Strategies to Suspension Serum-Free Adaptation of Mammalian Cell Lines for Recombinant Glycoprotein Production.

PubMed

Caron, Angelo Luis; Biaggio, Rafael Tagé; Swiech, Kamilla

2018-01-01

Serum-free suspension cultures are preferably required for recombinant protein production due to its readiness in upstream/downstream processing and scale-up, therefore increasing process productivity and competitiveness. This type of culture replaces traditional cell culturing as the presence of animal-derived components may introduce lot-a-lot variability and adventitious pathogens to the process. However, adapting cells to serum-free conditions is challenging, time-consuming, and cell line and medium dependent. In this chapter, we present different approaches that can be used to adapt mammalian cell lines from an anchorage-dependent serum supplemented culture to a suspension serum-free culture.

Pluripotent stem cells and reprogrammed cells in farm animals.

PubMed

Nowak-Imialek, Monika; Kues, Wilfried; Carnwath, Joseph W; Niemann, Heiner

2011-08-01

Pluripotent cells are unique because of their ability to differentiate into the cell lineages forming the entire organism. True pluripotent stem cells with germ line contribution have been reported for mice and rats. Human pluripotent cells share numerous features of pluripotentiality, but confirmation of their in vivo capacity for germ line contribution is impossible due to ethical and legal restrictions. Progress toward derivation of embryonic stem cells from domestic species has been made, but the derived cells were not able to produce germ line chimeras and thus are termed embryonic stem-like cells. However, domestic animals, in particular the domestic pig (Sus scrofa), are excellent large animals models, in which the clinical potential of stem cell therapies can be studied. Reprogramming technologies for somatic cells, including somatic cell nuclear transfer, cell fusion, in vitro culture in the presence of cell extracts, in vitro conversion of adult unipotent spermatogonial stem cells into germ line derived pluripotent stem cells, and transduction with reprogramming factors have been developed with the goal of obtaining pluripotent, germ line competent stem cells from domestic animals. This review summarizes the present state of the art in the derivation and maintenance of pluripotent stem cells in domestic animals.

Differential biological effects of dehydroepiandrosterone (DHEA) between mouse (B16F10) and human melanoma (BLM) cell lines.

PubMed

Joshi, Kumud; Hassan, Sherif S; Ramaraj, Pandurangan

2017-01-01

Dehydroepiandrosterone (DHEA) is a weak androgen and had been shown to have anti-cancer, anti-adipogenic and anti-inflammatory effects on mouse and other rodent models, but not on humans, suggesting a systemic level difference between mouse and human. Our previous study on DHEA biological functions involving a variety of cell lines, suggested that the functional differences between mouse and human existed even at the cellular level. Hence, using mouse and human melanoma cell models, in-vitro effects of DHEA on cell growth, mechanism of cell death and mechanism of DHEA action were studied. Results indicated a differential biological effects of DHEA between mouse and human melanoma cell lines. These in-vitro studies also suggested that the differential biological effects observed between these two cell lines could be due to the difference in the way DHEA was processed or metabolized inside the cell.

A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system.

PubMed

Aranda, Alejandro; Bezunartea, Jaione; Casales, Erkuden; Rodriguez-Madoz, Juan R; Larrea, Esther; Prieto, Jesus; Smerdou, Cristian

2014-12-01

We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. In the MCL, recombinant RNA levels increased >4,600-fold after induction, due to a doxycycline-dependent RNA amplification loop. The MCL maintained inducibility and expression during 50 passages, being more efficient for protein expression than a conventional cell line. To generate new cell lines, mutant LoxP sites were inserted into the MCL, allowing transgene and selection gene exchange by Cre-directed recombination, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest, like human cardiotrophin-1 and oncostatin-M at several mg/l/24 h. These proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.

Optimization of DNA Barcode Method to Assess Altered Chemical Toxicity due to CYP-mediated Metabolism.

EPA Science Inventory

A drawback of current in vitro chemical testing is that many commonly used cell lines lack chemical metabolism. To address this challenge, we present a method for assessing the impact of cellular metabolism on chemical-based cellular toxicity. A cell line with low endogenous meta...

CHARACTERIZATION OF AN EQUINE MACROPHAGE CELL LINE: APPLICATION TO STUDIES OF EIAV INFECTION

PubMed Central

Fidalgo-Carvalho, Isabel; Craigo, Jodi K.; Barnes, Shannon; Costa-Ramos, Carolina; Montelaro, Ronald C.

2009-01-01

EIAV is a monocyte/macrophage tropic virus. To date, even though EIAV has been under investigation for numerous years, very few details have been elucidated about EIAV/macrophage interactions. This is largely due to the absence of an equine macrophage cell line that would support viral replication. Herein we describe the spontaneous immortalization and generation of a clonal equine macrophage-like (EML) cell line with the functional and immunophenotype characteristics of differentiated equine monocyte derived macrophage(s) (eMDM(s)). These cells possess strong non-specific esterase (NSE) activity, are able to phagocytose fluorescent bioparticles, and produce nitrites in response to LPS. The EML-3C cell line expresses the EIAV receptor for cellular entry (ELR1) and supports replication of the virulent EIAVPV biological clone. Thus, EML-3C cells provide a useful cell line possessing equine macrophage related properties for the growth and study of EIAV infection as well as of other equine macrophage tropic viruses. PMID:19038510

Reliable in vitro studies require appropriate ovarian cancer cell lines

PubMed Central

2014-01-01

Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past, in vitro studies using cell line models have been an invaluable tool for basic, discovery-driven cancer research. However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future. PMID:24936210

The effect of syndecan-4 and glypican-1 knockdown on the proliferation and differentiation of turkey satellite cells differing in age and growth rates.

PubMed

Velleman, Sandra G; Clark, Daniel L; Tonniges, Jeffrey R

2018-09-01

Posthatch skeletal muscle growth requires myogenic satellite cells and the dynamic expression of cell membrane-associated proteins. The membrane associated heparan sulfate proteoglycans, syndecan-4 and glypican-1, link the satellite cell niche to the intracellular environment. Sydnecan-4 and glypican-1 are differentially expressed with age in turkey satellite cells and their over-expression impacts both satellite cell proliferation and differentiation, but their effect on satellite cells from lines with different growth potentials is not known. The objective of the current study was to determine if syndecan-4 and glypican-1 regulation of satellite cell proliferation and differentiation is affected by age and growth selection. Pectoralis major satellite cells isolated at 1 d, 7 and 16-wk of age from a Randombred Control 2 (RBC2) line and a 16-wk body weight (F) line selected from the RBC2 line turkeys were studied. Syndecan-4 and glypican-1 expression was knocked down in both lines. The F-line cells proliferated faster than RBC2 line cells regardless of age, while differentiation tended to be greater in RBC2 line cells than F-line cells at each age. Syndecan-4 knockdown decreased proliferation at 7- and 16-wk but not 1 d cells, and increased differentiation at 1 d and 7 wk but not 16 wk cells. Glypican-1 knockdown differentially affected proliferation depending on cell age, whereas differentiation was decreased for 7- and 16-wk but not 1 d cells. These data suggest syndecan-4 and glypican-1 differentially affected satellite cell function in an age-dependent manner, but had little impact on differences in proliferation and differentiation due to growth selection. Copyright © 2018. Published by Elsevier Inc.

Ligand activation of peroxisome proliferator-activated receptor-β/δ (PPAR β/δ) inhibits cell growth in a mouse mammary gland cancer cell line

PubMed Central

Foreman, Jennifer E.; Sharma, Arun K.; Amin, Shantu; Gonzalez, Frank J.; Peters, Jeffrey M.

2009-01-01

The effects of ligand activation of PPARβ/δ were examined in the mouse mammary tumor cell line (C20). Expression of PPARβ/δ was markedly lower in C20 cells as compared to the human non-tumorigenic mammary gland derived cell line (MCF10A) and mouse keratinocytes. Ligand activation of PPARβ/δ in C20 cells caused upregulation of the PPARβ/δ target gene angiopoietin-like 4 (Angptl4). Inhibition of C20 cell proliferation and clonogenicity was observed following treatment with GW0742 or GW501516, two highly specific PPARβ/δ ligands. In addition, an increase in apoptosis was observed in C20 cells cultured with 10 µM GW501516 that preceded the observed inhibition of cell proliferation. Results from this study show that proliferation of the C20 mouse mammary gland cancer cell line is inhibited by ligand activation of PPARβ/δ due in part to increased apoptosis. PMID:19660859

SENSORY HAIR CELL REGENERATION IN THE ZEBRAFISH LATERAL LINE

PubMed Central

Lush, Mark E.; Piotrowski, Tatjana

2014-01-01

Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. PMID:25045019

Identification of pyrogallol as an antiproliferative compound present in extracts from the medicinal plant Emblica officinalis: effects on in vitro cell growth of human tumor cell lines.

PubMed

Khan, Mahmud Tareq Hassan; Lampronti, Ilaria; Martello, Dino; Bianchi, Nicoletta; Jabbar, Shaila; Choudhuri, Mohammad Shahabuddin Kabir; Datta, Bidduyt Kanti; Gambari, Roberto

2002-07-01

In this study we compared the in vitro antiproliferative activity of extracts from medicinal plants toward human tumor cell lines, including human erythromyeloid K562, B-lymphoid Raji, T-lymphoid Jurkat, erythroleukemic HEL cell lines. Extracts from Emblica officinalis were the most active in inhibiting in vitro cell proliferation, after comparison to those from Terminalia arjuna, Aphanamixis polystachya, Oroxylum indicum, Cuscuta reflexa, Aegle marmelos, Saraca asoka, Rumex maritimus, Lagerstroemia speciosa, Red Sandalwood. Emblica officinalis extracts have been studied previously, due to their hepatoprotective, antioxidant, antifungal, antimicrobial and anti-inflammatory medicinal activities. Gas chromatography/mass spectrometry analyses allowed to identify pyrogallol as the common compound present both in unfractionated and n-butanol fraction of Emblica officinalis extracts. Antiproliferative effects of pyrogallol were therefore determined on human tumor cell lines thus identifying pyrogallol as an active component of Emblica officinalis extracts.

Rapid Progressive Disease After Nivolumab Therapy in Three Patients with Metastatic Renal Cell Carcinoma

PubMed Central

KOBARI, YUKI; KONDO, TSUNENORI; TAKAGI, TOSHIO; OMAE, KENJI; NAKAZAWA, HAYAKAZU; TANABE, KAZUNARI

2017-01-01

Background/Aim: Rapid progressive disease (RPD), accelerated tumour growth immediate after the initiation of immune checkpoint inhibitor therapy, has been reported in melanoma and lung cancer. Herein, we describe 3 cases of RPD during the initial phase of nivolumab treatment for metastatic renal cell carcinoma. Patients and Methods: The first and second patients received nivolumab in the fourth-line setting. The third patient received nivolumab therapy as third-line treatment. Results: The first patient developed severe respiratory failure due to carcinomatous lymphangiosis 14 days after initiation of nivolumab therapy. The second patient developed leg paraplegia due to rapid growth of the metastatic tumour at the sixth thoracic vertebrae 5 days later. The third patient developed grade 4 hypercalcemia due to RPD on day 3. Conclusion: Clinicians should be aware of RPD during the initial phase of nivolumab therapy, especially in patients with critical lesions in the late-line setting. PMID:28652455

Paclitaxel sensitivity in relation to ABCB1 expression, efflux and single nucleotide polymorphisms in ovarian cancer.

PubMed

Gao, Bo; Russell, Amanda; Beesley, Jonathan; Chen, Xiao Qing; Healey, Sue; Henderson, Michelle; Wong, Mark; Emmanuel, Catherine; Galletta, Laura; Johnatty, Sharon E; Bowtell, David; Haber, Michelle; Norris, Murray; Harnett, Paul; Chenevix-Trench, Georgia; Balleine, Rosemary L; deFazio, Anna

2014-05-09

ABCB1 (adenosine triphosphate-binding cassette transporter B1) mediates cellular elimination of many chemotherapeutic agents including paclitaxel, which is commonly used to treat ovarian cancer. A significant association between common single nucleotide polymorphisms (SNPs) in ABCB1 and progression-free survival has been reported in patients with ovarian cancer. Variable paclitaxel clearance due to genotype specific differences in ABCB1 activity in cancer cells and/or normal tissues may underlie the association. Using cell-based models, we evaluated the correlations between ABCB1 expression, polymorphisms, transporter activity and paclitaxel sensitivity in ovarian cancer (n = 10) and lymphoblastoid (n = 19) cell lines. Close associations between ABCB1 expression, transporter function and paclitaxel sensitivity were found in lymphoblastoid cell lines, although we could not demonstrate an association with common SNPs. In ovarian cancer cell lines, ABCB1 expression was low and the association between expression and function was lost. These results suggest that ABCB1 related survival difference in ovarian cancer patients is more likely to be due to differential whole body paclitaxel clearance mediated by normal cells rather than a direct effect on cancer cells.

Antiproliferation effect of imatinib mesylate on MCF7, T-47D tumorigenic and MCF 10A nontumorigenic breast cell lines via PDGFR-β, PDGF-BB, c-Kit and SCF genes

PubMed Central

Kadivar, Ali; Kamalidehghan, Behnam; Akbari Javar, Hamid; Karimi, Benyamin; Sedghi, Reihaneh; Noordin, Mohamed Ibrahim

2017-01-01

Recent cancer molecular therapies are targeting main functional molecules to control applicable process of cancer cells. Attractive targets are established by receptor tyrosine kinases, such as platelet-derived growth factor receptors (PDGFRs) and c-Kit as mostly irregular signaling, which is due to either over expression or mutation that is associated with tumorigenesis and cell proliferation. Imatinib mesylate is a selective inhibitor of receptor tyrosine kinase, including PDGFR-β and c-Kit. In this research, we studied how imatinib mesylate would exert effect on MCF7 and T-47D breast cancer and MCF 10A epithelial cell lines, the gene and protein expression of PDGFR-β, c-Kit and their relevant ligands platelet-derived growth factor (PDGF)-BB and stem cell factor (SCF). The MTS assay was conducted in therapeutic relevant concentration of 2–10 µM for 96, 120 and 144 h treatment. In addition, apoptosis induction and cytostatic activity of imatinib mesylate were investigated with the terminal deoxynucleotidyl transferase dUTP nick end labeling TUNEL and cell cycle assays, respectively, in a time-dependent manner. Comparative real-time PCR and Western blot analysis were conducted to evaluate the expression and regulation of imatinib target genes and proteins. Our finding revealed that imatinib mesylate antiproliferation effect, apoptosis induction and cytostatic activity were significantly higher in breast cancer cell lines compared to MCF 10A. This effect might be due to the expression of PDGFR-β, PDGF-BB, c-Kit and SCF, which was expressed by all examined cell lines, except the T-47D cell line which was not expressed c-Kit. However, examined gene and proteins expressed more in cancer cell lines. Therefore, imatinib mesylate was more effective on them. It is concluded that imatinib has at least two potential targets in both examined breast cancer cell lines and can be a promising drug for targeted therapy to treat breast cancer. PMID:28260860

Cytoarchitecture of Zika virus infection in human neuroblastoma and Aedes albopictus cell lines.

PubMed

Offerdahl, Danielle K; Dorward, David W; Hansen, Bryan T; Bloom, Marshall E

2017-01-15

The Zika virus (ZIKV) pandemic is a global concern due to its role in the development of congenital anomalies of the central nervous system. This mosquito-borne flavivirus alternates between mammalian and mosquito hosts, but information about the biogenesis of ZIKV is limited. Using a human neuroblastoma cell line (SK-N-SH) and an Aedes albopictus mosquito cell line (C6/36), we characterized ZIKV infection by immunofluorescence, transmission electron microscopy (TEM), and electron tomography (ET) to better understand infection in these disparate host cells. ZIKV replicated well in both cell lines, but infected SK-N-SH cells suffered a lytic crisis. Flaviviruses scavenge host cell membranes to serve as replication platforms and ZIKV showed the hallmarks of this process. Via TEM, we identified virus particles and 60-100nm spherular vesicles. ET revealed these vesicular replication compartments contain smaller 20-30nm spherular structures. Our studies indicate that SK-N-SH and C6/36 cells are relevant models for viral cytoarchitecture study. Published by Elsevier Inc.

Roscovitine strongly enhances the effect of olaparib on radiosensitivity for HPV neg. but not for HPV pos. HNSCC cell lines.

PubMed

Ziemann, Frank; Seltzsam, Steve; Dreffke, Kristin; Preising, Stefanie; Arenz, Andrea; Subtil, Florentine S B; Rieckmann, Thorsten; Engenhart-Cabillic, Rita; Dikomey, Ekkehard; Wittig, Andrea

2017-12-01

At present, advanced stage human Papillomavirus (HPV) negative and positive head and neck squamous cell carcinoma (HNSCC) are treated by intense multimodal therapy that includes radiochemotherapy, which are associated with relevant side effects. Patients with HPV positive tumors possess a far better prognosis than those with HPV negative cancers. Therefore, new therapeutic strategies are needed to improve the outcome especially of the latter one as well as quality of life for all HNSCC patients. Here we tested whether roscovitine, an inhibitor of cyclin-dependent kinases (CDKs), which hereby also blocks homologous recombination (HR), can be used to enhance the radiation sensitivity of HNSCC cell lines. In all five HPV negative and HPV positive cell lines tested, roscovitine caused inhibition of CDK1 and 2. Surprisingly, all HPV positive cell lines were found to be defective in HR. In contrast, HPV negative strains demonstrated efficient HR, which was completely suppressed by roscovitine. In line with this, for HPV negative but not for HPV positive cell lines, treatment with roscovitine resulted in a pronounced enhancement of the radiation-induced G2 arrest as well as a significant increase in radiosensitivity. Due to a defect in HR, all HPV positive cell lines were efficiently radiosensitized by the PARP-1 inhibitor olaparib. In contrast, in HPV negative cell lines a significant radiosensitization by olaparib was only achieved when combined with roscovitine.

Establishment and characterization of fetal and maternal mesenchymal stem/stromal cell lines from the human term placenta.

PubMed

Qin, Sharon Q; Kusuma, Gina D; Al-Sowayan, Batla; Pace, Rishika A; Isenmann, Sandra; Pertile, Mark D; Gronthos, Stan; Abumaree, Mohamed H; Brennecke, Shaun P; Kalionis, Bill

2016-03-01

Human placental mesenchymal stem/stromal cells (MSC) are an attractive source of MSC with great therapeutic potential. However, primary MSC are difficult to study in vitro due to their limited lifespan and patient-to-patient variation. Fetal and maternal MSC were prepared from cells of the chorionic and basal plates of the placenta, respectively. Fetal and maternal MSC were transduced with the human telomerase reverse transcriptase (hTERT). Conventional stem cell assays assessed the MSC characteristics of the cell lines. Functional assays for cell proliferation, cell migration and ability to form colonies in soft agar were used to assess the whether transduced cells retained properties of primary MSC. Fetal chorionic and maternal MSC were successfully transduced with hTERT to create the cell lines CMSC29 and DMSC23 respectively. The lifespans of CMSC29 and DMSC23 were extended in cell culture. Both cell lines retained important MSC characteristics including cell surface marker expression and multipotent differentiation potential. Neither of the cell lines was tumourigenic in vitro. Gene expression differences were observed between CMSC29 and DMSC23 cells and their corresponding parent, primary MSC. Both cell lines show similar migration potential to their corresponding primary, parent MSC. The data show that transduced MSC retained important functional properties of the primary MSC. There were gene expression and functional differences between cell lines CMSC29 and DMSC23 that reflect their different tissue microenvironments of the parent, primary MSC. CMSC29 and DMSC23 cell lines could be useful tools for optimisation and functional studies of MSC. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cell sources for in vitro human liver cell culture models.

PubMed

Zeilinger, Katrin; Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

2016-09-01

In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. © 2016 by the Society for Experimental Biology and Medicine.

Cell sources for in vitro human liver cell culture models

PubMed Central

Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

2016-01-01

In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro. However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro. Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. PMID:27385595

Establishment and characterization of a platinum- and paclitaxel-resistant high grade serous ovarian carcinoma cell line.

PubMed

Teng, Pang-Ning; Bateman, Nicholas W; Wang, Guisong; Litzi, Tracy; Blanton, Brian E; Hood, Brian L; Conrads, Kelly A; Ao, Wei; Oliver, Kate E; Darcy, Kathleen M; McGuire, William P; Paz, Keren; Sidransky, David; Hamilton, Chad A; Maxwell, G Larry; Conrads, Thomas P

2017-07-01

High grade serous ovarian cancer (HGSOC) patients have a high recurrence rate after surgery and adjuvant chemotherapy due to inherent or acquired drug resistance. Cell lines derived from HGSOC tumors that are resistant to chemotherapeutic agents represent useful pre-clinical models for drug discovery. Here, we describe establishment of a human ovarian carcinoma cell line, which we term WHIRC01, from a patient-derived mouse xenograft established from a chemorefractory HGSOC patient who did not respond to carboplatin and paclitaxel therapy. This newly derived cell line is platinum- and paclitaxel-resistant with cisplatin, carboplatin, and paclitaxel half-maximal lethal doses of 15, 130, and 20 µM, respectively. Molecular characterization of this cell line was performed using targeted DNA exome sequencing, transcriptomics (RNA-seq), and mass spectrometry-based proteomic analyses. Results from exomic sequencing revealed mutations in TP53 consistent with HGSOC. Transcriptomic and proteomic analyses of WHIRC01 showed high level of alpha-enolase and vimentin, which are associated with cell migration and epithelial-mesenchymal transition. WHIRC01 represents a chemorefractory human HGSOC cell line model with a comprehensive molecular profile to aid future investigations of drug resistance mechanisms and screening of chemotherapeutic agents.

Cellular characteristics of primary and immortal canine embryonic fibroblast cells.

PubMed

You, Seungkwon; Moon, Jai-Hee; Kim, Tae-Kyung; Kim, Sung-Chan; Kim, Jai-Woo; Yoon, Du-Hak; Kwak, Sungwook; Hong, Ki-Chang; Choi, Yun-Jaie; Kim, Hyunggee

2004-08-31

Using normal canine embryonic fibroblasts (CaEF) that were shown to be senescent at passages 7th-9th, we established two spontaneously immortalized CaEF cell lines (designated CGFR-Ca-1 and -2) from normal senescent CaEF cells, and an immortal CaEF cell line by exogenous introduction of a catalytic telomerase subunit (designated CGFR-Ca-3). Immortal CGFR- Ca-1, -2 and -3 cell lines grew faster than primary CaEF counterpart in the presence of either 0.1% or 10% FBS. Cell cycle analysis demonstrated that all three immortal CaEF cell lines contained a significantly high proportion of S-phase cells compared to primary CaEF cells. CGFR-Ca-1 and -3 cell lines showed a loss of p53 mRNA and protein expression leading to inactivation of p53 regulatory function, while the CGFR-Ca-2 cell line was found to have the inactive mutant p53. Unlike the CGFR-Ca-3 cell line that down-regulated p16INK4a mRNA due to its promoter methylation but had an intact p16INK4a regulatory function, CGFR-Ca-1 and -2 cell lines expressed p16INK4a mRNA but had a functionally inactive p16INK4a regulatory pathway as judged by the lack of obvious differences in cell growth and phenotype when reconstituted with wild-type p16INK4a. All CGFR-Ca-1, -2 and -3 cell lines were shown to be untransformed but immortal as determined by anchorage-dependent assay, while these cell lines were fully transformed when overexpressed oncogenic H-rasG12V. Taken together, similar to the nature of murine embryo fibroblasts, the present study suggests that normal primary CaEF cells have relatively short in vitro lifespans and should be spontaneously immortalized at high frequency.

Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants

PubMed Central

Gottschalk, Laura B.; Vecchio-Pagan, Briana; Sharma, Neeraj; Han, Sangwoo T.; Franca, Arianna; Wohler, Elizabeth S.; Batista, Denise A.S.; Goff, Loyal A.; Cutting, Garry R.

2016-01-01

Background Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells. Methods A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o−). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit current. RNA sequencing was used to compare the transcriptional profile of the resulting CF8Flp cell line to primary cells and tissues. Results Functional CFTR was expressed from integrated cDNA at the FRT site of the CF8Flp cell line at levels comparable to that seen in native airway cells. CF8Flp cells expressing WT-CFTR have a stable transcriptome comparable to that of primary cultured airway epithelial cells, including genes that play key roles in CFTR pathways. Conclusion CF8Flp cells provide a viable substitute for primary CF airway cells for the analysis of CFTR variants in a native context. PMID:26694805

Frequent biphasic cellular responses of permanent fish cell cultures to deoxynivalenol (DON)

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

Pietsch, Constanze, E-mail: constanze.pietsch@unibas.ch; Bucheli, Thomas D.; Wettstein, Felix E.

Contamination of animal feed with mycotoxins is a major problem for fish feed mainly due to usage of contaminated ingredients for production and inappropriate storage of feed. The use of cereals for fish food production further increases the risk of a potential contamination. Potential contaminants include the mycotoxin deoxynivalenol (DON) which is synthesized by globally distributed fungi of the genus Fusarium. The toxicity of DON is well recognized in mammals. In this study, we confirm cytotoxic effects of DON in established permanent fish cell lines. We demonstrate that DON is capable of influencing the metabolic activity and cell viability inmore » fish cells as determined by different assays to indicate possible cellular targets of this toxin. Evaluation of cell viability by measurement of membrane integrity, mitochondrial activity and lysosomal function after 24 h of exposure of fish cell lines to DON at a concentration range of 0-3000 ng ml{sup -1} shows a biphasic effect on cells although differences in sensitivity occur. The cell lines derived from rainbow trout are particularly sensitive to DON. The focus of this study lies, furthermore, on the effects of DON at different concentrations on production of reactive oxygen species (ROS) in the different fish cell lines. The results show that DON mainly reduces ROS production in all cell lines that were used. Thus, our comparative investigations reveal that the fish cell lines show distinct species-related endpoint sensitivities that also depend on the type of tissue from which the cells were derived and the severity of exposure. - Highlights: > DON uptake by cells is not extensive. > All fish cell lines are sensitive to DON. > DON is most cytotoxic to rainbow trout cells. > Biphasic cellular responses were frequently observed. > Our results are similar to studies on mammalian cell lines.« less

Inducible expression of photoacoustic reporter gene tyrosinase in cells using a single plasmid

NASA Astrophysics Data System (ADS)

Paproski, Robert J.; Zemp, Roger J.

2012-02-01

We have previously demonstrated that tyrosinase is a reporter gene for photoacoustic imaging since tyrosinase is the rate-limiting step in the synthesis of melanin, a pigment capable of producing strong photoacoustic signals. We previously created a cell line capable of inducible tyrosinase expression (important due to toxicity of melanin) by stably transfecting tyrosinase in MCF-7 Tet-OnR cell line (Clontech) which expresses a doxycycline-controlled transactivator. Unfortunately, Clontech provides few Tet-On Advanced cell lines making it difficult to have inducible tyrosinase expression in cell lines not provided by Clontech. In order to simplify the creation of cell lines with inducible expression of tyrosinase, we created a single plasmid that encodes both the transactivator as well as tyrosinase. PCR was used to amplify both the transactivator and tyrosinase from the Tet-OnR Advanced and pTRE-Tight-TYR plasmids, respectively. Both PCR products were cloned into the pEGFP-N1 plasmid and the newly created plasmid was transfected into ZR-75-1, MCF-7, and MIA PaCa-1 cells using lipofectamine. After several days, brown melanin was only observed in cells incubated with doxycycline, suggesting that the newly created single plasmid allowed inducible tyrosinase expression in many different cells lines.

Induction of DNA-strand breaks after X-irradiation in murine bone cells of various differentiation capacities

NASA Astrophysics Data System (ADS)

Lau, Patrick; Hellweg, Christine E.; Kirchner, Simone; Baumstark-Khan, Christa

During longterm space missions, astronauts suffer from the loss of minerals especially from weightbearing bones due to prolonged sojourn under microgravity. In addition to weightlessness, exposure to cosmic ionization radiation is another space related factor endangering health and productivity of astronauts. In order to elucidate changes in bone cell metabolism induced by ionizing radiation, ground-based bone cell models have been developed. The differentiation level of the bone cells may influence their radiation sensitivity. Therefore, our cell model comprises a collection of immortalized murine pre-osteoblast, osteoblast and osteocyte cell lines representing discrete stages of differentiation: the subclones 4 and 24 of the osteoblast cell line MC3T3-E1, the osteoblast cell line OCT-1 and the osteocyte cell line MLO-Y4 display varying potential to produce mineralized bone matrix upon incubation with ascorbic acid and β-glycerophosphate (osteogenic medium). The MLO-Y4 cells showed the highest and subclone 24 the lowest proliferation rate. The most intense von Kossa reaction after culture in osteogenic medium was observed in subclone 4, indicating mineralized bone matrix. The bone cell markers alkaline phosphatase and osteocalcin were determined to further characterize the differentiation stage. All cell lines expressed osteocalcin, as determined by reverse transcriptase polymerase chain reaction. The activity of alkaline phosphatase was highest in the cell line OCT-1 and very low in MLO-Y4 and S4. The peculiarity of the markers suggests a characterization of OCT-1 and S24 as preosteoblast, S4 as (mature) osteoblast, and MLO-Y4 as osteocyte. Survival after exposure to X-rays was determined using the colony forming ability test. The resulting dose-effect relationships revealed normal radiation sensitivity (compared to human fibroblasts). Cell clone specific variations (subclones 4 and 24) in the radiation sensitivity may be due to the differentiation level. The survival curve of MLO-Y4 shows a broad shoulder, suggesting a high repair capacity or a high DNA damage or misrepair tolerance. The quantitative acquisition of DNA-strand breaks was performed by fluorescent analysis of DNA unwinding and revealed a high level of DNA damage immediately after X-irradiation, which increases dose dependently. In conclusion, the cell line with the highest differentiation level (MLO-Y4) displays lower radiation sensitivity, regarding the shoulder width of the dose-effect curve, compared to the less differentiated osteoblast cell lines.

Establishment of a novel human medulloblastoma cell line characterized by highly aggressive stem-like cells.

PubMed

Silva, Patrícia Benites Gonçalves da; Rodini, Carolina Oliveira; Kaid, Carolini; Nakahata, Adriana Miti; Pereira, Márcia Cristina Leite; Matushita, Hamilton; Costa, Silvia Souza da; Okamoto, Oswaldo Keith

2016-08-01

Medulloblastoma is a highly aggressive brain tumor and one of the leading causes of morbidity and mortality related to childhood cancer. These tumors display differential ability to metastasize and respond to treatment, which reflects their high degree of heterogeneity at the genetic and molecular levels. Such heterogeneity of medulloblastoma brings an additional challenge to the understanding of its physiopathology and impacts the development of new therapeutic strategies. This translational effort has been the focus of most pre-clinical studies which invariably employ experimental models using human tumor cell lines. Nonetheless, compared to other cancers, relatively few cell lines of human medulloblastoma are available in central repositories, partly due to the rarity of these tumors and to the intrinsic difficulties in establishing continuous cell lines from pediatric brain tumors. Here, we report the establishment of a new human medulloblastoma cell line which, in comparison with the commonly used and well-established cell line Daoy, is characterized by enhanced proliferation and invasion capabilities, stem cell properties, increased chemoresistance, tumorigenicity in an orthotopic metastatic model, replication of original medulloblastoma behavior in vivo, strong chromosome structural instability and deregulation of genes involved in neural development. These features are advantageous for designing biologically relevant experimental models in clinically oriented studies, making this novel cell line, named USP-13-Med, instrumental for the study of medulloblastoma biology and treatment.

Biometric assessment of prostate cancer's metastatic potential.

PubMed

Cooper, C R; Emmett, N; Harris-Hooker, S; Patterson, R; Cooke, D B

1994-01-01

Currently, no protocol exists that can assess the metastatic potential of prostate adenocarcinoma. The reason for this is partly due to the lack of information on cellular changes that result in a tumor cell's becoming metastatic. In this investigation, attempts were made to devise a method that correlated with the metastatic potential of AT-1, Mat-Lu, and Mat-LyLu cell lines of the Dunning R-3327 rat prostatic adenocarcinoma system. To accomplish this, we applied BioQuant biometric parameters, i.e., area, shape factor, and cell motility. AT-1 had a lower shape factor and a greater area as compared with the more highly metastatic Mat-Lu subline. No significant difference in area or shape factor was detected between the AT-1 cell line and the highly metastatic Mat-LyLu line. However, the lowly metastatic AT-1 line had less motility as compared with the Mat-Lu and Mat-LyLu lines. This study revealed that metastatic potential could be partially predicted via area and shape factor and accurately predicted via cell motility.

Liquid Chromatography Mass Spectrometry Analysis and Cytotoxicity of Asparagus adscendens Roots against Human Cancer Cell Lines.

PubMed

Khan, Kashif Maqbool; Nahar, Lutfun; Mannan, Abdul; Arfan, Muhammad; Khan, Ghazanfar Ali; Al-Groshi, Afaf; Evans, Andrew; Dempster, Nicola M; Ismail, Fyaz M D; Sarker, Satyajit D

2018-01-01

Asparagus adscendens Roxb. (Asparagaceae), is native to the Himalayas. This plant has been used in the prevention and effective treatment of various forms of cancers. This paper reports, for the first time, on the cytotoxicity of the methanol (MeOH) extract of the roots of A. adscendens and its solid-phase extraction (SPE) fractions against four human carcinoma cell lines and LC-ESI-QTOF-MS analysis of the SPE fractions. Finely powdered roots of A. adscendens were macerated in methanol and extracted through SPE using gradient solvent system (water: methanol) proceeded for analysis on LC-ESI-QTOF-MS and cytotoxicity against four human carcinoma cell lines: breast (MCF7), liver (HEPG2), lung (A549), and urinary bladder (EJ138), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. The MeOH extract and four SPE fractions exhibited cytotoxicity against all cell lines with the IC 50 values ranging from 6 to 79 μg/mL. As observed in other Asparagus species, the presence of saponins and sapogenins in the SPE fractions was evident in the liquid chromatography-mass spectrometry data. It is reasonable to assume that the cytotoxicity of the MeOH extract of the roots of A. adscendens and its SPE fractions, at least partly, due to the presence of saponins and their aglycones. This suggests that A. adscendens could be exploited as a potential source of cytotoxic compounds with putative anticancer potential. The MeOH extract and all solid-phase extraction (SPE) fractions exhibited various levels of cytotoxicity against all cell lines with the IC 50 values ranging from 6 to 79 μg/mLThe presence of saponins and sapogenins in the SPE fractions was evident in the Liquid chromatography-mass spectrometry dataDue to the presence of saponins and their aglycones, suggest that A. adscendens could be exploited as a potential source of cytotoxic compounds with putative anticancer potential. Abbreviation used: SPE: Solid-phase extraction, MCF7: Breast cancer cell line, HEPG2: Liver cancer cell line, A549: Lung liver cancer cell line, EJ138: Urinary bladder cancer cell line, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide, LC-MS: Liquid chromatography-mass spectrometry.

Etoposide radiosensitizes p53-defective cholangiocarcinoma cell lines independent of their G2 checkpoint efficacies

PubMed Central

Hematulin, Arunee; Meethang, Sutiwan; Utapom, Kitsana; Wongkham, Sopit; Sagan, Daniel

2018-01-01

Radiotherapy has been accounted as the most comprehensive cancer treatment modality over the past few decades. However, failure of this treatment modality occurs in several malignancies due to the resistance of cancer cells to radiation. It was previously reported by the present authors that defective cell cycle checkpoints could be used as biomarkers for predicting the responsiveness to radiation in individual patients with cholangiocarcinoma (CCA). However, identification of functional defective cell cycle checkpoints from cells from a patient's tissues is cumbersome and not applicable in the clinic. The present study evaluated the radiosensitization potential of etoposide in p53-defective CCA KKU-M055 and KKU-M214 cell lines. Treatment with etoposide enhanced the responsiveness of two p53-defective CCA cell lines to radiation independent of G2 checkpoint function. In addition, etoposide treatment increased radiation-induced cell death without altering the dominant mode of cell death of the two cell lines. These findings indicate that etoposide could be used as a radiation sensitizer for p53-defective tumors, independent of the function of G2 checkpoint. PMID:29541168

Ophiobolin A, a sesterpenoid fungal phytotoxin, displays different mechanisms of cell death in mammalian cells depending upon the cancer cell origin.

PubMed

Morrison, Rachel; Lodge, Tiffany; Evidente, Antonio; Kiss, Robert; Townley, Helen

2017-03-01

Herein we have undertaken a systematic analysis of the effects of the fungal derivative ophiobolin A (OphA) on eight cancer cell lines from different tissue types. The LD50 for each cell line was determined and the change in cell size determined. Flow cytometric analysis and western blotting were used to assess the cell death markers for early apoptosis, late apoptosis and necrosis, and the involvement of the caspase signalling pathway. Alterations in calcium levels and reactive oxygen species were assessed due to their integral involvement in intracellular signalling. Subsequently, the endoplasmic reticulum (ER) and mitochondrial responses were investigated more closely. The extent of ER swelling, and the upregulation of proteins involved in the unfolded protein responses (UPR) were seen to vary according to cell line. The mitochondria were also shown to behave differently in response to the OphA in the different cell lines in terms of the change in membrane potential, the total area of mitochondria in the cell and the number of mitochondrial bifurcations. The data obtained in the present study indicate that the cancer cell lines tested are unable to successfully activate the ER stress/UPR responses, and that the mitochondria appear to be a central player in OphA-induced cancer cell death.

Preclinical Evaluation of Vemurafenib as Therapy for BRAFV600E Mutated Sarcomas.

PubMed

Gouravan, Sarina; Meza-Zepeda, Leonardo A; Myklebost, Ola; Stratford, Eva W; Munthe, Else

2018-03-23

The BRAF V600E mutation, which in melanoma is targetable with vemurafenib, is also found in sarcomas and we here evaluate the therapeutic potential in sarcoma cell lines. Four sarcoma cell lines harboring the BRAFV600E mutation, representing liposarcomas (SA-4 and SW872), Ewing sarcoma (A673) and atypical synovial sarcoma (SW982), were treated with vemurafenib and the effects on cell growth, apoptosis, cell cycle progression and cell signaling were determined. Vemurafenib induced a strong cytostatic effect in SA-4 cells, mainly due to cell cycle arrest, whereas only moderate levels of apoptosis were observed. However, a high dose was required compared to BRAF V600E mutated melanoma cells, and removal of vemurafenib demonstrated that the continuous presence of drug was required for sustained growth inhibition. A limited growth inhibition was observed in the other three cell lines. Protein analyses demonstrated reduced phosphorylation of ERK during treatment with vemurafenib in all the four sarcoma cell lines confirming that the MAPK pathway is active in these cell lines, and that the pathway can be inhibited by vemurafenib, but also that these cells can proliferate despite this. These findings indicate that vemurafenib alone would not be an efficient therapy against BRAF V600E mutated sarcomas. However, further investigations of combination with other drugs are warranted.

Effects of 1,3,5-triphenyl-4,5-dihydro-1H-pyrazole derivatives on cell-cycle and apoptosis in human acute leukemia cell lines.

PubMed

Santos Bubniak, Lorena Dos; Gaspar, Pâmela Cristina; de Moraes, Ana Carolina Rabello; Bigolin, Alisson; de Souza, Rubia Karine; Buzzi, Fátima Campos; Corrêa, Rogério; Filho, Valdir Cechinel; Bretanha, Lizandra Czermainski; Micke, Gustavo Amadeu; Nunes, Ricardo José; Santos-Silva, Maria Cláudia

2017-05-01

Pyrazoline is an important 5-membered nitrogen heterocycle that has been extensively researched. Ten derivatives were synthesized and tested for antileukemic effects on 2 human acute leukemia cell lines, K562 and Jurkat. The most cytotoxic of these derivatives, compound 21, was chosen for investigation of cytotoxicity mechanisms. The results obtained with selectivity calculations revealed that compound 21 is more selective for acute leukemia (K562 and Jurkat cell lines) than for other tumor cell lines. Moreover, compound 21 was not cytotoxic to normal cell lines, indicating a potential use in clinical tests. Compound 21 caused a significant cell cycle arrest in the S-phase in Jurkat cells and increased the proportion of cells in the sub G0/G1 phase in both cell lines. Cells treated with compound 21 demonstrated morphological changes characteristic of apoptosis in the EB/AO assay, confirmed by externalization of phosphatidylserine by the annexin V - fluorescein isothiocyanate method and by DNA fragmentation. An investigation of cytotoxicity mechanisms suggests the involvement of an intrinsic apoptosis pathway due to mitochondrial damage and an increase in the ratio of mitochondrial Bax/Bcl2. Pyrazoline 21 obeyed Lipinski's "rule of five" for drug-likeness. Based on these preliminary results, the antileukemic activity of compound 21 makes it a potential anticancer agent.

Comparative label-free LC-MS/MS analysis of colorectal adenocarcinoma and metastatic cells treated with 5-fluorouracil.

PubMed

Bauer, Kerry M; Lambert, Paul A; Hummon, Amanda B

2012-06-01

A label-free mass spectrometric strategy was used to examine the effect of 5-fluorouracil (5-FU) on the primary and metastatic colon carcinoma cell lines, SW480 and SW620, with and without treatment. 5-FU is the most common chemotherapeutic treatment for colon cancer. Pooled biological replicates were analyzed by nanoLC-MS/MS and protein quantification was determined via spectral counting. Phenotypic and proteomic changes were evident and often similar in both cell lines. The SW620 cells were more resistant to 5-FU treatment, with an IC(50) 2.7-fold higher than that for SW480. In addition, both cell lines showed pronounced abundance changes in pathways relating to antioxidative stress response and cell adhesion remodeling due to 5-FU treatment. For example, the detoxification enzyme NQO1 was increased with treatment in both cell lines, while disparate members of the peroxiredoxin family, PRDX2 or PRDX5 and PRDX6, were elevated with 5-FU exposure in either SW480 or SW620, respectively. Cell adhesion-associated proteins CTNNB1 and RhoA showed decreased expression with 5-FU treatment in both cell lines. The differential quantitative response in the proteomes of these patient-matched cell lines to drug treatment underscores the subtle molecular differences separating primary and metastatic cancer cells. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Utility Of Nitric Oxide And Hydrogen Sulfide-Releasing Chimeras As Anticancer Agents.

PubMed

Kashfi, Khosrow

2015-08-01

Aspirin is chemopreventive but has significant side effects. We developed NOSH-aspirin a safer, nitric oxide and hydrogen sulfide releasing hybrid. Here we report on NOSH-aspirin as an anti-inflammatory and its effects on human cancer cell kinetics and various cancer xenografts. Anti-inflammatory: Carageenan rat paw edema model. Cancer cell lines: Colon, HT-29, HCT 15, SW 480; breast, MCF-7, MDA-MB-231; pancreas, MIA PaCa2, BxPC3. Normal cell lines: human mammary, HMEpC; pancreatic epithelial, ACBRI 515. Xenografts: nude mice implanted with HT-29, MDA-MB-231, MIA PaCa2 cells, were treated with NOSH-aspirin (100mg/kg/d) or vehicle. After 3 weeks, mice were sacrificed, tumors excised, weighed, and fixed for IHC studies. NOSH-aspirin significantly reduced paw edema as function of time. NOSH-aspirin's IC 50 in nM at 24h for cell growth inhibition ranged from 50±2 to 250±10 in the cancer cell lines and about 400-fold higher in the normal cell lines. The cell growth inhibitory effects were due to a dose-dependent induction of apoptosis and cell cycle arrest (G0/G1), leading to reductions in cell proliferation. In xenografts, NOSH-aspirin had no effect on the weight of the mice. Tumor volume was reduced as a function of treatment time. At sacrifice, tumor mass reductions were colon: 89%, P=0.005; breast: 91%, P=0.006; pancreas: 75%, P=0.0031. Growth inhibition was due to reduced proliferation (decreased PCNA expression), and induction of apoptosis (increased TUNEL positive cells). NOSH-aspirin is a potent anti-inflammatory, preferentially affecting cancer cells and targets parameters important in determining cellular mass. Copyright © 2015. Published by Elsevier B.V.

Low concentrations of alendronate increase the local invasive potential of osteoblastic sarcoma cell lines via connexin 43 activation.

PubMed

Yoshitani, Kazuhiro; Kido, Akira; Honoki, Kanya; Akahane, Manabu; Fujii, Hiromasa; Tanaka, Yasuhito

2011-07-15

Bisphosphonates (BPs) are agents used for treating disorders of excessive bone resorption. In addition, due to their cell-killing activity, BPs were potent candidates for adjuvant cancer therapy. On the other hand, low-concentrations of BPs have been reported to increase cellular viability in several types of tumor cells. Therefore, we focused on the effect of BPs on cellular aggressiveness of malignant bone tumors at low concentrations. MTS assay was performed using osteosarcoma cell lines MG63 and HOS, fibrosarcoma cell line HT1080, and prostate cancer cell line PC3. All the cell lines showed toxicity at high concentrations. On the other hand, at lower concentrations, the cellular viabilities of HOS and MG63 were rather higher than those of untreated controls. Since this tendency was most evident, HOS was used for further assays, including cellular motility, bone resorption activity, and cathepsin K activity. The low-concentration of alendronate enhanced cellular viability and motility, which correlated with the expression of connexin 43 at the mRNA and protein levels. Interestingly, oleamide, a potent connexin 43 inhibitor, had an inhibitory effect on the enhanced proliferation. Our data suggest that alendronate may enhance the proliferation of osteoblastic cell line through connexin 43 activation. Copyright © 2011 Elsevier GmbH. All rights reserved.

Sensory hair cell regeneration in the zebrafish lateral line.

PubMed

Lush, Mark E; Piotrowski, Tatjana

2014-10-01

Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling, and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. Copyright © 2014 Wiley Periodicals, Inc.

Warburg and Crabtree Effects in Premalignant Barrett's Esophagus Cell Lines with Active Mitochondria

PubMed Central

Suchorolski, Martin T.; Paulson, Thomas G.; Sanchez, Carissa A.; Hockenbery, David; Reid, Brian J.

2013-01-01

Background Increased glycolysis is a hallmark of cancer metabolism, yet relatively little is known about this phenotype at premalignant stages of progression. Periodic ischemia occurs in the premalignant condition Barrett's esophagus (BE) due to tissue damage from chronic acid-bile reflux and may select for early adaptations to hypoxia, including upregulation of glycolysis. Methodology/Principal Findings We compared rates of glycolysis and oxidative phosphorylation in four cell lines derived from patients with BE (CP-A, CP-B, CP-C and CP-D) in response to metabolic inhibitors and changes in glucose concentration. We report that cell lines derived from patients with more advanced genetically unstable BE have up to two-fold higher glycolysis compared to a cell line derived from a patient with early genetically stable BE; however, all cell lines preserve active mitochondria. In response to the glycolytic inhibitor 2-deoxyglucose, the most glycolytic cell lines (CP-C and CP-D) had the greatest suppression of extra-cellular acidification, but were able to compensate with upregulation of oxidative phosphorylation. In addition, these cell lines showed the lowest compensatory increases in glycolysis in response to mitochondrial uncoupling by 2,4-dinitrophenol. Finally, these cell lines also upregulated their oxidative phosphorylation in response to glucose via the Crabtree effect, and demonstrate a greater range of modulation of oxygen consumption. Conclusions/Significance Our findings suggest that cells from premalignant Barrett's esophagus tissue may adapt to an ever-changing selective microenvironment through changes in energy metabolic pathways typically associated with cancer cells. PMID:23460817

Effect of different concentrations of oxygen on expression of sigma 1 receptor and superoxide dismutases in human colon adenocarcinoma cell lines.

PubMed

Skrzycki, Michał; Czeczot, Hanna; Mielczarek-Puta, Magdalena; Otto-Ślusarczyk, Dagmara; Graboń, Wojciech

2017-06-01

Tumor cells due to distance from capillary vessels exist in different oxygenation conditions (anoxia, hypoxia, normoxia). Changes in cell oxygenation lead to reactive oxygen species production and oxidative stress. Sigma 1 receptor (Sig1R) is postulated to be stress responding agent and superoxide dismutases (SOD1 and SOD2) are key antioxidant enzymes. It is possible that they participate in tumor cells adaptation to different concentrations of oxygen. Evaluation of Sig1R, SOD1, and SOD2 expression in different concentrations of oxygen (1%, 10%, 21%) in colon adenocarcinoma cell lines. SW480 (primary adenocarcinoma) and SW620 (metastatic) cell lines were cultured in standard conditions in Dulbecco's modified Eagle's medium for 5 days, and next cultured in Hypoxic Chamber in 1% O 2 , 10% O 2 , 21% O 2 . Number of living cells was determined by trypan blue assay. Level of mRNA for Sig1R, SOD1, and SOD2 was determined by standard PCR method. Statistical analysis was conducted using Statistica 10.1 software. We observed significant changes in expression of Sig1R, SOD1, SOD2 due to different oxygen concentrations. ANOVA analysis revealed significant interactions between studied parameters mainly in hypoxia conditions in SW480 cells and between Sig1R and SOD2 in SW620 cells. It also showed that changes in expression of studied proteins depend significantly on type of the cell line. Changes of Sig1R and SOD2 expression point to mitochondria as main organelle responsible for survival of tumor cells exposed to hypoxia or oxidative stress. Studied proteins are involved in intracellular response to stress related with different concentrations of oxygen.

Lapatinib sensitivities of two novel trastuzumab-resistant HER2 gene-amplified gastric cancer cell lines.

PubMed

Oshima, Yukiko; Tanaka, Harunari; Murakami, Hiroki; Ito, Yuichi; Furuya, Tomomi; Kondo, Eisaku; Kodera, Yasuhiro; Nakanishi, Hayao

2014-01-01

Trastuzumab (Tmab) resistance is a major clinical problem to be resolved in patients with HER2-positive gastric cancers. However, in contrast to the situation for HER2-positive breast cancer lines, the Tmab-resistant gastric cancer preclinical models that are needed to develop a new therapy to overcome this problem are not yet available. We developed three new cell lines from HER2 gene-amplified gastric cancer cell lines (GLM-1, GLM-4, NCI N-87) by a new in vivo selection method consisting of the repeated culture of small residual peritoneal metastasis but not subcutaneous tumor after Tmab treatment. We then evaluated the anti-tumor efficacy of lapatinib for these Tmab-resistant cells. We successfully isolated two Tmab-resistant cell lines (GLM1-HerR2(3), GLM4-HerR2) among the three tested cell lines. These resistant cells differed from the parental cells in their flat morphology and rapid growth in vitro, but HER2, P95HER2 expression, and Tmab binding were essentially the same for the parental and resistant cells. MUC4 expression was up- or downregulated depending on the cell line. These resistant cells were still sensitive to lapatinib, similar to the parental cells, in vitro. This growth inhibition of the Tmab-resistant cells by lapatinib was due to both G1 cell-cycle arrest and apoptosis induction via effective blockade of the PI3K/Akt and MAPK pathways. A preclinical study confirmed that the Tmab-resistant tumors are significantly susceptible to lapatinib. These results suggest that lapatinib has antitumor activity against the Tmab-resistant gastric cancer cell lines, and that these cell lines are useful for understanding the mechanism of Tmab resistance and for developing a new molecular therapy for Tmab-resistant HER2-positive gastric cancers.

Cationic Phosphorus Dendrimer Enhances Photodynamic Activity of Rose Bengal against Basal Cell Carcinoma Cell Lines.

PubMed

Dabrzalska, Monika; Janaszewska, Anna; Zablocka, Maria; Mignani, Serge; Majoral, Jean Pierre; Klajnert-Maculewicz, Barbara

2017-05-01

In the last couple of decades, photodynamic therapy emerged as a useful tool in the treatment of basal cell carcinoma. However, it still meets limitations due to unfavorable properties of photosensitizers such as poor solubility or lack of selectivity. Dendrimers, polymers widely studied in biomedical field, may play a role as photosensitizer carriers and improve the efficacy of photodynamic treatment. Here, we describe the evaluation of an electrostatic complex of cationic phosphorus dendrimer and rose bengal in such aspects as singlet oxygen production, cellular uptake, and phototoxicity against three basal cell carcinoma cell lines. Rose bengal-cationic dendrimer complex in molar ratio 5:1 was compared to free rose bengal. Obtained results showed that the singlet oxygen production in aqueous medium was significantly higher for the complex than for free rose bengal. The cellular uptake of the complex was 2-7-fold higher compared to a free photosensitizer. Importantly, rose bengal, rose bengal-dendrimer complex, and dendrimer itself showed no dark toxicity against all three cell lines. Moreover, we observed that phototoxicity of the complex was remarkably enhanced presumably due to high cellular uptake. On the basis of the obtained results, we conclude that rose bengal-cationic dendrimer complex has a potential in photodynamic treatment of basal cell carcinoma.

MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells

PubMed Central

Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

2015-01-01

Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach. PMID:25949869

MHC-I modulation due to changes in tumor cell metabolism regulates tumor sensitivity to CTL and NK cells.

PubMed

Catalán, Elena; Charni, Seyma; Jaime, Paula; Aguiló, Juan Ignacio; Enríquez, José Antonio; Naval, Javier; Pardo, Julián; Villalba, Martín; Anel, Alberto

2015-01-01

Tumor cells have a tendency to use glucose fermentation to obtain energy instead of mitochondrial oxidative phosphorylation (OXPHOS). We demonstrated that this phenotype correlated with loss of ERK5 expression and with reduced MHC class I expression. Consequently, tumor cells could evade cytotoxic T lymphocyte (CTL)-mediated immune surveillance, but also increase their sensitivity to natural killer (NK) cells. These outcomes were evaluated using two cellular models: leukemic EL4 cells and L929 transformed fibroblasts and their derived ρ° cell lines, which lack mitochondrial DNA. We have also used a L929 cell sub-line that spontaneously lost matrix attachment (L929dt), reminiscent of metastasis generation, that also downregulated MHC-I and ERK5 expression. MHC-I expression is lower in ρ° cells than in the parental cell lines, but they were equally sensitive to CTL. On the contrary, ρ° cells were more sensitive to activated NK cells than parental cells. On the other hand, L929dt cells were resistant to CTL and NK cells, showed reduced viability when forced to perform OXPHOS, and surviving cells increased MHC-I expression and became sensitive to CTL. The present results suggest that when the reduction in MHC-I levels in tumor cells due to glycolytic metabolism is partial, the increase in sensitivity to NK cells seems to predominate. However, when tumor cells completely lose MHC-I expression, the combination of treatments that increase OXPHOS with CTL-mediated immunotherapy could be a promising therapeutic approach.

Urtica dioica inhibits cell growth and induces apoptosis by targeting Ornithine decarboxylase and Adenosine deaminase as key regulatory enzymes in adenosine and polyamines homeostasis in human breast cancer cell lines.

PubMed

Fattahi, Sadegh; Ghadami, Elham; Asouri, Mohsen; Motevalizadeh Ardekanid, Ali; Akhavan-Niaki, Haleh

2018-02-28

Breast cancer is a heterogeneous and multifactorial disease with variable disease progression risk, and treatment response. Urtica dioica is a traditional herb used as an adjuvant therapeutic agent in cancer. In the present study, we have evaluated the effects of the aqueous extract of Urtica dioica on Adenosine deaminase (ADA) and Ornithine decarboxylase (ODC1) gene expression in MCF-7, MDA-MB-231, two breast cancer cell lines being estrogen receptor positive and estrogen receptor negative, respectively.  Cell lines were cultured in suitable media. After 24 h, different concentrations of the extract were added and after 72 h, ADA and ODC1 gene expression as well as BCL2 and BAX apoptotic genes were assessed by Taqman real time PCR assay. Cells viability was assessed by MTT assay, and apoptosis was also evaluated at cellular level. The intra and extracellular levels of ODC1 and ADA enzymes were evaluated by ELISA. Results showed differential expression of ADA and ODC1 genes in cancer cell lines. In MCF-7 cell line, the expression level of ADA was upregulated in a dose-dependent manner but its expression did not change in MDA-MB cell line. ODC1 expression was increased in both examined cell lines. Also, increased level of the apoptotic BAX/BCL-2 ratio was detected in MCF-7 cells. These results demonstrated that Urtica dioica induces apoptosis in breast cancer cells by influencing ODC1 and ADA genes expression, and estrogen receptors. The different responses observed with these cell lines could be due to the interaction of Urtica dioica as a phytoestrogen with the estrogen receptor.

Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines.

PubMed

Emadi Baygi, Modjtaba; Soheili, Zahra Soheila; Schmitz, Ingo; Sameie, Shahram; Schulz, Wolfgang A

2010-12-01

The epithelial-mesenchymal transition (EMT) is regarded as an important step in cancer metastasis. Snail, a master regulator of EMT, has been recently proposed to act additionally as a cell survival factor and inducer of motility. We have investigated the function of Snail (SNAI1) in prostate cancer cells by downregulating its expression via short (21-mer) interfering RNA (siRNA) and measuring the consequences on EMT markers, cell viability, death, cell cycle, senescence, attachment, and invasivity. Of eight carcinoma cell lines, the prostate carcinoma cell lines LNCaP and PC-3 showed the highest and moderate expression of SNAI1 mRNA, respectively, as measured by quantitative RT-PCR. Long-term knockdown of Snail induced a severe decline in cell numbers in LNCaP and PC-3 and caspase activity was accordingly enhanced in both cell lines. In addition, suppression of Snail expression induced senescence in LNCaP cells. SNAI1-siRNA-treated cells did not tolerate detachment from the extracellular matrix, probably due to downregulation of integrin α6. Expression of E-cadherin, vimentin, and fibronectin was also affected. Invasiveness of PC-3 cells was not significantly diminished by Snail knockdown. Our data suggest that Snail acts primarily as a survival factor and inhibitor of cellular senescence in prostate cancer cell lines. We therefore propose that Snail can act as early driver of prostate cancer progression.

A boosted negative bit-line SRAM with write-assisted cell in 45 nm CMOS technology

NASA Astrophysics Data System (ADS)

Bhatnagar, Vipul; Kumar, Pradeep; Pandey, Neeta; Pandey, Sujata

2018-02-01

A new 11 T SRAM cell with write-assist is proposed to improve operation at low supply voltage. In this technique, a negative bit-line voltage is applied to one of the write bit-lines, while a boosted voltage is applied to the other write bit-line where transmission gate access is used in proposed 11 T cell. Supply voltage to one of the inverters is interrupted to weaken the feedback. Improved write feature is attributed to strengthened write access devices and weakened feedback loop of cell at the same time. Amount of boosting required for write performance improvement is also reduced due to feedback weakening, solving the persistent problem of half-selected cells and reliability reduction of access devices with the other suggested boosted and negative bit-line techniques. The proposed design improves write time by 79%, 63% and slower by 52% with respect to LP 10 T, WRE 8 T and 6 T cells respectively. It is found that write margin for the proposed cell is improved by about 4×, 2.4× and 5.37× compared to WRE8 T, LP10 T and 6 T respectively. The proposed cell with boosted negative bit line (BNBL) provides 47%, 31%, and 68.4% improvement in write margin with respect to no write-assist, negative bit line (NBL) and boosted bit line (BBL) write-assist respectively. Also, new sensing circuit with replica bit-line is proposed to give a more precise timing of applying boosted voltages for improved results. All simulations are done on TSMC 45 nm CMOS technology.

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

Gopalan, Vinod; Smith, Robert A.; Lam, Alfred K.-Y., E-mail: a.lam@griffith.edu.au

miR-498 is a non-coding RNA located intergenically in 19q13.41. Due to its predicted targeting of several genes involved in control of cellular growth, we examined the expression of miR-498 in colon cancer cell lines and a large cohort of patients with colorectal adenocarcinoma. Two colon cancer cancer cell lines (SW480 and SW48) and one normal colonic epithelial cell line (FHC) were recruited. The expression of miR-498 was tested in these cell lines by using quantitative real-time polymerase chain reaction (qRT-PCR). Tissues from 80 patients with surgical resection of colorectum (60 adenocarcinomas and 20 non-neoplastic tissues) were tested for miR-498 expressionmore » by qRT-PCR. In addition, an exogenous miR-498 (mimic) was used to detect the miRNA's effects on cell proliferation and cell cycle events in SW480 using MTT calorimetric assay and flow cytometry respectively. The colon cancer cell lines showed reduced expression of miR-498 compared to a normal colonic epithelial cell line. Mimic driven over expression of miR-498 in the SW480 cell line resulted in reduced cell proliferation and increased proportions of G2-M phase cells. In tissues, miR-498 expression was too low to be detected in all colorectal adenocarcinoma compared to non-neoplastic tissues. This suggests that the down regulation of miR-498 in colorectal cancer tissues and the direct suppressive cellular effect noted in cancer cell lines implies that miR-498 has some direct or indirect role in the pathogenesis of colorectal adenocarcinomas. - Highlights: • miR-498 is a non-coding RNA located in 19q13.41. • Colon cancer cell lines showed reduced expression of miR-498. • Mimic driven over expression of miR-498 in colon cancer cells resulted in lower cell proliferation. • miR-498 expression was down regulated in all colorectal adenocarcinoma tissues.« less

[The influence of immobilized fibronectin on karyotypic variability of two rat kangaroo kidney cell lines].

PubMed

Polianskaia, G G; Goriachaia, T S; Pinaev, G P

2007-01-01

The numerical and structural karyotypic variability has been investigated in "markerless" Rat kangaroo kidney cell lines NBL-3-17 and NBL-3-11 when cultivating on a fibronectin-coated surface. In cell line NBL-3-17, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in frequency of cells with modal number of chromosomes, and an increase in frequency of cells with lower chromosomal number. Many new additional structural variants of the karyotype (SVK) appear. The observed alterations seem to be due preference adhesion of cells with lower chromosome number, disturbances of mitotic apparatus and selection of SVK, which are more adopted to changes in culture conditions. Detachment of cells from the fibronectin-coated surface, followed by 5 days cultivation on a hydrophilic surface restored control distribution. In cell line NBL-3-11, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of numerical karyotypic variability did not change compared to control variants. In cell line NBL-3-17 the frequency of chromosomal aberrations under cultivation on the fibronectin-coated surface for 1, 2, 4 and 8 days did not change relative to control variants. In cell line NBL-3-11 the frequency of chromosomal aberrations under the same conditions significantly increases, mainly at the expence of chromosomal, chromatid breaks and dicentrics (telomeric association) relative to control variants. We discuss possible reasons of differences in the character of numerical and structural karyotypic variability between cell lines NBL-3-17 (hypotriploid) and NBL-3-11 (hypodiploid) under cultivation on fibronectin. The reasons of the observed interline karyotypic differences possibly consist in peculiarity of karyotypic structure of cell line NBL-3-11 and in the change of gene expression, namely in a dose of certain functioning genes in the hypotryploid cell line NBL-3-17.

Three dimensional culture of the murine osteoblastic cell line OCT-1 on collagen coated microcarriers

NASA Astrophysics Data System (ADS)

Lau, P.; Hellweg, C. E.; Kirchner, S.; Baumstark-Khan, C.

2005-08-01

During long-term space missions, astronauts suffer from the loss of minerals especially from weightbearing bones due to prolonged sojourn under microgravity. Bone loss during space flight is about 1-2% per month. Bone is continually being remodelled under the influence of three types of highly specialized cells. Osteoblasts, the bone forming cells, osteoclasts, the bone resorbing cells and finally osteocytes preserve the homeostasis of bone formation and resorption. In vitro 3- dimensional cell culture of osteoblastic cell lines on microcarrier beads might be a better model to evaluate changes in bone cell morphology, function and differentiation under influence of spaceflight related factors than the conventional 2-D monolayer culture technique. Furthermore, it allows production of a greater amount of cells compared to the monolayer culture. Aim of this study is to examine the effects of culturing the immortalized murine osteoblastic cell line OCT-1 in a 3- dimensional environment on cell morphology and proliferation rate.

Ophiobolin A, a sesterpenoid fungal phytotoxin, displays different mechanisms of cell death in mammalian cells depending upon the cancer cell origin

PubMed Central

Morrison, Rachel; Lodge, Tiffany; Evidente, Antonio; Kiss, Robert; Townley, Helen

2017-01-01

Herein we have undertaken a systematic analysis of the effects of the fungal derivative ophiobolin A (OphA) on eight cancer cell lines from different tissue types. The LD50 for each cell line was determined and the change in cell size determined. Flow cytometric analysis and western blotting were used to assess the cell death markers for early apoptosis, late apoptosis and necrosis, and the involvement of the caspase signalling pathway. Alterations in calcium levels and reactive oxygen species were assessed due to their integral involvement in intracellular signalling. Subsequently, the endoplasmic reticulum (ER) and mitochondrial responses were investigated more closely. The extent of ER swelling, and the upregulation of proteins involved in the unfolded protein responses (UPR) were seen to vary according to cell line. The mitochondria were also shown to behave differently in response to the OphA in the different cell lines in terms of the change in membrane potential, the total area of mitochondria in the cell and the number of mitochondrial bifurcations. The data obtained in the present study indicate that the cancer cell lines tested are unable to successfully activate the ER stress/UPR responses, and that the mitochondria appear to be a central player in OphA-induced cancer cell death. PMID:28112374

Parallel RNAi screens across different cell lines identify generic and cell type-specific regulators of actin organization and cell morphology.

PubMed

Liu, Tao; Sims, David; Baum, Buzz

2009-01-01

In recent years RNAi screening has proven a powerful tool for dissecting gene functions in animal cells in culture. However, to date, most RNAi screens have been performed in a single cell line, and results then extrapolated across cell types and systems. Here, to dissect generic and cell type-specific mechanisms underlying cell morphology, we have performed identical kinome RNAi screens in six different Drosophila cell lines, derived from two distinct tissues of origin. This analysis identified a core set of kinases required for normal cell morphology in all lines tested, together with a number of kinases with cell type-specific functions. Most significantly, the screen identified a role for minibrain (mnb/DYRK1A), a kinase associated with Down's syndrome, in the regulation of actin-based protrusions in CNS-derived cell lines. This cell type-specific requirement was not due to the peculiarities in the morphology of CNS-derived cells and could not be attributed to differences in mnb expression. Instead, it likely reflects differences in gene expression that constitute the cell type-specific functional context in which mnb/DYRK1A acts. Using parallel RNAi screens and gene expression analyses across cell types we have identified generic and cell type-specific regulators of cell morphology, which include mnb/DYRK1A in the regulation of protrusion morphology in CNS-derived cell lines. This analysis reveals the importance of using different cell types to gain a thorough understanding of gene function across the genome and, in the case of kinases, the difficulties of using the differential gene expression to predict function.

BINDING OF SOLUBLE IMMUNE COMPLEXES TO HUMAN LYMPHOBLASTOID CELLS

PubMed Central

Theofilopoulos, Argyrios N.; Dixon, Frank J.; Bokisch, Viktor A.

1974-01-01

In the present work we studied the expression of membrane-bound Ig (MBIg) as well as receptors for IgG Fc and complement on nine human lymphoblastoid cell lines. When MBIg and receptors for IgG Fc were compared, four categories of cell lines could be distinguished: (a) cell lines having both MBIg and receptors for IgG Fc, (b) cell lines having MBIg but lacking receptors for IgG Fc, (c) cell lines lacking MBIg but having receptors for IgG Fc, and (d) cell lines lacking both MBIg and receptors for IgG Fc. Two types of receptors for complement could be detected on the cell lines studied, one for C3-C3b and one for C3d. When sensitized red cells carrying C3b or C3d were used for rosette tests, three categories of cell lines could be distinguished: (a) cell lines having receptors for C3b and C3d, (b) cell lines having receptors only for C3d and (c) cell lines lacking both receptors. However, when a more sensitive immunofluorescent method was used instead of the rosette technique, it was found that cell lines unable to form rosettes with EAC1423bhu were able to bind soluble C3 or C3b which indicated the presence of these receptors on the cell surface. Inhibition experiments showed that receptors for C3-C3b and receptors for C3d are distinct and that receptors for C3-C3b and C3d are different from receptors for IgG Fc. A cell line (Raji) without MBIg but with receptors for IgG Fc, C3-C3b, and C3d was selected for use in studying the binding mechanism of soluble immune complexes to cell surface membrane. Aggregated human gamma globulin was used in place of immune complexes. Immune complexes containing complement bind to Raji cells only via receptors for complement, namely receptors for C3-C3b and C3d. Binding of immune complexes containing complement to cells is much greater than that of complexes without complement. Immune complexes bound to cells via receptors for complement can be partially released from the cell surface by addition of normal human serum as well as isolated human C3 or C3b. We postulate that such release is due to competition of immune complex bound C3b and free C3 or C3b for the receptors on Raji cells. PMID:4139225

Phenotypic high-throughput screening elucidates target pathway in breast cancer stem cell-like cells.

PubMed

Carmody, Leigh C; Germain, Andrew R; VerPlank, Lynn; Nag, Partha P; Muñoz, Benito; Perez, Jose R; Palmer, Michelle A J

2012-10-01

Cancer stem cells (CSCs) are resistant to standard cancer treatments and are likely responsible for cancer recurrence, but few therapies target this subpopulation. Due to the difficulty in propagating CSCs outside of the tumor environment, previous work identified CSC-like cells by inducing human breast epithelial cells into an epithelial-to-mesenchymal transdifferentiated state (HMLE_sh_ECad). A phenotypic screen was conducted against HMLE_sh_ECad with 300 718 compounds from the Molecular Libraries Small Molecule Repository to identify selective inhibitors of CSC growth. The screen yielded 2244 hits that were evaluated for toxicity and selectivity toward an isogenic control cell line. An acyl hydrazone scaffold emerged as a potent and selective scaffold targeting HMLE_sh_ECad. Fifty-three analogues were acquired and tested; compounds ranged in potency from 790 nM to inactive against HMLE_sh_ECad. Of the analogues, ML239 was best-in-class with an IC(50)= 1.18 µM against HMLE_sh_ECad, demonstrated a >23-fold selectivity over the control line, and was toxic to another CSC-like line, HMLE_shTwist, and a breast carcinoma cell line, MDA-MB-231. Gene expression studies conducted with ML239-treated cells showed altered gene expression in the NF-κB pathway in the HMLE_sh_ECad line but not in the isogenic control line. Future studies will be directed toward the identification of ML239 target(s).

Low temperature caused modifications in the arrangement of cell wall pectins due to changes of osmotic potential of cells of maize leaves (Zea mays L.).

PubMed

Bilska-Kos, Anna; Solecka, Danuta; Dziewulska, Aleksandra; Ochodzki, Piotr; Jończyk, Maciej; Bilski, Henryk; Sowiński, Paweł

2017-03-01

The cell wall emerged as one of the important structures in plant stress responses. To investigate the effect of cold on the cell wall properties, the content and localization of pectins and pectin methylesterase (PME) activity, were studied in two maize inbred lines characterized by different sensitivity to cold. Low temperature (14/12 °C) caused a reduction of pectin content and PME activity in leaves of chilling-sensitive maize line, especially after prolonged treatment (28 h and 7 days). Furthermore, immunocytohistological studies, using JIM5 and JIM7 antibodies, revealed a decrease of labeling of both low- and high-methylesterified pectins in this maize line. The osmotic potential, quantified by means of incipient plasmolysis was lower in several types of cells of chilling-sensitive maize line which was correlated with the accumulation of sucrose. These studies present new finding on the effect of cold stress on the cell wall properties in conjunction with changes in the osmotic potential of maize leaf cells.

Evaluating hepatocellular carcinoma cell lines for tumour samples using within-sample relative expression orderings of genes.

PubMed

Ao, Lu; Guo, You; Song, Xuekun; Guan, Qingzhou; Zheng, Weicheng; Zhang, Jiahui; Huang, Haiyan; Zou, Yi; Guo, Zheng; Wang, Xianlong

2017-11-01

Concerns are raised about the representativeness of cell lines for tumours due to the culture environment and misidentification. Liver is a major metastatic destination of many cancers, which might further confuse the origin of hepatocellular carcinoma cell lines. Therefore, it is of crucial importance to understand how well they can represent hepatocellular carcinoma. The HCC-specific gene pairs with highly stable relative expression orderings in more than 99% of hepatocellular carcinoma but with reversed relative expression orderings in at least 99% of one of the six types of cancer, colorectal carcinoma, breast carcinoma, non-small-cell lung cancer, gastric carcinoma, pancreatic carcinoma and ovarian carcinoma, were identified. With the simple majority rule, the HCC-specific relative expression orderings from comparisons with colorectal carcinoma and breast carcinoma could exactly discriminate primary hepatocellular carcinoma samples from both primary colorectal carcinoma and breast carcinoma samples. Especially, they correctly classified more than 90% of liver metastatic samples from colorectal carcinoma and breast carcinoma to their original tumours. Finally, using these HCC-specific relative expression orderings from comparisons with six cancer types, we identified eight of 24 hepatocellular carcinoma cell lines in the Cancer Cell Line Encyclopedia (Huh-7, Huh-1, HepG2, Hep3B, JHH-5, JHH-7, C3A and Alexander cells) that are highly representative of hepatocellular carcinoma. Evaluated with a REOs-based prognostic signature for hepatocellular carcinoma, all these eight cell lines showed the same metastatic properties of the high-risk metastatic hepatocellular carcinoma tissues. Caution should be taken for using hepatocellular carcinoma cell lines. Our results should be helpful to select proper hepatocellular carcinoma cell lines for biological experiments. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Exposure to Cobalt Causes Transcriptomic and Proteomic Changes in Two Rat Liver Derived Cell Lines

PubMed Central

Permenter, Matthew G.; Dennis, William E.; Sutto, Thomas E.; Jackson, David A.; Lewis, John A.; Stallings, Jonathan D.

2013-01-01

Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies. PMID:24386269

Spontaneous lung metastasis formation of human Merkel cell carcinoma cell lines transplanted into scid mice.

PubMed

Knips, Jill; Czech-Sioli, Manja; Spohn, Michael; Heiland, Max; Moll, Ingrid; Grundhoff, Adam; Schumacher, Udo; Fischer, Nicole

2017-07-01

Merkel cell carcinoma (MCC) is an aggressive skin cancer entity that frequently leads to rapid death due to its high propensity to metastasize. The etiology of most MCC cases is linked to Merkel cell polyomavirus (MCPyV), a virus which is monoclonally integrated in up to 95% of tumors. While there are presently no animal models to study the role of authentic MCPyV infection on transformation, tumorigenesis or metastasis formation, xenograft mouse models employing engrafted MCC-derived cell lines (MCCL) represent a promising approach to study certain aspects of MCC pathogenesis. Here, the two MCPyV-positive MCC cell lines WaGa and MKL-1 were subcutaneously engrafted in scid mice. Engraftment of both MCC cell lines resulted in the appearance of circulating tumor cells and metastasis formation, with WaGa-engrafted mice showing a significantly shorter survival time as well as increased numbers of spontaneous lung metastases compared to MKL-1 mice. Interestingly, explanted tumors compared to parental cell lines exhibit an upregulation of MCPyV sT-Antigen expression in all tumors, with WaGa tumors showing significantly higher sT-Antigen expression than MKL-1 tumors. RNA-Seq analysis of explanted tumors and parental cell lines furthermore revealed that in the more aggressive WaGa tumors, genes involved in inflammatory response, growth factor activity and Wnt signalling pathway are significantly upregulated, suggesting that sT-Antigen is the driver of the observed differences in metastasis formation. © 2017 UICC.

The role of topotecan for extending the platinum-free interval in recurrent ovarian cancer: an in vitro model.

PubMed

Horowitz, Neil S; Hua, Jun; Gibb, Randall K; Mutch, David G; Herzog, Thomas J

2004-07-01

Topotecan, a novel topoisomerase-I inhibitor, is an active agent of second-line chemotherapy for extending the platinum-free interval (PFI) and improving the chances of a response to platinum in recurrent ovarian cancer patients. The aim of this study was to understand the molecular mechanism of topotecan-based second-line chemotherapy through an in vitro cell culture model and to gain clinical insight into sequencing issues for second-line treatment with novel agents versus retreatment with platinum. The human ovarian cancer cell line A2780 and the cisplatin resistance cell line A2780-CR were separately seeded in 6-well cell culture plates and then exposed to multiple concentrations of cisplatin plus paclitaxel or topotecan for 7 days. Surviving cells were recovered and cultured in drug-free media for 3 weeks and then replated in a 96-well microtiter plate. The LD(50) for these cells was determined by a cytotoxic MTT assay after exposure to multiple clinically relevant concentrations of cisplatin or topotecan. Surviving cells were cultured in drug-free media for an additional 4 weeks at which time the LD(50) was reassessed for each cell population by a second MTT assay. Using RT-PCR and Northern blot hybridization to measure mRNA expression, the molecular profile of these cells in terms of resistance was evaluated for the multidrug-resistant gene (MDR-1), multidrug-resistant protein (MRP), Topoisomerase-I, and beta-Actin. The LD(50) to cisplatin was unchanged in A2780-CR cells treated by topotecan. Those A2780-CR cells originally exposed to higher concentrations of cisplatin became more resistant to cisplatin in the MTT assays, while those A2780-CR cell lines treated with a combination of lower cisplatin concentrations and paclitaxel became more sensitive to cisplatin in the MTT assay (P < 0.01). The second MTT assay demonstrated that the LD(50) for cisplatin in every cell line decreased significantly after a 4-week drug-free interval (P < 0.01). There was no difference in the mRNA expression for MRP or topoisomerase-I regardless of cell line, or type or concentration of chemotherapeutic exposure. The mRNA for MDR-1 was uniquely overexpressed in the cisplatin-resistant cell line A2780-CR9 initially treated with low doses of cisplatin and paclitaxel, but was not amplified in A2780 (P < 0.01). The acquired resistance to cisplatin in A2780 is potentially due to P-glycoprotein-mediated multidrug resistance. This acquired resistance to cisplatin is an unstable phenotype in that some cell populations become sensitive after a drug-free interval and topotecan treatment. This reversal of resistance, however, does not appear to be simply due to loss of MDR-1 expression. While in vivo confirmation is required, agents with novel mechanisms of action offer a strategy to extend the platinum-free interval and thereby improve survival in patients with recurrent ovarian cancer.

Alpha-2 Heremans Schmid Glycoprotein (AHSG) Modulates Signaling Pathways in Head and Neck Squamous Cell Carcinoma Cell Line SQ20B

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

Thompson, Pamela D.; Sakwe, Amos; Koumangoye, Rainelli

2014-02-15

This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels ofmore » AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-β was examined to determine whether levels of the TGF-β binding AHSG influenced the effect of TGF-β on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-β influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis. Highlights: • Head and neck squamous cell carcinoma cell lines synthesize and secret AHSG. • AHSG depleted cell lines are significantly inhibited in their ability to proliferate, adhere, migrate, invade and protect MMP-9. • Human AHSG and bovine fetuin-A are functionally equivalent in regards to growth promotion of cancer cell lines.« less

Metastatic signet-ring cell cancer of the bladder responding to chemotherapy with capecitabine: case report and review of literature

PubMed Central

Michels, Jorg; Barbour, Sean; Cavers, Douglas; Chi, Kim N.

2010-01-01

Signet-ring cell cancers deriving from the bladder are rare entities and usually present with advanced incurable disease and associated poor outlook. No standard effective chemotherapeutic option has been described largely due to the rarity of this malignancy. We report a case of a patient with metastatic bladder cancer, signet-ring cell variant. The patient progressed rapidly on standard first-line bladder cancer chemotherapy with gemcitabine and carboplatin. He responded well to second-line capecitabine with a clinically meaningful progression-free survival. PMID:20368884

Development and characterization of a mouse floxed Bmp2 osteoblast cell line that retains osteoblast genotype and phenotype

PubMed Central

Wu, Li-an; Feng, Junsheng; Wang, Lynn; Mu, Yan-dong; Baker, Andrew; Donly, Kevin J.; Harris, Stephen E.; MacDougall, Mary; Chen, Shuo

2011-01-01

Bone morphogenetic protein 2 (Bmp2) is essential for osteoblast differentiation and osteogenesis. Generation of floxed Bmp2 osteoblast cell lines is a valuable tool for studying the effects of Bmp2 on osteoblast differentiation and its signaling pathways during skeletal metabolism. Due to relatively limited sources of primary osteoblasts, we have developed cell lines that serve as good surrogate models for the study of osteoblast cell differentiation and bone mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 osteoblast cell lines. Primary mouse floxed Bmp2 osteoblasts were transfected with pSV3-neo and clonally selected. These transfected cells were verified by PCR and immunohistochemistry. To determine the genotype and phenotype of the immortalized cells, cell morphology, proliferation, differentiation and mineralization were analyzed. Also, expression of osteoblast-related gene markers including Runx2, Osx, ATF4, Dlx3, bone sialoprotein, dentin matrix protein 1, osteonectin, osteocalcin and osteopontin were examined by quantitative RT-PCR and immunohistochemistry. These results showed that immortalized floxed Bmp2 osteoblasts had a higher proliferation rate but preserved their genotypic and phenotypic characteristics similar to the primary cells. Thus, we, for the first time, describe the development of immortalized mouse floxed Bmp2 osteoblast cell lines and present a useful model to study osteoblast biology mediated by BMP2 and its downstream signaling transduction pathways. PMID:21271257

Real-time measurement of hyperpolarized lactate production and efflux as a biomarker of tumor aggressiveness in an MR compatible 3D cell culture bioreactor.

PubMed

Sriram, Renuka; Van Criekinge, Mark; Hansen, Ailin; Wang, Zhen J; Vigneron, Daniel B; Wilson, David M; Keshari, Kayvan R; Kurhanewicz, John

2015-09-01

We have developed a 3D cell/tissue culture bioreactor compatible with hyperpolarized (HP) (13)C MR and interrogated HP [1-(13)C]lactate production and efflux in human renal cell carcinoma (RCC) cells. This platform is capable of resolving intracellular and extracellular HP lactate pools, allowing the kinetic measurement of lactate production and efflux in the context of cancer aggressiveness and response to therapy. HP (13)C MR studies were performed on three immortalized human renal cell lines: HK2, a normal renal proximal tubule cell line from which a majority of RCCs arise, UMRC6, a cell line derived from a localized RCC, and UOK262, an aggressive and metastatic RCC. The intra- (Lacin ) and extracellular (Lacex ) HP lactate signals were robustly resolved in dynamic (13)C spectra of the cell lines due to a very small but reproducible chemical shift difference (0.031 ± 0.0005 ppm). Following HP [1-(13)C]pyruvate delivery, the ratio of HP Lacin /Lacex was significantly lower for UOK262 cells compared with both UMRC6 and HK2 cells due to a significant (p < 0.05) increase in the Lacex pool size. Lacin /Lacex correlated with the MCT4 mRNA expression of the cell lines, and inhibition of MCT4 transport using DIDS resulted in a significant reduction in the HP Lacex pool size. The extension of these studies to living patient-derived RCC tissue slices using HP [1,2-(13)C2]pyruvate demonstrated a similarly split lactate doublet with a high Lacex pool fraction; in contrast, only a single NMR resonance is noted for HP [5-(13)C]glutamate, consistent with intracellular localization. These studies support the importance of lactate efflux as a biomarker of cancer aggressiveness and metastatic potential, and the utility of the MR compatible 3D cell/tissue culture bioreactor to study not only cellular metabolism but also transport. Additionally, this platform offers a sophisticated way to follow therapeutic interventions and screen novel therapies that target lactate export. Copyright © 2015 John Wiley & Sons, Ltd.

Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35.

PubMed

Zhao, Menglin; Wang, Jiaxian; Luo, Manyu; Luo, Han; Zhao, Meiqi; Han, Lei; Zhang, Mengxiao; Yang, Hui; Xie, Yueqing; Jiang, Hua; Feng, Lei; Lu, Huili; Zhu, Jianwei

2018-07-01

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable recombinant CHO cell line has always been a difficult task due to the heterogenic insertion and its caused requirement of multiple rounds of selection. Site-specific integration of transgenes into CHO hot spots is an ideal strategy to overcome these challenges since it can generate isogenic cell lines with consistent productivity and stability. In this study, we investigated three sites with potential high transcriptional activities: C12orf35, HPRT, and GRIK1, to determine the possible transcriptional hot spots in CHO cells, and further construct a reliable site-specific integration strategy to develop recombinant cell lines efficiently. Genes encoding representative proteins mCherry and anti-PD1 monoclonal antibody were targeted into these three loci respectively through CRISPR/Cas9 technology. Stable cell lines were generated successfully after a single round of selection. In comparison with a random integration control, all the targeted integration cell lines showed higher productivity, among which C12orf35 locus was the most advantageous in both productivity and cell line stability. Binding affinity and N-glycan analysis of the antibody revealed that all batches of product were of similar quality independent on integrated sites. Deep sequencing demonstrated that there was low level of off-target mutations caused by CRISPR/Cas9, but none of them contributed to the development process of transgene cell lines. Our results demonstrated the feasibility of C12orf35 as the target site for exogenous gene integration, and strongly suggested that C12orf35 targeted integration mediated by CRISPR/Cas9 is a reliable strategy for the rapid development of recombinant CHO cell lines.

The Dox-pDC - A murine conditionally immortalized plasmacytoid dendritic cell line with native immune profile

PubMed Central

Wiedemuth, Ralf; Binner, Aline; Navratiel, Katrin; Anastassiadis, Konstantinos; Brenner, Sebastian

2018-01-01

Plasmacytoid dendritic cells (pDC) constitute a very rare blood cell population and play a significant role in immune response and immune-mediated disorders. Investigations on primary pDCs are hindered not only due to their rarity but also because they represent a heterogeneous cell population which is difficult to culture ex vivo. We generated a conditionally immortalized pDC line (Dox-pDC) from mice with Doxycycline-inducible SV40 Large T Antigen with a comparable immune profile to primary pDCs. The Dox-pDC secrete pro- and anti-inflammatory cytokines upon Toll-like receptor 9 stimulation and upregulate their MHCI, MHCII and costimulatory molecules. Further, the Dox-pDC activate and polarize naïve T cells in vivo and in vitro in response to the model antigen Ovalbumin. Due to their long-term culture stability and their robust proliferation Dox-pDC represent a reliable alternative to primary mouse pDC. PMID:29489861

The Dox-pDC - A murine conditionally immortalized plasmacytoid dendritic cell line with native immune profile.

PubMed

Thieme, Sebastian; Holzbaur, Alexander; Wiedemuth, Ralf; Binner, Aline; Navratiel, Katrin; Anastassiadis, Konstantinos; Brenner, Sebastian; Richter, Cornelia

2018-01-01

Plasmacytoid dendritic cells (pDC) constitute a very rare blood cell population and play a significant role in immune response and immune-mediated disorders. Investigations on primary pDCs are hindered not only due to their rarity but also because they represent a heterogeneous cell population which is difficult to culture ex vivo. We generated a conditionally immortalized pDC line (Dox-pDC) from mice with Doxycycline-inducible SV40 Large T Antigen with a comparable immune profile to primary pDCs. The Dox-pDC secrete pro- and anti-inflammatory cytokines upon Toll-like receptor 9 stimulation and upregulate their MHCI, MHCII and costimulatory molecules. Further, the Dox-pDC activate and polarize naïve T cells in vivo and in vitro in response to the model antigen Ovalbumin. Due to their long-term culture stability and their robust proliferation Dox-pDC represent a reliable alternative to primary mouse pDC.

Non-p-glycoprotein-mediated multidrug resistance in detransformed rat cells selected for resistance to methylglyoxal bis(guanylhydrazone).

PubMed

Weber, J M; Sircar, S; Horvath, J; Dion, P

1989-11-01

Three independent variants (G2, G4, G5), resistant to methylglyoxal bis(guanylhydrazone), an anticancer drug, have been isolated by single step selection from an adenovirus-transformed rat brain cell line (1). These variants display selective cross-resistance to several natural product drugs of dissimilar structure and action. Multidrug resistance has recently been shown to be caused by overexpression of the membrane-associated p-glycoprotein, most often caused by amplification of the mdr gene. Several types of experiments were conducted to determine whether the observed drug resistance in our cell lines could be due to changes at the mdr locus. The following results were obtained: (a) the mdr locus was not amplified; (b) transcription of the mdr gene and p-glycoprotein synthesis were not increased; (c) multidrug resistance cell lines, which carry an amplified mdr locus, were not cross-resistant to methylglyoxal bis(guanylhydrazone); (d) verapamil did not reverse the resistance of G cells or mdr cells to methylglyoxal bis(guanylhydrazone), nor that of G cells to vincristine; and (e) methylglyoxal bis(guanylhydrazone) resistance was recessive and depended on a block to drug uptake, as opposed to mdr cells which are dominant and express increased drug efflux. The results obtained suggest that the drug resistance in the G2, G4, and G5 cells was atypical and may be due to a mechanism distinct from that mediated by the mdr locus.

