Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation

PubMed Central

Wang, Jing; Luo, Cheng; Shan, Changliang; You, Qiancheng; Lu, Junyan; Elf, Shannon; Zhou, Yu; Wen, Yi; Vinkenborg, Jan L.; Fan, Jun; Kang, Heebum; Lin, Ruiting; Han, Dali; Xie, Yuxin; Karpus, Jason; Chen, Shijie; Ouyang, Shisheng; Luan, Chihao; Zhang, Naixia; Ding, Hong; Merkx, Maarten; Liu, Hong; Chen, Jing; Jiang, Hualiang; He, Chuan

2016-01-01

Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies. PMID:26587712

Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation

NASA Astrophysics Data System (ADS)

Wang, Jing; Luo, Cheng; Shan, Changliang; You, Qiancheng; Lu, Junyan; Elf, Shannon; Zhou, Yu; Wen, Yi; Vinkenborg, Jan L.; Fan, Jun; Kang, Heebum; Lin, Ruiting; Han, Dali; Xie, Yuxin; Karpus, Jason; Chen, Shijie; Ouyang, Shisheng; Luan, Chihao; Zhang, Naixia; Ding, Hong; Merkx, Maarten; Liu, Hong; Chen, Jing; Jiang, Hualiang; He, Chuan

2015-12-01

Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.

Importance of inverse correlation between ALDH3A1 and PPARγ in tumor cells and tissue regeneration.

PubMed

Oraldi, M; Saracino, S; Maggiora, M; Chiaravalloti, A; Buemi, C; Martinasso, G; Paiuzzi, E; Thompson, D; Vasiliou, V; Canuto, R A

2011-05-30

Aldehyde dehydrogenase (ALDH) enzymes are involved in maintaining cellular homeostasis by metabolizing both endogenous and exogenous reactive aldehydes. They modulate several cell functions including proliferation, differentiation, survival as well as cellular response to oxidative stress. We previously reported that ALDH3A1 expression is inversely correlated with the activation of PPARs (Peroxisome Proliferators-Activated Receptors), a category of orphan nuclear hormone receptors, in both rat and human cells. PPARγ is involved in cell proliferation. In this study, we have used PPARγ transfection and inhibition to examine the relationship between ALDH3A1 and PPARγ and their role as regulators of cell proliferation. Induction of PPARγ in A549 and NCTC 2544 cells by transfection caused a decrease in ALDH3A1 and inhibition of cell proliferation, a result we obtained previously using ligands that induce PPARγ. A reduction of PPARγ expression using siRNA increased ALDH3A1 expression and cell proliferation. In cells induced to proliferate in a model of tissue regeneration, ALDH3A1 expression increased during the period of proliferation, whereas PPARγ expression decreased. In conclusion, through modulation of PPARγ or ALDH3A1, it may be possible to reduce cell proliferation in tumor cells or stimulate cell proliferation in normal cells during tissue regeneration. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Low doses of TiO2-polyethylene glycol nanoparticles stimulate proliferation of hepatocyte cells

NASA Astrophysics Data System (ADS)

Sun, Qingqing; Kanehira, Koki; Taniguchi, Akiyoshi

2016-01-01

This paper describes the effect of low concentrations of 100 nm polyethylene glycol-modified TiO2 nanoparticles (TiO2-PEG NPs) on HepG2 hepatocellular carcinoma cells. Proliferation of HepG2 cells increased significantly when the cells were exposed to low doses (<100 μg ml-1) of TiO2-PEG NPs. These results were further confirmed by cell counting experiments and cell cycle assays. Cellular uptake assays were performed to determine why HepG2 cells proliferate with low-dose exposure to TiO2-PEG NPs. The results showed that exposure to lower doses of NPs led to less cellular uptake, which in turn decreased cytotoxicity. We therefore hypothesized that TiO2-PEG NPs could affect the activity of hepatocyte growth factor receptors (HGFRs), which bind to hepatocyte growth factor and stimulate cell proliferation. The localization of HGFRs on the surface of the cell membrane was detected via immunofluorescence staining and confocal microscopy. The results showed that HGFRs aggregate after exposure to TiO2-PEG NPs. In conclusion, our results indicate that TiO2-PEG NPs have the potential to promote proliferation of HepG2 cells through HGFR aggregation and suggest that NPs not only exhibit cytotoxicity but also affect cellular responses.

P44/WDR77 restricts the sensitivity of proliferating cells to TGFβ signaling

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

Yi, Pengfei; Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030; Gao, Shen

2014-07-18

Highlights: • P44/WDR77 causes proliferating cells to become non-responsive to TGFβ signaling. • P44/WDR77 down-regulates TβRII and TβR2 expression. • P44/WDR77 down-regulated TGFβ signaling correlates with lung tumorigenesis. - Abstract: We previously reported that a novel WD-40 domain-containing protein, p44/WDR77, drives quiescent epithelial cells to re-enter the cell cycle and plays an essential role for growth of lung and prostate cancer cells. Transforming growth factor beta (TGFβ) signaling is important in the maintenance of non-transformed cells in the quiescent or slowly cycling stage. However, both non-transformed proliferating cells and human cancer cells are non-responsive to endogenous TGFβ signaling. The mechanismmore » by which proliferating cells become refractory to TGFβ inhibition is not well established. Here, we found that silencing p44/WDR77 increased cellular sensitivity to TGFβ signaling and that this was inversely correlated with decreased cell proliferation. Smad2 or 3 phosphorylation, TGFβ-mediated transcription, and TGFβ2 and TGFβ receptor type II (TβRII) expression were dramatically induced by silencing of p44/WDR77. These data support the hypothesis that p44/WDR77 down-regulates the expression of the TGFβ ligand and its receptor, thereby leading to a cellular non-response to TGFβ signaling. Finally, we found that p44/WDR77 expression was correlated with cell proliferation and decreased TGFβ signaling during lung tumorigenesis. Together, these results suggest that p44/WDR77 expression causes the non-sensitivity of proliferating cells to TGFβ signaling, thereby contributing to cellular proliferation during lung tumorigenesis.« less

Effect of Withaferin A on A549 cellular proliferation and apoptosis in non-small cell lung cancer.

PubMed

Cai, Yong; Sheng, Zhao-Ying; Chen, Yun; Bai, Chong

2014-01-01

To explore the effect of Withaferin A on A549 cellular proliferation and apoptosis in non-small cell lung cancer (NSCLC). NSCNC cell line A549 was selected to explore the effect of Withaferin A on A549 cellular proliferation, apoptosis and the PI3K/Akt signal pathway capable of regulating tumor biological behavior by assessment of cellular proliferation, cellular apoptotic rates and cellular cycling as well as by immuno-blotting. Withaferin A could inhibit A549 cellular proliferation and the control rate was dosage-dependent (P<0.05), which also increased time-dependently with the same dosage of Withaferin A (P<0.05). The apoptotic indexes in A549 cells treated with 0, 2.5, 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A for 48 h were significantly different (P<0.05). In addition, the apoptotic rates of each group in both early and advanced stages were higher than those in 0 μmol·L-1 (P<0.05), which were evidently higher after 48 h than those after 24 h (P<0.05). A549 cells treated by Withaferin A for 48 h were markedly lower in Bcl-2 level and obviously higher in Bax and cleaved caspase-3 levels than those treated by 0 μmol·L-1 Withaferin A (P<0.05), and there were significant differences among 5, 10 and 20 μmol·L-1 Withaferin A (P<0.05). The ratios of A549 cells treated by Withaferin A for 48 h in G0/G1 stage were higher than those in 0 μmol·L-1 , while those in S and G2/M stages were obviously lower than those in G2/M stage, and there were significant differences in 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A (P<0.05). Additionally, p-Akt/Akt values were in reverse association with dosage, and the differences were significant (P<0.05). Withaferin A can inhibit the proliferation and apoptosis of A549 cells by suppressing activation of the PI3K/Akt pathways.

Quantifying the correlation between spatially defined oxygen gradients and cell fate in an engineered three-dimensional culture model.

PubMed

Ardakani, Amir G; Cheema, Umber; Brown, Robert A; Shipley, Rebecca J

2014-09-06

A challenge in three-dimensional tissue culture remains the lack of quantitative information linking nutrient delivery and cellular distribution. Both in vivo and in vitro, oxygen is delivered by diffusion from its source (blood vessel or the construct margins). The oxygen level at a defined distance from its source depends critically on the balance of diffusion and cellular metabolism. Cells may respond to this oxygen environment through proliferation, death and chemotaxis, resulting in spatially resolved gradients in cellular density. This study extracts novel spatially resolved and simultaneous data on tissue oxygenation, cellular proliferation, viability and chemotaxis in three-dimensional spiralled, cellular collagen constructs. Oxygen concentration gradients drove preferential cellular proliferation rates and viability in the higher oxygen zones and induced chemotaxis along the spiral of the collagen construct; an oxygen gradient of 1.03 mmHg mm(-1) in the spiral direction induced a mean migratory speed of 1015 μm day(-1). Although this movement was modest, it was effective in balancing the system to a stable cell density distribution, and provided insights into the natural cell mechanism for adapting cell number and activity to a prevailing oxygen regime.

DNA mismatch-specific targeting and hypersensitivity of mismatch-repair-deficient cells to bulky rhodium(III) intercalators

PubMed Central

Hart, Jonathan R.; Glebov, Oleg; Ernst, Russell J.; Kirsch, Ilan R.; Barton, Jacqueline K.

2006-01-01

Mismatch repair (MMR) is critical to maintaining the integrity of the genome, and deficiencies in MMR are correlated with cancerous transformations. Bulky rhodium intercalators target DNA base mismatches with high specificity. Here we describe the application of bulky rhodium intercalators to inhibit cellular proliferation differentially in MMR-deficient cells compared with cells that are MMR-proficient. Preferential inhibition by the rhodium complexes associated with MMR deficiency is seen both in a human colon cancer cell line and in normal mouse fibroblast cells; the inhibition of cellular proliferation depends strictly on the MMR deficiency of the cell. Furthermore, our assay of cellular proliferation is found to correlate with DNA mismatch targeting by the bulky metallointercalators. It is the Δ-isomer that is active both in targeting base mismatches and in inhibiting DNA synthesis. Additionally, the rhodium intercalators promote strand cleavage at the mismatch site with photoactivation, and we observe that the cellular response is enhanced with photoactivation. Targeting DNA mismatches may therefore provide a cell-selective strategy for chemotherapeutic design. PMID:17030786

[Study on thaspine in inducing apoptosis of A549 cell].

PubMed

Zhang, Yan-min; He, Lang-chong

2007-04-01

To investigate the effect of thaspine on the cellular proliferation, apoptosis and cell cycle in A549 cell line. A549 cell was cultured with different concentrations of thaspine. Cellular proliferation was detected with MTT, apoptosis and cell cycle were checked with Flow Cytometer, and change of microstructure was observed by transmission electron microscope. Thaspine could inhibit the proliferation and induce apoptosis of A549 cell in a time-dose dependent manner. Cell cycle was significantly stopped at the S phase by thaspine with FCM technology. Under electronic microscope, the morphology of A549 cell showed nuclear karyopycnosis, chromatin agglutination and typical apoptotic body when the cell was treated with thaspine. Thaspine has the effects of anti-tumor and inducing apoptosis.

Expression and significance of Ki-67 in lung cancer.

PubMed

Folescu, Roxana; Levai, Codrina Mihaela; Grigoraş, Mirela Loredana; Arghirescu, Teodora Smaranda; Talpoş, Ioana Cristina; Gîndac, Ciprian Mihai; Zamfir, Carmen Lăcrămioara; Poroch, Vladimir; Anghel, Mirella Dorina

2018-01-01

Ki-67 parameter is a proliferation marker in malignant tumors. The increased proliferation activity and the decreased prognosis in lung cancer determined us to investigate different parameters connected to the tumor's aggression, such as cellularity, Ki-67 positivity rate, and proliferating cell nuclear antigen (PCNA). We evaluated the proliferative activity in 62 primary lung tumors by determining the cell's percentage of Ki-67 and immunoreactive PCNA (using MIB-1 and PCNA monoclonal antibodies), classifying Ki-67 and PCNA immunoreactivity into three score groups. The results obtained emphasized a linkage between Ki-67 score with the histological tumor subtype, tumor cellularity and degree of differentiation and with other proliferation immunohistochemistry (IHC) markers, such as p53 cellular tumor antigen. The tumor's cellularity, the Ki-67 positivity rate and PCNA, together with the clinical stage and the histological differentiation bring extra pieces of useful information in order to anticipate the evolution and the prognosis of lung cancer.

The MADS-box XAANTAL1 increases proliferation at the Arabidopsis root stem-cell niche and participates in transition to differentiation by regulating cell-cycle components

PubMed Central

García-Cruz, Karla V.; García-Ponce, Berenice; Garay-Arroyo, Adriana; Sanchez, María De La Paz; Ugartechea-Chirino, Yamel; Desvoyes, Bénédicte; Pacheco-Escobedo, Mario A.; Tapia-López, Rosalinda; Ransom-Rodríguez, Ivan; Gutierrez, Crisanto; Alvarez-Buylla, Elena R.

2016-01-01

Background Morphogenesis depends on the concerted modulation of cell proliferation and differentiation. Such modulation is dynamically adjusted in response to various external and internal signals via complex transcriptional regulatory networks that mediate between such signals and regulation of cell-cycle and cellular responses (proliferation, growth, differentiation). In plants, which are sessile, the proliferation/differentiation balance is plastically adjusted during their life cycle and transcriptional networks are important in this process. MADS-box genes are key developmental regulators in eukaryotes, but their role in cell proliferation and differentiation modulation in plants remains poorly studied. Methods We characterize the XAL1 loss-of-function xal1-2 allele and overexpression lines using quantitative cellular and cytometry analyses to explore its role in cell cycle, proliferation, stem-cell patterning and transition to differentiation. We used quantitative PCR and cellular markers to explore if XAL1 regulates cell-cycle components and PLETHORA1 (PLT1) gene expression, as well as confocal microscopy to analyse stem-cell niche organization. Key Results We previously showed that XAANTAL1 (XAL1/AGL12) is necessary for Arabidopsis root development as a promoter of cell proliferation in the root apical meristem. Here, we demonstrate that XAL1 positively regulates the expression of PLT1 and important components of the cell cycle: CYCD3;1, CYCA2;3, CYCB1;1, CDKB1;1 and CDT1a. In addition, we show that xal1-2 mutant plants have a premature transition to differentiation with root hairs appearing closer to the root tip, while endoreplication in these plants is partially compromised. Coincidently, the final size of cortex cells in the mutant is shorter than wild-type cells. Finally, XAL1 overexpression-lines corroborate that this transcription factor is able to promote cell proliferation at the stem-cell niche. Conclusion XAL1 seems to be an important component of the networks that modulate cell proliferation/differentiation transition and stem-cell proliferation during Arabidopsis root development; it also regulates several cell-cycle components. PMID:27474508

Low doses of TiO2-polyethylene glycol nanoparticles stimulate proliferation of hepatocyte cells

PubMed Central

Sun, Qingqing; Kanehira, Koki; Taniguchi, Akiyoshi

2016-01-01

Abstract This paper describes the effect of low concentrations of 100 nm polyethylene glycol-modified TiO2 nanoparticles (TiO2-PEG NPs) on HepG2 hepatocellular carcinoma cells. Proliferation of HepG2 cells increased significantly when the cells were exposed to low doses (<100 μg ml–1) of TiO2-PEG NPs. These results were further confirmed by cell counting experiments and cell cycle assays. Cellular uptake assays were performed to determine why HepG2 cells proliferate with low-dose exposure to TiO2-PEG NPs. The results showed that exposure to lower doses of NPs led to less cellular uptake, which in turn decreased cytotoxicity. We therefore hypothesized that TiO2-PEG NPs could affect the activity of hepatocyte growth factor receptors (HGFRs), which bind to hepatocyte growth factor and stimulate cell proliferation. The localization of HGFRs on the surface of the cell membrane was detected via immunofluorescence staining and confocal microscopy. The results showed that HGFRs aggregate after exposure to TiO2-PEG NPs. In conclusion, our results indicate that TiO2-PEG NPs have the potential to promote proliferation of HepG2 cells through HGFR aggregation and suggest that NPs not only exhibit cytotoxicity but also affect cellular responses. PMID:27877913

Positive and Negative Regulatory Mechanisms for Fine-Tuning Cellularity and Functions of Medullary Thymic Epithelial Cells.

PubMed

Akiyama, Taishin; Tateishi, Ryosuke; Akiyama, Nobuko; Yoshinaga, Riko; Kobayashi, Tetsuya J

2015-01-01

Self-tolerant T cells and regulatory T cells develop in the thymus. A wide variety of cell-cell interactions in the thymus is required for the differentiation, proliferation, and repertoire selection of T cells. Various secreted and cell surface molecules expressed in thymic epithelial cells (TECs) mediate these processes. Moreover, cytokines expressed by cells of hematopoietic origin regulate the cellularity of TECs. Tumor necrosis factor (TNF) family RANK ligand, lymphotoxin, and CD40 ligand, expressed in T cells and innate lymphoid cells (ILCs), promote the differentiation and proliferation of medullary TECs (mTECs) that play critical roles in the induction of immune tolerance. A recent study suggests that interleukin-22 (IL-22) produced by ILCs promotes regeneration of TECs after irradiation. Intriguingly, tumor growth factor-β and osteoprotegerin limit cellularity of mTECs, thereby attenuating regulatory T cell generation. We will review recent insights into the molecular basis for cell-cell interactions regulating differentiation and proliferation of mTECs and also discuss about a perspective on use of mathematical models for understanding this complicated system.

Cellular automata model for human articular chondrocytes migration, proliferation and cell death: An in vitro validation.

PubMed

Vaca-González, J J; Gutiérrez, M L; Guevara, J M; Garzón-Alvarado, D A

2017-01-01

Articular cartilage is characterized by low cell density of only one cell type, chondrocytes, and has limited self-healing properties. When articular cartilage is affected by traumatic injuries, a therapeutic strategy such as autologous chondrocyte implantation is usually proposed for its treatment. This approach requires in vitro chondrocyte expansion to yield high cell number for cell transplantation. To improve the efficiency of this procedure, it is necessary to assess cell dynamics such as migration, proliferation and cell death during culture. Computational models such as cellular automata can be used to simulate cell dynamics in order to enhance the result of cell culture procedures. This methodology has been implemented for several cell types; however, an experimental validation is required for each one. For this reason, in this research a cellular automata model, based on random-walk theory, was devised in order to predict articular chondrocyte behavior in monolayer culture during cell expansion. Results demonstrated that the cellular automata model corresponded to cell dynamics and computed-accurate quantitative results. Moreover, it was possible to observe that cell dynamics depend on weighted probabilities derived from experimental data and cell behavior varies according to the cell culture period. Thus, depending on whether cells were just seeded or proliferated exponentially, culture time probabilities differed in percentages in the CA model. Furthermore, in the experimental assessment a decreased chondrocyte proliferation was observed along with increased passage number. This approach is expected to having other uses as in enhancing articular cartilage therapies based on tissue engineering and regenerative medicine.

Immunostimulation by cytomegalovirus (CMV): helper T cell-dependent activation of immunoglobulin production in vitro by lymphocytes from CMV-immune donors

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

Yachie, A.; Tosato, G.; Straus, S.E.

1985-08-01

Cytomegalovirus (CMV) is the cause of a number of different diseases ranging from self-limited benign infections in healthy adults to life threatening illnesses among immunocompromised hosts and newborns. Suppression of cell-mediated immunity is often found in cases of acute CMV infection, and in addition, the virus may also be a potent stimulant of lymphoid cells in vivo. The authors studied cellular proliferation and immunoglobulin (Ig) production induced by CMV to determine its effect on human lymphocytes in vitro. The CMV that was added to cultures of lymphocytes from CMV-seronegative donors failed to induce either significant cellular proliferation or Ig production.more » By contrast, CMV-stimulated cultures from CMV-seropositive donors induced both prominent cellular proliferation and Ig production. B cell differentiation into Ig-secreting cells required the presence of T cells, and this T cell help was sensitive to irradiation with 2000 rad and to treatment with cyclosporin A. When T cells were depleted of OKT4+ cells with monoclonal antibody and complement, the co-cultured B cells failed to produce Ig, whereas the depletion of OKT8+ cells had no effect on the Ig-secreting cell response. Inactivation of CMV before culture did not result in a reduction of either cellular proliferation or Ig production. Thus, infection of target cells is not required for in vitro lymphocyte activation by CMV. These results demonstrate that CMV is a potent activator of B cells inducing Ig production in vitro, and that this process requires the presence of virus-specific memory T cells.« less

Tetraspanin CD9 modulates human lymphoma cellular proliferation via histone deacetylase activity

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

Herr, Michael J.; Department of Medicine, The University of Tennessee Health Science Center, Memphis, TN 38163; Department of Molecular Sciences, The University of Tennessee Health Science Center, Memphis, TN 38163

2014-05-16

Highlights: • CD9 is differentially expressed in human Burkitt’s lymphoma cells. • We found that CD9 expression promotes these cells proliferation. • CD9 expression also increases HDAC activity. • HDAC inhibition decreased both cell proliferation and importantly CD9 expression. • CD9 may dictate HDAC efficacy and play a role in HDAC regulation. - Abstract: Non-Hodgkin Lymphoma (NHL) is a type of hematological malignancy that affects two percent of the overall population in the United States. Tetraspanin CD9 is a cell surface protein that has been thoroughly demonstrated to be a molecular facilitator of cellular phenotype. CD9 expression varies in twomore » human lymphoma cell lines, Raji and BJAB. In this report, we investigated the functional relationship between CD9 and cell proliferation regulated by histone deacetylase (HDAC) activity in these two cell lines. Introduction of CD9 expression in Raji cells resulted in significantly increased cell proliferation and HDAC activity compared to Mock transfected Raji cells. The increase in CD9–Raji cell proliferation was significantly inhibited by HDAC inhibitor (HDACi) treatment. Pretreatment of BJAB cells with HDAC inhibitors resulted in a significant decrease in endogenous CD9 mRNA and cell surface expression. BJAB cells also displayed decreased cell proliferation after HDACi treatment. These results suggest a significant relationship between CD9 expression and cell proliferation in human lymphoma cells that may be modulated by HDAC activity.« less

Povidone-Iodine Has a Profound Effect on In Vitro Osteoblast Proliferation and Metabolic Function and Inhibits Their Ability to Mineralize and Form Bone.

PubMed

Newton Ede, Matthew P; Philp, Ashleigh M; Philp, Andrew; Richardson, Stephen M; Mohammad, Saeed; Jones, Simon W

2016-05-01

A study examining the clinical protocol of scoliosis wound irrigation, demonstrating povidone-iodine's (PVI) effect on human osteoblast cells. Primary and immortal cell line osteoblasts were treated with 0.35% PVI for 3 minutes, and analyzed for proliferation rate, oxidative capacity, and mineralization. To model spinal wound irrigation with dilute PVI in vitro, in order to investigate the effect of PVI on osteoblast proliferation, metabolism, and bone mineralization. Previously PVI irrigation has been proposed as a safe and effective practice to avoid bacterial growth after spinal surgery. However, recent evidence in multiple cell types suggests that PVI has a deleterious effect on cellular viability and cellular function. Primary and immortal human osteoblast cells were exposed to either phosphate buffered saline control or with 0.35% PVI for 3 minutes. Cellular proliferation was measured over the duration of 7 days by MTS assay. Oxygen consumption rate, extracellular acidification rate, and proton production rate were analyzed using a Seahorse XF24 Bioanalyzer. Protein expression of the electron transport chain subunits CII-SDHB, CIII-UQRCR2, and CV-ATP5A was measured via Western blotting. Mineralized bone nodules were stained with alizarin red. Expressed as a percentage of normal osteoblast proliferation, osteoblasts exposed to 0.35% PVI exhibited a significant 24% decrease in proliferation after 24 hours. This was a sustained response, resulting in a 72% decline in cellular proliferation at 1 week. There was a significant reduction in oxygen consumption rate, extracellular acidification rate, and proton production rate (P < 0.05), in osteoblasts that had been exposed to 0.35% PVI for 3 minutes, coupled with a marked reduction in the protein expression of CII-SDHB. Osteoblasts exposed to 0.35% PVI exhibited reduced bone nodule mineralization compared to control phosphate buffered saline exposed osteoblasts (P < 0.01). PVI has a rapid and detrimental effect on human osteoblast cellular proliferation, metabolic function, and bone nodule mineralization. NA.

Low Expression of lncRNA-GAS5 Is Implicated in Human Primary Varicose Great Saphenous Veins

PubMed Central

Yuan, Tian-You; Wang, Shi-Yi; Feng, Jing; Wang, Jing; Liu, Yuan; Wu, Ya-Han; Ma, Xiu-E; Ge, Jin; Cui, Ying-Yu; Jiang, Xiao-Yan

2015-01-01

The cellular mechanisms of primary varicose great saphenous veins (GSVs) involve inflammation, apoptosis, and proliferation of local cells and extracellular matrix degradation. Long non-coding RNAs (lncRNAs) play important roles in these cellular processes; however, which and how lncRNAs related to these mechanisms take effect on GSVs remain unclear. By screening lncRNAs that might experience changes in GSV varicosities, we selected the lower expressed lncRNA-GAS5 (growth arrest specific transcript 5) for functional assessments. Silencing of lncRNA-GAS5 promoted cell proliferation and migration, and cell cycle of the human saphenous vein smooth muscle cells (HSVSMCs), whereas overexpressing it inhibited these cellular behaviors and reduced apoptosis of HSVSMCs. RNA pull-down experiment revealed a direct bind of lncRNA-GAS5 to a Ca2+-dependent RNA-binding protein, Annexin A2. Further experiments showed that silencing of Annexin A2 reduced the HSVSMCs proliferation and vice versa. In the context of lncRNA-GAS5 knockdown, silencing of Annexin A2 reduced the proliferation of HSVSMCs while overexpression of Annexin A2 increased the proliferation. Thus, the low expression of lncRNA-GAS5 may facilitate HSVSMCs proliferation and migration through Annexin A2 and thereby the pathogenesis of GSV varicosities. PMID:25806802

Effect of heated naringenin on immunomodulatory properties and cellular antioxidant activity.

PubMed

Maatouk, Mouna; Elgueder, Dorra; Mustapha, Nadia; Chaaban, Hind; Bzéouich, Imen Mokdad; Loannou, Irina; Kilani, Soumaya; Ghoul, Mohamed; Ghedira, Kamel; Chekir-Ghedira, Leila

2016-11-01

Naringenin is one of the most popular flavonoids derived from citrus. It has been reported to be an effective anti-inflammatory compound. Citrus fruit may be used raw, cooked, stewed, or boiled. The present study was conducted to investigate the effect of thermal processes on naringenin in its immunomodulatory and cellular antioxidant activities. The effects of flavonoids on B and T cell proliferation were assessed on splenocytes stimulated or not with mitogens. However, their effects on cytotoxic T lymphocyte (CTL) and natural killer (NK) activities were assessed in splenocytes co-incubated with target cells. The amount of nitric oxide production and the lysosomal enzyme activity were evaluated in vitro on mouse peritoneal macrophages. Cellular antioxidant activity in splenocytes and macrophages was determined by measuring the fluorescence of the dichlorofluorescin (DCF). Our findings revealed that naringenin induces B cell proliferation and enhances NK activity. The highest concentration of native naringenin exhibits a significant proliferation of T cells, induces CTL activity, and inhibits cellular oxidation in macrophages. Conversely, it was observed that when heat-processed, naringenin improves the cellular antioxidant activity in splenocytes, increases the cytotoxic activity of NK cells, and suppresses the cytotoxicity of T cells. However, heat treatment maintains the anti-inflammatory potency of naringenin.

Understanding the Warburg effect: the metabolic requirements of cell proliferation.

PubMed

Vander Heiden, Matthew G; Cantley, Lewis C; Thompson, Craig B

2009-05-22

In contrast to normal differentiated cells, which rely primarily on mitochondrial oxidative phosphorylation to generate the energy needed for cellular processes, most cancer cells instead rely on aerobic glycolysis, a phenomenon termed "the Warburg effect." Aerobic glycolysis is an inefficient way to generate adenosine 5'-triphosphate (ATP), however, and the advantage it confers to cancer cells has been unclear. Here we propose that the metabolism of cancer cells, and indeed all proliferating cells, is adapted to facilitate the uptake and incorporation of nutrients into the biomass (e.g., nucleotides, amino acids, and lipids) needed to produce a new cell. Supporting this idea are recent studies showing that (i) several signaling pathways implicated in cell proliferation also regulate metabolic pathways that incorporate nutrients into biomass; and that (ii) certain cancer-associated mutations enable cancer cells to acquire and metabolize nutrients in a manner conducive to proliferation rather than efficient ATP production. A better understanding of the mechanistic links between cellular metabolism and growth control may ultimately lead to better treatments for human cancer.

Mequindox induced cellular DNA damage via generation of reactive oxygen species.

PubMed

Liu, Jing; Ouyang, Man; Jiang, Jun; Mu, Peiqiang; Wu, Jun; Yang, Qi; Zhang, Caihui; Xu, Weiying; Wang, Lijuan; Huen, Michael S Y; Deng, Yiqun

2012-01-24

Mequindox, a quinoxaline-N-dioxide derivative that possesses antibacterial properties, has been widely used as a feed additive in the stockbreeding industry in China. While recent pharmacological studies have uncovered potential hazardous effects of mequindox, exactly how mequindox induces pathological changes and the cellular responses associated with its consumption remain largely unexplored. In this study, we investigated the cellular responses associated with mequindox treatment. We report here that mequindox inhibits cell proliferation by arresting cells at the G2/M phase of the cell cycle. Interestingly, this mequindox-associated deleterious effect on cell proliferation was observed in human, pig as well as chicken cells, suggesting that mequindox acts on evolutionarily conserved target(s). To further understand the mequindox-host interaction and the mechanism underlying mequindox-induced cell cycle arrest, we measured the cellular content of DNA damage, which is known to perturb cell proliferation and compromise cell survival. Accordingly, using γ-H2AX as a surrogate marker for DNA damage, we found that mequindox treatment induced cellular DNA damage, which paralleled the chemical-induced elevation of reactive oxygen species (ROS) levels. Importantly, expression of the antioxidant enzyme catalase partially alleviated these mequindox-associated effects. Taken together, our results suggest that mequindox cytotoxicity is attributable, in part, to its role as a potent inducer of DNA damage via ROS. © 2011 Elsevier B.V. All rights reserved.

Low oxygen level increases proliferation and metabolic changes in bovine granulosa cells.

PubMed

Shiratsuki, Shogo; Hara, Tomotaka; Munakata, Yasuhisa; Shirasuna, Koumei; Kuwayama, Takehito; Iwata, Hisataka

2016-12-05

The present study addresses molecular backgrounds underlying low oxygen induced metabolic changes and 1.2-fold change in bovine granulosa cell (GCs) proliferation. RNA-seq revealed that low oxygen (5%) upregulated genes associated with HIF-1 and glycolysis and downregulated genes associated with mitochondrial respiration than that in high oxygen level (21%). Low oxygen level induced high glycolytic activity and low mitochondrial function and biogenesis. Low oxygen level enhanced GC proliferation with high expression levels of HIF-1, VEGF, AKT, mTOR, and S6RP, whereas addition of anti-VEGF antibody decreased cellular proliferation with low phosphorylated AKT and mTOR expression levels. Low oxygen level reduced SIRT1, whereas activation of SIRT1 by resveratrol increased mitochondrial replication and decreased cellular proliferation with reduction of phosphorylated mTOR. These results suggest that low oxygen level stimulates the HIF1-VEGF-AKT-mTOR pathway and up-regulates glycolysis, which contributes to GC proliferation, and downregulation of SIRT1 contributes to hypoxia-associated reduction of mitochondria and cellular proliferation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The MADS-box XAANTAL1 increases proliferation at the Arabidopsis root stem-cell niche and participates in transition to differentiation by regulating cell-cycle components.

PubMed

García-Cruz, Karla V; García-Ponce, Berenice; Garay-Arroyo, Adriana; Sanchez, María De La Paz; Ugartechea-Chirino, Yamel; Desvoyes, Bénédicte; Pacheco-Escobedo, Mario A; Tapia-López, Rosalinda; Ransom-Rodríguez, Ivan; Gutierrez, Crisanto; Alvarez-Buylla, Elena R

2016-07-29

Morphogenesis depends on the concerted modulation of cell proliferation and differentiation. Such modulation is dynamically adjusted in response to various external and internal signals via complex transcriptional regulatory networks that mediate between such signals and regulation of cell-cycle and cellular responses (proliferation, growth, differentiation). In plants, which are sessile, the proliferation/differentiation balance is plastically adjusted during their life cycle and transcriptional networks are important in this process. MADS-box genes are key developmental regulators in eukaryotes, but their role in cell proliferation and differentiation modulation in plants remains poorly studied. We characterize the XAL1 loss-of-function xal1-2 allele and overexpression lines using quantitative cellular and cytometry analyses to explore its role in cell cycle, proliferation, stem-cell patterning and transition to differentiation. We used quantitative PCR and cellular markers to explore if XAL1 regulates cell-cycle components and PLETHORA1 (PLT1) gene expression, as well as confocal microscopy to analyse stem-cell niche organization. We previously showed that XAANTAL1 (XAL1/AGL12) is necessary for Arabidopsis root development as a promoter of cell proliferation in the root apical meristem. Here, we demonstrate that XAL1 positively regulates the expression of PLT1 and important components of the cell cycle: CYCD3;1, CYCA2;3, CYCB1;1, CDKB1;1 and CDT1a In addition, we show that xal1-2 mutant plants have a premature transition to differentiation with root hairs appearing closer to the root tip, while endoreplication in these plants is partially compromised. Coincidently, the final size of cortex cells in the mutant is shorter than wild-type cells. Finally, XAL1 overexpression-lines corroborate that this transcription factor is able to promote cell proliferation at the stem-cell niche. XAL1 seems to be an important component of the networks that modulate cell proliferation/differentiation transition and stem-cell proliferation during Arabidopsis root development; it also regulates several cell-cycle components. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

An in vitro method to test the safety and efficacy of low-level laser therapy (LLLT) in the healing of a canine skin model.

PubMed

Gagnon, Dominique; Gibson, Thomas W G; Singh, Ameet; zur Linden, Alex R; Kazienko, Jaimie E; LaMarre, Jonathan

2016-04-08

Low-level laser therapy (LLLT) has been used clinically as a treatment modality for a variety of medical conditions including wound-healing processes. It is an attractive and emerging method to enhance wound healing and improve clinical outcomes both in human and veterinary medicine. Despite the fact that the use of LLLT continues to gain in popularity, there is no universally accepted theory that defends all its cellular effects and beneficial biological processes in tissue repair. The present study was designed to evaluate the effect of LLLT on cellular migration and proliferation of cultured canine epidermal keratinocytes (CPEK) in an in vitro wound healing model. Keratinocyte migration and proliferation were assessed using a scratch migration assay and a proliferation assay, respectively. Fifteen independent replicates were performed for each assay. Canine epidermal keratinocyte cells exposed to LLLT with 0.1, 0.2, and 1.2 J/cm(2) migrated significantly more rapidly (p < 0.03) and showed significantly higher rates of proliferation (p < 0.0001) compared to non-irradiated cells cultured in the same medium and cells exposed to the higher energy dose of 10 J/cm(2). Irradiation with 10 J/cm(2) was characterized by decreased cellular migration and proliferation. These results revealed that LLLT has a measurable, dose-dependent effect on two different aspects of keratinocyte biology in vitro. In this in vitro wound-healing model, LLLT increased cellular migration and proliferation at doses of 0.1, 0.2, and 1.2 J/cm(2) while exposure to 10 J/cm(2) decreased cellular migration and proliferation. These data suggest that the beneficial effects of LLLT in vivo may be due, in part, to effects on keratinocyte behavior.

Cellular proliferation in the urorectal septation complex of the human embryo at Carnegie stages 13-18: a nuclear area-based morphometric analysis.

PubMed

Nebot-Cegarra, Josep; Fàbregas, Pere Jordi; Sánchez-Pérez, Inma

2005-10-01

In order to analyse the patterns of cellular proliferation both in the mesenchyme of the urorectal septum (URS) and in the adjacent territories (posterior urogenital mesenchyme, anterior intestinal mesenchyme and cloacal folds mesenchyme), as well as their contribution to the process of cloacal division, a computer-assisted method was used to obtain the nuclear area of 3874 mesenchymal cells from camera lucida drawings of nuclear contours of selected sections of human embryos [Carnegie stages (CSs) 13-18]. Based on changes in the size of the nucleus during the cellular cycle, we considered proliferating cells in each territory to be those with a nuclear area over the 75th percentile. The URS showed increasing cell proliferation, with proliferation patterns that coincided closely with cloacal folds mesenchyme, and with less overall proliferation than urogenital and intestinal mesenchymes. Furthermore, at CS 18, we observed the beginning of the rupture in the cloacal membrane; however, no fusion has been demonstrated either between the URS and the cloacal membrane or between the cloacal folds. The results suggest that cloacal division depends on a morphogenetic complex where the URS adjacent territories could determine septal displacement at the time that their mesenchymes could be partially incorporated within the proliferating URS.

Cellular proliferation in the urorectal septation complex of the human embryo at Carnegie stages 13–18: a nuclear area-based morphometric analysis

PubMed Central

Nebot-Cegarra, Josep; Fàbregas, Pere Jordi; Sánchez-Pérez, Inma

2005-01-01

In order to analyse the patterns of cellular proliferation both in the mesenchyme of the urorectal septum (URS) and in the adjacent territories (posterior urogenital mesenchyme, anterior intestinal mesenchyme and cloacal folds mesenchyme), as well as their contribution to the process of cloacal division, a computer-assisted method was used to obtain the nuclear area of 3874 mesenchymal cells from camera lucida drawings of nuclear contours of selected sections of human embryos [Carnegie stages (CSs) 13–18]. Based on changes in the size of the nucleus during the cellular cycle, we considered proliferating cells in each territory to be those with a nuclear area over the 75th percentile. The URS showed increasing cell proliferation, with proliferation patterns that coincided closely with cloacal folds mesenchyme, and with less overall proliferation than urogenital and intestinal mesenchymes. Furthermore, at CS 18, we observed the beginning of the rupture in the cloacal membrane; however, no fusion has been demonstrated either between the URS and the cloacal membrane or between the cloacal folds. The results suggest that cloacal division depends on a morphogenetic complex where the URS adjacent territories could determine septal displacement at the time that their mesenchymes could be partially incorporated within the proliferating URS. PMID:16191164

Enhancing proliferation and optimizing the culture condition for human bone marrow stromal cells using hypoxia and fibroblast growth factor-2.

PubMed

Lee, Jung-Seok; Kim, Seul Ki; Jung, Byung-Joo; Choi, Seong-Bok; Choi, Eun-Young; Kim, Chang-Sung

2018-04-01

This study aimed to determine the cellular characteristics and behaviors of human bone marrow stromal cells (hBMSCs) expanded in media in a hypoxic or normoxic condition and with or without fibroblast growth factor-2 (FGF-2) treatment. hBMSCs isolated from the vertebral body and expanded in these four groups were evaluated for cellular proliferation/migration, colony-forming units, cell-surface characterization, in vitro differentiation, in vivo transplantation, and gene expression. Culturing hBMSCs using a particular environmental factor (hypoxia) and with the addition of FGF-2 increased the cellular proliferation rate while enhancing the regenerative potential, modulated the multipotency-related processes (enhanced chondrogenesis-related processes/osteogenesis, but reduced adipogenesis), and increased cellular migration and collagen formation. The gene expression levels in the experimental samples showed activation of the hypoxia-inducible factor-1 pathway and glycolysis in the hypoxic condition, with this not being affected by the addition of FGF-2. The concurrent application of hypoxia and FGF-2 could provide a favorable condition for culturing hBMSCs to be used in clinical applications associated with bone tissue engineering, due to the enhancement of cellular proliferation and regenerative potential. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Msx homeobox genes inhibit differentiation through upregulation of cyclin D1.

PubMed

Hu, G; Lee, H; Price, S M; Shen, M M; Abate-Shen, C

2001-06-01

During development, patterning and morphogenesis of tissues are intimately coordinated through control of cellular proliferation and differentiation. We describe a mechanism by which vertebrate Msx homeobox genes inhibit cellular differentiation by regulation of the cell cycle. We show that misexpression of Msx1 via retroviral gene transfer inhibits differentiation of multiple mesenchymal and epithelial progenitor cell types in culture. This activity of Msx1 is associated with its ability to upregulate cyclin D1 expression and Cdk4 activity, while Msx1 has minimal effects on cellular proliferation. Transgenic mice that express Msx1 under the control of the mouse mammary tumor virus long terminal repeat (MMTV LTR) display impaired differentiation of the mammary epithelium during pregnancy, which is accompanied by elevated levels of cyclin D1 expression. We propose that Msx1 gene expression maintains cyclin D1 expression and prevents exit from the cell cycle, thereby inhibiting terminal differentiation of progenitor cells. Our model provides a framework for reconciling the mutant phenotypes of Msx and other homeobox genes with their functions as regulators of cellular proliferation and differentiation during embryogenesis.

[Effects of ezrin silencing on pancreatic cancer cell line Panc-1].

PubMed

Meng, Yun-xiao; Yu, Shuang-ni; Lu, Zhao-hui; Chen, Jie

2012-12-01

To explore the effects of ezrin silencing on pancreatic cancer cell line Panc-1. Pancreatic cancer cell line Panc-1 was transfected with ezrin silencing plasmid. The proliferation and the cell cycle status were determined by CCK-8 assay and flow cytometry analysis, respectively. Cellular membrane protrusions/microvilli formation were visualized by scanning election microscopy. Colony formation assay was used to determine the cell anchor-independent growth ability in vitro. Trans-filter migration and invasion assays were performed with 8 µm pore inserts in a 24-well BioCoat chamber with/without Matrigel. Ezrin silencing decreased cellular protrusions/microvilli formation, anchorage-independent growth, cell migration and invasion, but had no effects on cell proliferation in vitro and cell cycle, in pancreatic cancer cell line Panc-1. Ezrin expression affects the cellular protrusions/microvilli formation, anchorage-independent growth, cell migration and invasion in pancreatic cancer cell line Panc-1.

Circulatory shear flow alters the viability and proliferation of circulating colon cancer cells

NASA Astrophysics Data System (ADS)

Fan, Rong; Emery, Travis; Zhang, Yongguo; Xia, Yuxuan; Sun, Jun; Wan, Jiandi

2016-06-01

During cancer metastasis, circulating tumor cells constantly experience hemodynamic shear stress in the circulation. Cellular responses to shear stress including cell viability and proliferation thus play critical roles in cancer metastasis. Here, we developed a microfluidic approach to establish a circulatory microenvironment and studied circulating human colon cancer HCT116 cells in response to a variety of magnitude of shear stress and circulating time. Our results showed that cell viability decreased with the increase of circulating time, but increased with the magnitude of wall shear stress. Proliferation of cells survived from circulation could be maintained when physiologically relevant wall shear stresses were applied. High wall shear stress (60.5 dyne/cm2), however, led to decreased cell proliferation at long circulating time (1 h). We further showed that the expression levels of β-catenin and c-myc, proliferation regulators, were significantly enhanced by increasing wall shear stress. The presented study provides a new insight to the roles of circulatory shear stress in cellular responses of circulating tumor cells in a physiologically relevant model, and thus will be of interest for the study of cancer cell mechanosensing and cancer metastasis.

Imaging of protein kinase C activation by FRET during proliferation induced by low-energy laser irradiation in living cells

NASA Astrophysics Data System (ADS)

Gao, Xuejuan; Chen, Tongsheng; Xing, Da; Wang, Fang

2005-01-01

Protein kinase Cs (PKCs) play an important role in cellular proliferation, and low-energy laser irradiation (LELI) can enhance cellular proliferation. The present work contributes to the understanding of the mechanisms of action by studying effects of LELI at the dose of 0.8 J/cm2 on PKCs activities in the single lung adenocarcinoma cell (ASTC-a-1) and in real time by fluorescence resonance energy transfer (FRET) technique. C-kinase activity reporter (CKAR), consisting of a cyan fluorescent protein (CFP), the FHA2 phosphothreonine-binding domain, a PKC substrate sequence, and a yellow fluorescent protein (YFP), was utilized. The living cell imaging showed a decrease in FRET in the cytosol and nucleus after the cells were treated with LELI. These results suggest that PKCs could be activated by LELI throughout the cell, and the proliferation of ASTC-a-1 cells could be modulated by the activated PKCs.

Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity

NASA Astrophysics Data System (ADS)

Chiu, Brian; Z-M Wan, Jim; Abley, Doris; Akabutu, John

2005-05-01

Recent studies have demonstrated that stem cells derived from adult hematopoietic tissues are capable of trans-differentiation into non-hematopoietic cells, and that the culture in microgravity ( μg) may modulate the proliferation and differentiation. We investigated the application of μg to human umbilical cord blood stem cells (CBSC) in the induction of vascular endothelial phenotype expression and cellular proliferation. CD34+ mononuclear cells were isolated from waste human umbilical cord blood samples and cultured in simulated μg for 14 days. The cells were seeded in rotary wall vessels (RWV) with or without microcarrier beads (MCB) and vascular endothelial growth factor was added during culture. Controls consisted of culture in 1 G. The cell cultures in RWV were examined by inverted microscopy. Cell counts, endothelial cell and leukocyte markers performed by flow-cytometry and FACS scan were assayed at days 1, 4, 7 and at the termination of the experiments. Culture in RWV revealed significantly increased cellular proliferation with three-dimensional (3D) tissue-like aggregates. At day 4, CD34+ cells cultured in RWV bioreactor without MCB developed vascular tubular assemblies and exhibited endothelial phenotypic markers. These data suggest that CD34+ human umbilical cord blood progenitors are capable of trans-differentiation into vascular endothelial cell phenotype and assemble into 3D tissue structures. Culture of CBSC in simulated μg may be potentially beneficial in the fields of stem cell biology and somatic cell therapy.

Engineering of Surface Functionality onto Polystyrene Microcarriers for the Attachment and Growth of Human Endothelial Cells

NASA Astrophysics Data System (ADS)

Xiong, Gordon M.; Foord, John S.; Griffiths, Jon-Paul; Parker, Emily M.; Moloney, Mark G.; Choong, Cleo

2014-08-01

This work reports the effects of introducing diverse chemical functionalities onto the surface of polystyrene microcarrier beads on their ability to function as injectable cell carriers. Cellular adhesion and proliferation, as well as cellular outgrowths from microcarrier surfaces, using human umbilical vein endothelial cells (HUVECs), were examined in detail. It was observed that initial cell adhesion appeared to be most significantly decreased by hydrophobicity, whilst cell proliferation appeared to be improved in most chemical functional groups over unmodified polystyrene. Overall, our study highlights the importance of surface chemistry in directing the growth and function of human endothelial cells.

Induction of sister chromatid exchanges and inhibition of cellular proliferation in vitro. I. Caffeine

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

Guglielmi, G.E.; Vogt, T.F.; Tice, R.R.

1982-01-01

While many agents have been examined for their ability to induce SCE's, complete dose-response information has often been lacking. We have reexamined the ability of one such compound - caffeine - to induce SCEs and also to inhibit cellular proliferation in human peripheral lymphocytes in vitro. An acute exposure to caffeine prior to the DNA synthetic period did not affect either SCE frequency or the rate of cellular proliferation. Chronic exposure to caffeine throughout the culture period lead to both a dose-dependent increase in SCEs (SCE/sub d/ or doubling dose = 2.4 mM; SCE/sub 10/ or the dose capable ofmore » inducing 10 SCE = 1.4 mM) and a dose-dependent inhibition of cellular proliferation (IC/sub 50/ or the 50% inhibition concentration = 2.6 mM). The relative proportion of first generation metaphase cells, an assessment of proliferative inhibiton, increased linearly with increasing caffeine concentrations. However, SCE frequency increased nonlinearly over the same range of caffeine concentrations. Examination of the ratio of nonsymmetrical to symmetrical SCEs in third generation metaphase cells indicated that caffeine induced SCEs in equal frequency in each of three successive generations. The dependency of SCE induction and cellular proliferative inhibition on caffeine's presence during the DNA synthetic period suggests that caffeine may act as an antimetabolite in normal human cells.« less

Treatment Analysis in a Cancer Stem Cell Context Using a Tumor Growth Model Based on Cellular Automata.

PubMed

Monteagudo, Ángel; Santos, José

2015-01-01

Cancer can be viewed as an emergent behavior in terms of complex system theory and artificial life, Cellular Automata (CA) being the tool most used for studying and characterizing the emergent behavior. Different approaches with CA models were used to model cancer growth. The use of the abstract model of acquired cancer hallmarks permits the direct modeling at cellular level, where a cellular automaton defines the mitotic and apoptotic behavior of cells, and allows for an analysis of different dynamics of the cellular system depending on the presence of the different hallmarks. A CA model based on the presence of hallmarks in the cells, which includes a simulation of the behavior of Cancer Stem Cells (CSC) and their implications for the resultant growth behavior of the multicellular system, was employed. This modeling of cancer growth, in the avascular phase, was employed to analyze the effect of cancer treatments in a cancer stem cell context. The model clearly explains why, after treatment against non-stem cancer cells, the regrowth capability of CSCs generates a faster regrowth of tumor behavior, and also shows that a continuous low-intensity treatment does not favor CSC proliferation and differentiation, thereby allowing an unproblematic control of future tumor regrowth. The analysis performed indicates that, contrary to the current attempts at CSC control, trying to make CSC proliferation more difficult is an important point to consider, especially in the immediate period after a standard treatment for controlling non-stem cancer cell proliferation.

Oral pathogens change proliferation properties of oral tumor cells by affecting gene expression of human defensins.

PubMed

Hoppe, T; Kraus, D; Novak, N; Probstmeier, R; Frentzen, M; Wenghoefer, M; Jepsen, S; Winter, J

2016-10-01

The impact of oral pathogens onto the generation and variability of oral tumors has only recently been investigated. To get further insights, oral cancer cells were treated with pathogens and additionally, as a result of this bacterial cellular infection, with human defensins, which are as anti-microbial peptide members of the innate immune system. After cell stimulation, proliferation behavior, expression analysis of oncogenic relevant defensin genes, and effects on EGFR signaling were investigated. The expression of oncogenic relevant anti-microbial peptides was analyzed with real-time PCR and immunohistochemistry. Cell culture experiments were performed to examine cellular impacts caused by stimulation, i.e., altered gene expression, proliferation rate, and EGF receptor-dependent signaling. Incubation of oral tumor cells with an oral pathogen (Porphyromonas gingivalis) and human α-defensins led to an increase in cell proliferation. In contrast, another oral bacterium used, Aggregatibacter actinomycetemcomitans, enhanced cell death. The bacteria and anti-microbial peptides exhibited diverse effects on the transcript levels of oncogenic relevant defensin genes and epidermal growth factor receptor signaling. These two oral pathogens exhibited opposite primary effects on the proliferation behavior of oral tumor cells. Nevertheless, both microbe species led to similar secondary impacts on the proliferation rate by modifying expression levels of oncogenic relevant α-defensin genes. In this respect, oral pathogens exerted multiplying effects on tumor cell proliferation. Additionally, human defensins were shown to differently influence epidermal growth factor receptor signaling, supporting the hypothesis that these anti-microbial peptides serve as ligands of EGFR, thus modifying the proliferation behavior of oral tumor cells.

Click Chemistry for Analysis of Cell Proliferation in Flow Cytometry.

PubMed

Clarke, Scott T; Calderon, Veronica; Bradford, Jolene A

2017-10-02

The measurement of cellular proliferation is fundamental to the assessment of cellular health, genotoxicity, and the evaluation of drug efficacy. Labeling, detection, and quantification of cells in the synthesis phase of cell cycle progression are not only important for characterizing basic biology, but also in defining cellular responses to drug treatments. Changes in DNA replication during S-phase can provide valuable insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation is the incorporation of a thymidine analog during DNA synthesis. This chapter presents a pulse labeling method using the thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU), with subsequent detection by click chemistry. EdU detection using click chemistry is bio-orthogonal to most living systems and does not non-specifically label other biomolecules. Live cells are first pulsed with EdU. After antibody labeling cell surface markers, fixation, and permeabilization, the incorporated EdU is covalently labeled using click chemistry thereby identifying proliferating cells. Improvements in click chemistry allow for labeling in the presence of fluorescent proteins and phycobiliproteins without quenching due to copper. Measuring DNA replication during cell cycle progression has cell health applications in flow cytometry, fluorescence microscopy, and high content imaging. This protocol has been developed and optimized for research use only and is not suitable for use in diagnostic procedures. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

[Cell signaling pathways interaction in cellular proliferation: Potential target for therapeutic interventionism].

PubMed

Valdespino-Gómez, Víctor Manuel; Valdespino-Castillo, Patricia Margarita; Valdespino-Castillo, Víctor Edmundo

2015-01-01

Nowadays, cellular physiology is best understood by analysing their interacting molecular components. Proteins are the major components of the cells. Different proteins are organised in the form of functional clusters, pathways or networks. These molecules are ordered in clusters of receptor molecules of extracellular signals, transducers, sensors and biological response effectors. The identification of these intracellular signaling pathways in different cellular types has required a long journey of experimental work. More than 300 intracellular signaling pathways have been identified in human cells. They participate in cell homeostasis processes for structural and functional maintenance. Some of them participate simultaneously or in a nearly-consecutive progression to generate a cellular phenotypic change. In this review, an analysis is performed on the main intracellular signaling pathways that take part in the cellular proliferation process, and the potential use of some components of these pathways as target for therapeutic interventionism are also underlined. Copyright © 2015 Academia Mexicana de Cirugía A.C. Published by Masson Doyma México S.A. All rights reserved.

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

Butyrate inhibits cancerous HCT116 cell proliferation but to a lesser extent in noncancerous NCM460 colon cells

USDA-ARS?s Scientific Manuscript database

Butyrate, an intestinal microbiota metabolite of dietary fiber, exhibits chemoprevention effects on colon cancer development. However, the mechanistic action of butyrate at the cellular level remains to be determined. We hypothesize that butyrate inhibits cancerous cell proliferation but to a lesser...

Systematic evaluation of sericin protein as a substitute for fetal bovine serum in cell culture.

PubMed

Liu, Liyuan; Wang, Jinhuan; Duan, Shengchang; Chen, Lei; Xiang, Hui; Dong, Yang; Wang, Wen

2016-08-17

Fetal bovine serum (FBS) shows obvious deficiencies in cell culture, such as low batch to batch consistency, adventitious biological contaminant risk, and high cost, which severely limit the development of the cell culture industry. Sericin protein derived from the silkworm cocoon has become increasingly popular due to its diverse and beneficial cell culture characteristics. However, systematic evaluation of sericin as a substitute for FBS in cell culture medium remains limited. In this study, we conducted cellular morphological, physiological, and transcriptomic evaluation on three widely used mammalian cells. Compared with cells cultured in the control, those cultured in sericin-substitute medium showed similar cellular morphology, similar or higher cellular overall survival, lower population doubling time (PDT), and a higher percentage of S-phase with similar G2/G1 ratio, indicating comparable or better cell growth and proliferation. At the transcriptomic level, differentially expressed genes between cells in the two media were mainly enriched in function and biological processes related to cell growth and proliferation, reflecting that genes were activated to facilitate cell growth and proliferation. The results of this study suggest that cells cultured in sericin-substituted medium perform as well as, or even better than, those cultured in FBS-containing medium.

Systematic evaluation of sericin protein as a substitute for fetal bovine serum in cell culture

PubMed Central

Liu, Liyuan; Wang, Jinhuan; Duan, Shengchang; Chen, Lei; Xiang, Hui; Dong, Yang; Wang, Wen

2016-01-01

Fetal bovine serum (FBS) shows obvious deficiencies in cell culture, such as low batch to batch consistency, adventitious biological contaminant risk, and high cost, which severely limit the development of the cell culture industry. Sericin protein derived from the silkworm cocoon has become increasingly popular due to its diverse and beneficial cell culture characteristics. However, systematic evaluation of sericin as a substitute for FBS in cell culture medium remains limited. In this study, we conducted cellular morphological, physiological, and transcriptomic evaluation on three widely used mammalian cells. Compared with cells cultured in the control, those cultured in sericin-substitute medium showed similar cellular morphology, similar or higher cellular overall survival, lower population doubling time (PDT), and a higher percentage of S-phase with similar G2/G1 ratio, indicating comparable or better cell growth and proliferation. At the transcriptomic level, differentially expressed genes between cells in the two media were mainly enriched in function and biological processes related to cell growth and proliferation, reflecting that genes were activated to facilitate cell growth and proliferation. The results of this study suggest that cells cultured in sericin-substituted medium perform as well as, or even better than, those cultured in FBS-containing medium. PMID:27531556

Podocytes populate cellular crescents in a murine model of inflammatory glomerulonephritis.

PubMed

Moeller, Marcus J; Soofi, Abdulsalaam; Hartmann, Inge; Le Hir, Michel; Wiggins, Roger; Kriz, Wilhelm; Holzman, Lawrence B

2004-01-01

Cellular crescents are a defining histologic finding in many forms of inflammatory glomerulonephritis. Despite numerous studies, the origin of glomerular crescents remains unresolved. A genetic cell lineage-mapping study with a novel transgenic mouse model was performed to investigate whether visceral glomerular epithelial cells, termed podocytes, are precursors of cells that populate cellular crescents. The podocyte-specific 2.5P-Cre mouse line was crossed with the ROSA26 reporter line, resulting in irreversible constitutive expression of beta-galactosidase in doubly transgenic 2.5P-Cre/ROSA26 mice. In these mice, crescentic glomerulonephritis was induced with a previously described rabbit anti-glomerular basement membrane antiserum nephritis approach. Interestingly, beta-galactosidase-positive cells derived from podocytes adhered to the parietal basement membrane and populated glomerular crescents during the early phases of cellular crescent formation, accounting for at least one-fourth of the total cell mass. In cellular crescents, the proliferation marker Ki-67 was expressed in beta-galactosidase-positive and beta-galactosidase-negative cells, indicating that both cell types contributed to the formation of cellular crescents through proliferation in situ. Podocyte-specific antigens, including WT-1, synaptopodin, nephrin, and podocin, were not expressed by any cells in glomerular crescents, suggesting that podocytes underwent profound phenotypic changes in this nephritis model.

Control of proliferation and cancer growth by the Hippo signaling pathway

PubMed Central

Ehmer, Ursula; Sage, Julien

2015-01-01

The control of cell division is essential for normal development and the maintenance of cellular homeostasis. Abnormal cell proliferation is associated with multiple pathological states, including cancer. While the Hippo/YAP signaling pathway was initially thought to control organ size and growth, increasing evidence indicates that this pathway also plays a major role in the control of proliferation independent of organ size control. In particular, accumulating evidence indicates that the Hippo/YAP signaling pathway functionally interacts with multiple other cellular pathways and serves as a central node in the regulation of cell division, especially in cancer cells. Here recent observations are highlighted that connect Hippo/YAP signaling to transcription, the basic cell cycle machinery, and the control of cell division. Furthermore, the oncogenic and tumor suppressive attributes of YAP/TAZ are reviewed which emphasizes the relevance of the Hippo pathway in cancer. PMID:26432795

Cellular modelling of secondary radial growth in conifer trees: application to Pinus radiata (D. Don).

PubMed

Forest, Loïc; Demongeot, Jacques; Demongeota, Jacques

2006-05-01

The radial growth of conifer trees proceeds from the dynamics of a merismatic tissue called vascular cambium or cambium. Cambium is a thin layer of active proliferating cells. The purpose of this paper was to model the main characteristics of cambial activity and its consecutive radial growth. Cell growth is under the control of the auxin hormone indole-3-acetic. The model is composed of a discrete part, which accounts for cellular proliferation, and a continuous part involving the transport of auxin. Cambium is modeled in a two-dimensional cross-section by a cellular automaton that describes the set of all its constitutive cells. Proliferation is defined as growth and division of cambial cells under neighbouring constraints, which can eliminate some cells from the cambium. The cell-growth rate is determined from auxin concentration, calculated with the continuous model. We studied the integration of each elementary cambial cell activity into the global coherent movement of macroscopic morphogenesis. Cases of normal and abnormal growth of Pinus radiata (D. Don) are modelled. Abnormal growth includes deformed trees where gravity influences auxin transport, producing heterogeneous radial growth. Cross-sectional microscopic views are also provided to validate the model's hypothesis and results.

Phototoxic effects of free phthalocyanine and phthalocyanine conjugated to gold nanoparticles for targeted photodynamic therapy of melanoma cancer

NASA Astrophysics Data System (ADS)

Manoto, Sello L.; Oluwole, David O.; Malabi, Rudzani; Maphanga, Charles; Ombinda-Lemboumba, Saturnin; Nyokong, Tebello; Mthunzi-Kufa, Patience

2017-02-01

Photodynamic therapy (PDT) has emerged as an effective treatment modality for various malignant neoplasia and diseases. In PDT, the photochemical interaction of photosensitizer (PS), light and molecular oxygen produces singlet oxygen which can lead to tumour cell apoptosis, necrosis or autophagy. The success of PDT is limited by the hydrophobic characteristic of the PS which hinders treatment administration and efficiency. To circumvent this limitation, PS can be incorporated in nanostructured drug delivery systems such as gold nanoparticles (AuNPs). In this study, we investigated the effectiveness of free zinc monocarboxyphenoxy phthalocyanine (ZnMCPPc) and ZnMCPPc conjugated to AuNPs. Commercially purchased melanoma cancer cells cultured as cell monolayers were used in this study. Changes in cellular response were evaluated using cellular morphology, viability, proliferation and cytotoxicity. Untreated cells showed no changes in cellular morphology, proliferation and cytotoxicity. However, photoactivated free ZnMCPPc and ZnMCPPc conjugated to AuNPs showed changes in cellular morphology and a dose dependent decrease in cellular viability and proliferation as well as an increase in cell membrane. ZnMCPPc conjugated to AuNPs showed an improved efficiency in PDT as compared to free ZnMCPPc, which might be as a result of the vehicle effect of AuNPs. Both PSs used in this study were effective in inducing cell death with ZnMCPPc conjugated to AuNPs showing great potential as an effective PS for PDT.

Histomorphometric analysis of collagen architecture of auricular keloids in an Asian population.

PubMed

Chong, Yosep; Park, Tae Hwan; Seo, Sang won; Chang, Choong Hyun

2015-03-01

Keloids are a pathologic condition of the reparative process, which present as excessive scar formation that involves various cells and cytokines. Many studies focusing on the histologic feature of keloids, however, have shown discordant results without consideration of architectural aspect of collagen structure. The purpose of this study was to demonstrate a schematic illustration of collagen architecture of keloids, specifically auricular keloids, and to analyze each part on the histomorphologic and morphometric basis. Thirty-nine surgically excised auricular keloids were retrieved from the file of Kangbuk Samsung Hospital. After exhaustive histomorphologic analysis, 3 distinctive structural parts, keloidal collagen, organizing collagen, and proliferating core collagen, were identified and mapped in every case. Cellularity of fibroblasts, blood vessel density, degree of inflammatory cell infiltration, and mast cells counts using Masson trichrome stain, Van Gieson stain, toluidine blue stain, and immunohistochemical stains for CD31 and smooth muscle actin were analyzed in each part of each case. Morphometric analysis on these parameters using ImageJ software was performed using 3 representative images of each part. Three parts were histomorphologically distinct by shape and array of collagen bundles, fibroblasts cellularity, blood vessel density, degree of inflammatory cells, and mast cell infiltration. Morphometric analysis revealed statistically significant difference between each part in fibroblasts cellularity, blood vessel density, degree of inflammatory cell infiltration, and mast cells count. All parameters were exceedingly high in whorling hypercellular fibrous nodules in proliferating core collagen showing simultaneous changes in other parts. Morphologically and morphometrically, 3 distinctive parts were identified in auricular keloids. Mast cell infiltrations, blood vessel density, and fibroblast cellularity are simultaneously increased or decreased according to these parts. Proliferating core collagen might serve as a proliferating center of keloids and might be a key portion for tumor growth and recurrence.

Prenatal Alcohol Exposure Affects Progenitor Cell Numbers in Olfactory Bulbs and Dentate Gyrus of Vervet Monkeys.

PubMed

Burke, Mark W; Inyatkin, Alexey; Ptito, Maurice; Ervin, Frank R; Palmour, Roberta M

2016-10-27

Fetal alcohol exposure (FAE) alters hippocampal cell numbers in rodents and primates, and this may be due, in part, to a reduction in the number or migration of neuronal progenitor cells. The olfactory bulb exhibits substantial postnatal cellular proliferation and a rapid turnover of newly formed cells in the rostral migratory pathway, while production and migration of postnatal neurons into the dentate gyrus may be more complex. The relatively small size of the olfactory bulb, compared to the hippocampus, potentially makes this structure ideal for a rapid analysis. This study used the St. Kitts vervet monkey ( Chlorocebus sabeus ) to (1) investigate the normal developmental sequence of post-natal proliferation in the olfactory bulb and dentate gyrus and (2) determine the effects of naturalistic prenatal ethanol exposure on proliferation at three different ages (neonate, five months and two years). Using design-based stereology, we found an age-related decrease of actively proliferating cells in the olfactory bulb and dentate gyrus for both control and FAE groups. Furthermore, at the neonatal time point, the FAE group had fewer actively proliferating cells as compared to the control group. These data are unique with respect to fetal ethanol effects on progenitor proliferation in the primate brain and suggest that the olfactory bulb may be a useful structure for studies of cellular proliferation.

The Lcn2-engineered HEK-293 cells show senescence under stressful condition

PubMed Central

Bahmani, Bahareh; Amiri, Fatemeh; Mohammadi Roushandeh, Amaneh; Bahadori, Marzie; Harati, Mozhgan Dehghan; Habibi Roudkenar, Mehryar

2015-01-01

Objective(s): Lipocalin2 (Lcn2) gene is highly expressed in response to various types of cellular stresses. The precise role of Lcn2 has not been fully understood yet. However, it plays a key role in controlling vital cellular processes such as proliferation, apoptosis and metabolism. Recently it was shown that Lcn2 decreases senescence and increases proliferation of mesenchymal stem cells (MSC) with finite life span under either normal or oxidative stress conditions. However, Lcn2 effects on immortal cell line with infinite proliferation are not defined completely. Materials and Materials and Methods: HEK-293 cells were transfected with recombinant pcDNA3.1 containing Lcn2 fragment (pcDNA3.1-Lcn2). Expression of lipocalin2 in transfected cells was evaluated by RT-PCR, real time RT-PCR, and ELISA. Different cell groups were treated with H2O2 and WST-1 assay was performed to determine their proliferation rate. Senescence was studied by β-galactosidase and gimsa staining methods as well as evaluation of the expression of senescence-related genes by real time RT-PCR. Results: Lcn2 increased cell proliferation under normal culture condition, while the proliferation slightly decreased under oxidative stress. This decrease was further found to be attributed to senescence. Conclusion: Our findings indicated that under harmful conditions, Lcn2 gene is responsible for the regulation of cell survival through senescence. PMID:26124931

The Effect of Skeletal Maturity on the Regenerative Function of Intrinsic ACL Cells

PubMed Central

Mastrangelo, Ashley N.; Magarian, Elise M.; Palmer, Matthew P.; Vavken, Patrick; Murray, Martha M.

2010-01-01

Anterior cruciate ligament (ACL) injuries are an important clinical problem, particularly for adolescent patients. The effect of skeletal maturity on the potential for ACL healing is as yet unknown. In this study, we hypothesized that fibroblastic cells from the ACLs of skeletally immature animals would proliferate and migrate more quickly than cells from adolescent and adult animals. ACL tissue from skeletally immature, adolescent, and adult pigs and sheep were obtained and cells obtained using explant culture. Cell proliferation within a collagen–platelet scaffold was measured at days 2, 7, and 14 of culture using AMMTT assay. Cellular migration was measured at 4 and 24 h using a modified Boyden chamber assay, and cell outgrowth from the explants also measured at 1 week. ACL cells from skeletally immature animals had higher proliferation between 7 and 14 days (p < 0.01 for all comparisons) and higher migration potential at all time points in both species (p < 0.01 for all comparisons).ACL cells from skeletally immature animals have greater cellular proliferation and migration potential than cells from adolescent or adult animals. These experiments suggest that skeletal maturity may influence the biologic repair capacity of intrinsic ACL cells. PMID:19890988

S-Nitrosation of monocarboxylate transporter 1: Inhibition of pyruvate-fueled respiration and proliferation of breast cancer cells

PubMed Central

Diers, Anne R.; Broniowska, Katarzyna A.; Chang, Ching-Fang; Hill, R. Blake; Hogg, Neil

2014-01-01

Summary Energy substrates metabolized through mitochondria (e.g., pyruvate, glutamine) are required for biosynthesis of macromolecules in proliferating cells. Since several mitochondrial proteins are known to be targets of S-nitrosation, we determined whether bioenergetics are modulated by S-nitrosation and defined the subsequent effects on proliferation. The nitrosating agent S-nitroso-L-cysteine (L-CysNO) was used to initiate intracellular S-nitrosation, and treatment decreased mitochondrial function and inhibited proliferation of MCF7 mammary adenocarcinoma cells. Surprisingly, the D isomer of CysNO (D-CysNO) which is not transported into cells also caused mitochondrial dysfunction and limited proliferation. Both L- and D-CysNO also inhibited cellular pyruvate uptake and caused S-nitrosation of thiol groups on monocarboxylate transporter 1, a proton-linked pyruvate transporter. These data demonstrate the importance of mitochondrial metabolism in proliferative responses in breast cancer and highlight a novel role for inhibition of metabolic substrate uptake through S-nitrosation of exofacial protein thiols in cellular responses to nitrosative stress. PMID:24486553

PDZ binding motif of HTLV-1 Tax promotes virus-mediated T-cell proliferation in vitro and persistence in vivo.

PubMed

Xie, Li; Yamamoto, Brenda; Haoudi, Abdelali; Semmes, O John; Green, Patrick L

2006-03-01

HTLV-1 cellular transformation and disease induction is dependent on expression of the viral Tax oncoprotein. PDZ is a modular protein interaction domain used in organizing signaling complexes in eukaryotic cells through recognition of a specific binding motif in partner proteins. Tax-1, but not Tax-2, contains a PDZ-binding domain motif (PBM) that promotes the interaction with several cellular PDZ proteins. Herein, we investigate the contribution of the Tax-1 PBM in HTLV-induced proliferation and immortalization of primary T cells in vitro and viral survival in an infectious rabbit animal model. We generated several HTLV-1 and HTLV-2 Tax viral mutants, including HTLV-1deltaPBM, HTLV-2+C22(+PBM), and HTLV-2+ C18(deltaPBM). All Tax mutants maintained the ability to significantly activate the CREB/ATF or NFkappaB signaling pathways. Microtiter proliferation assays revealed that the Tax-1 PBM significantly increases both HTLV-1- and HTLV-2-induced primary T-cell proliferation. In addition, Tax-1 PBM was responsible for the micronuclei induction activity of Tax-1 relative to that of Tax-2. Viral infection and persistence were severely attenuated in rabbits inoculated with HTLV-1deltaPBM. Our results provide the first direct evidence suggesting that PBM-mediated associations between Tax-1 and cellular proteins play a key role in HTLV-induced cell proliferation and genetic instability in vitro and facilitate viral persistence in vivo.

Contact Inhibition: Also a Control for Cell Proliferation in Unicellular Algae?

PubMed

Costas, E; Aguilera, A; Gonzalez-Gil, S; López-Rodas, V

1993-02-01

According to traditional views, the proliferation of unicellular algae is controlled primarily by environmental conditions. But as in mammalian cells, other biological mechanisms, such as growth factors, cellular aging, and contact inhibition, might also control algal proliferation. Here we ask whether contact inhibition regulates growth in several species of unicellular algae as it does in mammalian cells. Laboratory cultures of the dinoflagellate Prorocentrum lima (Ehrenberg) Dodge show contact inhibition at low cell density, so this would be an autocontrol mechanism of cell proliferation that could also act in natural populations of P. lima. But, Synechocystis spp., Phaeodactylum tricornutum (Bohlin), Skeletonema costatum (Greville), and Tetraselmis spp. do not exhibit contact inhibition in laboratory cultures because they are able to grow at high cellular density. Apparently their growth is limited by nutrient depletion or catabolite accumulation instead of contact inhibition. Spirogyra insignis (Hassall) Kutz, Prorocentrum triestinum Schiller, and Alexandrium tamarense (Halim) Balech show a complex response, as they are able to grow in both low and high cell density medium. These results suggest that contact inhibition is more adaptative in benthic unicellular algae.

Aspartate β-hydroxylase modulates cellular senescence via glycogen synthase kinase 3β in hepatocellular carcinoma

PubMed Central

Iwagami, Yoshifumi; Huang, Chiung-Kuei; Olsen, Mark J.; Thomas, John-Michael; Jang, Grace; Kim, Miran; Lin, Qiushi; Carlson, Rolf I.; Wagner, Carl E.; Dong, Xiaoqun; Wands, Jack R.

2015-01-01

Background & Aims Aspartate β-hydroxylase (ASPH) is an enzyme overexpressed in human hepatocellular carcinoma (HCC) tumors and participates in the malignant transformation process. We determined if ASPH was a therapeutic target by exerting effects on cellular senescence to retard HCC progression. Methods ASPH knockdown or knockout was achieved by shRNAs or CRISPR/Cas9 system, respectively, whereas enzymatic inhibition was rendered by a potent 2nd generation small molecule inhibitor (SMI) of ASPH. Alterations of cell proliferation, colony formation and cellular senescence were evaluated in human HCC cell lines. The potential mechanisms for activating cellular senescence were explored using murine subcutaneous and orthotopic xenograft models. Results Inhibition of ASPH expression and enzymatic activity significantly reduced cell proliferation and colony formation, but induced tumor cell senescence. Following inhibition of ASPH activity, phosphorylation of GSK3β and p16 expression were increased to promote senescence whereas cyclin D1 and PCNA were decreased to reduce cell proliferation. The mechanisms involved demonstrate that ASPH binds to GSK3β and inhibits its subsequent interactions with AKT and p38 upstream kinases as shown by co-immunoprecipitation. In vivo experiments demonstrated that the SMI treatment of HCC bearing mice resulted in significant dose-dependent reduced tumor growth, induced phosphorylation of GSK3β, enhanced p16 expression in tumor cells and promoted cellular senescence. Conclusions We have identified a new mechanism that promotes HCC growth and progression by modulating senescence of tumor cells. These findings suggest that ASPH enzymatic activity is a novel therapeutic target for HCC. PMID:26683595

A large shRNA library approach identifies lncRNA Ntep as an essential regulator of cell proliferation

PubMed Central

Beermann, Julia; Kirste, Dominique; Iwanov, Katharina; Lu, Dongchao; Kleemiß, Felix; Kumarswamy, Regalla; Schimmel, Katharina; Bär, Christian; Thum, Thomas

2018-01-01

The mammalian cell cycle is a complex and tightly controlled event. Myriads of different control mechanisms are involved in its regulation. Long non-coding RNAs (lncRNA) have emerged as important regulators of many cellular processes including cellular proliferation. However, a more global and unbiased approach to identify lncRNAs with importance for cell proliferation is missing. Here, we present a lentiviral shRNA library-based approach for functional lncRNA profiling. We validated our library approach in NIH3T3 (3T3) fibroblasts by identifying lncRNAs critically involved in cell proliferation. Using stringent selection criteria we identified lncRNA NR_015491.1 out of 3842 different RNA targets represented in our library. We termed this transcript Ntep (non-coding transcript essential for proliferation), as a bona fide lncRNA essential for cell cycle progression. Inhibition of Ntep in 3T3 and primary fibroblasts prevented normal cell growth and expression of key fibroblast markers. Mechanistically, we discovered that Ntep is important to activate P53 concomitant with increased apoptosis and cell cycle blockade in late G2/M. Our findings suggest Ntep to serve as an important regulator of fibroblast proliferation and function. In summary, our study demonstrates the applicability of an innovative shRNA library approach to identify long non-coding RNA functions in a massive parallel approach. PMID:29099486

HER4 selectively coregulates estrogen stimulated genes associated with breast tumor cell proliferation

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

Han, Wen; Jones, Frank E., E-mail: fjones3@tulane.edu

2014-01-10

Highlights: •HER4/4ICD is an obligate coactivator for 37% of estrogen regulated genes. •HER4/4ICD coactivated genes selectively regulate estrogen stimulated proliferation. •Estrogen stimulated tumor cell migration occurs independent of HER4/4ICD. •Disrupting HER4/4ICD and ER coactivated gene expression may suppress breast cancer. -- Abstract: The EGFR-family member HER4 undergoes regulated intramembrane proteolysis (RIP) to generate an intracellular domain (4ICD) that functions as a transcriptional coactivator. Accordingly, 4ICD coactivates the estrogen receptor (ER) and associates with ER at target gene promoters in breast tumor cells. However, the extent of 4ICD coactivation of ER and the functional significance of the 4ICD/ER transcriptional complex ismore » unclear. To identify 4ICD coactivated genes we performed a microarray gene expression analysis of β-estradiol treated cells comparing control MCF-7 breast cancer cells to MCF-7 cells where HER4 expression was stably suppressed using a shRNA. In the MCF-7 cell line, β-estradiol significantly stimulated or repressed by 2-fold or more 726 or 53 genes, respectively. Significantly, HER4/4ICD was an obligate coactivator for 277 or 38% of the β-estradiol stimulated genes. Ingenuity Pathway Analysis of β-estradiol regulated genes identified significant associations with multiple cellular functions regulating cellular growth and proliferation, cell cycle progression, cancer metastasis, decreased hypoplasia, tumor cell migration, apoptotic resistance of tumor cells, and increased transcription. Genes coactivated by 4ICD displayed functional specificity by only significantly contributing to cellular growth and proliferation, cell cycle progression, and decreased hypoplasia. In direct concordance with these in situ results we show that HER4 knockdown in MCF-7 cells results in a loss of estrogen stimulated tumor cell proliferation and cell cycle progression, whereas, estrogen stimulated tumor cell migration was unaffected by loss of HER4 expression. In summary, we demonstrate for the first time that a cell surface receptor functions as an obligate ER coactivator with functional specificity associated with breast tumor cell proliferation and cell cycle progression. Nearly 90% of ER positive tumors coexpress HER4, therefore we predict that the majority of breast cancer patients would benefit from a strategy to therapeutic disengage ER/4ICD coregulated tumor cell proliferation.« less

Local fibroblast proliferation but not influx is responsible for synovial hyperplasia in a murine model of rheumatoid arthritis

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

Matsuo, Yusuke; Mizoguchi, Fumitaka; Saito, Tetsuya

Synovial fibroblasts play crucial roles in inflammation and joint destruction in rheumatoid arthritis (RA). How they accumulate in the RA joints remains unclear. This study was conducted to discern whether cellular influx from the outside of the joints and local proliferation are responsible for synovial fibroblast accumulation in an animal model of RA. We found that synovial fibroblasts were identified as GFP+ cells using collagen type I alpha 2 (Col1a2)-GFP transgenic reporter mice. Then, bone marrow transplantation and parabiosis techniques were utilized to study the cellular influx. Irradiated wild-type mice were transplanted with bone marrow from Col1a2-GFP mice. Col1a2-GFP andmore » wild-type mice were conjoined for parabiosis. The transplanted mice and the parabionts were subjected to collagen antibody-induced arthritis (CAIA). We found no GFP+ cells in the hyperplastic synovial tissues from the transplanted mice with CAIA and from the wild-type parabionts with CAIA. Furthermore, normal and CAIA synovial tissues from Col1a2-GFP mice and from fluorescent ubiquitination-based cell cycle indicator (Fucci) transgenic mice, in which cells in S/G{sub 2}/M phases of the cell cycle express Azami-Green, were studied for Ki67, a cellular proliferation marker, and vimentin, a fibroblast marker, expression. The percentages of Ki67+/GFP+ and Azami-Green+/vimentin+ cells in the CAIA synovial tissues were higher than those in the untreated synovial tissues (34% vs. 0.40% and 19% vs. 0.26%, respectively). These findings indicate that local fibroblast proliferation but not cellular influx is responsible for the synovial hyperplasia in CAIA. Suppression of proliferation of the local synovial fibroblasts should be a promising treatment for RA. - Highlights: • We studied how synovial fibroblasts accumulate in joints in a murine model of RA. • Bone marrow-derived cells did not accumulate in arthritic joints. • Synovial fibroblasts did not accumulate in arthritic joints via circulation. • Local proliferation was responsible for the synovial fibroblast accumulation.« less

Simultaneous Analysis of P53 Protein Expression and Cell Proliferation in Irradiated Human Lymphocytes by Flow Cytometry

PubMed Central

de Freitas e Silva, Rafael; Gonçalves dos Santos, Neyliane Frassinetti; Pereira, Valéria Rěgo Alves; Amaral, Ademir

2014-01-01

P53 protein has an intrinsic role in modulating the cellular response against DNA radioinduced damages and has been pointed out as an indirect indicator of individual radiosensitivity. The rate of cell proliferation is also a parameter that has been related to tissue sensitivity to radiation. However, this feature is yet understudied. In this context, the aim of this work was to employ Flow Cytometry (FC) for simultaneously assessing of p53 protein expression levels together with cellular proliferation rate of irradiated human lymphocytes. From in vitro irradiated human blood samples, mononuclear cells were isolated and labeled with Carboxylfluorescein Diacetate Succinimidyl Ester (CFSE) prior to phytohaemagglutinin (PHA) stimulation in culture for 96 hours. Cells were also labeled with anti-p53 monoclonal antibody PE-conjugated in order to analyze either proliferation rate or p53 expression levels by FC. It was verified a reduction in the proliferation rate of irradiated lymphocytes and, in parallel, a rise in the p53 expression levels, similar for quiescent and proliferating lymphocytes. The results emphasize the importance of the use of CFSE-stained lymphocytes in assays associated to proliferation rate and the use of this methodology in several studies, such as for evaluating individual radiosensitivity. PMID:24659936

The thorny path linking cellular senescence to organismalaging

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

Patil, Christopher K.; Mian, Saira; Campisi, Judith

2005-08-09

Half a century is fast approaching since Hayflick and colleagues formally described the limited ability of normal human cells to proliferate in culture (Hayflick and Moorhead, 1961). This finding--that normal somatic cells, in contrast to cancer cells, cannot divide indefinitely--challenged the prevailing idea that cells from mortal multicellular organisms were intrinsically ''immortal'' (Carrell, 1912). It also spawned two hypotheses, essential elements of which persist today. The first held that the restricted proliferation of normal cells, now termed cellular senescence, suppresses cancer (Hayflick, 1965; Sager, 1991; Campisi, 2001). The second hypothesis, as explained in the article by Lorenzini et al., suggestedmore » that the limited proliferation of cells in culture recapitulated aspects of organismal aging (Hayflick, 1965; Martin, 1993). How well have these hypotheses weathered the ensuing decades? Before answering this question, we first consider current insights into the causes and consequences of cellular senescence. Like Lorenzini et al., we limit our discussion to mammals. We also focus on fibroblasts, the cell type studied by Lorenzini et al., but consider other types as well. We suggest that replicative capacity in culture is not a straightforward assessment, and that it correlates poorly with both longevity and body mass. We speculate this is due to the malleable and variable nature of replicative capacity, which renders it an indirect metric of qualitative and quantitative differences among cells to undergo senescence, a response that directly alters cellular phenotype and might indirectly alter tissue structure and function.« less

Biological behaviour of human umbilical artery smooth muscle cell grown on nickel-free and nickel-containing stainless steel for stent implantation

PubMed Central

Li, Liming; An, Liwen; Zhou, Xiaohang; Pan, Shuang; Meng, Xin; Ren, Yibin; Yang, Ke; Guan, Yifu

2016-01-01

To evaluate the clinical potential of high nitrogen nickel-free austenitic stainless steel (HNNF SS), we have compared the cellular and molecular responses of human umbilical artery smooth muscle cells (HUASMCs) to HNNF SS and 316L SS (nickel-containing austenitic 316L stainless steel). CCK-8 analysis and flow cytometric analysis were used to assess the cellular responses (proliferation, apoptosis, and cell cycle), and quantitative real-time PCR (qRT-PCR) was used to analyze the gene expression profiles of HUASMCs exposed to HNNF SS and 316L SS, respectively. CCK-8 analysis demonstrated that HUASMCs cultured on HNNF SS proliferated more slowly than those on 316L SS. Flow cytometric analysis revealed that HNNF SS could activate more cellular apoptosis. The qRT-PCR results showed that the genes regulating cell apoptosis and autophagy were up-regulated on HNNF SS. Thus, HNNF SS could reduce the HUASMC proliferation in comparison to 316L SS. The findings furnish valuable information for developing new biomedical materials for stent implantation. PMID:26727026

Biological behaviour of human umbilical artery smooth muscle cell grown on nickel-free and nickel-containing stainless steel for stent implantation

NASA Astrophysics Data System (ADS)

Li, Liming; An, Liwen; Zhou, Xiaohang; Pan, Shuang; Meng, Xin; Ren, Yibin; Yang, Ke; Guan, Yifu

2016-01-01

To evaluate the clinical potential of high nitrogen nickel-free austenitic stainless steel (HNNF SS), we have compared the cellular and molecular responses of human umbilical artery smooth muscle cells (HUASMCs) to HNNF SS and 316L SS (nickel-containing austenitic 316L stainless steel). CCK-8 analysis and flow cytometric analysis were used to assess the cellular responses (proliferation, apoptosis, and cell cycle), and quantitative real-time PCR (qRT-PCR) was used to analyze the gene expression profiles of HUASMCs exposed to HNNF SS and 316L SS, respectively. CCK-8 analysis demonstrated that HUASMCs cultured on HNNF SS proliferated more slowly than those on 316L SS. Flow cytometric analysis revealed that HNNF SS could activate more cellular apoptosis. The qRT-PCR results showed that the genes regulating cell apoptosis and autophagy were up-regulated on HNNF SS. Thus, HNNF SS could reduce the HUASMC proliferation in comparison to 316L SS. The findings furnish valuable information for developing new biomedical materials for stent implantation.

