed macrophages displayed increased chemotaxis toward CXCL4 compared with macrophages incubated with estrogen or progesterone. These data implicate CXCL4 in endometrial repair after menses. Reduced cortisol at the time of menses may contribute to delayed endometrial repair and HMB, in part by mechanisms involving aberrant expression of CXCL4. Copyright © 2017 by the Endocrine Society

Distribution of calmodulin in corn seedlings - Immunocytochemical localization in coleoptiles and root apices

NASA Technical Reports Server (NTRS)

Dauwalder, M.; Roux, S. J.

1986-01-01

Immunofluorescence techniques have been used to study the distribution of calmodulin in several tissues in etiolated corn (Zea mays, var. Bear Hybrid) seedlings. Uniform staining was seen in the background cytoplasm of most cell types. Cell walls and vacuoles were not stained. In coleoptile mesophyll cells the nucleoplasm of most nuclei was stained as was the stroma of most amyloplasts. The lumen border of mature tracheary elements in coleoptiles also stained. In the rootcap the most intensely stained regions were the cytoplasms of columella cells and of the outermost cells enmeshed in the layer of secreted slime. Nuclei in the rootcap cells did not stain distinctly, but those in all cell types of the root meristem did. Also in the root meristem, the cytoplasm of metaxylem elements stained brightly. These results are compared and contrasted with previous data on the localization of calmodulin in pea root apices and epicotyls and discussed in relation to current hypotheses on mechanisms of gravitropism.

Resveratrol induces cell cycle arrest and apoptosis in human eosinophils from asthmatic individuals.

PubMed

Hu, Xin; Wang, Jing; Xia, Yu; Simayi, Mihereguli; Ikramullah, Syed; He, Yuanbing; Cui, Shihong; Li, Shuang; Wushouer, Qimanguli

2016-12-01

Eosinophils exert a number of inflammatory effects through the degranulation and release of intracellular mediators, and are considered to be key effector cells in allergic disorders, including asthma. In order to investigate the regulatory effects of the natural polyphenol, resveratrol, on eosinophils derived from asthmatic individuals, the cell counting Kit‑8 assay and flow cytometry analysis were used to determine cell proliferation and cell cycle progression in these cells, respectively. Cellular apoptosis was detected using annexin V-fluorescein isothiocyanate/propidium iodide double‑staining. The protein expression levels of p53, p21, cyclin‑dependent kinase 2 (CDK2), cyclin A, cyclin E, Bim, B‑cell lymphoma (Bcl)‑2 and Bcl‑2‑associated X protein (Bax) were measured by western blot analysis following resveratrol treatment. The results indicated that resveratrol effectively suppressed the proliferation of eosinophils from asthmatic patients in a concentration‑ and time‑dependent manner. In addition, resveratrol was observed to arrest cell cycle progression in G1/S phase by increasing the protein expression levels of p53 and p21, and concurrently reducing the protein expression levels of CDK2, cyclin A and cyclin E. Furthermore, resveratrol treatment significantly induced apoptosis in eosinophils, likely through the upregulation of Bim and Bax protein expression levels and the downregulation of Bcl‑2 protein expression. These findings suggested that resveratrol may be a potential agent for the treatment of asthma by decreasing the number of eosinophils.

NMDAR-1 staining in the lateral geniculate nucleus of normal and visually deprived cats.

PubMed

Ziburkus, J; Bickford, M E; Guido, W

2000-01-01

In normal adult cats, a monoclonal antibody directed toward the NR-1 subunit of the N-methyl-D-aspartate (NMDA) receptor (Pharmingen, clone 54.1) produced dense cellular and neuropil labeling throughout all layers of the lateral geniculate nucleus (LGN) and adjacent thalamic nuclei, including the thalamic reticular, perigeniculate, medial intralaminar, and ventral lateral geniculate nuclei. Cellular staining revealed well-defined somata, and in some cases proximal dendrites. NMDAR-1 cell labeling was also evident in the LGN of early postnatal kittens, suggesting that developing LGN cells possess this receptor subunit at or before eye opening. Within the A-layers of the adult LGN, staining encompassed a wide range of soma sizes. Soma size comparisons of NMDAR-1 stained cells with those stained with an antibody directed toward a nonphosphorylated neurofilament protein (SMI-32), which selectively stains Y-relay cells (Bickford et al., 1998), or an antibody to glutamic acid decarboxylase (GAD), which stains for GABAergic interneurons, suggested that NMDA receptors are utilized by relay cells and interneurons. NMDAR-1 staining was also observed in the LGN of cats with early monocular lid suture. Although labeling was apparent in both deprived and nondeprived A-layers of LGN, the distribution of soma sizes was significantly different. In the deprived A-layers of LGN, staining was limited to small- and medium-sized cells. Cells with relatively large soma were lacking. However, cell density measurements as well as soma size comparisons with cells stained for Nissl substance suggested these differences were due to deprivation-induced cell shrinkage and not to a loss of NMDAR-1 staining in Y-cells. Taken together, these results suggest that NMDA receptors are utilized by both relay cells and interneurons in LGN and that alterations in early visual experience do not necessarily affect the expression of NMDA receptors in the LGN.

2',4'-Dihydroxychalcone-induced apoptosis of human gastric cancer MGC-803 cells via down-regulation of survivin mRNA.

PubMed

Lou, Chenghua; Yang, Guangming; Cai, Hao; Zou, Mingchang; Xu, Zisheng; Li, Yu; Zhao, Fengming; Li, Weidong; Tong, Li; Wang, Mingyan; Cai, Baochang

2010-08-01

2',4'-Dihydroxychalcone (TFC), a main component in Herba Oxytropis, is grouped under flavonoids, which are well known to have antitumor activities in vitro. In this study, the possible antitumor mechanism of TFC in human gastric cancer MGC-803 cells is examined. Hoechst 33258 staining analysis indicates that TFC causes MGC-803 cell shrinkage and apoptotic body formation, typical characteristics of apoptosis. Flow cytometric analysis demonstrates that TFC causes cell cycle arrest in the G2/M phase. Furthermore, TFC significantly increases caspase-3 activity but decreases survivin mRNA expression. Therefore, TFC can induce the apoptosis of MGC-803 cells via down-regulation of survivin mRNA expression. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

A new fluorescent test for cell vitality using calcofluor white M2R.

PubMed

Fischer, J M; Peterson, C A; Bols, N C

1985-03-01

The fluorescent fabric-brightener dye, Calcofluor white M2R (CFW), can be used to distinguish between living and dead cells from a variety of animal and plant sources. CFW does not stain living mouse fibroblasts or trout red blood cells and stains only the cell walls in living cells from the epidermis of onion bulb scale, staminal hairs of Tradescantia, and longitudinal sections of broad bean stems and roots. Heat-killed plant or animal cells are recognized by their lightly stained cytoplasm and brightly stained nuclei. The optimum staining concentrations were very low (0.01% to 0.03%) and nontoxic. Using onion scale epidermis in which some cells had been killed by heating as a test system, and the plasmolysis-deplasmolysis rection as the ultimate test for cell vitality, results from CFW staining correctly predicted cell vitality for about 98% of the cells tested. This success rate was comparable to those for Evans blue, uranin or neutral red in this test system.

Research on fluorescence detection method of Microcystis aeruginosa

NASA Astrophysics Data System (ADS)

Wang, Xiao-xiong

2017-07-01

The paper studied the viability determination of Microcystis aeruginosa by FDA and PI staining. The staining results were measured by fluorescence microscopy. The results indicated that viable and dead cells were stained as bright green and red fluorescent respectively by FDA and PI. Through PI-FDA dual color fluorescence staining, the color of green and red distinct obviously by fluorescence microscope. The staining rate has relation with the cell density. If the cell density of M. aeruginosa was 1.0×107-1.0×109 cell·mL-1, the staining rate would be 100.0% or 98.0% by PI and of FDA respectively.

Comparison of the effects of photon versus carbon ion irradiation when combined with chemotherapy in vitro.

PubMed

Schlaich, Fabian; Brons, Stephan; Haberer, Thomas; Debus, Jürgen; Combs, Stephanie E; Weber, Klaus-Josef

2013-11-06

Characterization of combination effects of chemotherapy drugs with carbon ions in comparison to photons in vitro. The human colon adenocarcinoma cell line WiDr was tested for combinations with camptothecin, cisplatin, gemcitabine and paclitaxel. In addition three other human tumour cell lines (A549: lung, LN-229: glioblastoma, PANC-1: pancreas) were tested for the combination with camptothecin. Cells were irradiated with photon doses of 2, 4, 6 and 8 Gy or carbon ion doses of 0.5, 1, 2 and 3 Gy. Cell survival was assessed using the clonogenic growth assay. Treatment dependent changes in cell cycle distribution (up to 12 hours post-treatment) were measured by FACS analysis after propidium-iodide staining. Apoptosis was monitored for up to 36 hours post-treatment by Nicoletti-assay (with qualitative verification using DAPI staining). All cell lines exhibited the well-known increase of killing efficacy per unit dose of carbon ion exposure, with relative biological efficiencies at 10% survival (RBE10) ranging from 2.3 to 3.7 for the different cell lines. In combination with chemotherapy additive toxicity was the prevailing effect. Only in combination with gemcitabine or cisplatin (WiDr) or camptothecin (all cell lines) the photon sensitivity was slightly enhanced, whereas purely independent toxicities were found with the carbon ion irradiation, in all cases. Radiation-induced cell cycle changes displayed the generally observed dose-dependent G2-arrest with little effect on S-phase fraction for all cell lines for photons and for carbon ions. Only paclitaxel showed a significant induction of apoptosis in WiDr cell line but independent of the used radiation quality. Combined effects of different chemotherapeutics with photons or with carbon ions do neither display qualitative nor substantial quantitative differences. Small radiosensitizing effects, when observed with photons are decreased with carbon ions. The data support the idea that a radiochemotherapy with common drugs and carbon ion irradiation might be as feasible as respective photon-based protocols. The present data serve as an important radiobiological basis for further combination experiments, as well as clinical studies on combination treatments.

[Artemisinin inhibits proliferation of gallbladder cancer cell lines through triggering cell cycle arrest and apoptosis].

PubMed

Jia, J G; Zhang, L G; Guo, C X; Wang, Y G; Chen, B L; Wang, Y M; Qian, J

2016-03-01

To evaluate the effects of artemisinin on proliferation, cell cycle and apoptosis of gallbladder cancer cells. Gallbladder carcinoma cell lines(GBC-SD and NOZ)were cultured in vitro. The effects of artemisinin in different concentration on proliferation of the two cell lines in vitro were examined using MTT assay. The cell cycle distribution of GBC-SD and NOZ cells 24 h after treatments with artemisinin(20 μmol/L) were examined using flow cytometry. The apoptosis of GBC-SD and NOZ cells 24 h after treatments with artemisinin (20 μmol/L) were examined using Annexin V/PI staining.The expressions of p-ERK1/2, CDK4, cyclin D1, p16, cytochrome C and caspase-3 were examined by Western blot assay. t-test and one way ANOVA were used to evaluate the differences between two groups and more than two groups, respectively. The cell proliferation was significantly inhibited by artemisinin, the IC50 of artemisinin against GBC-SD and NOZ cells were 14.05 μmol/L and 12.42 μmol/L, respectively.Artemisinin induced cycle arrest, and G1 population of GBC-SD and NOZ cells increased to 74.60% and 78.86%. Cell apoptosis and apoptotic population of GBC-SD and NOZ cells were increased to 15.67% and 16.51% after dealt with artemisinin, respectively. In addition, expression of p16 was increased, and expressions of p-ERK1/2, CDK4 and cyclin D1 were down-regulated by artemisinin(all P<0.05). Cytochrome C was released from mitochondria to cytoplasm leading to the activation of caspase-3 and PARP after dealt with artemisinin(P<0.05). The inhibition effect of artemisinin on the proliferation gallbladder cancer cells is accompanied by down-regulation of ERK1/2 signaling pathway, G1 phase arrest and triggering caspase-3-mediate apoptosis.

in Vitro and in Vivo Inhibitory Effects of α-Mangostin on Cholangiocarcinoma Cells and Allografts

PubMed Central

Aukkanimart, Ratchadawan; Boonmars, Thidarut; Sriraj, Pranee; Sripan, Panupan; Songsri, Jiraporn; Ratanasuwan, Panaratana; Laummaunwai, Porntip; Boueroy, Parichart; Khueangchaingkhwang, Sukhonthip; Pumhirunroj, Benjamabhorn; Artchayasawat, Atchara; Boonjaraspinyo, Sirintip; Wu, Zhiliang; Hahnvajanawong, Chariya; Vaeteewoottacharn, Kulthida; Wongkham, Sopit

2017-01-01

We investigated the anti-cholangiocarcinoma effect of α-mangostin from Garcinia mangostana pericarp extract (GM) in a human cholangiocarcinoma (CCA) cell line and a hamster CCA allograft model. In vitro, human CCA cells were treated with GM at various concentrations and for different time periods; then cell-cycle arrest and apoptosis were evaluated using flow cytometry, and metastatic potential with wound healing assays. In vivo, hamster allografts were treated with GM, gemcitabine (positive control) and a placebo (negative control) for 1 month; tumor weight and volume were then determined. Histopathological features and immunostaining (CK19 and PCNA) characteristics were examined by microscopy. The present study found that α-mangostin could: inhibit CCA cell proliferation by inducing apoptosis through the mitochondrial pathway; induce G1 cell-cycle arrest; and inhibit metastasis. Moreover, α-mangostin could inhibit CCA growth, i.e. reduce tumor mass (weight and size) and alter CCA pathology, as evidenced by reduced positive staining for CK19 and PCNA. The present study thus suggested that α-mangostin is a promising anti-CCA compound whose ready availability in tropical countries might indicate use for prevention and treatment of CCA. PMID:28441703

Combination of HDAC inhibitor TSA and silibinin induces cell cycle arrest and apoptosis by targeting survivin and cyclinB1/Cdk1 in pancreatic cancer cells.

PubMed

Feng, Wan; Cai, Dawei; Zhang, Bin; Lou, Guochun; Zou, Xiaoping

2015-08-01

Histone deacetylases (HDAC) are involved in diverse biological processes and therefore emerge as potential targets for pancreatic cancer. Silibinin, an active component of silymarin, is known to inhibit growth of pancreatic cancer in vivo and in vitro. Herein, we examined the cytotoxic effects of TSA in combination with silibinin and investigated the possible mechanism in two pancreatic cancer cell lines (Panc1 and Capan2). Our study found that combination treatment of HDAC inhibitor and silibinin exerted additive growth inhibitory effect on pancreatic cancer cell. Annexin V-FITC/PI staining and flow cytometry analysis demonstrated that combination therapy induced G2/M cell cycle arrest and apoptosis in Panc1and Capan2 cells. The induction of apoptosis was further confirmed by evaluating the activation of caspases. Moreover, treatment with TSA and silibinin resulted in a profound reduction in the expression of cyclinA2, cyclinB1/Cdk1 and survivin. Taken together, our study might indicate that the novel combination of HDAC inhibitor and silibinin could offer therapeutic potential against pancreatic cancer. Copyright © 2015. Published by Elsevier Masson SAS.

Novel Synthetic Mono-triazole Glycosides Induce G0/G1 Cell-cycle Arrest and Apoptosis in Cholangiocarcinoma Cells.

PubMed

Obchoei, Sumalee; Saeeng, Rungnapha; Wongkham, Chaisiri; Wongkham, Sopit

2016-11-01

The treatment of cholangiocarcinoma (CCA) is still ineffective and the search for a novel treatment is needed. In this study, eight novel mono-triazole glycosides (W1-W8) were synthesized and tested for their anticancer activities in CCA cell lines. The anti-proliferation effect and the underlying mechanisms of the triazole glycosides were explored. Viable cells were determined using the MTT test. Among glycosides tested, W4 and W5 exhibited the most potent anticancer activity in a dose- and time-dependent fashion. Flow cytometry and wstern blot analysis revealed that W4 and W5 induced G 0 /G 1 phase cell-cycle arrest through down-regulation of cyclin D1, cyclin E and induction of cyclin-dependent kinase inhibitors, p27 and p21 protein expression. Annexin V/propidium iodide (PI) staining demonstrated that W4 and W5 also induced apoptotic cells in a dose-dependent manner via caspase signaling cascade. Together, these findings imply that the novel synthetic glycosides might be a promising anticancer agent for CCA. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Does autophagy in the midgut epithelium of centipedes depend on the day/night cycle?

PubMed

Rost-Roszkowska, M M; Chajec, Ł; Vilimova, J; Tajovský, K; Kszuk-Jendrysik, M

2015-01-01

The midgut epithelium of two centipedes, Lithobius forficatus and Scolopendra cingulata, is composed of digestive, secretory and regenerative cells. In L. forficatus, the autophagy occurred only in the cytoplasm of the digestive cells as a sporadic process, while in S. cingulata, it occurred intensively in the digestive, secretory and regenerative cells of the midgut epithelium. In both of the species that were analyzed, this process proceeded in a continuous manner and did not depend on the day/night cycle. Ultrastructural analysis showed that the autophagosomes and autolysosomes were located mainly in the apical and perinuclear cytoplasm of the digestive cells in L. forficatus. However, in S. cingulata, the entire cytoplasm was filled with autophagosomes and autolysosomes. Initially the membranes of phagophores surround organelles during autophagosome formation. Autolysosomes result from the fusion of autophagosomes and lysosomes. Residual bodies which are the last stage of autophagy were released into the midgut lumen due to necrosis. Autophagy in the midgut epithelia that were analyzed was confirmed using acid phosphatase and mono-dansyl-cadaverine stainings. Copyright © 2014 Elsevier Ltd. All rights reserved.

Strigolactone analogues induce apoptosis through activation of p38 and the stress response pathway in cancer cell lines and in conditionally reprogrammed primary prostate cancer cells.

PubMed

Pollock, Claire B; McDonough, Sara; Wang, Victor S; Lee, Hyojung; Ringer, Lymor; Li, Xin; Prandi, Cristina; Lee, Richard J; Feldman, Adam S; Koltai, Hinanit; Kapulnik, Yoram; Rodriguez, Olga C; Schlegel, Richard; Albanese, Christopher; Yarden, Ronit I

2014-03-30

Strigolactones are a novel class of plant hormones produced in roots and regulate shoot and root development. We have previously shown that synthetic strigolactone analogues potently inhibit growth of breast cancer cells and breast cancer stem cells. Here we show that strigolactone analogues inhibit the growth and survival of an array of cancer-derived cell lines representing solid and non-solid cancer cells including: prostate, colon, lung, melanoma, osteosarcoma and leukemic cell lines, while normal cells were minimally affected. Treatment of cancer cells with strigolactone analogues was hallmarked by activation of the stress-related MAPKs: p38 and JNK and induction of stress-related genes; cell cycle arrest and apoptosis evident by increased percentages of cells in the sub-G1 fraction and Annexin V staining. In addition, we tested the response of patient-matched conditionally reprogrammed primary prostate normal and cancer cells. The tumor cells exhibited significantly higher sensitivity to the two most potent SL analogues with increased apoptosis confirmed by PARP1 cleavage compared to their normal counterpart cells. Thus, Strigolactone analogues are promising candidates for anticancer therapy by their ability to specifically induce cell cycle arrest, cellular stress and apoptosis in tumor cells with minimal effects on growth and survival of normal cells.

Phaleria macrocarpa (Boerl.) fruit induce G0/G1 and G2/M cell cycle arrest and apoptosis through mitochondria-mediated pathway in MDA-MB-231 human breast cancer cell.

PubMed

Kavitha, Nowroji; Ein Oon, Chern; Chen, Yeng; Kanwar, Jagat R; Sasidharan, Sreenivasan

2017-04-06

Phaleria macrocarpa (Scheff) Boerl, is a well-known folk medicinal plant in Indonesia. Traditionally, P. macrocarpa has been used to control cancer, impotency, hemorrhoids, diabetes mellitus, allergies, liver and hearth disease, kidney disorders, blood diseases, acne, stroke, migraine, and various skin diseases. The purpose of this study was to determine the in situ cytotoxicity effect P. macrocarpa fruit ethyl acetate fraction (PMEAF) and the underlying molecular mechanism of cell death. MDA-MB-231 cells were incubated with PMEAF for 24h. Cell cycle and viability were examined using flow cytometry analysis. Apoptosis was determined using the Annexin V assay and also by fluorescence microscopy. Apoptosis protein profiling was detected by RayBio® Human Apoptosis Array. The AO/PI staining and flow cytometric analysis of MDA-MB-231 cells treated with PMEAF were showed apoptotic cell death. The cell cycle analysis by flow cytometry analysis revealed that the accumulation of PMEAF treated MDA-MB-231 cells in G 0 /G 1 and G 2 /M-phase of the cell cycle. Moreover, the PMEAF exert cytotoxicity by increased the ROS production in MDA-MB-231 cells consistently stimulated the loss of mitochondrial membrane potential (∆ Ψm ) and induced apoptosis cell death by activation of numerous signalling proteins. The results from apoptosis protein profiling array evidenced that PMEAF stimulated the expression of 9 pro-apoptotic proteins (Bax, Bid, caspase 3, caspase 8, cytochrome c, p21, p27, p53 and SMAC) and suppressed the 4 anti-apoptotic proteins (Bcl-2, Bcl-w, XIAP and survivin) in MDA-MB-231 cells. The results indicated that PMEAF treatment induced apoptosis in MDA-MB-231 cells through intrinsic mitochondrial related pathway with the participation of pro and anti-apoptotic proteins, caspases, G 0 /G 1 and G 2 /M-phases cell cycle arrest by p53-mediated mechanism. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Effect of quercetin on apoptosis of PANC-1 cells

PubMed Central

Lee, Joo Hyun; Lee, Han-Beom; Jung, Gum O; Oh, Jung Taek; Park, Dong Eun

2013-01-01

Purpose To investigate the chemotherapeutic effect of quercetin against cancer cells, signaling pathway of apoptosis was explored in human pancreatic cells. Methods Various anticancer drugs including adriamycin, cisplatin, 5-fluorouracil (5-FU) and gemcitabine were used. Cell viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-nyltetra zolium bromide assay. Apoptosis was determined by 4'-6-diamidino-2-phenylindole nuclei staining and flow cytometry in PANC-1 cells treated with 50 µg/mL quercetin for 24 hours. Expression of endoplas mic reticulum (ER) stress mediators including, Grp78/Bip, p-PERK, PERK, ATF4, ATF6 and GADD153/CHOP proteins were measured by Western blot analysis. Mitochondrial membrane potential was measured by fluorescence staining with JC-1, rhodamine 123. Quercetin induced the apoptosis of PANC-1, which was characterized as nucleic acid and genomic DNA fragmentation, chromatin condensation, and sub-G0/G1 fraction of cell cycle increase. But not adriamycin, cisplatin, gemcitabine, and 5-FU. PANC-1 cells were markedly sensitive to quercetin. Results Treatment with quercetin resulted in the increased accumulation of intracellular Ca2+ ion. Treatment with quercetin also increased the expression of Grp78/Bip and GADD153/CHOP protein and induced mitochondrial dysfunction. Quercetin exerted cytotoxicity against human pancreatic cancer cells via ER stress-mediated apoptotic signaling including reactive oxygen species production and mitochondrial dysfunction. Conclusion These data suggest that quercetin may be an important modulator of chemosensitivity of cancer cells against anticancer chemotherapeutic agents. PMID:24368982

Effect of quercetin on apoptosis of PANC-1 cells.

PubMed

Lee, Joo Hyun; Lee, Han-Beom; Jung, Gum O; Oh, Jung Taek; Park, Dong Eun; Chae, Kwon Mook

2013-12-01

To investigate the chemotherapeutic effect of quercetin against cancer cells, signaling pathway of apoptosis was explored in human pancreatic cells. Various anticancer drugs including adriamycin, cisplatin, 5-fluorouracil (5-FU) and gemcitabine were used. Cell viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-nyltetra zolium bromide assay. Apoptosis was determined by 4'-6-diamidino-2-phenylindole nuclei staining and flow cytometry in PANC-1 cells treated with 50 µg/mL quercetin for 24 hours. Expression of endoplas mic reticulum (ER) stress mediators including, Grp78/Bip, p-PERK, PERK, ATF4, ATF6 and GADD153/CHOP proteins were measured by Western blot analysis. Mitochondrial membrane potential was measured by fluorescence staining with JC-1, rhodamine 123. Quercetin induced the apoptosis of PANC-1, which was characterized as nucleic acid and genomic DNA fragmentation, chromatin condensation, and sub-G0/G1 fraction of cell cycle increase. But not adriamycin, cisplatin, gemcitabine, and 5-FU. PANC-1 cells were markedly sensitive to quercetin. Treatment with quercetin resulted in the increased accumulation of intracellular Ca(2+) ion. Treatment with quercetin also increased the expression of Grp78/Bip and GADD153/CHOP protein and induced mitochondrial dysfunction. Quercetin exerted cytotoxicity against human pancreatic cancer cells via ER stress-mediated apoptotic signaling including reactive oxygen species production and mitochondrial dysfunction. These data suggest that quercetin may be an important modulator of chemosensitivity of cancer cells against anticancer chemotherapeutic agents.

Dimethylfumarate induces cell cycle arrest and apoptosis via regulating intracellular redox systems in HeLa cells.

PubMed

Han, Guocan; Zhou, Qiang

2016-12-01

Dimethylfumarate (DMF) is cytotoxic to several kinds of cells and serves as an anti-tumor drug. This study was designed to investigate the effects and underlying mechanism of DMF on cervical cancer cells. HeLa cells were cultured and treated with 0, 50, 100, 150, and 200 μM DMF, respectively. After 24 h, cell growth was evaluated using Cell Counting Kit-8 (CCK-8) assay and the cell cycle was examined using flow cytometry. In addition, cell apoptosis was detected by Annexin V/propidium iodide (PI) staining and the expressions of caspase-3 and poly-ADP-ribose polymerase (PARP) were detected using western blotting. The redox-related factors were then assessed. Furthermore, all of the indicators were detected in HeLa cells after combined treatment of DMF and N-acetyl-L-cysteine (NAC, an oxygen-free radical scavenger). The cell number and cell growth of HeLa were obviously inhibited by DMF in a dose-dependent manner, as the cell cycle was arrested at G0/G1 phase (P < 0.05). The apoptotic HeLa cells were markedly increased, and the expression levels of caspase-3 and PARP were significantly increased in a DMF concentration-dependent way (P < 0.05). Meanwhile, loss of △Ψm, increase in reactive oxygen species and O 2 ·- , and the decrease in catalase activity and glutathione (GSH) level were found after DMF treatment (P < 0.05). All these changes were significantly attenuated and even completely disappeared by adding NAC (P < 0.05). In conclusion, the cytotoxicity of DMF on cell proliferation and apoptosis of HeLa cells was mainly related to the intracellular redox systems by depletion of intracellular GSH.

Voltage-gated Na+ channel SCN5A is a key regulator of a gene transcriptional network that controls colon cancer invasion

PubMed Central

House, Carrie D.; Vaske, Charles J.; Schwartz, Arnold M.; Obias, Vincent; Frank, Bryan; Luu, Truong; Sarvazyan, Narine; Irby, Rosalyn; Strausberg, Robert L.; Hales, Tim G.; Stuart, Joshua M.; Lee, Norman H.

2010-01-01

Voltage-gated Na+ channels (VGSCs) have been implicated in the metastatic potential of human breast, prostate and lung cancer cells. Specifically, the SCN5A gene encoding the VGSC isotype Nav1.5 has been defined as a key driver of human cancer cell invasion. In this study, we examined the expression and function of VGSCs in a panel of colon cancer cell lines by electrophysiological recordings. Na+ channel activity and invasive potential were inhibited pharmacologically by tetrodotoxin or genetically by siRNAs specifically targeting SCN5A. Clinical relevance was established by immunohistochemistry of patient biopsies, where there was strong Nav1.5 protein staining in colon cancer specimens but little to no staining in matched-paired normal colon tissues. We explored the mechanism of VGSC-mediated invasive potential on the basis of reported links between VGSC activity and gene expression in excitable cells. Probabilistic modeling of loss-of-function screens and microarray data established an unequivocal role of VGSC SCN5A as a high level regulator of a colon cancer invasion network, involving genes that encompass Wnt signaling, cell migration, ectoderm development, response to biotic stimulus, steroid metabolic process and cell cycle control. siRNA-mediated knockdown of predicted downstream network components caused a loss of invasive behavior, demonstrating network connectivity and its function in driving colon cancer invasion. PMID:20651255

A mucin-like glycoprotein identified by MAG (mouse ascites Golgi) antibodies. Menstrual cycle-dependent localization in human endometrium.

PubMed Central

Kliman, H. J.; Feinberg, R. F.; Schwartz, L. B.; Feinman, M. A.; Lavi, E.; Meaddough, E. L.

1995-01-01

Human endometrial glands synthesize and secrete a high molecular weight mucin-like glycoprotein in a menstrual cycle-dependent fashion. A novel moiety within this Golgi-associated glycoprotein is strongly reactive with IgG antibodies in numerous murine ascites, and has been termed MAG (mouse ascites Golgi). Immunohistochemical staining of 201 endometrial biopsies revealed the following patterns: MAG first appeared in the Golgi on cycle day 5, peaked on day 15, was present on the surface of the luminal epithelium between days 17 and 19, and was no longer detectable after day 19. MAG was also present in cervical, prostate, seminal vesicle, and lacrimal glands, pancreatic acinar cells, gall bladder and bile duct epithelium, and certain cells of the salivary and sweat glands. Interestingly, only tissues from blood group A individuals exhibited this staining. As a common link among all these cell types is the expression of mucins, we speculated that the MAG epitope could be a mucin-associated blood group A-related epitope. This hypothesis was tested by absorption experiments with a variety of glycoconjugates and erythrocytes and by immunoblots of MAG-rich material. The absorption studies demonstrated that only type III porcine mucin (< 1% sialic acid) and blood type A or AB erythrocytes were able to absorb the anti-MAG antibody. Inasmuch as N-acetyl-galactosamine alone, the terminal blood group A carbohydrate, did not block MAG antibody binding, the MAG epitope appears to involve N-acetylgalactosamine plus other determinants. Immunoblots of endometrial extracts and saliva from blood type A individuals revealed MAG-reactive material with a molecular weight > 200 kd under reducing conditions. Because the MAG epitope appears on the endometrial surface during the purported implantation window, we speculate that mucin-like epitopes could play a role in the earliest apposition phases of conceptus-endometrial interaction. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 PMID:7531946

AMPK activators inhibit the proliferation of human melanomas bearing the activated MAPK pathway.

PubMed

Petti, Carlotta; Vegetti, Claudia; Molla, Alessandra; Bersani, Ilaria; Cleris, Loredana; Mustard, Kirsty J; Formelli, Franca; Hardie, Grahame D; Sensi, Marialuisa; Anichini, Andrea

2012-10-01

Raf/MEK/ERK signaling can inhibit the liver kinase B1-AMP-activated protein kinase (LKB1-AMPK) pathway, thus rendering melanoma cells resistant to energy stress conditions. We evaluated whether pharmacological reactivation of the AMPK function could exert antitumor effects on melanoma cells bearing this pathway constitutively active because of a mutation in NRAS or BRAF genes. Nine melanoma cell lines were treated with the AMPK activators 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and phenformin. The activation of AMPK enzymatic activity, phosphorylation of AMPK and acetyl-CoA carboxylase kinase, in-vitro proliferation, cell cycle, and in-vivo growth of xenografts in nude mice were evaluated. AICAR and phenformin promoted phosphorylation and enzymatic activity of AMPK, as well as phosphorylation of the AMPK downstream target acetyl-CoA carboxylase. Drug treatment of either BRAF-mutant or NRAS-mutant melanomas, at doses not inducing cell death, was accompanied by a dose-dependent decrease in melanoma cell proliferation because of cell cycle arrest in either the G0/G1 or the S phase, associated with an increased expression of the p21 cell cycle inhibitor. Melanomas isolated from subcutaneously implanted mice, 25 days from treatment with AICAR, showed increased staining of the senescence-associated marker β-galactosidase, high p21 expression, and evidence of necrosis. Altogether, these results indicate that pharmacological activators of AMPK-dependent pathways inhibit the cell growth of melanoma cells with active Raf/MEK/ERK signaling and provide a rationale for further investigation on their use in combination therapies.

[Pseudolaric acid B induces G2/M arrest and inhibits invasion and migration in HepG2 hepatoma cells].

PubMed

Li, Shuai; Guo, Lianyi

2018-01-01

Objective To investigate the mechanisms of pseudolaric acid B (PAB) blocks cell cycle and inhibits invasion and migration in human hepatoma HepG2 cells. Methods The proliferation effect of PAB on HepG2 cells was evaluated by MTT assay. The effect of PAB on the cell cycle of HepG2 cells was analyzed by flow cytometry. Immunofluorescence cytochemical staining was applied to observe the effect of PAB on the α-tubulin polymerization and expression in HepG2 cells. Transwell TM chamber invasion assay and wound healing assay were performed to detect the influence of PAB on the migration and invasion ability of HepG2 cells. Western blotting was used to determine the expressions of α-tubulin, E-cadherin and MMP-9 in HepG2 cells after treated with PAB. Results PAB inhibited the proliferation of HepG2 cells in a dose-dependent manner and blocked the cell cycle in G2/M phase. PAB significantly changed the polymerization and decreased the expression of α-tubulin. The capacities of invasion and migration of HepG2 cells treated by PAB were significantly depressed. The protein levels of α-tubulin and MMP-9 decreased while the E-cadherin protein level increased. Conclusion PAB can inhibits the proliferation of HepG2 cells by down-regulating the expression of α-tubulin and influencing its polymerization, arresting HepG2 cells in G2/M phase. Meanwhile, PAB also can inhibit the invasion and migration of HepG2 cells by lowering cytoskeleton α-tubulin and MMP-9, and increasing E-cadherin.

Protocol for vital dye staining of corneal endothelial cells.

PubMed

Park, Sunju; Fong, Alan G; Cho, Hyung; Zhang, Cheng; Gritz, David C; Mian, Gibran; Herzlich, Alexandra A; Gore, Patrick; Morganti, Ashley; Chuck, Roy S

2012-12-01

To describe a step-by-step methodology to establish a reproducible staining protocol for the evaluation of human corneal endothelial cells. Four procedures were performed to determine the best protocol. (1) To determine the optimal trypan blue staining method, goat corneas were stained with 4 dilutions of trypan blue (0.4%, 0.2%, 0.1%, and 0.05%) and 1% alizarin red. (2) To determine the optimal alizarin red staining method, goat corneas were stained with 2 dilutions of alizarin red (1% and 0.5%) and 0.2% trypan blue. (3) To ensure that trypan blue truly stains damaged cells, goat corneas were exposed to either 3% hydrogen peroxide or to balanced salt solution, and then stained with 0.2% trypan blue and 0.5% alizarin red. (4) Finally, fresh human corneal buttons were examined; 1 group was stained with 0.2% trypan blue and another group with 0.4% trypan blue. For the 4 procedures performed, the results are as follows: (1) trypan blue staining was not observed in any of the normal corneal samples; (2) 0.5% alizarin red demonstrated sharper cell borders than 1% alizarin red; (3) positive trypan blue staining was observed in the hydrogen peroxide exposed tissue in damaged areas; (4) 0.4% trypan blue showed more distinct positive staining than 0.2% trypan blue. We were able to determine the optimal vital dye staining conditions for human corneal endothelial cells using 0.4% trypan blue and 0.5% alizarin red.

Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

PubMed Central

Dang, Yalong; Waxman, Susannah; Wang, Chao; Jensen, Adrianna; Loewen, Ralitsa T.; Bilonick, Richard A.

2017-01-01

Objective The trabecular meshwork (TM) is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might be a viable therapeutic approach. Decellularized TM scaffolds have previously been produced by ablating cells with suicide gene therapy or saponin, which risks incomplete cell removal or dissolution of the extracellular matrix, respectively. We hypothesized that improved trabecular meshwork cell ablation would result from freeze-thaw cycles compared to chemical treatment. Materials and Methods We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion within two hours of sacrifice. Intraocular pressure (IOP) was recorded continuously by a pressure transducer system. After 72 h of IOP stabilization, eight eyes were assigned to freeze-thaw (F) ablation (−80 °C × 2), to 0.02% saponin (S) treatment, or the control group (C), respectively. The TM was transduced with an eGFP expressing feline immunodeficiency viral (FIV) vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture media for 180 h. TM histology was assessed by hematoxylin and eosin staining. TM viability was evaluated by a calcein AM/propidium iodide (PI) assay. The TM extracellular matrix was stained with Picro Sirius Red. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with five degrees of freedom. Results F and S experienced a similar IOP reduction of 30% from baseline (P = 0.64). IOP reduction of about 30% occurred in F within 24 h and in S within 48 h. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis and Picro Sirius staining confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed very low PI and no calcein staining in F in contrast to many PI-labeled, dead TM cells and calcein-labeled viable TM cells in S. Conclusion We developed a rapid TM ablation method that uses cyclic freezing that is free of biological or chemical agents and able to produce a decellularized TM scaffold with preserved TM extracellular matrix in an organotypic perfusion culture. PMID:28828244

Image analysis assisted study of mitotic figures in oral epithelial dysplasia and squamous cell carcinoma using differential stains.

PubMed

Tandon, Ankita; Singh, Narendra Nath; Brave, V R; Sreedhar, Gadiputi

2016-11-01

Mitosis is a process of cell division resulting in two genetically equivalent daughter cells. Excessive proliferation of cells due to mitosis is the hallmark in pre cancer and cancer. This study was conducted to count the number of mitotic figures in normal oral mucosa, oral epithelial dysplasia and squamous cell carcinoma in both Hematoxylin and Eosin (H&E) and Crystal Violet stained sections. Also the overall number of mitotic figures with both stains were compared along with the evaluation of staining efficacy of both the stains. The present study was conducted on 20 specimens each of the three categories. These were further divided into two groups for staining with H&E and with 1% Crystal Violet respectively. Images were captured and analyzed using image analysis software Dewinter Biowizard 4.1. Comparison of mitotic figure count in three categories in sections stained with both stains showed statistically significant difference ( p  < 0.001). The mean number of mitotic figures seen in Crystal Violet reagent were significantly higher as seen in H&E stain ( p  < 0.001). The overall diagnostic efficacy of Crystal Violet was 87.6%. Crystal Violet scored over H&E stain and also helped to better appreciate metaphases in Squamous cell carcinoma and telophases in dysplasia. Number of mitotic figures progressively increase with the advancement of the pathology. Use of 1% Crystal Violet provides better appreciation of mitotic figures and can be employed as a selective stain in routine histopathology.

Application of the thiocarbohydrazide method for vicinal glycol group detection to the study of gastric mucosa endocrine cells.

PubMed

Lefranc, G; Chung, Y T; Barrière, P; Pradal, G

1980-01-01

The thiocarbohydrazide-silver proteinate (TCH SP) method was applied to the study of cat, rabbit and mouse gastric mucosa endocrine cells. After 24-h treatment with thiocarbohydrazide (TCH), glycogen was seen in the hyaloplasm of X, D, P, A and O cells but not in EC, EC-like or D1 cells. With flotation times as short as 30 to 40 min glycogen was readily detected in X cells. Secretory granules of EC cells were constantly stained, while those of D1 cells failed to react. In most experiments granules of X, A and O cells showed peripheral "staining", while in others staining of variable intensity affected the entire granular cross-section in X, D and P cells. With 72-h exposure to TCH, EC and EC-like cells showed particles resembling glycogen, even staining or only peripheral staining of certain EC cell granules. From the results of this and previous studies, EC cell staining is believed to be due wholly or partly, according to exposure times, to the action of silver proteinate, while that of certain non-EC cells is probably a specific indicator of complexed carbohydrates.

Combined pitavastatin and dacarbazine treatment activates apoptosis and autophagy resulting in synergistic cytotoxicity in melanoma cells.

PubMed

Al-Qatati, Abeer; Aliwaini, Saeb

2017-12-01

Melanoma is an aggressive skin cancer and its incidence is increasing faster than any other type of cancer. Whilst dacarbazine (DTIC) is the standard chemotherapy for metastatic melanoma, it has limited success. Statins, including pitavastatin, have been demonstrated to have a range of anti-cancer effects in a number of human cancer cell lines. The present study therefore explored the anti-cancer activity of combined DTIC and pitavastatin in A375 and WM115 human melanoma cells. Cell survival assays demonstrated that combined DTIC and pitavastatin treatment resulted in synergistic cell death. Cell cycle analyses further revealed that this combined treatment resulted in a G1 cell cycle arrest, as well as a sub-G1 population, indicative of apoptosis. Activation of apoptosis was confirmed by Annexin V-fluorescein isothiocyanate/propidium iodide double-staining and an increase in the levels of active caspase 3 and cleaved poly (ADP-ribose) polymerase. Furthermore, it was demonstrated that apoptosis occurs through the intrinsic pathway, evident from the release of cytochrome c. Finally, combined DTIC and pitavastatin treatment was demonstrated to also activate autophagy as part of a cell death mechanism. The present study provides novel evidence to suggest that the combined treatment of DTIC and pitavastatin may be effective in the treatment of melanoma.

Combined pitavastatin and dacarbazine treatment activates apoptosis and autophagy resulting in synergistic cytotoxicity in melanoma cells

PubMed Central

Al-Qatati, Abeer; Aliwaini, Saeb

2017-01-01

Melanoma is an aggressive skin cancer and its incidence is increasing faster than any other type of cancer. Whilst dacarbazine (DTIC) is the standard chemotherapy for metastatic melanoma, it has limited success. Statins, including pitavastatin, have been demonstrated to have a range of anti-cancer effects in a number of human cancer cell lines. The present study therefore explored the anti-cancer activity of combined DTIC and pitavastatin in A375 and WM115 human melanoma cells. Cell survival assays demonstrated that combined DTIC and pitavastatin treatment resulted in synergistic cell death. Cell cycle analyses further revealed that this combined treatment resulted in a G1 cell cycle arrest, as well as a sub-G1 population, indicative of apoptosis. Activation of apoptosis was confirmed by Annexin V-fluorescein isothiocyanate/propidium iodide double-staining and an increase in the levels of active caspase 3 and cleaved poly (ADP-ribose) polymerase. Furthermore, it was demonstrated that apoptosis occurs through the intrinsic pathway, evident from the release of cytochrome c. Finally, combined DTIC and pitavastatin treatment was demonstrated to also activate autophagy as part of a cell death mechanism. The present study provides novel evidence to suggest that the combined treatment of DTIC and pitavastatin may be effective in the treatment of melanoma. PMID:29344241

6-Shogaol induces cell cycle arrest and apoptosis in human hepatoma cells through pleiotropic mechanisms.