Bone stroma-derived cells change coregulators recruitment to androgen receptor and decrease cell proliferation in androgen-sensitive and castration-resistant prostate cancer cells

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

Villagran, Marcelo A.; Gutierrez-Castro, Francisco A.; Pantoja, Diego F.

Prostate cancer (CaP) bone metastasis is an early event that remains inactive until later-stage progression. Reduced levels of circulating androgens, due to andropause or androgen deprivation therapies, alter androgen receptor (AR) coactivator expression. Coactivators shift the balance towards enhanced AR-mediated gene transcription that promotes progression to androgen-resistance. Disruptions in coregulators may represent a molecular switch that reactivates latent bone metastasis. Changes in AR-mediated transcription in androgen-sensitive LNCaP and androgen-resistant C4-2 cells were analyzed for AR coregulator recruitment in co-culture with Saos-2 and THP-1. The Saos-2 cell line derived from human osteosarcoma and THP-1 cell line representing human monocytes were usedmore » to display osteoblast and osteoclast activity. Increased AR activity in androgen-resistant C4-2 was due to increased AR expression and SRC1/TIF2 recruitment and decreased SMRT/NCoR expression. AR activity in both cell types was decreased over 90% when co-cultured with Saos-2 or THP-1 due to dissociation of AR from the SRC1/TIF2 and SMRT/NCoR coregulators complex, in a ligand-dependent and cell-type specific manner. In the absence of androgens, Saos-2 decreased while THP-1 increased proliferation of LNCaP cells. In contrast, both Saos-2 and THP-1 decreased proliferation of C4-2 in absence and presence of androgens. Global changes in gene expression from both CaP cell lines identified potential cell cycle and androgen regulated genes as mechanisms for changes in cell proliferation and AR-mediated transactivation in the context of bone marrow stroma cells. - Highlights: • Decreased corepressor expression change AR in androgen-resistance prostate cancer. • Bone stroma-derived cells change AR coregulator recruitment in prostate cancer. • Bone stroma cells change cell proliferation in androgen-resistant cancer cells. • Global gene expression in CaP cells is modified by bone stroma cells in co-cultures. • Potential new multi-subunit coactivator complexes for AR in CaP bone metastasis.« less

9-AAA inhibits growth and induces apoptosis in human melanoma A375 and rat prostate adenocarcinoma AT-2 and Mat-LyLu cell lines but does not affect the growth and viability of normal fibroblasts.

PubMed

Korohoda, Włodzimierz; Hapek, Anna; Pietrzak, Monika; Ryszawy, Damian; Madeja, Zbigniew

2016-11-01

The present study found that, similarly to 5-fluorouracil, low concentrations (1-10 µM) of 9-aminoacridine (9-AAA) inhibited the growth of the two rat prostate cancer AT-2 and Mat-LyLu cell lines and the human melanoma A375 cell line. However, at the same concentrations, 9-AAA had no effect on the growth and apoptosis of normal human skin fibroblasts (HSFs). The differences between the cellular responses of the AT-2 and Mat-LyLu cell lines, which differ in malignancy, were found to be relatively small compared with the differences between normal HSFs and the cancer cell lines. Visible effects on the cell growth and survival of tumor cell lines were observed after 24-48 h of treatment with 9-AAA, and increased over time. The inhibition of cancer cell growth was found to be due to the gradually increasing number of cells dying by apoptosis, which was observed using two methods, direct counting and FlowSight analysis. Simultaneously, cell motile activity decreased to the same degree in cancer and normal cells within the first 8 h of incubation in the presence of 9-AAA. The results presented in the current study suggest that short-lasting tests for potential anticancer substances can be insufficient; which may result in cell type-dependent differences in the responses of cells to tested compounds that act with a delay being overlooked. The observed differences in responses between normal human fibroblasts and cancer cells to 9-AAA show the requirement for additional studies to be performed simultaneously on differently reacting cancer and normal cells, to determine the molecular mechanisms responsible for these differences.

Lethality to leukemia cell lines of DNA interstrand cross-links generated by Cloretazine derived alkylating species

PubMed Central

Penketh, Philip G.; Baumann, Raymond P.; Ishiguro, Kimiko; Shyam, Krishnamurthy; Seow, Helen A.; Sartorelli, Alan C.

2010-01-01

Cloretazine [1, 2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]-hydrazine; VNP40101M; 101M] is a relatively new prodrug with activity in elderly acute myelogenous leukemia patients. Its therapeutic action is due largely to the production of 1-(3-cytosinyl),2-(1-guanyl)ethane cross-links (G-C ethane cross-links) in DNA. The number of cross-links produced in three experimental leukemia lines (L1210, U937 and HL-60) were fewer than 10 per genome at their respective LC50 concentrations. Only 1 in approximately 20,000 90CE molecules produce a cross-link in the AGT (O6-alkylguanine-DNA alkyltransferase) negative L1210 and U937 cell lines and 1 in 400,000 in the AGT positive HL-60 cell line. PMID:18479747

Preclinical optimization of a broad-spectrum anti-bladder cancer tri-drug regimen via the Feedback System Control (FSC) platform

NASA Astrophysics Data System (ADS)

Liu, Qi; Zhang, Cheng; Ding, Xianting; Deng, Hui; Zhang, Daming; Cui, Wei; Xu, Hongwei; Wang, Yingwei; Xu, Wanhai; Lv, Lei; Zhang, Hongyu; He, Yinghua; Wu, Qiong; Szyf, Moshe; Ho, Chih-Ming; Zhu, Jingde

2015-06-01

Therapeutic outcomes of combination chemotherapy have not significantly advanced during the past decades. This has been attributed to the formidable challenges of optimizing drug combinations. Testing a matrix of all possible combinations of doses and agents in a single cell line is unfeasible due to the virtually infinite number of possibilities. We utilized the Feedback System Control (FSC) platform, a phenotype oriented approach to test 100 options among 15,625 possible combinations in four rounds of assaying to identify an optimal tri-drug combination in eight distinct chemoresistant bladder cancer cell lines. This combination killed between 82.86% and 99.52% of BCa cells, but only 47.47% of the immortalized benign bladder epithelial cells. Preclinical in vivo verification revealed its markedly enhanced anti-tumor efficacy as compared to its bi- or mono-drug components in cell line-derived tumor xenografts. The collective response of these pathways to component drugs was both cell type- and drug type specific. However, the entire spectrum of pathways triggered by the tri-drug regimen was similar in all four cancer cell lines, explaining its broad spectrum killing of BCa lines, which did not occur with its component drugs. Our findings here suggest that the FSC platform holdspromise for optimization of anti-cancer combination chemotherapy.

In Silico Estimation of Translation Efficiency in Human Cell Lines: Potential Evidence for Widespread Translational Control

PubMed Central

Stevens, Stewart G.; Brown, Chris M

2013-01-01

Recently large scale transcriptome and proteome datasets for human cells have become available. A striking finding from these studies is that the level of an mRNA typically predicts no more than 40% of the abundance of protein. This correlation represents the overall figure for all genes. We present here a bioinformatic analysis of translation efficiency – the rate at which mRNA is translated into protein. We have analysed those human datasets that include genome wide mRNA and protein levels determined in the same study. The analysis comprises five distinct human cell lines that together provide comparable data for 8,170 genes. For each gene we have used levels of mRNA and protein combined with protein stability data from the HeLa cell line to estimate translation efficiency. This was possible for 3,990 genes in one or more cell lines and 1,807 genes in all five cell lines. Interestingly, our analysis and modelling shows that for many genes this estimated translation efficiency has considerable consistency between cell lines. Some deviations from this consistency likely result from the regulation of protein degradation. Others are likely due to known translational control mechanisms. These findings suggest it will be possible to build improved models for the interpretation of mRNA expression data. The results we present here provide a view of translation efficiency for many genes. We provide an online resource allowing the exploration of translation efficiency in genes of interest within different cell lines (http://bioanalysis.otago.ac.nz/TranslationEfficiency). PMID:23460887

Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

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

Sustarsic, Elahu G.; Department of Biological Sciences, Ohio University, Athens, OH; Junnila, Riia K.

2013-11-08

Highlights: •Most cancer types of the NCI60 have sub-sets of cell lines with high GHR expression. •GHR is highly expressed in melanoma cell lines. •GHR is elevated in advanced stage IV metastatic tumors vs. stage III. •GH treatment of metastatic melanoma cell lines alters growth and cell signaling. -- Abstract: Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includesmore » 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on this data, GH could be a new therapeutic target in melanoma.« less

High-resolution analysis of CpG methylation and in vivo protein-DNA interactions at the alternative Epstein-Barr virus latency promoters Qp and Cp in the nasopharyngeal carcinoma cell line C666-1.

PubMed

Bakos, Agnes; Banati, Ferenc; Koroknai, Anita; Takacs, Maria; Salamon, Daniel; Minarovits-Kormuta, Susanna; Schwarzmann, Fritz; Wolf, Hans; Niller, Hans Helmut; Minarovits, Janos

2007-10-01

Transcripts for the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNAs) are initiated at alternative promoters (Wp, Cp, for EBNA 1-6 transcripts and Qp, for EBNA 1 transcripts only) located in the BamHI W, C or Q fragment of the viral genome. To understand the host-cell dependent expression of EBNAs in EBV-associated tumors (lymphomas and carcinomas) and in vitro transformed cell lines, it is necessary to analyse the regulatory mechanisms governing the activity of the alternative promoters of EBNA transcripts. Such studies focused mainly on lymphoid cell lines carrying latent EBV genomes, due to the lack of EBV-associated carcinoma cell lines maintaining latent EBV genomes during cultivation in tissue culture. We took advantage of the unique nasopharyngeal carcinoma cell line, C666-1, harboring EBV genomes, and undertook a detailed analysis of CpG methylation patterns and in vivo protein-DNA interactions at the latency promoters Qp and Cp. We found that the active, unmethylated Qp was marked with strong footprints of cellular transcription factors and the viral protein EBNA 1. In contrast, we could not detect binding of relevant transcription factors to the methylated, silent Cp. We concluded that the epigenetic marks at Qp and Cp in C666-1 cells of epithelial origin resemble those of group I Burkitt's lymphoma cell lines.

Total triterpenoids from Ganoderma Lucidum suppresses prostate cancer cell growth by inducing growth arrest and apoptosis.

PubMed

Wang, Tao; Xie, Zi-ping; Huang, Zhan-sen; Li, Hao; Wei, An-yang; Di, Jin-ming; Xiao, Heng-jun; Zhang, Zhi-gang; Cai, Liu-hong; Tao, Xin; Qi, Tao; Chen, Di-ling; Chen, Jun

2015-10-01

In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.

Participation of an abnormality in the transforming growth factor-beta signaling pathway in resistance of malignant glioma cells to growth inhibition induced by that factor.

PubMed

Zhang, Lei; Sato, Eiji; Amagasaki, Kenichi; Nakao, Atsuhito; Naganuma, Hirofumi

2006-07-01

Malignant glioma cells secrete and activate transforming growth factor-beta (TGFbeta) and are resistant to growth inhibition by that factor. Nevertheless, the mechanism underlying this effect remains poorly understood. In this study, the mechanism of the resistance to growth inhibition induced by TGFbeta was investigated. The authors examined the expression of downstream components of the TGFbeta receptor, including Smad2, Smad3, Smad4, and Smad7, and the effect of TGFbeta1 treatment on the phosphorylation of Smad2 and the nuclear translocation of Smad2 and Smad3 by using 10 glioma cell lines and the A549 cell line, which is sensitive to TGFbeta-mediated growth inhibition. The expression of two transcriptional corepressor proteins, SnoN and Ski, and the effect of TGFbeta1 treatment on the expression of the SnoN protein and the cell cycle regulators p21, p15, cyclin-dependent kinase-4 (CDK4), and cyclin D1 were also examined. Expression of the Smad2 and Smad3 proteins was lower in the glioma cell lines than in the A549 cell line and in normal astrocytes. In particular, Smad3 expression was low or very low in nine of the 10 malignant glioma cell lines. Expression of Smad4 was low in four glioma cell lines, and expression of the Smad7 protein was similar when compared with protein expression in the A549 cell line and in normal astrocytes. The levels of Smad2 phosphorylation after TGFbeta1 treatment were lower in glioma cell lines than in the A549 cell line, except for one glioma cell line. Seven of the 10 glioma cell lines exhibited lower levels of nuclear translocation of Smad2 and Smad3, and two cell lines that expressed very low levels of Smad3 protein showed no nuclear translocation. All glioma cell lines expressed the SnoN protein and its expression was unaltered by treatment with TGFbeta1. Three glioma cell lines expressed high levels of the Ski protein. The expression of the p21(cip1), p15(INK4B), CDK4, and cyclin D1 proteins was not altered by TGFbeta1, treatment, except in one cell line that displayed a slight increase in p21 protein. Overall, the expression of the Smad2 and Smad3 proteins was low in the glioma cell lines, the phosphorylation and nuclear translocation of Smad2 and Smad3 were impaired, and the TGFbeta receptor signal did not affect the expression of the SnoN, p21, p15, cyclin D1, and CDK4 proteins. These results suggest that the ability to resist TGFbeta-mediated growth inhibition in malignant glioma cells is due to abnormalities in the TGFbeta signaling pathway.

Establishment of a novel and highly permissive cell line for the efficient replication of cyprinid herpesvirus 2 (CyHV-2).

PubMed

Ma, Jie; Jiang, Nan; LaPatra, Scott E; Jin, Ling; Xu, Jin; Fan, Yuding; Zhou, Yong; Zeng, Lingbing

2015-06-12

Haematopoietic necrosis of gibel carp (Carassius auratus gibelio) is caused by cyprinid herpesvirus 2 (CyHV-2) and has caused huge economic losses in aquaculture worldwide. Currently the isolation and propagation of CyHV-2 in vitro is very difficult due to the lack of permissive cell lines. Studies on the pathogenesis of CyHV-2 have been hampered because the virus has not been extensively characterized. In this study, a novel cell line from the brain of gibel carp, denoted GiCB, has been established and characterized. Sustainable propagation of CyHV-2 in the GiCB cell line has been confirmed by virus infection and titration, PCR, transmission electron microscopy, immunofluorescence assay and fluorescence in situ hybridization. The GiCB cells showed typical cytopathic effect by day 6 post-infection with CyHV-2 including cell shrinkage, rounding, and cell fusion with cytoplasmic vacuolization. The virus titer reached 10(7.5 ± 0.37)TCID₅₀/ml and has been successfully passaged over 50 times in the GiCB cell line. Electron microscopy analysis revealed the complete replication of CyHV-2 in GiCB cells. CyHV-2-infected GiCB cells reacted strongly with polyclonal antibodies against CyHV-2 and CyHV-2 RNA in cells hybridized specifically with the virus RNA probes. Additionally, an experimental infection demonstrated that CyHV-2 produced in GiCB cells caused 100% mortality in gibel carp. All the results provide solid evidence that the GiCB cell line is highly permissive for the isolation and propagation of CyHV-2. This is a significant advancement that will promote additional research on CyHV-2 infection in fish in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

Genistein modulates oxidative stress in breast cancer cell lines according to ERα/ERβ ratio: effects on mitochondrial functionality, sirtuins, uncoupling protein 2 and antioxidant enzymes.

PubMed

Nadal-Serrano, Mercedes; Pons, Daniel Gabriel; Sastre-Serra, Jorge; Blanquer-Rosselló, M del Mar; Roca, Pilar; Oliver, Jordi

2013-09-01

Genistein is a biologically active isoflavone with estrogenic activity and can be found in a variety of soy products. This natural compound displays a wide array of biological activities, but it is best known for its ability to inhibit cancer progression, especially for hormone-related ones such as breast cancer. Genistein has been shown to bind both the estrogen receptor alpha (ERα) and the estrogen receptor beta (ERβ), although it has a higher affinity for the ERβ. The ERα/ERβ ratio is a prognostic marker for breast tumors, and ERβ expression could indicate the presence of tumors more benign in state, whereas ERα indicates malignant tumors. The objective of the present study was to investigate the effects of genistein on oxidative stress and mitochondrial functionality through its interaction with the estrogen receptor in breast cancer cell lines with different ERα/ERβ ratios. The lower ERα/ERβ ratio T47D cell line showed lower oxidative stress and greater mitochondrial functionality, along with an up-regulation of uncoupling protein 2 and sirtuins. On the other hand, genistein-treated MCF-7 cell line, with the highest ERα/ERβ ratio, reported no changes for the control situation. On the whole, our results show different genistein effects depending on ERα/ERβ ratio for oxidative stress regulation, mitochondrial functionality, and modulation of UCPs, antioxidant enzymes and sirtuins in breast cancer cell lines. Effects of genistein on oxidative stress and mitochondria could be due at least in part, to a higher ERβ presence, but could also be due to up-regulation of ERβ caused by the genistein treatment. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cytoarchitecture of Zika virus infection in human neuroblastoma and Aedes albopictus cell lines

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

Offerdahl, Danielle K.

The Zika virus (ZIKV) pandemic is a global concern due to its role in the development of congenital anomalies of the central nervous system. This mosquito-borne flavivirus alternates between mammalian and mosquito hosts, but information about the biogenesis of ZIKV is limited. Using a human neuroblastoma cell line (SK-N-SH) and an Aedes albopictus mosquito cell line (C6/36), we characterized ZIKV infection by immunofluorescence, transmission electron microscopy (TEM), and electron tomography (ET) to better understand infection in these disparate host cells. ZIKV replicated well in both cell lines, but infected SK-N-SH cells suffered a lytic crisis. Flaviviruses scavenge host cell membranesmore » to serve as replication platforms and ZIKV showed the hallmarks of this process. Via TEM, we identified virus particles and 60–100 nm spherular vesicles. ET revealed these vesicular replication compartments contain smaller 20–30 nm spherular structures. Our studies indicate that SK-N-SH and C6/36 cells are relevant models for viral cytoarchitecture study. - Highlights: •First electron tomography of Zika virus cytoarchitecture. •Comparison of Zika virus infection in human neuroblastoma and mosquito cells. •Ultrastructure of Zika virus infection in human neuroblastoma and mosquito cells.« less

Selective cytotoxicity of transformed cells but not normal cells by a sialoglycopeptide growth regulator in the presence of tumor necrosis factor

NASA Technical Reports Server (NTRS)

Woods, K. M.; Fattaey, H.; Johnson, T. C.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

The tumor necrosis factor-alpha (TNF)-resistant, SV40-transformed, murine fibroblast cell lines, F5b and F5m, became sensitive to TNF-mediated cytolysis after treatment with a biologically active 18 kDa peptide fragment (SGP) derived from a 66-kDa parental cell surface sialoglycoprotein. Neither TNF nor the SGP alone exhibited cytotoxicity to the two SV40-transformed cell lines. However, Balb/c 3T3 cells, incubated with SGP alone or with SGP and TNF, were not killed. Therefore, SGP can selectively sensitize cells for TNF alpha-mediated cytotoxicity. This selective sensitization may be due to the previously documented ability of the SGP to selectively mediate cell cycle arrest.

Use of deferasirox, an iron chelator, to overcome imatinib resistance of chronic myeloid leukemia cells.

PubMed

Kim, Dae Sik; Na, Yoo Jin; Kang, Myoung Hee; Yoon, Soo-Young; Choi, Chul Won

2016-03-01

The treatment of chronic myeloid leukemia (CML) has achieved impressive success since the development of the Bcr-Abl tyrosine kinase inhibitor, imatinib mesylate. Nevertheless, resistance to imatinib has been observed, and a substantial number of patients need alternative treatment strategies. We have evaluated the effects of deferasirox, an orally active iron chelator, and imatinib on K562 and KU812 human CML cell lines. Imatinib-resistant CML cell lines were created by exposing cells to gradually increasing concentrations of imatinib. Co-treatment of cells with deferasirox and imatinib induced a synergistic dose-dependent inhibition of proliferation of both CML cell lines. Cell cycle analysis showed an accumulation of cells in the subG1 phase. Western blot analysis of apoptotic proteins showed that co-treatment with deferasirox and imatinib induced an increased expression of apoptotic proteins. These tendencies were clearly identified in imatinib-resistant CML cell lines. The results also showed that co-treatment with deferasirox and imatinib reduced the expression of BcrAbl, phosphorylated Bcr-Abl, nuclear factor-κB (NF-κB) and β-catenin. We observed synergistic effects of deferasirox and imatinib on both imatinib-resistant and imatinib-sensitive cell lines. These effects were due to induction of apoptosis and cell cycle arrest by down-regulated expression of NF-κB and β-catenin levels. Based on these results, we suggest that a combination treatment of deferasirox and imatinib could be considered as an alternative treatment option for imatinib-resistant CML.

Use of planar array electrophysiology for the development of robust ion channel cell lines.

PubMed

Clare, Jeffrey J; Chen, Mao Xiang; Downie, David L; Trezise, Derek J; Powell, Andrew J

2009-01-01

The tractability of ion channels as drug targets has been significantly improved by the advent of planar array electrophysiology platforms which have dramatically increased the capacity for electrophysiological profiling of lead series compounds. However, the data quality and through-put obtained with these platforms is critically dependent on the robustness of the expression reagent being used. The generation of high quality, recombinant cell lines is therefore a key step in the early phase of ion channel drug discovery and this can present significant challenges due to the diversity and organisational complexity of many channel types. This article focuses on several complex and difficult to express ion channels and illustrates how improved stable cell lines can be obtained by integration of planar array electrophysiology systems into the cell line generation process per se. By embedding this approach at multiple stages (e.g., during development of the expression strategy, during screening and validation of clonal lines, and during characterisation of the final cell line), the cycle time and success rate in obtaining robust expression of complex multi-subunit channels can be significantly improved. We also review how recent advances in this technology (e.g., population patch clamp) have further widened the versatility and applicability of this approach.

OutKnocker: a web tool for rapid and simple genotyping of designer nuclease edited cell lines.

PubMed

Schmid-Burgk, Jonathan L; Schmidt, Tobias; Gaidt, Moritz M; Pelka, Karin; Latz, Eicke; Ebert, Thomas S; Hornung, Veit

2014-10-01

The application of designer nucleases allows the induction of DNA double-strand breaks (DSBs) at user-defined genomic loci. Due to imperfect DNA repair mechanisms, DSBs can lead to alterations in the genomic architecture, such as the disruption of the reading frame of a critical exon. This can be exploited to generate somatic knockout cell lines. While high genome editing activities can be achieved in various cellular systems, obtaining cell clones that contain all-allelic frameshift mutations at the target locus of interest remains a laborious task. To this end, we have developed an easy-to-follow deep sequencing workflow and the evaluation tool OutKnocker (www.OutKnocker.org), which allows convenient, reliable, and cost-effective identification of knockout cell lines. © 2014 Schmid-Burgk et al.; Published by Cold Spring Harbor Laboratory Press.

Acute Exposure to High Dose γ-Radiation Results in Transient Activation of Bone Lining Cells

PubMed Central

Turner, Russell T.; Iwaniec, Urszula T.; Wong, Carmen P.; Lindenmaier, Laurence B.; Wagner, Lindsay A.; Branscum, Adam J.; Menn, Scott A.; Taylor, James; Zhang, Ye; Wu, Honglu; Sibonga, Jean D.

2014-01-01

The present studies investigated the cellular mechanisms for the detrimental effects of high dose whole body γ-irradiation on bone. In addition, radioadaptation and bone marrow transplantation were assessed as interventions to mitigate the skeletal complications of irradiation. Increased trabecular thickness and separation and reduced fractional cancellous bone volume, connectivity density, and trabecular number were detected in proximal tibia and lumbar vertebra 14 days following γ-irradiation with 6 Gy. To establish the cellular mechanism for the architectural changes, vertebrae were analyzed by histomorphometry 1, 3, and 14 days following irradiation. Marrow cell density decreased within 1 day (67% reduction, p<0.0001), reached a minimum value after 3 days (86% reduction, p<0.0001), and partially rebounded by 14 days (30% reduction, p=0.0025) following irradiation. In contrast, osteoblast-lined bone perimeter was increased by 290% (1 day, p=0.04), 1230% (3 days, p<0.0001), and 530% (14 days, p=0.003), respectively. There was a strong association between radiation-induced marrow cell death and activation of bone lining cells to express the osteoblast phenotype (Pearson correlation −0.85, p<0.0001). An increase (p=0.004) in osteoclast-lined bone perimeter was also detected with irradiation. A priming dose of γ-radiation (0.5 mGy), previously shown to reduce mortality, had minimal effect on the cellular responses to radiation and did not prevent detrimental changes in bone architecture. Bone marrow transplantation normalized marrow cell density, bone turnover, and most indices of bone architecture following irradiation. In summary, radiation-induced death of marrow cells is associated with 1) a transient increase in bone formation due, at least in part, to activation of bone lining cells, and 2) an increase in bone resorption due to increased osteoclast perimeter. Bone marrow transplantation is effective in mitigating the detrimental effects of acute exposure to high dose whole body γ-radiation on bone turnover. PMID:23954507

Optimization of DNA barcode method to assess altered chemical toxicity due to CYP-mediated metabolism

EPA Science Inventory

A drawback of current in vitro chemical testing is that many commonly used cell lines lack chemical metabolism. This hinders the use and relevance of cell culture in high throughput chemical toxicity screening. To address this challenge, we engineered HEK293T cells to overexpress...

Hyperosmolarity leads to an increase in derepressed system A activity in the renal epithelial cell line NBL-1.

PubMed Central

Soler, C; Felipe, A; Casado, F J; McGivan, J D; Pastor-Anglada, M

1993-01-01

Hyperosmolarity induced an increase in Na(+)-dependent L-alanine uptake in confluent monolayers of the established renal epithelial cell line NBL-1. This induction was attributable to system A and was only seen when the cells had been previously deprived of amino acids in the culture medium to derepress system A activity. It was additive to the adaptive regulation induction, and both were inhibited by cycloheximide. However, the hyperosmolarity effect was inhibited by colcemid (an inhibitor of microtubular function), but adaptive regulation was not. Otherwise, when cell monolayers were incubated in a control medium, basal Na(+)-dependent L-alanine uptake mediated by system B0 decreased. The results of this study show that: (i) system A activity was not induced by cell shrinkage and subsequent swelling due to extracellular hyperosmolarity when cells were incubated in control medium; (ii) previous expression of system A activity induced by amino acid starvation seems to be a prerequisite for further induction due to hyperosmolarity; and (iii) the effects of adaptive regulation and hyperosmotic stress are mediated by different mechanisms. PMID:8435065

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

PubMed Central

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

Impact of cell lines included in enterovirus isolation protocol on perception of nonpolio enterovirus species C diversity.

PubMed

Adeniji, Johnson Adekunle; Faleye, Temitope Oluwasegun Cephas

2014-10-01

There has been under-reporting of nonpolio enterovirus species Cs (NPESCs) in Nigeria despite the fact that most isolates recovered from the Nigerian vaccine derived poliovirus serotype 2 (VDPV2) outbreak were recombinants with nonstructural region of NPESC origin. It has been suggested that cell lines included in enterovirus isolation protocols might account for this phenomenon and this study examined this suggestion. Fifteen environmental samples concentrated previously and analysed using L20B and RD cell lines as part of the poliovirus environmental surveillance (ES) program in Nigeria were randomly selected and inoculated into two cell lines (MCF-7 and LLC-MK2). Isolates were identified as enteroviruses and species C members using different RT-PCR assays, culture in L20B cell line and sequencing of partial VP1. Forty-eight (48) isolates were recovered from the 15 samples, 47 (97.9%) of which were enteroviruses. Of the enteroviruses, 32 (68.1%) belonged to enterovirus species C (EC) of which 19 (40.4%) were polioviruses and 13 (27.7%) were NPESC members. All 13 NPESC isolates were recovered on MCF-7. Results of the study show that NPESCs are circulating in Nigeria and their under-reporting was due to the combination of cell lines used for enterovirus isolation in previous reports. Copyright © 2014 Elsevier B.V. All rights reserved.

Cell cycle arrest or survival signaling through αv integrins, activation of PKC and ERK1/2 lead to anoikis resistance of ovarian cancer spheroids.

PubMed

Carduner, Ludovic; Picot, Cédric R; Leroy-Dudal, Johanne; Blay, Lyvia; Kellouche, Sabrina; Carreiras, Franck

2014-01-15

Ovarian cancer is the most lethal gynecologic cancer mainly due to spheroids organization of cancer cells that disseminate within the peritoneal cavity. We have investigated the molecular mechanisms by which ovarian cancer spheroids resist anoikis, choosing as models the 2 well-characterized human ovarian cancer cell lines IGROV1 and SKOV3. These cell lines have the propensity to float as clusters, and were isolated from tumor tissue and ascites, respectively. To form spheroids, IGROV1 and SKOV3 ovarian adenocarcinoma cells were maintained under anchorage-independent culture conditions, in which both lines survive at least a week. A short apoptotic period prior to a survival signaling commitment was observed for IGROV1 cells whereas SKOV3 cells entered G0/G1 phase of the cell cycle. This difference in behavior was due to different signals. With regard to SKOV3 cells, activation of p38 and an increase in p130/Rb occurred once anchorage-independent culture was established. Analyses of the survival signaling pathway switched on by IGROV1 cells showed that activation of ERK1/2 was required to evade apoptosis, an effect partly dependent on PKC activation and αv integrins. αv-integrin expression is essential for survival through activation of ERK1/2 phosphorylation. The above data indicate that ovarian cancer cells can resist anoikis in the spheroid state by arrest in the cell cycle or through activation of αv-integrin-ERK-mediated survival signals. Such signaling might result in the selection of resistant cells within disseminating spheroids, favoring further relapse in ovarian cancers. © 2013 Elsevier Inc. All rights reserved.

[The level of superoxide dismutase expression in primary and metastatic colorectal cancer cells in hypoxia and tissue normoxia].

PubMed

Skrzycki, Michał; Czeczot, Hanna; Chrzanowska, Alicja; Otto-Ślusarczyk, Dagmara

2015-11-01

Superoxide oxidase (SOD) is a key antioxidant enzyme protecting cells against oxidative stress, which might induce cancerogenesis. In tumor cells SOD influences the level of the reactive oxygen species (ROS) allowing for survival and proliferation. High rate of cells proliferation in tumor leads to their temporary hypoxia due to lower rate of angiogenesis. Therefore during tumor development, cancer cells function in conditions of hypoxia or tissue normoxia. The aim of study was to evaluate of SOD isoenzymes (SOD1 and SOD2) expression level in cell lines of primary (SW 480) and metastatic (SW 620) colorectal cancer, cultured in hypoxia (1% oxygen), tissue normoxia (10% oxygen), and atmospheric normoxia (21% oxygen). Cells were cultured in MEM medium in different oxygen concentrations (1%, 10%, 21%) in hypoxic chamber with oxygenation regulator. The number of living cells in lines SW 480 and 620 was determined by trypan blue method. Expression of SOD1 and SOD2 at the mRNA level was determined by RT-PCR and PCR. In both studied cell lines (SW 480 and SW 620), the number of living cells (viability) was increased in hypoxia and atmospheric normoxia. The expression level of SOD1 and SOD2 in studied cell lines was different. The lowest level of expression of both SOD isoenzymes was observed in hypoxia. In conditions of atmospheric normoxia the expression level of SOD1 in SW480 cell line was increased, and similar in SW620 cell line comparing to tissue normoxia. Whereas the SOD2 expression level in atmospheric normoxia conditions in both cell lines was significantly increased. Observed differences were statistically significant (p ≤ 0,05). The profile of expression of SOD1 and SOD2 in cell lines SW480 and SW620 indicates differentiated response of tumor cells depending on access to oxygen. Low level of SOD isoenzymes expression in SW480 and SW620 cells in hypoxia indicates decreased production of ROS. Differences of SOD isoenzymes expression level in tissue normoxia indicate their compensatory action, allowing to maintain the balance between O₂- removal and H₂O₂production in studied tumor cells. In atmospheric normoxia conditions increased expression level of SOD1 and SOD2 observed in studied cell lines points to oxidative stress. © 2015 MEDPRESS.

Cytotoxicity of titanium and silicon dioxide nanoparticles

NASA Astrophysics Data System (ADS)

Wagner, Stefanie; Münzer, Simon; Behrens, Peter; Scheper, Thomas; Bahnemann, Detlef; Kasper, Cornelia

2009-05-01

Different TiO2 and SiO2 nanoparticles have been tested concerning their toxicity on selected mammalian cell lines. Various powders and suspensions, all of which consist of titanium or silicon dioxide nanoparticles have been examined. These particles differ in the crystal structure, the size and the BET-surface area. There was also a classification in fixed particles and in particles easily accessible in solution. With focus on the possible adsorption of the nanoparticles into the human organism, via skin and via respiratory tract, the effects on fibroblasts (NIH-3T3) and on a human lung adenocarcinoma epithelial cell line were examined. Additionally, the particles were tested with HEP-G2 cells, which are often used as model cell line for biocompatibility tests, and PC-12 cells, a rat adrenal pheochromocytoma cell line. The viability of the cells was examined by the MTT-test. The viability results were found to partly depend on the type of cells used. The experimental results show that the adhesion of the cells on the different powders strongly depends on the type of cell lines as well as on the type of powder. It was found that the lower viability of some cells on the powder coatings is not only caused by a cytotoxicity effect of the powders, but is also due to a lower adhesion of the cells on the particle surfaces. Furthermore, it could be shown that the physical properties of the powders cannot be easily correlated to any observed biological effect. While some powders show a significant suppression of the cell growth, others with similar physical properties indicate no toxic effect.

High-throughput microfluidic line scan imaging for cytological characterization

NASA Astrophysics Data System (ADS)

Hutcheson, Joshua A.; Powless, Amy J.; Majid, Aneeka A.; Claycomb, Adair; Fritsch, Ingrid; Balachandran, Kartik; Muldoon, Timothy J.

2015-03-01

Imaging cells in a microfluidic chamber with an area scan camera is difficult due to motion blur and data loss during frame readout causing discontinuity of data acquisition as cells move at relatively high speeds through the chamber. We have developed a method to continuously acquire high-resolution images of cells in motion through a microfluidics chamber using a high-speed line scan camera. The sensor acquires images in a line-by-line fashion in order to continuously image moving objects without motion blur. The optical setup comprises an epi-illuminated microscope with a 40X oil immersion, 1.4 NA objective and a 150 mm tube lens focused on a microfluidic channel. Samples containing suspended cells fluorescently stained with 0.01% (w/v) proflavine in saline are introduced into the microfluidics chamber via a syringe pump; illumination is provided by a blue LED (455 nm). Images were taken of samples at the focal plane using an ELiiXA+ 8k/4k monochrome line-scan camera at a line rate of up to 40 kHz. The system's line rate and fluid velocity are tightly controlled to reduce image distortion and are validated using fluorescent microspheres. Image acquisition was controlled via MATLAB's Image Acquisition toolbox. Data sets comprise discrete images of every detectable cell which may be subsequently mined for morphological statistics and definable features by a custom texture analysis algorithm. This high-throughput screening method, comparable to cell counting by flow cytometry, provided efficient examination including counting, classification, and differentiation of saliva, blood, and cultured human cancer cells.

Establishment and genetic characterization of an immortal tumor cell line derived from intestinal-type sinonasal adenocarcinoma.

PubMed

Pérez-Escuredo, Jhudit; García Martínez, Jorge; García-Inclán, Cristina; Vivanco, Blanca; Costales, María; Álvarez Marcos, César; Llorente, José Luis; Hermsen, Mario A

2011-02-01

Intestinal-type sinonasal adenocarcinoma (ITAC) is a rare tumor etiologically related to professional exposure to wood dust. The overall prognosis is poor, mainly due to the difficulty to resect the tumor completely in this anatomically complex region. Therefore, there is great need for alternative treatments. However, the lack of a good tumor model system for ITAC has hampered the development and testing of new therapeutic agents. Here, we report the establishment and characterization of the first human ITAC cell line named ITAC-3. The cell line was initiated from small explants of a T4bN0M0 colonic type ITAC from the ethmoid sinus. Growth and invasion parameters as well as genetic characteristics were analyzed. The population doubling time was 18 h and the cell line was capable of invasion in matrigel. Chromosomal analysis showed a tetraploid karyotype with both numerical and structural aberrations. High resolution microarray CGH analysis identified many copy number alterations, including homozygous deletions. TP53 carried a mutation c.818G>T in exon eight concurring with a strong nuclear protein overexpression. Immunohistochemical analysis showed protein overexpression of EGFR and normal expression of β-catenin and p16. This is the first report of the establishment of a cell line derived from a primary ITAC. The genomic profile of the cell line was the same as the primary tumor from which it was derived. This new cell line will be a useful tool for the development and testing of new therapeutic agents for this tumor type.

Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells.

PubMed

Rostovskaya, Maria; Bredenkamp, Nicholas; Smith, Austin

2015-10-19

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification. © 2015 The Authors.

The Metastatic Potential and Chemoresistance of Human Pancreatic Cancer Stem Cells

PubMed Central

Bhagwandin, Vikash J.; Bishop, J. Michael; Wright, Woodring E.; Shay, Jerry W.

2016-01-01

Cancer stem cells (CSCs) typically have the capacity to evade chemotherapy and may be the principal source of metastases. CSCs for human pancreatic ductal carcinoma (PDAC) have been identified, but neither the metastatic potential nor the chemoresistance of these cells has been adequately evaluated. We have addressed these issues by examining side-population (SP) cells isolated from the Panc-1 and BxPC3 lines of human PDAC cells, the oncogenotypes of which differ. SP cells could be isolated from monolayers of Panc-1, but only from spheroids of BxPC3. Using orthotopic xenografts into the severely immunocompromised NSG mouse, we found that SP cells isolated from both cell lines produced tumors that were highly metastatic, in contrast to previous experience with PDAC cell lines. SP cells derived from both cell lines expressed the ABCG2 transporter, which was demonstrably responsible for the SP phenotype. SP cells gave rise to non-SP (NSP) cells in vitro and in vivo, a transition that was apparently due to posttranslational inhibition of the ABCG2 transporter. Twenty-two other lines of PDAC cells also expressed ABCG2. The sensitivity of PDAC SP cells to the vinca alkaloid vincristine could be greatly increased by verapamil, a general inhibitor of transporters. In contrast, verapamil had no effect on the killing of PDAC cells by gemcitabine, the current first-line therapeutic for PDAC. We conclude that the isolation of SP cells can be a convenient and effective tool for the study of PDAC CSCs; that CSCs may be the principal progenitors of metastasis by human PDAC; that the ABCG2 transporter is responsible for the SP phenotype in human PDAC cells, and may be a ubiquitous source of drug-resistance in PDAC, but does not confer resistance to gemcitabine; and that inhibition of ABCG2 might offer a useful adjunct in a therapeutic attack on the CSCs of PDAC. PMID:26859746

Overexpression of miR-202 resensitizes imatinib resistant chronic myeloid leukemia cells through targetting Hexokinase 2

PubMed Central

Deng, Yingjun; Li, Xin; Feng, Jinxin; Zhang, Xiangliang

2018-01-01

Chronic myeloid leukemia (CML) is a myeloproliferative disease which uniquely expresses a constitutively active tyrosine kinase, BCR/ABL. As a specific inhibitor of the BCR-ABL tyrosine kinase, imatinib becomes the first choice for the treatment of CML due to its high efficacy and low toxicity. However, the development of imatinib resistance limits the long-term treatment benefits of it in CML patients. In the present study, we aimed to investigate the roles of miR-202 in the regulation of imatinib sensitivity in CML cell lines and the possible mechanisms involved in this process. We found miR-202 was down-regulated in seven CML cell lines by quantitative reverse-transcription PCR (qRT-PCR) analysis. Overexpression of miR-202 significantly suppressed proliferation rates of CML cells. By establishing imatinib resistant cell lines originating from K562 and KU812 cells, we observed expressions of miR-202 were down-regulated by imatinib treatments and imatinib resistant CML cell lines exhibited lower level of miR-202. On the contrary, imatinib resistant CML cell lines displayed up-regulated glycolysis rate than sensitive cells with the evidence that glucose uptake, lactate production, and key glycolysis enzymes were elevated in imatinib resistant cells. Importantly, the imatinib resistant CML cell lines were more sensitive to glucose starvation and glycolysis inhibitors. In addition, we identified Hexokinase 2 (HK2) as a direct target of miR-202 in CML cell lines. Overexpression of miR-202 sensitized imatinib resistant CML through the miR-202-mediated glycolysis inhibition by targetting HK2. Finally, we provided the clinical relevance that miR-202 was down-regulated in CML patients and patients with lower miR-202 expression displayed higher HK2 expression. The present study will provide new aspects on the miRNA-modulated tyrosine kinase inhibitor (TKI) sensitivity in CML, contributing to the development of new therapeutic anticancer drugs. PMID:29559564

High efficacy of the BCL-2 inhibitor ABT199 (venetoclax) in BCL-2 high-expressing neuroblastoma cell lines and xenografts and rational for combination with MCL-1 inhibition

PubMed Central

Bate-Eya, Laurel T.; den Hartog, Ilona J.M.; van der Ploeg, Ida; Schild, Linda; Koster, Jan; Santo, Evan E.; Westerhout, Ellen M.; Versteeg, Rogier; Caron, Huib N.; Molenaar, Jan J.; Dolman, M. Emmy M.

2016-01-01

The anti-apoptotic protein B cell lymphoma/leukaemia 2 (BCL-2) is highly expressed in neuroblastoma and plays an important role in oncogenesis. In this study, the selective BCL-2 inhibitor ABT199 was tested in a panel of neuroblastoma cell lines with diverse expression levels of BCL-2 and other BCL-2 family proteins. ABT199 caused apoptosis more potently in neuroblastoma cell lines expressing high BCL-2 and BIM/BCL-2 complex levels than low expressing cell lines. Effects on cell viability correlated with effects on BIM displacement from BCL-2 and cytochrome c release from the mitochondria. ABT199 treatment of mice with neuroblastoma tumors expressing high BCL-2 levels only resulted in growth inhibition, despite maximum BIM displacement from BCL-2 and the induction of a strong apoptotic response. We showed that neuroblastoma cells might survive ABT199 treatment due to its acute upregulation of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) and BIM sequestration by MCL-1. In vitro inhibition of MCL-1 sensitized neuroblastoma cell lines to ABT199, confirming the pivotal role of MCL-1 in ABT199 resistance. Our findings suggest that neuroblastoma patients with high BCL-2 and BIM/BCL-2 complex levels might benefit from combination treatment with ABT199 and compounds that inhibit MCL-1 expression. PMID:27056887

Expression of a fms-related oncogene in carcinogen-induced neoplastic epithelial cells

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

Walker, C.; Nettesheim, P.; Barrett, J.C.

1987-04-01

Following carcinogen exposure in vitro, normal rat tracheal epithelial cells are transformed in a multistage process in which the cultured cells become immortal and ultimately, neoplastic. Five cell lines derived from tumors produced by neoplastically transformed rat tracheal epithelial cells were examined for the expression of 11 cellular oncogenes previously implicated in pulmonary or epithelial carcinogenesis. RNA homologous to fms was expressed at a level 5-19 times higher than normal tracheal epithelial cells in three of five of the tumor-derived lines. All three lines expressing high levels of fms-related RNA gave rise to invasive tumors of epithelial origin when injectedmore » into nude mice. Increased expression of the fms-related mRNA was not due to gene amplification, and no gene rearrangement was detected by Southern analyses. RNA blot analysis using a 3' v-fms probe detected a 9.5-kilobase message in the three tumor-derived lines, whereas both normal rat aveolar macrophages and the human choriocarcinoma line BeWo expressed a fms transcript of approx. = 4 kilobases. The authors conclude from these data that the gene expressed as a 9.5-kilobase transcript in these neoplastic epithelial cells is a member of a fms-related gene family but may be distinct from the gene that encodes the macrophage colony-stimulating factor (CSF-1) receptor.« less

Hexavalent Chromium Induces Chromosome Instability in Human Urothelial Cells

PubMed Central

Wise, Sandra S.; Holmes, Amie L.; Liou, Louis; Adam, Rosalyn M.; Wise, John Pierce

2016-01-01

Numerous metals are well-known human bladder carcinogens. Despite the significant occupational and public health concern of metals and bladder cancer, the carcinogenic mechanisms remain largely unknown. Chromium, in particular, is a metal of concern as incidences of bladder cancer have been found elevated in chromate workers, and there is an increasing concern for patients with metal hip implants. However, the impact of Cr(VI) on bladder cells has not been studied. We compared chromate toxicity in two bladder cell lines; primary human urothelial cells and hTERT-immortalized human urothelial cells. Hexavalent chromium (Cr(VI)) induced a concentration- and time-dependent increase in chromosome damage in both cell lines, with the hTERT-immortalized cells exhibiting more chromosome damage than the primary cells. Chronic exposure to Cr(VI) also induced a concentration-dependent increase in aneuploid metaphases in both cell lines which was not observed after a 24 h exposure. Aneuploidy induction was higher in the hTERT-immortalized cells. When we correct for uptake, Cr(VI) induces a similar amount of chromosome damage and aneuploidy suggesting that the differences in Cr(VI) sensitivity between the two cells lines were due to differences in uptake. The increase in chromosome instability after chronic chromate treatment suggests this may be a mechanism for chromate-induced bladder cancer specifically and may be a mechanism for metal-induced bladder cancer in general. PMID:26908176

Bis-demethoxy curcumin analog nanoparticles: synthesis, characterization, and anticancer activity in vitro.

PubMed

Francis, Arul Prakash; Murthy, Prakhya Balakishna; Devas, Thiyagarajan

2014-07-01

We have optimized a protocol for the preparation of bisdemethoxy curcumin analog nanoparticles (BDMCA-NP) by the solvent assisted process. The structural similarities between bulk and nano BDMCA were determined by Co-TLC, NMR and F-TIR. This shows that our synthesis protocol enhanced the dispersibility and reduce the size of BDMCA without altering the integrity of functional moieties and structure, which is crucial for anticancer and antioxidant activities. The morphology and size of BDMCA-NP as determined by SEM, HRTEM and DLS was found to be around 80 nm. BDMCA-NP treated breast cancer cell lines (MCF 7) showed cell death as characterized by MTT assay. Flow cytometric analysis of BDMCA-NP treated MCF 7 cell lines showed an increase of cell count in G2/M phase indicates the cell cycle arrest. Western blot analysis revealed the presence of caspase 3, caspase 9, cleaved fragments of PARP and Bax proteins in the BDMCA-NP treated MCF 7 cell lines, but not in untreated cell lines. To recap, we have prepared BDMCA-NP by solvent assisted process, which exerted anticancer activity against breast cancer cells, which may be due to (i) enhanced dispersibility and surface: volume ratio, (ii) apoptosis (iii) mitochondrial pathway induced cell death, (iv) G2/M phase cell cycle arrest and (v) disassembly of mitotic spindle of the cancer cells. Thus, nano BDMCA can be used as a potent anticancer agent.

Inositol Pyrophosphate Profiling of Two HCT116 Cell Lines Uncovers Variation in InsP8 Levels

PubMed Central

Gu, Chunfang; Wilson, Miranda S. C.; Jessen, Henning J.; Saiardi, Adolfo; Shears, Stephen B.

2016-01-01

The HCT116 cell line, which has a pseudo-diploid karotype, is a popular model in the fields of cancer cell biology, intestinal immunity, and inflammation. In the current study, we describe two batches of diverged HCT116 cells, which we designate as HCT116NIH and HCT116UCL. Using both gel electrophoresis and HPLC, we show that HCT116UCL cells contain 6-fold higher levels of InsP8 than HCT116NIH cells. This observation is significant because InsP8 is one of a group of molecules collectively known as ‘inositol pyrophosphates’ (PP-InsPs)—highly ‘energetic’ and conserved regulators of cellular and organismal metabolism. Variability in the cellular levels of InsP8 within divergent HCT116 cell lines could have impacted the phenotypic data obtained in previous studies. This difference in InsP8 levels is more remarkable for being specific; levels of other inositol phosphates, and notably InsP6 and 5-InsP7, are very similar in both HCT116NIH and HCT116UCL lines. We also developed a new HPLC procedure to record 1-InsP7 levels directly (for the first time in any mammalian cell line); 1-InsP7 comprised <2% of total InsP7 in HCT116NIH and HCT116UCL lines. The elevated levels of InsP8 in the HCT116UCL lines were not due to an increase in expression of the PP-InsP kinases (IP6Ks and PPIP5Ks), nor to a decrease in the capacity to dephosphorylate InsP8. We discuss how the divergent PP-InsP profiles of the newly-designated HCT116NIH and HCT116UCL lines should be considered an important research opportunity: future studies using these two lines may uncover new features that regulate InsP8 turnover, and may also yield new directions for studying InsP8 function. PMID:27788189

Expression of cholecystokinin2-receptor in rat and human L cells and the stimulation of glucagon-like peptide-1 secretion by gastrin treatment.

PubMed

Cao, Yang; Cao, Xun; Liu, Xiao-Min

2015-03-01

Gastrin is a gastrointestinal hormone secreted by G cells. Hypergastrinemia can improve blood glucose and glycosylated hemoglobin levels. These positive effects are primarily due to the trophic effects of gastrin on β-cells. In recent years, many receptors that regulate secretion of glucagon-like peptide 1 (GLP-1) have been identified in enteroendocrine L cell lines. This led us to hypothesize that, in addition to the trophic effects of gastrin on β-cells, L cells also express cholecystokinin2-receptor (CCK2R), which may regulate GLP-1 secretion and have synergistic effects on glucose homeostasis. Our research provides a preliminary analysis of CCK2R expression and the stimulating effect of gastrin treatment on GLP-1 secretion in a human endocrine L cell line, using RT-PCR, Western blot, immunocytochemistry, and ELISA analyses. The expression of proglucagon and prohormone convertase 3, which regulate GLP-1 biosynthesis, were also analyzed by real-time PCR. Double immunofluorescence labeling was utilized to assess the intracellular localization of CCK2R and GLP-1 in L cells harvested from rat colon tissue. Our results showed that CCK2R was expressed in both the human L cell line and the rat L cells. We also showed that treatment with gastrin, a CCK2R agonist, stimulated the secretion of GLP-1, and that this effect was likely due to increased expression of proglucagon and PCSK1 (also known as prohormone convertase 3 (PC3 gene)). These results not only provide a basis for the role gastrin may play in intestinal L cells, and may also provide the basis for the development of a method of gastrin-mediated glycemic regulation. Copyright © 2014 Elsevier GmbH. All rights reserved.

Comparing Apoptosis and Necrosis Effects of Arctium Lappa Root Extract and Doxorubicin on MCF7 and MDA-MB-231 Cell Lines

PubMed Central

Ghafari, Fereshteh; Rajabi, Mohammad Reza; Mazoochi, Tahereh; Taghizadeh, Mohsen; Nikzad, Hossein; Atlasi, Mohammad Ali; Taherian, Aliakbar

2017-01-01

Objective: Breast cancer is a heterogeneous disease and very common malignancy in women worldwide. The efficacy of chemotherapy as an important part of breast cancer treatment is limited due to its side effects. While pharmaceutical companies are looking for better chemicals, research on traditional medicines that generally have fewer side effects is quite interesting. In this study, apoptosis and necrosis effect of Arctium lappa and doxorubicin was compared in MCF7, and MDA-MB-231 cell lines. Materials and Methods: MCF7 and MDA-MB-231 cells were cultured in RPMI 1640 containing 10% FBS and 100 U/ml penicillin/streptomycin. MTT assay and an annexin V/propidium iodide (AV/PI) kit were used respectively to compare the survival rate and apoptotic effects of different concentrations of doxorubicin and Arctium lappa root extract on MDA-MB-231 and MCF7 cells. Results: Arctium lappa root extract was able to reduce cell viability of the two cell lines in a dose and time dependent manner similar to doxorubicin. Flow cytometry results showed that similar to doxorubicin, Arctium Lappa root extract had a dose and time dependent apoptosis effect on both cell lines. 10µg/mL of Arctium lappa root extract and 5 µM of doxorubicin showed the highest anti-proliferative and apoptosis effect in MCF7 and MDA231 cells. Conclusion: The MCF7 (ER/PR-) and MDA-MB-231 (ER/PR+) cell lines represent two major breast cancer subtypes. The similar anti-proliferative and apoptotic effects of Arctium lappa root extract and doxorubicin (which is a conventional chemotherapy drug) on two different breast cancer cell lines strongly suggests its anticancer effects and further studies. PMID:28441789

Comparing Apoptosis and Necrosis Effects of Arctium Lappa Root Extract and Doxorubicin on MCF7 and MDA-MB-231 Cell Lines

PubMed

Ghafari, Fereshteh; Rajabi, Mohammad Reza; Mazoochi, Tahereh; Taghizadeh, Mohsen; Nikzad, Hossein; Atlasi, Mohammad Ali; Taherian, Aliakbar

2017-03-01

Objective: Breast cancer is a heterogeneous disease and very common malignancy in women worldwide. The efficacy of chemotherapy as an important part of breast cancer treatment is limited due to its side effects. While pharmaceutical companies are looking for better chemicals, research on traditional medicines that generally have fewer side effects is quite interesting. In this study, apoptosis and necrosis effect of Arctium lappa and doxorubicin was compared in MCF7, and MDA-MB-231 cell lines. Materials and Methods: MCF7 and MDA-MB-231 cells were cultured in RPMI 1640 containing 10% FBS and 100 U/ml penicillin/streptomycin. MTT assay and an annexin V/propidium iodide (AV/PI) kit were used respectively to compare the survival rate and apoptotic effects of different concentrations of doxorubicin and Arctium lappa root extract on MDA-MB-231 and MCF7 cells. Results: Arctium lappa root extract was able to reduce cell viability of the two cell lines in a dose and time dependent manner similar to doxorubicin. Flow cytometry results showed that similar to doxorubicin, Arctium Lappa root extract had a dose and time dependent apoptosis effect on both cell lines. 10μg/mL of Arctium lappa root extract and 5 μM of doxorubicin showed the highest anti-proliferative and apoptosis effect in MCF7 and MDA231 cells. Conclusion: The MCF7 (ER/PR-) and MDA-MB-231 (ER/PR+) cell lines represent two major breast cancer subtypes. The similar anti-proliferative and apoptotic effects of Arctium lappa root extract and doxorubicin (which is a conventional chemotherapy drug) on two different breast cancer cell lines strongly suggests its anticancer effects and further studies. Creative Commons Attribution License

Epigenetic Alterations Associated with CCCTC-Binding Factor Deregulation in Prostate Cancer

DTIC Science & Technology

2012-07-01

HPV16  E6  and/or  E7  prostate cell lines.  We have had to reestablish stable cell lines containing inducible multiple CTCF  shRNA in pTRIPZ vector in PPC...1, LNCaPs, 293T and non‐tumorigenic HPV16  E6   5 and/or  E7  prostate cell lines. We have had to rederive these due to leakage from the  promoter...empty pTRIPZ vector and control scrambled shRNA.  f. To test the tumorigenic ability of CTCF shRNA infected non‐tumorigenic  E6 / E7  cells using colony

Evaluation of in vitro anti-inflammatory effects of crude ginger and rosemary extracts obtained through supercritical CO2 extraction on macrophage and tumor cell line: the influence of vehicle type.

PubMed

Justo, Oselys Rodriguez; Simioni, Patricia Ucelli; Gabriel, Dirce Lima; Tamashiro, Wirla Maria da Silva Cunha; Rosa, Paulo de Tarso Vieira; Moraes, Ângela Maria

2015-10-29

Numerous plants from have been investigated due to their anti-inflammatory activity and, among then, extracts or components of ginger (Zingiber officinale Roscoe) and rosemary (Rosmarinus officinalis L.), sources of polyphenolic compounds. 6-gingerol from ginger rhizome and carnosic acid and carnosol from rosemary leaves present anti-tumor, anti-inflammatory and antioxidant activities. However, the evaluation of the mechanisms of action of these and other plant extracts is limited due to their high hydrophobicity. Dimethylsulfoxide (DMSO) is commonly used as a vehicle of liposoluble materials to mammalian cells in vitro, presenting enhanced cell penetration. Liposomes are also able to efficiently deliver agents to mammalian cells, being capable to incorporate in their structure not only hydrophobic molecules, but also hydrophilic and amphiphilic compounds. Another strategy is based on the use of Pluronic F-68, a biocompatible low-foaming, non-ionic surfactant, to disperse hydrophobic components. Here, these three delivery approaches were compared to analyze their influence on the in vitro anti-inflammatory effects of ginger and rosemary extracts, at different concentrations, on primary mammalian cells and on a tumor cell line. Ginger and rosemary extracts free of organic solvents were obtained by supercritical fluid extraction and dispersed in DMSO, Pluronic F-68 or liposomes, in variable concentrations. Cell viability, production of inflammatory mediators and nitric oxide (NO) release were measured in vitro on J774 cell line and murine macrophages primary culture stimulated with bacterial lipopolysaccharide and interferon-γ after being exposed or not to these extracts. Ginger and rosemary extracts obtained by supercritical CO2 extraction inhibited the production of pro-inflammatory cytokines and the release of NO by peritoneal macrophages and J774 cells. The delivery vehicles influenced the anti-inflammatory effects. Comparatively, the ginger extract showed the highest anti-inflammatory activity on the tumor cell line. Controversially, rosemary extract dispersed on DMSO induced a more significant IL-1 and TNF-α reduction than ginger extract in primary macrophages. Amongst the tested delivery vehicles, DMSO was the most suitable, presenting reduced cytotoxicity, followed by Pluronic F-68 and liposomes, provably due to differences in their form of absorption, distribution and cellular metabolism. Co-administration of liposomes and plant extracts may cause death of macrophages cells and induction of NO production. It can be concluded that some of the beneficial effects attributed to extracts of ginger and rosemary may be associated with the inhibition of inflammatory mediators due to their high antioxidant activity. However, these effects were influenced by the type of delivery vehicle.

Calcium influences sensitivity to growth inhibition induced by a cell surface sialoglycopeptide

NASA Technical Reports Server (NTRS)

Betz, N. A.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

1994-01-01

While studies concerning mitogenic factors have been an important area of research for many years, much less is understood about the mechanisms of action of cell surface growth inhibitors. We have purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) which can reversibly inhibit the proliferation of diverse cell types. The studies discussed in this article show that three mouse keratinocyte cell lines exhibit sixty-fold greater sensitivity than other fibroblasts and epithelial-like cells to CeReS-18-induced growth inhibition. Growth inhibition induced by CeReS-18 treatment is a reversible process, and the three mouse keratinocyte cell lines exhibited either single or multiple cell cycle arrest points, although a predominantly G0/G1 cell cycle arrest point was exhibited in Swiss 3T3 fibroblasts. The sensitivity of the mouse keratinocyte cell lines to CeReS-18-induced growth inhibition was not affected by the degree of tumorigenic progression in the cell lines and was not due to differences in CeReS-18 binding affinity or number of cell surface receptors per cell. However, the sensitivity of both murine fibroblasts and keratinocytes could be altered by changing the extracellular calcium concentration, such that increased extracellular calcium concentrations resulted in decreased sensitivity to CeReS-18-induced proliferation inhibition. Thus the increased sensitivity of the murine keratinocyte cell lines to CeReS-18 could be ascribed to the low calcium concentration used in their propagation. Studies are currently under way investigating the role of calcium in CeReS-18-induced growth arrest. The CeReS-18 may serve as a very useful tool to study negative growth control and the signal transduction events associated with cell cycling.

Macrolide Antibiotics Exhibit Cytotoxic Effect under Amino Acid-Depleted Culture Condition by Blocking Autophagy Flux in Head and Neck Squamous Cell Carcinoma Cell Lines

PubMed Central

Hirasawa, Kazuhiro; Moriya, Shota; Miyahara, Kana; Kazama, Hiromi; Hirota, Ayako; Takemura, Jun; Abe, Akihisa; Inazu, Masato; Hiramoto, Masaki; Tsukahara, Kiyoaki

2016-01-01

Autophagy, a self-digestive system for cytoplasmic components, is required to maintain the amino acid pool for cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy flux, and they potently enhance the cytocidal effect of various anticancer reagents in vitro. This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer cells require a larger metabolic demand than normal cells because of their exuberant growth, upregulated autophagy in tumor cells has now become the target for cancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic effect under an amino acid-starving condition in head and neck squamous cancer cell lines such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy in the central region owing to hypovascularity. AZM and CAM induced cell death under the amino acid-depleted (AAD) culture condition in these cell lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP knockdown by siRNA in the CAL 27 cells significantly suppressed macrolide-induced cell death under the AAD culture condition. CHOP-/- murine embryonic fibroblast (MEF) cell lines also attenuated AZM-induced cell death compared with CHOP+/+ MEF cell lines. Using a tet-off atg5 MEF cell line, knockout of atg5, an essential gene for autophagy, also induced cell death and CHOP in the AAD culture medium but not in the complete culture medium. This suggest that macrolide-induced cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely cancelled by the addition of amino acids in the culture medium, indicating that the cytotoxicity is due to the insufficient amino acid pool. These data suggest the possibility of using macrolides for “tumor-starving therapy”. PMID:27977675

Cancer-specific SNPs originate from low-level heteroplasmic variants in human mitochondrial genomes of a matched cell line pair.

PubMed

Hedberg, Annica; Knutsen, Erik; Løvhaugen, Anne Silje; Jørgensen, Tor Erik; Perander, Maria; Johansen, Steinar D

2018-04-19

Low-level mitochondrial heteroplasmy is a common phenomenon in both normal and cancer cells. Here, we investigate the link between low-level heteroplasmy and mitogenome mutations in a human breast cancer matched cell line by high-throughput sequencing. We identified 23 heteroplasmic sites, of which 15 were common between normal cells (Hs578Bst) and cancer cells (Hs578T). Most sites were clustered within the highly conserved Complex IV and ribosomal RNA genes. Two heteroplasmic variants in normal cells were found as fixed mutations in cancer cells. This indicates a positive selection of these variants in cancer cells. RNA-Seq analysis identified upregulated L-strand specific transcripts in cancer cells, which include three mitochondrial long non-coding RNA molecules. We hypothesize that this is due to two cancer cell-specific mutations in the control region.

Mir-30d suppresses cell proliferation of colon cancer cells by inhibiting cell autophagy and promoting cell apoptosis.

PubMed

Zhang, Rui; Xu, Jian; Zhao, Jian; Bai, Jinghui

2017-06-01

MiR-30 family plays an important role in the tumorigenesis of human cancers. The aim of the study is to investigate the role of miR-30d in human colon cancer cell lines and explore the molecular mechanism in the proliferation of colon cancer cells. The expression of miR-30d was determined by real-time polymerase chain reaction assay in colon cancer cell lines (HCT15, HCT116, HT-29, DLD-1, and SW480) and the results demonstrated that miR-30d level was significantly decreased in human colon cancer cell lines, compared with normal colon epithelial cell line. Transfection with miR-30d mimics inhibited cell proliferation, and transfection with miR-30d inhibitors significantly promoted cell viability of colon cancer cells. Furthermore, TargetScan analysis predicted that miR-30d interacted with messenger RNA on its 3' untranslated region of ATG5, phosphoinositide 3-kinase, and Beclin1 to negatively regulate cell autophagy in colon cancer cells. Moreover, transfection with miR-30d induced cell arrest at G2/M phase of HT-29 cells. Overexpression of miR-30d mimics inhibited cell viability probably due to the inhibition of cell autophagy and promotion of cell apoptosis. Thus, MiR-30d inhibited cell autophagy by directly targeting messenger RNA of ATG5, phosphoinositide 3-kinase, and Beclin1 and promoted cell apoptosis of human colon cancer cells. It is helpful to clarify the function of miR-30d in tumorigenesis of human cancers.

Effect of Physical Forces on the Metastatic Bone Microenvironment

DTIC Science & Technology

2013-10-01

G.R., et al., Cell proliferation of cultured human cancer cells are affected by the elevated tumor pressures that exist in vivo. Ann Biomed Eng, 2005... cell lines. In vitro experiments have shown that increased pressure leads to decreased PCa proliferation. Osteoblasts also have inhibited...applied to tumor cells . Novel candidate genes with altered expression due to pressure have been identified and are currently undergoing further

Use of deferasirox, an iron chelator, to overcome imatinib resistance of chronic myeloid leukemia cells

PubMed Central

Kim, Dae Sik; Na, Yoo Jin; Kang, Myoung Hee; Yoon, Soo-Young; Choi, Chul Won

2016-01-01

Background/Aims: The treatment of chronic myeloid leukemia (CML) has achieved impressive success since the development of the Bcr-Abl tyrosine kinase inhibitor, imatinib mesylate. Nevertheless, resistance to imatinib has been observed, and a substantial number of patients need alternative treatment strategies. Methods: We have evaluated the effects of deferasirox, an orally active iron chelator, and imatinib on K562 and KU812 human CML cell lines. Imatinib-resistant CML cell lines were created by exposing cells to gradually increasing concentrations of imatinib. Results: Co-treatment of cells with deferasirox and imatinib induced a synergistic dose-dependent inhibition of proliferation of both CML cell lines. Cell cycle analysis showed an accumulation of cells in the subG1 phase. Western blot analysis of apoptotic proteins showed that co-treatment with deferasirox and imatinib induced an increased expression of apoptotic proteins. These tendencies were clearly identified in imatinib-resistant CML cell lines. The results also showed that co-treatment with deferasirox and imatinib reduced the expression of BcrAbl, phosphorylated Bcr-Abl, nuclear factor-κB (NF-κB) and β-catenin. Conclusions: We observed synergistic effects of deferasirox and imatinib on both imatinib-resistant and imatinib-sensitive cell lines. These effects were due to induction of apoptosis and cell cycle arrest by down-regulated expression of NF-κB and β-catenin levels. Based on these results, we suggest that a combination treatment of deferasirox and imatinib could be considered as an alternative treatment option for imatinib-resistant CML. PMID:26874514

Prospects for the use of plant cell cultures in food biotechnology.

PubMed

Davies, Kevin M; Deroles, Simon C

2014-04-01

Plant cell cultures can offer continuous production systems for high-value food and health ingredients, independent of geographical or environmental variations and constraints. Yet despite many improvements in culture technologies, cell line selection, and bioreactor design, there are few commercial successes. This is principally due to the culture yield and market price of food products not being sufficient to cover the plant cell culture production costs. A better understanding of the underpinning biological mechanisms that control the target metabolite biosynthetic pathways may allow the metabolic engineering of cell lines to provide for economically competitive product yields. However, uncertainty around the regulatory and public acceptance of products derived from engineered cell cultures presents a barrier to the uptake of the technology by food product companies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cisplatin resistance induced in germ cell tumour cells is due to reduced susceptibility towards cell death but not to altered DNA damage induction or repair.

PubMed

Fenske, Annabelle E; Glaesener, Stephanie; Bokemeyer, Carsten; Thomale, Juergen; Dahm-Daphi, Jochen; Honecker, Friedemann; Dartsch, Dorothee C

2012-11-28

To identify factors involved in cisplatin (CDDP) resistance of germ cell tumours (GCTs), we exposed NTERA-2 cells, and the platinum-adapted subline NTERA-2R to CDDP and compared their response. While both cell lines showed comparable proliferation, NTERA-2R cells were clearly more resistant to the drug than the parental NTERA-2 cell line. Interestingly, the two lines showed identical extent of DNA adduct formation and elimination, indicating that neither changes in CDDP uptake, nor altered drug efflux, DNA binding, or repair caused the difference in resistance. Similarly, no difference occurred in the time-course of γH2AX formation, which was not linked to 53BP1 accumulation. In contrast, NTERA-2R cells showed a more pronounced dose-dependent S phase delay, a transient G(2)/M-block, and subsequent release into immediate cell death. We thus conclude that the enhanced resistance against CDDP is linked to reduced susceptibility to cell death rather than to an altered DNA adduct formation or adduct removal. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Effects of Cabazitaxel in Renal Cell Carcinoma Cell Lines.

PubMed

Mizutani, Kosuke; Tomoda, Masashi; Ohno, Yuta; Hayashi, Hideki; Fujita, Yasunori; Kawakami, Kyojiro; Kameyama, Koji; Kato, Taku; Sugiyama, Tadashi; Itoh, Yoshinori; Ito, Masafumi; Deguchi, Takashi

2015-12-01

Advanced renal cell carcinoma is treated with mammalian target of rapamycin (mTOR) inhibitors or tyrosine kinase inhibitors (TKIs). The effects of these drugs are, however, limited and novel treatment strategies are required. Clear-cell type renal cell carcinoma (ccRCC) is chemo-resistant, in part, due to expression of multidrug resistance proteins such as p-glycoprotein. Cabazitaxel, a tubulin-binding taxane drug used for castration-resistant prostate cancer, has less affinity for p-glycoprotein compared to docetaxel. In the current study, the effects of docetaxel and cabazitaxel on ccRCC cells were investigated. The expression of p-glycoprotein was evaluated in the ccRCC cell lines, Caki-1, KMRC-1 and OS-RC-2 by western blotting. Cells were treated with cabazitaxel or docetaxel, and growth kinetics and tubulin polymerization were determined by the WST-1 assay and cell-based tubulin polymerization assay, respectively. Intracellular drug concentrations were measured by chromatography. AKT activation after treatment was examined by western blotting. All ccRCC cell lines expressed p-glycoprotein. Cabazitaxel inhibited cell growth and induced tubulin polymerization more potently than docetaxel. The intracellular concentration of cabazitaxel was much higher than docetaxel in all cell lines. Both docetaxel and cabazitaxel inhibit AKT phosphorylation at 5 min among three cells. Cabazitaxel inhibits growth of ccRCC cells expressing p-glycoprotein and could thus be possibly used for advanced ccRCC patients in combination with targeted-therapy enhancing their effects. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Efficient PRNP deletion in bovine genome using gene-editing technologies in bovine cells

PubMed Central

Choi, WooJae; Kim, Eunji; Yum, Soo-Young; Lee, ChoongIl; Lee, JiHyun; Moon, JoonHo; Ramachandra, Sisitha; Malaweera, Buddika Oshadi; Cho, JongKi; Kim, Jin-Soo; Kim, SeokJoong; Jang, Goo

2015-01-01

abstract Even though prion (encoded by the PRNP gene) diseases like bovine spongiform encephalopathy (BSE) are fatal neurodegenerative diseases in cattle, their study via gene deletion has been limited due to the absence of cell lines or mutant models. In this study, we aim to develop an immortalized fibroblast cell line in which genome-engineering technology can be readily applied to create gene-modified clones for studies. To this end, this study is designed to 1) investigate the induction of primary fibroblasts to immortalization by introducing Bmi-1 and hTert genes; 2) investigate the disruption of the PRNP in those cells; and 3) evaluate the gene expression and embryonic development using knockout (KO) cell lines. Primary cells from a male neonate were immortalized with Bmi-1and hTert. Immortalized cells were cultured for more than 180 days without any changes in their doubling time and morphology. Furthermore, to knockout the PRNP gene, plasmids that encode transcription activator-like effector nuclease (TALEN) pairs were transfected into the cells, and transfected single cells were propagated. Mutated clonal cell lines were confirmed by T7 endonuclease I assay and sequencing. Four knockout cell lines were used for somatic cell nuclear transfer (SCNT), and the resulting embryos were developed to the blastocyst stage. The genes (CSNK2A1, FAM64A, MPG and PRND) were affected after PRNP disruption in immortalized cells. In conclusion, we established immortalized cattle fibroblasts using Bmi-1 and hTert genes, and used TALENs to knockout the PRNP gene in these immortalized cells. The efficient PRNP KO is expected to be a useful technology to develop our understanding of in vitro prion protein functions in cattle. PMID:26217959

Antigen-inducing ability of herpesvirus papio in human and baboon lymphoma lines, compared to Epstein-Barr virus.

PubMed

Klein, G; Falk, L; Falk, K

1978-01-01

Herpesvirus papio(HVP)-carrying baboon lymphoblastoid lines do not express a nuclear antigen like the Epstein-Barr virus(EBV)-determined nuclear antigen (EBNA), as judged by in situ anticomplement fluorescence staining, although the carry multiple viral genomes and, in the case of producerlines, early antigen (EA) and viral capsid antigen (VCA) that cross-react with the corresponding human EBV-determined antigens. To test whether the lack of in situ nuclear antigen expression is a property innate to the baboon virus or the baboon cell, nonproducer HVP-carrying baboon lymphoid cells of the 26 CB-1 line were superinfected with two human EBV strains. B95-8-derived EBV induced brilliant EBNA staining, proving that the baboon lymphoid cell was competent to synthesize EBNA. In the mirror experiment, HVP derived from the 9B or the 18C baboon line was added to the EBV-carrying Raji line, the EBV-negative Ramos and BJAB lines and the HVP-carrying nonproducer 26 CB-1 line, respectively. HVP induced EA and VCA in Raji, and EA in BJAB and 26 CB-1. EBNA was not induced in any of the three EBNA-negative lines, BJAB, Ramos and 26 CB-1. It is concluded that the lack of in situ nuclear staining in HVP-carrying baboon lines is a HVP-associated property and is not due to any innate inability of the baboon lymphoid cell to synthesize an antigen of the EBNA type.

Complexing Methylene Blue with Phosphorus Dendrimers to Increase Photodynamic Activity.

PubMed

Dabrzalska, Monika; Janaszewska, Anna; Zablocka, Maria; Mignani, Serge; Majoral, Jean Pierre; Klajnert-Maculewicz, Barbara

2017-02-23

The efficiency of photodynamic therapy is limited mainly due to low selectivity, unfavorable biodistribution of photosensitizers, and long-lasting skin sensitivity to light. However, drug delivery systems based on nanoparticles may overcome the limitations mentioned above. Among others, dendrimers are particularly attractive as carriers, because of their globular architecture and high loading capacity. The goal of the study was to check whether an anionic phosphorus dendrimer is suitable as a carrier of a photosensitizer-methylene blue (MB). As a biological model, basal cell carcinoma cell lines were used. We checked the influence of the MB complexation on its singlet oxygen production ability using a commercial fluorescence probe. Next, cellular uptake, phototoxicity, reactive oxygen species (ROS) generation, and cell death were investigated. The MB-anionic dendrimer complex (MB-1an) was found to generate less singlet oxygen; however, the complex showed higher cellular uptake and phototoxicity against basal cell carcinoma cell lines, which was accompanied with enhanced ROS production. Owing to the obtained results, we conclude that the photodynamic activity of MB complexed with an anionic dendrimer is higher than free MB against basal cell carcinoma cell lines.

Combined M-FISH and CGH analysis allows comprehensive description of genetic alterations in neuroblastoma cell lines.

PubMed

Van Roy, N; Van Limbergen, H; Vandesompele, J; Van Gele, M; Poppe, B; Salwen, H; Laureys, G; Manoel, N; De Paepe, A; Speleman, F

2001-10-01

Cancer cell lines are essential gene discovery tools and have often served as models in genetic and functional studies of particular tumor types. One of the future challenges is comparison and interpretation of gene expression data with the available knowledge on the genomic abnormalities in these cell lines. In this context, accurate description of these genomic abnormalities is required. Here, we show that a combination of M-FISH with banding analysis, standard FISH, and CGH allowed a detailed description of the genetic alterations in 16 neuroblastoma cell lines. In total, 14 cryptic chromosome rearrangements were detected, including a balanced t(2;4)(p24.3;q34.3) translocation in cell line NBL-S, with the 2p24 breakpoint located at about 40 kb from MYCN. The chromosomal origin of 22 marker chromosomes and 41 cytogenetically undefined translocated segments was determined. Chromosome arm 2 short arm translocations were observed in six cell lines (38%) with and five (31%) without MYCN amplification, leading to partial chromosome arm 2p gain in all but one cell line and loss of material in the various partner chromosomes, including 1p and 11q. These 2p gains were often masked in the GGH profiles due to MYCN amplification. The commonly overrepresented region was chromosome segment 2pter-2p22, which contains the MYCN gene, and five out of eleven 2p breakpoints clustered to the interface of chromosome bands 2p16 and 2p21. In neuroblastoma cell line SJNB-12, with double minutes (dmins) but no MYCN amplification, the dmins were shown to be derived from 16q22-q23 sequences. The ATBF1 gene, an AT-binding transcription factor involved in normal neurogenesis and located at 16q22.2, was shown to be present in the amplicon. This is the first report describing the possible implication of ATBF1 in neuroblastoma cells. We conclude that a combined approach of M-FISH, cytogenetics, and CGH allowed a more complete and accurate description of the genetic alterations occurring in the investigated cell lines. Copyright 2001 Wiley-Liss, Inc.

Researching glutamate – induced cytotoxicity in different cell lines: a comparative/collective analysis/study

PubMed Central

Kritis, Aristeidis A.; Stamoula, Eleni G.; Paniskaki, Krystallenia A.; Vavilis, Theofanis D.

2015-01-01

Although glutamate is one of the most important excitatory neurotransmitters of the central nervous system, its excessive extracellular concentration leads to uncontrolled continuous depolarization of neurons, a toxic process called, excitotoxicity. In excitotoxicity glutamate triggers the rise of intracellular Ca2+ levels, followed by up regulation of nNOS, dysfunction of mitochondria, ROS production, ER stress, and release of lysosomal enzymes. Excessive calcium concentration is the key mediator of glutamate toxicity through over activation of ionotropic and metabotropic receptors. In addition, glutamate accumulation can also inhibit cystine (CySS) uptake by reversing the action of the CySS/glutamate antiporter. Reversal of the antiporter action reinforces the aforementioned events by depleting neurons of cysteine and eventually glutathione’s reducing potential. Various cell lines have been employed in the pursuit to understand the mechanism(s) by which excitotoxicity affects the cells leading them ultimately to their demise. In some cell lines glutamate toxicity is exerted mainly through over activation of NMDA, AMPA, or kainate receptors whereas in other cell lines lacking such receptors, the toxicity is due to glutamate induced oxidative stress. However, in the greatest majority of the cell lines ionotropic glutamate receptors are present, co-existing to CySS/glutamate antiporters and metabotropic glutamate receptors, supporting the assumption that excitotoxicity effect in these cells is accumulative. Different cell lines differ in their responses when exposed to glutamate. In this review article the responses of PC12, SH-SY5Y, HT-22, NT-2, OLCs, C6, primary rat cortical neurons, RGC-5, and SCN2.2 cell systems are systematically collected and analyzed. PMID:25852482

DKK1 and Kremen Expression Predicts the Osteoblastic Response to Bone Metastasis.

PubMed

Clines, Katrina L; Clines, Gregory A

2018-05-14

Bone metastasis is a complication of advanced breast and prostate cancer. Tumor-secreted Dickkopf homolog 1 (DKK1), an inhibitor of canonical Wnt signaling and osteoblast differentiation, was proposed to regulate the osteoblastic response to metastatic cancer in bone. The objectives of this study were to compare DKK1 expression with the in vivo osteoblastic response in a panel of breast and prostate cancer cell lines, and to discover mechanisms that regulate cancer DKK1 expression. DKK1 expression was highest in MDA-MB-231 and PC3 cells that produce osteolytic lesions, and hence a suppressed osteoblastic response, in animal models of bone metastasis. LnCaP, C4-2B, LuCaP23.1, T47D, ZR-75-1, MCF-7, ARCaP and ARCaP M cancer cells that generate osteoblastic, mixed or no bone lesions had the lowest DKK1 expression. The cell lines with negligible expression, LnCaP, C4-2B and T47D, exhibited methylation of the DKK1 promoter. Canonical Wnt signaling activity was then determined and found in all cell lines tested, even in the MDA-MB-231 and PC3 cell lines despite sizeable amounts of DKK1 protein expression expected to block canonical Wnt signaling. A mechanism of DKK1 resistance in the osteolytic cell lines was investigated and determined to be at least partially due to down-regulation of the DKK1 receptors Kremen1 and Kremen2 in the MDA-MB-231 and PC3 cell lines. Combined DKK1 and Kremen expression in cancer cells may serve as predictive markers of the osteoblastic response of breast and prostate cancer bone metastasis. Published by Elsevier Inc.

Cytotoxic Activities of Physalis minima L. Chloroform Extract on Human Lung Adenocarcinoma NCI-H23 Cell Lines by Induction of Apoptosis

PubMed Central

Leong, Ooi Kheng; Muhammad, Tengku Sifzizul Tengku; Sulaiman, Shaida Fariza

2011-01-01

Physalis minima L. is reputed for having anticancer property. In this study, the chloroform extract of this plant exhibited remarkable cytotoxic activities on NCI-H23 (human lung adenocarcinoma) cell line at dose- and time-dependent manners (after 24, 48 and 72 h of incubation). Analysis of cell-death mechanism demonstrated that the extract exerted apoptotic programed cell death in NCI-H23 cells with typical DNA fragmentation, which is a biochemical hallmark of apoptosis. Morphological observation using transmission electron microscope (TEM) also displayed apoptotic characteristics in the treated cells, including clumping and margination of chromatins, followed by convolution of the nuclear and budding of the cells to produce membrane-bound apoptotic bodies. Different stages of apoptotic programed cell death as well as phosphatidylserine externalization were confirmed using annexin V and propidium iodide staining. Furthermore, acute exposure to the extract produced a significant regulation of c-myc, caspase-3 and p53 mRNA expression in this cell line. Due to its apoptotic effect on NCI-H23 cells, it is strongly suggested that the extract could be further developed as an anticancer drug. PMID:19541726

Manipulation of novel nano-prodrug composed of organic pigment-based hybrid network and its optical uses.

PubMed

Zhou, Zhan; Zheng, Yuhui; Cheng, Cheng Zhang; Wen, Jiajia; Wang, Qianming

2017-01-01

Here we developed the first case of pyropheophorbide-a-loaded PEGylated-hybrid carbon nanohorns (CNH-Pyro) to study tumor targeting therapy. During incubation with living cells, CNH-Pyro exhibited very intense red emissions. The intracellular imaging results were carried out by flow cytometry based on four different kinds of cell lines (including three adherent cell lines and one suspension cell line). Compared with free pyropheophorbide-a, CNH-Pyro demonstrated enhanced photodynamic tumor ablation efficiency during in vitro experiments due to improved biocompatibility of the hybrid nanomaterial and the photothermal therapy effect derived from carbon-network structure. Trypan blue staining experiments supported that the cell fate was dependent on the synergistic effects of both CNH-Pyro and laser irradiations. These results indicated that the chlorin-entrapped carbon nanohorns could provide powerful delivery vehicles for increasing photodynamic efficacy and possess early identification of the disease. Copyright © 2016 Elsevier B.V. All rights reserved.

Platelets Inhibit Migration of Canine Osteosarcoma Cells.

PubMed

Bulla, S C; Badial, P R; Silva, R C; Lunsford, K; Bulla, C

2017-01-01

The interaction between platelets and tumour cells is important for tumour growth and metastasis. Thrombocytopenia or antiplatelet treatment negatively impact on cancer metastasis, demonstrating potentially important roles for platelets in tumour progression. To our knowledge, there is no information regarding the role of platelets in cancer progression in dogs. This study was designed to test whether canine platelets affected the migratory behaviour of three canine osteosarcoma cell lines and to give insights of molecular mechanisms. Intact platelets, platelet lysate and platelet releasate inhibited the migration of canine osteosarcoma cell lines. Addition of blood leucocytes to the platelet samples did not alter the inhibitory effect on migration. Platelet treatment also significantly downregulated the transcriptional levels of SNAI2 and TWIST1 genes. The interaction between canine platelets or molecules released during platelet activation and these tumour cell lines inhibits their migration, which suggests that canine platelets might antagonize metastasis of canine osteosarcoma. This effect is probably due to, at least in part, downregulation of genes related to epithelial-mesenchymal transition. Copyright © 2016. Published by Elsevier Ltd.

Identification of two immortalized cell lines, ECV304 and bEnd3, for in vitro permeability studies of blood-brain barrier

PubMed Central

Mei, Shenghui; Jin, Hong; Zhu, Bin; Tian, Yue; Huo, Jiping; Cui, Xu; Guo, Anchen; Zhao, Zhigang

2017-01-01

To identify suitable cell lines for a mimetic system of in vivo blood-brain barrier (BBB) for drug permeability assessment, we characterized two immortalized cell lines, ECV304 and bEnd3 in the respect of the tightness, tight junction proteins, P-glycoprotein (P-gp) function and discriminative brain penetration. The ECV304 monoculture achieved higher transendothelial electrical resistance (TEER) and lower permeability to Lucifer yellow than bEnd3. However, co-culture with rat glioma C6 cells impaired the integrity of ECV304 and bEnd3 cell layers perhaps due to the heterogeneity among C6 cells in inducing BBB characteristics. The immunostaining of ZO-1 delivered distinct bands along cell borders on both cell lines while those of occludin and claudin-5 were diffused and weak. P-gp functionality was only proved in bEnd3 by Rhodamine 123 (R123) uptake assay. A permeability test of reference compounds displayed a similar rank order (digoxin < R123 < quinidine, verapamil < propranolol) in ECV304 and bEnd3 cells. In comparison with bEnd3, ECV304 developed tighter barrier for the passage of reference compounds and higher discrimination between transcellular and paracellular transport. However, the monoculture models of ECV304 and bEnd3 fail to achieve the sufficient tightness of in vitro BBB permeability models with high TEER and evident immunostaining of tight junction proteins. Further strategies to enhance the paracellular tightness of both cell lines to mimic in vivo BBB tight barrier deserve to be conducted. PMID:29059256

Identification of two immortalized cell lines, ECV304 and bEnd3, for in vitro permeability studies of blood-brain barrier.

PubMed

Yang, Shu; Mei, Shenghui; Jin, Hong; Zhu, Bin; Tian, Yue; Huo, Jiping; Cui, Xu; Guo, Anchen; Zhao, Zhigang

2017-01-01

To identify suitable cell lines for a mimetic system of in vivo blood-brain barrier (BBB) for drug permeability assessment, we characterized two immortalized cell lines, ECV304 and bEnd3 in the respect of the tightness, tight junction proteins, P-glycoprotein (P-gp) function and discriminative brain penetration. The ECV304 monoculture achieved higher transendothelial electrical resistance (TEER) and lower permeability to Lucifer yellow than bEnd3. However, co-culture with rat glioma C6 cells impaired the integrity of ECV304 and bEnd3 cell layers perhaps due to the heterogeneity among C6 cells in inducing BBB characteristics. The immunostaining of ZO-1 delivered distinct bands along cell borders on both cell lines while those of occludin and claudin-5 were diffused and weak. P-gp functionality was only proved in bEnd3 by Rhodamine 123 (R123) uptake assay. A permeability test of reference compounds displayed a similar rank order (digoxin < R123 < quinidine, verapamil < propranolol) in ECV304 and bEnd3 cells. In comparison with bEnd3, ECV304 developed tighter barrier for the passage of reference compounds and higher discrimination between transcellular and paracellular transport. However, the monoculture models of ECV304 and bEnd3 fail to achieve the sufficient tightness of in vitro BBB permeability models with high TEER and evident immunostaining of tight junction proteins. Further strategies to enhance the paracellular tightness of both cell lines to mimic in vivo BBB tight barrier deserve to be conducted.

Comparative evaluation of PLGA nanoparticle delivery system for 5-fluorouracil and curcumin on squamous cell carcinoma.

PubMed

Masloub, Shaimaa M; Elmalahy, Mohamed H; Sabry, Dina; Mohamed, Wael S; Ahmed, Sahar H

2016-04-01

The purpose of this study is to assess the effect of 5-fluorouracil nanoparticles and curcumin naoparticles on cell proliferation and the expression of the apoptotic marker (caspase 3) in squamous cell carcinoma cell line. PLGA 5-fluorouracil nanopartciles and PLGA curcumin nanoparticles were prepared and applied for 24 and 48h on human laryngeal squamous carcinoma cell line (Hep-2) as regard IC 50 concentration. MTT assay was used for evaluation of cytotoxicity of prepared nanoparticles. Quantitaive reverse transcriptase polymerase chain reaction (QRT-PCR) was used for the assessment of caspase-3 expression in the treated cell line. The drug release rate profiles was dependent upon polymer to drug ratio, noting that the higher PLGA polymer ratio to 5-fluprouracil or curcumin drug showed faster release rates. On the other hand, the least PLGA polymer ratio to 5-fluprouracil or curcumin drug showed the slowest release rates. MTT assay revelaed that 5-fluorouracil nanoparticels or curcumin nanoparticels showed a clear cytotoxic effect on Hep-2 cell line compared to non treated cancer cells. The RT-PCR assessment of caspase-3 expression revealed that there was a significant increase in caspase-3 expression in Hep-2 cell line treated with 5-fluorouracil nanoparticles or curcumin compared to non treated cancer cells. Curcumin nanoparticles could be more active in inducing apoptosis in short term assays (24h) than long term assays (48h) due to differential cellular uptake. While 5-fluorouracil nanoparticles induced higher significant apoptosis in long term (48h) compared to curcumin group. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cells for bioartificial liver devices: the human hepatoma-derived cell line C3A produces urea but does not detoxify ammonia.