Phototodynamic activity of zinc monocarboxyphenoxy phthalocyane (ZnMCPPc) conjugated to gold silver (AuAg) nanoparticles in melanoma cancer cells

NASA Astrophysics Data System (ADS)

Manoto, Sello L.; Oluwole, David O.; Malabi, Rudzani; Maphanga, Charles; Ombinda-Lemboumba, Saturnin; Nyokong, Tebello; Mthunzi-Kufa, Patience

2017-02-01

Photodynamic therapy (PDT) is a minimally invasive therapeutic modality for the treatment of neoplastic and non-neoplastic diseases. In PDT of cancer, irradiation with light of a specific wavelength leads to activation of a photosensitizer which results in generation of reactive oxygen species (ROS) which induces cell death. Many phthalocyanine photosensitizers are hydrophobic and insoluble in water, which limits their therapeutic efficiency. Consequently, advanced delivery systems and strategies are needed to improve the effectiveness of these photosensitizers. Nanoparticles have shown promising results in increasing aqueous solubility, bioavailability, stability and delivery of photosensitizers to their target. This study investigated the photodynamic activity of zinc monocarboxyphenoxy phthalocyanine (ZnMCPPc) conjugated to gold silver (AuAg) nanoparticles in melanoma cancer cells. The photodynamic activity of ZnMCPPc conjugated to AuAg nanoparticles were evaluated using cellular morphology, viability, proliferation and cytotoxicity. Untreated cells showed no changes in cellular morphology, proliferation and cytotoxicity. However, photoactivated ZnMCPPc conjugated to AuAg nanoparticles showed changes in cell morphology and a dose dependent decrease in cellular viability, proliferation and an increase in cell membrane damage. The ZnMCPPc conjugated to AuAg nanoparticles used in this study was highly effective in inducing cell death of melanoma cancer cells.

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.

Cellular automata and integrodifferential equation models for cell renewal in mosaic tissues

PubMed Central

Bloomfield, J. M.; Sherratt, J. A.; Painter, K. J.; Landini, G.

2010-01-01

Mosaic tissues are composed of two or more genetically distinct cell types. They occur naturally, and are also a useful experimental method for exploring tissue growth and maintenance. By marking the different cell types, one can study the patterns formed by proliferation, renewal and migration. Here, we present mathematical modelling suggesting that small changes in the type of interaction that cells have with their local cellular environment can lead to very different outcomes for the composition of mosaics. In cell renewal, proliferation of each cell type may depend linearly or nonlinearly on the local proportion of cells of that type, and these two possibilities produce very different patterns. We study two variations of a cellular automaton model based on simple rules for renewal. We then propose an integrodifferential equation model, and again consider two different forms of cellular interaction. The results of the continuous and cellular automata models are qualitatively the same, and we observe that changes in local environment interaction affect the dynamics for both. Furthermore, we demonstrate that the models reproduce some of the patterns seen in actual mosaic tissues. In particular, our results suggest that the differing patterns seen in organ parenchymas may be driven purely by the process of cell replacement under different interaction scenarios. PMID:20375040

Role of cell division and self-propulsion in self-organization of 2D cell co-cultures

NASA Astrophysics Data System (ADS)

Das, Moumita; Dey, Supravat; Wu, Mingming; Ma, Minglin

Self-organization of cells is a key process in developmental and cancer biology. The differential adhesion hypothesis (DAH), which assumes cells as equilibrium liquid droplets and relates the self-assembly of cells to differences in inter-cellular adhesiveness, has been very successful in explaining cellular organization during morphogenesis where neighboring cells have the same non-equilibrium properties (motility, proliferation rate). However, recently it has been experimentally shown that for a co-culture of two different cell types proliferating at different rates, the resulting spatial morphologies cannot be explained using the DAH alone. Motivated by this, we develop and study a two-dimensional model of a cell co-culture that includes cell division and self-propulsion in addition to cell-cell adhesion, and systemically study how cells with significantly different adhesion, motility, and proliferation rate dynamically organize themselves in a spatiotemporal and context-dependent manner. Our results may help to understand how differential equilibrium and non-equilibrium properties cooperate and compete leading to different morphologies during tumor development, with important consequences for invasion and metastasis

Histogram analysis parameters of apparent diffusion coefficient reflect tumor cellularity and proliferation activity in head and neck squamous cell carcinoma

PubMed Central

Winter, Karsten; Richter, Cindy; Hoehn, Anna-Kathrin

2018-01-01

Our purpose was to analyze associations between apparent diffusion coefficient (ADC) histogram analysis parameters and histopathologicalfeatures in head and neck squamous cell carcinoma (HNSCC). The study involved 32 patients with primary HNSCC. For every tumor, the following histogram analysis parameters were calculated: ADCmean, ADCmax, ADCmin, ADCmedian, ADCmode, P10, P25, P75, P90, kurtosis, skewness, and entropy. Furthermore, proliferation index KI 67, cell count, total and average nucleic areas were estimated. Spearman's correlation coefficient (p) was used to analyze associations between investigated parameters. In overall sample, all ADC values showed moderate inverse correlations with KI 67. All ADC values except ADCmax correlated inversely with tumor cellularity. Slightly correlations were identified between total/average nucleic area and ADCmean, ADCmin, ADCmedian, and P25. In G1/2 tumors, only ADCmode correlated well with Ki67. No statistically significant correlations between ADC parameters and cellularity were found. In G3 tumors, Ki 67 correlated with all ADC parameters except ADCmode. Cell count correlated well with all ADC parameters except ADCmax. Total nucleic area correlated inversely with ADCmean, ADCmin, ADCmedian, P25, and P90. ADC histogram parameters reflect proliferation potential and cellularity in HNSCC. The associations between histopathology and imaging depend on tumor grading. PMID:29805759

Mitogenic Effects of Phosphatidylcholine Nanoparticles on MCF-7 Breast Cancer Cells

PubMed Central

Gándola, Yamila B.; Pérez, Sebastián E.; Irene, Pablo E.; Sotelo, Ana I.; Miquet, Johanna G.; Corradi, Gerardo R.; Carlucci, Adriana M.; Gonzalez, Lorena

2014-01-01

Lecithins, mainly composed of the phospholipids phosphatidylcholines (PC), have many different uses in the pharmaceutical and clinical field. PC are involved in structural and biological functions as membrane trafficking processes and cellular signaling. Considering the increasing applications of lecithin-based nanosystems for the delivery of therapeutic agents, the aim of the present work was to determine the effects of phosphatidylcholine nanoparticles over breast cancer cellular proliferation and signaling. PC dispersions at 0.01 and 0.1% (w/v) prepared in buffer pH 7.0 and 5.0 were studied in the MCF-7 breast cancer cell line. Neutral 0.1% PC-derived nanoparticles induced the activation of the MEK-ERK1/2 pathway, increased cell viability and induced a 1.2 fold raise in proliferation. These biological effects correlated with the increase of epidermal growth factor receptor (EGFR) content and its altered cellular localization. Results suggest that nanoparticles derived from PC dispersion prepared in buffer pH 7.0 may induce physicochemical changes in the plasma membrane of cancer cells which may affect EGFR cellular localization and/or activity, increasing activation of the MEK-ERK1/2 pathway and inducing proliferation. Results from the present study suggest that possible biological effects of delivery systems based on lecithin nanoparticles should be taken into account in pharmaceutical formulation design. PMID:24772432

Girdin/GIV is upregulated by cyclic tension, propagates mechanical signal transduction, and is required for the cellular proliferation and migration of MG-63 cells

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

Hu, Jiang-Tian; Li, Yan; Yu, Bing

2015-08-21

To explore how Girdin/GIV is regulated by cyclic tension and propagates downstream signals to affect cell proliferation and migration. Human osteoblast-like MG-63 cells were exposed to cyclic tension force at 4000 μstrain and 0.5 Hz for 6 h, produced by a four-point bending system. Cyclic tension force upregulated Girdin and Akt expression and phosphorylation in cultured MG-63 cells. Girdin and Akt each promoted the phosphorylation of the other under stimulated tension. In vitro MTT and transwell assays showed that Girdin and Akt are required for cell proliferation and migration during cellular quiescence. Moreover, STAT3 was determined to be essential for Girdin expression undermore » stimulated tension force in the physiological condition, as well as for osteoblast proliferation and migration during quiescence. These findings suggest that the STAT3/Girdin/Akt pathway activates in osteoblasts in response to mechanical stimulation and may play a significant role in triggering osteoblast proliferation and migration during orthodontic treatment. - Highlights: • Tension force upregulates Girdin and Akt expression and phosphorylation. • Girdin and Akt promotes the phosphorylation of each other under tension stimulation. • Girdin and Akt are required for MG-63 cell proliferation and migration. • STAT3 is essential for Girdin expression after application of the tension forces.« less

Matrix stiffness reverses the effect of actomyosin tension on cell proliferation.

PubMed

Mih, Justin D; Marinkovic, Aleksandar; Liu, Fei; Sharif, Asma S; Tschumperlin, Daniel J

2012-12-15

The stiffness of the extracellular matrix exerts powerful effects on cell proliferation and differentiation, but the mechanisms transducing matrix stiffness into cellular fate decisions remain poorly understood. Two widely reported responses to matrix stiffening are increases in actomyosin contractility and cell proliferation. To delineate their relationship, we modulated cytoskeletal tension in cells grown across a physiological range of matrix stiffnesses. On both synthetic and naturally derived soft matrices, and across a panel of cell types, we observed a striking reversal of the effect of inhibiting actomyosin contractility, switching from the attenuation of proliferation on rigid substrates to the robust promotion of proliferation on soft matrices. Inhibiting contractility on soft matrices decoupled proliferation from cytoskeletal tension and focal adhesion organization, but not from cell spread area. Our results demonstrate that matrix stiffness and actomyosin contractility converge on cell spreading in an unexpected fashion to control a key aspect of cell fate.

Matrix stiffness reverses the effect of actomyosin tension on cell proliferation

PubMed Central

Mih, Justin D.; Marinkovic, Aleksandar; Liu, Fei; Sharif, Asma S.; Tschumperlin, Daniel J.

2012-01-01

Summary The stiffness of the extracellular matrix exerts powerful effects on cell proliferation and differentiation, but the mechanisms transducing matrix stiffness into cellular fate decisions remain poorly understood. Two widely reported responses to matrix stiffening are increases in actomyosin contractility and cell proliferation. To delineate their relationship, we modulated cytoskeletal tension in cells grown across a physiological range of matrix stiffnesses. On both synthetic and naturally derived soft matrices, and across a panel of cell types, we observed a striking reversal of the effect of inhibiting actomyosin contractility, switching from the attenuation of proliferation on rigid substrates to the robust promotion of proliferation on soft matrices. Inhibiting contractility on soft matrices decoupled proliferation from cytoskeletal tension and focal adhesion organization, but not from cell spread area. Our results demonstrate that matrix stiffness and actomyosin contractility converge on cell spreading in an unexpected fashion to control a key aspect of cell fate. PMID:23097048

P53 protein in proliferation, repair and apoptosis of cells.

PubMed

Wawryk-Gawda, Ewelina; Chylińska-Wrzos, Patrycja; Lis-Sochocka, Marta; Chłapek, Katarzyna; Bulak, Kamila; Jędrych, Marian; Jodłowska-Jędrych, Barbara

2014-05-01

The p53 protein is an important factor of many intra- and extracellular processes. This protein regulates the repair of cellular DNA and induces apoptosis. It is also responsible for the regulation of the senescence and the cell entering the subsequent stages of the cellular cycle. The protein p53 is also involved in inhibiting angiogenesis and the induction of oxidative shock. In our study, we examined the activity of p53 protein in the uterine epithelial cells in rats treated with cladribine. Its action is mainly based on apoptosis induction. We compared the activity of p53 protein in cells with a high apoptosis index and in cells with active repair mechanisms and high proliferation index. We observed stronger p53 protein expression in the epithelial cells of the materials taken 24 h after the last dose of 2-CdA associated with the active process of apoptosis and inhibition of proliferation. After 4 weeks from the last dose of cladribine, the stronger expression of p53 protein was associated with both the existing changes in the cell's genome, the effects of the ongoing repair mechanisms, as well as the high proliferation activity.

Effect of Porosity of Alumina and Zirconia Ceramics toward Pre-Osteoblast Response

PubMed Central

Hadjicharalambous, Chrystalleni; Prymak, Oleg; Loza, Kateryna; Buyakov, Ales; Kulkov, Sergei; Chatzinikolaidou, Maria

2015-01-01

It is acknowledged that cellular responses are highly affected by biomaterial porosity. The investigation of this effect is important for the development of implanted biomaterials that integrate with bone tissue. Zirconia and alumina ceramics exhibit outstanding mechanical properties and are among the most popular implant materials used in orthopedics, but few data exist regarding the effect of porosity on cellular responses to these materials. The present study investigates the effect of porosity on the attachment and proliferation of pre-osteoblastic cells on zirconia and alumina. For each composition, ceramics of three different porosities are fabricated by sintering, and characterized using scanning electron microscopy, energy dispersive X-ray spectroscopy and X-ray powder diffraction. Cell proliferation is quantified, and microscopy is employed to qualitatively support the proliferation results and evaluate cell morphology. Cell adhesion and metabolic activity are found comparable among low porosity zirconia and alumina. In contrast, higher porosity favors better cell spreading on zirconia and improves growth, but does not significantly affect cell response on alumina. Between the highest porosity materials, cell response on zirconia is found superior to alumina. Results show that an average pore size of ~150 μm and ~50% porosity can be considered beneficial to cellular growth on zirconia ceramics. PMID:26579516

A bioactive triphasic ceramic-coated hydroxyapatite promotes proliferation and osteogenic differentiation of human bone marrow stromal cells.

PubMed

Nair, Manitha B; Bernhardt, Anne; Lode, Anja; Heinemann, Christiane; Thieme, Sebastian; Hanke, Thomas; Varma, Harikrishna; Gelinsky, Michael; John, Annie

2009-08-01

Hydroxyapatite (HA) ceramics are widely used as bone graft substitutes because of their biocompatibility and osteoconductivity. However, to enhance the success of therapeutic application, many efforts are undertaken to improve the bioactivity of HA. We have developed a triphasic, silica-containing ceramic-coated hydroxyapatite (HASi) and evaluated its performance as a scaffold for cell-based tissue engineering applications. Human bone marrow stromal cells (hBMSCs) were seeded on both HASi and HA scaffolds and cultured with and without osteogenic supplements for a period of 4 weeks. Cellular responses were determined in vitro in terms of cell adhesion, viability, proliferation, and osteogenic differentiation, where both materials exhibited excellent cytocompatibility. Nevertheless, an enhanced rate of cell proliferation and higher levels of both alkaline phosphatase expression and activity were observed for cells cultured on HASi with osteogenic supplements. These findings indicate that the bioactivity of HA endowed with a silica-containing coating has definitely influenced the cellular activity, projecting HASi as a suitable candidate material for bone regenerative therapy.

Adsorption of enamel matrix proteins to a bovine-derived bone grafting material and its regulation of cell adhesion, proliferation, and differentiation.

PubMed

Miron, Richard J; Bosshardt, Dieter D; Hedbom, Erik; Zhang, Yufeng; Haenni, Beat; Buser, Daniel; Sculean, Anton

2012-07-01

The use of various combinations of enamel matrix derivative (EMD) and grafting materials has been shown to promote periodontal wound healing/regeneration. However, the downstream cellular behavior of periodontal ligament (PDL) cells and osteoblasts has not yet been studied. Furthermore, it is unknown to what extent the bleeding during regenerative surgery may influence the adsorption of exogenous proteins to the surface of bone grafting materials and the subsequent cellular behavior. In the present study, the aim is to test EMD adsorption to the surface of natural bone mineral (NBM) particles in the presence of blood and determine the effect of EMD coating to NBM particles on downstream cellular pathways, such as adhesion, proliferation, and differentiation of primary human osteoblasts and PDL cells. NBM particles were precoated in various settings with EMD or human blood and analyzed for protein adsorption patterns via fluorescent imaging and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using fluorescent double-stranded DNA-binding dye. Cell differentiation was analyzed using real-time polymerase chain reaction for genes encoding runt-related transcription factor 2, alkaline phosphatase (ALP), osteocalcin (OC), and collagen1α1 (COL1A1), and mineralization was assessed using red dye staining. Analysis of cell attachment and cell proliferation revealed significantly higher osteoblast and PDL cell attachment on EMD-coated surfaces when compared with control and blood-coated surfaces. EMD also stimulated release of growth factors and cytokines, including bone morphogenetic protein 2 and transforming growth factor β1. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers, including COL1A1, ALP, and OC, in osteoblasts and PDL cells cultured on EMD-coated NBM particles. The present results suggest that 1) EMD enhances osteoblast and PDL cell attachment, proliferation, and differentiation on NBM particles, and 2) blood contamination of the grafting material before mixing with EMD may inhibit EMD adsorption.

The Concerted Action of Type 2 and Type 3 Deiodinases Regulates the Cell Cycle and Survival of Basal Cell Carcinoma Cells.

PubMed

Miro, Caterina; Ambrosio, Raffaele; De Stefano, Maria Angela; Di Girolamo, Daniela; Di Cicco, Emery; Cicatiello, Annunziata Gaetana; Mancino, Giuseppina; Porcelli, Tommaso; Raia, Maddalena; Del Vecchio, Luigi; Salvatore, Domenico; Dentice, Monica

2017-04-01

Thyroid hormones (THs) mediate pleiotropic cellular processes involved in metabolism, cellular proliferation, and differentiation. The intracellular hormonal environment can be tailored by the type 1 and 2 deiodinase enzymes D2 and D3, which catalyze TH activation and inactivation respectively. In many cellular systems, THs exert well-documented stimulatory or inhibitory effects on cell proliferation; however, the molecular mechanisms by which they control rates of cell cycle progression have not yet been entirely clarified. We previously showed that D3 depletion or TH treatment influences the proliferation and survival of basal cell carcinoma (BCC) cells. Surprisingly, we also found that BCC cells express not only sustained levels of D3 but also robust levels of D2. The aim of the present study was to dissect the contribution of D2 to TH metabolism in the BCC context, and to identify the molecular changes associated with cell proliferation and survival induced by TH and mediated by D2 and D3. We used the CRISPR/Cas9 technology to genetically deplete D2 and D3 in BCC cells and studied the consequences of depletion on cell cycle progression and on cell death. Cell cycle progression was analyzed by fluorescence activated cell sorting analysis of synchronized cells, and the apoptosis rate by annexin V incorporation. Mechanistic investigations revealed that D2 inactivation accelerates cell cycle progression thereby enhancing the proportion of S-phase cells and cyclin D1 expression. Conversely, D3 mutagenesis drastically suppressed cell proliferation and enhanced apoptosis of BCC cells. Furthermore, the basal apoptotic rate was oppositely regulated in D2- and D3-depleted cells. Our results indicate that BCC cells constitute an example in which the TH signal is finely tuned by the concerted expression of opposite-acting deiodinases. The dual regulation of D2 and D3 expression plays a critical role in cell cycle progression and cell death by influencing cyclin D1-mediated entry into the G1-S phase. These findings reinforce the concept that TH is a potential therapeutic target in human BCC.

A Non-Cell-Autonomous Role of BEC-1/BECN1/Beclin1 in Coordinating Cell-Cycle Progression and Stem Cell Proliferation during Germline Development.

PubMed

Ames, Kristina; Da Cunha, Dayse S; Gonzalez, Brenda; Konta, Marina; Lin, Feng; Shechter, Gabriel; Starikov, Lev; Wong, Sara; Bülow, Hannes E; Meléndez, Alicia

2017-03-20

The decision of stem cells to proliferate and differentiate is finely controlled. The Caenorhabditis elegans germline provides a tractable system for studying the mechanisms that control stem cell proliferation and homeostasis [1-4]. Autophagy is a conserved cellular recycling process crucial for cellular homeostasis in many different contexts [5], but its function in germline stem cell proliferation remains poorly understood. Here, we describe a function for autophagy in germline stem cell proliferation. We found that autophagy genes such as bec-1/BECN1/Beclin1, atg-16.2/ATG16L, atg-18/WIPI1/2, and atg-7/ATG7 are required for the late larval expansion of germline stem cell progenitors in the C. elegans gonad. We further show that BEC-1/BECN1/Beclin1 acts independently of the GLP-1/Notch or DAF-7/TGF-β pathways but together with the DAF-2/insulin IGF-1 receptor (IIR) signaling pathway to promote germline stem cell proliferation. Similar to DAF-2/IIR, BEC-1/BECN1/Beclin1, ATG-18/WIPI1/2, and ATG-16.2/ATG16L all promote cell-cycle progression and are negatively regulated by the phosphatase and tensin homolog DAF-18/PTEN. However, whereas BEC-1/BECN1/Beclin1 acts through the transcriptional regulator SKN-1/Nrf1, ATG-18/WIPI1/2 and ATG-16.2/ATG16L exert their function through the DAF-16/FOXO transcription factor. In contrast, ATG-7 functions in concert with the DAF-7/TGF-β pathway to promote germline proliferation and is not required for cell-cycle progression. Finally, we report that BEC-1/BECN1/Beclin1 functions non-cell-autonomously to facilitate cell-cycle progression and stem cell proliferation. Our findings demonstrate a novel non-autonomous role for BEC-1/BECN1/Beclin1 in the control of stem cell proliferation and cell-cycle progression, which may have implications for the understanding and development of therapies against malignant cell growth in the future. Copyright © 2017 Elsevier Ltd. All rights reserved.

Rho-associated kinase (ROCK) inhibition reverses low cell activity on hydrophobic surfaces.

PubMed

Tian, Yu Shun; Kim, Hyun Jung; Kim, Hyun-Man

2009-08-28

Hydrophobic polymers do not offer an adequate scaffold surface for cells to attach, migrate, proliferate, and differentiate. Thus, hydrophobic scaffolds for tissue engineering have traditionally been physicochemically modified to enhance cellular activity. However, modifying the surface by chemical or physical treatment requires supplementary engineering procedures. In the present study, regulation of a cell signal transduction pathway reversed the low cellular activity on a hydrophobic surface without surface modification. Inhibition of Rho-associated kinase (ROCK) by Y-27632 markedly enhanced adhesion, migration, and proliferation of osteoblastic cells cultured on a hydrophobic polystyrene surface. ROCK inhibition regulated cell-cycle-related molecules on the hydrophobic surface. This inhibition also decreased expression of the inhibitors of cyclin-dependent kinases such as p21(cip1) and p27(kip1) and increased expression of cyclin A and D. These results indicate that defective cellular activity on the hydrophobic surface can be reversed by the control of a cell signal transduction pathway without physicochemical surface modification.

Epidermal Homeostasis and Radiation Responses in a Multiscale Tissue Modeling Framework

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Cucinotta, Francis A.

2013-01-01

The surface of skin is lined with several thin layers of epithelial cells that are maintained throughout life time by a small population of stem cells. High dose radiation exposures could injure and deplete the underlying proliferative cells and induce cutaneous radiation syndrome. In this work we propose a multiscale computational model for skin epidermal dynamics that links phenomena occurring at the subcellular, cellular, and tissue levels of organization, to simulate the experimental data of the radiation response of swine epidermis, which is closely similar to human epidermis. Incorporating experimentally measured histological and cell kinetic parameters, we obtain results of population kinetics and proliferation indexes comparable to observations in unirradiated and acutely irradiated swine experiments. At the sub-cellular level, several recently published Wnt signaling controlled cell-cycle models are applied and the roles of key components and parameters are analyzed. Based on our simulation results, we demonstrate that a moderate increase of proliferation rate for the survival proliferative cells is sufficient to fully repopulate the area denuded by high dose radiation, as long as the integrity of underlying basement membrane is maintained. Our work highlights the importance of considering proliferation kinetics as well as the spatial organization of tissues when conducting in vivo investigations of radiation responses. This integrated model allow us to test the validity of several basic biological rules at the cellular level and sub-cellular mechanisms by qualitatively comparing simulation results with published research, and enhance our understanding of the pathophysiological effects of ionizing radiation on skin.

Peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor suppressor in hepatocellular carcinoma.

PubMed

Liu, Rui; Zhang, Haiyang; Zhang, Yan; Li, Shuang; Wang, Xinyi; Wang, Xia; Wang, Cheng; Liu, Bin; Zen, Ke; Zhang, Chen-Yu; Zhang, Chunni; Ba, Yi

2017-04-01

Peroxisome proliferator-activated receptor gamma coactivator-1 alpha plays a crucial role in regulating the biosynthesis of mitochondria, which is closely linked to the energy metabolism in various tumors. This study investigated the regulatory role of peroxisome proliferator-activated receptor gamma coactivator-1 alpha in the pathogenesis of hepatocellular carcinoma. In this study, the changes of peroxisome proliferator-activated receptor gamma coactivator-1 alpha messenger RNA levels between normal human liver and hepatocellular carcinoma tissue were examined by quantitative reverse transcription polymerase chain reaction. Knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by RNA interference in the human liver cell line L02, while overexpression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha was conducted by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha complementary DNA in the human hepatocarcinoma cell line HepG2. Cellular morphological changes were observed via optical and electron microscopy. Cellular apoptosis was determined by Hoechst 33258 staining. In addition, the expression levels of 21,400 genes in tissues and cells were detected by microarray. It was shown that peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression was significantly downregulated in hepatocellular carcinoma compared with normal liver tissues. After knockdown of peroxisome proliferator-activated receptor gamma coactivator-1 alpha expression in L02 cells, cells reverted to immature and dedifferentiated morphology exhibiting cancerous tendency. Apoptosis occurred in the HepG2 cells after transfection by adenovirus encoding peroxisome proliferator-activated receptor gamma coactivator-1 alpha. Microarray analysis showed consistent results. The results suggest that peroxisome proliferator-activated receptor gamma coactivator-1 alpha acts as a tumor suppressor in the formation and development of hepatocellular carcinoma and that peroxisome proliferator-activated receptor gamma coactivator-1 alpha may be a potential therapeutic target for hepatocellular carcinoma.

Scaffold architecture and fibrin gels promote meniscal cell proliferation

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

Pawelec, K. M., E-mail: pawelec.km@gmail.com, E-mail: jw626@cam.ac.uk; Best, S. M.; Cameron, R. E.

2015-01-01

Stability of the knee relies on the meniscus, a complex connective tissue with poor healing ability. Current meniscal tissue engineering is inadequate, as the signals for increasing meniscal cell proliferation have not been established. In this study, collagen scaffold structure, isotropic or aligned, and fibrin gel addition were tested. Metabolic activity was promoted by fibrin addition. Cellular proliferation, however, was significantly increased by both aligned architectures and fibrin addition. None of the constructs impaired collagen type I production or triggered adverse inflammatory responses. It was demonstrated that both fibrin gel addition and optimized scaffold architecture effectively promote meniscal cell proliferation.

Iron depletion results in Src kinase inhibition with associated cell cycle arrest in neuroblastoma cells

PubMed Central

Siriwardana, Gamini; Seligman, Paul A

2015-01-01

Iron is required for cellular proliferation. Recently, using systematic time studies of neuroblastoma cell growth, we better defined the G1 arrest caused by iron chelation to a point in mid-G1, where cyclin E protein is present, but the cyclin E/CDK2 complex kinase activity is inhibited. In this study, we again used the neuroblastoma SKNSH cells lines to pinpoint the mechanism responsible for this G1 block. Initial studies showed in the presence of DFO, these cells have high levels of p27 and after reversal of iron chelation p27 is degraded allowing for CDK2 kinase activity. The initial activation of CDK2 kinase allows cells to exit G1 and enter S phase. Furthermore, we found that inhibition of p27 degradation by DFO is directly associated with inhibition of Src kinase activity measured by lack of phosphorylation of Src at the 416 residue. Activation of Src kinase occurs very early after reversal from the DFO G1 block and is temporally associated with initiation of cellular proliferation associated with entry into S phase. For the first time therefore we show that iron chelation inhibits Src kinase activity and this activity is a requirement for cellular proliferation. PMID:25825542

Enhancement of myocardial regeneration through genetic engineering of cardiac progenitor cells expressing Pim-1 kinase.

PubMed

Fischer, Kimberlee M; Cottage, Christopher T; Wu, Weitao; Din, Shabana; Gude, Natalie A; Avitabile, Daniele; Quijada, Pearl; Collins, Brett L; Fransioli, Jenna; Sussman, Mark A

2009-11-24

Despite numerous studies demonstrating the efficacy of cellular adoptive transfer for therapeutic myocardial regeneration, problems remain for donated cells with regard to survival, persistence, engraftment, and long-term benefits. This study redresses these concerns by enhancing the regenerative potential of adoptively transferred cardiac progenitor cells (CPCs) via genetic engineering to overexpress Pim-1, a cardioprotective kinase that enhances cell survival and proliferation. Intramyocardial injections of CPCs overexpressing Pim-1 were given to infarcted female mice. Animals were monitored over 4, 12, and 32 weeks to assess cardiac function and engraftment of Pim-1 CPCs with echocardiography, in vivo hemodynamics, and confocal imagery. CPCs overexpressing Pim-1 showed increased proliferation and expression of markers consistent with cardiogenic lineage commitment after dexamethasone exposure in vitro. Animals that received CPCs overexpressing Pim-1 also produced greater levels of cellular engraftment, persistence, and functional improvement relative to control CPCs up to 32 weeks after delivery. Salutary effects include reduction of infarct size, greater number of c-kit(+) cells, and increased vasculature in the damaged region. Myocardial repair is significantly enhanced by genetic engineering of CPCs with Pim-1 kinase. Ex vivo gene delivery to enhance cellular survival, proliferation, and regeneration may overcome current limitations of stem cell-based therapeutic approaches.

HBV core promoter mutations promote cellular proliferation through E2F1-mediated upregulation of S-phase kinase-associated protein 2 transcription.

PubMed

Huang, Yuehua; Tai, Andrew W; Tong, Shuping; Lok, Anna S F

2013-06-01

Hepatitis B virus (HBV) core promoter (CP) mutations have been associated with an increased risk of hepatocellular carcinoma (HCC) in clinical studies. We previously reported that a combination of CP mutations seen in HCC patients, expressed in HBx gene, increased SKP2 (S-phase kinase-associated protein 2) expression, thereby promoting cellular proliferation. Here, we investigate the possible mechanisms by which CP mutations upregulate SKP2. We used immunoblotting and ATPlite assay to validate the effect of CP mutations in full-length HBV genome on cell cycle regulator levels and cell proliferation. Activation of SKP2 mRNA was assessed by quantitative real-time PCR in primary human hepatocytes (PHH) and HCC cell lines. Effect of CP mutations on SKP2 promoter activity was determined by luciferase assay. Target regulation of E2F1 on SKP2 was analyzed by siRNAs. CP mutations in full-length HBV genome upregulated SKP2 expression, thereby downregulating cell cycle inhibitors and accelerating cellular proliferation. CP mutations enhanced SKP2 promoter activity but had no effect on SKP2 protein stability. Mapping of the SKP2 promoter identified a region necessary for activation by CP mutations that contains an E2F1 response element. Knocking down E2F1 reduced the effects of CP mutations on SKP2 and cellular proliferation. The effect of CP mutations on E2F1 might be mediated through hyperphosphorylation of RB. HBV CP mutations enhance SKP2 transcription by activating the E2F1 transcription factor and in turn downregulate cell cycle inhibitors, thus providing a potential mechanism for an association between CP mutations and HCC. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Pathophysiological hypoxia affects the redox state and IL-2 signalling of human CD4+ T cells and concomitantly impairs survival and proliferation.

PubMed

Gaber, Timo; Tran, Cam Loan; Schellmann, Saskia; Hahne, Martin; Strehl, Cindy; Hoff, Paula; Radbruch, Andreas; Burmester, Gerd-Rüdiger; Buttgereit, Frank

2013-06-01

Inflamed areas are characterized by infiltration of immune cells, local hypoxia and alterations of cellular redox states. We investigated the impact of hypoxia on survival, proliferation, cytokine secretion, intracellular energy and redox state of human CD4(+) T cells. We found that pathophysiological hypoxia (<2% O2 ) significantly decreased CD4(+) T-cell survival after mitogenic stimulation. This effect was not due to an increased caspase-3/7-mediated apoptosis or adenosine-5'-triphosphate (ATP) consumption/depletion. However, the ability of stimulated T cells to proliferate was reduced under hypoxic conditions, despite increased expression of CD25. Pathophysiological hypoxia was also found to modify intracellular ROS (iROS) levels in stimulated T cells over time as compared with levels found in normoxia. Physiological hypoxia (5% O2 ) did not decrease CD4(+) T-cell survival and proliferation or modify iROS levels as compared with normoxia. We conclude that pathophysiological hypoxia affects T-cell proliferation and viability via disturbed IL-2R signalling downstream of STAT5a phosphorylation, but not as a result of impaired cellular energy homeostasis. We suggest iROS links early events in T-cell stimulation to the inhibition of the lymphoproliferative response under pathophysiological hypoxic conditions. The level of iROS may therefore act as a mediator of immune functions leading to down-regulation of long-term T-cell activity in inflamed tissues. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Frontline Science: IL-18 primes murine NK cells for proliferation by promoting protein synthesis, survival, and autophagy.

PubMed

El-Darawish, Yosif; Li, Wen; Yamanishi, Kyosuke; Pencheva, Magdalena; Oka, Naoto; Yamanishi, Hiromichi; Matsuyama, Tomohiro; Tanaka, Yoshimasa; Minato, Nagahiro; Okamura, Haruki

2018-03-30

Combined stimulation by IL-2 and IL-18 effectively promotes proliferation of NK cells, whereas singular stimulation does not. In this study, synergistic effects of these cytokines on NK cells proliferation was analyzed, focusing on the roles of IL-18. In splenic resting NK cells from IL-18KO mice, IL-18 rapidly activated NF-κB independently of IL-2, and activated or up-regulated various molecules downstream of PI3K/AKT and mTOR, including S6, Bcl-XL, ATG5, and LC3II, accompanying increases in cell growth and survival. Thus, IL-18 alone was revealed to augment various cellular processes (gene transcription, protein synthesis, survival) in the absence or presence of IL-2. Notably, combined IL-18 and IL-2 promoted autophagosome formation. In addition, priming NK cells with IL-18 augmented IL-2R, especially CD25, and enabled cells to respond to IL-2, resulting in activation of STAT3 and STAT5, followed by increase of cyclin B1 leading to proliferation. However, IL-2 alone failed to activate STAT3 or STAT5 in resting IL18KO NK cells. These results clarify the distinct roles of IL-2 and IL-18 in NK cell proliferation, and the intrinsic roles of IL-18 in various cellular processes, suggesting a range of functions of IL-18 expressed in an array of nonhematopoietic cells. ©2018 Society for Leukocyte Biology.

Nicotinamide induces mitochondrial-mediated apoptosis through oxidative stress in human cervical cancer HeLa cells.

PubMed

Feng, Yi; Wang, Yonghua; Jiang, Chengrui; Fang, Zishui; Zhang, Zhiqiang; Lin, Xiaoying; Sun, Liwei; Jiang, Weiying

2017-07-15

Nicotinamide participates in energy metabolism and influences cellular redox status and modulates multiple pathways related with both cellular survival and death. Recent studies have shown that it induced proliferation inhibition and apoptosis in many cancer cells. However, little is known about the effects of nicotinamide on human cervical cancer cells. We aimed to evaluate the effects of the indicated concentrations nicotinamide on cell proliferation, apoptosis and redox-related parameters in HeLa cells and investigated the apoptotic mechanism. After the treatment of the indicated concentrations nicotinamide, HeLa cell proliferation was evaluated by the CCK-8 assay and the production of ROS (reactive oxygen species) was measured using 2',7'-Dichlorofluorescin diacetate. The apoptotic effect was confirmed by observing the cellular and nuclear morphologies with fluorescence microscope and apoptotic rate of HeLa cell apoptosis was measured by flow cytometry using Annexin-V method. Moreover, we examined the mitochondrial membrane potential by JC-1 method and measured the expression of apoptosis related genes using qRT-PCR and immunoblotting. Nicotinamide restrained the HeLa cell proliferation and significantly increased the accumulation of ROS and depletion of GSH at relatively high concentrations. Furthermore, nicotinamide promoted HeLa cell apoptosis via the intrinsic mitochondrial apoptotic pathway. Our study revealed that nicotinamide induced the apoptosis through oxidative stress and intrinsic mitochondrial apoptotic pathways in HeLa cell. The results emerge that nicotinamide may be an inexpensive, safe and promising therapeutic agent or a neoadjuvant chemotherapy for cervical cancer patients, as well useful to find new drugs for cervical cancer therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster

NASA Astrophysics Data System (ADS)

Kim, Miju; Kim, Tae-Jin; Kim, Hye Mi; Doh, Junsang; Lee, Kyung-Mi

2017-01-01

Multi-cellular cluster formation of natural killer (NK) cells occurs during in vivo priming and potentiates their activation to IL-2. However, the precise mechanism underlying this synergy within NK cell clusters remains unclear. We employed lymphocyte-laden microwell technologies to modulate contact-mediated multi-cellular interactions among activating NK cells and to quantitatively assess the molecular events occurring in multi-cellular clusters of NK cells. NK cells in social microwells, which allow cell-to-cell contact, exhibited significantly higher levels of IL-2 receptor (IL-2R) signaling compared with those in lonesome microwells, which prevent intercellular contact. Further, CD25, an IL-2R α chain, and lytic granules of NK cells in social microwells were polarized toward MTOC. Live cell imaging of lytic granules revealed their dynamic and prolonged polarization toward neighboring NK cells without degranulation. These results suggest that IL-2 bound on CD25 of one NK cells triggered IL-2 signaling of neighboring NK cells. These results were further corroborated by findings that CD25-KO NK cells exhibited lower proliferation than WT NK cells, and when mixed with WT NK cells, underwent significantly higher level of proliferation. These data highlights the existence of IL-2 trans-presentation between NK cells in the local microenvironment where the availability of IL-2 is limited.

TASK-3 Downregulation Triggers Cellular Senescence and Growth Inhibition in Breast Cancer Cell Lines.

PubMed

Zúñiga, Rafael; Valenzuela, Claudio; Concha, Guierdy; Brown, Nelson; Zúñiga, Leandro

2018-03-29

TASK-3 potassium channels are believed to promote proliferation and survival of cancer cells, in part, by augmenting their resistance to both hypoxia and serum deprivation. While overexpression of TASK-3 is frequently observed in cancers, the understanding of its role and regulation during tumorigenesis remains incomplete. Here, we evaluated the effect of reducing the expression of TASK-3 in MDA-MB-231 and MCF-10F human mammary epithelial cell lines through small hairpin RNA (shRNA)-mediated knockdown. Our results show that knocking down TASK-3 in fully transformed MDA-MB-231 cells reduces proliferation, which was accompanied by an induction of cellular senescence and cell cycle arrest, with an upregulation of cyclin-dependent kinase (CDK) inhibitors p21 and p27. In non-tumorigenic MCF-10F cells, however, TASK-3 downregulation did not lead to senescence induction, although cell proliferation was impaired and an upregulation of CDK inhibitors was also evident. Our observations implicate TASK-3 as a critical factor in cell cycle progression and corroborate its potential as a therapeutic target in breast cancer treatment.

Hypoxia-inducible factor-1α promotes cell survival during ammonia stress response in ovarian cancer stem-like cells

PubMed Central

Kitajima, Shojiro; Lee, Kian Leong; Hikasa, Hiroki; Sun, Wendi; Huang, Ruby Yun-Ju; Yang, Henry; Matsunaga, Shinji; Yamaguchi, Takehiro; Araki, Marito; Kato, Hiroyuki

2017-01-01

Ammonia is a toxic by-product of metabolism that causes cellular stresses. Although a number of proteins are involved in adaptive stress response, specific factors that counteract ammonia-induced cellular stress and regulate cell metabolism to survive against its toxicity have yet to be identified. We demonstrated that the hypoxia-inducible factor-1α (HIF-1α) is stabilized and activated by ammonia stress. HIF-1α activated by ammonium chloride compromises ammonia-induced apoptosis. Furthermore, we identified glutamine synthetase (GS) as a key driver of cancer cell proliferation under ammonia stress and glutamine-dependent metabolism in ovarian cancer stem-like cells expressing CD90. Interestingly, activated HIF-1α counteracts glutamine synthetase function in glutamine metabolism by facilitating glycolysis and elevating glucose dependency. Our studies reveal the hitherto unknown functions of HIF-1α in a biphasic ammonia stress management in the cancer stem-like cells where GS facilitates cell proliferation and HIF-1α contributes to the metabolic remodeling in energy fuel usage resulting in attenuated proliferation but conversely promoting cell survival. PMID:29383096

TASK-3 Downregulation Triggers Cellular Senescence and Growth Inhibition in Breast Cancer Cell Lines

PubMed Central

Zúñiga, Rafael; Valenzuela, Claudio; Concha, Guierdy; Brown, Nelson; Zúñiga, Leandro

2018-01-01

TASK-3 potassium channels are believed to promote proliferation and survival of cancer cells, in part, by augmenting their resistance to both hypoxia and serum deprivation. While overexpression of TASK-3 is frequently observed in cancers, the understanding of its role and regulation during tumorigenesis remains incomplete. Here, we evaluated the effect of reducing the expression of TASK-3 in MDA-MB-231 and MCF-10F human mammary epithelial cell lines through small hairpin RNA (shRNA)-mediated knockdown. Our results show that knocking down TASK-3 in fully transformed MDA-MB-231 cells reduces proliferation, which was accompanied by an induction of cellular senescence and cell cycle arrest, with an upregulation of cyclin-dependent kinase (CDK) inhibitors p21 and p27. In non-tumorigenic MCF-10F cells, however, TASK-3 downregulation did not lead to senescence induction, although cell proliferation was impaired and an upregulation of CDK inhibitors was also evident. Our observations implicate TASK-3 as a critical factor in cell cycle progression and corroborate its potential as a therapeutic target in breast cancer treatment. PMID:29596383

Promotion of chloroplast proliferation upon enhanced post-mitotic cell expansion in leaves.

PubMed

Kawade, Kensuke; Horiguchi, Gorou; Ishikawa, Naoko; Hirai, Masami Yokota; Tsukaya, Hirokazu

2013-09-28

Leaves are determinate organs; hence, precise control of cell proliferation and post-mitotic cell expansion is essential for their growth. A defect in cell proliferation often triggers enhanced post-mitotic cell expansion in leaves. This phenomenon is referred to as 'compensation'. Several lines of evidence from studies on compensation have shown that cell proliferation and post-mitotic cell expansion are coordinately regulated during leaf development. Therefore, compensation has attracted much attention to the mechanisms for leaf growth. However, our understanding of compensation at the subcellular level remains limited because studies of compensation have focused mainly on cellular-level phenotypes. Proper leaf growth requires quantitative control of subcellular components in association with cellular-level changes. To gain insight into the subcellular aspect of compensation, we investigated the well-known relationship between cell area and chloroplast number per cell in compensation-exhibiting lines, and asked whether chloroplast proliferation is modulated in response to the induction of compensation. We first established a convenient and reliable method for observation of chloroplasts in situ. Using this method, we analyzed Arabidopsis thaliana mutants fugu5 and angustifolia3 (an3), and a transgenic line KIP-RELATED PROTEIN2 overexpressor (KRP2 OE), which are known to exhibit typical features of compensation. We here showed that chloroplast number per cell increased in the subepidermal palisade tissue of these lines. We analyzed tetraploidized wild type, fugu5, an3 and KRP2 OE, and found that cell area itself, but not nuclear ploidy, is a key parameter that determines the activity of chloroplast proliferation. In particular, in the case of an3, we uncovered that promotion of chloroplast proliferation depends on the enhanced post-mitotic cell expansion. The expression levels of chloroplast proliferation-related genes are similar to or lower than that in the wild type during this process. This study demonstrates that chloroplast proliferation is promoted in compensation-exhibiting lines. This promotion of chloroplast proliferation takes place in response to cell-area increase in post-mitotic phase in an3. The expression of chloroplast proliferation-related genes were not promoted in compensation-exhibiting lines including an3, arguing that an as-yet-unknown mechanism is responsible for modulation of chloroplast proliferation in these lines.

Promotion of chloroplast proliferation upon enhanced post-mitotic cell expansion in leaves

PubMed Central

2013-01-01

Background Leaves are determinate organs; hence, precise control of cell proliferation and post-mitotic cell expansion is essential for their growth. A defect in cell proliferation often triggers enhanced post-mitotic cell expansion in leaves. This phenomenon is referred to as ‘compensation’. Several lines of evidence from studies on compensation have shown that cell proliferation and post-mitotic cell expansion are coordinately regulated during leaf development. Therefore, compensation has attracted much attention to the mechanisms for leaf growth. However, our understanding of compensation at the subcellular level remains limited because studies of compensation have focused mainly on cellular-level phenotypes. Proper leaf growth requires quantitative control of subcellular components in association with cellular-level changes. To gain insight into the subcellular aspect of compensation, we investigated the well-known relationship between cell area and chloroplast number per cell in compensation-exhibiting lines, and asked whether chloroplast proliferation is modulated in response to the induction of compensation. Results We first established a convenient and reliable method for observation of chloroplasts in situ. Using this method, we analyzed Arabidopsis thaliana mutants fugu5 and angustifolia3 (an3), and a transgenic line KIP-RELATED PROTEIN2 overexpressor (KRP2 OE), which are known to exhibit typical features of compensation. We here showed that chloroplast number per cell increased in the subepidermal palisade tissue of these lines. We analyzed tetraploidized wild type, fugu5, an3 and KRP2 OE, and found that cell area itself, but not nuclear ploidy, is a key parameter that determines the activity of chloroplast proliferation. In particular, in the case of an3, we uncovered that promotion of chloroplast proliferation depends on the enhanced post-mitotic cell expansion. The expression levels of chloroplast proliferation-related genes are similar to or lower than that in the wild type during this process. Conclusions This study demonstrates that chloroplast proliferation is promoted in compensation-exhibiting lines. This promotion of chloroplast proliferation takes place in response to cell-area increase in post-mitotic phase in an3. The expression of chloroplast proliferation-related genes were not promoted in compensation-exhibiting lines including an3, arguing that an as-yet-unknown mechanism is responsible for modulation of chloroplast proliferation in these lines. PMID:24074400

Cellular changes in the hamster testicular interstitium with ageing and after exposure to short photoperiod.

PubMed

Beltrán-Frutos, E; Seco-Rovira, V; Ferrer, C; Madrid, J F; Sáez, F J; Canteras, M; Pastor, L M

2016-04-01

The aim of this study was to evaluate the cellular changes that occur in the hamster testicular interstitium in two very different physiological situations involving testicular involution: ageing and exposure to a short photoperiod. The animals were divided into an 'age group' with three subgroups - young, adult and old animals - and a 'regressed group' with animals subjected to a short photoperiod. The testicular interstitium was characterised by light and electron microscopy. Interstitial cells were studied histochemically with regard to their proliferation, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end labelling (TUNEL+) and testosterone synthetic activity. We identified two types of Leydig cell: Type A cells showed a normal morphology, while Type B cells appeared necrotic. With ageing, pericyte proliferation decreased but there was no variation in the index of TUNEL-positive Leydig cells. In the regressed group, pericyte proliferation was greater and TUNEL-positive cells were not observed in the interstitium. The testicular interstitium suffered few ultrastructural changes during ageing and necrotic Leydig cells were observed. In contrast, an ultrastructural involution of Leydig cells with no necrosis was observed in the regressed group. In conclusion, the testicular interstitium of Mesocricetus auratus showed different cellular changes in the two groups (age and regressed), probably due to the irreversible nature of ageing and the reversible character of changes induced by short photoperiod.

Pink1 and Parkin regulate Drosophila intestinal stem cell proliferation during stress and aging.

PubMed

Koehler, Christopher L; Perkins, Guy A; Ellisman, Mark H; Jones, D Leanne

2017-08-07

Intestinal stem cells (ISCs) maintain the midgut epithelium in Drosophila melanogaster Proper cellular turnover and tissue function rely on tightly regulated rates of ISC division and appropriate differentiation of daughter cells. However, aging and epithelial injury cause elevated ISC proliferation and decreased capacity for terminal differentiation of daughter enteroblasts (EBs). The mechanisms causing functional decline of stem cells with age remain elusive; however, recent findings suggest that stem cell metabolism plays an important role in the regulation of stem cell activity. Here, we investigate how alterations in mitochondrial homeostasis modulate stem cell behavior in vivo via RNA interference-mediated knockdown of factors involved in mitochondrial dynamics. ISC/EB-specific knockdown of the mitophagy-related genes Pink1 or Parkin suppresses the age-related loss of tissue homeostasis, despite dramatic changes in mitochondrial ultrastructure and mitochondrial damage in ISCs/EBs. Maintenance of tissue homeostasis upon reduction of Pink1 or Parkin appears to result from reduction of age- and stress-induced ISC proliferation, in part, through induction of ISC senescence. Our results indicate an uncoupling of cellular, tissue, and organismal aging through inhibition of ISC proliferation and provide insight into strategies used by stem cells to maintain tissue homeostasis despite severe damage to organelles. © 2017 Koehler et al.

Substance P accelerates wound healing in type 2 diabetic mice through endothelial progenitor cell mobilization and Yes-associated protein activation

PubMed Central

Um, Jihyun; Yu, Jinyeong; Park, Ki-Sook

2017-01-01

Wound healing is delayed in diabetes due to a number of factors, including impaired angiogenesis and poor dermal healing. The present study demonstrated that subcutaneous administration of substance P (SP) accelerates wound healing in db/db type 2 diabetic mice (db/db mice). SP injection (10 nM/kg, subcutaneously) enhanced angiogenesis, induced the mobilization of endothelial progenitor cells (EPCs) and increased the number of EPC-colony forming units (EPC-CFUs) in the bone marrow of db/db mice. Immunohistochemistry was performed to check the effects of SP on the cellular proliferation and the subcellular localization of Yes-associated protein (YAP) in the wound dermis. SP also upregulated cellular proliferation in the injured dermis of db/db mice. Compared with the control group, an increased number of cells in the wound dermis of SP-treated mice exhibited nuclear localization of YAP, which induces cellular proliferation. The results of the current study indicate that subcutaneous administration of SP may be a promising therapeutic strategy to treat diabetic wounds exhibiting impaired angiogenesis and dysfunctional dermal wound healing. PMID:28339006

Cellular basis of neuroepithelial bending during mouse spinal neural tube closure

PubMed Central

McShane, Suzanne G.; Molè, Matteo A.; Savery, Dawn; Greene, Nicholas D. E; Tam, Patrick P.L.; Copp, Andrew J.

2015-01-01

Summary Bending of the neural plate at paired dorsolateral hinge points (DLHPs) is required for neural tube closure in the spinal region of the mouse embryo. As a step towards understanding the morphogenetic mechanism of DLHP development, we examined variations in neural plate cellular architecture and proliferation during closure. Neuroepithelial cells within the median hinge point (MHP) contain nuclei that are mainly basally located and undergo relatively slow proliferation, with a 7 h cell cycle length. In contrast, cells in the dorsolateral neuroepithelium, including the DLHP, exhibit nuclei distributed throughout the apico-basal axis and undergo rapid proliferation, with a 4 h cell cycle length. As the neural folds elevate, cell numbers increase to a greater extent in the dorsolateral neural plate that contacts the surface ectoderm, compared with the more ventromedial neural plate where cells contact paraxial mesoderm and notochord. This marked increase in dorsolateral cell number cannot be accounted for solely on the basis of enhanced cell proliferation in this region. We hypothesised that neuroepithelial cells may translocate in a ventral-to-dorsal direction as DLHP formation occurs, and this was confirmed by vital cell labelling in cultured embryos. The translocation of cells into the neural fold, together with its more rapid cell proliferation, leads to an increase in cell density dorsolaterally compared with the more ventromedial neural plate. These findings suggest a model in which DLHP formation may proceed through ‘buckling’ of the neuroepithelium at a dorso-ventral boundary marked by a change in cell-packing density. PMID:26079577

In-vitro analysis of Quantum Molecular Resonance effects on human mesenchymal stromal cells

PubMed Central

Sella, Sabrina; Adami, Valentina; Amati, Eliana; Bernardi, Martina; Chieregato, Katia; Gatto, Pamela; Menarin, Martina; Pozzato, Alessandro; Pozzato, Gianantonio; Astori, Giuseppe

2018-01-01

Electromagnetic fields play an essential role in cellular functions interfering with cellular pathways and tissue physiology. In this context, Quantum Molecular Resonance (QMR) produces waves with a specific form at high-frequencies (4–64 MHz) and low intensity through electric fields. We evaluated the effects of QMR stimulation on bone marrow derived mesenchymal stromal cells (MSC). MSC were treated with QMR for 10 minutes for 4 consecutive days for 2 weeks at different nominal powers. Cell morphology, phenotype, multilineage differentiation, viability and proliferation were investigated. QMR effects were further investigated by cDNA microarray validated by real-time PCR. After 1 and 2 weeks of QMR treatment morphology, phenotype and multilineage differentiation were maintained and no alteration of cellular viability and proliferation were observed between treated MSC samples and controls. cDNA microarray analysis evidenced more transcriptional changes on cells treated at 40 nominal power than 80 ones. The main enrichment lists belonged to development processes, regulation of phosphorylation, regulation of cellular pathways including metabolism, kinase activity and cellular organization. Real-time PCR confirmed significant increased expression of MMP1, PLAT and ARHGAP22 genes while A2M gene showed decreased expression in treated cells compared to controls. Interestingly, differentially regulated MMP1, PLAT and A2M genes are involved in the extracellular matrix (ECM) remodelling through the fibrinolytic system that is also implicated in embryogenesis, wound healing and angiogenesis. In our model QMR-treated MSC maintained unaltered cell phenotype, viability, proliferation and the ability to differentiate into bone, cartilage and adipose tissue. Microarray analysis may suggest an involvement of QMR treatment in angiogenesis and in tissue regeneration probably through ECM remodelling. PMID:29293552

Low level light promotes the proliferation and differentiation of bone marrow derived mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Ahn, Jin-Chul; Rhee, Yun-Hee; Choi, Sun-Hyang; Kim, Dae Yu; Chung, Phil-Sang

2015-03-01

Low-level light irradiation (LLLI) reported to stimulate the proliferation or differentiation of a variety of cell types. However, very little is known about the effect of light therapy on stem cells. The aim of the present study was to evaluate the effect of LLLI on the molecular physiological change of human bone marrow derived stem cells (hBMSC) by wavelength (470, 630, 660, 740 and 850, 50mW). The laser diode was performed with different time interval (0, 7.5, 15, 30J/cm2, 50mW) on hBMSC. To determine the molecular physiological changes of cellular level of hBMSC, the clonogenic assay, ATP assay, reactive oxygen species (ROS) detection, mitochondria membrane potential (MMPΦ) staining and calcium efflux assay were assessed after irradiation. There was a difference between with and without irradiation on hBMSCs. An energy density up to 30 J/cm² improved the cell proliferation in comparison to the control group. Among these irradiated group, 630 and 660nm were significantly increased the cell proliferation. The cellular level of ATP and calcium influx was increased with energy dose-dependent in all LLLI groups. Meanwhile, ROS and MMPΦ were also increased after irradiation except 470nm. It can be concluded that LLLI using infrared light and an energy density up to 30 J/cm² has a positive stimulatory effect on the proliferation or differentiation of hBMSCs. Our results suggest that LLLI may influence to the mitochondrial membrane potential activity through ATP synthesis and increased cell metabolism which leads to cell proliferation and differentiation.

Carbocysteine counteracts the effects of cigarette smoke on cell growth and on the SIRT1/FoxO3 axis in bronchial epithelial cells.

PubMed

Pace, E; Di Vincenzo, S; Ferraro, M; Bruno, A; Dino, P; Bonsignore, M R; Battaglia, S; Saibene, F; Lanata, L; Gjomarkaj, M

2016-08-01

Cigarette smoke may accelerate cellular senescence by increasing oxidative stress. Altered proliferation and altered expression of anti-aging factors, including SIRT1 and FoxO3, characterise cellular senescence. The effects of carbocysteine on the SIRT1/FoxO3 axis and on downstream molecular mechanisms in human bronchial epithelial cells exposed to cigarette smoke are largely unknown. Aim of this study was to explore whether carbocysteine modulated SIRT1/FoxO3 axis, and downstream molecular mechanisms associated to cellular senescence, in a bronchial epithelial cell line (16-HBE) exposed to cigarette smoke. 16HBE cells were stimulated with/without cigarette smoke extracts (CSE) and carbocysteine. Flow cytometry and clonogenic assay were used to assess cell proliferation; western blot analysis was used for assessing nuclear expression of SIRT1 and FoxO3. The nuclear co-localization of SIRT1 and FoxO3 was assessed by fluorescence microscopy. Beta galactosidase (a senescence marker) and SIRT1 activity were assessed by specific staining and colorimetric assays, respectively. ChiP Assay and flow cytometry were used for assessing survivin gene regulation and protein expression, respectively. CSE decreased cell proliferation, the nuclear expression of SIRT1 and FoxO3 and increased beta galactosidase staining. CSE, reduced SIRT1 activity and FoxO3 localization on survivin promoter thus increasing survivin expression. In CSE stimulated bronchial epithelial cells carbocysteine reverted these phenomena by increasing cell proliferation, and SIRT1 and FoxO3 nuclear expression, and by reducing beta galactosidase staining and survivin expression. The study shows for the first time that carbocysteine may revert some senescence processes induced by oxidative stress due to cigarette smoke exposure. Copyright © 2016 Elsevier Inc. All rights reserved.

Co-culture of clonal beta cells with GLP-1 and glucagon-secreting cell line impacts on beta cell insulin secretion, proliferation and susceptibility to cytotoxins.

PubMed

Green, Alastair D; Vasu, Srividya; Moffett, R Charlotte; Flatt, Peter R

2016-06-01

We investigated the direct effects on insulin releasing MIN6 cells of chronic exposure to GLP-1, glucagon or a combination of both peptides secreted from GLUTag L-cell and αTC1.9 alpha-cell lines in co-culture. MIN6, GLUTag and αTC1.9 cell lines exhibited high cellular hormone content and release of insulin, GLP-1 and glucagon, respectively. Co-culture of MIN6 cells with GLUTag cells significantly increased cellular insulin content, beta-cell proliferation, insulin secretory responses to a range of established secretogogues and afforded protection against exposure cytotoxic concentrations of glucose, lipid, streptozotocin or cytokines. Benefits of co-culture of MIN6 cells with αTC1.9 alphacells were limited to enhanced beta-cell proliferation with marginal positive actions on both insulin secretion and cellular protection. In contrast, co-culture of MIN6 with GLUTag cells plus αTC1.9 cells, markedly enhanced both insulin secretory responses and protection against beta-cell toxins compared with co-culture with GLUTag cells alone. These data indicate important long-term effects of conjoint GLP-1 and glucagon exposure on beta-cell function. This illustrates the possible functional significance of alpha-cell GLP-1 production as well as direct beneficial effects of dual agonism at beta-cell GLP-1 and glucagon receptors. Copyright © 2016 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

Expression and regulation of enzymes in the ceramide metabolic pathway in human retinal pigment epithelial cells and their relevance to retinal degeneration.

PubMed

Zhu, DanHong; Sreekumar, Parameswaran G; Hinton, David R; Kannan, Ram

2010-03-31

Ceramide and its metabolic derivatives are important modulators of cellular apoptosis and proliferation. Dysregulation or imbalance of their metabolic pathways may promote the development of retinal degeneration. The aim of this study was to identify the expression and regulation of key enzymes of the ceramide pathway in retinal pigment epithelial (RPE) cells. RT-PCR was used to screen the enzymes involved in ceramide metabolism that are expressed in RPE. Over-expression of neutral sphingomyelinase-2 (SMPD3) or sphingosine kinase 1 (Sphk1) in ARPE-19 cells was achieved by transient transfection of SMPD3 or Sphk1 cDNA subcloned into an expression vector. The number of apoptotic or proliferating cells was determined using TUNEL and BrdU assays, respectively. Neutral sphingomyelinase-1, neutral sphingomyelinase-2, acidic ceramidase, ceramide kinase, SphK1 and Sphk2 were expressed in both ARPE-19 and early passage human fetal RPE (fRPE) cells, while alkaline ceramidase 2 was only expressed in fRPE cells. Over-expression of SMPD3 decreased RPE cell proliferation and increased cell apoptosis. The percentage of apoptotic cells increased proportionally with the amount of transfected SMPD3 DNA. Over-expression of SphK1 promoted cell proliferation and protected ARPE-19 cells from ceramide-induced apoptosis. The effect of C(2) ceramide on induction of apoptosis was evaluated in polarized vs. non-polarized RPE cultures; polarization of RPE was associated with much reduced apoptosis in response to ceramide. In conclusion, RPE cells possess the synthetic machinery for the production of ceramide, sphingosine, ceramide-1-phosphate (C1P), and sphingosine-1-phosphate (S1P). Over-expression of SMPD3 may increase cellular ceramide levels, leading to enhanced cell death and arrested cell proliferation. The selective induction of apoptosis in non-polarized RPE cultures by C(2) ceramide suggests that increased ceramide levels will preferentially affect non-polarized RPE, as are found in late age-related macular degeneration lesions, and may spare the normal RPE monolayer. SphK1 over-expression increased cellular S1P, which promoted cell proliferation and protected RPE from ceramide-induced apoptosis. Understanding the relationship between the metabolism of sphingolipids and their effects in RPE cell survival/death may help us to develop effective and efficient therapies for retinal degeneration. Copyright 2009 Elsevier Ltd. All rights reserved.

Histogram analysis parameters of apparent diffusion coefficient reflect tumor cellularity and proliferation activity in head and neck squamous cell carcinoma.

PubMed

Surov, Alexey; Meyer, Hans Jonas; Winter, Karsten; Richter, Cindy; Hoehn, Anna-Kathrin

2018-05-04

Our purpose was to analyze associations between apparent diffusion coefficient (ADC) histogram analysis parameters and histopathologicalfeatures in head and neck squamous cell carcinoma (HNSCC). The study involved 32 patients with primary HNSCC. For every tumor, the following histogram analysis parameters were calculated: ADCmean, ADCmax, ADC min , ADC median , ADC mode , P10, P25, P75, P90, kurtosis, skewness, and entropy. Furthermore, proliferation index KI 67, cell count, total and average nucleic areas were estimated. Spearman's correlation coefficient (p) was used to analyze associations between investigated parameters. In overall sample, all ADC values showed moderate inverse correlations with KI 67. All ADC values except ADCmax correlated inversely with tumor cellularity. Slightly correlations were identified between total/average nucleic area and ADC mean , ADC min , ADC median , and P25. In G1/2 tumors, only ADCmode correlated well with Ki67. No statistically significant correlations between ADC parameters and cellularity were found. In G3 tumors, Ki 67 correlated with all ADC parameters except ADCmode. Cell count correlated well with all ADC parameters except ADCmax. Total nucleic area correlated inversely with ADC mean , ADC min , ADC median , P25, and P90. ADC histogram parameters reflect proliferation potential and cellularity in HNSCC. The associations between histopathology and imaging depend on tumor grading.

Femtosecond laser fabricated spike structures for selective control of cellular behavior.

PubMed

Schlie, Sabrina; Fadeeva, Elena; Koch, Jürgen; Ngezahayo, Anaclet; Chichkov, Boris N

2010-09-01

In this study we investigate the potential of femtosecond laser generated micrometer sized spike structures as functional surfaces for selective cell controlling. The spike dimensions as well as the average spike to spike distance can be easily tuned by varying the process parameters. Moreover, negative replications in soft materials such as silicone elastomer can be produced. This allows tailoring of wetting properties of the spike structures and their negative replicas representing a reduced surface contact area. Furthermore, we investigated material effects on cellular behavior. By comparing human fibroblasts and SH-SY5Y neuroblastoma cells we found that the influence of the material was cell specific. The cells not only changed their morphology, but also the cell growth was affected. Whereas, neuroblastoma cells proliferated at the same rate on the spike structures as on the control surfaces, the proliferation of fibroblasts was reduced by the spike structures. These effects can result from the cell specific adhesion patterns as shown in this work. These findings show a possibility to design defined surface microstructures, which could control cellular behavior in a cell specific manner.

A dynamic cellular vertex model of growing epithelial tissues

NASA Astrophysics Data System (ADS)

Lin, Shao-Zhen; Li, Bo; Feng, Xi-Qiao

2017-04-01

Intercellular interactions play a significant role in a wide range of biological functions and processes at both the cellular and tissue scales, for example, embryogenesis, organogenesis, and cancer invasion. In this paper, a dynamic cellular vertex model is presented to study the morphomechanics of a growing epithelial monolayer. The regulating role of stresses in soft tissue growth is revealed. It is found that the cells originating from the same parent cell in the monolayer can orchestrate into clustering patterns as the tissue grows. Collective cell migration exhibits a feature of spatial correlation across multiple cells. Dynamic intercellular interactions can engender a variety of distinct tissue behaviors in a social context. Uniform cell proliferation may render high and heterogeneous residual compressive stresses, while stress-regulated proliferation can effectively release the stresses, reducing the stress heterogeneity in the tissue. The results highlight the critical role of mechanical factors in the growth and morphogenesis of epithelial tissues and help understand the development and invasion of epithelial tumors.

Cell density and N-cadherin interactions regulate cell proliferation in the sensory epithelia of the inner ear.

PubMed

Warchol, Mark E

2002-04-01

Sensory hair cells in the inner ears of nonmammalian vertebrates can regenerate after injury. In many species, replacement hair cells are produced by the proliferation of epithelial supporting cells. Thus, the ability of supporting cells to undergo renewed proliferation is a key determinant of regenerative ability. The present study used cultures of isolated inner ear sensory epithelia to identify cellular signals that regulate supporting cell proliferation. Small pieces of sensory epithelia from the chicken utricle were cultured in glass microwells. Under those conditions, cell proliferation was inversely related to local cell density. The signaling molecules N-cadherin, beta-catenin, and focal adhesion kinase were immunolocalized in the cultured epithelial cells, and high levels of phosphotyrosine immunoreactivity were present at cell-cell junctions and focal contacts of proliferating cells. Binding of microbeads coated with a function-blocking antibody to N-cadherin inhibited ongoing proliferation. The growth of epithelial cells was also affected by the density of extracellular matrix molecules. The results suggest that cell density, cell-cell contact, and the composition of the extracellular matrix may be critical influences on the regulation of sensory regeneration in the inner ear.

Ultra-fast laser microprocessing of medical polymers for cell engineering applications.

PubMed

Ortiz, R; Moreno-Flores, S; Quintana, I; Vivanco, MdM; Sarasua, J R; Toca-Herrera, J L

2014-04-01

Picosecond laser micromachining technology (PLM) has been employed as a tool for the fabrication of 3D structured substrates. These substrates have been used as supports in the in vitro study of the effect of substrate topography on cell behavior. Different micropatterns were PLM-generated on polystyrene (PS) and poly-L-lactide (PLLA) and employed to study cellular proliferation and morphology of breast cancer cells. The laser-induced microstructures included parallel lines of comparable width to that of a single cell (which in this case is roughly 20μm), and the fabrication of square-like compartments of a much larger area than a single cell (250,000μm(2)). The results obtained from this in vitro study showed that though the laser treatment altered substrate roughness, it did not noticeably affect the adhesion and proliferation of the breast cancer cells. However, pattern direction directly affected cell proliferation, leading to a guided growth of cell clusters along the pattern direction. When cultured in square-like compartments, cells remained confined inside these for eleven incubation days. According to these results, laser micromachining with ultra-short laser pulses is a suitable method to directly modify the cell microenvironment in order to induce a predefined cellular behavior and to study the effect of the physical microenvironment on cell proliferation. Copyright © 2013 Elsevier B.V. All rights reserved.

Cell-based cytotoxicity assays for engineered nanomaterials safety screening: exposure of adipose derived stromal cells to titanium dioxide nanoparticles.

PubMed

Xu, Yan; Hadjiargyrou, M; Rafailovich, Miriam; Mironava, Tatsiana

2017-07-11

Increasing production of nanomaterials requires fast and proper assessment of its potential toxicity. Therefore, there is a need to develop new assays that can be performed in vitro, be cost effective, and allow faster screening of engineered nanomaterials (ENMs). Herein, we report that titanium dioxide (TiO 2 ) nanoparticles (NPs) can induce damage to adipose derived stromal cells (ADSCs) at concentrations which are rated as safe by standard assays such as measuring proliferation, reactive oxygen species (ROS), and lactate dehydrogenase (LDH) levels. Specifically, we demonstrated that low concentrations of TiO 2 NPs, at which cellular LDH, ROS, or proliferation profiles were not affected, induced changes in the ADSCs secretory function and differentiation capability. These two functions are essential for ADSCs in wound healing, energy expenditure, and metabolism with serious health implications in vivo. We demonstrated that cytotoxicity assays based on specialized cell functions exhibit greater sensitivity and reveal damage induced by ENMs that was not otherwise detected by traditional ROS, LDH, and proliferation assays. For proper toxicological assessment of ENMs standard ROS, LDH, and proliferation assays should be combined with assays that investigate cellular functions relevant to the specific cell type.

Cellular senescence and organismal aging.

PubMed

Jeyapalan, Jessie C; Sedivy, John M

2008-01-01

Cellular senescence, first observed and defined using in vitro cell culture studies, is an irreversible cell cycle arrest which can be triggered by a variety of factors. Emerging evidence suggests that cellular senescence acts as an in vivo tumor suppression mechanism by limiting aberrant proliferation. It has also been postulated that cellular senescence can occur independently of cancer and contribute to the physiological processes of normal organismal aging. Recent data have demonstrated the in vivo accumulation of senescent cells with advancing age. Some characteristics of senescent cells, such as the ability to modify their extracellular environment, could play a role in aging and age-related pathology. In this review, we examine current evidence that links cellular senescence and organismal aging.

Cellular senescence and organismal aging

PubMed Central

Jeyapalan, Jessie C.; Sedivy, John M.

2012-01-01

Cellular senescence, first observed and defined using in vitro cell culture studies, is an irreversible cell cycle arrest which can be triggered by a variety of factors. Emerging evidence suggests that cellular senescence acts as an in vivo tumor suppression mechanism by limiting aberrant proliferation. It has also been postulated that cellular senescence can occur independently of cancer and contribute to the physiological processes of normal organismal aging. Recent data have demonstrated the in vivo accumulation of senescent cells with advancing age. Some characteristics of senescent cells, such as the ability to modify their extracellular environment, could play a role in aging and age related pathology. In this review, we examine current evidence that links cellular senescence and organismal aging. PMID:18502472

DKK3 Overexpression Increases the Malignant Properties of Head and Neck Squamous Cell Carcinoma Cells.

PubMed

Katase, Naoki; Nishimatsu, Shin-Ichiro; Yamauchi, Akira; Yamamura, Masahiro; Terada, Kumiko; Itadani, Masumi; Okada, Naoko; Hassan, Nur Mohammad Monsur; Nagatsuka, Hitoshi; Ikeda, Tohru; Nohno, Tsutomu; Fujita, Shuichi

2018-01-19

DKK3, a member of the dickkopf Wnt signaling pathway inhibitor family, is believed to be a tumor suppressor because of its reduced expression in cancer cells. However, our previous studies have revealed that DKK3 expression is predominantly observed in head and neck/oral squamous cell carcinoma (HNSCC/OSCC). Interestingly, HNSCC/OSCC patients with DKK3 expression showed a high rate of metastasis and poorer survival, and siRNA-mediated knockdown of DKK3 in HNSCC-derived cancer cell lines resulted in reduced cellular migration and invasion. From these data, it was hypothesized that DKK3 might exert an oncogenic function specific to HNSCC. In the present research, the DKK3 overexpression model was established, and its influences were investigated, together with molecular mechanism studies. The DKK3 expression profile in cancer cell lines was investigated, including HNSCC/OSCC, esophageal, gastric, colorectal, pancreatic, prostatic, and lung cancers. DKK3 overexpression was performed in HNSCC-derived cells by transfection of expression plasmid. The effects of DKK3 overexpression were assessed on cellular proliferation, migration, invasion, and in vivo tumor growth. The molecular mechanism of DKK3 overexpression was investigated by Western blotting and microarray analysis. DKK3 overexpression significantly elevated cellular proliferation, migration, and invasion, as well as increased mRNA expression of cyclin D1 and c-myc. However, reporter assays did not show TCF/LEF activation, suggesting that the increased malignant property of cancer cells was not driven by the Wnt/β-catenin pathway. For the investigation of the pathways/molecules in DKK3-mediated signals, the Western blot analyses revealed that phosphorylation of Akt (S473) and c-Jun (Ser63) was elevated. The application of a PI3K kinase inhibitor, LY294002, on HSC-3 DKK3 cells significantly decreased tumor cell proliferation, migration, and invasion. From these results, we demonstrated that DKK3 might contribute to cellular proliferation, invasion, migration, and tumor cell survival in HNSCC cells through a mechanism other than the canonical Wnt signaling pathway, which might be attributed to PI3K-Akt signaling.

Investigation of the Causes of Breast Cancer at the Cellular Level: Isolation of In Vivo Binding Sites of the Human Origin Recognition Complex

DTIC Science & Technology

2000-08-01

The coordination between cellular DNA replication and mitosis is critical to ensure controlled cell proliferation and accurate transmission of the...proteins involved in the initiation of DNA replication . Preliminary results are presented....genetic information as cells divide -two aspects of cellular life tipically lost in cancer. In order to unravel the molecular mechanisms of human DNA

Progesterone and synthetic progestin, dienogest, induce apoptosis of human primary cultures of adenomyotic stromal cells.

PubMed

Yamanaka, Akiyoshi; Kimura, Fuminori; Kishi, Yohei; Takahashi, Kentaro; Suginami, Hiroshi; Shimizu, Yutaka; Murakami, Takashi

2014-08-01

To investigate the direct effects of progesterone receptor (PR) agonists on proliferation and apoptosis of human adenomyotic cells. Human primary cultures of adenomyotic stromal cells (ASCs) from 24 patients with adenomyosis were co-treated with estradiol (E2) plus the PR agonists, endogenous progesterone (P) or the synthetic progestin dienogest (DNG), which is used to treat endometriosis. In ASCs, anti-proliferative effects and induction of apoptosis were evaluated in the presence or absence of P (10(-8)-10(-6)M) or DNG (10(-8)-10(-6)M) in culture medium containing E2. Cellular proliferation was analyzed with bromodeoxyuridine incorporation and flow cytometry. Apoptosis was detected with annexin V/7-amino-actinomycin D (7-AAD) staining with flow cytometry and cellular caspase 3/7 activity. P and DNG significantly decreased the proportion of cells in the S phase. In addition, both P and DNG increased apoptosis as measured by annexin V-positive/7-AAD -negative cells and caspase 3/7 activity. Both endogenous P and synthetic progestin directly inhibited cellular proliferation and induced apoptosis in human ASCs. These pharmacological features of progestational compounds provide insight into the therapeutic strategy for the treatment of adenomyosis. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Cellular immune responses to platelet factor 4 and heparin complexes in patients with heparin-induced thrombocytopenia.