PubMed

Wu, Jung-Ju; Omar, Hany A; Lee, Ying-Ray; Teng, Yen-Ni; Chen, Pin-Shern; Chen, Yu-Chung; Huang, Hsiao-Shan; Lee, Kuan-Han; Hung, Jui-Hsiang

2015-09-05

Shogaols are a group of the active constituents of ginger that have been identified to have various biological activities. The aim of the current study was to investigate the antitumor activity of 6-shogaol in hepatocellular carcinoma (HCC) and the possible involvement of reactive oxygen species as a putative mechanism of action. HCC cell lines, HepG2 and Huh-7, were used to study the in vitro anti-cancer activity of 6-shogaol via the application of various molecular biology techniques. Results showed that 6-shogaol effectively inhibited the cell viability, caused cell cycle arrest at G2/M phase and induced apoptosis in HCC cells as indicated by MTT assay, DAPI nuclear staining, annexin V assay, cell cycle analysis, and activation of caspase-3. Western blot analysis revealed the ability of 6-shogaol to target cancer survival signaling pathways mediated by mitogen-activated protein kinase (MAPK), 5' AMP-activated protein kinase (AMPK) and Akt. In addition, 6-Shogaol induced alteration of cyclin proteins expression and caused cleavage of protein kinase C delta. Furthermore, 6-Shogaol was able to induce the production of reactive oxygen species and endoplasmic reticulum (ER) stress-associated proteins and the consequent activation of autophagy in HepG2 cells. Taken together, the current study highlights evidences that 6-shogaol induces apoptosis, modulates cyclins expression and targets cancer survival signaling pathways in HCC cell lines, at least in part, via the production of reactive oxygen species. These findings support 6-shogaol's clinical promise as a potential candidate for HCC therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Induction of G1 and G2/M cell cycle arrests by the dietary compound 3,3'-diindolylmethane in HT-29 human colon cancer cells.

PubMed

Choi, Hyun Ju; Lim, Do Young; Park, Jung Han Yoon

2009-05-29

3,3'-Diindolylmethane (DIM), an indole derivative produced in the stomach after the consumption of broccoli and other cruciferous vegetables, has been demonstrated to exert anti-cancer effects in both in vivo and in vitro models. We have previously determined that DIM (0 - 30 micromol/L) inhibited the growth of HT-29 human colon cancer cells in a concentration-dependent fashion. In this study, we evaluated the effects of DIM on cell cycle progression in HT-29 cells. HT-29 cells were cultured with various concentrations of DIM (0 - 30 micromol/L) and the DNA was stained with propidium iodide, followed by flow cytometric analysis. [3H]Thymidine incorporation assays, Western blot analyses, immunoprecipitation and in vitro kinase assays for cyclin-dependent kinase (CDK) and cell division cycle (CDC)2 were conducted. The percentages of cells in the G1 and G2/M phases were dose-dependently increased and the percentages of cells in S phase were reduced within 12 h in DIM-treated cells. DIM also reduced DNA synthesis in a dose-dependent fashion. DIM markedly reduced CDK2 activity and the levels of phosphorylated retinoblastoma proteins (Rb) and E2F-1, and also increased the levels of hypophosphorylated Rb. DIM reduced the protein levels of cyclin A, D1, and CDK4. DIM also increased the protein levels of CDK inhibitors, p21CIP1/WAF1 and p27KIPI. In addition, DIM reduced the activity of CDC2 and the levels of CDC25C phosphatase and cyclin B1. Here, we have demonstrated that DIM induces G1 and G2/M phase cell cycle arrest in HT-29 cells, and this effect may be mediated by reduced CDK activity.

The Expression Pattern of the Cell Cycle Inhibitor p19INK4d by Progenitor Cells of the Rat Embryonic Telencephalon and Neonatal Anterior Subventricular Zone

PubMed Central

Coskun, Volkan; Luskin, Marla B.

2014-01-01

In this study we investigated whether the pattern of expression of the cyclin-dependent kinase inhibitor p19INK4d by the unique progenitor cells of the neonatal anterior subventricular zone (SVZa) can account for their ability to divide even though they express phenotypic characteristics of differentiated neurons. p19INK4d was chosen for analysis because it usually acts to block permanently the cell cycle at the G1 phase. p19INK4d immunoreactivity and the incorporation of bromodeoxyuridine (BrdU) by SVZa cells were compared with that of the more typical progenitor cells of the prenatal telencephalic ventricular zone. In the developing telencephalon, p19INK4d is expressed by postmitotic cells and has a characteristic perinuclear distribution depending on the laminar position and state of differentiation of a cell. Moreover, the laminar-specific staining of the developing cerebral cortex revealed that the ventricular zone (VZ) is divided into p19INK4d(+) and p19INK4d(−) sublaminae, indicating that the VZ has a previously unrecognized level of functional organization. Furthermore, the rostral migratory stream, traversed by the SVZa-derived cells, exhibits an anteriorhigh–posteriorlow gradient of p19INK4d expression. On the basis of the p19INK4d immunoreactivity and BrdU incorporation, SVZa-derived cells appear to exit and reenter the cell cycle successively. Thus, in contrast to telencephalic VZ cells, SVZa cells continue to undergo multiple rounds of division and differentiation before becoming postmitotic. PMID:11312294

Wee1 Kinase Inhibitor AZD1775 Radiosensitizes Hepatocellular Carcinoma Regardless of TP53 Mutational Status Through Induction of Replication Stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cuneo, Kyle C., E-mail: kcuneo@umich.edu; Morgan, Meredith A.; Davis, Mary A.

2016-06-01

Purpose: Wee1 kinase inhibitors are effective radiosensitizers in cells lacking a G{sub 1} checkpoint. In this study we examined the potential effect of Wee1 kinase inhibition on inducing replication stress in hepatocellular carcinoma (HCC). Methods and Materials: Five independent datasets from the Oncomine database comparing gene expression in HCC compared to normal tissue were combined and specific markers associated with Wee1 sensitivity were analyzed. We then performed a series of in vitro experiments to study the effect of Wee1 inhibition on irradiated HCC cell lines with varying p53 mutational status. Clonogenic survival assays and flow cytometry using anti-γH2AX and phospho-histone H3more » antibodies with propidium iodide were performed to study the effect of AZD1775 on survival, cell cycle, and DNA repair. Additionally, nucleoside enriched medium was used to examine the effect of altering nucleotide pools on Wee1 targeted radiation sensitization. Results: Our analysis of the Oncomine database found high levels of CDK1 and other cell cycle regulators indicative of Wee1 sensitivity in HCC. In our in vitro experiments, treatment with AZD1775 radiosensitized and chemosensitized Hep3B, Huh7, and HepG2 cell lines and was associated with delayed resolution of γH2AX foci and the induction of pan-nuclear γH2AX staining. Wee1 inhibition attenuated radiation-induced G{sub 2} arrest in the Hep3B (TP53 null) and Huh7 (TP53 mutant) cell lines but not in the TP53 wild-type cell line HepG2. Supplementation with nucleosides reversed the radiation-sensitizing effect of AZD1775 and reduced the amount of cells with pan-nuclear γH2AX staining after radiation. Conclusions: Radiation sensitization with Wee1 inhibition occurs in cells regardless of their p53 mutational status. In this study we show for the first time that replication stress via the overconsumption of nucleotides plays an important role in AZD1775-induced radiation sensitization.« less

Activin B promotes initiation and development of hair follicles in mice.

PubMed

Jia, Qin; Zhang, Min; Kong, Yanan; Chen, Shixuan; Chen, Yinghua; Wang, Xueer; Zhang, Lei; Lang, Weiya; Zhang, Lu; Zhang, Lin

2013-01-01

Activin B has been reported to promote the regeneration of hair follicles during wound healing. However, its role in the development and life cycle of hair follicles has not been elucidated. In our study, the effect of activin B on mouse hair follicles of cultured and neonatal mouse skin was investigated. In these models, PBS or activin B (5, 10 or 50 ng/ml) was applied, and hair follicle development was monitored. Hair follicle initiation and development was examined using hematoxylin and eosin staining, alkaline phosphatase activity staining, Oil Red O+ staining, and the detection of TdT-mediated dUTP-biotin nick end-labeling cell apoptosis. Activin B was found to efficiently induce the initiation of hair follicles in the skin of both cultured and neonatal mice and to promote the development of hair follicles in neonatal mouse skin. Moreover, activin-B-treated hair follicles were observed to enter the anagen stage from the telogen stage and to remain in the anagen stage. These results demonstrate that activin B promotes the initiation and development of hair follicles in mice.

Bacillus Anthracis Spores of the bclA Mutant Exhibit Increased Adherence to Epithelial Cells, Fibroblasts, and Endothelial Cells but not to Macrophages

DTIC Science & Technology

2007-09-01

immunofluorescence (IFM) and light microscopy. Samples were fixed in forma- lin, stained with immunofluorescent dyes (as described below) or spore stain ( malachite ...BEC. Adherence was assessed by microscopic observation of the infected cells stained with malachite green and counterstaining of the BEC. For enzymatic...this significant difference, BEC infected with spores were stained with malachite green and counter- stained with Wright-Giemsa (Fig. 1B and C). This

The intermediate filament network protein, vimentin, is required for parvoviral infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fay, Nikta; Panté, Nelly, E-mail: pante@zoology.ubc.ca

Intermediate filaments (IFs) have recently been shown to serve novel roles during infection by many viruses. Here we have begun to study the role of IFs during the early steps of infection by the parvovirus minute virus of mice (MVM). We found that during early infection with MVM, after endosomal escape, the vimentin IF network was considerably altered, yielding collapsed immunofluorescence staining near the nuclear periphery. Furthermore, we found that vimentin plays an important role in the life cycle of MVM. The number of cells, which successfully replicated MVM, was reduced in infected cells in which the vimentin network wasmore » genetically or pharmacologically modified; viral endocytosis, however, remained unaltered. Perinuclear accumulation of MVM-containing vesicles was reduced in cells lacking vimentin. Our data suggests that vimentin is required for the MVM life cycle, presenting possibly a dual role: (1) following MVM escape from endosomes and (2) during endosomal trafficking of MVM. - Highlights: • MVM infection changes the distribution of the vimentin network to perinuclear regions. • Disrupting the vimentin network with acrylamide decreases MVM replication. • MVM replication is significantly reduced in vimentin-null cells. • Distribution of MVM-containing vesicles is affected in MVM infected vimentin-null cells.« less

One-carbon cycle support rescues sperm damage in experimentally induced varicocoele in rats.

PubMed

Mohammadi, Parisa; Hassani-Bafrani, Hassan; Tavalaee, Marziyeh; Dattilo, Maurizio; Nasr-Esfahani, Mohammad H

2018-05-11

To investigate whether micronutrients in support of the one-carbon cycle and glutathione synthesis are effective in improving sperm damage after surgical varicocoele induction in rats and whether any effect is achieved without a rebound reductive stress as seen with oral antioxidants. Surgical varicocoele was induced in adult male Wistar rats and resulted in significant damage to the testis and sperm cells measured at 2 and 4 months after surgery. At 2 months after surgery, rats received a 2-month oral supplementation in support of the one-carbon cycle containing B vitamins (B2, B3, B6, folic acid and B12), N-acetyl-cysteine, zinc, small amounts of vitamin E, and a natural source of betalains and quercetine (Condensyl ® ; Parthenogen SAGL, Lugano, Switzerland and Nurilia SARL, Lyon, France). One-carbon cycle supplementation, compared to untreated controls, significantly improved the morphometric characteristics of testis (P < 0.05), sperm concentration, motility and abnormal morphology (P < 0.001), sperm chromatin condensation (aniline blue staining, P < 0.05), sperm DNA damage (acridine orange staining, P < 0.05) and sperm lipid peroxidation (BODIPY C11, P < 0.001). The improvement in both nuclear condensation and DNA damage and the lack of excessive inhibition of lipid peroxidation confirmed that no reductive stress had occurred. Micronutrients in support of the one-carbon cycle are effective in the treatment of surgically induced varicocoele in rats, probably by activating natural antioxidant defences and epigenetics. These results support the idea that essential micronutrients including B vitamins may also have a positive influence in clinical varicocoele, which should be tested in prospective clinical trials. © 2018 The Authors BJU International © 2018 BJU International Published by John Wiley & Sons Ltd.

Frequency of satellite association of human chromosomes is correlated with amount of Ag-staining of the nucleolus organizer region.

PubMed Central

Miller, D A; Tantravahi, R; Dev, V G; Miller, O J

1977-01-01

Methaphase chromosomes from karyotypically normal adult humans (three males, six females) and one male with a 13p - chromosome were stained by quinacrine and then by the Ag-AS silver staining method to reveal nucleolus organizer regions (NORs). Each person had a characteristic number of Ag-stained chromosomes per cell, always fewer than 10. Determination of the mean Ag-size of each chromosome showed that each of the 10 individuals had a unique distribution of Ag-stain. Within each individual, there was some variation from cell to cell in the number of acrocentric chromosomes that were Ag-stained; this was not random, and the same chromosomes (those that had at most a small amount of Ag-stain) tended to be unstained in every cell. Satellite associations were scored on the same cells. Chromosomes that had no Ag-stain were involved in satellite association less than 20% as often as those that had some Ag-stain. Chromosomes that had a small amount of Ag-stain were involved in association about 50% as often as those that had a large amount of stain. Regression analysis of the 50 (of a total of 100) acrocentric chromosomes which could be individually identified by quinacrine markers showed that the frequency with which a chromosome was involved in satellite association was strongly correlated with the amount of Ag-stained material in the NOR. Images Fig. 1 Fig. 3 PMID:70995

Beta sitosterol and Daucosterol (phytosterols identified in Grewia tiliaefolia) perturbs cell cycle and induces apoptotic cell death in A549 cells.

PubMed

Rajavel, Tamilselvam; Mohankumar, Ramar; Archunan, Govindaraju; Ruckmani, Kandasamy; Devi, Kasi Pandima

2017-06-13

Lung cancer is the leading cause of cancer related deaths both in developed and developing countries. Since majority of the existing therapeutic methods harms both normal and malignant cells, a viable alternative is to switch into safe and beneficial traditional medicinal plants. Hence the present study was framed to identify selective anti-lung cancer agents from the medicinal plant Grewia tiliaefolia (GT). Cell viability experiments showed that benzene extract of GT (BGT) leaf effectively inhibited the growth of A549 cells, while being non-toxic to normal human lung L132 and PBMC cells. Ames and comet assays demonstrated that BGT is of non-mutagenic and non-genotoxic nature in untransformed cells. The hematological and histopathological profiles of the in vivo acute and sub-acute toxicity studies demonstrated that BGT is safe and tolerable. Importantly, western blot analysis and Annexin V-FITC staining confirmed that BGT promotes mitochondrial dependent apoptotic cell death in A549 cells by arresting cell cycle at G2/M phase. Bio-assay guided fractionation revealed the presence of phytosteols (β-sitosterol and daucosterol) which significantly inhibited the growth of A549 cells both alone and in combination. This study warrants that these phytosterols in alone or in combination can be considered as safe and potential drug candidates for lung cancer treatment.

Curcumin enhances the radiosensitivity of renal cancer cells by suppressing NF-κB signaling pathway.

PubMed

Li, Gang; Wang, Ziming; Chong, Tie; Yang, Jie; Li, Hongliang; Chen, Haiwen

2017-10-01

The radiation resistance of renal cell carcinoma (RCC) remains the primary obstacle to improve patient survival. This study aimed to investigate the effects of curcumin on the radiosensitivity of RCC cells. Human RCC cell (ACHN) was exposed to irradiation (IR) and/or curcumin treatment. Cell viability, DNA repair, cell cycle, and apoptosis, were evaluated by MTT, immunofluoresence staining and flow cytometry. Moreover, ACHN cells were xenografted into nude mice and subjected to IR and/or curcumin treatment. The expression of NF-κB signaling related proteins in ACHN cells and xenografts was detected by western blot analysis. The results showed that curcumin significantly increased radiosensitivity of ACHN cells by inhibiting the cell proliferation and DNA damage repair, causing cell cycle arrest at G2/M phase, inducing apoptosis in vitro, and suppressing the growth of xenografts in vivo. In addition, curcumin enhanced radiosensitivity was through markedly inhibiting IR-induced NF-κB signaling by modulating the related protein expressions including NF-κBP65, I-κB, VEGF, COX2, and Bcl-2 in ACHN cells, which was further strengthened by NF-κB inhibitor PDTC treatment. Thus, curcumin may confer radiosensitivity on RCC via inhibition of NF-κB activation and its downstream regulars, suggesting the potential application of curcumin as an adjuvant in radiotherapy of RCC. Copyright © 2017. Published by Elsevier Masson SAS.

The anticancer effect of saffron in two p53 isogenic colorectal cancer cell lines

PubMed Central

2012-01-01

Background Saffron extract, a natural product, has been shown to induce apoptosis in several tumor cell lines. Nevertheless, the p53-dependency of saffron’s mechanism of action in colon cancer remains unexplored. Material and methods In order to examine saffron’s anti-proliferative and pro-apoptotic effects in colorectal cancer cells, we treated two p53 isogenic HCT116 cell lines (HCT wildtype and HCT p53−/−) with different doses of the drug and analyzed cell proliferation and apoptosis in a time-dependent manner. MTT viability and crystal violet assays were performed in order to determine the effective dose of saffron on both cell lines. The cell cycle progress was examined by Flow cytometric analysis. Apoptosis was assessed using Annexin-PI-staining and Western Blotting for caspase 3 and PARP cleavage. Autophagy was determined by Western Blotting of the light chain 3 (LC3)-II and Beclin 1 proteins. The protein content of phospho-H2AX (γH2AX), a sensor of DNA double strand breaks, was also analyzed by Western Blotting. Results Saffron extract induced a p53-dependent pattern of cell cycle distribution with a full G2/M stop in HCT116 p53 wildtype cells. However, it induced a remarkable delay in S/G2 phase transit with entry into mitosis in HCT116 p53 −/− cells. The apoptotic Pre-G1 cell fraction as well as Annexin V staining and caspase 3 cleavage showed a more pronounced apoptosis induction in HCT116 p53 wildtype cells. Obviously, the significantly higher DNA-damage, reflected by ɣH2AX protein levels in cells lacking p53, was coped by up-regulation of autophagy. The saffron-induced LC3-II protein level was a remarkable indication of the accumulation of autophagosomes, a response to the cellular stress condition of drug treatment. Conclusions This is the first study showing the effect of saffron in HCT116 colorectal cancer cells with different p53 status. Saffron induced DNA-damage and apoptosis in both cell lines. However, autophagy has delayed the induction of apoptosis in HCT116 p53 −/− cells. Considering the fact that most tumors show a functional p53 inactivation, further research is needed to elucidate the long-term effects of saffron in p53 −/− tumors. PMID:22640402

Different cytokeratin and neuronal cell adhesion molecule staining patterns in focal nodular hyperplasia and hepatic adenoma and their significance

PubMed Central

Iyer, Anita; Robert, Marie E.; Bifulco, Carlo B.; Salem, Ronald R.; Jain, Dhanpat

2013-01-01

Summary Differentiating focal nodular hyperplasia from hepatic adenoma can be challenging. Cytokeratin 7, neuronal cell adhesion molecule, and cytokeratin 19 are differentially expressed in hepatocytes, biliary epithelium, and possibly hepatic progenitor/stem cells. CD34 is known to have altered expression patterns in the hepatic endothelium in conditions associated with abnormal perfusion and in hepatocellular carcinoma. The purpose of this study was to examine the expression pattern of these markers in focal nodular hyperplasia and hepatic adenoma and assess their diagnostic use. Ten resection specimens each of hepatic adenoma and focal nodular hyperplasia (including a case of telangiectatic focal nodular hyperplasia) were selected for the study. Immunohistochemical analysis was performed using antibodies against cytokeratin 7, cytokeratin 19, neuronal cell adhesion molecule, and CD34 on formalin-fixed, paraffin-embedded sections from each case. The staining patterns and intensity for each marker were analyzed. In hepatic adenoma, the cytokeratin 7 stain revealed strong positivity in hepatocytes in patches, with a gradual decrease in the staining intensity as the cells differentiated towards mature hepatocytes. Although bile ducts were typically absent in hepatic adenoma, occasional ductules could be identified with cytokeratin 7 stain. In focal nodular hyperplasia, cytokeratin 7 showed strong staining of the biliary epithelium within the fibrous septa and staining of the peripheral hepatocytes of most lobules that was focal and weaker than hepatic adenoma. Cytokeratin 19 and neuronal cell adhesion molecule showed patchy and moderate staining in the biliary epithelium of the ductules in focal nodular hyperplasia. While in the hepatic adenoma, cytokeratin 19 showed only rare positivity in occasional cells within ductules, and neuronal cell adhesion molecule marked occasional isolated cells in the lesion. CD34 showed staining of sinusoids in the inflow areas (periportal areas) in both focal nodular hyperplasia and hepatic adenoma. One case of telangiectatic focal nodular hyperplasia revealed both hepatic adenoma–like and focal nodular hyperplasia–like staining patterns. Distinct cytokeratin 7, cytokeratin 19, and neuronal cell adhesion molecule staining patterns are seen in hepatic adenoma and focal nodular hyperplasia possibly suggest activation of different subsets of hepatic progenitor/stem cell and can be diagnostically useful. PMID:18602664

Antitumor effects with apoptotic death in human promyelocytic leukemia HL-60 cells and suppression of leukemia xenograft tumor growth by irinotecan HCl.

PubMed

Chen, Yung-Liang; Chueh, Fu-Shin; Yang, Jai-Sing; Hsueh, Shu-Ching; Lu, Chi-Cheng; Chiang, Jo-Hua; Lee, Ching-Sung; Lu, Hsu-Feng; Chung, Jing-Gung

2015-07-01

Irinotecan HCl (CPT-11) is an anticancer prodrug, but there is no available information addressing CPT-11-inhibited leukemia cells in in vitro and in vivo studies. Therefore, we investigated the cytotoxic effects of CPT-11 in promyelocytic leukemia HL-60 cells and in vivo and tumor growth in a leukemia xenograft model. Effects of CPT-11 on HL-60 cells were determined using flow cytometry, immunofluorescence staining, comet assay, real-time PCR, and Western blotting. CPT-11 demonstrated a dose- and time-dependent inhibition of cell growth, induction of apoptosis, and cell-cycle arrest at G0/G1 phase in HL-60 cells. CPT-11 promoted the release of AIF from mitochondria and its translocation to the nucleus. Bid, Bax, Apaf-1, caspase-9, AIF, Endo G, caspase-12, ATF-6b, Grp78, CDK2, Chk2, and cyclin D were all significantly upregulated and Bcl-2 was down-regulated by CPT-11 in HL-60 cells. Induction of cell-cycle arrest by CPT-11 was associated with changes in expression of key cell-cycle regulators such as CDK2, Chk2, and cyclin D in HL-60 cells. To test whether CPT-11 could augment antitumor activity in vivo, athymic BALB/c(nu/nu) nude mice were inoculated with HL-60 cells, followed by treatment with either CPT-11. The treatments significantly inhibited tumor growth and reduced tumor weight and volume in the HL-60 xenograft mice. The present study demonstrates the schedule-dependent antileukemia effect of CPT-11 using both in vitro and in vivo models. CPT-11 could potentially be a promising agent for the treatment of promyelocytic leukemia and requires further investigation. © 2014 Wiley Periodicals, Inc.

Inhibition of NF-kappaB-mediated transcription and induction of apoptosis in human breast cancer cells by epoxypseudoisoeugenol-2-methyl butyrate.

PubMed

Ma, Guoyi; Tabanca, Nurhayat; Husnu Can Baser, K; Kirimer, Nese; Pasco, David S; Khan, Ikhlas A; Khan, Shabana I

2009-03-01

Breast cancer is one of the most prevalent woman cancers. Genomic instability, accumulative mutations, and subsequent changes in intracellular signaling cascades play key roles in the development of human breast cancers. Activation of nuclear factor-kappaB (NF-kappaB) has been implicated in oncogenesis of breast cancers and is known to be associated with resistance to anticancer agents and apoptosis. Blocking NF-kappaB signaling may represent a therapeutic strategy in breast cancer therapy. The objective of this study is to investigate the in vitro effects of epoxypseudoisoeugenol-2-methyl butyrate (EPB), a phenylpropranoid isolated from Pimpinella corymbosa, on the activation of NF-kappaB, cell growth, cell cycle progression and apoptosis in MCF-7 (estrogen-dependent) and BT-549 (estrogen-independent) breast cancer cells. Transcriptional activity of NF-kappaB was measured by cell based reporter gene assay. Cell proliferation was determined by MTT assay. Cell cycle analysis was carried out by flow cytometry and apoptosis was observed by DAPI staining assy. EPB inhibited the NF-kappaB-mediated transcription activity induced by tumor necrosis factor-alpha (TNF-alpha) and phorbol myristate acetate (PMA) in MCF-7 cells. EPB also inhibited constitutive NF-kappaB transcriptional activity in BT-549 cells. EPB inhibited the proliferation of both MCF-7 and BT-549 cells in a concentration- and time-dependent manner. EPB induced cell cycle arrest in G(1)/G(0) phase and apoptosis in both MCF-7 and BT 549 cells. These in vitro results indicated that EPB has a potential for use against both hormone-dependent and hormone-independent breast cancers and its effects seem to be mediated by inhibiting the NF-kappaB activity.

The effect of leukocyte interleukin injection (Multikine) treatment on the peritumoral and intratumoral subpopulation of mononuclear cells and on tumor epithelia: a possible new approach to augmenting sensitivity to radiation therapy and chemotherapy in oral cancer--a multicenter phase I/II clinical Trial.

PubMed

Tímár, József; Forster-Horváth, C; Lukits, J; Döme, B; Ladányi, A; Remenár, E; Kásler, M; Bencsik, M; Répássy, G; Szabó, G; Velich, N; Suba, Z; Elõ, J; Balatoni, Z; Bajtai, A; Chretien, P; Talor, Eyal

2003-12-01

The main objective of this study was to investigate the effect of the administration of a novel immunoadjuvant, leukocyte interleukin injection, as part of an immuno-augmenting treatment regimen on the peritumoral and intratumoral subpopulations of the tumor infiltrating mononuclear cells and on the epithelial and stromal components, when administered to patients with advanced primary oral squamous cell carcinoma classified as T2-3N0-2M0, as compared with disease-matched control patients (not treated with leukocyte interleukin injection). Multicenter Phase I/II clinical trial. Fifty-four patients from four clinical centers were included in the dose-escalating study (27 in each group [leukocyte interleukin injection-treated and control groups]). Cumulative leukocyte inter-leukin injection doses were 2400, 4800, and 8000 IU (as interleukin-2 equivalent). Paraffin-embedded tumor samples obtained at surgical resection of the residual tumor (between days 21 and 28 after treatment initiation) were used. Histological analysis, necrosis evaluation, and American Joint Committee on Cancer grading were performed from H&E-stained sections. Immunohistochemical analysis was performed on three different tumor regions (surface, zone 1; center, zone 2; and tumor-stroma interface, zone 3). Trichrome staining was used to evaluate connective tissue, and morphometric measurements were made using ImagePro analysis software. Cell cycling was determined by the use of Ki-67 marker. Leukocyte interleukin injection treatment induced a shift from stromal infiltrating T cells toward intraepithelial T cells and posted a significant (P <.05) increase in intraepithelial CD3-positive T cells independent of the leukocyte interleukin injection dose, whereas the increase in CD25 (interleukin-2 receptor alpha [IL-2Ralpha])-positive lymphoid cells was significant only at the lowest leukocyte interleukin injection dose (P <.05). Furthermore, both low- and medium-dose leukocyte interleukin injection treatment induced a significant (P <.05) increase in the number of cycling tumor cells, as compared with control values. The results could be highly beneficial for patients with oral squamous cell carcinoma. First, leukocyte interleukin injection treatment induces T-cell migration into cancer nests and, second, noncycling cancer cells may enter cell cycling on administration of leukocyte interleukin injection. This latter effect may modulate the susceptibility of cancer cells to radiation therapy and chemotherapy. The findings may indicate a need to re-evaluate the way in which follow-up treatment (with radiation therapy and chemotherapy) of patients with head and neck cancer is currently approached.

Minoxidil activates β-catenin pathway in human dermal papilla cells: a possible explanation for its anagen prolongation effect.

PubMed

Kwack, Mi Hee; Kang, Bo Mi; Kim, Moon Kyu; Kim, Jung Chul; Sung, Young Kwan

2011-06-01

It is believed that the length of the actively growing phase of the anagen hair cycle mainly contributes to hair length. Recent studies showed that maintenance of β-catenin activity in the dermal papilla cells (DPCs) enables hair follicles to keep actively growing. Topical minoxidil treatment promotes hair growth in men with androgenetic alopecia, suggesting that minoxidil may prolong the actively growing phase of the anagen hair cycle. To investigate whether minoxidil prolongs the anagen hair cycle in mice and, if so, to investigate whether minoxidil activates β-catenin pathway in human DPCs. Dorsal skins of C57BL/6 mice were depilated to synchronize the hair cycle. After 10 days, 3% minoxidil were topically applied daily for 10 days. Sections of back skins were stained with hematoxylin and eosin. Hair follicles were graded and hair cycle score (HCS) was calculated. Cultured human DPCs were transiently transfected with the β-catenin responsive TCF reporter plasmid (pTopflash) and corresponding negative control reporter (pFopflash) to assess the activity of β-catenin signaling by minoxidil. Immunofluorescence staining and immunoblot were performed to examine the expression and localization of β-catenin in the presence or absence of minoxidil. Phosphorylation of GSK3β, PKA and PKB were also examined by immunoblot after minoxidil treatment. RT-PCR analysis and immunoblot were employed to investigate the expression of β-catenin pathway targets in DPCs, such as Axin2, Lef-1, and EP2. Modest extension of anagen phase thereby delay of catagen progression was observed by application of minoxidil in mice. Minoxidil stimulated the transcriptional activity of pTopflash but not pFopflash. Nuclear accumulation of β-catenin was also observed after minoxidil treatment. Immunoblot further showed that minoxidil treatment increases the phosphorylation of GSK3β, PKA and PKB. Moreover, minoxidil induced Axin2, Lef-1, and EP2 expression. Our results strongly suggest that minoxidil extends the anagen phase by activating β-catenin activity in the DPCs. Copyright © 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Viscum articulatum Burm. f. aqueous extract exerts antiproliferative effect and induces cell cycle arrest and apoptosis in leukemia cells.

PubMed

Mishra, Ruchi; Sharma, Saurabh; Sharma, Radhey Shyam; Singh, Savita; Sardesai, Milind Madhav; Sharma, Sadhna; Mishra, Vandana

2018-06-12

Viscum articulatum Burm. f. (leafless mistletoe) has been used in traditional system of medicines in India, China, Taiwan, Cambodia, Laos, and Vietnam, to treat blood-related diseases and various inflammatory and degenerative diseases including cancer. Anticancer activities of some phytomolecules purified from Viscum articulatum Burm. f. have been tested. However scientific evidence for the anticancerous potential of aqueous extract of V. articularum (VAQE) used in traditional medicine is lacking. To study the antiproliferative and apoptotic effect of VAQE on Jurkat E6.1 and THP1 leukemia cells. The aqueous extract of the whole plant of Viscum articulatum Burm. f. was prepared in phosphate buffer saline. In VAQE, total soluble protein was estimated using Bradford's dye-binding assay; flavonoid content was determined using aluminum chloride colorimetric assay; and phenolic content was estimated following Folin-Ciocalteu colorimetric assay. XTT cell viability assay was used to test VAQE induced cytotoxicity in Jurkat E6.1 and THP1 leukemia cells and peripheral blood mononuclear cells (PBMC). The effect of VAQE on cell cycle progression was analyzed by PI staining using flow cytometry. Annexin-V-FITC/PI differential staining method was used for detecting the onset of apoptosis in leukemia cells. Rhodamine 123 dye was used to detect the change in mitochondrial membrane potential (MMP) using flow cytometry. DCF-DA fluorescence dye was used to estimate the level of reactive oxygen species (ROS). The ROS inhibitors were used to evaluate the role of ROS in mediating DNA degradation in VAQE-treated leukemia cells. The molecular mechanisms underlying VAQE induced apoptosis induction was studied by analyzing the expression of anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) proteins, caspase-8 and caspase-3 enzymes using western blot. Diphenylamine (DPA) assay was used to determine the DNA fragmentation and conclusion of apoptosis. VAQE triggered cytotoxic effect on Jurkat E6.1 (IC 50 -2.4 µg/ml; 24 h) and THP1 (IC 50 -1.0 µg/ml; 24 h) cells in a dose- and time-dependent manner. The apoptosis induction and G2/M arrest of the cell cycle are the cause of VAQE-induced cytotoxicity in leukemia cells. The apoptosis in VAQE-treated Jurkat E6.1 and THP1 cells was mediated via a reduction in MMP, elevation of intracellular ROS, decreased expression of the anti-apoptotic (Bcl-2) and increased expression of the pro-apoptotic (Bax) protein, activation of caspase-8 and caspase-3 and DNA fragmentation. VAQE has a high efficacy to exert a cytotoxic effect in Jurkat E6.1 and THP1 cells and to induce apoptosis and G2/M cell cycle arrest. VAQE induces extrinsic pathway of apoptosis in both the leukemia cell lines via disruption of MMP, intracellular ROS imbalance, increased ratio of Bax/Bcl-2, activation of caspase-8, caspase-3 and ROS-mediated DNA fragmentation. The knowledge gained from the outcomes of the study may encourage the identification of novel chemotherapeutic agent from Viscum articulatum Burm. f. to treat leukemia. Copyright © 2018 Elsevier B.V. All rights reserved.

Molecular assessment, characterization, and differentiation of theca stem cells imply the presence of mesenchymal and pluripotent stem cells in sheep ovarian theca layer.

PubMed

Adib, Samane; Valojerdi, Mojtaba Rezazadeh

2017-10-01

The ability of ovarian theca stem cells to differentiate into oocyte and theca cells may lead to a major advancement in reproductive biology and infertility treatments. However, there is little information about function, growth and differentiation potential of these immature cells. In this study adult sheep theca stem cells (TSCs) characteristics, and differentiation potential into osteocyte-like cells (OSLCs), adipocyte-like cells (ALCs), theca progenitor-like cells (TPCs), and oocyte-like cells (OLCs) were investigated. TSCs were isolated, cultured, and compared with mesenchymal stem cells (MSCs), fibroblast cells (FCs), and pluripotent embryonic ovarian cells (EO). Adherent TSCs were morphologically similar to FCs. Cell cycle analysis showed high proliferation capacity of TSCs. TSCs were positive for the mesenchymal cells surface markers, and also expressed POU5F1. Differentiation potential of TSCs into OSLCs and ALCs were confirmed by alizarin red and oil red staining respectively. OSTEOCALCIN and COL1 were expressed in OSLCs. ALCs were positive for PPARα and LPL. TPCs expressed theca specific genes (GLI2, GLI3, PTCH1, CYP17A1, 3β-HSD and LHR) and secreted testosterone, dehydroepiandrostenedione (DHEA), androstenedione, progesterone and estradiol. Lipid droplets in these steroid cells were viewed by oil red staining. OLCs expressed oocyte-specific marker genes including, ZP3, ZP2, GDF9, SYCP3, PRDM1, STELLA, FRAGILIS, DAZL, as well as POU5F1, and showed separated sphere structure. Our results indicated that TSCs derived from ovarian follicles contain MSCs and pluripotent stem cells (PSCs) that can be differentiated into lineages of mesenchymal origin and are capable of differentiation into TPCs and OLCs under in vitro conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Metabolism in the Uncultivated Giant Sulfide-Oxidizing Bacterium Thiomargarita Namibiensis Assayed Using a Redox-Sensitive Dye

NASA Astrophysics Data System (ADS)

Bailey, J.; Flood, B.; Ricci, E.

2014-12-01

The colorless sulfur bacteria are non-photosynthetic chemolithotrophs that live at interfaces between nitrate, or oxygen, and hydrogen sulfide. In sulfidic settings such as cold seeps and oxygen minimum zones, these bacteria are thought to constitute a critical node in the geochemical cycling of carbon, sulfur, nitrogen, and phosphorous. Many of these bacteria remain uncultivated and their metabolisms and physiologies are incompletely understood. Thiomargarita namibiensis is the largest of these sulfur bacteria, with individual cells reaching millimetric diameters. Despite the current inability to maintain a Thiomargarita culture in the lab, their large size allows for individual cells to be followed in time course experiments. Here we report on the novel use of a tetrazolium-based dye that measures the flux of NADH production from catabolic pathways via a colorimetric response. Staining with this dye allows for metabolism to be detected, even in the absence of observable cell division. When coupled to microscopy, this approach also allows for metabolism in Thiomargaritato be differentiated from that of epibionts or contaminants in xenic samples. The results of our tetrazolium dye-based assay suggests that Thiomargarita is the most metabolically versatile under anoxic conditions where it appears capable of using acetate, succinate, formate, thiosulfate, citrate, thiotaurine, hydrogen sulfide, and perhaps hydrogen as electron donors. Under hypoxic conditions, staining results suggest the utilization of acetate, citrate, and hydrogen sulfide. Cells incubated under oxic conditions showed the weakest tetrazolium staining response, and then only to hydrogen sulfide and questionably succinate. These initial results using a redox sensitive dye suggest that Thiomargarita is most metabolically versatile under anaerobic and hypoxic conditions. The results of this assay can be further evaluated using molecular approaches such as transcriptomics, as well as provide cultivation strategies.

Intrafibrillar-silicified collagen scaffolds enhance the osteogenic capacity of human dental pulp stem cells.

PubMed

Niu, Li-na; Sun, Jia-qi; Li, Qi-hong; Jiao, Kai; Shen, Li-juan; Wu, Dan; Tay, Franklin; Chen, Ji-hua

2014-07-01

The present study investigated the effects of intrafibrillar-silicified collagen scaffolds (ISCS) on the osteogenic differentiation of human dental pulp stem cells (hDPSCs) in vitro and in vivo. The hDPSCs were co-cultured with ISCS or nonsilicified collagen scaffolds (NCS) in control medium (CM) or osteogenic differentiation medium (ODM). Cell cycle and cell apoptosis were analyzed with flow cytometry to measure the viability of hDPSCs. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to evaluate the expression levels of osteogenic marker genes and proteins of hDPSCs. Alkaline phosphatase (ALP) staining and alizarin red S assay were used to evaluate the ALP activity of hDPSCs and their calcium deposition potential. In addition, hDPSCs and scaffolds were implanted subcutaneously in nude mice for 8 weeks. Harvested tissues were immunohistochemically stained for osteocalcin (OCN) expression from hDPSCs, and stained with alizarin red S for examination of their calcium deposition in vivo. The ISCS had no adverse effect on hDPSCs, promoted their proliferation, and significantly up-regulated the expression of osteogenesis-related genes and proteins. The hDPSCs co-cultured with ISCS in ODM exhibited the highest ALP activity and calcium deposition in vitro. The ISCS promoted the OCN expression and calcium deposition of hDPSCs after ectopic transplantation in vivo. Intrafibrillar-silicified collagen scaffolds significantly promoted the proliferation, osteogenic differentiation and mineralization of hDPSCs, when compared with NCS. This study demonstrates combining the use of hDPSCs and ISCS to promote bone-like tissue formation is a promising approach for clinical bone repair and regeneration. Copyright © 2014 Elsevier Ltd. All rights reserved.

Propidium iodide (PI) stains Nissl bodies and may serve as a quick marker for total neuronal cell count.

PubMed

Niu, Junfei; Li, Chunman; Wu, Haihui; Feng, Xianling; Su, Qingning; Li, Shihe; Zhang, Lihong; Yew, David Tai Wai; Cho, Eric Yu Pang; Sha, Ou

2015-03-01

Propidium iodide (PI) reacts with both DNA and RNA and is a commonly used fluorescent reagent for nucleic acid staining. The aim of the study was to compare the cellular staining patterns of PI with that of Nissl staining in rat nervous tissues and to report a modified staining method that selectively labels Nissl bodies in neurons. Cryosections and paraffin sections of different tissues of normal Sprague-Dawley rats, including trigeminal ganglia, dorsal root ganglia, spinal cord, liver, and small intestine, were stained by either PI or the hematoxylin and eosin method. Some sections were treated with RNase or DNase before the above staining, and some were double stained with PI and a Nissl stain. The sections were observed by light, fluorescence or confocal microscopy. Results showed strong PI signals detected as patterns of granules in the neuronal cytoplasm of all nervous tissues, whereas the staining of neuronal nuclei was weaker. In contrast, nuclei of neuroglial cells were strongly stained by PI, while the cytoplasm was not obviously stained. Pretreatment of the neural tissue with RNase abolished the PI signals. Furthermore, the PI positive granules in neuronal cytoplasm co-localized with Nissl bodies stained by the fluorescent Nissl stain. When the tissue was pretreated with DNase, PI only stained the cytoplasmic granules of neurons, but not that of glial cells. Our results show that PI stains Nissl bodies and may serve as an economical and convenient neuron marker for neuronal cell counting when specific neural markers such as antibodies are not readily available. Copyright © 2015. Published by Elsevier GmbH.

Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

PubMed Central

Macurek, Libor; Benada, Jan; Müllers, Erik; Halim, Vincentius A.; Krejčíková, Kateřina; Burdová, Kamila; Pecháčková, Sona; Hodný, Zdeněk; Lindqvist, Arne; Medema, René H.; Bartek, Jiri

2013-01-01

Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G1 phase to G2 and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G1 cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression. PMID:23255129

BTK suppresses myeloma cellular senescence through activating AKT/P27/Rb signaling.

PubMed

Gu, Chunyan; Peng, Hailin; Lu, Yue; Yang, Hongbao; Tian, Zhidan; Yin, Gang; Zhang, Wen; Lu, Sicheng; Zhang, Yi; Yang, Ye

2017-08-22

We previously explored the role of BTK in maintaining multiple myeloma stem cells (MMSCs) self-renewal and drug-resistance. Here we investigated the elevation of BTK suppressing MM cellular senescence, a state of irreversible cellular growth arrest. We firstly discovered that an increased expression of BTK in MM samples compared to normal controls by immunohistochemistry (IHC), and significant chromosomal gain in primary samples. In addition, BTK high-expressing MM patients are associated with poor outcome in both Total Therapy 2 (TT2) and TT3 cohorts. Knockdown BTK expression by shRNA induced MM cellular senescence using β-galactosidase (SA-b-gal) staining, cell growth arrest by cell cycle staining and decreased clonogenicity while forcing BTK expression in MM cells abrogated these characteristics. We also validated this feature in mouse embryonic fibroblast cells (MEFs), which showed that elevated BTK expression was resistant to MEF senescence after serial cultivation in vitro . Further mechanism study revealed that BTK activated AKT signaling leading to down-regulation of P27 expression and hindered RB activity while AKT inhibitor, LY294002, overcame BTK-overexpression induced cellular senescence resistance. Eventually we demonstrated that BTK inhibitor, CGI-1746, induced MM cellular senescence, colony reduction and tumorigenecity inhibition in vivo . Summarily, we designate a novel mechanism of BTK in mediating MM growth, and BTK inhibitor is of great potential in vivo and in vitro suggesting BTK is a promising therapeutic target for MM.

Genetic podocyte lineage reveals progressive podocytopenia with parietal cell hyperplasia in a murine model of cellular/collapsing focal segmental glomerulosclerosis.

PubMed

Suzuki, Taisei; Matsusaka, Taiji; Nakayama, Makiko; Asano, Takako; Watanabe, Teruo; Ichikawa, Iekuni; Nagata, Michio

2009-05-01

Focal segmental glomerulosclerosis (FSGS) is a progressive renal disease, and the glomerular visceral cell hyperplasia typically observed in cellular/collapsing FSGS is an important pathological factor in disease progression. However, the cellular features that promote FSGS currently remain obscure. To determine both the origin and phenotypic alterations in hyperplastic cells in cellular/collapsing FSGS, the present study used a previously described FSGS model in p21-deficient mice with visceral cell hyperplasia and identified the podocyte lineage by genetic tagging. The p21-deficient mice with nephropathy showed significantly higher urinary protein levels, extracapillary hyperplastic indices on day 5, and glomerular sclerosis indices on day 14 than wild-type controls. X-gal staining and immunohistochemistry for podocyte and parietal epithelial cell (PEC) markers revealed progressive podocytopenia with capillary collapse accompanied by PEC hyperplasia leading to FSGS. In our investigation, non-tagged cells expressed neither WT1 nor nestin. Ki-67, a proliferation marker, was rarely associated with podocytes but was expressed at high levels in PECs. Both terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and electron microscopy failed to show evidence of significant podocyte apoptosis on days 5 and 14. These findings suggest that extensive podocyte loss and simultaneous PEC hyperplasia is an actual pathology that may contribute to the progression of cellular/collapsing FSGS in this mouse model. Additionally, this is the first study to demonstrate the regulatory role of p21 in the PEC cell cycle.