PubMed

Mavri-Damelin, Demetra; Damelin, Leonard H; Eaton, Simon; Rees, Myrddin; Selden, Clare; Hodgson, Humphrey J F

2008-02-15

Extrahepatic bioartificial liver devices should provide an intact urea cycle to detoxify ammonia. The C3A cell line, a subclone of the hepatoma-derived HepG2 cell line, is currently used in this context as it produces urea, and this has been assumed to be reflective of ammonia detoxification via a functional urea cycle. However, based on our previous findings of perturbed urea-cycle function in the non-urea producing HepG2 cell line, we hypothesized that the urea produced by C3A cells was via a urea cycle-independent mechanism, namely, due to arginase II activity, and therefore would not detoxify ammonia. Urea was quantified using (15)N-ammonium chloride metabolic labelling with gas chromatography-mass spectrometry. Gene expression was determined by real-time reverse transcriptase-PCR, protein expression by western blotting, and functional activities with radiolabelling enzyme assays. Arginase inhibition studies used N(omega)-hydroxy-nor-L-arginine. Urea was detected in C3A conditioned medium; however, (15)N-ammonium chloride-labelling indicated that (15)N-ammonia was not incorporated into (15)N-labelled urea. Further, gene expression of two urea cycle genes, ornithine transcarbamylase and arginase I, were completely absent. In contrast, arginase II mRNA and protein was expressed at high levels in C3A cells and was inhibited by N(omega)-hydroxy-nor-L-arginine, which prevented urea production, thereby indicating a urea cycle-independent pathway. The urea cycle is non-functional in C3A cells, and their urea production is solely due to the presence of arginase II, which therefore cannot provide ammonia detoxification in a bioartificial liver system. This emphasizes the continued requirement for developing a component capable of a full repertoire of liver function. (c) 2007 Wiley Periodicals, Inc.

Sustainable production of azadirachtin from differentiated in vitro cell lines of neem (Azadirachta indica)

PubMed Central

Singh, Mithilesh; Chaturvedi, Rakhi

2013-01-01

Azadirachtin has high industrial demand due to its immediate application as an ecofriendly, biodegradable biopesticide and also due to its various other significant bioactivities. To date, the only commercially feasible way to produce azadirachtin is extraction from seeds, but their availability is very limited as the tree flowers only once a year and only one-third of the fruits are collected due to operational problems. Further, due to the strict out-breeding nature of the plant, the seeds are highly heterozygous, resulting in inconsistent metabolite production. Therefore, in the present study, to achieve sustainable production of azadirachtin, dedifferentiated and redifferentiated calli derived from various explants of neem—zygotic embryo, leaf and ovary—were investigated for their potential to biosynthesize azadirachtin. High-performance liquid chromatography analysis of the in vitro cell lines showed the presence of azadirachtin in all the samples tested, the content of which in cultured cells varied with explant source and cell differentiation response. The presence of azadirachtin in samples was further confirmed by positive electrospray ionization mass spectroscopy. The zygotic embryo cultures of neem accumulated much higher amounts of azadirachtin than leaf and ovary cultures. Furthermore, organized in vitro callus cultures (redifferentiated) supported higher azadirachtin biosynthesis, while unorganized callus cultures (dedifferentiated) supported the least. The maximum azadirachtin content of 2.33 mg g−1 dry weight was obtained from redifferentiated immature zygotic embryo cultures.

Procyanidin-rich extract of natural cocoa powder causes ROS-mediated caspase-3 dependent apoptosis and reduction of pro-MMP-2 in epithelial ovarian carcinoma cell lines.

PubMed

Taparia, Shruti Sanjay; Khanna, Aparna

2016-10-01

Over the last four centuries, cocoa and chocolate have been described as having potential medicinal value. As of today, Theobroma cacao L. (Sterculiaceae) and its products are consumed worldwide. They are of great research interest because of the concentration dependent antioxidant as well as pro-oxidant properties of some of their polyphenolic constituents, specially procyanidins and flavan-3-ols such as catechin. This study was aimed at investigating the cellular and molecular changes associated with cytotoxicity, caused due pro-oxidant activity of cocoa catechins and procyanidins, in ovarian cancer cell lines. Extract of non-alkalized cocoa powder enriched with catechins and procyanidins was used to treat human epithelial ovarian cancer cell lines OAW42 and OVCAR3 at various concentrations ≤1000μg/mL. The effect of treatment on intracellular reactive oxygen species (ROS) levels was determined. Apoptotic cell death, post treatment, was evaluated microscopically and using flow cytometry by means of annexin-propidium iodide (PI) dual staining. Levels of active caspase-3 as a pro-apoptotic marker and matrix metalloproteinase 2 (MMP2) as an invasive potential marker were detected using Western blotting and gelatin zymography. Treatment with extract caused an increase in intracellular ROS levels in OAW42 and OVCAR3 cell lines. Bright field and fluorescence microscopy of treated cells revealed apoptotic morphology and DNA damage. Increase in annexin positive cell population and dose dependent upregulation of caspase-3 confirmed apoptotic cell death. pro-MMP2 was found to be downregulated in a dose dependent manner in cells treated with the extract. Treated cells also showed a reduction in MMP2 activity. Our data suggests that cocoa catechins and procyanidins are cytotoxic to epithelial ovarian cancer, inducing apoptotic morphological changes, DNA damage and caspase-3 mediated cell death. Downregulation of pro-MMP2 and reduction in active MMP2 levels imply a decrease in invasive potential of the cells. Apoptosis and MMP2 downregulation appear to be linked to the increase in intracellular ROS levels, caused due to the prooxidant effect of cocoa procyanidin extract. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

A p53-dependent response limits the viability of mammalian haploid cells

PubMed Central

Olbrich, Teresa; Mayor-Ruiz, Cristina; Vega-Sendino, Maria; Gomez, Carmen; Ortega, Sagrario; Ruiz, Sergio; Fernandez-Capetillo, Oscar

2017-01-01

The recent development of haploid cell lines has facilitated forward genetic screenings in mammalian cells. These lines include near-haploid human cell lines isolated from a patient with chronic myelogenous leukemia (KBM7 and HAP1), as well as haploid embryonic stem cells derived from several organisms. In all cases, haploidy was shown to be an unstable state, so that cultures of mammalian haploid cells rapidly become enriched in diploids. Here we show that the observed diploidization is due to a proliferative disadvantage of haploid cells compared with diploid cells. Accordingly, single-cell–sorted haploid mammalian cells maintain the haploid state for prolonged periods, owing to the absence of competing diploids. Although the duration of interphase is similar in haploid and diploid cells, haploid cells spend longer in mitosis, indicative of problems in chromosome segregation. In agreement with this, a substantial proportion of the haploids die at or shortly after the last mitosis through activation of a p53-dependent cytotoxic response. Finally, we show that p53 deletion stabilizes haploidy in human HAP1 cells and haploid mouse embryonic stem cells. We propose that, similar to aneuploidy or tetraploidy, haploidy triggers a p53-dependent response that limits the fitness of mammalian cells. PMID:28808015

Possible stabilization of the frequency of a CO/sub 2/ laser using an external Stark cell containing 1-1 difluoroethane

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

Avtonomov, V.P.; Alexandrescu, R.; Dumitras, D.

1979-02-01

Results are presented of measurements of the Stark modulation index and absorption coefficient of CO/sub 2/ laser radiation due to the P (24) line by 1-1 difluorethane (C/sub 2/H/sub 4/F/sub 2/). The possibility of stabilizing the CO/sub 2/ laser frequency using a Stark cell is demonstrated and the laser frequency tuning efficiency within the P (24) line of the 00/sup 0/1--10/sup 0/0 transition is determined.

Derivation of feline vaccine-associated fibrosarcoma cell line and its growth on chick embryo chorioallantoic membrane - a new in vivo model for veterinary oncological studies.

PubMed

Zabielska, K; Lechowski, R; Król, M; Pawłowski, K M; Motyl, T; Dolka, I; Zbikowski, A

2012-12-01

Feline vaccine associated fibrosarcomas are the second most common skin tumor in cats. Methods of treatment are: surgery, chemotherapy and radiotherapy. Nevertheless, the usage of cytostatics in feline vaccine associated sarcoma therapy is limited due to their adverse side effects, high toxicity and low biodistribution after i.v. injection. Therefore, much research on new therapeutic drugs is being conducted. In human medicine, the chick embryo chorioallantoic membrane (CAM) model is used as a cheap and easy to perform assay to assess new drug effectiveness in cancer treatment. Various human cell lines have different tumors growth on CAM. In veterinary medicine such model has not been described yet. In the present article derivation of feline vaccine associated fibrosarcoma cell line and its growth on CAM is described. The cell line and the tumor grown were confirmed by histopathological and immunohistochemical examination. As far as we believe, this is the first attempt to create such model, which may be used for further in vivo studies in veterinary oncology.

Interaction of Electromagnetic Fields with Chondrocytes in Gel Culture

DTIC Science & Technology

1990-02-01

biosynthesis due to applied electromagnetic fields. The results suggest that both normal chondrocytes and swarm rat chondrosarcoma cells in agarose Culture...and Swarm rat chondrosarcoma cells in agarose iii culture can, under proper culture conditions, continue to synthesize ma- trix macromolecules at a...cartilagc, and in rat chondrosarcoma cells (a continuous cell line). The overt gene expression of chondrocytes results in the synthesis and deposition of a

Isolation and Characterization of Poliovirus in Cell Culture Systems.

PubMed

Thorley, Bruce R; Roberts, Jason A

2016-01-01

The isolation and characterization of enteroviruses by cell culture was accepted as the "gold standard" by clinical virology laboratories. Methods for the direct detection of all enteroviruses by reverse transcription polymerase chain reaction, targeting a conserved region of the genome, have largely supplanted cell culture as the principal diagnostic procedure. However, the World Health Organization's Global Polio Eradication Initiative continues to rely upon cell culture to isolate poliovirus due to the lack of a reliable sensitive genetic test for direct typing of enteroviruses from clinical specimens. Poliovirus is able to infect a wide range of mammalian cell lines, with CD155 identified as the primary human receptor for all three seroytpes, and virus replication leads to an observable cytopathic effect. Inoculation of cell lines with extracts of clinical specimens and subsequent passaging of the cells leads to an increased virus titre. Cultured isolates of poliovirus are suitable for testing by a variety of methods and remain viable for years when stored at low temperature.This chapter describes general procedures for establishing a cell bank and routine passaging of cell lines. While the sections on specimen preparation and virus isolation focus on poliovirus, the protocols are suitable for other enteroviruses.

Inhibition of endogenous hydrogen sulfide production in clear-cell renal cell carcinoma cell lines and xenografts restricts their growth, survival and angiogenic potential

PubMed Central

Sonke, Eric; Verrydt, Megan; Postenka, Carl O.; Pardhan, Siddika; Willie, Chantalle J.; Mazzola, Clarisse R.; Hammers, Matthew D.; Pluth, Michael D.; Lobb, Ian; Power, Nicholas E.; Chambers, Ann F.; Leong, Hon S.; Sener, Alp

2016-01-01

Clear cell renal cell carcinoma (ccRCC) is characterized by Von Hippel–Lindau (VHL)-deficiency, resulting in pseudohypoxic, angiogenic and glycolytic tumours. Hydrogen sulfide (H2S) is an endogenously-produced gasotransmitter that accumulates under hypoxia and has been shown to be pro-angiogenic and cytoprotective in cancer. It was hypothesized that H2S levels are elevated in VHL-deficient ccRCC, contributing to survival, metabolism and angiogenesis. Using the H2S-specific probe MeRhoAz, it was found that H2S levels were higher in VHL-deficient ccRCC cell lines compared to cells with wild-type VHL. Inhibition of H2S-producing enzymes could reduce the proliferation, metabolism and survival of ccRCC cell lines, as determined by live-cell imaging, XTT/ATP assay, and flow cytometry respectively. Using the chorioallantoic membrane angiogenesis model, it was found that systemic inhibition of endogenous H2S production was able to decrease vascularization of VHL-deficient ccRCC xenografts. Endogenous H2S production is an attractive new target in ccRCC due to its involvement in multiple aspects of disease. PMID:26068241

Raman spectroscopy differentiates between sensitive and resistant multiple myeloma cell lines

NASA Astrophysics Data System (ADS)

Franco, Domenico; Trusso, Sebastiano; Fazio, Enza; Allegra, Alessandro; Musolino, Caterina; Speciale, Antonio; Cimino, Francesco; Saija, Antonella; Neri, Fortunato; Nicolò, Marco S.; Guglielmino, Salvatore P. P.

2017-12-01

Current methods for identifying neoplastic cells and discerning them from their normal counterparts are often nonspecific and biologically perturbing. Here, we show that single-cell micro-Raman spectroscopy can be used to discriminate between resistant and sensitive multiple myeloma cell lines based on their highly reproducible biomolecular spectral signatures. In order to demonstrate robustness of the proposed approach, we used two different cell lines of multiple myeloma, namely MM.1S and U266B1, and their counterparts MM.1R and U266/BTZ-R subtypes, resistant to dexamethasone and bortezomib, respectively. Then, micro-Raman spectroscopy provides an easily accurate and noninvasive method for cancer detection for both research and clinical environments. Characteristic peaks, mostly due to different DNA/RNA ratio, nucleic acids, lipids and protein concentrations, allow for discerning the sensitive and resistant subtypes. We also explored principal component analysis (PCA) for resistant cell identification and classification. Sensitive and resistant cells form distinct clusters that can be defined using just two principal components. The identification of drug-resistant cells by confocal micro-Raman spectroscopy is thus proposed as a clinical tool to assess the development of resistance to glucocorticoids and proteasome inhibitors in myeloma cells.

HLA Engineering of Human Pluripotent Stem Cells

PubMed Central

Riolobos, Laura; Hirata, Roli K; Turtle, Cameron J; Wang, Pei-Rong; Gornalusse, German G; Zavajlevski, Maja; Riddell, Stanley R; Russell, David W

2013-01-01

The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their human leukocyte antigen (HLA) genes. This has led to the proposed use of histocompatible, patient-specific stem cells; however, the preparation of many different stem cell lines for clinical use is a daunting task. Here, we develop two distinct genetic engineering approaches that address this problem. First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line. A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population. Second, we derive HLA class I–negative cells by targeted disruption of both alleles of the Beta-2 Microglobulin (B2M) gene in ESCs. Mixed leukocyte reactions and peptide-specific HLA-restricted CD8+ T cell responses were reduced in class I–negative cells that had undergone differentiation in embryoid bodies. These B2M−/− ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes. Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines. PMID:23629003

HLA engineering of human pluripotent stem cells.

PubMed

Riolobos, Laura; Hirata, Roli K; Turtle, Cameron J; Wang, Pei-Rong; Gornalusse, German G; Zavajlevski, Maja; Riddell, Stanley R; Russell, David W

2013-06-01

The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their human leukocyte antigen (HLA) genes. This has led to the proposed use of histocompatible, patient-specific stem cells; however, the preparation of many different stem cell lines for clinical use is a daunting task. Here, we develop two distinct genetic engineering approaches that address this problem. First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line. A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population. Second, we derive HLA class I-negative cells by targeted disruption of both alleles of the Beta-2 Microglobulin (B2M) gene in ESCs. Mixed leukocyte reactions and peptide-specific HLA-restricted CD8(+) T cell responses were reduced in class I-negative cells that had undergone differentiation in embryoid bodies. These B2M(-/-) ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes. Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines.

Disrupting CCT-β : β-tubulin selectively kills CCT-β overexpressed cancer cells through MAPKs activation

PubMed Central

Liu, Yan-Jin; Kumar, Vathan; Lin, Yuan-Feng; Liang, Po-Huang

2017-01-01

We have previously demonstrated the ability of I-Trp to disrupt the protein–protein interaction of β-tubulin with chaperonin-containing TCP-1β (CCT-β). This caused more severe apoptosis in multidrug-resistant MES-SA/Dx5, compared to MES-SA, due to its higher CCT-β overexpression. In this study, we screened a panel of cancer cell lines, finding CCT-β overexpression in the triple-negative breast cancer cell line MDA-MB-231, colorectal cancer cell lines Colo205 and HCT116, and a gastric cancer cell line MKN-45. Thus, I-Trp killed these cancers with sub- to low-μM EC50, whereas it was non-toxic to MCF-10A. We then synthesized analogs of I-Trp and evaluated their cytotoxicity. Furthermore, apoptotic mechanism investigations revealed the activation of both protein ubiquitination/degradation and ER-associated protein degradation pathways. These pathways proceeded through activation of MAPKs at the onset of CCT-β : β-tubulin complex disruption. We thus establish an effective strategy to treat CCT-β overexpressed cancers by disrupting the CCT-β : β-tubulin complex. PMID:28906489

Reduction of ammonia and lactate through the coupling of glutamine synthetase selection and downregulation of lactate dehydrogenase-A in CHO cells.

PubMed

Noh, Soo Min; Park, Jin Hyoung; Lim, Myung Sin; Kim, Jong Won; Lee, Gyun Min

2017-02-01

Chinese hamster ovary (CHO) cell cultivation for production of therapeutic proteins is accompanied by production of metabolic wastes, mostly ammonia and lactate. To reduce ammonia production, the glutamine synthetase (GS) system was used to develop therapeutic monoclonal antibody (mAb)-producing CHO cells (SM-0.025). Additionally, the lactate dehydrogenase-A (LDH-A) was downregulated with shRNA to reduce lactate production in SM-0.025. The resulting mAb-producing cell lines (#2, #46, and #52) produced less ammonia than the host cell line during the exponential phase due to GS protein overexpression. LDH-A downregulation in SM-0.025 not only reduced lactate production but also further reduced ammonia production. Among the three LDH-A-downregulated clones, clone #2 had the highest mAb production along with significantly reduced specific lactate and ammonia production rates compared to those in SM-0.025. Waste reduction increased the galactosylation level of N-glycosylation, which improved mAb quality. LDH-A downregulation was also successfully applied to the host cell lines (CHO K1 and GS knockout CHO-K1). However, LDH-A downregulated host cells could not survive the pool-selection process wherein glutamine was excluded and methionine sulfoximine was added to the media. Taken together, LDH-A downregulation in the mAb-producing cell line generated with the GS system successfully reduced both ammonia and lactate levels, improving mAb galactosylation. However, LDH-A downregulation could not be applied to host cell lines because it hampered the selection process of the GS system.

Heat shock protein 72 expression allows permissive replication of oncolytic adenovirus dl1520 (ONYX-015) in rat glioblastoma cells

PubMed Central

Madara, Jonathan; Krewet, James A; Shah, Maulik

2005-01-01

In this study we have made novel observations with regards to potentiation of the tumoricidal activity of the oncolytic adenovirus, dl1520 (ONYX-015) in rat glioblastoma cell lines expressing heat shock protein 72 (HSP72) due to permissive virus replication. ONYX-015 is a conditionally replicating adenovirus that is deleted for the E1B 55 kDA gene product whose normal function is to interact with cell-cycle regulatory proteins to permit virus replication. However, many murine and rodent cell lines are not permissive for adenovirus replication. Previously, it has been reported that the heat shock response is necessary for adenovirus replication and that induction of heat shock proteins is mediated by E1 region gene products. Therefore, we hypothesized that HSP72 expression may allow for permissive replication of ONYX-015 in previously non-permissive cells. Rat glioma cell lines 9L and RT2 were transfected with a plasmids expressing HSP72 or GFP. After infection with ONYX-015, no tumoricidal activity is observed in GFP expressing cell lines despite adequate transduction. In contrast, HSP72 transfected cells show cytopathic effects by 72 hours and greater than 75% loss of viability by 96 hours. Burst assays show active virus replication in the HSP72 expressing cell lines. Therefore, 9L-HSP72 and RT2-HSP72 are ideal models to evaluate the efficacy of ONYX-015 in an immunocompetent rat model. Our study has implications for creating rodent tumor models for pre-clinical studies with E1 region deleted conditionally replicating adenovirus. PMID:15762988

Dacarbazine and the Agonistic TRAIL Receptor-2 Antibody Lexatumumab Induce Synergistic Anticancer Effects in Melanoma

PubMed Central

Engesæter, Birgit; Engebraaten, Olav; Flørenes, Vivi Ann; Mælandsmo, Gunhild Mari

2012-01-01

Mapatumumab and lexatumumab (targeting death receptor 4 (DR4) and 5 (DR5), respectively) are agonistic TRAIL receptor antibodies that induce apoptosis in a wide range of cancer cells. The potency of mapatumumab and lexatumumab was assessed in mono therapy protocols, and the ability to sensitize for dacarbazine (DTIC) treatment was explored in ten different melanoma cell lines. Our data indicated that melanoma cell lines tend to be resistant to mapatumumab, most likely due to low expression of DR4, while a dose dependent response to lexatumumab was observed. Combining DTIC and lexatumumab induced an additive or synergistic effect on cell death in the various melanoma cell lines. The synergistic effect observed in the FEMX-1 cell line was related to enhanced cleavage of Bid in parallel with elevated expression of the pro-apoptotic proteins Bim, Bax and Bak. Furthermore, the anti-apoptotic proteins Bcl-XL, cIAP-1, XIAP and livin were down regulated. Cleavage of Bid and down regulation of cIAP-2 and livin were observed in vivo. Altogether, these data suggest a change in the balance between pro- and anti-apoptotic proteins favoring induction of apoptosis. In the more therapy resistant cell line, HHMS, no changes in the pro- and anti-apoptotic proteins were observed. FEMX-1 xenografts treated with DTIC and lexatumumab showed reduced growth and increased level of apoptosis compared to the control groups, providing arguments for further evaluation of this combination in melanoma patients. PMID:23029050

Dacarbazine and the agonistic TRAIL receptor-2 antibody lexatumumab induce synergistic anticancer effects in melanoma.

PubMed

Engesæter, Birgit; Engebraaten, Olav; Flørenes, Vivi Ann; Mælandsmo, Gunhild Mari

2012-01-01

Mapatumumab and lexatumumab (targeting death receptor 4 (DR4) and 5 (DR5), respectively) are agonistic TRAIL receptor antibodies that induce apoptosis in a wide range of cancer cells. The potency of mapatumumab and lexatumumab was assessed in mono therapy protocols, and the ability to sensitize for dacarbazine (DTIC) treatment was explored in ten different melanoma cell lines. Our data indicated that melanoma cell lines tend to be resistant to mapatumumab, most likely due to low expression of DR4, while a dose dependent response to lexatumumab was observed. Combining DTIC and lexatumumab induced an additive or synergistic effect on cell death in the various melanoma cell lines. The synergistic effect observed in the FEMX-1 cell line was related to enhanced cleavage of Bid in parallel with elevated expression of the pro-apoptotic proteins Bim, Bax and Bak. Furthermore, the anti-apoptotic proteins Bcl-XL, cIAP-1, XIAP and livin were down regulated. Cleavage of Bid and down regulation of cIAP-2 and livin were observed in vivo. Altogether, these data suggest a change in the balance between pro- and anti-apoptotic proteins favoring induction of apoptosis. In the more therapy resistant cell line, HHMS, no changes in the pro- and anti-apoptotic proteins were observed. FEMX-1 xenografts treated with DTIC and lexatumumab showed reduced growth and increased level of apoptosis compared to the control groups, providing arguments for further evaluation of this combination in melanoma patients.

Re-evaluation of tumor-specific cytotoxicity of mitomycin C, bleomycin and peplomycin.

PubMed

Sasaki, Masahiro; Okamura, Masahiko; Ideo, Atsushi; Shimada, Jun; Suzuki, Fumika; Ishihara, Mariko; Kikuchi, Hirotaka; Kanda, Yumiko; Kunii, Shiro; Sakagami, Hiroshi

2006-01-01

Three antitumor antibiotics, mitomycin C, bleomycin sulfate and peplomycin sulfate, were compared for their tumor-specific cytotoxicity, using human oral squamous cell lines (HSC-2, HSC-3, HSC-4, Ca9-22 and NA), human promyelocytic leukemic cell line HL-60 and human normal oral cell types (gingival fibroblast HGF, pulp cell HPC and periodontal ligament fibroblast HPLF). Among these three compounds, mitomycin C showed the highest tumor-specificity, due to its higher cytotoxic activity against human oral tumor cell lines than bleomycin and peplomycin. However, there was considerable variation of drug sensitivity among the six tumor cell lines. Mitomycin C induced internucleosomal DNA fragmentation and caspase-3, -8 and -9 activation in HL-60 cells only after 24 h. On the other hand, mitomycin C induced no clear-cut DNA fragmentation in HCS-2 cells, although it activated caspase-3, -8 and -9 to a slightly higher extent. Western blot analysis demonstrated that mitomycin C did not induce any apparent change in the intracellular concentration of anti-apoptotic protein (Bcl-2) and pro-apoptotic proteins (Bax, Bad). Electron microscopy of mitomycin C-treated HL-60 cells showed intact mitochondria (as regards to integrity and size) and cell surface microvilli, without production of an apoptotic body or autophagosome, at an early stage after treatment. The present study suggests the incomplete induction of apoptosis or the induction of another type of cell death by mitomycin C treatment.

A computational integrative approach based on alternative splicing analysis to compare immortalized and primary cancer cells.

PubMed

Tripathi, Kumar Parijat; Granata, Ilaria; Guarracino, Mario Rosario

2017-10-01

Immortalized cell lines are widely used to study the effectiveness and toxicity of anti cancer drugs as well as to assess the phenotypic characteristics of cancer cells, such as proliferation and migration ability. Unfortunately, cell lines often show extremely different properties than tumor tissues. Also the primary cells, that are deprived of the in vivo environment, might adapt to artificial conditions, and differ from the tissue they should represent. Despite these considerations, cell lines are still one of the most used cancer models due to their availability and capability to expand without limitation, but the clinical relevance of their use is still a big issue in cancer research. Many studies tried to overcome this task, comparing cell lines and tumor samples through the definition of the genomic and transcriptomic differences. To this aim, most of them used nucleotide variation or gene expression data. Here we introduce a different strategy based on alternative splicing detection and integration of DNA and RNA sequencing data, to explore the differences between immortalized and tissue-derived cells at isoforms level. Furthermore, in order to better investigate the heterogeneity of both cell populations, we took advantage of a public available dataset obtained with a new simultaneous omics single cell sequencing methodology. The proposed pipeline allowed us to identify, through a computational and prediction approach, putative mutated and alternative spliced transcripts responsible for the dissimilarity between immortalized and primary hepato carcinoma cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antitumor killer lymphocytes in the peripheral blood of a patient with transitional cell carcinoma of the bladder.

PubMed

Kim, C J; Yuasa, T; Kushima, R; Tomoyoshi, T; Seto, A

1998-05-01

Peripheral blood lymphocytes (PBL) from patients with bladder cancer also contain cells possessing cytotoxic activity against autologous tumor cells. These cells are phenotypically heterogenous and include natural killer (NK) and cytotoxic T cells. This study investigated the role of cytotoxic lymphocytes directed against autologous bladder cancer cells. PBL were obtained at intervals before and after surgery and analyzed for cytotoxic activity against autologous bladder cancer cells in 4-hour 51Cr release assay. PBL stimulated with autologous tumor cells were also transformed with human T-lymphotropic virus type-1, establishing a cell line (KB31) which was analyzed for phenotype and cytotoxic activity against the autologous tumor cells. PBL preoperative cytotoxic activity was low, but increased after surgery. Cytotoxic activity was found not only against autologous bladder cancer cells, but also against heterologous bladder cancer (KK-47) and myeloid leukemia (K562) cells, with the highest activity against the heterologous cell lines. The cytotoxic activity of KB31 was 40% against autologous tumor cells 6 weeks after initiation of the cell line, but decreased to 5% by 6 months. This activity was lower than that against the other cell lines, and was similar to that of PBL in short-term culture. Fluorescence-activated cell sorter (FACS) analysis demonstrated that in KB31 cells at 6 weeks, CD8+ cells were dominant, but CD56+ cells predominated at 6 months. These results suggest that the presence of cytotoxic activity in the peripheral blood of the patient was due to both cytotoxic T cells and NK cells. The cytotoxic activity was lowest prior to surgery and increased postoperatively.

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

PubMed Central

Akopian, Veronika; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J.; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B.; Ludwig, Tenneille E.; McKay, Ronald D. G.; Miyazaki, Takamichi; Nakatsuji, Norio; Oh, Steve K. W.; Pera, Martin F.; Rossant, Janet; Stacey, Glyn N.; Suemori, Hirofumi

2010-01-01

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. PMID:20186512

Anti-tumor activity of the ATR inhibitor AZD6738 in HER2 positive breast cancer cells.

PubMed

Kim, Hee-Jun; Min, Ahrum; Im, Seock-Ah; Jang, Hyemin; Lee, Kyung Hun; Lau, Alan; Lee, Miso; Kim, Seongyeong; Yang, Yaewon; Kim, Jungeun; Kim, Tae Yong; Oh, Do-Youn; Brown, Jeffrey; O'Connor, Mark J; Bang, Yung-Jue

2017-01-01

Ataxia telangiectasia and Rad3-related (ATR) proteins are sensors of DNA damage, which induces homologous recombination (HR)-dependent repair. ATR is a master regulator of DNA damage repair (DDR), signaling to control DNA replication, DNA repair and apoptosis. Therefore, the ATR pathway might be an attractive target for developing new drugs. This study was designed to investigate the antitumor effects of the ATR inhibitor, AZD6738 and its underlying mechanism in human breast cancer cells. Growth inhibitory effects of AZD6738 against human breast cancer cell lines were studied using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (methyl thiazolyl tetrazolium, MTT) assay. Cell cycle analysis, Western blotting, immunofluorescence and comet assays were also performed to elucidate underlying mechanisms of AZD6738 action. Anti-proliferative and DDR inhibitory effects of AZD6738 were demonstrated in human breast cancer cell lines. Among 13 cell lines, the IC 50 values of nine cell lines were less than 1 μmol/L using MTT assay. Two cell lines, SK-BR-3 and BT-474, were chosen for further evaluation focused on human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells. Sensitive SK-BR-3 but not the less sensitive BT-474 breast cancer cells showed increased level of apoptosis and S phase arrest and reduced expression levels of phosphorylated check-point kinase 1 (CHK1) and other repair markers. Decreased functional CHK1 expression induced DNA damage accumulation due to HR inactivation. AZD6738 showed synergistic activity with cisplatin. Understanding the antitumor activity and mechanisms of AZD6738 in HER2-positive breast cancer cells creates the possibility for future clinical trials targeting DDR in HER2-positive breast cancer treatment. © 2016 UICC.

Leptin reverts pro-apoptotic and antiproliferative effects of α-linolenic acids in BCR-ABL positive leukemic cells: involvement of PI3K pathway.

PubMed

Beaulieu, Aurore; Poncin, Géraldine; Belaid-Choucair, Zakia; Humblet, Chantal; Bogdanovic, Gordana; Lognay, Georges; Boniver, Jacques; Defresne, Marie-Paule

2011-01-01

It is suspected that bone marrow (BM) microenvironmental factors may influence the evolution of chronic myeloid leukaemia (CML). In this study, we postulated that adipocytes and lipids could be involved in the progression of CML. To test this hypothesis, adipocytes were co-cultured with two BCR-ABL positive cell lines (PCMDS and K562). T cell (Jurkat) and stroma cell (HS-5) lines were used as controls. In the second set of experiments, leukemic cell lines were treated with stearic, oleic, linoleic or α-linolenic acids in presence or absence of leptin. Survival, proliferation, leptin production, OB-R isoforms (OB-Ra and OB-Rb), phosphoinositide 3-kinase (PI3k) and BCL-2 expression have been tested after 24h, 48h and 72h of treatment. Our results showed that adipocytes induced a decrease of CML proliferation and an increase in lipid accumulation in leukemic cells. In addition, CML cell lines induced adipocytes cell death. Chromatography analysis showed that BM microenvironment cells were full of saturated (SFA) and monounsaturated (MUFA) fatty acids, fatty acids that protect tumor cells against external agents. Stearic acid increased Bcl-2 expression in PCMDS, whereas oleic and linoleic acids had no effects. In contrast, α-linolenic acid decreased the proliferation and the survival of CML cell lines as well as BCL-2 and OB-R expression. The effect of α-linolenic acids seemed to be due to PI3K pathway and Bcl-2 inhibition. Leptin production was detected in the co-culture medium. In the presence of leptin, the effect of α-linolenic acid on proliferation, survival, OB-R and BCl-2 expression was reduced.

The cytotoxic action of the CD56+ fraction of cytokine-induced killer cells against a K562 cell line is mainly restricted to the natural killer cell subset.

PubMed

Chieregato, Katia; Zanon, Cristina; Castegnaro, Silvia; Bernardi, Martina; Amati, Eliana; Sella, Sabrina; Rodeghiero, Francesco; Astori, Giuseppe

2017-01-01

Cytokine-induced killer cells are polyclonal T cells generated ex vivo and comprise two main subsets: the CD56- fraction, possessing an alloreactive potential caused by T cells (CD3+CD56-), and the CD56+ fraction, characterised by a strong antitumour capacity induced by natural killer-like T cells (NK-like T, CD3+CD56+) and natural killer cells (NK, CD3-CD56+ bright). We investigated the cytotoxic action of selected CD56+ cell subpopulations against a human chronic myeloid leukaemia (K562) cell line. After immunomagnetic selection of the CD56+ cell fraction, NK bright cells (CD3-CD56+ bright) and two subsets of NK-like T cells (CD3+CD56+), called NK-like T CD56 dim and NK-like T CD56 bright, could be identified. The cytotoxic effect against K562 cells was mainly exerted by the NK bright subpopulation and resulted to be inversely correlated with the percentage of NK-like T CD56 dim cells in the culture. The lytic action appeared to be independent of cell degranulation as suggested by the lack of change in the expression of CD107a. We conclude that the cytotoxic action of CD56+ cells against a K562 cell line is mainly due to the NK cells.

Prediction of cellular radiosensitivity from DNA damage induced by gamma-rays and carbon ion irradiation in canine tumor cells.

PubMed

Wada, Seiichi; Van Khoa, Tran; Kobayashi, Yasuhiko; Funayama, Tomoo; Ogihara, Kikumi; Ueno, Shunji; Ito, Nobuhiko

2005-11-01

Diseases of companion animals are shifting from infectious diseases to neoplasms (cancer), and since radiation therapy is one of the effective choices available for cancer treatment, the application of radiotherapy in veterinary medicine is likely to increase. However tumor tissues have different radiosensitivities, and therefore it is important to determine the intrinsic radiosensitivity of tumors in individual patients in advance of radiotherapy. We have studied the relationship between the surviving cell fraction measured by a clonogenic assay and DNA double strand breaks detected by a comet assay under neutral conditions in three canine tumor cell lines, after gamma-ray and carbon ion irradiation. In all the cell lines, cell death assessed by the clonogenic assay was much higher following irradiation with carbon ions than with gamma-rays. The initial and residual (4 hr) DNA damage due to gamma-ray and carbon ion irradiation were higher in a radiosensitive cell line than in a radioresistant cell line. The surviving cell fraction at 2 Gy (SF2) showed a tendency for correlation with both the initial and residual DNA damage. In particular, the residual damage per Gy was significantly correlated with SF2, regardless of the type of radiation. This indicates that cellular radiosensitivity can be predicted by detection of radiation-induced residual DNA damage.

Uptake in melanoma cells of N-(2-diethylaminoethyl)-2-iodobenzamide (BZA2), an imaging agent for melanoma staging: relation to pigmentation.

PubMed

Mansard, Sandrine; Papon, Janine; Moreau, Marie-France; Miot-Noirault, Elisabeth; Labarre, Pierre; Bayle, Martine; Veyre, Annie; Madelmont, Jean-Claude; Moins, Nicole

2005-07-01

N-(2-diethylaminoethyl)-2-iodobenzamide (BZA(2)) has been singled out as the most efficacious melanoma scintigraphy imaging agent. Our work was designed to assess the mechanisms of the specific affinity of the radioiodinated iodobenzamide for melanoma tissue. We studied the cellular uptake and retention of [(125)I]-BZA(2) on various cell lines. In vitro, cellular [(125)I]-BZA(2) uptake was related to the pigmentation status of the cells: higher in pigmented melanoma cell lines (M4 Beu, IPC 227, B 16) than in a nonpigmented one (M3 Dau) and nonmelanoma cell lines (MCF 7 and L 929). Two mechanisms were assessed: binding of the tracer to melanin or to sigma receptors of melanoma cells. First, the uptake of [(125)I]-BZA(2) after melanogenesis stimulation by alpha-melanocyte-stimulating hormone and l-tyrosine increased in the B 16 melanoma cell line both in vitro and in vivo according to melanin concentration. Moreover, the binding of [(125)I]-BZA(2) to synthetic melanin was dependent on melanin concentration and could be saturated. Second, no competition was evidenced on M4 Beu cells between [(125)I]-BZA(2) and haloperidol, a sigma ligand, at concentrations < or =10(-6) M. We show that the specificity and sensibility of BZA(2) as a melanoma scintigraphic imaging agent are mostly due to interactions with melanic pigments.

High Antioxidant Activity Facilitates Maintenance of Cell Division in Leaves of Drought Tolerant Maize Hybrids

PubMed Central

Avramova, Viktoriya; AbdElgawad, Hamada; Vasileva, Ivanina; Petrova, Alexandra S.; Holek, Anna; Mariën, Joachim; Asard, Han; Beemster, Gerrit T. S.

2017-01-01

We studied the impact of drought on growth regulation in leaves of 13 maize varieties with different drought sensitivity and geographic origins (Western Europe, Egypt, South Africa) and the inbred line B73. Combining kinematic analysis of the maize leaf growth zone with biochemical measurements at a high spatial resolution allowed us to examine the correlation between the regulation of the cellular processes cell division and elongation, and the molecular redox-regulation in response to drought. Moreover, we demonstrated differences in the response of the maize lines to mild and severe levels of water deficit. Kinematic analysis indicated that drought tolerant lines experienced less impact on leaf elongation rate due to a smaller reduction of cell production, which, in turn, was due to a smaller decrease of meristem size and number of cells in the leaf meristem. Clear differences in growth responses between the groups of lines with different geographic origin were observed in response to drought. The difference in drought tolerance between the Egyptian hybrids was significantly larger than between the European and South-African hybrids. Through biochemical analyses, we investigated whether antioxidant activity in the growth zone, contributes to the drought sensitivity differences. We used a hierarchical clustering to visualize the patterns of lipid peroxidation, H2O2 and antioxidant concentrations, and enzyme activities throughout the growth zone, in response to stress. The results showed that the lines with different geographic region used different molecular strategies to cope with the stress, with the Egyptian hybrids responding more at the metabolite level and African and the European hybrids at the enzyme level. However, drought tolerance correlated with both, higher antioxidant levels throughout the growth zone and higher activities of the redox-regulating enzymes CAT, POX, APX, and GR specifically in leaf meristems. These findings provide evidence for a link between antioxidant regulation in the leaf meristem, cell division, and drought tolerance. PMID:28210264

In vitro long-term culture of human primitive hematopoietic cells supported by murine stromal cell line MS-5.

PubMed

Nishi, N; Ishikawa, R; Inoue, H; Nishikawa, M; Yoneya, T; Kakeda, M; Tsumura, H; Ohashi, H; Mori, K J

1997-04-01

When Lin-CD34+CD38- cells from normal human cord blood were cocultured with MS-5, colony forming cells were maintained for over 8 weeks. Prevention of contact between MS-5 and Lin-CD34+CD38- cells by using a membrane filter was negligible for this activity, indicating that the activity of MS-5 on human primitive hematopoietic cells may be due to soluble factor(s) secreted from MS-5. We tried to purify this activity by a [3H]TdR incorporation assay. The activity was found in 150 kD fraction and was neutralized with anti-mSCF (stem cell factor) antibody. Another 20-30 kD fraction synergized with mSCF to stimulate the growth of Lin-CD34+CD38- cells but failed alone. This fraction supported the growth of the G-CSF (granulocyte-colony stimulating factor)-dependent cell line FD/GR3, FDC-P2 transfected with mG-CSF receptor cDNA. This synergy was canceled in the presence of soluble mG-CSF receptor. Addition of anti-mSCF antibody and soluble mG-CSF receptor to the culture completely abrogated the activity of MS-5-culture supernatant. These results indicate the activity of MS-5 on Lin-CD34+CD38- cells is due to synergistic effect of mSCF and mG-CSF.

Antitumor Effects and Mechanism of Novel Emodin Rhamnoside Derivatives against Human Cancer Cells In Vitro

PubMed Central

Deng, Jun-peng; Jiang, Ling-zhi; Xiong, Ping; Yang, Bin-jie; Liu, Shan-shan

2015-01-01

A series of novel anthracene L-rhamnopyranosides compounds were designed and synthesized and their anti-proliferative activities on cancer cell lines were investigated. We found that one derivative S-8 (EM-d-Rha) strongly inhibited cell proliferation of a panel of different human cancer cell lines including A549, HepG2, OVCAR-3, HeLa and K562 and SGC-790 cell lines, and displayed IC50 values in low micro-molar ranges, which are ten folds more effective than emodin. In addition, we found EM-d-Rha (3-(2”,3”-Di-O-acetyl-α-L-rhamnopyranosyl-(1→4)-2’,3’-di-O-acetyl-α-L-rhamnopyranosyl)-emodin) substantially induced cellular apoptosis of HepG2 and OVCAR-3 cells in the early growth stage. Furthermore, EM-d-Rha led to the decrease of mitochondrial transmembrane potential, and up-regulated the express of cells apoptosis factors in a concentration- and time-dependent manner. The results indicated the EM-d-Rha may inhibit the growth and proliferation of HepG2 cells through the pathway of apoptosis induction, and the possible molecular mechanism may due to the activation of intrinsic apoptotic signal pathway. PMID:26682731

Usefulness of sural nerve biopsy in the genomic era.

PubMed

Kanda, Takashi

2009-08-01

The value of peripheral nerve biopsy is now sometimes questioned due to the high complication rate and the recent development of noninvasive molecular techniques for diagnosis of hereditary neuropathy. However, the disorders that can be diagnosed by genetic analysis are limited and sural nerve biopsy is still a powerful tool for making a correct diagnosis of peripheral neuropathy. Histological evaluation of the sural nerve has long focused on changes of the two major components of peripheral nerves, axons and myelin, as well as on the detection of diagnostic changes such as amyloid deposits, sarcoid tubercles, and vasculitis. In addition to these components, the sural nerve biopsy specimen contains various important cells, including perineurial cells, mast cells, endothelial cells, pericytes, and lymphocytes. Among these cells, the endothelial cells and pericytes form the blood-nerve barrier (BNB) and investigation of these cells can reveal important information, especially in inflammatory neuropathies. To better understand the biological basis of BNB, we established rat and human immortal cell lines from the endothelial cells and pericytes of endoneurial microvessels. Characterization of these cell lines is now underway at our laboratory. These BNB cell lines should provide useful information concerning the pathophysiology of peripheral neuropathy, and we should obtain a new perspective for the investigation of nerve biopsy specimens after understanding the molecular background of the BNB.

Slug/SNAI2 regulates cell proliferation and invasiveness of metastatic prostate cancer cell lines.

PubMed

Emadi Baygi, Modjtaba; Soheili, Zahra-Soheila; Essmann, Frank; Deezagi, Abdolkhaleg; Engers, Rainer; Goering, Wolfgang; Schulz, Wolfgang A

2010-08-01

Many metastatic cancers recapitulate the epithelial-to-mesenchymal transition (EMT) resulting in enhanced cell motility and invasiveness. The EMT is regulated by several transcription factors, including the zinc finger protein SNAI2, also named Slug, which appears to exert additional functions during development and cancer progression. We have studied the function of SNAI2 in prostate cancer cells. Quantitative RT-PCR analysis showed strong SNAI2 expression particularly in the PC-3 and PC3-16 prostate carcinoma cell lines. Knockdown of SNAI2 by specific siRNA induced changes in EMT markers and inhibited invasion of both cell lines into a matrigel matrix. SNAI2 siRNA-treated cells did not tolerate detachment from the culture plates, likely at least in part due to downregulation of integrin alpha6beta4. SNAI2 knockdown disturbed the microtubular and actin cytoskeletons, especially severely in PC-3 cells, resulting in grossly enlarged, flattened, and sometimes multinuclear cells. Knockdown also decreased cell proliferation, with a prominent G0/G1 arrest in PC3-16. Together, our data imply that SNAI2 exerts strong effects on the cytoskeleton and adhesion of those prostate cancer cells that express it and is necessary for their proliferation and invasiveness.

Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates.

PubMed

Ozgür, Burak; Saaby, Lasse; Langthaler, Kristine; Brodin, Birger

2018-01-15

Recently, we transfected the porcine intestinal cell line IPEC-J2, with human P-glycoprotein (P-gp, ABCB1). The resulting cell line, iP-gp, has a high expression of functional human P-gp in the apical membrane, and a low expression of nonhuman ATP-binding cassette (ABC) transporters. The aim of the present work was to investigate the usability of iP-gp cell line for determining transepithelial transport kinetics of the prototypical P-gp substrates digoxin and rhodamine 123. The cell line generated tight monolayers after 16days of culture, reflected by high transepithelial electrical resistance values (TEER>15,000Ω·cm 2 ), immunocytochemistry and low fluxes of the paracellular flux marker [ 14 C]-mannitol. Monolayer integrity was not affected the common solvents dimethyl sulfoxide (DMSO), methanol and ethanol in concentrations up to 2% (v/v). Transepithelial fluxes of [ 3 H]-labeled digoxin and rhodamine 123 were measured at varying donor concentrations, and kinetic parameters were estimated. K m and V max of P-gp mediated basolateral-to-apical (B-A) flux of rhodamine 123 were estimated to 332±124μM and 111±16pmol·cm -2 ·min -1 (n=3, total N=6), respectively. V max and K m of digoxin B-A flux could not be estimated due to the low aqueous solubility of digoxin. The half maximal inhibitory concentrations (IC 50 ) of the selective P-gp inhibitor, zosuquidar (LY-335979), were estimated to 0.05±0.01μM (n=3, total N=6) and 0.04±0.01μM (n=3, total N=6) in transport experiments with digoxin and rhodamine 123 as substrates, respectively. Bidirectional fluxes of digoxin and rhodamine 123 were measured in transfected Madin Darby canine kidney cells (MDCK II MDR1) and compared with the fluxes obtained with the iP-gp cell monolayers. Efflux ratios were highest in the iP-gp cells, due to a tighter paracellular pathway. In conclusion, both digoxin and rhodamine 123 could be used to obtain IC 50 values of inhibition, K i values were only possible to obtain using rhodamine 123. The observed tightness, robustness towards solvents and the high efflux ratios confirmed that the iP-gp cell line may serve as a useful screening tool for investigations of substrate-P-gp interactions and modulation of P-gp function. Copyright © 2017 Elsevier B.V. All rights reserved.

Establishment and characterisation of a novel bovine SV40 large T-antigen-transduced foetal hepatocyte-derived cell line.

PubMed

Gleich, Alexander; Kaiser, Bastian; Schumann, Julia; Fuhrmann, Herbert

2016-06-01

Due to lack of in vitro models for bovine hepatocytes apart from primary cells, there is demand for a bovine hepatocyte-derived cell line. Transduction of bovine foetal hepatocytes with SV40 large T-antigen was performed using the vector pRetro-E2 SV40. Phase contrast microscopy was carried out to evaluate morphology. Immunofluorescence staining was conducted to study expression of keratins, tight junction proteins zona occludens-1 and claudin-1, glucose transporter-2 and P-glycoprotein as well as phosphoenolpyruvate carboxykinase. Urea and triglyceride production was quantified photometrically. Histochemical staining of glycogen by Periodic acid-Schiff stain and of lipids with Oil red O was performed after 24 h incubation with 20 mM glucose and 85 μM palmitic acid, respectively. Gene expression analysis of hepatocyte-typical genes was conducted by reverse transcription PCR. We obtained a SV40LTAg-transduced extended passage cell line, referred to as BFH12. Polygonal growth, keratins, tight junction proteins zona occludens-1 and claudin-1 and glucose transporter-2 as well as P-glycoprotein and phosphoenolpyruvate carboxykinase were attested positively. Urea production calculated as cell-specific rate was 14.2 ± 2.0 fmol/h (early passage) and 17.6 ± 3.7 fmol/h (late passage). Cell-specific triglyceride production was 1.6 ± 0.5 fmol/h (early passage) and 2.1 ± 0.3 fmol/h (late passage). Additionally, cells were positive for glycogen and lipid storage and showed a gene expression pattern resembling foetal hepatocytes. With the properties described here, the novel cell line BFH12 is a hepatocyte-derived cell line which can be used as an in vitro whole cell model.

Pumpkin seed extract: Cell growth inhibition of hyperplastic and cancer cells, independent of steroid hormone receptors.

PubMed

Medjakovic, Svjetlana; Hobiger, Stefanie; Ardjomand-Woelkart, Karin; Bucar, Franz; Jungbauer, Alois

2016-04-01

Pumpkin seeds have been known in folk medicine as remedy for kidney, bladder and prostate disorders since centuries. Nevertheless, pumpkin research provides insufficient data to back up traditional beliefs of ethnomedical practice. The bioactivity of a hydro-ethanolic extract of pumpkin seeds from the Styrian pumpkin, Cucurbita pepo L. subsp. pepo var. styriaca, was investigated. As pumpkin seed extracts are standardized to cucurbitin, this compound was also tested. Transactivational activity was evaluated for human androgen receptor, estrogen receptor and progesterone receptor with in vitro yeast assays. Cell viability tests with prostate cancer cells, breast cancer cells, colorectal adenocarcinoma cells and a hyperplastic cell line from benign prostate hyperplasia tissue were performed. As model for non-hyperplastic cells, effects on cell viability were tested with a human dermal fibroblast cell line (HDF-5). No transactivational activity was found for human androgen receptor, estrogen receptor and progesterone receptor, for both, extract and cucurbitin. A cell growth inhibition of ~40-50% was observed for all cell lines, with the exception of HDF-5, which showed with ~20% much lower cell growth inhibition. Given the receptor status of some cell lines, a steroid-hormone receptor independent growth inhibiting effect can be assumed. The cell growth inhibition for fast growing cells together with the cell growth inhibition of prostate-, breast- and colon cancer cells corroborates the ethnomedical use of pumpkin seeds for a treatment of benign prostate hyperplasia. Moreover, due to the lack of androgenic activity, pumpkin seed applications can be regarded as safe for the prostate. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Isolation and characterization of an IGROV-1 human ovarian cancer cell line made resistant to Ecteinascidin-743 (ET-743)

PubMed Central

Erba, E; Bergamaschi, D; Bassano, L; Ronzoni, S; Liberti, G Di; Muradore, I; Vignati, S; Faircloth, G; Jimeno, J; D'Incalci, M

2000-01-01

By exposing Igrov-1 human ovarian cancer cells to increasing concentrations of Ecteinascidin-743 (ET-743), either for a short or prolonged time, we obtained sublines resistant to ET-743 which overexpress Pgp. The most resistant clone (Igrov-1/25 ET) was evaluated for biological and pharmacological characterizations. The increased Pgp levels of Igrov-1/25 ET were not due to amplification of the mdr-1 gene but to increased mRNA levels. No increase in other multidrug resistance-related proteins such as MRP or LRP was observed in Igrov-1/25 ET. The IC50values of ET-743 against Igrov-1/25 ET was approximately 50 times higher than the parental cell line. Resistance was not reversed while maintaining the cell line in drug-free medium for at least 24 months. Igrov-1/25 ET was cross-resistant to Doxorubicin and VP16 while it was equally sensitive to L-PAM, MNNG, CPT and only marginally less sensitive to Cis-DDP and Oxaliplatin compared to the parental cell line. Igrov-1/25 ET exposed to Doxorubicin retained this drug much less, mainly because of a more efficient drug efflux. The cyclosporine analogue SDZ PSC-833 reversed the resistance of Igrov-1/25 ET to ET-743, without any enhancement of the drug activity against the parental Igrov-1 cell line. Igrov-1/25 ET exhibits typical features of cell lines overexpressing the mdr-1 gene and can be a potentially useful tool in selecting ET-743 non-cross-resistant analogues as well as to investigate methods to counteract resistance to this drug. © 2000 Cancer Research Campaign PMID:10817511

Cytotoxicity and genotoxicity of coronaridine from Tabernaemontana catharinensis A.DC in a human laryngeal epithelial carcinoma cell line (Hep-2)

PubMed Central

Rizo, Walace Fraga; Ferreira, Luis Eduardo; Colnaghi, Vanessa; Martins, Juliana Simões; Franchi, Leonardo Pereira; Takahashi, Catarina Satie; Beleboni, Rene Oliveira; Marins, Mozart; Pereira, Paulo Sérgio; Fachin, Ana Lúcia

2013-01-01

Cancer has become a major public health problem worldwide and the number of deaths due to this disease is increasing almost exponentially. In the constant search for new treatments, natural products of plant origin have provided a variety of new compounds to be explored as antitumor agents. Tabernaemontana catharinensis is a medicinal plant that produces alkaloids with expressive antitumor activity, such as heyneanine, coronaridine and voacangine. The aim of present study was firstly to screen the cytotoxic activity of the indole alkaloids heyneanine, coronaridine and voacangine against HeLa (human cervix tumor), 3T3 (normal mouse embryo fibroblasts), Hep-2 (human laryngeal epithelial carcinoma) and B-16 (murine skin) cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); and secondly to analyze the apoptotic activity, cell membrane damage and genotoxicity of the compound that showed the best cytotoxic activity against the tumor cell lines tested. Coronaridine was the one that exhibited greater cytotoxic activity in the laryngeal carcinoma cell line Hep-2 (IC50 = 54.47 μg/mL) than the other alkaloids tested (voacangine IC50 = 159.33 g/mL, and heyneanine IC50 = 689.45 μg/mL). Coronaridine induced apoptosis in cell lines 3T3 and Hep-2, even at high concentrations. The evaluation of genotoxicity by comet assay showed further that coronaridine caused minimal DNA damage in the Hep-2 tumor cell line, and the LDH test showed that it did not affect the plasma membrane. These results suggest that further investigation of coronaridine as an antitumor agent has merit. PMID:23569415

Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines

PubMed Central

Godlewska, Marlena; Krasuska, Wanda

2018-01-01

Thyroid peroxidase (TPO) is an enzyme and autoantigen expressed in thyroid and breast tissues. Thyroid TPO undergoes a complex maturation process however, nothing is known about post-translational modifications of breast-expressed TPO. In this study, we have investigated the biochemical properties of TPO expressed in normal and cancerous human breast tissues, and the maturation process and antigenicity of TPO present in a panel of human breast tissue-derived cell lines. We found that the molecular weight of breast TPO was slightly lower than that of thyroid TPO due to decreased glycosylation and as suggest results of Western blot also shorter amino acid chain. Breast TPO exhibit enzymatic activity and isoelectric point comparable to that of thyroid TPO. The biochemical properties of TPO expressed in mammary cell lines and normal thyrocytes are similar regarding glycan content, molecular weight and isoelectric point. However, no peroxidase activity and dimer formation was detected in any of these cell lines since the majority of TPO protein was localized in the cytoplasmic compartment, and the TPO expression at the cell surface was too low to detect its enzymatic activity. Lactoperoxidase, a protein highly homologous to TPO expressed also in breast tissues, does not influence the obtained data. TPO expressed in the cell lines was recognized by a broad panel of TPO-specific antibodies. Although some differences in biochemical properties between thyroid and breast TPO were observed, they do not seem to be critical for the overall three-dimensional structure. This conclusion is supported by the fact that TPO expressed in breast tissues and cell lines reacts well with conformation-sensitive antibodies. Taking into account a close resemblance between both proteins, especially high antigenicity, future studies should investigate the potential immunotherapies directed against breast-expressed TPO and its specific epitopes. PMID:29513734

Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines.

PubMed

Godlewska, Marlena; Krasuska, Wanda; Czarnocka, Barbara

2018-01-01

Thyroid peroxidase (TPO) is an enzyme and autoantigen expressed in thyroid and breast tissues. Thyroid TPO undergoes a complex maturation process however, nothing is known about post-translational modifications of breast-expressed TPO. In this study, we have investigated the biochemical properties of TPO expressed in normal and cancerous human breast tissues, and the maturation process and antigenicity of TPO present in a panel of human breast tissue-derived cell lines. We found that the molecular weight of breast TPO was slightly lower than that of thyroid TPO due to decreased glycosylation and as suggest results of Western blot also shorter amino acid chain. Breast TPO exhibit enzymatic activity and isoelectric point comparable to that of thyroid TPO. The biochemical properties of TPO expressed in mammary cell lines and normal thyrocytes are similar regarding glycan content, molecular weight and isoelectric point. However, no peroxidase activity and dimer formation was detected in any of these cell lines since the majority of TPO protein was localized in the cytoplasmic compartment, and the TPO expression at the cell surface was too low to detect its enzymatic activity. Lactoperoxidase, a protein highly homologous to TPO expressed also in breast tissues, does not influence the obtained data. TPO expressed in the cell lines was recognized by a broad panel of TPO-specific antibodies. Although some differences in biochemical properties between thyroid and breast TPO were observed, they do not seem to be critical for the overall three-dimensional structure. This conclusion is supported by the fact that TPO expressed in breast tissues and cell lines reacts well with conformation-sensitive antibodies. Taking into account a close resemblance between both proteins, especially high antigenicity, future studies should investigate the potential immunotherapies directed against breast-expressed TPO and its specific epitopes.

CellTrans: An R Package to Quantify Stochastic Cell State Transitions.

PubMed

Buder, Thomas; Deutsch, Andreas; Seifert, Michael; Voss-Böhme, Anja

2017-01-01

Many normal and cancerous cell lines exhibit a stable composition of cells in distinct states which can, e.g., be defined on the basis of cell surface markers. There is evidence that such an equilibrium is associated with stochastic transitions between distinct states. Quantifying these transitions has the potential to better understand cell lineage compositions. We introduce CellTrans, an R package to quantify stochastic cell state transitions from cell state proportion data from fluorescence-activated cell sorting and flow cytometry experiments. The R package is based on a mathematical model in which cell state alterations occur due to stochastic transitions between distinct cell states whose rates only depend on the current state of a cell. CellTrans is an automated tool for estimating the underlying transition probabilities from appropriately prepared data. We point out potential analytical challenges in the quantification of these cell transitions and explain how CellTrans handles them. The applicability of CellTrans is demonstrated on publicly available data on the evolution of cell state compositions in cancer cell lines. We show that CellTrans can be used to (1) infer the transition probabilities between different cell states, (2) predict cell line compositions at a certain time, (3) predict equilibrium cell state compositions, and (4) estimate the time needed to reach this equilibrium. We provide an implementation of CellTrans in R, freely available via GitHub (https://github.com/tbuder/CellTrans).

Expression of Recombinant Antibodies

PubMed Central

Frenzel, André; Hust, Michael; Schirrmann, Thomas

2013-01-01

Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

Generation of iPS-derived model cells for analyses of hair shaft differentiation.

PubMed

Kido, Takumi; Horigome, Tomoatsu; Uda, Minori; Adachi, Naoki; Hirai, Yohei

2017-09-01

Biological evaluation of hair growth/differentiation activity in vitro has been a formidable challenge, primarily due to the lack of relevant model cell systems. To solve this problem, we generated a stable model cell line in which successive differentiation via epidermal progenitors to hair components is easily inducible and traceable. Mouse induced pluripotent stem (iPS) cell-derived cells were selected to stably express a tetracycline (Tet)-inducible bone morphogenic protein-4 (BMP4) expression cassette and a luciferase reporter driven by a hair-specific keratin 31 gene (krt31) promoter (Tet-BMP4-KRT31-Luc iPS). While Tet- BMP4-KRT31-Luc iPS cells could be maintained as stable iPS cells, the cells differentiated to produce luciferase luminescence in the presence of all-trans retinoic acid (RA) and doxycycline (Dox), and addition of a hair differentiation factor significantly increased luciferase fluorescence. Thus, this cell line may provide a reliable cell-based screening system to evaluate drug candidates for hair differentiation activity.

LRD-22, a novel dual dithiocarbamatic acid ester, inhibits Aurora-A kinase and induces apoptosis and cell cycle arrest in HepG2 cells

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

Wang, Huiling; Li, Ridong; Li, Li

In this study we investigated the antitumor activity of the novel dual dithiocarbamatic acid ester LRD-22 in vitro and in vivo. Several cancer cell lines were employed to determine the effect of LRD-22 on cell growth, and the MTT assay showed there was a significant decrease in viable tumor cell numbers in the presence of LRD-22, especially in the HepG2 cell line. Colony formation assay also showed LRD-22 strongly inhibits HepG2 cell growth. Evaluation of the mechanism involved showed that inhibitory effects of LRD-22 on cell growth are due to induction of apoptosis and G2/M arrest. LRD-22 inhibited Aurora-A phosphorylation at Thr{submore » 288} and subsequently impaired p53 phosphorylation at Ser{sub 315} which was associated with the proteasome degradation pathway. Tumor suppressor protein p53 is stabilized by this mechanism and accumulates through inhibition of Aurora-A kinase activity via treatment with LRD-22. In vivo study of HepG2 xenograft in nude mice also shows LRD-22 suppresses tumor growth at a concentration of 5 mg/kg without animals suffering loss of body weight. In conclusion, our results demonstrate LRD-22 acts as an Aurora-A kinase inhibitor to induce apoptosis and inhibit proliferation in HepG2 cells, and should be considered as a promising targeting agent for HCC therapy. - Highlights: • LRD-22 significantly inhibits cancer cell growth, especially in the HepG2 cell line. • The inhibitory effect of LRD-22 is due to induction of apoptosis and cell cycle arrest. • LRD-22 inhibits Aurora-A phosphorylation which results in subsequent impairment of the p53 pathway. • LRD-22 suppresses tumor growth in xenograft mice without body weight loss.« less

Selective antiproliferative and apoptotic effects of flavonoids purified from Rhus verniciflua Stokes on normal versus transformed hepatic cell lines.

PubMed

Son, Young-Ok; Lee, Kyung-Yeol; Lee, Jeong-Chae; Jang, Hyon-Seok; Kim, Jong-Ghee; Jeon, Young-Mi; Jang, Yong-Suk

2005-01-15

Considerable attention is being concentrated on dietary flavonoids in developing novel cancer-preventive approaches due to their potential ability to induce selective apoptosis of cancer cells. In this study, we prepared a flavonoid-containing fraction from a crude acetone extract of Rhus verniciflua Stokes (RVS), traditionally used as a food additive and as an herbal medicine, and named RVS chloroform-methanol fraction (RCMF). We evaluated the effects of RCMF on proliferation and apoptosis using mouse embryonic primary hepatic cells (MPHC), embryonic normal hepatic cell line (BNL CL.2), and its SV40-mediated transformed cell line (BNL SV A.8). We also investigated the effects of RCMF on the antioxidant defense system in those cells. This study demonstrated that RCMF exhibited a selective growth inhibition and apoptosis induction on transformed cells. BNL SV A.8 cells were more sensitive to RCMF-mediated cytotoxicity than were MPHC or BNL CL.2. RCMF-mediated reduction of MnSOD activity and glutathione (GSH) content in BNL SV A.8 cells is thought to be associated with RCMF-induced apoptosis. Our findings suggest that RCMF is an agent which may be capable of inducing growth inhibition and apoptosis of hepatic tumor cells.

Accumulation of prohibitin is a common cellular response to different stressing stimuli and protects melanoma cells from ER stress and chemotherapy-induced cell death

PubMed Central

Tortelli, Tharcisio Citrangulo; de Godoy, Lyris Martins Franco; de Souza, Gustavo Antonio; Bonatto, Diego; Otake, Andreia Hanada; de Freitas Saito, Renata; Rosa, Jose Cesar; Greene, Lewis Joel; Chammas, Roger

2017-01-01

Melanoma is responsible for most deaths among skin cancers and conventional and palliative care chemotherapy are limited due to the development of chemoresistance. We used proteomic analysis to identify cellular responses that lead to chemoresistance of human melanoma cell lines to cisplatin. A systems approach to the proteomic data indicated the participation of specific cellular processes such as oxidative phosphorylation, mitochondrial organization and homeostasis, as well as the unfolded protein response (UPR) to be required for the survival of cells treated with cisplatin. Prohibitin (PHB) was among the proteins consistently accumulated, interacting with the functional clusters associated with resistance to cisplatin. We showed PHB accumulated at different levels in melanoma cell lines under stressing stimuli, such as (i) treatment with temozolomide (TMZ), dacarbazine (DTIC) and cisplatin; (ii) serum deprivation; (iii) tunicamycin, an UPR inducer. Prohibitin accumulated in the mitochondria of melanoma cells after cisplatin and tunicamycin treatment and its de novo accumulation led to chemoresistance melanoma cell lines. In contrast, PHB knock-down sensitized melanoma cells to cisplatin and tunicamycin treatment. We conclude that PHB participates in the survival of cells exposed to different stress stimuli, and can therefore serve as a target for the sensitization of melanoma cells to chemotherapy. PMID:28562344

Quantitative High-throughput Luciferase Screening in Identifying CAR Modulators

PubMed Central

Lynch, Caitlin; Zhao, Jinghua; Wang, Hongbing; Xia, Menghang

2017-01-01

Summary The constitutive androstane receptor (CAR, NR1I3) is responsible for the transcription of multiple drug metabolizing enzymes and transporters. There are two possible methods of activation for CAR, direct ligand binding and a ligand-independent method, which makes this a unique nuclear receptor. Both of these mechanisms require translocation of CAR from the cytoplasm into the nucleus. Interestingly, CAR is constitutively active in immortalized cell lines due to the basal nuclear location of this receptor. This creates an important challenge in most in vitro assay models because immortalized cells cannot be used without inhibiting the basal activity. In this book chapter, we go into detail of how to perform quantitative high-throughput screens to identify hCAR1 modulators through the employment of a double stable cell line. Using this line, we are able to identify activators, as well as deactivators, of the challenging nuclear receptor, CAR. PMID:27518621

Quantitative High-Throughput Luciferase Screening in Identifying CAR Modulators.

PubMed

Lynch, Caitlin; Zhao, Jinghua; Wang, Hongbing; Xia, Menghang

2016-01-01

The constitutive androstane receptor (CAR, NR1I3) is responsible for the transcription of multiple drug metabolizing enzymes and transporters. There are two possible methods of activation for CAR, direct ligand binding and a ligand-independent method, which makes this a unique nuclear receptor. Both of these mechanisms require translocation of CAR from the cytoplasm into the nucleus. Interestingly, CAR is constitutively active in immortalized cell lines due to the basal nuclear location of this receptor. This creates an important challenge in most in vitro assay models because immortalized cells cannot be used without inhibiting the high basal activity. In this book chapter, we go into detail of how to perform quantitative high-throughput screens to identify hCAR1 modulators through the employment of a double stable cell line. Using this line, we are able to identify activators, as well as deactivators, of the challenging nuclear receptor, CAR.

Patau syndrome with long survival in a case of unusual mosaic trisomy 13.

PubMed

Fogu, Giuseppina; Maserati, Emanuela; Cambosu, Francesca; Moro, Maria Antonietta; Poddie, Fausto; Soro, Giovanna; Bandiera, Pasquale; Serra, Gigliola; Tusacciu, Gianni; Sanna, Giuseppina; Mazzarello, Vittorio; Montella, Andrea

2008-01-01

We report a 12-year-old patient with Patau syndrome, in whom two cell lines were present from birth, one with total trisomy 13 due to isochromosome (13q), and one with partial trisomy 13. A cytogenetic re-evaluation at 9 years of age brought to light in skin fibroblasts a third cell line, partially monosomic for chromosome 13. The derivatives (13) present in the three cell lines were characterized through fluorescence in situ hybridization (FISH) experiments with suitable probes; the results suggested a sequence of rearrangements which beginning from an isochromosome (13q) could have led to the other two derivatives. We report the clinical data at birth and at the age of 12; at this age pigmentary lesions with phylloid pattern were noted. Cytogenetic findings of the chromosomal analyses on different tissues, including skin fibroblasts from differently pigmented areas, are also reported.

Hybrid proline-rich proteins: novel players in plant cell elongation?

PubMed Central

Dvořáková, Lenka; Srba, Miroslav; Opatrny, Zdenek; Fischer, Lukas

2012-01-01

Background and Aims Hybrid proline-rich proteins (HyPRPs) represent a large family of putative cell-wall proteins characterized by the presence of a variable N-terminal domain and a conserved C-terminal domain that is related to non-specific lipid transfer proteins. The function of HyPRPs remains unclear, but their widespread occurrence and abundant expression patterns indicate that they may be involved in a basic cellular process. Methods To elucidate the cellular function of HyPRPs, we modulated the expression of three HyPRP genes in tobacco (Nicotiana tabacum) BY-2 cell lines and in potato (Solanum tuberosum) plants. Key Results In BY-2 lines, over-expression of the three HyPRP genes with different types of N-terminal domains resulted in similar phenotypic changes, namely increased cell elongation, both in suspension culture and on solid media where the over-expression resulted in enhanced calli size. The over-expressing cells showed increased plasmolysis in a hypertonic mannitol solution and accelerated rate of protoplast release, suggesting loosening of the cell walls. In contrast to BY-2 lines, no phenotypic changes were observed in potato plants over-expressing the same or analogous HyPRP genes, presumably due to more complex compensatory mechanisms in planta. Conclusions Based on the results from BY-2 lines, we propose that HyPRPs, more specifically their C-terminal domains, represent a novel group of proteins involved in cell expansion. PMID:22028464

Loss of the deubiquitylase BAP1 alters class I histone deacetylase expression and sensitivity of mesothelioma cells to HDAC inhibitors

PubMed Central

Sacco, Joseph J.; Kenyani, Jenna; Butt, Zohra; Carter, Rachel; Chew, Hui Yi; Cheeseman, Liam P.; Darling, Sarah; Denny, Michael; Urbé, Sylvie; Clague, Michael J.; Coulson, Judy M.

2015-01-01

Histone deacetylases are important targets for cancer therapeutics, but their regulation is poorly understood. Our data show coordinated transcription of HDAC1 and HDAC2 in lung cancer cell lines, but suggest HDAC2 protein expression is cell-context specific. Through an unbiased siRNA screen we found that BRCA1-associated protein 1 (BAP1) regulates their expression, with HDAC2 reduced and HDAC1 increased in BAP1 depleted cells. BAP1 loss-of-function is increasingly reported in cancers including thoracic malignancies, with frequent mutation in malignant pleural mesothelioma. Endogenous HDAC2 directly correlates with BAP1 across a panel of lung cancer cell lines, and is downregulated in mesothelioma cell lines with genetic BAP1 inactivation. We find that BAP1 regulates HDAC2 by increasing transcript abundance, rather than opposing its ubiquitylation. Importantly, although total cellular HDAC activity is unaffected by transient depletion of HDAC2 or of BAP1 due to HDAC1 compensation, this isoenzyme imbalance sensitizes MSTO-211H cells to HDAC inhibitors. However, other established mesothelioma cell lines with low endogenous HDAC2 have adapted to become more resistant to HDAC inhibition. Our work establishes a mechanism by which BAP1 loss alters sensitivity of cancer cells to HDAC inhibitors. Assessment of BAP1 and HDAC expression may ultimately help identify patients likely to respond to HDAC inhibitors. PMID:25970771

Oncolytic Activity of Avian Influenza Virus in Human Pancreatic Ductal Adenocarcinoma Cell Lines

PubMed Central

Pizzuto, Matteo S.; Silic-Benussi, Micol; Pavone, Silvia; Ciminale, Vincenzo; Capua, Ilaria

2014-01-01

ABSTRACT Pancreatic ductal adenocarcinoma (PDA) is the most lethal form of human cancer, with dismal survival rates due to late-stage diagnoses and a lack of efficacious therapies. Building on the observation that avian influenza A viruses (IAVs) have a tropism for the pancreas in vivo, the present study was aimed at testing the efficacy of IAVs as oncolytic agents for killing human PDA cell lines. Receptor characterization confirmed that human PDA cell lines express the alpha-2,3- and the alpha-2,6-linked glycan receptor for avian and human IAVs, respectively. PDA cell lines were sensitive to infection by human and avian IAV isolates, which is consistent with this finding. Growth kinetic experiments showed preferential virus replication in PDA cells over that in a nontransformed pancreatic ductal cell line. Finally, at early time points posttreatment, infection with IAVs caused higher levels of apoptosis in PDA cells than gemcitabine and cisplatin, which are the cornerstone of current therapies for PDA. In the BxPC-3 PDA cell line, apoptosis resulted from the engagement of the intrinsic mitochondrial pathway. Importantly, IAVs did not induce apoptosis in nontransformed pancreatic ductal HPDE6 cells. Using a model based on the growth of a PDA cell line as a xenograft in SCID mice, we also show that a slightly pathogenic avian IAV significantly inhibited tumor growth following intratumoral injection. Taken together, these results are the first to suggest that IAVs may hold promise as future agents of oncolytic virotherapy against pancreatic ductal adenocarcinomas. IMPORTANCE Despite intensive studies aimed at designing new therapeutic approaches, PDA still retains the most dismal prognosis among human cancers. In the present study, we provide the first evidence indicating that avian IAVs of low pathogenicity display a tropism for human PDA cells, resulting in viral RNA replication and a potent induction of apoptosis in vitro and antitumor effects in vivo. These results suggest that slightly pathogenic IAVs may prove to be effective for oncolytic virotherapy of PDA and provide grounds for further studies to develop specific and targeted viruses, with the aim of testing their efficacy in clinical contexts. PMID:24899201

Inhibition of latent membrane protein 1 impairs the growth and tumorigenesis of latency II Epstein-Barr virus-transformed T cells.

PubMed

Ndour, Papa Alioune; Brocqueville, Guillaume; Ouk, Tan-Sothéa; Goormachtigh, Gautier; Morales, Olivier; Mougel, Alexandra; Bertout, Julie; Melnyk, Oleg; Fafeur, Véronique; Feuillard, Jean; Coll, Jean; Adriaenssens, Eric

2012-04-01

Epstein-Barr virus (EBV) is a common human herpesvirus. Infection with EBV is associated with several human malignancies in which the virus expresses a set of latent proteins, among which is latent membrane protein 1 (LMP1). LMP1 is able to transform numerous cell types and is considered the main oncogenic protein of EBV. The mechanism of action is based on mimicry of activated members of the tumor necrosis factor (TNF) receptor superfamily, through the ability of LMP1 to bind similar adapters and to activate signaling pathways. We previously generated two unique models: a monocytic cell line and a lymphocytic (NC5) cell line immortalized by EBV that expresses the type II latency program. Here we generated LMP1 dominant negative forms (DNs), based on fusion between green fluorescent protein (GFP) and transformation effector site 1 (TES1) or TES2 of LMP1. Then we generated cell lines conditionally expressing these DNs. These DNs inhibit NF-κB and Akt pathways, resulting in the impairment of survival processes and increased apoptosis in these cell lines. This proapoptotic effect is due to reduced interaction of LMP1 with specific adapters and the recruitment of these adapters to DNs, which enable the generation of an apoptotic complex involving TRADD, FADD, and caspase 8. Similar results were obtained with cell lines displaying a latency III program in which LMP1-DNs decrease cell viability. Finally, we prove that synthetic peptides display similar inhibitory effects in EBV-infected cells. DNs derived from LMP1 could be used to develop therapeutic approaches for malignant diseases associated with EBV.

Inhibition of Latent Membrane Protein 1 Impairs the Growth and Tumorigenesis of Latency II Epstein-Barr Virus-Transformed T Cells

PubMed Central

Ndour, Papa Alioune; Brocqueville, Guillaume; Ouk, Tan-Sothéa; Goormachtigh, Gautier; Morales, Olivier; Mougel, Alexandra; Bertout, Julie; Melnyk, Oleg; Fafeur, Véronique; Feuillard, Jean; Coll, Jean

2012-01-01

Epstein-Barr virus (EBV) is a common human herpesvirus. Infection with EBV is associated with several human malignancies in which the virus expresses a set of latent proteins, among which is latent membrane protein 1 (LMP1). LMP1 is able to transform numerous cell types and is considered the main oncogenic protein of EBV. The mechanism of action is based on mimicry of activated members of the tumor necrosis factor (TNF) receptor superfamily, through the ability of LMP1 to bind similar adapters and to activate signaling pathways. We previously generated two unique models: a monocytic cell line and a lymphocytic (NC5) cell line immortalized by EBV that expresses the type II latency program. Here we generated LMP1 dominant negative forms (DNs), based on fusion between green fluorescent protein (GFP) and transformation effector site 1 (TES1) or TES2 of LMP1. Then we generated cell lines conditionally expressing these DNs. These DNs inhibit NF-κB and Akt pathways, resulting in the impairment of survival processes and increased apoptosis in these cell lines. This proapoptotic effect is due to reduced interaction of LMP1 with specific adapters and the recruitment of these adapters to DNs, which enable the generation of an apoptotic complex involving TRADD, FADD, and caspase 8. Similar results were obtained with cell lines displaying a latency III program in which LMP1-DNs decrease cell viability. Finally, we prove that synthetic peptides display similar inhibitory effects in EBV-infected cells. DNs derived from LMP1 could be used to develop therapeutic approaches for malignant diseases associated with EBV. PMID:22258264

PLGA-CTAB curcumin nanoparticles: Fabrication, characterization and molecular basis of anticancer activity in triple negative breast cancer cell lines (MDA-MB-231 cells).

PubMed

Meena, Ramovatar; Kumar, Sumit; Kumar, Raj; Gaharwar, Usha Singh; Rajamani, Paulraj

2017-10-01

Triple-negative breast cancers (TNBC) are aggressive cancers, which do not control by hormonal therapy or therapies that target HER-2 receptors. Curcumin (Cur) has shown cytotoxic effects in multiple cancer cell lines. However, its medical uses remain limited due to low aqueous solubility and poor bioavailability. Therefore, present study was aimed to fabricate the small positive charge curcumin nanoparticles (CN) by nanoprecipitation methods using PLGA and CTAB, and to evaluate its anticancer efficacy and underlying the mechanism in triple negative breast cancer cell lines (MDA-MB-231 cells). In in-vitro drug release assay, Cur was released from CN by flicking diffusion and anomalous transport process. CN showed a higher cellular incorporation than free Cur resulted in higher cytotoxicity. Checking the anticancer activity at the molecular level, Cur has shown to induce the reactive oxygen species production that subsequently causes the DNA damage and resulting in p38-MAPK activation. The p38-MAPK induce the expression of p16 /INKK4a , p21 /waf1/cip1 and p53 resulting in a reduction in the level of CDK2, CDK4, cyclin D1 and cyclin E and subsequently cell cycle arrest at G1/S and G2/M phase. It also reduces the expression of DNA repair gene, i.e. BRCA1, BRCA2, Rad51, Rad50, Mre11 and NBS1 resulting in apoptosis induction due to persistent DNA damage. This study presents an effective delivery of curcumin in TNBC cancer cells and it could open the new frontiers in clinical cancer chemotherapy. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Expression of recombinant sea urchin cellulase SnEG54 using mammalian cell lines.

PubMed

Okumura, Fumihiko; Kameda, Hiroyuki; Ojima, Takao; Hatakeyama, Shigetsugu

2010-05-07

We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci

PubMed Central

Philippe, Claude; Vargas-Landin, Dulce B; Doucet, Aurélien J; van Essen, Dominic; Vera-Otarola, Jorge; Kuciak, Monika; Corbin, Antoine; Nigumann, Pilvi; Cristofari, Gaël

2016-01-01

LINE-1 (L1) retrotransposons represent approximately one sixth of the human genome, but only the human-specific L1HS-Ta subfamily acts as an endogenous mutagen in modern humans, reshaping both somatic and germline genomes. Due to their high levels of sequence identity and the existence of many polymorphic insertions absent from the reference genome, the transcriptional activation of individual genomic L1HS-Ta copies remains poorly understood. Here we comprehensively mapped fixed and polymorphic L1HS-Ta copies in 12 commonly-used somatic cell lines, and identified transcriptional and epigenetic signatures allowing the unambiguous identification of active L1HS-Ta copies in their genomic context. Strikingly, only a very restricted subset of L1HS-Ta loci - some being polymorphic among individuals - significantly contributes to the bulk of L1 expression, and these loci are differentially regulated among distinct cell lines. Thus, our data support a local model of L1 transcriptional activation in somatic cells, governed by individual-, locus-, and cell-type-specific determinants. DOI: http://dx.doi.org/10.7554/eLife.13926.001 PMID:27016617

A panel of induced pluripotent stem cells from chimpanzees: a resource for comparative functional genomics

PubMed Central

Gallego Romero, Irene; Pavlovic, Bryan J; Hernando-Herraez, Irene; Zhou, Xiang; Ward, Michelle C; Banovich, Nicholas E; Kagan, Courtney L; Burnett, Jonathan E; Huang, Constance H; Mitrano, Amy; Chavarria, Claudia I; Friedrich Ben-Nun, Inbar; Li, Yingchun; Sabatini, Karen; Leonardo, Trevor R; Parast, Mana; Marques-Bonet, Tomas; Laurent, Louise C; Loring, Jeanne F; Gilad, Yoav

2015-01-01

Comparative genomics studies in primates are restricted due to our limited access to samples. In order to gain better insight into the genetic processes that underlie variation in complex phenotypes in primates, we must have access to faithful model systems for a wide range of cell types. To facilitate this, we generated a panel of 7 fully characterized chimpanzee induced pluripotent stem cell (iPSC) lines derived from healthy donors. To demonstrate the utility of comparative iPSC panels, we collected RNA-sequencing and DNA methylation data from the chimpanzee iPSCs and the corresponding fibroblast lines, as well as from 7 human iPSCs and their source lines, which encompass multiple populations and cell types. We observe much less within-species variation in iPSCs than in somatic cells, indicating the reprogramming process erases many inter-individual differences. The low within-species regulatory variation in iPSCs allowed us to identify many novel inter-species regulatory differences of small magnitude. DOI: http://dx.doi.org/10.7554/eLife.07103.001 PMID:26102527

Cell-SELEX-Based Identification of a Human and Mouse Cross-Reactive Endothelial Cell-Internalizing Aptamer.

PubMed

Dua, Pooja; Kang, Sinae; Shin, Hye-Soo; Kim, Soyoun; Lee, Dong-Ki

2018-04-02

Increased interest and insights gained by researchers on the roles of endothelial cells in the pathophysiology of cancer, inflammatory, and cardiovascular diseases have led to the design of pharmacological interventions aimed at the endothelium lining in the diseased sites. Toward this end, we used established brain microvascular endothelial cell lines mouse (bEND3), human (hCMEC/D3), and Toggle Cell-SELEX to identify a species cross-reactive, endothelial cell-internalizing aptamer R11-3. This 2'F-modified RNA aptamer is specific for endothelial cells as no internalization was seen with cells of nonendothelial origin. R11-3 was truncated in size, and its potential in endothelial targeted therapeutics was established using VEGFR2 targeting long interfering RNA (liRNA) aptamer chimera. Due to its specificity for both mouse and human endothelial cells, we believe that this aptamer not only fits for development of endothelial targeted drug development for human diseases but is also suitable for preclinical evaluation in mice.

Determination of the optimum conditions for lung cancer cells treatment using cold atmospheric plasma

NASA Astrophysics Data System (ADS)

Akhlaghi, Morteza; Rajaei, Hajar; Mashayekh, Amir Shahriar; Shafiae, Mojtaba; Mahdikia, Hamed; Khani, Mohammadreza; Hassan, Zuhair Mohammad; Shokri, Babak

2016-10-01

Cold atmospheric plasmas (CAPs) can affect live cells and organisms due to the production of different reactive species. In this paper, the effects of various parameters of the CAP such as the treatment time, gas mixture, gas flow rate, applied voltage, and distance from the nozzle on the LL/2 lung cancer cell line have been studied. The probable effect of UV radiation has also been investigated using an MgF2 filter. Besides the cancerous cells, the 3T3 fibroblast cell line as a normal cell has been treated with the CAP. The Methylthiazol Tetrazolium assay showed that all parameters except the gas flow rate could impress effectively on the cancer cell viability. The cell proliferation seemed to be stopped after plasma treatment. The flow cytometry assay revealed that apoptosis and necrosis were appreciable. It was also found that treating time up to 2 min will not exert any effect on the normal cells.