PubMed

Nazy, Ishac; Clare, Rumi; Staibano, Phillip; Warkentin, Theodore E; Larche, Mark; Moore, Jane C; Smith, James W; Whitlock, Richard P; Kelton, John G; Arnold, Donald M

2018-05-03

Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin characterized by thrombocytopenia and thrombotic complications. HIT is caused by pathogenic antibodies that bind to complexes of platelet factor 4 and heparin (PF4/heparin) leading to platelet activation and inducing a hypercoagulable state. Previous studies have shown immunity to PF4/heparin occurs early in life even before heparin exposure; however, the immunogenesis of HIT is not well characterized. The aim of this study was to investigate cellular proliferation in response to PF4/heparin complexes in patients with HIT. Peripheral blood mononuclear cells (PBMCs) from healthy controls (n = 30), postoperative cardiac surgery patients who underwent cardiopulmonary bypass (CPB, n = 17), and patients with confirmed HIT (n = 41) were cultured with PF4 and PF4/heparin. Cellular proliferation was assessed by 3 H-thymidine uptake and 5-ethynyl-2'-deoxyuridine (EdU) detection. PBMCs proliferated in the presence of PF4 and was enhanced by the addition of heparin in all study groups. CPB and HIT patients exhibited significantly higher proliferative responses compared to healthy controls. PBMC proliferation was antigen-specific, depended on the presence of platelets, and only CD14 + cells were identified as proliferating cells. Culture supernatants were tested for the levels of regulatory cytokines and both CPB and HIT patients produced significantly lower levels of IL-10 and TGF-β1 compared to healthy controls. These findings further demonstrate that cellular immune sensitization to PF4/heparin occurs before heparin exposure and suggests that immune dysregulation can contribute to the immunogenesis of HIT. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Short communication: Arginase inhibition reduces the synthesis of casein in bovine mammary epithelial cells.

PubMed

Wang, M Z; Ding, L Y; Wang, C; Chen, L M; Loor, J J; Wang, H R

2017-05-01

The main purpose of this work was to determine the effect of arginase inhibition via N ω -hydroxy-nor-l-arginine (nor-NOHA) on casein synthesis in bovine mammary epithelial cells (BMEC). Passage 2 BMEC isolated from dairy cows were seeded to 6-well plates and randomly divided into 4 treatments: (1) control [Dulbecco's modified Eagle medium:Nutrient Mixture F-12 medium (DMEM/F12)]; (2) nor-NOHA (DMEM/F12 + 1 mmol/L nor-NOHA); (3) nor-NOHA + arginine (DMEM/F12 + 1 mmol/L nor-NOHA + 3.2 mmol/L Arg); and (4) nor-NOHA + ornithine (DMEM/F12+ 1 mmol/L nor-NOHA + 1 mmol/L Orn). Then, we determined the activity of enzymes related to Arg metabolism and casein synthesis in BMEC and the proliferation of cells. The addition of nor-NOHA reduced the activity of arginase and ornithine decarboxylase but had no effect on the activity of nitric oxide synthase, and these responses were the same at the gene expression level. In addition, supplementation of nor-NOHA in BMEC reduced cellular proliferation and casein synthesis. Addition of Arg to nor-NOHA resulted in cellular proliferation and casein synthesis similar to that of nor-NOHA alone. In contrast, addition of Orn to the medium with nor-NOHA increased the synthesis of casein and cellular proliferation compared with Nor-NOHA. In conclusion, suppression of the Arg-arginase-Orn pathway reduced casein synthesis and cellular proliferation, which indicated that this pathway is an important regulator of the synthesis of casein in BMEC. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Human Platelet Lysate versus Fetal Calf Serum: These Supplements Do Not Select for Different Mesenchymal Stromal Cells.

PubMed

Fernandez-Rebollo, Eduardo; Mentrup, Birgit; Ebert, Regina; Franzen, Julia; Abagnale, Giulio; Sieben, Torsten; Ostrowska, Alina; Hoffmann, Per; Roux, Pierre-François; Rath, Björn; Goodhardt, Michele; Lemaitre, Jean-Marc; Bischof, Oliver; Jakob, Franz; Wagner, Wolfgang

2017-07-11

Culture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology are affected by these supplements - it is therefore important to determine if they favor outgrowth of different subpopulations and thereby impact on the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow-derived MSCs in parallel with HPL or FCS and demonstrated that HPL significantly increases proliferation and leads to dramatic differences in cellular morphology. Remarkably, global DNA-methylation profiles did not reveal any significant differences. Even at the transcriptomic level, there were only moderate changes in pairwise comparison. Furthermore, the effects on proliferation, cytoskeletal organization, and focal adhesions were reversible by interchanging to opposite culture conditions. These results indicate that cultivation of MSCs with HPL or FCS has no systematic bias for specific cell types.

Adhesion-mediated self-renewal abilities of Ph+ blastoma cells

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

Funayama, Keiji; Saito-Kurimoto, Yumi; Ebihara, Yasuhiro

2010-05-28

The Philadelphia chromosome-positive blastoma, maintained by serial subcutaneous transplantation in nude mice, is a highly proliferating biological mass consisting of homogenous CD34{sup +}CD38{sup -} myeloblastoid cells. These cells newly evolved from pluripotent leukemia stem cells of chronic myeloid leukemia in the chronic phase. Therefore, this mass may provide a unique tool for better understanding cellular and molecular mechanisms of self-renewal of leukemia stem cells. In this paper, we demonstrated that intravenously injected blastoma cells can cause Ph+ blastic leukemia with multiple invasive foci in NOD/SCID mice but not in nude mice. In addition, using an in vitro culture system, wemore » clearly showed that blastoma cell adhesion to OP9 stromal cells accelerates blastoma cell proliferation that is associated with up-regulation of BMI1 gene expression; increased levels of {beta}-catenin and the Notch1 intra-cellular domain; and changed the expression pattern of variant CD44 forms, which are constitutively expressed in these blastoma cells. These findings strongly suggest that adhesion of leukemic stem cells to stromal cells via CD44 might be indispensable for their cellular defense against attack by immune cells and for maintenance of their self-renewal ability.« less

Hypergravity Stimulates Osteoblast Proliferation Via Matrix-Integrin-Signaling Pathways

NASA Technical Reports Server (NTRS)

Vercoutere, W.; Parra, M.; Roden, C.; DaCosta, M.; Wing, A.; Damsky, C.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.

2003-01-01

Extensive characterizations of the physiologic consequences of microgravity and gravity indicate that lack of weight-bearing may cause tissue atrophy through cellular and subcellular level mechanisms. We hypothesize that gravity is needed for the efficient transduction of cell growth and survival signals from the extra-cellular matrix (ECM) in mechanosensitive tissues. Recent work from our laboratory and from others shows that an increase of gravity increases bone cell growth and survival. We found that 50-g hypergravity stimulation increased osteoblast proliferation for cells grown on Collagen Type I and Fibronectin, but not on Laminin or uncoated plastic. This may be a tissue-specific response, because 50-g hypergravity stimulation caused no increase in proliferation for primary rat fibroblasts. These results combined with RT-PCR for all possible integrins indicate that beta1 integrin subunit may be involved. The osteoblast proliferation response on Collagen Type I was greater at 25-g than at 10-g or 50-g; 24-h duration of hypergravity was necessary to see an increase in proliferation. Survival was enhanced during hypergravity stimulation by the presence of matrix. Flow cytometry analysis indicated that cell cycle may be altered; BrdU incorporation in proliferating cells showed an increase in the number of actively dividing cells from about 60% at 1-g to over 90% at 25-g. To further investigate the molecular components involved, we applied fluorescence labeling of cytoskeletal and signaling molecules to cells after 2 to 30 minutes of hypergravity stimulation. While structural components did not appear to be altered, phosphorylation increased, indicating that signaling pathways may be activated. These data indicate that gravity mechanostimulation of osteoblast proliferation involves specific matrix-integrin signaling pathways which are sensitive to duration and g-level.

In vitro effects of dental cements on hard and soft tissues associated with dental implants.

PubMed

Rodriguez, Lucas C; Saba, Juliana N; Chung, Kwok-Hung; Wadhwani, Chandur; Rodrigues, Danieli C

2017-07-01

Dental cements for cement-retained restorations are often chosen based on clinician preference for the product's material properties, mixing process, delivery mechanism, or viscosity. The composition of dental cement may play a significant role in the proliferation or inhibition of different bacterial strains associated with peri-implant disease, and the effect of dental cements on host cellular proliferation may provide further insight into appropriate cement material selection. The purpose of this in vitro study was to investigate the cellular host response of bone cells (osteoblasts) and soft tissue cells (gingival fibroblasts) to dental cements. Zinc oxide (eugenol and noneugenol), zinc phosphate, and acrylic resin cements were molded into pellets and directly applied to confluent preosteoblast (cell line MC3T3 E1) or gingival fibroblast cell cultures (cell line HGF) to determine cellular viability after exposure. Controls were defined as confluent cell cultures with no cement exposure. Direct contact cell culture testing was conducted following International Organization for Standardization 10993 methods, and all experiments were performed in triplicate. To compare either the MC3T3 E1 cell line, or the HGF cell line alone, a 1-way ANOVA test with multiple comparisons was used (α=.05). To compare the MC3T3 E1 cell line results and the HGF cell line results, a 2-way ANOVA test with multiple comparisons was used (α=.05). The results of this study illustrated that while both bone and soft tissue cell lines were vulnerable to the dental cement test materials, the soft tissue cell line (human gingival fibroblasts) was more susceptible to reduced cellular viability after exposure. The HGF cell line was much more sensitive to cement exposure. Here, the acrylic resin, zinc oxide (eugenol), and zinc phosphate cements significantly reduced cellular viability after exposure with respect to HGF cells only. Within the limitation of this in vitro cellular study, the results indicated that cell response to various implant cements varied significantly, with osteoblast proliferation much less affected than gingival fibroblast cells. Furthermore, the zinc oxide noneugenol dental cement appeared to affect the cell lines significantly less than the other test cements. Copyright © 2016 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.

Ultrastructural Characteristics of Rat Hepatic Oval Cells and Their Intercellular Contacts in the Model of Biliary Fibrosis: New Insights into Experimental Liver Fibrogenesis

PubMed Central

Lebensztejn, Dariusz Marek; Daniluk, Urszula; Sobaniec, Piotr; Sendrowski, Krzysztof; Daniluk, Jaroslaw; Debek, Wojciech

2017-01-01

Purpose Recently, it has been emphasized that hepatic progenitor/oval cells (HPCs) are significantly involved in liver fibrogenesis. We evaluated the multipotential population of HPCs by transmission electron microscope (TEM), including relations with adherent hepatic nonparenchymal cells (NPCs) in rats with biliary fibrosis induced by bile duct ligation (BDL). Methods The study used 6-week-old Wistar Crl: WI(Han) rats after BDL for 1, 6, and 8 weeks. Results Current ultrastructural analysis showed considerable proliferation of HPCs in experimental intensive biliary fibrosis. HPCs formed proliferating bile ductules and were scattered in periportal connective tissue. We distinguished 4 main types of HPCs: 0, I, II (bile duct-like cells; most common), and III (hepatocyte-like cells). We observed, very seldom presented in literature, cellular interactions between HPCs and adjacent NPCs, especially commonly found transitional hepatic stellate cells (T-HSCs) and Kupffer cells/macrophages. We showed the phenomenon of penetration of the basement membrane of proliferating bile ductules by cytoplasmic processes sent by T-HSCs and the formation of direct cell-cell contact with ductular epithelial cells related to HPCs. Conclusions HPC proliferation induced by BDL evidently promotes portal fibrogenesis. Better understanding of the complex cellular interactions between HPCs and adjacent NPCs, especially T-HSCs, may help develop antifibrotic therapies in the future. PMID:28769978

PROLIFERATION AS A KEY EVENT IN DEVELOPMENTAL TOXICITY: "CHEMICAL SCREENING IN HUMAN NEURAL STEM CELLS USING HIGH CONTENT IMAGING

EPA Science Inventory

New toxicity testing approaches will rely on in vitro assays to assess chemical effects at the cellular and molecular level. Cell proliferation is imperative to normal development, and chemical disruption of this process can be detrimental to the organism. As part of an effort to...

Immunobiological Aspects of erbB Receptors in Breast Cancer

DTIC Science & Technology

2000-08-01

receptor . The proliferation of cells expressing these chimeric receptors was EGF-dependent, and cells expressing EGFR/Y882F chimeric receptors were...determine Cells were washed twice with cold phosphate-buffered saline which cellular substrates couple with the receptor complex. (PBS) and lysed with 1...turnover, receptor proteins suggests that these substrates are properly lo- and cellular transformation in NEN757 cells (Qian et al., cated for

The effect of noncoherent red light irradiation on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

PubMed

Peng, Fei; Wu, Hua; Zheng, Yadong; Xu, Xiqiang; Yu, Jizhe

2012-05-01

Mesenchymal stem cells (MSCs) are promising for use in regenerative medicine. Low-level light irradiation (LLLI) has been shown to modulate various processes in different biological systems. The aim of our study was to investigate the effect of red light emitted from a light-emitting diode (LED) on bone marrow MSCs with or without osteogenic supplements. MSCs both with and without osteogenic supplements were divided into four groups, and each group was irradiated at doses of 0, 1, 2 and 4 J/cm(2). Cellular proliferation was evaluated using WST-8 and 5-ethynyl-2'-deoxyuridine (EdU) fluorescence staining. The alkaline phosphatase activity, mineralization, and expression of osteoblast master genes (Col1α1, Alpl, Bglap and Runx2) were monitored as indicators of MSC differentiation towards osteoblasts. In groups without osteogenic supplements, red light at all doses significantly stimulated cellular proliferation, whereas the osteogenic phenotype of the MSCs was not enhanced. In groups with osteogenic supplements, red light increased alkaline phosphatase activity and mineralized nodule formation, and stimulated the expression of Bglap and Runx2, but decreased cellular proliferation. In conclusion, nonconherent red light can promote proliferation but cannot induce osteogenic differentiation of MSCs in normal media, while it enhances osteogenic differentiation and decreases proliferation of MSCs in media with osteogenic supplements.

Effect of S-adenosyl-L-methionine (SAM), an allosteric activator of cystathionine-β-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro.

PubMed

Módis, Katalin; Coletta, Ciro; Asimakopoulou, Antonia; Szczesny, Bartosz; Chao, Celia; Papapetropoulos, Andreas; Hellmich, Mark R; Szabo, Csaba

2014-09-15

Recent data show that colon cancer cells selectively overexpress cystathionine-β-synthase (CBS), which produces hydrogen sulfide (H2S), to maintain cellular bioenergetics, support tumor growth and stimulate angiogenesis and vasorelaxation in the tumor microenvironment. The purpose of the current study was to investigate the effect of the allosteric CBS activator S-adenosyl-L-methionine (SAM) on the proliferation and bioenergetics of the CBS-expressing colon cancer cell line HCT116. The non-transformed, non-tumorigenic colon epithelial cell line NCM356 was used as control. For assessment of cell proliferation, the xCELLigence system was used. Bioenergetic function was measured by Extracellular Flux Analysis. Experiments using human recombinant CBS or HCT116 homogenates complemented the cell-based studies. SAM markedly enhanced CBS-mediated H2S production in vitro, especially when a combination of cysteine and homocysteine was used as substrates. Addition of SAM (0.1-3 mM) to HCT116 cells induced a concentration-dependent increase H2S production. SAM exerted time- and concentration-dependent modulatory effects on cell proliferation. At 0.1-1 mM SAM increased HCT116 proliferation between 0 and 12 h, while the highest SAM concentration (3 mM) inhibited proliferation. Over a longer time period (12-24 h), only the lowest concentration of SAM used (0.1 mM) stimulated cell proliferation; higher SAM concentrations produced a concentration-dependent inhibition. The short-term stimulatory effects of SAM were attenuated by the CBS inhibitor aminooxyacetic acid (AOAA) or by stable silencing of CBS. In contrast, the inhibitory effects of SAM on cell proliferation was unaffected by CBS inhibition or CBS silencing. In contrast to HCT116 cells, the lower rate of proliferation of the low-CBS expressor NCM356 cells was unaffected by SAM. Short-term (1 h) exposure of HCT116 cells to SAM induced a concentration-dependent increase in oxygen consumption and bioenergetic function at 0.1-1 mM, while 3 mM was inhibitory. Longer-term (72 h) exposure of HCT116 cells to all concentrations of SAM tested suppressed mitochondrial oxygen consumption rate, cellular ATP content and cell viability. The stimulatory effect of SAM on bioenergetics was attenuated in cells with stable CBS silencing, while the inhibitory effects were unaffected. In NCM356 cells SAM exerted smaller effects on cellular bioenergetics than in HCT116 cells. We have also observed a downregulation of CBS in response to prolonged exposure of SAM both in HCT116 and NCM356 cells. Taken together, the results demonstrate that H2S production in HCT116 cells is stimulated by the allosteric CBS activator, SAM. At low-to intermediate levels and early time periods the resulting H2S serves as an endogenous cancer cell growth and bioenergetic factor. In contrast, the inhibition of cell proliferation and bioenergetic function by SAM does not appear to relate to adverse autocrine effects of H2S resulting from CBS over-stimulation but, rather to CBS-independent pharmacological effects. Copyright © 2014 Elsevier Inc. All rights reserved.

Effects of exogenous vitamins A, C, and E and NADH supplementation on proliferation, cytokines release, and cell redox status of lymphocytes from healthy aged subjects.

PubMed

Bouamama, Samia; Merzouk, Hafida; Medjdoub, Amel; Merzouk-Saidi, Amel; Merzouk, Sid Ahmed

2017-06-01

Aging is an inevitable biological event that is associated with immune alterations. These alterations are related to increased cellular oxidative stress and micronutrient deficiency. Antioxidant supplementation could improve these age-related abnormalities. The aim of this study was to determine in vitro effects of vitamin A, vitamin C, vitamin E, and nicotinamide adenine dinucleotide (NADH) on T cell proliferation, cytokine release, and cell redox status in the elderly compared with young adults. Peripheral blood lymphocytes were isolated using a density gradient of Histopaque. They were cultured in vitro and stimulated with concanavalin A in the presence or absence of vitamins. Cell proliferation was determined by conducting MTT assays, and based on interleukin-2 and interleukin-4 secretions. Cell oxidant/antioxidant balance was assessed by assaying reduced glutathione (GSH), malondialdehyde, carbonyl protein levels, and catalase activity. The present study demonstrated that T-lymphocyte proliferation was decreased with aging and was associated with cytokine secretion alterations, GSH depletion, and intracellular oxidative stress. In the elderly, vitamin C, vitamin E, and NADH significantly improved lymphocyte proliferation and mitigated cellular oxidative stress, whereas vitamin A did not affect cell proliferation or cell redox status. In conclusion, vitamin C, vitamin E, and NADH supplementation improved T-lymphocytes response in the elderly, and could contribute to the prevention of age-related immune alterations. Consumption of food items containing these vitamins is recommended, and further investigation is necessary to evaluate the effect of vitamin supplementation in vivo.

Cell Proliferation, Reactive Oxygen and Cellular Glutathione

PubMed Central

Day, Regina M.; Suzuki, Yuichiro J.

2005-01-01

A variety of cellular activities, including metabolism, growth, and death, are regulated and modulated by the redox status of the environment. A biphasic effect has been demonstrated on cellular proliferation with reactive oxygen species (ROS)—especially hydrogen peroxide and superoxide—in which low levels (usually submicromolar concentrations) induce growth but higher concentrations (usually >10–30 micromolar) induce apoptosis or necrosis. This phenomenon has been demonstrated for primary, immortalized and transformed cell types. However, the mechanism of the proliferative response to low levels of ROS is not well understood. Much of the work examining the signal transduction by ROS, including H2O2, has been performed using doses in the lethal range. Although use of higher ROS doses have allowed the identification of important signal transduction pathways, these pathways may be activated by cells only in association with ROS-induced apoptosis and necrosis, and may not utilize the same pathways activated by lower doses of ROS associated with increased cell growth. Recent data has shown that low levels of exogenous H2O2 up-regulate intracellular glutathione and activate the DNA binding activity toward antioxidant response element. The modulation of the cellular redox environment, through the regulation of cellular glutathione levels, may be a part of the hormetic effect shown by ROS on cell growth. PMID:18648617

Tissue expander stimulated lengthening of arteries (TESLA) induces early endothelial cell proliferation in a novel rodent model.

PubMed

Potanos, Kristina; Fullington, Nora; Cauley, Ryan; Purcell, Patricia; Zurakowski, David; Fishman, Steven; Vakili, Khashayar; Kim, Heung Bae

2016-04-01

We examine the mechanism of aortic lengthening in a novel rodent model of tissue expander stimulated lengthening of arteries (TESLA). A rat model of TESLA was examined with a single stretch stimulus applied at the time of tissue expander insertion with evaluation of the aorta at 2, 4 and 7day time points. Measurements as well as histology and proliferation assays were performed and compared to sham controls. The aortic length was increased at all time points without histologic signs of tissue injury. Nuclear density remained unchanged despite the increase in length suggesting cellular hyperplasia. Cellular proliferation was confirmed in endothelial cell layer by Ki-67 stain. Aortic lengthening may be achieved using TESLA. The increase in aortic length can be achieved without tissue injury and results at least partially from cellular hyperplasia. Further studies are required to define the mechanisms involved in the growth of arteries under increased longitudinal stress. Copyright © 2015 Elsevier Inc. All rights reserved.

Stem cell factor and interleukin-2/15 combine to enhance MAPK-mediated proliferation of human natural killer cells

PubMed Central

Benson, Don M.; Yu, Jianhua; Becknell, Brian; Wei, Min; Freud, Aharon G.; Ferketich, Amy K.; Trotta, Rossana; Perrotti, Danilo; Briesewitz, Roger

2009-01-01

Stem cell factor (SCF) promotes synergistic cellular proliferation in combination with several growth factors, and appears important for normal natural killer (NK)–cell development. CD34+ hematopoietic precursor cells (HPCs) require interleukin-15 (IL-15) for differentiation into human NK cells, and this effect can be mimicked by IL-2. Culture of CD34+ HPCs or some primary human NK cells in IL-2/15 and SCF results in enhanced growth compared with either cytokine alone. The molecular mechanisms responsible for this are unknown and were investigated in the present work. Activation of NK cells by IL-2/15 increases expression of c-kit whose kinase activity is required for synergy with IL-2/15 signaling. Mitogen-activated protein kinase (MAPK) signaling intermediaries that are activated both by SCF and IL-2/15 are enhanced in combination to facilitate earlier cell-cycle entry. The effect results at least in part via enhanced MAPK-mediated modulation of p27 and CDK4. Collectively the data reveal a novel mechanism by which SCF enhances cellular proliferation in combination with IL-2/15 in primary human NK cells. PMID:19060242

Wilms' tumorigenesis is altered by misexpression of the transcriptional co-activator, CITED1

PubMed Central

Lovvorn, Harold N.; Boyle, Scott; Shi, Genbin; Shyr, Yu; Wills, Marcia L.; Perantoni, Alan O.; de Caestecker, Mark

2011-01-01

Purpose Wilms' tumors arise from arrested differentiation of renal progenitor cells. CITED1 is a transcriptional regulator that blocks the metanephric mesenchymal-to-epithelial conversion and is expressed in the blastema of both the developing kidney and Wilms' tumors. We hypothesized that alterations of CITED1-dependent signaling promote persistence of blastema and thereby subject these pluripotent cells to future oncogenic events. Methods We used a retroviral delivery system to overexpress the full-length CITED1 (F/L) protein and 2 deletion mutants lacking either of its known functional domains, ΔSID (Smad-4 Interacting Domain) and ΔCR2 (Conserved Region 2; the CITED1 transactivation domain), in a human Wilms' tumor cell line that endogenously expresses CITED1. In vitro effects on cellular proliferation and apoptosis were assayed. In vivo effects on tumorigenesis, growth, proliferation, and apoptosis were determined after heterotransplantation into immunodeficient mice (n = 15 per cell line). Results In vitro, overexpression of CITED1-F/L significantly increased, whereas overexpression of the functionally inactivating mutant, CITED1-ΔCR2, significantly reduced cellular proliferation relative to the other lines ( P <.0001). In vivo, Wilms' tumor incidence was significantly reduced in animals injected with cells overexpressing the mutant CITED1-ΔCR2 (7%) compared with CITED1-F/L (40%, P = .03) and CITED1-ΔSID (60%, P < .002). Similarly, mean tumor volume was least in the CITED1-ΔCR2 animals when compared with CITED1-F/L ( P = .03) and CITED1-ΔSID animals ( P <.005). Furthermore, the CITED1-ΔCR2 tumor showed the least cellular proliferation. Misexpression of CITED1 did not affect apoptosis either in vitro or in vivo. Conclusions Overexpression of CITED1 in a human Wilms' tumor cell line significantly increases proliferation in vitro, whereas mutation of its functionally critical transactivation domain (ΔCR2) significantly reduces proliferation. This mutation further perturbs tumorigenesis and tumor growth after heterotransplantation into immunodeficient mice. We speculate that overexpression of CITED1 promotes expansion of a rapidly proliferating population of blastema and thereby induces an unstable environment highly susceptible to future oncogenic events. PMID:17336183

PRODUCTION AND CHARACTERIZATION OF MULTIPLE-LAYERED POPULATIONS OF ANIMAL CELLS

PubMed Central

Kruse, Paul F.; Miedema, Ed

1965-01-01

Dense populations containing 129 x 106 Jensen sarcoma, 134 x 106 DON Chinese hamster, 28.9 x 106 WI-38 human diploid, 61.8 x 106 HEp-2 human carcinoma, and 67.4 x 106 WISH human amnion cells were produced from dilute inocula, 0.85 to 5.33 x 106, in 7 to 8 days in a perfusion system using replicate T-60 flasks. Perfusion rates as high as 560 ml medium/day/T-60 were required to maintain pH (to ca ±0.1 unit) and adequate nutrient supplies. The cell densities encountered are described by the term "monolayer equivalents" (M.E.), defined as number of cells per culture divided by number of cells in a monolayer. The M.E.'s for T-60 cultures containing unusually dense populations of 40 x 106 WI-38 and 250 x 106 DON cells (9-day perfusion) were 5 and 17, respectively, and numbers of cells in illustrations of stained cross-sections of membranes from these cultures were in excellent agreement. Threshold M.E.'s exist below which proliferation is the chief cellular activity and above which one or more cell functions may predominate even though proliferation persists. Cellular nutrition and metabolism may change with changes in M.E., as illustrated in different patterns of glutamic acid, proline, and glycine utilization or production in dense vs. dilute WI-38 cell populations. The results indicated that the role of contact inhibition phenomena in arresting cellular proliferation was diminished in perfusion system environments. PMID:5884626

Influence of beam shape on in-vitro cellular transformations in human skin fibroblasts

NASA Astrophysics Data System (ADS)

Mthunzi, Patience; Forbes, Andrew; Hawkins, Denise; Abrahamse, Heidi; Karsten, Aletta E.

2005-08-01

A variety of strategies have been utilised for prevention and treatment of chronic wounds such as leg ulcers, diabetic foot ulcers and pressure sores1. Low Level Laser Therapy (LLLT) has been reported to be an invaluable tool in the enhancement of wound healing through stimulating cell proliferation, accelerating collagen synthesis and increasing ATP synthesis in mitochondria to name but a few2. This study focused on an in-vitro analysis of the cellular responses induced by treatment with three different laser beam profiles namely, the Gaussian (G), Super Gaussian (SG) and Truncated Gaussian (TG), on normal wounded irradiated (WI) and wounded non-irradiated (WNI) human skin fibroblast cells (WS1), to test their influence in wound healing at 632.8 nm using a helium neon (HeNe) laser. For each beam profile, measurements were made using average energy densities over the sample ranging from 0.2 to 1 J, with single exposures on normal wounded cells. The cells were subjected to different post irradiation incubation periods, ranging from 0 to 24 hours to evaluate the duration (time) dependent effects resulting from laser irradiation. The promoted cellular alterations were measured by increase in cell viability, cell proliferation and cytotoxicity. The results obtained showed that treatment with the G compared to the SG and TG beams resulted in a marked increase in cell viability and proliferation. The data also showed that when cells undergo laser irradiation some cellular processes are driven by the peak energy density rather than the energy of the laser beam. We show that there exist threshold values for damage, and suggest optimal operating regimes for laser based wound healing.

CD10/NEP in non-small cell lung carcinomas. Relationship to cellular proliferation.

PubMed Central

Ganju, R K; Sunday, M; Tsarwhas, D G; Card, A; Shipp, M A

1994-01-01

The cell surface metalloproteinase CD10/neutral endopeptidase 24.11 (NEP) hydrolyzes a variety of peptide substrates and reduces cellular responses to specific peptide hormones. Because CD10/NEP modulates peptide-mediated proliferation of small cell carcinomas of the lung (SCLC) and normal fetal bronchial epithelium, we evaluated the enzyme's expression in non-small cell lung carcinomas (NSCLC). Bronchoalveolar and large cell carcinoma cell lines had low levels of CD10/NEP expression whereas squamous, adenosquamous, and adenocarcinoma cell lines had higher and more variable levels of the cell surface enzyme. Regional variations in CD10/NEP immunostaining in primary NSCLC specimens prompted us to correlate CD10/NEP expression with cell growth. In primary carcinomas of the lung, clonal NSCLC cell lines and SV40-transformed fetal airway epithelium, subsets of cells expressed primarily CD10/NEP or the proliferating cell nuclear antigen (PCNA). Cultured airway epithelial cells had the lowest levels of CD10/NEP expression when the highest percentage of cells were actively dividing; in addition, these cells grew more rapidly when cell surface CD10/NEP was inhibited. NSCLC cell lines had receptors for a variety of mitogenic peptides known to be CD10/NEP substrates, underscoring the functional significance of growth-related variability in CD10/NEP expression. Images PMID:7962523

Genetic analysis of indefinite division in human cells: Evidence for a cell senescence-related gene(s) on human chromosome 4

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

Yi Ning; Ledbetter, D.H.; Smith, J.R.

1991-07-01

Earlier studies had demonstrated that fusion of normal with immortal human cells yielded hybrids having limited division potential. This indicated that the phenotype of limited proliferation (cellular senescence) is dominant and that immortal cells result from recessive changes in normal growth-regulatory genes. In additional studies, the authors exploited the fact that the immortal phenotype is recessive and, by fusing various immortal human cell lines with each other, identified four complementation groups for indefinite division. Assignment of cell lines to specific groups allowed us to take a focused approach to identify the chromosomes and genes involved in growth regulation that havemore » been modified in immortal cells. They report here that introduction of a normal human chromosome 4 into three immortal cell lines (HeLa, J82, T98G) assigned to complementation group B resulted in loss of proliferation and reversal of the immortal phenotype. No effect on the proliferation potential of cell lines representative of the other complementation groups was observed. This result suggests that a gene(s) involved in cellular senescence and normal growth regulation resides on chromosome 4.« less

TopBP1 deficiency causes an early embryonic lethality and induces cellular senescence in primary cells.

PubMed

Jeon, Yoon; Ko, Eun; Lee, Kyung Yong; Ko, Min Ji; Park, Seo Young; Kang, Jeeheon; Jeon, Chang Hwan; Lee, Ho; Hwang, Deog Su

2011-02-18

TopBP1 plays important roles in chromosome replication, DNA damage response, and other cellular regulatory functions in vertebrates. Although the roles of TopBP1 have been studied mostly in cancer cell lines, its physiological function remains unclear in mice and untransformed cells. We generated conditional knock-out mice in which exons 5 and 6 of the TopBP1 gene are flanked by loxP sequences. Although TopBP1-deficient embryos developed to the blastocyst stage, no homozygous mutant embryos were recovered at E8.5 or beyond, and completely resorbed embryos were frequent at E7.5, indicating that mutant embryos tend to die at the peri-implantation stage. This finding indicated that TopBP1 is essential for cell proliferation during early embryogenesis. Ablation of TopBP1 in TopBP1(flox/flox) mouse embryonic fibroblasts and 3T3 cells using Cre recombinase-expressing retrovirus arrests cell cycle progression at the G(1), S, and G(2)/M phases. The TopBP1-ablated mouse cells exhibit phosphorylation of H2AX and Chk2, indicating that the cells contain DNA breaks. The TopBP1-ablated mouse cells enter cellular senescence. Although RNA interference-mediated knockdown of TopBP1 induced cellular senescence in human primary cells, it induced apoptosis in cancer cells. Therefore, TopBP1 deficiency in untransformed mouse and human primary cells induces cellular senescence rather than apoptosis. These results indicate that TopBP1 is essential for cell proliferation and maintenance of chromosomal integrity.

Iron depletion results in Src kinase inhibition with associated cell cycle arrest in neuroblastoma cells.

PubMed

Siriwardana, Gamini; Seligman, Paul A

2015-03-01

Iron is required for cellular proliferation. Recently, using systematic time studies of neuroblastoma cell growth, we better defined the G1 arrest caused by iron chelation to a point in mid-G1, where cyclin E protein is present, but the cyclin E/CDK2 complex kinase activity is inhibited. In this study, we again used the neuroblastoma SKNSH cells lines to pinpoint the mechanism responsible for this G1 block. Initial studies showed in the presence of DFO, these cells have high levels of p27 and after reversal of iron chelation p27 is degraded allowing for CDK2 kinase activity. The initial activation of CDK2 kinase allows cells to exit G1 and enter S phase. Furthermore, we found that inhibition of p27 degradation by DFO is directly associated with inhibition of Src kinase activity measured by lack of phosphorylation of Src at the 416 residue. Activation of Src kinase occurs very early after reversal from the DFO G1 block and is temporally associated with initiation of cellular proliferation associated with entry into S phase. For the first time therefore we show that iron chelation inhibits Src kinase activity and this activity is a requirement for cellular proliferation. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Tocotrienol-rich fraction prevents cellular aging by modulating cell proliferation signaling pathways.

PubMed

Khor, S C; Mohd Yusof, Y A; Wan Ngah, W Z; Makpol, S

Vitamin E has been suggested as nutritional intervention for the prevention of degenerative and age-related diseases. In this study, we aimed to elucidate the underlying mechanism of tocotrienol-rich fraction (TRF) in delaying cellular aging by targeting the proliferation signaling pathways in human diploid fibroblasts (HDFs). Tocotrienol-rich fraction was used to treat different stages of cellular aging of primary human diploid fibroblasts viz. young (passage 6), pre-senescent (passage 15) and senescent (passage 30). Several selected targets involved in the downstream of PI3K/AKT and RAF/MEK/ERK pathways were compared in total RNA and protein. Different transcriptional profiles were observed in young, pre-senescent and senescent HDFs, in which cellular aging increased AKT, FOXO3, CDKN1A and RSK1 mRNA expression level, but decreased ELK1, FOS and SIRT1 mRNA expression level. With tocotrienol-rich fraction treatment, gene expression of AKT, FOXO3, ERK and RSK1 mRNA was decreased in senescent cells, but not in young cells. The three down-regulated mRNA in cellular aging, ELK1, FOS and SIRT1, were increased with tocotrienol-rich fraction treatment. Expression of FOXO3 and P21Cip1 proteins showed up-regulation in senescent cells but tocotrienol-rich fraction only decreased P21Cip1 protein expression in senescent cells. Tocotrienol-rich fraction exerts gene modulating properties that might be responsible in promoting cell cycle progression during cellular aging.

Commonly consumed and specialty dietary mushrooms reduce cellular proliferation in MCF-7 human breast cancer cells.

PubMed

Martin, Keith R; Brophy, Sara K

2010-11-01

Worldwide, over one million women will be newly diagnosed with breast cancer in the next year. Moreover, breast cancer is the second leading cause of cancer death in the USA. An accumulating body of evidence suggests that consumption of dietary mushrooms can protect against breast cancer. In this study, we tested and compared the ability of five commonly consumed or specialty mushrooms to modulate cell number balance in the cancer process using MCF-7 human breast cancer cells. Hot water extracts (80°C for 2 h) of maitake (MT, Grifola frondosa), crimini (CRIM, Agaricus bisporus), portabella (PORT, Agaricus bisporus), oyster (OYS, Pleurotus ostreatus) and white button (WB, Agaricus bisporus) mushrooms or water alone (5% v/v) were incubated for 24 h with MCF-7 cells. Cellular proliferation determined by bromodeoxyuridine incorporation was significantly (P < 0.05) reduced up to 33% by all mushrooms, with MT and OYS being the most effective. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction, an often used mitochondrion-dependent marker of proliferation, was unchanged although decreased (P > 0.05) by 15% with OYS extract. Lactate dehydrogenase release, as a marker of necrosis, was significantly increased after incubation with MT but not with other test mushrooms. Furthermore, MT extract significantly increased apoptosis, or programmed cell death, as determined by terminal deoxynucleotidyl end labeling method, whereas other test mushrooms displayed trends of ∼15%. The total numbers of cells per flask, determined by hemacytometry, were not different from control cultures. Overall, all test mushrooms significantly suppressed cellular proliferation, with MT further significantly inducing apoptosis and cytotoxicity in human breast cancer cells. This suggests that both common and specialty mushrooms may be chemoprotective against breast cancer.

Differential cellular responses by oncogenic levels of c-Myc expression in long-term confluent retinal pigment epithelial cells.

PubMed

Wang, Yiping; Cheng, Xiangdong; Samma, Muhammad Kaleem; Kung, Sam K P; Lee, Clement M; Chiu, Sung Kay

2018-06-01

c-Myc is a highly pleiotropic transcription factor known to control cell cycle progression, apoptosis, and cellular transformation. Normally, ectopic expression of c-Myc is associated with promoting cell proliferation or triggering cell death via activating p53. However, it is not clear how the levels of c-Myc lead to different cellular responses. Here, we generated a series of stable RPE cell clones expressing c-Myc at different levels, and found that consistent low level of c-Myc induced cellular senescence by activating AP4 in post-confluent RPE cells, while the cells underwent cell death at high level of c-Myc. In addition, high level of c-Myc could override the effect of AP4 on cellular senescence. Further knockdown of AP4 abrogated senescence-like phenotype in cells expressing low level of c-Myc, and accelerated cell death in cells with medium level of c-Myc, indicating that AP4 was required for cellular senescence induced by low level of c-Myc.

Phospholipase C delta 4 (PLCδ4) is a nuclear protein involved in cell proliferation and senescence in mesenchymal stromal stem cells.

PubMed

Kunrath-Lima, Marianna; de Miranda, Marcelo Coutinho; Ferreira, Andrea da Fonseca; Faraco, Camila Cristina Fraga; de Melo, Mariane Izabella Abreu; Goes, Alfredo Miranda; Rodrigues, Michele Angela; Faria, Jerusa Araújo Quintão Arantes; Gomes, Dawidson Assis

2018-06-01

Ca 2+ is an important second messenger, and it is involved in many cellular processes such as cell death and proliferation. The rise in intracellular Ca 2+ levels can be due to the generation of inositol 1,4,5-trisphosphate (InsP 3 ), which is a product of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) hydrolysis by phospholipases C (PLCs), that leads to Ca 2+ release from endoplasmic reticulum by InsP 3 receptors (InsP 3 R). Ca 2+ signaling patterns can vary in different regions of the cell and increases in nuclear Ca 2+ levels have specific biological effects that differ from those of Ca 2+ increase in the cytoplasm. There are PLCs in the cytoplasm and nucleus, but little is known about the functions of nuclear PLCs. This work aimed to characterize phenotypically the human PLCδ4 (hPLCδ4) in mesenchymal stem cells. This nuclear isoform of PLC is present in different cell types and has a possible role in proliferative processes. In this work, hPLCδ4 was found to be mainly nuclear in human adipose-derived mesenchymal stem cells (hASC). PLCδ4 knockdown demonstrated that it is essential for hASC proliferation, without inducing cell death. An increase of cells in G1, and a reduction of cells on interphase and G2/M in knockdown cells were seen. Furthermore, PLCδ4 knockdown increased the percentage of senescent cells, p16 INK4A+ and p21 Cip1 mRNAs expression, which could explain the impaired cell proliferation. The results show that hPLCδ4 is in involved in cellular proliferation and senescence in hASC. Copyright © 2018 Elsevier Inc. All rights reserved.

Different toxic effects of YTX in tumor K-562 and lymphoblastoid cell lines

PubMed Central

Fernández-Araujo, Andrea; Sánchez, Jon A.; Alfonso, Amparo; Vieytes, Mercedes R.; Botana, Luis M.

2015-01-01

Yessotoxin (YTX) modulates cellular phosphodiesterases (PDEs). In this regard, opposite effects had been described in the tumor model K-562 cell line and fresh human lymphocytes in terms of cell viability, cyclic adenosine 3',5'-cyclic monophosphate (cAMP) production and protein expression after YTX treatment. Studies in depth of the pathways activated by YTX in K-562 cell line, have demonstrated the activation of two different cell death types, apoptosis, and autophagy after 24 and 48 h of treatment, respectively. Furthermore, the key role of type 4A PDE (PDE4A) in both pathways activated by YTX was demonstrated. Therefore, taking into account the differences between cellular lines and fresh cells, a study of cell death pathways activated by YTX in a non-tumor cell line with mitotic activity, was performed. The cellular model used was the lymphoblastoid cell line that represents a non-tumor model with normal apoptotic and mitotic machinery. In this context, cell viability and cell proliferation, expression of proteins involved in cell death activated by YTX and mitochondrial mass, were studied after the incubation with the toxin. Opposite to the tumor model, no cell death activation was observed in lymphoblastoid cell line in the presence of YTX. In this sense, variations in apoptosis hallmarks were not detected in the lymphoblastoid cell line after YTX incubation, whereas this type I of programmed cell death was observed in K-562 cells. On the other hand, autophagy cell death was triggered in this cellular line, while other autophagic process is suggested in lymphoblastoid cells. These YTX effects are related to PDE4A in both cellular lines. In addition, while cell death is triggered in K-562 cells after YTX treatment, in lymphoblastoid cells the toxin stops cellular proliferation. These results point to YTX as a specific toxic compound of tumor cells, since in the non-tumor lymphoblastoid cell line, no cell death hallmarks are observed. PMID:26136685

Phytocalpain controls the proliferation and differentiation fates of cells in plant organ development.

PubMed

Ahn, Joon-Woo; Kim, Moonil; Lim, Jeong Hwa; Kim, Gyung-Tae; Pai, Hyun-Sook

2004-06-01

Calpain, a calcium-dependent cysteine protease, plays an essential role in basic cellular processes in animal cells, including cell proliferation, apoptosis, and differentiation. NbDEK encodes the calpain homolog of N. benthamiana. In this study, virus-induced gene silencing (VIGS) of NbDEK resulted in arrested organ development and hyperplasia in all the major plant organs examined. The epidermal layers of the leaves and stems were covered with hyperproliferating cell masses, and stomata and trichome development was severely inhibited. During flower development, a single dome-like structure was grown from the flower meristem to generate a large cylinder-shaped flower lacking any floral organs. At the cellular level, cell division was sustained in tissues that were otherwise already differentiated, and cell differentiation was severely hampered. NbDEK is ubiquitously expressed in all the plant tissues examined. In the abnormal organs of the NbDEK VIGS lines, protein levels of D-type cyclins (CycD)2, CycD3, and proliferating cell nuclear antigen (PCNA) were greatly elevated, and transcription of E2F (E2 promoter binding factor), E2F-regulated genes, retinoblastoma (Rb), and KNOTTED1 (KN1)-type homeobox genes was also stimulated. These results suggest that phytocalpain is a key regulator of cell proliferation and differentiation during plant organogenesis, and that it acts partly by controlling the CycD/Rb pathway.

Resveratrol induces cellular senescence with attenuated mono-ubiquitination of histone H2B in glioma cells

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

Gao, Zhen; Xu, Michael S.; Barnett, Tamara L.

2011-04-08

Research highlights: {yields} Resveratrol induces cellular senescence in glioma cell. {yields} Resveratrol inhibits mono-ubiquitination of histone H2B at K120. {yields} Depletion of RNF20, phenocopies the inhibitory effects of resveratrol. {yields} Mono-ubiquitination of histone H2B at K120 is a novel target of resveratrol. {yields} RNF20 inhibits cellular senescence in proliferating glioma cells. -- Abstract: Resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenol naturally occurring in grapes and other plants, has cancer chemo-preventive effects and therapeutic potential. Although resveratrol modulates multiple pathways in tumor cells, how resveratrol or its affected pathways converge on chromatin to mediate its effects is not known. Using glioma cells as amore » model, we showed here that resveratrol inhibited cell proliferation and induced cellular hypertrophy by transforming spindle-shaped cells to enlarged, irregular and flatten-shaped ones. We further showed that resveratrol-induced hypertrophic cells expressed senescence-associated-{beta}-galactosidase, suggesting that resveratrol-induced cellular senescence in glioma cells. Consistent with these observations, we demonstrated that resveratrol inhibited clonogenic efficiencies in vitro and tumor growth in a xenograft model. Furthermore, we found that acute treatment of resveratrol inhibited mono-ubiquitination of histone H2B at K120 (uH2B) in breast, prostate, pancreatic, lung, brain tumor cells as well as primary human cells. Chronic treatment with low doses of resveratrol also inhibited uH2B in the resveratrol-induced senescent glioma cells. Moreover, we showed that depletion of RNF20, a ubiquitin ligase of histone H2B, inhibited uH2B and induced cellular senescence in glioma cells in vitro, thereby recapitulated the effects of resveratrol. Taken together, our results suggest that uH2B is a novel direct or indirect chromatin target of resveratrol and RNF20 plays an important role in inhibiting cellular senescence programs that are intact in glioma cells.« less

Interleukin-1, tumor necrosis factor-alpha, and transforming growth factor-beta 1 and integrative meniscal repair: influences on meniscal cell proliferation and migration

PubMed Central

2011-01-01

Introduction Interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) are up-regulated in injured and osteoarthritic knee joints. IL-1 and TNF-α inhibit integrative meniscal repair; however, the mechanisms by which this inhibition occurs are not fully understood. Transforming growth factor-β1 (TGF-β1) increases meniscal cell proliferation and accumulation, and enhances integrative meniscal repair. An improved understanding of the mechanisms modulating meniscal cell proliferation and migration will help to improve approaches for enhancing intrinsic or tissue-engineered repair of the meniscus. The goal of this study was to examine the hypothesis that IL-1 and TNF-α suppress, while TGF-β1 enhances, cellular proliferation and migration in cell and tissue models of meniscal repair. Methods A micro-wound assay was used to assess meniscal cell migration and proliferation in response to the following treatments for 0, 24, or 48 hours: 0 to 10 ng/mL IL-1, TNF-α, or TGF-β1, in the presence or absence of 10% serum. Proliferated and total cells were fluorescently labeled and imaged using confocal laser scanning microscopy and the number of proliferated, migrated, and total cells was determined in the micro-wound and edges of each image. Meniscal cell proliferation was also assessed throughout meniscal repair model explants treated with 0 or 10 ng/mL IL-1, TNF-α, or TGF-β1 for 14 days. At the end of the culture period, biomechanical testing and histological analyses were also performed. Statistical differences were assessed using an ANOVA and Newman-Keuls post hoc test. Results IL-1 and TNF-α decreased cell proliferation in both cell and tissue models of meniscal repair. In the presence of serum, TGF-β1 increased outer zone cell proliferation in the micro-wound and in the cross section of meniscal repair model explants. Both IL-1 and TNF-α decreased the integrative shear strength of repair and extracellular matrix deposition in the meniscal repair model system, while TGF-β1 had no effect on either measure. Conclusions Meniscal cell proliferation in vivo may be diminished following joint injury due to the up-regulation of inflammatory cytokines, thereby limiting native cellular repair of meniscal lesions. Therefore, therapies that can promote meniscal cell proliferation have promise to enhance meniscal repair and improve tissue engineering strategies. PMID:22087734

Osteoblastlike cell adhesion on titanium surfaces modified by plasma nitriding.

PubMed

da Silva, Jose Sandro Pereira; Amico, Sandro Campos; Rodrigues, Almir Olegario Neves; Barboza, Carlos Augusto Galvao; Alves, Clodomiro; Croci, Alberto Tesconi

2011-01-01

The aim of this study was to evaluate the characteristics of various titanium surfaces modified by cold plasma nitriding in terms of adhesion and proliferation of rat osteoblastlike cells. Samples of grade 2 titanium were subjected to three different surface modification processes: polishing, nitriding by plasma direct current, and nitriding by cathodic cage discharge. To evaluate the effect of the surface treatment on the cellular response, the adhesion and proliferation of osteoblastlike cells (MC3T3) were quantified and the results were analyzed by Kruskal-Wallis and Friedman statistical tests. Cellular morphology was observed by scanning electron microscopy. There was more MC3T3 cell attachment on the rougher surfaces produced by cathodic cage discharge compared with polished samples (P < .05). Plasma nitriding improves titanium surface roughness and wettability, leading to osteoblastlike cell adhesion.

Reversible effect of all-trans-retinoic acid on AML12 hepatocyte proliferation and cell cycle progression

EPA Science Inventory

The role of all-trans-retinoic acid (atRA) in the regulation of cellular proliferation and differentiation is well documented. Numerous studies have established the cancer preventive propertiesofatRAwhichfunctionstoregulate levels ofcellcycleproteinsessentialfortheGliS transition...

Loss of PTEN causes SHP2 activation, making lung cancer cells unresponsive to IFN-γ

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

Chen, Chia-Ling; Chiang, Tzu-Hui; Tseng, Po-Chun

Src homology-2 domain-containing phosphatase (SHP) 2, an oncogenic phosphatase, inhibits type II immune interferon (IFN)-γ signaling by subverting signal transducers and activators of transcription 1 tyrosine phosphorylation and activation. For cancer immunoediting, this study aimed to investigate the decrease of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein, leading to cellular impairment of IFN-γ signaling. In comparison with human lung adenocarcinoma A549 cells, the natural PTEN loss in another human lung adenocarcinoma line, PC14PE6/AS2 cells, presents reduced responsiveness in IFN-γ-induced IFN regulatory factor 1 activation and CD54 expression. Artificially silencing PTEN expression in A549 cellsmore » also caused cells to be unresponsive to IFN-γ without affecting IFN-γ receptor expression. IFN-γ-induced inhibition of cell proliferation and cytotoxicity were demonstrated in A549 cells but were defective in PC14PE6/AS2 cells and in PTEN-deficient A549 cells. Aberrant activation of SHP2 by ROS was specifically shown in PC14PE6/AS2 cells and PTEN-deficient A549 cells. Inhibiting ROS and SHP2 rescued cellular responses to IFN-γ-induced cytotoxicity and inhibition of cell proliferation in PC14PE6/AS2 cells. These results demonstrate that a decrease in PTEN facilitates ROS/SHP2 signaling, causing lung cancer cells to become unresponsive to IFN-γ. - Highlights: • This study demonstrates that PTEN decrease causes cellular unresponsive to IFN-γ. • Lung cancer cells with PTEN deficiency show unresponsive to IFN-γ signaling. • PTEN decrease inhibits IFN-γ-induced CD54, cell proliferation inhibition, and cytotoxicity. • ROS-mediated SHP2 activation makes PTEN-deficient cells unresponsive to IFN-γ.« less

ADAM-12 as a Diagnostic Marker for the Proliferation, Migration and Invasion in Patients with Small Cell Lung Cancer

PubMed Central

Shao, Shuhong; Li, Zunling; Gao, Wei; Yu, Guohua; Liu, Dexiang; Pan, Fang

2014-01-01

Small cell lung cancer (SCLC) is highly aggressive and is characterized by malignant metastasis. Approximately 90% of patients die due to extensive metastasis. The extracellular matrix (ECM) is a natural barrier that can prevent cellular invasion and metastasis. Therefore, degradation of the ECM must take place in order for extensive metastasis to occur. A disintegrin and metalloprotease (ADAM) is a multi-domain protease that plays an important role in tumorigenesis, as well as tumor development, invasion and metastasis. However, there have been few reports on the expression and role of ADAMs in SCLC. In the current study, the expression and role of ADAMs in SCLC proliferation, invasion and metastasis was investigated. A total of 150 SCLC tissue samples were examined by immunohistochemistry for ADAMs expression. ADAM-12 was found to be abundantly expressed in 72.67% samples and other ADAMs were found to be expressed in 10% to 40% of samples. ADAM-12 levels in serum and urine, from 70 SCLC patients and 40 normal controls, were also measured using ELISA. ADAM-12 expression was significantly higher in SCLC patients than in healthy controls and in patients with extensive disease compared to those with more limited disease. Silencing the expression of ADAM-12 in H1688 cells through the use of specific siRNA significantly reduced cellular proliferation, invasion and metastasis. Supplementing the expression of ADAM-12-L or -S in H345 cells, significantly enhanced cellular proliferation, invasion and metastasis. Animal models with metastatic SCLC also exhibited increased expression of ADAM-12 along with enhanced invasion and metastasis. In brief, ADAM-12 is an independent prognostic factor and diagnostic marker, and is involved in the proliferation, invasion and metastasis of SCLC. PMID:24465799

Cellular processes involved in human epidermal cells exposed to extremely low frequency electric fields.

PubMed

Collard, J-F; Hinsenkamp, M

2015-05-01

We observed on different tissues and organisms a biological response after exposure to pulsed low frequency and low amplitude electric or electromagnetic fields but the precise mechanism of cell response remains unknown. The aim of this publication is to understand, using bioinformatics, the biological relevance of processes involved in the modification of gene expression. The list of genes analyzed was obtained after microarray protocol realized on cultures of human epidermal explants growing on deepidermized human skin exposed to a pulsed low frequency electric field. The directed acyclic graph on a WebGestalt Gene Ontology module shows six categories under the biological process root: "biological regulation", "cellular process", "cell proliferation", "death", "metabolic process" and "response to stimulus". Enriched derived categories are coherent with the type of in vitro culture, the stimulation protocol or with the previous results showing a decrease of cell proliferation and an increase of differentiation. The Kegg module on WebGestalt has highlighted "cell cycle" and "p53 signaling pathway" as significantly involved. The Kegg website brings out interactions between FoxO, MAPK, JNK, p53, p38, PI3K/Akt, Wnt, mTor or NF-KappaB. Some genes expressed by the stimulation are known to have an exclusive function on these pathways. Analyses performed with Pathway Studio linked cell proliferation, cell differentiation, apoptosis, cell cycle, mitosis, cell death etc. with our microarrays results. Medline citation generated by the software and the fold change variation confirms a diminution of the proliferation, activation of the differentiation and a less well-defined role of apoptosis or wound healing. Wnt and DKK functional classes, DKK1, MACF1, ATF3, MME, TXNRD1, and BMP-2 genes proposed in previous publications after a manual analysis are also highlighted with other genes after Pathway Studio automatic procedure. Finally, an analysis conducted on a list of genes characterized by an accelerated regulation after extremely low frequency pulsed stimulation also confirms their role in the processes of cell proliferation and differentiation. Bioinformatics approach allows in-depth research, without the bias of pre-selection, on cellular processes involved in a huge gene list. Copyright © 2015 Elsevier Inc. All rights reserved.

Platelets and Plasma Stimulate Sheep Rotator Cuff Tendon Tenocytes When Cultured in an Extracellular Matrix Scaffold

PubMed Central

Kelly, Brian A.; Proffen, Benedikt L.; Haslauer, Carla M.; Murray, Martha M.

2015-01-01

The addition of platelet-rich plasma (PRP) to rotator cuff repair has not translated into improved outcomes after surgery. However, recent work stimulating ligament healing has demonstrated improved outcomes when PRP or whole blood is combined with an extracellular matrix carrier. The objective of this study was to evaluate the effect of three components of blood (plasma, platelets and macrophages) on the in vitro activity of ovine rotator cuff cells cultured in an extracellular matrix environment. Tenocytes were obtained from six ovine infraspinatus tendons and cultured over 14 days in an extracellular matrix scaffold with the following additives: 1) Plasma (PPP), 2) Plasma and platelets (PAP), 3) Plasma and macrophages (PPPM), 4) Plasma, platelets and macrophages (PAPM), 5) Phosphate buffered saline (PBS), and 6) PBS with macrophages (PBSM). Assays measuring cellular metabolism (AlamarBlue), proliferation (Quantitative DNA assay), synthesis of collagen and cytokines (SIRCOL, TNF-α and IL-10 ELISA, and MMP assay), and collagen gene expression (qPCR) were performed over the duration of the experiment, as well as histology at the conclusion. Plasma was found to stimulate cell attachment and spreading on the scaffold, as well as cellular proliferation. Platelets also stimulated cell proliferation, cellular metabolism, transition of cells to a myofibroblast phenotype and contraction of the scaffolds. The addition of macrophages did not have any significant effect on the sheep rotator cuff cells in vitro. In vivo studies are needed to determine if these changes in cellular function will translate into improved tendon healing. PMID:26419602

Platelets and plasma stimulate sheep rotator cuff tendon tenocytes when cultured in an extracellular matrix scaffold.

PubMed

Kelly, Brian A; Proffen, Benedikt L; Haslauer, Carla M; Murray, Martha M

2016-04-01

The addition of platelet-rich plasma (PRP) to rotator cuff repair has not translated into improved outcomes after surgery. However, recent work stimulating ligament healing has demonstrated improved outcomes when PRP or whole blood is combined with an extracellular matrix carrier. The objective of this study was to evaluate the effect of three components of blood (plasma, platelets, and macrophages) on the in vitro activity of ovine rotator cuff cells cultured in an extracellular matrix environment. Tenocytes were obtained from six ovine infraspinatus tendons and cultured over 14 days in an extracellular matrix scaffold with the following additives: (1) plasma (PPP), (2) plasma and platelets (PAP), (3) plasma and macrophages (PPPM), (4) plasma, platelets and macrophages (PAPM), (5) phosphate buffered saline (PBS), and (6) PBS with macrophages (PBSM). Assays measuring cellular metabolism (AlamarBlue), proliferation (Quantitative DNA assay), synthesis of collagen and cytokines (SIRCOL, TNF-α and IL-10 ELISA, and MMP assay), and collagen gene expression (qPCR) were performed over the duration of the experiment, as well as histology at the conclusion. Plasma was found to stimulate cell attachment and spreading on the scaffold, as well as cellular proliferation. Platelets also stimulated cell proliferation, cellular metabolism, transition of cells to a myofibroblast phenotype, and contraction of the scaffolds. The addition of macrophages did not have any significant effect on the sheep rotator cuff cells in vitro. In vivo studies are needed to determine whether these changes in cellular function will translate into improved tendon healing. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Evaluation of the Effects of Airborne Particulate Matter on Bone Marrow-Mesenchymal Stem Cells (BM-MSCs): Cellular, Molecular and Systems Biological Approaches

PubMed Central

Abu-Elmagd, Muhammad; Alghamdi, Mansour A.; Shamy, Magdy; Khoder, Mamdouh I.; Costa, Max; Assidi, Mourad; Kadam, Roaa; Alsehli, Haneen; Gari, Mamdooh; Pushparaj, Peter Natesan; Kalamegam, Gauthaman; Al-Qahtani, Mohammed H.

2017-01-01

Particulate matter (PM) contains heavy metals that affect various cellular functions and gene expression associated with a range of acute and chronic diseases in humans. However, the specific effects they exert on the stem cells remain unclear. Here, we report the effects of PM collected from the city of Jeddah on proliferation, cell death, related gene expression and systems of biological analysis in bone marrow mesenchymal stem cells (BM-MSCs), with the aim of understanding the underlying mechanisms. PM2.5 and PM10 were tested in vitro at various concentrations (15 to 300 µg/mL) and durations (24 to 72 h). PMs induced cellular stress including membrane damage, shrinkage and death. Lower concentrations of PM2.5 increased proliferation of BM-MSCs, while higher concentrations served to decrease it. PM10 decreased BM-MSCs proliferation in a concentration-dependent manner. The X-ray fluorescence spectrometric analysis showed that PM contains high levels of heavy metals. Ingenuity Pathway Analysis (IPA) and hierarchical clustering analyses demonstrated that heavy metals were associated with signaling pathways involving cell stress/death, cancer and chronic diseases. qRT-PCR results showed differential expression of the apoptosis genes (BCL2, BAX); inflammation associated genes (TNF-α and IL-6) and the cell cycle regulation gene (p53). We conclude that PM causes inflammation and cell death, and thereby predisposes to chronic debilitating diseases. PMID:28425934

Axl as a mediator of cellular growth and survival.

PubMed

Axelrod, Haley; Pienta, Kenneth J

2014-10-15

The control of cellular growth and proliferation is key to the maintenance of homeostasis. Survival, proliferation, and arrest are regulated, in part, by Growth Arrest Specific 6 (Gas6) through binding to members of the TAM receptor tyrosine kinase family. Activation of the TAM receptors leads to downstream signaling through common kinases, but the exact mechanism within each cellular context varies and remains to be completely elucidated. Deregulation of the TAM family, due to its central role in mediating cellular proliferation, has been implicated in multiple diseases. Axl was cloned as the first TAM receptor in a search for genes involved in the progression of chronic to acute-phase leukemia, and has since been established as playing a critical role in the progression of cancer. The oncogenic nature of Axl is demonstrated through its activation of signaling pathways involved in proliferation, migration, inhibition of apoptosis, and therapeutic resistance. Despite its recent discovery, significant progress has been made in the development of effective clinical therapeutics targeting Axl. In order to accurately define the role of Axl in normal and diseased processes, it must be analyzed in a cell type-specific context.

Different effects of 25-kDa amelogenin on the proliferation, attachment and migration of various periodontal cells

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

Li, Xiting; Shu, Rong, E-mail: shurong123@hotmail.com; Liu, Dali

Previous studies have assumed that amelogenin is responsible for the therapeutic effect of the enamel matrix derivative (EMD) in periodontal tissue healing and regeneration. However, it is difficult to confirm this hypothesis because both the EMD and the amelogenins are complex mixtures of multiple proteins. Further adding to the difficulties is the fact that periodontal tissue regeneration involves various types of cells and a sequence of associated cellular events including the attachment, migration and proliferation of various cells. In this study, we investigated the potential effect of a 25-kDa recombinant porcine amelogenin (rPAm) on primarily cultured periodontal ligament fibroblasts (PDLF),more » gingival fibroblasts (GF) and gingival epithelial cells (GEC). The cells were treated with 25-kDa recombinant porcine amelogenin at a concentration of 10 {mu}g/mL. We found that rPAm significantly promoted the proliferation and migration of PDLF, but not their adhesion. Similarly, the proliferation and adhesion of GF were significantly enhanced by treatment with rPAm, while migration was greatly inhibited. Interestingly, this recombinant protein inhibited the growth rate, cell adhesion and migration of GEC. These data suggest that rPAm may play an essential role in periodontal regeneration through the activation of periodontal fibroblasts and inhibition of the cellular behaviors of gingival epithelial cells.« less

Distinct gene regulatory programs define the inhibitory effects of liver X receptors and PPARG on cancer cell proliferation.

PubMed

Savic, Daniel; Ramaker, Ryne C; Roberts, Brian S; Dean, Emma C; Burwell, Todd C; Meadows, Sarah K; Cooper, Sara J; Garabedian, Michael J; Gertz, Jason; Myers, Richard M

2016-07-11

The liver X receptors (LXRs, NR1H2 and NR1H3) and peroxisome proliferator-activated receptor gamma (PPARG, NR1C3) nuclear receptor transcription factors (TFs) are master regulators of energy homeostasis. Intriguingly, recent studies suggest that these metabolic regulators also impact tumor cell proliferation. However, a comprehensive temporal molecular characterization of the LXR and PPARG gene regulatory responses in tumor cells is still lacking. To better define the underlying molecular processes governing the genetic control of cellular growth in response to extracellular metabolic signals, we performed a comprehensive, genome-wide characterization of the temporal regulatory cascades mediated by LXR and PPARG signaling in HT29 colorectal cancer cells. For this analysis, we applied a multi-tiered approach that incorporated cellular phenotypic assays, gene expression profiles, chromatin state dynamics, and nuclear receptor binding patterns. Our results illustrate that the activation of both nuclear receptors inhibited cell proliferation and further decreased glutathione levels, consistent with increased cellular oxidative stress. Despite a common metabolic reprogramming, the gene regulatory network programs initiated by these nuclear receptors were widely distinct. PPARG generated a rapid and short-term response while maintaining a gene activator role. By contrast, LXR signaling was prolonged, with initial, predominantly activating functions that transitioned to repressive gene regulatory activities at late time points. Through the use of a multi-tiered strategy that integrated various genomic datasets, our data illustrate that distinct gene regulatory programs elicit common phenotypic effects, highlighting the complexity of the genome. These results further provide a detailed molecular map of metabolic reprogramming in cancer cells through LXR and PPARG activation. As ligand-inducible TFs, these nuclear receptors can potentially serve as attractive therapeutic targets for the treatment of various cancers.

Mesenchymal Stem Cell-Derived Factors Restore Function to Human Frataxin-Deficient Cells.

PubMed

Kemp, Kevin; Dey, Rimi; Cook, Amelia; Scolding, Neil; Wilkins, Alastair

2017-08-01

Friedreich's ataxia is an inherited neurological disorder characterised by mitochondrial dysfunction and increased susceptibility to oxidative stress. At present, no therapy has been shown to reduce disease progression. Strategies being trialled to treat Friedreich's ataxia include drugs that improve mitochondrial function and reduce oxidative injury. In addition, stem cells have been investigated as a potential therapeutic approach. We have used siRNA-induced knockdown of frataxin in SH-SY5Y cells as an in vitro cellular model for Friedreich's ataxia. Knockdown of frataxin protein expression to levels detected in patients with the disorder was achieved, leading to decreased cellular viability, increased susceptibility to hydrogen peroxide-induced oxidative stress, dysregulation of key anti-oxidant molecules and deficiencies in both cell proliferation and differentiation. Bone marrow stem cells are being investigated extensively as potential treatments for a wide range of neurological disorders, including Friedreich's ataxia. The potential neuroprotective effects of bone marrow-derived mesenchymal stem cells were therefore studied using our frataxin-deficient cell model. Soluble factors secreted by mesenchymal stem cells protected against cellular changes induced by frataxin deficiency, leading to restoration in frataxin levels and anti-oxidant defences, improved survival against oxidative stress and stimulated both cell proliferation and differentiation down the Schwann cell lineage. The demonstration that mesenchymal stem cell-derived factors can restore cellular homeostasis and function to frataxin-deficient cells further suggests that they may have potential therapeutic benefits for patients with Friedreich's ataxia.

Transient inter-cellular polymeric linker.

PubMed

Ong, Siew-Min; He, Lijuan; Thuy Linh, Nguyen Thi; Tee, Yee-Han; Arooz, Talha; Tang, Guping; Tan, Choon-Hong; Yu, Hanry

2007-09-01

Three-dimensional (3D) tissue-engineered constructs with bio-mimicry cell-cell and cell-matrix interactions are useful in regenerative medicine. In cell-dense and matrix-poor tissues of the internal organs, cells support one another via cell-cell interactions, supplemented by small amount of the extra-cellular matrices (ECM) secreted by the cells. Here we connect HepG2 cells directly but transiently with inter-cellular polymeric linker to facilitate cell-cell interaction and aggregation. The linker consists of a non-toxic low molecular-weight polyethyleneimine (PEI) backbone conjugated with multiple hydrazide groups that can aggregate cells within 30 min by reacting with the aldehyde handles on the chemically modified cell-surface glycoproteins. The cells in the cellular aggregates proliferated; and maintained the cortical actin distribution of the 3D cell morphology while non-aggregated cells died over 7 days of suspension culture. The aggregates lost distinguishable cell-cell boundaries within 3 days; and the ECM fibers became visible around cells from day 3 onwards while the inter-cellular polymeric linker disappeared from the cell surfaces over time. The transient inter-cellular polymeric linker can be useful for forming 3D cellular and tissue constructs without bulk biomaterials or extensive network of engineered ECM for various applications.

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

Endele, Max; Etzrodt, Martin; Schroeder, Timm, E-mail: timm.schroeder@bsse.ethz.ch

Hematopoiesis is the cumulative consequence of finely tuned signaling pathways activated through extrinsic factors, such as local niche signals and systemic hematopoietic cytokines. Whether extrinsic factors actively instruct the lineage choice of hematopoietic stem and progenitor cells or are only selectively allowing survival and proliferation of already intrinsically lineage-committed cells has been debated over decades. Recent results demonstrated that cytokines can instruct lineage choice. However, the precise function of individual cytokine-triggered signaling molecules in inducing cellular events like proliferation, lineage choice, and differentiation remains largely elusive. Signal transduction pathways activated by different cytokine receptors are highly overlapping, but support themore » production of distinct hematopoietic lineages. Cellular context, signaling dynamics, and the crosstalk of different signaling pathways determine the cellular response of a given extrinsic signal. New tools to manipulate and continuously quantify signaling events at the single cell level are therefore required to thoroughly interrogate how dynamic signaling networks yield a specific cellular response. - Highlights: • Recent studies provided definite proof for lineage-instructive action of cytokines. • Signaling pathways involved in hematopoietic lineage instruction remain elusive. • New tools are emerging to quantitatively study dynamic signaling networks over time.« less

Cytoskeleton-interacting LIM-domain protein CRP1 suppresses cell proliferation and protects from stress-induced cell death

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

Latonen, Leena; Jaervinen, Paeivi M.; Haartman Institute, University of Helsinki, FIN-00014 Helsinki

2008-02-15

Members of the cysteine-rich protein (CRP) family are actin cytoskeleton-interacting LIM-domain proteins known to act in muscle cell differentiation. We have earlier found that CRP1, a founding member of this family, is transcriptionally induced by UV radiation in human diploid fibroblasts [M. Gentile, L. Latonen, M. Laiho, Cell cycle arrest and apoptosis provoked by UV radiation-induced DNA damage are transcriptionally highly divergent responses, Nucleic Acids Res. 31 (2003) 4779-4790]. Here we show that CRP1 is induced by growth-inhibitory signals, such as increased cellular density, and cytotoxic stress induced by UV radiation or staurosporine. We found that high levels of CRP1more » correlate with differentiation-associated morphology towards the myofibroblast lineage and that expression of ectopic CRP1 suppresses cell proliferation. Following UV- and staurosporine-induced stresses, expression of CRP1 provides a survival advantage evidenced by decreased cellular death and increased cellular metabolic activity and attachment. Our studies identify that CRP1 is a novel stress response factor, and provide evidence for its growth-inhibitory and cytoprotective functions.« less

Chemosensitizing effects of carbon-based nanomaterials in cancer cells: enhanced apoptosis and inhibition of proliferation as underlying mechanisms

NASA Astrophysics Data System (ADS)

Erdmann, Kati; Ringel, Jessica; Hampel, Silke; Rieger, Christiane; Huebner, Doreen; Wirth, Manfred P.; Fuessel, Susanne

2014-10-01

Recent studies have shown that carbon nanomaterials such as carbon nanofibres (CNFs) and multi-walled carbon nanotubes (CNTs) can exert antitumor activities themselves and sensitize cancer cells to conventional chemotherapeutics such as carboplatin and cisplatin. In the present study, the chemosensitizing effect of CNFs and CNTs on cancer cells of urological origin was investigated regarding the underlying mechanisms. Prostate cancer (DU-145, PC-3) and bladder cancer (EJ28) cells were treated with carbon nanomaterials (CNFs, CNTs) and chemotherapeutics (carboplatin, cisplatin) alone as well as in combination for 24 h. Forty-eight (EJ28) or 72 h (DU-145, PC-3) after the end of treatment the effects on cellular proliferation, clonogenic survival, cell death rate and cell cycle distribution were evaluated. Depending on the cell line, simultaneous administration of chemotherapeutics and carbon nanomaterials produced an additional inhibition of cellular proliferation and clonogenic survival of up to 77% and 98%, respectively, compared to the inhibitory effects of the chemotherapeutics alone. These strongly enhanced antiproliferative effects were accompanied by an elevated cell death rate, which was predominantly mediated via apoptosis and not by necrosis. The antitumor effects of combinations with CNTs were less pronounced than those with CNFs. The enhanced effects of the combinatory treatments on cellular function were mostly of additive to partly synergistic nature. Furthermore, cell cycle analysis demonstrated an arrest at the G2/M phase mediated by a monotreatment with chemotherapeutics. Following combinatory treatments, mostly less than or nearly additive increases of cell fractions in the G2/M phase could be observed. In conclusion, the pronounced chemosensitizing effects of CNFs and CNTs were mediated by an enhanced apoptosis and inhibition of proliferation. The combination of carbon-based nanomaterials and conventional chemotherapeutics represents a novel approach in cancer therapy to bypass chemoresistance by minimizing the chemotherapeutic dosing.

Genome-wide differential gene expression in immortalized DF-1 chicken embryo fibroblast cell line

PubMed Central

2011-01-01

Background When compared to primary chicken embryo fibroblast (CEF) cells, the immortal DF-1 CEF line exhibits enhanced growth rates and susceptibility to oxidative stress. Although genes responsible for cell cycle regulation and antioxidant functions have been identified, the genome-wide transcription profile of immortal DF-1 CEF cells has not been previously reported. Global gene expression in primary CEF and DF-1 cells was performed using a 4X44K chicken oligo microarray. Results A total of 3876 differentially expressed genes were identified with a 2 fold level cutoff that included 1706 up-regulated and 2170 down-regulated genes in DF-1 cells. Network and functional analyses using Ingenuity Pathways Analysis (IPA, Ingenuity® Systems, http://www.ingenuity.com) revealed that 902 of 3876 differentially expressed genes were classified into a number of functional groups including cellular growth and proliferation, cell cycle, cellular movement, cancer, genetic disorders, and cell death. Also, the top 5 gene networks with intermolecular connections were identified. Bioinformatic analyses suggested that DF-1 cells were characterized by enhanced molecular mechanisms for cell cycle progression and proliferation, suppressing cell death pathways, altered cellular morphogenesis, and accelerated capacity for molecule transport. Key molecules for these functions include E2F1, BRCA1, SRC, CASP3, and the peroxidases. Conclusions The global gene expression profiles provide insight into the cellular mechanisms that regulate the unique characteristics observed in immortal DF-1 CEF cells. PMID:22111699

Regulation of cellular growth by the Drosophila target of rapamycin dTOR

PubMed Central

Zhang, Hongbing; Stallock, James P.; Ng, Joyce C.; Reinhard, Christoph; Neufeld, Thomas P.

2000-01-01

The TOR protein kinases (TOR1 and TOR2 in yeast; mTOR/FRAP/RAFT1 in mammals) promote cellular proliferation in response to nutrients and growth factors, but their role in development is poorly understood. Here, we show that the Drosophila TOR homolog dTOR is required cell autonomously for normal growth and proliferation during larval development, and for increases in cellular growth caused by activation of the phosphoinositide 3-kinase (PI3K) signaling pathway. As in mammalian cells, the kinase activity of dTOR is required for growth factor-dependent phosphorylation of p70 S6 kinase (p70S6K) in vitro, and we demonstrate that overexpression of p70S6K in vivo can rescue dTOR mutant animals to viability. Loss of dTOR also results in cellular phenotypes characteristic of amino acid deprivation, including reduced nucleolar size, lipid vesicle aggregation in the larval fat body, and a cell type-specific pattern of cell cycle arrest that can be bypassed by overexpression of the S-phase regulator cyclin E. Our results suggest that dTOR regulates growth during animal development by coupling growth factor signaling to nutrient availability. PMID:11069888

Subtotal Ablation of Parietal Epithelial Cells Induces Crescent Formation

PubMed Central

Sicking, Eva-Maria; Fuss, Astrid; Uhlig, Sandra; Jirak, Peggy; Dijkman, Henry; Wetzels, Jack; Engel, Daniel R.; Urzynicok, Torsten; Heidenreich, Stefan; Kriz, Wilhelm; Kurts, Christian; Ostendorf, Tammo; Floege, Jürgen; Smeets, Bart

2012-01-01

Parietal epithelial cells (PECs) of the renal glomerulus contribute to the formation of both cellular crescents in rapidly progressive GN and sclerotic lesions in FSGS. Subtotal transgenic ablation of podocytes induces FSGS but the effect of specific ablation of PECs is unknown. Here, we established an inducible transgenic mouse to allow subtotal ablation of PECs. Proteinuria developed during doxycycline-induced cellular ablation but fully reversed 26 days after termination of doxycycline administration. The ablation of PECs was focal, with only 30% of glomeruli exhibiting histologic changes; however, the number of PECs was reduced up to 90% within affected glomeruli. Ultrastructural analysis revealed disruption of PEC plasma membranes with cytoplasm shedding into Bowman’s space. Podocytes showed focal foot process effacement, which was the most likely cause for transient proteinuria. After >9 days of cellular ablation, the remaining PECs formed cellular extensions to cover the denuded Bowman’s capsule and expressed the activation marker CD44 de novo. The induced proliferation of PECs persisted throughout the observation period, resulting in the formation of typical cellular crescents with periglomerular infiltrate, albeit without accompanying proteinuria. In summary, subtotal ablation of PECs leads the remaining PECs to react with cellular activation and proliferation, which ultimately forms cellular crescents. PMID:22282596

Assessment of the potential activity of major dietary compounds as selective estrogen receptor modulators in two distinct cell models for proliferation and differentiation

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

Lecomte, Sylvain; Lelong, Marie; Bourgine, Gaëlle

Estrogen receptors (ERs) α and β are distributed in most tissues of women and men. ERs are bound by estradiol (E2), a natural hormone, and mediate the pleiotropic and tissue-specific effects of E2, such as proliferation of breast epithelial cells or protection and differentiation of neuronal cells. Numerous environmental molecules, called endocrine disrupting compounds, also interact with ERs. Phytoestrogens belong to this large family and are considered potent therapeutic molecules that act through their selective estrogen receptor modulator (SERM) activity. Using breast cancer cell lines as a model of estrogen-dependent proliferation and a stably ER-expressing PC12 cell line as amore » model of neuronal differentiating cells, we studied the SERM activity of major dietary compounds, such as apigenin, liquiritigenin, daidzein, genistein, coumestrol, resveratrol and zearalenone. The ability of these compounds to induce ER-transactivation and breast cancer cell proliferation and enhance Nerve Growth Factor (NGF) -induced neuritogenesis was assessed. Surprisingly, although all compounds were able to activate the ER through an estrogen responsive element reporter gene, they showed differential activity toward proliferation or differentiation. Apigenin and resveratrol showed a partial or no proliferative effect on breast cancer cells but fully contributed to the neuritogenesis effect of NGF. However, daidzein and zearalenone showed full effects on cellular proliferation but did not induce cellular differentiation. In summary, our results suggest that the therapeutic potential of phytoestrogens can diverge depending on the molecule and the phenotype considered. Hence, apigenin and resveratrol might be used in the development of therapeutics for breast cancer and brain diseases. - Highlights: • SERM activity of dietary compounds on proliferation and differentiation is studied. • All the dietary compounds tested transactivate estrogen receptors. • Apigenin and resveratrol could be good candidates for future therapeutics. • Daidzein and zearalenone are to be avoided to maintain human health.« less

Production and characterization of multiple-layered populations of animal cells.

PubMed

Kruse, P F; Miedema, E

1965-11-01

Dense populations containing 129 x 10(6) Jensen sarcoma, 134 x 10(6) DON Chinese hamster, 28.9 x 10(6) WI-38 human diploid, 61.8 x 10(6) HEp-2 human carcinoma, and 67.4 x 10(6) WISH human amnion cells were produced from dilute inocula, 0.85 to 5.33 x 10(6), in 7 to 8 days in a perfusion system using replicate T-60 flasks. Perfusion rates as high as 560 ml medium/day/T-60 were required to maintain pH (to ca +/-0.1 unit) and adequate nutrient supplies. The cell densities encountered are described by the term "monolayer equivalents" (M.E.), defined as number of cells per culture divided by number of cells in a monolayer. The M.E.'s for T-60 cultures containing unusually dense populations of 40 x 10(6) WI-38 and 250 x 10(6) DON cells (9-day perfusion) were 5 and 17, respectively, and numbers of cells in illustrations of stained cross-sections of membranes from these cultures were in excellent agreement. Threshold M.E.'s exist below which proliferation is the chief cellular activity and above which one or more cell functions may predominate even though proliferation persists. Cellular nutrition and metabolism may change with changes in M.E., as illustrated in different patterns of glutamic acid, proline, and glycine utilization or production in dense vs. dilute WI-38 cell populations. The results indicated that the role of contact inhibition phenomena in arresting cellular proliferation was diminished in perfusion system environments.