Primary Ewing's sarcoma of vulva: a case report and a review of the literature.

PubMed

Che, Shao-Min; Cao, Pei-Long; Chen, Hong-Wei; Liu, Zi; Meng, Du

2013-03-01

Ewing sarcomas/peripheral primitive neuroectodermal tumors (ES/pPNET) are extremely rare in the vulva. A review of the literature reveals only 14 previously reported possible cases. Here we reported a case of primary extraskeletal Ewing's sarcoma (EES) of the vulva in a 37-year-old woman. Characteristic histologic features of ES/pPNET were present in this case, including a monomorphic population of small round blue cells with cytoplasmic glycogen confirmed by periodic acid-Schiff, membrane staining with CD99 and nuclear staining with FLI-1. After surgery, the patient was found to have pulmonary metastasis and then received six cycles of polychemotherapy. She is still alive with stable disease after 1 year of follow up. Our findings underline the crucial role of immunohistochemical techniques in the differential diagnosis of small round cell tumors in these unusual locations. We also give a summary about the clinical and pathological features of the primary ES/pPNET in the vulva reported previously in the literature. © 2012 The Authors. Journal of Obstetrics and Gynaecology Research © 2012 Japan Society of Obstetrics and Gynecology.

Calcium: A novel and efficient inducer of differentiation of adipose-derived stem cells into neuron-like cells.

PubMed

Goudarzi, Farjam; Tayebinia, Heidar; Karimi, Jamshid; Habibitabar, Elahe; Khodadadi, Iraj

2018-06-05

This study comparatively investigated the effectiveness of calcium and other well-known inducers such as isobutylmethylxanthine (IBMX) and insulin in differentiating human adipose-derived stem cells (ADSCs) into neuronal-like cells. ADSCs were immunophenotyped and differentiated into neuron-like cells with different combinations of calcium, IBMX, and insulin. Calcium mobilization across the membrane was determined. Differentiated cells were characterized by cell cycle profiling, staining of Nissl bodies, detecting the gene expression level of markers such as neuronal nuclear antigen (NeuN), microtubule associated protein 2 (MAP2), neuron-specific enolase (NSE), doublecortin, synapsin I, glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) by quantitative real-time polymerase chain reaction (quantitative real-time polymerase chain reaction (qRT-PCR) and protein level by the immunofluorescence technique. Treatment with Ca + IBMX + Ins induced neuronal appearance and projection of neurite-like processes in the cells, accompanied with inhibition of proliferation and halt in the cell cycle. A significantly higher expression of MBP, GFAP, NeuN, NSE, synapsin 1, doublecortin, and MAP2 was detected in differentiated cells, confirming the advantages of Ca + IBMX + Ins to the other combinations of inducers. Here, we showed an efficient protocol for neuronal differentiation of ADSCs, and calcium fostered differentiation by augmenting the number of neuron-like cells and instantaneous increase in the expression of neuronal markers. © 2018 Wiley Periodicals, Inc.

Anethum graveolens (dill) - A medicinal herb induces apoptosis and cell cycle arrest in HepG2 cell line.

PubMed

Mohammed, Furkhan Ahmed; Elkady, Ayman I; Syed, Fareeduddin Quadri; Mirza, Muqtadir Baig; Hakeem, Khalid Rehman; Alkarim, Saleh

2018-06-12

The medicinal herb, Anethum graveolens L. (dill) is one of the potent culinary herbs used as an alternative form of medicine worldwide. The unguent topical Oil from the aerial parts of A. graveolens was found to be effective in the management of uterus cancer in ethnomedicine has been reported. The incidence and mortality rates of Hepatocellular carcinoma (HCC) are steadily rising worldwide, especially, in underdeveloped and developing countries. Moreover, HCC develops rapidly in patients with chronic cirrhosis or hepatitis, where the solid tumours/malignancies coexist with the inflammation. Recent studies have shown that the medicinal herb, Anethum graveolens, holds anticancer potential, which could be a promising approach for the treatment of various tumours. In the current study, we have analysed the antiproliferative effect of ethyl acetate fraction of Dill Seeds (EAFD) on HepG2 cell line. Cell viability and proliferation were observed by MTT assay; Morphological changes were studied using fluorescent stains like Hoechst 33342, acridine orange/ethidium bromide and JC-1 dye. Further, the pro-apoptotic activity was demonstrated through Annexin-V-FITC/ PI assay and cell cycle analysis. Different concentrations (0.1, 0.2, 0.4, 0.6, 0.8 mg/ml) of EAFD were studied. EAFD markedly suppressed the proliferation of HepG2 cells in a dose and time-dependent manner. The phase contrast and fluorescence microscopy revealed the morphological alterations like disruption, shrinkage, detachment and blebbing of cell membrane accompanied by nuclear condensation after exposure to EAFD. Radical scavenging activity was evidenced by measurement of ROS levels post-treatment. Modulation of mitochondrial membrane potential was exhibited leading to the activation of caspases 3/7 and 9 which is a committed step towards apoptosis. Annexin V-FITC/ PI assay and cell cycle, later confirmed the apoptosis and cell cycle arrest in 'G2/M' phase through flow cytometric analysis. In conclusion, a significant apoptogenic effect was exhibited by EAFD against HepG2 cells in inducing apoptosis and cell cycle arrest. Our findings indicate that the medicinal herb- Anethum graveolens, holds potential in treating hepatocellular carcinoma effectively. Copyright © 2018 Elsevier B.V. All rights reserved.

Proliferation of Peripheral Blood Lymphocytes and Mesenchymal Stromal Cells Derived from Wharton's Jelly in Mixed and Membrane-Separated Cultures.

PubMed

Poltavtsev, A M; Poltavtseva, R A; Yushina, M N; Pavlovich, S V; Svirshchevskaya, E V

2017-08-01

We studied the effect of mesenchymal stromal cells on proliferation of CFSE-stained T cells in mixed and membrane-separated (Transwell) cultures and in 3D culture of mesenchymal stromal cells from Wharton's jelly. The interaction of mesenchymal stromal cells with mitogen-activated peripheral blood lymphocytes from an allogeneic donor was followed by suppression of T-cell proliferation in a wide range of cell proportions. Culturing in the Transwell system showed the absence of suppression assessed by the fraction of proliferating cells and by the cell cycle analysis. In 3D cultures, contact interaction of mesenchymal stromal cells and lymphocytes was demonstrated that led to accumulation of G2/M phase lymphocytes and G0/G1 phase mesenchymal stromal cells. The suppressive effect of mesenchymal stromal cells from Wharton's jelly is mediated by two mechanisms. The effects are realized within 6 days, which suggests that the therapeutic effects of mesenchymal stromal cells persist until their complete elimination from the body.

[Morphology, biology and life-cycle of Plasmodium parasites].

PubMed

Hommel, Marcel

2007-10-01

Laveran first discovered that an infectious agent was responsible for malaria by using a simple microscope, without the assistance of specific stains. Our knowledge of the Plasmodium life cycle and cellular biology has progressed with each technological advance, from Romanovsky staining and histology to electron microscopy, immunocytochemistry, molecular methods and modern imaging techniques. The use of bird, primate and rodent models also made a major contribution, notably in the development of antimalarial drugs that are still in use today.

Targeting a Newly Established Spontaneous Feline Fibrosarcoma Cell Line by Gene Transfer

PubMed Central

Nande, Rounak; De Carlo, Flavia; Carper, Miranda; Claudio, Charlene D.; Denvir, Jim; Valluri, Jagan; Duncan, Gary C.; Claudio, Pier Paolo

2012-01-01

Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas. PMID:22666387

Targeting a newly established spontaneous feline fibrosarcoma cell line by gene transfer.

PubMed

Nande, Rounak; Di Benedetto, Altomare; Aimola, Pierpaolo; De Carlo, Flavia; Carper, Miranda; Claudio, Charlene D; Denvir, Jim; Valluri, Jagan; Duncan, Gary C; Claudio, Pier Paolo

2012-01-01

Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas.

Platinum blue staining of cells grown in electrospun scaffolds.

PubMed

Yusuf, Mohammed; Millas, Ana Luiza G; Estandarte, Ana Katrina C; Bhella, Gurdeep K; McKean, Robert; Bittencourt, Edison; Robinson, Ian K

2014-01-01

Fibroblast cells grown in electrospun polymer scaffolds were stained with platinum blue, a heavy metal stain, and imaged using scanning electron microscopy. Good contrast on the cells was achieved compared with samples that were gold sputter coated. The cell morphology could be clearly observed, and the cells could be distinguished from the scaffold fibers. Here we optimized the required concentration of platinum blue for imaging cells grown in scaffolds and show that a higher concentration causes platinum aggregation. Overall, platinum blue is a useful stain for imaging cells because of its enhanced contrast using scanning electron microscopy (SEM). In the future it would be useful to investigate cell growth and morphology using three-dimensional imaging methods.

Alternate pathogenesis of systemic neoplasia in the bivalve mollusc Mytilus.

PubMed

Moore, J D; Elston, R A; Drum, A S; Wilkinson, M T

1991-09-01

The proliferative disease systemic neoplasia, also termed hemic neoplasia or disseminated sarcoma, was studied in four Puget Sound, Washington populations of the bay mussel (Mytilus sp.). Using flow cytometric measurement of DAPI-stained cells withdrawn from the hemolymph, DNA content frequency histograms were generated for 73 individuals affected by the disease. The cells manifesting systemic neoplasia were found to exist as either of two separate types, characterized by G0G1 phase nuclear DNA contents of either approximately 4.9 x haploid (pentaploid form) or approximately 3.8 x haploid (tetraploid form). The two disease forms were found to coexist in all four mussel populations sampled, with overall relative prevalences of 66% pentaploid form, 29% tetraploid form, and 5% exhibiting both disease forms simultaneously. These findings represent the first unequivocal demonstration of multiple cell types in a bivalve neoplasia. The two forms appear to represent separate pathogenetic processes rather than sequential stages of a single pathogenesis. Two cell cycling parameters associated with proliferative activity were employed to compare the alternate forms: (i) the percentage of cells assigned to the DNA Synthesis (S) phase of the neoplastic cell cycle, and (ii) the proportion of neoplastic cell mitotic figures in hemocytological preparations. Mean values for both parameters were significantly higher for mussels with the tetraploid form of the disease, suggesting a higher rate of proliferation relative to the pentaploid form. Qualitatively, cells of the tetraploid form contained slightly lower nuclear and cytoplasmic volumes compared to those of the pentaploid form. An observed wide variation in neoplastic cell nuclear size within either disease form may reflect the distribution of cells in the G0G1, S, and G2M phases of the cell cycle. Potential etiologic relationships between the two forms are discussed.

Endogenous Life Cycle of Eimeria melanomytis (Apicomplexa: Eimeriidae) from the Dusky Rice Rat, Melanomys caliginosus (Rodentia: Cricetidae: Sigmodontinae) in Costa Rica.

PubMed

Chinchilla, Misael; Valerio, Idalia; Sánchez, Ronald; Duszynski, Donald W

2017-02-01

Endogenous stages of the life cycle of Eimeria melanomytis, infecting the peripheral epithelial cells of villi of the small intestine of experimentally infected young dusky rice rats, Melanomys caliginosus , were studied. Giemsa-stained mucosal scrapings and histological sections were examined for all the stages. Eimeria melanomytis has 3 generations of meronts (M), different in size, shape, and number of merozoites (m); and in size, shape, and location of the nuclei within the cytoplasm of the meronts. The 3 meront types, M 1 -M 3 , respectively, had 11-14 (m 1 ), 7-10 (m 2 ), and 20-30 (m 3 ) merozoites. Macrogametocytes and microgametocytes, as well as macrogametes and microgametes, complete the sexual cycle forming the unsporulated oocysts. This parasite's endogenous development produced severe intestinal lesions in experimentally infected dusky rice rats.

Artemisinin reduces cell proliferation and induces apoptosis in neuroblastoma.

PubMed

Zhu, Shunqin; Liu, Wanhong; Ke, Xiaoxue; Li, Jifu; Hu, Renjian; Cui, Hongjuan; Song, Guanbin

2014-09-01

Artemisinin, a natural product from the Chinese medicinal plant, Artemisia annua L., is commonly used in the treatment of malaria, and has recently been reported to have potent anticancer activity in various types of human tumors. Yet, the effect of artemisinin on neuroblastoma is still unclear. In the present study, we aimed to investigate the effects of artemisinin on neuroblastoma cells. We observed that artemisinin significantly inhibited cell growth and proliferation, and caused cell cycle arrest in the G1 phase in neuroblastoma cell lines. Annexin V-FITC/PI staining assay revealed that artemisinin markedly induced apoptosis. Soft agar assays revealed that artemisinin suppressed the ability of clonogenic formation of neuroblastoma cells and a xenograft study in NOD/SCID mice showed that artemisinin inhibited tumor growth and development in vivo. Therefore, our results suggest that the Chinese medicine artemisinin could serve as a novel potential therapeutic agent in the treatment of neuroblastoma.

In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

PubMed Central

Soares, P.B.; Jeremias, T.S.; Alvarez-Silva, M.; Licínio, M.A.; Santos-Silva, M.C.; Vituri, C.L.

2012-01-01

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved. PMID:23011404

Optimal staining methods for delineation of cortical areas and neuron counts in human brains.

PubMed

Uylings, H B; Zilles, K; Rajkowska, G

1999-04-01

For cytoarchitectonic delineation of cortical areas in human brain, the Gallyas staining for somata with its sharp contrast between cell bodies and neuropil is preferable to the classical Nissl staining, the more so when an image analysis system is used. This Gallyas staining, however, does not appear to be appropriate for counting neuron numbers in pertinent brain areas, due to the lack of distinct cytological features between small neurons and glial cells. For cell counting Nissl is preferable. In an optimal design for cell counting at least both the Gallyas and the Nissl staining must be applied, the former staining for cytoarchitectural delineaton of cortical areas and the latter for counting the number of neurons in the pertinent cortical areas. Copyright 1999 Academic Press.

A comparative study for selectivity of micronuclei in oral exfoliated epithelial cells

PubMed Central

Grover, S; Mujib, ABR; Jahagirdar, A; Telagi, N; Kulkarni, PG

2012-01-01

Background: Micronucleus (MN) represents small, additional nuclei formed by the exclusion of chromosome fragments or whole chromosomes lagging at mitosis. MN rates, therefore, indirectly reflect chromosome breakage or impairment of the mitotic apparatus. During the last few decades, micronuclei (“MNi”) in oral exfoliated epithelial cells are widely used as biomarkers of chromosomal damage, genome instability and cancer risk in humans. However, until now only little attention has been given to the effect of different staining procedures on the results of these MN assays. Aim: To compare the MNi frequencies in oral exfoliated epithelial cells using three different stains, i.e.,Feulgen stain, Papanicolaou stain (Pap) and hemotoxylin and eosin stain (H and E). Materials and Methods: Oral exfoliated cells from 45 cases of potentially malignant disorders (15 oral submucous fibrosis, 15 lichen planus and 15 leukoplakia) and 15 controls with healthy mucosa, were taken and MNi frequencies (No. of MNi/1000 cells) were compared using three different stains. Results: Mean MNi frequency in cases was found to be 3.8 with Feulgen stain, 16.8 with PAP and 25.9 with H and E. In controls, mean MNi frequency was 1.6 with Feulgen stain, 7.7 with PAP and 9.6 with H and E stain. Statistically significant value (P < 0.01) were observed when the three stains were compared together using Kruskal Walli's ANOVA test. Conclusion: Feulgen being a DNA-specific stain gave the least counts, although statistically significant results from the comparison of MNi frequency between cases and controls were obtained with all the three stains. PMID:23326025

A comparative study for selectivity of micronuclei in oral exfoliated epithelial cells.

PubMed

Grover, S; Mujib, Abr; Jahagirdar, A; Telagi, N; Kulkarni, Pg

2012-10-01

Micronucleus (MN) represents small, additional nuclei formed by the exclusion of chromosome fragments or whole chromosomes lagging at mitosis. MN rates, therefore, indirectly reflect chromosome breakage or impairment of the mitotic apparatus. During the last few decades, micronuclei ("MNi") in oral exfoliated epithelial cells are widely used as biomarkers of chromosomal damage, genome instability and cancer risk in humans. However, until now only little attention has been given to the effect of different staining procedures on the results of these MN assays. To compare the MNi frequencies in oral exfoliated epithelial cells using three different stains, i.e.,Feulgen stain, Papanicolaou stain (Pap) and hemotoxylin and eosin stain (H and E). Oral exfoliated cells from 45 cases of potentially malignant disorders (15 oral submucous fibrosis, 15 lichen planus and 15 leukoplakia) and 15 controls with healthy mucosa, were taken and MNi frequencies (No. of MNi/1000 cells) were compared using three different stains. Mean MNi frequency in cases was found to be 3.8 with Feulgen stain, 16.8 with PAP and 25.9 with H and E. In controls, mean MNi frequency was 1.6 with Feulgen stain, 7.7 with PAP and 9.6 with H and E stain. Statistically significant value (P < 0.01) were observed when the three stains were compared together using Kruskal Walli's ANOVA test. Feulgen being a DNA-specific stain gave the least counts, although statistically significant results from the comparison of MNi frequency between cases and controls were obtained with all the three stains.

WE-G-BRE-08: Radiosensitization by Olaparib Eluting Nanospheres

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tangutoori, S; Kumar, R; Sridhar, S

2014-06-15

Purpose: Permanent prostate brachytherapy often uses inert bio-absorbable spacers to achieve the desired geometric distribution of sources within the prostate. Transforming these spacers into implantable nanoplatforms for chemo-radiation therapy (INCeRT) provides a means of providing sustained in-situ release of radiosensitizers in the prostate to enhance the therapeutic ratio of the procedure. Olaparib, a PARP inhibitor, suppresses DNA repair processes present during low dose rate continuous irradiation. This work investigates the radiosensitizing/DNA damage repair inhibition by NanoOlaparib eluting nanospheres. Methods: Human cell line PC3 (from ATCC), was maintained in F12-k medium supplemented with fetal bovine serum. Clonogenic assay kit (from Fischermore » Scientific) was used to fix and stain the cells to determine the long term effects of irradiation. Nanoparticle size and zeta potential of nanospheres were determined using a Zeta particle size analyzer. The incorporation of Olaparib in nanospheres was evaluated by HPLC. Irradiation was performed in a small animal irradiator operating at 220 KeV.The long term effects of radio-sensitization with olaparib and nanoolaparib was determined using the clonogenic assay at 2 Gy and 4 Gy doses. The cells were allowed to grow for around 10 doubling cycles, The colonies were fixed and stained using clonogenic assay kit. The excess stain was washed off using DI water and the images were taken using a digital camera. Results: Radiosensitization studies were carried out in prostate cancer cell line, PC3 radiation at 0, 2 and 4Gy doses. Strongest dose response was observed with nanoolaparib treated cells compared to untreated cells. Conclusion: A two stage drug release of drug eluting nanospheres from a biodegradable spacer has been suggested for sustained in-situ release of Olaparib to suppress DNA repair processes during prostate brachytherapy. The Olaparib eluting nanospheres had the same in-vitro radiosensitizing effect as free olaparib. DOD 1R21CA16977501, A. David Mazzone Awards Program 2012PD164.« less

Autophagy Induced by CX-4945, a Casein Kinase 2 Inhibitor, Enhances Apoptosis in Pancreatic Cancer Cell Lines.

PubMed

Hwang, Dae Wook; So, Kwang Sup; Kim, Song Cheol; Park, Kwang-Min; Lee, Young-Joo; Kim, Sun-Whe; Choi, Chang-Min; Rho, Jin Kyung; Choi, Yun Jung; Lee, Jae Cheol

2017-04-01

Pancreatic cancer is the most lethal malignancy with only a few effective chemotherapeutic drugs. Because the inhibition of casein kinase 2 (CK2) has been reported as a novel therapeutic strategy for many cancers, we investigated the effects of CK2 inhibitors in pancreatic cancer cell lines. The BxPC3, 8902, MIA PaCa-2 human pancreatic cancer cell lines, and CX-4945, a novel CK2 inhibitor, were used. Autophagy was analyzed by acridine orange staining, fluorescence microscope detection of punctuate patterns of GFP-tagged LC3 and immunoblotting for LC3. Cell survival, cell cycle, and apoptosis analysis was performed. CX-4945 induced significant inhibition of proliferation and triggered autophagy in pancreatic cancer cells. This suppression of proliferation was caused by the direct inhibition of CK2α, which was required for autophagy and apoptosis in pancreatic cancer cells. CX-4945 suppressed cell cycle progression in G2/M and induced apoptosis. The inhibition of CX-4945-induced autophagy was rescued by 3-methyladenine or small interfering RNA against Atg7, which attenuated apoptosis in pancreatic cancer cells. CX-4945, a potent and selective inhibitor of CK2, effectively induces autophagy and apoptosis in pancreatic cancer cells, indicating that the induction of autophagy by CX-4945 may have an important role in the treatment of pancreatic cancer.

Ferulic acid reverses ABCB1-mediated paclitaxel resistance in MDR cell lines.

PubMed

Muthusamy, Ganesan; Balupillai, Agilan; Ramasamy, Karthikeyan; Shanmugam, Mohana; Gunaseelan, Srithar; Mary, Beaulah; Prasad, N Rajendra

2016-09-05

Multidrug resistance (MDR) remains a major obstacle in cancer chemotherapy. The use of the dietary phytochemicals as chemosensitizing agents to enhance the efficacy of conventional cytostatic drugs has recently gained the attention as a plausible approach for overcoming the drug resistance. The aim of this study was to investigate whether a naturally occurring diet-based phenolic acid, ferulic acid, could sensitize paclitaxel efficacy in ABCB1 overexpressing (P-glycoprotein) colchicine selected KB Ch(R)8-5 cell line. In vitro drug efflux assays demonstrated that ferulic acid inhibits P-glycoprotein transport function in drug resistant KB Ch(R)8-5 cell lines. However, ferulic acid significantly downregulates ABCB1 expression in a concentration dependent manner. Cytotoxicity assay reveals that ferulic acid decreased paclitaxel resistance in KBCh(R)8-5 and HEK293/ABCB1 cells, which indicates its chemosensitizing potential. Clonogenic cell survival assay and apoptotic morphological staining further confirm the chemosensitizing potential of ferulic acid in drug resistant KB Ch(R)8-5 cell lines. Ferulic acid treatment enhances paclitaxel mediated cell cycle arrest and upregulates paclitaxel-induced apoptotic signaling in KB resistant cells. Hence, it has been concluded that downregulation of ABCB1 and subsequent induction of paclitaxel-mediated cell cycle arrest and apoptotic signaling may be the cause for the chemosensitizing potential of ferulic acid in P-gp overexpressing cell lines. Copyright © 2016 Elsevier B.V. All rights reserved.

Proflavine Hemisulfate as a Fluorescent Contrast Agent for Point-of-Care Cytology

PubMed Central

Prieto, Sandra P.; Powless, Amy J.; Boice, Jackson W.; Sharma, Shree G.; Muldoon, Timothy J.

2015-01-01

Proflavine hemisulfate, an acridine-derived fluorescent dye, can be used as a rapid stain for cytologic examination of biological specimens. Proflavine fluorescently stains cell nuclei and cytoplasmic structures, owing to its small amphipathic structure and ability to intercalate DNA. In this manuscript, we demonstrated the use of proflavine as a rapid cytologic dye on a number of specimens, including normal exfoliated oral squamous cells, cultured human oral squamous carcinoma cells, and leukocytes derived from whole blood specimens using a custom-built, portable, LED-illuminated fluorescence microscope. No incubation time was needed after suspending cells in 0.01% (w/v) proflavine diluted in saline. Images of proflavine stained oral cells had clearly visible nuclei as well as granular cytoplasm, while stained leukocytes exhibited bright nuclei, and highlighted the multilobar nature of nuclei in neutrophils. We also demonstrated the utility of quantitative analysis of digital images of proflavine stained cells, which can be used to detect significant morphological differences between different cell types. Proflavine stained oral cells have well-defined nuclei and cell membranes which allowed for quantitative analysis of nuclear to cytoplasmic ratios, as well as image texture analysis to extract quantitative image features. PMID:25962131

Proflavine Hemisulfate as a Fluorescent Contrast Agent for Point-of-Care Cytology.

PubMed

Prieto, Sandra P; Powless, Amy J; Boice, Jackson W; Sharma, Shree G; Muldoon, Timothy J

2015-01-01

Proflavine hemisulfate, an acridine-derived fluorescent dye, can be used as a rapid stain for cytologic examination of biological specimens. Proflavine fluorescently stains cell nuclei and cytoplasmic structures, owing to its small amphipathic structure and ability to intercalate DNA. In this manuscript, we demonstrated the use of proflavine as a rapid cytologic dye on a number of specimens, including normal exfoliated oral squamous cells, cultured human oral squamous carcinoma cells, and leukocytes derived from whole blood specimens using a custom-built, portable, LED-illuminated fluorescence microscope. No incubation time was needed after suspending cells in 0.01% (w/v) proflavine diluted in saline. Images of proflavine stained oral cells had clearly visible nuclei as well as granular cytoplasm, while stained leukocytes exhibited bright nuclei, and highlighted the multilobar nature of nuclei in neutrophils. We also demonstrated the utility of quantitative analysis of digital images of proflavine stained cells, which can be used to detect significant morphological differences between different cell types. Proflavine stained oral cells have well-defined nuclei and cell membranes which allowed for quantitative analysis of nuclear to cytoplasmic ratios, as well as image texture analysis to extract quantitative image features.

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA.

PubMed

Wang, Hongyang; Owens, James D; Shih, Joanna H; Li, Ming-Chung; Bonner, Robert F; Mushinski, J Frederic

2006-04-27

Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts.

Histological staining methods preparatory to laser capture microdissection significantly affect the integrity of the cellular RNA

PubMed Central

Wang, Hongyang; Owens, James D; Shih, Joanna H; Li, Ming-Chung; Bonner, Robert F; Mushinski, J Frederic

2006-01-01

Background Gene expression profiling by microarray analysis of cells enriched by laser capture microdissection (LCM) faces several technical challenges. Frozen sections yield higher quality RNA than paraffin-imbedded sections, but even with frozen sections, the staining methods used for histological identification of cells of interest could still damage the mRNA in the cells. To study the contribution of staining methods to degradation of results from gene expression profiling of LCM samples, we subjected pellets of the mouse plasma cell tumor cell line TEPC 1165 to direct RNA extraction and to parallel frozen sectioning for LCM and subsequent RNA extraction. We used microarray hybridization analysis to compare gene expression profiles of RNA from cell pellets with gene expression profiles of RNA from frozen sections that had been stained with hematoxylin and eosin (H&E), Nissl Stain (NS), and for immunofluorescence (IF) as well as with the plasma cell-revealing methyl green pyronin (MGP) stain. All RNAs were amplified with two rounds of T7-based in vitro transcription and analyzed by two-color expression analysis on 10-K cDNA microarrays. Results The MGP-stained samples showed the least introduction of mRNA loss, followed by H&E and immunofluorescence. Nissl staining was significantly more detrimental to gene expression profiles, presumably owing to an aqueous step in which RNA may have been damaged by endogenous or exogenous RNAases. Conclusion RNA damage can occur during the staining steps preparatory to laser capture microdissection, with the consequence of loss of representation of certain genes in microarray hybridization analysis. Inclusion of RNAase inhibitor in aqueous staining solutions appears to be important in protecting RNA from loss of gene transcripts. PMID:16643667

Endometrial Stromal Cells and Immune Cell Populations Within Lymph Nodes in a Nonhuman Primate Model of Endometriosis

PubMed Central

Fazleabas, A. T.; Braundmeier, A. G.; Markham, R.; Fraser, I. S.; Berbic, M.

2011-01-01

Mounting evidence suggests that immunological responses may be altered in endometriosis. The baboon (Papio anubis) is generally considered the best model of endometriosis pathogenesis. The objective of the current study was to investigate for the first time immunological changes within uterine and peritoneal draining lymph nodes in a nonhuman primate baboon model of endometriosis. Paraffin-embedded femoral lymph nodes were obtained from 22 normally cycling female baboons (induced endometriosis n = 11; control n = 11). Immunohistochemical staining was performed with antibodies for endometrial stromal cells, T cells, immature and mature dendritic cells, and B cells. Lymph nodes were evaluated using an automated cellular imaging system. Endometrial stromal cells were significantly increased in lymph nodes from animals with induced endometriosis, compared to control animals (P = .033). In animals with induced endometriosis, some lymph node immune cell populations including T cells, dendritic cells and B cells were increased, suggesting an efficient early response or peritoneal drainage. PMID:21617251

Effects of caffeic acid on cisplatin-induced hair cell damage in HEI-OC1 auditory cells.

PubMed

Choi, June; Kim, Shin Hye; Rah, Yoon Chan; Chae, Sung Won; Lee, Jong Dae; Md, Byung Don Lee; Park, Moo Kyun

2014-12-01

Cisplatin is a widely used anticancer chemotherapeutic agent. However, it is notorious for its ototoxicity and nephrotoxicity due to induction of reactive oxygen species (ROS). Caffeic acid is a naturally occurring polyphenol present in honey that is known to reduce the generation of oxygen-derived free radicals. The objective of the present study was to evaluate the protective effects and mechanism underlying the effect of caffeic acid on cisplatin-induced ototoxicity in HEI-OC1 auditory cell lines. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was determined by Hoechst 33258 staining and Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Cell cycle stages were analyzed by flow cytometry. The radical-scavenging activity of caffeic acid was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The expression levels of caspase-3, -8, and -9, as well as the activity of caspase-3, were evaluated. Caffeic acid showed a protective effect against cisplatin-induced HEI-OC1 cell damage as demonstrated by the MTT assay. Caffeic acid decreased cell death by apoptosis and necrosis. Caffeic acid showed strong scavenging activity against the radical DPPH and decreased intracellular ROS production. Caffeic acid decreased the expression of caspase-3 and -8 and increased the activity of caspase-3. Caffeic acid attenuated cisplatin-induced hair cell loss in HEI-OC1 cell lines; these effects were mediated by its radical scavenging activity and inhibition of apoptosis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Enhancer of zeste homolog 2 expression is associated with tumor cell proliferation and metastasis in gastric cancer.

PubMed

Choi, Jung Hye; Song, Young Soo; Yoon, Jin Sun; Song, Kang Won; Lee, Young Yiul

2010-03-01

The enhancer of zeste homolog 2 (EZH2), a member of the polycomb group of proteins, plays an important role in cell proliferation and cell cycle regulation. EZH2 is overexpressed in aggressive forms of prostate, breast, bladder, and endometrial cancers. However, the role of EZH2 expression in gastric cancer has not been fully determined. This study was conducted to investigate the correlation between EZH2 and cell cycle-related molecules, and the clinical value of EZH2 expression in gastric cancer. We analyzed EZH2 expression using Western blotting in AGS, MKN-28, SNU-16, SNU-484, SNU-601, and SNU-638 gastric cancer cell lines. After transfection of EZH2 siRNA into MKN-28 cells, the change in cell cycle-related molecules was assessed by Western blot analysis. Expression of EZH2, Ki-67, and p53 was determined by immunohistochemical staining of tissue microarrays from specimens of 137 cases of resected gastric cancer. We found high expressions of EZH2 in all of the tested gastric cancer cell lines. RNA interference of EZH2 induced upregulation of p53 and HDAC1 and downregulation of cyclin D1 and cyclin E. High EZH2 expression was observed in 60.6% of gastric cancers and in 6.7% of non-neoplastic gastric tissues (p < 0.01); 40.1% were positive for p53 in gastric cancers. High EZH2 expression was correlated with Ki-67 and p53 expressions and was significantly associated with distant metastases and non-signet ring cells. Our results suggest that high EZH2 expression is associated with tumor cell proliferation and metastasis in gastric cancer.

Semiquantitative immunohistochemical marker staining and localization in canine thyroid carcinoma and normal thyroid gland.

PubMed

Pessina, P; Castillo, V; Sartore, I; Borrego, J; Meikle, A

2016-09-01

Immunoreactive proteins in follicular cells, fibroblasts and endothelial cells were assessed in canine thyroid carcinomas and healthy thyroid glands. No differences were detected in thyrotropin receptor and thyroglobulin staining between cancer and normal tissues, but expression was higher in follicular cells than in fibroblasts. Fibroblast growth factor-2 staining was more intense in healthy follicular cells than in those of carcinomas. Follicular cells in carcinomas presented two- to three-fold greater staining intensity of thyroid transcription factor-1 and proliferating cell nuclear antigen, respectively, than healthy cells, and a similar trend was found for the latter antigen in fibroblasts. Vascular endothelial growth factor staining was more intense in the endothelial cells of tumours than in those of normal tissues. In conclusion, greater expression of factors related to proliferation and angiogenesis was demonstrated in several cell types within thyroid carcinomas compared to healthy tissues, which may represent mechanisms of tumour progression in this disease. © 2014 John Wiley & Sons Ltd.

Histochemical analysis of glycoconjugates in the skin of a catfish (arius tenuispinis, day).

PubMed

Al-Banaw, A; Kenngott, R; Al-Hassan, J M; Mehana, N; Sinowatz, F

2010-02-01

A histochemical study using conventional carbohydrate histochemistry (periodic-acid staining including diastase controls, alcian blue staining at pH 1 and 2.5) as well as using a battery of 14 fluorescein isothiocyanate (FITC)-labelled lectins to identify glycoconjugates present in 10 different areas of the skin of a catfish (Arius tenuispinis) was carried out. The lectins used were: mannose-binding lectins (Con A, LCA and PSA), galactose-binding lectins (PNA, RCA), N-acetylgalactosamine-binding lectins (DBA, SBA, SJA and GSL I), N-acetylglucosamine-binding lectins (WGA and WGAs), fucose-binding lectins (UEA) and lectins which bind to complex carbohydrate configurations (PHA E, PHA L). Conventional glycoconjugate staining (PAS staining, alcian blue at pH 1 and 2.5) showed that the mucous goblet cells contain a considerable amount of glycoconjugates in all locations of the skin, whereas the other unicellular gland type, the club cells, lacked these glycoconjugates. The glycoproteins found in goblet cells are neutral and therefore stain magenta when subjected to PAS staining. Alcian blue staining indicating acid glycoproteins was distinctly positive at pH 1, but gave only a comparable staining at pH 2.5. The mucus of the goblet cells therefore also contains acid glycoproteins rich in sulphate groups. Using FITC-labelled lectins, the carbohydrate composition of the glycoproteins of goblet cells could be more fully characterized. A distinct staining of the mucus of goblet cells was found with the mannose-binding lectins LCA and PSA; the galactosamine-binding lectins DBA, SBA and GLS I; the glucosamine-binding lectin WGA; and PHA E which stains glycoproteins with complex carbohydrate configurations. No reaction occurred with the fucose-binding lectin UEA and the sialic acid-specific lectin SNA. In addition, the galactose-binding lectins PNA and RCA showed only a weak or completely negative staining of the mucus in the goblet cells. The specificity of the lectin staining could be proved by inhibiting binding of the lectins by competitive inhibition with the corresponding sugars. From these data, we can conclude that the mucus produced by the epidermal goblet cells of A. tenuispinis is rich in mannose, N-acetylgalactosamine and N-acetylglucosamine residues.

Propidium iodide staining: a new application in fluorescence microscopy for analysis of cytoarchitecture in adult and developing rodent brain.

PubMed

Hezel, Marcus; Ebrahimi, Fahim; Koch, Marco; Dehghani, Faramarz

2012-10-01

Immunohistochemical visualization of antigens in specimen has evolved to an indispensable technique in biomedical research for investigations of cell morphology and pathology both in bright field and fluorescence microscopy. While there are couple of staining methods that reveal entire cytoarchitecture in bright field microscopy such as Nissl or hemalaun-eosin, there are still limitations in visualizations of cytoarchitecture in fluorescence microscopy. The present study reports a simple staining method that provides the required illustration of cell allocations and cellular composition in fluorescence microscopy in adult and in developing rodent central nervous system using the fluorophore propidium iodide (PI, 5μg/mL). PI is a well-accepted marker for degenerating cells when applied prior to fixation (pre-fixation PI staining). Here, PI was added to the sections after the fixation (post-fixation PI staining). This revised labeling procedure led to similar cytoarchitectural staining patterns in fluorescence microscopy as observed with hemalaun in bright field microscopy. This finding was proven in organotypic hippocampal slice cultures (OHSC) and brain sections obtained from different postnatal developmental stages. Excitotoxically lesioned OHSC subjected to pre-fixation PI staining merely showed brightly labeled condensed nuclei of degenerating neurons. In contrast, post-fixation PI staining additionally revealed extensive labeling of neuronal cell bodies and glial cells within the OHSC, thus allowing visualization of stratification of neuronal layers and cell morphology. Furthermore, post-fixation PI staining was combined with NeuN, calbindin, calretinin, glial fibrillary acidic protein or Griffonia simplicifolia isolectin B4 (IB(4)) in post natal (p1 and p9) and adult rats. In early post-natal brain sections almost all mentioned cellular markers led to an incomplete staining of the native cell organization and resulted in an inaccurate estimation of cell morphology when compared to adult brains. In contrast, post-fixation PI staining allowed investigation of the whole cytoarchitecture independent of the developmental stage. Taken together, post-fixation PI staining provides a detailed insight in the morphology of both developing and adult brain tissues in fluorescence microscopy. Copyright © 2012 Elsevier Ltd. All rights reserved.

Cell permeability and nuclear DNA staining by propidium iodide in basidiomycetous yeasts.

PubMed

Zhang, Ning; Fan, Yuxuan; Li, Chen; Wang, Qiming; Leksawasdi, Noppol; Li, Fuli; Wang, Shi'an

2018-05-01

Non-model yeasts within basidiomycetes have considerable importance in agriculture, industry, and environment, but they are not as well studied as ascomycetous yeasts. Serving as a basic technique, nuclear DNA staining is widely used in physiology, ecology, cell biology, and genetics. However, it is unclear whether the classical nuclear DNA staining method for ascomycetous yeasts is applicable to basidiomycetous yeasts. In this study, 5 yeasts ineffectively stained by the classical propidium iodide (PI) staining method were identified from 23 representative basidiomycetous yeasts. Pretreatment of cells using dimethyl sulfoxide (DMSO) or snailase markedly improved cell penetration to PI and thus enabled DNA content determination by flow cytometry on the recalcitrant yeasts. The pretreatments are efficient, simple, and fast, avoiding tedious mutagenesis or genetic engineering used in previous reports. The heterogeneity of cell penetration to PI among basidiomycetous yeasts was attributed to the discrepancy in cell wall polysaccharides instead of capsule or plasma membrane. This study also indicated that care must be taken in attributing PI-negative staining as viable cells when studying non-model microorganisms.

Endogenous Life Cycle of Eimeria marmosopos (Apicomplexa: Eimeriidae) from the Opossum, Didelphis marsupialis (Didelphimorphia: Didelphidae) in Costa Rica.

PubMed

Chinchilla, Misael; Valerio, Idalia; Duszynski, Donald

2015-08-01

The endogenous life cycle of Eimeria marmosopos was studied in experimentally infected young opossums, Didelphis marsupialis . All the endogenous stages were located in the epithelial cells of villi in the small intestine. Giemsa-stained mucosal scrapings and histological sections were studied for the diagnosis of all the life cycle stages. Eimeria marmosopos has 3 generations of meronts (M) that differ by size, shape, and number of merozoites (m), which also differ in their size, shape, and location of their nuclei within the cytoplasm of the meronts. The 3 meront types, M(1)-M(3), respectively, had 8-15 (m(1)), 4-9 (m(2)), and 22-30 (m(3)) merozoites. Macrogametocytes and microgametocytes, as well as macrogametes and microgametes, completed the sexual cycle, finishing with the formation of unsporulated oocysts. This parasite's endogenous development produced severe intestinal lesions in experimentally infected opossums. There are 56 Eimeria species known from all marsupials worldwide, but this is the first complete life cycle in which both the asexual and sexual stages have been documented.

[Simultaneous staining with fluorescein diacetate-propidium iodide to determine isolated cochlear outer hair cell viability of guinea pig].

PubMed

Yu, Q; Shi, H; Wang, J

1995-01-01

A simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is discribed for use in the determination of isolated cochlear outer hair cell viability. With exciter light, viable cells fluoresce bright green, while nonviable cells are bright red. In cell culture and cytotoxicity studies, double-staining with FDA-PI is a accurate method to discriminate between live and nonviable cells.

Retinal specializations and visual ecology in an animal with an extremely elaborate pupil shape: The Little skate Leucoraja (Raja) erinacea Mitchell, 1825.

PubMed

Jinson, S Terrell; Liebich, Jan; Senft, Stephen L; Mäthger, Lydia M

2018-05-14

Investigating retinal specializations offers insights into eye functionality. Using retinal wholemount techniques, we investigated the distribution of retinal ganglion cells in the Little skate Leucoraja erinacea by (1) dye-backfilling into the optic nerve prior to retinal wholemounting; (2) Nissl-staining of retinal wholemounts. Retinas were examined for regional specializations (higher numbers) of ganglion cells that would indicate higher visual acuity in those areas. Total ganglion cell number were low compared to other elasmobranchs (backfilled: average 49,713 total ganglion cells, average peak cell density 1,315 ganglion cells mm -2 ; Nissl-stained: average 47,791 total ganglion cells, average peak cell density 1,319 ganglion cells mm -2 ). Ganglion cells fit into three size categories: small (5-20µm); medium (20-30µm); large: (≥ 30µm), and they were not homogeneously distributed across the retina. There was a dorsally located horizontal visual streak with increased ganglion cell density; additionally, there were approximately 3 local maxima in ganglion cell distribution (potential areae centrales) within this streak in which densities were highest. Using computerized tomography (CT) and micro-CT, geometrical dimensions of the eye were obtained. Combined with ganglion cell distributions, spatial resolving power was determined to be between 1.21 to 1.37 cycles per degree. Additionally, photoreceptor sizes across different retinal areas varied; photoreceptors were longest within the horizontal visual streak. Variations in the locations of retinal specializations appear to be related to the animal's anatomy: shape of the head and eyes, position of eyes, location of tapetum, and shape of pupil, as well as the visual demands associated with lifestyle and habitat type. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Saccharomyces cerevisiae samples stained with FUN-1 dye can be stored at -20 degrees C for later observation.

PubMed

Eggleston, M D; Marshall, P A

2007-01-01

FUN-1, a fluorescent vital dye, has been observed to form cylindrical intravacuolar structures within the vacuoles of metabolically active yeast cells. FUN-1 staining, which begins as a diffuse pool of fluorescent cytoplasmic stain, uses an unknown endogenous biochemical processing mechanism to compact and form orange-red cylindrical intravacuolar structures within the cell vacuole. In the clinical setting, FUN-1 is primarily used for identification of fungal infection. FUN-1 is utilized in the laboratory to distinguish between metabolically active and dead fungal cells. Although this stain is useful for distinguishing between live and dead fungal dead cells, few studies have utilized this chemical. This lack of use in the scientific community may be due to the requirement that cells are visualized directly after staining. Thus, it would be of interest to be able to stain cells and store them for later use. Our lab examined the longevity of cylindrical intravacuolar structures in two strains of Saccharomyces cerevisiae stained with FUN-1 and stored at -20 degrees C. We found that cylindrical intravacuolar structures could be reliably observed and imaged utilizing differential interference contrast microscopy and fluorescence microscopy for 21 days. We also observed that cells stained with FUN-1 would resume propagation on yeast extract, peptone, dextrose (YPD) plates after being frozen at -20 degrees C for 21 days. These modifications to the published procedure for FUN-1 dye staining should allow for a more prevalent and less time sensitive use of this important biological tool.