Hexavalent chromium induces chromosome instability in human urothelial cells

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

Wise, Sandra S.; Holmes, Amie L.; Department of Radiation Oncology, Dana Farber Cancer Institute, 450 Brookline Ave., Boston, MA 02215

Numerous metals are well-known human bladder carcinogens. Despite the significant occupational and public health concern of metals and bladder cancer, the carcinogenic mechanisms remain largely unknown. Chromium, in particular, is a metal of concern as incidences of bladder cancer have been found elevated in chromate workers, and there is an increasing concern for patients with metal hip implants. However, the impact of hexavalent chromium (Cr(VI)) on bladder cells has not been studied. We compared chromate toxicity in two bladder cell lines; primary human urothelial cells and hTERT-immortalized human urothelial cells. Cr(VI) induced a concentration- and time-dependent increase in chromosome damagemore » in both cell lines, with the hTERT-immortalized cells exhibiting more chromosome damage than the primary cells. Chronic exposure to Cr(VI) also induced a concentration-dependent increase in aneuploid metaphases in both cell lines which was not observed after a 24 h exposure. Aneuploidy induction was higher in the hTERT-immortalized cells. When we correct for uptake, Cr(VI) induces a similar amount of chromosome damage and aneuploidy suggesting that the differences in Cr(VI) sensitivity between the two cells lines were due to differences in uptake. The increase in chromosome instability after chronic chromate treatment suggests this may be a mechanism for chromate-induced bladder cancer, specifically, and may be a mechanism for metal-induced bladder cancer, in general. - Highlights: • Hexavalent chromium is genotoxic to human urothelial cells. • Hexavalent chromium induces aneuploidy in human urothelial cells. • hTERT-immortalized human urothelial cells model the effects seen in primary urothelial cells. • Hexavalent chromium has a strong likelihood of being carcinogenic for bladder tissue.« less

RNA interference of argininosuccinate synthetase restores sensitivity to recombinant arginine deiminase (rADI) in resistant cancer cells

PubMed Central

2011-01-01

Background Sensitivity of cancer cells to recombinant arginine deiminase (rADI) depends on expression of argininosuccinate synthetase (AS), a rate-limiting enzyme in synthesis of arginine from citrulline. To understand the efficiency of RNA interfering of AS in sensitizing the resistant cancer cells to rADI, the down regulation of AS transiently and permanently were performed in vitro, respectively. Methods We studied the use of down-regulation of this enzyme by RNA interference in three human cancer cell lines (A375, HeLa, and MCF-7) as a way to restore sensitivity to rADI in resistant cells. The expression of AS at levels of mRNA and protein was determined to understand the effect of RNA interference. Cell viability, cell cycle, and possible mechanism of the restore sensitivity of AS RNA interference in rADI treated cancer cells were evaluated. Results AS DNA was present in all cancer cell lines studied, however, the expression of this enzyme at the mRNA and protein level was different. In two rADI-resistant cell lines, one with endogenous AS expression (MCF-7 cells) and one with induced AS expression (HeLa cells), AS small interference RNA (siRNA) inhibited 37-46% of the expression of AS in MCF-7 cells. ASsiRNA did not affect cell viability in MCF-7 which may be due to the certain amount of residual AS protein. In contrast, ASsiRNA down-regulated almost all AS expression in HeLa cells and caused cell death after rADI treatment. Permanently down-regulated AS expression by short hairpin RNA (shRNA) made MCF-7 cells become sensitive to rADI via the inhibition of 4E-BP1-regulated mTOR signaling pathway. Conclusions Our results demonstrate that rADI-resistance can be altered via AS RNA interference. Although transient enzyme down-regulation (siRNA) did not affect cell viability in MCF-7 cells, permanent down-regulation (shRNA) overcame the problem of rADI-resistance due to the more efficiency in AS silencing. PMID:21453546

Glioblastoma entities express subtle differences in molecular composition and response to treatment

PubMed Central

Balça-Silva, Joana; Matias, Diana; Do Carmo, Anália; Dubois, Luiz Gustavo; Gonçalves, Ana Cristina; Girão, Henrique; Silva Canedo, Nathalie Henriques; Correia, Ana Helena; De Souza, Jorge Marcondes; Sarmento-Ribeiro, Ana Bela; Lopes, Maria Celeste; Moura-Neto, Vivaldo

2017-01-01

Glioblastoma (GBM) is a grade IV astrocytoma. GBM patients show resistance to chemotherapy such as temozolomide (TMZ), the gold standard treatment. In order to simulate the molecular mechanisms behind the different chemotherapeutic responses in GBM patients we compared the cellular heterogeneity and chemotherapeutic resistance mechanisms in different GBM cell lines. We isolated and characterized a human GBM cell line obtained from a GBM patient, named GBM11. We studied the GBM11 behaviour when treated with Tamoxifen (TMX) that, among other functions, is a protein kinase C (PKC) inhibitor, alone and in combination with TMZ in comparison with the responses of U87 and U118 human GBM cell lines. We evaluated the cell death, cell cycle arrest and cell proliferation, mainly through PKC expression, by flow cytometry and western blot analysis and, ultimately, cell migration capability and F-actin filament disorganization by fluorescence microscopy. We demonstrated that the constitutive activation of p-PKC seems to be one of the main metabolic implicated on GBM malignancy. Despite of its higher resistance, possibly due to the overexpression of P-glycoprotein and stem-like cell markers, GBM11 cells presented a subtle different chemotherapeutic response compared to U87 and U118 cells. The GBM11, U87, U118 cell lines show subtle molecular differences, which clearly indicate the characterization of GBM heterogeneity, one of the main reasons for tumor resistance. The adding of cellular heterogeneity in molecular behaviour constitutes a step closer in the understanding of resistant molecular mechanisms in GBM, and can circumvents the eventual impaired therapy. PMID:28714013

Stromelysin-3 over-expression enhances tumourigenesis in MCF-7 and MDA-MB-231 breast cancer cell lines: involvement of the IGF-1 signalling pathway

PubMed Central

Kasper, Grit; Reule, Matthias; Tschirschmann, Miriam; Dankert, Niels; Stout-Weider, Karen; Lauster, Roland; Schrock, Evelin; Mennerich, Detlev; Duda, Georg N; Lehmann, Kerstin E

2007-01-01

Background Stromelysin-3 (ST-3) is over-expressed in the majority of human carcinomas including breast carcinoma. Due to its known effect in promoting tumour formation, but its impeding effect on metastasis, a dual role of ST-3 in tumour progression, depending on the cellular grade of dedifferentiation, was hypothesized. Methods The present study was designed to investigate the influence of ST-3 in vivo and in vitro on the oestrogen-dependent, non-invasive MCF-7 breast carcinoma cell line as well as on the oestrogen-independent, invasive MDA-MB-231 breast carcinoma cell line. Therefore an orthotopic human xenograft tumour model in nude mice, as well as a 3D matrigel cell culture system, were employed. Results Using both in vitro and in vivo techniques, we have demonstrated that over-expression of ST-3 in MCF-7 and MDA-MB-231 cells leads to both increased cell numbers and tumour volumes. This observation was dependent upon the presence of growth factors. In particular, the enhanced proliferative capacity was in MCF-7/ST-3 completely and in MDA-MB-231/ST-3 cells partially dependent on the IGF-1 signalling pathway. Microarray analysis of ST-3 over-expressing cells revealed that in addition to cell proliferation, further biological processes seemed to be affected, such as cell motility and stress response. The MAPK-pathway as well as the Wnt and PI3-kinase pathways, appear to also play a potential role. Furthermore, we have demonstrated that breast cancer cell lines of different differentiation status, as well as the non-tumourigenic cell line MCF-10A, have a comparable capability to induce endogenous ST-3 expression in fibroblasts. Conclusion These data reveal that ST-3 is capable of enhancing tumourigenesis in highly differentiated "early stage" breast cancer cell lines as well as in further progressed breast cancer cell lines that have already undergone epithelial-mesenchymal transition. We propose that ST-3 induction in tumour fibroblasts leads to the stimulation of the IGF-1R pathway in carcinoma cells, thus enhancing their proliferative capacity. In addition, further different cellular processes seem to be activated by ST-3, possibly accounting for the dual role of ST-3 in tumour progression and metastasis. PMID:17233884

Evaluation of anticancer potential of Bacopa monnieri L. against MCF-7 and MDA-MB 231 cell line

PubMed Central

Mallick, Md. Nasar; Akhtar, Md. Salman; Najm, Mohd. Zeeshan; Tamboli, E. T.; Ahmad, Sayeed; Husain, Syed Akhtar

2015-01-01

Background: The ethanolic extract of Bacopa monnieri contains bacoside A and B, brahmin, cucurbitacins, and betulinic acid. Currently, cucurbitacins have also been reported for their strong anti-tumorigenic and anti-proliferative activity by inducing cell cycle arrest at the G2/M phase and formation of multiplied cells. The present study was carried out to evaluate the in vitro cytotoxic activity of ethanolic extract of dichloromethane (DCM) fraction of B. monnieri on two different cell lines. Materials and Methods: The ethanolic extract of B. monnieri was prepared using soxhlet extraction method and different fractions (hexane, DCM, methanol, acetone, and water) of ethanolic extracts were prepared. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of ethanolic extract and of all fractions was carried out on MCF-7 and MDA-MB 231 cell lines. The presence of cucurbitacins and betulinic acid in these fractions was confirmed by high-performance thin layer chromatography. Results: The IC50 values of ethanolic extract of B. monnieri in MCF-7 and MDA-MB 231 cell lines were 72.0 μg/mL and 75.0 μg/mL, respectively. The DCM fraction of B. monnieri showed maximum cytotoxic activity among all fraction upto 72 h and was found to be 57.0 μg/mL and 42.0 μg/mL, respectively. Conclusion: The results showed good cytotoxic activity in DCM fraction in both the cell lines may be due to the presence of cucurbitacins and betulinic acid in DCM fraction. PMID:26681894

Evaluation of anticancer potential of Bacopa monnieri L. against MCF-7 and MDA-MB 231 cell line.

PubMed

Mallick, Md Nasar; Akhtar, Md Salman; Najm, Mohd Zeeshan; Tamboli, E T; Ahmad, Sayeed; Husain, Syed Akhtar

2015-01-01

The ethanolic extract of Bacopa monnieri contains bacoside A and B, brahmin, cucurbitacins, and betulinic acid. Currently, cucurbitacins have also been reported for their strong anti-tumorigenic and anti-proliferative activity by inducing cell cycle arrest at the G2/M phase and formation of multiplied cells. The present study was carried out to evaluate the in vitro cytotoxic activity of ethanolic extract of dichloromethane (DCM) fraction of B. monnieri on two different cell lines. The ethanolic extract of B. monnieri was prepared using soxhlet extraction method and different fractions (hexane, DCM, methanol, acetone, and water) of ethanolic extracts were prepared. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of ethanolic extract and of all fractions was carried out on MCF-7 and MDA-MB 231 cell lines. The presence of cucurbitacins and betulinic acid in these fractions was confirmed by high-performance thin layer chromatography. The IC50 values of ethanolic extract of B. monnieri in MCF-7 and MDA-MB 231 cell lines were 72.0 μg/mL and 75.0 μg/mL, respectively. The DCM fraction of B. monnieri showed maximum cytotoxic activity among all fraction upto 72 h and was found to be 57.0 μg/mL and 42.0 μg/mL, respectively. The results showed good cytotoxic activity in DCM fraction in both the cell lines may be due to the presence of cucurbitacins and betulinic acid in DCM fraction.

Fibronectin Modulates Cell Adhesion and Signaling to Promote Single Cell Migration of Highly Invasive Oral Squamous Cell Carcinoma

PubMed Central

Ramos, Grasieli de Oliveira; Bernardi, Lisiane; Lauxen, Isabel; Sant’Ana Filho, Manoel; Horwitz, Alan Rick; Lamers, Marcelo Lazzaron

2016-01-01

Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad) or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad), plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization. PMID:26978651

Aspects of Salt Tolerance in a NaCl-Selected Stable Cell Line of Citrus sinensis.

PubMed

Ben-Hayyim, G; Kochba, J

1983-07-01

A NaCl-tolerant cell line which was selected from ovular callus of ;Shamouti' orange (Citrus sinensis L. Osbeck) proved to be a true cell line variant. This conclusion is based on the following observations. (a) Cells which have been removed from the selection pressure for at least four passages retain the same NaCl tolerance as do cells which are kept constantly on 0.2 molar NaCl. (b) Na(+) and Cl(-) uptake are considerably lower in salt-tolerant cells (R-10) than in salt-sensitive cells (L-5) at a given external NaCl concentration. (c) Growth of salt-tolerant cells is markedly suppressed upon replacement of NaCl by KCl, whereas the growth of salt-sensitive cells is only slightly affected. Accumulation of K(+) and Cl(-) accompanies the inhibition of growth. Experiments carried out with sodium and potassium sulfate suggest that the toxic effect is due to the accumulated Cl(-). (d) Removal of Ca(2+) from the growth medium severely inhibits the growth of salt-tolerant cells in the presence of NaCl, while it has a minor effect on growth of salt-sensitive cells in the presence of NaCl. (e) Electron micrographs show that the salt-tolerant cells have very big vacuoles when exposed to salt, while the size of the vacuoles of the salt-sensitive cells does not change.

Aspects of Salt Tolerance in a NaCl-Selected Stable Cell Line of Citrus sinensis1

PubMed Central

Ben-Hayyim, Gozal; Kochba, Joshua

1983-01-01

A NaCl-tolerant cell line which was selected from ovular callus of `Shamouti' orange (Citrus sinensis L. Osbeck) proved to be a true cell line variant. This conclusion is based on the following observations. (a) Cells which have been removed from the selection pressure for at least four passages retain the same NaCl tolerance as do cells which are kept constantly on 0.2 molar NaCl. (b) Na+ and Cl− uptake are considerably lower in salt-tolerant cells (R-10) than in salt-sensitive cells (L-5) at a given external NaCl concentration. (c) Growth of salt-tolerant cells is markedly suppressed upon replacement of NaCl by KCl, whereas the growth of salt-sensitive cells is only slightly affected. Accumulation of K+ and Cl− accompanies the inhibition of growth. Experiments carried out with sodium and potassium sulfate suggest that the toxic effect is due to the accumulated Cl−. (d) Removal of Ca2+ from the growth medium severely inhibits the growth of salt-tolerant cells in the presence of NaCl, while it has a minor effect on growth of salt-sensitive cells in the presence of NaCl. (e) Electron micrographs show that the salt-tolerant cells have very big vacuoles when exposed to salt, while the size of the vacuoles of the salt-sensitive cells does not change. Images Fig. 3 PMID:16663067

Effect of the anti-neoplastic drug doxorubicin on XPD-mutated DNA repair-deficient human cells.

PubMed

Saffi, Jenifer; Agnoletto, Mateus H; Guecheva, Temenouga N; Batista, Luís F Z; Carvalho, Helotonio; Henriques, João A P; Stary, Anne; Menck, Carlos F M; Sarasin, Alain

2010-01-02

Doxorubicin (DOX), a member of the anthracycline group, is a widely used drug in cancer therapy. The mechanisms of DOX action include topoisomerase II-poisoning, free radical release, DNA adducts and interstrand cross-link (ICL) formation. Nucleotide excision repair (NER) is involved in the removal of helix-distorting lesions and chemical adducts, however, little is known about the response of NER-deficient cell lines to anti-tumoral drugs like DOX. Wild type and XPD-mutated cells, harbouring mutations in different regions of this gene and leading to XP-D, XP/CS or TTD diseases, were treated with this drug and analyzed for cell cycle arrest and DNA damage by comet assay. The formation of DSBs was also investigated by determination of gammaH2AX foci. Our results indicate that all three NER-deficient cell lines tested are more sensitive to DOX treatment, when compared to wild type cells or XP cells complemented by the wild type XPD cDNA, suggesting that NER is involved in the removal of DOX-induced lesions. The cell cycle analysis showed the characteristic G2 arrest in repair-proficient MRC5 cell line after DOX treatment, whereas the repair-deficient cell lines presented significant increase in sub-G1 fraction. The NER-deficient cell lines do not show different patterns of DNA damage formation as assayed by comet assay and phosphorylated H2AX foci formation. Knock-down of topoisomerase IIalpha with siRNA leads to increased survival in both MRC5 and XP cells, however, XP cell line still remained significantly more sensitive to the treatment by DOX. Our study suggests that the enhanced sensitivity is due to DOX-induced DNA damage that is subject to NER, as we observed decreased unscheduled DNA synthesis in XP-deficient cells upon DOX treatment. Furthermore, the complementation of the XPD-function abolished the observed sensitivity at lower DOX concentrations, suggesting that the XPD helicase activity is involved in the repair of DOX-induced lesions. Copyright (c) 2009 Elsevier B.V. All rights reserved.

MEK and TAK1 Regulate Apoptosis in Colon Cancer Cells with KRAS-Dependent Activation of Proinflammatory Signaling.

PubMed

McNew, Kelsey L; Whipple, William J; Mehta, Anita K; Grant, Trevor J; Ray, Leah; Kenny, Connor; Singh, Anurag

2016-12-01

MEK inhibitors have limited efficacy in treating RAS-RAF-MEK pathway-dependent cancers due to feedback pathway compensation and dose-limiting toxicities. Combining MEK inhibitors with other targeted agents may enhance efficacy. Here, codependencies of MEK, TAK1, and KRAS in colon cancer were investigated. Combined inhibition of MEK and TAK1 potentiates apoptosis in KRAS-dependent cells. Pharmacologic studies and cell-cycle analyses on a large panel of colon cancer cell lines demonstrate that MEK/TAK1 inhibition induces cell death, as assessed by sub-G 1 accumulation, in a distinct subset of cell lines. Furthermore, TAK1 inhibition causes G 2 -M cell-cycle blockade and polyploidy in many of the cell lines. MEK plus TAK1 inhibition causes reduced G 2 -M/polyploid cell numbers and additive cytotoxic effects in KRAS/TAK1-dependent cell lines as well as a subset of BRAF-mutant cells. Mechanistically, sensitivity to MEK/TAK1 inhibition can be conferred by KRAS and BMP receptor activation, which promote expression of NF-κB-dependent proinflammatory cytokines, driving tumor cell survival and proliferation. MEK/TAK1 inhibition causes reduced mTOR, Wnt, and NF-κB signaling in TAK1/MEK-dependent cell lines concomitant with apoptosis. A Wnt/NF-κB transcriptional signature was derived that stratifies primary tumors into three major subtypes: Wnt-high/NF-κB-low, Wnt-low/NF-κB-high and Wnt-high/NF-κB-high, designated W, N, and WN, respectively. These subtypes have distinct characteristics, including enrichment for BRAF mutations with serrated carcinoma histology in the N subtype. Both N and WN subtypes bear molecular hallmarks of MEK and TAK1 dependency seen in cell lines. Therefore, N and WN subtype signatures could be utilized to identify tumors that are most sensitive to anti-MEK/TAK1 therapeutics. This study describes a potential therapeutic strategy for a subset of colon cancers that are dependent on oncogenic KRAS signaling pathways, which are currently difficult to block with selective agents. Mol Cancer Res; 14(12); 1204-16. ©2016 AACR. ©2016 American Association for Cancer Research.

Monocrotophos Induces the Expression of Xenobiotic Metabolizing Cytochrome P450s (CYP2C8 and CYP3A4) and Neurotoxicity in Human Brain Cells.

PubMed

Tripathi, Vinay Kumar; Kumar, Vivek; Pandey, Ankita; Vatsa, Pankhi; Dhasmana, Anupam; Singh, Rajat Pratap; Appikonda, Sri Hari Chandan; Hwang, Inho; Lohani, Mohtashim

2017-07-01

Expression of various cytochrome P450s (CYPs) in mammalian brain cells is well documented. However, such studies are hampered in neural/glial cells of human origin due to nonavailability of human brain cells. To address this issue, we investigated the expression and inducibility of CYP2C8 and CYP3A4 and their responsiveness against cyclophosphamide (CPA) and organophosphorus pesticide monocrotophos (MCP), a known developmental neurotoxicant in human neural (SH-SY5Y) and glial (U373-MG) cell lines. CPA induced significant expression of CYP2C8 and CYP3A4 in both types of cells in a time-dependent manner. Neural cell line exhibited relatively higher constitutive and inducible expression of CYPs than the glial cell line. MCP exposure alone could not induce the significant expression of CYPs, whereas the cells preexposed to CPA showed a significant response to MCP. Similar to the case of CPA induced expressions, neural cells were found to be more vulnerable than glial cells. Our data indicate differential expressions of CYPs in cultured human neural and glial cell lines. The findings were synchronized with protein ligand docking studies, which showed a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR and PXR. Similarly, the known CYP inducer CPA has also shown significant high docking scores with the two studied CYP regulators. We also observed a significant induction in reactive oxygen species (ROS), lipid peroxides (LPO), micronucleus (MN), chromosomal aberration (CA), and reduction in reduced glutathione (GSH) and catalase following the exposure of MCP. Moreover, the expressions of apoptotic markers such as caspase-3, caspase-9, Bax, and p53 were significantly upregulated, whereas the levels of antiapoptotic marker, Bcl2, was downregulated after the exposure of MCP in both cell lines. These findings confirm the involvement of ROS-mediated oxidative stress, which subsequently triggers apoptosis pathways in both human neural (SH-SY5Y) and glial (U373-MG) cell lines following the exposure of MCP.

Capture of cell culture-derived influenza virus by lectins: strain independent, but host cell dependent.

PubMed

Opitz, Lars; Zimmermann, Anke; Lehmann, Sylvia; Genzel, Yvonne; Lübben, Holger; Reichl, Udo; Wolff, Michael W

2008-12-01

Strategies to control influenza outbreaks are focused mainly on prophylactic vaccination. Human influenza vaccines are trivalent blends of different virus subtypes. Therefore and due to frequent antigenic drifts, strain independent manufacturing processes are required for vaccine production. This study verifies the strain independency of a capture method based on Euonymus europaeus lectin-affinity chromatography (EEL-AC) for downstream processing of influenza viruses under various culture conditions propagated in MDCK cells. A comprehensive lectin binding screening was conducted for two influenza virus types from the season 2007/2008 (A/Wisconsin/67/2005, B/Malaysia/2506/2004) including a comparison of virus-lectin interaction by surface plasmon resonance technology. EEL-AC resulted in a reproducible high product recovery rate and a high degree of contaminant removal in the case of both MDCK cell-derived influenza virus types demonstrating clearly the general applicability of EEL-AC. In addition, host cell dependency of EEL-AC was studied with two industrial relevant cell lines: Vero and MDCK cells. However, the choice of the host cell lines is known to lead to different product glycosylation profiles. Hence, altered lectin specificities have been observed between the two cell lines, requiring process adaptations between different influenza vaccine production systems.

Enterolactone induces G1-phase cell cycle arrest in non-small cell lung cancer cells by down-regulating cyclins and cyclin-dependent kinases

PubMed Central

Chikara, Shireen; Lindsey, Kaitlin; Dhillon, Harsharan; Mamidi, Sujan; Kittilson, Jeffrey; Christofidou-Solomidou, Melpo; Reindl, Katie M.

2017-01-01

Flaxseed is a rich source of the plant lignan secoisolariciresinol diglucoside (SDG) which is metabolized into mammalian lignans enterodiol (ED) and enterolactone (EL) in the digestive tract. The anti-cancer properties of these lignans have been demonstrated for various cancer types, but have not been studied for lung cancer. In this study we investigated the anti-cancer effects of EL for several non-small cell lung cancer (NSCLC) cell lines of various genetic backgrounds. EL inhibited the growth of A549, H441, and H520 lung cancer cells in concentration- and time-dependent manners. The anti-proliferative effects of EL for lung cancer cells were not due to enhanced cell death, but rather due to G1-phase cell cycle arrest. Molecular studies revealed that EL- decreased mRNA or protein expression levels of the G1-phase promoters cyclin D1, cyclin E, cyclin-dependent kinases (CDK)-2, -4, and -6, and p-cdc25A; decreased phosphorylated retinoblastoma (p-pRb) protein levels; and simultaneously increased levels of p21WAF1/CIP1, a negative regulator of the G1-phase. The results suggest that EL inhibits the growth of NSCLC cell lines by down-regulating G1-phase cyclins and CDKs, and up-regulating p21WAF1/CIP1, which leads to G1-phase cell cycle arrest. Therefore, EL may hold promise as an adjuvant treatment for lung cancer therapy. PMID:28323486

Enterolactone Induces G1-phase Cell Cycle Arrest in Nonsmall Cell Lung Cancer Cells by Downregulating Cyclins and Cyclin-dependent Kinases.

PubMed

Chikara, Shireen; Lindsey, Kaitlin; Dhillon, Harsharan; Mamidi, Sujan; Kittilson, Jeffrey; Christofidou-Solomidou, Melpo; Reindl, Katie M

2017-01-01

Flaxseed is a rich source of the plant lignan secoisolariciresinol diglucoside (SDG), which is metabolized into mammalian lignans enterodiol (ED) and enterolactone (EL) in the digestive tract. The anticancer properties of these lignans have been demonstrated for various cancer types, but have not been studied for lung cancer. In this study, we investigated the anticancer effects of EL for several nonsmall cell lung cancer (NSCLC) cell lines of various genetic backgrounds. EL inhibited the growth of A549, H441, and H520 lung cancer cells in concentration- and time-dependent manners. The antiproliferative effects of EL for lung cancer cells were not due to enhanced cell death, but rather due to G 1 -phase cell cycle arrest. Molecular studies revealed that EL decreased mRNA or protein expression levels of the G 1 -phase promoters cyclin D1, cyclin E, cyclin-dependent kinases (CDK)-2, -4, and -6, and p-cdc25A; decreased phosphorylated retinoblastoma (p-pRb) protein levels; and simultaneously increased levels of p21 WAF1/CIP1 , a negative regulator of the G 1 phase. The results suggest that EL inhibits the growth of NSCLC cell lines by downregulating G 1 -phase cyclins and CDKs, and upregulating p21 WAF1/CIP1 , which leads to G 1 -phase cell cycle arrest. Therefore, EL may hold promise as an adjuvant treatment for lung cancer therapy.

Cajanus cajan- a source of PPARγ activators leading to anti-inflammatory and cytotoxic effects.

PubMed

Schuster, Roswitha; Holzer, Wolfgang; Doerfler, Hannes; Weckwerth, Wolfram; Viernstein, Helmut; Okonogi, Siriporn; Mueller, Monika

2016-09-14

Cajanus cajan is an important legume crop in the human diet in many parts of the world. Due to its pharmacological properties, C. cajan is, moreover, used in traditional medicine for treating skin diseases, diabetes, inflammatory disorders and various other dysfunctions. In this study, we focused on the role of peroxisome proliferator-activated receptor gamma (PPARγ) as a potential therapeutic target of Cajanus cajan and its main compounds for the treatment of cancer, inflammation and inflammation-related disorders. The anti-inflammatory potential of C. cajan and its bioactive compounds and their cytotoxicity on the human cervical adenocarcinoma cell line HeLa, the human colorectal adenocarcinoma cell line CaCo-2 and the human breast adenocarcinoma cell line MCF-7 were elucidated. C. cajan and its compounds exerted significant anti-inflammatory activity on lipopolysaccharide-stimulated macrophages, showed good cytotoxic effects on the 3 different cancer cell lines and proved PPARγ activity in vitro. The main active compounds were orientin, pinostrobin and vitexin. Cajaninstilbene acid and pinosylvin monomethylether were identified as novel PPARγ activators. Based on these data, C. cajan provides excellent beneficial medicinal attributes and may be used as a potential food or a pharmaceutical supplement.

Sensitivity of imatinib-resistant T315I BCR-ABL CML to a synergistic combination of ponatinib and forskolin treatment.

PubMed

Oaxaca, Derrick M; Yang-Reid, Sun Ah; Ross, Jeremy A; Rodriguez, Georgialina; Staniswalis, Joan G; Kirken, Robert A

2016-09-01

Tyrosine kinase inhibitors (TKIs) have dramatically improved the life expectancy of patients suffering from chronic myeloid leukemia (CML); however, patients will eventually develop resistance to TKI therapy or adverse side effects due to secondary off-target mechanisms associated with TKIs. CML patients exhibiting TKI resistance are at greater risk of developing an aggressive and drug-insensitive disease. Drug-resistant CML typically arises in response to spontaneous mutations within the drug binding sites of the targeted oncoproteins. To better understand the mechanism of drug resistance in TKI-resistant CML patients, the BCR-ABL transformed cell line KCL22 was grown with increasing concentrations of imatinib for a period of 6 weeks. Subsequently, a drug-resistant derivative of the parental KCL22 cell line harboring the T315I gatekeeper mutation was isolated and investigated for TKI drug sensitivity via multi-agent drug screens. A synergistic combination of ponatinib- and forskolin-reduced cell viability was identified in this clinically relevant imatinib-resistant CML cell line, which also proved efficacious in other CML cell lines. In summary, this study provides new insight into the biological underpinnings of BCR-ABL-driven CML and potential rationale for investigating novel treatment strategies for patients with T315I CML.

Bioengineered 2'-fucosyllactose and 3-fucosyllactose inhibit the adhesion of Pseudomonas aeruginosa and enteric pathogens to human intestinal and respiratory cell lines.

PubMed

Weichert, Stefan; Jennewein, Stefan; Hüfner, Eric; Weiss, Christel; Borkowski, Julia; Putze, Johannes; Schroten, Horst

2013-10-01

Human milk oligosaccharides help to prevent infectious diseases in breastfed infants. Larger scale testing, particularly in animal models and human clinical studies, is still limited due to shortened availability of more complex oligosaccharides. The purpose of this study was to evaluate 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL) synthesized by whole-cell biocatalysis for their biological activity in vitro. Therefore, we have tested these oligosaccharides for their inhibitory potential of pathogen adhesion in two different human epithelial cell lines. 2'-FL could inhibit adhesion of Campylobacter jejuni, enteropathogenic Escherichia coli, Salmonella enterica serovar fyris, and Pseudomonas aeruginosa to the intestinal human cell line Caco-2 (reduction of 26%, 18%, 12%, and 17%, respectively), as could be shown for 3-FL (enteropathogenic E coli 29%, P aeruginosa 26%). Furthermore, adherence of P aeruginosa to the human respiratory epithelial cell line A549 was significantly inhibited by 2'-FL and 3-FL (reduction of 24% and 23%, respectively). These results confirm the biological and functional activity of biotechnologically synthesized human milk oligosaccharides. Mass-tailored human milk oligosaccharides could be used in the future to supplement infant formula ingredients or as preventatives to reduce the impact of infectious diseases. © 2013 Elsevier Inc. All rights reserved.

Rapamycin and CHIR99021 Coordinate Robust Cardiomyocyte Differentiation From Human Pluripotent Stem Cells Via Reducing p53-Dependent Apoptosis.

PubMed

Qiu, Xiao-Xu; Liu, Yang; Zhang, Yi-Fan; Guan, Ya-Na; Jia, Qian-Qian; Wang, Chen; Liang, He; Li, Yong-Qin; Yang, Huang-Tian; Qin, Yong-Wen; Huang, Shuang; Zhao, Xian-Xian; Jing, Qing

2017-10-02

Cardiomyocytes differentiated from human pluripotent stem cells can serve as an unexhausted source for a cellular cardiac disease model. Although small molecule-mediated cardiomyocyte differentiation methods have been established, the differentiation efficiency is relatively unsatisfactory in multiple lines due to line-to-line variation. Additionally, hurdles including line-specific low expression of endogenous growth factors and the high apoptotic tendency of human pluripotent stem cells also need to be overcome to establish robust and efficient cardiomyocyte differentiation. We used the H9-human cardiac troponin T-eGFP reporter cell line to screen for small molecules that promote cardiac differentiation in a monolayer-based and growth factor-free differentiation model. We found that collaterally treating human pluripotent stem cells with rapamycin and CHIR99021 during the initial stage was essential for efficient and reliable cardiomyocyte differentiation. Moreover, this method maintained consistency in efficiency across different human embryonic stem cell and human induced pluripotent stem cell lines without specifically optimizing multiple parameters (the efficiency in H7, H9, and UQ1 human induced pluripotent stem cells is 98.3%, 93.3%, and 90.6%, respectively). This combination also increased the yield of cardiomyocytes (1:24) and at the same time reduced medium consumption by about 50% when compared with the previous protocols. Further analysis indicated that inhibition of the mammalian target of rapamycin allows efficient cardiomyocyte differentiation through overcoming p53-dependent apoptosis of human pluripotent stem cells during high-density monolayer culture via blunting p53 translation and mitochondrial reactive oxygen species production. We have demonstrated that mammalian target of rapamycin exerts a stage-specific and multifaceted regulation over cardiac differentiation and provides an optimized approach for generating large numbers of functional cardiomyocytes for disease modeling and in vitro drug screening. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Amine dependence of proliferative activity in two transplantable lines of mouse colonic carcinoma.

PubMed

Tutton, P J; Barkla, D H

1987-01-01

Serotonin, histamine and their antagonists have previously been shown to influence both the cell proliferation rate and the volumetric growth rate of colonic tumours. Of these earlier studies, those on cell proliferation could not distinguish between direct effects on tumour cells and indirect effects on the host, whereas those on the volumetric growth rate of tumours, whilst suggesting an outcome related to the individual properties of the tumour rather than the host, could not distinguish between influences on cell gain, cell loss or stromal changes. In an attempt to distinguish between these possibilities the current experiments on the mitotic rate in two lines of transplantable mouse colonic carcinoma were undertaken. One line of tumour proved to be sensitive to inhibition by a histamine H2 receptor antagonist and a dopamine D2 antagonist but resistant to serotonin antagonists; the inhibition by histamine antagonists was surmountable by co-administration of histamine. The other line proved to be highly sensitive to the inhibitory effects of serotonin antagonist and less so to antagonists of the other two amines and in this case the effect of serotonin antagonists was surmountable by serotonin. These results suggest that the variations between different colonic tumours in the response to amine antagonists is due to differences in the extent of inhibition of cell proliferation rather than differences in cell loss or stromal effects. Thus it appears likely that amine antagonists are able to directly interfere with the proliferation of some colonic tumour cells.

The synthetic peptide CIGB-300 modulates CK2-dependent signaling pathways affecting the survival and chemoresistance of non-small cell lung cancer cell lines.

PubMed

Cirigliano, Stéfano M; Díaz Bessone, María I; Berardi, Damián E; Flumian, Carolina; Bal de Kier Joffé, Elisa D; Perea, Silvio E; Farina, Hernán G; Todaro, Laura B; Urtreger, Alejandro J

2017-01-01

Lung cancer is the most frequently diagnosed cancer and the leading cause of cancer-related deaths worldwide. Up to 80% of cancer patients are classified as non-small-cell lung cancer (NSCLC) and cisplatin remains as the gold standard chemotherapy treatment, despite its limited efficacy due to both intrinsic and acquired resistance. The CK2 is a Ser/Thr kinase overexpressed in various types of cancer, including lung cancer. CIGB-300 is an antitumor peptide with a novel mechanism of action, since it binds to CK2 substrates thus preventing the enzyme activity. The aim of this work was to analyze the effects of CIGB-300 treatment targeting CK2-dependent signaling pathways in NSCLC cell lines and whether it may help improve current chemotherapy treatment. The human NSCLC cell lines NCI-H125 and NIH-A549 were used. Tumor spheroids were obtained through the hanging-drop method. A cisplatin resistant A549 cell line was obtained by chronic administration of cisplatin. Cell viability, apoptosis, immunoblotting, immunofluorescence and luciferase reporter assays were used to assess CIGB-300 effects. A luminescent assay was used to monitor proteasome activity. We demonstrated that CIGB-300 induces an anti-proliferative response both in monolayer- and three-dimensional NSCLC models, presenting rapid and complete peptide uptake. This effect was accompanied by the inhibition of the CK2-dependent canonical NF-κB pathway, evidenced by reduced RelA/p65 nuclear levels and NF-κB protein targets modulation in both lung cancer cell lines, as well as conditionally reduced NF-κB transcriptional activity. In addition, NF-κB modulation was associated with enhanced proteasome activity, possibly through its α7/C8 subunit. Neither the peptide nor a classical CK2 inhibitor affected cytoplasmic β-CATENIN basal levels. Given that NF-κB activation has been linked to cisplatin-induced resistance, we explored whether CIGB-300 could bring additional therapeutic benefits to the standard cisplatin treatment. We established a resistant cell line that showed higher p65 nuclear levels after cisplatin treatment as compared with the parental cell line. Remarkably, the cisplatin-resistant cell line became more sensitive to CIGB-300 treatment. Our data provide new insights into CIGB-300 mechanism of action and suggest clinical potential on current NSCLC therapy.

Towards gene banking amphibian maternal germ lines: short-term incubation, cryoprotectant tolerance and cryopreservation of embryonic cells of the frog, Limnodynastes peronii.

PubMed

Lawson, Bianca; Clulow, Simon; Mahony, Michael J; Clulow, John

2013-01-01

Gene banking is arguably the best method available to prevent the loss of genetic diversity caused by declines in wild populations, when the causes of decline cannot be halted or reversed. For one of the most impacted vertebrate groups, the amphibians, gene banking technologies have advanced considerably, and gametes from the male line can be banked successfully for many species. However, cryopreserving the female germ line remains challenging, with attempts at cryopreserving oocytes unsuccessful due to their large size and yolk content. One possible solution is to target cryopreservation of early embryos that contain the maternal germ line, but consist of smaller cells. Here, we investigate the short term incubation, cryoprotectant tolerance, and cryopreservation of dissociated early embryonic cells from gastrulae and neurulae of the Striped Marsh Frog, Limnodynastes peronii. Embryos were dissociated and cells were incubated for up to 24 hours in various media. Viability of both gastrula and neurula cells remained high (means up to 40-60%) over 24 hours of incubation in all media, although viability was maintained at a higher level in Ca(2+)-free Simplified Amphibian Ringer; low speed centrifugation did not reduce cell viability. Tolerance of dissociated embryonic cells was tested for two cryoprotectants, glycerol and dimethyl sulphoxide; dissociated cells of both gastrulae and neurulae were highly tolerant to both-indeed, cell viability over 24 hours was higher in media containing low-to-medium concentrations than in equivalent cryoprotectant-free media. Viability over 24 hours was lower in concentrations of cryoprotectant higher than 10%. Live cells were recovered following cryopreservation of both gastrula and neurula cells, but only at low rates. Optimal cryodiluents were identified for gastrula and neurula cells. This is the first report of a slow cooling protocol for cryopreservation of amphibian embryonic cells, and sets future research directions for cryopreserving amphibian maternal germ lines.

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

Rafat, M; Bazalova, M; Palma, B

Purpose: To characterize the effect of very rapid dose delivery as compared to conventional therapeutic irradiation times on clonogenic cell survival. Methods: We used a Varian Trilogy linear accelerator to deliver doses up to 10 Gy using a 6 MV SRS photon beam. We irradiated four cancer cell lines in times ranging from 30 sec to 30 min. We also used a Varian TrueBeam linear accelerator to deliver 9 MeV electrons at 10 Gy in 10 s to 30 min to determine the effect of irradiation time on cell survival. We then evaluated the effect of using 60 and 120more » MeV electrons on cell survival using the Next Linear Collider Test Accelerator (NLCTA) beam line at the SLAC National Accelerator Laboratory. During irradiation, adherent cells were maintained at 37oC with 20%O2/5%CO2. Clonogenic assays were completed following irradiation to determine changes in cell survival due to dose delivery time and beam quality, and the survival data were fitted with the linear-quadratic model. Results: Cell lines varied in radiosensitivity, ranging from two to four logs of cell kill at 10 Gy for both conventional and very rapid irradiation. Delivering radiation in shorter times decreased survival in all cell lines. Log differences in cell kill ranged from 0.2 to 0.7 at 10 Gy for the short compared to the long irradiation time. Cell kill differences between short and long irradiations were more pronounced as doses increased for all cell lines. Conclusion: Our findings suggest that shortening delivery of therapeutic radiation doses to less than 1 minute may improve tumor cell kill. This study demonstrates the potential advantage of technologies under development to deliver stereotactic ablative radiation doses very rapidly. Bill Loo and Peter Maxim have received Honoraria from Varian and Research Support from Varian and RaySearch.« less

A magnetic-dependent protein corona of tailor-made superparamagnetic iron oxides alters their biological behaviors

NASA Astrophysics Data System (ADS)

Liu, Ziyao; Zhan, Xiaohui; Yang, Minggang; Yang, Qi; Xu, Xianghui; Lan, Fang; Wu, Yao; Gu, Zhongwei

2016-03-01

In recent years, it is becoming increasingly evident that once nanoparticles come into contact with biological fluids, a protein corona surely forms and critically affects the biological behaviors of nanoparticles. Herein, we investigate whether the formation of protein corona on the surface of superparamagnetic iron oxides (SPIOs) is influenced by static magnetic field. Under static magnetic field, there is no obvious variation in the total amount of protein adsorption, but the proportion of adsorbed proteins significantly changes. Noticeably, certain proteins including apolipoproteins, complement system proteins and acute phase proteins, increase in the protein corona of SPIOs in the magnetic field. More importantly, the magnetic-dependent protein corona of SPIOs enhances the cellular uptake of SPIOs into the normal cell line (3T3 cells) and tumor cell line (HepG2 cells), due to increased adsorption of apolipoprotein. In addition, SPIOs with the magnetic-dependent protein corona cause high cytotoxicity to 3T3 cells and HepG2 cells. This work discloses that superparamagnetism as a key feature of SPIOs affects the composition of protein corona to a large extent, which further alters the biological behaviors of SPIOs.In recent years, it is becoming increasingly evident that once nanoparticles come into contact with biological fluids, a protein corona surely forms and critically affects the biological behaviors of nanoparticles. Herein, we investigate whether the formation of protein corona on the surface of superparamagnetic iron oxides (SPIOs) is influenced by static magnetic field. Under static magnetic field, there is no obvious variation in the total amount of protein adsorption, but the proportion of adsorbed proteins significantly changes. Noticeably, certain proteins including apolipoproteins, complement system proteins and acute phase proteins, increase in the protein corona of SPIOs in the magnetic field. More importantly, the magnetic-dependent protein corona of SPIOs enhances the cellular uptake of SPIOs into the normal cell line (3T3 cells) and tumor cell line (HepG2 cells), due to increased adsorption of apolipoprotein. In addition, SPIOs with the magnetic-dependent protein corona cause high cytotoxicity to 3T3 cells and HepG2 cells. This work discloses that superparamagnetism as a key feature of SPIOs affects the composition of protein corona to a large extent, which further alters the biological behaviors of SPIOs. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08447d

Comparative analysis of TGF-β/Smad signaling dependent cytostasis in human hepatocellular carcinoma cell lines.

PubMed

Dzieran, Johanna; Fabian, Jasmin; Feng, Teng; Coulouarn, Cédric; Ilkavets, Iryna; Kyselova, Anastasia; Breuhahn, Kai; Dooley, Steven; Meindl-Beinker, Nadja M

2013-01-01

Hepatocellular carcinoma (HCC) is a major public health problem due to increased incidence, late diagnosis and limited treatment options. TGF-β is known to provide cytostatic signals during early stages of liver damage and regeneration, but exerts tumor promoting effects in onset and progression of liver cancer. To understand the mechanistic background of such a switch, we systematically correlated loss of cytostatic TGF-β effects with strength and dynamics of its downstream signaling in 10 HCC cell lines. We demonstrate that TGF-β inhibits proliferation and induces apoptosis in cell lines with low endogenous levels of TGF-β and Smad7 and strong transcriptional Smad3 activity (PLC/PRF/5, HepG2, Hep3B, HuH7), previously characterized to express early TGF-β signatures correlated with better outcome in HCC patients. TGF-β dependent cytostasis is blunted in another group of cell lines (HLE, HLF, FLC-4) expressing high amounts of TGF-β and Smad7 and showing significantly reduced Smad3 signaling. Of those, HLE and HLF exhibit late TGF-β signatures, which is associated with bad prognosis in HCC patients. RNAi with Smad3 blunted cytostatic effects in PLC/PRF/5, Hep3B and HuH7. HCC-M and HCC-T represent a third group of cell lines lacking cytostatic TGF-β signaling despite strong and prolonged Smad3 phosphorylation and low Smad7 and TGF-β expression. Inhibitory linker phosphorylation, as in HCC-T, may disrupt C-terminally phosphorylated Smad3 function. In summary, we assort 10 HCC cell lines in at least two clusters with respect to TGF-β sensitivity. Cell lines responsive to the TGF-β cytostatic program, which recapitulate early stage of liver carcinogenesis exhibit transcriptional Smad3 activity. Those with disturbed TGF-β/Smad3 signaling are insensitive to TGF-β dependent cytostasis and might represent late stage of the disease. Regulation of this switch remains complex and cell line specific. These features may be relevant to discriminate stage dependent TGF-β functions for the design of efficient TGF-β directed therapy in liver cancer.

Inhibition of brain tumor cell proliferation by alternating electric fields

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

Jeong, Hyesun; Oh, Seung-ick; Hong, Sunghoi, E-mail: shong21@korea.ac.kr, E-mail: radioyoon@korea.ac.kr

2014-11-17

This study was designed to investigate the mechanism by which electric fields affect cell function, and to determine the optimal conditions for electric field inhibition of cancer cell proliferation. Low-intensity (<2 V/cm) and intermediate-frequency (100–300 kHz) alternating electric fields were applied to glioblastoma cell lines. These electric fields inhibited cell proliferation by inducing cell cycle arrest and abnormal mitosis due to the malformation of microtubules. These effects were significantly dependent on the intensity and frequency of applied electric fields.

CD133 antisense suppresses cancer cell growth and increases sensitivity to cisplatin in vitro.

PubMed

Blancas-Mosqueda, Marisol; Zapata-Benavides, Pablo; Zamora-Ávila, Diana; Saavedra-Alonso, Santiago; Manilla-Muñoz, Edgar; Franco-Molina, Moisés; DE LA Peña, Carmen Mondragón; Rodríguez-Padilla, Cristina

2012-11-01

The increased incidence of cancer in recent years is associated with a high rate of mortality. Numerous types of cancer have a low percentage of CD133(+) cells, which have similar features to stem cells. The CD133 molecule is involved in apoptosis and cell proliferation. The aim of this study was to determine the biological effect of CD133 suppression and its role in the chemosensitization of cancer cell lines. RT-PCR and immunocytochemical analyses indicated that CD133 was expressed in the cancer cell lines B16F10, MCF7 and INER51. Downregulation of CD133 by transfection with an antisense sequence (As-CD133) resulted in a decrease in cancer cell viability of up to 52, 47 and 22% in B16F10, MCF-7 and INER51 cancer cell lines, respectively. This decreased viability appeared to be due to the induction of apoptosis. In addition, treatment with As-CD133 in combination with cisplatin had a synergic effect in all of the cancer cell lines analyzed, and in particular, significantly decreased the viability of B16F10 cancer cells compared with each treatment separately (3.1% viability for the combined treatment compared with 48% for 0.4 μg As-CD133 and 25% for 5 ng/μl cisplatin; P<0.05). The results indicate that the downregulation of CD133 by antisense is a potential therapeutic target for cancer and has a synergistic effect when administered with minimal doses of the chemotherapeutic drug cisplatin, suggesting that this combination strategy may be applied in cancer treatment.

A novel system for the production of high levels of functional human therapeutic proteins in stable cells with a Semliki Forest virus noncytopathic vector.

PubMed

Casales, Erkuden; Aranda, Alejandro; Quetglas, Jose I; Ruiz-Guillen, Marta; Rodriguez-Madoz, Juan R; Prieto, Jesus; Smerdou, Cristian

2010-05-31

Semliki Forest virus (SFV) vectors lead to high protein expression in mammalian cells, but expression is transient due to vector cytopathic effects, inhibition of host cell proteins and RNA-based expression. We have used a noncytopathic SFV mutant (ncSFV) RNA vector to generate stable cell lines expressing two human therapeutic proteins: insulin-like growth factor I (IGF-I) and cardiotrophin-1 (CT-1). Therapeutic genes were fused at the carboxy-terminal end of Puromycin N-acetyl-transferase gene by using as a linker the sequence coding for foot-and-mouth disease virus (FMDV) 2A autoprotease. These cassettes were cloned into the ncSFV vector. Recombinant ncSFV vectors allowed rapid and efficient selection of stable BHK cell lines with puromycin. These cells expressed IGF-I and CT-1 in supernatants at levels reaching 1.4 and 8.6 microg/10(6)cells/24 hours, respectively. Two cell lines generated with each vector were passaged ten times during 30 days, showing constant levels of protein expression. Recombinant proteins expressed at different passages were functional by in vitro signaling assays. Stability at RNA level was unexpectedly high, showing a very low mutation rate in the CT-1 sequence, which did not increase at high passages. CT-1 was efficiently purified from supernatants of ncSFV cell lines, obtaining a yield of approximately 2mg/L/24 hours. These results indicate that the ncSFV vector has a great potential for the production of recombinant proteins in mammalian cells. 2010 Elsevier B.V. All rights reserved.

Predicting skin sensitization potential and inter-laboratory reproducibility of a human Cell Line Activation Test (h-CLAT) in the European Cosmetics Association (COLIPA) ring trials.

PubMed

Sakaguchi, Hitoshi; Ryan, Cindy; Ovigne, Jean-Marc; Schroeder, Klaus R; Ashikaga, Takao

2010-09-01

Regulatory policies in Europe prohibited the testing of cosmetic ingredients in animals for a number of toxicological endpoints. Currently no validated non-animal test methods exist for skin sensitization. Evaluation of changes in cell surface marker expression in dendritic cell (DC)-surrogate cell lines represents one non-animal approach. The human Cell Line Activation Test (h-CLAT) examines the level of CD86 and CD54 expression on the surface of THP-1 cells, a human monocytic leukemia cell line, following 24h of chemical exposure. To examine protocol transferability, between-lab reproducibility, and predictive capacity, the h-CLAT has been evaluated by five independent laboratories in several ring trials (RTs) coordinated by the European Cosmetics Association (COLIPA). The results of the first and second RTs demonstrated that the protocol was transferable and basically had good between-lab reproducibility and predictivity, but there were some false negative data. To improve performance, protocol and prediction model were modified. Using the modified prediction model in the first and second RT, accuracy was improved. However, about 15% of the outcomes were not correctly identified, which exposes some of the limitations of the assay. For the chemicals evaluated, the limitation may due to chemical being a weak allergen or having low solubility (ex. alpha-hexylcinnamaldehyde). The third RT evaluated the modified prediction model and satisfactory results were obtained. From the RT data, the feasibility of utilizing cell lines as surrogate DC in development of in vitro skin sensitization methods shows promise. The data also support initiating formal pre-validation of the h-CLAT in order to fully understand the capabilities and limitations of the assay. Copyright 2010 Elsevier Ltd. All rights reserved.

Properties of the tick-borne encephalitis virus population during persistent infection of ixodid ticks and tick cell lines.

PubMed

Belova, Oxana A; Litov, Alexander G; Kholodilov, Ivan S; Kozlovskaya, Liubov I; Bell-Sakyi, Lesley; Romanova, Lidiya Iu; Karganova, Galina G

2017-10-01

Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE), a vector-borne zoonotic neuroinfection. For successful circulation in natural foci the virus has to survive in the vector for a long period of time. Information about the effect of long-term infection of ticks on properties of the viral population is of great importance. In recent years, changes in the eco-epidemiology of TBEV due to changes in distribution of ixodid ticks have been observed. These changes in TBEV-endemic areas could result in a shift of the main tick vector species, which in turn may lead to changes in properties of the virus. In the present study we evaluated the selective pressure on the TBEV population during persistent infection of various species of ticks and tick cell lines. TBEV effectively replicated and formed persistent infection in ticks and tick cell lines of the vector species (Ixodes spp.), potential vectors (Dermacentor spp.) and non-vector ticks (Hyalomma spp.). During TBEV persistence in Ixodes and Dermacentor ticks, properties of the viral population remained virtually unchanged. In contrast, persistent TBEV infection of tick cell lines from both vector and non-vector ticks favoured selection of viral variants with low neuroinvasiveness for laboratory mice and substitutions in the E protein that increased local positive charge of the virion. Thus, selective pressure on viral population may differ in ticks and tick cell lines during persistent infection. Nevertheless, virus variants with properties of the original strain adapted to mouse CNS were not eliminated from the viral population during long-term persistence of TBEV in ticks and tick cell lines. Copyright © 2017 Elsevier GmbH. All rights reserved.

Decursin enhances TRAIL-induced apoptosis through oxidative stress mediated- endoplasmic reticulum stress signalling in non-small cell lung cancers.

PubMed

Kim, Jaekwang; Yun, Miyong; Kim, Eun-Ok; Jung, Deok-Beom; Won, Gunho; Kim, Bonglee; Jung, Ji Hoon; Kim, Sung-Hoon

2016-03-01

The TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent due to its remarkable ability to selectively kill tumour cells. However, because most tumours exhibit resistance to TRAIL-induced apoptosis, the development of combination therapies to overcome resistance to TRAIL is required for effective cancer therapy. Cell viability and possible synergy between the plant pyranocoumarin decursin and TRAIL was measured by MTT assay and calcusyn software. Reactive oxygen species (ROS) and apoptosis were measured using dichlorodihydrofluorescein and annexin/propidium iodide in cell flow cytometry. Changes in protein levels were assessed with Western blotting. Combining decursin and TRAIL markedly decreased cell viability and increased apoptosis in TRAIL-resistant non-small-cell lung cancer (NSCLC) cell lines. Decursin induced expression of the death receptor 5 (DR5). Inhibition of DR5 attenuated apoptotic cell death in decursin + TRAIL treated NSCLC cell lines. Interestingly, induction of DR5 and CCAAT/enhancer-binding protein homologues protein by decursin was mediated through selective induction of the pancreatic endoplasmic reticulum kinase (PERK)/activating transcription factor 4 (ATF4) branch of the endoplasmic reticulum stress response pathway. Furthermore, enhancement of PERK/ATF4 signalling by decursin was mediated by ROS generation in NSCLC cell lines, but not in normal human lung cells. Decursin also markedly down-regulated expression of survivin and Bcl-xL in TRAIL-resistant NSCLC cells. ROS generation by decursin selectively activated the PERK/ATF4 axis of the endoplasmic reticulum stress signalling pathway, leading to enhanced TRAIL sensitivity in TRAIL-resistant NSCLC cell lines, partly via up-regulation of DR5. © 2015 The British Pharmacological Society.

Decursin enhances TRAIL‐induced apoptosis through oxidative stress mediated‐ endoplasmic reticulum stress signalling in non‐small cell lung cancers

PubMed Central

Kim, Jaekwang; Yun, Miyong; Kim, Eun‐Ok; Jung, Deok‐Beom; Won, Gunho; Kim, Bonglee; Jung, Ji Hoon

2016-01-01

Background and Purpose The TNF‐related apoptosis‐inducing ligand (TRAIL) is a promising anticancer agent due to its remarkable ability to selectively kill tumour cells. However, because most tumours exhibit resistance to TRAIL‐induced apoptosis, the development of combination therapies to overcome resistance to TRAIL is required for effective cancer therapy. Experimental Approach Cell viability and possible synergy between the plant pyranocoumarin decursin and TRAIL was measured by MTT assay and calcusyn software. Reactive oxygen species (ROS) and apoptosis were measured using dichlorodihydrofluorescein and annexin/propidium iodide in cell flow cytometry. Changes in protein levels were assessed with Western blotting. Key Results Combining decursin and TRAIL markedly decreased cell viability and increased apoptosis in TRAIL‐resistant non‐small‐cell lung cancer (NSCLC) cell lines. Decursin induced expression of the death receptor 5 (DR5). Inhibition of DR5 attenuated apoptotic cell death in decursin + TRAIL treated NSCLC cell lines. Interestingly, induction of DR5 and CCAAT/enhancer‐binding protein homologues protein by decursin was mediated through selective induction of the pancreatic endoplasmic reticulum kinase (PERK)/activating transcription factor 4 (ATF4) branch of the endoplasmic reticulum stress response pathway. Furthermore, enhancement of PERK/ATF4 signalling by decursin was mediated by ROS generation in NSCLC cell lines, but not in normal human lung cells. Decursin also markedly down‐regulated expression of survivin and Bcl‐xL in TRAIL‐resistant NSCLC cells. Conclusions and Implications ROS generation by decursin selectively activated the PERK/ATF4 axis of the endoplasmic reticulum stress signalling pathway, leading to enhanced TRAIL sensitivity in TRAIL‐resistant NSCLC cell lines, partly via up‐regulation of DR5. PMID:26661339

PNIPAAm-MAA nanoparticles as delivery vehicles for curcumin against MCF-7 breast cancer cells.

PubMed

Zeighamian, Vahideh; Darabi, Masoud; Akbarzadeh, Abolfazl; Rahmati-Yamchi, Mohammad; Zarghami, Nosratollah; Badrzadeh, Fariba; Salehi, Roya; Mirakabad, Fatemeh Sadat Tabatabaei; Taheri-Anganeh, Mortaza

2016-01-01

Breast cancer is the most frequently occurring cancer among women throughout the world. Natural compounds such as curcumin hold promise to treat a variety of cancers including breast cancer. However, curcumin's therapeutic application is limited, due to its rapid degradation and poor aqueous solubility. On the other hand, previous studies have stated that drug delivery using nanoparticles might improve the therapeutic response to anticancer drugs. Poly(N-isopropylacrylamide-co-methacrylic acid) (PNIPAAm-MAA) is one of the hydrogel copolymers utilized in the drug delivery system for cancer therapy. The aim of this study was to examine the cytotoxic potential of curcumin encapsulated within the NIPAAm-MAA nanoparticle, on the MCF-7 breast cancer cell line. In this work, polymeric nanoparticles were synthesized through the free radical mechanism, and curcumin was encapsulated into NIPAAm-MAA nanoparticles. Then, the cytotoxic effect of curcumin-loaded NIPAAm-MAA on the MCF-7 breast cancer cell line was measured by MTT assays. The evaluation of the results showed that curcumin-loaded NIPAAm-MAA has more cytotoxic effect on the MCF-7 cell line and efficiently inhibited the growth of the breast cancer cell population, compared with free curcumin. In conclusion, this study indicates that curcumin-loaded NIPAAm-MAA suppresses the growth of the MCF-7 cell line. Overall, it is concluded that encapsulating curcumin into the NIPAAm-MAA copolymer could open up new avenues for breast cancer treatment.

Venetoclax Is Effective in Small-Cell Lung Cancers with High BCL-2 Expression.

PubMed

Lochmann, Timothy L; Floros, Konstantinos V; Naseri, Mitra; Powell, Krista M; Cook, Wade; March, Ryan J; Stein, Giovanna T; Greninger, Patricia; Maves, Yuki Kato; Saunders, Laura R; Dylla, Scott J; Costa, Carlotta; Boikos, Sosipatros A; Leverson, Joel D; Souers, Andrew J; Krystal, Geoffrey W; Harada, Hisashi; Benes, Cyril H; Faber, Anthony C

2018-01-15

Purpose: Small-cell lung cancer (SCLC) is an often-fatal neuroendocrine carcinoma usually presenting as extensive disease, carrying a 3% 5-year survival. Despite notable advances in SCLC genomics, new therapies remain elusive, largely due to a lack of druggable targets. Experimental Design: We used a high-throughput drug screen to identify a venetoclax-sensitive SCLC subpopulation and validated the findings with multiple patient-derived xenografts of SCLC. Results: Our drug screen consisting of a very large collection of cell lines demonstrated that venetoclax, an FDA-approved BCL-2 inhibitor, was found to be active in a substantial fraction of SCLC cell lines. Venetoclax induced BIM-dependent apoptosis in vitro and blocked tumor growth and induced tumor regressions in mice bearing high BCL-2-expressing SCLC tumors in vivo BCL-2 expression was a predictive biomarker for sensitivity in SCLC cell lines and was highly expressed in a subset of SCLC cell lines and tumors, suggesting that a substantial fraction of patients with SCLC could benefit from venetoclax. Mechanistically, we uncover a novel role for gene methylation that helped discriminate high BCL-2-expressing SCLCs. Conclusions: Altogether, our findings identify venetoclax as a promising new therapy for high BCL-2-expressing SCLCs. Clin Cancer Res; 24(2); 360-9. ©2017 AACR . ©2017 American Association for Cancer Research.

High frequency of loss of PTEN expression in human solid salivary adenoid cystic carcinoma and its implication for targeted therapy.

PubMed

Liu, Han; Du, Li; Wang, Ru; Wei, Chao; Liu, Bo; Zhu, Lei; Liu, Pixu; Liu, Qiang; Li, Jiang; Lu, Shi-Long; Xiao, Jing

2015-05-10

Salivary gland tumor (SGT) is one of the least studied cancers due to its rarity and heterogeneous histological types. Here, we reported that loss of PTEN expression was most frequently found in the poorly differentiated, high grade solid adenoid cystic carcinomas. Loss of PTEN expression correlated with activation of mTOR by increased phosphorylated S6 ribosome protein. We further functionally studied the role of PTEN in a pair of human SACC cell lines, SACC-83 and SACC-LM. Reduced PTEN level was correlated with the metastasis potential. When we knocked down PTEN in the SACC-83 cell line, we observed increased proliferation and enhanced migration/invasion in vitro, and increased tumor size in vivo. We further tested the therapeutical effect by applying a PI3K/mTOR inhibitor NVP-BEZ235 to both SACC cell lines. Decreased cell proliferation, increased apoptosis, as well as reduced cell migration/invasion were observed in both cell lines upon the NVP-BEZ235 treatment. Moreover, the NVP-BEZ235 treatment in a SGT xenograft mouse model significantly reduced primary tumor size and lung metastasis. Taken together, our results demonstrated that PTEN is a potent tumor suppressor in human SGTs, and targeting PI3K/mTOR pathway may be effective in the targeted therapy for human SGT patients with loss of PTEN expression.

Apoptosis Activation in Human Lung Cancer Cell Lines by a Novel Synthetic Peptide Derived from Conus californicus Venom

PubMed Central

Oroz-Parra, Irasema; Navarro, Mario; Cervantes-Luevano, Karla E.; Álvarez-Delgado, Carolina; Salvesen, Guy; Sanchez-Campos, Liliana N.; Licea-Navarro, Alexei F.

2016-01-01

Lung cancer is one of the most common types of cancer in men and women and a leading cause of death worldwide resulting in more than one million deaths per year. The venom of marine snails Conus contains up to 200 pharmacologically active compounds that target several receptors in the cell membrane. Due to their diversity and specific binding properties, Conus toxins hold great potential as source of new drugs against cancer. We analyzed the cytotoxic effect of a 17-amino acid synthetic peptide (s-cal14.1a) that is based on a native toxin (cal14.1a) isolated from the sea snail Conus californicus. Cytotoxicity studies in four lung cancer cell lines were complemented with measurement of gene expression of apoptosis-related proteins Bcl-2, BAX and the pro-survival proteins NFκB-1 and COX-2, as well as quantification of caspase activity. Our results showed that H1299 and H1437 cell lines treated with s-call4.1a had decreased cell viability, activated caspases, and reduced expression of the pro-survival protein NFκB-1. To our knowledge, this is the first report describing activation of apoptosis in human lung cancer cell lines by s-cal14.1a and we offer insight into the possible mechanism of action. PMID:26861394

Evaluation of genotoxic potential of avarol, avarone, and its methoxy and methylamino derivatives in prokaryotic and eukaryotic test models.

PubMed

Kolarević, Stoimir; Milovanović, Dragana; Kračun-Kolarević, Margareta; Kostić, Jovana; Sunjog, Karolina; Martinović, Rajko; Đorđević, Jelena; Novaković, Irena; Sladić, Dušan; Vuković-Gačić, Branka

2018-01-04

In this study, mutagenic and genotoxic potential of anti-tumor compounds avarol, avarone, and its derivatives 3'-methoxyavarone, 4'-(methylamino)avarone and 3'-(methylamino)avarone was evaluated and compared to cytostatics commonly used in chemotherapy (5-fluorouracil, etoposid, and cisplatin). Mutagenic potential of selected hydroquinone and quinones was assessed in prokaryotic model by the SOS/umuC assay in Salmonella typhimurium TA1535/pSK1002. Genotoxic potential was also assessed in eukaryotic models using comet assay in human fetal lung cell line (MRC-5), human adenocarcinoma epithelial cell line (A549), and in human peripheral blood cells (HPBC). The results indicated that avarol and avarone do not exert mutagenic/genotoxic potential. Among the studied avarone derivatives, mutagenic potential was detected by SOS/umuC test for 3'-(methylamino)avarone, but only after metabolic activation. The results of comet assay indicated that 3'-methoxyavarone and 3'-(methylamino)avarone have a significant impact on the level of DNA damage in the MRC-5 cell line. Genotoxic potential was not observed in A549 cells or HPBC probably due to a different uptake rate for the compounds and lower in metabolism rate within these cells.

Using in situ hybridization and PFGE Southern hybridization to detect translocation breakpoints in a BOR/TRPS patient cell line

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

Gu, J.Z.; Sapru, M.; Smith, D.

Branchio-oto-renal syndrome (BOR) is an autosomal dominant disorder characterized by ear malformations, cervical fistulae, hearing loss and renal abnormalities. We have integrated the Genethon YAC contig maps with additional markers in the chromosome 8q region genetically linked by a unique patient cell line. This cell line is from a patient who has both the branchio-oto-renal syndrome and tricho-rhino-phalangeal syndrome (TRPS). High resolution cytogenetics demonstrated a direct insertion of materials from 8q13.3q21.13 to 8q24.11. TRPS has been previously linked to deletions involving 8q24.11-q24.13. The rearrangement in this patient suggests that TRPS results from loss of gene function due to insertion atmore » the 8q24.11 breakpoint and the possible location for the BOR gene is at either of the two breakpoints of 8q13.3 and 8q21.13. We have constructed cosmid contigs in 8q24.11. In situ hybridization with cosmids mapped to these locations as probes has helped to narrow down the breakpoints. Combinations of cosmids on either side or overlapping the 8q24.11 breakpoint show split signals on one chromosome 8q arm due to insertion of the materials from the proximal region. Cosmids mapped to the TRPS deletion region have been used to hybridize to pulsed field gel genomic blots of DNA from the patient cell line and detected rearranged genomic fragments. Both in situ hybridization and genomic PFGE Southern blot will be used to precisely locate the breakpoints.« less

Quantifying folic acid-functionalized multi-walled carbon nanotubes bound to colorectal cancer cells for improved photothermal ablation

NASA Astrophysics Data System (ADS)

Graham, Elizabeth G.; MacNeill, Christopher M.; Levi-Polyachenko, Nicole H.

2013-05-01

Peritoneal metastases of colorectal cancer are a significant challenge in the field of medicine today due to poor results of systemic chemotherapy caused by the poor diffusion of drugs across the blood-peritoneal barrier. Multi-walled carbon nanotubes (MWNTs) are a biocompatible nanomaterial that strongly absorb near-infrared light to locally heat the surrounding area. Colorectal cancer is known to overexpress folate receptor; therefore, folic acid (FA) was covalently attached to MWNTs to target colorectal cancer cells. Results from real-time polymerase chain reaction found differing expression of folate receptor-α in two colorectal cancer cell lines, RKO and HCT116, as well as a healthy epithelial cell line, HEPM. A spectrophotometric method was developed to quantify the mass of MWNTs bound to cells, and it was determined that FA-targeted MWNTs resulted in a 400-500 % greater affinity for colorectal cancer cells than untargeted MWNTs. The non-cancerous cell line, HEPM, had higher non-specific MWNT interaction and similar MWNT-FA affinity. Stimulated by 1,064 nm light, FA-functionalized MWNTs caused a 50-60 % decrease in colorectal cancer cell viability compared to a 4-10 % decrease caused by untargeted MWNTs. Our results indicate that FA-targeted MWNTs may increase the therapeutic index of MWNT-induced photothermal therapy.

Resistance of a soybean cell line to oxyfluorfen by overproduction of mitochondrial protoporphyrinogen oxidase.

PubMed

Warabi, E; Usui, K; Tanaka, Y; Matsumoto, H

2001-08-01

The diphenyl ether herbicide oxyfluorfen (2-chloro-4-trifluoromethylphenyl 3-ethoxy-4-nitrophenyl ether) inhibits protoporphyrinogen oxidase (Protox) which catalyzes the oxidation of protoporphyrinogen IX (Protogen) to protoporphyrin IX (Proto IX), the last step of the common pathway to chlorophyll and haeme biosynthesis. We have selected an oxyfluorfen-resistant soybean cell line by stepwise selection methods, and the resistance mechanism has been investigated. No growth inhibition was observed in resistant cells at a concentration of 10(-7) M oxyfluorfen, a concentration at which normal cells did not survive. While the degree of inhibition of total extractable Protox by oxyfluorfen was the same in both cell types, the enzyme activity in the mitochondrial fraction from non-treated resistant cells was about nine-fold higher than that from normal cells. Northern analysis of mitochondrial Protox revealed that the concentration of mitochondrial Protox mRNA was much higher in resistant cells than that in normal cells. There were no differences in the absorption and metabolic breakdown of oxyfluorfen. The growth of resistant cells was also insensitive to oxadiazon [5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2-(3H)- one], the other chemical class of Protox inhibitor. Therefore, the resistance of the selected soybean cell line to oxyfluorfen is probably mainly due to the overproduction of mitochondrial Protox.

Differential proteomic analysis of cancer stem cell properties in hepatocellular carcinomas by isobaric tag labeling and mass spectrometry.

PubMed

Ko, Ching-Huai; Cheng, Chieh-Fang; Lai, Chin-Pen; Tzu, Te-Hui; Chiu, Chih-Wei; Lin, Mei-Wei; Wu, Si-Yuan; Sun, Chung-Yuan; Tseng, Hsiang-Wen; Wang, Chun-Chung; Kuo, Zong-Keng; Wang, Ling-Mei; Chen, Sung-Fang

2013-08-02

Malignant tumors are relatively resistant to treatment due to their heterogeneous nature, drug resistance, and tendency for metastasis. Recent studies suggest that a subpopulation of cancer cells is responsible for the malignant outcomes. These cells are considered as cancer stem cells (CSC). Although a number of molecules have been identified in different cancer cells as markers for cancer stem cells, no promising markers are currently available for hepatocellular carcinoma cells. In this study, two clones of Hep3B cell lines were functionally characterized as control or CSC-like cells, based on properties including spheroid formation, drug resistance, and tumor initiation. Furthermore, their protein expression profiles were investigated by isobaric tags for relative and absolute quantitation (iTRAQ), and a total of 1,127 proteins were identified and quantified from the combined fractions; 50 proteins exhibited at least 2-fold differences between these two clones. These 50 proteins were analyzed by GeneGo and were found to be associated with liver neoplasms, hepatocellular carcinoma (HCC), and liver diseases. They were also components of metabolic pathways, immune responses, and cytoskeleton remodeling. Among these proteins, the expressions of S100P, S100A14, and vimentin were verified in several HCC cell lines, and their expressions were correlated with tumorigenicity in HCC cell lines. The functional significance of vimentin and S100A14 were also investigated and verified.

Pharmacokinetic drug evaluation of osimertinib for the treatment of non-small cell lung cancer.

PubMed

Rossi, Antonio; Muscarella, Lucia Anna; Di Micco, Concetta; Carbonelli, Cristiano; D'alessandro, Vito; Notarangelo, Stefano; Palomba, Giuseppe; Sanpaolo, Gerardo; Taurchini, Marco; Graziano, Paolo; Maiello, Evaristo

2017-12-01

First- and second-generation epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, erlotinib, icotinib, and afatinib are the standard-of-care for first-line therapy of non-small-cell lung cancer (NSCLC) harboring activating EGFR mutations. Unfortunately, after initial activity of an average 9-13 months, disease progression has been reported in the majority of patients. In about 50% of cases the progression is due to the onset of the T790M mutation in exon 20 of the EGFR gene. Third-generation EGFR-TKIs targeting this mutation were investigated, with osimertinib the only reaching clinical practice. Areas covered: A structured search of bibliographic databases for peer-reviewed research literature and of main meetings using a focused review question addressing osimertinib, was undertaken. Expert opinion: Osimertinib is the standard-of-care for EGFR-mutated patients progressing to first-line EGFR-TKIs due to the acquired EGFR T790M mutation. Results from the head-to-head first-line trial comparing osimertinib versus gefitinib or erlotinib in activating EGFR mutations might change the front-line approach. Osimertinib in combination regimens, such as immunotherapy, and in adjuvant setting are ongoing. Thus, the strategic approach for the management of EGFR-mutated NSCLC patients will change further in the next few years.

Epigenetic Alterations Associated With CCCTC-Binding Factor Deregulation in Prostate Cancer

DTIC Science & Technology

2013-09-01

cancer cells. (Months 1‐12)  a. Establish cell cultures of human prostate cancer (PC‐3 and PPC‐1) cell  lines, HPECs, non‐tumorigenic HPV16  E6  and/or  E7 ...and non‐tumorigenic HPV16  E6   5 and/or  E7  prostate cell lines. We have had to rederive these due to leakage from the  promoter leading to clonal...and control scrambled shRNA.  f. To test the tumorigenic ability of CTCF shRNA infected non‐tumorigenic  E6 / E7  cells using colony forming assays and

Synthesis, DNA/RNA affinity and antitumour activity of new aromatic diamidines linked by 3,4-ethylenedioxythiophene.

PubMed

Stolić, Ivana; Mišković, Katarina; Piantanida, Ivo; Lončar, Mirela Baus; Glavaš-Obrovac, Ljubica; Bajić, Miroslav

2011-02-01

A series of novel 2,5-bis(amidinophenyl)-3,4-ethylenedioxythiophenes (5-10 and 15) has been synthesized. Compounds 5-10 bind to the DNA minor groove as the dominant binding site and strongly stabilize the double helix of ct-DNA. Surprisingly, the same compounds also thermally stabilize ds-RNA, whereby most of them form stacked dimers along the RNA double helix. The only exception is compound 15 which, due to its structural features, showed no interaction with DNA or RNA. Compounds 5-10 have shown a moderate to strong cytotoxic effect (GI50=1.5-9.0 μM) on a panel of seven tumour cell lines. The diimidazoline derivative 9, due to its highest inhibitory potential on the growth of all tested tumour cell lines, was investigated in more detail by testing its ability to enter into cells and influence the cell cycle. Compound 9 (5 μM) was internalized successfully in cell cytoplasm during a 30-min incubation period, followed by nuclear localization upon 90-min incubation. Significant arrest in HeLa cells in the G2/M phase, shown by cell cycle analysis at an equitoxic (50 μM) concentration, suggests interaction of a studied compound with cellular DNA as the main mode of biological action. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Paclitaxel loading in PLGA nanospheres affected the in vitro drug cell accumulation and antiproliferative activity

PubMed Central

Vicari, Luisa; Musumeci, Teresa; Giannone, Ignazio; Adamo, Luana; Conticello, Concetta; De Maria, Ruggero; Pignatello, Rosario; Puglisi, Giovanni; Gulisano, Massimo

2008-01-01

Background PTX is one of the most widely used drug in oncology due to its high efficacy against solid tumors and several hematological cancers. PTX is administered in a formulation containing 1:1 Cremophor® EL (polyethoxylated castor oil) and ethanol, often responsible for toxic effects. Its encapsulation in colloidal delivery systems would gain an improved targeting to cancer cells, reducing the dose and frequency of administration. Methods In this paper PTX was loaded in PLGA NS. The activity of PTX-NS was assessed in vitro against thyroid, breast and bladder cancer cell lines in cultures. Cell growth was evaluated by MTS assay, intracellular NS uptake was performed using coumarin-6 labelled NS and the amount of intracellular PTX was measured by HPLC. Results NS loaded with 3% PTX (w/w) had a mean size < 250 nm and a polydispersity index of 0.4 after freeze-drying with 0.5% HP-Cyd as cryoprotector. PTX encapsulation efficiency was 30% and NS showed a prolonged drug release in vitro. An increase of the cytotoxic effect of PTX-NS was observed with respect to free PTX in all cell lines tested. Conclusion These findings suggest that the greater biological effect of PTX-NS could be due to higher uptake of the drug inside the cells as shown by intracellular NS uptake and cell accumulation studies. PMID:18657273

FAS Death Receptor: A Breast Cancer Subtype-Specific Radiation Response Biomarker and Potential Therapeutic Target

PubMed Central

Horton, Janet K.; Siamakpour-Reihani, Sharareh; Lee, Chen-Ting; Zhou, Ying; Chen, Wei; Geradts, Joseph; Fels, Diane R.; Hoang, Peter; Ashcraft, Kathleen A.; Groth, Jeff; Kung, Hsiu-Ni; Dewhirst, Mark W.; Chi, Jen-Tsan A.

2015-01-01

Although a standardized approach to radiotherapy has been used to treat breast cancer, regardless of subtype (e.g., luminal, basal), recent clinical data suggest that radiation response may vary significantly among subtypes. We hypothesized that this clinical variability may be due, in part, to differences in cellular radiation response. In this study, we utilized RNA samples for microarray analysis from two sources: 1. Paired pre- and postirradiation breast tumor tissue from 32 early-stage breast cancer patients treated in our unique preoperative radiation Phase I trial; and 2. Sixteen biologically diverse breast tumor cell lines exposed to 0 and 5 Gy irradiation. The transcriptome response to radiation exposure was derived by comparing gene expression in samples before and after irradiation. Genes with the highest coefficient of variation were selected for further evaluation and validated at the RNA and protein level. Gene editing and agonistic antibody treatment were performed to assess the impact of gene modulation on radiation response. Gene expression in our cohort of luminal breast cancer patients was distinctly different before and after irradiation. Further, two distinct patterns of gene expression were observed in our biologically diverse group of breast cancer cell lines pre- versus postirradiation. Cell lines that showed significant change after irradiation were largely luminal subtype, while gene expression in the basal and HER2+ cell lines was minimally impacted. The 100 genes with the most significant response to radiation in patients were identified and analyzed for differential patterns of expression in the radiation-responsive versus nonresponsive cell lines. Fourteen genes were identified as significant, including FAS, a member of the tumor necrosis factor receptor family known to play a critical role in programed cell death. Modulation of FAS in breast cancer cell lines altered radiation response phenotype and enhanced radiation sensitivity in radioresistant basal cell lines. Our findings suggest that cell-type-specific, radiation-induced FAS contributes to subtype-specific breast cancer radiation response and that activation of FAS pathways may be exploited for biologically tailored radiotherapy. PMID:26488758

A high-throughput flow cytometry-on-a-CMOS platform for single-cell dielectric spectroscopy at microwave frequencies.

PubMed

Chien, Jun-Chau; Ameri, Ali; Yeh, Erh-Chia; Killilea, Alison N; Anwar, Mekhail; Niknejad, Ali M

2018-06-06

This work presents a microfluidics-integrated label-free flow cytometry-on-a-CMOS platform for the characterization of the cytoplasm dielectric properties at microwave frequencies. Compared with MHz impedance cytometers, operating at GHz frequencies offers direct intracellular permittivity probing due to electric fields penetrating through the cellular membrane. To overcome the detection challenges at high frequencies, the spectrometer employs on-chip oscillator-based sensors, which embeds simultaneous frequency generation, electrode excitation, and signal detection capabilities. By employing an injection-locking phase-detection technique, the spectrometer offers state-of-the-art sensitivity, achieving a less than 1 aFrms capacitance detection limit (or 5 ppm in frequency-shift) at a 100 kHz noise filtering bandwidth, enabling high throughput (>1k cells per s), with a measured cellular SNR of more than 28 dB. With CMOS/microfluidics co-design, we distribute four sensing channels at 6.5, 11, 17.5, and 30 GHz in an arrayed format whereas the frequencies are selected to center around the water relaxation frequency at 18 GHz. An issue in the integration of CMOS and microfluidics due to size mismatch is also addressed through introducing a cost-efficient epoxy-molding technique. With 3-D hydrodynamic focusing microfluidics, we perform characterization on four different cell lines including two breast cell lines (MCF-10A and MDA-MB-231) and two leukocyte cell lines (K-562 and THP-1). After normalizing the higher frequency signals to the 6.5 GHz ones, the size-independent dielectric opacity shows a differentiable distribution at 17.5 GHz between normal (0.905 ± 0.160, mean ± std.) and highly metastatic (1.033 ± 0.107) breast cells with p ≪ 0.001.

HD iPSC-derived neural progenitors accumulate in culture and are susceptible to BDNF withdrawal due to glutamate toxicity

PubMed Central

Mattis, Virginia B.; Tom, Colton; Akimov, Sergey; Saeedian, Jasmine; Østergaard, Michael E.; Southwell, Amber L.; Doty, Crystal N.; Ornelas, Loren; Sahabian, Anais; Lenaeus, Lindsay; Mandefro, Berhan; Sareen, Dhruv; Arjomand, Jamshid; Hayden, Michael R.; Ross, Christopher A.; Svendsen, Clive N.

2015-01-01

Huntington's disease (HD) is a fatal neurodegenerative disease, caused by expansion of polyglutamine repeats in the Huntingtin gene, with longer expansions leading to earlier ages of onset. The HD iPSC Consortium has recently reported a new in vitro model of HD based on the generation of induced pluripotent stem cells (iPSCs) from HD patients and controls. The current study has furthered the disease in a dish model of HD by generating new non-integrating HD and control iPSC lines. Both HD and control iPSC lines can be efficiently differentiated into neurons/glia; however, the HD-derived cells maintained a significantly greater number of nestin-expressing neural progenitor cells compared with control cells. This cell population showed enhanced vulnerability to brain-derived neurotrophic factor (BDNF) withdrawal in the juvenile-onset HD (JHD) lines, which appeared to be CAG repeat-dependent and mediated by the loss of signaling from the TrkB receptor. It was postulated that this increased death following BDNF withdrawal may be due to glutamate toxicity, as the N-methyl-d-aspartate (NMDA) receptor subunit NR2B was up-regulated in the cultures. Indeed, blocking glutamate signaling, not just through the NMDA but also mGlu and AMPA/Kainate receptors, completely reversed the cell death phenotype. This study suggests that the pathogenesis of JHD may involve in part a population of ‘persistent’ neural progenitors that are selectively vulnerable to BDNF withdrawal. Similar results were seen in adult hippocampal-derived neural progenitors isolated from the BACHD model mouse. Together, these results provide important insight into HD mechanisms at early developmental time points, which may suggest novel approaches to HD therapeutics. PMID:25740845

Establishment of Myotis myotis cell lines--model for investigation of host-pathogen interaction in a natural host for emerging viruses.

PubMed

He, Xiaocui; Korytář, Tomáš; Zhu, Yaqing; Pikula, Jiří; Bandouchova, Hana; Zukal, Jan; Köllner, Bernd

2014-01-01

Bats are found to be the natural reservoirs for many emerging viruses. In most cases, severe clinical signs caused by such virus infections are normally not seen in bats. This indicates differences in the virus-host interactions and underlines the necessity to develop natural host related models to study these phenomena. Due to the strict protection of European bat species, immortalized cell lines are the only alternative to investigate the innate anti-virus immune mechanisms. Here, we report about the establishment and functional characterization of Myotis myotis derived cell lines from different tissues: brain (MmBr), tonsil (MmTo), peritoneal cavity (MmPca), nasal epithelium (MmNep) and nervus olfactorius (MmNol) after immortalization by SV 40 large T antigen. The usefulness of these cell lines to study antiviral responses has been confirmed by analysis of their susceptibility to lyssavirus infection and the mRNA patterns of immune-relevant genes after poly I:C stimulation. Performed experiments indicated varying susceptibility to lyssavirus infection with MmBr being considerably less susceptible than the other cell lines. Further investigation demonstrated a strong activation of interferon mediated antiviral response in MmBr contributing to its resistance. The pattern recognition receptors: RIG-I and MDA5 were highly up-regulated during rabies virus infection in MmBr, suggesting their involvement in promotion of antiviral responses. The presence of CD14 and CD68 in MmBr suggested MmBr cells are microglia-like cells which play a key role in host defense against infections in the central nervous system (CNS). Thus the expression pattern of MmBr combined with the observed limitation of lyssavirus replication underpin a protective mechanism of the CNS controlling the lyssavirus infection. Overall, the established cell lines are important tools to analyze antiviral innate immunity in M. myotis against neurotropic virus infections and present a valuable tool for a broad spectrum of future investigations in cellular biology of M. myotis.

Establishment of Myotis myotis Cell Lines - Model for Investigation of Host-Pathogen Interaction in a Natural Host for Emerging Viruses

PubMed Central

He, Xiaocui; Korytář, Tomáš; Zhu, Yaqing; Pikula, Jiří; Bandouchova, Hana; Zukal, Jan; Köllner, Bernd

2014-01-01

Bats are found to be the natural reservoirs for many emerging viruses. In most cases, severe clinical signs caused by such virus infections are normally not seen in bats. This indicates differences in the virus-host interactions and underlines the necessity to develop natural host related models to study these phenomena. Due to the strict protection of European bat species, immortalized cell lines are the only alternative to investigate the innate anti-virus immune mechanisms. Here, we report about the establishment and functional characterization of Myotis myotis derived cell lines from different tissues: brain (MmBr), tonsil (MmTo), peritoneal cavity (MmPca), nasal epithelium (MmNep) and nervus olfactorius (MmNol) after immortalization by SV 40 large T antigen. The usefulness of these cell lines to study antiviral responses has been confirmed by analysis of their susceptibility to lyssavirus infection and the mRNA patterns of immune-relevant genes after poly I:C stimulation. Performed experiments indicated varying susceptibility to lyssavirus infection with MmBr being considerably less susceptible than the other cell lines. Further investigation demonstrated a strong activation of interferon mediated antiviral response in MmBr contributing to its resistance. The pattern recognition receptors: RIG-I and MDA5 were highly up-regulated during rabies virus infection in MmBr, suggesting their involvement in promotion of antiviral responses. The presence of CD14 and CD68 in MmBr suggested MmBr cells are microglia-like cells which play a key role in host defense against infections in the central nervous system (CNS). Thus the expression pattern of MmBr combined with the observed limitation of lyssavirus replication underpin a protective mechanism of the CNS controlling the lyssavirus infection. Overall, the established cell lines are important tools to analyze antiviral innate immunity in M. myotis against neurotropic virus infections and present a valuable tool for a broad spectrum of future investigations in cellular biology of M. myotis. PMID:25295526

Characterization of a hydroxyurea-resistant human KB cell line with supersensitivity to 6-thioguanine.

PubMed

Yen, Y; Grill, S P; Dutschman, G E; Chang, C N; Zhou, B S; Cheng, Y C

1994-07-15

Hydroxyurea (HU) is currently used in the clinic for the treatment of chronic myelogenous leukemia, head and neck carcinoma, and sarcoma. One of its drawbacks, however, is the development of HU resistance. To study this problem, we developed a HU-resistant human KB cell line which exhibits a 15-fold resistance to HU. The characterization of this HU-resistant phenotype revealed a gene amplification of the M2 subunit of ribonucleotide reductase (RR), increased levels of M2 mRNA and protein, and a 3-fold increase of RR activity. This HU-resistant cell line also expressed a "collateral sensitivity" to 6-thioguanine (6-TG), with a 10-fold decrease in the dose inhibiting cell growth by 50% as compared to the KB parental line. The mechanism responsible for this supersensitivity to 6-TG is believed to be related to an increasingly efficient conversion of 6-TG to its triphosphate form, which is subsequently incorporated into DNA. After passage of the resistant cells in the absence of HU, the cell line reverts. The revertant cells lose their resistance to HU and concomitantly their sensitivity to 6-TG. This phenomenon is due to the return of RR to levels comparable to that of the KB parental cell line. These observations and their relevance to cancer chemotherapy will be discussed in this paper. Our results suggest that a clinical protocol could be designed which would allow for a lower dose of 6-TG to be used by taking advantage of the increased RR activity in HU-refractory cancer patients. Two drugs which display collateral sensitivity are known as a "Ying-Yang" pair. Alternate treatment with two different Ying-Yang pairs is the rationale for the "Ying-Yang Ping-Pong" theory in cancer treatment. This rationale allows for effective cancer chemotherapy with reduced toxicity.

Increased acid ceramidase expression depends on upregulation of androgen-dependent deubiquitinases, USP2, in a human prostate cancer cell line, LNCaP.

PubMed

Mizutani, Naoki; Inoue, Minami; Omori, Yukari; Ito, Hiromi; Tamiya-Koizumi, Keiko; Takagi, Akira; Kojima, Tetsuhito; Nakamura, Mitsuhiro; Iwaki, Soichiro; Nakatochi, Masahiro; Suzuki, Motoshi; Nozawa, Yoshinori; Murate, Takashi

2015-10-01

Acid ceramidase (ACDase) metabolizes ceramide to sphingosine, leading to sphingosine 1-phosphate production. Reportedly, ACDase has been upregulated in prostate cancer. However, its regulatory mechanism remains unclear. LNCaP (androgen-sensitive prostate cancer cell line) but not PC3 and DU-145, (androgen-unresponsive cell lines) exhibited the highest ACDase protein. Among three cell lines, ASAH1 mRNA level was not correlated with ACDase protein expression, and the 5'-promoter activity did not show androgen dependency, suggesting the post-transcriptional regulation of ACDase in LNCaP cells. Based on these results, LNCaP was analysed further. Casodex, androgen receptor antagonist, and charcoal-stripped FCS (CS-FCS) decreased ACDase protein and activity, whereas dihydrotestosterone in CS-FCS culture increased ACDase protein and enzyme activity. MG132, a proteasome inhibitor, prevented the decrease of ACDase protein when cultured in CS-FCS, suggesting the involvement of ubiquitin/proteasome system. Reportedly, USP2, a deubiquitinase, plays an important role in LNCaP cells. USP2 siRNA decreased ACDase protein, whereas USP2 overexpression increased ACDase protein of LNCaP cells. However, SKP2, an ubiquitin E3 ligase known to be active in prostate cancer, did not affect androgen-dependent ACDase expression in LNCaP cells. Thus, ACDase regulation by androgen in androgen-sensitive LNCaP cells is mainly due to its prolonged protein half-life by androgen-stimulated USP2 expression. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

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

Ramos-Solano, Moisés, E-mail: mrsolano84@gmail.com; Programa de Doctorado en Ciencias Biomédica, Centro Universitario de Ciencias de la Salud; Meza-Canales, Ivan D., E-mail: imezacanales@ice.mpg.de

According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviralmore » gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration. - Highlights: • WNT7A is expressed in normal keratinocytes or cervical cells without lesion. • WNT7A is significantly reduced in cervical cancer-derived cells. • Restoration of WNT7A expression in HeLa decreases proliferation and cell migration. • Silencing of WNT7A in HaCaT induces an increased proliferation and migration rate. • Decreased WNT7A expression in this model is due to hypermethylation.« less

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

Baumann, Philipp; Mandl-Weber, Sonja; Oduncu, Fuat

NVP-BEZ235 is a new inhibitor of phosphoinositol-3-kinase (PI3 kinase) and mammalian target of rapamycin (mTOR) whose efficacy in advanced solid tumours is currently being evaluated in a phase I/II clinical trial. Here we show that NVP-BEZ235 inhibits growth in common myeloma cell lines as well as primary myeloma cells at nanomolar concentrations in a time and dose dependent fashion. Further experiments revealed induction of apoptosis in three of four cell lines. Inhibition of cell growth was mainly due to inhibition of myeloma cell proliferation, as shown by the BrdU assay. Cell cycle analysis revealed induction of cell cycle arrest inmore » the G1 phase, which was due to downregulation of cyclin D1, pRb and cdc25a. NVP-BEZ235 inhibited phosphorylation of protein kinase B (Akt), P70S6k and 4E-BP-1. Furthermore we show that the stimulatory effect of CD40-ligand (CD40L), insulin-like growth factor 1 (IGF-1), interleukin-6 (IL-6) and conditioned medium of HS-5 stromal cells on myeloma cell growth is completely abrogated by NVP-BEZ235. In addition, synergism studies revealed synergistic and additive activity of NVP-BEZ235 together with melphalan, doxorubicin and bortezomib. Taken together, inhibition of PI3 kinase/mTOR by NVP-BEZ235 is highly effective and NVP-BEZ235 represents a potential new candidate for targeted therapy in multiple myeloma.« less

Primitive erythrocytes are generated from hemogenic endothelial cells.