Mechanisms and kinetics of proliferation and fibrosis development in a mouse model of thyrocyte hyperplasia.

PubMed

Ciornei, Radu Tudor; Hong, So-Hee; Fang, Yujiang; Zhu, Ziwen; Braley-Mullen, Helen

2016-01-01

IFN-γ(-/-) NOD.H-2h4 mice develop autoimmune disease with extensive hyperplasia and proliferation of thyroid epithelial cells (TEC H/P) and fibrosis. Splenic T cells from donors with severe TEC H/P transfer TEC H/P to SCID recipients. The goal of this study was to determine what factors control TEC H/P development/progression by examining T cells, markers of apoptosis, senescence and proliferation in thyroids of SCID recipients over time. At 28days, T cell infiltration was maximal, thyrocytes were proliferating, and fibrosis was moderate. At days 60 and 90, thyroids were larger with more fibrosis. T cells, cytokines and thyrocyte proliferation decreased, and cell cycle inhibitor proteins, and anti-apoptotic molecules increased. T cells and thyrocytes had foci of phosphorylated histone protein H2A.X, indicative of cellular senescence, when TEC H/P progressed and thyrocyte proliferation declined. Some thyrocytes were regenerating at day 90, with irregularly shaped empty follicles and ciliated epithelium. Proliferating thyrocytes were thyroid transcription factor (TTF1)-positive, suggesting they derived from epithelial cells and not brachial cleft remnants. Copyright © 2016 Elsevier Inc. All rights reserved.

CD1d-dependent expansion of NKT follicular helper cells in vivo and in vitro is a product of cellular proliferation and differentiation.

PubMed

Rampuria, Pragya; Lang, Mark L

2015-05-01

NKT follicular helper cells (NKTfh cells) are a recently discovered functional subset of CD1d-restricted NKT cells. Given the potential for NKTfh cells to promote specific antibody responses and germinal center reactions, there is much interest in determining the conditions under which NKTfh cells proliferate and/or differentiate in vivo and in vitro. We confirm that NKTfh cells expressing the canonical semi-invariant Vα14 TCR were CXCR5(+)/ICOS(+)/PD-1(+)/Bcl6(+) and increased in number following administration of the CD1d-binding glycolipid α-galactosylceramide (α-GC) to C57Bl/6 mice. We show that the α-GC-stimulated increase in NKTfh cells was CD1d-dependent since the effect was diminished by reduced CD1d expression. In vivo and in vitro treatment with α-GC, singly or in combination with IL-2, showed that NKTfh cells increased in number to a greater extent than total NKT cells, but proliferation was near-identical in both populations. Acquisition of the NKTfh phenotype from an adoptively transferred PD-1-depleted cell population was also evident, showing that peripheral NKT cells differentiated into NKTfh cells. Therefore, the α-GC-stimulated, CD1d-dependent increase in peripheral NKTfh cells is a result of cellular proliferation and differentiation. These findings advance our understanding of the immune response following immunization with CD1d-binding glycolipids. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cytotoxicity of citral against melanoma cells: The involvement of oxidative stress generation and cell growth protein reduction.

PubMed

Sanches, Larissa Juliani; Marinello, Poliana Camila; Panis, Carolina; Fagundes, Tatiane Renata; Morgado-Díaz, José Andrés; de-Freitas-Junior, Julio Cesar Madureira; Cecchini, Rubens; Cecchini, Alessandra Lourenço; Luiz, Rodrigo Cabral

2017-03-01

Citral is a natural compound that has shown cytotoxic and antiproliferative effects on breast and hematopoietic cancer cells; however, there are few studies on melanoma cells. Oxidative stress is known to be involved in all stages of melanoma development and is able to modulate intracellular pathways related to cellular proliferation and death. In this study, we hypothesize that citral exerts its cytotoxic effect on melanoma cells by the modulation of cellular oxidative status and/or intracellular signaling. To test this hypothesis, we investigated the antiproliferative and cytotoxic effects of citral on B16F10 murine melanoma cells evaluating its effects on cellular oxidative stress, DNA damage, cell death, and important signaling pathways, as these pathways, namely, extracellular signal-regulated kinases 1/2 (ERK1/2), AKT, and phosphatidylinositol-3 kinase, are involved in cell proliferation and differentiation. The p53 and nuclear factor kappa B were also investigated due to their ability to respond to intracellular stress. We observed that citral exerted antiproliferative and cytotoxic effects in B16F10; induced oxidative stress, DNA lesions, and p53 nuclear translocation; and reduced nitric oxide levels and nuclear factor kappa B, ERK1/2, and AKT. To investigate citral specificity, we used non-neoplastic human and murine cells, HaCaT (human skin keratinocytes) and NIH-3T3 cells (murine fibroblasts), and observed that although citral effects were not specific for cancer cells, non-neoplastic cells were more resistant to citral than B16F10. These findings highlight the potential clinical utility of citral in melanoma, with a mechanism of action involving the oxidative stress generation, nitric oxide depletion, and interference in signaling pathways related to cell proliferation.

In vitro proliferation and osteogenic differentiation of mesenchymal stem cells on nanoporous alumina

PubMed Central

Song, Yuanhui; Ju, Yang; Song, Guanbin; Morita, Yasuyuki

2013-01-01

Cell adhesion, migration, and proliferation are significantly affected by the surface topography of the substrates on which the cells are cultured. Alumina is one of the most popular implant materials used in orthopedics, but few data are available concerning the cellular responses of mesenchymal stem cells (MSCs) grown on nanoporous structures. MSCs were cultured on smooth alumina substrates and nanoporous alumina substrates to investigate the interaction between surface topographies of nanoporous alumina and cellular behavior. Nanoporous alumina substrates with pore sizes of 20 nm and 100 nm were used to evaluate the effect of pore size on MSCs as measured by proliferation, morphology, expression of integrin β1, and osteogenic differentiation. An MTT assay was used to measure cell viability of MSCs on different substrates, and determined that cell viability decreased with increasing pore size. Scanning electron microscopy was used to investigate the effect of pore size on cell morphology. Extremely elongated cells and prominent cell membrane protrusions were observed in cells cultured on alumina with the larger pore size. The expression of integrin β1 was enhanced in MSCs cultured on porous alumina, revealing that porous alumina substrates were more favorable for cell growth than smooth alumina substrates. Higher levels of osteoblastic differentiation markers such as alkaline phosphatase, osteocalcin, and mineralization were detected in cells cultured on alumina with 100 nm pores compared with cells cultured on alumina with either 20 nm pores or smooth alumina. This work demonstrates that cellular behavior is affected by variation in pore size, providing new insight into the potential application of this novel biocompatible material for the developing field of tissue engineering. PMID:23935364

Thioredoxin (Trxo1) interacts with proliferating cell nuclear antigen (PCNA) and its overexpression affects the growth of tobacco cell culture.

PubMed

Calderón, Aingeru; Ortiz-Espín, Ana; Iglesias-Fernández, Raquel; Carbonero, Pilar; Pallardó, Federico Vicente; Sevilla, Francisca; Jiménez, Ana

2017-04-01

Thioredoxins (Trxs), key components of cellular redox regulation, act by controlling the redox status of many target proteins, and have been shown to play an essential role in cell survival and growth. The presence of a Trx system in the nucleus has received little attention in plants, and the nuclear targets of plant Trxs have not been conclusively identified. Thus, very little is known about the function of Trxs in this cellular compartment. Previously, we studied the intracellular localization of PsTrxo1 and confirmed its presence in mitochondria and, interestingly, in the nucleus under standard growth conditions. In investigating the nuclear function of PsTrxo1 we identified proliferating cellular nuclear antigen (PCNA) as a PsTrxo1 target by means of affinity chromatography techniques using purified nuclei from pea leaves. Such protein-protein interaction was corroborated by dot-blot and bimolecular fluorescence complementation (BiFC) assays, which showed that both proteins interact in the nucleus. Moreover, PsTrxo1 showed disulfide reductase activity on previously oxidized recombinant PCNA protein. In parallel, we studied the effects of PsTrxo1 overexpression on Tobacco Bright Yellow-2 (TBY-2) cell cultures. Microscopy and flow-cytometry analysis showed that PsTrxo1 overexpression increases the rate of cell proliferation in the transformed lines, with a higher percentage of the S phase of the cell cycle at the beginning of the cell culture (days 1 and 3) and at the G2/M phase after longer times of culture (day 9), coinciding with an upregulation of PCNA protein. Furthermore, in PsTrxo1 overexpressed cells there is a decrease in the total cellular glutathione content but maintained nuclear GSH accumulation, especially at the end of the culture, which is accompanied by a higher mitotic index, unlike non-overexpressing cells. These results suggest that Trxo1 is involved in the cell cycle progression of TBY-2 cultures, possibly through its link with cellular PCNA and glutathione. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Seed Germination and Seedling Growth under Simulated Microgravity Causes Alterations in Plant Cell Proliferation and Ribosome Biogenesis

NASA Astrophysics Data System (ADS)

Matía, Isabel; van Loon, Jack W. A.; Carnero-Díaz, Eugénie; Marco, Roberto; Medina, Francisco Javier

2009-01-01

The study of the modifications induced by altered gravity in functions of plant cells is a valuable tool for the objective of the survival of terrestrial organisms in conditions different from those of the Earth. We have used the system "cell proliferation-ribosome biogenesis", two inter-related essential cellular processes, with the purpose of studying these modifications. Arabidopsis seedlings belonging to a transformed line containing the reporter gene GUS under the control of the promoter of the cyclin gene CYCB1, a cell cycle regulator, were grown in a Random Positioning Machine, a device known to accurately simulate microgravity. Samples were taken at 2, 4 and 8 days after germination and subjected to biometrical analysis and cellular morphometrical, ultrastructural and immunocytochemical studies in order to know the rates of cell proliferation and ribosome biogenesis, plus the estimation of the expression of the cyclin gene, as an indication of the state of cell cycle regulation. Our results show that cells divide more in simulated microgravity in a Random Positioning Machine than in control gravity, but the cell cycle appears significantly altered as early as 2 days after germination. Furthermore, higher proliferation is not accompanied by an increase in ribosome synthesis, as is the rule on Earth, but the functional markers of this process appear depleted in simulated microgravity-grown samples. Therefore, the alteration of the gravitational environmental conditions results in a considerable stress for plant cells, including those not specialized in gravity perception.

Calcium signaling and cell proliferation.

PubMed

Pinto, Mauro Cunha Xavier; Kihara, Alexandre Hiroaki; Goulart, Vânia A M; Tonelli, Fernanda M P; Gomes, Katia N; Ulrich, Henning; Resende, Rodrigo R

2015-11-01

Cell proliferation is orchestrated through diverse proteins related to calcium (Ca(2+)) signaling inside the cell. Cellular Ca(2+) influx that occurs first by various mechanisms at the plasma membrane, is then followed by absorption of Ca(2+) ions by mitochondria and endoplasmic reticulum, and, finally, there is a connection of calcium stores to the nucleus. Experimental evidence indicates that the fluctuation of Ca(2+) from the endoplasmic reticulum provides a pivotal and physiological role for cell proliferation. Ca(2+) depletion in the endoplasmatic reticulum triggers Ca(2+) influx across the plasma membrane in an phenomenon called store-operated calcium entries (SOCEs). SOCE is activated through a complex interplay between a Ca(2+) sensor, denominated STIM, localized in the endoplasmic reticulum and a Ca(2+) channel at the cell membrane, denominated Orai. The interplay between STIM and Orai proteins with cell membrane receptors and their role in cell proliferation is discussed in this review. Copyright © 2015 Elsevier Inc. All rights reserved.

Butyrate Inhibits Cancerous HCT116 Colon Cell Proliferation but to a Lesser Extent in Noncancerous NCM460 Colon Cells.

PubMed

Zeng, Huawei; Taussig, David P; Cheng, Wen-Hsing; Johnson, LuAnn K; Hakkak, Reza

2017-01-01

Butyrate, an intestinal microbiota metabolite of dietary fiber, exhibits chemoprevention effects on colon cancer development. However, the mechanistic action of butyrate remains to be determined. We hypothesize that butyrate inhibits cancerous cell proliferation but to a lesser extent in noncancerous cells through regulating apoptosis and cellular-signaling pathways. We tested this hypothesis by exposing cancerous HCT116 or non-cancerous NCM460 colon cells to physiologically relevant doses of butyrate. Cellular responses to butyrate were characterized by Western analysis, fluorescent microscopy, acetylation, and DNA fragmentation analyses. Butyrate inhibited cell proliferation, and led to an induction of apoptosis, genomic DNA fragmentation in HCT116 cells, but to a lesser extent in NCM460 cells. Although butyrate increased H3 histone deacetylation and p21 tumor suppressor expression in both cell types, p21 protein level was greater with intense expression around the nuclei in HCT116 cells when compared with that in NCM460 cells. Furthermore, butyrate treatment increased the phosphorylation of extracellular-regulated kinase 1/2 (p-ERK1/2), a survival signal, in NCM460 cells while it decreased p-ERK1/2 in HCT116 cells. Taken together, the activation of survival signaling in NCM460 cells and apoptotic potential in HCT116 cells may confer the increased sensitivity of cancerous colon cells to butyrate in comparison with noncancerous colon cells.

Butyrate Inhibits Cancerous HCT116 Colon Cell Proliferation but to a Lesser Extent in Noncancerous NCM460 Colon Cells

PubMed Central

Zeng, Huawei; Taussig, David P.; Cheng, Wen-Hsing; Johnson, LuAnn K.; Hakkak, Reza

2017-01-01

Butyrate, an intestinal microbiota metabolite of dietary fiber, exhibits chemoprevention effects on colon cancer development. However, the mechanistic action of butyrate remains to be determined. We hypothesize that butyrate inhibits cancerous cell proliferation but to a lesser extent in noncancerous cells through regulating apoptosis and cellular-signaling pathways. We tested this hypothesis by exposing cancerous HCT116 or non-cancerous NCM460 colon cells to physiologically relevant doses of butyrate. Cellular responses to butyrate were characterized by Western analysis, fluorescent microscopy, acetylation, and DNA fragmentation analyses. Butyrate inhibited cell proliferation, and led to an induction of apoptosis, genomic DNA fragmentation in HCT116 cells, but to a lesser extent in NCM460 cells. Although butyrate increased H3 histone deacetylation and p21 tumor suppressor expression in both cell types, p21 protein level was greater with intense expression around the nuclei in HCT116 cells when compared with that in NCM460 cells. Furthermore, butyrate treatment increased the phosphorylation of extracellular-regulated kinase 1/2 (p-ERK1/2), a survival signal, in NCM460 cells while it decreased p-ERK1/2 in HCT116 cells. Taken together, the activation of survival signaling in NCM460 cells and apoptotic potential in HCT116 cells may confer the increased sensitivity of cancerous colon cells to butyrate in comparison with noncancerous colon cells. PMID:28045428

Carcinogenesis explained within the context of a theory of organisms.

PubMed

Sonnenschein, Carlos; Soto, Ana M

2016-10-01

For a century, the somatic mutation theory (SMT) has been the prevalent theory to explain carcinogenesis. According to the SMT, cancer is a cellular problem, and thus, the level of organization where it should be studied is the cellular level. Additionally, the SMT proposes that cancer is a problem of the control of cell proliferation and assumes that proliferative quiescence is the default state of cells in metazoa. In 1999, a competing theory, the tissue organization field theory (TOFT), was proposed. In contraposition to the SMT, the TOFT posits that cancer is a tissue-based disease whereby carcinogens (directly) and mutations in the germ-line (indirectly) alter the normal interactions between the diverse components of an organ, such as the stroma and its adjacent epithelium. The TOFT explicitly acknowledges that the default state of all cells is proliferation with variation and motility. When taking into consideration the principle of organization, we posit that carcinogenesis can be explained as a relational problem whereby release of the constraints created by cell interactions and the physical forces generated by cellular agency lead cells within a tissue to regain their default state of proliferation with variation and motility. Within this perspective, what matters both in morphogenesis and carcinogenesis is not only molecules, but also biophysical forces generated by cells and tissues. Herein, we describe how the principles for a theory of organisms apply to the TOFT and thus to the study of carcinogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Inhibition of B cell proliferation by antisense DNA to both alpha and beta forms of Fc epsilon R II.

PubMed

Bhatti, L; Behle, K; Stevens, R H

1992-10-01

Epstein-Barr Virus (EBV) infection activates B lymphocyte proliferation through partially understood mechanisms, resulting in phenotypic changes, including the appearance of new antigens. One such antigen is Fc epsilon R II/CD-23 which may be relevant for B cell proliferation. We have used anti-sense oligonucleotides to study the importance of the two forms of this molecule for proliferation in the EBV-transformed, Fc epsilon R II +ve lymphoblastoid B cell line, RPMI 8866. Anti-sense oligodeoxynucleotides were generated to the two forms of Fc epsilon R II; Fc epsilon R IIa (alpha) and IIb (beta) which differ only in their intracytoplasmic domains. Addition of increasing concentrations of anti-sense oligonucleotides, ranging from 1 to 30 microM, significantly decreased cellular proliferation as measured by the incorporation of [3H]thymidine (inhibition range 8-88%). Optimum inhibition of cellular proliferation was apparent at 15 microM concentration of both anti-sense Fc epsilon R IIa and IIb (Fc epsilon R IIa, mean +/- SE = 75 +/- 7% inhibition, p less than 0.001; Fc epsilon R IIb, mean +/- SE = 71 +/- 7% inhibition, p less than 0.001). Anti-sense oligonucleotides complementary to the common part of Fc epsilon R II resulted in a similar inhibition of proliferation. Sense oligonucleotides did not induce significant inhibition. Preincubation of sense and anti-sense oligonucleotides resulted in an abrogation of proliferation inhibition. Moreover, none of these oligonucleotides had any effect on a Fc epsilon R II -ve cell line. Incubation with both anti-sense IIa and IIb resulted in additive, but not synergistic inhibition of proliferation. Addition of soluble Fc epsilon R II did not reverse inhibition of proliferation, suggesting that membrane-bound or intracellular rather than soluble Fc epsilon R II was important for the induced proliferation. Analysis of cell surface expression for Fc epsilon II indicated that while there was a pronounced effect on cell number following incubation with anti-sense oligonucleotides, surface expression of Fc epsilon R II was consistent as measured over different time points. PCR analysis revealed that while most cells expressed either the alpha or the beta form of Fc epsilon R II, EBV-transformed cell lines, particularly RPMI 8866, were found to express both alpha and beta forms simultaneously. This may constitute a mechanism whereby EBV infection confers an immortal state to the cell, resulting in its uncontrolled proliferation. Cell lines expressing only one receptor form, either alpha or beta, were unaffected after incubation with anti-sense oligonucleotides.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhancing the Biological Relevance of Secretome-Based Proteomics by Linking Tumor Cell Proliferation and Protein Secretion.

PubMed

Gregori, Josep; Méndez, Olga; Katsila, Theodora; Pujals, Mireia; Salvans, Cándida; Villarreal, Laura; Arribas, Joaquin; Tabernero, Josep; Sánchez, Alex; Villanueva, Josep

2014-07-15

Secretome profiling has become a methodology of choice for the identification of tumor biomarkers. We hypothesized that due to the dynamic nature of secretomes cellular perturbations could affect their composition but also change the global amount of protein secreted per cell. We confirmed our hypothesis by measuring the levels of secreted proteins taking into account the amount of proteome produced per cell. Then, we established a correlation between cell proliferation and protein secretion that explained the observed changes in global protein secretion. Next, we implemented a normalization correcting the statistical results of secretome studies by the global protein secretion of cells into a generalized linear model (GLM). The application of the normalization to two biological perturbations on tumor cells resulted in drastic changes in the list of statistically significant proteins. Furthermore, we found that known epithelial-to-mesenchymal transition (EMT) effectors were only statistically significant when the normalization was applied. Therefore, the normalization proposed here increases the sensitivity of statistical tests by increasing the number of true-positives. From an oncology perspective, the correlation between protein secretion and cellular proliferation suggests that slow-growing tumors could have high-protein secretion rates and consequently contribute strongly to tumor paracrine signaling.

Comparative study of Hippo pathway genes in cellular conveyor belts of a ctenophore and a cnidarian.

PubMed

Coste, Alicia; Jager, Muriel; Chambon, Jean-Philippe; Manuel, Michaël

2016-01-01

The Hippo pathway regulates growth rate and organ size in fly and mouse, notably through control of cell proliferation. Molecular interactions at the heart of this pathway are known to have originated in the unicellular ancestry of metazoans. They notably involve a cascade of phosphorylations triggered by the kinase Hippo, with subsequent nuclear to cytoplasmic shift of Yorkie localisation, preventing its binding to the transcription factor Scalloped, thereby silencing proliferation genes. There are few comparative expression data of Hippo pathway genes in non-model animal species and notably none in non-bilaterian phyla. All core Hippo pathway genes could be retrieved from the ctenophore Pleurobrachia pileus and the hydrozoan cnidarian Clytia hemisphaerica, with the important exception of Yorkie in ctenophore. Expression study of the Hippo, Salvador and Scalloped genes in tentacle "cellular conveyor belts" of these two organisms revealed striking differences. In P. pileus, their transcripts were detected in areas where undifferentiated progenitors intensely proliferate and where expression of cyclins B and D was also seen. In C. hemisphaerica, these three genes and Yorkie are expressed not only in the proliferating but also in the differentiation zone of the tentacle bulb and in mature tentacle cells. However, using an antibody designed against the C. hemiphaerica Yorkie protein, we show in two distinct cell lineages of the medusa that Yorkie localisation is predominantly nuclear in areas of active cell proliferation and mainly cytoplasmic elsewhere. This is the first evidence of nucleocytoplasmic Yorkie shift in association with the arrest of cell proliferation in a cnidarian, strongly evoking the cell division-promoting role of this protein and its inhibition by the activated Hippo pathway in bilaterian models. Our results furthermore highlight important differences in terms of deployment and regulation of Hippo pathway genes between cnidarians and ctenophores.

Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis

PubMed Central

Watanabe, Miki; Muraleedharan, Ranjithmenon; Lambert, Paul F.; Lane, Andrew N.; Romick-Rosendale, Lindsey E.; Wells, Susanne I.

2017-01-01

The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos). To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA) that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth. PMID:28558019

Overexpression of the human DEK oncogene reprograms cellular metabolism and promotes glycolysis.

PubMed

Matrka, Marie C; Watanabe, Miki; Muraleedharan, Ranjithmenon; Lambert, Paul F; Lane, Andrew N; Romick-Rosendale, Lindsey E; Wells, Susanne I

2017-01-01

The DEK oncogene is overexpressed in many human malignancies including at early tumor stages. Our reported in vitro and in vivo models of squamous cell carcinoma have demonstrated that DEK contributes functionally to cellular and tumor survival and to proliferation. However, the underlying molecular mechanisms remain poorly understood. Based on recent RNA sequencing experiments, DEK expression was necessary for the transcription of several metabolic enzymes involved in anabolic pathways. This identified a possible mechanism whereby DEK may drive cellular metabolism to enable cell proliferation. Functional metabolic Seahorse analysis demonstrated increased baseline and maximum extracellular acidification rates, a readout of glycolysis, in DEK-overexpressing keratinocytes and squamous cell carcinoma cells. DEK overexpression also increased the maximum rate of oxygen consumption and therefore increased the potential for oxidative phosphorylation (OxPhos). To detect small metabolites that participate in glycolysis and the tricarboxylic acid cycle (TCA) that supplies substrate for OxPhos, we carried out NMR-based metabolomics studies. We found that high levels of DEK significantly reprogrammed cellular metabolism and altered the abundances of amino acids, TCA cycle intermediates and the glycolytic end products lactate, alanine and NAD+. Taken together, these data support a scenario whereby overexpression of the human DEK oncogene reprograms keratinocyte metabolism to fulfill energy and macromolecule demands required to enable and sustain cancer cell growth.

Humoral and cellular responses to a non-adjuvanted monovalent H1N1 pandemic influenza vaccine in hospital employees

PubMed Central

2013-01-01

Background The efficacy of the H1N1 influenza vaccine relies on the induction of both humoral and cellular responses. This study evaluated the humoral and cellular responses to a monovalent non-adjuvanted pandemic influenza A/H1N1 vaccine in occupationally exposed subjects who were previously vaccinated with a seasonal vaccine. Methods Sixty healthy workers from a respiratory disease hospital were recruited. Sera and peripheral blood mononuclear cells (PBMCs) were obtained prior to and 1 month after vaccination with a non-adjuvanted monovalent 2009 H1N1 vaccine (Influenza A (H1N1) 2009 Monovalent Vaccine Panenza, Sanofi Pasteur). Antibody titers against the pandemic A/H1N1 influenza virus were measured via hemagglutination inhibition (HI) and microneutralization assays. Antibodies against the seasonal HA1 were assessed by ELISA. The frequency of IFN-γ-producing cells as well as CD4+ and CD8+ T cell proliferation specific to the pandemic virus A/H1N peptides, seasonal H1N1 peptides and seasonal H3N2 peptides were assessed using ELISPOT and flow cytometry. Results At baseline, 6.7% of the subjects had seroprotective antibody titers. The seroconversion rate was 48.3%, and the seroprotection rate was 66.7%. The geometric mean titers (GMTs) were significantly increased (from 6.8 to 64.9, p < 0.05). Forty-nine percent of the subjects had basal levels of specific IFN-γ-producing T cells to the pandemic A/H1N1 peptides that were unchanged post-vaccination. CD4+ T cell proliferation in response to specific pandemic A/H1N1 virus peptides was also unchanged; in contrast, the antigen-specific proliferation of CD8+ T cells significantly increased post-vaccination. Conclusion Our results indicate that a cellular immune response that is cross-reactive to pandemic influenza antigens may be present in populations exposed to the circulating seasonal influenza virus prior to pandemic or seasonal vaccination. Additionally, we found that the pandemic vaccine induced a significant increase in CD8+ T cell proliferation. PMID:24238117

Elevated level of human RPA interacting protein α (hRIPα) in cervical tumor cells is involved in cell proliferation through regulating RPA transport.

PubMed

Namkoong, Sim; Lee, Eun-Ju; Jang, Ik-Soon; Park, Junsoo

2012-10-19

Replication protein A (RPA) is a eukaryotic single-stranded DNA binding protein that is essential for DNA replication, repair, and recombination, and human RPA interacting protein α (hRIPα) is the nuclear transporter of RPA. Here, we report the regulatory role of hRIPα protein in cell proliferation. Western blot analysis revealed that the level of hRIPα was frequently elevated in cervical tumors tissues and hRIPα knockdown by siRNA inhibited cellular proliferation through deregulation of the cell cycle. In addition, overexpression of hRIPα resulted in increased clonogenicity. These results indicate that hRIPα is involved in cell proliferation through regulation of RPA transport. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Simultaneous Study of Mechanical Stretch-Induced Cell Proliferation and Apoptosis on C2C12 Myoblasts.

PubMed

Feng, Yu; Tian, Xiang-Yang; Sun, Peng; Cheng, Ze-Peng; Shi, Reng-Fei

2018-06-27

Mechanical stretch may cause myoblasts to either proliferate or undergo apoptosis. Identifying the molecular events that switch the fate of a stretched cell from proliferation to apoptosis is practically important in the field of regenerative medicine. A recent study on vascular smooth muscle cells illustrated that identification of these events may be achieved by addressing the stretch-induced opposite cellular outcomes simultaneously within a single investigation. To define conditions or a model in which both proliferation and apoptosis can be studied at the same time, we exposed in vitro cultured C2C12 myoblasts to a cyclic mechanical stretch regimen of 15% elongation at a stretching frequency of 1 Hz for 0, 2, 4, 6, or 8 h every day, consecutively, for 3 days. Both proliferation and apoptosis were observed. Moreover, as the duration of the stretch was prolonged, cell proliferation increased until it peaked at the optimal stretching duration. Afterwards, apoptosis gradually prevailed. Therefore, we established a model in which stretch-induced cell proliferation and apoptosis can be studied simultaneously. © 2018 S. Karger AG, Basel.

Proliferation and morphological transformation of RMK cells exposed to hydroquinine containing ionomers.

PubMed

Harvey, Veronica; Benghuzzi, Hamed; Tucci, Michell; Puckett, Aaron; Cason, Zelma

2002-01-01

Recent research in our laboratories has been directed towards the development of ionomeric polymers and monomers for use in biomedical applications such as adhesives, drug delivery matrices and tissue scaffolds. The chemical Hydroquinone (HQ) aids as a stabilizer and represents a major component in the development of the ionomers. However, hydroquinone in high concentration has the potential to initiate carcinogenic effects on cells. The curing reactions are based on free radical chemistry that require a radical scavenger, ascorbic acid (Asc) to adjust working and setting times and shelf-life stability. The few studies published on HQ have suggested that high dosages of HQ may stimulate apoptosis as well as an increased cellular leakage, however the effect of HQ on the biocompatability is unknown. Therefore the objectives of this study were to measure the functional capacity, cell proliferation and structural integrity of Rhesus monkey kidney epithelial (RMK) cells exposed to ionomer formulations containing 4 different levels of HQ. A total of 90 tubes of RMK (40,000 cells per tube) cells were divided equally into five equal groups. Group I served as a control and group II-V were subjected to ionomers containing 0, 500, 1000, and 2000 ppm HQ. Cell numbers, morphology, cellular and supermatant MDA levels, and total protein analysis were performed. The results suggest: (I) All ionomer groups increased cellular proliferation except for the 2000 ppm HQ group, (II) MDA levels were increased in cells containing 2000 ppm HQ at 24 hours; and 0 ppm at 48 hours. It may be concluded that HQ concentrations over 1000 ppm may adversely affect biocompatability.

Elastic force restricts growth of the murine utricle

PubMed Central

Gnedeva, Ksenia; Jacobo, Adrian; Salvi, Joshua D; Petelski, Aleksandra A; Hudspeth, A J

2017-01-01

Dysfunctions of hearing and balance are often irreversible in mammals owing to the inability of cells in the inner ear to proliferate and replace lost sensory receptors. To determine the molecular basis of this deficiency we have investigated the dynamics of growth and cellular proliferation in a murine vestibular organ, the utricle. Based on this analysis, we have created a theoretical model that captures the key features of the organ’s morphogenesis. Our experimental data and model demonstrate that an elastic force opposes growth of the utricular sensory epithelium during development, confines cellular proliferation to the organ’s periphery, and eventually arrests its growth. We find that an increase in cellular density and the subsequent degradation of the transcriptional cofactor Yap underlie this process. A reduction in mechanical constraints results in accumulation and nuclear translocation of Yap, which triggers proliferation and restores the utricle’s growth; interfering with Yap’s activity reverses this effect. DOI: http://dx.doi.org/10.7554/eLife.25681.001 PMID:28742024

Vitamin K3 suppressed inflammatory and immune responses in a redox-dependent manner.

PubMed

Checker, Rahul; Sharma, Deepak; Sandur, Santosh K; Khan, Nazir M; Patwardhan, Raghavendra S; Kohli, Vineet; Sainis, Krishna B

2011-08-01

Recent investigations suggest that cellular redox status may play a key role in the regulation of several immune functions. Treatment of lymphocytes with vitamin K3 (menadione) resulted in a significant decrease in cellular GSH/GSSG ratio and concomitant increase in the ROS levels. It also suppressed Concanavalin A (Con A)-induced proliferation and cytokine production in lymphocytes and CD4 + T cells in vitro. Immunosuppressive effects of menadione were abrogated only by thiol containing antioxidants. Mass spectrometric analysis showed that menadione directly interacted with thiol antioxidant GSH. Menadione completely suppressed Con A-induced activation of ERK, JNK and NF-κB in lymphocytes. It also significantly decreased the homeostasis driven proliferation of syngeneic CD4 + T cells. Further, menadione significantly delayed graft-vs-host disease morbidity and mortality in mice. Menadione suppressed phytohemagglutinin-induced cytokine production in human peripheral blood mononuclear cells. These results reveal that cellular redox perturbation by menadione is responsible for significant suppression of lymphocyte responses.

Effect of borax on immune cell proliferation and sister chromatid exchange in human chromosomes

PubMed Central

Pongsavee, Malinee

2009-01-01

Background Borax is used as a food additive. It becomes toxic when accumulated in the body. It causes vomiting, fatigue and renal failure. Methods The heparinized blood samples from 40 healthy men were studied for the impact of borax toxicity on immune cell proliferation (lymphocyte proliferation) and sister chromatid exchange in human chromosomes. The MTT assay and Sister Chromatid Exchange (SCE) technic were used in this experiment with the borax concentrations of 0.1, 0.15, 0.2, 0.3 and 0.6 mg/ml. Results It showed that the immune cell proliferation (lymphocyte proliferation) was decreased when the concentrations of borax increased. The borax concentration of 0.6 mg/ml had the most effectiveness to the lymphocyte proliferation and had the highest cytotoxicity index (CI). The borax concentrations of 0.15, 0.2, 0.3 and 0.6 mg/ml significantly induced sister chromatid exchange in human chromosomes (P < 0.05). Conclusion Borax had effects on immune cell proliferation (lymphocyte proliferation) and induced sister chromatid exchange in human chromosomes. Toxicity of borax may lead to cellular toxicity and genetic defect in human. PMID:19878537

Effects of 5-fluorouracil in nuclear and cellular morphology, proliferation, cell cycle, apoptosis, cytoskeletal and caveolar distribution in primary cultures of smooth muscle cells.

PubMed

Filgueiras, Marcelo de Carvalho; Morrot, Alexandre; Soares, Pedro Marcos Gomes; Costa, Manoel Luis; Mermelstein, Cláudia

2013-01-01

Colon cancer is one of the most prevalent types of cancer in the world and is one of the leading causes of cancer death. The anti-metabolite 5- fluorouracil (5-FU) is widely used in the treatment of patients with colon cancer and other cancer types. 5-FU-based chemotherapy has been shown to be very efficient in the improvement of overall survival of the patients and for the eradication of the disease. Unfortunately, common side effects of 5-FU include severe alterations in the motility of the gastrointestinal tissues. Nevertheless, the molecular and cellular effects of 5-FU in smooth muscle cells are poorly understood. Primary smooth muscle cell cultures are an important tool for studies of the biological consequences of 5-FU at the cellular level. The avian gizzard is one of the most robust organs of smooth muscle cells. Here we studied the molecular and cellular effects of the chemotherapic drug 5-FU in a primary culture of chick gizzard smooth muscle cells. We found that treatment of smooth muscle cells with 5-FU inhibits cell proliferation by the arrest of cells in the G1 phase of cell cycle and induce apoptosis. 5-FU induced a decrease in the percentage of histone H3-positive cells. Treatment of cells with 5-FU induced changes in cellular and nuclear morphology, a decrease in the number of stress fibers and a major decrease in the number of caveolin-3 positive cells. Our results suggest that the disorganization of the actin cytoskeleton and the reduction of caveolin-3 expression could explain the alterations in contractility observed in patients treated with 5-FU. These findings might have an impact in the understanding of the cellular effects of 5-FU in smooth muscle tissues and might help the improvement of new therapeutic protocols for the treatment of colon cancer.

Effects of 5-Fluorouracil in Nuclear and Cellular Morphology, Proliferation, Cell Cycle, Apoptosis, Cytoskeletal and Caveolar Distribution in Primary Cultures of Smooth Muscle Cells

PubMed Central

Filgueiras, Marcelo de Carvalho; Morrot, Alexandre; Soares, Pedro Marcos Gomes; Costa, Manoel Luis; Mermelstein, Cláudia

2013-01-01

Colon cancer is one of the most prevalent types of cancer in the world and is one of the leading causes of cancer death. The anti-metabolite 5- fluorouracil (5-FU) is widely used in the treatment of patients with colon cancer and other cancer types. 5-FU-based chemotherapy has been shown to be very efficient in the improvement of overall survival of the patients and for the eradication of the disease. Unfortunately, common side effects of 5-FU include severe alterations in the motility of the gastrointestinal tissues. Nevertheless, the molecular and cellular effects of 5-FU in smooth muscle cells are poorly understood. Primary smooth muscle cell cultures are an important tool for studies of the biological consequences of 5-FU at the cellular level. The avian gizzard is one of the most robust organs of smooth muscle cells. Here we studied the molecular and cellular effects of the chemotherapic drug 5-FU in a primary culture of chick gizzard smooth muscle cells. We found that treatment of smooth muscle cells with 5-FU inhibits cell proliferation by the arrest of cells in the G1 phase of cell cycle and induce apoptosis. 5-FU induced a decrease in the percentage of histone H3-positive cells. Treatment of cells with 5-FU induced changes in cellular and nuclear morphology, a decrease in the number of stress fibers and a major decrease in the number of caveolin-3 positive cells. Our results suggest that the disorganization of the actin cytoskeleton and the reduction of caveolin-3 expression could explain the alterations in contractility observed in patients treated with 5-FU. These findings might have an impact in the understanding of the cellular effects of 5-FU in smooth muscle tissues and might help the improvement of new therapeutic protocols for the treatment of colon cancer. PMID:23646193

Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein.

PubMed

Oberst, Michael D; Augé, Catherine; Morris, Chad; Kentner, Stacy; Mulgrew, Kathy; McGlinchey, Kelly; Hair, James; Hanabuchi, Shino; Du, Qun; Damschroder, Melissa; Feng, Hui; Eck, Steven; Buss, Nicholas; de Haan, Lolke; Pierce, Andrew J; Park, Haesun; Sylwester, Andrew; Axthelm, Michael K; Picker, Louis; Morris, Nicholas P; Weinberg, Andrew; Hammond, Scott A

2018-05-01

Ligation of OX40 (CD134, TNFRSF4) on activated T cells by its natural ligand (OX40L, CD252, TNFSF4) enhances cellular survival, proliferation, and effector functions such as cytokine release and cellular cytotoxicity. We engineered a recombinant human OX40L IgG4P Fc fusion protein termed MEDI6383 that assembles into a hexameric structure and exerts potent agonist activity following engagement of OX40. MEDI6383 displayed solution-phase agonist activity that was enhanced when the fusion protein was clustered by Fc gamma receptors (FcγRs) on the surface of adjacent cells. The resulting costimulation of OX40 on T cells induced NFκB promoter activity in OX40-expressing T cells and induced Th1-type cytokine production, proliferation, and resistance to regulatory T cell (Treg)-mediated suppression. MEDI6383 enhanced the cytolytic activity of tumor-reactive T cells and reduced tumor growth in the context of an alloreactive human T cell:tumor cell admix model in immunocompromised mice. Consistent with the role of OX40 costimulation in the expansion of memory T cells, MEDI6383 administered to healthy nonhuman primates elicited peripheral blood CD4 and CD8 central and effector memory T-cell proliferation as well as B-cell proliferation. Together, these results suggest that OX40 agonism has the potential to enhance antitumor immunity in human malignancies. Mol Cancer Ther; 17(5); 1024-38. ©2018 AACR . ©2018 American Association for Cancer Research.

Analysis of the presence of cell proliferation-related molecules in the Tgf-β3 null mutant mouse palate reveals misexpression of EGF and Msx-1.

PubMed

del Río, A; Barrio, M C; Murillo, J; Maldonado, E; López-Gordillo, Y; Martínez-Sanz, E; Martínez, M L; Martínez-Álvarez, C

2011-01-01

The Tgf-β(3) null mutant mouse palate presents several cellular anomalies that lead to the appearance of cleft palate. One of them concerns the cell proliferation of both the palatal medial edge epithelium and mesenchyme. In this work, our aim was to determine whether there was any variation in the presence/distribution of several cell proliferation-related molecules that could be responsible for the cell proliferation defects observed in these palates. Our results showed no difference in the presence of EGF-R, PDGF-A, TGF-β(2), Bmp-2, and Bmp-4, and differences were minimal for FGF-10 and Shh. However, the expression of EGF and Msx-1 changed substantially. The shift of the EGF protein expression was the one that most correlated with that of cell proliferation. This molecule is regulated by TGF-β(3), and experiments blocking its activity in culture suggest that EGF misexpression in the Tgf-β(3) null mutant mouse palate plays a role in the cell proliferation defect observed. Copyright © 2010 S. Karger AG, Basel.

Human Herpesvirus-8-Transformed Endothelial Cells Have Functionally Activated Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor

PubMed Central

Masood, Rizwan; Cesarman, Ethel; Smith, D. Lynne; Gill, Parkash S.; Flore, Ornella

2002-01-01

Kaposi’s sarcoma is a vascular tumor commonly associated with human immunodeficiency virus (HIV)-1 and human herpesvirus (HHV-8) also known as Kaposi’s sarcoma-associated herpesvirus. The principal features of this tumor are abnormal proliferation of vascular structures lined with spindle-shaped endothelial cells. HHV-8 may transform a subpopulation of endothelial cells in vitro via viral and cellular gene expression. We hypothesized that among the cellular genes, vascular endothelial growth factors (VEGFs) and their cognate receptors may be involved in viral-mediated transformation. We have shown that HHV-8-transformed endothelial cells (EC-HHV-8) express higher levels of VEGF, VEGF-C, VEGF-D, and PlGF in addition to VEGF receptors-1, -2, and -3. Furthermore, antibodies to VEGF receptor-2 inhibited cell proliferation and viability. Similarly, inhibition of VEGF gene expression with antisense oligonucleotides inhibited EC-HHV-8 cell proliferation/viability. The growth and viability of primary endothelial cells and a fibroblast cell line however were unaffected by either the VEGF receptor-2 antibody or the VEGF antisense oligodeoxynucleotides. VEGF and VEGF receptors are thus induced in EC-HHV-8 and participate in the transformation. Inhibitors of VEGF may thus modulate the disease process during development and progression. PMID:11786394

Cellular viability and genetic expression of human gingival fibroblasts to zirconia with enamel matrix derivative (Emdogain®)

PubMed Central

Kwon, Yong-Dae; Choi, Hyun-jung; Lee, Heesu; Lee, Jung-Woo; Weber, Hans-Peter

2014-01-01

PURPOSE The objective of this study was to investigate the biologic effects of enamel matrix derivative (EMD) with different concentrations on cell viability and the genetic expression of human gingival fibroblasts (HGF) to zirconia surfaces. MATERIALS AND METHODS Immortalized human gingival fibroblasts (HGF) were cultured (1) without EMD, (2) with EMD 25 µg/mL, and (3) with EMD 100 µg/mL on zirconia discs. MTT assay was performed to evaluate the cell proliferation activity and SEM was carried out to examine the cellular morphology and attachment. The mRNA expression of collagen type I, osteopontin, fibronectin, and TGF-β1 was evaluated with the real-time polymerase chain reaction (RT-PCR). RESULTS From MTT assay, HGF showed more proliferation in EMD 25 µg/mL group than control and EMD 100 µg/mL group (P<.05). HGFs showed more flattened cellular morphology on the experimental groups than on the control group after 4h culture and more cellular attachments were observed on EMD 25 µg/mL group and EMD 100 µg/mL group after 24h culture. After 48h of culture, cellular attachment was similar in all groups. The mRNA expression of type I collagen increased in a concentration dependent manner. The genetic expression of osteopontin, fibronectin, and TGF-β1 was increased at EMD 100 µg/mL. However, the mRNA expression of proteins associated with cellular attachment was decreased at EMD 25 µg/mL. CONCLUSION Through this short term culture of HGF on zirconium discs, we conclude that EMD affects the proliferation, attachment, and cell morphology of HGF cells. Also, EMD stimulates production of extracellular matrix collagen, osteopontin, and TGF-β1 in high concentration levels. CLINICAL RELEVANCE With the use of EMD, protective barrier between attached gingiva and transmucosal zirconia abutment may be enhanced leading to final esthetic results with implants. PMID:25352963

Whole lesion histogram analysis of meningiomas derived from ADC values. Correlation with several cellularity parameters, proliferation index KI 67, nucleic content, and membrane permeability.

PubMed

Surov, Alexey; Hamerla, Gordian; Meyer, Hans Jonas; Winter, Karsten; Schob, Stefan; Fiedler, Eckhard

2018-09-01

To analyze several histopathological features and their possible correlations with whole lesion histogram analysis derived from ADC maps in meningioma. The retrospective study involved 36 patients with primary meningiomas. For every tumor, the following histogram analysis parameters of apparent diffusion coefficient (ADC) were calculated: ADC mean , ADC max , ADC min , ADC median , ADC mode , ADC percentiles: P10, P25, P75, P90, as well kurtosis, skewness, and entropy. All measures were performed by two radiologists. Proliferation index KI 67, minimal, maximal and mean cell count, total nucleic area, and expression of water channel aquaporin 4 (AQP4) were estimated. Spearman's correlation coefficient was used to analyze associations between investigated parameters. A perfect interobserver agreement for all ADC values (0.84-0.97) was identified. All ADC values correlated inversely with tumor cellularity with the strongest correlation between P10, P25 and mean cell count (-0.558). KI 67 correlated inversely with all ADC values except ADC min . ADC parameters did not correlate with total nucleic area. All ADC values correlated statistically significant with expression of AQP4. ADC histogram analysis is a valid method with an excellent interobserver agreement. Cellularity parameters and proliferation potential are associated with different ADC values. Membrane permeability may play a greater role for water diffusion than cell count and proliferation activity. Copyright © 2018 Elsevier Inc. All rights reserved.

HSF1 is activated as a consequence of lymphocyte activation and regulates a major proteostasis network in T cells critical for cell division during stress

PubMed Central

Gandhapudi, Siva K.; Murapa, Patience; Threlkeld, Zachary D.; Ward, Martin; Sarge, Kevin D.; Snow, Charles; Woodward, Jerold G.

2013-01-01

Heat Shock Transcription Factor 1 (HSF1) is a major transcriptional regulator of the heat shock response in eukaryotic cells. HSF1 is also evoked in response to a variety of cellular stressors including elevated temperatures, oxidative stress, and other proteotoxic stressors. Previously, we demonstrated that HSF1 is activated in naive T cells at fever range temperatures (39.5°C) and is critical for in vitro T cell proliferation at fever temperatures. In this study, we demonstrated thatmurine HSF1 became activated to the DNA-binding form and trans-activated a large number of genes in lymphoid cells strictly as a consequence of receptor activation in the absence of apparent cellular stress. Microarray analysis comparing HSF1+/+ and HSF1−/− gene expression in T cells activated at 37°C revealed a diverse set of 323 genes significantly regulated by HSF1 in non-stressed T cells. In vivo proliferation studies revealed a significant impairment of HSF1−/− T cell expansion under conditions mimicking a robust immune response (staphylococcal enterotoxin B induced T cell activation). This proliferation defect due to loss of HSF1 is observed even under non-febrile temperatures. HSF1−/− T cells activated at fever temperatures show a dramatic reduction in cyclin E and cyclin A proteins during the cell cycle, although the transcription of these genes was not affected. Finally, B cell, and hematopoietic stem cell proliferation from HSF1−/− mice, but not HSF1+/+ mice were also attenuated under stressful conditions, indicating that HSF1 is critical for the cell cycle progression of lymphoid cells activated under stressful conditions. PMID:24043900

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-10-01

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

Novel chlorinated dibenzofurans isolated from the cellular slime mold, Polysphondylium filamentosum, and their biological activities.

PubMed

Kikuchi, Haruhisa; Kubohara, Yuzuru; Nguyen, Van Hai; Katou, Yasuhiro; Oshima, Yoshiteru

2013-08-01

Cellular slime molds are expected to have the huge potential for producing secondary metabolites including polyketides, and we have studied the diversity of secondary metabolites of cellular slime molds for their potential utilization as new biological resources for natural product chemistry. From the methanol extract of fruiting bodies of Polysphondylium filamentosum, we obtained new chlorinated benzofurans Pf-1 (4) and Pf-2 (5) which display multiple biological activities; these include stalk cell differentiation-inducing activity in the well-studied cellular slime mold, Dictyostelium discoideum, and inhibitory activities on cell proliferation in mammalian cells and gene expression in Drosophila melanogaster. Copyright © 2013 Elsevier Ltd. All rights reserved.

Polyurethane/nano-hydroxyapatite composite films as osteogenic platforms.

PubMed

Jackson, Bailey K; Bow, Austin J; Kannarpady, Ganesh; Biris, Alexandru S; Anderson, David E; Dhar, Madhu; Bourdo, Shawn E

2018-08-01

A wide variety of biomaterials are utilized in tissue engineering to promote cell proliferations in vitro or tissue growth in vivo. The combination of cells, extracellular matrices, and biocompatible materials may make it possible to grow functional living tissues ranging from bone to nerve cells. In bone regeneration, polymeric scaffolds can be enhanced by the addition of bioactive materials. To this end, this study designed several ratios of polyurethane (PU) and nano-hydroxyapatite (nHA) composites (PU-nHA ratios: 100/0, 90/10, 80/20, 70/30, 60/40 w/w). The physical and mechanical properties of these composites and their relative cellular compatibility in vitro were determined. The chemical composition and crystallinity of the composites were confirmed using X-ray diffraction, X-ray photoelectron spectroscopy, and thermogravimetric analyses. Atomic force microscopy, nano-indentation, and contact angle measurements were used to evaluate surface properties. The results showed a significant increase in surface roughness and a decrease in contact angle when the nHA concentration increased above 20%, resulting in a significant increase in hydrophilicity. These surface property changes influenced cellular behavior when MC 3T3-E1 cells were seeded on the composites. All composites were cytocompatible. There was a linear increase in cell proliferation on the 80/20 and 70/30 composites only, whereas subjective evaluation demonstrated noticeable clusters or nodules of cells (considered hallmarks of osteogenic differentiation) in the absence of any osteogenic inducers only on the 90/10 and 80/20 composites. Cellular data suggests that the 80/20 composite was an optimal environment for cell adhesion, proliferation, and, potentially, osteogenic differentiation in vitro.

Cellular Mechanisms Underlying Bone-Forming Cell Proliferative Response to Hypergravity

NASA Technical Reports Server (NTRS)

Vercoutere, W.; Parra, M.; DaCosta, M.; Wing, A.; Roden, C.; Damsky, C.; Holton, E.; Searby, N.; Globus, R.; Almeida, E.

2004-01-01

Life on Earth has evolved under the continuous influence of gravity (1-g). As humans explore and develop space, however, we must learn to adapt to an environment with little or no gravity. Studies indicate that lack of weightbearing for vertebrates occurring with immobilization, paralysis, or in a microgravity environment may cause muscle and bone atrophy through cellular and subcellular level mechanisms. We hypothesize that gravity is needed for the efficient transduction of cell growth and survival signals from the extra-cellular matrix (ECM) (consisting of molecules such as collagen, fibronectin, and laminin) in mechanosensitive tissues. We test for the presence of gravity-sensitive pathways in bone-forming cells (osteoblasts) using hypergravity applied by a cell culture centrifuge. Stimulation of 50 times gravity (50-g) increased proliferation in primary rat osteoblasts for cells grown on collagen Type I and fibronectin, but not on laminin or uncoated surfaces. Survival was also enhanced during hypergravity stimulation by the presence of ECM. Bromodeoxyuridine incorporation in proliferating cells showed an increase in the number of actively dividing cells from about 60% at 1-g to over 90% at 25-g. Reverse transcription-polymerase chain reaction was used to test for all possible integrins. Our combined results indicate that beta1 and/or beta3 integrin subunits may be involved. These data indicate that gravity mechanostimulation of osteoblast proliferation involves specific matrix-integrin signalling pathways which are sensitive to g-level. Further research to define the mechanisms involved will provide direction so that we may better adapt and counteract bone atrophy caused by the lack of weightbearing.

Exosomes Secreted by Toxoplasma gondii-Infected L6 Cells: Their Effects on Host Cell Proliferation and Cell Cycle Changes

PubMed Central

Kim, Min Jae; Jung, Bong-Kwang; Cho, Jaeeun; Song, Hyemi; Pyo, Kyung-Ho; Lee, Ji Min; Kim, Min-Kyung; Chai, Jong-Yil

2016-01-01

Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle. PMID:27180572

Metabolic regulation of cellular plasticity in the pancreas.

PubMed

Ninov, Nikolay; Hesselson, Daniel; Gut, Philipp; Zhou, Amy; Fidelin, Kevin; Stainier, Didier Y R

2013-07-08

Obese individuals exhibit an increase in pancreatic β cell mass; conversely, scarce nutrition during pregnancy has been linked to β cell insufficiency in the offspring [reviewed in 1, 2]. These phenomena are thought to be mediated mainly through effects on β cell proliferation, given that a nutrient-sensitive β cell progenitor population in the pancreas has not been identified. Here, we employed the fluorescent ubiquitination-based cell-cycle indicator system to investigate β cell replication in real time and found that high nutrient concentrations induce rapid β cell proliferation. Importantly, we found that high nutrient concentrations also stimulate β cell differentiation from progenitors in the intrapancreatic duct (IPD). Furthermore, using a new zebrafish line where β cells are constitutively ablated, we show that β cell loss and high nutrient intake synergistically activate these progenitors. At the cellular level, this activation process causes ductal cell reorganization as it stimulates their proliferation and differentiation. Notably, we link the nutrient-dependent activation of these progenitors to a downregulation of Notch signaling specifically within the IPD. Furthermore, we show that the nutrient sensor mechanistic target of rapamycin (mTOR) is required for endocrine differentiation from the IPD under physiological conditions as well as in the diabetic state. Thus, this study reveals critical insights into how cells modulate their plasticity in response to metabolic cues and identifies nutrient-sensitive progenitors in the mature pancreas. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effects of SiO2 and ZnO doping on mechanical and biological properties of 3D printed TCP scaffolds

PubMed Central

Fielding, Gary A.; Bandyopadhyay, Amit; Bose, Susmita

2011-01-01

Objectives To evaluate the effects of SiO2 (0.5 wt %) and ZnO (0.25 wt %) dopants on the mechanical and biological properties of tricalcium phosphate (TCP) scaffolds with three dimensionally (3D) interconnected pores. Methods Scaffolds were created with a commercial 3D printer. Post sintering phase analysis was determined by x-ray diffraction. Surface morphology of the scaffolds was examined by field emission electron microscopy. Mechanical strength was evaluated with a screw driven universal testing machine. MTT assay was used for cellular proliferation characteristics and cellular morphology was examined by field emission electron microscopy. Results Addition of dopants into TCP increased the average density of pure TCP from 90.8 ± 0.8% to 94.1 ± 1.6% and retarded the β to α phase transformation at high sintering temperatures, which resulted in up to 2.5 fold increase in compressive strength. In vitro cell-materials interaction studies, carried out using hFOB cells, confirmed that the addition of SiO2 and ZnO to the scaffolds facilitates faster cell proliferation when compared to pure TCP scaffolds. Significance Addition of SiO2 and ZnO dopants to the TCP scaffolds showed increased mechanical strength as well as increased cellular proliferation. PMID:22047943

Homeobox A7 stimulates breast cancer cell proliferation by up-regulating estrogen receptor-alpha

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

Zhang, Yu; Department of Obstetrics and Gynaecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia V5Z 4H4; Cheng, Jung-Chien

2013-11-01

Highlights: •HOXA7 regulates MCF7 cell proliferation. •HOXA7 up-regulates ERα expression. •HOXA7 mediates estrogen-induced MCF7 cell proliferation. -- Abstract: Breast cancer is the most common hormone-dependent malignancy in women. Homeobox (HOX) transcription factors regulate many cellular functions, including cell migration, proliferation and differentiation. The aberrant expression of HOX genes has been reported to be associated with human reproductive cancers. Estradiol (E2) and its nuclear receptors, estrogen receptor (ER)-alpha and ER-beta, are known to play critical roles in the regulation of breast cancer cell growth. However, an understanding of the potential relationship between HOXA7 and ER in breast cancer cells is limited.more » In this study, our results demonstrate that knockdown of HOXA7 in MCF7 cells significantly decreased cell proliferation and ERα expression. In addition, HOXA7 knockdown attenuated E2-induced cell proliferation as well as progesterone receptor (PR) expression. The stimulatory effects of E2 on cell proliferation and PR expression were abolished by co-treatment with ICI 182780, a selective ERα antagonist. In contrast, overexpression of HOXA7 significantly stimulated cell proliferation and ERα expression. Moreover, E2-induced cell proliferation, as well as PR expression, was enhanced by the overexpression of HOXA7. Neither knockdown nor overexpression of HOXA7 affected the ER-beta levels. Our results demonstrate a novel mechanistic role for HOXA7 in modulating breast cancer cell proliferation via regulation of ERα expression. This finding contributes to our understanding of the role HOXA7 plays in regulating the proliferation of ER-positive cancer cells.« less

Different cellular effects of four anti-inflammatory eye drops on human corneal epithelial cells: independent in active components.

PubMed

Qu, Mingli; Wang, Yao; Yang, Lingling; Zhou, Qingjun

2011-01-01

To evaluate and compare the cellular effects of four commercially available anti-inflammatory eye drops and their active components on human corneal epithelial cells (HCECs) in vitro. The cellular effects of four eye drops (Bromfenac Sodium Hydrate Eye Drops, Pranoprofen Eye Drops, Diclofenac Sodium Eye Drops, and Tobramycin & Dex Eye Drops) and their corresponding active components were evaluated in an HCEC line with five in vitro assays. Cell proliferation and migration were measured using 3-(4,5)-dimethylthiahiazo (-z-y1)-3 5-di-phenytetrazoliumromide (MTT) assay and transwell migration assay. Cell damage was determined with the lactate dehydrogenase (LDH) assay. Cell viability and median lethal time (LT₅₀) were measured by 7-amino-actinomycin D (7-AAD) staining and flow cytometry analysis. Cellular effects after exposure of HCECs to the four anti-inflammatory eye drops were concentration dependent. The differences of cellular toxicity on cell proliferation became significant at lower concentrations (<0.002%). Diclofenac Sodium Eye Drops showed significant increasing effects on cell damage and viability when compared with the other three solutions. Tobramycin & Dex Eye Drops inhibited the migration of HCECs significantly. Tobramycin & Dex Eye Drops showed the quickest effect on cell viability: the LT₅₀ was 3.28, 9.23, 10.38, and 23.80 min for Tobramycin & Dex Eye Drops, Diclofenac Sodium Eye Drops, Pranoprofen Eye Drops, and Bromfenac Sodium Hydrate Eye Drops, respectively. However, the comparisons of cellular toxicity revealed significant differences between the eye drops and their active components under the same concentration. The corneal epithelial toxicity differences among the active components of the four eye drops became significant as higher concentration (>0.020%). The four anti-inflammatory eye drops showed different cellular effects on HCECs, and the toxicity was not related with their active components, which provides new reference for the clinical application and drug research and development.

Axl as a mediator of cellular growth and survival

PubMed Central

Axelrod, Haley; Pienta, Kenneth J.

2014-01-01

The control of cellular growth and proliferation is key to the maintenance of homeostasis. Survival, proliferation, and arrest are regulated, in part, by Growth Arrest Specific 6 (Gas6) through binding to members of the TAM receptor tyrosine kinase family. Activation of the TAM receptors leads to downstream signaling through common kinases, but the exact mechanism within each cellular context varies and remains to be completely elucidated. Deregulation of the TAM family, due to its central role in mediating cellular proliferation, has been implicated in multiple diseases. Axl was cloned as the first TAM receptor in a search for genes involved in the progression of chronic to acute-phase leukemia, and has since been established as playing a critical role in the progression of cancer. The oncogenic nature of Axl is demonstrated through its activation of signaling pathways involved in proliferation, migration, inhibition of apoptosis, and therapeutic resistance. Despite its recent discovery, significant progress has been made in the development of effective clinical therapeutics targeting Axl. In order to accurately define the role of Axl in normal and diseased processes, it must be analyzed in a cell type-specific context. PMID:25344858

Dexamethasone reduces side population fraction through downregulation of ABCG2 transporter in MCF-7 breast cancer cells.

PubMed

Kim, Jong Bin; Hwang, Sung Eun; Yoon, Sang-Pil

2017-07-01

Side population (SP) cells represent a rare population among breast cancer cells. SP cells have been reported to act as cancer stem‑like cells, and to participate in the development of multidrug resistance via modulating the expression of ATP-binding cassette subfamily G member 2 (ABCG2). Dexamethasone is a corticosteroid drug that has been used as an adjuvant treatment to enhance the efficacy of chemotherapeutic agents; however, its effects in breast cancer have yet to be thoroughly investigated. In the present study, the effects of dexamethasone were investigated using the human MCF‑7 breast cancer cell line, and SPs were examined in detail. Cellular proliferation, SP fractions and ABCG2 expression were examined following treatment of MCF‑7 cells with dexamethasone. Dexamethasone was revealed to cause a dose‑ and time‑dependent decrease in cancer cell proliferation, and it also decreased the size of the SP fraction of MCF‑7 cells and the expression of the ABCG2 transporter. The effects of dexamethasone on cellular proliferation, SP fraction and ABCG2 expression were abolished following the administration of the glucocorticoid antagonist RU486. These results suggested that dexamethasone may target breast cancer cell SPs and thus increase the sensitivity of tumor cells to chemotherapy. Therefore, it may be hypothesized that dexamethasone can be used as a chemosensitizer in the adjuvant treatment of patients with breast cancer.

Abrogation of E-cadherin-mediated cell-cell contact in mouse embryonic stem cells results in reversible LIF-independent self-renewal.

PubMed

Soncin, Francesca; Mohamet, Lisa; Eckardt, Dominik; Ritson, Sarah; Eastham, Angela M; Bobola, Nicoletta; Russell, Angela; Davies, Steve; Kemler, Rolf; Merry, Catherine L R; Ward, Christopher M

2009-09-01

We have previously demonstrated that differentiation of embryonic stem (ES) cells is associated with downregulation of cell surface E-cadherin. In this study, we assessed the function of E-cadherin in mouse ES cell pluripotency and differentiation. We show that inhibition of E-cadherin-mediated cell-cell contact in ES cells using gene knockout (Ecad(-/-)), RNA interference (EcadRNAi), or a transhomodimerization-inhibiting peptide (CHAVC) results in cellular proliferation and maintenance of an undifferentiated phenotype in fetal bovine serum-supplemented medium in the absence of leukemia inhibitory factor (LIF). Re-expression of E-cadherin in Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells restores cellular dependence to LIF supplementation. Although reversal of the LIF-independent phenotype in Ecad(-/-) ES cells is dependent on the beta-catenin binding domain of E-cadherin, we show that beta-catenin null (betacat(-/-)) ES cells also remain undifferentiated in the absence of LIF. This suggests that LIF-independent self-renewal of Ecad(-/-) ES cells is unlikely to be via beta-catenin signaling. Exposure of Ecad(-/-), EcadRNAi, and CHAVC-treated ES cells to the activin receptor-like kinase inhibitor SB431542 led to differentiation of the cells, which could be prevented by re-expression of E-cadherin. To confirm the role of transforming growth factor beta family signaling in the self-renewal of Ecad(-/-) ES cells, we show that these cells maintain an undifferentiated phenotype when cultured in serum-free medium supplemented with Activin A and Nodal, with fibroblast growth factor 2 required for cellular proliferation. We conclude that transhomodimerization of E-cadherin protein is required for LIF-dependent ES cell self-renewal and that multiple self-renewal signaling networks subsist in ES cells, with activity dependent upon the cellular context.

Oscillatory Protein Expression Dynamics Endows Stem Cells with Robust Differentiation Potential

PubMed Central

Kaneko, Kunihiko

2011-01-01

The lack of understanding of stem cell differentiation and proliferation is a fundamental problem in developmental biology. Although gene regulatory networks (GRNs) for stem cell differentiation have been partially identified, the nature of differentiation dynamics and their regulation leading to robust development remain unclear. Herein, using a dynamical system modeling cell approach, we performed simulations of the developmental process using all possible GRNs with a few genes, and screened GRNs that could generate cell type diversity through cell-cell interactions. We found that model stem cells that both proliferated and differentiated always exhibited oscillatory expression dynamics, and the differentiation frequency of such stem cells was regulated, resulting in a robust number distribution. Moreover, we uncovered the common regulatory motifs for stem cell differentiation, in which a combination of regulatory motifs that generated oscillatory expression dynamics and stabilized distinct cellular states played an essential role. These findings may explain the recently observed heterogeneity and dynamic equilibrium in cellular states of stem cells, and can be used to predict regulatory networks responsible for differentiation in stem cell systems. PMID:22073296

Resveratrol-induced transcriptional up-regulation of ASMase (SMPD1) of human leukemia and cancer cells

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

Mizutani, Naoki; College of Life and Health Sciences, Chubu University, Kasugai; Omori, Yukari

2016-02-19

Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increasedmore » by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5′-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment. - Highlights: • Resveratrol inhibited cell proliferation of K562 and HCT116 cells. • Resveratrol increased cellular ceramide and decreased sphingomyelin and S1P. • ASMase mRNA and activity were increased with resveratrol. • ASMase inhibition suppressed RSV-induced ceramide accumulation. • Increased ASMase transcription was at least partially due to EGR family proteins.« less

Dickkopf-3 maintains the PANC-1 human pancreatic tumor cells in a dedifferentiated state.

PubMed

Zenzmaier, Christoph; Hermann, Martin; Hengster, Paul; Berger, Peter

2012-01-01

Pancreatic cancer (PaCa) is the fourth leading cause of cancer deaths in Western societies, with pancreatic ductal adenocarcinomas (PDACs) accounting for >90% of such cases. PDAC is a heterogeneous disease that includes a subset showing overexpression of the secreted glycoprotein Dickkopf-related protein 3 (Dkk-3), a protein shown to be downregulated in various cancers of different tissues. The biological function of Dkk-3 in this subset was studied using the Dkk-3 expressing PANC-1 cell line as a model for PDACs. The influence of Dkk-3 overexpression and knockdown on cellular differentiation and proliferation of PANC-1 was investigated. Confocal microscopy showed that Dkk-3 was expressed in a fraction of PANC-1 cells. While lentiviral-mediated overexpression of DKK3 did not alter cellular proliferation, knockdown of DKK3 resulted in significant reduction of cellular proliferation and concomitant induction of cell cycle inhibitors CDKN2B (p15INK4b), CDKN1A (p21CIP1) and CDKN1B (p27KIP1). In parallel, pancreatic epithelial cell differentiation markers AMY2A, CELA1, CTRB1, GCG, GLB1 and INS were significantly upregulated. PANC-1 cells differentiated using exendin-4 showed analogous induction of cell cycle inhibitors and differentiation markers. Thus, we conclude that Dkk-3 is required to maintain a highly dedifferentiated and consequently proliferative state in PANC-1, indicating a similar function in the Dkk-3 overexpressing subset of PDACs. Therefore, Dkk-3 represents a potential target for the treatment of Dkk-3-positive subtypes of PaCa to drive cells into cell cycle arrest and differentiation.

Three dimensional multi-cellular muscle-like tissue engineering in perfusion-based bioreactors.

PubMed

Cerino, Giulia; Gaudiello, Emanuele; Grussenmeyer, Thomas; Melly, Ludovic; Massai, Diana; Banfi, Andrea; Martin, Ivan; Eckstein, Friedrich; Grapow, Martin; Marsano, Anna

2016-01-01

Conventional tissue engineering strategies often rely on the use of a single progenitor cell source to engineer in vitro biological models; however, multi-cellular environments can better resemble the complexity of native tissues. Previous described co-culture models used skeletal myoblasts, as parenchymal cell source, and mesenchymal or endothelial cells, as stromal component. Here, we propose instead the use of adipose tissue-derived stromal vascular fraction cells, which include both mesenchymal and endothelial cells, to better resemble the native stroma. Percentage of serum supplementation is one of the crucial parameters to steer skeletal myoblasts toward either proliferation (20%) or differentiation (5%) in two-dimensional culture conditions. On the contrary, three-dimensional (3D) skeletal myoblast culture often simply adopts the serum content used in monolayer, without taking into account the new cell environment. When considering 3D cultures of mm-thick engineered tissues, homogeneous and sufficient oxygen supply is paramount to avoid formation of necrotic cores. Perfusion-based bioreactor culture can significantly improve the oxygen access to the cells, enhancing the viability and the contractility of the engineered tissues. In this study, we first investigated the influence of different serum supplementations on the skeletal myoblast ability to proliferate and differentiate during 3D perfusion-based culture. We tested percentages of serum promoting monolayer skeletal myoblast-proliferation (20%) and differentiation (5%) and suitable for stromal cell culture (10%) with a view to identify the most suitable condition for the subsequent co-culture. The 10% serum medium composition resulted in the highest number of mature myotubes and construct functionality. Co-culture with stromal vascular fraction cells at 10% serum also supported the skeletal myoblast differentiation and maturation, hence providing a functional engineered 3D muscle model that resembles the native multi-cellular environment. © 2015 Wiley Periodicals, Inc.

A damaged DNA binding protein 2 mutation disrupting interaction with proliferating-cell nuclear antigen affects DNA repair and confers proliferation advantage.

PubMed

Perucca, Paola; Mocchi, Roberto; Guardamagna, Isabella; Bassi, Elisabetta; Sommatis, Sabrina; Nardo, Tiziana; Prosperi, Ennio; Stivala, Lucia Anna; Cazzalini, Ornella

2018-06-01

In mammalian cells, Nucleotide Excision Repair (NER) plays a role in removing DNA damage induced by UV radiation. In Global Genome-NER subpathway, DDB2 protein forms a complex with DDB1 (UV-DDB), recognizing photolesions. During DNA repair, DDB2 interacts directly with PCNA through a conserved region in N-terminal tail and this interaction is important for DDB2 degradation. In this work, we sought to investigate the role of DDB2-PCNA association in DNA repair and cell proliferation after UV-induced DNA damage. To this end, stable clones expressing DDB2 Wt and DDB2 PCNA- were used. We have found that cells expressing a mutant DDB2 show inefficient photolesions removal, and a concomitant lack of binding to damaged DNA in vitro. Unexpected cellular behaviour after DNA damage, such as UV-resistance, increased cell growth and motility were found in DDB2 PCNA- stable cell clones, in which the most significant defects in cell cycle checkpoint were observed, suggesting a role in the new cellular phenotype. Based on these findings, we propose that DDB2-PCNA interaction may contribute to a correct DNA damage response for maintaining genome integrity. Copyright © 2018 Elsevier B.V. All rights reserved.

Myostatin Suppression of Akirin1 Mediates Glucocorticoid-Induced Satellite Cell Dysfunction

PubMed Central

Dong, Yanjun; Pan, Jenny S.; Zhang, Liping

2013-01-01

Glucocorticoids production is increased in many pathological conditions that are associated with muscle loss, but their role in causing muscle wasting is not fully understood. We have demonstrated a new mechanism of glucocorticoid-induced muscle atrophy: Dexamethasone (Dex) suppresses satellite cell function contributing to the development of muscle atrophy. Specifically, we found that Dex decreases satellite cell proliferation and differentiation in vitro and in vivo. The mechanism involved Dex-induced upregulation of myostatin and suppression of Akirin1, a promyogenic gene. When myostatin was inhibited in Dex-treated mice, Akirin1 expression increased as did satellite cell activity, muscle regeneration and muscle growth. In addition, silencing myostatin in myoblasts or satellite cells prevented Dex from suppressing Akirin1 expression and cellular proliferation and differentiation. Finally, overexpression of Akirin1 in myoblasts increased their expression of MyoD and myogenin and improved cellular proliferation and differentiation, theses improvements were no longer suppressed by Dex. We conclude that glucocorticoids stimulate myostatin which inhibits Akirin1 expression and the reparative functions of satellite cells. These responses attribute to muscle atrophy. Thus, inhibition of myostatin or increasing Akirin1 expression could lead to therapeutic strategies for improving satellite cell activation and enhancing muscle growth in diseases associated with increased glucocorticoid production. PMID:23516508

Myostatin suppression of Akirin1 mediates glucocorticoid-induced satellite cell dysfunction.

PubMed

Dong, Yanjun; Pan, Jenny S; Zhang, Liping

2013-01-01

Glucocorticoids production is increased in many pathological conditions that are associated with muscle loss, but their role in causing muscle wasting is not fully understood. We have demonstrated a new mechanism of glucocorticoid-induced muscle atrophy: Dexamethasone (Dex) suppresses satellite cell function contributing to the development of muscle atrophy. Specifically, we found that Dex decreases satellite cell proliferation and differentiation in vitro and in vivo. The mechanism involved Dex-induced upregulation of myostatin and suppression of Akirin1, a promyogenic gene. When myostatin was inhibited in Dex-treated mice, Akirin1 expression increased as did satellite cell activity, muscle regeneration and muscle growth. In addition, silencing myostatin in myoblasts or satellite cells prevented Dex from suppressing Akirin1 expression and cellular proliferation and differentiation. Finally, overexpression of Akirin1 in myoblasts increased their expression of MyoD and myogenin and improved cellular proliferation and differentiation, theses improvements were no longer suppressed by Dex. We conclude that glucocorticoids stimulate myostatin which inhibits Akirin1 expression and the reparative functions of satellite cells. These responses attribute to muscle atrophy. Thus, inhibition of myostatin or increasing Akirin1 expression could lead to therapeutic strategies for improving satellite cell activation and enhancing muscle growth in diseases associated with increased glucocorticoid production.

Normal tubular regeneration and differentiation of the post-ischemic kidney in mice lacking vimentin.

PubMed Central

Terzi, F.; Maunoury, R.; Colucci-Guyon, E.; Babinet, C.; Federici, P.; Briand, P.; Friedlander, G.

1997-01-01

Proliferation and dedifferentiation of tubular cells are the hallmark of early regeneration after renal ischemic injury. Vimentin, a class III intermediate filament expressed only in mesenchymal cells of mature mammals, was shown to be transiently expressed in post-ischemic renal tubular epithelial cells. Vimentin re-expression was interpreted as a marker of cellular dedifferentiation, but its role in tubular regeneration after renal ischemia has also been hypothesized. This role was evaluated in mice bearing a null mutation of the vimentin gene. Expression of vimentin, proliferating cell nuclear antigen (a marker of cellular proliferation), and villin (a marker of differentiated brush-border membranes) was studied in wild-type (Vim+/+), heterozygous (Vim+/-), and homozygous (Vim-/-) mice subjected to transient ischemia of the left kidney. As expected, vimentin was detected by immunohistochemistry at the basal pole of proximal tubular cells from post-ischemic kidney in Vim+/+ and Vim+/- mice from day 2 to day 28. The expression of the reporter gene beta-galactosidase in Vim+/- and Vim-/- mice confirmed the tubular origin of vimentin. No compensatory expression of keratin could be demonstrated in Vim-/- mice. The intensity of proliferating cell nuclear antigen labeling and the pattern of villin expression were comparable in Vim-/-, Vim+/- and Vim+/+ mice at any time of the study. After 60 days, the structure of post-ischemic kidneys in Vim-/- mice was indistinguishable from that of normal non-operated kidneys in Vim+/+ mice. In conclusion, 1) the pattern of post-ischemic proximal tubular cell proliferation, differentiation, and tubular organization was not impaired in mice lacking vimentin and 2) these results suggest that the transient tubular expression of vimentin is not instrumental in tubular regeneration after renal ischemic injury. Images Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 Figure 7 PMID:9094992

Lack of effect of a granulocyte proliferation inhibitor or their committed precursor cells.

PubMed

Lord, B I; Testa, N G; Wright, E G; Banerjee, R K

1977-05-01

Using the agar culture technique, we have measured the effect of granulocyte extracts GCE (and of erythrocyte-RCE and lymph node extracts-LNE) on the growth and proliferation of the committed granulocytic precursor cells, CFU-C. In addition we have determined their effects on the proliferation of the developing colony cells and on the ultimate cell production in the colonies. The results show that GCE has no effect on the growth or proliferative activity on the CFU-C. It does, however, reduce both the autoradiographic labelling indices of the developing colony cells and the net colony cellularities, acting as a cell cycle modulator. These are effects specific to the GCE since at the dose levels used, neither RCE nor LNE affected these measurements.

Cutting the brakes and flooring the gas: how TMEPAI turns TGF-β into a tumor promoter.

PubMed

Cichon, Magdalena A; Radisky, Derek C

2014-09-01

In normal or nonmalignant cells, TGF-β inhibits cellular proliferation through activation of the SMAD-dependent canonical signaling pathway. Recent findings demonstrate that the protein TMEPAI1 can block the cytostatic effects of the canonical TGF-β signaling pathway, while activating cellular proliferation through the noncanonical, SMAD-independent TGF-β signaling pathway. As TMEPAI1 shows increased expression in the poor prognosis basal and HER2 intrinsic subtypes of breast cancer, these findings point to a new avenue of targeted therapy with considerable therapeutic potential.

Wnt signaling potentiates nevogenesis

PubMed Central

Pawlikowski, Jeff S.; McBryan, Tony; van Tuyn, John; Drotar, Mark E.; Hewitt, Rachael N.; Maier, Andrea B.; King, Ayala; Blyth, Karen; Wu, Hong; Adams, Peter D.

2013-01-01

Cellular senescence is a stable proliferation arrest associated with an altered secretory pathway (senescence-associated secretory phenotype). Cellular senescence is also a tumor suppressor mechanism, to which both proliferation arrest and senescence-associated secretory phenotype are thought to contribute. The melanocytes within benign human nevi are a paradigm for tumor-suppressive senescent cells in a premalignant neoplasm. Here a comparison of proliferating and senescent melanocytes and melanoma cell lines by RNA sequencing emphasizes the importance of senescence-associated proliferation arrest in suppression of transformation. Previous studies showed that activation of the Wnt signaling pathway can delay or bypass senescence. Consistent with this, we present evidence that repression of Wnt signaling contributes to melanocyte senescence in vitro. Surprisingly, Wnt signaling is active in many senescent human melanocytes in nevi, and this is linked to histological indicators of higher proliferative and malignant potential. In a mouse, activated Wnt signaling delays senescence-associated proliferation arrest to expand the population of senescent oncogene-expressing melanocytes. These results suggest that Wnt signaling can potentiate nevogenesis in vivo by delaying senescence. Further, we suggest that activated Wnt signaling in human nevi undermines senescence-mediated tumor suppression and enhances the probability of malignancy. PMID:24043806

Wnt signaling potentiates nevogenesis.

PubMed

Pawlikowski, Jeff S; McBryan, Tony; van Tuyn, John; Drotar, Mark E; Hewitt, Rachael N; Maier, Andrea B; King, Ayala; Blyth, Karen; Wu, Hong; Adams, Peter D

2013-10-01

Cellular senescence is a stable proliferation arrest associated with an altered secretory pathway (senescence-associated secretory phenotype). Cellular senescence is also a tumor suppressor mechanism, to which both proliferation arrest and senescence-associated secretory phenotype are thought to contribute. The melanocytes within benign human nevi are a paradigm for tumor-suppressive senescent cells in a premalignant neoplasm. Here a comparison of proliferating and senescent melanocytes and melanoma cell lines by RNA sequencing emphasizes the importance of senescence-associated proliferation arrest in suppression of transformation. Previous studies showed that activation of the Wnt signaling pathway can delay or bypass senescence. Consistent with this, we present evidence that repression of Wnt signaling contributes to melanocyte senescence in vitro. Surprisingly, Wnt signaling is active in many senescent human melanocytes in nevi, and this is linked to histological indicators of higher proliferative and malignant potential. In a mouse, activated Wnt signaling delays senescence-associated proliferation arrest to expand the population of senescent oncogene-expressing melanocytes. These results suggest that Wnt signaling can potentiate nevogenesis in vivo by delaying senescence. Further, we suggest that activated Wnt signaling in human nevi undermines senescence-mediated tumor suppression and enhances the probability of malignancy.

Ion channels involved in cell volume regulation: effects on migration, proliferation, and programmed cell death in non adherent EAT cells and adherent ELA cells.

PubMed

Hoffmann, Else Kay

2011-01-01

This mini review outlines studies of cell volume regulation in two closely related mammalian cell lines: nonadherent Ehrlich ascites tumour cells (EATC) and adherent Ehrlich Lettre ascites (ELA) cells. Focus is on the regulatory volume decrease (RVD) that occurs after cell swelling, the volume regulatory ion channels involved, and the mechanisms (cellular signalling pathways) that regulate these channels. Finally, I shall also briefly review current investigations in these two cell lines that focuses on how changes in cell volume can regulate cell functions such as cell migration, proliferation, and programmed cell death. Copyright © 2011 S. Karger AG, Basel.