Genetic interactions between the hedgehog co-receptors Gas1 and Boc regulate cell proliferation during murine palatogenesis

PubMed Central

Xavier, Guilherme M.; Seppala, Maisa; Papageorgiou, Spyridon N.; Fan, Chen-Ming; Cobourne, Martyn T.

2016-01-01

Abnormal regulation of Sonic hedgehog (Shh) signaling has been described in a variety of human cancers and developmental anomalies, which highlights the essential role of this signaling molecule in cell cycle regulation and embryonic development. Gas1 and Boc are membrane co-receptors for Shh, which demonstrate overlapping domains of expression in the early face. This study aims to investigate potential interactions between these co-receptors during formation of the secondary palate. Mice with targeted mutation in Gas1 and Boc were used to generate Gas1; Boc compound mutants. The expression of key Hedgehog signaling family members was examined in detail during palatogenesis via radioactive in situ hybridization. Morphometric analysis involved computational quantification of BrdU-labeling and cell packing; whilst TUNEL staining was used to assay cell death. Ablation of Boc in a Gas1 mutant background leads to reduced Shh activity in the palatal shelves and an increase in the penetrance and severity of cleft palate, associated with failed elevation, increased proliferation and reduced cell death. Our findings suggest a dual requirement for Boc and Gas1 during early development of the palate, mediating cell cycle regulation during growth and subsequent fusion of the palatal shelves. PMID:27811357

Prognostic significance of S-phase fractions in peritumoral invading zone analyzed by laser scanning cytometry in patients with high-grade glioma: A preliminary study.

PubMed

Nakajima, Syoichi; Morii, Ken; Takahashi, Hitoshi; Fujii, Yukihiko; Yamanaka, Ryuya

2016-03-01

The predominant characteristic of malignant glioma is the presence of invading tumor cells in the peritumoral zone. Distinguishing between tumor cells and normal cells in a peritumoral lesion is challenging. Therefore, the aim of the present study was to investigate the cell-cycle phase measurements of fixed paraffin-embedded specimens from the peritumoral invading zone of high-grade gliomas using laser scanning cytometry. A total of 12 high-grade gliomas (2 anaplastic astrocytomas and 10 glioblastomas) were studied. The tumor core and peritumoral invading zone of each tumor specimen were investigated. Tissue sections (50 µm) from the paraffin blocks were deparaffinized, rehydrated and enzymatically disintegrated, and the cells in suspension were stained with propidium iodide and placed on microscope slides. A slight trend for an increased S-phase fraction in the peritumoral invading zone compared with the tumor core was observed (P=0.24). Additionally, there was a trend for a decrease in the overall survival time of patients with increasing peritumoral invading zone S-phase fraction (P=0.12). These data suggest that laser scanning cytometry is a powerful and clinically relevant tool for the objective analysis of the cell cycle in malignant gliomas.

The metastasis suppressor SOX11 is an independent prognostic factor for improved survival in gastric cancer

PubMed Central

QU, YING; ZHOU, CHENFEI; ZHANG, JIANIAN; CAI, QU; LI, JIANFANG; DU, TAO; ZHU, ZHENGGANG; CUI, XIAOJIANG; LIU, BINGYA

2014-01-01

SOX11 is involved in gastrulation and in malignant diseases. The aim of this study was to investigate the role of SOX11 in gastric cancer and its expression pattern and clinical significance. SOX11 overexpression cell model was used to examine in vitro and in vivo the role of SOX11 in cell growth and metastasis. Cell cycle analysis and Annexin V/PI double staining were used to investigate the effect of SOX11 on cell cycle progression and apoptosis. The expression of SOX11 in human gastric cancer was examined by immunohistochemistry. The correlation of SOX11 expression with clinicopathological characteristics and survival of patients was analyzed by Pearson’s χ2 and Kaplan-Meier analyses, respectively. Cox’s proportional hazard model was employed in multivariate analysis. SOX11 overexpression did not inhibit cell growth but strongly suppressed cell migration/invasion in vitro and in vivo. We found a significant correlation between high SOX11 protein levels and Lauren’s classification (intestinal type), differentiation status (high and medium), and early TNM stage. SOX11 is an independent prognostic factor for improved survival in gastric cancer patients. SOX11 was a potential tumor-suppressor and an independent positive prognostic factor in gastric cancer patients with less advanced clinicopathological features. PMID:24604109

PDF cycling in the dorsal protocerebrum of the Drosophila brain is not necessary for circadian clock function.

PubMed

Kula, Elzbieta; Levitan, Edwin S; Pyza, Elzbieta; Rosbash, Michael

2006-04-01

In Drosophila, the neuropeptide pigment-dispersing factor (PDF) is a likely circadian molecule, secreted by central pacemaker neurons (LNvs). PDF is expressed in both small and large LNvs (sLNvs and lLNvs), and there are striking circadian oscillations of PDF staining intensity in the small cell termini, which require a functional molecular clock. This cycling may be relevant to the proposed role of PDF as a synchronizer of the clock system or as an output signal connecting pacemaker cells to locomotor activity centers. In this study, the authors use a generic neuropeptide fusion protein (atrial natriuretic factor-green fluorescent protein [ANF-GFP]) and show that it can be expressed in the same neurons as PDF itself. Yet, ANF-GFP as well as PDF itself does not manifest any cyclical accumulation in sLNv termini in adult transgenic flies. Surprisingly, the absence of detectable PDF cycling is not accompanied by any detectable behavioral pheno-type, since these transgenic flies have normal morning and evening anticipation in a light-dark cycle (LD) and are fully rhythmic in constant darkness (DD). The molecular clock is also not compromised. The results suggest that robust PDF cycling in sLNv termini plays no more than a minor role in the Drosophila circadian system and is apparently not even necessary for clock output function.

Effects of demethoxycurcumin on the viability and apoptosis of skin cancer cells.

PubMed

Wu, Yaoqun; Zhang, Pei; Yang, Hongyun; Ge, Yong; Xin, Yong

2017-07-01

The present study investigated the effects and mechanisms of demethoxycurcumin (DMC) on a human skin squamous cell carcinoma cell line, A431, and a human keratinocyte cell line, HaCaT. A431 and HaCaT cells were cultured in vitro. The effects of DMC treatment on cell viability were analyzed using the Cell Counting kit‑8 (CCK‑8) assay; cell cycle distribution was analyzed by flow cytometry; apoptosis was assessed by flow cytometry and Hoechst 33258 staining; and the protein expression levels of cytochrome c, B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (BAX), caspase‑9 and caspase‑3 were evaluated by western blotting. CCK‑8 assay results demonstrated that DMC treatment significantly inhibited viability of A431 and HaCaT cells in a dose‑dependent manner. Flow cytometric analysis confirmed that DMC treatment induced apoptosis in a dose‑dependent manner, and significantly increased the proportion of cells in G2/M phase. Western blot analysis indicated that the protein expression levels of Bcl‑2 were decreased, whereas the expression levels of BAX, caspase‑9, caspase‑3 and cytochrome c were increased following DMC treatment compared with in untreated cells. In conclusion, DMC treatment significantly inhibited viability of A431 and HaCaT cells, and induced cell cycle arrest in G2/M phase. The present study indicated that DMC may induce apoptosis of skin cancer cells through a caspase‑dependent pathway.

The Androgen-Regulated Calcium-Activated Nucleotidase 1 (CANT1) Is Commonly Overexpressed in Prostate Cancer and Is Tumor-Biologically Relevant in Vitro

PubMed Central

Gerhardt, Josefine; Steinbrech, Corinna; Büchi, Oralea; Behnke, Silvia; Bohnert, Annette; Fritzsche, Florian; Liewen, Heike; Stenner, Frank; Wild, Peter; Hermanns, Thomas; Müntener, Michael; Dietel, Manfred; Jung, Klaus; Stephan, Carsten; Kristiansen, Glen

2011-01-01

Previously, we identified the calcium-activated nucleotidase 1 (CANT1) transcript as up-regulated in prostate cancer. Now, we studied CANT1 protein expression in a large cohort of nearly 1000 prostatic tissue samples including normal tissue, prostatic intraepithelial neoplasia (PIN), primary carcinomas, metastases, and castrate-resistant carcinomas, and further investigated its functional relevance. CANT1 displayed predominantly a Golgi-type immunoreactivity with additional and variable cytoplasmic staining. In comparison to normal tissues, the staining intensity was significantly increased in PIN lesions and cancer. In cancer, high CANT1 levels were associated with a better prognosis, and castrate-resistant carcinomas commonly showed lower CANT1 levels than primary carcinomas. The functional role of CANT1 was investigated using RNA interference in two prostate cancer cell lines with abundant endogenous CANT1 protein. On CANT1 knockdown, a significantly diminished cell number and DNA synthesis rate, a cell cycle arrest in G1 phase, and a strong decrease of cell transmigration rate and wound healing capacity of CANT1 knockdown cells was found. However, on forced CANT1 overexpression, cell proliferation and migration remained unchanged. In summary, CANT1 is commonly overexpressed in the vast majority of primary prostate carcinomas and in the precursor lesion PIN and may represent a novel prognostic biomarker. Moreover, this is the first study to demonstrate a functional involvement of CANT1 in tumor biology. PMID:21435463

Venom present in sea anemone (Heteractis magnifica) induces apoptosis in non-small-cell lung cancer A549 cells through activation of mitochondria-mediated pathway.

PubMed

Ramezanpour, Mahnaz; da Silva, Karen Burke; Sanderson, Barbara J S

2014-03-01

Lung cancer is a major cause of cancer deaths throughout the world and the complexity of apoptosis resistance in lung cancer is apparent. Venom from Heteractis magnifica caused dose-dependent decreases in survival of the human non-small-cell lung cancer cell line, as determined by the MTT and Crystal Violet assays. The H. magnifica venom induced cell cycle arrest and induced apoptosis of A549 cells, as confirmed by annexin V/propidium iodide staining. The venom-induced apoptosis in A549 cells was characterized by cleavage of caspase-3 and a reduction in the mitochondrial membrane potential. Interestingly, crude extracts from H. magnifica had less effect on the survival of non-cancer cell lines. In the non-cancer cells, the mechanism via which cell death occurred was through necrosis not apoptosis. These findings are important for future work using H. magnifica venom for pharmaceutical development to treat human lung cancer.

Effects of spaceflight on levels and activity of immune cells

NASA Technical Reports Server (NTRS)

Sonnenfeld, Gerald; Berry, Wallace D.; Mandel, Adrian D.; Konstantinova, Irena V.; Taylor, Gerald R.

1990-01-01

Experiments were carried out on cells from rats that had been flown on Soviet Biosputnik Cosmos 1887 to explore the effects of speceflight on immune responses. Rat bone marrow cells were examined for their response to colony stimulating factor-M. Rat spleen and bone marrow cells were stained with antibodies directed against cell surface antigenic markers. The results of the studies indicate that bone marrow cells from flown rats showed a decreased response to colony stimulating factor. There was a higher percentage of spleen cells from flown rats staining positively for pan-T-cell, suppressor-T-cell, and interleukin-2 receptor cell surface antigens. A small increase in the percentage of cells staining positively for helper-T-cell antigens was also noted. In addition, a higher percentage of cells that appeared to be part of the myelogenous population of bone marrow cells from flown rats stained positively for surface immunoglobulin.

Protein kinase A inhibitor, H89, enhances survival and clonogenicity of dissociated human embryonic stem cells through Rho-associated coiled-coil containing protein kinase (ROCK) inhibition.

PubMed

Zhang, Liang; Xu, Yanqing; Xu, Jiandong; Wei, Yuping; Xu, Xia

2016-04-01

Can cell survival of dissociated human embryonic stem cells (hESCs) be increased during culture? A protein kinase A (PKA) inhibitor, H89, can significantly enhance survival and clonogenicity of dissociated hESCs without affecting their pluripotency. hESCs are vulnerable to massive cell death upon cellular detachment and dissociation. hESCs were dissociated into single cells and then cultured in feeder-dependent and -independent manners. H89 was added to the culture medium at different concentrations for 1 day. The statistical results were obtained from at least three independent experiments (n ≥ 4). The group without treatment was used as the negative control. 4 µM H89 was added in the culture medium to promote cell survival and colony formation of dissociated hESCs. MTT method and propidium iodide (PI) staining were used to determine cell proliferation, cell death and cell cycle, respectively. To count colony formation, alkaline phosphatase (AP) staining was carried out. Western blot was performed to determine protein expression. Except AP staining, immunofluorescence, RT-PCR and karyotype analysis were used to confirm the pluripotent state of H89 treated hESCs. H89 inhibits the dissociation-induced phosphorylation of PKA and two substrates of Rho-associated coiled-coil containing protein kinase (ROCK), myosin light chain (MLC2) and myosin phosphatase target subunit 1 (MYPT1), significantly increases cell survival and colony formation, and strongly depresses dissociation-induced cell death and cell blebbing without affecting the pluripotency of hESCs and their differentiation in vitro. Appropriate H89 concentration should be used and 1 day of H89 treatment is sufficient for promoting survival and colony formation of dissociated hESCs. These results provide an alternative for human pluripotent stem cell (hPSC) culture, broaden the scope of participants in the cell death of single hES cells after dissociation and further enlighten clues to understand the mechanism of dissociation-induced cell death. This research was supported by the National Natural Science Foundation of China (21176238, 21576266), and Chinese Academy of Sciences. There is no conflict of interest to declare. Nil. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Apoptosis in liver cancer (HepG2) cells induced by functionalized gold nanoparticles.

PubMed

Ashokkumar, Thirunavukkarasu; Prabhu, Durai; Geetha, Ravi; Govindaraju, Kasivelu; Manikandan, Ramar; Arulvasu, Chinnasamy; Singaravelu, Ganesan

2014-11-01

An ethnopharmacological approach for biosynthesis of gold nanoparticles is being demonstrated using seed coat of Cajanus cajan. Medicinal value of capping molecule investigated for anticancer activity and results disclose its greater potential. The active principle of the seed coat [3-butoxy-2-hydroxypropyl 2-(2,4-dihydroxyphenyl) acetate] is elucidated. Rapid one-step synthesis yields highly stable, monodisperse (spherical) gold nanoparticles in the size ranging from 9 to 41 nm. Anticancer activity has been studied using liver cancer cells and cytotoxic mechanism has been evaluated using MTT, Annexin-V/PI Double-Staining Assay, Cell cycle, Comet assay and Flow cytometric analysis for apoptosis. The present investigation will open up a new possibility of functionalizing gold nanoparticles for apoptosis studies in liver cancer cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Immunohistochemical Analysis of Human Vallate Taste Buds

PubMed Central

Tizzano, Marco; Grigereit, Laura; Shultz, Nicole; Clary, Matthew S.

2015-01-01

The morphology of the vallate papillae from postmortem human samples was investigated with immunohistochemistry. Microscopically, taste buds were present along the inner wall of the papilla, and in some cases in the outer wall as well. The typical taste cell markers PLCβ2, GNAT3 (gustducin) and the T1R3 receptor stain elongated cells in human taste buds consistent with the Type II cells in rodents. In the human tissue, taste bud cells that stain with Type II cell markers, PLCβ2 and GNAT3, also stain with villin antibody. Two typical immunochemical markers for Type III taste cells in rodents, PGP9.5 and SNAP25, fail to stain any taste bud cells in the human postmortem tissue, although these antibodies do stain numerous nerve fibers throughout the specimen. Car4, another Type III cell marker, reacted with only a few taste cells in our samples. Finally, human vallate papillae have a general network of innervation similar to rodents and antibodies directed against SNAP25, PGP9.5, acetylated tubulin and P2X3 all stain free perigemmal nerve endings as well as intragemmal taste fibers. We conclude that with the exception of certain molecular features of Type III cells, human vallate papillae share the structural, morphological, and molecular features observed in rodents. PMID:26400924

A Comparison of Heat versus Methanol Fixation for Gram Staining Bacteria

ERIC Educational Resources Information Center

Minnerath, Jeanne M.; Roland, Jenna M.; Rossi, Lucas C.; Weishalla, Steven R.; Wolf, Melissa M.

2009-01-01

Gram staining bacteria is a fundamental technique introduced in general biology and microbiology laboratory courses. Two common problems students encounter when Gram staining bacteria are (1) having a difficult time locating bacterial cells on the microscope slide and (2) over-decolorizing bacterial cells during the staining procedure such that…

An imidazole functionalized pentameric thiophene displays different staining patterns in normal and malignant cells

NASA Astrophysics Data System (ADS)

Nilsson, Peter; Magnusson, Karin; Appelqvist, Hanna; Cieslar-Pobuda, Artur; Bäck, Marcus; Kågedal, Bertil; Jonasson, Jon; Los, Marek

2015-10-01

Molecular tools for fluorescent imaging of cells and their components are vital for understanding the function and activity of cells. Here, we report an imidazole functionalized pentameric oligothiophene, p-HTIm, that can be utilized for fluorescent imaging of cells. p-HTIm fluorescence in normal cells appeared in a peripheral punctate pattern partially co-localized with lysosomes, whereas a one-sided perinuclear Golgi associated localization of the dye was observed in malignant cells. The uptake of p-HTIm was temperature dependent and the intracellular target was reached within 1 h after staining. The ability of p-HTIm to stain cells was reduced when the imidazole side chain was chemically altered, verifying that specific imidazole side-chain functionalities are necessary for achieving the observed cellular staining. Our findings confirm that properly functionalized oligothiophenes can be utilized as fluorescent tools for vital staining of cells and that the selectivity towards distinct intracellular targets are highly dependent on the side-chain functionalities along the conjugated thiophene backbone.

Picomonas judraskeda Gen. Et Sp. Nov.: The First Identified Member of the Picozoa Phylum Nov., a Widespread Group of Picoeukaryotes, Formerly Known as ‘Picobiliphytes’

PubMed Central

Seenivasan, Ramkumar; Sausen, Nicole; Medlin, Linda K.; Melkonian, Michael

2013-01-01

In 2007, a novel, putatively photosynthetic picoeukaryotic lineage, the ‘picobiliphytes’, with no known close eukaryotic relatives, was reported from 18S environmental clone library sequences and fluorescence in situ hybridization. Although single cell genomics later showed these organisms to be heterotrophic rather than photosynthetic, until now this apparently widespread group of pico-(or nano-)eukaryotes has remained uncultured and the organisms could not be formally recognized. Here, we describe Picomonas judraskeda gen. et sp. nov., from marine coastal surface waters, which has a ‘picobiliphyte’ 18S rDNA signature. Using vital mitochondrial staining and cell sorting by flow cytometry, a single cell-derived culture was established. The cells are biflagellate, 2.5–3.8×2–2.5 µm in size, lack plastids and display a novel stereotypic cycle of cell motility (described as the “jump, drag, and skedaddle”-cycle). They consist of two hemispherical parts separated by a deep cleft, an anterior part that contains all major cell organelles including the flagellar apparatus, and a posterior part housing vacuoles/vesicles and the feeding apparatus, both parts separated by a large vacuolar cisterna. From serial section analyses of cells, fixed at putative stages of the feeding cycle, it is concluded that cells are not bacterivorous, but feed on small marine colloids of less than 150 nm diameter by fluid-phase, bulk flow endocytosis. Based on the novel features of cell motility, ultrastructure and feeding, and their isolated phylogenetic position, we establish a new phylum, Picozoa, for Picomonas judraskeda, representing an apparently widespread and ecologically important group of heterotrophic picoeukaryotes, formerly known as ‘picobiliphytes’. PMID:23555709

Ovarian Expression, Localization, and Function of Tissue Inhibitor of Metalloproteinase 3 (TIMP3) During the Periovulatory Period of the Human Menstrual Cycle1

PubMed Central

Rosewell, Katherine L.; Li, Feixue; Puttabyatappa, Muraly; Akin, James W.; Brännström, Mats; Curry, Thomas E.

2013-01-01

ABSTRACT Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability. PMID:24048576

Ovarian expression, localization, and function of tissue inhibitor of metalloproteinase 3 (TIMP3) during the periovulatory period of the human menstrual cycle.

PubMed

Rosewell, Katherine L; Li, Feixue; Puttabyatappa, Muraly; Akin, James W; Brännström, Mats; Curry, Thomas E

2013-11-01

Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability.

Berberine and Evodiamine Act Synergistically Against Human Breast Cancer MCF-7 Cells by Inducing Cell Cycle Arrest and Apoptosis.

PubMed

Du, Jia; Sun, Yang; Lu, Yi-Yu; Lau, Eric; Zhao, Ming; Zhou, Qian-Mei; Su, Shi-Bing

2017-11-01

The synergistic combinations of natural products have long been the basis of Traditional Chinese herbal Medicine formulas. In this study, we investigated the synergistic effects of a combination of berberine and evodiamine against human breast cancer MCF-7 cells in vitro and in vivo, and explored its mechanism. Cell survival was measured using the MTT assay. Apoptosis-related proteins were observed using western blot analysis. Apoptosis was detected with flow cytometric analysis and by Hoechst 33258 staining. Tumor xenografts were used in vivo. Compared to berberine or evodiamine treatments alone, the combination treatment of berberine (25 μM) and evodiamine (15 μM) synergistically inhibited the proliferation of MCF-7 cells in a time-dependent manner and resulted in the G 0 /G 1 phase accumulation of cells that exhibited increased expression levels of the CDK inhibitors p21 and p27 with a concomitant reduction in the expression levels of cell-cycle checkpoint proteins cyclin D1, cyclin E, CDK4, and CDK6. Furthermore, the combination treatment induced apoptosis that was accompanied by increased expression levels of p53 and Bax, reduced expression levels of Bcl-2, activation of caspase-7, and caspase-9, and the cleavage of PARP. The combination of berberine and evodiamine synergistically inhibited tumor growth in vivo in MCF-7 human breast cancer xenografts. Combination of berberine and evodiamine acts synergistically to suppress the proliferation of MCF-7 cells by inducing cell cycle arrest and apoptosis, illustrating the potential synergistic and combinatorial application of bioactive natural products. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Peptide-MHC Class I Tetramers Can Fail To Detect Relevant Functional T Cell Clonotypes and Underestimate Antigen-Reactive T Cell Populations.

PubMed

Rius, Cristina; Attaf, Meriem; Tungatt, Katie; Bianchi, Valentina; Legut, Mateusz; Bovay, Amandine; Donia, Marco; Thor Straten, Per; Peakman, Mark; Svane, Inge Marie; Ott, Sascha; Connor, Tom; Szomolay, Barbara; Dolton, Garry; Sewell, Andrew K

2018-04-01

Peptide-MHC (pMHC) multimers, usually used as streptavidin-based tetramers, have transformed the study of Ag-specific T cells by allowing direct detection, phenotyping, and enumeration within polyclonal T cell populations. These reagents are now a standard part of the immunology toolkit and have been used in many thousands of published studies. Unfortunately, the TCR-affinity threshold required for staining with standard pMHC multimer protocols is higher than that required for efficient T cell activation. This discrepancy makes it possible for pMHC multimer staining to miss fully functional T cells, especially where low-affinity TCRs predominate, such as in MHC class II-restricted responses or those directed against self-antigens. Several recent, somewhat alarming, reports indicate that pMHC staining might fail to detect the majority of functional T cells and have prompted suggestions that T cell immunology has become biased toward the type of cells amenable to detection with multimeric pMHC. We use several viral- and tumor-specific pMHC reagents to compare populations of human T cells stained by standard pMHC protocols and optimized protocols that we have developed. Our results confirm that optimized protocols recover greater populations of T cells that include fully functional T cell clonotypes that cannot be stained by regular pMHC-staining protocols. These results highlight the importance of using optimized procedures that include the use of protein kinase inhibitor and Ab cross-linking during staining to maximize the recovery of Ag-specific T cells and serve to further highlight that many previous quantifications of T cell responses with pMHC reagents are likely to have considerably underestimated the size of the relevant populations. Copyright © 2018 The Authors.

Kinetic Modeling of ABCG2 Transporter Heterogeneity: A Quantitative, Single-Cell Analysis of the Side Population Assay

PubMed Central

Prasanphanich, Adam F.; White, Douglas E.; Gran, Margaret A.

2016-01-01

The side population (SP) assay, a technique used in cancer and stem cell research, assesses the activity of ABC transporters on Hoechst staining in the presence and absence of transporter inhibition, identifying SP and non-SP cell (NSP) subpopulations by differential staining intensity. The interpretation of the assay is complicated because the transporter-mediated mechanisms fail to account for cell-to-cell variability within a population or adequately control the direct role of transporter activity on staining intensity. We hypothesized that differences in dye kinetics at the single-cell level, such as ABCG2 transporter-mediated efflux and DNA binding, are responsible for the differential cell staining that demarcates SP/NSP identity. We report changes in A549 phenotype during time in culture and with TGFβ treatment that correlate with SP size. Clonal expansion of individually sorted cells re-established both SP and NSPs, indicating that SP membership is dynamic. To assess the validity of a purely kinetics-based interpretation of SP/NSP identity, we developed a computational approach that simulated cell staining within a heterogeneous cell population; this exercise allowed for the direct inference of the role of transporter activity and inhibition on cell staining. Our simulated SP assay yielded appropriate SP responses for kinetic scenarios in which high transporter activity existed in a portion of the cells and little differential staining occurred in the majority of the population. With our approach for single-cell analysis, we observed SP and NSP cells at both ends of a transporter activity continuum, demonstrating that features of transporter activity as well as DNA content are determinants of SP/NSP identity. PMID:27851764

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients.

PubMed

Ostrow, Kimberly Laskie; Donaldson, Katelyn; Blakeley, Jaishri; Belzberg, Allan; Hoke, Ahmet

2015-01-01

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening.

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients

PubMed Central

Ostrow, Kimberly Laskie; Donaldson, Katelyn; Blakeley, Jaishri; Belzberg, Allan; Hoke, Ahmet

2015-01-01

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening. PMID:26657314

Mitomycin C in dacryocystorhinostomy: the search for the right concentration and duration--a fundamental study on human nasal mucosa fibroblasts.

PubMed

Ali, Mohammad Javed; Mariappan, Indumathi; Maddileti, Savitri; Ali, Md Hasnat; Naik, Milind N

2013-01-01

To establish primary cultures of human nasal mucosal fibroblasts (HNMFs) and to test the effect of varying concentrations of mitomycin C (MMC) and treatment durations on cellular proliferation and viability of the fibroblasts. Laboratory investigation. Nasal mucosa harvested from patients undergoing a dacryocystorhinostomy was used to establish primary cultures by explant culture method. Cells were expanded and frozen at every passage, and passage 3 cells were used for further experiments. The cells were then treated with different concentrations of mitomycin C (0.1-0.5 mg/ml) for different time periods (3, 5, and 10 minutes). Cell viability was checked by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cellular proliferation index was determined with bromodeoxyuridine immunostaining. Apoptotic index was measured using annexin A5 affinity assay, propidium iodide staining, and 4',6-diamidino-2-phenylindole counterstaining. The actin cytoskeletons of fibroblasts were studied using phalloidin staining. The doubling time of cultured HNMFs is approximately 24 hours. Similarly, 0.4 mg/ml beyond 5 minutes and 0.5 mg/ml concentration at all time points were lethal and caused extensive cell death when compared with controls. A concentration of 0.2 mg/ml for 3 minutes of exposure prevented cell proliferation of HNMF cells by inducing cell cycle arrest, without causing extensive apoptosis. The minimum effective concentration appears to be 0.2 mg/ml for 3 minutes. This in vitro study could be the starting point for further clinical and histopathologic studies to validate its clinical usefulness.

Cytoplasmic asters are required for progression past the first cell cycle in cloned mouse embryos.

PubMed

Miki, Hiromi; Inoue, Kimiko; Ogonuki, Narumi; Mochida, Keiji; Nagashima, Hiroshi; Baba, Tadashi; Ogura, Atsuo

2004-12-01

Unlike the oocytes of most other animal species, unfertilized murine oocytes contain cytoplasmic asters, which act as microtubule-organizing centers following fertilization. This study examined the role of asters during the first cell cycle of mouse nuclear transfer (NT) embryos. NT was performed by intracytoplasmic injection of cumulus cells. Cytoplasmic asters were localized by staining with an anti-alpha-tubulin antibody. Enucleation of MII oocytes caused no significant change in the number of cytoplasmic asters. The number of asters decreased after transfer of the donor nuclei into these enucleated oocytes, probably because some of the asters participated in the formation of the spindle that anchors the donor chromosomes. The cytoplasmic asters became undetectable within 2 h of oocyte activation, irrespective of the presence or absence of the donor chromosomes. After the standard NT protocol, a spindle-like structure persisted between the pseudopronuclei of these oocytes throughout the pronuclear stage. The asters reappeared shortly before the first mitosis and formed the mitotic spindle. When the donor nucleus was transferred into preactivated oocytes (delayed NT) that were devoid of free asters, the microtubules and microfilaments were distributed irregularly in the ooplasm and formed dense bundles within the cytoplasm. Thereafter, all of the delayed NT oocytes underwent fragmentation and arrested development. Treatment of these delayed NT oocytes with Taxol, which is a microtubule-assembling agent, resulted in the formation of several aster-like structures and reduced fragmentation. Some Taxol-treated oocytes completed the first cell cycle and developed further. This study demonstrates that cytoplasmic asters play a crucial role during the first cell cycle of murine NT embryos. Therefore, in mouse NT, the use of MII oocytes as recipients is essential, not only for chromatin reprogramming as previously reported, but also for normal cytoskeletal organization in reconstructed oocytes.

Anticancer activity of polysaccharide from Glehnia littoralis on human lung cancer cell line A549.

PubMed

Wu, Jun; Gao, Weiping; Song, Zhuoyue; Xiong, Qingping; Xu, Yingtao; Han, Yun; Yuan, Jun; Zhang, Rong; Cheng, Yunbo; Fang, Jiansong; Li, Weirong; Wang, Qi

2018-01-01

The purpose of this study was to investigate the anticancer activity of polysaccharide (PGL) from Glehnia littoralis on human lung cancer cell line A549. Based on MTT assay, the results suggested that PGL could significantly reduce A549 cells proliferation in a time- and dose-dependent manner. In addition, PGL displayed an inhibitory activity for the A549 cells migration in Transwell migration assay. The results from both flow cytometry analysis and Hochst 3342 staining of apoptotic cells indicated that PGL could promote apoptosis, and induce cycle arrest of A549 cells. Moreover, immunofluorescence assay elucidated PGL could also down-regulate expression of proliferating cell nuclear antigen (PCNA). Overall, these results showed that PGL exerts a strong anticancer action through inhibiting the A549 cells migration, proliferation and inducing cell apoptosis. It could be a new source of natural anticancer agent against lung cancer with potential value in supplements and medicine. Copyright © 2017 Elsevier B.V. All rights reserved.

Nuclear envelope breakdown and mitosis in sand dollar embryos is inhibited by microinjection of calcium buffers in a calcium-reversible fashion, and by antagonists of intracellular Ca2+ channels.

PubMed

Silver, R B

1989-01-01

Transient elevations in intracellular free Ca2+ are believed to signal the initiation of mitosis. This model predicts that mitosis might be arrested prior to nuclear envelope breakdown (NEB) or anaphase onset if intracellular Ca2+ concentration is buffered or dampened. Microinjection of a discrete dose of Ca2+ into the cell might then release the cell to resume mitotic cycling. Experimentally, one blastomere of two cell sand dollar (Echinaracnius parma) embryos was microinjected with Ca2+ buffers, Ca2+ solutions, or Ca2+ channel antagonists; the uninjected blastomere was the control. Cells were loaded with 10 pl doses of the Ca2+ buffer antipyrylazo III (ApIII) at specific times in the cell cycle to attempt a competitive inhibition of Ca2+-dependent steps in NEB and initiation of mitosis. Injection of 50 microM ApIII 6 min prior to NEB blocked NEB and further cell cycling. Injections of solutions between 0 and 30 microM ApIII were without observable effect. Control injections had no observable effect on the injected cell. Cells injected with 50 microM ApIII 2 min prior to the onset of anaphase in control cells were blocked in metaphase. Cells were sensitive to Ca2+ buffer injections 6 min prior to NEB (with a 40- to 45-sec duration), and 2 min prior to anaphase onset (with a 10- to 20-sec duration). Vital staining of these cells with H33342 demonstrated that they contained only one nucleus that had the same fluorescence intensity as seen prior to microinjection, and thus did not undergo DNA synthesis following the imposition of the Ca2+ buffer block to mitosis. Cells arrested in this fashion did not spontaneously resume mitotic cycling. This Ca2+ buffer-induced mitotic arrest was, however, experimentally reversible. Cells arrested with 50 microM ApIII 6 min prior to NEB could be returned to mitotic activity by injecting 300 microM CaCl2 5 min after the ApIII injection. The double injected cells resumed cycling, NEB, and mitosis after a delay of one cell cycle period, and remained one cell cycle out of phase with the sister (control) cell. Microinjection of antagonists of endomembrane Ca2+ channels inhibited NEB and anaphase onset in a concentration- and time-dependent fashion. The effective doses of compounds tested were 7 micrograms/ml ryanodine and 500 micrograms/ml TMB-8. These results indicate that a transient elevation of intracellular Ca2+ from endomembrane stores is required to initiate mitotic events, namely NEB and anaphase onset.(ABSTRACT TRUNCATED AT 400 WORDS)

New in vitro insights on a cell death pathway induced by magnolol and honokiol in aristolochic acid tubulotoxicity.

PubMed

Bunel, Valérian; Antoine, Marie-Hélène; Stévigny, Caroline; Nortier, Joëlle; Duez, Pierre

2016-01-01

Aristolochic acids (AA) are nephrotoxic agents found in Aristolochia species whose consumption leads to the onset of a progressive tubulointerstitial fibrosis. This AA-nephropathy was first reported during the Belgian outbreak of the 1990's in which more than a hundred patients consumed slimming pills containing an Aristolochia species and Magnolia officinalis. The patients developed an end-stage kidney disease requiring dialysis or transplantation. Magnolol and honokiol are bioactive compounds from M. officinalis known for their potent antioxidant activity. As they can alleviate oxidative stress, we investigated their respective effects on AA-mediated tubulotoxicity using HK-2 cells. Magnolol and honokiol were able to reduce the oxidative stress associated with AA-treatment. Cytotoxicity alleviation was further investigated and overall cell viability measurements unexpectedly revealed that both compounds worsened the survival of AA-treated cells. Flow cytometry analyses of annexin V/PI stained cells indicated that the lignans efficiently prevented AA-induced apoptosis; but favored necrosis. Microscopy observations highlighted extensive vacuolization; other types of cell death, including autophagy, paraptosis or accelerated senescence were excluded. Ki-67 index and cell cycle analysis indicated that both magnolol and honokiol inhibited proliferation by blocking the cell cycle at the G1 phase; they also prevented the AA-induced G2/M arrest. Copyright © 2015 Elsevier Ltd. All rights reserved.

The anti-tumor effect and biological activities of the extract JMM6 from the stem-barks of the Chinese Juglans mandshurica Maxim on human hepatoma cell line BEL-7402.

PubMed

Zhang, Yongli; Cui, Yuqiang; Zhu, Jiayong; Li, Hongzhi; Mao, Jianwen; Jin, Xiaobao; Wang, Xiangsheng; Du, Yifan; Lu, Jiazheng

2013-01-01

Juglans mandshurica Maxim is a traditional herbal medicines in China, and its anti-tumor bioactivities are of research interest. Bioassay-guided fractionation method was employed to isolate anti-tumor compounds from the stem barks of the Juglans mandshurica Maxim. The anti-tumor effect and biological activities of the extracted compound JMM6 were studied in BEL-7402 cells by MTT, Cell cycle analysis, Hoechst 33342 staining, Annexin V-FITC/PI assay and Detection of mitochondrial membrane potential (ΔΨm). After treatment with the JMM6, the growth of BEL-7402 cells was inhibited and cells displayed typical morphological apoptotic characteristics. Further investigations revealed that treatment with JMM6 mainly caused G2/M cell cycle arrest and induced apoptosis in BEL-7402 cells. To evaluate the alteration of mitochondria in JMM6 induced apoptosis. The data showed that JMM6 decreased significantly the ΔΨm, causing the depolarization of the mitochondrial membrane. Our results show that the JMM6 will have a potential advantage of anti-tumor, less harmful to normal cells. This paper not only summarized the JMM6 pick-up technology from Juglans mandshurica Maxim and biological characteristic, but also may provide further evidence to exploit the potential medicine compounds from the stem-barks of the Chinese Juglans mandshurica Maxim.

[Effects of rapamycin on biological characteristics of bone marrow mesenchymal stem cells from patients with aplastic anemia].

PubMed

Wang, Xin; Ma, Feng-Xia; Lu, Shi-Hong; Chi, Ying; Chen, Fang; Li, Xue; Li, Juan-Juan; Du, Wen-Jing; Feng, Ying; Cui, Jun-Jie; Song, Bao-Quan; Han, Zhong-Chao

2014-06-01

This study was aimed to investigate the effects of rapamycin on biological function and autophagy of bone marrow mesenchymal stem cells (BM-MSC) from patients with aplastic anemia so as to provide experimental basis for the clinical treatment of aplastic anemia (AA) with rapamycin. BM-MSC were treated with different concentrations of rapamycin (0, 10, 50, 100 nmol/L) for 48 h, the expression of LC3B protein was detected by Western blot to observe the effect of rapamycin on cell autophagy; cell apoptosis and cell cycles were detected by flow cytometry; the proliferation of BM-MSC of AA patients was measured by cell counting kit-8; the adipogenic differentiation of BM-MSC were tested by oil red O staining after adipogenic induction for 2 weeks; the adipogenic related genes (LPL, CFD, PPARγ) were detected by real-time PCR. The results showed that the proliferation and adipogenesis of BM-MSC of AA patients were inhibited by rapamycin. Moreover, the autophagy and apoptosis of BM-MSC were increased by rapamycin in a dose-dependent way.Rapamycin arrested the BM-MSC in G0/G1 phase and prevented them into S phase (P < 0.05). It is concluded that rapamycin plays an critical role in inhibiting cell proliferation, cell cycles, and adipogenesis, these effects may be related with the autophagy activation and mTOR inhibition resulting from rapamycin.

Antibacterial and Cytotoxic Activity of Compounds Isolated from Flourensia oolepis.

PubMed

Joray, Mariana Belén; Trucco, Lucas Daniel; González, María Laura; Napal, Georgina Natalia Díaz; Palacios, Sara María; Bocco, José Luis; Carpinella, María Cecilia

2015-01-01

The antibacterial and cytotoxic effects of metabolites isolated from an antibacterial extract of Flourensia oolepis were evaluated. Bioguided fractionation led to five flavonoids, identified as 2',4'-dihydroxychalcone (1), isoliquiritigenin (2), pinocembrin (3), 7-hydroxyflavanone (4), and 7,4'-dihydroxy-3'-methoxyflavanone (5). Compound 1 showed the highest antibacterial effect, with minimum inhibitory concentration (MIC) values ranging from 31 to 62 and 62 to 250 μg/mL, against Gram-positive and Gram-negative bacteria, respectively. On further assays, the cytotoxic effect of compounds 1-5 was determined by MTT assay on acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) cell lines including their multidrug resistant (MDR) phenotypes. Compound 1 induced a remarkable cytotoxic activity toward ALL cells (IC50 = 6.6-9.9 μM) and a lower effect against CML cells (IC50 = 27.5-30.0 μM). Flow cytometry was used to analyze cell cycle distribution and cell death by PI-labeled cells and by Annexin V/PI staining, respectively. Upon treatment, 1 induced cell cycle arrest in the G2/M phase accompanied by a strong induction of apoptosis. These results describe for the first time the antibacterial metabolites of F. oolepis extract, with 1 being the most effective. This chalcone also emerges as a selective cytotoxic agent against sensitive and resistant leukemic cells, highlighting its potential as a lead compound.

Nitric Oxide Synthase and Neuronal NADPH Diaphorase are Identical in Brain and Peripheral Tissues

NASA Astrophysics Data System (ADS)

Dawson, Ted M.; Bredt, David S.; Fotuhi, Majid; Hwang, Paul M.; Snyder, Solomon H.

1991-09-01

NADPH diaphorase staining neurons, uniquely resistant to toxic insults and neurodegenerative disorders, have been colocalized with neurons in the brain and peripheral tissue containing nitric oxide synthase (EC 1.14.23.-), which generates nitric oxide (NO), a recently identified neuronal messenger molecule. In the corpus striatum and cerebral cortex, NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in medium to large aspiny neurons. These same neurons colocalize with somatostatin and neuropeptide Y immunoreactivity. NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in the pedunculopontine nucleus with choline acetyltransferase-containing cells and are also colocalized in amacrine cells of the inner nuclear layer and ganglion cells of the retina, myenteric plexus neurons of the intestine, and ganglion cells of the adrenal medulla. Transfection of human kidney cells with NO synthase cDNA elicits NADPH diaphorase staining. The ratio of NO synthase to NADPH diaphorase staining in the transfected cells is the same as in neurons, indicating that NO synthase fully accounts for observed NADPH staining. The identity of neuronal NO synthase and NADPH diaphorase suggests a role for NO in modulating neurotoxicity.

Thermal Inactivation of Bacillus Anthracis using Laser Irradiation of Micro-Etched Platforms

DTIC Science & Technology

2009-03-01

Application of Malachite Green Staining protocol for greater clarity of spore population; fluorescent staining techniques. Further assess toxicity or...to normal staining procedures. The primary stain in the endospore stain procedure, malachite green, is driven into the cells with heat. Malachite ...dimethyl-aniline is a toxic chemical primarily used as a dye. Since malachite green is water-soluble and does not adhere well to the cell, and since the

Whole blood staining in suspension for nonspecific esterase and alkaline phosphatase analyzed with a Technicon H-1.

PubMed

Ross, D W; Bishop, C; Henderson, A; Kaplow, L

1990-01-01

We adapted previously published methods for nonspecific esterase and alkaline phosphatase staining of white blood cells in suspension for use on a Technicon H-1 hematology analyzer. The objective was to develop a semiautomated method using whole blood that could be employed on a large scale for hematology laboratory applications, including toxicology studies, measurement of neutrophil left shift, and cytochemical classification of myeloid leukemias. The nonspecific esterase method uses the pararosaniline stain, generating the unstable substrate from two stable precursors. Whole blood is added to the substrate plus dye mix. Next, acid lysis and fixation steps destroy red cells and stabilize the monocyte staining. The alkaline phosphatase stain employs a stable naphthyl phosphate substrate and fast blue B coupling dye. The red cells are lysed with a pH 10.3 propanediol buffer, and the white blood cells are then stabilized with formalin fixation. For both methods the staining is performed off-line, and the sample is then diluted with propanediol to match the refractive index of the sheath on the H-1 analyzer, before aspiration into the direct cytometry port. A cytogram of scattered versus absorbed light is obtained. The number of cells staining and the intensity of the stain can be quantified from the cytogram.

Loss of histochemical identity in mast cells lacking carboxypeptidase A.