PubMed

Stefanska, Monika; Batta, Kiran; Patel, Rahima; Florkowska, Magdalena; Kouskoff, Valerie; Lacaud, Georges

2017-07-25

Primitive erythroblasts are the first blood cells generated during embryonic hematopoiesis. Tracking their emergence both in vivo and in vitro has remained challenging due to the lack of specific cell surface markers. To selectively investigate primitive erythropoiesis, we have engineered a new transgenic embryonic stem (ES) cell line, where eGFP expression is driven by the regulatory sequences of the embryonic βH1 hemoglobin gene expressed specifically in primitive erythroid cells. Using this ES cell line, we observed that the first primitive erythroblasts are detected in vitro around day 1.5 of blast colony differentiation, within the cell population positive for the early hematopoietic progenitor marker CD41. Moreover, we establish that these eGFP + cells emerge from a hemogenic endothelial cell population similarly to their definitive hematopoietic counterparts. We further generated a corresponding βH1-eGFP transgenic mouse model and demonstrated the presence of a primitive erythroid primed hemogenic endothelial cell population in the developing embryo. Taken together, our findings demonstrate that both in vivo and in vitro primitive erythrocytes are generated from hemogenic endothelial cells.

Grb2 regulates B-cell maturation, B-cell memory responses and inhibits B-cell Ca2+ signalling.

PubMed

Ackermann, Jochen A; Radtke, Daniel; Maurberger, Anna; Winkler, Thomas H; Nitschke, Lars

2011-04-20

Grb2 is a ubiquitously expressed adaptor protein, which activates Ras and MAP kinases in growth factor receptor signalling, while in B-cell receptor (BCR) signalling this role is controversial. In B cell lines it was shown that Grb2 can inhibit BCR-induced Ca(2+) signalling. Nonetheless, the physiological role of Grb2 in primary B cells is still unknown. We generated a B-cell-specific Grb2-deficient mouse line, which had a severe reduction of mature follicular B cells in the periphery due to a differentiation block and decreased B-cell survival. Moreover, we found several changes in important signalling pathways: enhanced BCR-induced Ca(2+) signalling, alterations in mitogen-activated protein kinase activation patterns and strongly impaired Akt activation, the latter pointing towards a defect in PI3K signalling. Interestingly, B-cell-specific Grb2-deficient mice showed impaired IgG and B-cell memory responses, and impaired germinal centre formation. Thus, Grb2-dependent signalling pathways are crucial for lymphocyte differentiation processes, as well as for control of secondary humoral immune responses.

Genetic resistance to rhabdovirus infection in teleost fish is paralleled to the derived cell resistance status.

PubMed

Verrier, Eloi R; Langevin, Christelle; Tohry, Corinne; Houel, Armel; Ducrocq, Vincent; Benmansour, Abdenour; Quillet, Edwige; Boudinot, Pierre

2012-01-01

Genetic factors of resistance and predisposition to viral diseases explain a significant part of the clinical variability observed within host populations. Predisposition to viral diseases has been associated to MHC haplotypes and T cell immunity, but a growing repertoire of innate/intrinsic factors are implicated in the genetic determinism of the host susceptibility to viruses. In a long-term study of the genetics of host resistance to fish rhabdoviruses, we produced a collection of double-haploid rainbow trout clones showing a wide range of susceptibility to Viral Hemorrhagic Septicemia Virus (VHSV) waterborne infection. The susceptibility of fibroblastic cell lines derived from these clonal fish was fully consistent with the susceptibility of the parental fish clones. The mechanisms determining the host resistance therefore did not associate with specific host immunity, but rather with innate or intrinsic factors. One cell line was resistant to rhabdovirus infection due to the combination of an early interferon IFN induction--that was not observed in the susceptible cells--and of yet unknown factors that hamper the first steps of the viral cycle. The implication of IFN was well consistent with the wide range of resistance of this genetic background to VSHV and IHNV, to the birnavirus IPNV and the orthomyxovirus ISAV. Another cell line was even more refractory to the VHSV infection through different antiviral mechanisms. This collection of clonal fish and isogenic cell lines provides an interesting model to analyze the relative contribution of antiviral pathways to the resistance to different viruses.

[Dependence of ion transport across the plasma membrane on the density of the cell culture. I. Ion flows and the potassium and sodium content in 3 Chinese hamster cell lines (CHO)].

PubMed

Marakhova, I I; Pospelova, T V; Vinogradova, T A; Vereninov, A A; Ignatova, T N

1985-09-01

Cation transport has been investigated in three lines of Chinese ovary cells CHO-K1 during the cell culture growth. With the increase in the cell density potassium and sodium contents decreased from 1.2 to 0.8-0.5 and from 0.5 to 0.15-0.1 mmole/g protein, respectively. The time courses of potassium and sodium changes were different, and the increase in intracellular K/Na ratio from 1.5-2.0 to 5-10 with the increase in cell density was revealed. The rubidium influx was found to decrease during the culture growth mainly due to the decrease in ouabain-inhibitable and (ouabain + furosemide)- non-inhibitable influxes. The changes in cation fluxes and cation contents were observed in transformed cells without contact inhibition of division and were considered as a manifestation of density-dependent alterations of plasma membrane.

The promise of human embryonic stem cells in aging-associated diseases

PubMed Central

Yabut, Odessa; Bernstein, Harold S.

2011-01-01

Aging-associated diseases are often caused by progressive loss or dysfunction of cells that ultimately affect the overall function of tissues and organs. Successful treatment of these diseases could benefit from cell-based therapy that would regenerate lost cells or otherwise restore tissue function. Human embryonic stem cells (hESCs) promise to be an important therapeutic candidate in treating aging-associated diseases due to their unique capacity for self-renewal and pluripotency. To date, there are numerous hESC lines that have been developed and characterized. We will discuss how hESC lines are derived, their molecular and cellular properties, and how their ability to differentiate into all three embryonic germ layers is determined. We will also outline the methods currently employed to direct their differentiation into populations of tissue-specific, functional cells. Finally, we will highlight the general challenges that must be overcome and the strategies being developed to generate highly-purified hESC-derived cell populations that can safely be used for clinical applications. PMID:21566262

The acoustic sensor for rapid analysis of bacterial cells in the conductive suspensions.

PubMed

Borodina, I A; Zaitsev, B D; Guliy, O; Teplykh, A A; Shikhabudinov, A M

2017-11-01

The possibility of using the acoustic sensor on the basis of a two-channel delay line for rapid analysis of bacterial cells in the conductive suspensions was investigated. The dependencies of change in phase and insertion loss of output signal of the sensor on conductivity of buffer solution with various concentrations of cells due to a specific interaction "bacterial cells - mini-antibodies" for electrically open and electrically shorted channels of delay line were measured. It has been found that these changes have the most values for the electrically open channel. It has been also shown that the sensor rapidly responds to the specific interaction and the time stabilization of the phase and insertion loss of output signal is less than 10min. Copyright © 2017 Elsevier B.V. All rights reserved.

Antitumoral activity of 1,2-diaminocyclohexane derivatives in breast, colon and skin human cancer cells.

PubMed

Morales, Fátima; Ramírez, Alberto; Morata-Tarifa, Cynthia; Navarro, Saúl A; Marchal, Juan A; Campos, Joaquín M; Conejo-García, Ana

2017-03-01

Cancer is among the leading causes of death worldwide. Medical interest has focused on macrocyclic polyamines because of their properties as antitumor agents. Results/Methodology: We have designed and synthesized a series of 1,2-diaminocyclohexane derivatives with notable in vitro antiproliferative activities against the MCF-7, HCT-116 and A375 cancer cell lines. Cell cycle and apoptosis analyses were also carried out. Our results show that all the compounds are potent cytotoxic agents, especially against the A375 cell line. The selective activity of the macrocyclic derivative against A375, via apoptosis, supposes a great advantage for future therapeutic use. This exemplifies the potential of 1,2-diaminocyclohexane derivatives to qualify as lead structures for future anticancer drug development due to their easy syntheses and noteworthy bioactivity.

Sers Imaging of Nasopharyngeal Carcinoma Markers Using an Antibody-Conjugated Gold Nanoparticles Probe

NASA Astrophysics Data System (ADS)

Li, J.-H.; Du, Y.; Feng, G.-K.; Du, Y.-B.; Zhou, Y.-Q.; Zeng, M.-S.

2017-11-01

Surface-enhanced Raman scattering (SERS) nanotags as an ultrasensitive nanoprobe is becoming popular for the detection of biomarkers. Herein, antibody-conjugated gold nanoparticles (AuNPs) were used to target LMP2A in an LMP2A-infected CNE2 cell line. SERS maps showed that the LMP2A was distributed around the cell, which was consistent with the results of immunofl uorescence staining in the previous report. This location could be due to the specific binding of the bioconjugated nanotags to the receptors on the cell surface. However, the CNE2 cell line without LMP2A-infected showed no detectable signal at 1044 cm-1. The results demonstrated the potential feasibility of AuNPs nanotags as highly sensitive probes conjugated at the subcellular level for detection and localization of cancer markers in nasopharyngeal carcinoma (NPC).

Oxygen consumption rate and mitochondrial density in human melanoma monolayer cultures and multicellular spheroids.

PubMed

Hystad, M E; Rofstad, E K

1994-05-15

Rate of oxygen consumption per cell has been shown in previous studies to decrease with increasing depth in the viable rim of multicellular spheroids initiated from rodent cells, human colon-carcinoma cells, and human glioma cells, due to progressive accumulation of quiescent cells during spheroid growth. The purpose of our work was to determine oxygen-consumption profiles in human melanoma spheroids. Monolayer cultures of 4 lines (BEX-c, COX-c, SAX-c, and WIX-c) and spheroid cultures of 2 lines (BEX-c and WIX-c) were subjected to investigation. Spheroids were initiated from monolayer cell cultures and grown in spinner flasks. Rate of oxygen consumption was measured with a Clarke-type electrode. Mitochondrial density was determined by stereological analysis of transmission electron micrographs. Thickness of viable rim and cell packing density were assessed by light microscopy of central spheroid sections. Cell-cycle distribution was determined by analysis of DNA histograms measured by flow cytometry. Cell volume was measured by an electronic particle counter. Rate of oxygen consumption per cell differed by a factor of approximately 1.8 between the 4 cell lines and was positively correlated to total volume of mitochondria per cell. Rate of oxygen consumption per cell and total volume of mitochondria per cell were equal for monolayer cell cultures, 600-microns spheroids and 1,200-microns spheroids of the same line. Mitochondrial density and location in the cell did not differ between cells at the spheroid surface, in the middle of the viable rim and adjacent to the central necrosis. Cell-cycle distribution, cell volume, and cell-packing density in the outer and inner halves of the viable rim were not significantly different. Consequently, the rate of oxygen consumption per cell in inner regions of the viable rim was probably equal to that at the spheroid surface, suggesting that oxygen diffusion distances may be shorter in some melanomas than in many other tumor types.

Early Molecular Events in Murine Gastric Epithelial Cells Mediated by Helicobacter pylori CagA.

PubMed

Banerjee, Aditi; Basu, Malini; Blanchard, Thomas G; Chintalacharuvu, Subba R; Guang, Wei; Lillehoj, Erik P; Czinn, Steven J

2016-10-01

Murine models of Helicobacter pylori infection are used to study host-pathogen interactions, but lack of severe gastritis in this model has limited its usefulness in studying pathogenesis. We compared the murine gastric epithelial cell line GSM06 to the human gastric epithelial AGS cell line to determine whether similar events occur when cultured with H. pylori. The lysates of cells infected with H. pylori isolates or an isogenic cagA-deficient mutant were assessed for translocation and phosphorylation of CagA and for activation of stress pathway kinases by immunoblot. Phosphorylated CagA was detected in both cell lines within 60 minutes. Phospho-ERK 1/2 was present within several minutes and distinctly present in GSM06 cells at 60 minutes. Similar results were obtained for phospho-JNK, although the 54 kDa phosphoprotein signal was dominant in AGS, whereas the lower molecular weight band was dominant in GSM06 cells. These results demonstrate that early events in H. pylori pathogenesis occur within mouse epithelial cells similar to human cells and therefore support the use of the mouse model for the study of acute CagA-associated host cell responses. These results also indicate that reduced disease in H. pylori-infected mice may be due to lack of the Cag PAI, or by differences in the mouse response downstream of the initial activation events. © 2016 John Wiley & Sons Ltd.

The expression of VE-cadherin in breast cancer cells modulates cell dynamics as a function of tumor differentiation and promotes tumor-endothelial cell interactions.

PubMed

Rezaei, Maryam; Cao, Jiahui; Friedrich, Katrin; Kemper, Björn; Brendel, Oliver; Grosser, Marianne; Adrian, Manuela; Baretton, Gustavo; Breier, Georg; Schnittler, Hans-Joachim

2018-01-01

The cadherin switch has profound consequences on cancer invasion and metastasis. The endothelial-specific vascular endothelial cadherin (VE-cadherin) has been demonstrated in diverse cancer types including breast cancer and is supposed to modulate tumor progression and metastasis, but underlying mechanisms need to be better understood. First, we evaluated VE-cadherin expression by tissue microarray in 392 cases of breast cancer tumors and found a diverse expression and distribution of VE-cadherin. Experimental expression of fluorescence-tagged VE-cadherin (VE-EGFP) in undifferentiated, fibroblastoid and E-cadherin-negative MDA-231 (MDA-VE-EGFP) as well as in differentiated E-cadherin-positive MCF-7 human breast cancer cell lines (MCF-VE-EGFP), respectively, displayed differentiation-dependent functional differences. VE-EGFP expression reversed the fibroblastoid MDA-231 cells to an epithelial-like phenotype accompanied by increased β-catenin expression, actin and vimentin remodeling, increased cell spreading and barrier function and a reduced migration ability due to formation of VE-cadherin-mediated cell junctions. The effects were largely absent in both MDA-VE-EGFP and in control MCF-EGFP cell lines. However, MCF-7 cells displayed a VE-cadherin-independent planar cell polarity and directed cell migration that both developed in MDA-231 only after VE-EGFP expression. Furthermore, VE-cadherin expression had no effect on tumor cell proliferation in monocultures while co-culturing with endothelial cells enhanced tumor cell proliferation due to integration of the tumor cells into monolayer where they form VE-cadherin-mediated cell contacts with the endothelium. We propose an interactive VE-cadherin-based crosstalk that might activate proliferation-promoting signals. Together, our study shows a VE-cadherin-mediated cell dynamics and an endothelial-dependent proliferation in a differentiation-dependent manner.

Discovery of specific ligands for oral squamous carcinoma to develop anti-cancer drug loaded precise targeting nanotherapeutics.

PubMed

Yang, Fan; Liu, Ruiwu; Kramer, Randall; Xiao, Wenwu; Jordan, Richard; Lam, Kit S

2012-12-01

Oral squamous cell carcinoma has a low five-year survival rate, which may be due to late detection and a lack of effective tumor-specific therapies. Using a high throughput drug discovery strategy termed one-bead one-compound combinatorial library, the authors identified six compounds with high binding affinity to different human oral squamous cell carcinoma cell lines but not to normal cells. Current work is under way to develop these ligands to oral squamous cell carcinoma specific imaging probes or therapeutic agents.

A perpetual source of DNA or something really different: ethical issues in the creation of cell lines for African genomics research.

PubMed

de Vries, Jantina; Abayomi, Akin; Brandful, James; Littler, Katherine; Madden, Ebony; Marshall, Patricia; Ouwe Missi Oukem-Boyer, Odile; Seeley, Janet

2014-08-07

The rise of genomic studies in Africa - not least due to projects funded under H3Africa - is associated with the development of a small number of biorepositories across Africa. For the ultimate success of these biorepositories, the creation of cell lines including those from selected H3Africa samples would be beneficial. In this paper, we map ethical challenges in the creation of cell lines. The first challenge we identified relates to the moral status of cells living in culture. There is no doubt that cells in culture are alive, and the question is how this characteristic is relevant to ethical decision-making. The second challenge relates to the fact that cells in culture are a source of cell products and mitochondrial DNA. In combination with other technologies, cells in culture could also be used to grow human tissue. Whilst on the one hand, this feature increases the potential utility of the sample and promotes science, on the other it also enables further scientific work that may not have been specifically consented to or approved. The third challenge relates to ownership over samples, particularly in cases where cell lines are created by a biobank, and in a different country than where samples were collected. Relevant questions here concern the export of samples, approval of secondary use and the acceptability of commercialisation. A fourth challenge relates to perceptions of blood and bodily integrity, which may be particularly relevant for African research participants from certain cultures or backgrounds. Finally, we discuss challenges around informed consent and ethical review. In this paper, we sought to map the myriad of ethical challenges that need to be considered prior to making cell line creation a reality in the H3Africa project. Considering the relative novelty of this practice in Africa, such challenges will need to be considered, discussed and potentially be resolved before cell line creation in Africa becomes financially feasible and sustainable. We suggest that discussions need to be undertaken between stakeholders internationally, considering the international character of the H3Africa project. We also map out avenues for empirical research.

Photodynamic killing of cancer cells by a Platinum(II) complex with cyclometallating ligand

NASA Astrophysics Data System (ADS)

Doherty, Rachel E.; Sazanovich, Igor V.; McKenzie, Luke K.; Stasheuski, Alexander S.; Coyle, Rachel; Baggaley, Elizabeth; Bottomley, Sarah; Weinstein, Julia A.; Bryant, Helen E.

2016-03-01

Photodynamic therapy that uses photosensitizers which only become toxic upon light-irradiation provides a strong alternative to conventional cancer treatment due to its ability to selectively target tumour material without affecting healthy tissue. Transition metal complexes are highly promising PDT agents due to intense visible light absorption, yet the majority are toxic even without light. This study introduces a small, photostable, charge-neutral platinum-based compound, Pt(II) 2,6-dipyrido-4-methyl-benzenechloride, complex 1, as a photosensitizer, which works under visible light. Activation of the new photosensitizer at low concentrations (0.1-1 μM) by comparatively low dose of 405 nm light (3.6 J cm-2) causes significant cell death of cervical, colorectal and bladder cancer cell lines, and, importantly, a cisplatin resistant cell line EJ-R. The photo-index of the complex is 8. We demonstrate that complex 1 induces irreversible DNA single strand breaks following irradiation, and that oxygen is essential for the photoinduced action. Neither light, nor compound alone led to cell death. The key advantages of the new drug include a remarkably fast accumulation time (diffusion-controlled, minutes), and photostability. This study demonstrates a highly promising new agent for photodynamic therapy, and attracts attention to photostable metal complexes as viable alternatives to conventional chemotherapeutics, such as cisplatin.

Generation of a human CDX2 knock-in reporter iPSC line (MHHi007-A-1) to model human trophoblast differentiation.

PubMed

Malysheva, Svitlana V; Wunderlich, Stephanie; Haase, Alexandra; Göhring, Gudrun; Martin, Ulrich; Merkert, Sylvia

2018-05-19

Caudal-type homeobox 2 (CDX2) transcription factor is an important marker for early trophoblast lineages and intestinal epithelium. Due to its nuclear expression the immunostaining and sorting of viable CDX2 pos cells is not possible. In this paper we report the generation and describe key characteristics of a CDX2 Venus knock-in reporter hiPSC-cell line (MHHi007-A-1) which can serve as an in vitro tool to study human trophoblast and intestinal differentiation. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Biochemical and morphological changes associated with macrophages and osteoclasts when challenged with infection - biomed 2011.

PubMed

Wiggers, Erin Callie; Johnson, William; Tucci, Michelle; Benghuzzi, Hamed

2011-01-01

Osteomyelitis is a bacterial infection of the bone that occurs frequently as a complication of open fractures and various kinds of orthopedic surgery. This infection can often lead to more extensive surgeries and even death of the patient. In animal models of osteomyelitis, the site of infection by Staphylococcus aureus was observed to have high numbers of both macrophages and osteoclasts, both of which may contribute to large amounts of osteolysis and tissue damage. In order to evaluate the immune response in both types of cells, two cells lines, a macrophage cell line and a macrophage cell line stimulated to become osteoclasts by the addition of receptor activator of nuclear-factor B (RANKL), were exposed to lipopolysaccharides, opsonized S. aureus, and unopsonized S. aureus. The results showed that both cell types activated a biochemical cascade that included the release of cytokines and nitric oxide associated with cell damage and death in response to infection. However, macrophages and osteoclasts differed in response magnitude, most likely due to differences in cell-membrane receptors. This data supports the growing body of research that links the immune and skeletal systems. Further understanding of biochemical pathways shared by the two systems could lead to significant advances in the treatment of osteomyelitis and the success of prostheses.

Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number

PubMed Central

Spadafora, Domenico; Kozhukhar, Natalia; Alexeyev, Mikhail F.

2016-01-01

Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion. PMID:27031233

Human CD4 T cell epitopes selective for Vaccinia versus Variola virus.

PubMed

Probst, Alicia; Besse, Aurore; Favry, Emmanuel; Imbert, Gilles; Tanchou, Valérie; Castelli, Florence Anne; Maillere, Bernard

2013-04-01

Due to the high degree of sequence identity between Orthopoxvirus species, the specific B and T cell responses raised against these viruses are largely cross-reactive and poorly selective. We therefore searched for CD4 T cell epitopes present in the conserved parts of the Vaccinia genome (VACV) but absent from Variola viruses (VARV), with a view to identifying immunogenic sequences selective for VACV. We identified three long peptide fragments from the B7R, B10R and E7R proteins by in silico comparisons of the poxvirus genomes, and evaluated the recognition of these fragments by VACV-specific T cell lines derived from healthy donors. For the 12 CD4 T cell epitopes identified, we assessed their binding to common HLA-DR allotypes and their capacity to induce peptide-specific CD4 T-cell lines. Four peptides from B7R and B10R displayed a broad binding specificity for HLA-DR molecules and induced multiple T cell lines from healthy donors. Besides their absence from VARV, the two B10R peptide sequences were mutated in the Cowpox virus and completely absent from the Monkeypox genome. This work contributes to the development of differential diagnosis of poxvirus infections. Copyright © 2012 Elsevier Ltd. All rights reserved.

Release behavior and toxicity profiles towards A549 cell lines of ciprofloxacin from its layered zinc hydroxide intercalation compound.

PubMed

Abdul Latip, Ahmad Faiz; Hussein, Mohd Zobir; Stanslas, Johnson; Wong, Charng Choon; Adnan, Rohana

2013-01-01

Layered hydroxides salts (LHS), a layered inorganic compound is gaining attention in a wide range of applications, particularly due to its unique anion exchange properties. In this work, layered zinc hydroxide nitrate (LZH), a family member of LHS was intercalated with anionic ciprofloxacin (CFX), a broad spectrum antibiotic via ion exchange in a mixture solution of water:ethanol. Powder x-ray diffraction (XRD), Fourier transform infrared (FTIR) and thermogravimetric analysis (TGA) confirmed the drug anions were successfully intercalated in the interlayer space of LZH. Specific surface area of the obtained compound was increased compared to that of the host due to the different pore textures between the two materials. CFX anions were slowly released over 80 hours in phosphate-buffered saline (PBS) solution due to strong interactions that occurred between the intercalated anions and the host lattices. The intercalation compound demonstrated enhanced antiproliferative effects towards A549 cancer cells compared to the toxicity of CFX alone. Strong host-guest interactions between the LZH lattice and the CFX anion give rise to a new intercalation compound that demonstrates sustained release mode and enhanced toxicity effects towards A549 cell lines. These findings should serve as foundations towards further developments of the brucite-like host material in drug delivery systems.

Zinc in human health: effect of zinc on immune cells.

PubMed

Prasad, Ananda S

2008-01-01

Although the essentiality of zinc for plants and animals has been known for many decades, the essentiality of zinc for humans was recognized only 40 years ago in the Middle East. The zinc-deficient patients had severe immune dysfunctions, inasmuch as they died of intercurrent infections by the time they were 25 years of age. In our studies in an experimental human model of zinc deficiency, we documented decreased serum testosterone level, oligospermia, severe immune dysfunctions mainly affecting T helper cells, hyperammonemia, neurosensory disorders, and decreased lean body mass. It appears that zinc deficiency is prevalent in the developing world and as many as two billion subjects may be growth retarded due to zinc deficiency. Besides growth retardation and immune dysfunctions, cognitive impairment due to zinc deficiency also has been reported recently. Our studies in the cell culture models showed that the activation of many zinc-dependent enzymes and transcription factors were adversely affected due to zinc deficiency. In HUT-78 (T helper 0 [Th(0)] cell line), we showed that a decrease in gene expression of interleukin-2 (IL-2) and IL-2 receptor alpha(IL-2Ralpha) were due to decreased activation of nuclear factor-kappaB (NF-kappaB) in zinc deficient cells. Decreased NF-kappaB activation in HUT-78 due to zinc deficiency was due to decreased binding of NF-kappaB to DNA, decreased level of NF-kappaB p105 (the precursor of NF-kappaB p50) mRNA, decreased kappaB inhibitory protein (IkappaB) phosphorylation, and decreased Ikappa kappa. These effects of zinc were cell specific. Zinc also is an antioxidant and has anti-inflammatory actions. The therapeutic roles of zinc in acute infantile diarrhea, acrodermatitis enteropathica, prevention of blindness in patients with age-related macular degeneration, and treatment of common cold with zinc have been reported. In HL-60 cells (promyelocytic leukemia cell line), zinc enhances the up-regulation of A20 mRNA, which, via TRAF pathway, decreases NF-kappaB activation, leading to decreased gene expression and generation of tumor necrosis factor-alpha (TNF-alpha), IL-1beta, and IL-8. We have reported recently that in both young adults and elderly subjects, zinc supplementation decreased oxidative stress markers and generation of inflammatory cytokines.

Human induced pluripotent stem cell-derived hepatic cell lines as a new model for host interaction with hepatitis B virus

PubMed Central

Kaneko, Shun; Kakinuma, Sei; Asahina, Yasuhiro; Kamiya, Akihide; Miyoshi, Masato; Tsunoda, Tomoyuki; Nitta, Sayuri; Asano, Yu; Nagata, Hiroko; Otani, Satoshi; Kawai-Kitahata, Fukiko; Murakawa, Miyako; Itsui, Yasuhiro; Nakagawa, Mina; Azuma, Seishin; Nakauchi, Hiromitsu; Nishitsuji, Hironori; Ujino, Saneyuki; Shimotohno, Kunitada; Iwamoto, Masashi; Watashi, Koichi; Wakita, Takaji; Watanabe, Mamoru

2016-01-01

Hepatitis B virus (HBV) is not eradicated by current antiviral therapies due to persistence of HBV covalently closed circular DNA (cccDNA) in host cells, and thus development of novel culture models for productive HBV infection is urgently needed, which will allow the study of HBV cccDNA eradication. To meet this need, we developed culture models of HBV infection using human induced pluripotent stem cell-derived hepatocyte lineages, including immature proliferating hepatic progenitor-like cell lines (iPS-HPCs) and differentiated hepatocyte-like cells (iPS-Heps). These cells were susceptible to HBV infection, produced HBV particles, and maintained innate immune responses. The infection efficiency of HBV in iPS-HPCs predominantly depended on the expression levels of sodium taurocholate cotransporting polypeptide (NTCP), and was low relative to iPS-Heps: however, long-term culture of iPS-Heps was difficult. To provide a model for HBV persistence, iPS-HPCs overexpressing NTCP were established. The long-term persistence of HBV cccDNA was detected in iPS-HPCs overexpressing NTCP, and depended on the inhibition of the Janus-kinase signaling pathway. In conclusion, this study provides evidence that iPS-derived hepatic cell lines can be utilized for novel HBV culture models with genetic variation to investigate the interactions between HBV and host cells and the development of anti-HBV strategies. PMID:27386799

Documentation of normal and leukemic myelopoietic progenitor cells with high-resolution phase-contrast time-lapse cinematography.

PubMed

Boll, I T

2001-08-01

The high-resolution phase-contrast, time-lapse cinematography using oil immersion lenses and 16-mm film demonstrates the kinetic cell events as maturation, locomotion, mitosis, and apoptosis of cells cultivated at 37 degrees C for up to 10 days. 0.5 v/v frozen-thawed sera with presumably high cytokine concentrations were added to the plasma or agar clot. Vital progenitor cells from human bone marrow and blood have a large, bright, unstructured nucleus with a large nucleolus and a narrow rim of cytoplasm (nuclear/cytoplasmic volume ratio = 0.7). Their nuclei are 6-14 micrometer in diameter and double their volume within 8 h. Many (70%) move at a mean speed of 2 micrometer/min, and many (30%) multiply with alpha-2alpha mitoses, generating progenitor cell families. Various disturbances during the course of mitosis lead to the formation of polyploid cells, thereby yielding the megakaryocytic cell line. Some of the progenitor cells undergo asymmetric alpha-alphan mitoses: One of the two initially identical daughter cells remains a progenitor cell in the morphological sense, whereas the other daughter cell - depending on the size of its mother cell - matures in the same culture medium to form a granulocytopoietic, monocytopoietic or erythrocytopoietic cell line. - In acute myeloid leukemias (AML), the blasts and their nuclei are slightly larger than the corresponding progenitor cells and move faster (5 micrometer/min). Symmetric alpha-2alpha mitoses permit unlimited multiplication of the leukemic blasts if contact with cytotoxic lymphocytes does not render them apoptotic. This results in more stromal cells than normal. Granulocytopenia, monocytopenia, and anemia occur due to the genetic impairment of signaling control for asymmetric alpha-alphan mitoses, and thrombocytopenia occurs due to the reduction in polyploidization. Copyright 2001 S. Karger GmbH, Freiburg

Genotoxic and endocrine activities of bis(hydroxyphenyl)methane (bisphenol F) and its derivatives in the HepG2 cell line.

PubMed

Cabaton, Nicolas; Dumont, Coralie; Severin, Isabelle; Perdu, Elisabeth; Zalko, Daniel; Cherkaoui-Malki, Mustapha; Chagnon, Marie-Christine

2009-01-08

Human can be exposed to bis(hydroxyphenyl)methane (bisphenol F or BPF) and its derivatives as environment and food's contaminants. This study was investigated to identify and to compare toxic potency of BPF, BFDGE, and two of BPF metabolites using in vitro methods. BPF did not induce any genic mutation in bacteria when the Ames test was performed according to the OECD guideline. In contrast, using Human cell lines and Comet assay, we demonstrated that BPF and Bisphenol F Diglycidyl Ether (BFDGE) were effective on HepG2 cell DNA fragmentation at non-cytotoxic concentrations. DHB was also positive but at higher concentrations, near its limit of solubility. Neither BPF, nor DHB induced a positive response in the micronucleus assay. The increase of micronuclei observed when cells were exposed to BFDGE was mostly due to a cytotoxic effect. Concerning endocrine activities, BPF increased the luciferase activity in HepG2 cells transiently transfected with a concentration dependant pattern, DHB also induced a positive response but at highest concentrations. Estrogenic responses in the HepG2 cells differed with the estrogen receptor (ER) involved. Using MDA-kb2 cell line stably transfected with pMMTV-neo-Luc, only BPF was anti-androgenic at the highest concentration (10(-5)M). Then, we demonstrated using human cell lines, especially HepG2, BPF was the most toxic compound in term of genotoxicity and endocrine activities compared to DHB and BPF-OH, the free metabolites identified in rat urine when BPF was administrated to rats.

Studies of Pressure-Broadening of Alkali Atom Resonance Lines for Modeling Atmospheres of Extrasolar Giant Planets and Brown Dwarfs

NASA Technical Reports Server (NTRS)

Kirby, Kate; Babb, J.; Yoshino, K.

2004-01-01

In L-dwarfs and T-dwarfs the resonance lines of sodium and potassium are so profoundly pressure-broadened that their wings extend several hundred nanometers from line center. With accurate knowledge of the line profiles as a function of temperature and pressure: such lines can prove to be valuable diagnostics of the atmospheres of such objects. We have initiated a joint program of theoretical and experimental research to study the line-broadening of alkali atom resonance lines due to collisions with species such as helium and molecular hydrogen. Although potassium and sodium are the alkali species of most interest in the atmospheres of cool brown dwarfs and extrasolar giant planets, some of our theoretical focus this year has involved the calculation of pressure-broadening of lithium resonance lines by He, as a test of a newly developed suite of computer codes. In addition, theoretical calculations have been carried out to determine the leading long range van der Waals coefficients for the interactions of ground and excited alkali metal atoms with helium atoms, to within a probable error of 2%. Such data is important in determining the behavior of the resonance line profiles in the far wings. Important progress has been made on the experimental aspects of the program since the arrival of a postdoctoral fellow in September. A new absorption cell has been designed, which incorporates a number of technical improvements over the previous cell, including a larger cell diameter to enhance the signal, and fittings which allow for easier cleaning, thereby significantly reducing the instrument down-time.

Evidence of a cellular immune response against sialyl-Tn in breast and ovarian cancer patients after high-dose chemotherapy, stem cell rescue, and immunization with Theratope STn-KLH cancer vaccine.

PubMed

Sandmaier, B M; Oparin, D V; Holmberg, L A; Reddish, M A; MacLean, G D; Longenecker, B M

1999-01-01

Seven ovarian and 33 breast high-risk stage II/III and stage IV cancer patients received high-dose chemotherapy followed by stem cell rescue. Thirty to 151 days after stem cell transplantation, the patients received their first immunotherapy treatment with Theratope STn-KLH cancer vaccine. Most patients developed increasing IgG anti-STn titers to a sustained peak after the fourth or fifth immunizations. Only one patient had elevated CA27.29 (MUC1 mucin) serum levels at trial entry. Five of the seven patients with preimmunotherapy elevated serum CA125 levels demonstrated decreasing CA125 levels during immunotherapy, consistent with an antitumor response. Evidence of STn antigen-specific T-cell proliferation was obtained from 17 of the 27 evaluable patients who received at least three immunotherapy treatments. Eleven of the 26 patients tested had evidence of an anti-STn TH1 antigen-specific T-cell response as determined by interferon-gamma, but not interleukin (IL)-4, production. After immunization, lytic activity of peripheral blood lymphocytes (PBLs) tested against a lymphokine activated killer (LAK)-sensitive cell line, a natural killer (NK)-sensitive cell line, and an STn-expressing cancer cell line (OVCAR) increased significantly. In vitro IL-2 treatment of the PBLs after vaccination greatly enhanced killing of the STn+ cancer cell line. Evidence of the development of OVCAR specific killing activity, over and above that seen due to LAK or NK killing, is presented. These studies provide the strongest evidence in humans of the development of an antitumor T-cell response after immunization with a cancer-associated carbohydrate antigen.

Interleukin-6 expression under gravitational stress due to vibration and hypergravity in follicular thyroid cancer cells.

PubMed

Ma, Xiao; Wehland, Markus; Aleshcheva, Ganna; Hauslage, Jens; Waßer, Kai; Hemmersbach, Ruth; Infanger, Manfred; Bauer, Johann; Grimm, Daniela

2013-01-01

It is known that exposing cell lines in vitro to parabolic flights changes their gene expression and protein production patterns. Parabolic flights and spaceflight in general are accompanied by transient hypergravity and vibration, which may impact the cells and therefore, have to be considered too. To estimate the possible impact of transient hypergravity and vibration, we investigated the effects of these forces separately using dedicated ground-based facilities. We placed follicular thyroid ML-1 and CGTH W-1 cancer cells in a specific centrifuge (MuSIC Multi Sample Incubator Centrifuge; SAHC Short Arm Human Centrifuge) simulating the hypergravity phases that occur during one (P1) and 31 parabolas (P31) of parabolic flights, respectively. On the Vibraplex device, the same cell lines were treated with vibration waves corresponding to those that occur during a whole parabolic flight lasting for two hours. After the various treatments, cells were harvested and analyzed by quantitative real-time PCR, focusing on the genes involved in forming (ACTB, MYO9, TUBB, VIM, TLN1, and ITGB1) and modulating (EZR, RDX, and MSN) the cytoskeleton, as well as those encoding growth factors (EGF, CTGF, IL6, and IL8) or protein kinases (PRKAA1 and PRKCA). The analysis revealed alterations in several genes in both cell lines; however, fewer genes were affected in ML-1 than CGTH W-1 cells. Interestingly, IL6 was the only gene whose expression was changed in both cell lines by each treatment, while PKCA transcription remained unaffected in all experiments. We conclude that a PKCa-independent mechanism of IL6 gene activation is very sensitive to physical forces in thyroid cells cultured in vitro as monolayers.

Interleukin-6 Expression under Gravitational Stress Due to Vibration and Hypergravity in Follicular Thyroid Cancer Cells

PubMed Central

Ma, Xiao; Wehland, Markus; Aleshcheva, Ganna; Hauslage, Jens; Waßer, Kai; Hemmersbach, Ruth; Infanger, Manfred; Bauer, Johann; Grimm, Daniela

2013-01-01

It is known that exposing cell lines in vitro to parabolic flights changes their gene expression and protein production patterns. Parabolic flights and spaceflight in general are accompanied by transient hypergravity and vibration, which may impact the cells and therefore, have to be considered too. To estimate the possible impact of transient hypergravity and vibration, we investigated the effects of these forces separately using dedicated ground-based facilities. We placed follicular thyroid ML-1 and CGTH W-1 cancer cells in a specific centrifuge (MuSIC Multi Sample Incubator Centrifuge; SAHC Short Arm Human Centrifuge) simulating the hypergravity phases that occur during one (P1) and 31 parabolas (P31) of parabolic flights, respectively. On the Vibraplex device, the same cell lines were treated with vibration waves corresponding to those that occur during a whole parabolic flight lasting for two hours. After the various treatments, cells were harvested and analyzed by quantitative real-time PCR, focusing on the genes involved in forming (ACTB, MYO9, TUBB, VIM, TLN1, and ITGB1) and modulating (EZR, RDX, and MSN) the cytoskeleton, as well as those encoding growth factors (EGF, CTGF, IL6, and IL8) or protein kinases (PRKAA1 and PRKCA). The analysis revealed alterations in several genes in both cell lines; however, fewer genes were affected in ML-1 than CGTH W-1 cells. Interestingly, IL6 was the only gene whose expression was changed in both cell lines by each treatment, while PKCA transcription remained unaffected in all experiments. We conclude that a PKCa-independent mechanism of IL6 gene activation is very sensitive to physical forces in thyroid cells cultured in vitro as monolayers. PMID:23844163

CD133 antisense suppresses cancer cell growth and increases sensitivity to cisplatin in vitro

PubMed Central

BLANCAS-MOSQUEDA, MARISOL; ZAPATA-BENAVIDES, PABLO; ZAMORA-ÁVILA, DIANA; SAAVEDRA-ALONSO, SANTIAGO; MANILLA-MUÑOZ, EDGAR; FRANCO-MOLINA, MOISÉS; DE LA PEÑA, CARMEN MONDRAGÓN; RODRÍGUEZ-PADILLA, CRISTINA

2012-01-01

The increased incidence of cancer in recent years is associated with a high rate of mortality. Numerous types of cancer have a low percentage of CD133+ cells, which have similar features to stem cells. The CD133 molecule is involved in apoptosis and cell proliferation. The aim of this study was to determine the biological effect of CD133 suppression and its role in the chemosensitization of cancer cell lines. RT-PCR and immunocytochemical analyses indicated that CD133 was expressed in the cancer cell lines B16F10, MCF7 and INER51. Downregulation of CD133 by transfection with an antisense sequence (As-CD133) resulted in a decrease in cancer cell viability of up to 52, 47 and 22% in B16F10, MCF-7 and INER51 cancer cell lines, respectively. This decreased viability appeared to be due to the induction of apoptosis. In addition, treatment with As-CD133 in combination with cisplatin had a synergic effect in all of the cancer cell lines analyzed, and in particular, significantly decreased the viability of B16F10 cancer cells compared with each treatment separately (3.1% viability for the combined treatment compared with 48% for 0.4 μg As-CD133 and 25% for 5 ng/μl cisplatin; P<0.05). The results indicate that the downregulation of CD133 by antisense is a potential therapeutic target for cancer and has a synergistic effect when administered with minimal doses of the chemotherapeutic drug cisplatin, suggesting that this combination strategy may be applied in cancer treatment. PMID:23226746

Synthesis and biological evaluation of pyrimidine bridged combretastatin derivatives as potential anticancer agents and mechanistic studies.

PubMed

Kumar, Bhupinder; Sharma, Praveen; Gupta, Vivek Prakash; Khullar, Madhu; Singh, Sandeep; Dogra, Nilambra; Kumar, Vinod

2018-08-01

A number of pyrimidine bridged combretastatin derivatives were designed, synthesized and evaluated for anticancer activities against breast cancer (MCF-7) and lung cancer (A549) cell lines using MTT assays. Most of the synthesized compounds displayed good anticancer activity with IC 50 values in low micro-molar range. Compounds 4a and 4p were found most potent in the series with IC 50 values of 4.67 µM & 3.38 µM and 4.63 µM & 3.71 µM against MCF7 and A549 cancer cell lines, respectively. Biological evaluation of these compounds showed that selective cancer cell toxicity (in vitro using human lung and breast cancer cell lines) might be due to the inhibition of antioxidant enzymes instigating elevated ROS levels which triggers intrinsic apoptotic pathways. These compounds were found nontoxic to the normal human primary cells. Compound 4a, was found to be competitive inhibitor of colchicine and in the tubulin binding assay it showed tubulin polymerization inhibition potential comparable to colchicine. The molecular modeling studies also showed that the synthesized compounds fit well in the colchicine-binding pocket. Copyright © 2018 Elsevier Inc. All rights reserved.

Effect of Anacardium occidentale leaf extract on human acute lymphoblastic leukaemia cell lines.

PubMed

Santos, Janaína M; Cury, Nathalia M; Yunes, José A; López, Jorge A; Hernández-Macedo, Maria L

2018-01-16

Anacardium occidentale leaves are used in folk medicine due its therapeutic properties attributed to phenolic compounds. Therefore, this study was undertaken on its hydroethanolic leaf extract (AoHE) to evaluate cytotoxicity and apoptosis induction on acute lymphoblastic leukaemia cells. Results indicated that AoHE interfered in the cell cycle progression, inducing apoptosis by activation of casp3 at lower concentrations, thence, a promising candidate for the development of new cancer drugs.

Proteomic analysis of effects by x-rays and heavy ion in HeLa cells.

PubMed

Bing, Zhitong; Yang, Guanghui; Zhang, Yanan; Wang, Fengling; Ye, Caiyong; Sun, Jintu; Zhou, Guangming; Yang, Lei

2014-06-01

Carbon ion therapy may be better against cancer than the effects of a photon beam. To investigate a biological advantage of carbon ion beam over X-rays, the radioresistant cell line HeLa cells were used. Radiation-induced changes in the biological processes were investigated post-irradiation at 1 h by a clinically relevant radiation dose (2 Gy X-ray and 2 Gy carbon beam). The differential expression proteins were collected for analysing biological effects. The radioresistant cell line Hela cells were used. In our study, the stable isotope labelling with amino acids (SILAC) method coupled with 2D-LC-LTQ Orbitrap mass spectrometry was applied to identity and quantify the differentially expressed proteins after irradiation. The Western blotting experiment was used to validate the data. A total of 123 and 155 significantly changed proteins were evaluated with treatment of 2 Gy carbon and X-rays after radiation 1 h, respectively. These deregulated proteins were found to be mainly involved in several kinds of metabolism processes through Gene Ontology (GO) enrichment analysis. The two groups perform different response to different types of irradiation. The radioresistance of the cancer cells treated with 2 Gy X-rays irradiation may be largely due to glycolysis enhancement, while the greater killing effect of 2 Gy carbon may be due to unchanged glycolysis and decreased amino acid metabolism.

Microvesicle removal of anticancer drugs contributes to drug resistance in human pancreatic cancer cells

PubMed Central

Muralidharan-Chari, Vandhana; Kohan, Hamed Gilzad; Asimakopoulos, Alexandros G.; Sudha, Thangirala; Sell, Stewart; Kannan, Kurunthachalam; Boroujerdi, Mehdi; Davis, Paul J.; Mousa, Shaker A.

2016-01-01

High mortality in pancreatic cancer patients is partly due to resistance to chemotherapy. We describe that human pancreatic cancer cells acquire drug resistance by a novel mechanism in which they expel and remove chemotherapeutic drugs from the microenvironment via microvesicles (MVs). Using human pancreatic cancer cells that exhibit varied sensitivity to gemcitabine (GEM), we show that GEM exposure triggers the cancer cells to release MVs in an amount that correlates with that cell line's sensitivity to GEM. The importance of MV-release in gaining drug resistance in GEM-resistant pancreatic cancer cells was confirmed when the inhibition of MV-release sensitized the cells to GEM treatment, both in vitro and in vivo. Mechanistically, MVs remove drugs that are internalized into the cells and that are in the microenvironment. The differences between the drug-resistant and drug-sensitive pancreatic cancer cell lines tested here are explained based on the variable content of influx/efflux proteins present on MVs, which directly dictates the ability of MVs either to trap GEM or to allow GEM to flow back to the microenvironment. PMID:27391262

A highly reproducible quantitative viral outgrowth assay for the measurement of the replication-competent latent HIV-1 reservoir.

PubMed

Fun, Axel; Mok, Hoi Ping; Wills, Mark R; Lever, Andrew M

2017-02-24

Cure of Human Immunodeficiency Virus (HIV) infection remains elusive due to the persistence of HIV in a latent reservoir. Strategies to eradicate latent infection can only be evaluated with robust, sensitive and specific assays to quantitate reactivatable latent virus. We have taken the standard peripheral blood mononuclear cell (PBMC) based viral outgrowth methodology and from it created a logistically simpler and more highly reproducible assay to quantify replication-competent latent HIV in resting CD4 + T cells, both increasing accuracy and decreasing cost and labour. Purification of resting CD4 + T cells from whole PBMC is expedited and achieved in 3 hours, less than half the time of conventional protocols. Our indicator cell line, SupT1-CCR5 cells (a clonal cell line expressing CD4, CXCR4 and CCR5) provides a readily available standardised readout. Reproducibility compares favourably to other published assays but with reduced cost, labour and assay heterogeneity without compromising sensitivity.

Pyrimethamine as a Potent and Selective Inhibitor of Acute Myeloid Leukemia Identified by High-throughput Drug Screening.

PubMed

Sharma, Amit; Jyotsana, Nidhi; Lai, Courteney K; Chaturvedi, Anuhar; Gabdoulline, Razif; Görlich, Kerstin; Murphy, Cecilia; Blanchard, Jan E; Ganser, Arnold; Brown, Eric; Hassell, John A; Humphries, R Keith; Morgan, Michael; Heuser, Michael

2016-01-01

Hematopoietic stem and progenitor cell differentiation are blocked in acute myeloid leukemia (AML) resulting in cytopenias and a high risk of death. Most patients with AML become resistant to treatment due to lack of effective cytotoxic and differentiation promoting compounds. High MN1 expression confers poor prognosis to AML patients and induces resistance to cytarabine and alltrans-retinoic acid (ATRA) induced differentiation. Using a high-throughput drug screening, we identified the dihydrofolate reductase (DHFR) antagonist pyrimethamine to be a potent inducer of apoptosis and differentiation in several murine and human leukemia cell lines. Oral pyrimethamine treatment was effective in two xenograft mouse models and specifically targeted leukemic cells in human AML cell lines and primary patient cells, while CD34+ cells from healthy donors were unaffected. The antileukemic effects of PMT could be partially rescued by excess folic acid, suggesting an oncogenic function of folate metabolism in AML. Thus, our study identifies pyrimethamine as a candidate drug that should be further evaluated in AML treatment.

Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility

PubMed Central

2014-01-01

Background Alfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production. Results In this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line. Conclusion Modifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines. PMID:24650274

One- and two-photon states for quantum information

NASA Astrophysics Data System (ADS)

Peters, Nicholas A.

To find expression stability among transgenic lines, the Recombinase Mediated Transgene Integration (RMTI) technology using the Cre/ lox-mediated site-specific gene integration system was used. The objectives were to develop an efficient method of site-specific transgene integration and to test the effectiveness of this method by assaying transgene expression in the RMTI lines. The RMTI technology allows the precise integration of a transgene in a previously placed target genomic location containing a lox site. The efficiency of CRE-mediated site-specific integration in rice by particle bombardment was found to vary from 3 to 28% in nine different experiments. Some hemizygous site-specific integration plants that were derived from homozygous target locus were found to undergo CRE-mediated reversion of the integration locus. No reversion was observed in callus; however, reverting cells may have been excluded due to selection pressure. The expression of the transgene gus was studied in all 40 callus lines, 12 regenerated T0 plants and the T1 and T2 progenies of 5 lines. The isogenic SC lines had an average expression level based on the activity of beta-glucuronidase of 158 +/- 9 units/mg protein (mean +/- SEM; n=3; variance within SC lines are expressed as standard error of the mean SEM) indicating a significantly higher level of expression, as compared to MC lines that had a much lower expression level 44 +/- 8 units/mg protein (mean +/- SEM; n=3) and the imprecise lines that had 22 +/- 8 units/mg protein (mean +/- SEM; n=3). Transgene expression in the callus cells of precise single copy lines varied by ˜3 fold, whereas that in multi-copy lines varied by ˜30 fold. Furthermore, precise single copy lines, on an average, contained ˜3.5 fold higher expression than multi-copy lines. Transgene expression in the plants of precise single-copy lines was highly variable, which was found to be due to the loss of the integration because of CRE-mediated reversion in the locus. (Abstract shortened by UMI.)

Biocompatibility of Tygon® tubing in microfluidic cell culture.

PubMed

Jiang, Xiao; Jeffries, Rex E; Acosta, Miguel A; Tikunov, Andrey P; Macdonald, Jeffrey M; Walker, Glenn M; Gamcsik, Michael P

2015-02-01

Growth of the MDA-MB-231 breast cancer cell line in microfluidic channels was inhibited when culture media was delivered to the channels via microbore Tygon® tubing. Culture media incubated within this tubing also inhibited growth of these cells in conventional 96-well plates. These detrimental effects were not due to depletion of critical nutrients due to adsorption of media components onto the tubing surface. A pH change was also ruled out as a cause. Nuclear magnetic resonance spectroscopy of the cell growth media before and after incubation in the tubing confirmed no detectable loss of media components but did detect the presence of additional unidentified signals in the aliphatic region of the spectrum. These results indicate leaching of a chemical species from microbore Tygon® tubing that can affect cell growth in microfluidic devices.

Single-frequency TEA CO2 laser with a bleaching spectral intracavity filter

NASA Astrophysics Data System (ADS)

Sorochenko, V. R.

2017-02-01

The regime of single-frequency operation is realised in a TEA CO2 laser with a spectral filter inside the cavity (a cell filled with SF6) on P(12)-P(24) lines of the 10P band. The minimal scatter of the peak powers of the laser pulses in a series of ‘shots’ and the maximal ratio of the output energies in the single-frequency and free running regimes (greater than 0.84) are obtained on the P(16) line at an optimal SF6 pressure in the cell. Experimental results qualitatively agree with the absorption spectrum of SF6 calculated from the SPECTRA information-analytical system. It is shown that the high ratio of energies in two regimes is achived due to gas bleaching in the cell.

Novel in vivo model of inducible multidrug resistance in acute lymphoblastic leukemia with chromosomal translocation t(4;11)

USDA-ARS?s Scientific Manuscript database

Acute lymphoblastic leukemia (ALL) with translocation t(4;11) is found in 60-85% of infants with ALL and is classified as high-risk due to the generally poor prognosis for survival. Using the SEM cell line established from a patient with t(4;11) ALL, we evaluated the resistance of these cells to the...

Alterations of mitochondrial DNA in CEM cells selected for resistance toward ddC toxicity.

PubMed

Bjerke, M; Franco, M; Johansson, M; Balzarini, J; Karlsson, A

2006-01-01

2 ',3 '-dideoxycytidine (ddC) is a nucleoside analog that has been shown to produce a delayed toxicity which may be due to the depletion of mitochondrial DNA (mtDNA). In order to gain further understanding of the events involved in mitochondrial toxicity, two different CEM cell lines were selected for resistance to the delayed ddC toxicity.

Differential endocytosis of CD4 in lymphocytic and nonlymphocytic cells

PubMed Central

1991-01-01

The endocytosis of the T cell differentiation antigen CD4 has been investigated in CD4-transfected HeLa cells, the promyelocytic HL-60 cell line, and in a number of leukemia- or lymphoma-derived T cell lines. CD4 internalization was followed using radioiodinated antibodies in an acid-elution endocytosis assay, or by covalently modifying cell surface proteins with biotin and analyzing CD4 distributions by immunoprecipitation; both approaches gave equivalent results. The assays demonstrated that in transfected HeLa cells and in HL-60 cells CD4 was constitutively internalized and recycled in the absence of ligand. Immunogold labeling and electron microscopy demonstrated that CD4 enters cells through coated pits. In contrast to the nonlymphocytic cells, T cell lines showed very little endocytosis of CD4. Measurements of fluid phase endocytosis and morphometric analysis of the endosome compartment indicated that the endocytic capacities of HeLa and lymphoid cells are equivalent and suggested that the low level of CD4 uptake in lymphocytic cells is due to exclusion of CD4 from coated pits. This conclusion was supported by experiments using truncated CD4 molecules, lacking the bulk of the cytoplasmic domain, which were internalized equally efficiently in both transfected lymphocytes and HeLa cells. Together, these results indicate that the cytoplasmic domain of CD4 mediates the different interactions with the endocytic apparatus in lymphoid and nonlymphoid cells. We suggest that the CD4- associated lymphocyte-specific protein tyrosine kinase p56lck may be involved in preventing CD4 endocytosis in T cells. PMID:1900077

MIA PaCa-2 and PANC-1 - pancreas ductal adenocarcinoma cell lines with neuroendocrine differentiation and somatostatin receptors.

PubMed

Gradiz, Rui; Silva, Henriqueta C; Carvalho, Lina; Botelho, Maria Filomena; Mota-Pinto, Anabela

2016-02-17

Studies using cell lines should always characterize these cells to ensure that the results are not distorted by unexpected morphological or genetic changes possibly due to culture time or passage number. Thus, the aim of this study was to describe those MIA PaCa-2 and PANC-1 cell line phenotype and genotype characteristics that may play a crucial role in pancreatic cancer therapeutic assays, namely neuroendocrine chemotherapy and peptide receptor radionuclide therapy. Epithelial, mesenchymal, endocrine and stem cell marker characterization was performed by immunohistochemistry and flow cytometry, and genotyping by PCR, gene sequencing and capillary electrophoresis. MIA PaCa-2 (polymorphism) expresses CK5.6, AE1/AE3, E-cadherin, vimentin, chromogranin A, synaptophysin, SSTR2 and NTR1 but not CD56. PANC-1 (pleomorphism) expresses CK5.6, MNF-116, vimentin, chromogranin A, CD56 and SSTR2 but not E-cadherin, synaptophysin or NTR1. MIA PaCA-1 is CD24(-), CD44(+/++), CD326(-/+) and CD133/1(-), while PANC-1 is CD24(-/+), CD44(+), CD326(-/+) and CD133/1(-). Both cell lines have KRAS and TP53 mutations and homozygous deletions including the first 3 exons of CDKN2A/p16(INK4A), but no SMAD4/DPC4 mutations or microsatellite instability. Both have neuroendocrine differentiation and SSTR2 receptors, precisely the features making them suitable for the therapies we propose to assay in future studies.

Sustainable one-step synthesis of hierarchical microspheres of PEGylated MoS2 nanosheets and MoO3 nanorods: Their cytotoxicity towards lung and breast cancer cells

NASA Astrophysics Data System (ADS)

Kumar, Neeraj; George, Blassan Plackal Adimuriyil; Abrahamse, Heidi; Parashar, Vyom; Ngila, Jane Catherine

2017-02-01

Nanotechnology provides an emerging potent alternate mode of cancer therapy. Nanomaterials dispersion or solubility is of particular concern in utilising their full potential applications in biomedical fields. PEGylation of nanomaterials is considered to provide products with stealth properties, and physiological environment with no obvious adverse effects. The purpose of this work was to develop a sustainable one-step method for fabrication of hierarchical microspheres of PEGylated MoS2 nanosheets using a stoichiometric ratio of Mo(VI) and thiourea. This study further investigated the cytotoxicity of the PEGylated MoS2 nanosheets towards lung (A549) and breast cancer (MCF-7) cell lines by analysing morphological changes and performing dose-dependent cell proliferation, and cytotoxicity analysis using adenosine 5‧-triphosphate (ATP), and lactate dehydrogenase (LDH) assay. For comparison, MoO3 nanorods were synthesised by simple chemical route and their cytotoxicity towards lung (A549) and breast cancer (MCF-7) cell lines were checked. The findings suggested that PEGylated MoS2 nanosheets have excellent cytotoxicity towards breast cancer (MCF-7) cell lines, and MoO3 have better cytotoxicity towards lung (A549) cancer cell lines. This work envisages an accessible foundation for engineering sophisticated biomolecule-MoS2 nanosheets conjugation due to the defect-rich biocompatible surface, to achieve great versatility, additional functions, and further advances in the biomedical field.

Augmenting Chinese hamster genome assembly by identifying regions of high confidence.

PubMed

Vishwanathan, Nandita; Bandyopadhyay, Arpan A; Fu, Hsu-Yuan; Sharma, Mohit; Johnson, Kathryn C; Mudge, Joann; Ramaraj, Thiruvarangan; Onsongo, Getiria; Silverstein, Kevin A T; Jacob, Nitya M; Le, Huong; Karypis, George; Hu, Wei-Shou

2016-09-01

Chinese hamster Ovary (CHO) cell lines are the dominant industrial workhorses for therapeutic recombinant protein production. The availability of genome sequence of Chinese hamster and CHO cells will spur further genome and RNA sequencing of producing cell lines. However, the mammalian genomes assembled using shot-gun sequencing data still contain regions of uncertain quality due to assembly errors. Identifying high confidence regions in the assembled genome will facilitate its use for cell engineering and genome engineering. We assembled two independent drafts of Chinese hamster genome by de novo assembly from shotgun sequencing reads and by re-scaffolding and gap-filling the draft genome from NCBI for improved scaffold lengths and gap fractions. We then used the two independent assemblies to identify high confidence regions using two different approaches. First, the two independent assemblies were compared at the sequence level to identify their consensus regions as "high confidence regions" which accounts for at least 78 % of the assembled genome. Further, a genome wide comparison of the Chinese hamster scaffolds with mouse chromosomes revealed scaffolds with large blocks of collinearity, which were also compiled as high-quality scaffolds. Genome scale collinearity was complemented with EST based synteny which also revealed conserved gene order compared to mouse. As cell line sequencing becomes more commonly practiced, the approaches reported here are useful for assessing the quality of assembly and potentially facilitate the engineering of cell lines. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Radiosensitivity and effect of hypoxia in HPV positive head and neck cancer cells.

PubMed

Sørensen, Brita Singers; Busk, Morten; Olthof, Nadine; Speel, Ernst-Jan; Horsman, Michael R; Alsner, Jan; Overgaard, Jens

2013-09-01

HPV associated Head and Neck Squamous Cell Carcinoma (HNSCC) represents a distinct subgroup of HNSCC characterized by a favorable prognosis and a distinct molecular biology. Previous data from the randomized DAHANCA 5 trial indicated that HPV positive tumors did not benefit from hypoxic modifications by Nimorazole during radiotherapy, whereas a significant benefit was observed in the HPV negative tumors. However, more studies have demonstrated equal frequencies of hypoxic tumors among HPV-positive and HPV-negative tumors. The aim of the present study was to determine radiosensitivity, the impact of hypoxia and the effect of Nimorazole in HPV positive and HPV negative cell lines. The used cell lines were: UDSCC2, UMSCC47 and UPCISCC90 (HPV positive) and FaDuDD, UTSCC33 and UTSCC5 (HPV negative). Cells were cultured under normoxic or hypoxic conditions, and gene expression levels of previously established hypoxia induced genes were assessed by qPCR. Cells were irradiated with various doses under normoxia, hypoxia or hypoxia +1mM Nimorazole, and the clonogenic survival was determined. The HPV positive and HPV negative cell lines exhibited similar patterns of upregulation of hypoxia induced genes in response to hypoxia. The HPV positive cell lines were up to 2.4 times more radiation sensitive than HPV negative cell lines. However, all HPV positive cells displayed the same response to hypoxia in radiosensitivity, with an OER in the range 2.3-2.9, and a sensitizer effect of Nimorazole of 1.13-1.29, similar to HPV negative cells. Although HPV positive cells had a markedly higher radiosensitivity compared to HPV negative cells, they displayed the same relative radioresistance under hypoxia and the same relative sensitizer effect of Nimorazole. The clinical observation that HPV positive patients do not seem to benefit from Nimorazole treatment is not due to inherent differences in hypoxia sensitivity or response to Nimorazole, but can be accounted for by the overall higher radiosensitivity of HPV positive cells. Copyright © 2013. Published by Elsevier Ireland Ltd.

Stress Modulus of Cancer Cells

NASA Astrophysics Data System (ADS)

Bonin, Keith; Guthold, Martin; Guo, Xinyi; Sigley, Justin

2012-02-01

Our main goal is to study the different physical and mechanical properties of cells as they advance through different stages of neoplastic transformation from normal to the metastatic state. Since recent reports indicate there is significant ambiguity about how these properties change for different cancer cells, we plan to measure these properties for a single line of cells, and to determine whether the changes vary for different cellular components: i.e. whether the change in physical properties is due to a change in the cytoskeleton, the cell membrane, the cytoplasm, or a combination of these elements. Here we expect to present data on the stress modulus of cancer cells at different stages: normal, mortal cancerous, immortal cancerous, and tumorigenic. The cells are Weinberg cell line Human Mammary Epithelial (HME) cells. Atomic force microscope (AFM) probes with different diameters are used to push on the cell membrane to measure the local, regional and global cell stress modulus. Preliminary results on normal HME cells suggests a stress modulus of 1.5 ± 0.8 kPa when pushing with 7 μm spherical probes. We anticipate reporting an improved value for the modulus as well as results for some of the Weinberg cancer cells.

High-speed video capillaroscopy method for imaging and evaluation of moving red blood cells

NASA Astrophysics Data System (ADS)

Gurov, Igor; Volkov, Mikhail; Margaryants, Nikita; Pimenov, Aleksei; Potemkin, Andrey

2018-05-01

The video capillaroscopy system with high image recording rate to resolve moving red blood cells with velocity up to 5 mm/s into a capillary is considered. Proposed procedures of the recorded video sequence processing allow evaluating spatial capillary area, capillary diameter and central line with high accuracy and reliability independently on properties of individual capillary. Two-dimensional inter frame procedure is applied to find lateral shift of neighbor images in the blood flow area with moving red blood cells and to measure directly the blood flow velocity along a capillary central line. The developed method opens new opportunities for biomedical diagnostics, particularly, due to long-time continuous monitoring of red blood cells velocity into capillary. Spatio-temporal representation of capillary blood flow is considered. Experimental results of direct measurement of blood flow velocity into separate capillary as well as capillary net are presented and discussed.

Process analytical technology (PAT) in insect and mammalian cell culture processes: dielectric spectroscopy and focused beam reflectance measurement (FBRM).

PubMed

Druzinec, Damir; Weiss, Katja; Elseberg, Christiane; Salzig, Denise; Kraume, Matthias; Pörtner, Ralf; Czermak, Peter

2014-01-01

Modern bioprocesses demand for a careful definition of the critical process parameters (CPPs) already during the early stages of process development in order to ensure high-quality products and satisfactory yields. In this context, online monitoring tools can be applied to recognize unfavorable changes of CPPs during the production processes and to allow for early interventions in order to prevent losses of production batches due to quality issues. Process analytical technologies such as the dielectric spectroscopy or focused beam reflectance measurement (FBRM) are possible online monitoring tools, which can be applied to monitor cell growth as well as morphological changes. Since the dielectric spectroscopy only captures cells with intact cell membranes, even information about dead cells with ruptured or leaking cell membranes can be derived. The following chapter describes the application of dielectric spectroscopy on various virus-infected and non-infected cell lines with respect to adherent as well as suspension cultures in common stirred tank reactors. The adherent mammalian cell lines Vero (African green monkey kidney cells) and hMSC-TERT (telomerase-immortalized human mesenchymal stem cells) are thereby cultured on microcarrier, which provide the required growth surface and allow the cultivation of these cells even in dynamic culture systems. In turn, the insect-derived cell lines S2 and Sf21 are used as examples for cells typically cultured in suspension. Moreover, the FBRM technology as a further monitoring tool for cell culture applications has been included in this chapter using the example of Drosophila S2 insect cells.

Oleanolic acid induces p53-dependent apoptosis via the ERK/JNK/AKT pathway in cancer cell lines in prostatic cancer xenografts in mice.

PubMed

Kim, Gyeong-Ji; Jo, Hyeon-Ju; Lee, Kwon-Jai; Choi, Jeong Woo; An, Jeung Hee

2018-05-29

We evaluated oleanolic acid (OA)-induced anti-cancer activity, apoptotic mechanism, cell cycle status, and MAPK kinase signaling in DU145 (prostate cancer), MCF-7 (breast cancer), U87 (human glioblastoma), normal murine liver cell (BNL CL.2) and human foreskin fibroblast cell lines (Hs 68). The IC50 values for OA-induced cytotoxicity were 112.57 in DU145, 132.29 in MCF-7, and 163.60 in U87 cells, respectively. OA did not exhibit toxicity in BNL CL. 2 and Hs 68 cell lines in our experiments. OA, at 100 µg/mL, increased the number of apoptotic cells to 27.0% in DU145, 27.0% in MCF-7, and 15.7% in U87, when compared to control cells. This enhanced apoptosis was due to increases in p53, cytochrome c, Bax, PARP-1 and caspase-3 expression in DU145, MCF-7 and U87 cell lines. OA-treated DU145 cells were arrested in G2 because of the activation of p-AKT, p-JNK, p21 and p27, and the decrease in p-ERK, cyclin B1 and CDK2 expression; OA-treated MCF-7 cells were arrested in G1 owing to the activation of p-JNK, p-ERK, p21, and p27, and the decrease in p-AKT, cyclin D1, CDK4, cyclin E, and CDK2; and OA-treated U87 cells also exhibited G1 phase arrest caused by the increase in p-ERK, p-JNK, p-AKT, p21, and p27, and the decrease in cyclin D1, CDK4, cyclin E and CDK2. Thus, OA arrested the cell cycle at different phases and induced apoptosis in cancer cells. These results suggested that OA possibly altered the expression of the cell cycle regulatory proteins differently in varying types of cancer.

Sodium phenylbutyrate antagonizes prostate cancer through the induction of apoptosis and attenuation of cell viability and migration.

PubMed

Xu, Yawen; Zheng, Shaobo; Chen, Binshen; Wen, Yong; Zhu, Shanwen

2016-01-01

Prostate cancer (PCa) is a leading cause of cancer-related death in men. Sodium phenylbutyrate (SPB) has shown its potential as an anticancer therapy in numerous cancer types. In the present study, we attempted to assess the effect of SPB against PCa and whether this treatment was associated with the regulation of survivin. Two human PCa cancer cell lines, DU145 and PC3, were used in the present study. Cell Counting Kit-8 (CCK-8) assay was conducted to measure the proliferation of PCa cells incubated with SPB. The effect of SPB on the cell apoptosis, cell colony formation ability, and cell morphological change was also assessed. Transwell experiment and Western blotting assay were performed to determine the effect of SPB on the migration and invasion ability of both cell types. Moreover, the expression pattern of survivin and MAPK members in both cell types after the treatment of SPB was also detected. Additionally, an in vivo tumor formation assay was performed to evaluate the treatment potential of SPB against PCa. We found that the viability of PCa cells was significantly inhibited by SPB treatment. As illustrated by flow cytometry, for DU145 cell line the average apoptotic rate of SPB-treated cells was significantly lower than that of the control group (P<0.05); similar results were also seen for PC3 (P<0.05). SPB administration also attenuated the colony formation and migration abilities in both cell lines. The expression level of survivin in SPB-treated cells was significantly downregulated, while the phosphorylation of p-38 and ERK was enhanced. Furthermore, in vivo tumor formation of both cell lines was suppressed by SPB as well. The above results confirmed the potential of SPB as an effective therapeutic agent for the prevention or treatment of PCa. This amelioration might be due to the blockade of the survivin pathway.

Targeting mitochondrial respiration as a therapeutic strategy for cervical cancer.

PubMed

Tian, Shenglan; Chen, Heng; Tan, Wei

2018-05-23

Targeting mitochondrial respiration has been documented as an effective therapeutic strategy in cancer. However, the impact of mitochondrial respiration inhibition on cervical cancer cells are not well elucidated. Using a panel of cervical cancer cell lines, we show that an existing drug atovaquone is active against the cervical cancer cells with high profiling of mitochondrial biogenesis. Atovaquone inhibited proliferation and induced apoptosis with varying efficacy among cervical cancer cell lines regardless of HPV infection, cellular origin and their sensitivity to paclitaxel. We further demonstrated that atovaquone acts on cervical cancer cells via inhibiting mitochondrial respiration. In particular, atovaquone specifically inhibited mitochondrial complex III but not I, II or IV activity, leading to respiration inhibition and energy crisis. Importantly, we found that the different sensitivity of cervical cancer cell lines to atovaquone were due to their differential level of mitochondrial biogenesis and dependency to mitochondrial respiration. In addition, we demonstrated that the in vitro observations were translatable to in vivo cervical cancer xenograft mouse model. Our findings suggest that the mitochondrial biogenesis varies among patients with cervical cancer. Our work also suggests that atovaquone is a useful addition to cervical cancer treatment, particularly to those with high dependency on mitochondrial respiration. Copyright © 2018 Elsevier Inc. All rights reserved.

Belinostat and vincristine demonstrate mutually synergistic cytotoxicity associated with mitotic arrest and inhibition of polyploidy in a preclinical model of aggressive diffuse large B cell lymphoma.

PubMed

Havas, Aaron P; Rodrigues, Kameron B; Bhakta, Anvi; Demirjian, Joseph A; Hahn, Seongmin; Tran, Jack; Scavello, Margarethakay; Tula-Sanchez, Ana A; Zeng, Yi; Schmelz, Monika; Smith, Catharine L

2016-12-01

Diffuse Large B-cell lymphoma (DLBCL) is an aggressive malignancy that has a 60 percent 5-year survival rate, highlighting a need for new therapeutic approaches. Histone deacetylase inhibitors (HDACi) are novel therapeutics being clinically-evaluated in combination with a variety of other drugs. However, rational selection of companion therapeutics for HDACi is difficult due to their poorly-understood, cell-type specific mechanisms of action. To address this, we developed a pre-clinical model system of sensitivity and resistance to the HDACi belinostat using DLBCL cell lines. In the current study, we demonstrate that cell lines sensitive to the cytotoxic effects of HDACi undergo early mitotic arrest prior to apoptosis. In contrast, HDACi-resistant cell lines complete mitosis after a short delay and arrest in G1. To force mitotic arrest in HDACi-resistant cell lines, we used low dose vincristine or paclitaxel in combination with belinostat and observed synergistic cytotoxicity. Belinostat curtails vincristine-induced mitotic arrest and triggers a strong apoptotic response associated with downregulated MCL-1 expression and upregulated BIM expression. Resistance to microtubule targeting agents (MTAs) has been associated with their propensity to induce polyploidy and thereby increase the probability of genomic instability that enables cancer progression. Co-treatment with belinostat effectively eliminated a vincristine-induced, actively cycling polyploid cell population. Our study demonstrates that vincristine sensitizes DLBCL cells to the cytotoxic effects of belinostat and that belinostat prevents polyploidy that could cause vincristine resistance. Our findings provide a rationale for using low dose MTAs in conjunction with HDACi as a potential therapeutic strategy for treatment of aggressive DLBCL.

Increased reprogramming of human fetal hepatocytes compared with adult hepatocytes in feeder-free conditions.

PubMed

Hansel, Marc C; Gramignoli, Roberto; Blake, William; Davila, Julio; Skvorak, Kristen; Dorko, Kenneth; Tahan, Veysel; Lee, Brian R; Tafaleng, Edgar; Guzman-Lepe, Jorge; Soto-Gutierrez, Alejandro; Fox, Ira J; Strom, Stephen C

2014-01-01

Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus, hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these, 37 hiPSC lines were generated from fetal hepatocytes, 2 hiPSC lines from normal hepatocytes, and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar syndrome, type 1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression, flow cytometry, immunocytochemistry, and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with six factors, while fetal hepatocytes could be reprogrammed with three (OCT4, SOX2, NANOG) or four factors (OCT4, SOX2, NANOG, LIN28 or OCT4, SOX2, KLF4, C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult hepatocytes, although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation.

Copper (II) complexes of bidentate ligands exhibit potent anti-cancer activity regardless of platinum sensitivity status.

PubMed

Wehbe, Mohamed; Lo, Cody; Leung, Ada W Y; Dragowska, Wieslawa H; Ryan, Gemma M; Bally, Marcel B

2017-12-01

Insensitivity to platinum, either through inherent or acquired resistance, is a major clinical problem in the treatment of many solid tumors. Here, we explored the therapeutic potential of diethyldithiocarbamate (DDC), pyrithione (Pyr), plumbagin (Plum), 8-hydroxyquinoline (8-HQ), clioquinol (CQ) copper complexes in a panel of cancer cell lines that differ in their sensitivity to platins (cisplatin/carboplatin) using a high-content imaging system. Our data suggest that the copper complexes were effective against both platinum sensitive (IC 50  ~ 1 μM platinum) and insensitive (IC 50  > 5 μM platinum) cell lines. Furthermore, copper complexes of DDC, Pyr and 8-HQ had greater therapeutic activity compared to the copper-free ligands in all cell lines; whereas the copper-dependent activities of Plum and CQ were cell-line specific. Four of the copper complexes (Cu(DDC) 2 , Cu(Pyr) 2 , Cu(Plum) 2 and Cu(8-HQ) 2 ) showed IC 50 values less than that of cisplatin in all tested cell lines. The complex copper DDC (Cu(DDC) 2 ) was selected for in vivo evaluation due to its low nano-molar range activity in vitro and the availability of an injectable liposomal formulation. Liposomal (Cu(DDC) 2 ) was tested in a fast-growing platinum-resistant A2780-CP ovarian xenograft model and was found to achieve a statistically significant reduction (50%; p < 0.05) in tumour size. This work supports the potential use of copper-based therapeutics to treat cancers that are insensitive to platinum drugs.

A HISTOLOGICAL STUDY OF TYPHOID FEVER

PubMed Central

Mallory, F. B.

1898-01-01

The typhoid bacillus produces a mild diffusible toxine, partly within the intestinal tract, partly within the blood and organs of the body. This toxine produces proliferation of endothelial cells which acquire for a certain length of time malignant properties. The new-formed cells are epithelioid in character, have irregular, lightly staining, eccentrically situated nuclei, abundant, sharply defined, acidophilic protoplasm, and are characterized by marked phagocytic properties. These phagocytic cells are produced most abundantly along the line of absorption from the intestinal tract, both in the lymphatic apparatus and in the blood-vessels. They are also produced by distribution of the toxine through the general circulation, in greatest numbers where the circulation is slowest. Finally, they are produced all over the body in the lymphatic spaces and vessels by absorption of the toxine eliminated from the blood-vessels. The swelling of the intestinal lymphoid tissue of the mesenteric lymph nodes, and of the spleen is due almost entirely to the formation of phagocytic cells. The necrosis of the intestinal lymphoid tissue is accidental in nature and is caused through occlusion of the veins and capillaries by fibrinous thrombi, which owe their origin to degeneration of phagocytic cells beneath the lining endothelium of the vessels. Two varieties of focal lesions occur in the liver: one consists of the formation of phagocytic cells in the lymph spaces and vessels around the portal vessels under the action of the toxine absorbed by the lymphatics; the other is due to obstruction of liver capillaries by phagocytic cells derived in small part from the lining endothelium of the liver capillaries, but chiefly by embolism through the portal circulation of cells originating from the endothelium of the blood-vessels of the intestine and spleen. The liver cells lying between the occluded capillaries undergo necrosis and disappear. Later the foci of cells degenerate and fibrin forms between them. Invasion with polymorphonuclear leucocytes is rare. Many of the phagocytic cells pass through the liver and lungs, and get into the general circulation. A few come from the abdominal lymphatics through the thoracic duct. PMID:19866884

Epigenetic alterations are involved in the overexpression of glutathione S-transferase π-1 in human colorectal cancers.

PubMed

Zhang, Rui; Kang, Kyoung Ah; Piao, Mei Jing; Kim, Ki Cheon; Zheng, Jian; Yao, Cheng Wen; Cha, Ji Won; Maeng, Young Hee; Chang, Weon Young; Moon, Pyong-Gon; Baek, Moon-Chang; Hyun, Jin Won

2014-09-01

Glutathione S-transferase π-1 (GSTP-1) is a member of the glutathione S-transferase enzyme superfamily, which catalyzes the conjugation of electrophiles to glutathione during the process of detoxification. In this study, the epigenetic alterations of GSTP-1 expression in human colorectal cancers and the underlying mechanisms were investigated. In 10 colon cancer patients, proteomic analysis revealed that expression of GSTP-1 protein was higher in tumor tissues than in paired adjacent normal tissues. Likewise, in 7 of 10 colon cancer patients, GSTP-1 protein expression was more than 1.5-fold higher in tumor tissues than in adjacent normal tissues, as determined by western blotting. Immunohistochemical data confirmed that GSTP-1 protein was expressed at higher levels in colon cancer tissues compared to normal mucosa. GSTP-1 enzyme activity was closely correlated with GSTP-1 protein expression in colon cancer patients. Consistent with this, GSTP-1 mRNA, protein and activity levels were higher in the colorectal cancer cell lines Caco-2, HCT-116, HT-29, SNU-407 and SNU-1033 compared to the normal colon cell line FHC. Methylation-specific PCR results indicated that the high levels of GSTP-1 in human colorectal cancer cell lines were likely due to the lower degree of promoter methylation in colon cancer cell lines compared to the normal colon cell line, consistent with findings in colon cancer patients. Moreover, the levels of specific activator-protein complexes and histone marks were higher in human colorectal cancer cells compared to the normal human colon cell line, whereas the repressor protein complexes exhibited the opposite pattern. Furthermore, chromatin immunoprecipitation assays demonstrated that expression levels of the transcription factors AP-1 and SP-1 were correlated with the upregulation of GSTP-1 expression in colorectal cancer cells. Finally, knockdown of GSTP-1 promoted the sensitivity of SNU-407 cells to the anticancer agent 5-fluorouracil. These data indicate that GSTP-1 may serve as a clinically useful biomarker of colon cancer and a target for anti-colon cancer drugs.

Mesenchymal-Epithelial Transition and Circulating Tumor Cells in Small Cell Lung Cancer.

PubMed

Hamilton, Gerhard; Rath, Barbara

2017-01-01

Cancer patients die of metastatic disease but knowledge regarding individual steps of this complex process of intravasation, spread and extravasation leading to secondary lesions is incomplete. Subpopulations of tumor cells are supposed to undergo an epithelial-mesenchymal transition (EMT), to enter the bloodstream and eventually establish metastases in a reverse process termed mesenchymal-epithelial transition (MET). Small cell lung cancer (SCLC) represents a unique model to study metastatic spread due to early dissemination and relapse, as well as availability of a panel of circulating cancer cell (CTC) lines recently. Additionally, chemosensitive SCLC tumor cells switch to a completely resistant phenotype during cancer recurrence. In advanced disease, SCLC patients display extremely high blood counts of CTCs in contrast to other tumors, like breast, prostate and colon cancer. Local inflammatory conditions at the primary tumor site and recruitment of macrophages seem to increase the shedding of tumor cells into the circulation in processes which may proceed independently of EMT. Since millions of cells are released by tumors into the circulation per day, analysis of a limited number of CTCs at specific time points are difficult to be related to the development of metastatic lesions which may occur approximately one year later. We have obtained a panel of SCLC CTC cell line from patients with relapsing disease, which share characteristic markers of this malignancy and a primarily epithelial phenotype with unique formation of large tumorospheres, containing quiescent and hypoxic cells. Although smoking and inflammation promote EMT, partial expression of vimentin indicates a transitional state with partial EMT in these cell lines at most. The CTC lines exhibit high expression of EpCAM , absent phosphorylation of β-catenin and background levels of Snail. Provided that these tumor cells had ever undergone EMT, here in advanced disease MET seem to have occurred already in the peripheral circulation. Alternative explanations for the expression of mesenchymal markers of the CTC lines are the heterogeneity of SCLC cells, cooperative migration or altered gene expression in response to the inflammatory tumor microenvironment allowing for tumor spread without EMT/MET.

Cytokine-mediated FOXO3a phosphorylation suppresses FasL expression in hemopoietic cell lines: investigations of the role of Fas in apoptosis due to cytokine starvation.

PubMed

Behzad, Hayedeh; Jamil, Sarwat; Denny, Trisha A; Duronio, Vincent

2007-05-01

We have investigated phosphatidylinositol 3-kinase (PI3K)-dependent survival signalling pathways using several cytokines in three different hemopoietic cell lines, MC/9, FDC-P1, and TF-1. Cytokines caused PI3K- and PKB-dependent phosphorylation of FOXO3a (previously known as FKHRL1) at three distinct sites. Following cytokine withdrawal or PI3K inhibition, both of which are known to lead to apoptosis, there was a loss of FOXO3a phosphorylation, and a resulting increase in forkhead transcriptional activity, along with increased expression of Fas Ligand (FasL), which could be detected at the cell surface. Concurrently, an increase in cell surface expression of Fas was also detected. Despite the presence of both FasL and Fas, there was no detectable evidence that activation of Fas-mediated apoptotic events was contributing to apoptosis resulting from cytokine starvation or inhibition of PI3K activity. Thus, inhibition of FOXO3a activity is mediated by the PI3K-PKB pathway, but regulation of FasL is not the primary means by which cell survival is regulated in cytokine-dependent hemopoietic cells. We were also able to confirm increased expression of known FOXO3a targets, Bim and p27kip1. Together, these results support the conclusion that mitochondrial-mediated signals play the major role in apoptosis of hemopoietic cells due to loss of cytokine signalling.

Genetic Resistance to Rhabdovirus Infection in Teleost Fish Is Paralleled to the Derived Cell Resistance Status

PubMed Central

Verrier, Eloi R.; Langevin, Christelle; Tohry, Corinne; Houel, Armel; Ducrocq, Vincent; Benmansour, Abdenour; Quillet, Edwige; Boudinot, Pierre

2012-01-01

Genetic factors of resistance and predisposition to viral diseases explain a significant part of the clinical variability observed within host populations. Predisposition to viral diseases has been associated to MHC haplotypes and T cell immunity, but a growing repertoire of innate/intrinsic factors are implicated in the genetic determinism of the host susceptibility to viruses. In a long-term study of the genetics of host resistance to fish rhabdoviruses, we produced a collection of double-haploid rainbow trout clones showing a wide range of susceptibility to Viral Hemorrhagic Septicemia Virus (VHSV) waterborne infection. The susceptibility of fibroblastic cell lines derived from these clonal fish was fully consistent with the susceptibility of the parental fish clones. The mechanisms determining the host resistance therefore did not associate with specific host immunity, but rather with innate or intrinsic factors. One cell line was resistant to rhabdovirus infection due to the combination of an early interferon IFN induction - that was not observed in the susceptible cells - and of yet unknown factors that hamper the first steps of the viral cycle. The implication of IFN was well consistent with the wide range of resistance of this genetic background to VSHV and IHNV, to the birnavirus IPNV and the orthomyxovirus ISAV. Another cell line was even more refractory to the VHSV infection through different antiviral mechanisms. This collection of clonal fish and isogenic cell lines provides an interesting model to analyze the relative contribution of antiviral pathways to the resistance to different viruses. PMID:22514610

Timing of pulsed electromagnetic field stimulation does not affect the promotion of bone cell development.

PubMed

Hannay, Gwynne; Leavesley, David; Pearcy, Mark

2005-12-01

Pulsed electromagnetic field (PEMF) devices have been used clinically to promote the healing of surgically resistant fractures in vivo. However, there is a sparsity of data on how the timing of an applied PEMF effects the osteogenic cells that would be present within the fracture gap. The purpose of this study was to examine the response of osteoblast-like cells to a PEMF stimulus, mimicking that of a clinically available device, using four protocols for the timing of the stimulus. The PEMF signal consisted of a 5 ms pulse burst (containing 20 pulses) repeated at 15 Hz. Cultures of a human osteosarcoma cell line, SaOS-2, were exposed to the four timing protocols, each conducted over 3 days. Protocol one stimulated the cells for 8 h each day, protocol two stimulated the cells for 24 h on the first day, protocol three stimulated the cells for 24 h on the second day, and protocol four stimulated the cells for 24 h on the third day. Cells were seeded with either 25,000 or 50,000 cells/well (24-well cell culture plates). All assays showed reduced proliferation and increased differentiation (alkaline phosphatase activity) in the PEMF stimulated cultures compared with the control cultures, except for protocol four alkaline phosphatase measurements. No clear trend was observed between the four protocols; however this may be due to cell density. The results indicated that an osteoblast-like cell line is responsive to a 15 Hz PEMF stimulus, which will stimulate the cell line to into an increasing state of maturity. Bioelectromagnetics (c) 2005 Wiley-Liss, Inc

Lightning propagation and flash density in squall lines as determined with radar

NASA Technical Reports Server (NTRS)

Mazur, V.; Rust, W. D.

1983-01-01

Lightning echo rise times and range-time variations due to discharge propagation are determined using S and L band radars, and the evolution of precipitation reflectivity and the associated lightning activity in squall lines is investigated using VHF and L band radars. The rise time of radar echoes can be explained by ionized channel propagation through the radar beams. Speeds of at least 250,000 m/s are found from measurements of the radial velocity of streamer propagation along the antenna beam. The range-time variations in lightning echoes indicate that either new ionization occurs as streamers develop into different parts of the cloud, channel delay occurs during which adequate ionization exists for radar detection, or continuing current occurs. Determinations of the lightning flash density for a squall line in the U.S. show that the maximum lightning density tends to be near the leading edge of the precipitation cores in developing cells. Long discharges are produced as a cell in the squall line develops and the total lightning density increases, although short discharges predominate. As the cell dissipates, short flashes diminish or cease and the long flashes dominate the lightning activity.

Differential effects of a complex organochlorine mixture on the proliferation of breast cancer cell lines

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

Aube, Michel, E-mail: 4aubem@videotron.ca; Larochelle, Christian, E-mail: christian.larochelle@inspq.qc.ca; Ayotte, Pierre, E-mail: pierre.ayotte@inspq.qc.ca

2011-04-15

Organochlorine compounds (OCs) are a group of persistent chemicals that accumulate in fatty tissues with age. Although OCs has been tested individually for their capacity to induce breast cancer cell proliferation, few studies examined the effect of complex mixtures that comprise compounds frequently detected in the serum of women. We constituted such an OC mixture containing 15 different components in environmentally relevant proportions and assessed its proliferative effects in four breast cancer cell lines (MCF-7, T47D, CAMA-1, MDAMB231) and in non-cancerous CV-1 cells. We also determined the capacity of the mixture to modulate cell cycle stage of breast cancer cellsmore » and to induce estrogenic and antiandrogenic effects using gene reporter assays. We observed that low concentrations of the mixture (100x10{sup 3} and 50x10{sup 3} dilutions) stimulated the proliferation of MCF-7 cells while higher concentrations (10x10{sup 3} and 5x10{sup 3} dilutions) had the opposite effect. In contrast, the mixture inhibited the proliferation of non-hormone-dependent cell lines. The mixture significantly increased the number of MCF-7 cells entering the S phase, an effect that was blocked by the antiestrogen ICI 182,780. Low concentrations of the mixture also caused an increase in CAMA-1 cell proliferation but only in the presence estradiol and dihydrotestosterone (p<0.05 at the 50x10{sup 3} dilution). DDT analogs and polychlorinated biphenyls all had the capacity to stimulate the proliferation of CAMA-1 cells in the presence of sex steroids. Reporter gene assays further revealed that the mixture and several of its constituents (DDT analogs, aldrin, dieldrin, {beta}-hexachlorocyclohexane, toxaphene) induced estrogenic effects, whereas the mixture and several components (DDT analogs, aldrin, dieldrin and PCBs) inhibited the androgen signaling pathway. Our results indicate that the complex OC mixture increases the proliferation of MCF-7 cells due to its estrogenic potential. The proliferative effect of the mixture on CAMA-1 cells in the presence of sex steroids appears mostly due to the antiandrogenic properties of p,p'-DDE, a major constituent of the mixture. Other mixtures of contaminants that include emerging compounds of interest such as brominated flame retardants and perfluoroalkyl compounds should be tested for their capacity to induce breast cancer cell proliferation. - Research highlights: {yields} We studied effects of a complex organochlorine mixture on breast cancer cell growth. {yields} Weak xenoestrogens in the mixture stimulated the proliferation of MCF-7 cells. {yields} Antiandrogens increased the proliferation CAMA-1 cells grown with sex steroids. {yields} High concentrations of the mixture decreased the proliferation of all cell lines.« less

The photocytotoxicity of different lights on mammalian cells in interior lighting system.