Pirin Inhibits Cellular Senescence in Melanocytic Cells

PubMed Central

Licciulli, Silvia; Luise, Chiara; Scafetta, Gaia; Capra, Maria; Giardina, Giuseppina; Nuciforo, Paolo; Bosari, Silvano; Viale, Giuseppe; Mazzarol, Giovanni; Tonelli, Chiara; Lanfrancone, Luisa; Alcalay, Myriam

2011-01-01

Cellular senescence has been widely recognized as a tumor suppressing mechanism that acts as a barrier to cancer development after oncogenic stimuli. A prominent in vivo model of the senescence barrier is represented by nevi, which are composed of melanocytes that, after an initial phase of proliferation induced by activated oncogenes (most commonly BRAF), are blocked in a state of cellular senescence. Transformation to melanoma occurs when genes involved in controlling senescence are mutated or silenced and cells reacquire the capacity to proliferate. Pirin (PIR) is a highly conserved nuclear protein that likely functions as a transcriptional regulator whose expression levels are altered in different types of tumors. We analyzed the expression pattern of PIR in adult human tissues and found that it is expressed in melanocytes and has a complex pattern of regulation in nevi and melanoma: it is rarely detected in mature nevi, but is expressed at high levels in a subset of melanomas. Loss of function and overexpression experiments in normal and transformed melanocytic cells revealed that PIR is involved in the negative control of cellular senescence and that its expression is necessary to overcome the senescence barrier. Our results suggest that PIR may have a relevant role in melanoma progression. PMID:21514450

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

Hatkevich, Talia; Ramos, Joseph; Santos-Sanchez, Idalys

Since over 60% of breast cancers are estrogen receptor positive (ER+), many therapies have targeted the ER. The ER is activated by both estrogen binding and phosphorylation. While anti-estrogen therapies, such as tamoxifen (Tam) have been successful they do not target the growth factor promoting phosphorylation of the ER. Other proliferation pathways such as the phosphatidylinositol-3 kinase, (PI3K) and the mitogen-activated protein kinase (MAPK) pathways are activated in breast cancer cells and are associated with poor prognosis. Thus targeting multiple cellular proliferation and survival pathways at the onset of treatment is critical for the development of more effective therapies. Themore » grapefruit flavanone naringenin (Nar) is an inhibitor of both the PI3K and MAPK pathways. Previous studies examining either Nar or Tam used charcoal-stripped serum which removed estrogen as well as other factors. We wanted to use serum containing medium in order to retain all the potential inducers of cell proliferation so as not to exclude any targets of Nar. Here we show that a Nar–Tam combination is more effective than either Tam alone or Nar alone in MCF-7 breast cancer cells. We demonstrate that a Nar–Tam combination impaired cellular proliferation and viability to a greater extent than either component alone in MCF-7 cells. Furthermore, the use of a Nar–Tam combination requires lower concentrations of both compounds to achieve the same effects on proliferation and viability. Nar may function by inhibiting both PI3K and MAPK pathways as well as localizing ERα to the cytoplasm in MCF-7 cells. Our results demonstrate that a Nar–Tam combination induces apoptosis and impairs proliferation signaling to a greater extent than either compound alone. These studies provide critical information for understanding the molecular mechanisms involved in cell proliferation and apoptosis in breast cancer cells. - Highlights: • Nar–Tam impairs cell viability more effectively than either compound alone. • Lower concentrations of Nar and Tam are required when used in combination. • Nar localizes ERα to a perinuclear region of the cell. • Nar reduces protein levels of ERK1/2 and AKT.« less

Pneumolysin induces cellular senescence by increasing ROS production and activation of MAPK/NF-κB signal pathway in glial cells.

PubMed

Kwon, Ii-Seul; Kim, Jinwook; Rhee, Dong-Kwon; Kim, Byung-Oh; Pyo, Suhkneung

2017-04-01

Senescence is an irreversible proliferation arrest that is induced by various stress stimuli including genotoxin. Pneumolysin (PLY) is a pathogenicity factor unique to Streptococcus pneumoniae that is important in pneumococcal-induced diseases such as otitis media, meningitis and pneumonia. However, the cell fate response to the toxin is mechanistically unclear. We investigated the effect of PLY on cellular senescence in BV-2 microglial cells. Exposure to PLY resulted in changes in the expression of phospho-p53, p21, p16, pRb and CDK2 and increased the number of senescence associated β-gal positive cells. PLY-treatment also increased PAI-1 expression and cell proliferation arrest in concentration- and time-dependent manners. PLY induced NF-κB activation and phosphorylation of SIRT-1, ERK1/2, JNK, and p38 MAPK. In addition, PLY increased the production of reactive oxygen species. Overall, the results suggest that PLY regulates microglial cellular senescence by enhancing production of reactive oxygen species, activation of MAPK and NF-κB, and phosphorylation of SIRT-1. Copyright © 2017 Elsevier Ltd. All rights reserved.

CRISPR-Cas9 Mediated Telomere Removal Leads to Mitochondrial Stress and Protein Aggregation.

PubMed

Kim, Hyojung; Ham, Sangwoo; Jo, Minkyung; Lee, Gum Hwa; Lee, Yun-Song; Shin, Joo-Ho; Lee, Yunjong

2017-10-03

Aging is considered the major risk factor for neurodegenerative diseases including Parkinson's disease (PD). Telomere shortening is associated with cellular senescence. In this regard, pharmacological or genetic inhibition of telomerase activity has been used to model cellular aging. Here, we employed CRISPR-Cas9 technology to instantly remove the telomere to induce aging in a neuroblastoma cell line. Expression of both Cas9 and guide RNA targeting telomere repeats ablated the telomere, leading to retardation of cell proliferation. Instant deletion of telomere in SH-SY5Y cells impaired mitochondrial function with diminished mitochondrial respiration and cell viability. Supporting the pathological relevance of cell aging by CRISPR-Cas9 mediated telomere removal, alterations were observed in the levels of PD-associated proteins including PTEN-induced putative kinase 1, peroxisome proliferator-activated receptor γ coactivator 1-α, nuclear respiratory factor 1, parkin, and aminoacyl tRNA synthetase complex interacting multifunctional protein 2. Significantly, α-synuclein expression in the background of telomere removal led to the enhancement of protein aggregation, suggesting positive feed-forward interaction between aging and PD pathogenesis. Collectively, our results demonstrate that CRISPR-Cas9 can be used to efficiently model cellular aging and PD.

Chemical synthesis, characterisation, and biocompatibility of nanometre scale porous anodic aluminium oxide membranes for use as a cell culture substrate for the vero cell line: a preliminary study.

PubMed

Poinern, Gérrard Eddy Jai; Le, Xuan Thi; O'Dea, Mark; Becker, Thomas; Fawcett, Derek

2014-01-01

In this preliminary study we investigate for the first time the biomedical potential of using porous anodic aluminium oxide (AAO) membranes as a cell substrate for culturing the Cercopithecus aethiops (African green monkey) Kidney (Vero) epithelial cell line. One advantage of using the inorganic AAO membrane is the presence of nanometre scale pore channels that allow the exchange of molecules and nutrients across the membrane. The size of the pore channels can be preselected by adjusting the controlling parameters of a temperature controlled two-step anodization process. The cellular interaction and response of the Vero cell line with an in-house synthesised AAO membrane, a commercially available membrane, and a glass control were assessed by investigating cell adhesion, morphology, and proliferation over a 72 h period. The number of viable cells proliferating over the respective membrane surfaces revealed that the locally produced in-house AAO membrane had cells numbers similar to the glass control. The study revealed evidence of focal adhesion sites over the surface of the nanoporous membranes and the penetration of cellular extensions into the pore structure as well. The outcome of the study has revealed that nanometre scale porous AAO membranes have the potential to become practical cell culture scaffold substrates with the capability to enhance adhesion and proliferation of Vero cells.

Chemical Synthesis, Characterisation, and Biocompatibility of Nanometre Scale Porous Anodic Aluminium Oxide Membranes for Use as a Cell Culture Substrate for the Vero Cell Line: A Preliminary Study

PubMed Central

Poinern, Gérrard Eddy Jai; Le, Xuan Thi; Becker, Thomas; Fawcett, Derek

2014-01-01

In this preliminary study we investigate for the first time the biomedical potential of using porous anodic aluminium oxide (AAO) membranes as a cell substrate for culturing the Cercopithecus aethiops (African green monkey) Kidney (Vero) epithelial cell line. One advantage of using the inorganic AAO membrane is the presence of nanometre scale pore channels that allow the exchange of molecules and nutrients across the membrane. The size of the pore channels can be preselected by adjusting the controlling parameters of a temperature controlled two-step anodization process. The cellular interaction and response of the Vero cell line with an in-house synthesised AAO membrane, a commercially available membrane, and a glass control were assessed by investigating cell adhesion, morphology, and proliferation over a 72 h period. The number of viable cells proliferating over the respective membrane surfaces revealed that the locally produced in-house AAO membrane had cells numbers similar to the glass control. The study revealed evidence of focal adhesion sites over the surface of the nanoporous membranes and the penetration of cellular extensions into the pore structure as well. The outcome of the study has revealed that nanometre scale porous AAO membranes have the potential to become practical cell culture scaffold substrates with the capability to enhance adhesion and proliferation of Vero cells. PMID:24579077

Non-viral gene delivery regulated by stiffness of cell adhesion substrates.

PubMed

Kong, Hyun Joon; Liu, Jodi; Riddle, Kathryn; Matsumoto, Takuya; Leach, Kent; Mooney, David J

2005-06-01

Non-viral gene vectors are commonly used for gene therapy owing to safety concerns with viral vectors. However, non-viral vectors are plagued by low levels of gene transfection and cellular expression. Current efforts to improve the efficiency of non-viral gene delivery are focused on manipulations of the delivery vector, whereas the influence of the cellular environment in DNA uptake is often ignored. The mechanical properties (for example, rigidity) of the substrate to which a cell adheres have been found to mediate many aspects of cell function including proliferation, migration and differentiation, and this suggests that the mechanics of the adhesion substrate may regulate a cell's ability to uptake exogeneous signalling molecules. In this report, we present a critical role for the rigidity of the cell adhesion substrate on the level of gene transfer and expression. The mechanism relates to material control over cell proliferation, and was investigated using a fluorescent resonance energy transfer (FRET) technique. This study provides a new material-based control point for non-viral gene therapy.

Fibroblasts Cultured on Nanowires Exhibit Low Motility, Impaired Cell Division, and DNA Damage

PubMed Central

Persson, Henrik; Købler, Carsten; Mølhave, Kristian; Samuelson, Lars; Tegenfeldt, Jonas O; Oredsson, Stina; Prinz, Christelle N

2013-01-01

Nanowires are commonly used as tools for interfacing living cells, acting as biomolecule-delivery vectors or electrodes. It is generally assumed that the small size of the nanowires ensures a minimal cellular perturbation, yet the effects of nanowires on cell migration and proliferation remain largely unknown. Fibroblast behaviour on vertical nanowire arrays is investigated, and it is shown that cell motility and proliferation rate are reduced on nanowires. Fibroblasts cultured on long nanowires exhibit failed cell division, DNA damage, increased ROS content and respiration. Using focused ion beam milling and scanning electron microscopy, highly curved but intact nuclear membranes are observed, showing no direct contact between the nanowires and the DNA. The nanowires possibly induce cellular stress and high respiration rates, which trigger the formation of ROS, which in turn results in DNA damage. These results are important guidelines to the design and interpretation of experiments involving nanowire-based transfection and electrical characterization of living cells. PMID:23813871

The yeast DNA ligase gene CDC9 is controlled by six orientation specific upstream activating sequences that respond to cellular proliferation but which alone cannot mediate cell cycle regulation.

PubMed Central

White, J H; Johnson, A L; Lowndes, N F; Johnston, L H

1991-01-01

By fusing the CDC9 structural gene to the PGK upstream sequences and the CDC9 upstream to lacZ, we showed that the cell cycle expression of CDC9 is largely due to transcriptional regulation. To investigate the role of six ATGATT upstream repeats in CDC9 regulation, synthetic copies of the sequence were attached to a heterologous gene. The repeats stimulated transcription strongly and additively, but, unlike conventional yeast UAS elements, only when present in one orientation. Transcription driven by the repeats declines in cells held at START of the cell cycle or in stationary phase, as occurs with CDC9. However, the repeats by themselves cannot impart cell cycle regulation to a heterologous gene. CDC9 may therefore be controlled by an activating system operating through the repeats that is sensitive to cellular proliferation and a separate mechanism that governs the periodic expression in the cell cycle. Images PMID:1901644

Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis.

PubMed

Kümper, Sandra; Mardakheh, Faraz K; McCarthy, Afshan; Yeo, Maggie; Stamp, Gordon W; Paul, Angela; Worboys, Jonathan; Sadok, Amine; Jørgensen, Claus; Guichard, Sabrina; Marshall, Christopher J

2016-01-14

Rho-associated kinases 1 and 2 (ROCK1/2) are Rho-GTPase effectors that control key aspects of the actin cytoskeleton, but their role in proliferation and cancer initiation or progression is not known. Here, we provide evidence that ROCK1 and ROCK2 act redundantly to maintain actomyosin contractility and cell proliferation and that their loss leads to cell-cycle arrest and cellular senescence. This phenotype arises from down-regulation of the essential cell-cycle proteins CyclinA, CKS1 and CDK1. Accordingly, while the loss of either Rock1 or Rock2 had no negative impact on tumorigenesis in mouse models of non-small cell lung cancer and melanoma, loss of both blocked tumor formation, as no tumors arise in which both Rock1 and Rock2 have been genetically deleted. Our results reveal an indispensable role for ROCK, yet redundant role for isoforms 1 and 2, in cell cycle progression and tumorigenesis, possibly through the maintenance of cellular contractility.

Low-dose radiation modulates human mesenchymal stem cell proliferation through regulating CDK and Rb.

PubMed

Yang, Lei; Liu, Ziling; Chen, Chen; Cong, Xiaofeng; Li, Zhi; Zhao, Shasha; Ren, Meng

2017-01-01

Low-dose radiation (LDR) has been known to stimulate cell proliferation. The effect of LDR on human bone marrow mesenchymal stem cells (BMSCs), however, remains to be determined. The current study, therefore, aimed to investigate the effect of LDR on human BMSC proliferation and its mechanisms. To accomplish this, human BMSCs were isolated from ribs and cultured with or without exposition to LDR (75 mGy) for 24 h. Cell proliferation was assessed by MTT assay, the cytokines secreted by the BMSCs were quantified by ELISA, and the proteins associated with cell proliferation and cell cycle were evaluated by immunoblot analysis. BMSCs isolated from human ribs were capable of differentiating into osteoblasts and adipocytes. LDR stimulated human BMSC proliferation (0.580 ± 0.106 vs 0.419 ± 0.026 on day 4, P < 0.05; 0.794 ± 0.025 vs 0.689 ± 0.047 on day 7, P < 0.05) and increased S-phase proportion. LDR significantly enhanced the production of SCF, GM-CSF, and IL-11. Moreover, BMSCs modulated T-cell proliferation, and LDR further augmented the modulatory effect of BMSCs on T-cell proliferation. Cell cycle-associated proteins, such as Rb, CDK1, and CDC25B, appeared to mediate the stimulatory effect of LDR on BMSC proliferation. The findings of the current study indicate that physical stimulants, such as LDR, could be used for the large-scale expansion of human BMSCs, and thus may be used for MSC cellular therapy in clinic.

Cellular and multicellular form and function.

PubMed

Liu, Wendy F; Chen, Christopher S

2007-11-10

Engineering artificial tissue constructs requires the appropriate spatial arrangement of cells within scaffolds. The introduction of microengineering tools to the biological community has provided a valuable set of techniques to manipulate the cellular environment, and to examine how cell structure affects cellular function. Using micropatterning techniques, investigators have found that the geometric presentation of cell-matrix adhesions are important regulators of various cell behaviors including cell growth, proliferation, differentiation, polarity and migration. Furthermore, the presence of neighboring cells in multicellular aggregates has a significant impact on the proliferative and differentiated state of cells. Using microengineering tools, it will now be possible to manipulate the various environmental factors for practical applications such as engineering tissue constructs with greater control over the physical structure and spatial arrangement of cells within their surrounding microenvironment.

Radiosensitization of HNSCC cells by EGFR inhibition depends on the induction of cell cycle arrests

PubMed Central

Kriegs, Malte; Kasten-Pisula, Ulla; Riepen, Britta; Hoffer, Konstantin; Struve, Nina; Myllynen, Laura; Braig, Friederike; Binder, Mascha; Rieckmann, Thorsten; Grénman, Reidar; Petersen, Cordula; Dikomey, Ekkehard; Rothkamm, Kai

2016-01-01

The increase in cellular radiosensitivity by EGF receptor (EGFR) inhibition has been shown to be attributable to the induction of a G1-arrest in p53-proficient cells. Because EGFR targeting in combination with radiotherapy is used to treat head and neck squamous cell carcinomas (HNSCC) which are predominantly p53 mutated, we tested the effects of EGFR targeting on cellular radiosensitivity, proliferation, apoptosis, DNA repair and cell cycle control using a large panel of HNSCC cell lines. In these experiments EGFR targeting inhibited signal transduction, blocked proliferation and induced radiosensitization but only in some cell lines and only under normal (pre-plating) conditions. This sensitization was not associated with impaired DNA repair (53BP1 foci) or induction of apoptosis. However, it was associated with the induction of a lasting G2-arrest. Both, the radiosensitization and the G2-arrest were abrogated if the cells were re-stimulated (delayed plating) with actually no radiosensitization being detectable in any of the 14 tested cell lines. Therefore we conclude that EGFR targeting can induce a reversible G2 arrest in p53 deficient HNSCC cells, which does not consequently result in a robust cellular radiosensitization. Together with recent animal and clinical studies our data indicate that EGFR inhibition is no effective strategy to increase the radiosensitivity of HNSCC cells. PMID:27281611

Cellular stress induced by resazurin leads to autophagy and cell death via production of reactive oxygen species and mitochondrial impairment.

PubMed

Erikstein, Bjarte S; Hagland, Hanne R; Nikolaisen, Julie; Kulawiec, Mariola; Singh, Keshav K; Gjertsen, Bjørn T; Tronstad, Karl J

2010-10-15

Mitochondrial bioenergetics and reactive oxygen species (ROS) often play important roles in cellular stress mechanisms. In this study we investigated how these factors are involved in the stress response triggered by resazurin (Alamar Blue) in cultured cancer cells. Resazurin is a redox reactive compound widely used as reporter agent in assays of cell biology (e.g. cell viability and metabolic activity) due to its colorimetric and fluorimetric properties. In order to investigate resazurin-induced stress mechanisms we employed cells affording different metabolic and regulatory phenotypes. In HL-60 and Jurkat leukemia cells resazurin caused mitochondrial disintegration, respiratory dysfunction, reduced proliferation, and cell death. These effects were preceded by a burst of ROS, especially in HL-60 cells which were also more sensitive and contained autophagic vesicles. Studies in Rho(0) cells (devoid of mitochondrial DNA) indicated that the stress response does not depend on the rates of mitochondrial respiration. The anti-proliferative effect of resazurin was confirmed in native acute myelogenous leukemia (AML) blasts. In conclusion, the data suggest that resazurin triggers cellular ROS production and thereby initiates a stress response leading to mitochondrial dysfunction, reduced proliferation, autophagy, and cell degradation. The ability of cells to tolerate this type of stress may be important in toxicity and chemoresistance. © 2010 Wiley-Liss, Inc.

Effects of Ionizing Radiation on Human Adipose Derived Mesenchymal Stem Cells and their Differentiation towards the Osteoblastic Lineage

NASA Astrophysics Data System (ADS)

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

Radiation exposure and musculoskeletal disuse are among the major challenges during space missions. Astronauts face the problem to lose bone calcium due to uncoupling of bone formation and resorption. Bone forming osteoblasts can be derived from the undifferentiated mesenchymal stem cell compartment (MSC). In this study, the ability of human adipose tissue derived stem cells (ATSC) to differentiate into the osteoblastic lineage was examined after radiation exposure in presence of medium supplementation with osteogenic additives (ß-glycerophosphate, ascorbic acid and dexamethasone). The SAOS-2 cell line (human osteosarcoma cell line) was used as control for osteoblastic differentiation. Changes in cellular morphology, cell cycle progression, as well as cellular radiation sensitivity were characterized after ionizing radiation exposure with X-rays and heavy ions (Ti). Rapidly proliferating SAOS-2 cells are less radiation-sensitive than slowly proliferating ATSC cells after X-ray (CFA: dose effect curves show D0 values of 1 Gy and 0.75 Gy for SAOS-2 and ATSC, respectively) exposure. Heavy ion (Ti) exposure resulted in a greater extent of cells accumulating in the G2/M phase of the cell cycle in a dose-dependent manner when compared to X-ray exposure. Differentiation of cells towards the osteoblastic lineage was quantified by hydroxyapatite (HA) deposition using Lonza OsteoImageTM mineralization assay. The deposition of HA after X- and Ti-irradiation for highly proliferating SAOS-2 cells showed a dose-dependent time delay while slowly proliferating ATSC showed no effect from radiation exposure. More detailed investigation is required to reveal the radiation dependent mechanism of bone loss in astronauts.

Long noncoding RNA MINCR regulates cellular proliferation, migration, and invasion in hepatocellular carcinoma.

PubMed

Cao, Jinyu; Zhang, Deyuan; Zeng, Liangtao; Liu, Fanrong

2018-06-01

Accumulating evidence indicates that long noncoding RNAs (lncRNAs) are aberrantly expressed in many cancer types, including hepatocellular carcinoma (HCC). lncRNA MYC-induced long non-coding RNA (MINCR) were revealed to be markedly up-regulated in gallbladder cancer and Burkitt lymphoma cells. However, the biological role and function of MINCR in HCC progression are still unknown. The expression of MINCR in HCC tissues and cell lines was determined using quantitative real-time polymerase chain reaction assays. The effects of MINCR in HCC cell proliferation, migration, and invasion were determined using cell-counting kit 8 (CCK8) assay, wound healing assay, and Transwell assays in vitro. MINCR expression was up-regulated in HCC tissues and cell lines as compared with that in the negative control. The decreased expression of MINCR in vitro markedly inhibited HCC cell proliferation, migration, and invasion. Our results showed that MINCR is important in HCC development and may act as a therapeutic target that regulates HCC cellular proliferation, migration, and invasion, which are involved in HCC tumorigenesis. To the best of our know ledge, MINCR in HCC has not been studied. Our findings showed that this study is the first to reveal that MINCR may act as a therapeutic target in HCC. The in-depth exploration of the molecular mechanism is required to illuminate the molecular mechanisms of MINCR in HCC development. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

The effect of burn injury on CD8+ and CD4+ T cells in an irradiation model of homeostatic proliferation.

PubMed

Buchanan, Ian B; Maile, Robert; Frelinger, Jeffrey A; Fair, Jeffrey H; Meyer, Anthony A; Cairns, Bruce A

2006-11-01

Homeostatic proliferation of T cells has recently been shown to be an important mechanism in the host response to infection. However, its role in the T cell response to burn injury is unknown. In this study, we examine the effect of burn injury on CD4+ and CD8+ T cell homeostatic proliferation after irradiation. Wild-type C57BL/6 female mice were irradiated with six grays ionizing radiation and 48 hours later, syngeneic whole splenocytes or purified CD4+ or CD8+ T cells labeled with carboxy-fluorescein diacetate, succinimidyl ester were adoptively transferred. Two days later, mice underwent a 20% burn injury, followed by splenocyte harvest 3 and 10 days after injury. Burn mice demonstrate increased splenic cellularity and CD8+ T cell proliferation after adoptive transfer of either purified CD8+ cells or whole spleen populations compared with unburned (sham) mice. In contrast, CD4+ T cell proliferation after burn injury is unchanged after adoptive transfer of whole spleen cells and drastically decreased after adoptive transfer of a purified CD4+ population compared with sham mice. Ten days after burn injury CD8+ T cells continue to demonstrate greater proliferation than CD4+ T cells. CD8+ T cells are more robust than CD4+ T cells in their proliferative response after burn injury. In addition, CD8+ T cell proliferation appears less reliant on other immune cells than purified CD4+ T cell proliferation. These data reiterate the importance of CD8+ T cells in the initial immune response to burn injury.

Immunomodulatory and cellular anti-oxidant activities of caffeic, ferulic, and p-coumaric phenolic acids: a structure-activity relationship study.

PubMed

Kilani-Jaziri, Soumaya; Mokdad-Bzeouich, Imen; Krifa, Mounira; Nasr, Nouha; Ghedira, Kamel; Chekir-Ghedira, Leila

2017-10-01

Many studies have been performed to assess the potential utility of natural products as immunomodulatory agents to enhance host responses and to reduce damage to the human body. To determine whether phenolic compounds (caffeic, ferulic, and p-coumaric acids) have immunomodulatory effects and clarify which types of immune effector cells are stimulated in vitro, we evaluated their effect on splenocyte proliferation and lysosomal enzyme activity. We also investigated the activity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL). In addition, induction of the cellular antioxidant activity in splenocytes, macrophages, and red blood cells was determined by measuring the fluorescence of the DCF product. The study first results indicated that caffeic, ferulic, and p-coumaric acids significantly promote LPS-stimulated splenocyte proliferation, suggesting a potential activation of B cells, and enhanced humoral immune response in hosts treated by the tested natural products. Phenolic acids significantly enhanced the killing activity of isolated NK and CTL cells but had negligible effects on mitogen-induced proliferation of splenic T cells. We showed that caffeic acid enhances lysosomal enzyme activity in murine peritoneal macrophages, suggesting a potential role in activating such cells. Immunomodulatory activity was concomitant with the cellular antioxidant effect in macrophages and splenocytes of caffeic and ferulic acids. We conclude from this study that caffeic, ferulic, and p-coumaric acids exhibited an immunomodulatory effect which could be ascribed, in part, to their cytoprotective effect via their antioxidant capacity. Furthermore, these results suggest that these natural products could be potentially used to modulate immune cell functions in physiological and pathological conditions.

Induction of human breast cell carcinogenesis by triclocarban and intervention by curcumin

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

Sood, Shilpa; Choudhary, Shambhunath; Wang, Hwa-Chain Robert, E-mail: hcrwang@utk.edu

Highlights: •Triclocarban exposure induces breast epithelial cell carcinogenesis. •Triclocarban induces the Erk–Nox pathway, ROS elevation, and DNA damage. •Physiological doses of triclocarban induce cellular carcinogenesis. •Non-cytotoxic curcumin blocks triclocarban-induced carcinogenesis and pathways. -- Abstract: More than 85% of breast cancers are sporadic and attributable to long-term exposure to environmental carcinogens and co-carcinogens. To identify co-carcinogens with abilities to induce cellular pre-malignancy, we studied the activity of triclocarban (TCC), an antimicrobial agent commonly used in household and personal care products. Here, we demonstrated, for the first time, that chronic exposure to TCC at physiologically-achievable nanomolar concentrations resulted in progressive carcinogenesis ofmore » human breast cells from non-cancerous to pre-malignant. Pre-malignant carcinogenesis was measured by increasingly-acquired cancer-associated properties of reduced dependence on growth factors, anchorage-independent growth and increased cell proliferation, without acquisition of cellular tumorigenicity. Long-term TCC exposure also induced constitutive activation of the Erk–Nox pathway and increases of reactive oxygen species (ROS) in cells. A single TCC exposure induced transient induction of the Erk–Nox pathway, ROS elevation, increased cell proliferation, and DNA damage in not only non-cancerous breast cells but also breast cancer cells. Using these constitutively- and transiently-induced changes as endpoints, we revealed that non-cytotoxic curcumin was effective in intervention of TCC-induced cellular pre-malignancy. Our results lead us to suggest that the co-carcinogenic potential of TCC should be seriously considered in epidemiological studies to reveal the significance of TCC in the development of sporadic breast cancer. Using TCC-induced transient and constitutive endpoints as targets will likely help identify non-cytotoxic preventive agents, such as curcumin, effective in suppressing TCC-induced cellular pre-malignancy.« less

Induction of human breast cell carcinogenesis by triclocarban and intervention by curcumin

PubMed Central

Sood, Shilpa; Choudhary, Shambhunath; Wang, Hwa-Chain Robert

2013-01-01

More than 85% of breast cancers are sporadic and attributable to long-term exposure to environmental carcinogens and co-carcinogens. To identify co-carcinogens with abilities to induce cellular pre-malignancy, we studied the activity of triclocarban (TCC), an antimicrobial agent commonly used in household and personal care products. Here, we demonstrated, for the first time, that chronic exposure to TCC at physiologically-achievable nanomolar concentrations resulted in progressive carcinogenesis of human breast cells from non-cancerous to pre-malignant. Pre-malignant carcinogenesis was measured by increasingly-acquired cancer-associated properties of reduced dependence on growth factors, anchorage- independent growth and increased cell proliferation, without acquisition of cellular tumorigenicity. Long-term TCC exposure also induced constitutive activation of the Erk–Nox pathway and increases of reactive oxygen species (ROS) in cells. A single TCC exposure induced transient induction of the Erk–Nox pathway, ROS elevation, increased cell proliferation, and DNA damage in not only non-cancerous breast cells but also breast cancer cells. Using these constitutively- and transiently-induced changes as endpoints, we revealed that non-cytotoxic curcumin was effective in intervention of TCC-induced cellular pre-malignancy. Our results lead us to suggest that the co-carcinogenic potential of TCC should be seriously considered in epidemiological studies to reveal the significance of TCC in the development of sporadic breast cancer. Using TCC-induced transient and constitutive endpoints as targets will likely help identify non-cytotoxic preventive agents, such as curcumin, effective in suppressing TCC-induced cellular pre-malignancy. PMID:23942114

Distinct functional outputs of PTEN signalling are controlled by dynamic association with β-arrestins

PubMed Central

Lima-Fernandes, Evelyne; Enslen, Hervé; Camand, Emeline; Kotelevets, Larissa; Boularan, Cédric; Achour, Lamia; Benmerah, Alexandre; Gibson, Lucien C D; Baillie, George S; Pitcher, Julie A; Chastre, Eric; Etienne-Manneville, Sandrine; Marullo, Stefano; Scott, Mark G H

2011-01-01

The tumour suppressor PTEN (phosphatase and tensin deleted on chromosome 10) regulates major cellular functions via lipid phosphatase-dependent and -independent mechanisms. Despite its fundamental pathophysiological importance, how PTEN's cellular activity is regulated has only been partially elucidated. We report that the scaffolding proteins β-arrestins (β-arrs) are important regulators of PTEN. Downstream of receptor-activated RhoA/ROCK signalling, β-arrs activate the lipid phosphatase activity of PTEN to negatively regulate Akt and cell proliferation. In contrast, following wound-induced RhoA activation, β-arrs inhibit the lipid phosphatase-independent anti-migratory effects of PTEN. β-arrs can thus differentially control distinct functional outputs of PTEN important for cell proliferation and migration. PMID:21642958

Structural Development, Cellular Differentiation and Proliferation of the Respiratory Epithelium in the Bovine Fetal Lung.

PubMed

Drozdowska, J; Cousens, C; Finlayson, J; Collie, D; Dagleish, M P

2016-01-01

Fetal bovine lung samples of 11 different gestational ages were assigned to a classical developmental stage based on histological morphology. Immunohistochemistry was used to characterize the morphology of forming airways, proliferation rate of airway epithelium and the presence of epithelial cell types (i.e. ciliated cells, club cells, neuroepithelial cells (NECs) and type II pneumocytes). Typical structural organization of pseudoglandular (84-98 days gestational age [DGA]), canalicular (154-168 DGA) and alveolar (224-266 DGA) stages was recognized. In addition, transitional pseudoglandular-canalicular (112-126 DGA) and canalicular-saccular (182 DGA) morphologies were present. The embryonic stage was not observed. A significantly (P <0.05) higher proliferation rate of pulmonary epithelium, on average 5.5% and 4.4% in bronchi and bronchioles, respectively, was present in the transitional pseudoglandular-canalicular phase (112-126 DGA) compared with all other phases, while from 8 weeks before term (224-266 DGA) proliferation had almost ceased. The first epithelial cells identified by specific marker proteins in the earliest samples available for study (84 DGA) were ciliated cells and NECs. Club cells were present initially at 112 DGA and type II pneumocytes at 224 DGA. At the latest time points (224-226 DGA) these latter cell types were still present at a much lower percentage compared with adult cattle. This study characterized bovine fetal lung development by histological morphology and cellular composition of the respiratory epithelium and suggests that the apparent structural anatomical maturity of the bovine lung at term is not matched by functional maturity of the respiratory epithelium. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modulation of Estrogen Response Element-Driven Gene Expressions and Cellular Proliferation with Polar Directions by Designer Transcription Regulators

PubMed Central

Muyan, Mesut; Güpür, Gizem; Yaşar, Pelin; Ayaz, Gamze; User, Sırma Damla; Kazan, Hasan Hüseyin; Huang, Yanfang

2015-01-01

Estrogen receptor α (ERα), as a ligand-dependent transcription factor, mediates 17β-estradiol (E2) effects. ERα is a modular protein containing a DNA binding domain (DBD) and transcription activation domains (AD) located at the amino- and carboxyl-termini. The interaction of the E2-activated ERα dimer with estrogen response elements (EREs) of genes constitutes the initial step in the ERE-dependent signaling pathway necessary for alterations of cellular features. We previously constructed monomeric transcription activators, or monotransactivators, assembled from an engineered ERE-binding module (EBM) using the ERα-DBD and constitutively active ADs from other transcription factors. Monotransactivators modulated cell proliferation by activating and repressing ERE-driven gene expressions that simulate responses observed with E2-ERα. We reasoned here that integration of potent heterologous repression domains (RDs) into EBM could generate monotransrepressors that alter ERE-bearing gene expressions and cellular proliferation in directions opposite to those observed with E2-ERα or monotransactivators. Consistent with this, monotransrepressors suppressed reporter gene expressions that emulate the ERE-dependent signaling pathway. Moreover, a model monotransrepressor regulated DNA synthesis, cell cycle progression and proliferation of recombinant adenovirus infected ER-negative cells through decreasing as well as increasing gene expressions with polar directions compared with E2-ERα or monotransactivator. Our results indicate that an ‘activator’ or a ‘repressor’ possesses both transcription activating/enhancing and repressing/decreasing abilities within a chromatin context. Offering a protein engineering platform to alter signal pathway-specific gene expressions and cell growth, our approach could also be used for the development of tools for epigenetic modifications and for clinical interventions wherein multigenic de-regulations are an issue. PMID:26295471

Global transcriptomic analysis of model human cell lines exposed to surface-modified gold nanoparticles: the effect of surface chemistry

NASA Astrophysics Data System (ADS)

Grzincic, E. M.; Yang, J. A.; Drnevich, J.; Falagan-Lotsch, P.; Murphy, C. J.

2015-01-01

Gold nanoparticles (Au NPs) are attractive for biomedical applications not only for their remarkable physical properties, but also for the ease of which their surface chemistry can be manipulated. Many applications involve functionalization of the Au NP surface in order to improve biocompatibility, attach targeting ligands or carry drugs. However, changes in cells exposed to Au NPs of different surface chemistries have been observed, and little is known about how Au NPs and their surface coatings may impact cellular gene expression. The gene expression of two model human cell lines, human dermal fibroblasts (HDF) and prostate cancer cells (PC3) was interrogated by microarray analysis of over 14 000 human genes. The cell lines were exposed to four differently functionalized Au NPs: citrate, poly(allylamine hydrochloride) (PAH), and lipid coatings combined with alkanethiols or PAH. Gene functional annotation categories and weighted gene correlation network analysis were used in order to connect gene expression changes to common cellular functions and to elucidate expression patterns between Au NP samples. Coated Au NPs affect genes implicated in proliferation, angiogenesis, and metabolism in HDF cells, and inflammation, angiogenesis, proliferation apoptosis regulation, survival and invasion in PC3 cells. Subtle changes in surface chemistry, such as the initial net charge, lability of the ligand, and underlying layers greatly influence the degree of expression change and the type of cellular pathway affected.Gold nanoparticles (Au NPs) are attractive for biomedical applications not only for their remarkable physical properties, but also for the ease of which their surface chemistry can be manipulated. Many applications involve functionalization of the Au NP surface in order to improve biocompatibility, attach targeting ligands or carry drugs. However, changes in cells exposed to Au NPs of different surface chemistries have been observed, and little is known about how Au NPs and their surface coatings may impact cellular gene expression. The gene expression of two model human cell lines, human dermal fibroblasts (HDF) and prostate cancer cells (PC3) was interrogated by microarray analysis of over 14 000 human genes. The cell lines were exposed to four differently functionalized Au NPs: citrate, poly(allylamine hydrochloride) (PAH), and lipid coatings combined with alkanethiols or PAH. Gene functional annotation categories and weighted gene correlation network analysis were used in order to connect gene expression changes to common cellular functions and to elucidate expression patterns between Au NP samples. Coated Au NPs affect genes implicated in proliferation, angiogenesis, and metabolism in HDF cells, and inflammation, angiogenesis, proliferation apoptosis regulation, survival and invasion in PC3 cells. Subtle changes in surface chemistry, such as the initial net charge, lability of the ligand, and underlying layers greatly influence the degree of expression change and the type of cellular pathway affected. Electronic supplementary information (ESI) available: UV-Vis spectra of Au NPs, the most significantly changed genes of HDF cells after Au NP incubation under GO accession number GO:0007049 ``cell cycle'', detailed information about the primer/probe sets used for RT-PCR validation of results. See DOI: 10.1039/c4nr05166a

Ghrelin inhibits proliferation and increases T-type Ca{sup 2+} channel expression in PC-3 human prostate carcinoma cells

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

Diaz-Lezama, Nundehui; Hernandez-Elvira, Mariana; Sandoval, Alejandro

Research highlights: {yields} Ghrelin decreases prostate carcinoma PC-3 cells proliferation. {yields} Ghrelin favors apoptosis in PC-3 cells. {yields} Ghrelin increase in intracellular free Ca{sup 2+} levels in PC-3 cells. {yields} Grelin up-regulates expression of T-type Ca{sup 2+} channels in PC-3 cells. {yields} PC-3 cells express T-channels of the Ca{sub V}3.1 and Ca{sub V}3.2 subtype. -- Abstract: Ghrelin is a multifunctional peptide hormone with roles in growth hormone release, food intake and cell proliferation. With ghrelin now recognized as important in neoplastic processes, the aim of this report is to present findings from a series of in vitro studies evaluating themore » cellular mechanisms involved in ghrelin regulation of proliferation in the PC-3 human prostate carcinoma cells. The results showed that ghrelin significantly decreased proliferation and induced apoptosis. Consistent with a role in apoptosis, an increase in intracellular free Ca{sup 2+} levels was observed in the ghrelin-treated cells, which was accompanied by up-regulated expression of T-type voltage-gated Ca{sup 2+} channels. Interestingly, T-channel antagonists were able to prevent the effects of ghrelin on cell proliferation. These results suggest that ghrelin inhibits proliferation and may promote apoptosis by regulating T-type Ca{sup 2+} channel expression.« less

Apoptosis triggered by pyropheophorbide-α methyl ester-mediated photodynamic therapy in a giant cell tumor in bone

NASA Astrophysics Data System (ADS)

Li, K.-T.; Zhang, J.; Duan, Q.-Q.; Bi, Y.; Bai, D.-Q.; Ou, Y.-S.

2014-06-01

A giant cell tumor in bone is the common primary bone tumor with aggressive features, occurring mainly in young adults. Photodynamic therapy is a new therapeutic technique for tumors. In this study, we investigated the effects of Pyropheophorbide-α methyl ester (MPPa)-mediated photodynamic therapy on the proliferation of giant cell tumor cells and its mechanism of action. Cell proliferation was evaluated using an MTT assay. Cellular apoptosis was detected by Hoechst nuclear staining, and flow cytometric assay. Mitochondrial membrane potential changes and cytochrome c, caspase-9, caspase-3, and Bcl-2 expression was assessed. Finally, we found that MPPa-mediated photodynamic therapy could effectively suppress the proliferation of human giant cell tumor cells and induce apoptosis. The mitochondrial pathway was involved in the MPPa-photodynamic therapy-induced apoptosis.

Immortalization of canine adipose-derived mesenchymal stem cells and their seminiferous tubule transplantation.

PubMed

Fang, Jia; Wei, Yudong; Teng, Xin; Zhao, Shanting; Hua, Jinlian

2018-04-01

Adipose-derived mesenchymal stem cells (ADSCs) are proven to provide good effects in numerous tissue engineering application and other cell-based therapies. However, the difficulty in the proliferation of ADSCs, known as the "Hayflick limit" in vitro, limits their clinical application. Here, we immortalized canine ADSCs (cADSCs) with SV40 gene and transplanted them into busulfan-induced seminiferous tubules of infertile mice. The proliferation of these immortalized cells was improved significantly. Then, cellular differentiation assays showed that the immortalized cADSCs could differentiate into three-germ-layer cells, osteogenesis, chondrogenesis, adipogenesis phenotypes, and primordial germ cell-like cells (PGCLCs). In addition, the immortalized cADSCs can proliferate in the busulfan-induced seminiferous tubules of infertile mice. These findings confirmed that the immortalized cADSCs maintain the criteria of cADSCs. © 2017 Wiley Periodicals, Inc.

Sensitivity to methylmercury toxicity is enhanced in oxoguanine glycosylase 1 knockout murine embryonic fibroblasts and is dependent on cellular proliferation capacity

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

Ondovcik, Stephanie L.; Tamblyn, Laura; McPherson, John Peter

2013-07-01

Methylmercury (MeHg) is a persistent environmental contaminant with potent neurotoxic action for which the underlying molecular mechanisms remain to be conclusively delineated. Our objectives herein were twofold: first, to corroborate our previous findings of an increased sensitivity of spontaneously-immortalized oxoguanine glycosylase 1-null (Ogg1{sup −/−}) murine embryonic fibroblasts (MEFs) to MeHg through generation of Simian virus 40 (SV40) large T antigen-immortalized wild-type and Ogg1{sup −/−} MEFs; and second, to determine whether MeHg toxicity is proliferation-dependent. As with the spontaneously-immortalized cells used previously, the SV40 large T antigen-immortalized cells exhibited similar tendencies to undergo MeHg-initiated cell cycle arrest, with increased sensitivity inmore » the Ogg1{sup −/−} MEFs as measured by clonogenic survival and DNA damage. Compared to exponentially growing cells, those seeded at a higher density exhibited compromised proliferation, which proved protective against MeHg-mediated cell cycle arrest and induction of DNA double strand breaks (DSBs), measured by phosphorylation of the core histone H2A variant (H2AX) on serine 139 (γH2AX), and by its functional confirmation by micronucleus assessment. This enhanced sensitivity of Ogg1{sup −/−} MEFs to MeHg toxicity using discrete SV40 immortalization corroborates our previous studies, and suggests a novel role for OGG1 in minimizing MeHg-initiated DNA lesions that trigger replication-associated DSBs. Furthermore, proliferative capacity may determine MeHg toxicity in vivo and in utero. Accordingly, variations in cellular proliferative capacity and interindividual variability in repair activity may modulate the risk of toxicological consequences following MeHg exposure. - Highlights: • SV40 large T antigen-immortalized Ogg1{sup −/−} cells are more sensitive to MeHg. • Sensitivity to MeHg is dependent on cellular proliferation capacity. • OGG1 maintains genomic integrity following MeHg-initiated DNA damage. • OGG1 may limit MeHg-initiated DNA lesions that trigger replication-associated DSBs. • Variations in proliferation and repair activity may modulate toxicological risk.« less

Tobacco components stimulate Akt-dependent proliferation and NFkappaB-dependent survival in lung cancer cells.

PubMed

Tsurutani, Junji; Castillo, S Sianna; Brognard, John; Granville, Courtney A; Zhang, Chunyu; Gills, Joell J; Sayyah, Jacqueline; Dennis, Phillip A

2005-07-01

Retrospective studies have shown that patients with tobacco-related cancers who continue to smoke after their diagnoses have lower response rates and shorter median survival compared with patients who stop smoking. To provide insight into the biologic basis for these clinical observations, we tested whether two tobacco components, nicotine or the tobacco-specific carcinogen, 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), could activate the Akt pathway and increase lung cancer cell proliferation and survival. Nicotine or NNK, rapidly and potently, activated Akt in non-small cell lung cancer (NSCLC) or small cell lung cancer (SCLC) cells. Nicotinic activation of Akt increased phosphorylation of multiple downstream substrates of Akt in a time-dependent manner, including GSK-3, FKHR, tuberin, mTOR and S6K1. Since nicotine or NNK bind to cell surface nicotinic acetylcholine receptors (nAchR), we used RT-PCR to assess expression of nine alpha and three beta nAchR subunits in five NSCLC cell lines and two types of primary lung epithelial cells. NSCLC cells express multiple nAchR subunits in a cell line-specific manner. Agonists of alpha3/alpha4 or alpha7 subunits activated Akt in a time-dependent manner, suggesting that tobacco components utilize these subunits to activate Akt. Cellular outcomes after nicotine or NNK administration were also assessed. Nicotine or NNK increased proliferation of NSCLC cells in an Akt-dependent manner that was closely linked with changes in cyclin D1 expression. Despite similar induction of proliferation, only nicotine decreased apoptosis caused by serum deprivation and/or chemotherapy. Protection conferred by nicotine was NFkappaB-dependent. Collectively, these results identify tobacco component-induced, Akt-dependent proliferation and NFkappaB-dependent survival as cellular processes that could underlie the detrimental effects of smoking in cancer patients.

TORC1 is required to balance cell proliferation and cell death in planarians

PubMed Central

Tu, Kimberly C.; Pearson, Bret J.; Alvarado, Alejandro Sánchez

2012-01-01

Multicellular organisms are equipped with cellular mechanisms that enable them to replace differentiated cells lost to normal physiological turnover, injury, and for some such as planarians, even amputation. This process of tissue homeostasis is generally mediated by adult stem cells (ASCs), tissue-specific stem cells responsible for maintaining anatomical form and function. To do so, ASCs must modulate the balance between cell proliferation, i.e. in response to nutrients, and that of cell death, i.e. in response to starvation or injury. But how these two antagonistic processes are coordinated remains unclear. Here, we explore the role of the core components of the TOR pathway during planarian tissue homeostasis and regeneration and identified an essential function for TORC1 in these two processes. RNAi-mediated silencing of TOR in intact animals resulted in a significant increase in cell death, whereas stem cell proliferation and stem cell maintenance were unaffected. Amputated animals failed to increase stem cell proliferation after wounding and displayed defects in tissue remodeling. Together, our findings suggest two distinct roles for TORC1 in planarians. TORC1 is required to modulate the balance between cell proliferation and cell death during normal cell turnover and in response to nutrients. In addition, it is required to initiate appropriate stem cell proliferation during regeneration and for proper tissue remodeling to occur to maintain scale and proportion. PMID:22445864

Cellular pressure and volume regulation and implications for cell mechanics

NASA Astrophysics Data System (ADS)

Jiang, Hongyuan; Sun, Sean

2013-03-01

In eukaryotic cells, small changes in cell volume can serve as important signals for cell proliferation, death and migration. Volume and shape regulation also directly impacts the mechanics of the cell and multi-cellular tissues. Recent experiments found that during mitosis, eukaryotic cells establish a preferred steady volume and pressure, and the steady volume and pressure can robustly adapt to large osmotic shocks. Here we develop a mathematical model of cellular pressure and volume regulation, incorporating essential elements such as water permeation, mechano-sensitive channels, active ion pumps and active stresses in the actomyosin cortex. The model can fully explain the available experimental data, and predicts the cellular volume and pressure for several models of cell cortical mechanics. Furthermore, we show that when cells are subjected to an externally applied load, such as in an AFM indentation experiment, active regulation of volume and pressure leads to complex cellular response. We found the cell stiffness highly depends on the loading rate, which indicates the transport of water and ions might contribute to the observed viscoelasticity of cells.

Proliferating cellular nuclear antigen expression as a marker of perivascular macrophages in simian immunodeficiency virus encephalitis.

PubMed

Williams, Kenneth; Schwartz, Annette; Corey, Sarah; Orandle, Marlene; Kennedy, William; Thompson, Brendon; Alvarez, Xavier; Brown, Charlie; Gartner, Suzanne; Lackner, Andrew

2002-08-01

Brain perivascular macrophages are a major target of simian immunodeficiency virus (SIV) infection in rhesus macaques and HIV infection in humans. Perivascular macrophages are distinct from parenchymal microglia in their location, morphology, expression of myeloid markers, and turnover in the CNS. In contrast to parenchymal microglia, perivascular macrophages are continuously repopulated by blood monocytes, which undergo maturation to macrophages on entering the central nervous system (CNS). We studied differences in monocyte/macrophages in vivo that might account for preferential infection of perivascular macrophages by SIV. In situ hybridization for SIV and proliferating cellular nuclear antigen (PCNA) immunohistochemistry demonstrated that SIV-infected and PCNA-positive cells were predominantly found in perivascular cuffs of viremic animals and in histopathological lesions that characterize SIV encephalitis (SIVE) in animals with AIDS. Multilabel techniques including double-label immunohistochemistry and combined in situ hybridization and immunofluorescence confocal microscopy revealed numerous infected perivascular macrophages that were PCNA-positive. Outside the CNS, SIV-infected, PCNA-expressing macrophage subpopulations were found in the small intestine and lung of animals with AIDS. While PCNA is used as a marker of cell proliferation it is also strongly expressed in non-dividing cells undergoing DNA synthesis and repair. Therefore, more specific markers for cell proliferation including Ki-67, topoisomerase IIalpha, and bromodeoxyuridine (BrdU) incorporation were used which indicated that PCNA-positive cells within SIVE lesions were not proliferating. These observations are consistent with perivascular macrophages as terminally differentiated, non-dividing cells and underscores biological differences that could potentially define mechanisms of preferential, productive infection of perivascular macrophages in the rhesus macaque model of neuroAIDS. These studies suggest that within CNS and non-CNS tissues there exist subpopulations of macrophages that are SIV-infected and express PCNA.

Comparison of cellular responses of mesenchymal stem cells derived from bone marrow and synovium on combined silk scaffolds.

PubMed

Liu, Haifeng; Wei, Xing; Ding, Xili; Li, Xiaoming; Zhou, Gang; Li, Ping; Fan, Yubo

2015-01-01

As a brand new member in mesenchymal stem cells (MSCs) families, synovium-derived mesenchymal stem cells (SMSCs) have been increasingly regarded as a promising therapeutic cell species for musculoskeletal regeneration. However, there are few reports mentioning ligamentogenesis of SMSCs and especially null for their engineering use towards ligament regeneration. The aim of this study was to investigate and compare the cellular responses of MSCs derived from bone marrow and synovium on combined silk scaffolds that can be used to determine the cell source most appropriate for tissue-engineered ligament. Rabbit SMSCs and bone marrow-derived mesenchymal stem cells (BMSCs) were isolated and cultured in vitro for two weeks after seeding on the combined silk scaffolds. Samples were studied and compared for their cellular morphology, proliferation, collagen production, gene, and protein expression of ligament-related extracellular matrix (ECM) markers. In addition, the two cell types were transfected with green fluorescent protein to evaluate their fate after implantation in an intraarticular environment of the knee joint. After 14 days of culturing, SMSCs showed a significant increase in proliferation as compared with BMSCs. The transcript and protein expression levels of ligament-related ECM markers in SMSCs were significantly higher than those in BMSCs. Moreover, 6 weeks postoperatively, more viable cells were presented in SMSC-loaded constructs than in BMSC-loaded constructs. Therefore, based on the cellular response in vitro and in vivo, SMSCs may represent a more suitable cell source than BMSCs for further study and development of tissue-engineered ligament. © 2014 Wiley Periodicals, Inc.

Expression of R132H mutational IDH1 in human U87 glioblastoma cells affects the SREBP1a pathway and induces cellular proliferation.

PubMed

Zhu, Jian; Cui, Gang; Chen, Ming; Xu, Qinian; Wang, Xiuyun; Zhou, Dai; Lv, Shengxiang; Fu, Linshan; Wang, Zhong; Zuo, Jianling

2013-05-01

Sterol regulatory element-binding protein-1a (SREBP1a) is a member of the SREBP family of transcription factors, which mainly controls homeostasis of lipids. SREBP1a can also activate the transcription of isocitrate dehydrogenase 1 (IDH1) by binding to its promoter region. IDH1 mutations, especially R132H mutation of IDH1, are a common feature of a major subset of human gliomas. There are few data available on the relationship between mutational IDH1 expression and SREBP1a pathway. In this study, we investigated cellular effects and SREBP1a pathway alterations caused by R132H mutational IDH1 expression in U87 cells. Two glioma cell lines, stably expressing mutational (U87/R132H) or wild type (U87/wt) IDH1, were established. A cell line, stably transfected with pcDNA3.1(+) (U87/vector), was generated as a control. Click-iT EdU assay, sulforhodamine B assay, and wound healing assay respectively showed that the expression of R132H induced cellular proliferation, cell growth, and cell migration. Western blot revealed that SREBP1 was increased in U87/R132H compared with that in U87/wt. Elevated SREBP1a and several its target genes, but not SREBP1c, were detected by real-time polymerase chain reaction in U87/R132H. All these findings indicated that R132H mutational IDH1 is involved in the regulation of proliferation, growth, and migration of glioma cells. These effects may partially be mediated by SREBP1a pathway.

Cannabinoid receptor signaling in progenitor/stem cell proliferation and differentiation.

PubMed

Galve-Roperh, Ismael; Chiurchiù, Valerio; Díaz-Alonso, Javier; Bari, Monica; Guzmán, Manuel; Maccarrone, Mauro

2013-10-01

Cannabinoids, the active components of cannabis (Cannabis sativa) extracts, have attracted the attention of human civilizations for centuries, much earlier than the discovery and characterization of their substrate of action, the endocannabinoid system (ECS). The latter is an ensemble of endogenous lipids, their receptors [in particular type-1 (CB1) and type-2 (CB2) cannabinoid receptors] and metabolic enzymes. Cannabinoid signaling regulates cell proliferation, differentiation and survival, with different outcomes depending on the molecular targets and cellular context involved. Cannabinoid receptors are expressed and functional from the very early developmental stages, when they regulate embryonic and trophoblast stem cell survival and differentiation, and thus may affect the formation of manifold adult specialized tissues derived from the three different germ layers (ectoderm, mesoderm and endoderm). In the ectoderm-derived nervous system, both CB1 and CB2 receptors are present in neural progenitor/stem cells and control their self-renewal, proliferation and differentiation. CB1 and CB2 show opposite patterns of expression, the former increasing and the latter decreasing along neuronal differentiation. Recently, endocannabinoid (eCB) signaling has also been shown to regulate proliferation and differentiation of mesoderm-derived hematopoietic and mesenchymal stem cells, with a key role in determining the formation of several cell types in peripheral tissues, including blood cells, adipocytes, osteoblasts/osteoclasts and epithelial cells. Here, we will review these new findings, which unveil the involvement of eCB signaling in the regulation of progenitor/stem cell fate in the nervous system and in the periphery. The developmental regulation of cannabinoid receptor expression and cellular/subcellular localization, together with their role in progenitor/stem cell biology, may have important implications in human health and disease. Copyright © 2013 Elsevier Ltd. All rights reserved.

Thickness sensing of hMSCs on collagen gel directs stem cell fate

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

Leong, Wen Shing; Tay, Chor Yong; Yu, Haiyang

Research highlights: {yields} hMSCs appeared to sense thin collagen gel (130 {mu}m) with higher effective modulus as compared to thick gel (1440 {mu}m). {yields} Control of collagen gel thickness can modulate cellular behavior, even stem cell fate (neuronal vs. Quiescent). {yields} Distinct cellular behavior of hMSCs on thin and thick collagen gel suggests long range interaction of hMSCs with collagen gel. -- Abstract: Mechanically compliant substrate provides crucial biomechanical cues for multipotent stem cells to regulate cellular fates such as differentiation, proliferation and maintenance of their phenotype. Effective modulus of which cells sense is not only determined by intrinsic mechanicalmore » properties of the substrate, but also the thickness of substrate. From our study, it was found that interference from underlying rigid support at hundreds of microns away could induce significant cellular response. Human mesenchymal stem cells (hMSCs) were cultured on compliant biological gel, collagen type I, of different thickness but identical ECM composition and local stiffness. The cells sensed the thin gel (130 {mu}m) as having a higher effective modulus than the thick gel (1440 {mu}m) and this was reflected in their changes in morphology, actin fibers structure, proliferation and tissue specific gene expression. Commitment into neuronal lineage was observed on the thin gel only. Conversely, the thick gel (1440 {mu}m) was found to act like a substrate with lower effective modulus that inhibited actin fiber polymerization. Stem cells on the thick substrate did not express tissue specific genes and remained at their quiescent state. This study highlighted the need to consider not only the local modulus but also the thickness of biopolymer gel coating during modulation of cellular responses.« less

Altered proliferation and networks in neural cells derived from idiopathic autistic individuals.

PubMed

Marchetto, Maria C; Belinson, Haim; Tian, Yuan; Freitas, Beatriz C; Fu, Chen; Vadodaria, Krishna; Beltrao-Braga, Patricia; Trujillo, Cleber A; Mendes, Ana P D; Padmanabhan, Krishnan; Nunez, Yanelli; Ou, Jing; Ghosh, Himanish; Wright, Rebecca; Brennand, Kristen; Pierce, Karen; Eichenfield, Lawrence; Pramparo, Tiziano; Eyler, Lisa; Barnes, Cynthia C; Courchesne, Eric; Geschwind, Daniel H; Gage, Fred H; Wynshaw-Boris, Anthony; Muotri, Alysson R

2017-06-01

Autism spectrum disorders (ASD) are common, complex and heterogeneous neurodevelopmental disorders. Cellular and molecular mechanisms responsible for ASD pathogenesis have been proposed based on genetic studies, brain pathology and imaging, but a major impediment to testing ASD hypotheses is the lack of human cell models. Here, we reprogrammed fibroblasts to generate induced pluripotent stem cells, neural progenitor cells (NPCs) and neurons from ASD individuals with early brain overgrowth and non-ASD controls with normal brain size. ASD-derived NPCs display increased cell proliferation because of dysregulation of a β-catenin/BRN2 transcriptional cascade. ASD-derived neurons display abnormal neurogenesis and reduced synaptogenesis leading to functional defects in neuronal networks. Interestingly, defects in neuronal networks could be rescued by insulin growth factor 1 (IGF-1), a drug that is currently in clinical trials for ASD. This work demonstrates that selection of ASD subjects based on endophenotypes unraveled biologically relevant pathway disruption and revealed a potential cellular mechanism for the therapeutic effect of IGF-1.

Immunomodulator 'mushroom beta glucan' induces Wnt/β catenin signalling and improves wound recovery in tilapia and rat skin: a histopathological study.

PubMed

Hsiao, Chien-Mei; Wu, Yu-Sheng; Nan, Fan-Hua; Huang, Shih-Ling; Chen, Lynette; Chen, Shiu-Nan

2016-12-01

The present study aims to investigate the effects of mushroom beta glucan (MBG) on wound recovery in partial hepatectomy (PH) in Nile tilapia (Oreochromis niloticus) and in rat skin wound healing examination. Following PH, we focussed on the effects on liver repair ability using in vitro and in vivo tests. In vitro, we examined whether the MBG has an impact on liver cell proliferation, mainly through 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assays and bromodeoxyuridine (BrdU) cell proliferation assay detection method. Results showed that MBG treatment was remarkable in enhancing cell proliferation of hepatocytes and in maintaining the cellular viability. Immunohistochemical staining to analyse Wnt/β-catenin signalling also showed that MBG has the effect of promoting cell proliferation of liver tissues after PH surgery. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

Effect of molecular weight and concentration of hyaluronan on cell proliferation and osteogenic differentiation in vitro

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

Zhao, Ningbo, E-mail: curl-zhao@163.com; Wang, Xin, E-mail: 394041230@qq.com; Qin, Lei, E-mail: qinlei30@126.com

Hyaluronan (HA), the simplest glycosaminoglycan and a major component of the extracellular matrix, exists in various tissues. It is involved in some critical biological procedures, including cellular signaling, cell adhesion and proliferation, and cell differentiation. The effect of molecular weight (MW) and concentration of HA on cell proliferation and differentiation was controversial. In this study, we investigated the effect of MW and concentration of HA on the proliferation and osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro. Results showed that high MW HA decreased the cell adhesion rate in a concentration-dependant manner. The cell adhesion rate was decreased bymore » increasing MW of HA. Cell proliferation was significantly enhanced by low MW HA (P < 0.05). The factorial analysis indicated that MW and concentration had an interactive effect on the cell adhesion rate and cell proliferation (P < 0.05). High MW HA increased the mRNA expressions of ALP, RUNX-2 and OCN. The higher the MW was, the higher the mRNA expressions were. The factorial analysis indicated that MW and concentration had an interactive effect on ALP mRNA expression (P < 0.05). HA of higher MW and higher concentration promoted bone formation. These findings provide some useful information in understanding the mechanism underlying the effect of MW and concentration of HA on cell proliferation and differentiation. - Highlights: • Effect of hyaluronan on cell proliferation and differentiation is evaluated in vitro. • Hyaluronan of low molecular weight increases cell proliferation. • Hyaluronan of high molecular weight promotes cell osteogenic differentiation. • Molecular weight and concentration of hyaluronan show interactive effect.« less

Primordial odontogenic tumor: Subepithelial expression of Syndecan-1 and Ki-67 suggests origin during early odontogenesis.

PubMed

Bologna-Molina, R; Mikami, T; Pereira-Prado, V; Tapia-Repetto, G; Pires, F R; Carlos, R; Mosqueda-Taylor, A

2018-03-01

Primordial odontogenic tumor (POT) is composed of variably cellular myxoid connective tissue, surrounded by cuboidal to columnar odontogenic epithelium resembling the inner epithelium of the enamel organ, which often invaginates into the underlying connective tissue. The tumor is delimited at least partially by a thin fibrous capsule. It derives from the early stages of tooth development. Syndecan-1 is a heparan sulfate proteoglycan that has a physiological role in several cellular functions, including maintenance of the epithelial architecture, cell-to-cell adhesion and interaction of cells with extracellular matrix, and with diverse growth factors, stimulating cell proliferation. Ki-67 is considered the gold standard as a cell proliferation marker. The aim of this study was to examine the expression of Syndecan-1 and Ki-67 proliferation index in POT and normal tooth germs to better understand the biological behavior of this tumor. Results showed that Syndecan-1 was more intensely expressed in subepithelial mesenchymal areas of POT, in a pattern that resembles the early stages of tooth development. The cell proliferation index (4.1%) suggests that POT is a slow growing tumor. Syndecan-1 expression in tooth germs in late cap and early bell stages was similar to POT, showing immunopositivity in subepithelial mesenchymal condensed areas. The immunohistochemical findings showed a pattern in which the population of subepithelial mesenchymal cells exhibited greater proliferative activity than the central portion of the dental papilla. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.

CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells.

PubMed

Jafari, Naser; Kim, Hyunju; Park, Rackhyun; Li, Liqing; Jang, Minsu; Morris, Andrew J; Park, Junsoo; Huang, Cai

2017-01-01

Increased expression of NOX4 protein is associated with cancer progression and metastasis but the role of NOX4 in cell proliferation and invasion is not fully understood. We generated NOX4 knockout HeLa cell lines using the CRISPR-Cas9 gene editing system to explore the cellular functions of NOX4. After transfection of CRISPR-Cas9 construct, we performed T7 endonuclease 1 assays and DNA sequencing to generate and identify insertion and deletion of the NOX4 locus. We confirmed the knockout of NOX4 by Western blotting. NOX4 knockout cell lines showed reduced cell proliferation with an increase of sub-G1 cell population and the decrease of S/G2/M population. Moreover, NOX4 deficiency resulted in a dramatic decrease in invadopodium formation and the invasive activity. In addition, NOX4 deficiency also caused a decrease in focal adhesions and cell migration in HeLa cells. These results suggest that NOX4 is required for both efficient proliferation and invasion of HeLa cells.

Age-related islet inflammation marks the proliferative decline of pancreatic beta-cells in zebrafish

PubMed Central

Tsakmaki, Anastasia; Mousavy Gharavy, S Neda; Murawala, Priyanka; Konantz, Judith; Birke, Sarah; Hodson, David J; Rutter, Guy A; Bewick, Gavin A

2018-01-01

The pancreatic islet, a cellular community harboring the insulin-producing beta-cells, is known to undergo age-related alterations. However, only a handful of signals associated with aging have been identified. By comparing beta-cells from younger and older zebrafish, here we show that the aging islets exhibit signs of chronic inflammation. These include recruitment of tnfα-expressing macrophages and the activation of NF-kB signaling in beta-cells. Using a transgenic reporter, we show that NF-kB activity is undetectable in juvenile beta-cells, whereas cells from older fish exhibit heterogeneous NF-kB activity. We link this heterogeneity to differences in gene expression and proliferation. Beta-cells with high NF-kB signaling proliferate significantly less compared to their neighbors with low activity. The NF-kB signalinghi cells also exhibit premature upregulation of socs2, an age-related gene that inhibits beta-cell proliferation. Together, our results show that NF-kB activity marks the asynchronous decline in beta-cell proliferation with advancing age. PMID:29624168

Invasion of Epithelial Cells and Proteolysis of Cellular Focal Adhesion Components by Distinct Types of Porphyromonas gingivalis Fimbriae

PubMed Central

Nakagawa, Ichiro; Inaba, Hiroaki; Yamamura, Taihei; Kato, Takahiro; Kawai, Shinji; Ooshima, Takashi; Amano, Atsuo

2006-01-01

Porphyromonas gingivalis fimbriae are classified into six types (types I to V and Ib) based on the fimA genes encoding FimA (a subunit of fimbriae), and they play a critical role in bacterial interactions with host tissues. In this study, we compared the efficiencies of P. gingivalis strains with distinct types of fimbriae for invasion of epithelial cells and for degradation of cellular focal adhesion components, paxillin, and focal adhesion kinase (FAK). Six representative strains with the different types of fimbriae were tested, and P. gingivalis with type II fimbriae (type II P. gingivalis) adhered to and invaded epithelial cells at significantly greater levels than the other strains. There were negligible differences in gingipain activities among the six strains; however, type II P. gingivalis apparently degraded intracellular paxillin in association with a loss of phosphorylation 30 min after infection. Degradation was blocked with cytochalasin D or in mutants with fimA disrupted. Paxillin was degraded by the mutant with Lys-gingipain disrupted, and this degradation was prevented by inhibition of Arg-gingipain activity by Nα-p-tosyl-l-lysine chloromethyl ketone. FAK was also degraded by type II P. gingivalis. Cellular focal adhesions with green fluorescent protein-paxillin macroaggregates were clearly destroyed, and this was associated with cellular morphological changes and microtubule disassembly. In an in vitro wound closure assay, type II P. gingivalis significantly inhibited cellular migration and proliferation compared to the cellular migration and proliferation observed with the other types. These results suggest that type II P. gingivalis efficiently invades epithelial cells and degrades focal adhesion components with Arg-gingipain, which results in cellular impairment during wound healing and periodontal tissue regeneration. PMID:16790749

Long noncoding RNA PANDA and scaffold-attachment-factor SAFA control senescence entry and exit.

PubMed

Puvvula, Pavan Kumar; Desetty, Rohini Devi; Pineau, Pascal; Marchio, Agnés; Moon, Anne; Dejean, Anne; Bischof, Oliver

2014-11-19

Cellular senescence is a stable cell cycle arrest that limits the proliferation of pre-cancerous cells. Here we demonstrate that scaffold-attachment-factor A (SAFA) and the long noncoding RNA PANDA differentially interact with polycomb repressive complexes (PRC1 and PRC2) and the transcription factor NF-YA to either promote or suppress senescence. In proliferating cells, SAFA and PANDA recruit PRC complexes to repress the transcription of senescence-promoting genes. Conversely, the loss of SAFA-PANDA-PRC interactions allows expression of the senescence programme. Accordingly, we find that depleting either SAFA or PANDA in proliferating cells induces senescence. However, in senescent cells where PANDA sequesters transcription factor NF-YA and limits the expression of NF-YA-E2F-coregulated proliferation-promoting genes, PANDA depletion leads to an exit from senescence. Together, our results demonstrate that PANDA confines cells to their existing proliferative state and that modulating its level of expression can cause entry or exit from senescence.

mTORC1 and p53

PubMed Central

Hasty, Paul; Sharp, Zelton Dave; Curiel, Tyler J.; Campisi, Judith

2013-01-01

A balance must be struck between cell growth and stress responses to ensure that cells proliferate without accumulating damaged DNA. This balance means that optimal cell proliferation requires the integration of pro-growth and stress-response pathways. mTOR (mechanistic target of rapamycin) is a pleiotropic kinase found in complex 1 (mTORC1). The mTORC1 pathway governs a response to mitogenic signals with high energy levels to promote protein synthesis and cell growth. In contrast, the p53 DNA damage response pathway is the arbiter of cell proliferation, restraining mTORC1 under conditions of genotoxic stress. Recent studies suggest a complicated integration of these pathways to ensure successful cell growth and proliferation without compromising genome maintenance. Deciphering this integration could be key to understanding the potential clinical usefulness of mTORC1 inhibitors like rapamycin. Here we discuss how these p53-mTORC1 interactions might play a role in the suppression of cancer and perhaps the development of cellular senescence and organismal aging. PMID:23255104

Possible influence of infrasound on glioma cell response to chemotherapy: a pilot study.

PubMed

Yount, Garret; Taft, Ryan; West, Jeremy; Moore, Dan

2004-04-01

To assess the response of cultured human tumor cells to infrasound in combination with conventional anticancer agents using an infrasound-emitting apparatus marketed as a therapeutic device. Two pilot experiments measured proliferation of cultured brain tumor cells exposed to three treatment conditions: infrasound emission alone, infrasound in combination with the chemotherapy 5-fluorouracil (5-FU), and infrasound in combination with ionizing radiation. Results from each experimental condition were compared to those from appropriate control conditions. A standard colony-forming efficiency assay was used to assess tumor cell proliferation. Tumor cell proliferation was not significantly altered by treatment with infrasound alone, nor did infrasound appear to influence cellular response to x-rays. There was a significant interaction between 5-FU and infrasound (P < 0.0001), however, evident in decreased colony formation. Further research is warranted to assess potential synergism between infrasound and 5-FU against tumor cell proliferation, and to investigate the possible therapeutic use of infrasound.

Effects of organophosphates on the regulation of mesenchymal stem cell proliferation and differentiation.

PubMed

Prugh, Amber M; Cole, Stephanie D; Glaros, Trevor; Angelini, Daniel J

2017-03-25

Mesenchymal stem cells (MSCs) are multipotent cells located within various adult tissues. Recent literature has reported that human bone marrow-derived MSCs express active acetylcholinesterase (AChE) and that disruption of AChE activity by organophosphate (OP) chemicals decreases the ability of MSCs to differentiate into osteoblasts. The potential role of AChE in regulating MSC proliferation and differentiation is currently unknown. In the present study, we demonstrate that MSCs exposed to OPs have both decreased AChE activity and abundance. In addition, exposure to these OPs induced cellular death while decreasing cellular proliferation. Exposures to these compounds also reduced the adipogenic/osteogenic differentiation potentials of the MSCs. To elucidate the possible role of AChE in MSCs signaling following OP exposure, we captured potential AChE binding partners by performing polyhistidine (His 8 )-tagged AChE pulldowns, followed by protein identification using liquid chromatography mass spectrometry (LC-MS). Using this method, we determined that the focal adhesion protein, vinculin, is a potential binding partner with AChE in MSCs and these initial findings were confirmed with follow-up co-immunoprecipitation experiments. Identifying AChE binding partners helps to determine potential pathways associated with MSC proliferation and differentiation, and this understanding could lead to the development of future MSC-based tissue repair therapies. Published by Elsevier B.V.

Relationship between peroxisome proliferator-activated receptor alpha activity and cellular concentration of 14 perfluoroalkyl substances in HepG2 cells.

PubMed

Rosenmai, Anna Kjerstine; Ahrens, Lutz; le Godec, Théo; Lundqvist, Johan; Oskarsson, Agneta

2018-02-01

Peroxisome proliferator-activated receptor alpha (PPARα) is a molecular target for perfluoroalkyl substances (PFASs). Little is known about the cellular uptake of PFASs and how it affects the PPARα activity. We investigated the relationship between PPARα activity and cellular concentration in HepG2 cells of 14 PFASs, including perfluoroalkyl carboxylates (PFCAs), perfluoroalkyl sulfonates and perfluorooctane sulfonamide (FOSA). Cellular concentrations were determined by high-performance liquid chromatography-tandem mass spectrometry and PPARα activity was determined in transiently transfected cells by reporter gene assay. Cellular uptake of the PFASs was low (0.04-4.1%) with absolute cellular concentrations in the range 4-2500 ng mg -1 protein. Cellular concentration of PFCAs increased with perfluorocarbon chain length up to perfluorododecanoate. PPARα activity of PFCAs increased with chain length up to perfluorooctanoate. The maximum induction of PPARα activity was similar for short-chain (perfluorobutanoate and perfluoropentanoate) and long-chain PFCAs (perfluorododecanoate and perfluorotetradecanoate) (approximately twofold). However, PPARα activities were induced at lower cellular concentrations for the short-chain homologs compared to the long-chain homologs. Perfluorohexanoate, perfluoroheptanoate, perfluorooctanoate, perfluorononanoate (PFNA) and perfluorodecanoate induced PPARα activities >2.5-fold compared to controls. The concentration-response relationships were positive for all the tested compounds, except perfluorooctane sulfonate PFOS and FOSA, and were compound-specific, as demonstrated by differences in the estimated slopes. The relationships were steeper for PFCAs with chain lengths up to and including PFNA than for the other studied PFASs. To our knowledge, this is the first report establishing relationships between PPARα activity and cellular concentration of a broad range of PFASs. Copyright © 2017 John Wiley & Sons, Ltd.

Fabrication of mediator-free hybrid nano-interfaced electrochemical biosensor for monitoring cancer cell proliferation.

PubMed

Madhurantakam, Sasya; Jayanth Babu, K; Balaguru Rayappan, John Bosco; Krishnan, Uma Maheswari

2017-01-15

Glucose, a chief energy source in cellular metabolism, has a significant role in cell proliferation. Cancer cells utilize more glucose than normal cells to meet the energy demand arising due to their uncontrolled proliferation. The present work reports the development of a nano-interfaced amperometric biosensor for rapid and accurate monitoring of glucose utilization by cancer cells. A hybrid nano-interface comprising a blend of carbon nanotubes (CNTs) and graphene (GR) was employed to enhance the surface area of the working electrode and favour direct electron transfer. Glucose oxidase (GOx) immobilized on the interface serves as the sensing element due to its high selectivity and sensitivity towards glucose. Utilization of glucose was monitored at pre-determined time intervals in MiaPaCa-2 cancer cells. The results obtained from the amperometric technique were compared with the values obtained from a commercial glucometer. Alamar blue assay was performed to check the proliferation rate of the cells. A good correlation was obtained between the proliferation rate and glucose utilization. The designed biosensor was found to be unaffected by the presence of potential interferents and hence may serve as a novel in vitro tool to rapidly quantify the proliferation rates of cancer cells in response to different treatment strategies. Copyright © 2016 Elsevier B.V. All rights reserved.

Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts

PubMed Central

Gupta, Manoj K.; Teo, Adrian Kee Keong; Rao, Tata Nageswara; Bhatt, Shweta; Kleinridders, Andre; Shirakawa, Jun; Takatani, Tomozumi; Hu, Jiang; De Jesus, Dario F.; Windmueller, Rebecca; Wagers, Amy J.

2015-01-01

The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. Significance The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells. PMID:26253715

Estradiol induces endothelial cell migration and proliferation through estrogen receptor-enhanced RhoA/ROCK pathway.

PubMed

Oviedo, Pilar J; Sobrino, Agua; Laguna-Fernandez, Andrés; Novella, Susana; Tarín, Juan J; García-Pérez, Miguel-Angel; Sanchís, Juan; Cano, Antonio; Hermenegildo, Carlos

2011-03-30

Migration and proliferation of endothelial cells are involved in re-endothelialization and angiogenesis, two important cardiovascular processes that are increased in response to estrogens. RhoA, a small GTPase which controls multiple cellular processes, is involved in the control of cell migration and proliferation. Our aim was to study the role of RhoA on estradiol-induced migration and proliferation and its dependence on estrogen receptors activity. Human umbilical vein endothelial cells were stimulated with estradiol, in the presence or absence of ICI 182780 (estrogen receptors antagonist) and Y-27632 (Rho kinase inhibitor). Estradiol increased Rho GEF-1 gene expression and RhoA (gene and protein expression and activity) in an estrogen receptor-dependent manner. Cell migration, stress fiber formation and cell proliferation were increased in response to estradiol and were also dependent on the estrogen receptors and RhoA activation. Estradiol decreased p27 levels, and significantly raised the expression of cyclins and CDK. These effects were counteracted by the use of either ICI 182780 or Y-27632. In conclusion, estradiol enhances the RhoA/ROCK pathway and increases cell cycle-related protein expression by acting through estrogen receptors. This results in an enhanced migration and proliferation of endothelial cells. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Involvement of glutathione/glutathione S-transferase antioxidant system in butyrate-inhibited vascular smooth muscle cell proliferation.

PubMed

Ranganna, Kasturi; Mathew, Omana P; Yatsu, Frank M; Yousefipour, Zivar; Hayes, Barbara E; Milton, Shirlette G

2007-11-01

Vascular smooth muscle cell (VSMC) proliferation is an important etiological factor in vascular proliferative diseases such as primary atherosclerosis, hypertension, arterial and in-stent restenosis, and transplant vasculopathy. Our studies established that butyrate, a bacterial fermentation product of dietary fiber and a chromatin modulator, is a potent inhibitor of VSMC proliferation. The cardiovascular health benefits of a high-fiber diet, the principle source of butyrate in the body, have been known for a long time, however, very little is known about the antiatherogenic potential of butyrate. Because oxidative stress plays an important role in the pathogenesis of atherosclerosis, we examined involvement of the glutathione/glutathione S-transferase (GST) antioxidant system in butyrate's inhibition of VSMC proliferation. Treatment of proliferating VSMCs with butyrate leads to the induction of several GSTs. Interestingly, our study also demonstrated the nuclear localization of GST-P1 (GST-7-7), which is considered to be a cytosolic protein; this was demonstrated using immunostaining and was corroborated by western blotting. Also, the butyrate-induced antiproliferative action, and the induction of GST-P1 and its nuclear localization are downregulated when butyrate is withdrawn. Furthermore, assessment of intracellular glutathione levels reveals their augmentation by butyrate. Conversely, butyrate treatment reduces the levels of reactive oxygen species in VSMCs. Collectively, the butyrate-treatment-related increase in glutathione content, the reduction in reactive oxygen species, the upregulation of GST and the nuclear localization of GST-P1 in growth-arrested VSMCs imply that butyrate's antiproliferative action involves modulation of the cellular redox state. Thus, induction of the glutathione/GST antioxidant system appears to have other regulatory role(s) besides detoxification and regulation of the cellular redox state, for example, cell-cycle control and cell proliferation, which are both critical to atherogenesis.

Nanofiber Orientation and Surface Functionalization Modulate Human Mesenchymal Stem Cell Behavior In Vitro

PubMed Central

Kolambkar, Yash M.; Bajin, Mehmet; Wojtowicz, Abigail; Hutmacher, Dietmar W.; García, Andrés J.

2014-01-01

Electrospun nanofiber meshes have emerged as a new generation of scaffold membranes possessing a number of features suitable for tissue regeneration. One of these features is the flexibility to modify their structure and composition to orchestrate specific cellular responses. In this study, we investigated the effects of nanofiber orientation and surface functionalization on human mesenchymal stem cell (hMSC) migration and osteogenic differentiation. We used an in vitro model to examine hMSC migration into a cell-free zone on nanofiber meshes and mitomycin C treatment to assess the contribution of proliferation to the observed migration. Poly (ɛ-caprolactone) meshes with oriented topography were created by electrospinning aligned nanofibers on a rotating mandrel, while randomly oriented controls were collected on a stationary collector. Both aligned and random meshes were coated with a triple-helical, type I collagen-mimetic peptide, containing the glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine (GFOGER) motif. Our results indicate that nanofiber GFOGER peptide functionalization and orientation modulate cellular behavior, individually, and in combination. GFOGER significantly enhanced the migration, proliferation, and osteogenic differentiation of hMSCs on nanofiber meshes. Aligned nanofiber meshes displayed increased cell migration along the direction of fiber orientation compared to random meshes; however, fiber alignment did not influence osteogenic differentiation. Compared to each other, GFOGER coating resulted in a higher proliferation-driven cell migration, whereas fiber orientation appeared to generate a larger direct migratory effect. This study demonstrates that peptide surface modification and topographical cues associated with fiber alignment can be used to direct cellular behavior on nanofiber mesh scaffolds, which may be exploited for tissue regeneration. PMID:24020454

Effects of silica and zinc oxide doping on mechanical and biological properties of 3D printed tricalcium phosphate tissue engineering scaffolds.