PubMed

Feyerabend, Thorsten B; Hausser, Heinz; Tietz, Annette; Blum, Carmen; Hellman, Lars; Straus, Anita H; Takahashi, Hélio K; Morgan, Ellen S; Dvorak, Ann M; Fehling, Hans Jörg; Rodewald, Hans-Reimer

2005-07-01

Mast cell carboxypeptidase A (Mc-cpa) is a highly conserved secretory granule protease. The onset of expression in mast cell progenitors and lineage specificity suggest an important role for Mc-cpa in mast cells. To address the function of Mc-cpa, we generated Mc-cpa-null mice. Mc-cpa-/- mast cells lacked carboxypeptidase activity, revealing that Mc-cpa is a nonredundant enzyme. While Mc-cpa-/- peritoneal mast cells were ultrastructurally normal and synthesized normal amounts of heparin, they displayed striking histochemical and biochemical hallmarks of immature mast cells. Wild-type peritoneal mast cells had a mature phenotype characterized by differential histochemical staining with proteoglycan-reactive dyes (cells do not stain with alcian blue but stain with safranin and with berberine) and a high side scatter to forward scatter ratio by flow cytometry and were detergent resistant. In contrast, Mc-cpa-/- peritoneal mast cells, like immature bone marrow-derived cultured mast cells, stained with alcian blue normally or weakly and either did not stain with safranin and berberine or stained weakly, had a low side scatter to forward scatter ratio, and were detergent sensitive. This phenotype was partially ameliorated with age. Thus, histochemistry and flow cytometry, commonly used to measure mast cell maturation, deviated from morphology in Mc-cpa-/- mice. The Mc-cpa-/- mast cell phenotype was not associated with defects in degranulation in vitro or passive cutaneous anaphylaxis in vivo. Collectively, Mc-cpa plays a crucial role for the generation of phenotypically mature mast cells.

Loss of Histochemical Identity in Mast Cells Lacking Carboxypeptidase A

PubMed Central

Feyerabend, Thorsten B.; Hausser, Heinz; Tietz, Annette; Blum, Carmen; Hellman, Lars; Straus, Anita H.; Takahashi, Hélio K.; Morgan, Ellen S.; Dvorak, Ann M.; Fehling, Hans Jörg; Rodewald, Hans-Reimer

2005-01-01

Mast cell carboxypeptidase A (Mc-cpa) is a highly conserved secretory granule protease. The onset of expression in mast cell progenitors and lineage specificity suggest an important role for Mc-cpa in mast cells. To address the function of Mc-cpa, we generated Mc-cpa-null mice. Mc-cpa−/− mast cells lacked carboxypeptidase activity, revealing that Mc-cpa is a nonredundant enzyme. While Mc-cpa−/− peritoneal mast cells were ultrastructurally normal and synthesized normal amounts of heparin, they displayed striking histochemical and biochemical hallmarks of immature mast cells. Wild-type peritoneal mast cells had a mature phenotype characterized by differential histochemical staining with proteoglycan-reactive dyes (cells do not stain with alcian blue but stain with safranin and with berberine) and a high side scatter to forward scatter ratio by flow cytometry and were detergent resistant. In contrast, Mc-cpa−/− peritoneal mast cells, like immature bone marrow-derived cultured mast cells, stained with alcian blue normally or weakly and either did not stain with safranin and berberine or stained weakly, had a low side scatter to forward scatter ratio, and were detergent sensitive. This phenotype was partially ameliorated with age. Thus, histochemistry and flow cytometry, commonly used to measure mast cell maturation, deviated from morphology in Mc-cpa−/− mice. The Mc-cpa−/− mast cell phenotype was not associated with defects in degranulation in vitro or passive cutaneous anaphylaxis in vivo. Collectively, Mc-cpa plays a crucial role for the generation of phenotypically mature mast cells. PMID:15988029

Alkaline Phosphatase-Positive Immortal Mouse Embryo Fibroblasts Are Cells in a Transitional Reprogramming State Induced to Face Environmental Stresses

PubMed Central

Evangelista, Monica; Baroudi, Mariama El; Rizzo, Milena; Tuccoli, Andrea; Poliseno, Laura; Pellegrini, Marco; Rainaldi, Giuseppe

2015-01-01

In this study, we report that immortal mouse embryonic fibroblasts (I-MEFs) have a baseline level of cells positive for alkaline phosphatase (AP+) staining. Environmental stresses, including long-lasting growth in the absence of expansion and treatment with drugs, enhance the frequency of AP+ I-MEFs. By adapting fast red AP staining to the sorting procedure, we separated AP+ and AP− I-MEFs and demonstrated that the differentially expressed genes are consistent with a reprogrammed phenotype. In particular, we found that sestrin 1 is upregulated in AP+ I-MEFs. We focused on this gene and demonstrated that increased sestrin 1 expression is accompanied by the growth of I-MEFs in the absence of expansion and occurs before the formation of AP+ I-MEFs. Together with sestrin 1 upregulation, we found that AP+ I-MEFs accumulated in the G1 phase of the cell cycle, suggesting that the two events are causally related. Accordingly, we found that silencing sestrin 1 expression reduced the frequency and G1 accumulation of AP+ I-MEFs. Taken together, our data suggested that I-MEFs stressed by environmental changes acquire the AP+ phenotype and achieve a quiescent state characterized by a new transcriptional network. PMID:26740745

Alkaline Phosphatase-Positive Immortal Mouse Embryo Fibroblasts Are Cells in a Transitional Reprogramming State Induced to Face Environmental Stresses.

PubMed

Evangelista, Monica; Baroudi, Mariama El; Rizzo, Milena; Tuccoli, Andrea; Poliseno, Laura; Pellegrini, Marco; Rainaldi, Giuseppe

2015-01-01

In this study, we report that immortal mouse embryonic fibroblasts (I-MEFs) have a baseline level of cells positive for alkaline phosphatase (AP(+)) staining. Environmental stresses, including long-lasting growth in the absence of expansion and treatment with drugs, enhance the frequency of AP(+) I-MEFs. By adapting fast red AP staining to the sorting procedure, we separated AP(+) and AP(-) I-MEFs and demonstrated that the differentially expressed genes are consistent with a reprogrammed phenotype. In particular, we found that sestrin 1 is upregulated in AP(+) I-MEFs. We focused on this gene and demonstrated that increased sestrin 1 expression is accompanied by the growth of I-MEFs in the absence of expansion and occurs before the formation of AP(+) I-MEFs. Together with sestrin 1 upregulation, we found that AP(+) I-MEFs accumulated in the G1 phase of the cell cycle, suggesting that the two events are causally related. Accordingly, we found that silencing sestrin 1 expression reduced the frequency and G1 accumulation of AP(+) I-MEFs. Taken together, our data suggested that I-MEFs stressed by environmental changes acquire the AP(+) phenotype and achieve a quiescent state characterized by a new transcriptional network.

Anti-tumor effect of evodiamine by inducing Akt-mediated apoptosis in hepatocellular carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Fan; Shi, Le; Liang, Tao

Background: Evodiamine is an alkaloid extracted from Euodia rutaecarpa (Juss.) Benth. There is little information about the mechanisms of evodiamine on the apoptosis of hepatocellular carcinoma (HCC). Materials and methods: A xenograft model and CCK8 assay were used to investigate the anti-HCC effect of evodiamine. The effect of evodiamine on apoptosis was evaluated by DAPI staining and flow cytometry. Western blot analyses and immunohistochemistry were processed to assess the protein expressions of Akt and apoptotic proteins. Results: Evodiamine suppressed tumor growth, improved the expression of cleaved-caspase3 and decreased tumor specific growth factor (TSGF) and alpha fetoprotein (AFP) activities. Furthermore, evodiaminemore » inhibited cell viability and induced cell cycle arrest. DAPI staining revealed nuclear condensation in evodiamine-treated groups. Meanwhile, evodiamine increased the number of apoptotic cells. Furthermore, evodiamine suppressed Akt and regulated apoptotic proteins in HepG2 cells. Evodiamine decreased p-Akt levels activated by SC79, which led to the increase of bax/bcl-2 and cleaved-caspase3. Conclusions: Our findings suggested that evodiamine could exert anti-HCC effect through inducing Akt-mediated apoptosis. Evodiamine has the potential to be a therapeutic medicine for HCCs. - Highlights: • Anti-tumor effect of evodiamine in hepatocellular carcinoma. • Evodiamine induces apoptosis in hepatocellular carcinoma. • The correlation between induction of apoptosis and Akt expression.« less

Human corneal epithelial cell shedding and fluorescein staining in response to silicone hydrogel lenses and contact lens disinfecting solutions.

PubMed

Gorbet, Maud; Peterson, Rachael; McCanna, David; Woods, Craig; Jones, Lyndon; Fonn, Desmond

2014-03-01

A pilot study was conducted to evaluate human corneal epithelial cell shedding in response to wearing a silicone hydrogel contact lens/solution combination inducing corneal staining. The nature of ex vivo collected cells staining with fluorescein was also examined. A contralateral eye study was conducted in which up to eight participants were unilaterally exposed to a multipurpose contact lens solution/silicone hydrogel lens combination previously shown to induce corneal staining (renu® fresh™ and balafilcon A; test eye), with the other eye using a combination of balafilcon A soaked in a hydrogen peroxide care system (Clear Care®; control eye). Lenses were worn for 2, 4 or 6 hours. Corneal staining was graded after lens removal. The Ocular Surface Cell Collection Apparatus was used to collect cells from the cornea and the contact lens. In the test eye, maximum solution-induced corneal staining (SICS) was observed after 2 hours of lens wear (reducing significantly by 4 hours; p < 0.001). There were significantly more cells collected from the test eye after 4 hours of lens wear when compared to the control eye and the collection from the test eye after 2 hours (for both; n = 5; p < 0.001). The total cell yield at 4 hours was 813 ± 333 and 455 ± 218 for the test and control eyes, respectively (N = 5, triplicate, p = 0.003). A number of cells were observed to have taken up the fluorescein dye from the initial fluorescein instillation. Confocal microscopy of fluorescein-stained cells revealed that fluorescein was present throughout the cell cytoplasm and was retained in the cells for many hours after recovery from the corneal surface. This pilot study indicates that increased epithelial cell shedding was associated with a lens-solution combination which induces SICS. Our data provides insight into the transient nature of the SICS reaction and the nature of fluorescein staining observed in SICS.

Retinal ganglion cell topography and spatial resolving power in penguins.

PubMed

Coimbra, João Paulo; Nolan, Paul M; Collin, Shaun P; Hart, Nathan S

2012-01-01

Penguins are a group of flightless seabirds that exhibit numerous morphological, behavioral and ecological adaptations to their amphibious lifestyle, but little is known about the topographic organization of neurons in their retinas. In this study, we used retinal wholemounts and stereological methods to estimate the total number and topographic distribution of retinal ganglion cells in addition to an anatomical estimate of spatial resolving power in two species of penguins: the little penguin, Eudyptula minor, and the king penguin, Aptenodytes patagonicus. The total number of ganglion cells per retina was approximately 1,200,000 in the little penguin and 1,110,000 in the king penguin. The topographic distribution of retinal ganglion cells in both species revealed the presence of a prominent horizontal visual streak with steeper gradients in the little penguin. The little penguin retinas showed ganglion cell density peaks of 21,867 cells/mm², affording spatial resolution in water of 17.07-17.46 cycles/degree (12.81-13.09 cycles/degree in air). In contrast, the king penguin showed a relatively lower peak density of ganglion cells of 14,222 cells/mm², but--due to its larger eye--slightly higher spatial resolution in water of 20.40 cycles/degree (15.30 cycles/degree in air). In addition, we mapped the distribution of giant ganglion cells in both penguin species using Nissl-stained wholemounts. In both species, topographic mapping of this cell type revealed the presence of an area gigantocellularis with a concentric organization of isodensity contours showing a peak in the far temporal retina of approximately 70 cells/mm² in the little penguin and 39 cells/mm² in the king penguin. Giant ganglion cell densities gradually fall towards the outermost isodensity contours revealing the presence of a vertically organized streak. In the little penguin, we confirmed our cytological characterization of giant ganglion cells using immunohistochemistry for microtubule-associated protein 2. This suite of retinal specializations, which is also observed in the closely related procellariiform seabirds, affords the eyes of the little and king penguins panoramic surveillance of the horizon and motion detection in the frontal visual field. Copyright © 2012 S. Karger AG, Basel.

Shade changing effectiveness of plasdone and blue covarine-based whitening toothpaste on teeth stained with chlorhexidine and black tea.

PubMed

Bergesch, Vania; Baggio Aguiar, Flávio Henrique; Turssi, Cecilia Pedroso; Gomes França, Fabiana Mantovani; Basting, Roberta Tarkany; Botelho Amaral, Flávia Lucisano

2017-01-01

This study evaluated the effectiveness of toothbrushing with whitening toothpaste in altering the shade of stained human enamel. Thirty fragments of human enamel, stained with chlorhexidine/black tea underwent 1000 and 5000 brushing cycles (BC) with ( n = 10): PLS (Gel Dental Day, Bitufo), Close Up White Now, Unilever (COVB) and regular (Gel Dental Night, Bitufo) toothpaste. Images were taken before staining (baseline), after staining (STN) and following 1000 and 5000 BC and were analyzed using the CIELAB parameters (ΔE, Δb and ΔL). Data were submitted to two-way ANOVA (α = 0.05). ΔE was higher from STN to baseline; 1000 BC to STN and 5000 BC to STN ( P < 0.001). Significant differences in Δb values were found from 1000 BC to STN and 5000 BC to STN. For COVB, greater ΔL was observed from 1000 BC to STN, what differed statistically from the regular toothpaste ( P < 0.05). There was no difference between toothpaste when ΔL was calculated from 5000 CB to STN. Toothpaste containing COVB or PLS in association with 5000 BCs showed similar effectiveness in changing enamel shade; but after the first 1000 toothbrushing cycles, the use of COVB toothpaste promoted higher lightness in stained enamel.

Differential staining of bacteria: gram stain.

PubMed

Moyes, Rita B; Reynolds, Jackie; Breakwell, Donald P

2009-11-01

In 1884, Hans Christian Gram, a Danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. This multistep, sequential staining protocol separates bacteria into four groups based on cell morphology and cell wall structure: Gram-positive cocci, Gram-negative cocci, Gram-positive rods, and Gram-negative rods. The Gram stain is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures. (c) 2009 by John Wiley & Sons, Inc.

Mechanism for ginkgolic acid (15 : 1)-induced MDCK cell necrosis: Mitochondria and lysosomes damages and cell cycle arrest.

PubMed

Yao, Qing-Qing; Liu, Zhen-Hua; Xu, Ming-Cheng; Hu, Hai-Hong; Zhou, Hui; Jiang, Hui-Di; Yu, Lu-Shan; Zeng, Su

2017-05-01

Ginkgolic acids (GAs), primarily found in the leaves, nuts, and testa of ginkgo biloba, have been identified with suspected allergenic, genotoxic and cytotoxic properties. However, little information is available about GAs toxicity in kidneys and the underlying mechanism has not been thoroughly elucidated so far. Instead of GAs extract, the renal cytotoxicity of GA (15 : 1), which was isolated from the testa of Ginkgo biloba, was assessed in vitro by using MDCK cells. The action of GA (15 : 1) on cell viability was evaluated by the MTT and neutral red uptake assays. Compared with the control, the cytotoxicity of GA (15 : 1) on MDCK cells displayed a time- and dose-dependent manner, suggesting the cells mitochondria and lysosomes were damaged. It was confirmed that GA (15 : 1) resulted in the loss of cells mitochondrial trans-membrane potential (ΔΨm). In propidium iodide (PI) staining analysis, GA (15 : 1) induced cell cycle arrest at the G0/G1 and G2/M phases, influencing on the DNA synthesis and cell mitosis. Characteristics of necrotic cell death were observed in MDCK cells at the experimental conditions, as a result of DNA agarose gel electrophoresis and morphological observation of MDCK cells. In conclusion, these findings might provide useful information for a better understanding of the GA (15 : 1) induced renal toxicity. Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

[Histological structure of the trabecular meshwork in the eyeball: challenging the traditional concept and preliminary findings in rabbits, rats and mice].

PubMed

Shi, Yun; Zhou, Fan-Qi; Luo, Zhou-Cai; Chen, Ying-Hua; Chen, Yu; Dong, Wei-Ren

2017-10-20

To verify that the trabecular meshwork (TM) in the wall of the eyeball consists of smooth muscle fibers instead of collagen fibers or endothelial cells. Eighteen fresh eyeballs from 3 rabbits, 3 SD rats and 3 mice were sectioned along the sagittal plane and sliced after paraffin embedding for HE staining, VG staining, Masson staining, α-SMA immunohistochemistry or CD31 immunohistochemistry. These slices were observed under microscope and the structure of the TM was compared with those of scleral collagen fibers, ciliary muscles and endothelial cells. HE staining of the eyeball slices from the 3 animal species resulted in purplish red staining of the TM, which was highly consistent with ciliary muscle fibers. The cell?like structures on the surface of the TM were not clearly outlined, with flat nuclei showing a dark purple staining; these structures did not show obvious boundaries from the TM. Ciliary muscle fibers, which were smooth muscle cells in nature, were aligned in bundles in various directions. The longitudinally sectioned cells were flat and contained purplish cytoplasm and highly flattened nuclei. Scleral collagen fibers were stained dark red with a few fibroblasts sandwiched among them. The long axis of the fibroblasts was in parallel with that of the collagen fibers. The outline of the fibroblast was not clear and the nucleus was flat in dark blue. The vascular endothelial cells presented with different morphologies and contained light purplish cytoplasm and dark nuclei, protruding into the vascular cavity. VG staining of the TM revealed a pale red filamentous structure, and the collagen fibers were stained bright red. Masson staining of the TM showed a reticular structure consisting mainly of dark red fibers intermingled with thin green fibers. Scleral collagen fibers presented with a cord?like green wavy structure. The endothelial cells were green and flat, while the ciliary smooth muscle fibers were purple. In immunohistochemistry for α?SMA, the TM and the ciliary smooth muscle fibers showed a strong positivity in the cytoplasm, while the scleral collagen fibers and vascular endothelial cells showed negative staining; immunohistochemistry for CD31 showed no obvious positive staining in the TM, collagen fibers or ciliary smooth muscle cells from all the animals in spite of slight differences among them. The TM consists mainly of smooth muscle fibers with a thin layer of peripheral endomysium without endothelial cells.

SKLB060 Reversibly Binds to Colchicine Site of Tubulin and Possesses Efficacy in Multidrug-Resistant Cell Lines.

PubMed

Yan, Wei; Yang, Tao; Yang, Jianhong; Wang, Taijin; Yu, Yamei; Wang, Yuxi; Chen, Qiang; Bai, Peng; Li, Dan; Ye, Haoyu; Qiu, Qiang; Zhou, Yongzhao; Hu, Yiguo; Yang, Shengyong; Wei, Yuquan; Li, Weimin; Chen, Lijuan

2018-05-22

Many tubulin inhibitors are in clinical use as anti-cancer drugs. In our previous study, a novel series of 4-substituted coumarins derivatives were identified as novel tubulin inhibitors. Here, we report the anti-cancer activity and underlying mechanism of one of the 4-substituted coumarins derivatives (SKLB060). The anti-cancer activity of SKLB060 was tested on 13 different cancer cell lines and four xenograft cancer models. Immunofluorescence staining, cell cycle analysis, and tubulin polymerization assay were employed to study the inhibition of tubulin. N, N '-Ethylenebis(iodoacetamide) assay was used to measure binding to the colchicine site. Wound-healing migration and tube formation assays were performed on human umbilical vascular endothelial cells to study anti-vascular activity (the ability to inhibit blood vessel growth). Mitotic block reversibility and structural biology assays were used to investigate the SKLB060-tubulin bound model. SKLB060 inhibited tubulin polymerization and subsequently induced G2/M cell cycle arrest and apoptosis in cancer cells. SKLB060 bound to the colchicine site of β-tubulin and showed antivascular activity in vitro. Moreover, SKLB060 induced reversible cell cycle arrest and reversible inhibition of tubulin polymerization. A mitotic block reversibility assay showed that the effects of SKLB060 have greater reversibility than those of colcemid (a reversible tubulin inhibitor), indicating that SKLB060 binds to tubulin in a totally reversible manner. The crystal structures of SKLB060-tubulin complexes confirmed that SKLB060 binds to the colchicine site, and the natural coumarin ring in SKLB060 enables reversible binding. These results reveal that SKLB060 is a powerful and reversible microtubule inhibitor that binds to the colchicine site and is effective in multidrug-resistant cell lines. © 2018 The Author(s). Published by S. Karger AG, Basel.

Seasonal morphological variations and age-related changes of the seminal vesicle of viscacha (Lagostomus maximus maximus): an ultrastructural and immunohistochemical study.

PubMed

Chaves, Eduardo M; Aguilera-Merlo, Claudia; Cruceño, Albana; Fogal, Teresa; Piezzi, Ramón; Scardapane, Luis; Dominguez, Susana

2012-05-01

The viscacha is a seasonal rodent that exhibit an annual reproductive cycle with periods of maximum reproductive activity and gonadal regression. We studied seasonal variations in the morphology and cellular population of the seminal vesicles (SVs) during both periods and in impuber animals. Seminal vesicles were studied by light and electronic microscopy. Measurements of epithelial height, nuclear diameter, luminal diameter, and muscular layer were performed. Also, we studied the distribution of androgen receptors (AR) in this gland during the reproductive cycle and in impuber animal. During gonadal regression, principal and clear cells showed signs of reduced functional activity. These were characterized by an epithelium of smaller height, irregular nuclei, and cytoplasm with few organelles, dilated cisterns, and glycogen granules. In impuber animals, the principal cells showed large nuclei with chromatin lax and cytoplasm with small mitochondria, poorly developed Golgi apparatus, and granules of glycogen. On the other hand, the cells exhibited seasonal variations in the distribution and percentage of immunolabeled cells to AR throughout the annual reproductive cycle. During the gonadal regression period, glandular mucosa exhibited numerous epithelial cells with intense nuclear staining. However, fibromuscular stromal cells were weakly positive for AR in contrast to what was observed during the activity period. Considering that testosterone values are lower in adult animals during the period of gonadal regression and in impuber animals, our immunohistochemical results show a significant correlation with the percentage of AR-immunopositive cells. In conclusion, these results demonstrate that the structure of the SVs changes in the activity period of viscacha, probably because of elevated levels of testosterone leading to an increase in the secretory activity of epithelial cells. Copyright © 2012 Wiley Periodicals, Inc.

Silencing of the integrin-linked kinase gene suppresses the proliferation, migration and invasion of pancreatic cancer cells (Panc-1).

PubMed

Zhu, Xiang-Yu; Liu, Ning; Liu, Wei; Song, Shao-Wei; Guo, Ke-Jian

2012-04-01

Integrin-linked kinase (ILK) is an ankyrin repeat-containing serine-threonine protein kinase that is involved in the regulation of integrin-mediated processes such as cancer cell proliferation, migration and invasion. In this study, we examined the effect of a lentivirus-mediated knockdown of ILK on the proliferation, migration and invasion of pancreatic cancer (Panc-1) cells. Immunohistochemical staining showed that ILK expression was enhanced in pancreatic cancer tissue. The silencing of ILK in human Panc-1 cells led to cell cycle arrest in the G0/G1 phase and delayed cell proliferation, in addition to down-regulating cell migration and invasion. The latter effects were mediated by up-regulating the expression of E-cadherin, a key protein in cell adhesion. These findings indicate that ILK may be a new diagnostic marker for pancreatic cancer and that silencing ILK could be a potentially useful therapeutic approach for treating pancreatic cancer.

Effect of taxol from Pestalotiopsis mangiferae on A549 cells-In vitro study

PubMed Central

Kathiravan, Govindarajan; Sureban, Sripathi M.

2009-01-01

Pestalotiopsis mangiferae Coelomycete fungi were used to examine the production of taxol. The taxol isolated from this fungus is biologically active against cancer cell lines were investigated for its antiproliferative activity in human Non Small Cell Lung Cancer A549 cells. The results showed that the methylene chloride extraction of Pestalotiopsis mangiferae inhibited the proliferation of A 549 cells as measured by MTT and Trypan blue assay. Flow cytometric analysis showed that methylene chloride extraction of Pestalotiopsis mangiferae blocked cell cycle progression in G0/G1 phase. In addition fungal taxol induced A549 cell apoptosis as determined by propidium iodide staining. Further the percentage of LDH release was increased at increasing concentrations which is a measure of cell death. The levels of sialic acid levels and DNA, RNA and protein levels were decreased after treatment with methylene chloride extraction of Pestalotiopsis mangiferae. We suggests that methylene chloride extraction of Pestalotiopsis mangiferae might be considered for future therapeutic application with further studies against lung cancer. PMID:25206246

Effect of taxol from Pestalotiopsis mangiferae on A549 cells-In vitro study.

PubMed

Kathiravan, Govindarajan; Sureban, Sripathi M

2009-12-01

Pestalotiopsis mangiferae Coelomycete fungi were used to examine the production of taxol. The taxol isolated from this fungus is biologically active against cancer cell lines were investigated for its antiproliferative activity in human Non Small Cell Lung Cancer A549 cells. The results showed that the methylene chloride extraction of Pestalotiopsis mangiferae inhibited the proliferation of A 549 cells as measured by MTT and Trypan blue assay. Flow cytometric analysis showed that methylene chloride extraction of Pestalotiopsis mangiferae blocked cell cycle progression in G0/G1 phase. In addition fungal taxol induced A549 cell apoptosis as determined by propidium iodide staining. Further the percentage of LDH release was increased at increasing concentrations which is a measure of cell death. The levels of sialic acid levels and DNA, RNA and protein levels were decreased after treatment with methylene chloride extraction of Pestalotiopsis mangiferae. We suggests that methylene chloride extraction of Pestalotiopsis mangiferae might be considered for future therapeutic application with further studies against lung cancer.

[Isolation and identification of human periodontal ligament stem cells in vitro].

PubMed

Shen, Tao; Chang, Hui-jun; Jian, Cong-xiang; Yang, Yan-chun; Zhou, Ji-xiang

2011-02-01

To isolate and identify human periodontal ligament stem cells (PDLSC) by improved methods and assess the characteristics of PDLSC ex vivo. The periodontal ligament cells were obtained from the healthy impacted third molars and teeth extracted for orthodontic purposes and used to isolate PDLSC by limiting dilution assay. PDLSC were cultured and expanded in alpha-MEM supplemented with 10% FBS. Colony-forming assay, immunohistochemistry, flow cytometry, osteogenic and adipogenic induction were used to identify PDLSC. The obtained cells had high colony-forming efficiency and were positive staining for vimentin and negative for pancytokeratin. Flow cytometry revealed that the isolated cells were positive for STRO-1 and CD146 antibodies and most were in the G0/G1 phase of cell cycle. Under specific conditions, they could differentiate to the osteoblast and adipocyte lineages in vitro. Limiting dilution assay is an effective method to isolate PDLSC and the single-cell-derived colonies demonstrate the properties of stem cells in vitro.

The aflatoxin B1 -fumonisin B1 toxicity in BRL-3A hepatocytes is associated to induction of cytochrome P450 activity and arachidonic acid metabolism.

PubMed

Mary, Verónica S; Arias, Silvina L; Otaiza, Santiago N; Velez, Pilar A; Rubinstein, Héctor R; Theumer, Martín G

2017-06-01

Human oral exposure to aflatoxin B 1 (AFB 1 ) and fumonisin B 1 (FB 1 ) is associated with increased hepatocellular carcinoma. Although evidence suggested interactive AFB 1 -FB 1 hepatotoxicity, the underlying mechanisms remain mostly unidentified. This work was aimed at evaluating the possible AFB 1 -FB 1 interplay to induce genetic and cell cycle toxicities in BRL-3A rat hepatocytes, reactive oxygen species (ROS) involvement, and the AFB 1 metabolizing pathways cytochrome P450 (CYP) and arachidonic acid (ArAc) metabolism as ROS contributors. Flow cytometry of stained BRL-3A hepatocytes was used to study the cell cycle (propidium iodide), ROS intracellular production (DCFH-DA, HE, DAF-2 DA), and phospholipase A activity (staining with bis-BODIPY FL C11-PC). The CYP1A activity was assessed by the 7-ethoxyresorufin-O-deethylase (EROD) assay. Despite a 48-h exposure to FB 1 (30 μM) not being genotoxic, the AFB 1 (20 μM)-induced micronucleus frequency was overcome by the AFB 1 -FB 1 mixture (MIX), presumably showing toxin interaction. The mycotoxins blocked G1/S-phase, but only MIX caused cell death. Overall, the oxidative stress led these alterations as the pretreatment with N-acetyl-l-cysteine reduced such toxic effects. While AFB 1 had a major input to the MIX pro-oxidant activity, with CYP and ArAc metabolism being ROS contributors, these pathways were not involved in the FB 1 -elicited weak oxidative stress. The MIX-induced micronucleus frequency in N-acetyl-l-cysteine pretreated cells was greater than that caused by AFB 1 without antioxidants, suggesting enhanced AFB 1 direct genotoxicity probably owing to the higher CYP activity and ArAc metabolism found in MIX. The metabolic pathways modulation by AFB 1 -FB 1 mixtures could raise its hepatocarcinogenic properties. © 2017 Wiley Periodicals, Inc.

Immunohistochemical Analysis of Human Vallate Taste Buds.

PubMed

Tizzano, Marco; Grigereit, Laura; Shultz, Nicole; Clary, Matthew S; Finger, Thomas E

2015-11-01

The morphology of the vallate papillae from postmortem human samples was investigated with immunohistochemistry. Microscopically, taste buds were present along the inner wall of the papilla, and in some cases in the outer wall as well. The typical taste cell markers PLCβ2, GNAT3 (gustducin) and the T1R3 receptor stain elongated cells in human taste buds consistent with the Type II cells in rodents. In the human tissue, taste bud cells that stain with Type II cell markers, PLCβ2 and GNAT3, also stain with villin antibody. Two typical immunochemical markers for Type III taste cells in rodents, PGP9.5 and SNAP25, fail to stain any taste bud cells in the human postmortem tissue, although these antibodies do stain numerous nerve fibers throughout the specimen. Car4, another Type III cell marker, reacted with only a few taste cells in our samples. Finally, human vallate papillae have a general network of innervation similar to rodents and antibodies directed against SNAP25, PGP9.5, acetylated tubulin and P2X3 all stain free perigemmal nerve endings as well as intragemmal taste fibers. We conclude that with the exception of certain molecular features of Type III cells, human vallate papillae share the structural, morphological, and molecular features observed in rodents. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

[Osteogenesis of human adipose-derived mesenchymal stem cells-biomaterial mixture in vivo after 3D bio-printing].

PubMed

Song, Yang; Wang, Xiao-fei; Wang, Yu-guang; Sun, Yu-chun; Lv, Pei-jun

2016-02-18

To construct human adipose-derived mesenchymal stem cells (hASCs)-biomaterial mixture 3D bio-printing body and detect its osteogenesis in vivo, and to establish a guideline of osteogenesis in vivo by use of 3D bio-printing technology preliminarily. P4 hASCs were used as seed cells, whose osteogenic potential in vitro was tested by alkaline phosphatase (ALP) staining and alizarin red staining after 14 d of osteogenic induction. The cells were added into 20 g/L sodium alginate and 80 g/L gelatin mixture (cell density was 1 × 10(6)/mL), and the cell-sodium alginate-gelatin mixture was printed by Bioplotter 3D bio-printer (Envision company, Germany), in which the cells'survival rate was detected by live- dead cell double fluorescence staining. Next, the printing body was osteogenically induced for 1 week to gain the experimental group; and the sodium alginate-gelatin mixture without cells was also printed to gain the control group. Both the experimental group and the control group were implanted into the back of the nude mice. After 6 weeks of implantation, the samples were collected, HE staining, Masson staining, immunohistochemical staining and Inveon Micro CT test were preformed to analyze their osteogenic capability. The cells'survival rate was 89%± 2% after printing. Six weeks after implantation, the samples of the control group were mostly degraded, whose shape was irregular and gel-like; the samples of the experimental group kept their original size and their texture was tough. HE staining and Masson staining showed that the bone-like tissue and vessel in-growth could be observed in the experimental group 6 weeks after implantation, immunohistochemical staining showed that the result of osteocalcin was positive, and Micro CT results showed that samples of the experimental group had a higher density and the new bone volume was 18% ± 1%. hASCs -biomaterial mixture 3D bio-printing body has capability of ectopic bone formation in nude mice, and it is feasible to apply cells-biomaterial mixture 3D bio-printing technology in the area of bone formation in vivo.

Histopathological findings in immunohistological staining of the granulomatous tissue reaction associated with tuberculosis.

PubMed

Karimi, Shirin; Shamaei, Masoud; Pourabdollah, Mihan; Sadr, Makan; Karbasi, Mehrdad; Kiani, Arda; Bahadori, Moslem

2014-01-01

Purpose. The histological diagnosis of Mycobacterium tuberculosis (MTB) remains a diagnostic challenge despite different methods. Immunohistochemistry (IHC) not only could confirm granulomatous tissue involvement but also can demonstrate MTB antigen immunolocalization. This study tries to clarify the details of immunohistochemical staining for MTB with pAbBCG. Materials/Methods. Twenty-three confirmed TB granulomatous tissue samples were studied by Ziehl-Neelsen and immunohistochemistry (IHC) staining with pAbBCG. Samples were selected from the archive of the Department of Pathology, National Research Institute of Tuberculosis and Lung Disease, Tehran, Iran. Results. IHC staining was positive in all samples, whereas Ziehl-Neelsen was positive in 9 cases out of 23 (39.1%). Tissue types used were pleural tissue, lymph nodes, and lung tissue. IHC showed positive coarse granular cytoplasmic and round, fragmented bacillary staining. In this study, epithelioid cells clearly showed more positive staining at the periphery of the granuloma rather than the center of granuloma. There is also positive staining in endothelial cells, fibroblasts, plasma cells, lymphocytes, and macrophages outside the granuloma. Conclusion. Considering the criteria of positive immunohistochemical staining of TB granulomatous reactions, this stain not only highlights the presence of mycobacterial antigens for tissue diagnosis, but also could morphologically localize its distribution in different cells.

Histopathological Findings in Immunohistological Staining of the Granulomatous Tissue Reaction Associated with Tuberculosis

PubMed Central

Karimi, Shirin; Pourabdollah, Mihan; Sadr, Makan; Karbasi, Mehrdad; Bahadori, Moslem

2014-01-01

Purpose. The histological diagnosis of Mycobacterium tuberculosis (MTB) remains a diagnostic challenge despite different methods. Immunohistochemistry (IHC) not only could confirm granulomatous tissue involvement but also can demonstrate MTB antigen immunolocalization. This study tries to clarify the details of immunohistochemical staining for MTB with pAbBCG. Materials/Methods. Twenty-three confirmed TB granulomatous tissue samples were studied by Ziehl-Neelsen and immunohistochemistry (IHC) staining with pAbBCG. Samples were selected from the archive of the Department of Pathology, National Research Institute of Tuberculosis and Lung Disease, Tehran, Iran. Results. IHC staining was positive in all samples, whereas Ziehl-Neelsen was positive in 9 cases out of 23 (39.1%). Tissue types used were pleural tissue, lymph nodes, and lung tissue. IHC showed positive coarse granular cytoplasmic and round, fragmented bacillary staining. In this study, epithelioid cells clearly showed more positive staining at the periphery of the granuloma rather than the center of granuloma. There is also positive staining in endothelial cells, fibroblasts, plasma cells, lymphocytes, and macrophages outside the granuloma. Conclusion. Considering the criteria of positive immunohistochemical staining of TB granulomatous reactions, this stain not only highlights the presence of mycobacterial antigens for tissue diagnosis, but also could morphologically localize its distribution in different cells. PMID:24511393

Molecular response to phototoxic stress of UVB-irradiated ketoprofen through arresting cell cycle in G2/M phase and inducing apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Shicheng; Mizu, Hideo; Yamauchi, Hitoshi

The phototoxicity of ketoprofen (KP), a non-steroidal anti-inflammatory drug, has recently attracted considerable attention, because it is photolabile and undergoes degradation when irradiated by sunlight to induce various skin diseases. The present study shows that combination of UVB irradiation with KP induced the cytotoxicity and suppressed DNA synthesis in HaCaT cells in a concentration-dependent manner. UVB-irradiated KP inhibited the cell growth and induced G2/M cell cycle arrest by modulating the levels of cdc2, cyclin B1, Chk1, Tyr15-phosphorylated cdc2 and p21. It also provoked a striking accumulation of cyclin B1-cdc2-p21 complexes, concomitantly with an increase in the levels of Tyr15-phosphorylated cdc2more » and p21 protein. The presence of KP accentuated the apoptotic response to UVB radiation in HaCaT cells as evidenced by DAPI staining. The apoptotic process was associated with activation of caspase-9, caspase-3 and cleavage of PARP, and this activation could be prevented by a specific caspase-3 inhibitor. Taken together, our results suggest that KP-photoinduced apoptosis may be a useful approach to reduce or prevent skin carcinogenesis.« less

Single step modified ink staining for Tzanck test: quick detection of herpetic giant cells in Tzanck smear.

PubMed

Mizutani, Hitoshi; Akeda, Tomoko; Yamanaka, Kei-Ichi; Isoda, Kenichi; Gabazza, Esteban C

2012-02-01

Tzanck test has been recently re-evaluated as a method for the diagnosis of herpes virus infection. Giemsa staining for the Tzanck test is time-consuming and laborious. There is a need to develop simple and quick staining methods for bedside diagnosis of this disease. We report a single step and quick method for staining herpes giant cells in Tzanck smears using routinely available inks and physiological saline. A keratinocyte cell line (HaCaT) was cultured on a slide glass and stained with various commercially available blue, blue-black and black inks serially diluted with physiological saline. Clinical smear samples from herpes lesions were also stained with these solutions without specific pretreatment. The nuclei of HaCaT were clearly stained showing high contrast with the cytoplasm using 5% Parker-Quink blue-black ink saline solution. Concentration of ink solution higher or lower than 5% resulted in less contrast. Blue or black inks or other manufacturers' inks can also be used, but staining of the cultured keratinocytes was less clear. Smear of clinical samples from herpes lesions were also stained with 5% ink solution. The nuclei of the multinucleated giant cells were clearly stained, and the sample could be immediately used for microscopic examination. One step staining of Tzanck smear using this diluted ink solution is an inexpensive and a convenient bedside diagnostic tool for the dermatologist. © 2011 Japanese Dermatological Association.

GATA3: a promising marker for metastatic breast carcinoma in serous effusion specimens.

PubMed

Shield, Paul W; Papadimos, David J; Walsh, Michael D

2014-04-01

The usefulness of GATA3 (GATA-binding protein 3 to DNA sequence [A/T]GATA[A/G]) as a marker for metastatic breast carcinoma in serous effusion specimens was investigated. Cell block sections from 74 serous effusion specimens (32 ascitic, 2 pericardial, and 40 pleural fluids) were stained with an anti-GATA3 murine monoclonal antibody. The specimens included 62 confirmed metastatic carcinomas from the breast (30 specimens), female genital tract (13 specimens), gastrointestinal tract (7 specimens), lung adenocarcinoma (9 specimens), pancreas (1 specimen), kidney (1 specimen), and bladder (1 specimen). The breast carcinoma cases included 15 ductal carcinomas and 8 lobular carcinomas; the histology subtype was not available for 7 specimens. Twelve cases containing florid reactive mesothelial cells were also stained. The breast carcinoma cases were also stained for mammaglobin and gross cystic disease fluid protein of 15 kilodaltons (GCDFP-15) to compare their sensitivity with GATA3. Positive nuclear staining for GATA3 was found to be present in 90% of metastatic breast carcinoma specimens (27 of 30 specimens). All nonbreast metastatic carcinomas tested were negative with the exception of the single case of metastatic urothelial carcinoma. No staining was observed in any of the benign reactive cases or in benign mesothelial cells present in the malignant cell block preparations. Two cases demonstrated weak positivity of benign lymphoid cells. Staining results were unambiguous because all positive cases demonstrated intense nuclear staining in > 50% of tumor cells. Mammaglobin (57% staining; 17 of 30 cases) and GCDFP-15 (33% staining; 10 of 30 cases) were found to be less sensitive markers of breast carcinoma. If used in a panel, mammaglobin and GCFP-15 staining would have identified only 1 additional case compared with those stained with GATA3. GATA3 may be a useful addition to immunostaining panels for serous effusion specimens when metastatic breast carcinoma is a consideration. © 2014 American Cancer Society.

Cellient™ automated cell block versus traditional cell block preparation: a comparison of morphologic features and immunohistochemical staining.

PubMed

Wagner, David G; Russell, Donna K; Benson, Jenna M; Schneider, Ashley E; Hoda, Rana S; Bonfiglio, Thomas A

2011-10-01

Traditional cell block (TCB) sections serve as an important diagnostic adjunct to cytologic smears but are also used today as a reliable preparation for immunohistochemical (IHC) studies. There are many ways to prepare a cell block and the methods continue to be revised. In this study, we compare the TCB with the Cellient™ automated cell block system. Thirty-five cell blocks were obtained from 16 benign and 19 malignant nongynecologic cytology specimens at a large university teaching hospital and prepared according to TCB and Cellient protocols. Cell block sections from both methods were compared for possible differences in various morphologic features and immunohistochemical staining patterns. In the 16 benign cases, no significant morphologic differences were found between the TCB and Cellient cell block sections. For the 19 malignant cases, some noticeable differences in the nuclear chromatin and cellularity were identified, although statistical significance was not attained. Immunohistochemical or special stains were performed on 89% of the malignant cases (17/19). Inadequate cellularity precluded full evaluation in 23% of Cellient cell block IHC preparations (4/17). Of the malignant cases with adequate cellularity (13/17), the immunohistochemical staining patterns from the different methods were identical in 53% of cases. The traditional and Cellient cell block sections showed similar morphologic and immunohistochemical staining patterns. The only significant difference between the two methods concerned the lower overall cell block cellularity identified during immunohistochemical staining in the Cellient cell block sections. Copyright © 2010 Wiley-Liss, Inc.

Symptomatic candidiasis: Using self sampled vaginal smears to establish the presence of Candida, lactobacilli, and Gardnerella vaginalis.

PubMed

Engberts, M K; Boon, M E; van Haaften, M; Heintz, A P M

2007-10-01

In a prospective cohort study, 10 symptomatic women with recurrent vulvovaginal candidiasis were taught how to prepare vaginal smears of their own vaginal fluids on days 7, 14, 21, and 28. The 40 smears were stained with the PAS-method and examined by three different cytopathologists for presence of Candida. Thereafter, the smears were restained with Giemsa-stain to determine presence of lactobacilli, Gardnerella vaginalis ("clue cells") and neutrophils. All three cytopathologists unequivocally established Candida blastospores and (pseudo)hyphae in 27 out of the 40 PAS-stained vaginal smears, whereas in the remaining 13 smears Candida was not found. All 10 patients had Candida in their smears during the second half of their menstrual cycle.Self sampled smears prove to be reliable for establishing the presence of Candida in symptomatic patients with candidiasis. Candida is associated with a lactobacillus-predominated vaginal flora, but with the absence of Gardnerella vaginalis. Further studies may be directed towards the interaction between the various members of the vaginal flora. This study should open molecular methodology for determining the possible interactions of lactobacilli and Candida. (c) 2007 Wiley-Liss, Inc.

A karyometric note on nucleoli in human early granulocytic precursors.