PubMed

Song, Jiayin; Gao, Tingting; Ye, Maole; Bi, Hongtao; Liu, Gang

2012-12-05

In the present paper, two light sources commonly used in interior lighting system: incandescent light and light emitting diode (LED) were chosen to evaluate their influences on three kinds of mammalian cells, together with UVA and UVB, and the mechanism of the photocytotoxicity was investigated in terms of intracellular ROS production, lipid peroxidation, SOD activity and GSH level assays. The results showed that LED and incandescent light both had some photocytotoxicities. In the interior lighting condition (100lx-250lx), the cytotoxicities of LED and incandescent lamp on RF/6A cells (rhesus retinal pigment epithelium cell line) were stronger than that on two fibroblast cell lines, while the cytotoxicity of UVA and UVB on HS68 cells (fibroblast cell line) was highest in the tests. The mechanism analysis revealed that the photocytotoxicities of LED and incandescent lamp were both caused by cell lipid peroxidation. LED and incandescent light could promote the production of ROS, raise lipid peroxidation level and lower the activity of the antioxidant key enzymes in mammalian cells, and finally cause a number of cells death. However, the negative function of LED was significantly smaller than incandescent light and ultraviolet in daily interior lighting condition. And the significantly lower photocytotoxicity of LED might be due to the less existence of ultraviolet. Therefore, LED is an efficient and relative safe light source in interior lighting system, which should be widely used instead of traditional light source. Copyright © 2012 Elsevier B.V. All rights reserved.

Ultrasound-mediated structural changes in cells revealed by FTIR spectroscopy: A contribution to the optimization of gene and drug delivery

NASA Astrophysics Data System (ADS)

Grimaldi, Paola; Di Giambattista, Lucia; Giordani, Serena; Udroiu, Ion; Pozzi, Deleana; Gaudenzi, Silvia; Bedini, Angelico; Giliberti, Claudia; Palomba, Raffaele; Congiu Castellano, Agostina

2011-12-01

Ultrasound effects on biological samples are gaining a growing interest concerning in particular, the intracellular delivery of drugs and genes in a safe and in a efficient way. Future progress in this field will require a better understanding of how ultrasound and acoustic cavitation affect the biological system properties. The morphological changes of cells due to ultrasound (US) exposure have been extensively studied, while little attention has been given to the cells structural changes. We have exposed two different cell lines to 1 MHz frequency ultrasound currently used in therapy, Jurkat T-lymphocytes and NIH-3T3 fibroblasts, both employed as models respectively in the apoptosis and in the gene therapy studies. The Fourier Transform Infrared (FTIR) Spectroscopy was used as probe to reveal the structural changes in particular molecular groups belonging to the main biological systems. The genotoxic damage of cells exposed to ultrasound was ascertained by the Cytokinesis-Block Micronucleus (CBMN) assay. The FTIR spectroscopy results, combined with multivariate statistical analysis, regarding all cellular components (lipids, proteins, nucleic acids) of the two cell lines, show that Jurkat cells are more sensitive to therapeutic ultrasound in the lipid and protein regions, whereas the NIH-3T3 cells are more sensitive in the nucleic acids region; a meaningful genotoxic effect is present in both cell lines only for long sonication times while in the Jurkat cells also a significant cytotoxic effect is revealed for long times of exposure to ultrasound.

Lethality of radiation-induced chromosome aberrations in human tumour cell lines with different radiosensitivities.

PubMed

Coco-Martin, J M; Ottenheim, C P; Bartelink, H; Begg, A C

1996-03-01

In order to find an explanation for the eventual disappearance of all chromosome aberrations in two radiosensitive human tumour cell lines, the type and stability of different aberration types was investigated in more detail. To classify the aberrations into unstable and stable types, three-colour fluorescence in situ hybridization was performed, including a whole-chromosome probe, a pancentromere probe, and a stain for total DNA. This technique enables the appropriate classification of the aberrations principally by the presence (stable) or not (unstable) of a single centromere per chromosome. Unstable-type aberrations were found to disappear within 7 days (several divisions) in the two radiosensitive and the two radioresistant tumour lines investigated. Stable-type aberrations were found to remain at an approximately constant level over the duration of the experiment (14 days; 8-10 divisions) in the two radioresistant lines. In contrast, the majority of these stable-type aberrations had disappeared by 14 days in the two radiosensitive lines. The previous findings of disappearance of total aberrations in radiosensitive cells was therefore not due to a reduced induction of stable-type aberrations, but the complete disappearance of cells with this aberration type. These results could not be explained by differences in apoptosis or G1 blocks. Two possible explanations for these unexpected findings involve non-random induction of unstable-type aberrations, or lethality of stable-type aberrations. The results suggest caution in the use of stable-type aberration numbers as a predictor for radiosensitivity.

Curcumin conjugated silica nanoparticles for improving bioavailability and its anticancer applications.

PubMed

Gangwar, Rajesh K; Tomar, Geetanjali B; Dhumale, Vinayak A; Zinjarde, Smita; Sharma, Rishi B; Datar, Suwarna

2013-10-09

Curcumin, a yellow bioactive component of Indian spice turmeric, is known to have a wide spectrum of biological applications. In spite of various astounding therapeutic properties, it lacks in bioavailability mainly due to its poor solubility in water. In this work, we have conjugated curcumin with silica nanoparticles to improve its aqueous solubility and hence to make it more bioavailable. Conjugation and loading of curcumin with silica nanoparticles was further examined with transmission electron microscope (TEM) and thermogravimetric analyzer. Cytotoxicity analysis of synthesized silica:curcumin conjugate was studied against HeLa cell lines as well as normal fibroblast cell lines. This study shows that silica:curcumin conjugate has great potential for anticancer application.

An Adult Mouse Thyroid Side Population Cell Line that Exhibits Enriched Epithelial–Mesenchymal Transition

PubMed Central

Murata, Tsubasa; Iwadate, Manabu; Takizawa, Yoshinori; Miyakoshi, Masaaki; Hayase, Suguru; Yang, Wenjing; Cai, Yan; Yokoyama, Shigetoshi; Nagashima, Kunio; Wakabayashi, Yoshiyuki; Zhu, Jun

2017-01-01

Background: Studies of thyroid stem/progenitor cells have been hampered due to the small organ size and lack of tissue, which limits the yield of these cells. A continuous source that allows the study and characterization of thyroid stem/progenitor cells is desired to push the field forward. Method: A cell line was established from Hoechst-resistant side population cells derived from mouse thyroid that were previously shown to contain stem/progenitor-like cells. Characterization of these cells were carried out by using in vitro two- and three-dimensional cultures and in vivo reconstitution of mice after orthotopic or intravenous injection, in conjunction with quantitative reverse transcription polymerase chain reaction, Western blotting, immunohisto(cyto)chemistry/immunofluorescence, and RNA seq analysis. Results: These cells were named SPTL (side population cell-derived thyroid cell line). Under low serum culturing conditions, SPTL cells expressed the thyroid differentiation marker NKX2-1, a transcription factor critical for thyroid differentiation and function, while no expression of other thyroid differentiation marker genes were observed. SPTL cells formed follicle-like structures in Matrigel® cultures, which did not express thyroid differentiation marker genes. In mouse models of orthotopic and intravenous injection, the latter following partial thyroidectomy, a few SPTL cells were found in part of the follicles, most of which expressed NKX2-1. SPTL cells highly express genes involved in epithelial–mesenchymal transition, as demonstrated by RNA seq analysis, and exhibit a gene-expression pattern similar to anaplastic thyroid carcinoma. Conclusion: These results demonstrate that SPTL cells have the capacity to differentiate into thyroid to a limited degree. SPTL cells may provide an excellent tool to study stem cells, including cancer stem cells of the thyroid. PMID:28125936

Use of Stirred Suspension Bioreactors for Male Germ Cell Enrichment.

PubMed

Sakib, Sadman; Dores, Camila; Rancourt, Derrick; Dobrinski, Ina

2016-01-01

Spermatogenesis is a stem cell based system. Both therapeutic and biomedical research applications of spermatogonial stem cells require a large number of cells. However, there are only few germ line stem cells in the testis, contained in the fraction of undifferentiated spermatogonia. The lack of specific markers makes it difficult to isolate these cells. The long term maintenance and proliferation of nonrodent germ cells in culture has so far been met with limited success, partially due to the lack of highly enriched starting populations. Differential plating, which depends on the differential adhesion properties of testicular somatic and germ cells to tissue culture dishes, has been the method of choice for germ cell enrichment, especially for nonrodent germ cells. However, for large animals, this process becomes labor intensive and increases variability due to the need for extensive handling. Here, we describe the use of stirred suspension bioreactors, as a novel system for enriching undifferentiated germ cells from 1-week-old pigs. This method capitalizes on the adherent properties of somatic cells within a controlled environment, thus promoting the enrichment of progenitor cells with minimal handling and variability.

A simple non-perturbing cell migration assay insensitive to proliferation effects.

PubMed

Glenn, Honor L; Messner, Jacob; Meldrum, Deirdre R

2016-08-18

Migration is a fundamental cellular behavior that plays an indispensable role in development and homeostasis, but can also contribute to pathology such as cancer metastasis. Due to its relevance to many aspects of human health, the ability to accurately measure cell migration is of broad interest, and numerous approaches have been developed. One of the most commonly employed approaches, because of its simplicity and throughput, is the exclusion zone assay in which cells are allowed to migrate into an initially cell-free region. A major drawback of this assay is that it relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration. We report here a simple modification to the exclusion zone migration assay that exclusively measures cell migration and is not affected by proliferation. This approach makes use of a lineage-tracing vital stain that is retained through cell generations and effectively reads out migration relative to the original, parental cell population. This modification is simple, robust, non-perturbing, and inexpensive. We validate the method in a panel of cell lines under conditions that inhibit or promote migration and demonstrate its use in normal and cancer cell lines as well as primary cells.

p21Waf1/Cip1/Sdi1 Prevents Apoptosis as Well as Stimulates Growth in Cells Transformed or Immortalized by Human T-Cell Leukemia Virus Type 1-Encoded Tax

PubMed Central

Kawata, Sanae; Ariumi, Yasuo; Shimotohno, Kunitada

2003-01-01

Human T-cell leukemia virus type 1 (HTLV-1) Tax regulates the expression of virally encoded genes, as well as various endogenous host genes in trans. Tax-mediated regulation of gene expression is important for the immortalization of normal human T lymphocytes and the transformation of fibroblast cells, such as Rat-1 cells. Tax has the ability to transactivate p21Waf1/Cip1/Sdi1, resulting in high expression levels in HTLV-1-immortalized cells. Since p21 expression is suppressed due to methylation of the promoter region in Rat-l cell line, p21 may not be critical for the transformation of this cell line by Tax. To further understand the role of p21 for the proliferation of Tax-transformed Rat-1 cells, we examined the effect of ectopic expression of p21 in these cells. Here, we observed that p21 expression enhanced the transformation of this cell line via at least two mechanisms: (i) the enhancement of NF-κB activation and/or CREB signaling and (ii) the excitation of antiapoptotic machinery. To analyze the role of p21 that is overexpressed in HTLV-1-immortalized lymphocytes, p21 expression was suppressed by using an antisense oligonucleotide specific for p21 mRNA; these cells then became sensitive to apoptotic induction. These results suggest that p21 plays an important role in the proliferation of Tax-expressing cells through the regulation of at least two independent mechanisms. PMID:12805427

Transduction of human IL-9 receptor cDNA into TF1 cells induces IL-9 dependency and erythroid differentiation.

PubMed

Xiao, M; Luo, Z; Mantel, C; Broxmeyer, H E; Lu, L

2000-02-01

Human growth factor-dependent cell line TF1, which lacks interleukin (IL)-9 receptors (R) and does not grow in IL-9, was transduced with a retroviral vector containing human IL-9R cDNA and a selection marker. An IL-9-dependent TF1 cell line, which could also grow in other cytokines, was established after selection in G418 and could produce mature RBC in response to cytokine stimulation. TF1 cells transduced with the same viral vector without the IL-9R insert cDNA (mock control) and then selected responded the same as nontransduced TF1 cells. They failed to grow in response to IL-9 and did not generate RBC. An increased number and size of burst-forming units-erythroid (BFU-E)-like colonies were detected from IL-9R-transduced TF1 cells, compared with mock-transduced cells, in response to erythropoietin (EPO) and IL-9. To evaluate self-renewal and differentiation capacity, colony-replating assays were performed in the presence of IL-3, GM-CSF, IL-9, and EPO. After four replatings, the cloning efficiency of IL-9R-transduced TF1 cells decreased from 98% to 38%, most likely due to terminal erythroid cell differentiation. In contrast, no change in replating efficiency was detected in mock-transduced cells. TF1 cells stably expressing IL-9R and responding to IL-9 can serve as a cell line model to study the intracellular signals mediating IL-9-induced erythroid cell proliferation and differentiation.

Failure of matrix metalloproteinase-9 dimer induction by phorbol 12-myristate 13-acetate in normal human cell lines.

PubMed

Waheed Roomi, Mohd; Kalinovsky, Tatiana; Rath, Matthias; Niedzwiecki, Aleksandra

2015-06-01

Increasing experimental and clinical data has identified an association between increased levels of matrix metalloproteinase (MMP)-9 and shortened patient survival, cancer progression and metastasis. MMP-9 has a significant role in tumor cell invasion and metastasis, as it digests the basement membrane and components of the extracellular matrix. MMP-9 is secreted in either a monomeric or dimeric form. Although limited evidence exists concerning MMP-9 dimers, certain studies have demonstrated that the dimer is associated with aggressive tumor progression. This is believed to be due to the fact that cellular migration depends upon the MMP-9 dimer, and not the monomer. Our previous study revealed that cancer cell MMP-9 dimer secretion patterns could be divided into different categories, and that high MMP-9 and MMP-9 dimer secretion levels were correlated with the most aggressive cancer cell lines. It has been established that signal transduction pathways and cytokines, including those activated by phorbol 12-myristate 13-acetate (PMA), regulate the expression of MMPs. The aim of the present study was to analyze the expression patterns of MMP-2, MMP-9 and MMP-9 dimer in normal human cells from a number of tissues treated with PMA. Muscle, epithelial and connective tissues were selected for use in the present study, since adenosarcomas, carcinomas and sarcomas are derived from these tissue types, respectively. The cell lines were first cultured in 24-well tissue culture plates containing recommended media that was supplemented with 10% fetal bovine serum and antibiotics. When at confluency, the cells were washed and fresh medium was added. In addition, a parallel set of cultures was treated with PMA. Subsequent to a 24-h incubation period, the media were collected and analyzed using gelatinase zymography for the expression of MMP-2 and MMP-9 monomer and dimer forms. The results revealed that the cellular expression of MMP-2 and MMP-9 was dependent upon the primary tissue subtype. All cell lines, regardless of tissue origin, expressed MMP-2. PMA induced the expression of MMP-9 in muscle tissue, glandular epithelia and supportive connective tissue cell lines. By contrast, cell lines of endothelial origin and proper connective tissue were insensitive to treatment with PMA. MMP-9 dimer secretion was not observed in any of the cell lines, which indicated that cellular migration is not supported by these cells.

Development of patient-derived xenograft models from a spontaneously immortal low-grade meningioma cell line, KCI-MENG1.

PubMed

Michelhaugh, Sharon K; Guastella, Anthony R; Varadarajan, Kaushik; Klinger, Neil V; Parajuli, Prahlad; Ahmad, Aamir; Sethi, Seema; Aboukameel, Amro; Kiousis, Sam; Zitron, Ian M; Ebrahim, Salah A; Polin, Lisa A; Sarkar, Fazlul H; Bollig-Fischer, Aliccia; Mittal, Sandeep

2015-07-15

There is a paucity of effective therapies for recurrent/aggressive meningiomas. Establishment of improved in vitro and in vivo meningioma models will facilitate development and testing of novel therapeutic approaches. A primary meningioma cell line was generated from a patient with an olfactory groove meningioma. The cell line was extensively characterized by performing analysis of growth kinetics, immunocytochemistry, telomerase activity, karyotype, and comparative genomic hybridization. Xenograft models using immunocompromised SCID mice were also developed. Histopathology of the patient tumor was consistent with a WHO grade I typical meningioma composed of meningothelial cells, whorls, and occasional psammoma bodies. The original tumor and the early passage primary cells shared the standard immunohistochemical profile consistent with low-grade, good prognosis meningioma. Low passage KCI-MENG1 cells were composed of two cell types with spindle and round morphologies, showed linear growth curve, had very low telomerase activity, and were composed of two distinct unrelated clones on cytogenetic analysis. In contrast, high passage cells were homogeneously round, rapidly growing, had high telomerase activity, and were composed of a single clone with a near triploid karyotype containing 64-66 chromosomes with numerous aberrations. Following subcutaneous and orthotopic transplantation of low passage cells into SCID mice, firm tumors positive for vimentin and progesterone receptor (PR) formed, while subcutaneous implant of high passage cells yielded vimentin-positive, PR-negative tumors, concordant with a high-grade meningioma. Although derived from a benign meningioma specimen, the newly-established spontaneously immortal KCI-MENG1 meningioma cell line can be utilized to generate xenograft tumor models with either low- or high-grade features, dependent on the cell passage number (likely due to the relative abundance of the round, near-triploid cells). These human meningioma mouse xenograft models will provide biologically relevant platforms from which to investigate differences in low- vs. high-grade meningioma tumor biology and disease progression as well as to develop novel therapies to improve treatment options for poor prognosis or recurrent meningiomas.

Gallium, a promising candidate to disrupt the vicious cycle driving osteolytic metastases.

PubMed

Strazic-Geljic, Ivana; Guberovic, Iva; Didak, Blanka; Schmid-Antomarchi, Heidy; Schmid-Alliana, Annie; Boukhechba, Florian; Bouler, Jean-Michel; Scimeca, Jean-Claude; Verron, Elise

2016-09-15

Bone metastases of breast cancer typically lead to a severe osteolysis due to an excessive osteoclastic activity. On the other hand, the semi-metallic element gallium (Ga) displays an inhibitory action on osteoclasts, and therefore on bone resorption, as well as antitumour properties. Thus, we explored in vitro Ga effects on osteoclastogenesis in an aggressive bone metastatic environment based on the culture of pre-osteoclast RAW 264.7 cells with conditioned medium from metastatic breast tumour cells, i.e. the breast tumour cell line model MDA-MB-231 and its bone-seeking clone MDA-231BO. We first observed that Ga dose-dependently inhibited the tumour cells-induced osteoclastic differentiation of RAW 264.7 cells. To mimic a more aggressive environment where pro-tumourigenic factors are released from bone matrix due to osteoclastic resorption, metastatic breast tumour cells were stimulated with TGF-β, a mayor cytokine in bone metastasis vicious cycle. In these conditions, we observed that Ga still inhibited cancer cells-driven osteoclastogenesis. Lastly, we evidenced that Ga affected directly and strongly the proliferation/viability of both cancer cell lines, as well as the expression of major osteolytic factors in MDA-231BO cells. With the exception of two small scale clinical studies from 1980s, this is the first time that antitumour properties of Ga have been specifically studied in the context of bone metastases. Our data strongly suggest that, through its action against the vicious cycle involving bone cells and tumour cells, Ga represents a relevant and promising candidate for the local treatment of bone metastases in patients with breast cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Review: Endogenously Produced Volatiles for In Vitro Toxicity Testing Using Cell Lines

EPA Science Inventory

Due to the approximately 86,000 chemicals registered under the Toxic Substances Control Act and increasing ethical concerns regarding animal testing, it is not economically or technically feasible to screen every registered chemical for toxicity using animal-based toxicity assays...

Genotoxicity of 11 heavy metals detected as food contaminants in two human cell lines.

PubMed

Kopp, B; Zalko, D; Audebert, M

2018-04-01

Heavy metals, such as arsenic (As), antimony (Sb), barium (Ba), cadmium (Cd), cobalt (Co), germanium (Ge), lead (Pb), nickel (Ni), tellurium (Te), and vanadium (V) are widely distributed in the environment and in the food chain. Human exposure to heavy metals through water and food has been reported by different international agencies. Although some of these heavy metals are essential elements for human growth and development, they may also be toxic at low concentrations due to indirect mechanisms. In this study, the genotoxic and cytotoxic properties of 15 different oxidation statuses of 11 different heavy metals were investigated using high-throughput screening (γH2AX assay) in two human cell lines (HepG2 and LS-174T) representative of target organs (liver and colon) for food contaminants. Base on their lowest observed adverse effect concentration, the genotoxic potency of each heavy metal in each cell line was ranked in decreasing order, NaAsO 2  > CdCl 2  > PbCl 2 (only in LS-174T cells) > As 2 O 5  > SbCl 3  > K 2 TeO 3  > As 2 O 3 . No significant genotoxicity was observed with the other heavy metals tested. Cell viability data indicate that several heavy metals (As, Cd, Co, Ni, Sb, and Te) induce cytotoxicity at high concentrations, whereas an increase in the number of cells was observed for lead concentrations >100 µM in both cell lines tested, suggesting that lead stimulates cell growth. All these results highlight the possible human health hazards associated with the presence of heavy metals present in food. Environ. Mol. Mutagen. 59:202-210, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Evaluating the uptake and intracellular fate of polystyrene nanoparticles by primary and hepatocyte cell lines in vitro

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

Johnston, Helinor J., E-mail: h.johnston@napier.ac.u; Semmler-Behnke, Manuela; Brown, David M.

2010-01-01

Nanoparticles (NPs) are being used within diverse applications such as medicines, clothing, cosmetics and food. In order to promote the safe development of such nanotechnologies it is essential to assess the potential adverse health consequences associated with human exposure. The liver is recognised as a target site for NP toxicity, due to NP accumulation within this organ subsequent to injection, inhalation or instillation. The uptake of fluorescent polystyrene carboxylated particles (20 nm or 200 nm diameter) by hepatocytes was determined using confocal microscopy; with cells imaged 'live' during particle exposure or after exposure within fixed cells. Comparisons between the uptakemore » of polystyrene particles by primary rat hepatocytes, and human hepatocyte cell lines (C3A and HepG2) were made. Uptake of particles by hepatocytes was size, time, and serum dependent. Specifically, the uptake of 200 nm particles was limited, but 20 nm NPs were internalised by all cell types from 10 min onwards. At 10 min, 20 nm NP fluorescence co-localised with the tubulin cytoskeleton staining; after 30 min NP fluorescence compartmentalised into structures located within and/or between cells. The fate of internalised NPs was considered and they were not contained within early endosomes or lysosomes, but within mitochondria of cell lines. NPs accumulated within bile canaliculi to a limited extent, which suggests that NPs can be eliminated within bile. This is in keeping with the finding that gold NPs were eliminated in bile following intravenous injection into rats. The findings were, in the main, comparable between primary rat hepatocytes and the different human hepatocyte cell lines.« less

Effects of Plants and Isolates of Celastraceae Family on Cancer Pathways.

PubMed

Bukhari, Syed Nasir Abbas; Jantan, Ibrahim; Seyed, Mohamed Ali

2015-01-01

The evaluation of crude drugs of natural origin as sources of new effective anticancer agents continues to be important due to the lack of effective anticancer drugs currently used in practice which are generally accompanied with adverse effects at different levels of severity. The aim of this concise review is to gather existing literature on anticancer potential of extracts and compounds isolated from Celastraceae species. This review covers six genera (Maytenus, Tripterygium, Hippocratea, Gymnosporia, Celastrus and Austroplenckia) belonging to this family and their 33 isolates. Studies carried out by using different cell lines have shown remarkable indication of anticancer activity, however, only a restricted number of studies have been reported using in vivo tumor models. Some of the compounds, such as triptolide, celastrol and demethylzeylasteral from T. wilfordii, have been extensively studied on their mechanisms of action due to their potent activity on various cancer cell lines. Such promising lead compounds should generate considerable interest among scientists to improve their therapeutic potential with fewer side effects by molecular modification.

BEAM TRANSPORT LINES FOR THE BSNS.

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

WEI, J.

2006-06-26

This paper presents the design of two beam transport lines at the BSNS: one is the injection line from the Linac to the RCS and the other is the target line from the RCS to the target station. In the injection beam line, space charge effects, transverse halo collimation, momentum tail collimation and debunching are the main concerned topics. A new method of using triplet cells and stripping foils is used to collimate transverse halo. A long straight section is reserved for the future upgrading linac and debuncher. In the target beam line, large halo emittance, beam stability at themore » target due to kicker failures and beam jitters, shielding of back-scattering neutrons from the target are main concerned topics. Special bi-gap magnets will be used to reduce beam losses in the collimators in front of the target.« less

A novel imidazopyridine PI3K inhibitor with anticancer activity in non-small cell lung cancer cells.

PubMed

Lee, Hyunseung; Kim, Soo Jung; Jung, Kyung Hee; Son, Mi Kwon; Yan, Hong Hua; Hong, Sungwoo; Hong, Soon-Sun

2013-08-01

Lung cancer is the leading cause of cancer-related mortality in the world, and non-small cell lung cancer (NSCLC) accounts for approximately 85% of all cases. Since more than 60% of NSCLC cases express the epidermal growth factor receptor (EGFR), EGFR tyrosine kinase inhibitors are used to treat NSCLC. However, due to the acquired resistance associated with EGFR-targeted therapy, other strategies for the treatment of NSCLC are urgently needed. Therefore, we investigated the anticancer effects of a novel phosphatidylinositol 3-kinase α (PI3Kα) inhibitor, HS-173, in human NSCLC cell lines. HS-173 demonstrated anti-proliferative effects in NSCLC cells and effectively inhibited the PI3K signaling pathway in a dose‑dependent manner. In addition, it induced cell cycle arrest at G2/M phase as well as apoptosis. Taken together, our results demonstrate that HS-173 exhibits anticancer activities, including the induction of apoptosis, by blocking the PI3K/Akt/mTOR pathway in human NSCLC cell lines. We, therefore, suggest that this novel drug could potentially be used for targeted NSCLC therapy.

KML001 and doxercalciferol induce synergistic antileukemic effect in acute lymphoid leukemia cells.

PubMed

Liu, Yang; Shin, Dong-Yeop; Oh, Somi; Kim, Sujong; Koh, Youngil; Kim, Inho

2017-07-01

KML001 (NaAsO2, sodium metaarsenite, KOMINOX), a kind of arsenic compound, that has shown promising efficacy in non-Hodgkin's lymphoma (NHL) both in vitro and in vivo. In our study, the antileukemic effect of KML001 on acute lymphoid leukemia (ALL) and its mechanism of action were investigated. The results showed that KML001 inhibited cell proliferation in two types of ALL cell lines, CCRF-CEM and Molt-4. Exposure of ALL cells to KML001 induced apoptosis in a time-dependent manner. KML001 caused cell cycle arrest at G2/M phase instead of G0/G1 phase shown in other leukemia cells. In addition, we also tested the possibility of synergy of KML001 with doxercalciferol, a vitamin D2 derivative. Also, we found that a combination of KML001 with doxercalciferol showed a synergistic effect on ALL cell lines and this could be due to its different mechanism of action. Overall, our findings demonstrated KML001 could be a promising antileukemic agent especially when it is combined with doxercalciferol in ALL treatment.

Human Embryonic Kidney 293 Cells: A Vehicle for Biopharmaceutical Manufacturing, Structural Biology, and Electrophysiology.

PubMed

Hu, Jianwen; Han, Jizhong; Li, Haoran; Zhang, Xian; Liu, Lan Lan; Chen, Fei; Zeng, Bin

2018-01-01

Mammalian cells, e.g., CHO, BHK, HEK293, HT-1080, and NS0 cells, represent important manufacturing platforms in bioengineering. They are widely used for the production of recombinant therapeutic proteins, vaccines, anticancer agents, and other clinically relevant drugs. HEK293 (human embryonic kidney 293) cells and their derived cell lines provide an attractive heterologous system for the development of recombinant proteins or adenovirus productions, not least due to their human-like posttranslational modification of protein molecules to provide the desired biological activity. Secondly, they also exhibit high transfection efficiency yielding high-quality recombinant proteins. They are easy to maintain and express with high fidelity membrane proteins, such as ion channels and transporters, and thus are attractive for structural biology and electrophysiology studies. In this article, we review the literature on HEK293 cells regarding their origins but also stress their advancements into the different cell lines engineered and discuss some significant aspects which make them versatile systems for biopharmaceutical manufacturing, drug screening, structural biology research, and electrophysiology applications. © 2018 S. Karger AG, Basel.

Accumulation and altered localization of telomere-associated protein TRF2 in immortally transformed and tumor-derived human breast cells

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

Nijjar, Tarlochan; Bassett, Ekaterina; Garbe, James

2004-12-23

We have used cultured human mammary epithelial cells (HMEC) and breast tumor-derived lines to gain information on defects that occur during breast cancer progression. HMEC immortalized by a variety of agents (the chemical carcinogen benzo(a)pyrene, oncogenes c-myc and ZNF217, and/or dominant negative p53 genetic suppressor element GSE22) displayed marked up regulation (10-15 fold) of the telomere binding protein, TRF2. Up-regulation of TRF2 protein was apparently due to differences in post-transcriptional regulation, as mRNA levels remained comparable in finite life span and immortal HMEC. TRF2 protein was not up-regulated by the oncogenic agents alone in the absence of immortalization, nor bymore » expression of exogenously introduced hTERT genes. We found TRF2 levels to be at least 2-fold higher than in control cells in 11/15 breast tumor cell lines, suggesting that elevated TRF2 levels are a frequent occurrence during the transformation of breast tumor cells in vivo. The dispersed distribution of TRF2 throughout the nuclei in some immortalized and tumor-derived cells indicated that not all the TRF2 was associated with telomeres in these cells. The process responsible for accumulation of TRF2 in immortalized HMEC and breast tumor-derived cell lines may promote tumorigenesis by contributing to the cells ability to maintain an indefinite life span.« less

Comparison of tumor biology of two distinct cell sub-populations in lung cancer stem cells.

PubMed

Wang, Jianyu; Sun, Zhiwei; Liu, Yongli; Kong, Liangsheng; Zhou, Shixia; Tang, Junlin; Xing, Hongmei Rosie

2017-11-14

Characterization of the stem-like properties of cancer stem cells (CSCs) remain indirect and qualitative, especially the ability of CSCs to undergo asymmetric cell division for self renewal and differentiation, a unique property of cells of stem origin. It is partly due to the lack of stable cellular models of CSCs. In this study, we developed a new approach for CSC isolation and purification to derive a CSC-enriched cell line (LLC-SE). By conducting five consecutive rounds of single cell cloning using the LLC-SE cell line, we obtained two distinct sub-population of cells within the Lewis lung cancer CSCs that employed largely symmetric division for self-renewal (LLC-SD) or underwent asymmetric division for differentiation (LLC-ASD). LLC-SD and LLC-ASD cell lines could be stably passaged in culture and be distinguished by cell morphology, stem cell marker, spheroid formation and subcutaneous tumor initiation efficiency, as well as orthotopic lung tumor growth, progression and survival. The ability LLC-ASD cells to undergo asymmetric division was visualized and quantified by the asymmetric segregation of labeled BrdU and NUMB to one of the two daughter cells in anaphase cell division. The more stem-like LLC-SD cells exhibited higher capacity for tumorigenesis and progression and shorter survival. As few as 10 LLC-SD could initiate subcutaneous tumor growth when transplanted to the athymic mice. Collectively, these observations suggest that the SD-type of cells appear to be on the top of the hierarchical order of the CSCs. Furthermore, they have lead to generated cellular models of CSC self-renewal for future mechanistic investigations.

Efficacy and mechanism of action of Proellex, an antiprogestin in aromatase overexpressing and Letrozole resistant T47D breast cancer cells.

PubMed

Gupta, Akash; Mehta, Rajeshwari; Alimirah, Fatouma; Peng, Xinjian; Murillo, Genoveva; Wiehle, Ronald; Mehta, Rajendra G

2013-01-01

Aromatase inhibitors (AI) are considered as a first line therapy for ER+PR+ breast cancers. However, many patients acquire resistance to AI. In this study, we determined the response of antiprogestin CDB-4124 (Proellex) on the aromatase overexpressing and Letrozole resistant cell lines and also studies its mechanism of action in inhibition of breast cancer cell proliferation. For these studies we generated aromatase overexpressing T47D (T47Darom) and respective control (T47Dcon) breast cancer cell lines by stable transfection with plasmid containing CYP19A1 gene, or empty vector respectively. Letrozole resistant cell line (T47DaromLR) was generated by incubating T47Darom for 75 weeks in the presence of 10 μM Letrozole. Cell proliferation was determined by MTT or crystal violet assays. Gene expressions were quantified by QRT-PCR whereas proteins were identified by western blot analyses, flow cytometry and immunofluorescence staining. Aromatase activity was determined by estradiol ELISA. The effects of Proellex on the anchorage independent growth were measured by soft agar colony formation. Statistical differences between the various groups were determined by Student's 't' test or ANOVA followed by Bonferroni's post hoc test. Results showed that T47Darom and T47DaromLR cell lines had significantly higher aromatase expression (mRNA; 80-90 fold and protein) and as a result exhibited increased aromatization of testosterone to estradiol as compared to T47Dcon. Both these cell lines showed enhanced growth in the presence of Testosterone (50-60%). In T47DaromLR cells increased PR-B and EGFR expression as compared to T47Dcon cells was observed. Proellex and other known aromatase inhibitors (Letrozole, Anastrozole, and Exemestane) inhibited testosterone induced cell proliferation and anchorage independent growth of T47Darom cells. Cell growth inhibition was significantly greater when cells were treated with Proellex alone or in combination with other AIs as compared to AIs alone. Proellex inhibited mRNA and protein levels of PR-B, reduced PRB/p300 complex formation in the nuclei and significantly reduced EGFR expression in T47Darom cells. Our results in the present study indicate that antiproliferative effect of Proellex is probably due to PR-B/EGFR modulation in ER+PR+, aromatase expressing cells. Overall these results suggest that antiprogestin, Proellex can be developed as a possible treatment strategy for aromatase overexpressing ER+/PR+ breast cancer patients as well as for aromatase inhibitor resistant breast cancer patients. Copyright © 2012 Elsevier Ltd. All rights reserved.

Characterization of Apoptosis Signaling Cascades During the Differentiation Process of Human Neural ReNcell VM Progenitor Cells In Vitro.

PubMed

Jaeger, Alexandra; Fröhlich, Michael; Klum, Susanne; Lantow, Margareta; Viergutz, Torsten; Weiss, Dieter G; Kriehuber, Ralf

2015-11-01

Apoptosis is an essential physiological process accompanying the development of the central nervous system and human neurogenesis. However, the time scale and the underlying molecular mechanisms are yet poorly understood. Due to this fact, we investigated the functionality and general inducibility of apoptosis in the human neural ReNcell VM progenitor cell line during differentiation and also after exposure to staurosporine (STS) and ultraviolet B (UVB) irradiation. Transmission light microscopy, flow cytometry, and Western-/Immunoblot analysis were performed to compare proliferating and differentiating, in addition to STS- and UVB-treated cells. In particular, from 24 to 72 h post-initiation of differentiation, G0/G1 cell cycle arrest, increased loss of apoptotic cells, activation of pro-apoptotic BAX, Caspase-3, and cleavage of its substrate PARP were observed during cell differentiation and, to a higher extent, after treatment with STS and UVB. We conclude that redundant or defective cells are eliminated by apoptosis, while otherwise fully differentiated cells were less responsive to apoptosis induction by STS than proliferating cells, likely as a result of reduced APAF-1 expression, and increased levels of BCL-2. These data provide the evidence that apoptotic mechanisms in the neural ReNcell VM progenitor cell line are not only functional, but also inducible by external stimuli like growth factor withdrawal or treatment with STS and UVB, which marks this cell line as a suitable model to investigate apoptosis signaling pathways in respect to the differentiation processes of human neural progenitor cells in vitro.

Activation of apoptotic pathways in the absence of cell death in an inner-ear immortomouse cell line

PubMed Central

Chen, Fu-Quan; Hill, Kayla; Guan, Ya-Jun; Schacht, Jochen; Sha, Su-Hua

2012-01-01

Aminoglycoside antibiotics and cisplatin (CDDP) are the major ototoxins of clinical medicine due to their capacity to cause significant, as well as permanent hearing loss by targeting the mammalian sensory cells. Understanding the pathogenesis of damage is the first step in designing effective prevention of drug-induced hearing loss. In-vitro systems greatly enhance the efficiency of biochemical and molecular investigations through ease of access and manipulation. HEI-OC1, an inner ear cell line derived from the immortomouse, expresses markers for auditory sensory cells and, therefore, is a potential tool to study the ototoxic mechanisms of drugs like aminoglycoside antibiotics and CDDP. HEI-OC1 cells (and also HeLa cells) efficiently take up fluorescently tagged gentamicin and respond to drug treatment with changes in cell death and survival signaling pathways. Within hours, the C-jun N-terminal kinase pathway and the transcription factor AP-1 were activated and at later times, the “executioner caspase”, caspase-3. These responses were robust and elicited by both gentamicin and kanamycin. However, despite the initiation of apoptotic pathways and transient changes in nuclear morphology, cell death was not observed following aminoglycoside treatment, while administration of CDDP lead to significant cell death as determined by flow cytometric measurements; β-galactosidase analysis ruled out senescence in gentamicin-treated cells. The ability to withstand treatment with aminoglycosides but not with CDDP suggests that this cell line might be helpful in providing some insight into the differential actions of the two ototoxic drugs. PMID:22240458

Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion

PubMed Central

Hussein, Deema; Punjaruk, Wiyada; Storer, Lisa C.D.; Shaw, Lucy; Ottoman, Ramadan; Peet, Andrew; Miller, Suzanne; Bandopadhyay, Gagori; Heath, Rachel; Kumari, Rajendra; Bowman, Karen J.; Braker, Paul; Rahman, Ruman; Jones, George D.D.; Watson, Susan; Lowe, James; Kerr, Ian D.; Grundy, Richard G.; Coyle, Beth

2011-01-01

Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3–4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents. PMID:20978004

Pediatric brain tumor cancer stem cells: cell cycle dynamics, DNA repair, and etoposide extrusion.

PubMed

Hussein, Deema; Punjaruk, Wiyada; Storer, Lisa C D; Shaw, Lucy; Othman, Ramadhan; Ottoman, Ramadan; Peet, Andrew; Miller, Suzanne; Bandopadhyay, Gagori; Heath, Rachel; Kumari, Rajendra; Bowman, Karen J; Braker, Paul; Rahman, Ruman; Jones, George D D; Watson, Susan; Lowe, James; Kerr, Ian D; Grundy, Richard G; Coyle, Beth

2011-01-01

Reliable model systems are needed to elucidate the role cancer stem cells (CSCs) play in pediatric brain tumor drug resistance. The majority of studies to date have focused on clinically distinct adult tumors and restricted tumor types. Here, the CSC component of 7 newly established primary pediatric cell lines (2 ependymomas, 2 medulloblastomas, 2 gliomas, and a CNS primitive neuroectodermal tumor) was thoroughly characterized. Comparison of DNA copy number with the original corresponding tumor demonstrated that genomic changes present in the original tumor, typical of that particular tumor type, were retained in culture. In each case, the CSC component was approximately 3-4-fold enriched in neurosphere culture compared with monolayer culture, and a higher capacity for multilineage differentiation was observed for neurosphere-derived cells. DNA content profiles of neurosphere-derived cells expressing the CSC marker nestin demonstrated the presence of cells in all phases of the cell cycle, indicating that not all CSCs are quiescent. Furthermore, neurosphere-derived cells demonstrated an increased resistance to etoposide compared with monolayer-derived cells, having lower initial DNA damage, potentially due to a combination of increased drug extrusion by ATP-binding cassette multidrug transporters and enhanced rates of DNA repair. Finally, orthotopic xenograft models reflecting the tumor of origin were established from these cell lines. In summary, these cell lines and the approach taken provide a robust model system that can be used to develop our understanding of the biology of CSCs in pediatric brain tumors and other cancer types and to preclinically test therapeutic agents.

Immortalization of human prostate epithelial cells by HPV 16 E6/E7 open reading frames.

PubMed

Choo, C K; Ling, M T; Chan, K W; Tsao, S W; Zheng, Z; Zhang, D; Chan, L C; Wong, Y C

1999-08-01

The exact pathogenesis for prostate cancer is not known. Progress made in prostate cancer research has been slow, largely due to the lack of suitable in vitro models. Here, we report our work on the immortalization of a human prostate epithelial cell line and show that it can be used as a model to study prostate tumorigenesis. Replication-defective retrovirus harboring the human papillomavirus (HPV) type 16 E6 and E7 open reading frames was used to infect primary human prostate epithelial cells. Polymerase chain reaction, followed by Southern hybridization for the HPV 16 E6/E7, Western blot for prostatic acid phosphatase, telomeric repeat amplification protocol assay for telomerase activity, two-dimensional gels for cytokeratins, and cytogenetic analysis were undertaken to characterized the infected cells. The retrovirus-infected cell line, HPr-1, continued to grow in culture for more than 80 successive passages. Normal primary cells failed to proliferate after passage 6. HPr-1 cells bore close resemblance to normal primary prostate epithelial cells, both morphologically and biochemically. However, they possessed telomerase activity and proliferated indefinitely. Cytogenetic analysis of HPr-1 cells revealed a human male karyotype with clonal abnormalities and the appearance of multiple double minutes. The HPr-1 cells expressed prostatic acid phosphatase and cytokeratins K8 and K18, proving that they were prostate epithelial cells. They were benign in nude mice tumor formation and soft agar colony formation assay. The HPr-1 cell line is an in vitro representation of early prostate neoplastic progression. Copyright 1999 Wiley-Liss, Inc.

First-line treatment of EGFR-mutant non-small-cell lung cancer: the role of erlotinib and other tyrosine kinase inhibitors

PubMed Central

Nguyen, Kim-Son H; Neal, Joel W

2012-01-01

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) were initially established as second- or third-line treatment of advanced non-small-cell lung cancer (NSCLC). Subsequent studies, including IPASS, OPTIMAL, and EURTAC, have demonstrated that these TKIs are effective first-line therapeutic options in patients with tumors harboring activating mutations in the EGFR gene. The TKIs are better tolerated than conventional chemotherapy, with frequent yet mild side effects such as rash and diarrhea, and rarely interstitial lung disease. Because most patients on TKIs develop resistance due to a variety of mechanisms, the use of TKIs in the acquired-resistance setting and in the setting of earlier-staged cancers is being extensively studied. Here we review the major trials leading to the established use of EGFR TKIs in NSCLC, followed by discussion of recently completed and ongoing trials using the next-generation EGFR inhibitor afatinib. PMID:23055691

Damage and Recovery of Hair Cells in Fish Canal (But Not Superficial) Neuromasts after Gentamicin Exposure

NASA Technical Reports Server (NTRS)

Song, Jiakun; Yan, Hong Young; Popper, Arthur N.

1995-01-01

Recent evidence demonstrating the presence of two types of sensory hair cells in the ear of a telcost fish (Astronotus ocellatus, the oscar) indicates that hair cell heterogeneity may exist not only in amniotic vertebrates but also in anamniotes. Here we report that a similar heterogeneity between hair cell types may also occur in the other mechanosensory organ of the oscar, the lateral line. We exposed oscars to the aminoglycoside (ototoxic) antibiotic gentamicin sulfate and found damaged sensory hair cells in one class of the lateral line receptors, the canal neuromasts, but not in the other class, the superficial neuromasts. This effect was not due to the canal environment. Moreover, new ciliary bundles on hair cells of the canal neuromasts were found after, and during, gentamicin exposure. The pattern of hair cell destruction and recovery in canal neuromasts is similar to that of type 1-like hair cells found in the striolar region of the utricle and lagena of the oscar after gentamicin treatment. These results suggest that the hair cells in the canal and superficial neuromasts may be similar to type 1-like and type 2 hair cells, respectively, in the fish ear.

Release behavior and toxicity profiles towards A549 cell lines of ciprofloxacin from its layered zinc hydroxide intercalation compound

PubMed Central

2013-01-01

Background Layered hydroxides salts (LHS), a layered inorganic compound is gaining attention in a wide range of applications, particularly due to its unique anion exchange properties. In this work, layered zinc hydroxide nitrate (LZH), a family member of LHS was intercalated with anionic ciprofloxacin (CFX), a broad spectrum antibiotic via ion exchange in a mixture solution of water:ethanol. Results Powder x-ray diffraction (XRD), Fourier transform infrared (FTIR) and thermogravimetric analysis (TGA) confirmed the drug anions were successfully intercalated in the interlayer space of LZH. Specific surface area of the obtained compound was increased compared to that of the host due to the different pore textures between the two materials. CFX anions were slowly released over 80 hours in phosphate-buffered saline (PBS) solution due to strong interactions that occurred between the intercalated anions and the host lattices. The intercalation compound demonstrated enhanced antiproliferative effects towards A549 cancer cells compared to the toxicity of CFX alone. Conclusions Strong host-guest interactions between the LZH lattice and the CFX anion give rise to a new intercalation compound that demonstrates sustained release mode and enhanced toxicity effects towards A549 cell lines. These findings should serve as foundations towards further developments of the brucite-like host material in drug delivery systems. PMID:23849189

Archaeal viruses at the cell envelope: entry and egress

PubMed Central

Quemin, Emmanuelle R. J.; Quax, Tessa E. F.

2015-01-01

The cell envelope represents the main line of host defense that viruses encounter on their way from one cell to another. The cytoplasmic membrane in general is a physical barrier that needs to be crossed both upon viral entry and exit. Therefore, viruses from the three domains of life employ a wide range of strategies for perforation of the cell membrane, each adapted to the cell surface environment of their host. Here, we review recent insights on entry and egress mechanisms of viruses infecting archaea. Due to the unique nature of the archaeal cell envelope, these particular viruses exhibit novel and unexpected mechanisms to traverse the cellular membrane. PMID:26097469

Clearance of Human IgG1-Sensitised Red Blood Cells In Vivo in Humans Relates to the In Vitro Properties of Antibodies from Alternative Cell Lines

PubMed Central

Armour, Kathryn L.; Smith, Cheryl S.; Ip, Natasha C. Y.; Ellison, Cara J.; Kirton, Christopher M.; Wilkes, Anthony M.; Williamson, Lorna M.; Clark, Michael R.

2014-01-01

We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance. PMID:25302805

Mutation site and context dependent effects of ESR1 mutation in genome-edited breast cancer cell models.

PubMed

Bahreini, Amir; Li, Zheqi; Wang, Peilu; Levine, Kevin M; Tasdemir, Nilgun; Cao, Lan; Weir, Hazel M; Puhalla, Shannon L; Davidson, Nancy E; Stern, Andrew M; Chu, David; Park, Ben Ho; Lee, Adrian V; Oesterreich, Steffi

2017-05-23

Mutations in the estrogen receptor alpha (ERα) 1 gene (ESR1) are frequently detected in ER+ metastatic breast cancer, and there is increasing evidence that these mutations confer endocrine resistance in breast cancer patients with advanced disease. However, their functional role is not well-understood, at least in part due to a lack of ESR1 mutant models. Here, we describe the generation and characterization of genome-edited T47D and MCF7 breast cancer cell lines with the two most common ESR1 mutations, Y537S and D538G. Genome editing was performed using CRISPR and adeno-associated virus (AAV) technologies to knock-in ESR1 mutations into T47D and MCF7 cell lines, respectively. Various techniques were utilized to assess the activity of mutant ER, including transactivation, growth and chromatin-immunoprecipitation (ChIP) assays. The level of endocrine resistance was tested in mutant cells using a number of selective estrogen receptor modulators (SERMs) and degraders (SERDs). RNA sequencing (RNA-seq) was employed to study gene targets of mutant ER. Cells with ESR1 mutations displayed ligand-independent ER activity, and were resistant to several SERMs and SERDs, with cell line and mutation-specific differences with respect to magnitude of effect. The SERD AZ9496 showed increased efficacy compared to other drugs tested. Wild-type and mutant cell co-cultures demonstrated a unique evolution of mutant cells under estrogen deprivation and tamoxifen treatment. Transcriptome analysis confirmed ligand-independent regulation of ERα target genes by mutant ERα, but also identified novel target genes, some of which are involved in metastasis-associated phenotypes. Despite significant overlap in the ligand-independent genes between Y537S and D538G, the number of mutant ERα-target genes shared between the two cell lines was limited, suggesting context-dependent activity of the mutant receptor. Some genes and phenotypes were unique to one mutation within a given cell line, suggesting a mutation-specific effect. Taken together, ESR1 mutations in genome-edited breast cancer cell lines confer ligand-independent growth and endocrine resistance. These biologically relevant models can be used for further mechanistic and translational studies, including context-specific and mutation site-specific analysis of the ESR1 mutations.

Clearance of human IgG1-sensitised red blood cells in vivo in humans relates to the in vitro properties of antibodies from alternative cell lines.

PubMed

Armour, Kathryn L; Smith, Cheryl S; Ip, Natasha C Y; Ellison, Cara J; Kirton, Christopher M; Wilkes, Anthony M; Williamson, Lorna M; Clark, Michael R

2014-01-01

We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance.

Sox2 Is an Androgen Receptor-Repressed Gene That Promotes Castration-Resistant Prostate Cancer

PubMed Central

Kregel, Steven; Kiriluk, Kyle J.; Rosen, Alex M.; Cai, Yi; Reyes, Edwin E.; Otto, Kristen B.; Tom, Westin; Paner, Gladell P.; Szmulewitz, Russell Z.; Vander Griend, Donald J.

2013-01-01

Despite advances in detection and therapy, castration-resistant prostate cancer continues to be a major clinical problem. The aberrant activity of stem cell pathways, and their regulation by the Androgen Receptor (AR), has the potential to provide insight into novel mechanisms and pathways to prevent and treat advanced, castrate-resistant prostate cancers. To this end, we investigated the role of the embryonic stem cell regulator Sox2 [SRY (sex determining region Y)-box 2] in normal and malignant prostate epithelial cells. In the normal prostate, Sox2 is expressed in a portion of basal epithelial cells. Prostate tumors were either Sox2-positive or Sox2-negative, with the percentage of Sox2-positive tumors increasing with Gleason Score and metastases. In the castration-resistant prostate cancer cell line CWR-R1, endogenous expression of Sox2 was repressed by AR signaling, and AR chromatin-IP shows that AR binds the enhancer element within the Sox2 promoter. Likewise, in normal prostate epithelial cells and human embryonic stem cells, increased AR signaling also decreases Sox2 expression. Resistance to the anti-androgen MDV3100 results in a marked increase in Sox2 expression within three prostate cancer cell lines, and in the castration-sensitive LAPC-4 prostate cancer cell line ectopic expression of Sox2 was sufficient to promote castration-resistant tumor formation. Loss of Sox2 expression in the castration-resistant CWR-R1 prostate cancer cell line inhibited cell growth. Up-regulation of Sox2 was not associated with increased CD133 expression but was associated with increased FGF5 (Fibroblast Growth Factor 5) expression. These data propose a model of elevated Sox2 expression due to loss of AR-mediated repression during castration, and consequent castration-resistance via mechanisms not involving induction of canonical embryonic stem cell pathways. PMID:23326489

Generation and Characterization of the First Immortalized Alpaca Cell Line Suitable for Diagnostic and Immunization Studies

PubMed Central

Franceschi, Valentina; Jacca, Sarah; Sassu, Elena L.; Stellari, Fabio F.; van Santen, Vicky L.; Donofrio, Gaetano

2014-01-01

Raising of alpacas as exotic livestock for wool and meat production and as companion animals is growing in importance in the United States, Europe and Australia. Furthermore the alpaca, as well as the rest of the camelids, possesses the peculiarity of producing single-chain antibodies from which nanobodies can be generated. Nanobodies, due to their structural simplicity and reduced size, are very versatile in terms of manipulation and bio-therapeutic exploitation. In fact the biotech companies involved in nanobody production and application continue to grow in number and size. Hence, the development of reagents and tools to assist in the further growth of this new scientific and entrepreneurial reality is becoming a necessity. These are needed mainly to address alpaca disease diagnosis and prophylaxis, and to develop alpaca immunization strategies for nanobody generation. For instance an immortalized alpaca cell line would be extremely valuable. In the present work the first stabilized alpaca cell line from alpaca skin stromal cells (ASSCs) was generated and characterized. This cell line was shown to be suitable for replication of viruses bovine herpesvirus-1, bovine viral diarrhea virus and caprine herpesvirus-1 and the endocellular parasite Neospora caninum. Moreover ASSCs were easy to transfect and transduce by several methods. These two latter characteristics are extremely useful when recombinant antigens need to be produced in a host homologous system. This work could be considered as a starting point for the expansion of the biotechnologies linked to alpaca farming and industry. PMID:25140515

Generation and characterization of the first immortalized alpaca cell line suitable for diagnostic and immunization studies.

PubMed

Franceschi, Valentina; Jacca, Sarah; Sassu, Elena L; Stellari, Fabio F; van Santen, Vicky L; Donofrio, Gaetano

2014-01-01

Raising of alpacas as exotic livestock for wool and meat production and as companion animals is growing in importance in the United States, Europe and Australia. Furthermore the alpaca, as well as the rest of the camelids, possesses the peculiarity of producing single-chain antibodies from which nanobodies can be generated. Nanobodies, due to their structural simplicity and reduced size, are very versatile in terms of manipulation and bio-therapeutic exploitation. In fact the biotech companies involved in nanobody production and application continue to grow in number and size. Hence, the development of reagents and tools to assist in the further growth of this new scientific and entrepreneurial reality is becoming a necessity. These are needed mainly to address alpaca disease diagnosis and prophylaxis, and to develop alpaca immunization strategies for nanobody generation. For instance an immortalized alpaca cell line would be extremely valuable. In the present work the first stabilized alpaca cell line from alpaca skin stromal cells (ASSCs) was generated and characterized. This cell line was shown to be suitable for replication of viruses bovine herpesvirus-1, bovine viral diarrhea virus and caprine herpesvirus-1 and the endocellular parasite Neospora caninum. Moreover ASSCs were easy to transfect and transduce by several methods. These two latter characteristics are extremely useful when recombinant antigens need to be produced in a host homologous system. This work could be considered as a starting point for the expansion of the biotechnologies linked to alpaca farming and industry.

Enhancing wear resistance of working bodies of grinder through lining crushed material

NASA Astrophysics Data System (ADS)

Romanovich, A. A.; Annenko, D. M.; Romanovich, M. A.; Apukhtina, I. V.

2018-03-01

The article presents the analysis of directions of increasing wear resistance of working surfaces of rolls. A technical solution developed at the level of the invention is proposed, which is simple to implement in production conditions and which makes it possible to protect the roll surface from heavy wear due to surfacing of wear-resistant mesh material, cells of which are filling with grinding material in the process of work. Retaining them enables one to protect the roll surface from wear. The paper dwells on conditions of pressing materials in cells of eccentric rolls on the working surface with a grid of rectangular shape. The paper presents an equation for calculation of the cell dimension that provides the lining of the working surface by a mill material with respect to its properties. The article presents results of comparative studies on the grinding process of a press roller grinder (PRG) between rolls with and without a fusion-bonded mesh. It is clarified that the lining of rolls working surface slightly reduces the quality of the grinding, since the material thickness in the cell is small and has a finely divided and compacted structure with high strength.

Methoxyacetic acid suppresses prostate cancer cell growth by inducing growth arrest and apoptosis

PubMed Central

Parajuli, Keshab R; Zhang, Qiuyang; Liu, Sen; Patel, Neil K; Lu, Hua; Zeng, Shelya X; Wang, Guangdi; Zhang, Changde; You, Zongbing

2014-01-01

Methoxyacetic acid (MAA) is a primary metabolite of ester phthalates that are used in production of consumer products and pharmaceutical products. MAA causes embryo malformation and spermatocyte death through inhibition of histone deacetylases (HDACs). Little is known about MAA’s effects on cancer cells. In this study, two immortalized human normal prostatic epithelial cell lines (RWPE-1 and pRNS-1-1) and four human prostate cancer cell lines (LNCaP, C4-2B, PC-3, and DU-145) were treated with MAA at different doses and for different time periods. Cell viability, apoptosis, and cell cycle analysis were performed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR, Western blot, and chromatin immunoprecipitation analyses. We found that MAA dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. MAA-induced apoptosis was due to down-regulation of the anti-apoptotic gene baculoviral inhibitor of apoptosis protein repeat containing 2 (BIRC2, also named cIAP1), leading to activation of caspases 7 and 3 and turning on the downstream apoptotic events. MAA-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and CDK2 expression at the late time. MAA up-regulated p21 expression through inhibition of HDAC activities, independently of p53/p63/p73. These findings demonstrate that MAA suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which suggests that MAA could be used as a potential therapeutic drug for prostate cancer. PMID:25606576

Comparative analysis of 3D culture methods on human HepG2 cells.

PubMed

Luckert, Claudia; Schulz, Christina; Lehmann, Nadja; Thomas, Maria; Hofmann, Ute; Hammad, Seddik; Hengstler, Jan G; Braeuning, Albert; Lampen, Alfonso; Hessel, Stefanie

2017-01-01

Human primary hepatocytes represent a gold standard in in vitro liver research. Due to their low availability and high costs alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. The human hepatocarcinoma cell line HepG2 is often used as a liver model for toxicity studies. However, under two-dimensional (2D) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers such as albumin is very low. Cultivation for 21 days in a three-dimensional (3D) Matrigel culture system has been reported to strongly increase the metabolic competence of HepG2 cells. In our present study we further compared HepG2 cell cultivation in three different 3D systems: collagen, Matrigel and Alvetex culture. Cell morphology, albumin secretion, cytochrome P450 monooxygenase enzyme activities, as well as gene expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after 3, 7, 14, and 21 days of cultivation. Our results show that the previously reported increase of metabolic competence of HepG2 cells is not primarily the result of 3D culture but a consequence of the duration of cultivation. HepG2 cells grown for 21 days in 2D monolayer exhibit comparable biochemical characteristics, CYP activities and gene expression patterns as all 3D culture systems used in our study. However, CYP activities did not reach the level of HepaRG cells. In conclusion, the increase of metabolic competence of the hepatocarcinoma cell line HepG2 is not due to 3D cultivation but rather a result of prolonged cultivation time.

The development of methods for primary mast cells in vitro and ex vivo: An historical review.

PubMed

Yu, Tian-Yu; He, Zhi-Gang; Yang, Mu-Qing; Song, Jian; Ma, Cheng; Ma, Sun-Qiang; Feng, Jun-Lan; Bin Liu; Wang, Xiao-Dong; Li, Ji-Yu; Wei, Zhu-Bo

2018-05-26

Mast cells (MCs) are tissue-based stationary effector cells that form the immune system's first-line defense against various challenges. They are developed from the bone marrow-derived progenitors to complete their differentiation and maturation in the tissues where they eventually establish residence. MCs have been implicated in many diseases, such as allergy, parasitic infection, and neoplastic disorders. Immortalized MC lines, such as RBL-2H3, HMC-1, and LAD-2, are useful for investigating the biological functions of MC only to some extents due to the restriction of degranulation evaluation, in vivo injection and other factors. Over the past few decades, technologies for acquiring primarily MCs have been continually optimized, and novel protocols have been proposed. However, no relevant publications have analyzed and summarized these techniques. In this review, the classical approaches for extracting MCs are generalized, and new methods with potential values are introduced. We also evaluate the advantages and applicability of diverse MC models. Since MCs exhibit substantial plasticity and functional diversity due to different origins, it is both necessary and urgent to select a reliable and suitable source of MCs for a particular study. Copyright © 2018 Elsevier Inc. All rights reserved.

Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane

PubMed Central

Fujiwara, Takahiro K.; Iwasawa, Kokoro; Kalay, Ziya; Tsunoyama, Taka A.; Watanabe, Yusuke; Umemura, Yasuhiro M.; Murakoshi, Hideji; Suzuki, Kenichi G. N.; Nemoto, Yuri L.; Morone, Nobuhiro; Kusumi, Akihiro

2016-01-01

The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed “hop diffusion”) for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion. PMID:26864625

Variation in cisplatinum sensitivity is not associated with Fanconi Anemia/BRCA pathway inactivation in head and neck squamous cell carcinoma cell lines.

PubMed

Snyder, Eric R; Ricker, Justin L; Chen, Zhong; Waes, Carter Van

2007-01-08

Fanconi Anemia has recently been associated with a high risk of head and neck squamous cell carcinoma (HNSCC). Inactivation of the Fanconi Anemia (FANC-BRCA) pathway via promoter methylation of the FANCF gene has been proposed to be responsible for variation in cisplatinum (CDDP) sensitivity seen in ovarian and HNSCCs. Promoter methylation of the FANCF gene has been observed in 15% of HNSCC specimens, but the relationship to FANC pathway activation and CDDP sensitivity has not been reported. In the present study, 10 HNSCC cell lines were examined for expression of nine genes involved in the FANC-BRCA pathway by RT-PCR: FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL, BRCA1 and BRCA2. FANC pathway function was evaluated by western blotting for FANCD2 mono-ubiquitination. All of the cell lines were also analyzed for variation in CDDP cytotoxicity. While significant differences were found in CDDP cytotoxicity, Fanconi pathway defects are an infrequent cause, as no evidence of transcriptional down-regulation of FANCF or other FANC mRNAs, or functional FANC-BRCA pathway defects were observed. These findings suggest that the variation in CDDP sensitivity of many HNSCCs is most frequently due to factors other than FANC-BRCA pathway inactivation.

An assay for lateral line regeneration in adult zebrafish.

PubMed

Pisano, Gina C; Mason, Samantha M; Dhliwayo, Nyembezi; Intine, Robert V; Sarras, Michael P

2014-04-08

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al. that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Identification of cytotoxic mediators and their putative role in the signaling pathways during docosahexaenoic acid (DHA)-induced apoptosis of cancer cells.

PubMed

Das, Moitreyi; Das, Sumantra

2016-12-01

Docosahexaenoic acid (DHA), an important w-3 fatty acid exhibits differential behavior in cancer cells of neural origin when compared to that in normal healthy astrocytes. Treatment of C6 glioma and SH-SY5Y cell lines and primary astrocytes, representing the neoplastic cells and normal healthy cells respectively, with 100 µM DHA for 24 h showed significant loss of cell viability in the both the cancer cells as determined by MTT assay, whereas the primary astrocytes cultures were unaffected. Such loss of cell viability was due to apoptosis as confirmed by TUNEL staining and caspase-3 activation in cancer cells. Proteomic approach, employing 2-dimensional gel electrophoresis (2DE), difference gel electrophoresis (DIGE), and MALDI-TOF-TOF analysis identified six proteins which unlike in the astrocytes, were differently altered in the cancer cells upon exposure to DHA, suggesting their putative contribution in causing apoptosis in these cells. Of these, annexin A2, calumenin, pyruvate kinase M2 isoform, 14-3-3ζ were downregulated while aldo keto reductase-1B8 (AKR1B8) and glutathione-S-transferase P1 subunit (GSTP1) showed upregulation by DHA in the cancer cells. siRNA-mediated knockdown of AKR1B8 and GSTP1 inhibit DHA-induced apoptosis confirming their role in apoptotic process. Furthermore, western blot analysis identified upregulation of PPARα and the MAP kinases, JNK and p38 as well as increased ROS production selectively in the cell lines. Results suggest that DHA selectively induces apoptosis in the neural cell lines by regulating the expression of the above proteins to activate multiple apoptotic pathways which in association with excess ROS and activated MAPKs promote cell death.

Biodegradable charged polyester-based vectors (BCPVs) as an efficient non-viral transfection nanoagent for gene knockdown of the BCR-ABL hybrid oncogene in a human chronic myeloid leukemia cell line

NASA Astrophysics Data System (ADS)

Yang, Chengbin; Panwar, Nishtha; Wang, Yucheng; Zhang, Butian; Liu, Maixian; Toh, Huiting; Yoon, Ho Sup; Tjin, Swee Chuan; Chong, Peter Han Joo; Law, Wing-Cheung; Chen, Chih-Kuang; Yong, Ken-Tye

2016-04-01

First-line therapy of chronic myelogenous leukemia (CML) has always involved the use of BCR-ABL tyrosine-kinase inhibitors which is associated with an abnormal chromosome called Philadelphia chromosome. Although the overall survival rate has been improved by the current therapeutic regime, the presence of resistance has resulted in limited efficacy. In this study, an RNA interference (RNAi)-based therapeutic regime is proposed with the aim to knockdown the BCR-ABL hybrid oncogene using small interfering RNA (siRNA). The siRNA transfection rates have usually been limited due to the declining contact probability among polyplexes and the non-adherent nature of leukemic cells. Our work aims at addressing this limitation by using a biodegradable charged polyester-based vector (BCPV) as a nanocarrier for the delivery of BCR-ABL-specific siRNA to the suspension culture of a K562 CML cell line. BCR-ABL siRNAs were encapsulated in the BCPVs by electrostatic force. Cell internalization was facilitated by the BCPV and assessed by confocal microscopy and flow cytometry. The regulation of the BCR-ABL level in K562 cells as a result of RNAi was analyzed by real-time polymerase chain reaction (RT-PCR). We observed that BCPV was able to form stable nanoplexes with siRNA molecules, even in the presence of fetal bovine serum (FBS), and successfully assisted in vitro siRNA transfection in the non-adherent K562 cells. As a consequence of downregulation of BCR-ABL, BCPV-siRNA nanoplexes inhibited cell proliferation and promoted cell apoptosis. All results were compared with a commercial transfection reagent, Lipofectamine2000™, which served as a positive control. More importantly, this class of non-viral vector exhibits biodegradable features and negligible cytotoxicity, thus providing a versatile platform to deliver siRNA to non-adherent leukemia cells with high transfection efficiency by effectively overcoming extra- and intra-cellular barriers. Due to the excellent in vitro transfection results from BCPV-siRNA, a newly developed biodegradable transfection agent, BCPV, is being probed for transfection performance in an animal model.

Hepatocytes and IL-15: a favorable microenvironment for T cell survival and CD8+ T cell differentiation.

PubMed

Correia, Margareta P; Cardoso, Elsa M; Pereira, Carlos F; Neves, Rui; Uhrberg, Markus; Arosa, Fernando A

2009-05-15

Human intrahepatic lymphocytes are enriched in CD1d-unrestricted T cells coexpressing NKR. Although the origin of this population remains controversial, it is possible to speculate that the hepatic microenvironment, namely epithelial cells or the cytokine milieu, may play a role in its shaping. IL-15 is constitutively expressed in the liver and has a key role in activation and survival of innate and tissue-associated immune cells. In this in vitro study, we examined whether hepatocyte cell lines and/or IL-15 could play a role in the generation of NK-like T cells. The results show that both HepG2 cells and a human immortalized hepatocyte cell line increase survival and drive basal proliferation of T cells. In addition, IL-15 was capable of inducing Ag-independent up-regulation of NKR, including NKG2A, Ig-like receptors, and de novo expression of CD56 and NKp46 in CD8(+)CD56(-) T cells. In conclusion, our study suggests that hepatocytes and IL-15 create a favorable microenvironment for T cells to growth and survive. It can be proposed that the increased percentage of intrahepatic nonclassical NKT cells could be in part due to a local CD8(+) T cell differentiation.

Transfected MDCK cell line with enhanced expression of CYP3A4 and P-glycoprotein as a model to study their role in drug transport and metabolism.

PubMed

Kwatra, Deep; Budda, Balasubramanyam; Vadlapudi, Aswani Dutt; Vadlapatla, Ramya Krishna; Pal, Dhananjay; Mitra, Ashim K

2012-07-02

The aim of this study was to characterize and utilize MDCK cell line expressing CYP3A4 and P-glycoprotein as an in vitro model for evaluating drug-herb and drug-drug of abuse interactions. MDCK cell line simultaneously expressing P-gp and CYP3A4 (MMC) was developed and characterized by using expression and activity studies. Cellular transport study of 200 μM cortisol was performed to determine their combined activity. The study was carried across MDCK-WT, MDCK-MDR1 and MMC cell lines. Similar studies were also carried out in the presence of 50 μM naringin and 3 μM morphine. Samples were analyzed by HPLC for drug and its CYP3A4 metabolite. PCR, qPCR and Western blot studies confirmed the enhanced expression of the proteins in the transfected cells. The Vivid CYP3A4 assay and ketoconazole inhibition studies further confirmed the presence of active protein. Apical to basal transport of cortisol was found to be 10- and 3-fold lower in MMC as compared to MDCK-WT and MDCK-MDR1 respectively. Higher amount of metabolite was formed in MMC than in MDCK-WT, indicating enhanced expression of CYP3A4. Highest cortisol metabolite formation was observed in MMC cell line due to the combined activities of CYP3A4 and P-gp. Transport of cortisol increased 5-fold in the presence of naringin in MMC and doubled in MDCK-MDR1. Cortisol transport in MMC was significantly lower than that in MDCK-WT in the presence of naringin. The permeability increased 3-fold in the presence of morphine, which is a weaker inhibitor of CYP3A4. Formation of 6β-hydroxy cortisol was found to decrease in the presence of morphine and naringin. This new model cell line with its enhanced CYP3A4 and P-gp levels in addition to short culture time can serve as an invaluable model to study drug-drug interactions. This cell line can also be used to study the combined contribution of efflux transporter and metabolizing enzymes toward drug-drug interactions.

TRANSFECTED MDCK CELL LINE WITH ENHANCED EXPRESSION OF CYP3A4 AND P-GLYCOPROTEIN AS A MODEL TO STUDY THEIR ROLE IN DRUG TRANSPORT AND METABOLISM

PubMed Central

Kwatra, Deep; Budda, Balasubramanyam; Vadlapudi, Aswani Dutt; Vadlapatla, Ramya Krishna; Pal, Dhananjay; Mitra, Ashim K.

2012-01-01

The aim of this study was to characterize and utilize MDCK cell line expressing CYP3A4 and P-glycoprotein as an in vitro model for evaluating drug-herb and drug-drugs of abuse interactions. MDCK cell line simultaneously expressing P-gp and CYP3A4 (MMC) was developed and characterized by using expression and activity studies. Cellular transport study of 200 μM cortisol was performed to determine their combined activity. The study was carried across MDCK-WT, MDCK-MDR1 and MMC cell lines. Similar studies were also carried out in the presence of 50 μM naringin and 3 μM morphine. Samples were analyzed by HPLC for drug and its CYP3A4 metabolite. PCR, qPCR and western blot studies confirmed the enhanced expression of the proteins in the transfected cells. The vivid CYP3A4 assay and ketoconazole inhibition studies further confirmed the presence of active protein. Apical to basal transport of cortisol was found to be ten and three fold lower in MMC as compared to WT and MDCKMDR1 respectively. Higher amount of metabolite was formed in MMC than in MDCK-WT indicating enhanced expression of CYP3A4. Highest cortisol metabolite formation was observed in MMC cell line due to the combined metabolic activities of CYP3A4 and P-gp. Transport of cortisol increased fivefold in presence of naringin in MMC and doubled in MDCKMDR1. Cortisol transport in MMC was significantly lower than that in WT in presence of naringin. The permeability increased three fold in presence of morphine which is a weaker inhibitor of CYP3A4. Formation of 6β-hydroxy cortisol was found to decrease in presence of morphine and naringin. This new model cell line with its enhanced CYP3A4 and P-gp levels in addition to short culture time can serve as an invaluable model to study drug-drug interactions. This cell line can also be used to study the combined contribution of efflux transporter and metabolizing enzymes towards drug-drug interactions. PMID:22676443

Significant differences in genotoxicity induced by retrovirus integration in human T cells and induced pluripotent stem cells.

PubMed

Zheng, Weiyan; Wang, Yingjia; Chang, Tammy; Huang, He; Yee, Jiing-Kuan

2013-04-25

Retrovirus is frequently used in the genetic modification of mammalian cells and the establishment of induced pluripotent stem cells (iPSCs) via cell reprogramming. Vector-induced genotoxicity could induce profound effect on the physiology and function of these stem cells and their differentiated progeny. We analyzed retrovirus-induced genotoxicity in somatic cell Jurkat and two iPSC lines. In Jurkat cells, retrovirus frequently activated host gene expression and gene activation was not dependent on the distance between the integration site and the transcription start site of the host gene. In contrast, retrovirus frequently down-regulated host gene expression in iPSCs, possibly due to the action of chromatin silencing that spreads from the provirus to the nearby host gene promoter. Our data raises the issue that some of the phenotypic variability observed among iPSC clones derived from the same parental cell line may be caused by retrovirus-induced gene expression changes rather than by the reprogramming process itself. It also underscores the importance of characterizing retrovirus integration and carrying out risk assessment of iPSCs before they can be applied in basic research and clinics. Copyright © 2013 Elsevier B.V. All rights reserved.

Proteomic Comparison and MRM-Based Comparative Analysis of Metabolites Reveal Metabolic Shift in Human Prostate Cancer Cell Lines.

PubMed

Shu, Qingbo; Cai, Tanxi; Chen, Xiulan; Zhu, Helen He; Xue, Peng; Zhu, Nali; Xie, Zhensheng; Wei, Shasha; Zhang, Qing; Niu, Lili; Gao, Wei-Qiang; Yang, Fuquan

2015-08-07

One of the major challenges in prostate cancer therapy remains the development of effective treatments for castration-resistant prostate cancer (CRPC), as the underlying mechanisms for its progression remain elusive. Previous studies showed that androgen receptor (AR) is crucially involved in regulation of metabolism in prostate cancer (PCa) cells throughout the transition from early stage, androgen-sensitive PCa to androgen-independent CRPC. AR achieves such metabolic rewiring directively either via its transcriptional activity or via interactions with AMP-activated protein kinase (AMPK). However, due to the heterogeneous expression and activity status of AR in PCa cells, it remains a challenge to investigate the links between AR status and metabolic alterations. To this end, we compared the proteomes of three pairs of androgen-sensitive (AS) and androgen-independent (AI) PCa cell lines, namely, PC3-AR(+)/PC3, 22Rv1/Du145, and LNCaP/C42B, using an iTRAQ labeling approach. Our results revealed that most of the differentially expressed proteins between each pair function in metabolism, indicating a metabolic shift between AS and AI cells, as further validated by multiple reaction monitoring (MRM)-based quantification of nucleotides and relative comparison of fatty acids between these cell lines. Furthermore, increased adenylate kinase isoenzyme 1 (AK1) in AS relative to AI cells may result in activation of AMPK, representing a major regulatory factor involved in the observed metabolic shift in PCa cells.

A Low Power Linear Phase Programmable Long Delay Circuit.

PubMed

Rodriguez-Villegas, Esther; Logesparan, Lojini; Casson, Alexander J

2014-06-01

A novel linear phase programmable delay is being proposed and implemented in a 0.35 μm CMOS process. The delay line consists of N cascaded cells, each of which delays the input signal by Td/N, where Td is the total line delay. The delay generated by each cell is programmable by changing a clock frequency and is also fully independent of the frequency of the input signal. The total delay hence depends only on the chosen clock frequency and the total number of cascaded cells. The minimum clock frequency is limited by the maximum time a voltage signal can effectively be held by an individual cell. The maximum number of cascaded cells will be limited by the effects of accumulated offset due to transistor mismatch, which eventually will affect the operating mode of the individual transistors in a cell. This latter limitation has however been dealt with in the topology by having an offset compensation mechanism that makes possible having a large number of cascaded cells and hence a long resulting delay. The delay line has been designed for scalp-based neural activity analysis that is predominantly in the sub-100 Hz frequency range. For these signals, the delay generated by a 31-cell cascade has been demonstrated to be programmable from 30 ms to 3 s. Measurement results demonstrate a 31 stage, 50 Hz bandwidth, 0.3 s delay that operates from a 1.1 V supply with power consumption of 270 nW.

Protective Role of Selenium Compounds on the Proliferation, Apoptosis, and Angiogenesis of a Canine Breast Cancer Cell Line.

PubMed

Liu, Yuzhi; Li, Wenyu; Guo, Mengyao; Li, Chengye; Qiu, Changwei

2016-01-01

We herein examined the effects of different doses, forms, and compatibilities of selenium on a canine mammary gland tumor cell line, CTM1211, and explored the related mechanisms. Three selenium compounds, sodium selenite (SSE), methylseleninic acid (MSA), and methylselenocysteine (MSC), were selected for these experiments, and cyclophosphamide (CTX) served as a positive control. In the cell viability assay, the cell viability of each group at 48/72 h decreased significantly compared with the control group (p < 0.05), and the cell viability of the CTX + MSA group was lower than that of CTX and MSA groups (p < 0.05). Moreover, the inhibitory effect of selenium on cell proliferation was time-dependent but not concentration-dependent. In the cell apoptosis assay, the apoptosis values of each group increased significantly compared with the control group, and the apoptosis values of the CTX + MSA group increased the most significantly (p < 0.01). The protein and mRNA expression levels of vascular endothelial growth factor-alpha (VEGF-alpha), angiopoietin-2 (Ang-2), and hypoxia inducible factor-1 alpha (HIF-1 alpha) were downregulated in each group, while that of phosphatase and tensin homolog (PTEN) were upregulated (p < 0.05). In conclusion, these three selenium compounds, especially MSA, could significantly inhibit the viability and growth of the CTM1211 cell line, which is partly due to the induction of apoptosis and regulation of tumor angiogenesis.

FogBank: a single cell segmentation across multiple cell lines and image modalities.

PubMed

Chalfoun, Joe; Majurski, Michael; Dima, Alden; Stuelten, Christina; Peskin, Adele; Brady, Mary

2014-12-30

Many cell lines currently used in medical research, such as cancer cells or stem cells, grow in confluent sheets or colonies. The biology of individual cells provide valuable information, thus the separation of touching cells in these microscopy images is critical for counting, identification and measurement of individual cells. Over-segmentation of single cells continues to be a major problem for methods based on morphological watershed due to the high level of noise in microscopy cell images. There is a need for a new segmentation method that is robust over a wide variety of biological images and can accurately separate individual cells even in challenging datasets such as confluent sheets or colonies. We present a new automated segmentation method called FogBank that accurately separates cells when confluent and touching each other. This technique is successfully applied to phase contrast, bright field, fluorescence microscopy and binary images. The method is based on morphological watershed principles with two new features to improve accuracy and minimize over-segmentation. First, FogBank uses histogram binning to quantize pixel intensities which minimizes the image noise that causes over-segmentation. Second, FogBank uses a geodesic distance mask derived from raw images to detect the shapes of individual cells, in contrast to the more linear cell edges that other watershed-like algorithms produce. We evaluated the segmentation accuracy against manually segmented datasets using two metrics. FogBank achieved segmentation accuracy on the order of 0.75 (1 being a perfect match). We compared our method with other available segmentation techniques in term of achieved performance over the reference data sets. FogBank outperformed all related algorithms. The accuracy has also been visually verified on data sets with 14 cell lines across 3 imaging modalities leading to 876 segmentation evaluation images. FogBank produces single cell segmentation from confluent cell sheets with high accuracy. It can be applied to microscopy images of multiple cell lines and a variety of imaging modalities. The code for the segmentation method is available as open-source and includes a Graphical User Interface for user friendly execution.

56Co-labelled radioactive Fe3O4 nanoparticles for in vitro uptake studies on Balb/3T3 and Caco-2 cell lines

NASA Astrophysics Data System (ADS)

Marmorato, P.; Simonelli, F.; Abbas, K.; Kozempel, J.; Holzwarth, U.; Franchini, F.; Ponti, J.; Gibson, N.; Rossi, F.

2011-12-01

Magnetite nanoparticles (Fe3O4 NPs) are manufactured nanomaterials increasingly used in healthcare for different medical applications ranging from diagnosis to therapy. This study deals with the irradiation of Fe3O4 NPs with a proton beam in order to produce 56Co as radiolabel and also with the possible use of nuclear techniques for the quantification of Fe3O4 NPs in biological systems. Particular attention has been focused on the size distribution (in the range of 100 nm) and the surface charge of the NPs characterizing them before and after the irradiation process in order to verify if these essential properties would be preserved during irradiation. Moreover, X-ray diffraction studies have been performed on radioactive and non-radioactive NPs, to assess if major changes in NPs structure might occur due to thermal and/or radiation effects. The radiation emitted from the radiolabels has been used to quantify the cellular uptake of the NPs in in vitro studies. As for the biological applications two cell lines have been selected: immortalized mouse fibroblast cell line (Balb/3T3) and human epithelial colorectal adenocarcinoma cell line (Caco-2). The cell uptake has been quantified by radioactivity measurements of the 56Co radioisotope performed with high resolution γ-ray spectrometry equipment. This study has showed that, under well-established irradiation conditions, Fe3O4 NPs do not undergo significant structural modifications and thus the obtained results are in line with the uptake studies carried out with the same non-radioactive nanomaterials (NMs). Therefore, the radiolabelling method can be fruitfully applied to uptake studies because of the low-level exposure where higher sensitivity is required.

HD iPSC-derived neural progenitors accumulate in culture and are susceptible to BDNF withdrawal due to glutamate toxicity.

PubMed

Mattis, Virginia B; Tom, Colton; Akimov, Sergey; Saeedian, Jasmine; Østergaard, Michael E; Southwell, Amber L; Doty, Crystal N; Ornelas, Loren; Sahabian, Anais; Lenaeus, Lindsay; Mandefro, Berhan; Sareen, Dhruv; Arjomand, Jamshid; Hayden, Michael R; Ross, Christopher A; Svendsen, Clive N

2015-06-01

Huntington's disease (HD) is a fatal neurodegenerative disease, caused by expansion of polyglutamine repeats in the Huntingtin gene, with longer expansions leading to earlier ages of onset. The HD iPSC Consortium has recently reported a new in vitro model of HD based on the generation of induced pluripotent stem cells (iPSCs) from HD patients and controls. The current study has furthered the disease in a dish model of HD by generating new non-integrating HD and control iPSC lines. Both HD and control iPSC lines can be efficiently differentiated into neurons/glia; however, the HD-derived cells maintained a significantly greater number of nestin-expressing neural progenitor cells compared with control cells. This cell population showed enhanced vulnerability to brain-derived neurotrophic factor (BDNF) withdrawal in the juvenile-onset HD (JHD) lines, which appeared to be CAG repeat-dependent and mediated by the loss of signaling from the TrkB receptor. It was postulated that this increased death following BDNF withdrawal may be due to glutamate toxicity, as the N-methyl-d-aspartate (NMDA) receptor subunit NR2B was up-regulated in the cultures. Indeed, blocking glutamate signaling, not just through the NMDA but also mGlu and AMPA/Kainate receptors, completely reversed the cell death phenotype. This study suggests that the pathogenesis of JHD may involve in part a population of 'persistent' neural progenitors that are selectively vulnerable to BDNF withdrawal. Similar results were seen in adult hippocampal-derived neural progenitors isolated from the BACHD model mouse. Together, these results provide important insight into HD mechanisms at early developmental time points, which may suggest novel approaches to HD therapeutics. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Role of epigenetic factors in the selection of the alternative splicing isoforms of human KRAS in colorectal cancer cell lines.

PubMed

Riffo-Campos, Ángela L; Gimeno-Valiente, Francisco; Rodríguez, Fernanda M; Cervantes, Andrés; López-Rodas, Gerardo; Franco, Luis; Castillo, Josefa

2018-04-17

Mutation-driven activation of KRAS is crucial to cancer development. The human gene yields four mRNA splicing isoforms, 4A and 4B being translated to protein. Their different properties and oncogenic potential have been studied, but the mechanisms deciding the ratio 4A/4B are not known. To address this issue, the expression of the four KRAS isoforms was determined in 9 human colorectal cancer cell lines. HCT116 and SW48 were further selected because they present the highest difference in the ratio 4A/4B (twice as much in HCT116 than in SW48). Chromatin structure was analysed at the exon 4A, characteristic of isoform 4A, at its intronic borders and at the two flanking exons. The low nucleosome occupancy at exon 4A in both cell lines may result in a fast transcriptional rate, which would explain the general lower abundance of isoform 4A, also found in cells and tissues by other authors, but due to its similarity between both cell lines, chromatin structure does not influence alternative splicing. DNA methylation downstream exon 4A significantly differs in HCT116 and SW48 cells, but the CCCTC-binding factor, which affects the processivity of RNA polymerase and the alternative splicing, does not bind the differentially methylated sequences. Quantitative epigenetic analysis at mononucleosomal level revealed significant differences between both cell lines in H3K4me3, H3K27me3, H3K36me3, H3K9ac, H3K27ac and H4K20me1, and the inhibition of some histone-modifying enzymes alters the ratio 4A/4B. It can be concluded that the epigenetic modification of histones has an influence on the selection of isoforms 4A and 4B.

In-situ gamma-ray assay of the east cell line in the 235-F Plutonium fuel form facility

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

Diprete, D.

On September 17th -19th , 2013, scientists from SRNL took a series of in-situ gamma-ray measurements in the maintenance trench beneath Cells 1-5 on the east line of the PuFF facility using a well-collimated, high-purity germanium detector. The cell interiors were assayed along with the furnaces and storage coolers that protrude beneath the cells. The detector efficiency was estimated using a combination of MCNP simulations and empirical measurements. Data analysis was performed using three gamma-rays emitted by Pu-238 (99.85 keV, 152.7 keV, and 766.4 keV) providing three independent estimates of the mass of Pu-238 holdup in each of the cells.more » The weighted mean of these three results was used as the best estimate of Pu-238 holdup in the East Cell Line of PuFF. The results of the assay measurements are found in the table on the following page along with the results from the scoping assay performed in 2006. All uncertainties in this table (as well as the rest of the report) are reported at 1σ. Summing the assay results and treating MDAs as M 238Pu= 0 ± MDA, the total holdup in the East Cell Line was 240 ± 40 grams. This result is 100 grams lower than the previous estimate, a 0.55σ difference. The uncertainty in the Pu-238 holdup is also reduced substantially relative to the 2006 scoping assay. However, the current assay results are in agreement with the 2006 scoping assay results due to the large uncertainty associated with the 2006 scoping assays. The current assay results support the conclusion that the 2006 results bound the Pu-238 mass in Cells 1-5. These results should be considered preliminary since additional measurements of the East Cell line are scheduled for 2017 and 2018. Those measurements will provide detailed information about the distribution of Pu-238 in the cells to be used to refine the results of the current assay.« less

OsDOG, a gibberellin-induced A20/AN1 zinc-finger protein, negatively regulates gibberellin-mediated cell elongation in rice.

PubMed

Liu, Yaju; Xu, Yunyuan; Xiao, Jun; Ma, Qibin; Li, Dan; Xue, Zhen; Chong, Kang

2011-07-01

The A20/AN1 zinc-finger proteins (ZFPs) play pivotal roles in animal immune responses and plant stress responses. From previous gibberellin (GA) microarray data and A20/AN1 ZFP family member association, we chose Oryza sativa dwarf rice with overexpression of gibberellin-induced gene (OsDOG) to examine its function in the GA pathway. OsDOG was induced by gibberellic acid (GA(3)) and repressed by the GA-synthesis inhibitor paclobutrazol. Different transgenic lines with constitutive expression of OsDOG showed dwarf phenotypes due to deficiency of cell elongation. Additional GA(1) and real-time PCR quantitative assay analyses confirmed that the decrease of GA(1) in the overexpression lines resulted from reduced expression of GA3ox2 and enhanced expression of GA2ox1 and GA2ox3. Adding exogenous GA rescued the constitutive expression phenotypes of the transgenic lines. OsDOG has a novel function in regulating GA homeostasis and in negative maintenance of plant cell elongation in rice. Copyright © 2011 Elsevier GmbH. All rights reserved.