PubMed

Fielding, Gary A; Bandyopadhyay, Amit; Bose, Susmita

2012-02-01

To evaluate the effects of silica (SiO(2)) (0.5 wt%) and zinc oxide (ZnO) (0.25 wt%) dopants on the mechanical and biological properties of tricalcium phosphate (TCP) scaffolds with three dimensionally (3D) interconnected pores. Scaffolds were created with a commercial 3D printer. Post sintering phase analysis was determined by X-ray diffraction. Surface morphology of the scaffolds was examined by field emission scanning electron microscopy (FESEM). Mechanical strength was evaluated with a screw driven universal testing machine. MTT assay was used for cellular proliferation characteristics and cellular morphology was examined by FESEM. Addition of dopants into TCP increased the average density of pure TCP from 90.8 ± 0.8% to 94.1 ± 1.6% and retarded the β to α phase transformation at high sintering temperatures, which resulted in up to 2.5 fold increase in compressive strength. In vitro cell-materials interaction studies, carried out using hFOB cells, confirmed that the addition of SiO(2) and ZnO to the scaffolds facilitated faster cell proliferation when compared to pure TCP scaffolds. Addition of SiO(2) and ZnO dopants to the TCP scaffolds showed increased mechanical strength as well as increased cellular proliferation. Copyright © 2011 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Dual effect of LPS on murine myeloid leukemia cells: Pro-proliferation and anti-proliferation

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

Yu, Lingling; Noncoding RNA Center, Yangzhou University, Yangzhou 225001; Zhao, Yingmin

Modification of the bone marrow microenvironment is considered as a promising strategy to control leukemic cell proliferation, diseases progression and relapse after treatment. However, due to the diversity and complexity of the cellular and molecular compartments in the leukemic microenvironment, it is extremely difficult to dissect the role of each individual molecule or cell type in vivo. Here we established an in vitro system to dissect the role of lipopolysaccharide (LPS), stromal cells and endothelial cells in the growth of mouse myeloid tumor cells and B-lymphoma cells. We found that either LPS or bone marrow stromal cells as a feedermore » layer in culture is required for the proliferation of myeloid tumor cells. Surprisingly, the growth of myeloid leukemic cells on stromal cells is strongly inhibited when coupled with LPS in culture. This opposing effect of LPS, a complete switch from pro-proliferation to antitumor growth is due, at least in part, to the rapidly increased production of interleukin 12, Fas ligand and tissue inhibitor of metalloproteinases-2 from stromal cells stimulated by LPS. These results demonstrate that LPS can either facilitate or attenuate tumor cell proliferation, thus changing the disease course of myeloid leukemias through its direct effect or modulation of the tumor microenvironment. - Highlights: • LPS alone in culture is required for the proliferation of murine myeloid tumor cells. • Bone marrow stromal cells as a feeder layer is also required for the proliferation of myeloid tumor cells. • However, the growth of myeloid tumor cells is inhibited when LPS and stromal cells are both available in culture. • Thus LPS can either facilitate or attenuate tumor growth through its direct effect or modulation of tumor microenvironment.« less

Design and evaluation of a novel subatmospheric pressure bioreactor for the preconditioning of tissue-engineered vascular constructs.

PubMed

Coakley, Daniel N; Shaikh, Faisal M; O'Sullivan, Kathleen; Kavanagh, Eamon G; Grace, Pierce A; McGloughlin, Tim M

2016-02-01

The pre-conditioning of tissue-engineered vascular scaffolds with mechanical stimuli is being recognised as an essential step in producing a functional vascular construct. In this study we design and evaluate a novel bioreactor, which exerts a mechanical strain on developing vascular scaffolds via subatmospheric pressure. We design and construct a bioreactor, which exerts subatmospheric pressure via a vacuum assisted closure unit. Vascular scaffolds seeded with human umbilical endothelial cells were evaluated for structural integrity, microbial contamination, cellular viability, von Willebrand factor (VWF) production, cell proliferation and morphology under a range of subatmospheric pressures (75-200mmHg). The bioreactor produced sustained subatmospheric pressures, which exerted a mechanical strain on the vascular scaffold. No microbial contamination was found during the study. The structural integrity of the vascular construct was maintained. There was no difference in cellular viability between control or subatmospheric pressure groups (p = 0.817). Cells continued to produce VWF under a range of subatmospheric pressures. Cells subjected to subatmospheric pressures of 125mmHg and 200mmHg exhibited higher levels of growth than cells in atmospheric pressure at 24 (p≤0.016) and 48 hour (p≤0.001). Negative pressure affected cellular morphology, which were more organised, elongated and expanded when exposed to subatmospheric pressure. We have constructed and validated a novel subatmospheric bioreactor. The bioreactor maintained a continuous subatmospheric pressure to the vascular scaffolds in a stable, sterile and constant environment. The bioreactor exerted a strain on the vascular sheets, which was shown to alter cellular morphology and enhance cellular proliferation.

Fisetin and hesperetin induced apoptosis and cell cycle arrest in chronic myeloid leukemia cells accompanied by modulation of cellular signaling.

PubMed

Adan, Aysun; Baran, Yusuf

2016-05-01

Fisetin and hesperetin, naturally occurring flavonoids, have been reported as novel antioxidants with chemopreventive/chemotherapeutic potential against various types of cancer. However, their mechanism of action in CML is still unknown. This particular study aims to evaluate the therapeutic potentials of fisetin and hesperetin and their effects on cell proliferation, apoptosis, and cell cycle progression in human K562 CML cells. The results indicated that fisetin and hesperetin inhibited cell proliferation and triggered programmed cell death in these cells. The latter was confırmed by mitochondrial membrane depolarization and an increase in caspase-3 activation. In addition to that, we have detected S and G2/M cell cycle arrests and G0/G1 arrest upon fisetin and hesperetin treatment, respectively. To identify the altered genes and genetic networks in response to fisetin and hesperetin, whole-genome microarray analysis was performed. The microarray gene profiling analysis revealed some important signaling pathways including JAK/STAT pathway, KIT receptor signaling, and growth hormone receptor signaling that were altered upon fisetin and hesperetin treatment. Moreover, microarray data suggested potential candidate genes for targeted CML therapy. Fisetin and hesperetin significantly modulated the expression of genes involved in cell proliferation and division, apoptosis, cell cycle regulation, and other significant cellular processes such as replication, transcription, and translation. In conclusion, our results suggest that fisetin and hesperetin as potential natural agents for CML therapy.

[The role of cellular senescence in carcinogenesis and antitumor therapy].

PubMed

Mosieniak, Grazyna; Strzeszewska, Anna

2014-01-01

Cellular senescence is the process that lead to terminal growth arrest induced by unrepairable double strand DNA damage (DSB). Moreover, activation of the oncogenes as well as inhibition of the tumor suppressor genes were shown to contribute to senescence induction and the senescent cells were identified in the premalignant lesions. Thus senescence is considered as an natural antitumor barrier that act at the early stages of cancerogenesis to stop the proliferation of transformed cells. Interestingly, the premalignant cells that escaped senescence and progress into full blown tumor cells still remain sensitive to induction of senescence, for example during chemio- or radiotherapy. Thus, induction of cancer cell senescence, similarly to apoptosis, is considered to restrain tumor growth and thus contribute to effectiveness of anticancer therapy. The senescent cells, although do not proliferate, remain viable and metabolically active. They secret a lot of cytokines, mitogens as well as enzymes degrading extracellular matrix. These factors can have opposing effect on neighboring cells, leading to senescence induction or stimulation of proliferation. Thus, senescence can act as an double edge sword that inhibit the propagation of potentially dangerous, transformed cells on one hand or induce cell division of the same cell on the other. Presently a lot of work is focused on finding new therapeutic strategies that would involve the tumor targeted senescence induction in both early late stages of cancer development. Nevertheless, the unwanted influence of the senescent cells on the microenvironment, requires careful monitoring the effects of pro-senescent therapies in each case.

Butyrate and deoxycholic acid play common and distinct roles in HCT116 human colon cell proliferation.

PubMed

Zeng, Huawei; Claycombe, Kate J; Reindl, Katie M

2015-10-01

Consumption of a high-fat diet causes an increase in bile acid deoxycholic acid (DCA) in colon lumen and colon cancer risk, while butyrate, an intestinal microbiota metabolite of dietary fiber, has been shown to exhibit colon cancer-preventive effects. To distinguish these opposing effects of DCA and butyrate (two major metabolites in colon lumen), we examined the effects of physiologically relevant doses of butyrate (0.5-2 mmol/l) and DCA (0.05-0.3 mmol/l) on colon cell proliferation. We hypothesize that butyrate and DCA each modulates the cell cycle and apoptosis via common and distinct cellular signaling targets. In this study, we demonstrated that both butyrate and DCA inhibited cell proliferation by up to 89% and 92% and increased cell apoptosis rate by up to 3.1- and 4.5-fold, respectively. Cell cycle analyses revealed that butyrate led to an increase in G1 and G2 fractions with a concomitant drop in the S-phase fraction, but DCA induced an increase in only G1 fraction with a concomitant drop in the S-phase fraction when compared with the untreated cells. The examination of early cellular signaling revealed that DCA but not butyrate increased intracellular reactive oxygen species, genomic DNA breakage, the activation of ERK1/2, caspase-3 and PARP. In contrast, DCA decreased activated Rb protein level, and butyrate but not DCA increased p21 expression. Collectively, although both butyrate and DCA inhibit colonic cell proliferation, butyrate increases tumor suppressor gene expression, whereas DCA decreases tumor suppressor activation in cell cycle and apoptosis pathways. Published by Elsevier Inc.

Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

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

Cunha, Elizabeth S.; Kawahara, Rebeca; Kadowaki, Marina K.

Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cellmore » cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis. -- Highlights: Black-Right-Pointing-Pointer Melanogenesis stimulation by L-tyrosine+NH{sub 4}Cl in B16-F10 melanoma cells increases ROS levels. Black-Right-Pointing-Pointer Melanogenesis inhibits cell proliferation, and induced cell cycle arrest in the G1 phase. Black-Right-Pointing-Pointer Proteomic analysis showed alterations in proteins of the cell cycle and glucose metabolism. Black-Right-Pointing-Pointer RT-qPCR analysis confirmed alterations of metabolic targets after melanogenesis stimulation.« less

Differential roles of SS18-SSX fusion gene and insulin-like growth factor-1 receptor in synovial sarcoma cell growth

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

Toernkvist, Maria; Natalishvili, Natalia; Xie Yuntao

2008-04-11

Recently we demonstrated that the synovial sarcoma specific fusion gene SS18-SSX is crucial for cyclin D1 expression and is linked to cell proliferation. In this report we explore the role of SS18-SSX and IGF-1R for their potential functions in cellular proliferation and survival in cultured synovial sarcoma cells. We found that targeting of SS18-SSX mRNA by antisense oligonucleotide treatment drastically and rapidly decreased cell proliferation but caused only a slight increase of apoptosis. The synovial sarcoma cells were confirmed to express IGF-1R, and treatment with an IGF-1R inhibitor resulted in substantially reduced cell viability by inducing apoptosis in these cells.more » Conversely, inhibition of the IGF-1R resulted only in a slight to moderate decrease in DNA synthesis. In conclusion, SS18-SSX and IGF-1R seem to play important but different roles in maintaining malignant growth of synovial sarcoma cells. Whereas SS18-SSX maintains cyclin D1 and cell proliferation, IGF-1R protects from apoptosis.« less

Conditional Deletion of Pten Causes Bronchiolar Hyperplasia

PubMed Central

Davé, Vrushank; Wert, Susan E.; Tanner, Tiffany; Thitoff, Angela R.; Loudy, Dave E.; Whitsett, Jeffrey A.

2008-01-01

Tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase that regulates multiple cellular processes including cell polarity, migration, proliferation, and carcinogenesis. In this work, we demonstrate that conditional deletion of Pten (PtenΔ/Δ) in the respiratory epithelial cells of the developing mouse lung caused epithelial cell proliferation and hyperplasia as early as 4 to 6 weeks of age. While bronchiolar cell differentiation was normal, as indicated by β-tubulin and FOXJ1 expression in ciliated cells and by CCSP expression in nonciliated cells, cell proliferation (detected by expression of Ki-67, phospho-histone-H3, and cyclin D1) was increased and associated with activation of the AKT/mTOR survival pathway. Deletion of Pten caused papillary epithelial hyperplasia characterized by a hypercellular epithelium lining papillae with fibrovascular cores that protruded into the airway lumens. Cell polarity, as assessed by subcellular localization of cadherin, β-catenin, and zonula occludens-1, was unaltered. PTEN is required for regulation of epithelial cell proliferation in the lung and for the maintenance of the normal simple columnar epithelium characteristics of bronchi and bronchioles. PMID:17921358

Conditional deletion of Pten causes bronchiolar hyperplasia.

PubMed

Davé, Vrushank; Wert, Susan E; Tanner, Tiffany; Thitoff, Angela R; Loudy, Dave E; Whitsett, Jeffrey A

2008-03-01

Tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase that regulates multiple cellular processes including cell polarity, migration, proliferation, and carcinogenesis. In this work, we demonstrate that conditional deletion of Pten (Pten(Delta/Delta)) in the respiratory epithelial cells of the developing mouse lung caused epithelial cell proliferation and hyperplasia as early as 4 to 6 weeks of age. While bronchiolar cell differentiation was normal, as indicated by beta-tubulin and FOXJ1 expression in ciliated cells and by CCSP expression in nonciliated cells, cell proliferation (detected by expression of Ki-67, phospho-histone-H3, and cyclin D1) was increased and associated with activation of the AKT/mTOR survival pathway. Deletion of Pten caused papillary epithelial hyperplasia characterized by a hypercellular epithelium lining papillae with fibrovascular cores that protruded into the airway lumens. Cell polarity, as assessed by subcellular localization of cadherin, beta-catenin, and zonula occludens-1, was unaltered. PTEN is required for regulation of epithelial cell proliferation in the lung and for the maintenance of the normal simple columnar epithelium characteristics of bronchi and bronchioles.

Activation of cellular death programs associated with immunosenescence-like phenotype in TPPII knockout mice

PubMed Central

Huai, Jisen; Firat, Elke; Nil, Ahmed; Million, Daniele; Gaedicke, Simone; Kanzler, Benoit; Freudenberg, Marina; van Endert, Peter; Kohler, Gabriele; Pahl, Heike L.; Aichele, Peter; Eichmann, Klaus; Niedermann, Gabriele

2008-01-01

The giant cytosolic protease tripeptidyl peptidase II (TPPII) has been implicated in the regulation of proliferation and survival of malignant cells, particularly lymphoma cells. To address its functions in normal cellular and systemic physiology we have generated TPPII-deficient mice. TPPII deficiency activates cell type-specific death programs, including proliferative apoptosis in several T lineage subsets and premature cellular senescence in fibroblasts and CD8+ T cells. This coincides with up-regulation of p53 and dysregulation of NF-κB. Prominent degenerative alterations at the organismic level were a decreased lifespan and symptoms characteristic of immunohematopoietic senescence. These symptoms include accelerated thymic involution, lymphopenia, impaired proliferative T cell responses, extramedullary hematopoiesis, and inflammation. Thus, TPPII is important for maintaining normal cellular and systemic physiology, which may be relevant for potential therapeutic applications of TPPII inhibitors. PMID:18362329

CDKL5, a novel MYCN-repressed gene, blocks cell cycle and promotes differentiation of neuronal cells

PubMed Central

Valli, Emanuele; Trazzi, Stefania; Fuchs, Claudia; Erriquez, Daniela; Bartesaghi, Renata; Perini, Giovanni; Ciani, Elisabetta

2012-01-01

Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene are associated with a severe epileptic encephalopathy (early infantile epileptic encephalopathy type 2, EIEE2) characterized by early-onset intractable seizures, infantile spasms, severe developmental delay, intellectual disability, and Rett syndrome (RTT)-like features. Despite the clear involvement of CDKL5 mutations in intellectual disability, the function of this protein during brain development and the molecular mechanisms involved in its regulation are still unknown. Using human neuroblastoma cells as a model system we found that an increase in CDKL5 expression caused an arrest of the cell cycle in the G0/G1 phases and induced cellular differentiation. Interestingly, CDKL5 expression was inhibited by MYCN, a transcription factor that promotes cell proliferation during brain development and plays a relevant role in neuroblastoma biology. Through a combination of different and complementary molecular and cellular approaches we could show that MYCN acts as a direct repressor of the CDKL5 promoter. Overall our findings unveil a functional axis between MYCN and CDKL5 governing both neuron proliferation rate and differentiation. The fact that CDKL5 is involved in the control of both neuron proliferation and differentiation may help understand the early appearance of neurological symptoms in patients with mutations in CDKL5. PMID:22921766

Developmental Regulation of Nucleolus Size during Drosophila Eye Differentiation

PubMed Central

Baker, Nicholas E.

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals. PMID:23472166

Developmental regulation of nucleolus size during Drosophila eye differentiation.

PubMed

Baker, Nicholas E

2013-01-01

When cell cycle withdrawal accompanies terminal differentiation, biosynthesis and cellular growth are likely to change also. In this study, nucleolus size was monitored during cell fate specification in the Drosophila eye imaginal disc using fibrillarin antibody labeling. Nucleolus size is an indicator of ribosome biogenesis and can correlate with cellular growth rate. Nucleolar size was reduced significantly during cell fate specification and differentiation, predominantly as eye disc cells entered a cell cycle arrest that preceded cell fate specification. This reduction in nucleolus size required Dpp and Hh signaling. A transient enlargement of the nucleolus accompanied cell division in the Second Mitotic Wave. Nucleoli continued to diminish in postmitotic cells following fate specification. These results suggest that cellular growth is regulated early in the transition from proliferating progenitor cells to terminal cell fate specification, contemporary with regulation of the cell cycle, and requiring the same extracellular signals.

The effects of selected drugs and dietary compounds on proliferation and apoptosis in colorectal carcinoma.

PubMed

Kiedrowski, Miroslaw; Mroz, Andrzej

2014-01-01

Like many malignancies, the development of colorectal carcinoma (CRC) can be considered as an imbalance between the compromised process of programmed cell death (apoptosis) and excessive, uncontrolled proliferation. Several mutations and epigenetic alterations are acquired during colorectal carcinogenesis. These are responsible for the cell cycle regulation, cellular sensitivity to pro- and antiapoptotic factors, cell proliferation, angiogenesis, invasiveness, as well as metastatic potential. The molecular alterations, along with their morphological expressions, have been recognised in detail, and most of the CRC cases can be attributed to either adenoma-carcinoma or serrated neoplasia pathways: in the first, the antiapoptotic features prevail; while in the second, the proliferative activity is of the utmost importance. The aim of the work is to discuss the influence of selected drugs and dietary compounds on the proliferation and apoptosis in CRC.

Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor

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

Tsai, M.; Takeishi, Takashi; Geissler, E.N.

1991-07-15

The authors investigated the effects of a newly recognized multifunctional growth factor, the c-kit ligand stem cell factor (SCF), on mouse mast cell proliferation and phenotype. Recombinant rat SCF{sup 164} (rrSCF{sup 164}) induced the development of large numbers of dermal mast cells in normal mice in vivo. Many of these mast cells had features of connective tissue-type mast cells (CTMC), in that they were reactive both with the heparin-binding fluorescent dye berberine sulfate and with safranin. In vitro, rrSCF{sup 164} induced the proliferation of cloned interleukin 3 (IL-3)-dependent mouse mast cells and primary populations of IL-3-dependent, bone marrow-derived cultured mastmore » cells (BMCMC), which represent immature mast cells, and purified peritoneal mast cells, which represent a type of mature CTMC> BMCMC maintained in rrSCF{sup 164} not only proliferated but also matured. These findings identify SCF as a single cytokine that can induce immature, IL-3-dependent mast cells to mature and to acquire multiple characteristics of CTMC. These findings also directly demonstrate that SCF can regulate the development of a cellular lineage expressing c-kit through effects on both proliferation and maturation.« less

Synergistic inhibition of cancer cell proliferation with a combination of δ-tocotrienol and ferulic acid

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

Eitsuka, Takahiro, E-mail: eitsuka@nupals.ac.jp; Tatewaki, Naoto; Nishida, Hiroshi

2014-10-24

Highlights: • δ-Tocotrienol (δ-T3) and ferulic acid (FA) synergistically inhibit cancer cell growth. • The combination of δ-T3 and FA induces G1 arrest by up-regulating p21. • The synergy is attributed to an increase in the cellular concentration of δ-T3 by FA. - Abstract: Rice bran consists of many functional compounds and thus much attention has been focused on the health benefits of its components. Here, we investigated the synergistic inhibitory effects of its components, particularly δ-tocotrienol (δ-T3) and ferulic acid (FA), against the proliferation of an array of cancer cells, including DU-145 (prostate cancer), MCF-7 (breast cancer), and PANC-1more » (pancreatic cancer) cells. The combination of δ-T3 and FA markedly reduced cell proliferation relative to δ-T3 alone, and FA had no effect when used alone. Although δ-T3 induced G1 arrest by up-regulating p21 in PANC-1 cells, more cells accumulated in G1 phase with the combination of δ-T3 and FA. This synergistic effect was attributed to an increase in the cellular concentration of δ-T3 by FA. Our results suggest that the combination of δ-T3 and FA may present a new strategy for cancer prevention and therapy.« less

Defective Wound-healing in Aging Gingival Tissue.

PubMed

Cáceres, M; Oyarzun, A; Smith, P C

2014-07-01

Aging may negatively affect gingival wound-healing. However, little is known about the mechanisms underlying this phenomenon. The present study examined the cellular responses associated with gingival wound-healing in aging. Primary cultures of human gingival fibroblasts were obtained from healthy young and aged donors for the analysis of cell proliferation, cell invasion, myofibroblastic differentiation, and collagen gel remodeling. Serum from young and old rats was used to stimulate cell migration. Gingival repair was evaluated in Sprague-Dawley rats of different ages. Data were analyzed by the Mann-Whitney and Kruskal-Wallis tests, with a p value of .05. Fibroblasts from aged donors showed a significant decrease in cell proliferation, migration, Rac activation, and collagen remodeling when compared with young fibroblasts. Serum from young rats induced higher cell migration when compared with serum from old rats. After TGF-beta1 stimulation, both young and old fibroblasts demonstrated increased levels of alpha-SMA. However, alpha-SMA was incorporated into actin stress fibers in young but not in old fibroblasts. After 7 days of repair, a significant delay in gingival wound-healing was observed in old rats. The present study suggests that cell migration, myofibroblastic differentiation, collagen gel remodeling, and proliferation are decreased in aged fibroblasts. In addition, altered cell migration in wound-healing may be attributable not only to cellular defects but also to changes in serum factors associated with the senescence process. © International & American Associations for Dental Research.

Essential Dosage-Dependent Functions of the Transcription Factor Yin Yang 1 in Late Embryonic Development and Cell Cycle Progression†

PubMed Central

Affar, El Bachir; Gay, Frédérique; Shi, Yujiang; Liu, Huifei; Huarte, Maite; Wu, Su; Collins, Tucker; Li, En; Shi, Yang

2006-01-01

Constitutive ablation of the Yin Yang 1 (YY1) transcription factor in mice results in peri-implantation lethality. In this study, we used homologous recombination to generate knockout mice carrying yy1 alleles expressing various amounts of YY1. Phenotypic analysis of yy1 mutant embryos expressing ∼75%, ∼50%, and ∼25% of the normal complement of YY1 identified a dosage-dependent requirement for YY1 during late embryogenesis. Indeed, reduction of YY1 levels impairs embryonic growth and viability in a dose-dependent manner. Analysis of the corresponding mouse embryonic fibroblast cells also revealed a tight correlation between YY1 dosage and cell proliferation, with a complete ablation of YY1 inducing cytokinesis failure and cell cycle arrest. Consistently, RNA interference-mediated inhibition of YY1 in HeLa cells prevents cytokinesis, causes proliferative arrest, and increases cellular sensitivity to various apoptotic agents. Genome-wide expression profiling identified a plethora of YY1 target genes that have been implicated in cell growth, proliferation, cytokinesis, apoptosis, development, and differentiation, suggesting that YY1 coordinates multiple essential biological processes through a complex transcriptional network. These data not only shed new light on the molecular basis for YY1 developmental roles and cellular functions, but also provide insight into the general mechanisms controlling eukaryotic cell proliferation, apoptosis, and differentiation. PMID:16611997

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles.

PubMed

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel; Hilfiker, Andres

2016-01-01

Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle-cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN.

Induction of intracellular Ca2+ and pH changes in Sf9 insect cells by rhodojaponin-III, a natural botanic insecticide isolated from Rhododendron molle.

PubMed

Cheng, Xing-An; Xie, Jian-Jun; Hu, Mei-Ying; Zhang, Yan-Bo; Huang, Jing-Fei

2011-04-15

Many studies on intracellular calcium ([Ca2+](i)) and intracellular pH (pH(i)) have been carried out due to their importance in regulation of different cellular functions. However, most of the previous studies are focused on human or mammalian cells. The purpose of the present study was to characterize the effect of Rhodojaponin-III (R-III) on [Ca2+](i) and pH(i) and the proliferation of Sf9 cells. R-III strongly inhibited Sf9 cells proliferation with a time- and dose-dependent manner. Flow cytometry established that R-III interfered with Sf9 cells division and arrested them in G2/M. By using confocal scanning technique, effects of R-III on intracellular free calcium ([Ca2+](i)) and intracellular pH (pH(i)) in Sf9 cells were determined. R-III induced a significant dose-dependent (1, 10, 100, 200 μg/mL) increase in [Ca2+](i) and pH(i) of Sf9 cells in presence of Ca2+-containing solution (Hanks) and an irreversible decrease in the absence of extra cellular Ca2+. We also found that both extra cellular Ca2+ and intracellular Ca2+ stores contributed to the increase of [Ca2+](i), because completely treating Sf9 cells with CdCl(2) (5 mM), a Ca2+ channels blocker, R-III (100 μg/mL) induced a transient elevation of [Ca2+](i) in case of cells either in presence of Ca2+ containing or Ca2+ free solution. In these conditions, pH(i) showed similar changes with that of [Ca2+](i) on the whole. Accordingly, we supposed that there was a certain linkage for change of [Ca2+](i), cell cycle arrest, proliferation inhibition in Sf9 cells induced by R-III.

Current views on the role of Notch signaling and the pathogenesis of human leukemia

PubMed Central

2011-01-01

The Notch signaling pathway is highly conserved from Drosophila to humans and plays an important role in the regulation of cellular proliferation, differentiation and apoptosis. Constitutive activation of Notch signaling has been shown to result in excessive cellular proliferation and a wide range of malignancies, including leukemia, glioblastoma and lung and breast cancers. Notch can also act as a tumor suppressor, and its inactivation has been associated with an increased risk of spontaneous squamous cell carcinoma. This minireview focuses on recent advances related to the mechanisms and roles of activated Notch1, Notch2, Notch3 and Notch4 signaling in human lymphocytic leukemia, myeloid leukemia and B cell lymphoma, as well as their significance, and recent advances in Notch-targeted therapies. PMID:22128846

Menin determines K-RAS proliferative outputs in endocrine cells

PubMed Central

Chamberlain, Chester E.; Scheel, David W.; McGlynn, Kathleen; Kim, Hail; Miyatsuka, Takeshi; Wang, Juehu; Nguyen, Vinh; Zhao, Shuhong; Mavropoulos, Anastasia; Abraham, Aswin G.; O’Neill, Eric; Ku, Gregory M.; Cobb, Melanie H.; Martin, Gail R.; German, Michael S.

2014-01-01

Endocrine cell proliferation fluctuates dramatically in response to signals that communicate hormone demand. The genetic alterations that override these controls in endocrine tumors often are not associated with oncogenes common to other tumor types, suggesting that unique pathways govern endocrine proliferation. Within the pancreas, for example, activating mutations of the prototypical oncogene KRAS drive proliferation in all pancreatic ductal adenocarcimomas but are never found in pancreatic endocrine tumors. Therefore, we asked how cellular context impacts K-RAS signaling. We found that K-RAS paradoxically suppressed, rather than promoted, growth in pancreatic endocrine cells. Inhibition of proliferation by K-RAS depended on antiproliferative RAS effector RASSF1A and blockade of the RAS-activated proproliferative RAF/MAPK pathway by tumor suppressor menin. Consistent with this model, a glucagon-like peptide 1 (GLP1) agonist, which stimulates ERK1/2 phosphorylation, did not affect endocrine cell proliferation by itself, but synergistically enhanced proliferation when combined with a menin inhibitor. In contrast, inhibition of MAPK signaling created a synthetic lethal interaction in the setting of menin loss. These insights suggest potential strategies both for regenerating pancreatic β cells for people with diabetes and for targeting menin-sensitive endocrine tumors. PMID:25133424

Inducible indoleamine 2,3-dioxygenase 1 and programmed death ligand 1 expression as the potency marker for mesenchymal stromal cells.

PubMed

Guan, Qingdong; Li, Yun; Shpiruk, Tanner; Bhagwat, Swaroop; Wall, Donna A

2018-05-01

Establishment of a potency assay in the manufacturing of clinical-grade mesenchymal stromal cells (MSCs) has been a challenge due to issues of relevance to function, timeline and variability of responder cells. In this study, we attempted to develop a potency assay for MSCs. Clinical-grade bone marrow-derived MSCs were manufactured. The phenotype and immunosuppressive functions of the MSCs were evaluated based on the International Society for Cellular Therapy guidelines. Resting MSCs licensed by interferon (IFN)-γ exposure overnight were evaluated for changes in immune suppression and immune-relevant proteins. The relationship of immune-relevant protein expression with immunosuppression of MSCs was analyzed. MSC supressed third-party T-lymphocyte proliferation with high inter-donor and inter-test variability. The suppression of T-lymphocyte proliferation by IFN-γ-licensed MSCs correlated with that by resting MSCs. Many cellular proteins were up-regulated after IFN-γ exposure, including indoleamine 2,3-dioxygenase 1 (IDO-1), programmed death ligand 1 (PD-L1), vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1) and bone marrow stromal antigen 2 (BST-2). The expression levels of IDO-1 and PD-L1 on licensed MSCs, not VCAM-1, ICAM-1 or BST-2 on licensed MSCs, correlated with MSC suppression of third-party T-cell proliferation. A flow cytometry-based assay of MSCs post-IFN-γ exposure measuring expression of intracellular protein IDO-1 and cell surface protein PD-L1 captures two mechanisms of suppression and offers the potential of a relevant, rapid assay for MSC-mediated immune suppression that would fit with the manufacturing process. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Properties of a non-bioactive fluorescent derivative of differentiation-inducing factor-3, an anti-tumor agent found in Dictyostelium discoideum

PubMed Central

Kubohara, Yuzuru; Kikuchi, Haruhisa; Matsuo, Yusuke; Oshima, Yoshiteru; Homma, Yoshimi

2014-01-01

ABSTRACT Differentiation-inducing factor-3 (DIF-3), found in the cellular slime mold Dictyostelium discoideum, and its derivatives, such as butoxy-DIF-3 (Bu-DIF-3), are potent anti-tumor agents. To investigate the activity of DIF-like molecules in tumor cells, we recently synthesized a green fluorescent DIF-3 derivative, BODIPY-DIF-3G, and analyzed its bioactivity and cellular localization. In this study, we synthesized a red (orange) fluorescent DIF-3 derivative, BODIPY-DIF-3R, and compared the cellular localization and bioactivities of the two BODIPY-DIF-3s in HeLa human cervical cancer cells. Both fluorescent compounds penetrated the extracellular membrane within 0.5 h and localized mainly to the mitochondria. In formalin-fixed cells, the two BODIPY-DIF-3s also localized to the mitochondria, indicating that the BODIPY-DIF-3s were incorporated into mitochondria independently of the mitochondrial membrane potential. After treatment for 3 days, BODIPY-DIF-3G, but not BODIPY-DIF-3R, induced mitochondrial swelling and suppressed cell proliferation. Interestingly, the swollen mitochondria were stainable with BODIPY-DIF-3G but not with BODIPY-DIF-3R. When added to isolated mitochondria in vitro, BODIPY-DIF-3G increased dose-dependently the rate of O2 consumption, but BODIPY-DIF-3R did not. These results suggest that the bioactive BODIPY-DIF-3G suppresses cell proliferation, at least in part, by altering mitochondrial activity, whereas the non-bioactive BODIPY-DIF-3R localizes to the mitochondria but does not affect mitochondrial activity or cell proliferation. PMID:24682009

The use of the tyrosine phosphatase antagonist orthovanadate in the study of a cell proliferation inhibitor

NASA Technical Reports Server (NTRS)

Enebo, D. J.; Hanek, G.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

1993-01-01

Incubation of murine fibroblasts with orthovanadate, a global tyrosine phosphatase inhibitor, was shown to confer a "pseudo-transformed" phenotype with regard to cell morphology and growth characteristics. This alteration was manifested by both an increasing refractile appearance of the cells, consistent with many transformed cell lines, as well as an increase in maximum cell density was attained. Despite the abrogation of cellular tyrosine phosphatase activity, orthovanadate-treated cells remained sensitive to the biological activity of a naturally occurring sialoglycopeptide (SGP) cell surface proliferation inhibitor. The results indicated that tyrosine phosphatase activity, inhibited by orthovanadate, was not involved in the signal transduction pathway of the SGP.

Regulation of proliferation and functioning of transplanted cells by using herpes simplex virus thymidine kinase gene in mice.

PubMed

Tsujimura, Mari; Kusamori, Kosuke; Oda, Chihiro; Miyazaki, Airi; Katsumi, Hidemasa; Sakane, Toshiyasu; Nishikawa, Makiya; Yamamoto, Akira

2018-04-10

Though cell transplantation is becoming an attractive therapeutic method, uncontrolled cell proliferation or overexpression of cellular functions could cause adverse effects. These unfavorable outcomes could be avoided by regulating the proliferation or functioning of transplanted cells. In this study, we used a combination of the herpes simplex virus thymidine kinase (HSVtk) gene, a suicide gene, and ganciclovir (GCV) to control the proliferation and functioning of insulin-secreting cells after transplantation in diabetic mice. Mouse pancreatic β cell line MIN6 cells were selected as insulin-secreting cells for transfection with the HSVtk gene to obtain MIN6/HSVtk cells. Proliferation of MIN6/HSVtk cells was suppressed by GCV in a concentration-dependent manner; 0.25 μg/mL GCV maintained a constant number of MIN6/HSVtk cells for at least 16 days. MIN6 or MIN6/HSVtk cells were then transplanted to streptozotocin-induced diabetic mice. Mice transplanted with MIN6 cells exhibited hypoglycemia irrespective of GCV administration. In contrast, normal (around 150 mg/dL) blood glucose levels were maintained in mice transplanted with MIN6/HSVtk cells by a daily administration of 50 mg/kg of GCV. These results indicate that controlling the proliferation and functioning of HSVtk gene-expressing cells by GCV could greatly improve the usefulness and safety of cell-based therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Cervical cancer cells (HeLa) response to photodynamic therapy using a zinc phthalocyanine photosensitizer.

PubMed

Hodgkinson, Natasha; Kruger, Cherie Ann; Mokwena, Mpho; Abrahamse, Heidi

2017-12-01

Cervical cancer is the most common gynecological malignancy worldwide, and the leading cause of cancer related deaths among females. Conventional treatment for early cervical cancer is radical hysterectomy. In locally advanced cancer the treatment of choice is concurrent chemo radiation. Although such treatment methods show promise, they do have adverse side effects. To minimize these effects, as well as prevent cancer re-occurrence, new treatment methods are being investigated. Photodynamic therapy (PDT) involves the selective uptake of a photosensitizer (PS) by cancer cells, illumination with light of an appropriate wavelength that triggers a photochemical reaction leading to the generation of reactive oxygen and subsequent tumor regression. The effect of PDT on a cervical cancer cell line (HeLa) was assessed by exposing cultured cells to a sulphonated zinc phthalocyanine PS (ZnPcS mix ) and irradiating the cells using a 673nm diode laser. The effects were measured using the Trypan blue viability assay, adenosine triphosphate assay (ATP) luminescence assay for proliferation, Lactate Dehydrogenase (LDH) membrane integrity cytotoxicity assay, and fluorescent microscopy to assess PS cellular localization and nuclear damage. Fluorescent microscopy revealed localization of the PS in the cytoplasm and perinuclear region of HeLa cells. PDT treated cellular responses showed dose dependent structural changes, with decreased cell viability and proliferation, as well as considerable membrane damage. Hoechst stained cells also revealed DNA damage in PDT treated cells. The final findings from this study suggest that ZnPcS mix is a promising PS for the PDT treatment of cervical cancer in vitro, where a significant 85% cellular cytotoxicity with only 25% cellular viability was noted in cells which received 1μM ZnPcS mix when an 8J/cm 2 fluence was applied. Copyright © 2017 Elsevier B.V. All rights reserved.

MYC/MIZ1-dependent gene repression inversely coordinates the circadian clock with cell cycle and proliferation.

PubMed

Shostak, Anton; Ruppert, Bianca; Ha, Nati; Bruns, Philipp; Toprak, Umut H; Eils, Roland; Schlesner, Matthias; Diernfellner, Axel; Brunner, Michael

2016-06-24

The circadian clock and the cell cycle are major cellular systems that organize global physiology in temporal fashion. It seems conceivable that the potentially conflicting programs are coordinated. We show here that overexpression of MYC in U2OS cells attenuates the clock and conversely promotes cell proliferation while downregulation of MYC strengthens the clock and reduces proliferation. Inhibition of the circadian clock is crucially dependent on the formation of repressive complexes of MYC with MIZ1 and subsequent downregulation of the core clock genes BMAL1 (ARNTL), CLOCK and NPAS2. We show furthermore that BMAL1 expression levels correlate inversely with MYC levels in 102 human lymphomas. Our data suggest that MYC acts as a master coordinator that inversely modulates the impact of cell cycle and circadian clock on gene expression.

The effect of RO3201195 and a pyrazolyl ketone P38 MAPK inhibitor library on the proliferation of Werner syndrome cells.

PubMed

Bagley, Mark C; Dwyer, Jessica E; Baashen, Mohammed; Dix, Matthew C; Murziani, Paola G S; Rokicki, Michal J; Kipling, David; Davis, Terence

2016-01-21

Microwave-assisted synthesis of the pyrazolyl ketone p38 MAPK inhibitor RO3201195 in 7 steps and 15% overall yield, and the comparison of its effect upon the proliferation of Werner Syndrome cells with a library of pyrazolyl ketones, strengthens the evidence that p38 MAPK inhibition plays a critical role in modulating premature cellular senescence in this progeroid syndrome and the reversal of accelerated ageing observed in vitro on treatment with SB203580.

Proliferation of prostate cancer cells and activity of neutral endopeptidase is regulated by bombesin and IL-1beta with IL-1beta acting as a modulator of cellular differentiation.

PubMed

Albrecht, Martin; Doroszewicz, Jolanta; Gillen, Sonja; Gomes, Iara; Wilhelm, Beate; Stief, Thomas; Aumüller, Gerhard

2004-01-01

Neutral endopeptidase (NEP) is a cell-surface bound enzyme that cleaves and inactivates neuropeptides such as bombesin and substance P and is involved in the transition from hormonally regulated androgen-dependent prostate cancer (PC) to androgen-independent PC. Neuropeptides are implicated in growth regulation of different cell types and function as transmitters between the neuroendocrine and the immune system. NEP-expression, enzymatic activity of the membrane bound protein, cell proliferation, procalcitonin (PCT) production, and secretion as well as changes in cell morphology of prostatic cells were evaluated after treatment with the immunomodulatory cytokine interleukin-1beta (IL-1beta), neuropeptides (bombesin, substance P), and neuropeptide-conditioned media derived from a human neuroendocrine cell line. Incubation of LNCaP tumor cells with IL-1beta resulted in a diminished proliferative activity, induction of neurite-like outgrowth which was accompanied by the formation of tubular-type mitochondria typical for neuronal/neuroendocrine cells, and an increased production and secretion of PCT. Conversely, proliferation of prostatic stromal cells was enhanced by the cytokine coming along with an increased number of Golgi-apparatuses and ER-cisternae. Bombesin had an antimitotic effect on LNCaP, but not on stromal cells. Substance P did not influence the growth of any of the cell types investigated, whereas neuropeptide-conditioned media exerted a slightly mitogenic effect on both cell types. The activity of LNCaP cell-surface bound NEP was enhanced by bombesin, but was diminished by substance P and neuropeptide-conditioned media. Proliferation and activity of neuropeptide degrading NEP is regulated differently by immunomodulatory substances in PC cells and cells derived from the prostatic stroma with IL-1beta being a potent modulator of cellular differentiation and a potential target for anticancer drug design in PC cells. Copyright 2003 Wiley-Liss, Inc.

A SHH-FOXF1-BMP4 signaling axis regulating growth and differentiation of epithelial and mesenchymal tissues in ureter development.

PubMed

Bohnenpoll, Tobias; Wittern, Anna B; Mamo, Tamrat M; Weiss, Anna-Carina; Rudat, Carsten; Kleppa, Marc-Jens; Schuster-Gossler, Karin; Wojahn, Irina; Lüdtke, Timo H-W; Trowe, Mark-Oliver; Kispert, Andreas

2017-08-01

The differentiated cell types of the epithelial and mesenchymal tissue compartments of the mature ureter of the mouse arise in a precise temporal and spatial sequence from uncommitted precursor cells of the distal ureteric bud epithelium and its surrounding mesenchyme. Previous genetic efforts identified a member of the Hedgehog (HH) family of secreted proteins, Sonic hedgehog (SHH) as a crucial epithelial signal for growth and differentiation of the ureteric mesenchyme. Here, we used conditional loss- and gain-of-function experiments of the unique HH signal transducer Smoothened (SMO) to further characterize the cellular functions and unravel the effector genes of HH signaling in ureter development. We showed that HH signaling is not only required for proliferation and SMC differentiation of cells of the inner mesenchymal region but also for survival of cells of the outer mesenchymal region, and for epithelial proliferation and differentiation. We identified the Forkhead transcription factor gene Foxf1 as a target of HH signaling in the ureteric mesenchyme. Expression of a repressor version of FOXF1 in this tissue completely recapitulated the mesenchymal and epithelial proliferation and differentiation defects associated with loss of HH signaling while re-expression of a wildtype version of FOXF1 in the inner mesenchymal layer restored these cellular programs when HH signaling was inhibited. We further showed that expression of Bmp4 in the ureteric mesenchyme depends on HH signaling and Foxf1, and that exogenous BMP4 rescued cell proliferation and epithelial differentiation in ureters with abrogated HH signaling or FOXF1 function. We conclude that SHH uses a FOXF1-BMP4 module to coordinate the cellular programs for ureter elongation and differentiation, and suggest that deregulation of this signaling axis occurs in human congenital anomalies of the kidney and urinary tract (CAKUT).

Endoglin inhibits ERK-induced c-Myc and cyclin D1 expression to impede endothelial cell proliferation

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

Pan, Christopher C.; Bloodworth, Jeffrey C.; Mythreye, Karthikeyan

2012-08-03

Highlights: Black-Right-Pointing-Pointer Endoglin inhibits ERK activation in endothelial cells. Black-Right-Pointing-Pointer Endoglin is a regulator of c-Myc and cyclin D1 expression. Black-Right-Pointing-Pointer {beta}-arrestin2 interaction with endoglin is required for ERK/c-Myc repression. Black-Right-Pointing-Pointer Endoglin impedes cellular proliferation by targeting ERK-induced mitogenic signaling. -- Abstract: Endoglin is an endothelial-specific transforming growth factor beta (TGF-{beta}) co-receptor essential for angiogenesis and vascular remodeling. Endoglin regulates a wide range of cellular processes, including cell adhesion, migration, and proliferation, through TGF-{beta} signaling to canonical Smad and Smad-independent pathways. Despite its overall pro-angiogenic role in the vasculature, the underlying mechanism of endoglin action is poorly characterized. We previouslymore » identified {beta}-arrestin2 as a binding partner that causes endoglin internalization from the plasma membrane and inhibits ERK signaling towards endothelial migration. In the present study, we examined the mechanistic role of endoglin and {beta}-arrestin2 in endothelial cell proliferation. We show that endoglin impedes cell growth through sustained inhibition of ERK-induced c-Myc and cyclin D1 expression in a TGF-{beta}-independent manner. The down-regulation of c-Myc and cyclin D1, along with growth-inhibition, are reversed when the endoglin/{beta}-arrestin2 interaction is disrupted. Given that TGF-{beta}-induced Smad signaling potently represses c-Myc in most cell types, our findings here show a novel mechanism by which endoglin augments growth-inhibition by targeting ERK and key downstream mitogenic substrates.« less

Effect of hyaluronic acid in bone formation and its applications in dentistry.

PubMed

Zhao, Ningbo; Wang, Xin; Qin, Lei; Zhai, Min; Yuan, Jing; Chen, Ji; Li, Dehua

2016-06-01

Hyaluronic acid (HA), the simplest glycosaminoglycan, participates in several important biological procedures, including mediation of cellular signaling, regulation of cell adhesion and proliferation, and manipulation of cell differentiation. The effect of HA on cell proliferation and differentiation depends on its molecular weight (MW) and concentration. Moreover, the properties of high viscosity, elasticity, highly negative charge, biocompatibility, biodegradability, and nonimmunogenicity make HA attractive in tissue engineering and disease treatment. This review comprises an overview of the effect of HA on cell proliferation and differentiation in vitro, the role of HA in bone regeneration in vivo, and the clinical applications of HA in dentistry, focusing on the mechanism underlining the effect of MW and concentration of HA on cell proliferation and osteogenic differentiation. It is expected that practical progress of HA both in laboratory-based experiments and clinical applications will be achieved in the next few years. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1560-1569, 2016. © 2016 Wiley Periodicals, Inc.

Effects of electrical stimulation on cell proliferation and apoptosis.

PubMed

Love, Maria R; Palee, Siripong; Chattipakorn, Siriporn C; Chattipakorn, Nipon

2018-03-01

The application of exogenous electrical stimulation (ES) to cells in order to manipulate cell apoptosis and proliferation has been widely investigated as a possible method of treatment in a number of diseases. Alteration of the transmembrane potential of cells via ES can affect various intracellular signaling pathways which are involved in the regulation of cellular function. Controversially, several types of ES have proved to be effective in both inhibiting or inducing apoptosis, as well as increasing proliferation. However, the mechanisms through which ES achieves this remain fairly unclear. The aim of this review was to comprehensively summarize current findings from in vitro and in vivo studies on the effects of different types of ES on cell apoptosis and proliferation, highlighting the possible mechanisms through which ES induced these effects and define the optimum parameters at which ES can be used. Through this we hope to provide a greater insight into how future studies can most effectively use ES at the clinical trial stage. © 2017 Wiley Periodicals, Inc.

Oncogene miR-187-5p is associated with cellular proliferation, migration, invasion, apoptosis and an increased risk of recurrence in bladder cancer.

PubMed

Li, Zuwei; Lin, Canbin; Zhao, Liwen; Zhou, Liang; Pan, Xiang; Quan, Jing; Peng, Xiqi; Li, Weiqing; Li, Hang; Xu, Jinling; Xu, Weijie; Guan, Xin; Chen, Yun; Lai, Yongqing

2018-06-05

Bladder cancer, the ninth-most-common malignancy worldwide with an estimated 356,000 new cases and 145,000 deaths annually, has a propensity to relapse, requiring lifelong monitoring after diagnosis. 75% patients diagnosed with BC are non-muscle invasive BC and over 50% of them experience recurrences within 6-12 years of initial diagnosis. miRNA are small, noncoding RNA and shown to be oncogenes or anti-oncogenes in bladder cancer, contributing to numerous BC cell processes, including cell proliferation, differentiation, migration and apoptosis. RT-qPCR were performed to detect the expression of miR-187-5p in tissues and cell lines, After which, clinicopathological variables and the prognostic value of altered miR-187-5p expression in BC was analyzed with the 48 formalin-fixed paraffin-embedded BC samples. Moreover, Cell functional assays (wound healing assay, CCK-8 assay, transwell assay and flow cytometry assay) were performed to explore the relationship between miR-187-5p expression and cell proliferation, migration, invasion and apoptosis in BC. Up-regulation of miR-187-5p was observed in BC tissues and BC cell lines. Cox proportional hazard regression analysis demonstrated that the patients with low expression of miR-187-5p experience lower risks of recurrence in the univariate and multivariate analysis. The Kaplan-Meier recurrence-free curves suggested that the patients with low expression of miR-187-5p experience lower risks of recurrence. Up-regulation of miR-187-5p promotes cell proliferation and mobility and inhibits the apoptosis of 5637 and UM-UC-3 cell, while down-regulation of miR-187-5p reverses these effects. The results of our study demonstrated that oncogene miR-187-5p is associated with cellular proliferation, migration, invasion, apoptosis and an increased risk of recurrence in bladder cancer. Copyright © 2018 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

Intermediate frequency magnetic field generated by a wireless power transmission device does not cause genotoxicity in vitro.

PubMed

Shi, Dejing; Zhu, Chunbo; Lu, Rengui; Mao, Shitong; Qi, Yanhua

2014-10-01

The aim of this study was to evaluate effects of intermediate frequency magnetic fields (IFMF) generated by a wireless power transmission (WPT) based on magnetic resonance from the perspective of cellular genotoxicity on cultured human lens epithelial cells (HLECs). We evaluated the effects of exposure to 90 kHz magnetic fields at 93.36 µT on cellular genotoxicity in vitro for 2 and 4 h. The magnetic flux density is approximately 3.5 times higher than the reference level recommended by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. For assessment of genotoxicity, we studied cellular proliferation, apoptosis and DNA damage by Cell Counting Kit-8 (CCK-8) assay, flow cytometry analysis, alkaline comet assay and phosphorylated histone H2AX (γH2AX) foci formation test. We did not detect any effect of a 90 kHz IFMF generated by WPT based on magnetic resonance on cell proliferation, apoptosis, comet assay, and γH2AX foci formation test. Our results indicated that exposure to 90 kHz IFMF generated by WPT based on magnetic resonance at 93.36 µT for 2 and 4 h does not cause detectable cellular genotoxicity. © 2014 Wiley Periodicals, Inc.

Designer self-assembling hydrogel scaffolds can impact skin cell proliferation and migration

PubMed Central

Bradshaw, Michael; Ho, Diwei; Fear, Mark W.; Gelain, Fabrizio; Wood, Fiona M.; Iyer, K. Swaminathan

2014-01-01

There is a need to develop economical, efficient and widely available therapeutic approaches to enhance the rate of skin wound healing. The optimal outcome of wound healing is restoration to the pre-wound quality of health. In this study we investigate the cellular response to biological stimuli using functionalized nanofibers from the self-assembling peptide, RADA16. We demonstrate that adding different functional motifs to the RADA16 base peptide can influence the rate of proliferation and migration of keratinocytes and dermal fibroblasts. Relative to unmodified RADA16; the Collagen I motif significantly promotes cell migration, and reduces proliferation. PMID:25384420

Role of cellular bioenergetics in smooth muscle cell proliferation induced by platelet-derived growth factor.

PubMed

Perez, Jessica; Hill, Bradford G; Benavides, Gloria A; Dranka, Brian P; Darley-Usmar, Victor M

2010-05-13

Abnormal smooth muscle cell proliferation is a hallmark of vascular disease. Although growth factors are known to contribute to cell hyperplasia, the changes in metabolism associated with this response, particularly mitochondrial respiration, remain unclear. Given the increased energy requirements for proliferation, we hypothesized that PDGF (platelet-derived growth factor) would stimulate glycolysis and mitochondrial respiration and that this elevated bioenergetic capacity is required for smooth muscle cell hyperplasia. To test this hypothesis, cell proliferation, glycolytic flux and mitochondrial oxygen consumption were measured after treatment of primary rat aortic VSMCs (vascular smooth muscle cells) with PDGF. PDGF increased basal and maximal rates of glycolytic flux and mitochondrial oxygen consumption; enhancement of these bioenergetic pathways led to a substantial increase in the mitochondrial reserve capacity. Interventions with the PI3K (phosphoinositide 3-kinase) inhibitor LY-294002 or the glycolysis inhibitor 2-deoxy-D-glucose abrogated PDGF-stimulated proliferation and prevented augmentation of glycolysis and mitochondrial reserve capacity. Similarly, when L-glucose was substituted for D-glucose, PDGF-dependent proliferation was abolished, as were changes in glycolysis and mitochondrial respiration. Interestingly, LDH (lactate dehydrogenase) protein levels and activity were significantly increased after PDGF treatment. Moreover, substitution of L-lactate for D-glucose was sufficient to increase mitochondrial reserve capacity and cell proliferation after treatment with PDGF; these effects were inhibited by the LDH inhibitor oxamate. These results suggest that glycolysis, by providing substrates that enhance the mitochondrial reserve capacity, plays an essential role in PDGF-induced cell proliferation, underscoring the integrated metabolic response required for proliferation of VSMCs in the diseased vasculature.

Functional response of osteoblasts in functionally gradient titanium alloy mesh arrays processed by 3D additive manufacturing.

PubMed

Nune, K C; Kumar, A; Misra, R D K; Li, S J; Hao, Y L; Yang, R

2017-02-01

We elucidate here the osteoblasts functions and cellular activity in 3D printed interconnected porous architecture of functionally gradient Ti-6Al-4V alloy mesh structures in terms of cell proliferation and growth, distribution of cell nuclei, synthesis of proteins (actin, vinculin, and fibronectin), and calcium deposition. Cell culture studies with pre-osteoblasts indicated that the interconnected porous architecture of functionally gradient mesh arrays was conducive to osteoblast functions. However, there were statistically significant differences in the cellular response depending on the pore size in the functionally gradient structure. The interconnected porous architecture contributed to the distribution of cells from the large pore size (G1) to the small pore size (G3), with consequent synthesis of extracellular matrix and calcium precipitation. The gradient mesh structure significantly impacted cell adhesion and influenced the proliferation stage, such that there was high distribution of cells on struts of the gradient mesh structure. Actin and vinculin showed a significant difference in normalized expression level of protein per cell, which was absent in the case of fibronectin. Osteoblasts present on mesh struts formed a confluent sheet, bridging the pores through numerous cytoplasmic extensions. The gradient mesh structure fabricated by electron beam melting was explored to obtain fundamental insights on cellular activity with respect to osteoblast functions. Copyright © 2016 Elsevier B.V. All rights reserved.

Alterations to the protein profile of bladder carcinoma cell lines induced by plant extract MINA-05 in vitro.

PubMed

Nguyen-Khuong, Terry; White, Melanie Y; Hung, Tzong-Tyng; Seeto, Shona; Thomas, Melissa L; Fitzgerald, Anna M; Martucci, Carlos E; Luk, Sharon; Pang, Shiu-Fu; Russell, Pamela J; Walsh, Bradley J

2009-04-01

Bladder cancer (BLCa) is a severe urological cancer of both men and women that commonly recurs and once invasive, is difficult to treat. MINA-05 (CK Life Sciences Int'l, Hong Kong) is a derivative of complex botanical extracts, shown to reduce cellular proliferation of bladder and prostate carcinomas. We tested the effects of MINA-05 against human BLCa cell sublines, B8, B8-RSP-GCK, B8-RSP-LN and C3, from a transitional cell carcinoma, grade IV, to determine the molecular targets of treatment by observing the cellular protein profile. Cells were acclimatised for 48 h then treated for 72 h with concentrations of MINA-05 reflecting 1/2 IC(50), IC(50) and 2 x IC(50) (n = 3) or with vehicle, (0.5% DMSO). Dose-dependant changes in protein abundance were detected and characterised using 2-dimensional electrophoresis and MS. We identified 10 proteins that underwent changes in abundance, pI and/or molecular mass in response to treatment. MINA-05 was shown to influence proteins across numerous functional classes including cytoskeletal proteins, energy metabolism proteins, protein degradation proteins and tumour suppressors, suggesting a global impact on these cell lines. This study implies that the ability of MINA-05 to retard cellular proliferation is attributed to its ability to alter cell cycling, metabolism, protein degradation and the cancer cell environment.

Adenylate kinase 2 (AK2) promotes cell proliferation in insect development

PubMed Central

2012-01-01

Background Adenylate kinase 2 (AK2) is a phosphotransferase that catalyzes the reversible reaction 2ADP(GDP) ↔ ATP(GTP) + AMP and influences cellular energy homeostasis. However, the role of AK2 in regulating cell proliferation remains unclear because AK2 has been reported to be involved in either cell proliferation or cell apoptosis in different cell types of various organisms. Results This study reports AK2 promotion of cell proliferation using the lepidopteran insect Helicoverpa armigera and its epidermal cell line HaEpi as models. Western blot analysis indicates that AK2 constitutively expresses in various tissues during larval development. Immunocytochemistry analysis indicates that AK2 localizes in the mitochondria. The recombinant expressed AK2 in E. coli promotes cell growth and viability of HaEpi cell line by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. AK2 knockdown in larvae by RNA interference causes larval growth defects, including body weight decrease and development delay. AK2 knockdown in larvae also decreases the number of circulating haemocytes. The mechanism for such effects might be the suppression of gene transcription involved in insect development caused by AK2 knockdown. Conclusion These results show that AK2 regulates cell growth, viability, and proliferation in insect growth and development. PMID:23020757

Roles of Stat3 and ERK in G-CSF signaling.

PubMed

Kamezaki, Kenjirou; Shimoda, Kazuya; Numata, Akihiko; Haro, Takashi; Kakumitsu, Haruko; Yoshie, Masumi; Yamamoto, Masahiro; Takeda, Kiyoshi; Matsuda, Tadashi; Akira, Shizuo; Ogawa, Katsuhiro; Harada, Mine

2005-02-01

G-CSF specifically stimulates the proliferation and differentiation of cells that are committed to the neutrophil-granulocyte lineage. Although Stat3 was thought to be essential for the transduction of G-CSF-induced cell proliferation and differentiation signals, mice deficient for Stat3 in hematopoietic cells show neutrocytosis and infiltration of cells into the digestive tract. The number of progenitor cells in the neutrophil lineage is not changed, and G-CSF-induced proliferation of progenitor cells and prolonged neutrophil survival were observed in Stat3-deficient mice. In hematopoietic cells from Stat3-deficient mice, trace levels of SOCS3, a negative regulator of granulopoiesis, were observed, and SOCS3 expression was not induced by G-CSF stimulation. Stat3-null bone marrow cells displayed a significant activation of extra-cellular regulated kinase 1 (ERK1)/ERK2 under basal conditions, and the activation of ERK was enhanced and sustained by G-CSF stimulation. Furthermore, the augmented proliferation of Stat3-deficient bone marrow cells in response to G-CSF was dramatically decreased by addition of a MEK1 inhibitor. These results indicate that Stat3 functions as a negative regulator of G-CSF signaling by inducing SOCS3 expression and that ERK activation is the major factor responsible for inducing the proliferation of hematopoietic cells in response to G-CSF.

Complete horizontal skin cell resurfacing and delayed vertical cell infiltration into porcine reconstructive tissue matrix compared to bovine collagen matrix and human dermis.

PubMed

Mirastschijski, Ursula; Kerzel, Corinna; Schnabel, Reinhild; Strauss, Sarah; Breuing, Karl-Heinz

2013-10-01

Xenogenous dermal matrices are used for hernia repair and breast reconstruction. Full-thickness skin replacement is needed after burn or degloving injuries with exposure of tendons or bones. The authors used a human skin organ culture model to study whether porcine reconstructive tissue matrix (Strattice) is effective as a dermal tissue replacement. Skin cells or split-thickness skin grafts were seeded onto human deepidermized dermis, Strattice, and Matriderm. Cellular resurfacing and matrix infiltration were monitored by live fluorescence imaging, histology, and electron microscopy. Proliferation, apoptosis, cell differentiation, and adhesion were analyzed by immunohistochemistry. Epithelial resurfacing and vertical proliferation were reduced and delayed with both bioartificial matrices compared with deepidermized dermis; however, no differences in apoptosis, cell differentiation, or basement membrane formation were found. Vertical penetration was greatest on Matriderm, whereas no matrix infiltration was found on Strattice in the first 12 days. Uncompromised horizontal resurfacing was greatest with Strattice but was absent with Matriderm. Strattice showed no stimulatory effect on cellular inflammation. Matrix texture and surface properties governed cellular performance on tissues. Although dense dermal compaction delayed vertical cellular ingrowth for Strattice, it allowed uncompromised horizontal resurfacing. Dense dermal compaction may slow matrix decomposition and result in prolonged biomechanical stability of the graft. Reconstructive surgeons should choose the adequate matrix substitute depending on biomechanical requirements at the recipient site. Strattice may be suitable as a dermal replacement at recipient sites with high mechanical load requirements.

In vitro characterization of MG-63 osteoblast-like cells cultured on organic-inorganic lyophilized gelatin sponges for early bone healing.

PubMed

Rodriguez, Isaac A; Saxena, Gunjan; Hixon, Katherine R; Sell, Scott A; Bowlin, Gary L

2016-08-01

The development of three-dimensional porous scaffolds with enhanced osteogenic and angiogenic potential would be beneficial for inducing early-stage bone regeneration. Previous studies have demonstrated the advantages of mineralized and nonmineralized acellular 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) cross-linked gelatin sponges enhanced with preparations rich in growth factors, hydroxyapatite, and chitin whiskers. In this study, those same scaffolds were mineralized and dynamically seeded with MG-63 cells. Cell proliferation, protein/cytokine secretion, and compressive mechanical properties of scaffolds were evaluated. It was found that mineralization and the addition of growth factors increased cell proliferation compared to gelatin controls. Cells on all scaffolds responded in an appropriate bone regenerative fashion as shown through osteocalcin secretion and little to no secretion of bone resorbing markers. However, compressive mechanical properties of cellularized scaffolds were not significantly different from acellular scaffolds. The combined results of increased cellular attachment, infiltration, and bone regenerative protein/cytokine secretion on scaffolds support the need for the addition of a bone-like mineral surface. Cellularized scaffolds containing growth factors reported similar advantages and mechanical values in the range of native tissues present in the early stages of bone healing. These results suggest that the developed composite sponges exhibited cellular responses and mechanical properties appropriate for promoting early bone healing in various applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2011-2019, 2016. © 2016 Wiley Periodicals, Inc.

Adipose tissue-derived stem cells enhance bioprosthetic mesh repair of ventral hernias.

PubMed

Altman, Andrew M; Abdul Khalek, Feras J; Alt, Eckhard U; Butler, Charles E

2010-09-01

Bioprosthetic mesh used for ventral hernia repair becomes incorporated into the musculofascial edge by cellular infiltration and vascularization. Adipose tissue-derived stem cells promote tissue repair and vascularization and may increase the rate or degree of tissue incorporation. The authors hypothesized that introducing these cells into bioprosthetic mesh would result in adipose tissue-derived stem cell engraftment and proliferation and enhance incorporation of the bioprosthetic mesh. Adipose tissue-derived stem cells were isolated from the subcutaneous adipose tissue of syngeneic Brown Norway rats, expanded in vitro, and labeled with green fluorescent protein. Thirty-six additional rats underwent inlay ventral hernia repair with porcine acellular dermal matrix. Two 12-rat groups had the cells (1.0 x 10(6)) injected directly into the musculofascial/porcine acellular dermal matrix interface after repair or received porcine acellular dermal matrix on which the cells had been preseeded; the 12-rat control group received no stem cells. At 2 weeks, adipose tissue-derived stem cells in both stem cell groups engrafted, survived, migrated, and proliferated. Mean cellular infiltration into porcine acellular dermal matrix at the musculofascial/graft interface was significantly greater in the preseeded and injected stem cell groups than in the control group. Mean vascular infiltration of the porcine acellular dermal matrix was significantly greater in both stem cell groups than in the control group. Preseeded and injected adipose tissue-derived stem cells engraft, migrate, proliferate, and enhance the vascularity of porcine acellular dermal matrix grafts at the musculofascial/graft interface. These cells can thus enhance incorporation of porcine acellular dermal matrix into the abdominal wall after repair of ventral hernias.

Arabidopsis and Tobacco SUPERMAN regulate hormone signalling and mediate cell proliferation and differentiation

PubMed Central

Nibau, Candida; Di Stilio, Verónica S.; Wu, Hen-ming; Cheung, Alice Y.

2011-01-01

Arabidopsis thaliana SUPERMAN (SUP) plays an important role during flower development by maintaining the boundary between stamens and carpels in the inner two whorls. It was proposed that SUP maintains this boundary by regulating cell proliferation in both whorls, as loss-of-function superman mutants produce more stamens at the expense of carpels. However, the cellular mechanism that underlies SUP function remains unknown. Here Arabidopsis or tobacco (Nicotiana tabacum) SUP was overexpressed in tobacco plants to substantiate SUP's role as a regulator of cell proliferation and boundary definition and provide evidence that its biological role may be mediated via hormonal changes. It was found that moderate levels of SUP stimulated cell growth and proliferation, whereas high levels were inhibitory. SUP stimulated auxin- and cytokinin-regulated processes, and cells overexpressing SUP displayed reduced hormone dependency for proliferation and regeneration into plants. SUP also induced proliferation of female traits in the second and third flower whorls and promoted differentiation of petaloid properties in sepals, further supporting a role for SUP as a boundary regulator. Moreover, cytokinin suppressed stamen development and promoted differentiation of carpeloid tissues, suggesting that SUP may regulate male and female development via its effect on cytokinin signalling. Taken together, these observations suggest a model whereby the effect of SUP on cell growth and proliferation involves the modulation of auxin- and cytokinin-regulated processes. Furthermore, differential SUP expression or different sensitivities of different cell types to SUP may determine whether SUP stimulates or suppresses their proliferation. PMID:20980362

Simulated predator stimuli reduce brain cell proliferation in two electric fish species, Brachyhypopomus gauderio and Apteronotus leptorhynchus.

PubMed

Dunlap, Kent D; Keane, Geoffrey; Ragazzi, Michael; Lasky, Elise; Salazar, Vielka L

2017-07-01

The brain structure of many animals is influenced by their predators, but the cellular processes underlying this brain plasticity are not well understood. Previous studies showed that electric fish ( Brachyhypopomus occidentalis ) naturally exposed to high predator ( Rhamdia quelen ) density and tail injury had reduced brain cell proliferation compared with individuals facing few predators and those with intact tails. However, these field studies described only correlations between predator exposure and cell proliferation. Here, we used a congener Brachyhypopomus gauderio and another electric fish Apteronotus leptorhynchus to experimentally test the hypothesis that exposure to a predator stimulus and tail injury causes alterations in brain cell proliferation. To simulate predator exposure, we either amputated the tail followed by short-term (1 day) or long-term (17-18 days) recovery or repeatedly chased intact fish with a plastic rod over a 7 day period. We measured cell proliferation (PCNA+ cell density) in the telencephalon and diencephalon, and plasma cortisol, which commonly mediates stress-induced changes in brain cell proliferation. In both species, either tail amputation or simulated predator chase decreased cell proliferation in the telencephalon in a manner resembling the effect of predators in the field. In A. leptorhynchus , cell proliferation decreased drastically in the short term after tail amputation and partially rebounded after long-term recovery. In B. gauderio , tail amputation elevated cortisol levels, but repeated chasing had no effect. In A. leptorhynchus , tail amputation elevated cortisol levels in the short term but not in the long term. Thus, predator stimuli can cause reductions in brain cell proliferation, but the role of cortisol is not clear. © 2017. Published by The Company of Biologists Ltd.

Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis

PubMed Central

Kümper, Sandra; Mardakheh, Faraz K; McCarthy, Afshan; Yeo, Maggie; Stamp, Gordon W; Paul, Angela; Worboys, Jonathan; Sadok, Amine; Jørgensen, Claus; Guichard, Sabrina

2016-01-01

Rho-associated kinases 1 and 2 (ROCK1/2) are Rho-GTPase effectors that control key aspects of the actin cytoskeleton, but their role in proliferation and cancer initiation or progression is not known. Here, we provide evidence that ROCK1 and ROCK2 act redundantly to maintain actomyosin contractility and cell proliferation and that their loss leads to cell-cycle arrest and cellular senescence. This phenotype arises from down-regulation of the essential cell-cycle proteins CyclinA, CKS1 and CDK1. Accordingly, while the loss of either Rock1 or Rock2 had no negative impact on tumorigenesis in mouse models of non-small cell lung cancer and melanoma, loss of both blocked tumor formation, as no tumors arise in which both Rock1 and Rock2 have been genetically deleted. Our results reveal an indispensable role for ROCK, yet redundant role for isoforms 1 and 2, in cell cycle progression and tumorigenesis, possibly through the maintenance of cellular contractility. DOI: http://dx.doi.org/10.7554/eLife.12203.001 PMID:26765561

Time-lapse analysis of potential cellular responsiveness to Johrei, a Japanese healing technique

PubMed Central

Taft, Ryan; Moore, Dan; Yount, Garret

2005-01-01

Background Johrei is an alternative healing practice which involves the channeling of a purported universal healing energy to influence the health of another person. Despite little evidence to support the efficacy of such practices the use of such treatments is on the rise. Methods We assessed cultured human cancer cells for potential responsiveness to Johrei treatment from a short distance. Johrei treatment was delivered by practitioners who participated in teams of two, alternating every half hour for a total of four hours of treatment. The practitioners followed a defined set of mental procedures to minimize variability in mental states between experiments. An environmental chamber maintained optimal growth conditions for cells throughout the experiments. Computerized time-lapse microscopy allowed documentation of cancer cell proliferation and cell death before, during and after Johrei treatments. Results Comparing eight control experiments with eight Johrei intervention experiments, we found no evidence of a reproducible cellular response to Johrei treatment. Conclusion Cell death and proliferation rates of cultured human cancer cells do not appear responsive to Johrei treatment from a short distance. PMID:15667653

Myocilin Regulates Cell Proliferation and Survival*

PubMed Central

Joe, Myung Kuk; Kwon, Heung Sun; Cojocaru, Radu; Tomarev, Stanislav I.

2014-01-01

Myocilin, a causative gene for open angle glaucoma, encodes a secreted glycoprotein with poorly understood functions. To gain insight into its functions, we produced a stably transfected HEK293 cell line expressing myocilin under an inducible promoter and compared gene expression profiles between myocilin-expressing and vector control cell lines by a microarray analysis. A significant fraction of differentially expressed genes in myocilin-expressing cells was associated with cell growth and cell death, suggesting that myocilin may have a role in the regulation of cell growth and survival. Increased proliferation of myocilin-expressing cells was demonstrated by the WST-1 proliferation assay, direct cell counting, and immunostaining with antibodies against Ki-67, a cellular proliferation marker. Myocilin-containing conditioned medium also increased proliferation of unmodified HEK293 cells. Myocilin-expressing cells were more resistant to serum starvation-induced apoptosis than control cells. TUNEL-positive apoptotic cells were dramatically decreased, and two apoptotic marker proteins, cleaved caspase 7 and cleaved poly(ADP-ribose) polymerase, were significantly reduced in myocilin-expressing cells as compared with control cells under apoptotic conditions. In addition, myocilin-deficient mesenchymal stem cells exhibited reduced proliferation and enhanced susceptibility to serum starvation-induced apoptosis as compared with wild-type mesenchymal stem cells. Phosphorylation of ERK1/2 and its upstream kinases, c-Raf and MEK, was increased in myocilin-expressing cells compared with control cells. Elevated phosphorylation of ERK1/2 was also observed in the trabecular meshwork of transgenic mice expressing 6-fold higher levels of myocilin when compared with their wild-type littermates. These results suggest that myocilin promotes cell proliferation and resistance to apoptosis via the ERK1/2 MAPK signaling pathway. PMID:24563482

Docosahexaenoic acid (DHA) effects on proliferation and steroidogenesis of bovine granulosa cells.

PubMed

Maillard, Virginie; Desmarchais, Alice; Durcin, Maeva; Uzbekova, Svetlana; Elis, Sebastien

2018-04-26

Docosahexaenoic acid (DHA) is a n-3 polyunsaturated fatty acid (PUFA) belonging to a family of biologically active fatty acids (FA), which are known to have numerous health benefits. N-3 PUFAs affect reproduction in cattle, and notably directly affect follicular cells. In terms of reproduction in cattle, n-3 PUFA-enriched diets lead to increased follicle size or numbers. The objective of the present study was to analyze the effects of DHA (1, 10, 20 and 50 μM) on proliferation and steroidogenesis (parametric and/or non parametric (permutational) ANOVA) of bovine granulosa cells in vitro and mechanisms of action through protein expression (Kruskal-Wallis) and signaling pathways (non parametric ANOVA) and to investigate whether DHA could exert part of its action through the free fatty acid receptor 4 (FFAR4). DHA (10 and 50 μM) increased granulosa cell proliferation and DHA 10 μM led to a corresponding increase in proliferating cell nuclear antigen (PCNA) expression level. DHA also increased progesterone secretion at 1, 20 and 50 μM, and estradiol secretion at 1, 10 and 20 μM. Consistent increases in protein levels were also reported for the steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), and of the cholesterol transporter steroidogenic acute regulatory protein (StAR), which are necessary for production of progesterone or androstenedione. FFAR4 was expressed in all cellular types of bovine ovarian follicles, and in granulosa cells it was localized close to the cellular membrane. TUG-891 treatment (1 and 50 μM), a FFAR4 agonist, increased granulosa cell proliferation and MAPK14 phosphorylation in a similar way to that observed with DHA treatment. However, TUG-891 treatment (1, 10 and 50 μM) showed no effect on progesterone or estradiol secretion. These data show that DHA stimulated proliferation and steroidogenesis of bovine granulosa cells and led to MAPK14 phosphorylation. FFAR4 involvement in DHA effects requires further investigation, even if our data might suggest FFAR4 role in DHA effects on granulosa cell proliferation. Other mechanisms of DHA action should be investigated as the steroidogenic effects seemed to be independent of FFAR4 activation.

Understanding the cancer cell phenotype beyond the limitations of current omics analyses.

PubMed

Moreno-Sánchez, Rafael; Saavedra, Emma; Gallardo-Pérez, Juan Carlos; Rumjanek, Franklin D; Rodríguez-Enríquez, Sara

2016-01-01

Efforts to understand the mechanistic principles driving cancer metabolism and proliferation have been lately governed by genomic, transcriptomic and proteomic studies. This paper analyzes the caveats of these approaches. As molecular biology's central dogma proposes a unidirectional flux of information from genes to mRNA to proteins, it has frequently been assumed that monitoring the changes in the gene sequences and in mRNA and protein contents is sufficient to explain complex cellular processes. Such a stance commonly disregards that post-translational modifications can alter the protein function/activity and also that regulatory mechanisms enter into action, to coordinate the protein activities of pathways/cellular processes, in order to keep the cellular homeostasis. Hence, the actual protein activities (as enzymes/transporters/receptors) and their regulatory mechanisms ultimately dictate the final outcomes of a pathway/cellular process. In this regard, it is here documented that the mRNA levels of many metabolic enzymes and transcriptional factors have no correlation with the respective protein contents and activities. The validity of current clinical mRNA-based tests and proposed metabolite biomarkers for cancer detection/prognosis is also discussed. Therefore, it is proposed that, to achieve a thorough understanding of the modifications undergone by proliferating cancer cells, it is mandatory to experimentally analyze the cellular processes at the functional level. This could be achieved (a) locally, by examining the actual protein activities in the cell and their kinetic properties (or at least kinetically characterize the most controlling steps of the pathway/cellular process); (b) systemically, by analyzing the main fluxes of the pathway/cellular process, and how they are modulated by metabolites, all which should contribute to comprehending the regulatory mechanisms that have been altered in cancer cells. By adopting a more holistic approach it may become possible to improve the design of therapeutic strategies that would target cancer cells more specifically. © 2015 FEBS.

Estradiol and corticosterone stimulate the proliferation of a GH cell line, MtT/S: Proliferation of growth hormone cells.

PubMed

Nogami, Haruo; Hiraoka, Yoshiki; Aiso, Sadakazu

2016-08-01

Estrogens are known as a potent growth-stimulator of the anterior pituitary cells such as prolactin cells and somatomammotroph cell lines, while glucocorticoids often inhibit cellular proliferation in the pituitary gland as well as in the extra-pituitary tissues. In this study, the involvement of these steroid hormones in the regulation of proliferation was examined in the MtT/S cells, secreting growth hormone (GH). Effects of estrogens and glucocorticoids were examined in MtT/S cells grown in the medium containing dextran-coated charcoal treated serum. The relative cell density after culture was estimated by the Cell Titer-Glo Luminescent Cell Viability Assay System, and the proliferation rate was determined by the BrdU incorporation method. The mRNA levels were determined by real-time PCR. Estradiol and the specific agonist for both estrogen receptor (ER) α and ERβ stimulated MtT/S growth at a dose dependent manner. The membrane impermeable estrogen, 17β-estradiol-bovine serum albumin conjugate also stimulated the MtT/S proliferation. The effects of all estrogens were inhibited by an estrogen receptor antagonist, ICI182780. Corticosterone stimulated the proliferation of MtT/S cells at doses lower than 10nM without stimulating GH gene transcription, whereas it did not change the proliferation rate at 1μM. The effects of corticosterone were inhibited by glucocorticoid receptor inhibitor, RU486, but not by the mineralocorticoid receptor antagonist, spironolactone. Both estrogens and glucocorticoids were found to stimulate the proliferation of MtT/S, increasing the mRNA expression of cyclins D1, D3, and E. The results suggest that estrogens and glucocorticoids may be involved in the mechanisms responsible for the proliferation of GH cells in the course of pituitary development, to maintain the population of GH cells in the adult pituitary gland, and also in the promotion of GH cell tumors. Copyright © 2016 Elsevier Ltd. All rights reserved.

A comprehensive model of the spatio-temporal stem cell and tissue organisation in the intestinal crypt.

PubMed

Buske, Peter; Galle, Jörg; Barker, Nick; Aust, Gabriela; Clevers, Hans; Loeffler, Markus

2011-01-06

We introduce a novel dynamic model of stem cell and tissue organisation in murine intestinal crypts. Integrating the molecular, cellular and tissue level of description, this model links a broad spectrum of experimental observations encompassing spatially confined cell proliferation, directed cell migration, multiple cell lineage decisions and clonal competition.Using computational simulations we demonstrate that the model is capable of quantitatively describing and predicting the dynamic behaviour of the intestinal tissue during steady state as well as after cell damage and following selective gain or loss of gene function manipulations affecting Wnt- and Notch-signalling. Our simulation results suggest that reversibility and flexibility of cellular decisions are key elements of robust tissue organisation of the intestine. We predict that the tissue should be able to fully recover after complete elimination of cellular subpopulations including subpopulations deemed to be functional stem cells. This challenges current views of tissue stem cell organisation.

Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks.

PubMed

Polchow, Bianca; Kebbel, Kati; Schmiedeknecht, Gerno; Reichardt, Anne; Henrich, Wolfgang; Hetzer, Roland; Lueders, Cora

2012-05-16

In vitro fabricated tissue engineered vascular constructs could provide an alternative to conventional substitutes. A crucial factor for tissue engineering of vascular constructs is an appropriate cell source. Vascular cells from the human umbilical cord can be directly isolated and cryopreserved until needed. Currently no cell bank for human vascular cells is available. Therefore, the establishment of a future human vascular cell bank conforming to good manufacturing practice (GMP) conditions is desirable for therapeutic applications such as tissue engineered cardiovascular constructs. A fundamental step was the adaption of conventional research and development starting materials to GMP compliant starting materials. Human umbilical cord artery derived cells (HUCAC) and human umbilical vein endothelial cells (HUVEC) were isolated, cultivated, cryopreserved (short- and long-term) directly after primary culture and recultivated subsequently. Cell viability, expression of cellular markers and proliferation potential of fresh and cryopreserved cells were studied using trypan blue staining, flow cytometry analysis, immunofluorescence staining and proliferation assays. Statistical analyses were performed using Student's t-test. Sufficient numbers of isolated cells with acceptable viabilities and homogenous expression of cellular markers confirmed that the isolation procedure was successful using GMP compliant starting materials. The influence of cryopreservation was marginal, because cryopreserved cells mostly maintain phenotypic and functional characteristics similar to those of fresh cells. Phenotypic studies revealed that fresh cultivated and cryopreserved HUCAC were positive for alpha smooth muscle actin, CD90, CD105, CD73, CD29, CD44, CD166 and negative for smoothelin. HUVEC expressed CD31, CD146, CD105 and CD144 but not alpha smooth muscle actin. Functional analysis demonstrated acceptable viability and sufficient proliferation properties of cryopreserved HUCAC and HUVEC. Adaptation of cell isolation, cultivation and cryopreservation to GMP compliant starting materials was successful. Cryopreservation did not influence cell properties with lasting impact, confirming that the application of vascular cells from the human umbilical cord is feasible for cell banking. A specific cellular marker expression profile was established for HUCAC and HUVEC using flow cytometry analysis, applicable as a GMP compliant quality control. Use of these cells for the future fabrication of advanced therapy medicinal products GMP conditions are required by the regulatory authority.