PubMed

Smetana, K; Mikulenková, D; Jirásková, I; Klamová, H

2006-01-01

The diameter of nucleoli was measured in human bone marrow early granulocytic precursors after visualization by a simple cytochemical method for demonstration of RNA. Such method facilitated to clearly see nucleolar bodies without perinucleolar chromatin, including those of micronucleoli. The bone marrow of patients suffering from chronic myeloid leukaemia (untreated with cytostatics) provided a satisfactory number of both myeloblasts and promyelocytes for nucleolar measurements because of prevailing granulopoiesis. The direct nucleolar measurement was carried out on digitized and processed images on the screen at magnification 4,300x. It seems to be likely that the nucleolar size is directly related to the number of nucleoli per cell. The largest nucleoli were present in both myeloblasts and promyelocytes that possessed a single nucleolus. In contrast, the nucleolar diameter was significantly smaller in cells with multiple nucleoli. However, in cells with small multiple nucleoli, one of them was always larger and dominant with a large number of AgNORs. Such large nucleoli are possibly visible in specimens stained with panoptic procedures or methods staining nuclear chromatin or DNA. It should also be mentioned that both myeloblasts and promyelocytes mostly possessed two nucleoli with the mean diameter close to 1.5 microm. The incidence of early granulocytic precursors classified according to the nucleolar number and size strongly suggested that the various nucleolar number and nucleolar size in these cells might be related to the different stage of the cell cycle and might also explain their heterogeneity.

BTK suppresses myeloma cellular senescence through activating AKT/P27/Rb signaling

PubMed Central

Lu, Yue; Yang, Hongbao; Tian, Zhidan; Yin, Gang; Zhang, Wen; Lu, Sicheng; Zhang, Yi; Yang, Ye

2017-01-01

We previously explored the role of BTK in maintaining multiple myeloma stem cells (MMSCs) self-renewal and drug-resistance. Here we investigated the elevation of BTK suppressing MM cellular senescence, a state of irreversible cellular growth arrest. We firstly discovered that an increased expression of BTK in MM samples compared to normal controls by immunohistochemistry (IHC), and significant chromosomal gain in primary samples. In addition, BTK high-expressing MM patients are associated with poor outcome in both Total Therapy 2 (TT2) and TT3 cohorts. Knockdown BTK expression by shRNA induced MM cellular senescence using β-galactosidase (SA-b-gal) staining, cell growth arrest by cell cycle staining and decreased clonogenicity while forcing BTK expression in MM cells abrogated these characteristics. We also validated this feature in mouse embryonic fibroblast cells (MEFs), which showed that elevated BTK expression was resistant to MEF senescence after serial cultivation in vitro. Further mechanism study revealed that BTK activated AKT signaling leading to down-regulation of P27 expression and hindered RB activity while AKT inhibitor, LY294002, overcame BTK-overexpression induced cellular senescence resistance. Eventually we demonstrated that BTK inhibitor, CGI-1746, induced MM cellular senescence, colony reduction and tumorigenecity inhibition in vivo. Summarily, we designate a novel mechanism of BTK in mediating MM growth, and BTK inhibitor is of great potential in vivo and in vitro suggesting BTK is a promising therapeutic target for MM. PMID:28915637

NAD+ maintenance attenuates light induced photoreceptor degeneration Δ

PubMed Central

Bai, Shi; Sheline, Christian T.

2013-01-01

Light-induced retinal damage (LD) occurs after surgery or sun exposure. We previously showed that zinc (Zn2+) accumulated in photoreceptors and RPE cells after LD but prior to cell death, and pyruvate or nicotinamide attenuated the resultant death perhaps by restoring nicotinamide adenine dinucleotide (NAD+) levels. We first examined the levels of NAD+ and the efficacy of pyruvate or nicotinamide in oxidative toxicities using primary retinal cultures. We next manipulated NAD+ levels in vivo and tested the affect on LD to photoreceptors and RPE. NAD+ levels cycle with a 24-h rhythm in mammals, which is affected by the feeding schedule. Therefore, we tested the affect of increasing NAD+ levels on LD by giving nicotinamide, inverting the feeding schedule, or using transgenic mice which overexpress cytoplasmic nicotinamide mononucleotide adenyl-transferase-1 (cytNMNAT1), an NAD+ synthetic enzyme. Zn2+ accumulation was also assessed in culture and in retinal sections. Retinas of light damaged animals were examined by OCT and plastic sectioning, and retinal NAD levels were measured. Day fed, or nicotinamide treated rats showed less NAD+ loss, and LD compared to night fed rats or untreated rats without changing the Zn2+ staining pattern. CytNMNAT1 showed less Zn2+ staining, NAD+ loss, and cell death after LD. In conclusion, intense light, Zn2+ and oxidative toxicities caused an increase in Zn2+, NAD+ loss, and cell death which were attenuated by NAD+ restoration. Therefore, NAD+ levels play a protective role in LD-induced death of photoreceptors and RPE cells. PMID:23274583

In-vitro antitumor activity evaluation of hyperforin derivatives.

PubMed

Sun, Feng; Liu, Jin-Yun; He, Feng; Liu, Zhong; Wang, Rui; Wang, Dong-Mei; Wang, Yi-Fei; Yang, De-Po

2011-08-01

The derivatives of hyperforin, namely hyperforin acetate (2), 17,18,22,23,27,28,32,33-octahydrohyperforin acetate (3), and N,N-dicyclohexylamine salt of hyperforin (4), have been investigated for their antitumor properties. In-vitro studies demonstrated that 2 and 4 were active against HeLa (human cervical cancer), A375 (human malignant melanoma), HepG2 (human hepatocellular carcinoma), MCF-7 (human breast cancer), A549 (human nonsmall cell lung cancer), K562 (human chronic myeloid leukemia), and K562/ADR (human adriamycin-resistant K562) cell lines with IC(50) values in the range of 3.2-64.1 μM. The energy differences between highest occupied molecular orbital and lowest unoccupied molecular orbital of 2-4 were calculated to be 0.39778, 0.43106, and 0.30900 a.u., respectively, using the Gaussian 03 software package and ab initio method with the HF/6-311 G* basis set. The result indicated that the biological activity of 4 might be the strongest and that of 3 might be the weakest, which was in accordance with their corresponding antiproliferative effects against the tested tumor cell lines. Compound 4 caused cell cycle arrest at G2/M phase in flow cytometry experiment and induced apoptosis by 4',6-diamidino-2-phenylindole staining and Annexin V-FITC/PI (propidium iodide) double-labeled staining in HepG2 cells. The results indicated a potential for N,N-dicyclohexylamine salt of hyperforin as a new antitumor drug.

Validation of 31 of the most commonly used immunohistochemical antibodies in cytology prepared using the Cellient(®) automated cell block system.

PubMed

Montgomery, Eric; Gao, Chen; de Luca, Julie; Bower, Jessie; Attwood, Kristropher; Ylagan, Lourdes

2014-12-01

The Cellient(®) cell block system has become available as an alternative, partially automated method to create cell blocks in cytology. We sought to show a validation method for immunohistochemical (IHC) staining on the Cellient cell block system (CCB) in comparison with the formalin fixed paraffin embedded traditional cell block (TCB). Immunohistochemical staining was performed using 31 antibodies on 38 patient samples for a total of 326 slides. Split samples were processed using both methods by following the Cellient(®) manufacturer's recommendations for the Cellient cell block (CCB) and the Histogel method for preparing the traditional cell block (TCB). Interpretation was performed by three pathologists and two cytotechnologists. Immunohistochemical stains were scored as: 0/1+ (negative) and 2/3+ (positive). Inter-rater agreement for each antibody was evaluated for CCB and TCB, as well as the intra-rater agreement between TCB and CCB between observers. Interobserver staining concordance for the TCB was obtained with statistical significance (P < 0.05) in 24 of 31 antibodies. Interobserver staining concordance for the CCB was obtained with statistical significance in 27 of 31 antibodies. Intra-observer staining concordance between TCB and CCB was obtained with statistical significance in 24 of 31 antibodies tested. In conclusions, immunohistochemical stains on cytologic specimens processed by the Cellient system are reliable and concordant with stains performed on the same split samples processed via a formalin fixed-paraffin embedded (FFPE) block. The Cellient system is a welcome adjunct to cytology work-flow by producing cell block material of sufficient quality to allow the use of routine IHC. © 2014 Wiley Periodicals, Inc.

Wnt modulates MCL1 to control cell survival in triple negative breast cancer

PubMed Central

2014-01-01

Background Triple negative breast cancer (TNBC) has higher rates of recurrence and distant metastasis, and poorer outcome as compared to non-TNBC. Aberrant activation of WNT signaling has been detected in TNBC, which might be important for triggering oncogenic conversion of breast epithelial cell. Therefore, we directed our focus on identifying the WNT ligand and its underlying mechanism in TNBC cells. Methods We performed large-scale analysis of public microarray data to screen the WNT ligands and the clinical significance of the responsible ligand in TNBC. WNT5B was identified and its overexpression in TNBC was confirmed by immunohistochemistry staining, Western blot and ELISA. ShRNA was used to knockdown WNT5B expression (shWNT5B). Cellular functional alteration with shWNT5B treatment was determined by using wound healing assay, mammosphere assay; while cell cycle and apoptosis were examined by flowcytometry. Mitochondrial morphology was photographed by electron microscope. Biological change of mitochondria was detected by RT-PCR and oxygen consumption assay. Activation of WNT pathway and its downstream targets were evaluated by liciferase assay, immunohistochemistry staining and immunoblot analysis. Statistical methods used in the experiments besides microarray analysis was two-tailed t-test. Results WNT5B was elevated both in the tumor and the patients’ serum. Suppression of WNT5B remarkably impaired cell growth, migration and mammosphere formation. Additionally, G0/G1 cell cycle arrest and caspase-independent apoptosis was observed. Study of the possible mechanism indicated that these effects occurred through suppression of mitochondrial biogenesis, as evidenced by reduced mitochondrial DNA (MtDNA) and compromised oxidative phosphorylation (OXPHOS). In Vivo and in vitro data uncovered that WNT5B modulated mitochondrial physiology was mediated by MCL1, which was regulated by WNT/β-catenin responsive gene, Myc. Clinic data analysis revealed that both WNT5B and MCL1 are associated with enhanced metastasis and decreased disease-free survival. Conclusions All our findings suggested that WNT5B/MCL1 cascade is critical for TNBC and understanding its regulatory apparatus provided valuable insight into the pathogenesis of the tumor development and the guidance for targeting therapeutics. PMID:24564888

Detection of alkaline phosphatase in canine cells previously stained with Wright-Giemsa and its utility in differentiating osteosarcoma from other mesenchymal tumors.

PubMed

Ryseff, Julia K; Bohn, Andrea A

2012-09-01

Osteosarcoma (OSA) is a common primary bone tumor in dogs. Demonstration of alkaline phosphatase (ALP) reactivity by tumor cells on unstained slides is useful in differentiating osteosarcoma from other types of sarcoma. However, unstained slides are not always available. The objectives of this study were to evaluate the diagnostic utility of detecting ALP expression in differentiating osteosarcoma from other sarcomas in dogs using cytologic material previously stained with Wright-Giemsa stain and to assess the sensitivity and specificity of ALP expression for diagnosing osteosarcoma using a specific protocol. Archived aspirates of histologically confirmed sarcomas in dogs that had been previously stained with Wright-Giemsa stain were treated with 5-bromo, 4-chloro, 3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) as a substrate for ALP. Cells were evaluated for expression of ALP after incubation with BCIP/NBT for 1 hour. Sensitivity and specificity of ALP expression for diagnosis of OSA were calculated. In samples from 83 dogs, cells from 15/17 OSAs and from 4/66 tumors other than OSA (amelanotic melanoma, gastrointestinal stromal tumor, collision tumor, and anaplastic sarcoma) expressed ALP. Sensitivity and specificity of ALP expression detected using BCIP/NBT substrate applied to cells previously stained with Wright-Giemsa stain for OSA were 88 and 94%, respectively. ALP expression detected using BCIP/NBT substrate applied to previously stained cells is useful in differentiating canine OSA from other mesenchymal neoplasms. © 2012 American Society for Veterinary Clinical Pathology.

Studies of the endothelial origin of cells in systemic angioendotheliomatosis and other vascular lesions of the brain and meninges using ulex europaeus lectin stains.

PubMed

Schelper, R L; Olson, S P; Carroll, T J; Hart, M N; Witters, E

1986-01-01

Ulex europaeus agglutinin I (UEA-I) is a plant lectin which binds specifically to alpha-L-fucose moieties on the surface glycoproteins of human endothelial cells. The binding is completely inhibited by preincubation of the lectin with fucose. UEA-I can be conjugated directly to fluorescein or peroxidase and can be used to stain endothelium of paraffin embedded tissues. UEA-I staining was evaluated on normal and infarcted brain, systemic angioendotheliomatosis, metastatic epidural angiosarcoma, hemangioendothelioma, hemangioblastoma, angioblastic meningioma of both the hemangioblastic and hemangiopericytic types, and vascular meningioma. The endothelium, but not neuropil of normal and infarcted brain was positive for UEA-I. The tumor cells of hemangioendothelioma and angiosarcoma also stained. However, no staining was seen in malignant intravascular cells of angioendotheliomatosis, the stromal cells of hemangioblastoma, or pericytes of angioblastic meningioma. It is concluded that the malignant cells in angioendotheliomatosis, the stromal cells of hemangioblastoma and the pericytes of angioblastic meningioma do not produce surface glycoproteins characteristic of endothelial cells.

Utility of Fite-Faraco stain for both mast cell count and bacillary index in skin biopsies of leprosy patients.

PubMed

Chatura, K R; Sangeetha, S

2012-01-01

To assess the utility of a single stain for both mast cell count and bacillary index (BI), 50 skin-biopsie patients were stained with Fite-Faraco (FF) stain, viewed under oil immersion and BI calculated using the Ridley's logarithmic scale, and mast cells counted as the number of cells per mm2. Mean mast cell count per mm2 at the tuberculoid pole was lowest in TT 7.9 and highest in BT 14.23. At the lepromatous end, it was highest in BL 9.21, while in LL it was 8.23. Highest counts were seen in the borderline types overall. The correlation coefficient between histopathological diagnosis and BI is 0.822 which is a positive correlation to a significant degree. The correlation coefficient between histopathological diagnosis and mast cell count was found to be -0.17, which is a negative correlation but not to a significant degree. FF stain was utilised to visualise both bacilli for estimation of BI and mast cells for mast cell count, a seldom attempted feature in literature.

Gap Junction Protein Connexin 43 Serves as a Negative Marker for a Stem Cell-Containing Population of Human Limbal Epithelial Cells

PubMed Central

Chen, Zhuo; Evans, W. Howard; Pflugfelder, Stephen C.; Li, De-Quan

2010-01-01

This study evaluated whether the gap junction protein connexin (Cx) 43 could serve as a negative cell surface marker for human corneal epithelial stem cells. Cx43 expression was evaluated in corneo-limbal tissue and primary limbal epithelial cultures. Immunofluorescent staining and laser scanning confocal microscopy showed that Cx43 was strongly expressed in the corneal and limbal suprabasal epithelial cells, but the basal cells of the limbal epithelium were negative. Cx43 antibody stained mainly large cells but not small cells in primary limbal epithelial cultures. As determined by semiquantitative reverse transcription polymerase chain reaction (PCR) and real-time PCR, Cx43 mRNA was more abundant in the corneal than limbal epithelia, and it was expressed in higher levels in mature limbal epithelial cultures. Using GAP11, a rabbit polyclonal antibody against the Cx32 extracellular loop 2 (151–187), a sequence that is highly homologous in Cx43, the Cx43dim and Cx43bright cells were selected from primary limbal epithelial cultures by fluorescence-activated cell sorting and were evaluated for stem cell properties. These Cx43dim and Cx43bright cells were confirmed by their expression levels of Cx43 protein and mRNA. The Cx43dim cells were found to contain higher percentages of slow-cycling bromodeoxyuridine (BrdU)-label retaining cells and the cells that were positive for stem cell-associated markers p63, ABCG2, and integrin β1 and negative for differentiation markers K3 and involucrin. The Cx43dim cells possessed a greater proliferative potential than Cx43bright cells and nonfractionated cells as evaluated by BrdU incorporation, colony-forming efficiency, and growth capacity. Our findings suggest that human limbal basal cells do not express connexin 43, which could serve as a negative cell surface marker for the stem cell-containing population of human limbal epithelial cells. PMID:16424398

Assessment of the in vitro cytotoxicity and in vivo anti-tumor activity of the alcoholic stem bark extract/fractions of Mimusops elengi Linn.

PubMed

Kumar, Harish; Savaliya, Mihir; Biswas, Subhankar; Nayak, Pawan G; Maliyakkal, Naseer; Manjunath Setty, M; Gourishetti, Karthik; Pai, K Sreedhara Ranganath

2016-08-01

Various parts of Mimusops elengi Linn. (Sapotaceae) have been used widely in traditional Indian medicine for the treatment of pain, inflammation and wounds. The study was conducted to explore the use of stem bark of M. elengi on pharmacological grounds and to evaluate the scientific basis of cytotoxic and anti-tumor activity. Extract/fractions were prepared and in vitro cytotoxicity was assessed using SRB assay. Most effective fractions were subjected to fluorescence microscopy based acridine orange/ethidium bromide (AO/EB) and Hoechst 33342 staining to determine apoptosis induction and DNA fragmentation assay. Comet and micronuclei assay were performed to assess genotoxicity. Cell cycle analysis was also performed. In vivo anti-tumor potential was evaluated by Ehrlich ascites carcinoma (EAC) model in mice. The alcoholic stem bark extract of M. elengi along with four fractions showed potential in vitro cytotoxicity in SRB assay. Of these, dichloromethane and ethyl acetate fractions were selected for further studies. The fractions revealed apoptosis inducing potential in AO/EB and Hoechst 33342 staining, which was further confirmed by DNA fragmentation assay. Genotoxic potential was revealed by comet and micronuclei assay. Fractions also exhibited specific cell cycle inhibition in G0/G1 phase. In EAC model, ethyl acetate fraction along with the standard (cisplatin) effectively reduced the increase in body weight compared to control and improved mean survival time. Both fractions were able to restore the altered hematological and biochemical parameters. Hence, M. elengi stem bark may be a possible therapeutic candidate having cytotoxic and anti-tumor potential.

Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells.

PubMed

Buckley, Alysia G; Looi, Kevin; Iosifidis, Thomas; Ling, Kak-Ming; Sutanto, Erika N; Martinovich, Kelly M; Kicic-Starcevich, Elizabeth; Garratt, Luke W; Shaw, Nicole C; Lannigan, Francis J; Larcombe, Alexander N; Zosky, Graeme; Knight, Darryl A; Rigby, Paul J; Kicic, Anthony; Stick, Stephen M

2018-01-01

Apically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host. Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type of cells used to achieve this. Here, we assessed four fixation methods including; (i) 4% ( v /v) paraformaldehyde; (ii) 100% methanol; (iii) acetone or; (iv) 1:1 methanol: acetone. Pre-extraction with Triton X-100 was also performed and assessed on cells prior to fixation with either methanol or paraformaldehyde. Cells were also permeabilized with 0.1% (v/v) Saponin in 1× TBS following fixation and subsequently stained for tight junction proteins. Confocal microscopy was then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order to determine a standardised workflow of reproducible staining. Positive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins but no staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone also produced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessed using primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed following methanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorial fixation with methanol and acetone. The present study demonstrates the importance of a personalised approach to optimise staining for the visualisation of different tight junction proteins. Of significance, the workflow, once optimised, can readily be translated into primary airway epithelial cell air-liquid interface cultures where it can be used to assess barrier integrity in chronic lung diseases.

Evaluation of intranuclear BrdU detection procedures for use in multicolor flow cytometry*

PubMed Central

Rothaeusler, Kristina; Baumgarth, Nicole

2010-01-01

Background Measurement of cell proliferation via BrdU incorporation in combination with multicolor cell surface staining would facilitate studies on cell subsets that require multiple markers for their identification. However, the extent to which the often harsh cell preparation procedures required affect the staining quality of more recently developed fluorescent dyes has not been assessed. Methods Three cell preparation protocols for BrdU measurement were compared for their ability to maintain fluorescent surface staining and scatter parameters of in vivo BrdU-labeled cells by flow cytometry. A 10-color fluorescent panel was developed to test the quality of surface staining following cell treatment and the ability to perform BrdU measurements on even small B lymphocyte subsets. Results All cell preparation procedures affected the quality of fluorescent and/or scatter parameters to varying degrees. Paraformaldehyde / saponin-based procedures preserved sufficient fluorescent surface staining to determine BrdU incorporation rates among all splenic B cell subsets, including B-1a cells, which constitute roughly 0.5% of cells. Turnover rates of B-1a cells were similar to immature B cells and higher than those of the other mature B cell subsets. Conclusion Paraformaldehyde / saponin-based cell preparation procedures facilitate detailed cell turnover studies on small cell subsets in vivo, revealing new functional information on rare cell populations. PMID:16538653

Development of hepatitis C virus genotype 3a cell culture system.

PubMed

Kim, Sulyi; Date, Tomoko; Yokokawa, Hiroshi; Kono, Tamaki; Aizaki, Hideki; Maurel, Patrick; Gondeau, Claire; Wakita, Takaji

2014-12-01

Hepatitis C virus (HCV) genotype 3a infection poses a serious health problem worldwide. A significant association has been reported between HCV genotype 3a infections and hepatic steatosis. Nevertheless, virological characterization of genotype 3a HCV is delayed due to the lack of appropriate virus cell culture systems. In the present study, we established the first infectious genotype 3a HCV system by introducing adaptive mutations into the S310 strain. HCV core proteins had different locations in JFH-1 and S310 virus-infected cells. Furthermore, the lipid content in S310 virus-infected cells was higher than Huh7.5.1 cells and JFH-1 virus-infected cells as determined by the lipid droplet staining area. This genotype 3a infectious cell culture system may be a useful experimental model for studying genotype 3a viral life cycles, molecular mechanisms of pathogenesis, and genotype 3a-specific antiviral drug development. © 2014 by the American Association for the Study of Liver Diseases.

Autophagic Cell Death, Polyploidy and Senescence Induced in Breast Tumor Cells by the Substituted Pyrrole JG-03-14, a Novel Microtubule Poison

PubMed Central

Arthur, Christopher R.; Gupton, John T.; Kellogg, Glen E.; Yeudall, W. Andrew; Cabot, Myles C.; Newsham, Irene; Gewirtz, David A.

2007-01-01

JG-03-14, a substituted pyrrole that inhibits microtubule polymerization, was screened against MCF-7 (p53 wild type), MDA-MB 231 (p53 mutant), MCF-7/caspase 3 and MCF-7/ADR (multidrug resistant) breast tumor cell lines. Cell viability and growth inhibition were assessed by the crystal violet dye assay. Apoptosis was evaluated by the TUNEL assay, cell cycle distribution by flow cytometry, autophagy by acridine orange staining of vesicle formation, and senescence based on β-galactosidase staining and cell morphology. Our studies indicate that exposure to JG-03-14, at a concentration of 500 nM, induces time dependent cell death in the MCF-7 and MDA-MB 231 cell lines. In MCF-7 cells, a residual surviving cell population was found to be senescent; in contrast, there was no surviving senescent population in treated MDA-MB 231 cells. No proliferative recovery was detected over a period of 15 days post-treatment in either cell line. Both the TUNEL assay and FLOW cytometry indicated a relatively limited degree of apoptosis (< 10%) in response to drug treatment in MCF-7 cells with more extensive apoptosis (but < 20%) in MDA-MB231 cells; acidic vacuole formation indicative of autophagic cell death was relatively extensive in both MCF-7 and MDA-MB231 cells. In addition, JG-03-14 induced the formation of a large hyperdiploid cell population in MDA-MB231 cells. JG-03-14 also demonstrated pronounced anti-proliferative activity in MCF-7/caspase 3 cells and in the MCF-7/ADR cell line. The observation that JG-03-14 promotes autophagic cell death and also retains activity in tumor cells expressing the multidrug resistance pump indicates that novel microtubule poisons of the substituted pyrroles class may hold promise in the treatment of breast cancer. PMID:17692290

p21 is Responsible for Ionizing Radiation-induced Bypass of Mitosis.

PubMed

Zhang, Xu Rui; Liu, Yong Ai; Sun, Fang; Li, He; Lei, Su Wen; Wang, Ju Fang

2016-07-01

To explore the role of p21 in ionizing radiation-induced changes in protein levels during the G2/M transition and long-term G2 arrest. Protein expression levels were assessed by western blot in the human uveal melanoma 92-1 cells after treatment with ionizing radiation. Depletion of p21 was carried out by employing the siRNA technique. Cell cycle distribution was determined by flow cytometry combined with histone H3 phosphorylation at Ser28, an M-phase marker. Senescence was assessed by senescence- associated-β-galactosidase (SA-β-gal) staining combined with Ki67 staining, a cell proliferation marker. Accompanying increased p21, the protein levels of G2/M transition genes declined significantly in 92-1 cells irradiated with 5 Gy of X-rays. Furthermore, these irradiated cells were blocked at the G2 phase followed by cellular senescence. Depletion of p21 rescued radiation-induced G2 arrest as demonstrated by the upregulation of G2/M transition kinases, as well as the high expression of histone H3 phosphorylated at Ser28. Knockdown of p21 resulted in entry into mitosis of irradiated 92-1 cells. However, cells with serious DNA damage failed to undergo cytokinesis, leading to the accumulation of multinucleated cells. Our results indicated that p21 was responsible for the downregulation of G2/M transition regulatory proteins and the bypass of mitosis induced by irradiation. Downregulation of p21 by siRNA resulted in G2-arrested cells entering into mitosis with serious DNA damage. This is the first report on elucidating the role of p21 in the bypass of mitosis. Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Use of DNA stains in immunophenotyping by slide-based cytometry

NASA Astrophysics Data System (ADS)

Gerstner, Andreas O. H.; Laffers, Wiebke; Bootz, Friedrich; Tarnok, Attila

2003-06-01

Immunophenotyping of peripheral blood leukocytes (PBL) is a very well documented application of Slide Based Cytometry (SBC). As for any other assay it is of highest importance to ensure that all cells which are relevant for an analysis are recognized. Unlike assays for cultured cells which have homogenous morphology immunophenotyping of PBLs is performed on cells with heterogeneous size and shape. Therefore, triggering on parameters related to cell morphology might lead to an incomplete analysis of just a subset of cells especially in pathological conditions. Several dyes stain DNA specifically in a wide variety of emission spectra. Many of them show some influence of the chromatin condensation and organization on the staining intensity. DNA dyes therefore can be used to differentiate between cell types having the same ploidy. This can be exploited for immunophenotyping since some dyes therefore can partially replace antibody staining. The concept of using DNA dyes in the setting of immunostaining has the following advantages: (1) nuclear staining provides a stable and easy triggering signal that guarantees both, that neither cells are excluded nor that debris or polluting particles are included into the analysis; (2) some DNA dyes differentiate between mononuclear and polymorphonuclear cells. A disadvantage of DNA dyes is that mostly cells have to be permeabilized. Because of this only one set of immunophenotypic markers can be stained, cells are fixed and permeabilized, and then nuclei are stained with the appropriate DNA dye. In the study we demonstrate the use of the most commonly available DNA dyes (7-AAD, To-Pro, To-To, PI etc.) in their applicability in immunophenotyping. An overview of spectral properties, fluorescence spill-over and optimal combinations with surface antigen staining will be shown. As in general for SBC only very small sample volumes are needed. This allows to serially analyze PBL in clinical settings that up to now could not be studied in detail such as in the critical ill patient, during major surgery, and in new-borns and infants.

Celecoxib prevents colitis associated colon carcinogenesis: an upregulation of apoptosis.

PubMed

Setia, Shruti; Nehru, Bimla; Sanyal, Sankar N

2014-12-01

Uncontrolled cell proliferation and suppressed apoptosis are the critical events transforming a normal cell to a cancerous one wherein the inflammatory microenvironment supports this oncogenic transformation. The process of colon carcinogenesis may be aggravated in chronic inflammatory conditions such as ulcerative colitis where non-steroidal anti-inflammatory drugs (NSAIDs) may effectively prevent the cellular and molecular events. Western blots and immunofluorescent analysis of DNA mismatch repair enzymes, cell cycle regulators and pro- and anti-apoptotic proteins were performed in dextran sulfate sodium (DSS)-induced ulcerative colitis and 1,2-dimethyl benz(a)anthracene (DMH)-induced colon cancer. Also, apoptotic studies were done in isolated colonocytes using fluorescent staining and in paraffin sections using TUNEL assay. An upregulation of cell cycle regulators: cyclin D1/cdk4 and cyclin E/cdk2 and anti-apoptotic Bcl-2, along with the suppression of DNA repair enzymes: MLH1 and MSH2; tumour suppressors: p53, p21and Rb and pro-apoptotic proteins: Bax and Bad were observed in the DSS, DMH and DSS+DMH groups. Proliferating cell nuclear antigen (PCNA) was also overexpressed in these groups. The ultimate executioner of the apoptotic pathway; caspase-3, was suppressed in these groups. Apoptotic studies in colonocytes and paraffin sections revealed suppressed apoptosis in these groups. These effects were corrected with the administration of a second generation NSAID, celecoxib along with the treatment of DSS and DMH. The chemopreventive action of celecoxib in colitis mediated colon carcinogenesis may include the regulation of DNA mismatch repair enzymes, cell cycle check points, cell proliferation and apoptosis. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Quantification of Intracellular Ice Formation and Recrystallization During Freeze-Thaw Cycles and Their Relationship with the Viability of Pig Iliac Endothelium Cells.

PubMed

Liu, Xiaoli; Zhao, Gang; Shu, Zhiquan; Niu, Dan; Zhang, Zhiguo; Zhou, Ping; Cao, Yunxia; Gao, Dayong

2016-12-01

Quantitative evaluation of the inherent correlation between cell cryoinjuries and intracellular ice formation (IIF) together with recrystallization (IIR) is of primary importance for both optimization of biopreservation and cryotherapy. The objective of this study is to thoroughly explore the roles of IIF on cell viability by using pig iliac endothelium cells (PIECs) as model cells during freezing and thawing. The experimental results indicated that both the probabilities of IIF (PIF) and IIR (PIR) increased along with the increase of cooling rates (p < 0.05) during the freeze-thaw cycles at cooling rates of 40, 60, 80, 100, and 150°C/min and the same warming rates of 100°C/min in phosphate-buffered saline-based solutions with or without 1 M DMSO. Viability evaluation with Hoechst 33342/propidium iodide double staining showed that most of the cells were killed (viability <20%) by the abovementioned freeze-thaw cycles, which indicated that the cooling rates investigated were all too rapid since large amounts of IIF and IIR were introduced. Another interesting phenomenon is that the presence of a low concentration of DMSO (1 M) tends to improve cell viability while increasing the PIF and PIR during freezing/thawing, contrary to the common belief that larger PIF corresponds to greater cryoinjury. This may be attributed to the intrinsic protection effect of DMSO by reduction of solution injury or other potential injuries. These findings may be of potential application value for both cryopreservation and cryosurgery by providing helpful additions to the existing studies on investigation of cryoinjuries of PIECs.

Autoantibodies in melanoma-associated retinopathy target TRPM1 cation channels of retinal ON bipolar cells.

PubMed

Dhingra, Anuradha; Fina, Marie E; Neinstein, Adam; Ramsey, David J; Xu, Ying; Fishman, Gerald A; Alexander, Kenneth R; Qian, Haohua; Peachey, Neal S; Gregg, Ronald G; Vardi, Noga

2011-03-16

Melanoma-associated retinopathy (MAR) is characterized by night blindness, photopsias, and a selective reduction of the electroretinogram b-wave. In certain cases, the serum contains autoantibodies that react with ON bipolar cells, but the target of these autoantibodies has not been identified. Here we show that the primary target of autoantibodies produced in MAR patients with reduced b-wave is the TRPM1 cation channel, the newly identified transduction channel in ON bipolar cells. Sera from two well characterized MAR patients, but not from a control subject, stained human embryonic kidney cells transfected with the TRPM1 gene, and Western blots probed with these MAR sera showed the expected band size (∼180 kDa). Staining of mouse and primate retina with MAR sera revealed immunoreactivity in all types of ON bipolar cells. Similar to staining for TRPM1, staining with the MAR sera was strong in dendritic tips and somas and was weak or absent in axon terminals. This staining colocalized with GFP in Grm6-GFP transgenic mice, where GFP is expressed in all and only ON bipolar cells, and also colocalized with Gα(o), a marker for all types of ON bipolar cells. The staining in ON bipolar cells was confirmed to be specific to TRPM1 because MAR serum did not stain these cells in a Trpm1(-/-) mouse. Evidence suggests that the recognized epitope is likely intracellular, and the sera can be internalized by retinal cells. We conclude that the vision of at least some patients with MAR is compromised due to autoantibody-mediated inactivation of the TRPM1 channel.

5-(2-Carboxyethenyl) isatin derivative induces G{sub 2}/M cell cycle arrest and apoptosis in human leukemia K562 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Yao; Zhao, Hong-Ye; Han, Kai-Lin

2014-08-08

Highlights: • 5-(2-Carboxyethenyl) isatin derivative (HKL 2H) inhibited K562’s proliferation. • HKL 2H caused the morphology change of G{sub 2}/M phase arrest and typical apoptosis. • HKL 2H induced G2/M cell cycle phase arrest in K562 cells. • HKL 2H induced apoptosis in K562 cells through the mitochondrial pathway. - Abstract: Our previous study successfully identified that the novel isatin derivative (E)-methyl 3-(1-(4-methoxybenzyl)-2,3-dioxoindolin-5-yl) acrylate (HKL 2H) acts as an anticancer agent at an inhibitory concentration (IC{sub 50}) level of 3 nM. In this study, the molecular mechanism how HKL 2H induces cytotoxic activity in the human chronic myelogenous leukemia K562more » cells was investigated. Flow cytometric analysis showed that the cells were arrested in the G{sub 2}/M phase and accumulated subsequently in the sub-G{sub 1} phase in the presence of HKL 2H. HKL 2H treatment down-regulated the expressions of CDK1 and cyclin B but up-regulated the level of phosphorylated CDK1. Annexin-V staining and the classic DNA ladder studies showed that HKL 2H induced the apoptosis of K562 cells. Our study further showed that HKL 2H treatment caused the dissipation of mitochondrial membrane potential, activated caspase-3 and lowered the Bcl-2/Bax ratio in K562 cells, suggesting that the HKL 2H-causing programmed cell death of K562 cells was caused via the mitochondrial apoptotic pathway. Taken together, our data demonstrated that HKL 2H, a 5-(2-carboxyethenyl) isatin derivative, notably induces G{sub 2}/M cell cycle arrest and mitochondrial-mediated apoptosis in K562 cells, indicating that this compound could be a promising anticancer candidate for further investigation.« less

Cellular effect of styrene substituted biscoumarin caused cellular apoptosis and cell cycle arrest in human breast cancer cells.

PubMed

Perumalsamy, Haribalan; Sankarapandian, Karuppasamy; Kandaswamy, Narendran; Balusamy, Sri Renukadevi; Periyathambi, Dhaiveegan; Raveendiran, Nanthini

2017-11-01

Coumarins occurs naturally across plant kingdoms exhibits significant pharmacological properties and pharmacokinetic activity. The conventional, therapeutic agents are often associated with poor stability, absorption and increased side effects. Therefore, identification of a drug that has little or no-side effect on humans is consequential. Here, we investigated the antiproliferative activity of styrene substituted biscoumarin against various human breast cancer cell lines, such as MCF-7, (ER-) MDA-MB-231 and (AR+) MDA-MB-453. Styrene substituted biscoumarin induced cell death by apoptosis in MDA-MB-231 cell line was analyzed. Antiproliferative activity of Styrene substituted biscoumarin was performed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Styrene substituted biscoumarin induced apoptosis was assessed by Hoechst staining, Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining and flow cytometric analysis. Migratory and proliferating characteristic of breast cancer cell line MDA-MB-231 was also analyzed by wound healing and colony formation assay. Furthermore, mRNA expression of BAX and BCL-2 were quantified using qRT-PCR and protein expression level analyzed by Western blot. The inhibition concentration (IC 50 ) of styrene substituted biscoumarin was assayed against three breast cancer cell lines. The inhibition concentration (IC 50 ) value of styrene substituted biscoumarin toward MDA-MB-231, MDA-MB-453 and MCF-7 cell lines was 5.63, 7.30 and 10.84μg/ml respectively. Styrene substituted biscoumarin induced apoptosis was detected by Hoechst staining, DAPI/PI analysis and flow-cytometric analysis. The migration and proliferative efficiency of MDA-MB-231 cells were completely arrested upon styrene substituted biscoumarin treatment. Also, mRNA gene expression and protein expression of pro-apoptotic (BAX) and anti-apoptotic (BCL-2) genes were analyzed by qRT-PCR and western blot analysis upon styrene substituted biscoumarin treatment to MDA-MB-231 cells. Our results showed that styrene substituted biscoumarin downregulated BCL-2 gene expression and upregulated BAX gene expression to trigger apoptotic process. Styrene substituted biscoumarin could induce apoptosis through intrinsic mitochondrial pathway in breast cancer cell lines, particularly in MDA-MB-231. Our data suggest that styrene substituted biscoumarin may act as a potential chemotherapeutic agent against breast cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate.

PubMed

Morono, Yuki; Takano, Suguru; Miyanaga, Kazuhiko; Tanji, Yasunori; Unno, Hajime; Hori, Katsutoshi

2004-03-01

Staining of esterase-active bacteria with carboxyfluorescein diacetate (CFDA) has been used to evaluate the viability of various types of cell. However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell. Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells. We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining. We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.

Antiproliferative effects of cinobufacini on human hepatocellular carcinoma HepG2 cells detected by atomic force microscopy

PubMed Central

Wu, Qing; Lin, Wei-Dong; Liao, Guan-Qun; Zhang, Li-Guo; Wen, Shun-Qian; Lin, Jia-Ying

2015-01-01

AIM: To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action. METHODS: HepG2 cells were treated with different concentrations of cinobufacini. Cell viability was measured by methylthiazolyl tetrazolium (MTT) assay. Cell cycle distribution was analyzed by flow cytometry (FCM). Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope. Changes in morphology and ultrastructure of cells were detected by atomic force microscopy (AFM) at the nanoscale level. RESULTS: MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dose-dependent manner. With the concentration of cinobufacini increasing from 0 to 0.10 mg/mL, the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1% (P < 0.05). FCM analysis demonstrated cell cycle arrest at S phase induced by cinobufacini. The immunofluorescence studies of cytoskeletal and nuclear morphology showed that after cinobufacini treatment, the regular reorganization of actin filaments in HepG2 cells become chaotic, while the nuclei were not damaged seriously. Additionally, high-resolution AFM imaging revealed that cell morphology and ultrastructure changed a lot after treatment with cinobufacini. It appeared as significant shrinkage and deep pores in the cell membrane, with larger particles and a rougher cell surface. CONCLUSION: Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity. PMID:25624718

Heparanase expression in periapical granulomas and radicular cysts.

PubMed

Elad, S; Sherman, Y; Palmon, A; Vlodavsky, I; Or, R

2013-01-01

Heparanase is an endo-β-D-glucuronidase enzyme which degrades heparan sulfate glycosaminoglycan side chains of proteoglycans in the extracellular matrix and in basement membranes. The aim of this study was to evaluate the expression of heparanase in periapical granulomas (PGs) and radicular cysts (RCs). Immunohistochemistry was used to assess heparanase expression in PGs and RCs. Parameters including stain intensity, location and cell type were used to characterize heparanase expression in the periapical lesions. Ordered categories (from weak to strong) were used to compare the level of heparanase staining in the PG and RC groups. Both epithelial cells and inflammatory cells were positive for heparanase. The relative staining of the epithelial cells was strong, whereas the relative staining of the inflammatory cells was weak. Significant differences in immunohistochemical staining of epithelial cells were observed between RCs and PGs (p = 0.002). The relative expression of heparanase in epithelial cells in RCs was strong. In PGs, lesions with few or no epithelial cells, heparanase was predominantly expressed weakly by inflammatory cells. PGs and RCs have the same infectious origin. Therefore, the different cellular sources of heparanase in these periapical lesions may imply that this enzyme has specific pathogenetic functions in RCs and PGs.

Limitations in the Use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and Cell Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans.

PubMed

Boyd, Vinc; Cholewa, Olivia Maria; Papas, Klearchos K

2008-03-01

BACKGROUND: A review of current literature shows that the combined use of the cell permeable esterase-substrate fluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide (PI) to be one of the most common fluorescence-based methods to assess the viability of isolated islets of Langerhans, and it is currently used for islet product release prior to transplantation in humans. However, results from this assay do not correlate with islet viability and function or islet transplantation success in animals or humans (Eckhard et al. 2004; Ricordi et al. 2001). This may be in part attributed to considerable differences as well as discrepancies in the use of these reagents on islets. We critically surveyed the literature and evaluated the impact of a number of variables associated with the use of FDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated other fluorescent stains, such as SYTO(R)13, SYTO(R)24 and SYBR(R)14 as possible alternatives to FDA. RESULTS: We found that the stability of stains in storage and stock solutions, the number of islets stained, concentration of stains, staining incubation time, the buffer/media used, and the method of examining islets were significant in the final scoring of viability. For archival file photos, the exposure time and camera/software settings can also impact interpretation of viability. Although our results show that FDA does detect intracellular esterase activity and staining with PI does assess cell membrane integrity, the results obtained from using these stains did not correlate directly with expected islet function and viability per transplantation into diabetic athymic nude mice (Papas et al. 2007). In addition, the use of two nucleic acid stains, such as SYTO(R)13 and PI, for live/dead scoring exhibited staining anomalies which limit their accuracy in assessing islet viability. CONCLUSIONS: From a review of the literature and from our observations on the impact of reagent handling and various staining and imaging parameters used to visually evaluate islets, consistent interpretation of islet cell membrane integrity and viability is dependent upon a number of factors. We discuss the utility and limitations of these reagents in evaluating islet cell membrane integrity and viability.

Limitations in the Use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and Cell Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans

PubMed Central

Boyd, Vinc; Cholewa, Olivia Maria; Papas, Klearchos K.