Nuclear Motility in Glioma Cells Reveals a Cell-Line Dependent Role of Various Cytoskeletal Components

PubMed Central

Kiss, Alexa; Horvath, Peter; Rothballer, Andrea; Kutay, Ulrike; Csucs, Gabor

2014-01-01

Nuclear migration is a general term for the movement of the nucleus towards a specific site in the cell. These movements are involved in a number of fundamental biological processes, such as fertilization, cell division, and embryonic development. Despite of its importance, the mechanism of nuclear migration is still poorly understood in mammalian cells. In order to shed light on the mechanical processes underlying nuclear movements, we adapted a micro-patterning based assay. C6 rat and U87 human glioma cells seeded on fibronectin patterns - thereby forced into a bipolar morphology - displayed oscillatory movements of the nucleus or the whole cell, respectively. We found that both the actomyosin system and microtubules are involved in the nuclear/cellular movements of both cell lines, but their contributions are cell-/migration-type specific. Dynein activity was necessary for nuclear migration of C6 cells but active myosin-II was dispensable. On the other hand, coupled nuclear and cellular movements of U87 cells were driven by actomyosin contraction. We explain these cell-line dependent effects by the intrinsic differences in the overall mechanical tension due to the various cytoskeletal elements inside the cell. Our observations showed that the movements of the nucleus and the centrosome are strongly correlated and display large variation, indicating a tight but flexible coupling between them. The data also indicate that the forces responsible for nuclear movements are not acting directly via the centrosome. Based on our observations, we propose a new model for nuclear oscillations in C6 cells in which dynein and microtubule dynamics are the main drivers of nuclear movements. This mechanism is similar to the meiotic nuclear oscillations of Schizosaccharomyces pombe and may be evolutionary conserved. PMID:24691067

JNK1 Inhibition Attenuates Hypoxia-Induced Autophagy and Sensitizes to Chemotherapy.

PubMed

Vasilevskaya, Irina A; Selvakumaran, Muthu; Roberts, David; O'Dwyer, Peter J

2016-08-01

Inhibition of hypoxia-induced stress signaling through JNK potentiates the effects of oxaliplatin. The JNK pathway plays a role in both autophagy and apoptosis; therefore, it was determined how much of the effect of JNK inhibition on oxaliplatin sensitivity is dependent on its effect on autophagy. We studied the impact of JNK isoform downregulation in the HT29 colon adenocarcinoma cell line on hypoxia- and oxaliplatin-induced responses. Electron microscopic analyses demonstrated that both oxaliplatin- and hypoxia-induced formations of autophagosomes were reduced significantly in HT29 cells treated with the JNK inhibitor SP600125. The role of specific JNK isoforms was defined using HT29-derived cell lines stably expressing dominant-negative constructs for JNK1 and JNK2 (HTJ1.3 and HTJ2.2, respectively). These cell lines demonstrated that functional JNK1 is required for hypoxia-induced autophagy and that JNK2 does not substitute for it. Inhibition of autophagy in HTJ1.3 cells also coincided with enhancement of intrinsic apoptosis. Analysis of Bcl2-family proteins revealed hyperphosphorylation of Bcl-XL in the HTJ1.3 cell line, but this did not lead to the expected dissociation from Beclin 1. Consistent with this, knockdown of Bcl-XL in HT29 cells did not significantly affect the induction of autophagy, but abrogated hypoxic resistance to oxaliplatin due to the faster and more robust activation of apoptosis. These data suggest that balance between autophagy and apoptosis is shifted toward apoptosis by downregulation of JNK1, contributing to oxaliplatin sensitization. These findings further support the investigation of JNK inhibition in colorectal cancer treatment. Mol Cancer Res; 14(8); 753-63. ©2016 AACR. ©2016 American Association for Cancer Research.

Analysis of the Glucocorticoid Receptor in Spontaneous and Drug-induced Steroid Resistant Mutants Isolated from the Human Leukemic Cell Line CEM-C7

DTIC Science & Technology

1991-01-04

be due to defects in GR function. However, the loss of GR function in x" S49 cells is paralleled by the loss of GR mRNA which is not seen in x’ OEM ...in the protein transcription factor IIIA from Xepopus oocytes. EMBO J. 4: 1609-1614. Milgrom. E.; Atger, M.; Baulieu, E.-E. 1973 Acidophilic

Magnolol inhibits growth of gallbladder cancer cells through the p53 pathway

PubMed Central

Li, Maolan; Zhang, Fei; Wang, Xu’an; Wu, Xiangsong; Zhang, Bingtai; Zhang, Ning; Wu, Wenguang; Wang, Zheng; Weng, Hao; Liu, Shibo; Gao, Guofeng; Mu, Jiasheng; Shu, Yijun; Bao, Runfa; Cao, Yang; Lu, Jianhua; Gu, Jun; Zhu, Jian; Liu, Yingbin

2015-01-01

Magnolol, the major active compound found in Magnolia officinalis has a wide range of clinical applications due to its anti-inflammation and anti-oxidation effects. This study investigated the effects of magnolol on the growth of human gallbladder carcinoma (GBC) cell lines. The results indicated that magnolol could significantly inhibit the growth of GBC cell lines in a dose- and time-dependent manner. Magnolol also blocked cell cycle progression at G0/G1 phase and induced mitochondrial-related apoptosis by upregulating p53 and p21 protein levels and by downregulating cyclin D1, CDC25A, and Cdk2 protein levels. When cells were pretreated with a p53 inhibitor (pifithrin-a), followed by magnolol treatment, pifithrin-a blocked magnolol-induced apoptosis and G0/G1 arrest. In vivo, magnolol suppressed tumor growth and activated the same mechanisms as were activated in vitro. In conclusion, our study is the first to report that magnolol has an inhibitory effect on the growth of GBC cells and that this compound may have potential as a novel therapeutic agent for the treatment of GBC. PMID:26250568

Human Corneal Limbal-Epithelial Cell Response to Varying Silk Film Geometric Topography In Vitro

PubMed Central

Lawrence, Brian D.; Pan, Zhi; Liu, Aihong; Kaplan, David L.; Rosenblatt, Mark I.

2012-01-01

Silk fibroin films are a promising class of biomaterials that have a number of advantages for use in ophthalmic applications due to their transparent nature, mechanical properties and minimal inflammatory response upon implantation. Freestanding silk films with parallel line and concentric ring topographies were generated for in vitro characterization of human corneal limbal-epithelial (HCLE) cell response upon differing geometric patterned surfaces. Results indicated that silk film topography significantly affected initial HCLE culture substrate attachment, cellular alignment, cell-to-cell contact formation, actin cytoskeleton alignment, and focal adhesion (FA) localization. Most notably, parallel line patterned surfaces displayed a 36%–54% increase on average in initial cell attachment, which corresponded to an over 2-fold increase in FA localization when compared to other silk film surfaces and controls. In addition, distinct localization of FA formation was observed along the edges for all patterned silk film topographies. In conclusion, silk film feature topography appears to help direct corneal epithelial cell response and cytoskeleton development, especially in regards to FA distribution, in vitro. PMID:22705042

Exploiting persistent infection for selection of bovine herpesvirus 4 recombinants.

PubMed

Donofrio, G; Martignani, E; Cavirani, S; Flammini, C F

2005-09-01

Bovine herpesvirus 4 (BoHV-4) is a gamma-herpesvirus with no clear disease association, and due to its biological characteristics, has been suggested as a gene delivery vector. It was demonstrated previously that recombinant BoHV-4 carrying a neomycin-resistance gene was able to infect a human rhabdomyosarcoma cell line (RD-4), resulting in no detectable cytopathic effect (CPE) and allowing selection of G418-resistant persistently-infected cells containing circular episomal viral DNA [Donofrio, G., Cavirani, S., van Santen, V.L., 2000a. Establishment of a cell line persistently infected with recombinant BoHV-4. J. Gen. Virol. 81, 1807-1814.]. Those cells produce infectious virus and infection is predominantly non-permissive and non-cytopathic. Starting from these results, the ability of RD-4 cells to sustain persistent infection was combined with positive selection activity conferred by the neomycin-expression cassette insert, as an easier way to select recombinants of BoHV-4 following homologous recombination in permissive cells. A tool for selecting BoHV-4 recombinants was developed by drug positive selection.

Interstitial telomeric repeats are not preferentially involved in radiation-induced chromosome aberrations in human cells.

PubMed

Desmaze, C; Pirzio, L M; Blaise, R; Mondello, C; Giulotto, E; Murnane, J P; Sabatier, L

2004-01-01

Telomeric repeat sequences, located at the end of eukaryotic chromosomes, have been detected at intrachromosomal locations in many species. Large blocks of telomeric sequences are located near the centromeres in hamster cells, and have been reported to break spontaneously or after exposure to ionizing radiation, leading to chromosome aberrations. In human cells, interstitial telomeric sequences (ITS) can be composed of short tracts of telomeric repeats (less than twenty), or of longer stretches of exact and degenerated hexanucleotides, mainly localized at subtelomeres. In this paper, we analyzed the radiation sensitivity of a naturally occurring short ITS localized in 2q31 and we found that this region is not a hot spot of radiation-induced chromosome breaks. We then selected a human cell line in which approximately 800 bp of telomeric DNA had been introduced by transfection into an internal euchromatic chromosomal region in chromosome 4q. In parallel, a cell line containing the plasmid without telomeric sequences was also analyzed. Both regions containing the transfected plasmids showed a higher frequency of radiation-induced breaks than expected, indicating that the instability of the regions containing the transfected sequences is not due to the presence of telomeric sequences. Taken together, our data show that ITS themselves do not enhance the formation of radiation-induced chromosome rearrangements in these human cell lines. Copyright 2003 S. Karger AG, Basel

MicroRNA-211 Regulates Oxidative Phosphorylation and Energy Metabolism in Human Vitiligo.

PubMed

Sahoo, Anupama; Lee, Bongyong; Boniface, Katia; Seneschal, Julien; Sahoo, Sanjaya K; Seki, Tatsuya; Wang, Chunyan; Das, Soumen; Han, Xianlin; Steppie, Michael; Seal, Sudipta; Taieb, Alain; Perera, Ranjan J

2017-09-01

Vitiligo is a common chronic skin disorder characterized by loss of epidermal melanocytes and progressive depigmentation. Vitiligo has complex immune, genetic, environmental, and biochemical causes, but the exact molecular mechanisms of vitiligo development and progression, particularly those related to metabolic control, are poorly understood. In this study we characterized the human vitiligo cell line PIG3V and the normal human melanocyte line HEM-l by RNA sequencing, targeted metabolomics, and shotgun lipidomics. Melanocyte-enriched microRNA-211, a known metabolic switch in nonpigmented melanoma cells, was severely down-regulated in vitiligo cell line PIG3V and skin biopsy samples from vitiligo patients, whereas its predicted targets PPARGC1A, RRM2, and TAOK1 were reciprocally up-regulated. microRNA-211 binds to PGC1-α 3' untranslated region locus and represses it. Although mitochondrial numbers were constant, mitochondrial complexes I, II, and IV and respiratory responses were defective in vitiligo cells. Nanoparticle-coated microRNA-211 partially augmented the oxygen consumption rate in PIG3V cells. The lower oxygen consumption rate, changes in lipid and metabolite profiles, and increased reactive oxygen species production observed in vitiligo cells appear to be partly due to abnormal regulation of microRNA-211 and its target genes. These genes represent potential biomarkers and therapeutic targets in human vitiligo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Bog bilberry (Vaccinium uliginosum L.) extract reduces cultured Hep-G2, Caco-2, and 3T3-L1 cell viability, affects cell cycle progression, and has variable effects on membrane permeability.

PubMed

Liu, Jia; Zhang, Wei; Jing, Hao; Popovich, David G

2010-04-01

Bog bilberry (Vaccinium uliginosum L.) is a blue-pigmented edible berry related to bilberry (Vaccinium myrtillus L.) and the common blueberry (Vaccinium corymbosum). The objective of this study was to investigate the effect of a bog bilberry anthocyanin extract (BBAE) on cell growth, membrane permeability, and cell cycle of 2 malignant cancer cell lines, Caco-2 and Hep-G2, and a nonmalignant murine 3T3-L1 cell line. BBAE contained 3 identified anthocyanins. The most abundant anthocyanin was cyanidin-3-glucoside (140.9 +/- 2.6 microg/mg of dry weight), followed by malvidin-3-glucoside (10.3 +/- 0.3 microg/mg) and malvidin-3-galactoside (8.1 +/- 0.4 microg/mg). Hep-G2 LC50 was calculated to be 0.563 +/- 0.04 mg/mL, Caco-2 LC50 was 0.390 +/- 0.30 mg/mL and 0.214 +/- 0.02 mg/mL for 3T3-L1 cells. LDH release, a marker of membrane permeability, was significantly increased in Hep-G2 cells and Caco-2 cells after 48 and 72 h compared to 24 h. The increase was 21% at 48 h and 57% at 72 h in Caco-2 cells and 66% and 139% in Hep-G2 cells compared to 24 h. However, 3T3-L1 cells showed an unexpected significant lower LDH activity (P < or = 0.05) after 72 h of exposure corresponding to a 21% reduction in LDH release. BBAE treatment increased sub-G1 in all 3 cell lines without influencing cells in the G2/M phase. BBAE treatment reduced the growth and increased the accumulation of sub-G1 cells in 2 malignant and 1 nonmalignant cell line; however, the effect on membrane permeability differs considerably between the malignant and nonmalignant cells and may in part be due to differences in cellular membrane composition.

Clozapine protects PC-12 cells from death due to oxidative stress induced by hydrogen peroxide via a cell-type specific mechanism involving inhibition of extracellular signal-regulated kinase phosphorylation.

PubMed

Magliaro, Brian C; Saldanha, Colin J

2009-08-04

Recent evidence suggests that some atypical antipsychotic drugs may protect against oxidative stress and consequent neurodegeneration by mechanisms that remain unclear. Using the neuron-like rat pheochromocytoma (PC-12) cell line, Clozapine and N-desmethylclozapine were tested for their ability to protect against cell death due to oxidative stress induced by hydrogen peroxide (H(2)O(2)). These drugs demonstrated significant protection of PC-12 cells, as measured by both the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) and Alamar Blue cell viability assays. However, neither viability assay detected a protective effect of Clozapine on human embryonic kidney (HEK293), rat primary cortical neurons, or human neuroblastoma (SH-SY5Y) exposed to H(2)O(2) treatment. The mechanism of protection involves a PC-12 cell-specific differential response to H(2)O(2) treatment vs. the other cell lines. Pre-treatment with 250 microM or 125 microM diethyldithiocarbamate (DETC), a superoxide dismutase (SOD) inhibitor, unexpectedly showed protection of the PC-12 cells from H(2)O(2) treatment. Western blots revealed that Clozapine, N-desmethylclozapine, and DETC reduce the phosphorylation of extracellular signal-regulated kinase (ERK) that is caused by H(2)O(2) exposure in PC-12 cells. In both HEK293 and SH-SY5Y cells, H(2)O(2) exposure did not increase ERK phosphorylation over control, demonstrating a different response to H(2)O(2) vs. PC-12 cells, and explaining why Clozapine could not protect these cells. Also, U0126, a specific MEK inhibitor, was able to protect PC-12 cells from H(2)O(2) exposure, showing that inhibiting ERK phosphorylation is sufficient to provide protection. Cumulatively, these results indicate that Clozapine, N-desmethylclozapine, DETC, and U0126 protect PC-12 cells by blocking the cell-type specific H(2)O(2) induced increase in ERK phosphorylation.

Folic acid induces cell type-specific changes in the transcriptome of breast cancer cell lines: a proof-of-concept study.

PubMed

Price, R Jordan; Lillycrop, Karen A; Burdge, Graham C

2016-01-01

The effect of folic acid (FA) on breast cancer (BC) risk is uncertain. We hypothesised that this uncertainty may be due, in part, to differential effects of FA between BC cells with different phenotypes. To test this we investigated the effect of treatment with FA concentrations within the range of unmetabolised FA reported in humans on the expression of the transcriptome of non-transformed (MCF10A) and cancerous (MCF7 and Hs578T) BC cells. The total number of transcripts altered was: MCF10A, seventy-five (seventy up-regulated); MCF7, twenty-four (fourteen up-regulated); and Hs578T, 328 (156 up-regulated). Only the cancer-associated gene TAGLN was altered by FA in all three cell lines. In MCF10A and Hs578T cells, FA treatment decreased pathways associated with apoptosis, cell death and senescence, but increased those associated with cell proliferation. The folate transporters SLC19A1, SLC46A1 and FOLR1 were differentially expressed between cell lines tested. However, the level of expression was not altered by FA treatment. These findings suggest that physiological concentrations of FA can induce cell type-specific changes in gene regulation in a manner that is consistent with proliferative phenotype. This has implications for understanding the role of FA in BC risk. In addition, these findings support the suggestion that differences in gene expression induced by FA may involve differential activities of folate transporters. Together these findings indicate the need for further studies of the effect of FA on BC.

Ultrasound-mediated structural changes in cells revealed by FTIR spectroscopy: a contribution to the optimization of gene and drug delivery.

PubMed

Grimaldi, Paola; Di Giambattista, Lucia; Giordani, Serena; Udroiu, Ion; Pozzi, Deleana; Gaudenzi, Silvia; Bedini, Angelico; Giliberti, Claudia; Palomba, Raffaele; Castellano, Agostina Congiu

2011-12-15

Ultrasound effects on biological samples are gaining a growing interest concerning in particular, the intracellular delivery of drugs and genes in a safe and in a efficient way. Future progress in this field will require a better understanding of how ultrasound and acoustic cavitation affect the biological system properties. The morphological changes of cells due to ultrasound (US) exposure have been extensively studied, while little attention has been given to the cells structural changes. We have exposed two different cell lines to 1 MHz frequency ultrasound currently used in therapy, Jurkat T-lymphocytes and NIH-3T3 fibroblasts, both employed as models respectively in the apoptosis and in the gene therapy studies. The Fourier Transform Infrared (FTIR) Spectroscopy was used as probe to reveal the structural changes in particular molecular groups belonging to the main biological systems. The genotoxic damage of cells exposed to ultrasound was ascertained by the Cytokinesis-Block Micronucleus (CBMN) assay. The FTIR spectroscopy results, combined with multivariate statistical analysis, regarding all cellular components (lipids, proteins, nucleic acids) of the two cell lines, show that Jurkat cells are more sensitive to therapeutic ultrasound in the lipid and protein regions, whereas the NIH-3T3 cells are more sensitive in the nucleic acids region; a meaningful genotoxic effect is present in both cell lines only for long sonication times while in the Jurkat cells also a significant cytotoxic effect is revealed for long times of exposure to ultrasound. Copyright © 2011 Elsevier B.V. All rights reserved.

In vitro antiplasmodial and cytotoxic activities of sesquiterpene lactones from Vernonia fimbrillifera Less. (Asteraceae).

PubMed

Bordignon, Annélise; Frédérich, Michel; Ledoux, Allison; Campos, Pierre-Eric; Clerc, Patricia; Hermann, Thomas; Quetin-Leclercq, Joëlle; Cieckiewicz, Ewa

2018-06-01

Due to the in vitro antiplasmodial activity of leaf extracts from Vernonia fimbrillifera Less. (Asteraceae), a bioactivity-guided fractionation was carried out. Three sesquiterpene lactones were isolated, namely 8-(4'-hydroxymethacrylate)-dehydromelitensin (1), onopordopicrin (2) and 8α-[4'-hydroxymethacryloyloxy]-4-epi-sonchucarpolide (3). Their structures were elucidated by spectroscopic methods (1D and 2D NMR and MS analyses) and by comparison with published data. The isolated compounds exhibited antiplasmodial activity with IC 50 values ≤ 5 μg/mL. Cytotoxicity of the compounds against a human cancer cell line (HeLa) and a mouse lung epithelial cell line (MLE12) was assessed to determine selectivity. Compound 3 displayed promising selective antiplasmodial activity (SI > 10).

CLO: The cell line ontology

PubMed Central

2014-01-01

Background Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO consortium, new cell line cells, upper level alignment with the Cell Ontology (CL) and the Ontology for Biomedical Investigation, and logical extensions. Construction and content Collaboration among the CLO, CL, and OBI has established consensus definitions of cell line-specific terms such as ‘cell line’, ‘cell line cell’, ‘cell line culturing’, and ‘mortal’ vs. ‘immortal cell line cell’. A cell line is a genetically stable cultured cell population that contains individual cell line cells. The hierarchical structure of the CLO is built based on the hierarchy of the in vivo cell types defined in CL and tissue types (from which cell line cells are derived) defined in the UBERON cross-species anatomy ontology. The new hierarchical structure makes it easier to browse, query, and perform automated classification. We have recently added classes representing more than 2,000 cell line cells from the RIKEN BRC Cell Bank to CLO. Overall, the CLO now contains ~38,000 classes of specific cell line cells derived from over 200 in vivo cell types from various organisms. Utility and discussion The CLO has been applied to different biomedical research studies. Example case studies include annotation and analysis of EBI ArrayExpress data, bioassays, and host-vaccine/pathogen interaction. CLO’s utility goes beyond a catalogue of cell line types. The alignment of the CLO with related ontologies combined with the use of ontological reasoners will support sophisticated inferencing to advance translational informatics development. PMID:25852852

Enhanced Cell-Specific Ablation in Zebrafish Using a Triple Mutant of Escherichia Coli Nitroreductase

PubMed Central

Mathias, Jonathan R.; Zhang, Zhanying; Saxena, Meera T.

2014-01-01

Abstract Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology. PMID:24428354

Enhanced cell-specific ablation in zebrafish using a triple mutant of Escherichia coli nitroreductase.

PubMed

Mathias, Jonathan R; Zhang, Zhanying; Saxena, Meera T; Mumm, Jeff S

2014-04-01

Transgenic expression of bacterial nitroreductase (NTR) facilitates chemically-inducible targeted cell ablation. In zebrafish, the NTR system enables studies of cell function and cellular regeneration. Metronidazole (MTZ) has become the most commonly used prodrug substrate for eliciting cell loss in NTR-expressing transgenic zebrafish due to the cell-specific nature of its cytotoxic derivatives. Unfortunately, MTZ treatments required for effective cell ablation border toxic effects, and, thus, likely incur undesirable nonspecific effects. Here, we tested whether a triple mutant variant of NTR, previously shown to display improved activity in bacterial assays, can solve this issue by promoting cell ablation in zebrafish using reduced prodrug treatment regimens. We generated several complementary transgenic zebrafish lines expressing either wild-type or mutant NTR (mutNTR) in specific neural cell types, and assayed prodrug-induced cell ablation kinetics using confocal time series imaging and plate reader-based quantification of fluorescent reporters expressed in targeted cell types. The results show that cell ablation can be achieved in mutNTR expressing transgenic lines with markedly shortened prodrug exposure times and/or at lower prodrug concentrations. The mutNTR variant characterized here can circumvent problematic nonspecific/toxic effects arising from low prodrug conversion efficiency, thus increasing the effectiveness and versatility of this selective cell ablation methodology.

Expression of WNT genes in cervical cancer-derived cells: Implication of WNT7A in cell proliferation and migration.

PubMed

Ramos-Solano, Moisés; Meza-Canales, Ivan D; Torres-Reyes, Luis A; Alvarez-Zavala, Monserrat; Alvarado-Ruíz, Liliana; Rincon-Orozco, Bladimiro; Garcia-Chagollan, Mariel; Ochoa-Hernández, Alejandra B; Ortiz-Lazareno, Pablo C; Rösl, Frank; Gariglio, Patricio; Jave-Suárez, Luis F; Aguilar-Lemarroy, Adriana

2015-07-01

According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviral gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration. Copyright © 2015 Elsevier Inc. All rights reserved.

Mitochondrial fluctuations as a measure of active biomechanical properties of mammalian cells

NASA Astrophysics Data System (ADS)

Xu, Wenlong; Alizadeh, Elaheh; Castle, Jordan; Prasad, Ashok

A single-cell assay of mechanical properties would give significant insights into cellular processes. Force spectrum microscopy is one such technique, which involves both active and passive particle tracking microrheology on the same cells. Since active microrheology requires expensive instruments, it is of great interest to develop simpler alternatives. Here we study an alternative using endogenous mitochondrial fluctuations, rather than fluorescent beads, in particle tracking microrheology. Mitochondria of the C3H-10T1/2 cell line are labeled and tracked using confocal microscopy, their mean square displacement (MSD) measured, and mechanical parameters calculated. Active fluctuations are distinguished from passive fluctuations by treatment with ATP synthesis inhibitors. We find that the MSD of mitochondria resembles that of particles in viscoelastic media. However, comparisons of MSD between controls and cells disrupted in the actin or microtubule network showed surprisingly small effects, while ATP-depleted cells showed significantly decreased MSD, and characteristics of thermally driven fluctuations. Both active and ATP-depleted parameters showed heterogeneity among cells and between cell lines. This method is potentially very useful due to its simplicity. We gratefully acknowledge support from NSF CAREER Grant PHY-1151454 awarded to Ashok Prasad.

Inheritance of gene density–related higher order chromatin arrangements in normal and tumor cell nuclei

PubMed Central

Cremer, Marion; Küpper, Katrin; Wagler, Babett; Wizelman, Leah; Hase, Johann v.; Weiland, Yanina; Kreja, Ludwika; Diebold, Joachim; Speicher, Michael R.; Cremer, Thomas

2003-01-01

A gene density–related difference in the radial arrangement of chromosome territories (CTs) was previously described for human lymphocyte nuclei with gene-poor CT #18 located toward the nuclear periphery and gene-dense CT #19 in the nuclear interior (Croft, J.A., J.M. Bridger, S. Boyle, P. Perry, P. Teague, and W.A. Bickmore. 1999. J. Cell Biol. 145:1119–1131). Here, we analyzed the radial distribution of chromosome 18 and 19 chromatin in six normal cell types and in eight tumor cell lines, some of them with imbalances and rearrangements of the two chromosomes. Our findings demonstrate that a significant difference in the radial distribution of #18 and #19 chromatin is a common feature of higher order chromatin architecture in both normal and malignant cell types. However, in seven of eight tumor cell lines, the difference was less pronounced compared with normal cell nuclei due to a higher fraction of nuclei showing an inverted CT position, i.e., a CT #18 located more internally than a CT #19. This observation emphasizes a partial loss of radial chromatin order in tumor cell nuclei. PMID:12952935

MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells | Office of Cancer Genomics

Cancer.gov

The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor suppressor gene, CDKN2A.

Loss of PTEN as a Predictive Biomarker of Response to Lithium Chloride, A Potential Targeted Treatment for Breast Cancer

DTIC Science & Technology

2012-06-01

infected cells, we were unable to produce HCC712 and HCC1187 cell lines with knocked out PTEN. We hypothesize that this is due to the high level of...Growth Factor Receptor in MCF-10A human breast epithelial cells. Western blot demonstrating levels of total EGFR in parental MCF-10A, and three stably...overexpression of EGFR. We performed western blot analyses to determine the degree of MAPK and PI3K pathway activation by comparing relative levels of

The participation of singlet oxygen in a photocitotoxicity of extract from amazon plant to cancer cells

NASA Astrophysics Data System (ADS)

Tcibulnikova, Anna V.; Degterev, Igor A.; Bryukhanov, Valery V.; Roberto, Mantuanelly M.; Campos Pereira, F. D.; Marin-Morales, M. A.; Slezhkin, Vasily A.; Samusev, Ilya G.

2018-01-01

We have been searching for new photosensitizers (PS) for photodynamic therapy (PDT) of cancer based on extracts from Amazonian plants since 2009. In this paper, we demonstrate that, under certain conditions, the extract from fruits of the Amazonian palm Euterpe oleraceae (popular name Açaí) can serve as a PS for PDT treatment of murine breast cancer cells (4T1 cell line). We have been first to show directly that the photodynamic effect of plant PS is due to singlet oxygen.

Genetic engineering of CHO cells for viral resistance to minute virus of mice.

PubMed

Mascarenhas, Joaquina X; Korokhov, Nikolay; Burger, Lisa; Kassim, Ademola; Tuter, Jason; Miller, Daniel; Borgschulte, Trissa; George, Henry J; Chang, Audrey; Pintel, David J; Onions, David; Kayser, Kevin J

2017-03-01

Contamination by the parvovirus minute virus of mice (MVM) remains a challenge in Chinese hamster ovary (CHO) biopharmaceutical production processes. Although infrequent, infection of a bioreactor can be catastrophic for a manufacturer, can impact patient drug supply and safety, and can have regulatory implications. We evaluated engineering a CHO parental cell line (CHOZN ® GS -/- ) to create a new host cell line that is resistant to MVM infection by modifying the major receptors used by the virus to enter cells. Attachment to a cell surface receptor is a key first step in the infection cycle for many viruses. While the exact functional receptor for MVM binding to CHO cell surface is unknown, sialic acid on the cell surface has been implicated. In this work, we used the zinc finger nuclease gene editing technology to validate the role of sialic acid on the cell surface in the binding and internalization of the MVM virus. Our approach was to systematically mutate genes involved in cell surface sialylation and then challenge each cell line for their ability to resist viral entry and propagation. To test the importance of sialylation, the following genes were knocked out: the CMP-sialic acid transporter, solute carrier family 35A1 (Slc35a1), the core 1-β-1,3-galactosyltransferase-1 specific chaperone (Cosmc), and mannosyl (α-1,3-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase (Mgat1) as well as members of the sialyltransferase family. Slc35a1 is responsible for transporting sialic acid into the Golgi. Knocking out function of this gene in a cell results in asialylated glycan structures, thus eliminating the ability of MVM to bind to and enter the cell. The complete absence of sialic acid on the Slc35a1 knockout cell line led to complete resistance to MVM infection. The Cosmc and Mgat1 knockouts also show significant inhibition of infection likely due to their effect on decreasing cell surface sialic acid. Previously in vitro glycan analysis has been used to elucidate the precise sialic acid structures required for MVM binding and internalization. In this work, we performed the sequential knockout of various sialyltransferases that add terminal sialic acid to glycans with different linkage specificities. Cell lines with modifications of the various genes included in this study resulted in varying effects on MVM infection expanding on the knowledge of MVM receptors. MVM resistant host cell lines were also tested for the production of model recombinant proteins. Our data demonstrate that resistance against the MVM virus can be incorporated into CHO production cell lines, adding another level of defense against the devastating financial consequences of MVM infection without compromising recombinant protein yield or quality. Biotechnol. Bioeng. 2017;114: 576-588. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Hematopoietic stem cell transplantation for acquired aplastic anemia

PubMed Central

Georges, George E.; Storb, Rainer

2016-01-01

Purpose of review There has been steady improvement in outcomes with allogeneic bone marrow transplantation (BMT) for severe aplastic anemia (SAA), due to progress in optimization of the conditioning regimens, donor hematopoietic cell source and supportive care. Here we review recently published data that highlight the improvements and current issues in the treatment of SAA. Recent findings Approximately one-third of AA patients treated with immune suppression therapy (IST) have acquired mutations in myeloid cancer candidate genes. Because of the greater probability for eventual failure of IST, human leukocyte antigen (HLA)-matched sibling donor BMT is the first-line of treatment for SAA. HLA-matched unrelated donor (URD) BMT is generally recommended for patients who have failed IST. However, in younger patients for whom a 10/10-HLA-allele matched URD can be rapidly identified, there is a strong rationale to proceed with URD BMT as first-line therapy. HLA-haploidentical BMT using post-transplant cyclophosphamide (PT-CY) conditioning regimens, is now a reasonable second-line treatment for patients who failed IST. Summary Improved outcomes have led to an increased first-line role of BMT for treatment of SAA. The optimal cell source from an HLA-matched donor is bone marrow. Additional studies are needed to determine the optimal conditioning regimen for HLA-haploidentical donors. PMID:27607445

Thyroid cell lines in research on goitrogenesis.

PubMed

Gerber, H; Peter, H J; Asmis, L; Studer, H

1991-12-01

Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes.

ATM inhibitor KU-55933 increases the TMZ responsiveness of only inherently TMZ sensitive GBM cells.

PubMed

Nadkarni, Aditi; Shrivastav, Meena; Mladek, Ann C; Schwingler, Paul M; Grogan, Patrick T; Chen, Junjie; Sarkaria, Jann N

2012-12-01

Ataxia telangiectasia mutated (ATM) kinase is critical in sensing and repairing DNA double-stranded breaks (DSBs) such as those induced by temozolomide (TMZ). ATM deficiency increases TMZ sensitivity, which suggests that ATM inhibitors may be effective TMZ sensitizing agents. In this study, the TMZ sensitizing effects of 2 ATM specific inhibitors were studied in established and xenograft-derived glioblastoma (GBM) lines that are inherently sensitive to TMZ and derivative TMZ-resistant lines. In parental U251 and U87 glioma lines, the addition of KU-55933 to TMZ significantly increased cell killing compared to TMZ alone [U251 survival: 0.004 ± 0.0015 vs. 0.08 ± 0.01 (p < 0.001), respectively, and U87 survival: 0.02 ± 0.005 vs. 0.04 ± 0.002 (p < 0.001), respectively] and also elevated the fraction of cells arrested in G2/M [U251 G2/M fraction: 61.8 ± 1.1 % vs. 35 ± 0.8 % (p < 0.001), respectively, and U87 G2/M fraction 25 ± 0.2 % vs.18.6 ± 0.4 % (p < 0.001), respectively]. In contrast, KU-55933 did not sensitize the resistant lines to TMZ, and neither TMZ alone or combined with KU-55933 induced a G2/M arrest. While KU-55933 did not enhance TMZ induced Chk1/Chk2 activation, it increased TMZ-induced residual γ-H2AX foci in the parental cells but not in the TMZ resistant cells. Similar sensitization was observed with either KU-55933 or CP-466722 combined with TMZ in GBM12 xenograft line but not in GBM12TMZ, which is resistant to TMZ due to MGMT overexpression. These findings are consistent with a model where ATM inhibition suppresses the repair of TMZ-induced DSBs in inherently TMZ-sensitive tumor lines, which suggests an ATM inhibitor potentially could be deployed with an improvement in the therapeutic window when combined with TMZ.

Mitigating the risk of Zika virus contamination of raw materials and cell lines in the manufacture of biologicals.

PubMed

Zmurko, Joanna; Vasey, Douglas B; Donald, Claire L; Armstrong, Alison A; McKee, Marian L; Kohl, Alain; Clayton, Reginald F

2018-02-01

Ensuring the virological safety of biologicals is challenging due to the risk of viral contamination of raw materials and cell banks, and exposure during in-process handling to known and/or emerging viral pathogens. Viruses may contaminate raw materials and biologicals intended for human or veterinary use and remain undetected until appropriate testing measures are employed. The outbreak and expansive spread of the mosquito-borne flavivirus Zika virus (ZIKV) poses challenges to screening human- and animal -derived products used in the manufacture of biologicals. Here, we report the results of an in vitro study where detector cell lines were challenged with African and Asian lineages of ZIKV. We demonstrate that this pathogen is robustly detectable by in vitro assay, thereby providing assurance of detection of ZIKV, and in turn underpinning the robustness of in vitro virology assays in safety testing of biologicals.

Synthesis of curcumin-loaded chitosan phosphate nanoparticle and study of its cytotoxicity and antimicrobial activity.

PubMed

Deka, C; Aidew, L; Devi, N; Buragohain, A K; Kakati, D K

2016-11-01

Curcumin has acquired an important position in the treatment of various diseases. But its use, as a chemotherapeutic agent, is limited due to its low water solubility, poor bioavailability, and its sensitive nature at the physiological pH. To overcome this, curcumin was loaded into chitosan phosphate nanoparticles (CPNs). The loading efficiency was found to be 84%. DLS studies revealed the average particle size of CPNs and curcumin-loaded CPNs as 53 and 91 nm, respectively, and TEM results supplemented these values. A sustained release pattern was noticed and the amount of curcumin released in acidic pH was higher than at physiological pH. The curcumin nanoformulation exhibited proficient activity against both Gram-positive and Gram-negative bacteria as well as fungus. Cytocompatibility of the nanoformulations against peripheral blood mononuclear cells (PBMCs) and murine monocyte-macrophage cell line was confirmed by incubating with PBMCs and murine monocyte-macrophage cell line.

Synthesis, molecular docking and anticancer studies of peptides and iso-peptides.

PubMed

Jabeen, Farukh; Panda, Siva S; Kondratyuk, Tamara P; Park, Eun-Jung; Pezzuto, John M; Ihsan-ul-Haq; Hall, C Dennis; Katritzky, Alan R

2015-08-01

Chiral peptides and iso-peptides were synthesized in excellent yield by using benzotriazole mediated solution phase synthesis. Benzotriazole acted both as activating and leaving group, eliminating frequent use of protection and subsequent deprotection. The procedure was based on the hypothesis that epimerization should be suppressed in solution due to a faster coupling rate than SPPS. All the synthesized peptides complied with Lipinski's Ro5 except for the rotatable bonds. Inhibition of cell proliferation of cancer cell lines is one of the most commonly used methods to study the effectiveness of any anticancer agents. Synthesized peptides and iso-peptides were tested against three cancer cell lines (MCF-7, MDA-MB 231) to determine their anti-proliferative potential. NFkB was also determined. Molecular docking studies were also carried out to complement the experimental results. Copyright © 2015 Elsevier Ltd. All rights reserved.

Analysis of renal cancer cell lines from two major resources enables genomics-guided cell line selection

NASA Astrophysics Data System (ADS)

Sinha, Rileen; Winer, Andrew G.; Chevinsky, Michael; Jakubowski, Christopher; Chen, Ying-Bei; Dong, Yiyu; Tickoo, Satish K.; Reuter, Victor E.; Russo, Paul; Coleman, Jonathan A.; Sander, Chris; Hsieh, James J.; Hakimi, A. Ari

2017-05-01

The utility of cancer cell lines is affected by the similarity to endogenous tumour cells. Here we compare genomic data from 65 kidney-derived cell lines from the Cancer Cell Line Encyclopedia and the COSMIC Cell Lines Project to three renal cancer subtypes from The Cancer Genome Atlas: clear cell renal cell carcinoma (ccRCC, also known as kidney renal clear cell carcinoma), papillary (pRCC, also known as kidney papillary) and chromophobe (chRCC, also known as kidney chromophobe) renal cell carcinoma. Clustering copy number alterations shows that most cell lines resemble ccRCC, a few (including some often used as models of ccRCC) resemble pRCC, and none resemble chRCC. Human ccRCC tumours clustering with cell lines display clinical and genomic features of more aggressive disease, suggesting that cell lines best represent aggressive tumours. We stratify mutations and copy number alterations for important kidney cancer genes by the consistency between databases, and classify cell lines into established gene expression-based indolent and aggressive subtypes. Our results could aid investigators in analysing appropriate renal cancer cell lines.

Novel urea and bis-urea primaquine derivatives with hydroxyphenyl or halogenphenyl substituents: Synthesis and biological evaluation.

PubMed

Perković, I; Antunović, M; Marijanović, I; Pavić, K; Ester, K; Kralj, M; Vlainić, J; Kosalec, I; Schols, D; Hadjipavlou-Litina, D; Pontiki, E; Zorc, B

2016-11-29

A series of novel compounds 3a-j and 6a-j with primaquine and hydroxyl or halogen substituted benzene moieties bridged by urea or bis-urea functionalities were designed, synthesized and evaluated for biological activity. The title compounds were prepared using benzotriazole as the synthon, through several synthetic steps. 3-[3,5-Bis(trifluoromethyl)phenyl]-1-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}urea (3j) was the most active urea and 1-[({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)amino]-3-[3-(trifluoromethyl)phenyl]urea (6h) the most active bis-urea derivative in antiproliferative screening in vitro against eight tested cancer cell lines. Urea derivatives 3a-g with hydroxy group or one halogen atom showed moderate antiproliferative effects against all the tested cell lines, but stronger activity against breast carcinoma MCF-7 cell line, while trifluoromethyl derivatives 3h-j showed antiproliferative effects against all the tested cell lines in low micromolar range. Finally, bis-ureas with hydroxy and fluoro substituents 6a-d showed extreme selectivity and chloro or bromo derivatives 6e-g high selectivity against MCF-7 cells (IC 50 0.1-2.6 μM). p-Fluoro derivative 6d, namely 3-(4-fluorophenyl)-1-[({4-[(6-methoxyquinolin-8-yl)amino]pentyl}carbamoyl)amino]urea, is the most promising compound. Further biological experiments showed that 6d affected cell cycle and induced cell death of MCF-7 cell line. Due to its high activity against MCF-7 cell line (IC 50 0.31 μM), extreme selectivity and full agreement with the Lipinski's and Gelovani's rules for prospective small molecular drugs, 6d may be considered as a lead compound in development of breast carcinoma drugs. Urea 3b and almost all bis-ureas showed high antioxidant activity in DPPH assay, but urea derivatives were more active in lipid peroxidation test. Only few compounds exhibited weak inhibition of soybean lipoxygenase. Compound 3j exhibited the strongest antimicrobial activity in susceptibility assay in vitro (MIC = 1.6-12.5 μg ml -1 ). Copyright © 2016 Elsevier Masson SAS. All rights reserved.

In vitro antiproliferative/cytotoxic activity on cancer cell lines of a cardanol and a cardol enriched from Thai Apis mellifera propolis.

PubMed

Teerasripreecha, Dungporn; Phuwapraisirisan, Preecha; Puthong, Songchan; Kimura, Kiyoshi; Okuyama, Masayuki; Mori, Haruhide; Kimura, Atsuo; Chanchao, Chanpen

2012-03-30

Propolis is a complex resinous honeybee product. It is reported to display diverse bioactivities, such as antimicrobial, anti-inflammatory and anti-tumor properties, which are mainly due to phenolic compounds, and especially flavonoids. The diversity of bioactive compounds depends on the geography and climate, since these factors affect the floral diversity. Here, Apis mellifera propolis from Nan province, Thailand, was evaluated for potential anti-cancer activity. Propolis was sequentially extracted with methanol, dichloromethane and hexane and the cytotoxic activity of each crude extract was assayed for antiproliferative/cytotoxic activity in vitro against five human cell lines derived from duet carcinoma (BT474), undifferentiated lung (Chaco), liver hepatoblastoma (Hep-G(2)), gastric carcinoma (KATO-III) and colon adenocarcinoma (SW620) cancers. The human foreskin fibroblast cell line (Hs27) was used as a non-transformed control. Those crude extracts that displayed antiproliferative/cytotoxic activity were then further fractionated by column chromatography using TLC-pattern and MTT-cytotoxicity bioassay guided selection of the fractions. The chemical structure of each enriched bioactive compound was analyzed by nuclear magnetic resonance and mass spectroscopy. The crude hexane and dichloromethane extracts of propolis displayed antiproliferative/cytotoxic activities with IC(50) values across the five cancer cell lines ranging from 41.3 to 52.4 μg/ml and from 43.8 to 53.5 μg/ml, respectively. Two main bioactive components were isolated, one cardanol and one cardol, with broadly similar in vitro antiproliferation/cytotoxicity IC(50) values across the five cancer cell lines and the control Hs27 cell line, ranging from 10.8 to 29.3 μg/ml for the cardanol and < 3.13 to 5.97 μg/ml (6.82 - 13.0 μM) for the cardol. Moreover, both compounds induced cytotoxicity and cell death without DNA fragmentation in the cancer cells, but only an antiproliferation response in the control Hs27 cells However, these two compounds did not account for the net antiproliferation/cytotoxic activity of the crude extracts suggesting the existence of other potent compounds or synergistic interactions in the propolis extracts. This is the first report that Thai A. mellifera propolis contains at least two potentially new compounds (a cardanol and a cardol) with potential anti-cancer bioactivity. Both could be alternative antiproliferative agents for future development as anti-cancer drugs.

A simple non-perturbing cell migration assay insensitive to proliferation effects

PubMed Central

Glenn, Honor L.; Messner, Jacob; Meldrum, Deirdre R.

2016-01-01

Migration is a fundamental cellular behavior that plays an indispensable role in development and homeostasis, but can also contribute to pathology such as cancer metastasis. Due to its relevance to many aspects of human health, the ability to accurately measure cell migration is of broad interest, and numerous approaches have been developed. One of the most commonly employed approaches, because of its simplicity and throughput, is the exclusion zone assay in which cells are allowed to migrate into an initially cell-free region. A major drawback of this assay is that it relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration. We report here a simple modification to the exclusion zone migration assay that exclusively measures cell migration and is not affected by proliferation. This approach makes use of a lineage-tracing vital stain that is retained through cell generations and effectively reads out migration relative to the original, parental cell population. This modification is simple, robust, non-perturbing, and inexpensive. We validate the method in a panel of cell lines under conditions that inhibit or promote migration and demonstrate its use in normal and cancer cell lines as well as primary cells. PMID:27535324

Large-scale culture of a megakaryocytic progenitor cell line with a single-use bioreactor system.

PubMed

Nurhayati, Retno Wahyu; Ojima, Yoshihiro; Dohda, Takeaki; Kino-Oka, Masahiro

2018-03-01

The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single-use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large-scale production and feasibility of meeting clinical-grade standards. The current work evaluated the capacity of a single-use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (k L a) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h -1 , respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7-fold with a total cell number of 1.2 ± 0.2 × 10 9 cells L -1 . In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single-use bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:362-369, 2018. © 2017 American Institute of Chemical Engineers.

Supportive care in the era of immunotherapies for advanced non-small-cell lung cancer.

PubMed

Awada, Gil; Klastersky, Jean

2018-03-01

The therapeutic armamentarium for advanced non-small-cell lung cancer has evolved considerably over the past years. Immune checkpoint inhibitors targeting programmed cell death-1 such as pembrolizumab and nivolumab or programmed cell death ligand 1 such as atezolizumab, durvalumab and avelumab have shown favorable efficacy results in this patient population in the first-line and second-line setting. These immunotherapies are associated with a distinct toxicity profile based on autoimmune organ toxicity which is a new challenge for supportive care during treatment with these drugs. The differential diagnosis of events occurring during immune checkpoint inhibitor treatment is broad: they can be due to immune-related or nonimmune-related adverse events, atypical tumor responses (pseudoprogression or hyperprogression) or events related to comorbidities or other treatments. The management of these patients includes a thorough baseline clinical, biological and radiologic evaluation, patient education, correct follow-up and management by a multidisciplinary team with a central role for the medical oncologist. Immune-related toxicities should be managed according to available guidelines.

A novel cell model to study the function of the adrenoleukodystrophy-related protein

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

Gueugnon, Fabien; Volodina, Natalia; Taouil, Jaoued Et

2006-03-03

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder due to mutations in the ABCD1 (ALD) gene. ALDRP, the closest homolog of ALDP, has been shown to have partial functional redundancy with ALDP and, when overexpressed, can compensate for the loss-of-function of ALDP. In order to characterize the function of ALDRP and to understand the phenomenon of gene redundancy, we have developed a novel system that allows the controlled expression of the ALDRP-EGFP fusion protein (normal or non-functional mutated ALDRP) using the Tet-On system in H4IIEC3 rat hepatoma cells. The generated stable cell lines express negligible levels of endogenous ALDRP and doxycyclinemore » dosage-dependent levels of normal or mutated ALDRP. Importantly, the ALDRP-EGFP protein is targeted correctly to peroxisome and is functional. The obtained cell lines will be an indispensable tool in our further studies aimed at the resolution of the function of ALDRP to characterize its potential substrates in a natural context.« less

Inferring genome-wide interplay landscape between DNA methylation and transcriptional regulation.

PubMed

Tang, Binhua; Wang, Xin

2015-01-01

DNA methylation and transcriptional regulation play important roles in cancer cell development and differentiation processes. Based on the currently available cell line profiling information from the ENCODE Consortium, we propose a Bayesian inference model to infer and construct genome-wide interaction landscape between DNA methylation and transcriptional regulation, which sheds light on the underlying complex functional mechanisms important within the human cancer and disease context. For the first time, we select all the currently available cell lines (>=20) and transcription factors (>=80) profiling information from the ENCODE Consortium portal. Through the integration of those genome-wide profiling sources, our genome-wide analysis detects multiple functional loci of interest, and indicates that DNA methylation is cell- and region-specific, due to the interplay mechanisms with transcription regulatory activities. We validate our analysis results with the corresponding RNA-sequencing technique for those detected genomic loci. Our results provide novel and meaningful insights for the interplay mechanisms of transcriptional regulation and gene expression for the human cancer and disease studies.

PCI-24781 (abexinostat), a novel histone deacetylase inhibitor, induces reactive oxygen species-dependent apoptosis and is synergistic with bortezomib in neuroblastoma

PubMed Central

Sholler, Giselle Saulnier; Currier, Erika A.; Dutta, Akshita; Slavik, Marni A.; Illenye, Sharon A.; Mendonca, Maria Cecilia F.; Dragon, Julie; Roberts, Stephen S.; Bond, Jeffrey P.

2014-01-01

In this study, we investigated the cytotoxic effects of a broad-spectrum histone deacetylase (HDAC) inhibitor, PCI-24781, alone and in combination with the proteasome inhibitor bortezomib in neuroblastoma cell lines. The combination was shown to induce synergistic cytotoxity involving the formation of reactive oxygen species. The cleavage of caspase-3 and PARP, as determined by western blotting, indicated that cell death was primarily due to apoptosis. Xenograft mouse models indicated increased survival among animals treated with this combination. The Notch signaling pathway and MYCN gene expression were quantified by reverse transcription-polymerase chain reaction (PCR) in cells treated with PCI-24781 and bortezomib, alone and in combination. Notch pathway expression increased in response to an HDAC inhibitor. NFKB1 and MYCN were both significantly down regulated. Our results suggest that PCI-24781 and bortezomib are synergistic in neuroblastoma cell lines and may be a new therapeutic strategy for this disease. PMID:25520806

Radioimmunotargeting of human tumour cells in immunocompetent animals.

PubMed Central

Fjeld, J. G.; Bruland, O. S.; Benestad, H. B.; Schjerven, L.; Stigbrand, T.; Nustad, K.

1990-01-01

A tumour model system is reported that for many purposes may be an alternative to xenografted nude mice. The model allows immunotargeting of human tumour cells in immunocompetent animals. The target cells are contained in i.p. diffusion chambers (DC) with micropore membrane walls that are permeable to molecules, including the cell specific monoclonal antibodies (MoAb), but impermeable to cells. Thus, the tumour cells are protected from the host immunocompetent cells. In the work here presented the model was tested in immunocompetent mice and pigs, with tumour cells and antibody preparations that had demonstrated specific targeting in the nude mouse xenograft model. Hence, the DC were filled with cells from the human cell lines Hep-2 (expressing placental alkaline phosphatase, PLALP), or OHS (a sarcoma cell line), and the MoAb preparations injected i.v. were a 125I-labelled Fab fragment of the PLALP specific antibody H7, or a 125I-labelled F(ab')2 fragment of the sarcoma specific antibody TP-1. Specific targeting of the human tumour cells was demonstrated in both mice and pigs. The target: blood ratios were comparable in the two species, reaching a maximum of about 15 after 24 h with the Fab preparation, and a ratio of 25 after 72 h with the F(ab')2. The target uptake relative to injected dose was lower in pigs than in mice, but the difference between the two species was smaller than expected, presumably due to a slower antibody clearance in the pigs than in the mice. An artificial cell targeting system like this has several advantages in the search for solutions to many of the fundamental problems experienced in immunotargeting. Firstly, parallel binding experiments can be carried out in vitro with the same target. Because in vitro results are only influenced by the diffusion into the DC and the immunological binding characteristics of the antibodies, targeting differences between antibody preparations due to these factors can then be distinguished from differences due to pharmacokinetical properties. Secondly, the animals can be implanted with any type and number of target cells, or with antigen negative control cells. Thirdly, and perhaps most important, the system opens a possibility for evaluation of the murine MoAb in xenogenic species, and this may predict the clinical targeting potential better than experiments on mice. PMID:2223574

Growth-inhibitory and antiangiogenic activity of the MEK inhibitor PD0325901 in malignant melanoma with or without BRAF mutations.

PubMed

Ciuffreda, Ludovica; Del Bufalo, Donatella; Desideri, Marianna; Di Sanza, Cristina; Stoppacciaro, Antonella; Ricciardi, Maria Rosaria; Chiaretti, Sabina; Tavolaro, Simona; Benassi, Barbara; Bellacosa, Alfonso; Foà, Robin; Tafuri, Agostino; Cognetti, Francesco; Anichini, Andrea; Zupi, Gabriella; Milella, Michele

2009-08-01

The Raf/MEK/ERK pathway is an important mediator of tumor cell proliferation and angiogenesis. Here, we investigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC(50) in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G(1)-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27(KIP1)) and apoptosis (Bcl-2 and survivin) regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma.

Growth-Inhibitory and Antiangiogenic Activity of the MEK Inhibitor PD0325901 in Malignant Melanoma with or without BRAF Mutations12

PubMed Central

Ciuffreda, Ludovica; Del Bufalo, Donatella; Desideri, Marianna; Di Sanza, Cristina; Stoppacciaro, Antonella; Ricciardi, Maria Rosaria; Chiaretti, Sabina; Tavolaro, Simona; Benassi, Barbara; Bellacosa, Alfonso; Foà, Robin; Tafuri, Agostino; Cognetti, Francesco; Anichini, Andrea; Zupi, Gabriella; Milella, Michele

2009-01-01

The Raf/MEK/ERK pathway is an important mediator of tumor cell proliferation and angiogenesis. Here, we investigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC50 in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G1-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27KIP1) and apoptosis (Bcl-2 and survivin) regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma. PMID:19649202

The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines.

PubMed

Rojnik, Matija; Jevnikar, Zala R; Doljak, Bojan; Turk, Samo; Zidar, Nace; Kos, Janko

2012-10-01

Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type. Copyright © 2012 Elsevier GmbH. All rights reserved.

Amniotic fluid stem cells: a promising therapeutic resource for cell-based regenerative therapy.

PubMed

Antonucci, Ivana; Pantalone, Andrea; Tete, Stefano; Salini, Vincenzo; Borlongan, Cesar V; Hess, David; Stuppia, Liborio

2012-01-01

Stem cells have been proposed as a powerful tool in the treatment of several human diseases, both for their ability to represent a source of new cells to replace those lost due to tissue injuries or degenerative diseases, and for the ability of produce trophic molecules able to minimize damage and promote recovery in the injured tissue. Different cell types, such as embryonic, fetal or adult stem cells, human fetal tissues and genetically engineered cell lines, have been tested for their ability to replace damaged cells and to restore the tissue function after transplantation. Amniotic fluid -derived Stem cells (AFS) are considered a novel resource for cell transplantation therapy, due to their high renewal capacity, the "in vitro" expression of embryonic cell lineage markers, and the ability to differentiate in tissues derived from all the three embryonic layers. Moreover, AFS do not produce teratomas when transplanted into animals and are characterized by a low antigenicity, which could represent an advantage for cell transplantation or cell replacement therapy. The present review focuses on the biological features of AFS, and on their potential use in the treatment of pathological conditions such as ischemic brain injury and bone damages.

Bio optofluidics cell sorter: cell-BOCS concept and applications

NASA Astrophysics Data System (ADS)

Roth, Tue; Glückstad, Jesper

2012-03-01

The cell-BOCS is a novel microfluidics based cell-sorting instrument utilizing next generation optical trapping technology developed at the Technical University of Denmark. It is targeted emerging bio-medical research and diagnostics markets where it for certain applications offers a number of advantages over conventional fluorescence activated cell-sorting (FACSTM) technology. Advantages include gentle handling of cells, sterile sorting, easy operation, small footprint and lower cost allowing out-of-core-facility use. Application examples are found within sorting of fragile transfected cells, high value samples and primary cell lines, where traditional FACS technology has limited application due to it's droplet-based approach to cell-sorting. In the diagnostics field, in particular applying the cell-BOCS for isolating pure populations of circulating tumor cells is an area that has generated a lot of interest.

Regulation of apoptosis in human melanoma and neuroblastoma cells by statins, sodium arsenite and TRAIL: a role of combined treatment versus monotherapy

PubMed Central

Ivanov, Vladimir N.; Hei, Tom K.

2015-01-01

Treatment of melanoma cells by sodium arsenite or statins (simvastatin and lovastatin) dramatically modified activities of the main cell signaling pathways resulting in the induction of heme oxygenase-1 (HO-1) and in a downregulation of cyclooxygenase-2 (COX-2) protein levels. Through heme degradation and the production of carbon monoxide and biliverdin, HO-1 plays a protective role in different scenario of oxidative stress followed by mitochondrial apoptosis. Both sodium arsenite and statins could be efficient inducers of apoptosis in some melanoma cell lines, but often exhibited only modest proapoptotic activity in others, due to numerous protective mechanisms. We demonstrated in the present study that treatment by sodium arsenite or statins with an additional inhibition of HO-1 expression (or activation) caused a substantial upregulation of apoptosis in melanoma cells. Sodium arsenite- or statin-induced apoptosis was independent of BRAF status (wild type versus V600E) in melanoma lines. Monotreatment required high doses of statins (20–40 μM) for effective induction of apoptosis. As an alternative approach, pretreatment of melanoma cells with statin at decreased doses (5–20 μM) dramatically enhanced TRAIL-induced apoptosis, due to suppression of the NF-κB and STAT3-transcriptional targets (including COX-2) and downregulation of cFLIP-L (a caspase-8 inhibitor) protein levels. Furthermore, combined treatment with sodium arsenite and TRAIL or simvastatin and TRAIL efficiently induced apoptotic commitment in human neuroblastoma cells. In summary, our findings on enhancing effects of combined treatment of cancer cells using statin and TRAIL provide the rationale for further preclinical evaluation. PMID:21910007

Evaluating cell lines as tumour models by comparison of genomic profiles

PubMed Central

Domcke, Silvia; Sinha, Rileen; Levine, Douglas A.; Sander, Chris; Schultz, Nikolaus

2013-01-01

Cancer cell lines are frequently used as in vitro tumour models. Recent molecular profiles of hundreds of cell lines from The Cancer Cell Line Encyclopedia and thousands of tumour samples from the Cancer Genome Atlas now allow a systematic genomic comparison of cell lines and tumours. Here we analyse a panel of 47 ovarian cancer cell lines and identify those that have the highest genetic similarity to ovarian tumours. Our comparison of copy-number changes, mutations and mRNA expression profiles reveals pronounced differences in molecular profiles between commonly used ovarian cancer cell lines and high-grade serous ovarian cancer tumour samples. We identify several rarely used cell lines that more closely resemble cognate tumour profiles than commonly used cell lines, and we propose these lines as the most suitable models of ovarian cancer. Our results indicate that the gap between cell lines and tumours can be bridged by genomically informed choices of cell line models for all tumour types. PMID:23839242

An autologous dendritic cell canine mammary tumor hybrid-cell fusion vaccine.

PubMed

Bird, R Curtis; Deinnocentes, Patricia; Church Bird, Allison E; van Ginkel, Frederik W; Lindquist, Joni; Smith, Bruce F

2011-01-01

Mammary cancer is among the most prevalent canine tumors and frequently resulting in death due to metastatic disease that is highly homologous to human breast cancer. Most canine tumors fail to raise effective immune reactions yet, some spontaneous remissions do occur. Hybrid canine dendritic cell-tumor cell fusion vaccines were designed to enhance antigen presentation and tumor immune recognition. Peripheral blood-derived autologous dendritic cell enriched populations were isolated from dogs based on CD11c(+) expression and fused with canine mammary tumor (CMT) cells for vaccination of laboratory Beagles. These hybrid cells were injected into popliteal lymph nodes of normal dogs, guided by ultrasound, and included CpG-oligonucleotide adjuvants. Three rounds of vaccination were delivered. Significant IgG responses were observed in all vaccinated dogs compared to vehicle-injected controls. Canine IgG antibodies recognized shared CMT antigens as was demonstrated by IgG-recognition of three unrelated/independently derived CMT cell lines, and recognition of freshly isolated, unrelated, primary biopsy-derived CMT cells. A bias toward an IgG2 isotype response was observed after two vaccinations in most dogs. Neither significant cytotoxic T cell responses were detected, nor adverse or side-effects due to vaccination or due to the induced immune responses noted. These data provide proof-of-principle for this cancer vaccine strategy and demonstrate the presence of shared CMT antigens that promote immune recognition of mammary cancer.

Lining seam elimination algorithm and surface crack detection in concrete tunnel lining

NASA Astrophysics Data System (ADS)

Qu, Zhong; Bai, Ling; An, Shi-Quan; Ju, Fang-Rong; Liu, Ling

2016-11-01

Due to the particularity of the surface of concrete tunnel lining and the diversity of detection environments such as uneven illumination, smudges, localized rock falls, water leakage, and the inherent seams of the lining structure, existing crack detection algorithms cannot detect real cracks accurately. This paper proposed an algorithm that combines lining seam elimination with the improved percolation detection algorithm based on grid cell analysis for surface crack detection in concrete tunnel lining. First, check the characteristics of pixels within the overlapping grid to remove the background noise and generate the percolation seed map (PSM). Second, cracks are detected based on the PSM by the accelerated percolation algorithm so that the fracture unit areas can be scanned and connected. Finally, the real surface cracks in concrete tunnel lining can be obtained by removing the lining seam and performing percolation denoising. Experimental results show that the proposed algorithm can accurately, quickly, and effectively detect the real surface cracks. Furthermore, it can fill the gap in the existing concrete tunnel lining surface crack detection by removing the lining seam.

Continuous hematopoietic cell lines as model systems for leukemia-lymphoma research.

PubMed

Drexler, H G; Matsuo, A Y; MacLeod, R A

2000-11-01

Along with other improvements, the advent of continuous human leukemia-lymphoma (LL) cell lines as a rich resource of abundant, accessible and manipulable living cells has contributed significantly to a better understanding of the pathophysiology of hematopoietic tumors. The first LL cell lines, Burkitt's lymphoma-derived lines, were established in 1963. Since then, more than 1000 cell lines have been described, although not all of them in full detail. The major advantages of continuous cell lines is the unlimited supply and worldwide availability of identical cell material, and the infinite viable storability in liquid nitrogen. LL cell lines are characterized generally by monoclonal origin and differentiation arrest, sustained proliferation in vitro under preservation of most cellular features, and specific genetic alterations. The most practical classification of LL cell lines assigns them to one of the physiologically occurring cell lineages, based on their immunophenotype, genotype and functional features. Truly malignant cell lines must be discerned from Epstein-Barr virus (EBV)-immortalized normal cells, using various distinguishing parameters. However, the picture is not quite so straightforward, as some types of LL cell lines are indeed EBV+, and some EBV+ normal cell lines carry also genetic aberrations and may mimic malignancy-associated features. Apart from EBV and human T-cell leukemia virus in some lines, the majority of wild-type LL cell lines are virus-negative. The efficiency of cell line establishment is rather low and the deliberate establishment of new LL cell lines remains by and large an unpredictable random process. Difficulties in establishing continuous cell lines may be caused by the inappropriate selection of nutrients and growth factors for these cells. Clearly, a generally suitable microenvironment for hematopoietic cells, either malignant or normal, cannot yet be created in vitro. The characterization and publication of new LL cell lines should provide important and informative core data, attesting to their scientific significance. Large percentages of LL cell lines are contaminated with mycoplasma (about 30%) or are cross-contaminated with other cell lines (about 15-20%). Solutions to these problems are sensitive detection, effective elimination and rigorous prevention of mycoplasma infection, and proper, regular authentication of cell lines. The underlying cause, however, appears to be negligent cell culture practice. The willingness of investigators to make their LL cell lines available to others is all too often limited. There is a need in the scientific community for clean and authenticated high-quality LL cell lines to which every scientist has access. These are offered by various institutionalized public cell line banks. It has been argued that LL cell lines are genetically unstable (both cytogenetically and molecular genetically). For instance, cell lines are supposed to acquire numerical and structural chromosomal alterations and various types of mutations (e.g. point mutations) in vitro. We present evidence that while nearly 100% of all LL cell lines indeed carry genetic alterations, these alterations appear to be stable rather than unstable. As an example of the practical utility of LL cell lines, the recent advances in studies of classical and molecular cytogenetics, which in large part were made possible by cell lines, are highlighted. A list of the most useful, robust and publicly available reference cell lines that may be used for a variety of experimental purposes is proposed. Clearly, by opening new avenues for investigation, studies of LL cell lines have provided seminal insights into the biology of hematopoietic neoplasia. Over a period of nearly four decades, these initially rather exotic cell cultures, known only to a few specialists, have become ubiquitous powerful research tools that are available to every investigator.

The phytoestrogen genistein enhances multidrug resistance in breast cancer cell lines by translational regulation of ABC transporters.

PubMed

Rigalli, Juan Pablo; Tocchetti, Guillermo Nicolás; Arana, Maite Rocío; Villanueva, Silvina Stella Maris; Catania, Viviana Alicia; Theile, Dirk; Ruiz, María Laura; Weiss, Johanna

2016-06-28

Breast cancer is the most frequent malignancy in women. Multidrug resistance due to overexpression of ABC drug transporters is a common cause of chemotherapy failure and disease recurrence. Genistein (GNT) is a phytoestrogen present in soybeans and hormone supplements. We investigated the effect of GNT on the expression and function of ABC transporters in MCF-7 and MDA-MB-231 breast cancer cell lines. Results demonstrated an induction at the protein level of ABCC1 and ABCG2 and of ABCC1 in MCF-7 and MDA-MB-231, respectively. MCF-7 cells showed a concomitant increase in doxorubicin and mitoxantrone efflux and resistance, dependent on ABCG2 activity. ABCC1 induction by GNT in MDA-MB-231 cells modified neither drug efflux nor chemoresistance due to simultaneous acute inhibition of the transporter activity by GNT. All inductions took place at the translational level, as no increment in mRNA was observed and protein increase was prevented by cycloheximide. miR-181a, already demonstrated to inhibit ABCG2 translation, was down-regulated by GNT, explaining translational induction. Effects were independent of classical estrogen receptors. Results suggest potential nutrient-drug interactions that could threaten chemotherapy efficacy, especially in ABCG2-expressing tumors treated with substrates of this transporter. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Kinetics of ROS generation induced by polycyclic aromatic hydrocarbons and organic extracts from ambient air particulate matter in model human lung cell lines.

PubMed

Libalova, Helena; Milcova, Alena; Cervena, Tereza; Vrbova, Kristyna; Rossnerova, Andrea; Novakova, Zuzana; Topinka, Jan; Rossner, Pavel

2018-03-01

Polycyclic aromatic hydrocarbons (PAHs) associated with particulate matter (PM) may induce oxidative damage via reactive oxygen species (ROS) generation. However, the kinetics of ROS production and the link with antioxidant response induction has not been well studied. To elucidate the differences in oxidative potential of individual PAH compounds and extractable organic matter (EOM) from PM containing various PAH mixtures, we studied ROS formation and antioxidant response [total antioxidant capacity (TAC) and expression of HMOX1 and TXNRD1] in human alveolar basal epithelial cells (A549 cells) and human embryonic lung fibroblasts (HEL12469 cells). We treated the cells with three concentrations of model PAHs (benzo[a]pyrene, B[a]P; 3-nitrobenzanthrone, 3-NBA) and EOM from PM <2.5 μm (PM2.5). ROS levels were evaluated at 8 time intervals (30 min-24 h). In both cell lines, B[a]P treatment was associated with a time-dependent decrease of ROS levels. This trend was more pronounced in HEL12469 cells and was accompanied by increased TAC. A similar response was observed upon 3-NBA treatment in HEL12469 cells. In A549 cells, however, this compound significantly increased superoxide levels. This response was accompanied by the decrease of TAC as well as HMOX1 and TXNRD1 expression. In both cell lines, a short-time exposure to EOMs tended to increase ROS levels, while a marked decrease was observed after longer treatment periods. This was accompanied by the induction of HMOX1 and TXNRD1 expression in HEL12469 cells and increased TAC in A549 cells. In summary, our data indicate that in the studied cell lines B[a]P and EOMs caused a time-dependent decrease of intracellular ROS levels, probably due to the activation of the antioxidant response. This response was not detected in A549 cells following 3-NBA treatment, which acted as a strong superoxide inducer. Pro-oxidant properties of EOMs are limited to short-time exposure periods. Copyright © 2018 Elsevier B.V. All rights reserved.

The anti-proliferative and apoptotic effects of crocin on chemosensitive and chemoresistant cervical cancer cells.

PubMed

Mollaei, Homa; Safaralizadeh, Reza; Babaei, Esmaeil; Abedini, Mohamad Reza; Hoshyar, Reyhane

2017-10-01

Cervical cancer is the fourth cause of cancer-related mortality among females worldwide. Although current therapies reduce disease symptoms, resistance of tumor cells to chemotherapy agents after a while is a serious problem. Therefore, utilization of novel adjuvant agents to increase efficiency of chemotherapy is essential. In the last two decades, botanicals with effective anticancer activities have been studied. Among them, the anticancer properties of crocin have been more attended. In this study, the molecular mechanism of crocin action was investigated in sensitive human cervical cancer cell line (OV2008) in comparison with the resistant one (C13). A 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay showed that crocin inhibits proliferation of sensitive cells (OV2008) at a time- and dose-dependent manner at 48 and 72h. Also, this inhibitory effect has been shown on resistant cells (C13) at 72h. Hoechst staining and flow cytometry assay also confirmed these results and revealed that antiproliferative effect of crocin might be due to the induction of apoptosis. Moreover, the genetic mechanism of crocin-induced apoptosis was accomplished by studying the relative expressions of P53, Bax, Bcl2 and miR-365, an upstream regulator of the last two ones. Real-time PCR analysis indicated that 1.5 and 3mg/ml crocin led to up-regulation of Bax and P53 and down-regulation of Bcl2 and miR-365 at all time intervals in both two cell lines. However, OV2008 cell line was more sensitive to crocin, and alternation of gene expretion was more obvious in this cell line. In this regard, the present study demonstrated the anti-proliferative and apoptotic activities of crocin against both sensitive and resistant cervical cancer cells that may benefit cervical cancer treatment as an adjuvant agent to decrease chemoresistance and increase the efficiency of therapy. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

An intergenic non-coding rRNA correlated with expression of the rRNA and frequency of an rRNA single nucleotide polymorphism in lung cancer cells.

PubMed

Shiao, Yih-Horng; Lupascu, Sorin T; Gu, Yuhan D; Kasprzak, Wojciech; Hwang, Christopher J; Fields, Janet R; Leighty, Robert M; Quiñones, Octavio; Shapiro, Bruce A; Alvord, W Gregory; Anderson, Lucy M

2009-10-19

Ribosomal RNA (rRNA) is a central regulator of cell growth and may control cancer development. A cis noncoding rRNA (nc-rRNA) upstream from the 45S rRNA transcription start site has recently been implicated in control of rRNA transcription in mouse fibroblasts. We investigated whether a similar nc-rRNA might be expressed in human cancer epithelial cells, and related to any genomic characteristics. Using quantitative rRNA measurement, we demonstrated that a nc-rRNA is transcribed in human lung epithelial and lung cancer cells, starting from approximately -1000 nucleotides upstream of the rRNA transcription start site (+1) and extending at least to +203. This nc-rRNA was significantly more abundant in the majority of lung cancer cell lines, relative to a nontransformed lung epithelial cell line. Its abundance correlated negatively with total 45S rRNA in 12 of 13 cell lines (P = 0.014). During sequence analysis from -388 to +306, we observed diverse, frequent intercopy single nucleotide polymorphisms (SNPs) in rRNA, with a frequency greater than predicted by chance at 12 sites. A SNP at +139 (U/C) in the 5' leader sequence varied among the cell lines and correlated negatively with level of the nc-rRNA (P = 0.014). Modelling of the secondary structure of the rRNA 5'-leader sequence indicated a small increase in structural stability due to the +139 U/C SNP and a minor shift in local configuration occurrences. The results demonstrate occurrence of a sense nc-rRNA in human lung epithelial and cancer cells, and imply a role in regulation of the rRNA gene, which may be affected by a +139 SNP in the 5' leader sequence of the primary rRNA transcript.

Overcoming Acquired Resistance to AZD9291, A Third-Generation EGFR Inhibitor, through Modulation of MEK/ERK-Dependent Bim and Mcl-1 Degradation.

PubMed

Shi, Puyu; Oh, You-Take; Deng, Liang; Zhang, Guojing; Qian, Guoqing; Zhang, Shuo; Ren, Hui; Wu, Grant; Legendre, Benjamin; Anderson, Emily; Ramalingam, Suresh S; Owonikoko, Taofeek K; Chen, Mingwei; Sun, Shi-Yong

2017-11-01

Purpose: The mechanisms accounting for anticancer activity of AZD9291 (osimertinib or TAGRISSO), an approved third-generation EGFR inhibitor, in EGFR-mutant non-small cell lung cancer (NSCLC) cells and particularly for the subsequent development of acquired resistance are unclear and thus are the focus of this study. Experimental Design: AZD9219-resistant cell lines were established by exposing sensitive cell lines to AZD9291. Protein alterations were detected with Western blotting. Apoptosis was measured with annexin V/flow cytometry. Growth-inhibitory effects of tested drugs were evaluated in vitro with cell number estimation and colony formation assay and in vivo with mouse xenograft models. Protein degradation was determined by comparing protein half-lives and inhibiting proteasome. Gene knockdown were achieved with siRNA or shRNA. Results: AZD9291 potently induced apoptosis in EGFR-mutant NSCLC cell lines, in which ERK phosphorylation was suppressed accompanied with Bim elevation and Mcl-1 reduction likely due to enhanced Mcl-1 degradation and increased Bim stability. Blocking Bim elevation by gene knockdown or enforcing Mcl-1 expression attenuated or abolished AZD9291-induced apoptosis. Moreover, AZD9291 lost its ability to modulate Bim and Mcl-1 levels in AZD9291-resistant cell lines. The combination of a MEK inhibitor with AZD9291 restores the sensitivity of AZD9291-resistant cells including those with C797S mutation to undergo apoptosis and growth regression in vitro and in vivo Conclusions: Modulation of MEK/ERK-dependent Bim and Mcl-1 degradation critically mediates sensitivity and resistance of EGFR-mutant NSCLC cells to AZD9291 and hence is an effective strategy to overcome acquired resistance to AZD9291. Clin Cancer Res; 23(21); 6567-79. ©2017 AACR . ©2017 American Association for Cancer Research.

5-alpha-reductase type I (SRD5A1) is up-regulated in non-small cell lung cancer but does not impact proliferation, cell cycle distribution or apoptosis

PubMed Central

2012-01-01

Background Non-small cell lung cancer (NSCLC) is one of the most frequent malignancies and has a high mortality rate due to late detection and lack of efficient treatments. Identifying novel drug targets for this indication may open the way for new treatment strategies. Comparison of gene expression profiles of NSCLC and normal adjacent tissue (NAT) allowed to determine that 5-alpha-reductase type I (SRD5A1) was up-regulated in NSCLC compared to NAT. This raised the question whether SRD5A1 was involved in sustained proliferation and survival of NSCLC. Methods siRNA-mediated silencing of SRD5A1 was performed in A549 and NCI-H460 lung cancer cell lines in order to determine the impact on proliferation, on distribution during the different phases of the cell cycle, and on apoptosis/necrosis. In addition, lung cancer cell lines were treated with 4-azasteroids, which specifically inhibit SRD5A1 activity, and the effects on proliferation were measured. Statistical analyses using ANOVA and post-hoc Tamhane-T2-test were performed. In the case of non-parametric data, the Kruskal-Wallis test and the post-hoc Mann-Whitney-U-test were used. Results The knock-down of SRDA51 expression was very efficient with the SRD5A1 transcripts being reduced to 10% of control levels. Knock-down efficiency was furthermore confirmed at the protein level. However, no effect of SRD5A1 silencing was observed in the proliferation assay, the cell cycle analysis, and the apoptosis/necrosis assay. Treatment of lung cancer cell lines with 4-azasteroids did not significantly inhibit proliferation. Conclusions In summary, the results suggest that SRD5A1 is not a crucial enzyme for the sustained proliferation of NSCLC cell lines. PMID:22257483

5-alpha-reductase type I (SRD5A1) is up-regulated in non-small cell lung cancer but does not impact proliferation, cell cycle distribution or apoptosis.

PubMed

Kapp, Friedrich G; Sommer, Anette; Kiefer, Thomas; Dölken, Gottfried; Haendler, Bernard

2012-01-18

Non-small cell lung cancer (NSCLC) is one of the most frequent malignancies and has a high mortality rate due to late detection and lack of efficient treatments. Identifying novel drug targets for this indication may open the way for new treatment strategies. Comparison of gene expression profiles of NSCLC and normal adjacent tissue (NAT) allowed to determine that 5-alpha-reductase type I (SRD5A1) was up-regulated in NSCLC compared to NAT. This raised the question whether SRD5A1 was involved in sustained proliferation and survival of NSCLC. siRNA-mediated silencing of SRD5A1 was performed in A549 and NCI-H460 lung cancer cell lines in order to determine the impact on proliferation, on distribution during the different phases of the cell cycle, and on apoptosis/necrosis. In addition, lung cancer cell lines were treated with 4-azasteroids, which specifically inhibit SRD5A1 activity, and the effects on proliferation were measured. Statistical analyses using ANOVA and post-hoc Tamhane-T2-test were performed. In the case of non-parametric data, the Kruskal-Wallis test and the post-hoc Mann-Whitney-U-test were used. The knock-down of SRDA51 expression was very efficient with the SRD5A1 transcripts being reduced to 10% of control levels. Knock-down efficiency was furthermore confirmed at the protein level. However, no effect of SRD5A1 silencing was observed in the proliferation assay, the cell cycle analysis, and the apoptosis/necrosis assay. Treatment of lung cancer cell lines with 4-azasteroids did not significantly inhibit proliferation. In summary, the results suggest that SRD5A1 is not a crucial enzyme for the sustained proliferation of NSCLC cell lines.

Telomere dynamics in an immortal human cell line.

PubMed Central

Murnane, J P; Sabatier, L; Marder, B A; Morgan, W F

1994-01-01

The integration of transfected plasmid DNA at the telomere of chromosome 13 in an immortalized simian virus 40-transformed human cell line provided the first opportunity to study polymorphism in the number of telomeric repeat sequences on the end of a single chromosome. Three subclones of this cell line were selected for analysis: one with a long telomere on chromosome 13, one with a short telomere, and one with such extreme polymorphism that no distinct band was discernible. Further subcloning demonstrated that telomere polymorphism resulted from both gradual changes and rapid changes that sometimes involved many kilobases. The gradual changes were due to the shortening of telomeres at a rate similar to that reported for telomeres of somatic cells without telomerase, eventually resulting in the loss of nearly all of the telomere. However, telomeres were not generally lost completely, as shown by the absence of polymorphism in the subtelomeric plasmid sequences. Instead, telomeres that were less than a few hundred base pairs in length showed a rapid, highly heterogeneous increase in size. Rapid changes in telomere length also occurred on longer telomeres. The frequency of this type of change in telomere length varied among the subclones and correlated with chromosome fusion. Therefore, the rapid changes in telomere length appeared occasionally to result in the complete loss of telomeric repeat sequences. Rapid changes in telomere length have been associated with telomere loss and chromosome instability in yeast and could be responsible for the high rate of chromosome fusion observed in many human tumor cell lines. Images PMID:7957062

The epigenetically regulated miR-494 associates with stem-cell phenotype and induces sorafenib resistance in hepatocellular carcinoma.

PubMed

Pollutri, Daniela; Patrizi, Clarissa; Marinelli, Sara; Giovannini, Catia; Trombetta, Elena; Giannone, Ferdinando A; Baldassarre, Maurizio; Quarta, Santina; Vandewynckel, Y P; Vandierendonck, A; Van Vlierberghe, H; Porretti, Laura; Negrini, Massimo; Bolondi, Luigi; Gramantieri, Laura; Fornari, Francesca

2018-01-05

Hepatocellular carcinoma (HCC) represents the second cause of cancer-related mortality worldwide and is associated with poor prognosis, especially in patients not amenable for curative treatments. The multi-kinase inhibitor sorafenib represents the first-line treatment option for advanced HCC; nevertheless, its effectiveness is limited due to tumor heterogeneity as well as innate or acquired drug resistance, raising the need for new therapeutic strategies. MicroRNAs (miRNAs) involvement in treatment response as well as their safety and efficacy in preclinical models and clinical trials have been widely documented in the oncologic field, including HCC. Here, we identified miR-494 upregulation in a subgroup of human and rat HCCs with stem cell-like characteristics, as well as multiple epigenetic mechanisms involved in its aberrant expression in HCC cell lines and patients. Moreover, we identified p27, puma and pten among miR-494 targets, contributing to speed up cell cycle progression, enhance survival potential in stressful conditions and increase invasive and clonogenic capabilities. MiR-494 overexpression increased sorafenib resistance via mTOR pathway activation in HCC cell lines and, in line, high miR-494 levels associated with decreased sorafenib response in two HCC animal models. A sorafenib-combined anti-miR-494-based strategy revealed an enhanced anti-tumor potential with respect to sorafenib-only treatment in our HCC rat model. In conclusion, our findings suggested miR-494 as a possible therapeutic target as well as a candidate biomarker for patient stratification in advanced HCC.

Reduction of T cell receptor diversity in NOD mice prevents development of type 1 diabetes but not Sjögren's syndrome.

PubMed

Kern, Joanna; Drutel, Robert; Leanhart, Silvia; Bogacz, Marek; Pacholczyk, Rafal

2014-01-01

Non-obese diabetic (NOD) mice are well-established models of independently developing spontaneous autoimmune diseases, Sjögren's syndrome (SS) and type 1 diabetes (T1D). The key determining factor for T1D is the strong association with particular MHCII molecule and recognition by diabetogenic T cell receptor (TCR) of an insulin peptide presented in the context of I-Ag7 molecule. For SS the association with MHCII polymorphism is weaker and TCR diversity involved in the onset of the autoimmune phase of SS remains poorly understood. To compare the impact of TCR diversity reduction on the development of both diseases we generated two lines of TCR transgenic NOD mice. One line expresses transgenic TCRβ chain originated from a pathogenically irrelevant TCR, and the second line additionally expresses transgenic TCRαmini locus. Analysis of TCR sequences on NOD background reveals lower TCR diversity on Treg cells not only in the thymus, but also in the periphery. This reduction in diversity does not affect conventional CD4+ T cells, as compared to the TCRmini repertoire on B6 background. Interestingly, neither transgenic TCRβ nor TCRmini mice develop diabetes, which we show is due to lack of insulin B:9-23 specific T cells in the periphery. Conversely SS develops in both lines, with full glandular infiltration, production of autoantibodies and hyposalivation. It shows that SS development is not as sensitive to limited availability of TCR specificities as T1D, which suggests wider range of possible TCR/peptide/MHC interactions driving autoimmunity in SS.

Agronomic performance of Populus deltoides trees engineered for biofuel production

DOE PAGES

Macaya-Sanz, David; Chen, Jin?Gui; Kalluri, Udaya C.; ...

2017-11-30

Background: One of the major barriers to the development of lignocellulosic feedstocks is the recalcitrance of plant cell walls to deconstruction and saccharification. Recalcitrance can be reduced by targeting genes involved in cell wall biosynthesis, but this can have unintended consequences that compromise the agronomic performance of the trees under field conditions. Here we report the results of a field trial of fourteen distinct transgenic Populus deltoides lines that had previously demonstrated reduced recalcitrance without yield penalties under greenhouse conditions.Results: Survival and productivity of the trial were excellent in the first year, and there was little evidence for reduced performancemore » of the transgenic lines with modified target gene expression. Surprisingly, the most striking phenotypic effects in this trial were for two empty-vector control lines that had modified bud set and bud flush. This is most likely due to somaclonal variation or insertional mutagenesis. Traits related to yield, crown architecture, herbivory, pathogen response, and frost damage showed few significant differences between target gene transgenics and empty vector controls. However, there were a few interesting exceptions. Lines overexpressing the DUF231 gene, a putative O-acetyltransferase, showed early bud flush and marginally increased height growth. Lines overexpressing the DUF266 gene, a putative glycosyltransferase, had significantly decreased stem internode length and slightly higher volume index. Finally, lines overexpressing the PFD2 gene, a putative member of the prefoldin complex, had a slightly reduced volume index.Conclusions: This field trial demonstrates that these cell wall modifications, which decreased cell wall recalcitrance under laboratory conditions, did not seriously compromise first-year performance in the field, despite substantial challenges, including an outbreak of a stem boring insect (Gypsonoma haimbachiana), attack by a leaf rust pathogen (Melampsora spp.), and a late frost event. This bodes well for the potential utility of these lines as advanced biofuels feedstocks.« less

Agronomic performance of Populus deltoides trees engineered for biofuel production

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

Macaya-Sanz, David; Chen, Jin?Gui; Kalluri, Udaya C.

Background: One of the major barriers to the development of lignocellulosic feedstocks is the recalcitrance of plant cell walls to deconstruction and saccharification. Recalcitrance can be reduced by targeting genes involved in cell wall biosynthesis, but this can have unintended consequences that compromise the agronomic performance of the trees under field conditions. Here we report the results of a field trial of fourteen distinct transgenic Populus deltoides lines that had previously demonstrated reduced recalcitrance without yield penalties under greenhouse conditions.Results: Survival and productivity of the trial were excellent in the first year, and there was little evidence for reduced performancemore » of the transgenic lines with modified target gene expression. Surprisingly, the most striking phenotypic effects in this trial were for two empty-vector control lines that had modified bud set and bud flush. This is most likely due to somaclonal variation or insertional mutagenesis. Traits related to yield, crown architecture, herbivory, pathogen response, and frost damage showed few significant differences between target gene transgenics and empty vector controls. However, there were a few interesting exceptions. Lines overexpressing the DUF231 gene, a putative O-acetyltransferase, showed early bud flush and marginally increased height growth. Lines overexpressing the DUF266 gene, a putative glycosyltransferase, had significantly decreased stem internode length and slightly higher volume index. Finally, lines overexpressing the PFD2 gene, a putative member of the prefoldin complex, had a slightly reduced volume index.Conclusions: This field trial demonstrates that these cell wall modifications, which decreased cell wall recalcitrance under laboratory conditions, did not seriously compromise first-year performance in the field, despite substantial challenges, including an outbreak of a stem boring insect (Gypsonoma haimbachiana), attack by a leaf rust pathogen (Melampsora spp.), and a late frost event. This bodes well for the potential utility of these lines as advanced biofuels feedstocks.« less

Novel piplartine-containing ruthenium complexes: synthesis, cell growth inhibition, apoptosis induction and ROS production on HCT116 cells.

PubMed

D'Sousa Costa, Cinara O; Araujo Neto, João H; Baliza, Ingrid R S; Dias, Rosane B; Valverde, Ludmila de F; Vidal, Manuela T A; Sales, Caroline B S; Rocha, Clarissa A G; Moreira, Diogo R M; Soares, Milena B P; Batista, Alzir A; Bezerra, Daniel P

2017-11-28

Piplartine (piperlongumine) is a plant-derived molecule that has been receiving intense interest due to its anticancer characteristics that target the oxidative stress. In the present paper, two novel piplartine-containing ruthenium complexes [Ru(piplartine)(dppf)(bipy)](PF 6 ) 2 (1) and [Ru(piplartine)(dppb)(bipy)](PF 6 ) 2 (2) were synthesized and investigated for their cellular and molecular responses on cancer cell lines. We found that both complexes are more potent than metal-free piplartine in a panel of cancer cell lines on monolayer cultures, as well in 3D model of cancer multicellular spheroids formed from human colon carcinoma HCT116 cells. Mechanistic studies uncovered that the complexes reduced the cell growth and caused phosphatidylserine externalization, internucleosomal DNA fragmentation, caspase-3 activation and loss of the mitochondrial transmembrane potential on HCT116 cells. Moreover, the pre-treatment with Z-VAD(OMe)-FMK, a pan-caspase inhibitor, reduced the complexes-induced apoptosis, indicating cell death by apoptosis through caspase-dependent and mitochondrial intrinsic pathways. Treatment with the complexes also caused a marked increase in the production of reactive oxygen species (ROS), including hydrogen peroxide, superoxide anion and nitric oxide, and decreased reduced glutathione levels. Application of N-acetyl-cysteine, an antioxidant, reduced the ROS levels and apoptosis induced by the complexes, indicating activation of ROS-mediated apoptosis pathway. RNA transcripts of several genes, including gene related to the cell cycle, apoptosis and oxidative stress, were regulated under treatment. However, the complexes failed to induce DNA intercalation. In conclusion, the complexes are more potent than piplartine against different cancer cell lines and are able to induce caspase-dependent and mitochondrial intrinsic apoptosis on HCT116 cells by ROS-mediated pathway.