Substrate effect modulates adhesion and proliferation of fibroblast on graphene layer.

PubMed

Lin, Feng; Du, Feng; Huang, Jianyong; Chau, Alicia; Zhou, Yongsheng; Duan, Huiling; Wang, Jianxiang; Xiong, Chunyang

2016-10-01

Graphene is an emerging candidate for biomedical applications, including biosensor, drug delivery and scaffold biomaterials. Cellular functions and behaviors on different graphene-coated substrates, however, still remain elusive to a great extent. This paper explored the functional responses of cells such as adhesion and proliferation, to different kinds of substrates including coverslips, silicone, polydimethylsiloxane (PDMS) with different curing ratios, PDMS treated with oxygen plasma, and their counterparts coated with single layer graphene (SLG). Specifically, adherent cell number, spreading area and cytoskeleton configuration were exploited to characterize cell-substrate adhesion ability, while MTT assay was employed to test the proliferation capability of fibroblasts. Experimental outcome demonstrated graphene coating had excellent cytocompatibility, which could lead to an increase in early adhesion, spreading, proliferation, and remodeling of cytoskeletons of fibroblast cells. Notably, it was found that the underlying substrate effect, e.g., stiffness of substrate materials, could essentially regulate the adhesion and proliferation of cells cultured on graphene. The stiffer the substrates were, the stronger the abilities of adhesion and proliferation of fibroblasts were. This study not only deepens our understanding of substrate-modulated interfacial interactions between live cells and graphene, but also provides a valuable guidance for the design and application of graphene-based biomaterials in biomedical engineering. Copyright © 2016 Elsevier B.V. All rights reserved.

CD83 Antibody Inhibits Human B Cell Responses to Antigen as well as Dendritic Cell-Mediated CD4 T Cell Responses.

PubMed

Wong, Kuan Y; Baron, Rebecca; Seldon, Therese A; Jones, Martina L; Rice, Alison M; Munster, David J

2018-05-15

Anti-CD83 Ab capable of Ab-dependent cellular cytotoxicity can deplete activated CD83 + human dendritic cells, thereby inhibiting CD4 T cell-mediated acute graft-versus-host disease. As CD83 is also expressed on the surface of activated B lymphocytes, we hypothesized that anti-CD83 would also inhibit B cell responses to stimulation. We found that anti-CD83 inhibited total IgM and IgG production in vitro by allostimulated human PBMC. Also, Ag-specific Ab responses to immunization of SCID mice xenografted with human PBMC were inhibited by anti-CD83 treatment. This inhibition occurred without depletion of all human B cells because anti-CD83 lysed activated CD83 + B cells by Ab-dependent cellular cytotoxicity and spared resting (CD83 - ) B cells. In cultured human PBMC, anti-CD83 inhibited tetanus toxoid-stimulated B cell proliferation and concomitant dendritic cell-mediated CD4 T cell proliferation and expression of IFN-γ and IL-17A, with minimal losses of B cells (<20%). In contrast, the anti-CD20 mAb rituximab depleted >80% of B cells but had no effect on CD4 T cell proliferation and cytokine expression. By virtue of the ability of anti-CD83 to selectively deplete activated, but not resting, B cells and dendritic cells, with the latter reducing CD4 T cell responses, anti-CD83 may be clinically useful in autoimmunity and transplantation. Advantages might include inhibited expansion of autoantigen- or alloantigen-specific B cells and CD4 T cells, thus preventing further production of pathogenic Abs and inflammatory cytokines while preserving protective memory and regulatory cells. Copyright © 2018 by The American Association of Immunologists, Inc.

Goat milk with and without increased concentrations of lysozyme improves repair of intestinal cell damage induced by enteroaggregative Escherichia coli.

PubMed

Carvalho, Eunice B; Maga, Elizabeth A; Quetz, Josiane S; Lima, Ila F N; Magalhães, Hemerson Y F; Rodrigues, Felipe A R; Silva, Antônio V A; Prata, Mara M G; Cavalcante, Paloma A; Havt, Alexandre; Bertolini, Marcelo; Bertolini, Luciana R; Lima, Aldo A M

2012-08-11

Enteroaggregative Escherichia coli (EAEC) causes diarrhea, malnutrition and poor growth in children. Human breast milk decreases disease-causing bacteria by supplying nutrients and antimicrobial factors such as lysozyme. Goat milk with and without human lysozyme (HLZ) may improve the repair of intestinal barrier function damage induced by EAEC. This work investigates the effect of the milks on intestinal barrier function repair, bacterial adherence in Caco-2 and HEp-2 cells, intestinal cell proliferation, migration, viability and apoptosis in IEC-6 cells in the absence or presence of EAEC. Rat intestinal epithelial cells (IEC-6, ATCC, Rockville, MD) were used for proliferation, migration and viability assays and human colon adenocarcinoma (Caco-2, ATCC, Rockville, MD) and human larynx carcinoma (HEp-2, ATCC, Rockville, MD) cells were used for bacterial adhesion assays. Goats expressing HLZ in their milk were generated and express HLZ in milk at concentration of 270 μg/ml. Cells were incubated with pasteurized milk from either transgenic goats expressing HLZ or non-transgenic control goats in the presence and absence of EAEC strain 042 (O44:H18). Cellular proliferation was significantly greater in the presence of both HLZ transgenic and control goat milk compared to cells with no milk. Cellular migration was significantly decreased in the presence of EAEC alone but was restored in the presence of milk. Milk from HLZ transgenic goats had significantly more migration compared to control milk. Both milks significantly reduced EAEC adhesion to Caco-2 cells and transgenic milk resulted in less colonization than control milk using a HEp-2 assay. Both milks had significantly increased cellular viability as well as less apoptosis in both the absence and presence of EAEC. These data demonstrated that goat milk is able to repair intestinal barrier function damage induced by EAEC and that goat milk with a higher concentration of lysozyme offers additional protection.

Permeabilization of the plasma membrane of L1210 mouse leukemia cells using lithotripter shock waves.

PubMed

Gambihler, S; Delius, M; Ellwart, J W

1994-09-01

Permeabilization of L1210 cells by lithotripter shock waves in vitro was monitored by evaluating the accumulation of fluorescein-labeled dextrans with a relative molecular mass ranging from 3,900-2,000,000. Incubation with labeled dextran alone caused a dose- and time-dependent increase in cellular fluorescence as determined by flow cytometry, with a vesicular distribution pattern in the cells consistent with endocytotic uptake. Shock wave exposure prior to incubation with labeled dextran revealed similar fluorescence intensities compared to incubation with labeled dextran alone. When cells were exposed to shock waves in the presence of labeled dextran, mean cellular fluorescence was further increased, indicating additional internalization of the probe. Confocal laser scanning microscopy confirmed intracellular fluorescence of labeled dextran with a diffuse distribution pattern. Fluorescence-activated cell sorting with subsequent determination of proliferation revealed that permeabilized cells were viable and able to proliferate. Permeabilization of the membrane of L1210 cells by shock waves in vitro allowed loading of dextrans with a relative molecular mass up to 2,000,000. Permeabilization of tumor cells by shock waves provides a useful tool for introducing molecules into cells which might be of interest for drug targeting in tumor therapy in vivo.

Cell death and renewal during prey capture and digestion in the carnivorous sponge Asbestopluma hypogea (Porifera: Poecilosclerida).

PubMed

Martinand-Mari, Camille; Vacelet, Jean; Nickel, Michael; Wörheide, Gert; Mangeat, Paul; Baghdiguian, Stephen

2012-11-15

The sponge Asbestopluma hypogea is unusual among sponges due to its peculiar carnivorous feeding habit. During various stages of its nutrition cycle, the sponge is subjected to spectacular morphological modifications. Starved animals are characterized by many elongated filaments, which are crucial for the capture of prey. After capture, and during the digestion process, these filaments actively regress before being regenerated during a subsequent period of starvation. Here, we demonstrate that these morphological events rely on a highly dynamic cellular turnover, implying a coordinated sequence of programmed cell death (apoptosis and autophagy), cell proliferation and cell migration. A candidate niche for cell renewal by stem cell proliferation and differentiation was identified at the base of the sponge peduncle, characterized by higher levels of BrdU/EdU incorporation. Therefore, BrdU/EdU-positive cells of the peduncle base are candidate motile cells responsible for the regeneration of the prey-capturing main sponge body, i.e. the dynamic filaments. Altogether, our results demonstrate that dynamics of cell renewal in sponge appear to be regulated by cellular mechanisms as multiple and complex as those already identified in bilaterian metazoans.

Three-dimensional printing of Hela cells for cervical tumor model in vitro.

PubMed

Zhao, Yu; Yao, Rui; Ouyang, Liliang; Ding, Hongxu; Zhang, Ting; Zhang, Kaitai; Cheng, Shujun; Sun, Wei

2014-09-01

Advances in three-dimensional (3D) printing have enabled the direct assembly of cells and extracellular matrix materials to form in vitro cellular models for 3D biology, the study of disease pathogenesis and new drug discovery. In this study, we report a method of 3D printing for Hela cells and gelatin/alginate/fibrinogen hydrogels to construct in vitro cervical tumor models. Cell proliferation, matrix metalloproteinase (MMP) protein expression and chemoresistance were measured in the printed 3D cervical tumor models and compared with conventional 2D planar culture models. Over 90% cell viability was observed using the defined printing process. Comparisons of 3D and 2D results revealed that Hela cells showed a higher proliferation rate in the printed 3D environment and tended to form cellular spheroids, but formed monolayer cell sheets in 2D culture. Hela cells in 3D printed models also showed higher MMP protein expression and higher chemoresistance than those in 2D culture. These new biological characteristics from the printed 3D tumor models in vitro as well as the novel 3D cell printing technology may help the evolution of 3D cancer study.

HIRA orchestrates a dynamic chromatin landscape in senescence and is required for suppression of neoplasia

PubMed Central

Cole, John J.; Nelson, David M.; Dikovskaya, Dina; Faller, William J.; Vizioli, Maria Grazia; Hewitt, Rachael N.; Anannya, Orchi; McBryan, Tony; Manoharan, Indrani; van Tuyn, John; Morrice, Nicholas; Pchelintsev, Nikolay A.; Ivanov, Andre; Brock, Claire; Drotar, Mark E.; Nixon, Colin; Clark, William; Sansom, Owen J.; Anderson, Kurt I.; King, Ayala; Blyth, Karen

2014-01-01

Cellular senescence is a stable proliferation arrest that suppresses tumorigenesis. Cellular senescence and associated tumor suppression depend on control of chromatin. Histone chaperone HIRA deposits variant histone H3.3 and histone H4 into chromatin in a DNA replication-independent manner. Appropriately for a DNA replication-independent chaperone, HIRA is involved in control of chromatin in nonproliferating senescent cells, although its role is poorly defined. Here, we show that nonproliferating senescent cells express and incorporate histone H3.3 and other canonical core histones into a dynamic chromatin landscape. Expression of canonical histones is linked to alternative mRNA splicing to eliminate signals that confer mRNA instability in nonproliferating cells. Deposition of newly synthesized histones H3.3 and H4 into chromatin of senescent cells depends on HIRA. HIRA and newly deposited H3.3 colocalize at promoters of expressed genes, partially redistributing between proliferating and senescent cells to parallel changes in expression. In senescent cells, but not proliferating cells, promoters of active genes are exceptionally enriched in H4K16ac, and HIRA is required for retention of H4K16ac. HIRA is also required for retention of H4K16ac in vivo and suppression of oncogene-induced neoplasia. These results show that HIRA controls a specialized, dynamic H4K16ac-decorated chromatin landscape in senescent cells and enforces tumor suppression. PMID:25512559

HIRA orchestrates a dynamic chromatin landscape in senescence and is required for suppression of neoplasia.

PubMed

Rai, Taranjit Singh; Cole, John J; Nelson, David M; Dikovskaya, Dina; Faller, William J; Vizioli, Maria Grazia; Hewitt, Rachael N; Anannya, Orchi; McBryan, Tony; Manoharan, Indrani; van Tuyn, John; Morrice, Nicholas; Pchelintsev, Nikolay A; Ivanov, Andre; Brock, Claire; Drotar, Mark E; Nixon, Colin; Clark, William; Sansom, Owen J; Anderson, Kurt I; King, Ayala; Blyth, Karen; Adams, Peter D

2014-12-15

Cellular senescence is a stable proliferation arrest that suppresses tumorigenesis. Cellular senescence and associated tumor suppression depend on control of chromatin. Histone chaperone HIRA deposits variant histone H3.3 and histone H4 into chromatin in a DNA replication-independent manner. Appropriately for a DNA replication-independent chaperone, HIRA is involved in control of chromatin in nonproliferating senescent cells, although its role is poorly defined. Here, we show that nonproliferating senescent cells express and incorporate histone H3.3 and other canonical core histones into a dynamic chromatin landscape. Expression of canonical histones is linked to alternative mRNA splicing to eliminate signals that confer mRNA instability in nonproliferating cells. Deposition of newly synthesized histones H3.3 and H4 into chromatin of senescent cells depends on HIRA. HIRA and newly deposited H3.3 colocalize at promoters of expressed genes, partially redistributing between proliferating and senescent cells to parallel changes in expression. In senescent cells, but not proliferating cells, promoters of active genes are exceptionally enriched in H4K16ac, and HIRA is required for retention of H4K16ac. HIRA is also required for retention of H4K16ac in vivo and suppression of oncogene-induced neoplasia. These results show that HIRA controls a specialized, dynamic H4K16ac-decorated chromatin landscape in senescent cells and enforces tumor suppression. © 2014 Rai et al.; Published by Cold Spring Harbor Laboratory Press.

Proliferation and differentiation of brown adipocytes from interstitial cells during cold acclimation

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

Bukowiecki, L.J.; Geloeen, A.; Collet, A.J.

1986-06-01

The mechanisms of brown adipocyte proliferation and differentiation during cold acclimation (and/or adaptation to hyperphagia) have been studied by quantitative photonic radioautography. (/sup 3/H)thymidine was injected to warm-acclimated (25/sup 0/C) rats and to animals exposed to 5/sup 0/C for 2 days. Samples of interscapular brown adipose tissue were collected for quantitative analysis of mitotic frequencies at various periods of time (4 h-15 days) after the injection of (/sup 3/H)thymidine, the rats being maintained at the temperatures to which they were initially exposed. It was found that cold exposure for 2 days markedly enhanced mitotic activity in endothelial cells, interstitial cells,more » and brown preadipocytes rather than in fully differentiated brown adipocytes. The total tissue labeling index (percent of labeled nuclei) increased approx.70 times over control values. The authors now report that cellular labeling progressively increased in mature brown adipocytes during cold acclimation, whereas it correspondingly decreased in interstitial cells and brown preadipocytes. This indicates that the sequence of events for cellular differentiation is interstitial cells ..-->.. brown preadipocytes ..-->.. mature brown adipocytes. Remarkable, labeling frequency did not change in endothelial cells during cold acclimation demonstrating that these cells cannot be considered as progenitors of brown adipocytes. It is suggested that brown adipocyte proliferation and differentiation from interstitial cells represent the fundamental phenomena explaining the enhanced capacity of cold-acclimated and/or hyperphagic rats to respond calorigenically to catecholamines.« less

EBV-driven B-cell lymphoproliferative disorders: from biology, classification and differential diagnosis to clinical management

PubMed Central

Ok, Chi Young; Li, Ling; Young, Ken H

2015-01-01

Epstein–Barr virus (EBV) is a ubiquitous herpesvirus, affecting >90% of the adult population. EBV targets B-lymphocytes and achieves latent infection in a circular episomal form. Different latency patterns are recognized based on latent gene expression pattern. Latent membrane protein-1 (LMP-1) mimics CD40 and, when self-aggregated, provides a proliferation signal via activating the nuclear factor-kappa B, Janus kinase/signal transducer and activator of transcription, phosphoinositide 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase pathways to promote cellular proliferation. LMP-1 also induces BCL-2 to escape from apoptosis and gives a signal for cell cycle progression by enhancing cyclin-dependent kinase 2 and phosphorylation of retinoblastoma (Rb) protein and by inhibiting p16 and p27. LMP-2A blocks the surface immunoglobulin-mediated lytic cycle reactivation. It also activates the Ras/PI3K/Akt pathway and induces Bcl-xL expression to promote B-cell survival. Recent studies have shown that ebv-microRNAs can provide extra signals for cellular proliferation, cell cycle progression and anti-apoptosis. EBV is well known for association with various types of B-lymphocyte, T-lymphocyte, epithelial cell and mesenchymal cell neoplasms. B-cell lymphoproliferative disorders encompass a broad spectrum of diseases, from benign to malignant. Here we review our current understanding of EBV-induced lymphomagenesis and focus on biology, diagnosis and management of EBV-associated B-cell lymphoproliferative disorders. PMID:25613729

Influence of inhibitors of serotonin uptake on intestinal epithelium and colorectal carcinomas.

PubMed

Tutton, P J; Barkla, D H

1982-08-01

Previous studies have shown that in certain tissues, including colonic carcinomas, cell proliferation may be promoted by serotonin, and indirect evidence suggests that the effects of this amine on colonic tumours involves a cellular-uptake mechanism. In the present study, two specific inhibitors of serotonin uptake, Citalopram and Fluoxetine, are examined for their effects on cell proliferation and tumour growth. Each of the agents was found to suppress cell division in dimethylhydrazine-induced colonic tumours in rats, and to retard the growth of 2 out of 3 lines of human colonic tumours propagated as xenografts in immune-deprived mice.

On the mechanism of Cr (VI)-induced carcinogenesis: dose dependence of uptake and cellular responses.

PubMed

Liu, K; Husler, J; Ye, J; Leonard, S S; Cutler, D; Chen, F; Wang, S; Zhang, Z; Ding, M; Wang, L; Shi, X

2001-06-01

Cr (VI) compounds are widely used industrial chemicals and are recognized human carcinogens. The mechanisms of carcinogenesis associated with these compounds remain to be investigated. The present study focused on dose-dependence of Cr (VI)-induced uptake and cellular responses. The results show that Cr (VI) is able to enter the cells (human lung epithelial cell line A549) at low concentration (< 10 microM) and that the Cr (VI) uptake appears to be a combination of saturable transport and passive diffusion. Electron spin resonance (ESR) trapping measurements showed that upon stimulation with Cr (VI), A549 cells were able to generate reactive oxygen species (ROS). The amount of ROS generated depended on the Cr (VI) concentration. ROS generation involved NADPH-dependent flavoenzymes. Cr (VI) affected the following cellular parameters in a dose-dependent manner, (a) activation of nuclear transcription factors NF-kappaB, and p53, (b) DNA damage, (c) induction of cell apoptosis, and (d) inhibition of cell proliferation. The activation of transcription factors was assessed by electrophoretic mobility shift assay and western blot analysis, DNA damage by single cell gel electrophoresis assay, cell apoptosis by DNA fragmentation assay, and cell proliferation by a non-radioactive ELISA kit. At the concentration range used in the present study, no thresholds were found in all of these cell responses to Cr (VI). The results may guide further research to better understand and evaluate the risk of Cr (VI)-induced carcinogenesis at low levels of exposure.

Bmi1 overexpression in the cerebellar granule cell lineage of mice affects cell proliferation and survival without initiating medulloblastoma formation

PubMed Central

Behesti, Hourinaz; Bhagat, Heeta; Dubuc, Adrian M.; Taylor, Michael D.; Marino, Silvia

2013-01-01

SUMMARY BMI1 is a potent inducer of neural stem cell self-renewal and neural progenitor cell proliferation during development and in adult tissue homeostasis. It is overexpressed in numerous human cancers – including medulloblastomas, in which its functional role is unclear. We generated transgenic mouse lines with targeted overexpression of Bmi1 in the cerebellar granule cell lineage, a cell type that has been shown to act as a cell of origin for medulloblastomas. Overexpression of Bmi1 in granule cell progenitors (GCPs) led to a decrease in cerebellar size due to decreased GCP proliferation and repression of the expression of cyclin genes, whereas Bmi1 overexpression in postmitotic granule cells improved cell survival in response to stress by altering the expression of genes in the mitochondrial cell death pathway and of Myc and Lef-1. Although no medulloblastomas developed in ageing cohorts of transgenic mice, crosses with Trp53−/− mice resulted in a low incidence of medulloblastoma formation. Furthermore, analysis of a large collection of primary human medulloblastomas revealed that tumours with a BMI1high TP53low molecular profile are significantly enriched in Group 4 human medulloblastomas. Our data suggest that different levels and timing of Bmi1 overexpression yield distinct cellular outcomes within the same cellular lineage. Importantly, Bmi1 overexpression at the GCP stage does not induce tumour formation, suggesting that BMI1 overexpression in GCP-derived human medulloblastomas probably occurs during later stages of oncogenesis and might serve to enhance tumour cell survival. PMID:23065639

Constitutive expression of tdTomato protein as a cytotoxicity and proliferation marker for space radiation biology

NASA Astrophysics Data System (ADS)

Chishti, Arif A.; Hellweg, Christine E.; Berger, Thomas; Baumstark-Khan, Christa; Feles, Sebastian; Kätzel, Thorben; Reitz, Günther

2015-01-01

The radiation risk assessment for long-term space missions requires knowledge on the biological effectiveness of different space radiation components, e.g. heavy ions, on the interaction of radiation and other space environmental factors such as microgravity, and on the physical and biological dose distribution in the human body. Space experiments and ground-based experiments at heavy ion accelerators require fast and reliable test systems with an easy readout for different endpoints. In order to determine the effect of different radiation qualities on cellular proliferation and the biological depth dose distribution after heavy ion exposure, a stable human cell line expressing a novel fluorescent protein was established and characterized. tdTomato, a red fluorescent protein of the new generation with fast maturation and high fluorescence intensity, was selected as reporter of cell proliferation. Human embryonic kidney (HEK/293) cells were stably transfected with a plasmid encoding tdTomato under the control of the constitutively active cytomegalovirus (CMV) promoter (ptdTomato-N1). The stably transfected cell line was named HEK-ptdTomato-N1 8. This cytotoxicity biosensor was tested by ionizing radiation (X-rays and accelerated heavy ions) exposure. As biological endpoints, the proliferation kinetics and the cell density reached 100 h after irradiation reflected by constitutive expression of the tdTomato were investigated. Both were reduced dose-dependently after radiation exposure. Finally, the cell line was used for biological weighting of heavy ions of different linear energy transfer (LET) as space-relevant radiation quality. The relative biological effectiveness of accelerated heavy ions in reducing cellular proliferation peaked at an LET of 91 keV/μm. The results of this study demonstrate that the HEK-ptdTomato-N1 reporter cell line can be used as a fast and reliable biosensor system for detection of cytotoxic damage caused by ionizing radiation.

Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma.

PubMed

Suzuki, Shigeki; Hoshino, Hiroaki; Yoshida, Kazuma; Nakanishi, Jun; Tsuchiya-Hirata, Shizu; Kobuke, Seiji; Haruyama, Naoto; Nishimura, Fusanori; Shiba, Hideki

2018-01-15

Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC. Copyright © 2017 Elsevier Inc. All rights reserved.

Cell proliferation assessment in oncology.

PubMed

Hofstädter, F; Knüchel, R; Rüschoff, J

1995-01-01

A review of the current knowledge on cell cycle control and the techniques used to assess proliferation of normal and neoplastic cells was the focus of a workshop in Regensburg, Germany, held under the joint auspices of the Graduiertenkolleg: Therapieforschung Onkologie and the Committee on AgNOR Quantification. An overview of the recently discovered group of cyclins and their specific kinases, and of other proliferation-associated antigens, such as Ki67, PCNA and topoiseromase II alpha, was given. The topics continued with a reappraisal of modern imaging and flow-cytometric techniques. An update of the relation of AgNORs to cellular proliferation and differentiation was the link to presentations on clinical data, problems and strategies for standardization, as well as guidelines to establish the prognostic value of marker molecules. These lectures were supported by posters. Bringing together researchers from life sciences, technically oriented workers, pathologists, and clinicians resulted in a lively and constructive discussion, which is briefly summarized in the Concluding remarks.

E2F mediates enhanced alternative polyadenylation in proliferation.

PubMed

Elkon, Ran; Drost, Jarno; van Haaften, Gijs; Jenal, Mathias; Schrier, Mariette; Oude Vrielink, Joachim A F; Agami, Reuven

2012-07-02

The majority of mammalian genes contain multiple poly(A) sites in their 3' UTRs. Alternative cleavage and polyadenylation are emerging as an important layer of gene regulation as they generate transcript isoforms that differ in their 3' UTRs, thereby modulating genes' response to 3' UTR-mediated regulation. Enhanced cleavage at 3' UTR proximal poly(A) sites resulting in global 3' UTR shortening was recently linked to proliferation and cancer. However, mechanisms that regulate this enhanced alternative polyadenylation are unknown. Here, we explored, on a transcriptome-wide scale, alternative polyadenylation events associated with cellular proliferation and neoplastic transformation. We applied a deep-sequencing technique for identification and quantification of poly(A) sites to two human cellular models, each examined under proliferative, arrested and transformed states. In both cell systems we observed global 3' UTR shortening associated with proliferation, a link that was markedly stronger than the association with transformation. Furthermore, we found that proliferation is also associated with enhanced cleavage at intronic poly(A) sites. Last, we found that the expression level of the set of genes that encode for 3'-end processing proteins is globally elevated in proliferation, and that E2F transcription factors contribute to this regulation. Our results comprehensively identify alternative polyadenylation events associated with cellular proliferation and transformation, and demonstrate that the enhanced alternative polyadenylation in proliferative conditions results not only in global 3' UTR shortening but also in enhanced premature cleavage in introns. Our results also indicate that E2F-mediated co-transcriptional regulation of 3'-end processing genes is one of the mechanisms that links enhanced alternative polyadenylation to proliferation.

Dendritic cells modulate burn wound healing by enhancing early proliferation.

PubMed

Vinish, Monika; Cui, Weihua; Stafford, Eboni; Bae, Leon; Hawkins, Hal; Cox, Robert; Toliver-Kinsky, Tracy

2016-01-01

Adequate wound healing is vital for burn patients to reduce the risk of infections and prolonged hospitalization. Dendritic cells (DCs) are antigen presenting cells that release cytokines and are central for the activation of innate and acquired immune responses. Studies have showed their presence in human burn wounds; however, their role in burn wound healing remains to be determined. This study investigated the role of DCs in modulating healing responses within the burn wound. A murine model of full-thickness contact burns was used to study wound healing in the absence of DCs (CD11c promoter-driven diphtheria toxin receptor transgenic mice) and in a DC-rich environment (using fms-like tyrosine kinase-3 ligand, FL- a DC growth factor). Wound closure was significantly delayed in DC-deficient mice and was associated with significant suppression of early cellular proliferation, granulation tissue formation, wound levels of TGFβ1 and formation of CD31+ vessels in healing wounds. In contrast, DC enhancement significantly accelerated early wound closure, associated with increased and accelerated cellular proliferation, granulation tissue formation, and increased TGFβ1 levels and CD31+ vessels in healing wounds. We conclude that DCs play an important role in the acceleration of early wound healing events, likely by secreting factors that trigger the proliferation of cells that mediate wound healing. Therefore, pharmacological enhancement of DCs may provide a therapeutic intervention to facilitate healing of burn wounds. © 2016 by the Wound Healing Society.

Targeted inhibition of EG-1 blocks breast tumor growth.

PubMed

Lu, Ming; Sartippour, Maryam R; Zhang, Liping; Norris, Andrew J; Brooks, Mai N

2007-06-01

EG-1 is a gene product that is significantly elevated in human breast cancer tissues. Previously, we have shown that EG-1 overexpression stimulates cellular proliferation both in vitro and in vivo. Here, we ask whether this molecule can be targeted for experimental therapeutic purpose. siRNA lentivirus and polyclonal antibodies were designed to suppress EG-1 expression. These agents were then used in cell culture proliferation assays and breast tumor xenograft models. Serum and urine from breast cancer patients were also analyzed for the presence of EG-1 peptide. We report here for the first time that endogenous EG-1 can be targeted to inhibit breast tumor growth. This inhibition, whether delivered via siRNA lentivirus or polyclonal antibody, resulted in decreased cellular proliferation in culture and smaller xenografts in mice. The effects were shown in both ER (estrogen receptor)-positive human breast cancer MCF-7 cells, as well as in ER-negative MDA-MB-231 cells. Furthermore, we detected soluble EG-1 in serum and urine of breast cancer patients. These observations demonstrate that EG-1 is relevant to human breast cancer, and is a molecular target worthy of translational efforts into effective breast cancer therapy.

GPER mediates estrogen-induced signaling and proliferations in human breast epithelial cells, and normal and malignant breast

PubMed Central

Scaling, Allison L.

2014-01-01

17β-estradiol (estrogen), through receptor binding and activation, is required for mammary gland development. Estrogen stimulates epithelial proliferation in the mammary gland, promoting ductal elongation and morphogenesis. In addition to a developmental role, estrogen promotes proliferation in tumorigenic settings, particularly breast cancer. The proliferative effects of estrogen in the normal breast and breast tumors are attributed to estrogen receptor α. Although in vitro studies have demonstrated that the G protein-coupled estrogen receptor (GPER, previously called GPR30) can modulate proliferation in breast cancer cells both positively and negatively depending on cellular context, its role in proliferation in the intact normal or malignant breast remains unclear. Estrogen-induced GPER-dependent proliferation was assessed in the immortalized non-tumorigenic human breast epithelial cell line, MCF10A, and an ex vivo organ culture model employing human breast tissue from reduction mammoplasty or tumor resections. Stimulation by estrogen and the GPER-selective agonist G-1 increased the mitotic index in MCF10A cells and proportion of cells in the cell cycle in human breast and breast cancer explants, suggesting increased proliferation. Inhibition of candidate signaling pathways that may link GPER activation to proliferation revealed a dependence on Src, epidermal growth factor receptor transactivation by heparin-bound EGF and subsequent ERK phosphorylation. Proliferation was not dependent on matrix metalloproteinase cleavage of membrane bound pro-HB-EGF. The contribution of GPER to estrogen-induced proliferation in MCF10A cells and breast tissue was confirmed by the ability of GPER-selective antagonist G36 to abrogate estrogen- and G-1-induced proliferation, and the ability of siRNA knockdown of GPER to reduce estrogen- and G-1-induced proliferation in MCF10A cells. This is the first study to demonstrate GPER-dependent proliferation in primary normal and malignant human tissue, revealing a role for GPER in estrogen-induced breast physiology and pathology. PMID:24718936

LAMP-3 (Lysosome-Associated Membrane Protein 3) Promotes the Intracellular Proliferation of Salmonella typhimurium.

PubMed

Lee, Eun-Ju; Park, Kwan-Sik; Jeon, In-Sook; Choi, Jae-Woon; Lee, Sang-Jeon; Choy, Hyun E; Song, Ki-Duk; Lee, Hak-Kyo; Choi, Joong-Kook

2016-07-01

Lysosomes are cellular organelles containing diverse classes of catabolic enzymes that are implicated in diverse cellular processes including phagocytosis, autophagy, lipid transport, and aging. Lysosome-associated membrane proteins (LAMP-1 and LAMP-2) are major glycoproteins important for maintaining lysosomal integrity, pH, and catabolism. LAMP-1 and LAMP-2 are constitutively expressed in Salmonella-infected cells and are recruited to Salmonella-containing vacuoles (SCVs) as well as Salmonella-induced filaments (Sifs) that promote the survival and proliferation of the Salmonella. LAMP-3, also known as DC-LAMP/CD208, is a member of the LAMP family of proteins, but its role during Salmonella infection remains unclear. DNA microarray analysis identified LAMP-3 as one of the genes responding to LPS stimulation in THP-1 macrophage cells. Subsequent analyses reveal that LPS and Salmonella induced the expression of LAMP-3 at both the transcriptional and translational levels. Confocal Super resolution N-SIM imaging revealed that LAMP-3, like LAMP-2, shifts its localization from the cell surface to alongside Salmonella. Knockdown of LAMP-3 by specific siRNAs decreased the number of Salmonella recovered from the infected cells. Therefore, we conclude that LAMP-3 is induced by Salmonella infection and recruited to the Salmonella pathogen for intracellular proliferation.

LAMP-3 (Lysosome-Associated Membrane Protein 3) Promotes the Intracellular Proliferation of Salmonella typhimurium

PubMed Central

Lee, Eun-Ju; Park, Kwan-Sik; Jeon, In-Sook; Choi, Jae-Woon; Lee, Sang-Jeon; Choy, Hyun E.; Song, Ki-Duk; Lee, Hak-Kyo; Choi, Joong-Kook

2016-01-01

Lysosomes are cellular organelles containing diverse classes of catabolic enzymes that are implicated in diverse cellular processes including phagocytosis, autophagy, lipid transport, and aging. Lysosome-associated membrane proteins (LAMP-1 and LAMP-2) are major glycoproteins important for maintaining lysosomal integrity, pH, and catabolism. LAMP-1 and LAMP-2 are constitutively expressed in Salmonella-infected cells and are recruited to Salmonella-containing vacuoles (SCVs) as well as Salmonella-induced filaments (Sifs) that promote the survival and proliferation of the Salmonella. LAMP-3, also known as DC-LAMP/CD208, is a member of the LAMP family of proteins, but its role during Salmonella infection remains unclear. DNA microarray analysis identified LAMP-3 as one of the genes responding to LPS stimulation in THP-1 macrophage cells. Subsequent analyses reveal that LPS and Salmonella induced the expression of LAMP-3 at both the transcriptional and translational levels. Confocal Super resolution N-SIM imaging revealed that LAMP-3, like LAMP-2, shifts its localization from the cell surface to alongside Salmonella. Knockdown of LAMP-3 by specific siRNAs decreased the number of Salmonella recovered from the infected cells. Therefore, we conclude that LAMP-3 is induced by Salmonella infection and recruited to the Salmonella pathogen for intracellular proliferation. PMID:27329040

CAS role in the brain apoptosis of Bufo arenarum induced by cypermethrin.

PubMed

Izaguirre, M F; Vergara, M N; Casco, V H

2006-08-01

CAS might have a key role in the apoptosis induced by toxins, acting as anti-apoptotic factor, stimulating the cellular proliferation and the cell contact stabilization. To start to elucidate their role in the brain apoptosis of Bufo arenarum induced by cypermethrin (CY), the expression patterns of CAS and several cell adhesion molecules (CAMs) were established. Bufo arenarum tadpoles of the control and acute bioassay survival at different doses (39, 156, 625 and 2,500 microg CY/L) and times (24, 48, 72 and 96 h) of CY treatment were fixed in Carnoy, embedded in paraffin and sectioned. CAS and CAMs expression was determined by immunofluorescence and immunohistochemistry, respectively. When the bioassay starts, CAS increases suggesting a proliferative or regenerative effect, but decreases when the doses and/or the biocide exposure time increases, suggesting compromise of the cellular cycle control and trigger of an apoptotic wave. However, these neurotoxic mechanisms should not involve degradation of N-cadherin and alpha-catenin, in contrast of beta-catenin and axonal N-CAM180, at least in the initial apoptotic phase. Additionally, an adhesion compensatory mechanism by N-CAM180 is observed in the neuron cell body. These results suggest a dual role of CAS in the cellular cycle control during the CY-induced apoptosis: induction of cell proliferation and stabilization of the cell-cell junctions by modulating CAMs expression.

Peroxiredoxin Expression of Human Osteosarcoma Cells Is Influenced by Cold Atmospheric Plasma Treatment.

PubMed

Gümbel, Denis; Gelbrich, Nadine; Napp, Matthias; Daeschlein, Georg; Kramer, Axel; Sckell, Axel; Burchardt, Martin; Ekkernkamp, Axel; Stope, Matthias B

2017-03-01

To evaluate the potential involvement of redox-specific signalling pathways in cold atmospheric plasma (CAP)-induced apoptosis on human osteosarcoma cells. Osteosarcoma cell lines were treated with CAP with or without antioxidative agents and seeded in cell culture plates. Cell proliferation was determined by counting viable cells. Carrier gas-treated cells served as control. Peroxiredoxin (PRX) 1-3 expression and secretion were assessed. CAP treatment exhibited strongly attenuated proliferation rates. This effect was significantly attenuated by the addition of N-acetylcysteine (NAC). CAP-treated cells exhibited an increase of PRX 1 and 2 10 sec after treatment. The ratio of oxidized to reduced PRX1 and PRX2 was significantly altered with increasing cellular concentration of the oxidized dimer. Antioxidant supplementation with NAC increases proliferation of CAP-treated osteosarcoma cells, implicating an involvement of redox signalling. Activation of PRX1 and -2 indicate CAP affects redox homeostasis. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

The contribution of B-cell proliferation to spleen enlargement in Babesia microti-infected mice.

PubMed Central

Inchley, C J

1987-01-01

Flow cytofluorimetric analysis showed that B-cell proliferation makes a major contribution to the enlargement and increased cellularity of the spleen, which are characteristic of Babesia microti infections in mice. Expansion of the B-cell population was accompanied by modulation of the cell surface, which affected most B lymphocytes, and which was detected as a reduction in the density of surface immunoglobulin. This effect was noted as early as Day 7, shortly after the appearance of parasites in the circulation and the onset of gross spleen changes. In contrast to the results for B cells, the frequency of splenic T cells declined, and when the data were transformed into absolute numbers it became clear that only limited T-cell proliferation had occurred. There was no evidence to suggest that the balance of T-cell subsets was shifted in favour of suppressor T cells. The relationships of these results to reports of immunosuppression by this parasite are discussed. Images Figure 2 Figure 5 PMID:3493207

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

Andersson, Olov, E-mail: olov.andersson@ki.se

Glucose homeostasis, which is controlled by the endocrine cells of the pancreas, is disrupted in both type I and type II diabetes. Deficiency in the number of insulin-producing β cells – a primary cause of type I diabetes and a secondary contributor of type II diabetes – leads to hyperglycemia and hence an increase in the need for insulin. Although diabetes can be controlled with insulin injections, a curative approach is needed. A potential approach to curing diabetes involves regenerating the β-cell mass, e.g. by increasing β-cell proliferation, survival, neogenesis or transdifferentiation. The nucleoside adenosine and its cognate nucleotide ATPmore » have long been known to affect insulin secretion, but have more recently been shown to increase β-cell proliferation during homeostatic control and regeneration of the β-cell mass. Adenosine is also known to have anti-inflammatory properties, and agonism of adenosine receptors can promote the survival of β-cells in an inflammatory microenvironment. In this review, both intracellular and extracellular mechanisms of adenosine and ATP are discussed in terms of their established and putative effects on β-cell regeneration. - Highlights: • A potential way to cure diabetes is to regenerate the β-cell mass by promoting cell survival, proliferation or neogenesis. • Adenosine may promote β-cell regeneration through several cellular mechanisms. • Adenosine and its cognate nucleotide ATP can each promote β-cell proliferation. • Do adenosine and ATP interact in promoting β-cell proliferation?.« less

VEGF improves survival of mesenchymal stem cells in infarcted hearts

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

Pons, Jennifer; Huang Yu; Arakawa-Hoyt, Janice

2008-11-14

Bone marrow-derived mesenchymal stem cells (MSC) are a promising source for cell-based treatment of myocardial infarction (MI), but existing strategies are restricted by low cell survival and engraftment. We examined whether vascular endothelial growth factor (VEGF) improve MSC viability in infracted hearts. We found long-term culture increased MSC-cellular stress: expressing more cell cycle inhibitors, p16{sup INK}, p21 and p19{sup ARF}. VEGF treatment reduced cellular stress, increased pro-survival factors, phosphorylated-Akt and Bcl-xL expression and cell proliferation. Co-injection of MSCs with VEGF to MI hearts increased cell engraftment and resulted in better improvement of cardiac function than that injected with MSCs ormore » VEGF alone. In conclusion, VEGF protects MSCs from culture-induce cellular stress and improves their viability in ischemic myocardium, which results in improvements of their therapeutic effect for the treatment of MI.« less

O-linked β-N-acetylglucosamine transferase directs cell proliferation in idiopathic pulmonary arterial hypertension.

PubMed

Barnes, Jarrod W; Tian, Liping; Heresi, Gustavo A; Farver, Carol F; Asosingh, Kewal; Comhair, Suzy A A; Aulak, Kulwant S; Dweik, Raed A

2015-04-07

Idiopathic pulmonary arterial hypertension (IPAH) is a cardiopulmonary disease characterized by cellular proliferation and vascular remodeling. A more recently recognized characteristic of the disease is the dysregulation of glucose metabolism. The primary link between altered glucose metabolism and cell proliferation in IPAH has not been elucidated. We aimed to determine the relationship between glucose metabolism and smooth muscle cell proliferation in IPAH. Human IPAH and control patient lung tissues and pulmonary artery smooth muscle cells (PASMCs) were used to analyze a specific pathway of glucose metabolism, the hexosamine biosynthetic pathway. We measured the levels of O-linked β-N-acetylglucosamine modification, O-linked β-N-acetylglucosamine transferase (OGT), and O-linked β-N-acetylglucosamine hydrolase in control and IPAH cells and tissues. Our data suggest that the activation of the hexosamine biosynthetic pathway directly increased OGT levels and activity, triggering changes in glycosylation and PASMC proliferation. Partial knockdown of OGT in IPAH PASMCs resulted in reduced global O-linked β-N-acetylglucosamine modification levels and abrogated PASMC proliferation. The increased proliferation observed in IPAH PASMCs was directly impacted by proteolytic activation of the cell cycle regulator, host cell factor-1. Our data demonstrate that hexosamine biosynthetic pathway flux is increased in IPAH and drives OGT-facilitated PASMC proliferation through specific proteolysis and direct activation of host cell factor-1. These findings establish a novel regulatory role for OGT in IPAH, shed a new light on our understanding of the disease pathobiology, and provide opportunities to design novel therapeutic strategies for IPAH. © 2015 American Heart Association, Inc.

Protein phosphatase 2A in stretch-induced endothelial cell proliferation

NASA Technical Reports Server (NTRS)

Murata, K.; Mills, I.; Sumpio, B. E.

1996-01-01

We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459-466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic stain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C.

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

PubMed

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

2017-01-01

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

Cell proliferation and apoptosis in the anterior intestine of an amphibious, euryhaline mudskipper (Periophthalmus modestus).

PubMed

Takahashi, H; Sakamoto, T; Narita, K

2006-06-01

In order to replace the diffusive loss of water to the surrounding environment, seawater (SW)-acclimated euryhaline fishes have gastrointestinal tracts with higher ion/water flux in concert with greater permeability, and contrast that to freshwater (FW)-acclimated fish. To understand the cellular basis for these differences, we examined cell proliferation and apoptosis in the anterior intestine of mudskipper transferred from one-third SW to FW or to SW for 1 and 7 days, and those kept out of water for 1 day. The intestinal apoptosis (indicated by DNA laddering) increased during seawater acclimation. TUNEL staining detected numerous apoptotic cells over the epithelium of SW-acclimated fish. Cell proliferation ([3H]thymidine incorporation) in the FW fish was greater than those in SW 7 days after transfer. Labeling with a Proliferating cell nuclear antigen (PCNA) antibody indicated that proliferating cells were greater in number and randomly distributed in the epithelium of FW fish, whereas in SW fish they were almost entirely in the troughs of the intestinal folds. There were no changes in cell turnover in fish kept out of water. During acclimation to different salinities, modification of the cell turnover and abundance may play an important role in regulating the permeability (and transport capacity) of the gastrointestinal tract of fish.

The Mitotic and Metabolic Effects of Phosphatidic Acid in the Primary Muscle Cells of Turbot (Scophthalmus maximus).

PubMed

Wang, Tingting; Wang, Xuan; Zhou, Huihui; Jiang, Haowen; Mai, Kangsen; He, Gen

2018-01-01

Searching for nutraceuticals and understanding the underlying mechanism that promote fish growth is at high demand for aquaculture industry. In this study, the modulatory effects of soy phosphatidic acids (PA) on cell proliferation, nutrient sensing, and metabolic pathways were systematically examined in primary muscle cells of turbot ( Scophthalmus maximus ). PA was found to stimulate cell proliferation and promote G1/S phase transition through activation of target of rapamycin signaling pathway. The expression of myogenic regulatory factors, including myoD and follistatin , was upregulated, while that of myogenin and myostatin was downregulated by PA. Furthermore, PA increased intracellular free amino acid levels and enhanced protein synthesis, lipogenesis, and glycolysis, while suppressed amino acid degradation and lipolysis. PA also was found to increased cellular energy production through stimulated tricarboxylic acid cycle and oxidative phosphorylation. Our results identified PA as a potential nutraceutical that stimulates muscle cell proliferation and anabolism in fish.

FUNCTIONAL DEREGULATION OF KIT: LINK TO MAST CELL PROLIFERATIVE DISEASES AND OTHER NEOPLASMS

PubMed Central

Cruse, Glenn; Metcalfe, Dean D.; Olivera, Ana

2014-01-01

SYNOPSIS Signaling through the receptor tyrosine kinase KIT mediates differentiation, proliferation and survival of hematopoietic precursor cells and mast cells. Constitutive KIT signaling due to somatic point mutations in c-Kit is an important occurrence in the development of mast cell proliferation disorders and other hematological malignancies. In this review, we discuss the common gain-of-function mutations found in these malignancies, particularly in mast cell proliferation disorders, and summarize the current understanding of the molecular mechanisms by which transforming point mutations in KIT may affect KIT structure and function and lead to altered downstream signaling and cellular transformation. Drugs targeting KIT have shown mixed success in the treatment of these diseases. A brief overview of the most common KIT inhibitors currently used, the reasons for the varied clinical results of such inhibitors and a discussion of potential new strategies are provided. PMID:24745671

A New Model for the Estimation of Cell Proliferation Dynamics Using CFSE Data

PubMed Central

Banks, H.T.; Sutton, Karyn L.; Thompson, W. Clayton; Bocharov, Gennady; Doumic, Marie; Schenkel, Tim; Argilaguet, Jordi; Giest, Sandra; Peligero, Cristina; Meyerhans, Andreas

2011-01-01

CFSE analysis of a proliferating cell population is a popular tool for the study of cell division and division-linked changes in cell behavior. Recently [13, 43, 45], a partial differential equation (PDE) model to describe lymphocyte dynamics in a CFSE proliferation assay was proposed. We present a significant revision of this model which improves the physiological understanding of several parameters. Namely, the parameter γ used previously as a heuristic explanation for the dilution of CFSE dye by cell division is replaced with a more physical component, cellular autofluorescence. The rate at which label decays is also quantified using a Gompertz decay process. We then demonstrate a revised method of fitting the model to the commonly used histogram representation of the data. It is shown that these improvements result in a model with a strong physiological basis which is fully capable of replicating the behavior observed in the data. PMID:21889510

Substance P enhances proliferation and paracrine potential of adipose-derived stem cells in vitro

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

Kim, Suna; Piao, Jiyuan; Son, Youngsook

Stem cells have tremendous promise to treat intractable diseases. Notably, adipose-derived stem cells (ADSCs) are actively being investigated because of ease of sampling and high repopulation capacity in vitro. ADSCs can exert a therapeutic effect through differentiation and paracrine potential, and these actions have been proven in many diseases, including cutaneous and inflammatory diseases. Transplantation of ADSCs necessitates therapeutic quantities and thus, long term ex vivo culture of ADSCs. However, this procedure can impair the activity of ADSCs and provoke cellular senescence, leading to low efficacy in vivo. Accordingly, strategies to restore cellular activity and inhibit senescence of stem cells during ex vivo culturemore » are needed for stem cell-based therapies. This study evaluated a potential supplementary role of Substance P (SP) in ADSC ex vivo culture. After confirming that the ADSC cell cycle was damaged by passage 6 (p6), ADSCs at p6 were cultured with SP, and their proliferation rates, cumulative cell numbers, cytokine profiles, and impact on T/endothelial cells were assessed. Long-term culture weakened proliferation ability and secretion of the cytokines, transforming growth factor-beta 1 (TGF-beta1), vascular endothelial growth factor (VEGF), and stromal cell derived factor-1 alpha (SDF-1alpha) in ADSCs. However, SP treatment reduced the population doubling time (PDT), enabling gain of a sufficient number of ADSCs at early passages. In addition, SP restored cytokine secretion, enhancing the ADSC-mediated paracrine effect on T cell and human umbilical vein endothelial cells (HUVECs). Taken together, these results suggest that SP can retain the therapeutic effect of ADSCs by elevating their proliferative and paracrine potential in ex vivo culture. - Highlights: • Long-term culture of ADSCs leads to cell senescence. • Paracrine potential of ADSC decreases as passage number increases. • SP enhances the weakened proliferation capacity of ADSCs. • SP stimulates cytokine secretion from ADSC with impaired paracrine potential.« less

Local bone marrow renin-angiotensin system in the genesis of leukemia and other malignancies.

PubMed

Haznedaroglu, I C; Malkan, U Y

2016-10-01

The existence of a local renin-angiotensin system (RAS) specific to the hematopoietic bone marrow (BM) microenvironment had been proposed two decades ago. Most of the RAS molecules including ACE, ACE2, AGT, AGTR1, AGTR2, AKR1C4, AKR1D1, ANPEP, ATP6AP2, CMA1, CPA3, CTSA, CTSD, CTSG, CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP21A2, DPP3, EGFR, ENPEP, GPER, HSD11B1, HSD11B2, IGF2R, KLK1, LNPEP, MAS1, MME, NR3C1, NR3C2, PREP, REN, RNPEP, and THOP1 are locally present in the BM microenvironment. Local BM RAS peptides control the hematopoietic niche, myelopoiesis, erythropoiesis, thrombopoiesis and the development of other cellular lineages. Local BM RAS is important in hematopoietic stem cell biology and microenvironment. Angiotensin II regulates the proliferation, differentiation, and engraftment of hematopoietic stem cells. Activation of Mas receptor or ACE2 promotes proliferation of CD34+ cells. BM contains a progenitor that expresses renin throughout development. Angiotensin II attenuates the migration and proliferation of CD34+ Cells and promotes the adhesion of both MNCs and CD34+ cells. Renin cells in hematopoietic organs are precursor B cells. The renin cell requires RBP-J to differentiate. Mutant renin-expressing hematopoietic precursors can cause leukemia. Deletion of RBP-J in the renin-expressing progenitors enriches the precursor B-cell gene programme. Mutant cells undergo a neoplastic transformation, and mice develop a highly penetrant B-cell leukemia with multi-organ infiltration and early death. Many biological conditions during the development and function of blood cells are mediated by RAS, such as apoptosis, cellular proliferation, intracellular signaling, mobilization, angiogenesis, and fibrosis. The aim of this paper is to review recent developments regarding the actions of local BM RAS in the genesis of leukemia and other malignancies molecules.

CD147/basigin promotes progression of malignant melanoma and other cancers.

PubMed

Kanekura, Takuro; Chen, Xiang

2010-03-01

CD147/basigin, a transmembrane protein belonging to the immunoglobulin super family, was originally cloned as a carrier of Lewis X carbohydrate antigen. CD147 is strongly related to cancer progression; it is highly expressed by various cancer cells including malignant melanoma (MM) cells and it plays important roles in tumor invasiveness, metastasis, cellular proliferation, and in vascular endothelial growth factor (VEGF) production, tumor cell glycolysis, and multi-drug resistance (MDR). CD147 on cancer cells induces matrix metalloproteinase expression by neighboring fibroblasts, leading to tumor cell invasion. In a nude mouse model of pulmonary metastasis from MM, the metastatic potential of CD147-expressing MM cells injected into the tail vein is abolished by CD147 silencing. CD147 enhances cellular proliferation and VEGF production by MM cells; it promotes tumor cell glycolysis by facilitating lactate transport in combination with monocarboxylate transporters, resulting in tumor progression. CD147 is responsible for the MDR phenotype via P-glycoprotein expression. These findings strongly suggest CD147 as a possible therapeutic target for overcoming metastasis and MDR, major obstacles to the effective treatment of malignant cancers. 2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Resveratrol Overcomes Cellular Resistance to Vemurafenib Through Dephosphorylation of AKT in BRAF-mutated Melanoma Cells.

PubMed

Luo, Hao; Umebayashi, Masayo; Doi, Keiko; Morisaki, Takashi; Shirasawa, Senji; Tsunoda, Toshiyuki

2016-07-01

The serine/threonine-protein kinase B-Raf (BRAF) V600E mutant (BRAF(V600E)) inhibitor vemurafenib, has improved clinical outcomes for patients with BRAF(V600E) melanoma, but acquired cellular resistance mediated by AKT serine/threonine kinase 1 (AKT) phosphorylation limits its efficacy. We examined the effect of resveratrol on vemurafenib-resistant melanoma cells. A vemurafenib-resistant human metastatic melanoma cell line positive for the BRAF V600E mutation was established. The anti-tumorigenic effects of vemurafenib and resveratrol, both alone and in combination, were examined through analysis of cell proliferation and protein expression. The level of phosphorylated AKT (p-AKT) was increased in the primary melanoma cells after treatment with vemurafenib, and the basal level of p-AKT was increased in vemurafenib-resistant melanoma cells. Notably, resveratrol both alone and in combination with vemurafenib effectively suppressed cell proliferation and AKT phosphorylation in both parental and vemurafenib-resistant melanoma cells. Vemurafenib resistance can be reversed by addition of resveratrol in patients undergoing treatment with BRAF inhibitors. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Induction of type 1 iodothyronine deiodinase expression inhibits proliferation and migration of renal cancer cells.

PubMed

Poplawski, Piotr; Rybicka, Beata; Boguslawska, Joanna; Rodzik, Katarzyna; Visser, Theo J; Nauman, Alicja; Piekielko-Witkowska, Agnieszka

2017-02-15

Type 1 iodothyronine deiodinase (DIO1) regulates peripheral metabolism of thyroid hormones that control cellular proliferation, differentiation and metabolism. The significance of DIO1 in cancer is unknown. In this study we hypothesized that diminished expression of DIO1, observed in renal cancer, contributes to the carcinogenic process in the kidney. Here, we demonstrate that ectopic expression of DIO1 in renal cancer cells changes the expression of genes controlling cell cycle, including cyclin E1 and E2F5, and results in inhibition of proliferation. The expression of genes encoding collagens (COL1A1, COL4A2, COL5A1), integrins (ITGA4, ITGA5, ITGB3) and transforming growth factor-β-induced (TGFBI) is significantly altered in renal cancer cells with induced expression of DIO1. Finally, we show that overexpression of DIO1 inhibits migration of renal cancer cells. In conclusion, we demonstrate for the first time that loss of DIO1 contributes to renal carcinogenesis and that its induced expression protects cells against cancerous proliferation and migration. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Foxp1 Regulates the Proliferation of Hair Follicle Stem Cells in Response to Oxidative Stress during Hair Cycling

PubMed Central

Zhao, Jianzhi; Li, Hanjun; Zhou, Rujiang; Ma, Gang; Dekker, Joseph D.; Tucker, Haley O.; Yao, Zhengju; Guo, Xizhi

2015-01-01

Hair follicle stem cells (HFSCs) in the bugle circularly generate outer root sheath (ORS) through linear proliferation within limited cycles during anagen phases. However, the mechanisms controlling the pace of HFSC proliferation remain unclear. Here we revealed that Foxp1, a transcriptional factor, was dynamically relocated from the nucleus to the cytoplasm of HFSCs in phase transitions from anagen to catagen, coupled with the rise of oxidative stress. Mass spectrum analyses revealed that the S468 phosphorylation of Foxp1 protein was responsive to oxidative stress and affected its nucleocytoplasmic translocation. Foxp1 deficiency in hair follicles led to compromised ROS accrual and increased HFSC proliferation. And more, NAC treatment profoundly elongated the anagen duration and HFSC proliferation in Foxp1-deficient background. Molecularly, Foxp1 augmented ROS levels through suppression of Trx1-mediated reductive function, thereafter imposing the cell cycle arrest by modulating the activity of p19/p53 pathway. Our findings identify a novel role for Foxp1 in controlling HFSC proliferation with cellular dynamic location in response to oxidative stress during hair cycling. PMID:26171970

Effects of nicotine on cellular proliferation, macromolecular synthesis and cell cycle phase distribution in human and murine cells

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

Konno, S.; Chiao, J.; Rossi, J.

1986-05-01

Addition of nicotine causes a dose- and time-dependent inhibition of cell growth in established human and murine cells. In the human promyelocytic HL-60 leukemic cells, 3 mM nicotine results in a 50% inhibition of cellular proliferation after 80 h. Nicotine was also found to affect the cell cycle distribution of HL-60 cells. Treatment with 4 mM nicotine for 20 h causes an increase in proportion of Gl-phase cells (from 49% to 57%) and a significant decrease in the proportion of S-phase cells (from 41% to 32%). These results suggest that nicotine causes cell arrest in the Gl-phase which may inmore » part account for its effects on cell growth. To determine whether nicotine has a primary effect on the uptake/transport of macromolecular precursors into cells, HL-60 cells were treated with 2-6 mM nicotine for 30 h/sub 3/ at the end of which time cells were labeled with (/sup 3/H)thymidine, (/sup 3/H)uridine, (/sup 14/C)lysine and (/sup 35/S)methionine, the trichloroacetic acid (TCA) soluble and insoluble radioactivities from each of the labeling conditions were determined. These studies show that nicotine primarily affect the synthesis of proteins.« less

The Effect of Laser Irradiation on Adipose Derived Stem Cell Proliferation and Differentiation

NASA Astrophysics Data System (ADS)

Abrahamse, H.; de Villiers, J.; Mvula, B.

2009-06-01

There are two fundamental types of stem cells: Embryonic Stem cells and Adult Stem cells. Adult Stem cells have a more restricted potential and can usually differentiate into a few different cell types. In the body these cells facilitate the replacement or repair of damaged or diseased cells in organs. Low intensity laser irradiation was shown to increase stem cell migration and stimulate proliferation and it is thought that treatment of these cells with laser irradiation may increase the stem cell harvest and have a positive effect on the viability and proliferation. Our research is aimed at determining the effect of laser irradiation on differentiation of Adipose Derived Stem Cells (ADSCs) into different cell types using a diode laser with a wavelength of 636 nm and at 5 J/cm2. Confirmation of stem cell characteristics and well as subsequent differentiation were assessed using Western blot analysis and cellular morphology supported by fluorescent live cell imaging. Functionality of subsequent differentiated cells was confirmed by measuring adenosine triphosphate (ATP) production and cell viability.

The BRG1 chromatin remodeling enzyme links cancer cell metabolism and proliferation

PubMed Central

Wu, Qiong; Madany, Pasil; Dobson, Jason R.; Schnabl, Jake M.; Sharma, Soni; Smith, Tara C.; van Wijnen, Andre J.; Stein, Janet L.; Lian, Jane B.; Stein, Gary S.; Muthuswami, Rohini; Imbalzano, Anthony N.; Nickerson, Jeffrey A.

2016-01-01

Cancer cells reprogram cellular metabolism to meet the demands of growth. Identification of the regulatory machinery that regulates cancer-specific metabolic changes may open new avenues for anti-cancer therapeutics. The epigenetic regulator BRG1 is a catalytic ATPase for some mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is a well-characterized tumor suppressor in some human cancers, but is frequently overexpressed without mutation in other cancers, including breast cancer. Here we demonstrate that BRG1 upregulates de novo lipogenesis and that this is crucial for cancer cell proliferation. Knockdown of BRG1 attenuates lipid synthesis by impairing the transcription of enzymes catalyzing fatty acid and lipid synthesis. Remarkably, exogenous addition of palmitate, the key intermediate in fatty acid synthesis, rescued the cancer cell proliferation defect caused by BRG1 knockdown. Our work suggests that targeting BRG1 to reduce lipid metabolism and, thereby, to reduce proliferation, has promise for epigenetic therapy in triple negative breast cancer. PMID:27223259

The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration

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

Blom, Magdalena; Reis, Katarina; Heldin, Johan

RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as corticalmore » actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration. - Highlights: • Increased RhoD expression leads to loss of actin structures, e.g. stress fibers and gives rise to decreased actin dynamics. • RhoD knockdown induces various actin-containing structures such as edge ruffles, stress fibers and cortical actin, in a cell-type specific manner. • RhoD induces specific actin rearrangements depending on its subcellular localization. • RhoD knockdown has effects on cellular processes, such as directed cell migration and proliferation.« less

[Effects of three Wenyang Jianpi Tang on cell proliferation and apoptosis of nonalcoholic fatty liver cells].

PubMed

Yang, Jia-Yao; Tao, Dong-Qing; Liu, Song; Zhang, Shu; Ma, Wei; Shi, Zhao-Hong

2017-04-01

To investigate the effects of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang on the cell proliferation and apoptosis of nonalcoholic fatty liver cells through the nonalcoholic fatty liver cell model established by inducing L02 cells with oleic acid. Different concentrations of oleic acid were added into L02 cells to induce the nonalcoholic fatty liver cell model. Oil red O staining was used to observe fatty droplets of fatty liver cells. Automatic biochemical analyzer was used to detect the levels of aspartic transaminase(AST), alanine aminotransferase(ALT), total cholesterol(TC), and triglyceride(TG) in the cell supernatants. There were five groups, namely normal group, model group, model and Sijunzi Tang group, model and Lizhong Tang group, and model and Fuzi Lizhong Tang group. The cell proliferation and apoptosis of the five groups were detected by MTT colorimetry test and flow cytometer. The expressions of PCNA, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax and Bcl-2 proteins of the five groups were detected by Western blot. The oil red O staining results showed that the optimum concentration of oleic acid that was used to induce nonalcoholic fatty liver cell models was 80 mg•L-1. The levels of AST, ALT, TC and TG in the nonalcoholic fatty liver cell supernatants were higher than that in normal liver cell supernatants(P<0.01). MTT colorimetry test and flow cytometer results showed that all of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells(P<0.01). And Fuzi Lizhong Tang showed the best effect. Western blot results showed that Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could down-regulate the expressions of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9 and Bax proteins, and up-regulate the expressions of PCNA and Bcl-2 proteins of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. In conclusion, all of Sijunzi Tang, Lizhong Tang and Fuzi Lizhong Tang could effectively promote the cell proliferation, and inhibit the cellular apoptosis of nonalcoholic fatty liver cells. And Fuzi Lizhong Tang showed the best effect. The pharmacodynamic mechanism may be related to the expressions of key factors in pathways related with proliferation and apoptosis mediated by the three decoctions. Copyright© by the Chinese Pharmaceutical Association.

Slug is upregulated during wound healing and regulates cellular phenotypes in corneal epithelial cells.

PubMed

Aomatsu, Keiichi; Arao, Tokuzo; Abe, Kosuke; Kodama, Aya; Sugioka, Koji; Matsumoto, Kazuko; Kudo, Kanae; Kimura, Hideharu; Fujita, Yoshihiko; Hayashi, Hidetoshi; Nagai, Tomoyuki; Shimomura, Yoshikazu; Nishio, Kazuto

2012-02-16

The involvement of the epithelial mesenchymal transition (EMT) in the process of corneal wound healing remains largely unclear. The purpose of the present study was to gain insight into Slug expression and corneal wound healing. Slug expression during wound healing in the murine cornea was evaluated using fluorescence staining in vivo. Slug or Snail was stably introduced into human corneal epithelial cells (HCECs). These stable transfectants were evaluated for the induction of the EMT, cellular growth, migration activity, and expression changes in differentiation-related molecules. Slug, but not Snail, was clearly expressed in the nuclei of corneal epithelial cells in basal lesion of the corneal epithelium during wound healing in vivo. The overexpression of Slug or Snail induced an EMT-like cellular morphology and cadherin switching in HCECs, indicating that these transcription factors were able to mediate the typical EMT in HCECs. The overexpression of Slug or Snail suppressed cellular proliferation but enhanced the migration activity. Furthermore, ABCG2, TP63, and keratin 19, which are known as stemness-related molecules, were downregulated in these transfectants. It was found that Slug is upregulated during corneal wound healing in vivo. The overexpression of Slug mediated a change in the cellular phenotype affecting proliferation, migration, and expression levels of differentiation-related molecules. This is the first evidence that Slug is regulated during the process of corneal wound healing in the corneal epithelium in vivo, providing a novel insight into the EMT and Slug expression in corneal wound healing.

The silencing of Pokemon attenuates the proliferation of hepatocellular carcinoma cells in vitro and in vivo by inhibiting the PI3K/Akt pathway.

PubMed

Lin, Chan-Chan; Zhou, Jing-Ping; Liu, Yun-Peng; Liu, Jing-Jing; Yang, Xiao-Ning; Jazag, Amarsanaa; Zhang, Zhi-Ping; Guleng, Bayasi; Ren, Jian-Lin

2012-01-01

Pokemon (POK erythroid myeloid ontogenic factor), which belongs to the POK protein family, is also called LRF, OCZF and FBI-1. As a transcriptional repressor, Pokemon assumes a critical function in cellular differentiation and oncogenesis. Our study identified an oncogenic role for Pokemon in human hepatocellular carcinoma (HCC). We successfully established human HepG2 and Huh-7 cell lines in which Pokemon was stably knocked down. We demonstrated that Pokemon silencing inhibited cell proliferation and migration. Pokemon knockdown inhibited the PI3K/Akt and c-Raf/MEK/ERK pathways and modulated the expression of various cell cycle regulators in HepG2 and Huh-7 cells. Therefore, Pokemon may also be involved in cell cycle progression in these cells. We confirmed that Pokemon silencing suppresses hepatocellular carcinoma growth in tumor xenograft mice. These results suggest that Pokemon promotes cell proliferation and migration in hepatocellular carcinoma and accelerates tumor development in an Akt- and ERK-signaling-dependent manner.

Targeting cancer by binding iron: Dissecting cellular signaling pathways

PubMed Central

Lui, Goldie Y.L.; Kovacevic, Zaklina; Richardson, Vera; Merlot, Angelica M.; Kalinowski, Danuta S.; Richardson, Des R.

2015-01-01

Newer and more potent therapies are urgently needed to effectively treat advanced cancers that have developed resistance and metastasized. One such strategy is to target cancer cell iron metabolism, which is altered compared to normal cells and may facilitate their rapid proliferation. This is supported by studies reporting the anti-neoplastic activities of the clinically available iron chelators, desferrioxamine and deferasirox. More recently, ligands of the di-2-pyridylketone thiosemicarbazone (DpT) class have demonstrated potent and selective anti-proliferative activity across multiple cancer-types in vivo, fueling studies aimed at dissecting their molecular mechanisms of action. In the past five years alone, significant advances have been made in understanding how chelators not only modulate cellular iron metabolism, but also multiple signaling pathways implicated in tumor progression and metastasis. Herein, we discuss recent research on the targeting of iron in cancer cells, with a focus on the novel and potent DpT ligands. Several key studies have revealed that iron chelation can target the AKT, ERK, JNK, p38, STAT3, TGF-β, Wnt and autophagic pathways to subsequently inhibit cellular proliferation, the epithelial-mesenchymal transition (EMT) and metastasis. These developments emphasize that these novel therapies could be utilized clinically to effectively target cancer. PMID:26125440

Intestinal Cell Proliferation and Senescence Are Regulated by Receptor Guanylyl Cyclase C and p21*

PubMed Central

Basu, Nirmalya; Saha, Sayanti; Khan, Imran; Ramachandra, Subbaraya G.; Visweswariah, Sandhya S.

2014-01-01

Guanylyl cyclase C (GC-C) is expressed in intestinal epithelial cells and serves as the receptor for bacterial heat-stable enterotoxin (ST) peptides and the guanylin family of gastrointestinal hormones. Activation of GC-C elevates intracellular cGMP, which modulates intestinal fluid-ion homeostasis and differentiation of enterocytes along the crypt-villus axis. GC-C activity can regulate colonic cell proliferation by inducing cell cycle arrest, and mice lacking GC-C display increased cell proliferation in colonic crypts. Activation of GC-C by administration of ST to wild type, but not Gucy2c−/−, mice resulted in a reduction in carcinogen-induced aberrant crypt foci formation. In p53-deficient human colorectal carcinoma cells, ST led to a transcriptional up-regulation of p21, the cell cycle inhibitor, via activation of the cGMP-responsive kinase PKGII and p38 MAPK. Prolonged treatment of human colonic carcinoma cells with ST led to nuclear accumulation of p21, resulting in cellular senescence and reduced tumorigenic potential. Our results, therefore, identify downstream effectors for GC-C that contribute to regulating intestinal cell proliferation. Thus, genomic responses to a bacterial toxin can influence intestinal neoplasia and senescence. PMID:24217248

New Treatment Options for Osteosarcoma - Inactivation of Osteosarcoma Cells by Cold Atmospheric Plasma.

PubMed

Gümbel, Denis; Gelbrich, Nadine; Weiss, Martin; Napp, Matthias; Daeschlein, Georg; Sckell, Axel; Ender, Stephan A; Kramer, Axel; Burchardt, Martin; Ekkernkamp, Axel; Stope, Matthias B

2016-11-01

Cold atmospheric plasma has been shown to inhibit tumor cell growth and induce tumor cell death. The aim of the study was to investigate the effects of cold atmospheric plasma treatment on proliferation of human osteosarcoma cells and to characterize the underlying cellular mechanisms. Human osteosarcoma cells (U2-OS and MNNG/HOS) were treated with cold atmospheric plasma and seeded in culture plates. Cell proliferation, p53 and phospho-p53 protein expression and nuclear morphology were assessed. The treated human osteosarcoma cell lines exhibited attenuated proliferation rates by up to 66%. The cells revealed an induction of p53, as well as phospho-p53 expression, by 2.3-fold and 4.5-fold, respectively, compared to controls. 4',6-diamidino-2-phenylindole staining demonstrated apoptotic nuclear condensation following cold atmospheric plasma treatment. Cold atmospheric plasma treatment significantly attenuated cell proliferation in a preclinical in vitro osteosarcoma model. The resulting increase in p53 expression and phospho-activation in combination with characteristic nuclear changes indicate this was through induction of apoptosis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Local cellular neighborhood controls proliferation in cell competition

PubMed Central

Bove, Anna; Gradeci, Daniel; Fujita, Yasuyuki; Banerjee, Shiladitya; Charras, Guillaume; Lowe, Alan R.

2017-01-01

Cell competition is a quality-control mechanism through which tissues eliminate unfit cells. Cell competition can result from short-range biochemical inductions or long-range mechanical cues. However, little is known about how cell-scale interactions give rise to population shifts in tissues, due to the lack of experimental and computational tools to efficiently characterize interactions at the single-cell level. Here, we address these challenges by combining long-term automated microscopy with deep-learning image analysis to decipher how single-cell behavior determines tissue makeup during competition. Using our high-throughput analysis pipeline, we show that competitive interactions between MDCK wild-type cells and cells depleted of the polarity protein scribble are governed by differential sensitivity to local density and the cell type of each cell’s neighbors. We find that local density has a dramatic effect on the rate of division and apoptosis under competitive conditions. Strikingly, our analysis reveals that proliferation of the winner cells is up-regulated in neighborhoods mostly populated by loser cells. These data suggest that tissue-scale population shifts are strongly affected by cellular-scale tissue organization. We present a quantitative mathematical model that demonstrates the effect of neighbor cell–type dependence of apoptosis and division in determining the fitness of competing cell lines. PMID:28931601

CDKL5, a novel MYCN-repressed gene, blocks cell cycle and promotes differentiation of neuronal cells.

PubMed

Valli, Emanuele; Trazzi, Stefania; Fuchs, Claudia; Erriquez, Daniela; Bartesaghi, Renata; Perini, Giovanni; Ciani, Elisabetta

2012-01-01

Mutations in the CDKL5 (cyclin-dependent kinase-like 5) gene are associated with a severe epileptic encephalopathy (early infantile epileptic encephalopathy type 2, EIEE2) characterized by early-onset intractable seizures, infantile spasms, severe developmental delay, intellectual disability, and Rett syndrome (RTT)-like features. Despite the clear involvement of CDKL5 mutations in intellectual disability, the function of this protein during brain development and the molecular mechanisms involved in its regulation are still unknown. Using human neuroblastoma cells as a model system we found that an increase in CDKL5 expression caused an arrest of the cell cycle in the G(0)/G(1) phases and induced cellular differentiation. Interestingly, CDKL5 expression was inhibited by MYCN, a transcription factor that promotes cell proliferation during brain development and plays a relevant role in neuroblastoma biology. Through a combination of different and complementary molecular and cellular approaches we could show that MYCN acts as a direct repressor of the CDKL5 promoter. Overall our findings unveil a functional axis between MYCN and CDKL5 governing both neuron proliferation rate and differentiation. The fact that CDKL5 is involved in the control of both neuron proliferation and differentiation may help understand the early appearance of neurological symptoms in patients with mutations in CDKL5. Copyright © 2012 Elsevier B.V. All rights reserved.

Melatonin antagonizes interleukin-18-mediated inhibition on neural stem cell proliferation and differentiation.

PubMed

Li, Zheng; Li, Xingye; Chan, Matthew T V; Wu, William Ka Kei; Tan, DunXian; Shen, Jianxiong

2017-09-01

Neural stem cells (NSCs) are self-renewing, pluripotent and undifferentiated cells which have the potential to differentiate into neurons, oligodendrocytes and astrocytes. NSC therapy for tissue regeneration, thus, gains popularity. However, the low survivals rate of the transplanted cell impedes its utilities. In this study, we tested whether melatonin, a potent antioxidant, could promote the NSC proliferation and neuronal differentiation, especially, in the presence of the pro-inflammatory cytokine interleukin-18 (IL-18). Our results showed that melatonin per se indeed exhibited beneficial effects on NSCs and IL-18 inhibited NSC proliferation, neurosphere formation and their differentiation into neurons. All inhibitory effects of IL-18 on NSCs were significantly reduced by melatonin treatment. Moreover, melatonin application increased the production of both brain-derived and glial cell-derived neurotrophic factors (BDNF, GDNF) in IL-18-stimulated NSCs. It was observed that inhibition of BDNF or GDNF hindered the protective effects of melatonin on NSCs. A potentially protective mechanism of melatonin on the inhibition of NSC's differentiation caused IL-18 may attribute to the up-regulation of these two major neurotrophic factors, BNDF and GNDF. The findings indicate that melatonin may play an important role promoting the survival of NSCs in neuroinflammatory diseases. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Adrenaline stimulates the proliferation and migration of mesenchymal stem cells towards the LPS-induced lung injury.

PubMed

Wu, Xiaodan; Wang, Zhiming; Qian, Mengjia; Wang, Lingyan; Bai, Chunxue; Wang, Xiangdong

2014-08-01

Bone marrow-derived mesenchymal stem cells (BMSCs) could modulate inflammation in experimental lung injury. On the other hand, adrenergic receptor agonists could increase DNA synthesis of stem cells. Therefore, we investigated the therapeutic role of adrenaline-stimulated BMSCs on lipopolysaccharide (LPS)-induced lung injury. BMSCs were cultured with adrenergic receptor agonists or antagonists. Suspensions of lung cells or sliced lung tissue from animals with or without LPS-induced injury were co-cultured with BMSCs. LPS-stimulated alveolar macrophages were co-cultured with BMSCs (with adrenaline stimulation or not) in Transwell for 6 hrs. A preliminary animal experiment was conducted to validate the findings in ex vivo study. We found that adrenaline at 10 μM enhanced proliferation of BMSCs through both α- and β-adrenergic receptors. Adrenaline promoted the migration of BMSCs towards LPS-injured lung cells or lung tissue. Adrenaline-stimulated BMSCs decreased the inflammation of LPS-stimulated macrophages, probably through the expression and secretion of several paracrine factors. Adrenaline reduced the extent of injury in LPS-injured rats. Our data indicate that adrenaline-stimulated BMSCs might contribute to the prevention from acute lung injury through the activation of adrenergic receptors, promotion of proliferation and migration towards injured lung, and modulation of inflammation. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

3D Non-Woven Polyvinylidene Fluoride Scaffolds: Fibre Cross Section and Texturizing Patterns Have Impact on Growth of Mesenchymal Stromal Cells

PubMed Central

Schellenberg, Anne; Ross, Robin; Abagnale, Giulio; Joussen, Sylvia; Schuster, Philipp; Arshi, Annahit; Pallua, Norbert; Jockenhoevel, Stefan; Gries, Thomas; Wagner, Wolfgang

2014-01-01

Several applications in tissue engineering require transplantation of cells embedded in appropriate biomaterial scaffolds. Such structures may consist of 3D non-woven fibrous materials whereas little is known about the impact of mesh size, pore architecture and fibre morphology on cellular behavior. In this study, we have developed polyvinylidene fluoride (PVDF) non-woven scaffolds with round, trilobal, or snowflake fibre cross section and different fibre crimp patterns (10, 16, or 28 needles per inch). Human mesenchymal stromal cells (MSCs) from adipose tissue were seeded in parallel on these scaffolds and their growth was compared. Initial cell adhesion during the seeding procedure was higher on non-wovens with round fibres than on those with snowflake or trilobal cross sections. All PVDF non-woven fabrics facilitated cell growth over a time course of 15 days. Interestingly, proliferation was significantly higher on non-wovens with round or trilobal fibres as compared to those with snowflake profile. Furthermore, proliferation increased in a wider, less dense network. Scanning electron microscopy (SEM) revealed that the MSCs aligned along the fibres and formed cellular layers spanning over the pores. 3D PVDF non-woven scaffolds support growth of MSCs, however fibre morphology and mesh size are relevant: proliferation is enhanced by round fibre cross sections and in rather wide-meshed scaffolds. PMID:24728045

MicroRNA-24 promotes 3T3-L1 adipocyte differentiation by directly targeting the MAPK7 signaling

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

Jin, Min, E-mail: min_jin@zju.edu.cn; Wu, Yutao; Wang, Jing

Over the past years, MicroRNAs (miRNAs) act as a vital role in harmony with gene regulation and maintaining cellular homeostasis. It is well testified that miRNAshave been involved in numerous physiological and pathological processes, including embryogenesis, cell fate decision, and cellular differentiation. Adipogenesis is an organized process of cellular differentiation by which pre-adipocytes differentiate towards mature adipocytes, and it is tightly modulated by a series of transcription factors such as peroxisome proliferator-activated receptor γ (PPAR-γ) and sterol regulatory-element binding proteins 1 (SREBP1). However, the molecular mechanisms underlying the connection between miRNAs and adipogenesis-related transcription factors remain obscure. In this study,more » we unveiled that miR- 24 was remarkably upregulated during 3T3-L1 adipogenesis. Overexpression of miR-24 significantly promoted 3T3-L1 adipogenesis, as evidenced by its ability to increase the expression of PPAR-γ and SREBP1, lipid droplet formation and triglyceride (TG) accumulation. Furthermore, we found that neither ectopic expression of miR-24nor miR-24 inhibitor affect cell proliferation and cell cycle progression. Finally, we demonstrated that miR-24 plays the modulational role by directly repressing MAPK7, a key number in the MAPK signaling pathway. These data indicate that miR-24 is a novel positive regulator of adipocyte differentiation by targeting MAPK7, which provides new insights into the molecular mechanism of miRNA-mediated cellular differentiation. -- Highlights: •We firstly found miR-24 was upregulated in 3T3-L1 pre-adipocytes differentiation. •miR-24 promoted 3T3-L1 pre-adipocytes differentiation while silencing the expression of miR-24 had an opposite function. •miR-24 regulated 3T3-L1 differentiation by directly targeting MAPK7 signaling pathway. •miR-24did not affect 3T3-L1 pre-adipocytes cellular proliferation.« less

Leucine Promotes Proliferation and Differentiation of Primary Preterm Rat Satellite Cells in Part through mTORC1 Signaling Pathway

PubMed Central

Dai, Jie-Min; Yu, Mu-Xue; Shen, Zhen-Yu; Guo, Chu-Yi; Zhuang, Si-Qi; Qiu, Xiao-Shan

2015-01-01

Signaling through the mammalian target of rapamycin (mTOR) in response to leucine modulates many cellular and developmental processes. However, in the context of satellite cell proliferation and differentiation, the role of leucine and mTORC1 is less known. This study investigates the role of leucine in the process of proliferation and differentiation of primary preterm rat satellite cells, and the relationship with mammalian target of rapamycin complex 1 (mTORC1) activation. Dissociation of primary satellite cells occurred with type I collagenase and trypsin, and purification, via different speed adherence methods. Satellite cells with positive expression of Desmin were treated with leucine and rapamycin. We observed that leucine promoted proliferation and differentiation of primary satellite cells and increased the phosphorylation of mTOR. Rapamycin inhibited proliferation and differentiation, as well as decreased the phosphorylation level of mTOR. Furthermore, leucine increased the expression of MyoD and myogenin while the protein level of MyoD decreased due to rapamycin. However, myogenin expressed no affect by rapamycin. In conclusion, leucine may up-regulate the activation of mTORC1 to promote proliferation and differentiation of primary preterm rat satellite cells. We have shown that leucine promoted the differentiation of myotubes in part through the mTORC1-MyoD signal pathway. PMID:26007333

Fetal Allostimulation of Maternal Cells: A Potential Mechanism for Perinatal HIV Transmission following Obstetrical Hemorrhage

PubMed Central

Wang, Guangwu; Izadpanah, Nazanin; Kitchen, Christina M.R.