2010-01-01

Background A review of current literature shows that the combined use of the cell permeable esterase-substrate fluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide (PI) to be one of the most common fluorescence-based methods to assess the viability of isolated islets of Langerhans, and it is currently used for islet product release prior to transplantation in humans. However, results from this assay do not correlate with islet viability and function or islet transplantation success in animals or humans (Eckhard et al. 2004; Ricordi et al. 2001). This may be in part attributed to considerable differences as well as discrepancies in the use of these reagents on islets. We critically surveyed the literature and evaluated the impact of a number of variables associated with the use of FDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated other fluorescent stains, such as SYTO®13, SYTO®24 and SYBR®14 as possible alternatives to FDA. Results We found that the stability of stains in storage and stock solutions, the number of islets stained, concentration of stains, staining incubation time, the buffer/media used, and the method of examining islets were significant in the final scoring of viability. For archival file photos, the exposure time and camera/software settings can also impact interpretation of viability. Although our results show that FDA does detect intracellular esterase activity and staining with PI does assess cell membrane integrity, the results obtained from using these stains did not correlate directly with expected islet function and viability per transplantation into diabetic athymic nude mice (Papas et al. 2007). In addition, the use of two nucleic acid stains, such as SYTO®13 and PI, for live/dead scoring exhibited staining anomalies which limit their accuracy in assessing islet viability. Conclusions From a review of the literature and from our observations on the impact of reagent handling and various staining and imaging parameters used to visually evaluate islets, consistent interpretation of islet cell membrane integrity and viability is dependent upon a number of factors. We discuss the utility and limitations of these reagents in evaluating islet cell membrane integrity and viability. PMID:20814586

The effect of well-characterized, very low-dose x-ray radiation on fibroblasts

PubMed Central

Truong, Katelyn; Bradley, Suzanne; Baginski, Bryana; Wilson, Joseph R.; Medlin, Donald; Zheng, Leon; Wilson, R. Kevin; Rusin, Matthew; Takacs, Endre

2018-01-01

The purpose of this study is to determine the effects of low-dose radiation on fibroblast cells irradiated by spectrally and dosimetrically well-characterized soft x-rays. To achieve this, a new cell culture x-ray irradiation system was designed. This system generates characteristic fluorescent x-rays to irradiate the cell culture with x-rays of well-defined energies and doses. 3T3 fibroblast cells were cultured in cups with Mylar® surfaces and were irradiated for one hour with characteristic iron (Fe) K x-ray radiation at a dose rate of approximately 550 μGy/hr. Cell proliferation, total protein analysis, flow cytometry, and cell staining were performed on fibroblast cells to determine the various effects caused by the radiation. Irradiated cells demonstrated increased proliferation and protein production compared to control samples. Flow cytometry revealed that a higher percentage of irradiated cells were in the G0/G1 phase of the cell cycle compared to control counterparts, which is consistent with other low-dose studies. Cell staining results suggest that irradiated cells maintained normal cell functions after radiation exposure, as there were no qualitative differences between the images of the control and irradiated samples. The result of this study suggest that low-dose soft x-ray radiation might cause an initial pause, followed by a significant increase, in proliferation. An initial “pause” in cell proliferation could be a protective mechanism of the cells to minimize DNA damage caused by radiation exposure. The new cell irradiation system developed here allows for unprecedented control over the properties of the x-rays given to the cell cultures. This will allow for further studies on various cell types with known spectral distribution and carefully measured doses of radiation, which may help to elucidate the mechanisms behind varied cell responses to low-dose x-rays reported in the literature. PMID:29300773

Senescence-like Phenotypes in Human Nevi

PubMed Central

Joselow, Andrew; Lynn, Darren; Terzian, Tamara; Box, Neil F.

2016-01-01

Summary Cellular senescence is an irreversible arrest of cell proliferation at the G1 stage of the cell cycle in which cells become refractory to growth stimuli. Senescence is a critical and potent defense mechanism that mammalian cells have to suppress tumors. While there are many ways to induce a senescence response, oncogene-induced senescence (OIS) remains key to inhibiting progression of cells that have acquired oncogenic mutations. In primary cells in culture, OIS induces a set of measurable phenotypic and behavioral changes, in addition to cell cycle exit. Senescence-associated β-Galactosidase (SA-β-Gal) activity is a main hallmark of senescent cells, along with morphological changes that may depend on the oncogene that is activated, or on the primary cell type. Characteristic cellular changes of senescence include increased size, flattening, multi-nucleation, and extensive vacuolation. At the molecular level, tumor suppressor genes such as p53 and p16INK4a may play a role in initiation or maintenance of OIS. Activation of a DNA damage response and a senescence-associated secretory phenotype could delineate the onset of senescence. Despite advances in our understanding of how OIS suppresses some tumor types, the in vivo role of OIS in melanocytic nevi and melanoma remains poorly understood and not validated. In an effort to stimulate research in this field, we review in this chapter the known markers of senescence and provide experimental protocols for their identification by immunofluorescent staining in melanocytic nevi and malignant melanoma. PMID:27812879

Sulfamic acid promoted one-pot synthesis of phenanthrene fused-dihydrodibenzo-quinolinones: Anticancer activity, tubulin polymerization inhibition and apoptosis inducing studies.

PubMed

Kumar, Niggula Praveen; Thatikonda, Sowjanya; Tokala, Ramya; Kumari, S Sujana; Lakshmi, Uppu Jaya; Godugu, Chandraiah; Shankaraiah, Nagula; Kamal, Ahmed

2018-05-01

A facile one-pot method for the synthesis of new phenanthrene fused-dihydrodibenzo-quinolinone derivatives has been successfully accomplished by employing sulfamic acid as catalyst. These new compounds were evaluated for their in vitro cytotoxic potential against human lung (A549), prostate (PC-3 and DU145), breast (MCF-7) and colon (HT-29 and HCT-116) cancer cell lines. Among all the tested compounds, one of the derivatives 8p showed good anti-proliferative activity against A549 lung cancer cell line with an IC 50 of 3.17 ± 0.52 µM. Flow cytometric analyses revealed that compound 8p arrested both Sub G1 and G2/M phases of cell cycle in a dose dependent manner. The compound 8p also displayed significant inhibition of tubulin polymerization and disruption of microtubule network (IC 50 of 5.15 ± 0.15 µM). Molecular docking studies revealed that compound 8p efficiently interacted with critical amino acid Cys241 of the α/β-tubulin by a hydrogen bond (SH…O = 2.4 Å). Further, the effect of 8p on cell viability was also studied by AO/EB, DCFDA and DAPI staining. The apoptotic characteristic features revealed that 8p inhibited cell proliferation effectively through apoptosis by inducing the ROS generation. Analysis of mitochondrial membrane potential through JC-1 staining and annexin V binding assay indicated the extent of apoptosis in A549 cancer cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Recruitment of single muscle fibers during submaximal cycling exercise.

PubMed

Altenburg, T M; Degens, H; van Mechelen, W; Sargeant, A J; de Haan, A

2007-11-01

In literature, an inconsistency exists in the submaximal exercise intensity at which type II fibers are activated. In the present study, the recruitment of type I and II fibers was investigated from the very beginning and throughout a 45-min cycle exercise at 75% of the maximal oxygen uptake, which corresponded to 38% of the maximal dynamic muscle force. Biopsies of the vastus lateralis muscle were taken from six subjects at rest and during the exercise, two at each time point. From the first biopsy single fibers were isolated and characterized as type I and II, and phosphocreatine-to-creatine (PCr/Cr) ratios and periodic acid-Schiff (PAS) stain intensities were measured. Cross sections were cut from the second biopsy, individual fibers were characterized as type I and II, and PAS stain intensities were measured. A decline in PCr/Cr ratio and in PAS stain intensity was used as indication of fiber recruitment. Within 1 min of exercise both type I and, although to a lesser extent, type II fibers were recruited. Furthermore, the PCr/Cr ratio revealed that the same proportion of fibers was recruited during the whole 45 min of exercise, indicating a rather constant recruitment. The PAS staining, however, proved inadequate to fully demonstrate fiber recruitment even after 45 min of exercise. We conclude that during cycling exercise a greater proportion of type II fibers is recruited than previously reported for isometric contractions, probably because of the dynamic character of the exercise. Furthermore, the PCr/Cr ratio method is more sensitive in determining fiber activation than the PAS stain intensity method.

Heme oxygenase-1 protects INF-gamma primed endothelial cells from Jurkat T-cell adhesion.

PubMed

Du, D; Chang, S; Chen, B; Zhou, H; Chen, Z K

2007-12-01

The heme oxygenase-1 (HO-1) system is associated with the rate-limiting step of conversion of heme, one of the most critical roles in cytoprotective mechanisms. Our study investigated its potential role in protection of endothelial cells from T cells. The recombinant plasmid pcDNA3-HO-1 was transfected into endothelial cells. Indirect fluorescent staining was used to examine the expression of HO-1 protein. Then endothelial cells primed by INF-gamma were mixed in culture with Jurkat T cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). The number of adhesive Jurkat T cells was determined using FACS to evaluate the adhesion effect. After being cultured with endothelial cells, the cell cycle of Jurkat T cells was detected using FACS. Expression of HO-1 on endothelial cells conferred significant protection against Jurkat T-cell-mediated adhesion. The rate of Jurkat T-cell adhesions was reduced to 19.06%, in contrast with 31.42% in the control group (P<.05). After using ZnPP, an inhibitor of HO-1, the rate of Jurkat T-cell adhesion recovered to 29.08%. The binding activities between endothelial cells and Jurkat T cells was blocked by HO-1 expression. The proliferation of Jurkat T cells was inhibited after culture with endothelial cells, which had been transfected with HO-1, which blocked cell cycle entry of T cells. More than 60% of Jurkat T cells remained in G0/G1 compared with 40% among the control group. HO-1 directly protected endothelial cells primed by INF-gamma from Jurkat T cells and down-regulated the expression of HLA-DR on the surface of endothelial cells. These results indicated that transgenic expression of HO-1 may be useful to prevent lymphocytes from responding to endothelial cells.

Histochemical and immunohistochemical characterization of chordoma in ferrets.

PubMed

Yui, Takeshi; Ohmachi, Tetsuo; Matsuda, Kazuya; Okamoto, Minoru; Taniyama, Hiroyuki

2015-04-01

Chordomas of the tip of the tail in 6 ferrets were examined using histopathological, histochemical and immunohistochemical procedures. Histopathologically, round neoplastic cells containing numerous cytoplasmic vacuoles of varying sizes, categorized as "physaliphorous cells", were observed in the amorphous eosinophilic or pale basophilic myxoid stroma. Physaliphorous cells were arranged in lobules and in a "chordoid" or "cobblestone" manner. The neoplasms were diagnosed as benign chordoma without local invasion and metastasis. Histochemically, the cytoplasm of small neoplastic cells was positive for periodic acid-Schiff stain and alcian blue (AB) pH 2.5 and pH 1.0 stains, but negative for hyaluronidase digestion-AB pH 2.5 stain. All neoplastic cells were strongly stained with colloidal ion, negative for high iron diamine AB pH 2.5 and toluidine blue pH 2.5 stains, and positive for Mayer's mucicarmine stain. Immunohistochemistry using antibodies directed against low-molecular-weight cytokeratins (CK18, CK19 and CK20), vimentin and mucin core protein (MUC5AC) revealed that neoplastic cells had both epithelial and mesenchymal elements. The expression of low-molecular-weight cytokeratins suggests that neoplastic cells acquired the properties of glandular epithelial cells and produced epithelial mucus. Furthermore, the expression of cytokeratins, vimentin, S100 protein, brachyury and epithelial membrane antigen indicates that the neoplasms were equivalent to the classic type of human chordoma. Therefore, immunohistochemistry using these antibodies can be useful for the characterization of ferret chordoma.

Al adjuvants can be tracked in viable cells by lumogallion staining.

PubMed

Mile, Irene; Svensson, Andreas; Darabi, Anna; Mold, Matthew; Siesjö, Peter; Eriksson, Håkan

2015-07-01

The mechanism behind the adjuvant effect of aluminum salts is poorly understood notwithstanding that aluminum salts have been used for decades in clinical vaccines. In an aqueous environment and at a nearly neutral pH, the aluminum salts form particulate aggregates, and one plausible explanation of the lack of information regarding the mechanisms could be the absence of an efficient method of tracking phagocytosed aluminum adjuvants and thereby the intracellular location of the adjuvant. In this paper, we want to report upon the use of lumogallion staining enabling the detection of phagocytosed aluminum adjuvants inside viable cells. Including micromolar concentrations of lumogallion in the culture medium resulted in a strong fluorescence signal from cells that had phagocytosed the aluminum adjuvant. The fluorescence appeared as spots in the cytoplasm and by confocal microscopy and co-staining with probes presenting fluorescence in the far-red region of the spectrum, aluminum adjuvants could to a certain extent be identified as localized in acidic vesicles, i.e., lysosomes. Staining and detection of intracellular aluminum adjuvants was achieved not only by diffusion of lumogallion into the cytoplasm, thereby highlighting the presence of the adjuvant, but also by pre-staining the aluminum adjuvant prior to incubation with cells. Pre-staining of aluminum adjuvants resulted in bright fluorescent particulate aggregates that remained fluorescent for weeks and with only a minor reduction of fluorescence upon extensive washing or incubation with cells. Both aluminum oxyhydroxide and aluminum hydroxyphosphate, two of the most commonly used aluminum adjuvants in clinical vaccines, could be pre-stained with lumogallion and were easily tracked intracellularly after incubation with phagocytosing cells. Staining of viable cells using lumogallion will be a useful method in investigations of the mechanisms behind aluminum adjuvants' differentiation of antigen-presenting cells into inflammatory cells. Information will be gained regarding the phagosomal pathways and the events inside the phagosomes, and thereby the ultimate fate of phagocytosed aluminum adjuvants could be resolved. Copyright © 2015 Elsevier B.V. All rights reserved.

High- and low-LET induced chromosome damage in human lymphocytes: a time-course of aberrations in metaphase and interphase

NASA Technical Reports Server (NTRS)

George, K.; Wu, H.; Willingham, V.; Furusawa, Y.; Kawata, T.; Cucinotta, F. A.; Dicello, J. F. (Principal Investigator)

2001-01-01

PURPOSE: To investigate how cell-cycle delays in human peripheral lymphocytes affect the expression of complex chromosome damage in metaphase following high- and low-LET radiation exposure. MATERIALS AND METHODS: Whole blood was irradiated in vitro with a low and a high dose of 1 GeV u(-1) iron particles, 400MeV u(-1) neon particles or y-rays. Lymphocytes were cultured and metaphase cells were collected at different time points after 48-84h in culture. Interphase chromosomes were prematurely condensed using calyculin-A, either 48 or 72 h after exposure to iron particles or gamma-rays. Cells in first division were analysed using a combination of FISH whole-chromosome painting and DAPI/ Hoechst 33258 harlequin staining. RESULTS: There was a delay in expression of chromosome damage in metaphase that was LET- and dose-dependant. This delay was mostly related to the late emergence of complex-type damage into metaphase. Yields of damage in PCC collected 48 h after irradiation with iron particles were similar to values obtained from cells undergoing mitosis after prolonged incubation. CONCLUSION: The yield of high-LET radiation-induced complex chromosome damage could be underestimated when analysing metaphase cells collected at one time point after irradiation. Chemically induced PCC is a more accurate technique since problems with complicated cell-cycle delays are avoided.

p27Kip1 is expressed in proliferating cells in its form phosphorylated on threonine 187

PubMed Central

Troncone, Giancarlo; Martinez, Juan C; Iaccarino, Antonino; Zeppa, Pio; Caleo, Alessia; Russo, Maria; Migliaccio, Ilenia; Motti, Maria L; Califano, Daniela; Palmieri, Emiliano A; Palombini, Lucio

2005-01-01

Background G1/S cell cycle progression requires p27Kip1 (p27) proteolysis, which is triggered by its phosphorylation on threonine (Thr) 187. Since its levels are abundant in quiescent and scarce in cycling cells, p27 is an approved marker for quiescent cells, extensively used in histopathology and cancer research. Methods However here we showed that by using a specific phosphorylation site (pThr187) antibody, p27 is detectable also in proliferative compartments of normal, dysplastic and neoplastic tissues. Results In fact, whereas un-phosphorylated p27 and MIB-1 showed a significant inverse correlation (Spearman R = -0.55; p < 0,001), pThr187-p27 was positively and significantly correlated with MIB-1 expression (Spearman R = 0.88; p < 0,001). Thus proliferating cells only stain for pThr187-p27, whereas they are un-reactive with the regular p27 antibodies. However increasing the sensitivity of the immunocytochemistry (ICH) by the use of an ultra sensitive detection system based on tiramide signal amplification, simultaneous expression and colocalisation of both forms of p27 was shown in proliferating compartments nuclei by double immunofluorescence and laser scanning confocal microscopy studies. Conclusion Overall, our data suggest that p27 expression also occurs in proliferating cells compartments and the combined use of both regular and phospho- p27 antibodies is suggested. PMID:15725363

HU content and dynamics in Escherichia coli during the cell cycle and at different growth rates.

PubMed

Abebe, Anteneh Hailu; Aranovich, Alexander; Fishov, Itzhak

2017-10-16

DNA-binding proteins play an important role in maintaining bacterial chromosome structure and functions. Heat-unstable (HU) histone-like protein is one of the most abundant of these proteins and participates in all major chromosome-related activities. Owing to its low sequence specificity, HU fusions with fluorescent proteins were used for general staining of the nucleoid, aiming to reveal its morphology and dynamics. We have exploited a single chromosomal copy of hupA-egfp fusion under the native promoter and used quantitative microscopy imaging to investigate the amount and dynamics of HUα in Escherichia coli cells. We found that in steady-state growing populations the cellular HUα content is proportional to the cell size, whereas its concentration is size independent. Single-cell live microscopy imaging confirmed that the amount of HUα exponentially increases during the cell cycle, but its concentration is maintained constant. This supports the existence of an auto-regulatory mechanism underlying the HUα cellular level, in addition to reflecting the gene copy number. Both the HUα amount and concentration strongly increase with the cell growth rate in different culture media. Unexpectedly, the HU/DNA stoichiometry also remarkably increases with the growth rate. This last finding may be attributed to a higher requirement for maintaining the chromosome structure in nucleoids with higher complexity. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Suppressive activities and mechanisms of ugonin J on vascular smooth muscle cells and balloon angioplasty-induced neointimal hyperplasia.

PubMed

Pan, Chun-Hsu; Li, Pei-Chuan; Chien, Yi-Chung; Yeh, Wan-Ting; Liaw, Chih-Chuang; Sheu, Ming-Jyh; Wu, Chieh-Hsi

2018-02-01

Neointimal hyperplasia (or restenosis) is primarily attributed to excessive proliferation and migration of vascular smooth muscle cells (VSMCs). In this study, we investigated the inhibitory effects and mechanisms of ugonin J on VSMC proliferation and migration as well as neointimal formation. Cell viability and the cell-cycle distribution were, respectively, analyzed using an MTT assay and flow cytometry. Cell migration was examined using a wound-healing analysis and a transwell assay. Protein expressions and gelatinase activities were, respectively, measured using Western blot and gelatin zymography. Balloon angioplasty-induced neointimal formation was induced in a rat carotid artery model and then examined using immunohistochemical staining. Ugonin J induced cell-cycle arrest at the G 0 /G 1 phase and apoptosis to inhibit VSMC growth. Ugonin J also exhibited marked suppressive activity on VSMC migration. Ugonin J significantly reduced activations of focal adhesion kinase, phosphoinositide 3-kinase, v-akt murine thymoma viral oncogene homolog 1, and extracellular signal-regulated kinase 1/2 proteins. Moreover, ugonin J obviously reduced expressions and activity levels of matrix metalloproteinase-2 and matrix metalloproteinase-9. In vivo data indicated that ugonin J prevented balloon angioplasty-induced neointimal hyperplasia. Our study suggested that ugonin J has the potential for application in the prevention of balloon injury-induced neointimal formation. Copyright © 2017 John Wiley & Sons, Ltd.

High- and low-LET induced chromosome damage in human lymphocytes: a time-course of aberrations in metaphase and interphase.

PubMed

George, K; Wu, H; Willingham, V; Furusawa, Y; Kawata, T; Cucinotta, F A

2001-02-01

To investigate how cell-cycle delays in human peripheral lymphocytes affect the expression of complex chromosome damage in metaphase following high- and low-LET radiation exposure. Whole blood was irradiated in vitro with a low and a high dose of 1 GeV u(-1) iron particles, 400MeV u(-1) neon particles or y-rays. Lymphocytes were cultured and metaphase cells were collected at different time points after 48-84h in culture. Interphase chromosomes were prematurely condensed using calyculin-A, either 48 or 72 h after exposure to iron particles or gamma-rays. Cells in first division were analysed using a combination of FISH whole-chromosome painting and DAPI/ Hoechst 33258 harlequin staining. There was a delay in expression of chromosome damage in metaphase that was LET- and dose-dependant. This delay was mostly related to the late emergence of complex-type damage into metaphase. Yields of damage in PCC collected 48 h after irradiation with iron particles were similar to values obtained from cells undergoing mitosis after prolonged incubation. The yield of high-LET radiation-induced complex chromosome damage could be underestimated when analysing metaphase cells collected at one time point after irradiation. Chemically induced PCC is a more accurate technique since problems with complicated cell-cycle delays are avoided.

Apigetrin inhibits adipogenesis in 3T3-L1 cells by downregulating PPARγ and CEBP-α.

PubMed

Hadrich, Fatma; Sayadi, Sami

2018-04-25

Apigetrin, a flavonoid found in many plant leaves and seeds, has been known to possess antimutagenic, anti-cancer, antioxidant and anti-inflammatory properties. Here, we are investigating the effect of the apigetrin on adipocytes differentiation in 3T3-L1 adipocytes, and elucidating the mechanism of its action. Lipids accumulation was measured by Oil Red O staining and cell cycle was analyzed by flow cytometry. The antioxidant effect of apigetrin was evaluated against hydrogen peroxide. The expression of various genes, involved in adipogenesis and inflammation, was studied by real-time PCR. Our results showed that apigterin treatment inhibited significantly lipid accumulation without effect on cell viability at 100 μM, and it exerted the anti-adipogenic effect during the early stages of differentiation. Flow cytometry analysis showed that apigenin-7-O-glucoside (Ap7G) inhibited cell proliferation during mitotic clonal expansion and caused cell cycle delay. Quantitative PCR analysis revealed that the mRNA levels of C/EBP-α, PPAR-γ, SREBP-1c and FAS were suppressed after apigetrin treatment at 100 μM. Moreover, the mRNA level of pro-inflammatory genes (TNF-α and IL-6) were suppressed after apigterin treatment, at high concentration preadipocyte cells. Taken together, these results indicated that apigenin-7-O-glucoside inhibits adipogenesis of 3T3-L1 preadipocytes at early stage of adipogenesis.

Expression of LIM kinase 1 is associated with reversible G1/S phase arrest, chromosomal instability and prostate cancer.

PubMed

Davila, Monica; Jhala, Darshana; Ghosh, Debashis; Grizzle, William E; Chakrabarti, Ratna

2007-06-08

LIM kinase 1 (LIMK1), a LIM domain containing serine/threonine kinase, modulates actin dynamics through inactivation of the actin depolymerizing protein cofilin. Recent studies have indicated an important role of LIMK1 in growth and invasion of prostate and breast cancer cells; however, the molecular mechanism whereby LIMK1 induces tumor progression is unknown. In this study, we investigated the effects of ectopic expression of LIMK1 on cellular morphology, cell cycle progression and expression profile of LIMK1 in prostate tumors. Ectopic expression of LIMK1 in benign prostatic hyperplasia cells (BPH), which naturally express low levels of LIMK1, resulted in appearance of abnormal mitotic spindles, multiple centrosomes and smaller chromosomal masses. Furthermore, a transient G1/S phase arrest and delayed G2/M progression was observed in BPH cells expressing LIMK1. When treated with chemotherapeutic agent Taxol, no metaphase arrest was noted in these cells. We have also noted increased nuclear staining of LIMK1 in tumors with higher Gleason Scores and incidence of metastasis. Our results show that increased expression of LIMK1 results in chromosomal abnormalities, aberrant cell cycle progression and alteration of normal cellular response to microtubule stabilizing agent Taxol; and that LIMK1 expression may be associated with cancerous phenotype of the prostate.

Co-targeting deoxyribonucleic acid-dependent protein kinase and poly(adenosine diphosphate-ribose) polymerase-1 promotes accelerated senescence of irradiated cancer cells.

PubMed

Azad, Arun; Bukczynska, Patricia; Jackson, Susan; Haupt, Ygal; Haput, Ygal; Cullinane, Carleen; McArthur, Grant A; Solomon, Benjamin

2014-02-01

To examine the effects of combined blockade of DNA-dependent protein kinase (DNA-PK) and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) on accelerated senescence in irradiated H460 and A549 non-small cell lung cancer cells. The effects of KU5788 and AG014699 (inhibitors of DNA-PK and PARP-1, respectively) on clonogenic survival, DNA double-strand breaks (DSBs), apoptosis, mitotic catastrophe, and accelerated senescence in irradiated cells were examined in vitro. For in vivo experiments, H460 xenografts established in athymic nude mice were treated with BEZ235 (a DNA-PK, ATM, and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) and AG014699 to determine effects on proliferation, DNA DSBs, and accelerated senescence after radiation. Compared with either inhibitor alone, combination treatment with KU57788 and AG014699 reduced postradiation clonogenic survival and significantly increased persistence of Gamma-H2AX (γH2AX) foci in irradiated H460 and A549 cells. Notably, these effects coincided with the induction of accelerated senescence in irradiated cells as reflected by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression, and senescence-associated cytokine secretion. In irradiated H460 xenografts, concurrent therapy with BEZ235 and AG014699 resulted in sustained Gamma-H2AX (γH2AX) staining and prominent β-galactosidase activity. Combined DNA-PK and PARP-1 blockade increased tumor cell radiosensitivity and enhanced the prosenescent properties of ionizing radiation in vitro and in vivo. These data provide a rationale for further preclinical and clinical testing of this therapeutic combination. Copyright © 2014. Published by Elsevier Inc.

Impact of lysosomal storage disorders on biology of mesenchymal stem cells: Evidences from in vitro silencing of glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes.

PubMed

Squillaro, Tiziana; Antonucci, Ivana; Alessio, Nicola; Esposito, Anna; Cipollaro, Marilena; Melone, Mariarosa Anna Beatrice; Peluso, Gianfranco; Stuppia, Liborio; Galderisi, Umberto

2017-12-01

Lysosomal storage disorders (LDS) comprise a group of rare multisystemic diseases resulting from inherited gene mutations that impair lysosomal homeostasis. The most common LSDs, Gaucher disease (GD), and Fabry disease (FD) are caused by deficiencies in the lysosomal glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes, respectively. Given the systemic nature of enzyme deficiency, we hypothesized that the stem cell compartment of GD and FD patients might be also affected. Among stem cells, mesenchymal stem cells (MSCs) are a commonly investigated population given their role in hematopoiesis and the homeostatic maintenance of many organs and tissues. Since the impairment of MSC functions could pose profound consequences on body physiology, we evaluated whether GBA and GLA silencing could affect the biology of MSCs isolated from bone marrow and amniotic fluid. Those cell populations were chosen given the former's key role in organ physiology and the latter's intriguing potential as an alternative stem cell model for human genetic disease. Our results revealed that GBA and GLA deficiencies prompted cell cycle arrest along with the impairment of autophagic flux and an increase of apoptotic and senescent cell percentages. Moreover, an increase in ataxia-telangiectasia-mutated staining 1 hr after oxidative stress induction and a return to basal level at 48 hr, along with persistent gamma-H2AX staining, indicated that MSCs properly activated DNA repair signaling, though some damages remained unrepaired. Our data therefore suggest that MSCs with reduced GBA or GLA activity are prone to apoptosis and senescence due to impaired autophagy and DNA repair capacity. © 2017 Wiley Periodicals, Inc.

Classification of phytoplankton cells as live or dead using the vital stains fluorescein diacetate and 5-chloromethylfluorescein diacetate.

PubMed

MacIntyre, Hugh L; Cullen, John J

2016-08-01

Regulations for ballast water treatment specify limits on the concentrations of living cells in discharge water. The vital stains fluorescein diacetate (FDA) and 5-chloromethylfluorescein diacetate (CMFDA) in combination have been recommended for use in verification of ballast water treatment technology. We tested the effectiveness of FDA and CMFDA, singly and in combination, in discriminating between living and heat-killed populations of 24 species of phytoplankton from seven divisions, verifying with quantitative growth assays that uniformly live and dead populations were compared. The diagnostic signal, per-cell fluorescence intensity, was measured by flow cytometry and alternate discriminatory thresholds were defined statistically from the frequency distributions of the dead or living cells. Species were clustered by staining patterns: for four species, the staining of live versus dead cells was distinct, and live-dead classification was essentially error free. But overlap between the frequency distributions of living and heat-killed cells in the other taxa led to unavoidable errors, well in excess of 20% in many. In 4 very weakly staining taxa, the mean fluorescence intensity in the heat-killed cells was higher than that of the living cells, which is inconsistent with the assumptions of the method. Applying the criteria of ≤5% false negative plus ≤5% false positive errors, and no significant loss of cells due to staining, FDA and FDA+CMFDA gave acceptably accurate results for only 8-10 of 24 species (i.e., 33%-42%). CMFDA was the least effective stain and its addition to FDA did not improve the performance of FDA alone. © 2016 The Authors. Journal of Phycology published by Wiley Periodicals, Inc. on behalf of Phycological Society of America.

Co-expression of p53 and MDM2 in human atherosclerosis: implications for the regulation of cellularity of atherosclerotic lesions.

PubMed

Ihling, C; Haendeler, J; Menzel, G; Hess, R D; Fraedrich, G; Schaefer, H E; Zeiher, A M

1998-07-01

Atherosclerosis is a fibroproliferative disease of the arterial intima. It was recently found that wild-type p53 (wt p53) accumulates in human atherosclerotic tissue. Wt p53 is a cell cycle regulator involved in DNA repair, DNA synthesis, cell differentiation, and apoptosis and might therefore make an important contribution to the cellularity of atherosclerotic plaques. The product of the MDM2 gene is a nuclear protein which forms a complex with p53, thereby inhibiting the negative regulatory effects of wt p53 on cell cycle progression. In order to address a potential role of the interaction of p53 with MDM2 for the regulation of cellularity in atherosclerotic tissue, 22 carotid atheromatous plaques from patients undergoing endarterectomy were studied to determine the presence of p53 immunoreactivity (IR), MDM2 IR, cell proliferation as evidenced by MIB1/Ki-67 IR and DNA fragmentation by in situ terminal transferase-mediated dUTP 3' end labelling (TUNEL), as a marker for apoptosis. p53 IR localized to areas with evidence of chronic inflammation (22/22) and was observed in virtually all cell types in 68.79 +/- 7.51 per cent of the nuclei. p53 staining in the control tissue from human internal mammary arteries was present in 0.2 +/- 0.29 per cent of the cells (P < or = 0.002). MDM2 IR was present in all cases (22/22) in macrophages and smooth muscle cells (SMCs) in 60.53 +/- 8.32 per cent of the nuclei (controls: 0.8 +/- 0.65 per cent, P < or = 0.002) and co-localized with p53 IR as shown by examination of adjacent sections and by double immunofluorescence labelling. Importantly, co-immunoprecipitation and western blot analysis revealed that p53 and MDM2 were physically associated, indicating that MDM2-p53 complex formation takes place in vivo in human atherosclerotic tissue. Positive TUNEL staining and MIB1/Ki-67 IR present in 3.01 +/- 1.27 per cent of the nuclei (controls: 0 per cent, P < or = 0.002) localized to the same plaque compartments as p53 IR and MDM2 IR. Thus, the fate of cells with p53 accumulation may depend on the interaction and the stoichiometry of the p53 and MDM2 proteins. Cells were indeed found with strong p53 accumulation and nuclear morphology typical for apoptosis and there were a few MIB1/Ki-67-positive cells with co-expression of MDM2, indicating a possible role for MDM2 in reversing the negative regulatory effects of p53 for cell cycle progression. The nuclear co-localization of p53 IR with MDM2 IR and the co-immunoprecipitation assay indicate the presence of p53-MDM2 complex formation in vivo in human atherosclerotic tissue. The destiny of individual p53 and MDM2-co-expressing cells either to undergo p53-dependent apoptosis or to re-enter the cycle of cell proliferation may depend on the relative ratios of the two proteins. p53 and MDM2 may therefore play an important role in regulating cellularity and inflammatory activity in human atherosclerotic plaques.

Nanog interact with CDK6 to regulates astrocyte cells proliferation following spinal cord injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gu, Jun; Department of Orthopaedics, Xishan People's Hospital, Wuxi, Jiangsu; Ni, Yingjie

2016-01-22

Previous research had reported transcription factors Nanog expressed in pluripotent embryonic stem cells (ESCS) that played an important role in regulating the cell proliferation. Nanog levels are frequently elevated in ESCS, but the role in the spinal cord was not clear. To examine the biological relevance of Nanog, we studied its properties in spinal cord injury model. The expression of Nanog and PCNA was gradually increased and reached a peak at 3 day by western blot analysis. The expression of Nanog was further analyzed by immunohistochemistry. Double immunofluorescent staining uncovered that Nanog can co-labeled with PCNA and GFAP in themore » spinal cord tissue. In vitro, Nanog can promote the proliferation of astrocyte cell by Fluorescence Activating Cell Sorter (FACS) and CCK8. Meanwhile, the cell-cycle protein CDK6 could interact with Nanog in the spinal cord tissue. Taken together, these data suggested that both Nanog may play important roles in spinal cord pathophysiology via interact with CDK6.« less

Arctigenin enhances chemosensitivity to cisplatin in human nonsmall lung cancer H460 cells through downregulation of survivin expression.

PubMed

Wang, Huan-qin; Jin, Jian-jun; Wang, Jing

2014-01-01

Arctigenin, a dibenzylbutyrolactone lignan, enhances cisplatin-mediated cell apoptosis in cancer cells. Here, we sought to investigate the effects of arctigenin on cisplatin-treated non-small-cell lung cancer (NSCLC) H460 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and annexin-V/propidium iodide staining were performed to analyze the proliferation and apoptosis of H460 cells. Arctigenin dose-dependently suppressed cell proliferation and potentiated cell apoptosis, coupled with increased cleavage of caspase-3 and poly(ADP-ribose) polymerase. Moreover, arctigenin sensitized H460 cells to cisplatin-induced proliferation inhibition and apoptosis. Arctigenin alone or in combination with cisplatin had a significantly lower amount of survivin. Ectopic expression of survivin decreased cell apoptosis induced by arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01). Moreover, arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01) induced G1/G0 cell-cycle arrest. Our data provide evidence that arctigenin has a therapeutic potential in combina-tion with chemotherapeutic agents for NSLC. © 2013 Wiley Periodicals, Inc.

Effects of cold light bleaching on the color stability of composite resins

PubMed Central

Cao, Liqun; Huang, Lijuan; Wu, Meisheng; Wei, Hua; Zhao, Shouliang

2015-01-01

To evaluate the effects of cold light bleaching on the color stability of four restorations using a thermocycling stain challenge. 160 specimens (10 mm in diameter and 2 mm thick) were fabricated from 4 composite resins (Gradia Direct-A, Z350XT, Premisa, and Précis) and divided into 4 subgroups. Color was assessed according to the CIEL*a*b* color scale at baseline, after the first cycle of bleaching, after thermocycling stain challenges, and after the second cycle of bleaching. Mean values were compared using three-way analysis of variance, and multiple comparisons of the mean values were performed using the Tukey-Kramer test. All groups showed significant color changes after stain challenge, the color change was more significant in Gradia Direct and Z350XT than in Premisa and Précis. After the second cycle of bleaching, color mostly recovered to its original values. The color stability of Gradia Direct and Z350XT was inferior to that of Premisa and Précis. The discoloration of composite resin materials can be partly removed after cold light bleaching. PMID:26309549

Preferential invasion of mitotic cells by Salmonella reveals that cell surface cholesterol is maximal during metaphase.

PubMed

Santos, António J M; Meinecke, Michael; Fessler, Michael B; Holden, David W; Boucrot, Emmanuel

2013-07-15

Cell surface-exposed cholesterol is crucial for cell attachment and invasion of many viruses and bacteria, including the bacterium Salmonella, which causes typhoid fever and gastroenteritis. Using flow cytometry and 3D confocal fluorescence microscopy, we found that mitotic cells, although representing only 1-4% of an exponentially growing population, were much more efficiently targeted for invasion by Salmonella. This targeting was not dependent on the spherical shape of mitotic cells, but was instead SipB and cholesterol dependent. Thus, we measured the levels of plasma membrane and cell surface cholesterol throughout the cell cycle using, respectively, brief staining with filipin and a fluorescent ester of polyethylene glycol-cholesterol that cannot flip through the plasma membrane, and found that both were maximal during mitosis. This increase was due not only to the rise in global cell cholesterol levels along the cell cycle but also to a transient loss in cholesterol asymmetry at the plasma membrane during mitosis. We measured that cholesterol, but not phosphatidylserine, changed from a ∼2080 outerinner leaflet repartition during interphase to ∼5050 during metaphase, suggesting this was specific to cholesterol and not due to a broad change of lipid asymmetry during metaphase. This explains the increase in outer surface levels that make dividing cells more susceptible to Salmonella invasion and perhaps to other viruses and bacteria entering cells in a cholesterol-dependent manner. The change in cholesterol partitioning also favoured the recruitment of activated ERM (Ezrin, Radixin, Moesin) proteins at the plasma membrane and thus supported mitotic cell rounding.

Analysis of the C. elegans Nucleolus by Immuno-DNA FISH.

PubMed

Lanctôt, Christian

2016-01-01

Caenorhabditis elegans is a well-established model organism which allows, among others, to investigate the link between nucleolar structure/function on the one hand and cell fate choices and cellular differentiation on the other. In addition, C. elegans can be used to study the role of the nucleolus in processes that can be difficult to faithfully reproduce in vitro, such as gametogenesis, disease development, and aging. Here I present two complementary techniques, immunofluorescent staining and DNA fluorescence in situ hybridization, that have been adapted to label nucleolar components at various stages of the life cycle of the worm.

Discriminative staining methods for the nervous system: luxol fast blue--periodic acid-Schiff--hematoxylin triple stain and subsidiary staining methods.

PubMed

Goto, N

1987-09-01

This paper describes a new series of staining methods which can discriminatively demonstrate every structure of the nervous system, including axons and capillaries, in animal and human materials. Methods described in this paper consist of one primary stain, luxol fast blue-periodic acid Schiff-hematoxylin (LPH) and six different subsidiary staining methods. The LPH triple stain can precisely differentiate the following structures: neurons (Nissl bodies, cytoplasm, nuclear membrane and nucleolus), various kinds of nuclei (glia, ependyma, endothelium, leucocyte, connective tissue, etc.), myelin sheaths, neuronal processes (axons and dendrites), reacted glial cell bodies (protoplasmic astrocytes, foamy cells, etc.), blood vessels (arteries, veins and capillaries), meninges, intervening connective tissue, erythrocytes, lipofuscin granules, amyloid bodies, and others. Subsidiary staining methods are also described briefly. Applications are discussed in the context of staining technology and neuromorphological research.

Curcuma longa extract as a histological dye for collagen fibres and red blood cells

PubMed Central

Avwioro, O G; Onwuka, S K; Moody, J O; Agbedahunsi, J M; Oduola, T; Ekpo, O E; Oladele, A A

2007-01-01

Crude ethanolic extract and column chromatographic fractions of the Allepey cultivar of Curcuma longa Roxb, commonly called turmeric (tumeric) in commerce, were used as a stain for tissue sections. Staining was carried out under basic, acidic and neutral media conditions. Inorganic and organic dissolution solvents were used. The stain was used as a counterstain after alum and iron haematoxylins. C. longa stained collagen fibres, cytoplasm, red blood cells and muscle cells yellow. It also stained in a fashion similar to eosin, except for its intense yellow colour. Preliminary phytochemical evaluation of the active column fraction revealed that it contained flavonoids, free anthraquinone and deoxy sugar. A cheap, natural dye can thus be obtained from C. longa. PMID:17451535

Paranuclear blue inclusions in metastatic undifferentiated small cell carcinoma in the bone marrow.

PubMed

Wittchow, R; Laszewski, M; Walker, W; Dick, F

1992-09-01

Paranuclear blue inclusions (PBIs) are frequently identified within metastatic undifferentiated small cell carcinoma (SCC) cells on air-dried bone marrow aspirates stained with Wright's stain. To determine the sensitivity and specificity of this finding, 116 bone marrow aspirates containing metastatic neoplasms were evaluated for the presence and frequency of PBIs. Bone marrow specimens included 47 cases of metastatic SCC of the lung, 13 cases of large cell lymphoma, 19 cases of neuroblastoma, five cases of small, noncleaved cell lymphoma, seven cases of rhabdomyosarcoma, three cases of Ewing's sarcoma, three cases of other sarcomas, and 19 cases of non-small cell carcinoma (adenocarcinoma). PBIs were identified in 40 of 47 (85%) cases of SCC and their frequency varied from 0 to 24% of tumor cells among different cases. In approximately half the cases of SCC, PBIs were identified in 1 to 4% tumor cells; and in eight cases, PBIs were present in 5% or more of tumor cells. PBIs were also identified in two of seven (29%) cases of rhabdomyosarcoma and one case of malignant peripheral nerve sheath tumor, but they were not seen in Ewing's sarcoma, small non-cleaved cell lymphoma, large cell lymphoma, neuroblastoma, or non-small cell carcinoma. In addition, PBIs were not seen in alcohol-fixed, Papanicolaou-stained cytology specimens containing SCC. Ultrastructurally, PBIs may represent phagocytized nuclear/cellular material. PBIs are a feature of small cell carcinoma on air-dried, cytologic material stained with Romanowsky type stains. Their presence may provide diagnostic information with regard to the differential diagnosis of metastatic SCC in the bone marrow. Future studies evaluating non-bone marrow Wright's stained fine-needle aspiration specimens are needed to determine if PBIs are useful in distinguishing SCC from other poorly differentiated tumors in the cytology laboratory.

Glutamine synthetase, heat shock protein-70, and glypican-3 in intrahepatic cholangiocarcinoma and tumors metastatic to liver.

PubMed

Lagana, Stephen M; Moreira, Roger K; Remotti, Helen E; Bao, Fei

2013-05-01

Glutamine synthetase (GS), heat shock protein-70 (HSP-70), and glypican-3 (GPC-3) are markers best characterized in hepatocellular lesions, where they are useful in distinguishing hepatocellular carcinoma from dysplastic nodules. Their staining patterns in intrahepatic cholangiocarcinoma (IH-ChCa) and metastatic tumors in liver are not well described. Tissue microarrays containing 41 IH-ChCa and 24 metastatic tumors in liver were stained with commercially available antibodies to GS, HSP-70, and GPC-3. Five percent staining of tumor cells was considered positive for HSP-70 and GPC-3. For GS, 50% was the cut-off. GS reactivity was present in 31 of 41 IH-ChCa (76%), with the median amount of staining being 65% of tumor cells. HSP-70 reactivity was present in 36 of 41 IH-ChCa (88%) with the median amount of staining being 75% of tumor cells. GPC-3 reactivity was absent from all IH-ChCa. Twenty-seven of 41 IH-ChCa cases were positive for both GS and HSP-70 (66%). GS reactivity was present in 17 of 24 tumors metastatic to liver (71%), with the median amount of staining being 50% of tumor cells. HSP-70 reactivity was present in 21 of 24 tumors metastatic to liver (88%) with the median amount of staining being 80% of tumor cells. GPC-3 reactivity was present in 2 of 24 tumors metastatic to liver (8%) with one showing 5% staining and the other showing 50% staining of tumor cells. Fifteen of 24 cases were positive for both GS and HSP-70 (63%), and 2 cases were positive for all 3 markers (8%). Of the panel of immunostains currently commonly used to distinguish hepatocellular carcinoma from dysplastic hepatocytic nodules, only GPC-3 did not react frequently with metastatic tumors and IH-ChCa, although there was staining in 2 metastatic tumors. GS and HSP-70 are typically positive in IH-ChCa and metastatic tumors. Nothing should be inferred about the histogenesis of a tumor based on positive staining with either of these 2 markers, which currently have no role in tumor of unknown origin panels.

Virus replication cycle of white spot syndrome virus in secondary cell cultures from the lymphoid organ of Litopenaeus vannamei.