Silibinin inhibits migration and invasion of the rhabdoid tumor G401 cell line via inactivation of the PI3K/Akt signaling pathway.

PubMed

Li, Yumei; Zhang, Chunmei; Cai, Danfeng; Chen, Congde; Mu, Dongmei

2017-12-01

Rhabdoid tumors, which tend to occur prior to the age of 2 years, are one of the most aggressive malignancies and have a poor prognosis due to the frequency of metastasis. Silibinin, a natural extract, has been approved as a potential tumor suppressor in various studies, however, whether or not it also exerts its antitumor capacity in rhabdoid tumors, particularly with regards to tumor migration and invasion, is unclear. The rhabdoid tumor G401 cell line was used in the present in vitro study. An MTT assay was used to assess the cytotoxicity of silibinin on G401 cells, cell migration was studied using a wound healing assay and a Transwell migration assay, and cell invasion was determined using a Transwell invasion assay. The underlying mechanism in silibinin inhibited cell migration and invasion was investigated by western blot analysis and further confirmed using a specific inhibitor. Experimental results demonstrated that high doses of silibinin suppressed cell viability, and that low doses of silibinin inhibited cell migration and invasion without affecting cell proliferation. The phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway was involved in the silibinin-induced inhibition of metastasis. Silibinin inactivated the PI3K/Akt pathway, and inhibited cell migration and invasion, an effect that was further enhanced when LY294002, a classic PI3K inhibitor, was used concurrently. In general, silibinin inhibits migration and invasion of the rhabdoid tumor G401 cell line via inactivation of the PI3K/Akt signaling pathway and may be a potential chemotherapeutic drug to combat rhabdoid tumors in the future.

Resistance to etoposide-induced apoptosis in a Burkitt's lymphoma cell line.

PubMed

Zhao, E G; Song, Q; Cross, S; Misko, I; Lees-Miller, S P; Lavin, M F

1998-08-31

Burkitt's lymphoma cells that vary in their phenotypic characteristics show significantly different degrees of susceptibility to radiation-induced apoptosis. Propensity to undergo apoptosis is reflected in the degradation of substrates such as DNA-dependent protein kinase but the status of bcl-2, c-myc and p53 has been uninformative. In this study, we have focused on 2 Epstein-Barr virus (EBV)-associated Burkitt's cell lines, one (WW2) susceptible and the other (BL29) resistant to etoposide-induced apoptosis. Differences in expression of BHRF1, an EBV gene that is homologous to the Bcl-2 proto-oncogene and known to inhibit apoptosis, or changes in apoptosis inhibitory proteins (IAPs), did not appear to account for the difference in susceptibility in the 2 cell lines. Cytoplasmic extracts from etoposide-treated WW2 cells caused apoptotic changes in nuclei isolated from either BL29 or WW2 cells, whereas extracts from BL29 cells failed to do so. In addition, extracts from etoposide-treated WW2 cells degraded the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an important indicator of apoptosis, but this protein was resistant to degradation by BL29 extracts. It appears likely that caspase 3 (CPP32) is involved in this degradation since it was activated only in the apoptosis susceptible cells and the pattern of cleavage of DNA-PKcs was similar to that reported previously with recombinant caspase 3. As observed previously, addition of caspase 3 to nuclei failed to induce morphological changes indicative of apoptosis, but addition of caspase 3 to nuclei in the presence of extract from the resistant cells led to apoptotic changes. We conclude that resistance to apoptosis in BL29 cells is due to a failure of etoposide to activate upstream effectors of caspase activity.

Synthesis and biological evaluation of some novel triazole hybrids of curcumin mimics and their selective anticancer activity against breast and prostate cancer cell lines.

PubMed

Mandalapu, Dhanaraju; Saini, Karan S; Gupta, Sonal; Sharma, Vikas; Yaseen Malik, Mohd; Chaturvedi, Swati; Bala, Veenu; Hamidullah; Thakur, Subhadra; Maikhuri, Jagdamba P; Wahajuddin, Muhammad; Konwar, Rituraj; Gupta, Gopal; Sharma, Vishnu Lal

2016-09-01

The anti-cancer property of curcumin, an active component of turmeric, is limited due to its poor solubility, stability and bioavailability. To enhance its efficacy, we designed a novel series of twenty-four monocarbonyl curcumin analogue-1,2,3-triazole conjugates and evaluated their anti-cancer activity towards endocrine related cancers. The new compounds (17-40) were synthesized through CuAAC click reaction and SAR analysis carried out. Out of these all, compound 17 showed most significant anti-cancer activity against prostate cancer cells with IC50 values of 8.8μM and 9.5μM in PC-3 and DU-145 cells, respectively. Another compound 26 showed significant anti-cancer activity against breast cancer cells with IC50 of 6μM, 10μM and 6.4μM in MCF-7, MDA-MB-231 and 4T1 cells, respectively while maintaining low toxicity towards non-cancer originated cell line, HEK-293. Compounds 17 and 26 arrested cell cycle and induced mitochondria-mediated apoptosis in cancer cells. Further, both of these compounds significantly down-regulated cell proliferation marker (PCNA), inhibited activation of cell survival protein (Akt phosphorylation), upregulated pro-apoptotic protein (Bax) and down-regulated anti-apoptotic protein (Bcl-2) in their respective cell lines. In addition, in vitro stability, solubility and plasma binding studies of the compounds 17 and 26 showed them to be metabolically stable. Thus, this study identified two new curcumin monocarbonyl-1,2,3-triazole conjugate compounds with more potent activity than curcumin against breast and prostate cancers. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tolcapone induces oxidative stress leading to apoptosis and inhibition of tumor growth in Neuroblastoma.

PubMed

Maser, Tyler; Rich, Maria; Hayes, David; Zhao, Ping; Nagulapally, Abhinav B; Bond, Jeffrey; Saulnier Sholler, Giselle

2017-06-01

Catechol-O-methyltransferase (COMT) is an enzyme that inactivates dopamine and other catecholamines by O-methylation. Tolcapone, a drug commonly used in the treatment of Parkinson's disease, is a potent inhibitor of COMT and previous studies indicate that Tolcapone increases the bioavailability of dopamine in cells. In this study, we demonstrate that Tolcapone kills neuroblastoma (NB) cells in preclinical models by inhibition of COMT. Treating four established NB cells lines (SMS-KCNR, SH-SY5Y, BE(2)-C, CHLA-90) and two primary NB cell lines with Tolcapone for 48 h decreased cell viability in a dose-dependent manner, with IncuCyte imaging and Western blotting indicating that cell death was due to caspase-3-mediated apoptosis. Tolcapone also increased ROS while simultaneously decreasing ATP-per-cell in NB cells. Additionally, COMT was inhibited by siRNA in NB cells and showed similar increases in apoptotic markers compared to Tolcapone. In vivo xenograft models displayed inhibition of tumor growth and a significant decrease in time-to-event in mice treated with Tolcapone compared to untreated mice. These results indicate that Tolcapone is cytotoxic to neuroblastoma cells and invite further studies into Tolcapone as a promising novel therapy for the treatment of neuroblastoma. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Drug loaded silica coated MnFe2O4 ferromagnetic biomaterials for targeted cancer treatment

NASA Astrophysics Data System (ADS)

Anand, Vikas; Singh, K. J.; Kaur, Kulwinder; Bhatia, Gaurav

2017-05-01

Magnetically attracted silica coated MnFe2O4 samples have been prepared by using co-precipitation method. Structural changes have been confirmed from XRD spectra. Ferromagnetic behavior of samples has been studied by using vibration sample magnetometer. Cytotoxicity and cell culture of samples have been investigated by using human MG63 cell line and found that sample provide a healthy environment to the growth of cell lines. Drug carrier ability of sample has been checked with gentamycin as an antibiotic and results show that sample can be used as excellent drug carriers. Drug loaded samples can be easily targeted to specific area due to their attractive nature towards external magnetic field. Moreover, magnetic nanoparticles can also be used to kill the cancer cells by using hyperthermia technique. Hyperthermia is a process to raise the temperature of surrounding cells in the presence of external AC magnetic field above the maximum temperature limit for surviving of the cancer cells. Cancer cell can survive only up to 47°C. After that cancer cells start to die, but healthy cells can easily survive up to higher temperature. Our results indicate that prepared samples possess good drug carrier ability and hence, can be potential candidates for cancer cell treatment.

Oxygen-Glucose Deprivation (OGD) Modulates the Unfolded Protein Response (UPR) and Inflicts Autophagy in a PC12 Hypoxia Cell Line Model.

PubMed

Vavilis, Theofanis; Delivanoglou, Nikoleta; Aggelidou, Eleni; Stamoula, Eleni; Mellidis, Kyriakos; Kaidoglou, Aikaterini; Cheva, Angeliki; Pourzitaki, Chryssa; Chatzimeletiou, Katerina; Lazou, Antigone; Albani, Maria; Kritis, Aristeidis

2016-07-01

Hypoxia is the lack of sufficient oxygenation of tissue, imposing severe stress upon cells. It is a major feature of many pathological conditions such as stroke, traumatic brain injury, cerebral hemorrhage, perinatal asphyxia and can lead to cell death due to energy depletion and increased free radical generation. The present study investigates the effect of hypoxia on the unfolded protein response of the cell (UPR), utilizing a 16-h oxygen-glucose deprivation protocol (OGD) in a PC12 cell line model. Expression of glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94), key players of the UPR, was studied along with the expression of glucose-regulated protein 75 (GRP75), heat shock cognate 70 (HSC70), and glyceraldehyde 3-phosphate dehydrogenase, all with respect to the cell death mechanism(s). Cells subjected to OGD displayed upregulation of GRP78 and GRP94 and concurrent downregulation of GRP75. These findings were accompanied with minimal apoptotic cell death and induction of autophagy. The above observation warrants further investigation to elucidate whether autophagy acts as a pro-survival mechanism that upon severe and prolonged hypoxia acts as a concerted cell response leading to cell death. In our OGD model, hypoxia modulates UPR and induces autophagy.

Carboplatin resistant human laryngeal carcinoma cells are cross resistant to curcumin due to reduced curcumin accumulation.

PubMed

Rak, Sanjica; Cimbora-Zovko, Tamara; Gajski, Goran; Dubravčić, Klara; Domijan, Ana-Marija; Delaš, Ivančica; Garaj-Vrhovac, Verica; Batinić, Drago; Sorić, Jasna; Osmak, Maja

2013-03-01

Curcumin is a natural compound that exhibits a wide range of beneficial effects, among them the anti-tumor activity. Recently it was shown that curcumin may be efficient against drug resistant tumor cells. The goal of our investigation was to examine if human laryngeal carcinoma cells resistant to carboplatin display sensitivity to curcumin, as compared to parental cells, and if this sensitivity is altered, to determine the molecular mechanisms that are responsible for it. We found that carboplatin resistant 7T cells were also cross resistant to curcumin. After the treatment with equimolar concentration of curcumin, 7T cells exhibited lower intracellular accumulation of curcumin which coincided with reduced formation of reactive oxygen species (ROS), diminished lipid and DNA damage followed by reduced induction of apoptosis and expression of heat shock protein 70 (Hsp70), as compared to parental HEp-2 cells. However, after the treatment with equitoxic concentration of curcumin, intracellular accumulation and all the explored downstream effects were similar in both cell lines suggesting that resistance of 7T cells to curcumin was based on its reduced intracellular accumulation. Since curcumin accumulates mostly in the membranes, we explored the fatty acid composition of both cell lines, but we did not find any difference between them. Copyright © 2012 Elsevier Ltd. All rights reserved.

Measurement of DNA repair deficiency in workers exposed to benzene

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

Hallberg, L.M.; Au, W.W.; El Zein, R.

1996-05-01

We hypothesize that chronic exposure to environmental toxicants can induce genetic damage causing DNA repair deficiencies and leading to the postulated mutator phenotype of carcinogenesis. To test our hypothesis, a host cell reactivation (HCR) assay was used in which pCMVcat plasmids were damaged with UV light (175, 350 J/m{sup 2} UV light), inactivating the chloramphenicol acetyltransferase reporter gene, and then transfected into lymphocytes. Transfected lymphocytes were therefore challenged to repair the damaged plasmids, reactivating the reporter gene. Xeroderma pigmentosum (XP) and Gaucher cell lines were used as positive and negative controls for the HCR assay. The Gaucher cell line repairedmore » normally but XP cell lines demonstrated lower repair activity. Additionally, the repair activity of the XP heterozygous cell line showed intermediate repair compared to the homozygous XP and Gaucher cells. We used HCR to measure the effects of benzene exposure on 12 exposed and 8 nonexposed workers from a local benzene plant. Plasmids 175 J/m{sup 2} and 350 J/m{sup 2} were repaired with a mean frequency of 66% and 58%, respectively, in control workers compared to 71% and 62% in exposed workers. Conversely, more of the exposed workers were grouped into the reduced repair category than controls. These differences in repair capacity between exposed and control workers were, however, not statistically significant. The lack of significant differences between the exposed and control groups may be due to extremely low exposure to benzene (<0.3 ppm), small population size, or a lack of benzene genotoxicity at these concentrations. These results are consistent with a parallel hprt gene mutation assay. 26 refs., 4 figs., 2 tabs.« less

MicroRNAs associated with the efficacy of photodynamic therapy in biliary tract cancer cell lines.

PubMed

Wagner, Andrej; Mayr, Christian; Bach, Doris; Illig, Romana; Plaetzer, Kristjan; Berr, Frieder; Pichler, Martin; Neureiter, Daniel; Kiesslich, Tobias

2014-11-05

Photodynamic therapy (PDT) is a palliative treatment option for unresectable hilar biliary tract cancer (BTC) showing a considerable benefit for survival and quality of life with few side effects. Currently, factors determining the cellular response of BTC cells towards PDT are unknown. Due to their multifaceted nature, microRNAs (miRs) are a promising analyte to investigate the cellular mechanisms following PDT. For two photosensitizers, Photofrin® and Foscan®, the phototoxicity was investigated in eight BTC cell lines. Each cell line (untreated) was profiled for expression of n=754 miRs using TaqMan® Array Human MicroRNA Cards. Statistical analysis and bioinformatic tools were used to identify miRs associated with PDT efficiency and their putative targets, respectively. Twenty miRs correlated significantly with either high or low PDT efficiency. PDT was particularly effective in cells with high levels of clustered miRs 25-93*-106b and (in case of miR-106b) a phenotype characterized by high expression of the mesenchymal marker vimentin and high proliferation (cyclinD1 and Ki67 expression). Insensitivity towards PDT was associated with high miR-200 family expression and (for miR-cluster 200a/b-429) expression of differentiation markers Ck19 and Ck8/18. Predicted and validated downstream targets indicate plausible involvement of miRs 20a*, 25, 93*, 130a, 141, 200a, 200c and 203 in response mechanisms to PDT, suggesting that targeting these miRs could improve susceptibility to PDT in insensitive cell lines. Taken together, the miRNome pattern may provide a novel tool for predicting the efficiency of PDT and-following appropriate functional verification-may subsequently allow for optimization of the PDT protocol.

Hepatoprotective activity of punarnavashtak kwath, an Ayurvedic formulation, against CCl4-induced hepatotoxicity in rats and on the HepG2 cell line.

PubMed

Shah, Vaishali N; Shah, Mamta B; Bhatt, Parloop A

2011-04-01

Punarnavashtak kwath (PNK) is a classical Ayurvedic formulation, mentioned in Ayurvedic literature Bhaishajya Ratnavali, for hepatic disorders and asthma. This study investigated the hepatoprotective activity of PNK to validate the traditional use of this formulation. PNK was prepared in the laboratory according to the method given in Ayurvedic literature. Phytochemical screening was performed to determine the presence of phytoconstituents. Hepatoprotective activity was evaluated against CCl(4)-induced hepatotoxicity in rats and by its effect on the HepG2 cell line. Preliminary phytochemical screening revealed the presence of alkaloids, tannins, flavonoids, saponins, and a bitter principle in PNK. Administration of PNK produced significant hepatoprotective effect as demonstrated by decreased levels of serum liver marker enzymes such as aspartate transaminase, serum alanine transaminase, serum alkaline phosphatase, and serum bilirubin and an increase in protein level. Thiopentone-induced sleeping time was also decreased in the PNK-treated animals compared with the CCl(4)-treated group. It also showed antioxidant activity by increase in activity of glutathione, superoxide dismutase, and catalase and by a decrease in thiobarbituric acid reactive substance level compared with the CCl(4)-treated group. Results of a histopathological study also support the hepatoprotective activity of PNK. Investigation carried out on the HepG2 cell line depicted significant increase in viability of cells exposed to PNK as compared with CCl(4)-treated cells. It can be concluded that PNK protects hepatocytes from CCl(4)-induced liver damages due to its antioxidant effect on hepatocytes. An in vitro study on HepG2 cell lines also supports its protective effect.

Molecular and cytogenetic analysis of human diploid fibroblast cells transformed by simian virus 40: What causes immortalization

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

Patsalis, P.C.

1993-01-01

Transformation of human diploid fibroblasts (HF) with SV40 can result in extension of life span beyond the normal limit of senescence and in a minority of cases, immortalization. This study used comparison of matched parental (diploid) and immortalized cell lines to determine if any single genetic factor could be related to the immortalization phenomena. The integration site of SV40 was shown to be at chromosome 5q21 by cytologic hybridization. A comparison of mortal and immortal cells showed no alterations involving the integrated SV40 genome per se. Karyotypic analysis of matched cell lines identified a specific chromosomal breakpoint (6q21) in immortalizedmore » cells that was not present in the parental line. Hybridization analysis confirmed that sequences on the distal portion of 6q are lost in immortalized cells. Two single copy DNAs which flank the breakpoint were identified and used to further define the exact breakpoint on 6q13. FISH analysis demonstrated the region 6q13 [yields] 21 as belonging to another chromosome and confirmed the 6q13 breakpoint. A survey of random translocations and other anomalies occurring in the immortalized lines was also made. Some of these are regions known to contain oncogenes and transforming proteins. The MCC tumor suppressor gene was rearranged and deregulated. The genes DCC, Bel-2, APC were also found to be deregulated. The authors propose that deletion of specific sequences due to breakage of chromosome 6q represent one of the mutational events responsible for immortalization of SV40 transformed HF. In addition, the MCC and possibly other genes are involved in the progression of immortalization.« less

[Tumor cells transfer between the patient and laboratory animal as a basic methodological approach to the study of cancerogenesis and identification of biomarkers].

PubMed

Klos, D; Stašek, M; Loveček, M; Skalický, P; Vrba, R; Aujeský, R; Havlík, R; Neoral, Č; Varanashi, L; Hajdúch, M; Vrbková, J; Džubák, P

The investigation of prognostic and predictive factors for early diagnosis of tumors, their surveillance and monitoring of the impact of therapeutic modalities using hybrid laboratory models in vitro/in vivo is an experimental approach with a significant potential. It is preconditioned by the preparation of in vivo tumor models, which may face a number of potential technical difficulties. The assessment of technical success of grafting and xenotransplantation based on the type of the tumor or cell line is important for the preparation of these models and their further use for proteomic and genomic analyses. Surgically harvested gastrointestinal tract tumor tissue was processed or stable cancer cell lines were cultivated; the viability was assessed, and subsequently the cells were inoculated subcutaneously to SCID mice with an individual duration of tumor growth, followed by its extraction. We analysed 140 specimens of tumor tissue including 17 specimens of esophageal cancer (viability 13/successful inoculations 0), 13 tumors of the cardia (11/0), 39 gastric tumors (24/4), 47 pancreatic tumors (34/1) and 24 specimens of colorectal cancer (22/9). 3 specimens were excluded due to histological absence of the tumor (complete remission after neoadjuvant therapy in 2 cases of esophageal carcinoma, 1 case of chronic pancreatitis). We observed successful inoculation in 17 of 28 tumor cell lines. The probability of successful grafting to the mice model in tumors of the esophagus, stomach and pancreas is significantly lower in comparison with colorectal carcinoma and cell lines generated tumors. The success rate is enhanced upon preservation of viability of the harvested tumor tissue, which depends on the sequence of clinical and laboratory algorithms with a high level of cooperation.Key words: proteomic analysis - xenotransplantation - prognostic and predictive factors - gastrointestinal tract tumors.

Lipophilicity-dependent ruthenium N-heterocyclic carbene complexes as potential anticancer agents.

PubMed

Lv, Gaochao; Guo, Liubin; Qiu, Ling; Yang, Hui; Wang, Tengfei; Liu, Hong; Lin, Jianguo

2015-04-28

Five Ru(II)-N-heterocyclic carbenes (NHC) (1-5) were synthesized by reacting the appropriately substituted imidazolium chlorides with Ag2O, forming the NHC-silver chloride in situ followed by transmetalation with dimeric p-cymene ruthenium(II) dichloride. All the complexes were characterized by NMR and ESI-MS, and complex 1 was also characterized by single-crystal X-ray diffraction. The IC50 values of these five complexes were determined by the MTT-based assay on four human cancer cell lines, SKOV-3 (ovarian), PC-3 (prostate), MDA-MB-231 (breast) and EC109 (esophagus). The cytotoxicities of these complexes changed from a moderate effect to a fine one, corresponding to the increasing lipophilicity order of the complex of 2 < 1 < 3 < 4 < 5 (0.91, 0.88, 1.36, 1.85 and 2.62 for 1–5 respectively). Complex 5 showed the most cytotoxicity with the IC50 values 10.3 ± 0.3 μM for SKOV-3, 2.9 ± 0.1 μM for PC-3, 8.2 ± 0.6 μM for MDA-MB-231, 6.4 ± 0.2 μM for EC109 cell lines. Due to the superior cytotoxicity of complex 5 against the PC-3 cell lines, further biological evaluations were carried out to elucidate its action mechanism. The morphologic changes and cell cycle analysis showed that complex 5 can inhibit PC-3 cell lines by inducing cell cycle arrest at the G2/M phase. The DNA binding experiments further demonstrate that complex 5 has a better binding ability for DNA (Kb = 2.2 × 10(6) M(-1)) than complexes 1-4 (3.8 × 10(5), 7.0 × 10(5), 5.7 × 10(5), and 1.9 × 10(5) respectively).

MicroRNAs Associated with the Efficacy of Photodynamic Therapy in Biliary Tract Cancer Cell Lines

PubMed Central

Wagner, Andrej; Mayr, Christian; Bach, Doris; Illig, Romana; Plaetzer, Kristjan; Berr, Frieder; Pichler, Martin; Neureiter, Daniel; Kiesslich, Tobias

2014-01-01

Photodynamic therapy (PDT) is a palliative treatment option for unresectable hilar biliary tract cancer (BTC) showing a considerable benefit for survival and quality of life with few side effects. Currently, factors determining the cellular response of BTC cells towards PDT are unknown. Due to their multifaceted nature, microRNAs (miRs) are a promising analyte to investigate the cellular mechanisms following PDT. For two photosensitizers, Photofrin® and Foscan®, the phototoxicity was investigated in eight BTC cell lines. Each cell line (untreated) was profiled for expression of n = 754 miRs using TaqMan® Array Human MicroRNA Cards. Statistical analysis and bioinformatic tools were used to identify miRs associated with PDT efficiency and their putative targets, respectively. Twenty miRs correlated significantly with either high or low PDT efficiency. PDT was particularly effective in cells with high levels of clustered miRs 25-93*-106b and (in case of miR-106b) a phenotype characterized by high expression of the mesenchymal marker vimentin and high proliferation (cyclinD1 and Ki67 expression). Insensitivity towards PDT was associated with high miR-200 family expression and (for miR-cluster 200a/b-429) expression of differentiation markers Ck19 and Ck8/18. Predicted and validated downstream targets indicate plausible involvement of miRs 20a*, 25, 93*, 130a, 141, 200a, 200c and 203 in response mechanisms to PDT, suggesting that targeting these miRs could improve susceptibility to PDT in insensitive cell lines. Taken together, the miRNome pattern may provide a novel tool for predicting the efficiency of PDT and—following appropriate functional verification—may subsequently allow for optimization of the PDT protocol. PMID:25380521

Thymol Elicits HCT-116 Colorectal Carcinoma Cell Death Through Induction of Oxidative Stress.

PubMed

Chauhan, Anil Kumar; Bahuguna, Ashutosh; Paul, Souren; Kang, Sun Chul

2018-02-07

Colon cancer is one of the most deadly and common carcinomas occurring worldwide and there have been many attempts to treat this cancer. The present work was designed in order to evaluate thymol as a potent drug against colon cancer. Cytotoxicity of thymol at different concentrations was evaluated against a human colon carcinoma cell line (HCT-116 cells). Fluorescent staining was carried out to evaluate the level of ROS as well as mitochondrial and DNA fragmentation and immunoblot analysis were performed to confirm apoptosis and mitoptosis. Results of the study demonstrated that thymol efficiently created an oxidative stress environment inside HCT-116 cells, a colorectal carcinoma cell line, through induction of ROS production along with intense damage to DNA and mitochondria, as observed through Hoechst and rhodamine 123 staining, respectively. Moreover, expression of PARP-1, p-JNK, cytochrome-C and caspase-3 proteins was up-regulated, suggesting HCT-116 cells underwent mitoptotic cell death. Therefore, thymol could be used as a potent drug against colon cancer due to its lower toxicity and prevalence in natural medicinal plants. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Speed genome editing by transient CRISPR/Cas9 targeting and large DNA fragment deletion.

PubMed

Luo, Jing; Lu, Liaoxun; Gu, Yanrong; Huang, Rong; Gui, Lin; Li, Saichao; Qi, Xinhui; Zheng, Wenping; Chao, Tianzhu; Zheng, Qianqian; Liang, Yinming; Zhang, Lichen

2018-06-07

Genetic engineering of cell lines and model organisms has been facilitated enormously by the CRISPR/Cas9 system. However, in cell lines it remains labor intensive and time consuming to obtain desirable mutant clones due to the difficulties in isolating the mutated clones and sophisticated genotyping. In this study, we have validated fluorescent protein reporter aided cell sorting which enables the isolation of maximal diversity in mutant cells. We further applied two spectrally distinct fluorescent proteins DsRed2 and ECFP as reporters for independent CRISPR/Cas9 mediated targeting, which allows for one-cell-one-well sorting of the mutant cells. Because of ultra-high efficiency of the CRISPR/Cas9 system with dual reporters and large DNA fragment deletion resulting from independent loci cleavage, monoclonal mutant cells could be easily identified by conventional PCR. In the speed genome editing method presented here, sophisticated genotyping methods are not necessary to identify loss of function mutations after CRISPR/Cas9 genome editing, and desirable loss of function mutant clones could be obtained in less than one month following transfection. Copyright © 2018 Elsevier B.V. All rights reserved.

Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A)-Mediated changes in Fas Expression and Fas-Dependent Apoptosis: Role of Lyn/Syk activation

PubMed Central

Incrocci, Ryan; Hussain, Samira; Stone, Amanda; Bieging, Kathryn; Alt, Lauren A.C.; Fay, Michael J.; Swanson-Mungerson, Michelle

2015-01-01

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, −3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies. PMID:26255694

Establishment of nude mice with complete loss of lymphocytes and NK cells and application for in vivo bio-imaging.

PubMed

Kariya, Ryusho; Matsuda, Kouki; Gotoh, Kumiko; Vaeteewoottacharn, Kulthida; Hattori, Shinichiro; Okada, Seiji

2014-01-01

Nude mice are used in human xenograft research; however, only 25-35% of human tumors have been successfully transplanted into nude mice and their application is limited due to high natural killer (NK) cell activity. More severely immunodeficient mice with loss of NK activity are needed to overcome this limitation. Balb/c nude Rag-2(-/-)Jak3(-/-) (Nude-RJ) mice were established by crossing Rag-2(-/-)Jak3(-/-) mice and nude mice. The K562 cell line was implanted subcutaneously to compare tumorigenicity between Nude-RJ mice and Nude mice. The cholangiocarcinoma mCherry expressing cell line (KKU-M213) was implanted subcutaneously, and fluorescence intensity and tumor weight were measured. Nude R/J mice showed complete loss of lymphocytes and NK cells. Xeno-transplantation of K562 cells showed higher proliferation in Nude R/J mice than nude mice. Subcutaneously-transplanted mCherry-transduced KKU-M213 cells were successfully detected with a fluorescence imager. Nude-R/J mice are valuable tools for in vivo imaging studies in biomedical research. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Bone formation in vitro and in nude mice by human osteosarcoma cells.

PubMed

Ogose, A; Motoyama, T; Hotta, T; Watanabe, H; Takahashi, H E

1995-01-01

Osteosarcomas contain variable amounts of bony tissue, but the mechanism of bone formation by osteosarcoma is not well understood. While a number of cultured human osteosarcoma cell lines have been established, they are maintained by different media and differ qualitatively with regard to bone formation. We examined different media for their ability to support bone formation in vitro and found the alpha-modification of Eagle's minimal essential medium supplemented with beta glycerophosphate was best for this purpose, because it contained the proper calcium and phosphate concentrations. Subsequently, we compared seven human osteosarcoma cell lines under the same experimental conditions to clarify their ability to induce bone formation. NOS-1 cells most frequently exhibited features of bone formation in vitro and in nude mice. Collagen synthesis by tumour cells themselves seemed to be the most important factor for bone volume. However, even HuO9 cells, which lacked collagen synthesis and failed to form bone in vitro, successfully formed tumours containing bone in nude mice. Histological analysis of HuO9 cells in diffusion chambers implanted in nude mice and the findings of polymerase chain reaction indicated that the phenomenon was probably due to bone morphogenetic protein.

Drug screening of cancer cell lines and human primary tumors using droplet microfluidics.

PubMed

Wong, Ada Hang-Heng; Li, Haoran; Jia, Yanwei; Mak, Pui-In; Martins, Rui Paulo da Silva; Liu, Yan; Vong, Chi Man; Wong, Hang Cheong; Wong, Pak Kin; Wang, Haitao; Sun, Heng; Deng, Chu-Xia

2017-08-22

Precision Medicine in Oncology requires tailoring of therapeutic strategies to individual cancer patients. Due to the limited quantity of tumor samples, this proves to be difficult, especially for early stage cancer patients whose tumors are small. In this study, we exploited a 2.4 × 2.4 centimeters polydimethylsiloxane (PDMS) based microfluidic chip which employed droplet microfluidics to conduct drug screens against suspended and adherent cancer cell lines, as well as cells dissociated from primary tumor of human patients. Single cells were dispersed in aqueous droplets and imaged within 24 hours of drug treatment to assess cell viability by ethidium homodimer 1 staining. Our results showed that 5 conditions could be screened for every 80,000 cells in one channel on our chip under current circumstances. Additionally, screening conditions have been adapted to both suspended and adherent cancer cells, giving versatility to potentially all types of cancers. Hence, this study provides a powerful tool for rapid, low-input drug screening of primary cancers within 24 hours after tumor resection from cancer patients. This paves the way for further technological advancement to cutting down sample size and increasing drug screening throughput in advent to personalized cancer therapy.

Increased mitochondrial-encoded gene transcription in immortal DF-1 cells.

PubMed

Kim, H; You, S; Kim, I J; Farris, J; Foster, L K; Foster, D N

2001-05-01

We have established, in continuous cell culture, a spontaneously immortalized chicken embryo fibroblast (CEF) cell line (DF-1) as well as several other immortal CEF cell lines. The immortal DF-1 cells divided more rapidly than primary and other immortal CEF cells. To identify the genes involved in rapidly dividing DF-1 cells, we have used differential display RT-PCR. Of the numerous genes analyzed, three mitochondrial-encoded genes (ATPase 8/6, 16S rRNA, and cytochrome b) were shown to express at higher levels in DF-1 cells compared to primary and other immortal CEF cells. The inhibition of mitochondrial translation by treatment with chloramphenicol markedly decreased ATP production and cell proliferation in DF-1 cells, while not affecting growth in either primary or other immortal CEF cells. This result suggests a correlation between rapid cell proliferation and the increased mitochondrial respiratory functions. We also determined that the increased transcription of mitochondrial-encoded genes in DF-1 cells is due to increased de novo transcript synthesis as shown by mitochondrial run-on assays, and not the result of either increased mitochondrial biogenesis or mitochondrial transcript half-lives. Together, the present studies suggest that the transcriptional activation of mitochondrial-encoded genes and the elevated respiratory function should be one of the characteristics of rapidly dividing immortal cells. Copyright 2001 Academic Press.

Proteomic characterization of paired non-malignant and malignant African-American prostate epithelial cell lines distinguishes them by structural proteins.

PubMed

Myers, Jennifer S; Vallega, Karin A; White, Jason; Yu, Kaixian; Yates, Clayton C; Sang, Qing-Xiang Amy

2017-07-11

While many factors may contribute to the higher prostate cancer incidence and mortality experienced by African-American men compared to their counterparts, the contribution of tumor biology is underexplored due to inadequate availability of African-American patient-derived cell lines and specimens. Here, we characterize the proteomes of non-malignant RC-77 N/E and malignant RC-77 T/E prostate epithelial cell lines previously established from prostate specimens from the same African-American patient with early stage primary prostate cancer. In this comparative proteomic analysis of RC-77 N/E and RC-77 T/E cells, differentially expressed proteins were identified and analyzed for overrepresentation of PANTHER protein classes, Gene Ontology annotations, and pathways. The enrichment of gene sets and pathway significance were assessed using Gene Set Enrichment Analysis and Signaling Pathway Impact Analysis, respectively. The gene and protein expression data of age- and stage-matched prostate cancer specimens from The Cancer Genome Atlas were analyzed. Structural and cytoskeletal proteins were differentially expressed and statistically overrepresented between RC-77 N/E and RC-77 T/E cells. Beta-catenin, alpha-actinin-1, and filamin-A were upregulated in the tumorigenic RC-77 T/E cells, while integrin beta-1, integrin alpha-6, caveolin-1, laminin subunit gamma-2, and CD44 antigen were downregulated. The increased protein level of beta-catenin and the reduction of caveolin-1 protein level in the tumorigenic RC-77 T/E cells mirrored the upregulation of beta-catenin mRNA and downregulation of caveolin-1 mRNA in African-American prostate cancer specimens compared to non-malignant controls. After subtracting race-specific non-malignant RNA expression, beta-catenin and caveolin-1 mRNA expression levels were higher in African-American prostate cancer specimens than in Caucasian-American specimens. The "ECM-Receptor Interaction" and "Cell Adhesion Molecules", and the "Tight Junction" and "Adherens Junction" pathways contained proteins are associated with RC-77 N/E and RC-77 T/E cells, respectively. Our results suggest RC-77 T/E and RC-77 N/E cell lines can be distinguished by differentially expressed structural and cytoskeletal proteins, which appeared in several pathways across multiple analyses. Our results indicate that the expression of beta-catenin and caveolin-1 may be prostate cancer- and race-specific. Although the RC-77 cell model may not be representative of all African-American prostate cancer due to tumor heterogeneity, it is a unique resource for studying prostate cancer initiation and progression.

Real-life effectiveness of erlotinib as second-line treatment of stage IIIB/IV squamous non-small cell lung cancer: Results of the PEPiTA observational study.

PubMed

Monnet, Isabelle; Audigier-Valette, Clarisse; Girard, Nicolas; Vergnenègre, Alain; Molinier, Olivier; Souquet, Pierre Jean; Blanchon, François; Bonnetain, Franck; Taguieva-Pioger, Naila; Lamour, Corinne; Wislez, Marie

2016-08-01

Erlotinib, an inhibitor of tyrosine kinase activity of the epidermal growth factor receptor, is effective in non-small cell lung cancer (NSCLC). Data on erlotinib use in squamous NSCLC are limited. This observational study aimed at evaluating the efficacy and safety of second-line erlotinib in patients with stage IIIB/IV squamous NSCLC in a real-life setting. Patients with predominantly squamous stage IIIB/IV NSCLC, who failed first-line platinum-based therapy, were recruited and followed-up for 12 months. Patients underwent visits each trimester. Data were derived from case report forms, and functional assessment of cancer therapy-lung (FACT-L) questionnaires. A total of 152 patients were enrolled; the majority were males (90%) and mean age was 67.7 years. All patients had squamous (97%) or predominantly squamous (3%) NSCLC, of stage IIIB (21%) or IV (79%). Median progression free survival (PFS) and overall survival were 3 and 5.8 months, respectively. Disease progression was observed in the majority of the patients, mostly due to progression of primary tumour and/or metastatic sites, and led to death in 91/107 of patients. Of the 107 deaths reported, none were due to erlotinib. FACT-L questionnaires were interpretable up to the first visit and were in line with PFS data, showing a relatively good quality of life up to Month 3 (mean total score=78.8). No new or unexpected safety issues were reported. The results of this real-life cohort study like those of previous phase III/IV subgroups study analyses indicate that erlotinib is a valuable option for second-line treatment of stage IIIB/IV squamous NSCLC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Ontological representation, integration, and analysis of LINCS cell line cells and their cellular responses.

PubMed

Ong, Edison; Xie, Jiangan; Ni, Zhaohui; Liu, Qingping; Sarntivijai, Sirarat; Lin, Yu; Cooper, Daniel; Terryn, Raymond; Stathias, Vasileios; Chung, Caty; Schürer, Stephan; He, Yongqun

2017-12-21

Aiming to understand cellular responses to different perturbations, the NIH Common Fund Library of Integrated Network-based Cellular Signatures (LINCS) program involves many institutes and laboratories working on over a thousand cell lines. The community-based Cell Line Ontology (CLO) is selected as the default ontology for LINCS cell line representation and integration. CLO has consistently represented all 1097 LINCS cell lines and included information extracted from the LINCS Data Portal and ChEMBL. Using MCF 10A cell line cells as an example, we demonstrated how to ontologically model LINCS cellular signatures such as their non-tumorigenic epithelial cell type, three-dimensional growth, latrunculin-A-induced actin depolymerization and apoptosis, and cell line transfection. A CLO subset view of LINCS cell lines, named LINCS-CLOview, was generated to support systematic LINCS cell line analysis and queries. In summary, LINCS cell lines are currently associated with 43 cell types, 131 tissues and organs, and 121 cancer types. The LINCS-CLO view information can be queried using SPARQL scripts. CLO was used to support ontological representation, integration, and analysis of over a thousand LINCS cell line cells and their cellular responses.

Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines.

PubMed

Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

2017-03-01

Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.

Histopathologic and Myogenic Gene Expression Changes Associated with Wooden Breast in Broiler Breast Muscles.

PubMed

Velleman, Sandra G; Clark, Daniel L

2015-09-01

The wooden breast condition is a myopathy affecting the pectoralis major (p. major) muscle in fast-growing commercial broiler lines. Currently, wooden breast-affected birds are phenotypically detected by palpation of the breast area, with affected birds having a very hard p. major muscle that is of lower value. The objective of this study was to compare the wooden breast myopathy in two fast-growing broiler lines (Lines A and B) with incidence of wooden breast to a slower growing broiler Line C with no phenotypically observable wooden breast. One of the characteristics of the wooden breast condition is fibrosis of the p. major muscle. Morphologic assessment of Lines A and B showed significant fibrosis in both lines, but the collagen distribution and arrangement of the collagen fibrils was different. In Line A, the collagen fibrils were tightly packed, whereas in Line B the collagen fibrils were diffuse. This difference in collagen organization may be due to the expression of the extracellular matrix proteoglycan decorin. Decorin is a regulator of collagen crosslinking and is expressed at significantly higher levels in Line A wooden breast-affected p. major muscle, which would lead to tightly packed collagen fibers due to high levels of collagen crosslinking. Furthermore, expression of the muscle-specific transcriptional regulatory factors for proliferation and differentiation of muscle cells leading to the regeneration of muscle in response to muscle damage was significantly elevated in Line A, and only the factor for differentiation, myogenin, was increased in Line B. The results from this study provide initial evidence that the etiology of the wooden breast myopathy may vary between fast-growing commercial broiler lines.

Oncolytic adenovirus encoding tumor necrosis factor-related apoptosis inducing ligand (TRAIL) inhibits the growth and metastasis of triple-negative breast cancer

PubMed Central

Zhu, Wei; Zhang, Hongwei; Shi, Yi; Song, Mangen; Zhu, Bijun; Wei, Lai

2013-01-01

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a promising cancer therapeutic target due to its selective apoptosis-inducing effect in cancer cells. To efficiently deliver TRAIL to the tumor cells, an oncolytic adenovirus (p55-hTERT-HRE-TRAIL) carrying the TRAIL coding sequence was constructed. In the present study, we aimed to investigate the effect of p55-hTERT-HRE-TRAIL on the growth and metastasis of triple-negative breast cancer (TNBC). We observed that infection of the recombinant adenovirus resulted in expression of TRAIL and massive cell death in a TNBC cell line MDA-MB-231. This effect is much weaker in MCF-10A, which is a normal breast cell line. Administration of P55-HTERT-HRE-TRAIL significantly reduced orthotopic breast tumor growth and extended survival in a metastatic model. Our results suggest the oncolytic adenovirus armed with P55-HTERT-HRE-TRAIL, which exhibited enhanced anti-tumor activity and improved survival, is a promising candidate for virotherapy of TNBC. PMID:24025362

In vitro antioxidant and antiproliferative activities of six international basil cultivars.

PubMed

Elansary, Hosam O; Mahmoud, Eman A

2015-01-01

The total phenolic, flavonoid and tannin contents in leaves extracts of Ocimum basilicum (OB) (Lamiaceae) international cultivars, as well as their overall antioxidant activities using DPPH and linoleic acid assays, were investigated. Furthermore, the antiproliferative and cytotoxic activities against line HeLa, MCF-7, Jurkat, HT-29, T24, MIAPaCa-2 cancer cells and one normal human cell line HEK-293 were examined. DPPH and linoleic acid assays ranged from 75.8% to 93.3% and from 74.5% to 97.1%; respectively. O. b. 'purple ruffle', O. b. 'dark opale', O. b. 'genovese', O. b. 'anise', O. b. 'bush green' and O. b. L. (OBL) varied in their antiproliferative and cytotoxic activities, influenced cell cycle progression and stimulated apoptosis in most cancer cells. OBL exhibited the highest antioxidant and antiproliferative activities. OB extracts not only improve taste but also have certain anticancer activity against diverse cancer cells due to the presence of compounds such as rosmarinic acid, chicoric acid and caftaric acid. Thus, OB represents a potent source of anticancer materials.

Benzodiazepines modulate the A2 adenosine binding sites on 108CC15 neuroblastoma X glioma hybrid cells.

PubMed Central

Snell, C. R.; Snell, P. H.

1984-01-01

We have demonstrated high affinity diazepam binding sites of the Ro5-4864 benzodiazepine receptor subtype on 108CC15 neuroblastoma X glioma hybrid cells. These cells were previously shown to have purinoceptors of the A2 adenosine subtype and we have now found that [3H]-adenosine can be displaced from this binding site by the benzodiazepines and related compounds that can also bind to the Ro5-4864 site. Diazepam was found to have no intrinsic activity at the A2-receptor as measured by the stimulation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) production in this cell line. At concentrations sufficient to compete for the A2-receptor, diazepam was shown to facilitate, by approximately 2 fold, the stimulation of cyclic AMP by adenosine. These effects are not due to inhibition of adenosine uptake or phosphodiesterase activity, but are probably a consequence of modulation of the coupling of the A2-receptor to cyclic AMP production in this hybrid cell line. PMID:6150742

Mutation in GNE Downregulates Peroxiredoxin IV Altering ER Redox Homeostasis.

PubMed

Chanana, Pratibha; Padhy, Gayatri; Bhargava, Kalpana; Arya, Ranjana

2017-12-01

GNE myopathy is a rare neuromuscular genetic disorder characterized by early adult onset and muscle weakness due to mutation in sialic acid biosynthetic enzyme, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). More than 180 different GNE mutations are known all over the world with unclear pathomechanism. Although hyposialylation of glycoproteins is speculated to be the major cause, but cellular mechanism leading to loss of muscle mass has not yet been deciphered. Besides sialic acid biosynthesis, GNE affects other cellular functions such as cell adhesion and apoptosis. In order to understand the effect of mutant GNE protein on cellular functions, differential proteome profile of HEK293 cells overexpressing pathologically relevant recombinant mutant GNE protein (D207V and V603L) was analyzed. These cells, along with vector control and wild-type GNE-overexpressing cells, were subjected to two-dimensional gel electrophoresis coupled with mass spectrometry (MALDI-TOF/TOF MS/MS). In the study, 10 differentially expressed proteins were identified. Progenesis same spots software revealed downregulation of peroxiredoxin IV (PrdxIV), an ER-resident H 2 O 2 sensor that regulates neurogenesis. Significant reduction in mRNA and protein levels of PrdxIV was observed in GNE mutant cell lines compared with vector control. However, neither total reactive oxygen species was altered nor H 2 O 2 accumulation was observed in GNE mutant cell lines. Interestingly, ER redox state was significantly affected due to reduced normal GNE enzyme activity. Our study indicates that downregulation of PrdxIV affects ER redox state that may contribute to misfolding and aggregation of proteins in GNE myopathy.

Hypusine modification in eukaryotic initiation factor 5A in rodent cells selected for resistance to growth inhibition by ornithine decarboxylase-inhibiting drugs.

PubMed Central

Tome, M E; Gerner, E W

1996-01-01

Selection of HTC cells in drugs that inhibit ornithine decarboxylase (ODC) has produced two cell lines, HMOA and DH23A/b, that contain increased amounts of more stable ODC. In addition to alterations in ODC, these cells appear to produce modified eukaryotic initiation factor 5A (eIF-5A) at different rates, a reaction that both requires spermidine and is essential for proliferation. Alterations to the modification of eIF-5A by spermidine cannot be accounted for by changes in eIF-5A protein or modified eIF-5A turnover. Deoxyhypusine synthetase activity is similar in the parental and variant cell lines and is unaltered by growth into plateau phase or by spermidine depletion. The increased rate of eIF-5A modification in DH23A/b cells is due to an increased accumulation of the unmodified eIF-5A precursor. Increased precursor accumulation is not due to increased eIF-5A transcription, but rather it can be attributed to a metabolic accumulation caused by growth under conditions of chronically limiting spermidine. Selection using drugs that inhibit ODC apparently does not cause alterations in the eIF-5A modification pathway. These data support the hypothesis that one of the main effects of spermidine depletion is depletion of the modified eIF-5A pool, and that this is a critical factor in the cytostasis often observed after depletion of cellular polyamines. PMID:8947467

Establishment and characterization of three immortal bovine muscular epithelial cell lines.

PubMed

Jin, Xun; Lee, Joong-Seob; Kwak, Sungwook; Lee, Soo-Yeon; Jung, Ji-Eun; Kim, Tae-Kyung; Xu, Chenxiong; Hong, Zhongshan; Li, Zhehu; Kim, Sun-Myung; Pian, Xumin; Lee, Dong-Hee; Yoon, Jong-Taek; You, Seungkwon; Choi, Yun-Jaie; Kim, Huunggee

2006-02-28

We have established three immortal bovine muscular epithelial (BME) cell lines, one spontaneously immortalized (BMES), the second SV40LT-mediated (BMEV) and the third hTERT-mediated (BMET). The morphology of the three immortal cell lines was similar to that of early passage primary BME cells. Each of the immortal cell lines made cytokeratin, a typical epithelial marker. BMET grew faster than the other immortal lines and the BME cells, in 10% FBS-DMEM medium, whereas neither the primary cells nor the three immortal cell lines grew in 0.5% FBS-DMEM. The primary BME cells and the immortal cell lines, with the exception of BMES, made increasing amounts of p53 protein when treated with doxorubicin, a DNA damaging agent. On the other hand, almost half of the cells in populations of the three immortal cell lines may lack p16(INK4a) regulatory function, compared to primary BME cells that were growth arrested by enforced expression of p16(INK4a). In soft-agar assays, the primary cells and immortal cell lines proved to be less transformed in phenotype than HeLa cells. The three immortal epithelial-type cell lines reported here are the first cell lines established from muscle tissue of bovine or other species.

Investigation of the expression of RIF1 gene on head and neck, pancreatic and brain cancer and cancer stem cells.

PubMed

GursesCila, Hacer E; Acar, Muradiye; Barut, Furkan B; Gunduz, Mehmet; Grenman, Reidar; Gunduz, Esra

2016-12-01

Recent studies have shown that cancer stem cells are resistant to chemotherapy. The aim of this study was to compare RIF1 gene expression in head and neck, pancreatic cancer and glioma cell lines and the cancer stem cells isolated from these cell lines. UT-SCC-74 from Turku University and UT-SCC-74B primary tumor metastasis and neck cancer cell lines, YKG-1 glioma cancer cell line from RIKEN, pancreatic cancer cell lines and ASPC-1 cells from ATCC were grown in cell culture. To isolate cancer stem cells, ALDH-1 for UT-SCC-74 and UT-SCC-74B cell line, CD-133 for YKG-1 cell line and CD-24 for ASPC-1 cell line, were used as markers of cancer stem cells. RNA isolation was performed for both cancer lines and cancer stem cells. RNAs were converted to cDNA. RIF1 gene expression was performed by qRT-PCR analysis. RIF1 gene expression was compared with cancer cell lines and cancer stem cells isolated from these cell lines. The possible effect of RIF1 gene was evaluated. In the pancreatic cells, RIF1 gene expression in the stem cell-positive cell line was 256 time that seen in the stem cell-negative cell line. Considering the importance of RIF1 in NHEJ and of NHEJ in pancreatic cancer, RIF1 may be one of the genes that plays an important role in the diagnoses and therapeutic treatment of pancreatic cancer. The results of head and neck and brain cancers are inconclusive and further studies are required to elucidate the connection between RIF1 gene and these other types of cancers.

Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

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

Lazarus, Kyren A.; Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122; Zhao, Zhe

2013-08-30

Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels highermore » in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.« less

Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31

USDA-ARS?s Scientific Manuscript database

The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the subclonal cell line, PICM-31A1. P...

Biofabrication of morphology improved cadmium sulfide nanoparticles using Shewanella oneidensis bacterial cells and ionic liquid: For toxicity against brain cancer cell lines.

PubMed

Wang, Li; Chen, Siyuan; Ding, Yiming; Zhu, Qiang; Zhang, Nijia; Yu, Shuqing

2018-01-01

The present work determines the anticancer activity of bio-mediated synthesized cadmium sulfide nanoparticles using the ionic liquid and bacterial cells (Shewanella oneidensis). Bacterial cells have been exposed to be important resources that hold huge potential as ecofriendly, cost-effective, evading toxic of dangerous chemicals and the alternative of conventional physiochemical synthesis. The Shewanella oneidensis is an important kind of metal reducing bacterium, known as its special anaerobic respiratory and sulfate reducing capacity. The crystalline nature, phase purity and surface morphology of biosynthesized cadmium sulfide nanoparticles were analyzed by Fourier transform infrared spectroscopy, X-ray diffraction, Field emission scanning electron microscopy, Energy dispersive spectroscopy and Transmission electron microscopy. The use of imidazolium based ionic liquids as soft templating agent for controlling self-assembly and crystal growth direction of metal sulfide nanoparticles has also advanced as an important method. The microscopic techniques showed that the nanoparticles are designed on the nano form and have an excellent spherical morphology, due to the self-assembled mechanism of ionic liquid assistance. The antitumor efficiency of the cadmium sulfide nanoparticles was investigated against brain cancer cell lines using rat glioma cell lines. The effectively improved nano-crystalline and morphological structure of CdS nanoparticles in the presence of IL exhibit excellent cytotoxicity and dispersion ability on the cell shape is completely spread out showing a nice toxic environment against cancer cells. The cytotoxicity effect of cadmium sulfide nanoparticles was discussed with a diagrammatic representation. Copyright © 2017. Published by Elsevier B.V.

Modulation of AKR1C2 by curcumin decreases testosterone production in prostate cancer.

PubMed

Ide, Hisamitsu; Lu, Yan; Noguchi, Takahiro; Muto, Satoru; Okada, Hiroshi; Kawato, Suguru; Horie, Shigeo

2018-04-01

Intratumoral androgen biosynthesis has been recognized as an essential factor of castration-resistant prostate cancer. The present study investigated the effects of curcumin on the inhibition of intracrine androgen synthesis in prostate cancer. Human prostate cancer cell lines, LNCaP and 22Rv1 cells were incubated with or without curcumin after which cell proliferation was measured at 0, 24, 48 and 72 hours, respectively. Prostate tissues from the transgenic adenocarcinoma of the mouse prostate (TRAMP) model were obtained after 1-month oral administration of 200 mg/kg/d curcumin. Testosterone and dihydrotestosterone concentrations in LNCaP prostate cancer cells were determined through LC-MS/MS assay. Curcumin inhibited cell proliferation and induced apoptosis of prostate cancer cells in a dose-dependent manner. Curcumin decreased the expression of steroidogenic acute regulatory proteins, CYP11A1 and HSD3B2 in prostate cancer cell lines, supporting the decrease of testosterone production. After 1-month oral administration of curcumin, Aldo-Keto reductase 1C2 (AKR1C2) expression was elevated. Simultaneously, decreased testosterone levels in the prostate tissues were observed in the TRAMP mice. Meanwhile, curcumin treatments considerably increased the expression of AKR1C2 in prostate cancer cell lines, supporting the decrease of dihydrotestosterone. Taken together, these results suggest that curcumin's natural bioactive compounds could have potent anticancer properties due to suppression of androgen production, and this could have therapeutic effects on prostate cancer. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest.

PubMed

Bortolotto, Luis Felipe Buso; Barbosa, Flávia Regina; Silva, Gabriel; Bitencourt, Tamires Aparecida; Beleboni, Rene Oliveira; Baek, Seung Joon; Marins, Mozart; Fachin, Ana Lúcia

2017-01-01

Chalcones are precursors of flavonoids that exhibit structural heterogeneity and potential antitumor activity. The objective of this study was to characterize the cytotoxicity of trans-chalcone and licochalcone A (LicoA 1 ) against a breast cancer cell line (MCF-7) and normal murine fibroblasts (3T3). Also the mechanisms of the anti-cancer activity of these two compounds were studied. The alkaline comet assay revealed dose-dependent genotoxicity, which was more responsive against the tumor cell line, compared to the 3T3 mouse fibroblast cell line. Flow cytometry showed that the two chalcones caused the cell cycle arrest in the G1 phase and induced apoptosis in MCF-7 cells. Using PCR Array, we found that trans-chalcone and LicoA trigger apoptosis mediated by the intrinsic pathway as demonstrated by the inhibition of Bcl-2 and induction of Bax. In western blot assay, the two chalcones reduced the expression of cell death-related proteins such as Bcl-2 and cyclin D1 and promoted the cleavage of PARP. However, only trans-chalcone induced the expression of the CIDEA gene and protein in these two experiments. Furthermore, transient transfections of MCF-7 using a construction of a promoter-luciferase vector showed that trans-chalcone induced the expression of the CIDEA promoter activity in 24 and 48h. In conclusion, the results showed that trans-chalcone promoted high induction of the CIDEA promoter gene and protein, which is related to DNA fragmentation during apoptosis. Copyright © 2016. Published by Elsevier Masson SAS.

Hsp70- and p53-reponses after heat treatment and/or X-irradiation mediate the susceptibility of hematopoietic cells to undergo apoptosis.

PubMed

Nijhuis, E H A; Poot, A A; Feijen, J; Vermes, I

2008-02-01

The effect of heat treatment in combination with X-irradiation was examined with regard to expression of p53, a tumor suppressor gene product, and Hsp70, a heat-shock protein, in association with the occurrence of programmed cell death (apoptosis). Three hematopoietic cell lines (HSB2, HL60 and Kasumi-1), which differ in p53 status, were exposed to 42.5 degrees C during one hour and/or X-radiation (total dose 8 Gy). After exposure, both mRNA and protein expression levels of Hsp70 and p53 were investigated by real-time PCR (polymerase chain reaction) and Western blotting. Apoptosis was simultaneously analyzed by observation of cell morphology as well as flowcytometric determination of Annexin V binding to phosphatidylserine and propidium iodide exclusion. Both HL60 and HSB2 cell lines with a low p53 status and a quick response to heat treatment with Hsp70 over-expression are less susceptible to heat-induced apoptosis compared to Kasumi-1 cells with wild-type p53 protein and no Hsp70 response. The combination of first applying X-irradiation followed by heat treatment resulted in the most effective induction of apoptosis due to impairment of the Hsp70 response in all three cell lines. These results indicate that the Hsp70 response and p53 status mediate the susceptibility of hematopoietic cells to undergo heat-induced apoptosis. Therefore, these parameters can be used as markers to predict the effectiveness of hyperthermia in cancer treatment.

Development and Preclinical Application of an Immunocompetent Transplant Model of Basal Breast Cancer with Lung, Liver and Brain Metastases

PubMed Central

Hoenerhoff, Mark; Hixon, Julie A.; Durum, Scott K.; Qiu, Ting-hu; He, Siping; Burkett, Sandra; Liu, Zi-Yao; Swanson, Steven M.; Green, Jeffrey E.

2016-01-01

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer that is associated with a poor prognosis and for which no targeted therapies currently exist. In order to improve preclinical testing for TNBC that relies primarily on using human xenografts in immunodeficient mice, we have developed a novel immunocompetent syngeneic murine tumor transplant model for basal-like triple-negative breast cancer. The C3(1)/SV40-T/t-antigen (C3(1)/Tag) mouse mammary tumor model in the FVB/N background shares important similarities with human basal-like TNBC. However, these tumors or derived cell lines are rejected when transplanted into wt FVB/N mice, likely due to the expression of SV40 T-antigen. We have developed a sub-line of mice (designated REAR mice) that carry only one copy of the C3(1)/Tag-antigen transgene resulting from a spontaneous transgene rearrangement in the original founder line. Unlike the original C3(1)/Tag mice, REAR mice do not develop mammary tumors or other phenotypes observed in the original C3(1)/Tag transgenic mice. REAR mice are more immunologically tolerant to SV40 T-antigen driven tumors and cell lines in an FVB/N background (including prostate tumors from TRAMP mice), but are otherwise immunologically intact. This transplant model system offers the ability to synchronously implant the C3(1)/Tag tumor-derived M6 cell line or individual C3(1)/Tag tumors from various stages of tumor development into the mammary fat pads or tail veins of REAR mice. C3(1)/Tag tumors or M6 cells implanted into the mammary fat pads spontaneously metastasize at a high frequency to the lung and liver. M6 cells injected by tail vein can form brain metastases. We demonstrate that irradiated M6 tumor cells or the same cells expressing GM-CSF can act as a vaccine to retard tumor growth of implanted tumor cells in the REAR model. Preclinical studies performed in animals with an intact immune system should more authentically replicate treatment responses in human patients. PMID:27171183

Differential Effects of Histone Acetyltransferase GCN5 or PCAF Knockdown on Urothelial Carcinoma Cells

PubMed Central

Koutsogiannouli, Evangelia A.; Hader, Christiane; Pinkerneil, Maria; Hoffmann, Michèle J.; Schulz, Wolfgang A.

2017-01-01

Disturbances in histone acetyltransferases (HATs) are common in cancers. In urothelial carcinoma (UC), p300 and CBP are often mutated, whereas the GNAT family HATs GCN5 and PCAF (General Control Nonderepressible 5, p300/CBP-Associated Factor) are often upregulated. Here, we explored the effects of specific siRNA-mediated knockdown of GCN5, PCAF or both in four UC cell lines (UCCs). Expression of various HATs and marker proteins was measured by qRT-PCR and western blot. Cellular effects of knockdowns were analyzed by flow cytometry and ATP-, caspase-, and colony forming-assays. GCN5 was regularly upregulated in UCCs, whereas PCAF was variable. Knockdown of GCN5 or both GNATs, but not of PCAF alone, diminished viability and inhibited clonogenic growth in 2/4 UCCs, inducing cell cycle changes and caspase-3/7 activity. PCAF knockdown elicited GCN5 mRNA upregulation. Double knockdown increased c-MYC and MDM2 (Mouse Double Minute 2) in most cell lines. In conclusion, GCN5 upregulation is especially common in UCCs. GCN5 knockdown impeded growth of specific UCCs, whereas PCAF knockdown elicited minor effects. The limited sensitivity towards GNAT knockdown and its variation between the cell lines might be due to compensatory effects including HAT, c-MYC and MDM2 upregulation. Our results predict that developing drugs targeting individual HATs for UC treatment may be challenging. PMID:28678170

Yessotoxin, a Marine Toxin, Exhibits Anti-Allergic and Anti-Tumoural Activities Inhibiting Melanoma Tumour Growth in a Preclinical Model

PubMed Central

Tobío, Araceli; Alfonso, Amparo; Madera-Salcedo, Iris; Botana, Luis M.

2016-01-01

Yessotoxins (YTXs) are a group of marine toxins produced by the dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera. They may have medical interest due to their potential role as anti-allergic but also anti-cancer compounds. However, their biological activities remain poorly characterized. Here, we show that the small molecular compound YTX causes a slight but significant reduction of the ability of mast cells to degranulate. Strikingly, further examination revealed that YTX had a marked and selective cytotoxicity for the RBL-2H3 mast cell line inducing apoptosis, while primary bone marrow derived mast cells were highly resistant. In addition, YTX exhibited strong cytotoxicity against the human B-chronic lymphocytic leukaemia cell line MEC1 and the murine melanoma cell line B16F10. To analyse the potential role of YTX as an anti-cancer drug in vivo we used the well-established B16F10 melanoma preclinical mouse model. Our results demonstrate that a few local application of YTX around established tumours dramatically diminished tumour growth in the absence of any significant toxicity as determined by the absence of weight loss and haematological alterations. Our data support that YTX may have a minor role as an anti-allergic drug, but reveals an important potential for its use as an anti-cancer drug. PMID:27973568

Macroalgae Extracts From Antarctica Have Antimicrobial and Anticancer Potential

PubMed Central

Martins, Rosiane M.; Nedel, Fernanda; Guimarães, Victoria B. S.; da Silva, Adriana F.; Colepicolo, Pio; de Pereira, Claudio M. P.; Lund, Rafael G.

2018-01-01

Background: Macroalgae are sources of bioactive compounds due to the large number of secondary metabolites they synthesize. The Antarctica region is characterized by extreme weather conditions and abundant aggregations of macroalgae. However, current knowledge on their biodiversity and their potential for bio-prospecting is still fledging. This study evaluates the antimicrobial and cytotoxic activity of different extracts of four macroalgae (Cystosphaera jacquinotii, Iridaea cordata, Himantothallus grandifolius, and Pyropia endiviifolia) from the Antarctic region against cancer and non-cancer cell lines. Methods: The antimicrobial activity of macroalgae was evaluated by the broth microdilution method. Extracts were assessed against Staphylococcus aureus ATCC 19095, Enterococcus faecalis ATCC 4083, Escherichia coli ATCC29214, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 62342, and the clinical isolates from the human oral cavity, namely, C. albicans (3), C. parapsilosis, C. glabrata, C. lipolytica, and C. famata. Cytotoxicity against human epidermoid carcinoma (A-431) and mouse embryonic fibroblast (NIH/3T3) cell lines was evaluated with MTT colorimetric assay. Results: An ethyl acetate extract of H. grandifolius showed noticeable antifungal activity against all fungal strains tested, including fluconazole-resistant samples. Cytotoxicity investigation with a cancer cell line revealed that the ethyl acetate extract of I. cordata was highly cytotoxic against A-431 cancer cell line, increasing the inhibitory ratio to 91.1 and 95.6% after 24 and 48 h exposure, respectively, for a concentration of 500 μg mL−1. Most of the algal extracts tested showed little or no cytotoxicity against fibroblasts. Conclusion: Data suggest that macroalgae extracts from Antarctica may represent a source of therapeutic agents. HIGHLIGHTS Different macroalgae samples from Antarctica were collected and the lyophilized biomass of each macroalgae was extracted sequentially with different solventsThe antimicrobial and anticancer potential of macroalgae extracts were evaluatedEthyl acetate extract of H. grandifolius showed noticeable antifungal activity against all the fungal strains tested, including fluconazole-resistant samplesEthyl acetate extract of I. cordata was highly cytotoxic against the A-431 cancer cell lineMost of the algal extracts tested showed little or no cytotoxicity against normal cell lines PMID:29568291

Treatment decision-making for advanced non-small cell lung cancer and differences among European countries: 1st AIOT-ETOP meeting.

PubMed

Gridelli, Cesare; Stahel, Rolf; Besse, Benjamin; Ciardiello, Fortunato; Felip, Enriqueta; Gasparini, Stefano; Graziano, Paolo; Rossi, Antonio; de Marinis, Filippo

2011-12-01

The Italian Association of Thoracic Oncology (AIOT) and the European Thoracic Oncology Platform (ETOP) realized the first conjunct educational meeting, open to European oncologists involved in the treatment of thoracic malignancies, entitled "Advanced non-small cell lung cancer: new perspectives in first-line setting". The educational meeting included 8 interactive talks, held by European key opinion leaders, and 5 related clinical cases in which the attendees, divided in working tables based on their country origin, were involved for interactive discussion. The aim of this course was to elucidate the differences or similarities among the European countries in the first-line treatment of patients affected by advanced non-small cell lung cancer (NSCLC). Twenty-two attendees of the following countries participated: Austria, France, Italy, Spain, Swiss, and UK. As expected, some discrepancies between the groups were identified concerning the approach to the diagnostic phase, the choice of first-line regimen, the duration of treatment and the use of maintenance therapy. These discrepancies were mainly due to familiarity with specific therapies and lack of access to certain therapies due to local regulatory issues. The European Medicine Agency grants marketing of drugs in all Europe, regulatory agency of each country can register the drug, but can also deny public reimbursement thus restricting the options of the oncologist. The European Oncology Associations should join to their effort to achieve a uniform access to the cancer therapy for all patients in Europe. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Molecularly Targeted Dose-Enhancement Radiotherapy Using Gold and Luminescent Nanoparticles in an Orthotopic Human Prostate Cancer Rat Model

DTIC Science & Technology

2013-10-01

cell lines, such as cervix cancer cell line (HeLa) and breast cancer cell line (MDA-MB-231), were also employed. The experiments with other cell lines...breast cancer cell line (MDA-MB- 231), and cervix cancer cell line (HeLa). Different from our hypothesis, prostate cancer cell lines did not present...Radiotherapy Using Gold and Luminescent Nanoparticles in an Orthotopic Human Prostate Cancer Rat Model PRINCIPAL INVESTIGATOR: Kwang Song

[The factors involved in invasive ability of endometrial carcinoma cells].

PubMed

Mori, Y; Mizuuchi, H; Sato, K; Okamura, N; Kudo, R

1994-06-01

The in vitro invasive ability, the expression of cell adhesion molecule E-cadherin, activity of matrix metalloproteinase (MMP) and K-ras point mutation were investigated in eight human endometrial carcinoma cell lines. 1) In vitro invasive abilities of endometrial carcinoma cell lines depend on the degree of cell differentiation and the origin of cell lines. A poorly-differentiated carcinoma cell line (NUE-1) and a cell line derived from metastatic lymph node (SNG-M) were more invasive than moderately-(HEC-1A, HEC-1BE) and well-differentiated (HEC-6, Ishikawa) cell lines. 2) Immunohistochemically, less or non-invasive cell lines expressed E-cadherin strongly, whereas a highly invasive cell line (NUE-1) expressed E-cadherin weakly. 3) When cultured on Matrigel-coated dishes, the tumor cells derived from moderately- and well-differentiated carcinoma aggregated with each other and did not invade Matrigel in the invasion assay. The aggregated cells expressed E-cadherin more strongly when cultured on Matrigel. 4) 72-kD gelatinase (MMP-2) was secreted in serum-free conditioned medium of all cell lines. In an invasive cell line (NUE-1,SNG-M), the activity of MMP-2 was stronger than in other cell lines. And the activity of 92-kDa gelatinase (MMP-9) was detected in most invasive cell line (NUE-1). 5) Point mutation of K-ras codon 12 was detected in four of eight (50%) cell lines by the PCR-RFLP method. The changes in the DNA sequence were identified, but K-ras point mutation was not correlated with in vitro invasiveness of the tumor cells.

Loss of MLH1 sensitizes colon cancer cells to DNA-PKcs inhibitor KU60648.

PubMed

Hinrichsen, Inga; Ackermann, Anne; Düding, Tonja; Graband, Annika; Filmann, Natalie; Plotz, Guido; Zeuzem, Stefan; Brieger, Angela

2017-07-01

Germline mutations of MLH1 are responsible for tumor generation in nearly 50% of patients with Lynch Syndrome, and around 15% of sporadic colorectal cancers show MLH1-deficiency due to promotor hypermethylation. Although these tumors are of lower aggressiveness the benefit for these patients from standard chemotherapy is still under discussion. Recently, it was shown that the sensitivity to the DNA-PKcs inhibitor KU60648 is linked to loss of the MMR protein MSH3. However, loss of MSH3 is rather secondary, as a consequence of MMR-deficiency, and frequently detectable in MLH1-deficient tumors. Therefore, we examined the expression of MLH1, MSH2, MSH6, and MSH3 in different MMR-deficient and proficient cell lines and determined their sensitivity to KU60648 by analyzing cell viability and survival. MLH1-dependent ability of double strand break (DSB) repair was monitored after irradiation via γH2AX detection. A panel of 12 colon cancer cell lines, two pairs of cells, where MLH1 knock down was compared to controls with the same genetic background, and one MLH1-deficient cell line where MLH1 was overexpressed, were included. In summary, we found that MLH1 and/or MSH3-deficient cells exhibited a significantly higher sensitivity to KU60648 than MMR-proficient cells and that overexpression of MLH1 in MLH1-deficient cells resulted in a decrease of cell sensitivity. KU60648 efficiency seems to be associated with reduced DSB repair capacity. Since the molecular testing of colon tumors for MLH1 expression is a clinical standard we believe that MLH1 is a much better marker and a greater number of patients would benefit from KU60648 treatment. © 2017 Wiley Periodicals, Inc.

Undecylprodigiosin selectively induces apoptosis in human breast carcinoma cells independent of p53

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

Ho, T.-F.; Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan; Department of Medical Technology, Central Taiwan University of Science and Technology, Taichung 40605, Taiwan

2007-12-15

Undecylprodigiosin (UP) is a bacterial bioactive metabolite produced by Streptomyces and Serratia. In this study, we explored the anticancer effect of UP. Human breast carcinoma cell lines BT-20, MCF-7, MDA-MB-231 and T47D and one nonmalignant human breast epithelial cell line, MCF-10A, were tested in this study. We found that UP exerted a potent cytotoxicity against all breast carcinoma cell lines in a dose- and time-dependent manner. In contrast, UP showed limited toxicity to MCF-10A cells, indicating UP's cytotoxic effect is selective for malignant cells. UP's cytotoxic effect was due to apoptosis, as confirmed by positive TUNEL signals, annexin V-binding, caspasemore » 9 activation and PARP cleavage. Notably, UP-induced apoptosis was blocked by the pan-caspase inhibitor z-VAD.fmk, further indicating the involvement of caspase activity. Moreover, UP caused a marked decrease of the levels of antiapoptotic BCL-X{sub L}, Survivin and XIAP while enhancing the levels of proapoptotic BIK, BIM, MCL-1S and NOXA, consequently favoring induction of apoptosis. Additionally, we found that cells with functional p53 (MCF-7, T47D) or mutant p53 (BT-20, MDA-MB-231) were both susceptible to UP's cytotoxicity. Importantly, UP was able to induce apoptosis in MCF-7 cells with p53 knockdown by RNA interference, confirming the dispensability of p53 in UP-induced apoptosis. Overall, our results establish that UP induces p53-independent apoptosis in breast carcinoma cells with no marked toxicity to nonmalignant cells, raising the possibility of its use as a new chemotherapeutic drug for breast cancer irrespective of p53 status.« less

Mutation spectrum of MSH3-deficient HHUA/chr.2 cells reflects in vivo activity of the MSH3 gene product in mismatch repair.

PubMed

Tauchi, H; Komatsu, K; Ishizaki, K; Yatagai, F; Kato, T

2000-02-14

The endometrial tumor cell line HHUA carries mutations in two mismatch repair (MMR) genes MSH3 and MSH6. We have established an MSH3-deficient HHUA/chr.2 cell line by introducing human chromosome 2, which carries wild-type MSH6 and MSH2 genes, to HHUA cells. Introduction of chromosome 2 to HHUA cells partially restored G:G MMR activity to the cell extract and reduced the frequency of mutation at the hypoxanthine-guanine phosphoribosyltransferase (hprt*) locus to about 3% that of the parental HHUA cells, which is five-fold the frequency in MMR-proficient cells, indicating that the residual mutator activity in HHUA/chr.2 is due to an MSH3-deficiency in these cells. The spectrum of mutations occurring at the HPRT locus of HHUA/chr.2 was determined with 71 spontaneous 6TG(r) clones. Base substitutions and +/-1 bp frameshifts were the major mutational events constituting, respectively, 54% and 42% of the total mutations, and more than 70% of them occurred at A:T sites. A possible explanation for the apparent bias of mutations to A:T sites in HHUA/chr.2 is haploinsufficiency of the MSH6 gene on the transferred chromosome 2. Comparison of the mutation spectra of HHUA/chr.2 with that of the MSH6-deficient HCT-15 cell line [S. Ohzeki, A. Tachibana, K. Tatsumi, T. Kato, Carcinogenesis 18 (1997) 1127-1133.] suggests that in vivo the MutSalpha (MSH2:MSH6) efficiently repairs both mismatch and unpaired extrahelical bases, whereas MutSbeta (MSH2:MSH3) efficiently repairs extrahelical bases and repairs mismatch bases to a limited extent.

[Effect of sodium phenylbutyrate on the apoptosis of human tongue squamous cancer cell line and expression of p21 and survivin genes].

PubMed

Chen, Wei-qiang; Feng, Feng-lan; Gu, Hong-biao; Pan, De-shun

2010-07-01

To examine the effects of sodium phenylbutyrate on the apoptosis of human tongue squamous cancer cell line and expression of p21 and survivin genes. The inhibition effects of sodium phenylbutyrate on Tca8113 and human tongue squamous cell carcinoma (TCSSA) cell lines were detected by methyl thiazoly terazolium (MTT) and the apoptosis of the cancer cells after being induced by sodium phenylbutyrate examined by flow cytometry (FCM). The expression of p21 and survivin genes were observed with Western blotting and RT-PCR. Compared with control group, the level of p21 mRNA and protein of Tca8113 cellline increased to 0.09 ± 0.08 and increased 0.72 ± 0.10, that of TCSSA cellline increased 1.34 ± 0.12 and 1.56 ± 0.09 (P < 0.05). Compared with control group, the level of surrive mRNA and protein of Tca8113 cellline decreased to 1.10 ± 0.05 and 1.14 ± 1.10, that of TCSSA cellline decreased to 0.12 ± 0.08 and 0.94 ± 0.09 (P < 0.05). Sodium phenylbutyrate inhibited the cell proliferation, promoted cell apoptosis and arrested the cells in G₁/G₀ phase. The amount of p21 mRNA and protein were increased, and the expression of survivin gene was decreased. Sodium phenylbutyrate exhibited remarkable inhibitory effects on human tongue squamous cancer cell proliferation and induced cancer cell apoptosis. The mechanism may be due to up-regulation of p21 gene and down-regulation of survivin gene. The mRNA level of p21 gene and survivin gene showed a strong correlation.

Graphene oxide and reduced graphene oxide induced neural pheochromocytoma-derived PC12 cell lines apoptosis and cell cycle alterations via the ERK signaling pathways.

PubMed

Kang, Yiyuan; Liu, Jia; Wu, Junrong; Yin, Qian; Liang, Huimin; Chen, Aijie; Shao, Longquan

2017-01-01

Given the novel applications of graphene materials in biomedical and electronics industry, the health hazards of these particles have attracted extensive worldwide attention. Although many studies have been performed on graphene material-induced toxic effects, toxicological data for the effect of graphene materials on the nervous system are lacking. In this study, we focused on the biological effects of graphene oxide (GO) and reduced graphene oxide (rGO) materials on PC12 cells, a type of traditional neural cell line. We found that GO and rGO exerted significant toxic effects on PC12 cells in a dose- and time-dependent manner. Moreover, apoptosis appeared to be a response to toxicity. A potent increase in the number of PC12 cells at G0/G1 phase after GO and rGO exposure was detected by cell cycle analysis. We found that phosphorylation levels of ERK signaling molecules, which are related to cell cycle regulation and apoptosis, were significantly altered after GO and rGO exposure. In conclusion, our results show that GO has more potent toxic effects than rGO and that apoptosis and cell cycle arrest are the main toxicity responses to GO and rGO treatments, which are likely due to ERK pathway regulation.

Radiation induction of drug resistance in RIF-1: Correlation of tumor and cell culture results

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

Moulder, J.E.; Hopwood, L.E.; Volk, D.M.

1991-02-01

The RIF-1 tumor line contains cells that are resistant to various anti-neoplastic drugs, including 5-fluorouracil (5FU), methotrexate (MTX), adriamycin (ADR), and etoposide (VP16). The frequency of these drug-resistant cells is increased after irradiation. The frequency of drug-resistant cells and the magnitude of radiation-induced drug resistance are different in cell culture than in tumors. The dose-response and expression time relationships for radiation induction of drug resistance observed in RIF-1 tumors are unusual.We hypothesize that at high radiation doses in vivo, we are selecting for cells that are both drug resistant and radiation resistant due to microenvironmental factors, whereas at low radiationmore » doses in vivo and all radiation doses in vitro, we are observing true mutants. These studies indicate that there can be significant differences in drug-resistance frequencies between tumors and their cell lines of origin, and that radiation induction of drug resistance depends significantly on whether the induction is done in tumors or in cell culture. These results imply that theories about the induction of drug resistance that are based on cell culture studies may be inapplicable to the induction of drug resistance in tumors.« less

Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

PubMed Central

Park, Jin Hyoung; Jin, Jong Hwa; Lim, Myung Sin; An, Hyun Joo; Kim, Jong Won; Lee, Gyun Min

2017-01-01

Chinese hamster ovary (CHO) cells are the most common cell line used for the production of therapeutic proteins including monoclonal antibodies (mAbs). Host cell proteins (HCPs), secreted and released from lysed cells, accumulate extracellularly during the cultures of recombinant CHO (rCHO) cells, potentially impairing product quality. In an effort to maintain good mAb quality during the cultures, HCPs accumulated extracellularly in batch and fed-batch cultures of a mAb-producing rCHO cell line were identified and quantified by nanoflow liquid chromatography-tandem mass spectrometry, followed by their gene ontology and functional analysis. Due to higher cell concentration and longer culture duration, more HCPs were identified and quantitated in fed-batch culture (2145 proteins identified and 1673 proteins quantified) than in batch culture (1934 proteins identified and 1486 proteins quantified). Clustering analysis of HCPs showed that the concentration profiles of HCPs affecting mAb quality (Lgmn, Ctsd, Gbl1, and B4galt1) correlated with changes in mAb quality attributes such as aggregation, charge variants, and N-glycosylation during the cultures. Taken together, the dataset of HCPs obtained in this study provides insights into determining the appropriate target proteins to be removed during both the cultures and purification steps for ensuring good mAb quality. PMID:28281648

Quercetin conjugated with silica nanoparticles inhibits tumor growth in MCF-7 breast cancer cell lines.

PubMed

Aghapour, Fahimeh; Moghadamnia, Ali Akbar; Nicolini, Andrea; Kani, Seydeh Narges Mousavi; Barari, Ladan; Morakabati, Payam; Rezazadeh, Leyla; Kazemi, Sohrab

2018-06-12

Quercetin is a plant polyphenol from the flavonoid group that plays a fundamental role in controlling homeostasis due to its potent antioxidant properties. However, quercetin has extremely low water solubility, which is a major challenge in drug absorption. In this study, we described a simple method for the synthesis of quercetin nanoparticles. The quercetin nanoparticles had an average diameter of 82 nm and prominent yellow emission under UV irradiation. Therefore, we used an in vitro model treated with quercetin and quercetin nanoparticles to investigate the effects of quercetin nanoparticles on MCF-7 breast cancer cell line. MCF-7 cells were cultured with different concentrations (1-100 μM) of quercetin nanoparticles at the 24th, 48th and 72 nd hours, and cell cycle and apoptosis assays were detected by flow cytometry (FCM). In this study, we found that quercetin nanoparticles (1-100 μM) could significantly reduce cell vitality, growth rate and colony formation of MCF-7 cells. Quercetin nanoparticles can inhibit cell growth by blocking the cell cycle and promoting apoptosis in MCF-7 cells more than quercetin. As a result, quercetin nanoparticles may be useful therapy or prevention on breast cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

31P-nuclear magnetic resonance spectroscopy in vivo of six human melanoma xenograft lines: tumour bioenergetic status and blood supply.

PubMed Central

Lyng, H.; Olsen, D. R.; Southon, T. E.; Rofstad, E. K.

1993-01-01

Six human melanoma xenograft lines grown s.c. in BALB/c-nu/nu mice were subjected to 31P-nuclear magnetic resonance (31P-NMR) spectroscopy in vivo. The following resonances were detected: phosphomonoesters (PME), inorganic phosphate (Pi), phosphodiesters (PDE), phosphocreatine (PCr) and nucleoside triphosphate gamma, alpha and beta (NTP gamma, alpha and beta). The main purpose of the work was to search for possible relationships between 31P-NMR resonance ratios and tumour pH on the one hand and blood supply per viable tumour cell on the other. The latter parameter was measured by using the 86Rb uptake method. Tumour bioenergetic status [the (PCr + NTP beta)/Pi resonance ratio], tumour pH and blood supply per viable tumour cell decreased with increasing tumour volume for five of the six xenograft lines. The decrease in tumour bioenergetic status was due to a decrease in the (PCr + NTP beta)/total resonance ratio as well as an increase in the Pi/total resonance ratio. The decrease in the (PCr + NTP beta)/total resonance ratio was mainly a consequence of a decrease in the PCr/total resonance ratio for two lines and mainly a consequence of a decrease in the NTP beta/total resonance ratio for three lines. The magnitude of the decrease in the (PCr + NTP beta)/total resonance ratio and the magnitude of the decrease in tumour pH were correlated to the magnitude of the decrease in blood supply per viable tumour cell. Tumour pH decreased with decreasing tumour bioenergetic status, and the magnitude of this decrease was larger for the tumour lines showing a high than for those showing a low blood supply per viable tumour cell. No correlations across the tumour lines were found between tumour pH and tumour bioenergetic status or any other resonance ratio on the one hand and blood supply per viable tumour cell on the other. The differences in the 31P-NMR spectrum between the tumour lines were probably caused by differences in the intrinsic biochemical properties of the tumour cells rather than by the differences in blood supply per viable tumour cell. Biochemical properties of particular importance included rate of respiration, glycolytic capacity and tolerance to hypoxic stress. On the other hand, tumour bioenergetic status and tumour pH were correlated to blood supply per viable tumour cell within individual tumour lines. These observations suggest that 31P-NMR spectroscopy may be developed to be a clinically useful method for monitoring tumour blood supply and parameters related to tumour blood supply during and after physiological intervention and tumour treatment. However, clinically useful parameters for prediction of tumour treatment resistance caused by insufficient blood supply can probably not be derived from a single 31P-NMR spectrum since correlations across tumour lines were not detected; additional information is needed. PMID:8260356

In vitro Development of Chemotherapy and Targeted Therapy Drug-Resistant Cancer Cell Lines: A Practical Guide with Case Studies

PubMed Central

McDermott, Martina; Eustace, Alex J.; Busschots, Steven; Breen, Laura; Crown, John; Clynes, Martin; O’Donovan, Norma; Stordal, Britta

2014-01-01

The development of a drug-resistant cell line can take from 3 to 18 months. However, little is published on the methodology of this development process. This article will discuss key decisions to be made prior to starting resistant cell line development; the choice of parent cell line, dose of selecting agent, treatment interval, and optimizing the dose of drug for the parent cell line. Clinically relevant drug-resistant cell lines are developed by mimicking the conditions cancer patients experience during chemotherapy and cell lines display between two- and eight-fold resistance compared to their parental cell line. Doses of drug administered are low, and a pulsed treatment strategy is often used where the cells recover in drug-free media. High-level laboratory models are developed with the aim of understanding potential mechanisms of resistance to chemotherapy agents. Doses of drug are higher and escalated over time. It is common to have difficulty developing stable clinically relevant drug-resistant cell lines. A comparative selection strategy of multiple cell lines or multiple chemotherapeutic agents mitigates this risk and gives insight into which agents or type of cell line develops resistance easily. Successful selection strategies from our research are presented. Pulsed-selection produced platinum or taxane-resistant large cell lung cancer (H1299 and H460) and temozolomide-resistant melanoma (Malme-3M and HT144) cell lines. Continuous selection produced a lapatinib-resistant breast cancer cell line (HCC1954). Techniques for maintaining drug-resistant cell lines are outlined including; maintaining cells with chemotherapy, pulse treating with chemotherapy, or returning to master drug-resistant stocks. The heterogeneity of drug-resistant models produced from the same parent cell line with the same chemotherapy agent is explored with reference to P-glycoprotein. Heterogeneity in drug-resistant cell lines reflects the heterogeneity that can occur in clinical drug resistance. PMID:24639951

Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

PubMed Central

Warleta, Fernando; Quesada, Cristina Sánchez; Campos, María; Allouche, Yosra; Beltrán, Gabriel; Gaforio, José J.

2011-01-01

Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A) or breast cancer cells (MDA-MB-231 and MCF7). We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS) level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells. PMID:22254082

Atovaquone for Prophylaxis of Toxoplasmosis after Allogeneic Hematopoietic Stem Cell Transplantation.

PubMed

Mendorf, Alexander; Klyuchnikov, Evgeny; Langebrake, Claudia; Rohde, Holger; Ayuk, Francis; Regier, Marc; Christopeit, Maximilian; Zabelina, Tatjana; Bacher, Adelbert; Stübig, Thomas; Wolschke, Christine; Bacher, Ulrike; Kröger, Nicolaus

2015-01-01

Toxoplasmosis and infections by other opportunistic agents such as Pneumocystis jirovecii constitute life-threatening risks for patients after allogeneic hematopoietic stem cell transplantation. Trimethoprim/sulfamethoxazole (TMP-SMX) has been well established for post-transplant toxoplasmosis and pneumocystis prophylaxis, but treatment may be limited due to toxicity. We explored atovaquone as an alternative and compared it with TMP-SMX regarding toxicity and efficacy during the first 100 days after transplantation in 155 consecutive adult stem cell recipients. Eight patients with a prior history of TMP-SMX intolerance received atovaquone as first-line prophylaxis. TMP-SMX was used for 141 patients as first-line strategy, but 13 patients (9.2%) were later switched to atovaquone due to TMP-SMX toxicity or gastrointestinal symptoms. No active toxoplasmosis or active P. jirovecii infection developed under continued prophylaxis with either TMP-SMX or atovaquone. However, for reasons of TMP-SMX and/or atovaquone toxicity, 7 patients were unable to tolerate any efficacious toxoplasmosis prophylaxis and therefore obtained inhalative pentamidine as P. jirovecii prophylaxis but no toxoplasmosis prophylaxis. Importantly, 2 of these patients developed severe toxoplasmosis. In summary, atovaquone appears as a valid alternative for at least some post-transplant patients who cannot tolerate TMP-SMX. This should be further confirmed by multicenter trials. © 2015 S. Karger AG, Basel.

Two-dimensional particle-in-cell plasma source ion implantation of a prolate spheroid target

NASA Astrophysics Data System (ADS)

Liu, Cheng-Sen; Han, Hong-Ying; Peng, Xiao-Qing; Chang, Ye; Wang, De-Zhen

2010-03-01

A two-dimensional particle-in-cell simulation is used to study the time-dependent evolution of the sheath surrounding a prolate spheroid target during a high voltage pulse in plasma source ion implantation. Our study shows that the potential contour lines pack more closely in the plasma sheath near the vertex of the major axis, i.e. where a thinner sheath is formed, and a non-uniform total ion dose distribution is incident along the surface of the prolate spheroid target due to the focusing of ions by the potential structure. Ion focusing takes place not only at the vertex of the major axis, where dense potential contour lines exist, but also at the vertex of the minor axis, where sparse contour lines exist. This results in two peaks of the received ion dose, locating at the vertices of the major and minor axes of the prolate spheroid target, and an ion dose valley, staying always between the vertices, rather than at the vertex of the minor axis.