PubMed

Li, Wenfeng; Desmarets, Lowiese M B; De Gryse, Gaëtan M A; Theuns, Sebastiaan; Van Tuan, Vo; Van Thuong, Khuong; Bossier, Peter; Nauwynck, Hans J

2015-09-01

The replication cycle of white spot syndrome virus (WSSV) was investigated in secondary cell cultures from the lymphoid organ of Litopenaeus vannamei. The secondary cells formed a confluent monolayer at 24 h post-reseeding, and this monolayer could be maintained for 10 days with a viability of 90 %. Binding of WSSV to cells reached a maximum (73 ± 3 % of cells and 4.84 ± 0.2 virus particles per virus-binding cell) at 120 min at 4 °C. WSSV entered cells by endocytosis. The co-localization of WSSV and early endosomes was observed starting from 30 min post-inoculation (p.i.). Double indirect immunofluorescence staining showed that all cell-bound WSSV particles entered these cells in the period between 0 and 60 min p.i. and that the uncoating of WSSV occurred in the same period. After 1 h inoculation at 27 °C, the WSSV nucleocapsid protein VP664 and envelope protein VP28 started to be synthesized in the cytoplasm from 1 and 3 h p.i., and were transported into nuclei from 3 and 6 h p.i., respectively. The percentage of cells that were VP664- and VP28-positive in their nuclei peaked (50 ± 4 %) at 12 h p.i. Quantitative PCR showed that WSSV DNA started to be synthesized from 6 h p.i. In vivo titration of the supernatants showed that the progeny WSSV were released from 12 h p.i. and peaked at 18 h p.i. In conclusion, the secondary cell cultures from the lymphoid organ were proven to be ideal for examination of the replication cycle of WSSV.

Determination of oestrous cycle of the rats by direct examination: how reliable?

PubMed

Yener, T; Turkkani Tunc, A; Aslan, H; Aytan, H; Cantug Caliskan, A

2007-02-01

For determination of the oestrous cycle in rats classical Papanicolaou technique has long been used successfully. Instead of using many stains in Papanicolaou, staining the vaginal secretions with only methylene blue has also been defined. Recently a new technique in which vaginal samples are directly examined under light microscope has been introduced. The aim of this study was to assess the reliability of this new technique by comparing it with the classical staining techniques. From 20 Wistar rats 60 vaginal samples were collected with a micropipette, three from each. Briefly, the vagina was flushed two to three times then the fluid was placed onto a glass slide. The fluid was equally distributed onto three glass slides. The glass slides were coded. Two samples were stained with Papanicolaou and methylene blue while the other one was examined directly. Determination of the phases of the oestrous cycle was made by the same histologist who was blinded to the groups and coding system. After determination of the oestrous phase in all samples, the results were compared and it was found that the results were matching. In conclusion, the same results can be obtained with the direct examination technique and this technique is reliable, so there is no need to use relatively time-consuming, less practical and more expensive techniques such as Papanicolaou or methylene blue.

Knockdown of Pentraxin 3 suppresses tumorigenicity and metastasis of human cervical cancer cells.

PubMed

Ying, Tsung-Ho; Lee, Chien-Hsing; Chiou, Hui-Ling; Yang, Shun-Fa; Lin, Chu-Liang; Hung, Chia-Hung; Tsai, Jen-Pi; Hsieh, Yi-Hsien

2016-07-05

Pentraxin 3 (PTX3) as an inflammatory molecule has been shown to be involved in immune response, inflammation, and cancer. However, the effects of PTX3 on the biological features of cervical cancer cells in vitro and in vivo have not been delineated. Immunohistochemical staining showed that increased PTX3 expression was significantly associated with tumor grade (P < 0.011) and differentiation (P < 0.019). Knocking down PTX3 with lentivirus-mediated small hairpin RNA (shRNA) in cervical cancer cell lines resulted in inhibited cell viability, diminished colony-forming ability, and induced cell cycle arrest at the G2/M phase of the cell cycle, along with downregulated expression of cyclin B1, cdc2, and cdc25c, and upregulated expression of p-cdc2, p-cdc25c, p21, and p27. Furthermore, knockdown of PTX3 significantly decreased the potential of migration and invasion of cervical cancer cells by inhibiting matrix metalloproteidase-2 (MMP-2), MMP-9, and urokinase plasminogen activator (uPA). Moreover, in vivo functional studies showed PTX3-knockdown in mice suppressed tumorigenicity and lung metastatic potential. Conversely, overexpression of PTX3 enhanced proliferation and invasion both in vitro and in vivo. Our results demonstrated that PTX3 contributes to tumorigenesis and metastasis of human cervical cancer cells. Further studies are warranted to demonstrate PTX3 as a novel therapeutic biomarker for human cervical cancer.

Antibacterial and Cytotoxic Activity of Compounds Isolated from Flourensia oolepis

PubMed Central

Joray, Mariana Belén; Trucco, Lucas Daniel; González, María Laura; Napal, Georgina Natalia Díaz; Palacios, Sara María; Bocco, José Luis; Carpinella, María Cecilia

2015-01-01

The antibacterial and cytotoxic effects of metabolites isolated from an antibacterial extract of Flourensia oolepis were evaluated. Bioguided fractionation led to five flavonoids, identified as 2′,4′-dihydroxychalcone (1), isoliquiritigenin (2), pinocembrin (3), 7-hydroxyflavanone (4), and 7,4′-dihydroxy-3′-methoxyflavanone (5). Compound 1 showed the highest antibacterial effect, with minimum inhibitory concentration (MIC) values ranging from 31 to 62 and 62 to 250 μg/mL, against Gram-positive and Gram-negative bacteria, respectively. On further assays, the cytotoxic effect of compounds 1–5 was determined by MTT assay on acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) cell lines including their multidrug resistant (MDR) phenotypes. Compound 1 induced a remarkable cytotoxic activity toward ALL cells (IC50 = 6.6–9.9 μM) and a lower effect against CML cells (IC50 = 27.5–30.0 μM). Flow cytometry was used to analyze cell cycle distribution and cell death by PI-labeled cells and by Annexin V/PI staining, respectively. Upon treatment, 1 induced cell cycle arrest in the G2/M phase accompanied by a strong induction of apoptosis. These results describe for the first time the antibacterial metabolites of F. oolepis extract, with 1 being the most effective. This chalcone also emerges as a selective cytotoxic agent against sensitive and resistant leukemic cells, highlighting its potential as a lead compound. PMID:26819623

A phase IIa study of HA-irinotecan, formulation of hyaluronic acid and irinotecan targeting CD44 in extensive-stage small cell lung cancer.

PubMed

Alamgeer, Muhammad; Neil Watkins, D; Banakh, Ilia; Kumar, Beena; Gough, Daniel J; Markman, Ben; Ganju, Vinod

2018-04-01

Preclinical studies in small cell lung cancer (SCLC) have shown that hyaluronic acid (HA) can be effectively used to deliver chemotherapy and selectively decrease CD44 expressing (stem cell-like) tumour cells. The current study aimed to replicate these findings and obtain data on safety and activity of HA-irinotecan (HA-IR). Eligible patients with extensive stage SCLC were consented. A safety cohort (n = 5) was treated with HA-IR and Carboplatin (C). Subsequently, the patients were randomised 1:1 to receive experimental (HA-IR + C) or standard (IR + C) treatment, to a maximum of 6 cycles. The second line patients were added to the study and treated with open label HA-IR + C. Tumour response was measured after every 2 cycles. Baseline tumour specimens were stained for CD44s and CD44v6 expression. Circulating tumour cells (CTCs) were enumerated before each treatment cycle. Out of 39 patients screened, 34 were evaluable for the study. The median age was 66 (range 39-83). The overall response rates were 69% and 75% for experimental and standard arms respectively. Median progression free survival was 42 and 28 weeks, respectively (p = 0.892). The treatments were well tolerated. The incidence of grade III/IV diarrhea was more common in the standard arm, while anaemia was more common in the experimental arm. IHC analysis suggested that the patients with CD44s positive tumours may gain survival benefit from HA-IR. HA-IR is well tolerated and active in ES-SCLC. The effect of HA-IR on CD44s + cancer stem-like cells provide an early hint towards a potential novel target.

Cell cycle synchronization and analysis of apoptosis-related gene in skin fibroblasts from domestic cat (Felis silvestris catus) and kodkod (Leopardus guigna).

PubMed

Veraguas, D; Gallegos, P F; Castro, F O; Rodriguez-Alvarez, L

2017-10-01

The kodkod population is in constant decrease and the somatic cell nuclear transfer (SCNT) might help to preserve the genetic pool of this species. The cell cycle synchronization of donor cells plays a crucial role in SCNT. The objective of this research was to evaluate two different methods for quiescence induction, serum starvation (SS) and contact inhibition (CI), both for 1, 3 and 5 days, on skin fibroblast from domestic cat and kodkod. Flow cytometry analysis revealed that in domestic cat, SS and CI, both at 3 and 5 days, increased the percentage of fibroblasts in G0/G1 compared to growing cells (GC) (p < .05). In kodkod, only SS for 3 and 5 days and CI for 1 and 3 days increased the percentage of fibroblasts in G0/G1 compared to GC (p < .05). Viability analysis by differential staining revealed that SS for 5 days decreased the proportion of live fibroblasts in domestic cat and kodkod (p < .05). Regarding gene expression analysis, in domestic cat fibroblasts, no differences were found in the BAX/BCL2 ratio in SS and CI (both at 1, 3 and 5 days) compared to GC. In kodkod fibroblasts, BAX/BCL2 ratio was increased in CI at 3 and 5 days compared to SS at 3 and 5 days (p < .05). In conclusion, in kodkod fibroblasts SS for 5 days and CI after 3 days might have a negative impact on cellular viability. According to these results, we suggest SS for 3 days for cell cycle synchronization in kodkod fibroblasts. © 2017 Blackwell Verlag GmbH.

Defense cells profile of cervical mucous during follicular and luteal phases of estrus cycle in river buffalo

PubMed Central

Ayen, Esmail; Hasanzadeh, Shapour; Tabatabaei, Saleh

2012-01-01

The aim of this study was to evaluate the defense cells changes of cervical mucous during follicular and luteal phases of estrus cycle in river buffalo. Reproductive organs of the adult and apparently healthy female buffaloes were collected from the slaughterhouse. By visual investigation of both the ovaries for presence of corpus luteum and growing follicles, the luteal and follicular phase of each buffalo was specified. Cervical discharge samples were collected by sterile swabs and then spread over the glass slides, dried and fixed with methanol. The specimens were undergone Giemsa staining. The percentage of lymphocytes, neutrophils, monocytes (macrophages), eosinophils and basophils in each case (for both the follicular and luteal phases) were obtained at 20 microscopic fields. The percentage of lymphocytes, neutrophils and basophils in luteal phase were higher than the follicular phase. The percentage of eosinophils in follicular phase was higher than the luteal phase. The percentage of monocytes (macrophages) in luteal and follicular phases was nearly equal. The statistical analysis showed that the differences of all cells between follicular and luteal phase were not significant (P > 0.05). The most defense cells in discharges of external os of cervix (both follicular and luteal phases) were neutrophils and lymphocytes. PMID:25653745

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy†

PubMed Central

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-soo; Torelli, Marco D.; Hamers, Robert J.; Murhpy, Catherine J.; Orr, Galya

2015-01-01

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate eficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells. PMID:24816810

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localizationmore » patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.« less

Apoptosis- and differentiation-inducing activities of jacaric acid, a conjugated linolenic acid isomer, on human eosinophilic leukemia EoL-1 cells.

PubMed

Liu, Wai-Nam; Leung, Kwok-Nam

2014-11-01

Conjugated linolenic acids (CLNAs) are a group of naturally occurring positional and geometrical isomers of the C18 polyunsaturated essential fatty acid, linolenic acid (LNA), with three conjugated double bonds (C18:3). Although previous research has demonstrated the growth-inhibitory effects of CLNA on a wide variety of cancer cell lines in vitro, their action mechanisms and therapeutic potential on human myeloid leukemia cells remain poorly understood. In the present study, we found that jacaric acid (8Z,10E,12Z-octadecatrienoic acid), a CLNA isomer which is present in jacaranda seed oil, inhibited the in vitro growth of human eosinophilic leukemia EoL-1 cells in a time- and concentration-dependent manner. Mechanistic studies showed that jacaric acid triggered cell cycle arrest of EoL-1 cells at the G0/G1 phase and induced apoptosis of the EoL-1 cells, as measured by the Cell Death Detection ELISAPLUS kit, Annexin V assay and JC-1 dye staining. Notably, the jacaric acid-treated EoL-1 cells also underwent differentiation as revealed by morphological and phenotypic analysis. Collectively, our results demonstrated the capability of jacaric acid to inhibit the growth of EoL-1 cells in vitro through triggering cell cycle arrest and by inducing apoptosis and differentiation of the leukemia cells. Therefore, jacaric acid might be developed as a potential candidate for the treatment of certain forms of myeloid leukemia with minimal toxicity and few side effects.

Multimodal confocal mosaicing microscopy: an emphasis on squamous cell carcinoma

NASA Astrophysics Data System (ADS)

Chen, Nathaniel W.; Sensibaugh, Jordan; Ardeshiri, Ardaland; Blanchard, Adam; Jacques, Steven; Gareau, Daniel

2010-02-01

Our previous study reported a sensitivity of 96.6% and a specificity of 89.2% in rapidly detecting Basal Cell Carcinomas (BCCs) when nuclei were stained with acridine orange. Squamous Cell Carcinomas (SCCs) and infiltrative BCCs remain difficult to detect. More complete screening can be achieved utilizing both acridine orange for nuclei staining and eosin for cytoplasmic contrast, using two lasers to excite the two stains independently. Nuclear fluorescence is achieved by staining with acridine orange (0.5mM, 60 s), and cytoplasmic fluorescence is achieved by staining with eosin working solution (30 s). This work shows good morphological contrast of SCC and infiltrative BCC with eosin, acridine orange, and reflectance, and presents a means for rapid SCC and infiltrative BCC detection in fresh skin excisions using multimodal confocal microscopy. In addition, digital staining is shown to effectively simulate hematoxylin and eosin (H&E) histology with confocal mosaics.

[Technical recommendations and best practice guidelines for May-Grünwald-Giemsa staining: literature review and insights from the quality assurance].

PubMed

Piaton, Eric; Fabre, Monique; Goubin-Versini, Isabelle; Bretz-Grenier, Marie-Françoise; Courtade-Saïdi, Monique; Vincent, Serge; Belleannée, Geneviève; Thivolet, Françoise; Boutonnat, Jean; Debaque, Hervé; Fleury-Feith, Jocelyne; Vielh, Philippe; Cochand-Priollet, Béatrix; Egelé, Caroline; Bellocq, Jean-Pierre; Michiels, Jean-François

2015-08-01

May-Grünwald-Giemsa (MGG) stain is a Romanowsky-type, polychromatic stain as those of Giemsa, Leishman and Wright. Apart being the reference method of haematology, it has become a routine stain of diagnostic cytopathology for the study of air-dried preparations (lymph node imprints, centrifuged body fluids and fine needle aspirations). In the context of their actions of promoting the principles of quality assurance in cytopathology, the French Association for Quality Assurance in Anatomic and Cytologic Pathology (AFAQAP) and the French Society of Clinical Cytology (SFCC) conducted a proficiency test on MGG stain in 2013. Results from the test, together with the review of literature data allow pre-analytical and analytical steps of MGG stain to be updated. Recommendations include rapid air-drying of cell preparations/imprints, fixation using either methanol or May-Grünwald alone for 3-10minutes, two-step staining: 50% May-Grünwald in buffer pH 6.8 v/v for 3-5minutes, followed by 10% buffered Giemsa solution for 10-30minutes, and running water for 1-3minutes. Quality evaluation must be performed on red blood cells (RBCs) and leukocytes, not on tumour cells. Under correct pH conditions, RBCs must appear pink-orange (acidophilic) or buff-coloured, neither green nor blue. Leukocyte cytoplasm must be almost transparent, with clearly delineated granules. However, staining may vary somewhat and testing is recommended for automated methods (slide stainers) which remain the standard for reproducibility. Though MGG stain remains the reference stain, Diff-Quik(®) stain can be used for the rapid evaluation of cell samples. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Localization of alpha-uterine protein in human endometrium.

PubMed

Horne, C H; Paterson, W F; Sutcliffe, R G

1982-07-01

Immunoperoxidase staining was used to investigate the origin of human alpha-uterine protein (AUP). Specific staining was observed in the glandular epithelium of the endometrium during the secretory phase of the menstrual cycle and during pregnancy, and in a patient on an oestrogen-progestagen contraceptive pill. The pattern of staining strongly suggests that AUP is secreted into the uterine lumen. The location and concentration of AUP in the uterus may explain the relative concentrations of AUP in amniotic fluid and maternal serum.

A Cell Programmable Assay (CPA) chip.

PubMed

Ju, Jongil; Warrick, Jay; Beebe, David J

2010-08-21

This article describes two kinds of "Cell Programmable Assay" (CPA) chips that utilize passive pumping for the culture and autonomous staining of cells to simply common protocols. One is a single timer channel CPA (sCPA) chip that has one timer channel and one main channel containing a cell culture chamber. The sCPA is used to culture and stain cells using Hoechst nuclear staining dye (a 2 step staining process). The other is a dual timer channel CPA (dCPA) chip that has two timer channels and one main channel with a chamber for cell culture. The dCPA is used here to culture, fix, permeablize, and stain cells using DAPI. The additional timer channel of the dCPA chip allows for automation of 3 steps. The CPA chips were successfully evaluated using HEK 293 cells. In addition, we provide a simplified equation for tuning or redesigning CPA chips to meet the needs of a variety of protocols that may require different timings. The equation is easy to use as it only depends upon the dimensions of microchannel and the volume of the reagent drops. The sCPA and dCPA chips can be readily modified to apply to a wide variety of common cell culture methods and procedures.

Overexpression of heat shock protein 27 (HSP27) increases gemcitabine sensitivity in pancreatic cancer cells through S-phase arrest and apoptosis

PubMed Central

Guo, Yang; Ziesch, Andreas; Hocke, Sandra; Kampmann, Eric; Ochs, Stephanie; De Toni, Enrico N; Göke, Burkhard; Gallmeier, Eike

2015-01-01

We previously established a role for HSP27 as a predictive marker for therapeutic response towards gemcitabine in pancreatic cancer. Here, we investigate the underlying mechanisms of HSP27-mediated gemcitabine sensitivity. Utilizing a pancreatic cancer cell model with stable HSP27 overexpression, cell cycle arrest and apoptosis induction were analysed by flow cytometry, nuclear staining, immunoblotting and mitochondrial staining. Drug sensitivity studies were performed by proliferation assays. Hyperthermia was simulated using mild heat shock at 41.8°C. Upon gemcitabine treatment, HSP27-overexpressing cells displayed an early S-phase arrest subsequently followed by a strongly increased sub-G1 fraction. Apoptosis was characterized by PARP-, CASPASE 3-, CASPASE 8-, CASPASE 9- and BIM- activation along with a mitochondrial membrane potential loss. It was reversible through chemical caspase inhibition. Importantly, gemcitabine sensitivity and PARP cleavage were also elicited by heat shock-induced HSP27 overexpression, although to a smaller extent, in a panel of pancreatic cancer cell lines. Finally, HSP27-overexpressing pancreatic cancer cells displayed an increased sensitivity also towards death receptor-targeting agents, suggesting another pro-apoptotic role of HSP27 along the extrinsic apoptosis pathway. Taken together, in contrast to the well-established anti-apoptotic properties of HSP27 in cancer, our study reveals novel pro-apoptotic functions of HSP27—mediated through both the intrinsic and the extrinsic apoptotic pathways—at least in pancreatic cancer cells. HSP27 could represent a predictive marker of therapeutic response towards specific drug classes in pancreatic cancer and provides a novel molecular rationale for current clinical trials applying the combination of gemcitabine with regional hyperthermia in pancreatic cancer patients. PMID:25331547

DOE Office of Scientific and Technical Information (OSTI.GOV)

Azad, Arun, E-mail: arun.azad@bccancer.bc.ca; Department of Pathology, St. Vincent's Hospital, University of Melbourne, Parkville, Victoria; Bukczynska, Patricia

Purpose: To examine the effects of combined blockade of DNA-dependent protein kinase (DNA-PK) and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) on accelerated senescence in irradiated H460 and A549 non-small cell lung cancer cells. Methods and Materials: The effects of KU5788 and AG014699 (inhibitors of DNA-PK and PARP-1, respectively) on clonogenic survival, DNA double-strand breaks (DSBs), apoptosis, mitotic catastrophe, and accelerated senescence in irradiated cells were examined in vitro. For in vivo experiments, H460 xenografts established in athymic nude mice were treated with BEZ235 (a DNA-PK, ATM, and phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor) and AG014699 to determine effects on proliferation, DNA DSBs,more » and accelerated senescence after radiation. Results: Compared with either inhibitor alone, combination treatment with KU57788 and AG014699 reduced postradiation clonogenic survival and significantly increased persistence of Gamma-H2AX (γH2AX) foci in irradiated H460 and A549 cells. Notably, these effects coincided with the induction of accelerated senescence in irradiated cells as reflected by positive β-galactosidase staining, G2-M cell-cycle arrest, enlarged and flattened cellular morphology, increased p21 expression, and senescence-associated cytokine secretion. In irradiated H460 xenografts, concurrent therapy with BEZ235 and AG014699 resulted in sustained Gamma-H2AX (γH2AX) staining and prominent β-galactosidase activity. Conclusion: Combined DNA-PK and PARP-1 blockade increased tumor cell radiosensitivity and enhanced the prosenescent properties of ionizing radiation in vitro and in vivo. These data provide a rationale for further preclinical and clinical testing of this therapeutic combination.« less

Long Term Culture of the A549 Cancer Cell Line Promotes Multilamellar Body Formation and Differentiation towards an Alveolar Type II Pneumocyte Phenotype

PubMed Central

Cooper, James Ross; Abdullatif, Muhammad Bilal; Burnett, Edward C.; Kempsell, Karen E.; Conforti, Franco; Tolley, Howard; Collins, Jane E.; Davies, Donna E.

2016-01-01

Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 ‘alveolar’ cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham’s F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line. PMID:27792742

Daoy medulloblastoma cells that express CD133 are radioresistant relative to CD133- cells, and the CD133+ sector is enlarged by hypoxia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blazek, Ed R.; Foutch, Jennifer L.; Maki, Guitta

2007-01-01

Purpose: Primary medulloblastoma and glioblastoma multiforme tumor cells that express the surface marker CD133 are believed to be enriched for brain tumor stem cells because of their unique ability to initiate or reconstitute tumors in immunodeficient mice. This study sought to characterize the radiobiological properties and marker expression changes of CD133+ vs. CD133- cells of an established medulloblastoma cell line. Methods and Materials: Daoy and D283 Med cell lines were stained with fluorescently labeled anti-CD133 antibody and sorted into CD133+ and CD133- populations. The effect of oxygen (2% vs. 20%) on CD133 expression was measured. Both populations were analyzed formore » marker stability, cell cycle distribution, and radiosensitivity. Results: CD133+ Daoy cells restored nearly native CD133+ and CD133- populations within 18 days, whereas CD133- cells remained overwhelmingly CD133-. Culturing Daoy cells in 2% oxygen rather than the standard 20% oxygen increased their CD133 expression 1.6-fold. CD133+ Daoy cells were radioresistant via the {beta}-parameter of the linear-quadratic model relative to CD133- Daoy cells, although their {alpha}-parameters and cell cycle distributions were identical. Conclusions: Restoration of the original CD133+ and CD133- populations from CD133+ Daoy cells in serum is further evidence that CD133+ cells are functionally distinct from CD133- cells. The radioresistance of CD133+ compared with CD133- Daoy cells is consistent with better repair of sublethal damage. Enlargement of the CD133+ sector is a new feature of the hypoxic response.« less

The crude extract of Corni Fructus induces apoptotic cell death through reactive oxygen species-modulated pathways in U-2 OS human osteosarcoma cells.

PubMed

Liao, Ching-Lung; Hsu, Shu-Chun; Yu, Chien-Chih; Yang, Jai-Sing; Tang, Nou-Ying; Wood, Wellington Gibson; Lin, Jaung-Geng; Chung, Jing-Gung

2014-09-01

Crude extract of Corni Fructus (CECF) has been used in Traditional Chinese medicine for the treatment of different diseases for hundreds of years. The purpose of this study was to investigate the cytotoxic effects of CECF on U-2 OS human osteosarcoma cells. Flow cytometry was used for measuring the percentage of viable cells, cell-cycle distribution, apoptotic cells in sub-G1 phase, reactive oxygen species (ROS), Ca(2+) levels, and mitochondrial membrane potential (ΔΨm ). Comet assay and 4'-6-diamidino-2-phenylindole staining were used for examining DNA damage and condensation. Western blotting was used to examine apoptosis-associated protein levels in U-2 OS cells after exposed to CECF. Immunostaining and confocal laser system microscope were used to examine protein translocation after CECF incubation. CECF decreased the percentage of viability, induced DNA damage and DNA condensation, G₀/G₁ arrest, and apoptosis in U-2 OS cells. CECF-stimulated activities of caspase-8, caspase-9, and caspase-3, ROS, and Ca(2+) production, decreased ΔΨm levels of in U-2 OS cells. CECF increased protein levels of caspase-3, caspase-9, Bax, cytochrome c, GRP78, AIF, ATF-6α, Fas, TRAIL, p21, p27, and p16 which were associated with cell-cycle arrest and apoptosis. These findings suggest that CECF triggers apoptosis in U-2 OS cells via ROS-modulated caspase-dependent and -independent pathways. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

Immunohistochemical characterization of prototypical endometrial clear cell carcinoma--diagnostic utility of HNF-1β and oestrogen receptor.

PubMed

Hoang, Lien N; Han, Guangming; McConechy, Melissa; Lau, Sherman; Chow, Christine; Gilks, C Blake; Huntsman, David G; Köbel, Martin; Lee, Cheng-Han

2014-03-01

The great majority of ovarian clear cell carcinomas have a hepatocyte nuclear factor 1 homeobox B (HNF-1β)-positive and oestrogen receptor (ER)-negative immunoprofile. However, the pattern of HNF-1β and ER immunostaining in clear cell carcinomas of the endometrium and the usefulness of this panel in distinguishing clear cell carcinoma from other histological types of endometrial carcinoma have yet to be well defined. We examined the immunostaining patterns of HNF-1β, ER and p53 in 15 morphologically classic pure endometrial clear cell carcinomas, and compared these patterns with 15 endometrioid and 15 serous carcinomas of the endometrium. We observed the presence of diffuse (>70%) moderate to strong nuclear HNF-1β staining and negative ER staining in 14 of 15 clear cell carcinomas, with the remaining case showing both diffuse strong nuclear HNF-1β staining and focal ER staining. In comparison, only one of 15 serous carcinomas and none of 15 endometrioid carcinomas showed a combination of diffuse moderate to strong HNF-1β nuclear staining and negative ER staining. Aberrant p53 immunostaining was observed in five of 15 (33%) clear cell carcinomas. Overall, our findings demonstrate that, similarly to the situation for the ovary, a diagnostic panel of HNF-1β and ER may be considered for separating clear cell carcinoma from endometrioid and serous carcinoma of the endometrium. © 2013 John Wiley & Sons Ltd.

A Historical Perspective on the Identification of Cell Types in Pancreatic Islets of Langerhans by Staining and Histochemical Techniques

PubMed Central

2015-01-01

Before the middle of the previous century, cell types of the pancreatic islets of Langerhans were identified primarily on the basis of their color reactions with histological dyes. At that time, the chemical basis for the staining properties of islet cells in relation to the identity, chemistry and structure of their hormones was not fully understood. Nevertheless, the definitive islet cell types that secrete glucagon, insulin, and somatostatin (A, B, and D cells, respectively) could reliably be differentiated from each other with staining protocols that involved variations of one or more tinctorial techniques, such as the Mallory-Heidenhain azan trichrome, chromium hematoxylin and phloxine, aldehyde fuchsin, and silver impregnation methods, which were popularly used until supplanted by immunohistochemical techniques. Before antibody-based staining methods, the most bona fide histochemical techniques for the identification of islet B cells were based on the detection of sulfhydryl and disulfide groups of insulin. The application of the classical islet tinctorial staining methods for pathophysiological studies and physiological experiments was fundamental to our understanding of islet architecture and the physiological roles of A and B cells in glucose regulation and diabetes. PMID:26216133

Characterization of Cytokinetic Mutants Using Small Fluorescent Probes.

PubMed

Smertenko, Andrei; Moschou, Panagiotis; Zhang, Laining; Fahy, Deirdre; Bozhkov, Peter

2016-01-01

Cytokinesis is a powerful paradigm for addressing fundamental questions of plant biology including molecular mechanisms of development, cell division, cell signaling, membrane trafficking, cell wall synthesis, and cytoskeletal dynamics. Genetics was instrumental in identification of proteins regulating cytokinesis. Characterization of mutant lines generated using forward or reverse genetics includes microscopic analysis for defects in cell division. Typically, failure of cytokinesis results in appearance of multinucleate cells, formation of cell wall stubs, and isotropic cell expansion in the root elongation zone. Small fluorescent probes served as a very effective tool for the detection of cytokinetic defects. Such probes stain living or formaldehyde-fixed specimens avoiding complex preparatory steps. Although resolution of the fluorescence probes is inferior to electron microscopy, the procedure is fast, easy, and does not require expensive materials or equipment. This chapter describes techniques for staining DNA with the probes DAPI and SYTO82, for staining membranes with FM4-64, and for staining cell wall with propidium iodide.

Acridine orange staining reaction as an index of physiological activity in Escherichia coli

NASA Technical Reports Server (NTRS)

McFeters, G. A.; Singh, A.; Byun, S.; Callis, P. R.; Williams, S.

1991-01-01

The assumption that the acridine orange (AO) color reaction may be used as an index of physiological activity was investigated in laboratory grown Escherichia coli. Spectrofluorometric observations of purified nucleic acids, ribosomes and the microscopic color of bacteriophage-infected cells stained with AO confirmed the theory that single-stranded nucleic acids emit orange to red fluorescence while those that are double-stranded fluoresce green in vivo. Bacteria growing actively in a rich medium could be distinguished from cells in stationary phase by the AO reaction. Cells from log phase appeared red, whereas those in stationary phase were green. However, this differentiation was not seen when the bacteria were grown in a minimal medium or when a variation of the staining method was used. Also, shifting bacteria in stationary phase to starvation conditions rapidly changed their AO staining reaction. Boiling and exposure to lethal concentrations of azide and formalin resulted in stationary-phase cells that appeared red after staining but bacteria killed with chlorine remained green. These findings indicate that the AO staining reaction may be suggestive of physiological activity under defined conditions. However, variables in staining and fixation procedures as well as uncertainties associated with mixed bacterial populations in environmental samples may produce results that are not consistent with the classical interpretation of this reaction. The importance of validating the putative physiological implications of this staining reaction is stressed.

Predominant mucosal expression of 5-HT4(+h) receptor splice variants in pig stomach and colon

PubMed Central

Priem, Evelien KV; De Maeyer, Joris H; Vandewoestyne, Mado; Deforce, Dieter; Lefebvre, Romain A

2013-01-01

AIM: To investigate cellular 5-HT4(-h/+h) receptor distribution, particularly in the epithelial layer, by laser microdissection and polymerase chain reaction (PCR) in porcine gastrointestinal (GI) tissues. METHODS: A stepwise approach was used to evaluate RNA quality and to study cell-specific 5-HT4 receptor mRNA expression in the porcine gastric fundus and colon descendens. After freezing, staining and laser microdissection and pressure catapulting (LMPC), RNA quality was evaluated by the Experion automated electrophoresis system. 5-HT4 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expressions were examined by endpoint reverse transcription (RT)-PCR in mucosal and muscle-myenteric plexus (MMP) tissue fractions, in mucosal and MMP parts of hematoxylin and eosin (HE) stained tissue sections and in microdissected patches of the epithelial and circular smooth muscle cell layer in these sections. Pig gastric fundus tissue sections were also stained immunohistochemically (IHC) for enterochromaffin cells (EC cells; MAB352); these cells were isolated by LMPC and examined by endpoint RT-PCR. RESULTS: After HE staining, the epithelial and circular smooth muscle cell layer of pig colon descendens and the epithelial cell layer of gastric fundus were identified morphologically and isolated by LMPC. EC cells of pig gastric fundus were successfully stained by IHC and isolated by LMPC. Freezing, HE and IHC staining, and LMPC had no influence on RNA quality. 5-HT4 receptor and GAPDH mRNA expressions were detected in mucosa and MMP tissue fractions, and in mucosal and MMP parts of HE stained tissue sections of pig colon descendens and gastric fundus. In the mucosa tissue fractions of both GI regions, the expression of h-exon containing receptor [5-HT4(+h) receptor] mRNA was significantly higher (P < 0.01) compared to 5-HT4(-h) receptor expression, and a similar trend was obtained in the mucosal part of HE stained tissue sections. Large microdissected patches of the epithelial and circular smooth muscle cell layer of pig colon descendens and of the epithelial cell layer of pig gastric fundus, also showed 5-HT4 receptor and GAPDH mRNA expression. No 5-HT4 receptor mRNA expression was detected in gastric LMPC-isolated EC cells from IHC stained tissues, which cells were positive for GAPDH. CONCLUSION: Porcine GI mucosa predominantly expresses 5-HT4(+h) receptor splice variants, suggesting their contribution to the 5-HT4 receptor-mediated mucosal effects of 5-HT. PMID:23840113

Localization of binding sites of Ulex europaeus I, Helix pomatia and Griffonia simplicifolia I-B4 lectins and analysis of their backbone structures by several glycosidases and poly-N-acetyllactosamine-specific lectins in human breast carcinomas.

PubMed

Ito, N; Imai, S; Haga, S; Nagaike, C; Morimura, Y; Hatake, K

1996-09-01

Several studies have shown the deletion of blood group A or B antigens and the accumulation of H antigens in human breast carcinomas. Other studies have independently demonstrated that the binding sites of lectins such as Helix pomatia agglutinin (HPA) and Griffonia simplicifolia agglutinin I-B4 (GSAI-B4) are highly expressed in these cells. In order to clarify the molecular mechanisms of malignant transformation and metastasis of carcinoma cells, it is important to understand the relationship between such phenotypically distinct events. For this purpose, we examined whether the binding sites of these lectins and Ulex europaeus agglutinin I (UEA-I) are expressed concomitantly in the same carcinoma cells and analyzed their backbone structures. The expression of the binding sites of these lectins was observed independently of the blood group (ABO) of the patients and was not affected by the histological type of the carcinomas. Observation of serial sections stained with these lectins revealed that the distribution of HPA binding sites was almost identical to that of GSAI-B4 in most cases. Furthermore, in some cases, UEA-I binding patterns were similar to those of HPA and GSAI-B4 but in other cases, mosaic staining patterns with these lectins were also observed, i.e., some cell clusters were stained with both HPA and GSAI-B4 but not with UEA-I and adjacent cell clusters were stained only with UEA-I. Digestion with endo-beta-galactosidase or N-glycosidase F markedly reduced the staining intensity of these lectins. Together with the reduction of staining by these lectins, reactivity with Griffonia simplicifolia agglutinin II appeared in carcinoma cells following endo-beta-galactosidase digestion. Among the lectins specific to poly-N-acetyllactosamine, Lycopersicon esculentum agglutinin (LEA) most vividly and consistently stained the cancer cells. Next to LEA, pokeweed mitogen agglutinin was also effective in staining these cells. Carcinoma cells reactive with these lectins corresponded well to those stained with both HPA and GSAI-B4, and in some cases, with UEA-I. These results demonstrate that the binding sites of UEA-I, HPA, and GSAI-B4 are expressed concomitantly in the same carcinoma cells and all carry linear and branched poly-N-acetyllactosamine on N-glycans, suggesting that the synthesis of this complex carbohydrate is one of the most important and basic processes leading to the malignant transformation of cells, invasion, and metastasis of carcinoma cells.

Two-color immunostaining of liver fine needle aspiration biopsies with CD34 and carcinoembryonic antigen. Potential utilization in the diagnosis of primary hepatocellular carcinoma vs. metastatic tumor.

PubMed

Yoder, Michael; Zimmerman, Robert L; Bibbo, Marluce

2004-04-01

To examine immunohistochemical staining of cell block material with antibodies against vascular marker CD34 and polyclonal carcinoembryonic antigen (pCEA) for their clinical utility as part of a 2-color staining protocol in fine needle aspiration (FNA) biopsy of liver masses to distinguish metastases from primary hepatocellular carcinoma (HCC). The authors obtained cell block material from 96 liver FNAs and performed simultaneous (i.e., "dual-color") immunohistochemical staining utilizing antibodies against vascular marker CD34 and pCEA. Cases were blinded and evaluated by the authors for staining pattern and intensity. A consensus was obtained, the results were unblinded, and the diagnoses were correlated. After staining, 89 cases had sufficient tissue for evaluation. Of the 19 HCC cases, 16 (84%) showed peripheral staining with CD34, and 13 (68%) showed a canalicular or mixed canalicular-cytoplasmic staining pattern for pCEA. Thirteen cases (68%) showed staining for both antigens. All HCC exhibited immunostaining for at least 1 antibody in an appropriate staining pattern. Of the 67 cases of metastatic malignancy, 5 (7%) showed a predominantly transgressing pattern of CD34 staining, 43 (64%) showed a predominantly cytoplasmic or mixed cytoplasmic-canalicular pattern of pCEA staining, and 2 cases (3%) showed staining for both antigens in a transgressing CD34 pattern and cytoplasmic pCEA pattern. None of the 3 normal liver tissue blocks showed staining with either antigen. Two-color immunohistochemical staining of liver cell block material obtained by FNA with antibodies to CD34 and pCEA can be helpful in differentiating metastatic tumors vs. primary HCC.

A combined Bodian-Nissl stain for improved network analysis in neuronal cell culture.

PubMed

Hightower, M; Gross, G W

1985-11-01

Bodian and Nissl procedures were combined to stain dissociated mouse spinal cord cells cultured on coverslips. The Bodian technique stains fine neuronal processes in great detail as well as an intracellular fibrillar network concentrated around the nucleus and in proximal neurites. The Nissl stain clearly delimits neuronal cytoplasm in somata and in large dendrites. A combination of these techniques allows the simultaneous depiction of neuronal perikarya and all afferent and efferent processes. Costaining with little background staining by either procedure suggests high specificity for neurons. This procedure could be exploited for routine network analysis of cultured neurons.

Utility of Modified Ultrafast Papanicolaou Stain in Cytological Diagnosis

PubMed Central

Arakeri, Surekha Ulhas

2017-01-01

Introduction Need for minimal turnaround time for assessing Fine Needle Aspiration Cytology (FNAC) has encouraged innovations in staining techniques that require lesser staining time with unequivocal cell morphology. The standard protocol for conventional Papanicolaou (PAP) stain requires about 40 minutes. To overcome this, Ultrafast Papanicolaou (UFP) stain was introduced which reduces staining time to 90 seconds and also enhances the quality. However, reagents required for this were not easily available hence, Modified Ultrafast Papanicolaou (MUFP) stain was introduced subsequently. Aim To assess the efficacy of MUFP staining by comparing the quality of MUFP stain with conventional PAP stain. Materials and Methods FNAC procedure was performed by using 10 ml disposable syringe and 22-23 G needle. Total 131 FNAC cases were studied which were lymph node (30), thyroid (38), breast (22), skin and soft tissue (24), salivary gland (11) and visceral organs (6). Two smears were prepared and stained by MUFP and conventional PAP stain. Scores were given on four parameters: background of smears, overall staining pattern, cell morphology and nuclear staining. Quality Index (QI) was calculated from ratio of total score achieved to maximum score possible. Statistical analysis using chi square test was applied to each of the four parameters before obtaining the QI in both stains. Students t-test was applied to evaluate the efficacy of MUFP in comparison with conventional PAP stain. Results The QI of MUFP for thyroid, breast, lymph node, skin and soft tissue, salivary gland and visceral organs was 0.89, 0.85, 0.89, 0.83, 0.92, and 0.78 respectively. Compared to conventional PAP stain QI of MUFP smears was better in all except visceral organ cases and was statistically significant. MUFP showed clear red blood cell background, transparent cytoplasm and crisp nuclear features. Conclusion MUFP is fast, reliable and can be done with locally available reagents with unequivocal morphology which is the need of the hour for a cytopathology set-up. PMID:28511391

Utility of Modified Ultrafast Papanicolaou Stain in Cytological Diagnosis.

PubMed

Sinkar, Prachi; Arakeri, Surekha Ulhas

2017-03-01

Need for minimal turnaround time for assessing Fine Needle Aspiration Cytology (FNAC) has encouraged innovations in staining techniques that require lesser staining time with unequivocal cell morphology. The standard protocol for conventional Papanicolaou (PAP) stain requires about 40 minutes. To overcome this, Ultrafast Papanicolaou (UFP) stain was introduced which reduces staining time to 90 seconds and also enhances the quality. However, reagents required for this were not easily available hence, Modified Ultrafast Papanicolaou (MUFP) stain was introduced subsequently. To assess the efficacy of MUFP staining by comparing the quality of MUFP stain with conventional PAP stain. FNAC procedure was performed by using 10 ml disposable syringe and 22-23 G needle. Total 131 FNAC cases were studied which were lymph node (30), thyroid (38), breast (22), skin and soft tissue (24), salivary gland (11) and visceral organs (6). Two smears were prepared and stained by MUFP and conventional PAP stain. Scores were given on four parameters: background of smears, overall staining pattern, cell morphology and nuclear staining. Quality Index (QI) was calculated from ratio of total score achieved to maximum score possible. Statistical analysis using chi square test was applied to each of the four parameters before obtaining the QI in both stains. Students t-test was applied to evaluate the efficacy of MUFP in comparison with conventional PAP stain. The QI of MUFP for thyroid, breast, lymph node, skin and soft tissue, salivary gland and visceral organs was 0.89, 0.85, 0.89, 0.83, 0.92, and 0.78 respectively. Compared to conventional PAP stain QI of MUFP smears was better in all except visceral organ cases and was statistically significant. MUFP showed clear red blood cell background, transparent cytoplasm and crisp nuclear features. MUFP is fast, reliable and can be done with locally available reagents with unequivocal morphology which is the need of the hour for a cytopathology set-up.

Osteogenic Differentiation of Three-Dimensional Bioprinted Constructs Consisting of Human Adipose-Derived Stem Cells In Vitro and In Vivo.

PubMed

Wang, Xiao-Fei; Song, Yang; Liu, Yun-Song; Sun, Yu-Chun; Wang, Yu-Guang; Wang, Yong; Lyu, Pei-Jun

2016-01-01

Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3D) bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN). Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E) staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM) compared with that in normal proliferation medium (PM). Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects.

Osteogenic Differentiation of Three-Dimensional Bioprinted Constructs Consisting of Human Adipose-Derived Stem Cells In Vitro and In Vivo

PubMed Central

Liu, Yun-Song; Sun, Yu-chun; Wang, Yu-guang; Wang, Yong; Lyu, Pei-Jun

2016-01-01

Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3D) bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN). Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E) staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM) compared with that in normal proliferation medium (PM). Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects. PMID:27332814

New method for estimating digestion of Paracoccidioides brasiliensis by phagocytic cells in vitro.

PubMed Central

Goihman-Yahr, M; Essenfeld-Yahr, E; Albornoz, M C; Yarzábal, L; de Gómez, M H; San Martín, B; Ocanto, A; Convit, J

1979-01-01

We describe a method by which phagocytosis and digestion of Paracoccidioides brasiliensis yeast cells by polymorphonuclear leukocytes or other phagocytic cells may be estimated. Suspensions of P. brasiliensis in its yeastlike phase were sonicated, counted, and incubated with known numbers of peripheral blood polymorphonuclear leukocytes. At given intervals, cytocentrifuge droplets were stained by a variation of Papanicolaou's method. Stained preparations were examined with phase-contrast optics. Digested organisms showed total or partial disappearance of protoplasm. Green-stained cell walls resisted digestion. The proportion of digested cells as a function of time was estimated. Images PMID:90683