2008-01-01

Abstract Our aim was to elucidate the mechanism by which HIV transmission is increased following obstetrical hemorrhage. We investigated whether fetal allostimulation of maternal cells, which could occur following fetal-to-maternal hemorrhage, increases proliferation, HIV replication, and cellular activation. Peripheral blood mononuclear cells (PBMCs) were collected from HIV-infected mothers and their infants to assess maternal-fetal allostimulation. Responses were compared to allostimulation with unrelated donors. Maternal and fetal cells were cocultured to assess allogeneic stimulation. Cell proliferation was measured by [3H]thymidine incorporation and cell activation was assessed via fluorochrome-labeled antibody staining and flow cytometric analysis. Virus production from HIV-infected maternal cells was quantitated by p24 enzyme-linked immunosorbent assay or by branched chain DNA assay. Allostimulation with fetal cells led to maternal cell proliferation. In women with unsuppressed viral loads, virus release was also enhanced following allostimulation of maternal cells with fetal cells. Fetal cells are capable of allogeneically stimulating maternal cells, with responses comparable to those seen following allostimulation with unrelated donors. Allostimulation of maternal cells by fetal cells results in statistically significant increases in proliferation and enhanced HIV replication, suggesting a possible physiological mechanism for mother-to-child transmission of HIV in women with obstetrical hemorrhage. PMID:19102686

Yeast Methylotrophy and Autophagy in a Methanol-Oscillating Environment on Growing Arabidopsis thaliana Leaves

PubMed Central

Kawaguchi, Kosuke; Yurimoto, Hiroya; Oku, Masahide; Sakai, Yasuyoshi

2011-01-01

The yeast Candida boidinii capable of growth on methanol proliferates and survives on the leaves of Arabidopsis thaliana. The local methanol concentration at the phyllosphere of growing A. thaliana exhibited daily periodicity, and yeast cells responded by altering both the expression of methanol-inducible genes and peroxisome proliferation. Even under these dynamically changing environmental conditions, yeast cells proliferated 3 to 4 times in 11 days. Among the C1-metabolic enzymes, enzymes in the methanol assimilation pathway, but not formaldehyde dissimilation or anti-oxidizing enzymes, were necessary for yeast proliferation at the phyllosphere. Furthermore, both peroxisome assembly and pexophagy, a selective autophagy pathway that degrades peroxisomes, were necessary for phyllospheric proliferation. Thus, the present study sheds light on the life cycle and physiology of yeast in the natural environment at both the molecular and cellular levels. PMID:21966472

Cell behavior related to implant surfaces with different microstructure and chemical composition: an in vitro analysis.

PubMed

Conserva, Enrico; Lanuti, Anna; Menini, Maria

2010-01-01

This paper reports on an in vitro comparison of osteoblast and mesenchymal stem cell (MSC) adhesion, proliferation, and differentiation related to two different surface treatments applied to the same implant design to determine whether the interaction between cells and implants is influenced by surface structure and chemical composition of the implants. Thirty-nine implants with a sandblasted (SB) surface and 39 implants with a grit-blasted and high-temperature acid-etched (GBAE) surface were used. The implant macrostructures and microstructures were analyzed by high- and low-voltage scanning electron microscopy (SEM) and by stereo-SEM. The surface chemical composition was investigated by energy dispersive analysis and x-ray photoemission spectroscopy. SaOS-2 osteoblasts and human MSCs were used for the evaluation of cell proliferation and alkaline phosphatase enzymatic activity in contact with the two surfaces. The GBAE surface showed fewer contaminants and a very high percentage of titanium (19.7%) compared to the SB surface (14.2%). The two surfaces showed similar mean roughness (Ra), but the depth (Rz) and density (RSm) of the porosity were significantly increased in the GBAE surface. The GBAE surface presented more osteoblast and MSC proliferation than the SB surface. No statistically significant differences in alkaline phosphatase activity were found between surfaces for either cellular line. The GBAE surface showed less surface contaminants and a higher percentage of titanium (19.7%) than the SB surface. The macro/micropore structured design and chemical composition of the GBAE surface allowed greater cell adhesion and proliferation and an earlier cell spreading but did not play an obvious role in in vitro cellular differentiation.

Exploring the effects of low-level laser therapy on fibroblasts and tumor cells following gamma radiation exposure.

PubMed

Ramos Silva, Camila; Cabral, Fernanda Viana; de Camargo, Claudinei Francisco Morais; Núñez, Silvia Cristina; Mateus Yoshimura, Tania; de Lima Luna, Arthur Cássio; Maria, Durvanei Augusto; Ribeiro, Martha Simões

2016-12-01

Ionizing radiation (IR) induces DNA damage and low-level laser therapy (LLLT) has been investigated to prevent or repair detrimental outcomes resulting from IR exposure. Few in vitro studies, however, explore the biological mechanisms underlying those LLLT benefits. Thus, in this work, fibroblasts and tumor cells are submitted to IR with doses of 2.5 Gy and 10 Gy. After twenty-four-h, the cells are exposed to LLLT with fluences of 30 J cm -2 , 90 J cm -2 , and 150 J cm -2 . Cellular viability, cell cycle phases, cell proliferation index and senescence are evaluated on days 1 and 4 after LLLT irradiation. For fibroblasts, LLLT promotes - in a fluence-dependent manner - increments in cell viability and proliferation, while a reduction in the senescence was observed. Regarding tumor cells, no influences of LLLT on cell viability are noticed. Whereas LLLT enhances cell populations in S and G 2 /M cell cycle phases for both cellular lines, a decrease in proliferation and increase in senescence was verified only for tumor cells. Putting together, the results suggest that fibroblasts and tumor cells present different responses to LLLT following exposure to gamma-radiation, and these promising results should stimulate further investigations. Senescence of tumor cells and fibroblasts on the 4 th day after ionizing radiation (IR) and low-level laser therapy (LLLT) exposures. The number of senescent cells increased significantly for tumor cells (a) while for fibroblasts no increment was observed (b). The blue collor indicates senescence activity. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of brevetoxins on murine myeloma SP2/O cells: Aberrant cellular division

USGS Publications Warehouse

Han, T.K.; Derby, M.; Martin, D.F.; Wright, S.D.; Dao, M.L.

2003-01-01

Massive deaths of manatees (Trichechus manatus latirostris) during the red tide seasons have been attributed to brevetoxins produced by the dinoflagellate Karenia brevis (formerly Ptychodiscus breve and Gymnodinium breve). Although these toxins have been found in macrophages and lymphocytes in the lung, liver, and secondary lymphoid tissues of these animals, the molecular mechanisms of brevetoxicosis have not yet been identified. To investigate the effects of brevetoxins on immune cells, a murine myeloma cell line (SP2/O) was used as a model for in vitro studies. By adding brevetoxins to cultures of the SP2/O cells at concentrations ranging from 20 to 600 ng/ml, an apparent increase in proliferation was observed at around 2 hours post challenge as compared to the unchallenged cell cultures. This was followed by a drop in cell number at around 3 hours, suggesting an aberrant effect of brevetoxins on cellular division, the cells generated at 2 hours being apparently short-lived. In situ immunochemical staining of the SP2/O cells at 1 and 2 hour post challenge showed an accumulation of the toxins in the nucleus. A 21-kDa protein was subsequently isolated from the SP2/O cells as having brevetoxin-binding properties, and immunologically identified as p21, a nuclear factor known to down-regulate cellular proliferation through inhibition of cyclin-dependent kinases. These data are the first on a possible effect of brevetoxins on the cell cycle via binding to p21, a phenomenon that needs to be further investigated and validated in normal immune cells.

Protein kinase C activation is required for the lead-induced inhibition of proliferation and differentiation of cultured oligodendroglial progenitor cells.

PubMed

Deng, Wenbin; Poretz, Ronald D

2002-03-01

Lead (Pb) is a common neurotoxicant of major public health concern. Previous studies revealed that cultured oligodendrocyte progenitor cells (OPCs) are highly vulnerable to Pb toxicity. The present study examines the effect of Pb on the survival, proliferation and differentiation of OPCs in vitro. Dose-response studies showed that> or = l5-10 microM Pb is cytotoxic to OPCs within 24 h. However, 1 microM of Pb was found to inhibit the proliferation and differentiation of OPCs without affecting cell viability. Pb markedly decreased the proliferative capability of OPCs and inhibited cell-intrinsic lineage progression of OPCs at a late progenitor stage. The Pb-induced decrease of proliferation and differentiation was abolished by inhibition of protein kinase C (PKC) with bisindolylmaleimide I, while the effect of the PKC-activating agent phorbol-12,13-didecanoate was potentiated by Pb. Furthermore, Pb exposure of OPCs caused the translocation of PKC from the cytoplasm to membrane without an increase in total cellular PKC enzymic activity. These results indicate that Pb inhibits the proliferation and differentiation of oligodendrocyte lineage cells in vitro through a mechanism requiring PKC activation.

Transgenic Overexpression of the Transcription Factor Nkx6.1 in β-Cells of Mice Does Not Increase β-Cell Proliferation, β-Cell Mass, or Improve Glucose Clearance

PubMed Central

Schaffer, Ashleigh E.; Yang, Almira J.; Thorel, Fabrizio; Herrera, Pedro L.

2011-01-01

The loss or dysfunction of the pancreatic endocrine β-cell results in diabetes. Recent innovative therapeutic approaches for diabetes aim to induce β-cell proliferation in vivo by pharmacological intervention. Based on the finding that overexpression of the transcription factor Nkx6.1 in islets in vitro increases β-cell proliferation while maintaining β-cell function, Nkx6.1 has been proposed as a potential target for diabetes therapy. However, it is unknown whether elevated Nkx6.1 levels in β-cells in vivo have similar effects as observed in isolated islets. To this end, we sought to investigate whether overexpression of Nkx6.1 in β-cells in vivo could increase β-cell mass and/or improve β-cell function in normal or β-cell-depleted mice. Using a bigenic inducible Cre-recombinase-based transgenic model, we analyzed the effects of Nkx6.1 overexpression on β-cell proliferation, β-cell mass, and glucose metabolism. We found that mice overexpressing Nkx6.1 in β-cells displayed similar β-cell proliferation rates and β-cell mass as control mice. Furthermore, after partial β-cell ablation, Nkx6.1 overexpression was not sufficient to induce β-cell regeneration under either nondiabetic or diabetic conditions. Together these results demonstrate that sustained Nkx6.1 overexpression in vivo does not stimulate β-cell proliferation, expand β-cell mass, or improve glucose metabolism in either normal or β-cell-depleted pancreata. Thus, raising cellular Nkx6.1 levels in β-cells in vivo is unlikely to have a positive impact on type 2 diabetes. PMID:21964593

Blocking Glycolytic Metabolism Increases Memory T Cells and Antitumor Function | Center for Cancer Research

Cancer.gov

CD8+ T cells are a major component of the cellular immune response, which is necessary to control a variety of bacterial and viral infections. CD8+ T cells also play a major role in the cell-mediated antitumor immune response. After encountering antigen, naïve CD8+ T cells undergo an extensive period of proliferation and expansion, and differentiate into effector cells and

Selenium in bone health: roles in antioxidant protection and cell proliferation.

PubMed

Zeng, Huawei; Cao, Jay J; Combs, Gerald F

2013-01-10

Selenium (Se) is an essential trace element for humans and animals, and several findings suggest that dietary Se intake may be necessary for bone health. Such findings may relate to roles of Se in antioxidant protection, enhanced immune surveillance and modulation of cell proliferation. Elucidation of the mechanisms by which Se supports these cellular processes can lead to a better understanding of the role of this nutrient in normal bone metabolism. This article reviews the current knowledge concerning the molecular functions of Se relevant to bone health.

Selenium in Bone Health: Roles in Antioxidant Protection and Cell Proliferation

PubMed Central

Zeng, Huawei; Cao, Jay J.; Combs, Gerald F.

2013-01-01

Selenium (Se) is an essential trace element for humans and animals, and several findings suggest that dietary Se intake may be necessary for bone health. Such findings may relate to roles of Se in antioxidant protection, enhanced immune surveillance and modulation of cell proliferation. Elucidation of the mechanisms by which Se supports these cellular processes can lead to a better understanding of the role of this nutrient in normal bone metabolism. This article reviews the current knowledge concerning the molecular functions of Se relevant to bone health. PMID:23306191

Induction of Cell Death through Alteration of Oxidants and Antioxidants in Epithelial Cells Exposed to High Energy Protons

NASA Technical Reports Server (NTRS)

Ramesh, Govindarajan; Wu, Honglu

2012-01-01

Radiation affects several cellular and molecular processes including double strand breakage, modifications of sugar moieties and bases. In outer space, protons are the primary radiation source which poses a range of potential health risks to astronauts. On the other hand, the use of proton radiation for tumor radiation therapy is increasing as it largely spares healthy tissues while killing tumor tissues. Although radiation related research has been conducted extensively, the molecular toxicology and cellular mechanisms affected by proton radiation remain poorly understood. Therefore, in the present study, we irradiated rat epithelial cells (LE) with different doses of protons and investigated their effects on cell proliferation and cell death. Our data showed an inhibition of cell proliferation in proton irradiated cells with a significant dose dependent activation and repression of reactive oxygen species (ROS) and antioxidants, glutathione and superoxide dismutase respectively as compared to control cells. In addition, apoptotic related genes such as caspase-3 and -8 activities were induced in a dose dependent manner with corresponding increased levels of DNA fragmentation in proton irradiated cells than control cells. Together, our results show that proton radiation alters oxidant and antioxidant levels in the cells to activate apoptotic pathway for cell death.

Human fetal bone cells in delivery systems for bone engineering.

PubMed

Tenorio, Diene M H; Scaletta, Corinne; Jaccoud, Sandra; Hirt-Burri, Nathalie; Pioletti, Dominique P; Jaques, Bertrand; Applegate, Lee Ann

2011-11-01

The aim of this study was to culture human fetal bone cells (dedicated cell banks of fetal bone derived from 14 week gestation femurs) within both hyaluronic acid gel and collagen foam, to compare the biocompatibility of both matrices as potential delivery systems for bone engineering and particularly for oral application. Fetal bone cell banks were prepared from one organ donation and cells were cultured for up to 4 weeks within hyaluronic acid (Mesolis®) and collagen foams (TissueFleece®). Cell survival and differentiation were assessed by cell proliferation assays and histology of frozen sections stained with Giemsa, von Kossa and ALP at 1, 2 and 4 weeks of culture. Within both materials, fetal bone cells could proliferate in three-dimensional structure at ∼70% capacity compared to monolayer culture. In addition, these cells were positive for ALP and von Kossa staining, indicating cellular differentiation and matrix production. Collagen foam provides a better structure for fetal bone cell delivery if cavity filling is necessary and hydrogels would permit an injectable technique for difficult to treat areas. In all, there was high biocompatibility, cellular differentiation and matrix deposition seen in both matrices by fetal bone cells, allowing for easy cell delivery for bone stimulation in vivo. Copyright © 2011 John Wiley & Sons, Ltd.

Activation of cutaneous immune responses in complex regional pain syndrome

PubMed Central

Birklein, Frank; Drummond, Peter D.; Li, Wenwu; Schlereth, Tanja; Albrecht, Nahid; Finch, Philip M.; Dawson, Linda F.; Clark, J. David; Kingery, Wade S.

2014-01-01

The pathogenesis of complex regional pain syndrome (CRPS) is unresolved, but TNF-α and IL-6 are elevated in experimental skin blister fluid from CRPS affected limbs, as is tryptase, a marker for mast cells. In the rat fracture model of CRPS exaggerated sensory and sympathetic neural signaling stimulate keratinocyte and mast cell proliferation, causing the local production of high levels of inflammatory cytokines leading to pain behavior. The current investigation used CRPS patient skin biopsies to determine whether keratinocyte and mast cell proliferation occur in CRPS skin and to identify the cellular source of the up-regulated TNF-α, IL-6, and tryptase observed in CRPS experimental skin blister fluid. Skin biopsies were collected from the affected skin and the contralateral mirror site in 55 CRPS patients and the biopsy sections were immunostained for keratinocyte, cell proliferation, mast cell markers, TNF-α, and IL-6. In early CRPS keratinocytes were activated in the affected skin, resulting in proliferation, epidermal thickening, and up-regulated TNF-α and IL-6 expression. In chronic CRPS there was reduced keratinocyte proliferation with epidermal thinning in the affected skin. Acute CRPS patients also had increased mast cell accumulation in the affected skin, but there was no increase in mast cell numbers in chronic CRPS. PMID:24462502

Higher Molecular Weight Polyethylene Glycol Increases Cell Proliferation While Improving Barrier Function in an In Vitro Colon Cancer Model

PubMed Central

Bharadwaj, Shruthi; Vishnubhotla, Ramana; Shan, Sun; Chauhan, Chinmay; Cho, Michael; Glover, Sarah C.

2011-01-01

Polyethylene glycol (PEG) has been previously shown to protect against enteric pathogens and prevent colon cancer invasion. To determine if PEG could indeed protect against previously observed pro-invasive effects of commensal E. coli and EPEC, Caco-2 cells grown in an in vitro model of colon cancer were infected with strains of human commensal E. coli or EPEC and treated with 10% PEG 3350, PEG 8000, and PEG 20,000, respectively. At 24 hours after infection, MMP-1 and MMP-13 activities, cell cluster thickness, depth of invasion, and proliferation were determined using standard molecular biology techniques and advanced imaging. We found that higher molecular weight PEG, especially PEG 8000 and 20,000, regardless of bacterial infection, increased proliferation and depth of invasion although a decrease in cellular density and MMP-1 activity was also noted. Maximum proliferation and depth of invasion of Caco-2 cells was observed in scaffolds treated with a combination of commensal E. coli strain, HS4 and PEG 8000. In conclusion, we found that PEG 8000 increased cell proliferation and led to the preservation of cell density in cells treated with commensal bacteria. This is important, because the preservation of a proliferative response in colon cancer results in a more chemo-responsive tumor. PMID:21976966

Higher molecular weight polyethylene glycol increases cell proliferation while improving barrier function in an in vitro colon cancer model.

PubMed

Bharadwaj, Shruthi; Vishnubhotla, Ramana; Shan, Sun; Chauhan, Chinmay; Cho, Michael; Glover, Sarah C

2011-01-01

Polyethylene glycol (PEG) has been previously shown to protect against enteric pathogens and prevent colon cancer invasion. To determine if PEG could indeed protect against previously observed pro-invasive effects of commensal E. coli and EPEC, Caco-2 cells grown in an in vitro model of colon cancer were infected with strains of human commensal E. coli or EPEC and treated with 10% PEG 3350, PEG 8000, and PEG 20,000, respectively. At 24 hours after infection, MMP-1 and MMP-13 activities, cell cluster thickness, depth of invasion, and proliferation were determined using standard molecular biology techniques and advanced imaging. We found that higher molecular weight PEG, especially PEG 8000 and 20,000, regardless of bacterial infection, increased proliferation and depth of invasion although a decrease in cellular density and MMP-1 activity was also noted. Maximum proliferation and depth of invasion of Caco-2 cells was observed in scaffolds treated with a combination of commensal E. coli strain, HS4 and PEG 8000. In conclusion, we found that PEG 8000 increased cell proliferation and led to the preservation of cell density in cells treated with commensal bacteria. This is important, because the preservation of a proliferative response in colon cancer results in a more chemo-responsive tumor.

Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

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

Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling

As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-inducedmore » inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.« less

Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib.

PubMed

Zhai, Yang; Zhang, Yanjun; Nan, Kejun; Liang, Xuan

2017-05-01

The clinical resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been linked to EGFR T790M resistance mutations or MET amplifications. Additional mechanisms underlying EGFR-TKI drug resistance remain unclear. The present study demonstrated that icotinib significantly inhibited the proliferation and increased the apoptosis rate of HCC827 cells; the cellular mRNA and protein expression levels of phosphatase and tensin homolog (PTEN) were also significantly downregulated. To investigate the effect of PTEN expression levels on the sensitivity of HCC827 cells to icotinib, PTEN expression was silenced using a PTEN-specific small interfering RNA. The current study identified that the downregulation of PTEN expression levels may promote cellular proliferation in addition to decreasing the apoptosis of HCC827 cells, and may reduce the sensitivity of HCC827 cells to icotinib. These results suggested that reduced PTEN expression levels were associated with the decreased sensitivity of HCC827 cells to icotinib. Furthermore, PTEN expression levels may be a useful marker for predicting icotinib resistance and elucidating the resistance mechanisms underlying EGFR-mutated NSCLC.

Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib

PubMed Central

Zhai, Yang; Zhang, Yanjun; Nan, Kejun; Liang, Xuan

2017-01-01

The clinical resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been linked to EGFR T790M resistance mutations or MET amplifications. Additional mechanisms underlying EGFR-TKI drug resistance remain unclear. The present study demonstrated that icotinib significantly inhibited the proliferation and increased the apoptosis rate of HCC827 cells; the cellular mRNA and protein expression levels of phosphatase and tensin homolog (PTEN) were also significantly downregulated. To investigate the effect of PTEN expression levels on the sensitivity of HCC827 cells to icotinib, PTEN expression was silenced using a PTEN-specific small interfering RNA. The current study identified that the downregulation of PTEN expression levels may promote cellular proliferation in addition to decreasing the apoptosis of HCC827 cells, and may reduce the sensitivity of HCC827 cells to icotinib. These results suggested that reduced PTEN expression levels were associated with the decreased sensitivity of HCC827 cells to icotinib. Furthermore, PTEN expression levels may be a useful marker for predicting icotinib resistance and elucidating the resistance mechanisms underlying EGFR-mutated NSCLC. PMID:28521430

Cancer cell metabolism: one hallmark, many faces.

PubMed

Cantor, Jason R; Sabatini, David M

2012-10-01

Cancer cells must rewire cellular metabolism to satisfy the demands of growth and proliferation. Although many of the metabolic alterations are largely similar to those in normal proliferating cells, they are aberrantly driven in cancer by a combination of genetic lesions and nongenetic factors such as the tumor microenvironment. However, a single model of altered tumor metabolism does not describe the sum of metabolic changes that can support cell growth. Instead, the diversity of such changes within the metabolic program of a cancer cell can dictate by what means proliferative rewiring is driven, and can also impart heterogeneity in the metabolic dependencies of the cell. A better understanding of this heterogeneity may enable the development and optimization of therapeutic strategies that target tumor metabolism.

Hsp90 Is a Novel Target Molecule of CDDO-Me in Inhibiting Proliferation of Ovarian Cancer Cells.

PubMed

Qin, Dong-Jun; Tang, Cai-Xia; Yang, Li; Lei, Hu; Wei, Wei; Wang, Ying-Ying; Ma, Chun-Min; Gao, Feng-Hou; Xu, Han-Zhang; Wu, Ying-Li

2015-01-01

Synthetic triterpenoid methyl-2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oate (CDDO-Me) has been shown as a promising agent against ovarian cancer. However, the underlying mechanism is not well understood. Here, we demonstrate that CDDO-Me directly interacts with Hsp90 in cells by cellular thermal shift assay. CDDO-Me treatment leads to upregulation of Hsp70 and degradation of Hsp90 clients (ErbB2 and Akt), indicating the inhibition of Hsp90 by CDDO-Me in cells. Knockdown of Hsp90 significantly inhibits cell proliferation and enhances the anti-proliferation effect of CDDO-Me in H08910 ovarian cancer cells. Dithiothreitol inhibits the interaction of CDDO-Me with Hsp90 in cells and abrogates CDDO-Me induced upregulation of Hsp70, degradation of Akt and cell proliferation inhibition. This suggests the anti-ovarian cancer effect of CDDO-Me is possibly mediated by the formation of Michael adducts between CDDO-Me and reactive nucleophiles on Hsp90. This study identifies Hsp90 as a novel target protein of CDDO-Me, and provides a novel insight into the mechanism of action of CDDO-Me in ovarian cancer cells.

Effect of extracorporeal shock wave on proliferation and differentiation of equine adipose tissue-derived mesenchymal stem cells in vitro

PubMed Central

Raabe, O; Shell, K; Goessl, A; Crispens, C; Delhasse, Y; Eva, A; Scheiner-Bobis, G; Wenisch, S; Arnhold, S

2013-01-01

Mesenchymal stem cells are regarded as common cellular precursors of the musculoskeletal tissue and are responsible for tissue regeneration in the course of musculoskeletal disorders. In equine veterinary medicine extracorporeal shock wave therapy (ESWT) is used to optimize healing processes of bone, tendon and cartilage. Nevertheless, little is known about the effects of the shock waves on cells and tissues. Thus, the aim of this study was to investigate the influence of focused ESWT on the viability, proliferation, and differentiation capacity of adipose tissue-derived mesenchymal stem cells (ASCs) and to explore its effects on gap junctional communication and the activation of signalling cascades associated with cell proliferation and differentiation. ASCs were treated with different pulses of focused ESWT. Treated cells showed increased proliferation and expression of Cx43, as detected by means of qRT-PCR, histological staining, immunocytochemistry and western blot. At the same time, cells responded to ESWT by significant activation (phosphorylation) of Erk1/2, detected in western blots. No significant effects on the differentiation potential of the ASCs were evident. Taken together, the present results show significant effects of shock waves on stem cells in vitro. PMID:23671817

Ghrelin promotes oral tumor cell proliferation by modifying GLUT1 expression.

PubMed

Kraus, Dominik; Reckenbeil, Jan; Wenghoefer, Matthias; Stark, Helmut; Frentzen, Matthias; Allam, Jean-Pierre; Novak, Natalija; Frede, Stilla; Götz, Werner; Probstmeier, Rainer; Meyer, Rainer; Winter, Jochen

2016-03-01

In our study, ghrelin was investigated with respect to its capacity on proliferative effects and molecular correlations on oral tumor cells. The presence of all molecular components of the ghrelin system, i.e., ghrelin and its receptors, was analyzed and could be detected using real-time PCR and immunohistochemistry. To examine cellular effects caused by ghrelin and to clarify downstream-regulatory mechanisms, two different oral tumor cell lines (BHY and HN) were used in cell culture experiments. Stimulation of either cell line with ghrelin led to a significantly increased proliferation. Signal transduction occurred through phosphorylation of GSK-3β and nuclear translocation of β-catenin. This effect could be inhibited by blocking protein kinase A. Glucose transporter1 (GLUT1), as an important factor for delivering sufficient amounts of glucose to tumor cells having high requirements for this carbohydrate (Warburg effect) was up-regulated by exogenous and endogenous ghrelin. Silencing intracellular ghrelin concentrations using siRNA led to a significant decreased expression of GLUT1 and proliferation. In conclusion, our study describes the role for the appetite-stimulating peptide hormone ghrelin in oral cancer proliferation under the particular aspect of glucose uptake: (1) tumor cells are a source of ghrelin. (2) Ghrelin affects tumor cell proliferation through autocrine and/or paracrine activity. (3) Ghrelin modulates GLUT1 expression and thus indirectly enhances tumor cell proliferation. These findings are of major relevance, because glucose uptake is assumed to be a promising target for cancer treatment.

Mesenchymal stem cells do not suppress lymphoblastic leukemic cell line proliferation.

PubMed

Mousavi Niri, Neda; Jaberipour, Mansooreh; Razmkhah, Mahboobeh; Ghaderi, Abbas; Habibagahi, Mojtaba

2009-12-01

Several studies have demonstrated the immunosuppresive effects of mesenchymal stem cells (MSCs) in allogeneic or mitogenic interactions. Cell-cell contact inhibition and secretion of suppressive soluble factors have been suggested in this regard. To investigate if adipose derived MSCs could inhibit Jurkat lymphoblastic leukemia T cell proliferation during coculture. Adherent cells with the ability of cellular growth were isolated from normal adipose tissues. Initial characterization of growing cells by flow cytometry suggested their mesenchymal stem cell characteristics. Cells were maintained in culture and used during third to fifth culture passages. Jurkat or allogeneic peripheral blood mononuclear cells (PBMCs) were labeled with carboxy fluorescein diacetate succinimidyl ester and cocultured with increasing doses of MSCs or MSC culture supernatant. Proliferation of PBMCs or Jurkat cells under these conditions was assessed by flow cytometry after 2 and 3 days of coculture, respectively. Results showed the expression of CD105, CD166 and CD44, and the absence of CD45, CD34 and CD14 on the surface of MSC like cells. Moreover, initial differentiation studies showed the potential of cell differentiation into hepatocytes. Comparison of Jurkat cell proliferation in the presence and absence of MSCs showed no significant difference, with 70% of cells displaying signs of at least one cell division. Similarly, the highest concentration of MSC culture supernatant (50% vol/vol) had no significant effect on Jurkat cell proliferation (p>0.6). The same MSC lots significantly suppressed the allogeneic PHA activated PBMCs under similar culture conditions. Using Jurkat cells as a model of leukemia T cells, our results indicated an uncertainty about the suppressive effect of MSCs and their inhibitory metabolites on tumor or leukemia cell proliferation. Additional systematic studies with MSCs of different sources are needed to fully characterize the immunological properties of MSCs before planning clinical applications.

Viability and proliferation of endothelial cells upon exposure to GaN nanoparticles

PubMed Central

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Cebotari, Serghei; Lux, Marco; Haverich, Axel

2016-01-01

Summary Nanotechnology is a rapidly growing and promising field of interest in medicine; however, nanoparticle–cell interactions are not yet fully understood. The goal of this work was to examine the interaction between endothelial cells and gallium nitride (GaN) semiconductor nanoparticles. Cellular viability, adhesion, proliferation, and uptake of nanoparticles by endothelial cells were investigated. The effect of free GaN nanoparticles versus the effect of growing endothelial cells on GaN functionalized surfaces was examined. To functionalize surfaces with GaN, GaN nanoparticles were synthesized on a sacrificial layer of zinc oxide (ZnO) nanoparticles using hydride vapor phase epitaxy. The uptake of GaN nanoparticles by porcine endothelial cells was strongly dependent upon whether they were fixed to the substrate surface or free floating in the medium. The endothelial cells grown on surfaces functionalized with GaN nanoparticles demonstrated excellent adhesion and proliferation, suggesting good biocompatibility of the nanostructured GaN. PMID:27826507

Premature aging/senescence in cancer cells facing therapy: good or bad?

PubMed

Gonzalez, Llilians Calvo; Ghadaouia, Sabrina; Martinez, Aurélie; Rodier, Francis

2016-02-01

Normal and cancer cells facing their demise following exposure to radio-chemotherapy can actively participate in choosing their subsequent fate. These programmed cell fate decisions include true cell death (apoptosis-necroptosis) and therapy-induced cellular senescence (TIS), a permanent "proliferative arrest" commonly portrayed as premature cellular aging. Despite a permanent loss of proliferative potential, senescent cells remain viable and are highly bioactive at the microenvironment level, resulting in a prolonged impact on tissue architecture and functions. Cellular senescence is primarily documented as a tumor suppression mechanism that prevents cellular transformation. In the context of normal tissues, cellular senescence also plays important roles in tissue repair, but contributes to age-associated tissue dysfunction when senescent cells accumulate. Theoretically, in multi-step cancer progression models, cancer cells have already bypassed cellular senescence during their immortalization step (see hallmarks of cancer). It is then perhaps surprising to find that cancer cells often retain the ability to undergo TIS, or premature aging. This occurs because cellular senescence results from multiple signalling pathways, some retained in cancer cells, aiming to prevent cell cycle progression in damaged cells. Since senescent cancer cells persist after therapy and secrete an array of cytokines and growth factors that can modulate the tumor microenvironment, these cells may have beneficial and detrimental effects regarding immune modulation and survival of remaining proliferation-competent cancer cells. Similarly, while normal cells undergoing senescence are believed to remain indefinitely growth arrested, whether this is true for senescent cancer cells remains unclear, raising the possibility that these cells may represent a reservoir for cancer recurrence after treatment. This review discusses our current knowledge on cancer cell senescence and highlight questions that must be addressed to fully understand the beneficial and detrimental impacts of cellular senescence during cancer therapy.

In Vitro Modeling of Repetitive Motion Injury and Myofascial Release

PubMed Central

Meltzer, Kate R.; Cao, Thanh V.; Schad, Joseph F.; King, Hollis; Stoll, Scott T.; Standley, Paul R.

2010-01-01

Objective In this study we modeled repetitive motion strain (RMS) and myofascial release (MFR) in vitro to investigate possible cellular and molecular mechanisms to potentially explain the immediate clinical outcomes associated with RMS and MFR. Method Cultured human fibroblasts were strained with 8 hours RMS, 60 seconds MFR and combined treatment; RMS+MFR. Fibroblasts were immediately sampled upon cessation of strain and evaluated for cell morphology, cytokine secretions, proliferation, apoptosis, and potential changes to intracellular signaling molecules. Results RMS induced fibroblast elongation of lameopodia, cellular decentralization, reduction of cell to cell contact and significant decreases in cell area to perimeter ratios compared to all other experimental groups (p<0.0001). Cellular proliferation indicated no change among any treatment group; however RMS resulted in a significant increase in apoptosis rate (p<0.05) along with increases in death-associated protein kinase (DAPK) and focal adhesion kinase (FAK) phosphorylation by 74% and 58% respectively, when compared to control. These responses were not observed in the MFR and RMS+MFR group. Of the twenty cytokines measured there was a significant increase in GRO secretion in the RMS+MFR group when compared to control and MFR alone. Conclusion Our modeled injury (RMS) appropriately displayed enhanced apoptosis activity and loss of intercellular integrity that is consistent with pro-apoptotic DAPK2 and FAK signaling. Treatment with MFR following RMS resulted in normalization in apoptotic rate and cell morphology both consistent with changes observed in DAPK2. These in vitro studies build upon the cellular evidence base needed to fully explain clinical efficacy of manual manipulative therapies. PMID:20226363

Adenovirus type 5 exerts genome-wide control over cellular programs governing proliferation, quiescence, and survival

PubMed Central

Miller, Daniel L; Myers, Chad L; Rickards, Brenden; Coller, Hilary A; Flint, S Jane

2007-01-01

Background Human adenoviruses, such as serotype 5 (Ad5), encode several proteins that can perturb cellular mechanisms that regulate cell cycle progression and apoptosis, as well as those that mediate mRNA production and translation. However, a global view of the effects of Ad5 infection on such programs in normal human cells is not available, despite widespread efforts to develop adenoviruses for therapeutic applications. Results We used two-color hybridization and oligonucleotide microarrays to monitor changes in cellular RNA concentrations as a function of time after Ad5 infection of quiescent, normal human fibroblasts. We observed that the expression of some 2,000 genes, about 10% of those examined, increased or decreased by a factor of two or greater following Ad5 infection, but were not altered in mock-infected cells. Consensus k-means clustering established that the temporal patterns of these changes were unexpectedly complex. Gene Ontology terms associated with cell proliferation were significantly over-represented in several clusters. The results of comparative analyses demonstrate that Ad5 infection induces reversal of the quiescence program and recapitulation of the core serum response, and that only a small subset of the observed changes in cellular gene expression can be ascribed to well characterized functions of the viral E1A and E1B proteins. Conclusion These findings establish that the impact of adenovirus infection on host cell programs is far greater than appreciated hitherto. Furthermore, they provide a new framework for investigating the molecular functions of viral early proteins and information relevant to the design of conditionally replicating adenoviral vectors. PMID:17430596

Heparin concentration is critical for cell culture with human platelet lysate.

PubMed

Hemeda, Hatim; Kalz, Jana; Walenda, Gudrun; Lohmann, Michael; Wagner, Wolfgang

2013-09-01

Culture media for mesenchymal stromal cells (MSCs) are generally supplemented with fetal bovine serum. Human platelet lysate (hPL) has been proven to be a very effective alternative without the risk of xenogeneic infections or immune reactions. In contrast to fetal bovine serum, hPL comprises plasma, and anticoagulants-usually unfractionated heparin (UFH)-need to be added to prevent gel formation. Cultures of MSCs in hPL media with various concentrations of UFH and enoxaparin, a low-molecular-weight heparin (LMWH), were systematically compared with regard to proliferation, fibroblastoid colony-forming unit frequency, immunophenotype and in vitro differentiation. At least 0.61 IU/mL UFH or 0.024 mg/mL LMWH was necessary for reliable prevention of coagulation of hPL pools used in this study. Higher concentrations impaired cellular proliferation in a dose-dependent manner even without benzyl alcohol, which is commonly added to heparins as a bacteriostatic agent. Colony-forming unit frequency was also reduced at higher heparin concentrations, particularly with LMWH, whereas no significant effect was observed on cellular morphology or immunophenotype. High concentrations of heparins reduced the in vitro differentiation toward adipogenic and osteogenic lineages. Heparin concentration is critical for culture of MSCs in hPL media; this is of particular relevance for cellular therapy where cell culture procedures need to be optimized and standardized. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

High-Content, High-Throughput Screening for the Identification of Cytotoxic Compounds Based on Cell Morphology and Cell Proliferation Markers

PubMed Central

Martin, Heather L.; Adams, Matthew; Higgins, Julie; Bond, Jacquelyn; Morrison, Ewan E.; Bell, Sandra M.; Warriner, Stuart; Nelson, Adam; Tomlinson, Darren C.

2014-01-01

Toxicity is a major cause of failure in drug discovery and development, and whilst robust toxicological testing occurs, efficiency could be improved if compounds with cytotoxic characteristics were identified during primary compound screening. The use of high-content imaging in primary screening is becoming more widespread, and by utilising phenotypic approaches it should be possible to incorporate cytotoxicity counter-screens into primary screens. Here we present a novel phenotypic assay that can be used as a counter-screen to identify compounds with adverse cellular effects. This assay has been developed using U2OS cells, the PerkinElmer Operetta high-content/high-throughput imaging system and Columbus image analysis software. In Columbus, algorithms were devised to identify changes in nuclear morphology, cell shape and proliferation using DAPI, TOTO-3 and phosphohistone H3 staining, respectively. The algorithms were developed and tested on cells treated with doxorubicin, taxol and nocodazole. The assay was then used to screen a novel, chemical library, rich in natural product-like molecules of over 300 compounds, 13.6% of which were identified as having adverse cellular effects. This assay provides a relatively cheap and rapid approach for identifying compounds with adverse cellular effects during screening assays, potentially reducing compound rejection due to toxicity in subsequent in vitro and in vivo assays. PMID:24505478

γ-Oryzanol Enhances Adipocyte Differentiation and Glucose Uptake

PubMed Central

Jung, Chang Hwa; Lee, Da-Hye; Ahn, Jiyun; Lee, Hyunjung; Choi, Won Hee; Jang, Young Jin; Ha, Tae-Youl

2015-01-01

Recent studies show that brown rice improves glucose intolerance and potentially the risk of diabetes, although the underlying molecular mechanisms remain unclear. One of the phytochemicals found in high concentration in brown rice is γ-oryzanol (Orz), a group of ferulic acid esters of phytosterols and triterpene alcohols. Here, we found that Orz stimulated differentiation of 3T3-L1 preadipocytes and increased the protein expression of adipogenic marker genes such as peroxisome proliferator-activated receptor gamma (PPAR-γ) and CCAAT/enhanced binding protein alpha (C/EBPα). Moreover, Orz significantly increased the glucose uptake in insulin-resistant cells and translocation of glucose transporter type 4 (GLUT4) from the cytosol to the cell surface. To investigate the mechanism by which Orz stimulated cell differentiation, we examined its effects on cellular signaling of the mammalian target of rapamycin complex 1 (mTORC1), a central mediator of cellular growth and proliferation. The Orz treatment increased mTORC1 kinase activity based on phosphorylation of 70-kDa ribosomal S6 kinase 1 (S6K1). The effect of Orz on adipocyte differentiation was dependent on mTORC1 activity because rapamycin blocks cell differentiation in Orz-treated cells. Collectively, our results indicate that Orz stimulates adipocyte differentiation, enhances glucose uptake, and may be associated with cellular signaling mediated by PPAR-γ and mTORC1. PMID:26083118

γ-Oryzanol Enhances Adipocyte Differentiation and Glucose Uptake.

PubMed

Jung, Chang Hwa; Lee, Da-Hye; Ahn, Jiyun; Lee, Hyunjung; Choi, Won Hee; Jang, Young Jin; Ha, Tae-Youl

2015-06-15

Recent studies show that brown rice improves glucose intolerance and potentially the risk of diabetes, although the underlying molecular mechanisms remain unclear. One of the phytochemicals found in high concentration in brown rice is γ-oryzanol (Orz), a group of ferulic acid esters of phytosterols and triterpene alcohols. Here, we found that Orz stimulated differentiation of 3T3-L1 preadipocytes and increased the protein expression of adipogenic marker genes such as peroxisome proliferator-activated receptor gamma (PPAR-γ) and CCAAT/enhanced binding protein alpha (C/EBPα). Moreover, Orz significantly increased the glucose uptake in insulin-resistant cells and translocation of glucose transporter type 4 (GLUT4) from the cytosol to the cell surface. To investigate the mechanism by which Orz stimulated cell differentiation, we examined its effects on cellular signaling of the mammalian target of rapamycin complex 1 (mTORC1), a central mediator of cellular growth and proliferation. The Orz treatment increased mTORC1 kinase activity based on phosphorylation of 70-kDa ribosomal S6 kinase 1 (S6K1). The effect of Orz on adipocyte differentiation was dependent on mTORC1 activity because rapamycin blocks cell differentiation in Orz-treated cells. Collectively, our results indicate that Orz stimulates adipocyte differentiation, enhances glucose uptake, and may be associated with cellular signaling mediated by PPAR-γ and mTORC1.

Anti-tumor response with immunologically modified carbon nanotubes and phototherapy

NASA Astrophysics Data System (ADS)

Acquaviva, Joseph T.; Zhou, Feifan; Boarman, Ellen; Chen, Wei R.

2013-02-01

While successes of different cancer therapies have been achieved in various degrees a systemic immune response is needed to effectively treat late-stage, metastatic cancers, and to establish long-term tumor resistance in the patients. A novel method for combating metastatic cancers has been developed using immunologically modified carbon nanotubes in conjunction with phototherapy. Glycated chitosan (GC) is a potent immunological adjuvant capable of increasing host immune responses, including antigen presentation by activation of dendritic cells (DCs) and causing T cell proliferation. GC is also an effective surfactant for nanomaterials. By combining single-walled carbon nanotubes (SWNTs) and GC, immunologically modified carbon nanotubes (SWNT-GC) were constructed. The SWNT-GC suspension retains the enhanced light absorption properties in the near infrared (NIR) region and the ability to enter cells, which are characteristic of SWNTs. The SWNT-GC also retains the immunological properties of GC. Cellular SWNT-GC treatments increased macrophage activity, DC activation and T cell proliferation. When cellular SWNT-GC was irradiated with a laser of an appropriate wavelength, these immune activities could be enhanced. The combination of laser irradiation and SWNT-GC induced cellular toxicity in targeted tumor cells, leading to a systemic antitumor response. Immunologically modified carbon nanotubes in conjunction with phototherapy is a novel and promising method to produce a systemic immune response for the treatment of metastatic cancers.

From single cells to tissue architecture-a bottom-up approach to modelling the spatio-temporal organisation of complex multi-cellular systems.

PubMed

Galle, J; Hoffmann, M; Aust, G

2009-01-01

Collective phenomena in multi-cellular assemblies can be approached on different levels of complexity. Here, we discuss a number of mathematical models which consider the dynamics of each individual cell, so-called agent-based or individual-based models (IBMs). As a special feature, these models allow to account for intracellular decision processes which are triggered by biomechanical cell-cell or cell-matrix interactions. We discuss their impact on the growth and homeostasis of multi-cellular systems as simulated by lattice-free models. Our results demonstrate that cell polarisation subsequent to cell-cell contact formation can be a source of stability in epithelial monolayers. Stroma contact-dependent regulation of tumour cell proliferation and migration is shown to result in invasion dynamics in accordance with the migrating cancer stem cell hypothesis. However, we demonstrate that different regulation mechanisms can equally well comply with present experimental results. Thus, we suggest a panel of experimental studies for the in-depth validation of the model assumptions.

Evaluation of Simulated Microgravity Environments Induced by Diamagnetic Levitation of Plant Cell Suspension Cultures

NASA Astrophysics Data System (ADS)

Kamal, Khaled Y.; Herranz, Raúl; van Loon, Jack J. W. A.; Christianen, Peter C. M.; Medina, F. Javier

2016-06-01

Ground-Based Facilities (GBF) are essetial tools to understand the physical and biological effects of the absence of gravity and they are necessary to prepare and complement space experiments. It has been shown previously that a real microgravity environment induces the dissociation of cell proliferation from cell growth in seedling root meristems, which are limited populations of proliferating cells. Plant cell cultures are large and homogeneous populations of proliferating cells, so that they are a convenient model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of the Arabidopsis thaliana cell line MM2d were exposed to four altered gravity and magnetic field environments in a magnetic levitation facility for 3 hours, including two simulated microgravity and Mars-like gravity levels obtained with different magnetic field intensities. Samples were processed either by quick freezing, to be used in flow cytometry for cell cycle studies, or by chemical fixation for microscopy techniques to measure parameters of the nucleolus. Although the trend of the results was the same as those obtained in real microgravity on meristems (increased cell proliferation and decreased cell growth), we provide a technical discussion in the context of validation of proper conditions to achieve true cell levitation inside a levitating droplet. We conclude that the use of magnetic levitation as a simulated microgravity GBF for cell suspension cultures is not recommended.

Cyclin-dependent kinase inhibitor p21(Waf1): contemporary view on its role in senescence and oncogenesis.

PubMed

Romanov, V S; Pospelov, V A; Pospelova, T V

2012-06-01

p21(Waf1) was identified as a protein suppressing cyclin E/A-CDK2 activity and was originally considered as a negative regulator of the cell cycle and a tumor suppressor. It is now considered that p21(Waf1) has alternative functions, and the view of its role in cellular processes has begun to change. At present, p21(Waf1) is known to be involved in regulation of fundamental cellular programs: cell proliferation, differentiation, migration, senescence, and apoptosis. In fact, it not only exhibits antioncogenic, but also oncogenic properties. This review provides a contemporary understanding of the functions of p21(Waf1) depending on its intracellular localization. On one hand, when in the nucleus, it serves as a negative cell cycle regulator and tumor suppressor, in particular by participating in the launch of a senescence program. On the other hand, when p21(Waf1) is localized in the cytoplasm, it acts as an oncogene by regulating migration, apoptosis, and proliferation.

Metabolic cooperation between cancer and non-cancerous stromal cells is pivotal in cancer progression.

PubMed

Lopes-Coelho, Filipa; Gouveia-Fernandes, Sofia; Serpa, Jacinta

2018-02-01

The way cancer cells adapt to microenvironment is crucial for the success of carcinogenesis, and metabolic fitness is essential for a cancer cell to survive and proliferate in a certain organ/tissue. The metabolic remodeling in a tumor niche is endured not only by cancer cells but also by non-cancerous cells that share the same microenvironment. For this reason, tumor cells and stromal cells constitute a complex network of signal and organic compound transfer that supports cellular viability and proliferation. The intensive dual-address cooperation of all components of a tumor sustains disease progression and metastasis. Herein, we will detail the role of cancer-associated fibroblasts, cancer-associated adipocytes, and inflammatory cells, mainly monocytes/macrophages (tumor-associated macrophages), in the remodeling and metabolic adaptation of tumors.

Small extracellular vesicles secreted from senescent cells promote cancer cell proliferation through EphA2

PubMed Central

Takasugi, Masaki; Okada, Ryo; Takahashi, Akiko; Virya Chen, David; Watanabe, Sugiko; Hara, Eiji

2017-01-01

Cellular senescence prevents the proliferation of cells at risk for neoplastic transformation. However, the altered secretome of senescent cells can promote the growth of the surrounding cancer cells. Although extracellular vesicles (EVs) have emerged as new players in intercellular communication, their role in the function of senescent cell secretome has been largely unexplored. Here, we show that exosome-like small EVs (sEVs) are important mediators of the pro-tumorigenic function of senescent cells. sEV-associated EphA2 secreted from senescent cells binds to ephrin-A1, that is, highly expressed in several types of cancer cells and promotes cell proliferation through EphA2/ephrin-A1 reverse signalling. sEV sorting of EphA2 is increased in senescent cells because of its enhanced phosphorylation resulting from oxidative inactivation of PTP1B phosphatase. Our results demonstrate a novel mechanism of reactive oxygen species (ROS)-regulated cargo sorting into sEVs, which is critical for the potentially deleterious growth-promoting effect of the senescent cell secretome. PMID:28585531

Thyroid Hormone Induces Apoptosis in Primary Cell Cultures of Tadpole Intestine: Cell Type Specificity and Effects of Extracellular Matrix

PubMed Central

Su, Yuan; Shi, Yufang; Stolow, Melissa A.; Shi, Yun-Bo

1997-01-01

Thyroid hormone (T3 or 3,5,3′-triiodothyronine) plays a causative role during amphibian metamorphosis. To investigate how T3 induces some cells to die and others to proliferate and differentiate during this process, we have chosen the model system of intestinal remodeling, which involves apoptotic degeneration of larval epithelial cells and proliferation and differentiation of other cells, such as the fibroblasts and adult epithelial cells, to form the adult intestine. We have established in vitro culture conditions for intestinal epithelial cells and fibroblasts. With this system, we show that T3 can enhance the proliferation of both cell types. However, T3 also concurrently induces larval epithelial apoptosis, which can be inhibited by the extracellular matrix (ECM). Our studies with known inhibitors of mammalian cell death reveal both similarities and differences between amphibian and mammalian cell death. These, together with gene expression analysis, reveal that T3 appears to simultaneously induce different pathways that lead to specific gene regulation, proliferation, and apoptotic degeneration of the epithelial cells. Thus, our data provide an important molecular and cellular basis for the differential responses of different cell types to the endogenous T3 during metamorphosis and support a role of ECM during frog metamorphosis. PMID:9396758

Feasibility of obtaining breast epithelial cells from healthy women for studies of cellular proliferation.

PubMed

Miller, N A; Thomas, M; Martin, L J; Hedley, D W; Michal, S; Boyd, N F

1997-05-01

Increased dietary fat intake and rate of breast epithelial cell proliferation have each been associated with the development of breast cancer. The goal of this study was to measure the effect of a low fat, high carbohydrate diet on the rate of breast epithelial cell proliferation in women at high risk for breast cancer. Women were recruited from the intervention and control groups of a randomized low fat dietary intervention trial, breast epithelial cells were obtained by fine needle aspiration, and cell proliferation was assessed in these samples using immunofluorescent detection of Ki-67 and PCNA. The effects of needle size and study group on cell yield and cytologic features of the cells were also examined. Fifty three women (20 in the intervention group and 33 in the control group) underwent the biopsy procedure. Slides from 38 subjects were stained for Ki-67 and from 14 subjects for PCNA. No cell proliferation (fluorescence) was detected for either Ki-67 or PCNA in any of the slides. Epithelial cell yield and number of stromal fragments were greater with a larger needle size. Numbers of stromal fragments and bipolar naked nuclei were greater in the low fat as compared to the control group but no differences in epithelial cell yield were observed between the two groups. This study confirms that fine needle aspiration biopsy is a feasible method of obtaining epithelial cells from women without discrete breast masses, but suggests that cell proliferation cannot be assessed using Ki-67 and PCNA in such samples.

Downregulation of p16(ink4a) inhibits cell proliferation and induces G1 cell cycle arrest in cervical cancer cells.

PubMed

Zhang, Chu-Yue; Bao, Wei; Wang, Li-Hua

2014-06-01

Studies have suggested that p16(ink4a) may be a surrogate biomarker for the diagnosis of cervical cancer; however, the function of p16(ink4a) in human cervical cancer cells remains largely unknown. Therefore, in this study, we aimed to investigate the role of p16(ink4a) in human cervical cancer cells. Immunocytochemistry was used to examine invasive squamous cell carcinoma and its precancerous lesions. p16(ink4a)-siRNA was transfected into SiHa and HeLa cells to deplete its expression. The cellular levels of p16(ink4a) mRNA and protein were detected by qRT-PCR and western blot analysis. Proliferation rates were assessed by methyl thiazolyl tetrazolium (MTT) and plate colony formation assays. Cellular migration and invasion ability were assessed by a wound healing assay and Transwell assay. Cellular apoptosis and the cell cycle were measured by flow cytometry. The protein levels of retinoblastoma (Rb), phosphorylated Rb (phospho-Rb), cyclin D1 and caspase-3 were determined by western blot analysis. The results revealed that p16(ink4a) was overexpressed in the cervical cancer and precancerous lesions (P<0.05). The downregulation of p16(ink4a) in the SiHa and HeLa cells inhibited their proliferation, migration and invasion. In the SiHa cells, p16(ink4a)-siRNA also induced G1 cell cycle arrest and apoptosis. Western blot analysis revealed that the downregulation of p16(ink4a) in the SiHa cells markedly induced caspase-3 activation and decreased cyclin D1 expression. These data suggest that the overexpression of p16(ink4a) appears to be useful in monitoring cervical precancerous lesions, which supports that the hypothesis that p16(ink4a) is a surrogate biomarker for the diagnosis of cervical cancer. The therapeutic targeting of overexpressed p16(ink4a) in the p16(ink4a)-cyclin-Rb pathway may be a useful strategy in the treatment of cervical cancer.

In vitro analysis of equine, bone marrow-derived mesenchymal stem cells demonstrates differences within age- and gender-matched horses.

PubMed

Carter-Arnold, J L; Neilsen, N L; Amelse, L L; Odoi, A; Dhar, M S

2014-09-01

Stem cell therapies are used routinely in equine practice. Most published reports characterise stem cells derived from younger horses; however, middle-aged horses are often in athletic performance, and experience degenerative medical conditions. Thus, mesenchymal stem cells (MSCs) from this group should be investigated. To describe differences in in vitro adherence, proliferation and potential for differentiation of equine bone marrow-derived MSCs (equine BMMSCs) harvested from middle-aged (10-13 years old) female donors. Descriptive study of stem cell characteristics. Equine BMMSCs from 6 horses were cultured in vitro and evaluated for viability, proliferation, osteogenesis, chondrogenesis, adipogenesis, cluster-of-differentiation markers and gene expression. Equine BMMSCs from all 6 donors demonstrated fibroblastic, cellular morphology, adherence to plastic and expression of cluster-of-differentiation markers. They varied in their rate of proliferation and trilineage differentiation. The equine BMMSCs of one of 6 donors demonstrated a higher rate of proliferation, enhanced ability for cell passaging and a more robust in vitro differentiation. Comparatively, equine BMMSCs from 2 donors demonstrated a lower rate of proliferation and lack of osteogenic and chondrogenic differentiation. The results of this study confirm that donor-to-donor variation in equine BMMSCs exists and this variation can be documented using in vitro assays. Subjective assessment suggests that the rate of proliferation tends to correlate with differentiation potential. © 2013 EVJ Ltd.

The molecular responses of skeletal muscle satellite cells to continuous expression of IGF-1: implications for the rescue of induced muscular atrophy in aged rats

NASA Technical Reports Server (NTRS)

Chakravarthy, M. V.; Booth, F. W.; Spangenburg, E. E.

2001-01-01

Approximately 50% of humans older than 85 years have physical frailty due to weak skeletal muscles. This indicates a need for determining mechanisms to combat this problem. A critical cellular factor for postnatal muscle growth is a population of myogenic precursor cells called satellite cells. Given the complex process of sarcopenia, it has been postulated that, at some point in this process, a limited satellite cell proliferation potential could become rate-limiting to the regrowth of old muscles. It is conceivable that if satellite cell proliferative capacity can be maintained or enhanced with advanced age, sarcopenia could potentially be delayed or prevented. Therefore, the purposes of this paper are to describe whether IGF-I can prevent muscular atrophy induced by repeated cycles of hindlimb immobilization, increase the in vitro proliferation in satellite cells from these muscles and, if so, the molecular mechanisms by which IGF-I mediates this increased proliferation. Our results provide evidence that IGF-I can enhance aged muscle regrowth possibly through increased satellite cell proliferation. The results also suggest that IGF-I enhances satellite cell proliferation by decreasing the cell cycle inhibitor, p27Kip1, through the PI3'-K/Akt pathway. These data provide molecular evidence for IGF-I's rescue effect upon aging-associated skeletal muscle atrophy.

E2F mediates enhanced alternative polyadenylation in proliferation

PubMed Central

2012-01-01

Background The majority of mammalian genes contain multiple poly(A) sites in their 3' UTRs. Alternative cleavage and polyadenylation are emerging as an important layer of gene regulation as they generate transcript isoforms that differ in their 3' UTRs, thereby modulating genes' response to 3' UTR-mediated regulation. Enhanced cleavage at 3' UTR proximal poly(A) sites resulting in global 3' UTR shortening was recently linked to proliferation and cancer. However, mechanisms that regulate this enhanced alternative polyadenylation are unknown. Results Here, we explored, on a transcriptome-wide scale, alternative polyadenylation events associated with cellular proliferation and neoplastic transformation. We applied a deep-sequencing technique for identification and quantification of poly(A) sites to two human cellular models, each examined under proliferative, arrested and transformed states. In both cell systems we observed global 3' UTR shortening associated with proliferation, a link that was markedly stronger than the association with transformation. Furthermore, we found that proliferation is also associated with enhanced cleavage at intronic poly(A) sites. Last, we found that the expression level of the set of genes that encode for 3'-end processing proteins is globally elevated in proliferation, and that E2F transcription factors contribute to this regulation. Conclusions Our results comprehensively identify alternative polyadenylation events associated with cellular proliferation and transformation, and demonstrate that the enhanced alternative polyadenylation in proliferative conditions results not only in global 3' UTR shortening but also in enhanced premature cleavage in introns. Our results also indicate that E2F-mediated co-transcriptional regulation of 3'-end processing genes is one of the mechanisms that links enhanced alternative polyadenylation to proliferation. PMID:22747694

Distinct Responses of Stem Cells to Telomere Uncapping-A Potential Strategy to Improve the Safety of Cell Therapy.

PubMed

Liu, Chang Ching; Ma, Dong Liang; Yan, Ting-Dong; Fan, XiuBo; Poon, Zhiyong; Poon, Lai-Fong; Goh, Su-Ann; Rozen, Steve G; Hwang, William Ying Khee; Tergaonkar, Vinay; Tan, Patrick; Ghosh, Sujoy; Virshup, David M; Goh, Eyleen L K; Li, Shang

2016-10-01

In most human somatic cells, the lack of telomerase activity results in progressive telomere shortening during each cell division. Eventually, DNA damage responses triggered by critically short telomeres induce an irreversible cell cycle arrest termed replicative senescence. However, the cellular responses of human pluripotent stem cells to telomere uncapping remain unknown. We generated telomerase knockout human embryonic stem (ES) cells through gene targeting. Telomerase inactivation in ES cells results in progressive telomere shortening. Telomere DNA damage in ES cells and neural progenitor cells induces rapid apoptosis when telomeres are uncapped, in contrast to fibroblast cells that enter a state of replicative senescence. Significantly, telomerase inactivation limits the proliferation capacity of human ES cells without affecting their pluripotency. By targeting telomerase activity, we can functionally separate the two unique properties of human pluripotent stem cells, namely unlimited self-renewal and pluripotency. We show that the potential of ES cells to form teratomas in vivo is dictated by their telomere length. By controlling telomere length of ES cells through telomerase inactivation, we can inhibit teratoma formation and potentially improve the safety of cell therapies involving terminally differentiated cells as well as specific progenitor cells that do not require sustained cellular proliferation in vivo, and thus sustained telomerase activity. Stem Cells 2016;34:2471-2484. © 2016 AlphaMed Press.

Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor

PubMed Central

Krall, Abigail S.; Xu, Shili; Graeber, Thomas G.; Braas, Daniel; Christofk, Heather R.

2016-01-01

Cellular amino acid uptake is critical for mTOR complex 1 (mTORC1) activation and cell proliferation. However, the regulation of amino acid uptake is not well-understood. Here we describe a role for asparagine as an amino acid exchange factor: intracellular asparagine exchanges with extracellular amino acids. Through asparagine synthetase knockdown and altering of media asparagine concentrations, we show that intracellular asparagine levels regulate uptake of amino acids, especially serine, arginine and histidine. Through its exchange factor role, asparagine regulates mTORC1 activity and protein synthesis. In addition, we show that asparagine regulation of serine uptake influences serine metabolism and nucleotide synthesis, suggesting that asparagine is involved in coordinating protein and nucleotide synthesis. Finally, we show that maintenance of intracellular asparagine levels is critical for cancer cell growth. Collectively, our results indicate that asparagine is an important regulator of cancer cell amino acid homeostasis, anabolic metabolism and proliferation. PMID:27126896

Influence of inhibitors of serotonin uptake on intestinal epithelium and colorectal carcinomas.

PubMed Central

Tutton, P. J.; Barkla, D. H.

1982-01-01

Previous studies have shown that in certain tissues, including colonic carcinomas, cell proliferation may be promoted by serotonin, and indirect evidence suggests that the effects of this amine on colonic tumours involves a cellular-uptake mechanism. In the present study, two specific inhibitors of serotonin uptake, Citalopram and Fluoxetine, are examined for their effects on cell proliferation and tumour growth. Each of the agents was found to suppress cell division in dimethylhydrazine-induced colonic tumours in rats, and to retard the growth of 2 out of 3 lines of human colonic tumours propagated as xenografts in immune-deprived mice. PMID:6983886

Down-regulation of TCF21 by hypermethylation induces cell proliferation, migration and invasion in colorectal cancer

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

Dai, Youyi; Duan, Huaxin; The First Affiliated Hospital of Hunan Normal University

Epigenetic alteration induced loss function of the transcription factor 21 (TCF21) has been associated with different types of human cancers. However, the epigenetic regulation and molecular functions of TCF21 in colorectal cancer (CRC) remain unknown. In this study, TCF21 expression levels and methylation status of its promoter region in CRC cell lines (n = 5) and CRC tissues (n = 151) as well as normal colorectal mucosa (n = 30) were assessed by RTq-PCR and methylation analysis (methylation specific PCR, MSP and bisulfite sequencing PCR, BSP), respectively. The cellular functions of TCF21 on CRC cell proliferation, apoptosis, invasion and migration were investigated in vitro. Our data revealedmore » that TCF21 was frequently silenced by promoter hypermethylation in both tested CRC cell lines and primary CRC, and correlation analysis between methylation status and clinicopathologic parameters found that TCF21 methylation was significantly correlated with lymph node invasion (P = 0.013), while no significant correlation was found in other parameters. In addition, demethylation treatment resulted in re-expression of TCF21 in CRC cell lines, and cellular function experiments revealed that restoration of TCF21 inhibited CRC cell proliferation, promoted apoptosis and suppressed cell invasion and migration, suggesting that TCF21 may function as a tumor suppressor gene, which is downregulated through promoter hypermethylation in CRC development. - Highlights: • TCF21 was frequently silenced by promoter DNA methylation in CRC cells. • TCF21 was frequently methylated in primary CRC and significantly correlated with metastasis. • Restoration of TCF21 promotes cell apoptosis of CRC cells. • Restoration of TCF21 inhibits cell invasion and migration of CRC cells.« less

Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

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

Kelly, Catriona, E-mail: catriona.kelly@qub.ac.uk; Flatt, Peter R.; McClenaghan, Neville H.

2010-08-20

Research highlights: {yields} TGP52 cells display enhanced functionality in pseudoislet form. {yields} Somatostatin content was reduced, but secretion increased in high glucose conditions. {yields} Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mMmore » glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.« less

Early effects of altered gravity environments on plant cell growth and cell proliferation: Characterization of morphofunctional nucleolar types in an Arabidopsis cell culture system

NASA Astrophysics Data System (ADS)

Manzano, Ana Isabel; Herranz, Raul; Manzano, Aránzazu; Van Loon, Jack; Medina, Francisco Javier

2016-02-01

Changes in the cell growth rate of an in vitro cellular system in Arabidopsis thaliana induced by short exposure to an altered gravity environment have been estimated by a novel approach. The method consisted of defining three structural nucleolar types which are easy and reliable indicators of the ribosome biogenesis activity and, consequently, of protein biosynthesis, a parameter strictly correlated to cell growth in this cellular system. The relative abundance of each nucleolar type was statistically assessed in different conditions of gravity. Samples exposed to simulated microgravity for 200 min showed a significant decrease in nucleolar activity compared to 1g controls, whereas samples exposed to hypergravity (2g) for the same period showed nucleolar activity slightly increased,. These effects could be considered as an early cellular response to the environmental alteration, given the short duration of the treatment. The functional significance of the structural data was validated by a combination of several different well-known parameters, using microscopical, flow cytometry, qPCR and proteomic approaches, which showed that the decreased cell growth rate was decoupled from an increased cell proliferation rate under simulated microgravity, and the opposite trend was observed under hypergravity. Actually, not all parameters tested showed the same quantitative changes, indicating that the response to the environmental alteration is time-dependent. These results are in agreement with previous observations in root meristematic cells and they show the ability of plant cells to produce a response to gravity changes, independently of their integration into plant organs.

Cell Patterns Emerge from Coupled Chemical and Physical Fields with Cell Proliferation Dynamics: The Arabidopsis thaliana Root as a Study System

PubMed Central

Barrio, Rafael A.; Romero-Arias, José Roberto; Noguez, Marco A.; Azpeitia, Eugenio; Ortiz-Gutiérrez, Elizabeth; Hernández-Hernández, Valeria; Cortes-Poza, Yuriria; Álvarez-Buylla, Elena R.

2013-01-01

A central issue in developmental biology is to uncover the mechanisms by which stem cells maintain their capacity to regenerate, yet at the same time produce daughter cells that differentiate and attain their ultimate fate as a functional part of a tissue or an organ. In this paper we propose that, during development, cells within growing organs obtain positional information from a macroscopic physical field that is produced in space while cells are proliferating. This dynamical interaction triggers and responds to chemical and genetic processes that are specific to each biological system. We chose the root apical meristem of Arabidopsis thaliana to develop our dynamical model because this system is well studied at the molecular, genetic and cellular levels and has the key traits of multicellular stem-cell niches. We built a dynamical model that couples fundamental molecular mechanisms of the cell cycle to a tension physical field and to auxin dynamics, both of which are known to play a role in root development. We perform extensive numerical calculations that allow for quantitative comparison with experimental measurements that consider the cellular patterns at the root tip. Our model recovers, as an emergent pattern, the transition from proliferative to transition and elongation domains, characteristic of stem-cell niches in multicellular organisms. In addition, we successfully predict altered cellular patterns that are expected under various applied auxin treatments or modified physical growth conditions. Our modeling platform may be extended to explicitly consider gene regulatory networks or to treat other developmental systems. PMID:23658505

Mitomycin C in dacryocystorhinostomy: the search for the right concentration and duration--a fundamental study on human nasal mucosa fibroblasts.

PubMed

Ali, Mohammad Javed; Mariappan, Indumathi; Maddileti, Savitri; Ali, Md Hasnat; Naik, Milind N

2013-01-01

To establish primary cultures of human nasal mucosal fibroblasts (HNMFs) and to test the effect of varying concentrations of mitomycin C (MMC) and treatment durations on cellular proliferation and viability of the fibroblasts. Laboratory investigation. Nasal mucosa harvested from patients undergoing a dacryocystorhinostomy was used to establish primary cultures by explant culture method. Cells were expanded and frozen at every passage, and passage 3 cells were used for further experiments. The cells were then treated with different concentrations of mitomycin C (0.1-0.5 mg/ml) for different time periods (3, 5, and 10 minutes). Cell viability was checked by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cellular proliferation index was determined with bromodeoxyuridine immunostaining. Apoptotic index was measured using annexin A5 affinity assay, propidium iodide staining, and 4',6-diamidino-2-phenylindole counterstaining. The actin cytoskeletons of fibroblasts were studied using phalloidin staining. The doubling time of cultured HNMFs is approximately 24 hours. Similarly, 0.4 mg/ml beyond 5 minutes and 0.5 mg/ml concentration at all time points were lethal and caused extensive cell death when compared with controls. A concentration of 0.2 mg/ml for 3 minutes of exposure prevented cell proliferation of HNMF cells by inducing cell cycle arrest, without causing extensive apoptosis. The minimum effective concentration appears to be 0.2 mg/ml for 3 minutes. This in vitro study could be the starting point for further clinical and histopathologic studies to validate its clinical usefulness.

Epidermal Growth Factor Signaling towards Proliferation: Modeling and Logic Inference Using Forward and Backward Search

PubMed Central

Riesco, Adrián; Santos-Buitrago, Beatriz; De Las Rivas, Javier; Knapp, Merrill; Talcott, Carolyn

2017-01-01

In biological systems, pathways define complex interaction networks where multiple molecular elements are involved in a series of controlled reactions producing responses to specific biomolecular signals. These biosystems are dynamic and there is a need for mathematical and computational methods able to analyze the symbolic elements and the interactions between them and produce adequate readouts of such systems. In this work, we use rewriting logic to analyze the cellular signaling of epidermal growth factor (EGF) and its cell surface receptor (EGFR) in order to induce cellular proliferation. Signaling is initiated by binding the ligand protein EGF to the membrane-bound receptor EGFR so as to trigger a reactions path which have several linked elements through the cell from the membrane till the nucleus. We present two different types of search for analyzing the EGF/proliferation system with the help of Pathway Logic tool, which provides a knowledge-based development environment to carry out the modeling of the signaling. The first one is a standard (forward) search. The second one is a novel approach based on narrowing, which allows us to trace backwards the causes of a given final state. The analysis allows the identification of critical elements that have to be activated to provoke proliferation. PMID:28191459

Epidermal Growth Factor Signaling towards Proliferation: Modeling and Logic Inference Using Forward and Backward Search.

PubMed

Riesco, Adrián; Santos-Buitrago, Beatriz; De Las Rivas, Javier; Knapp, Merrill; Santos-García, Gustavo; Talcott, Carolyn

2017-01-01

In biological systems, pathways define complex interaction networks where multiple molecular elements are involved in a series of controlled reactions producing responses to specific biomolecular signals. These biosystems are dynamic and there is a need for mathematical and computational methods able to analyze the symbolic elements and the interactions between them and produce adequate readouts of such systems. In this work, we use rewriting logic to analyze the cellular signaling of epidermal growth factor (EGF) and its cell surface receptor (EGFR) in order to induce cellular proliferation. Signaling is initiated by binding the ligand protein EGF to the membrane-bound receptor EGFR so as to trigger a reactions path which have several linked elements through the cell from the membrane till the nucleus. We present two different types of search for analyzing the EGF/proliferation system with the help of Pathway Logic tool, which provides a knowledge-based development environment to carry out the modeling of the signaling. The first one is a standard (forward) search. The second one is a novel approach based on narrowing , which allows us to trace backwards the causes of a given final state. The analysis allows the identification of critical elements that have to be activated to provoke proliferation.

Rapid construction of mechanically- confined multi- cellular structures using dendrimeric intercellular linker.

PubMed

Mo, Xuejun; Li, Qiushi; Yi Lui, Lena Wai; Zheng, Baixue; Kang, Chiang Huen; Nugraha, Bramasta; Yue, Zhilian; Jia, Rui Rui; Fu, Hong Xia; Choudhury, Deepak; Arooz, Talha; Yan, Jie; Lim, Chwee Teck; Shen, Shali; Hong Tan, Choon; Yu, Hanry

2010-10-01

Tissue constructs that mimic the in vivo cell-cell and cell-matrix interactions are especially useful for applications involving the cell- dense and matrix- poor internal organs. Rapid and precise arrangement of cells into functional tissue constructs remains a challenge in tissue engineering. We demonstrate rapid assembly of C3A cells into multi- cell structures using a dendrimeric intercellular linker. The linker is composed of oleyl- polyethylene glycol (PEG) derivatives conjugated to a 16 arms- polypropylenimine hexadecaamine (DAB) dendrimer. The positively charged multivalent dendrimer concentrates the linker onto the negatively charged cell surface to facilitate efficient insertion of the hydrophobic oleyl groups into the cellular membrane. Bringing linker- treated cells into close proximity to each other via mechanical means such as centrifugation and micromanipulation enables their rapid assembly into multi- cellular structures within minutes. The cells exhibit high levels of viability, proliferation, three- dimensional (3D) cell morphology and other functions in the constructs. We constructed defined multi- cellular structures such as rings, sheets or branching rods that can serve as potential tissue building blocks to be further assembled into complex 3D tissue constructs for biomedical applications. 2010 Elsevier Ltd. All rights reserved.

Quercetin prevents protein nitration and glycolytic block of proliferation in hydrogen peroxide insulted cultured neuronal precursor cells (NPCs): Implications on CNS regeneration.

PubMed

Sajad, Mir; Zargan, Jamil; Zargar, Mohammad Afzal; Sharma, Jyoti; Umar, Sadiq; Arora, Rajesh; Khan, Haider A

2013-05-01

Survival along with optimal proliferation of neuronal precursors determines the outcomes of the endogenous cellular repair in CNS. Cellular-oxidation based cell death has been described in several neurodegenerative disorders. Therefore, this study was aimed at the identification of the potent targets of oxidative damage to the neuronal precursors and its effective prevention by a natural flavonoid, Quercetin. Neuronal precursor cells (NPCs), Nestin+ and GFAP (Glial fibrillary acidic protein)+ were isolated and cultured from adult rat SVZ (subventricular zone). These cells were challenged with a single dose of H2O2 (50μM) and/or pre-treated with different concentrations of Quercetin. H2O2 severely limited the cellular viability and expansion of the neurospheres. Cellular-oxidation studies revealed reduction in glutathione dependent redox buffering along with depletion of enzymatic cellular antioxidants that might potentiate the nitrite (NO2(-)) and superoxide anion (O2(-)) mediated peroxynitrite (ONOO(-)) formation and irreversible protein nitration. We identified depleted PK-M2 (M2 isoform of pyruvate kinase) activity and apoptosis of NPCs revealed by the genomic DNA fragmentation and elevated PARP (poly ADP ribose polymerase) activity along with increased Caspase activity initiated by severely depolarised mitochondrial membranes. However, the pre-treatment of Quercetin in a dose-response manner prevented these changes and restored the expansion of neurospheres preferably by neutralizing the oxidative conditions and thereby reducing peroxynitrite formation, protein nitration and PK-M2 depletion. Our results unravel the potential interactions of oxidative environment and respiration in the survival and activation of precursors and offer a promise shown by a natural flavonoid in the protective strategy for neuronal precursors of adult brain. Copyright © 2013 Elsevier Inc. All rights reserved.

Parasagittal solitary fibrous tumor resembling hemangiopericytoma.

PubMed

Shidoh, Satoka; Yoshida, Kazunari; Takahashi, Satoshi; Mikami, Shuji; Mukai, Makio; Kawase, Takeshi

2010-04-01

Solitary fibrous tumor (SFT) is a rare mesenchymal tumor in the central nervous system, and the clinical behavior of this tumor is similar to that of meningioma. We report the case of a Japanese woman with parasagittal SFT that resembled hemangiopericytoma (HPC). Histological examination revealed that the tumor was highly cellular, with cells containing oval- or spindle-shaped nuclei arranged in sheets or a pattern-less growth mode. Focal vascular proliferation was also observed. Some areas showed intercellular stroma containing remarkable eosinophilic collagens. Tumor cells showed a strong immunoreactivity for CD34 but were negative for S-100 protein and epithelial membrane antigen. MIB-1 labeling index of the tumor was 6.6%. Owing to the high cellularity, high MIB-1 labeling index, and focal vascular proliferation, it was difficult to distinguish this lesion from HPC. However, the tumor was finally diagnosed as SFT on the basis of the strong immunostaining for CD34 and absence of pericellular reticulin.

Vitamin D protects endothelial cells from irradiation-induced senescence and apoptosis by modulating MAPK/SirT1 axis.

PubMed

Marampon, F; Gravina, G L; Festuccia, C; Popov, V M; Colapietro, E A; Sanità, P; Musio, D; De Felice, F; Lenzi, A; Jannini, E A; Di Cesare, E; Tombolini, V

2016-04-01

Radiotherapy toxicity is related to oxidative stress-mediated endothelial dysfunction. Here, we investigated on radioprotective properties of Vitamin D (Vit.D) on human endothelial cells (HUVEC). HUVEC, pre-treated with Vit.D, were exposed to ionizing radiation (IR): ROS production, cellular viability, apoptosis, senescence and western blot for protein detection were performed. The role of MAPKs pathway was investigated by using U0126 (10 μM) MEKs/ERKs-, SB203580 (2.5 μM) p38-inhibitor or by over/expressing MKK6 p38-upstream activator. Vit.D reduced IR-induced ROS production protecting proliferating and quiescent HUVEC from cellular apoptosis or senescence, respectively, by regulating MAPKs pathways. In proliferating HUVEC, Vit.D prevented IR-induced apoptosis by activating ERKs while in quiescent HUVEC counteracted IR-induced senescence by inhibiting the p38-IR-induced activation. MEKs&ERKs inhibition in proliferating or MKK6/mediated p38 activation in quiescent HUVEC, respectively, reverted anti-apoptotic or anti-senescent Vit.D properties. SirT1 protein expression levels were up-regulated by Vit.D. ERKs inhibition blocked Vit.D-induced SirT1 protein up-regulation in proliferating cells. In quiescent HUVEC cells, p38 inhibition counteracted the IR-induced SirT1 protein down-regulation, while MKK6 transfection abrogated the Vit.D positive effects on SirT1 protein levels after irradiation. SirT1 inhibition by sirtinol blocked the Vit.D radioprotective effects. Vit.D protects HUVEC from IR induced/oxidative stress by positively regulating the MAPKs/SirT1 axis.

Synthesis and SAR of novel imidazoquinoxaline-based Lck inhibitors: improvement of cell potency.

PubMed

Chen, Ping; Iwanowicz, Edwin J; Norris, Derek; Gu, Henry H; Lin, James; Moquin, Robert V; Das, Jagabandhu; Wityak, John; Spergel, Steven H; de Fex, Henry; Pang, Suhong; Pitt, Sydney; Shen, Ding Ren; Schieven, Gary L; Barrish, Joel C

2002-11-04

A series of anilino(imidazoquinoxaline) analogues bearing solubilizing side chains at the 6- and 7-positions of the fused phenyl ring has been prepared and evaluated for inhibition against Lck enzyme and of T-cell proliferation. Significant improvement of the cellular activity was achieved over the initial lead, compound 2.

Malignancy without immortality? Cellular immortalization as a possible late event in melanoma progression

PubMed Central

Soo, Julia K; MacKenzie Ross, Alastair D; Kallenberg, David M; Milagre, Carla; Heung Chong, W; Chow, Jade; Hill, Lucy; Hoare, Stacey; Collinson, Rebecca S; Hossain, Mehnaz; Keith, W Nicol; Marais, Richard; Bennett, Dorothy C

2011-01-01

Cell senescence is a permanent growth arrest following extended proliferation. Cultured cancer cells including metastatic melanoma cells often appear immortal (proliferate indefinitely), while uncultured benign nevi (moles) show senescence markers. Here, with new explantation methods, we investigated which classes of primary pigmented lesions are typically immortal. Nevi yielded a few proliferating cells, consistent with most nevus cells being senescent. No nevus culture (0/28) appeared immortal. Some thin and thick melanoma cultures proved immortal under these conditions, but surprisingly few (4/37). All arrested cultures displayed three senescence markers in some cells: β-galactosidase, nuclear p16, and heterochromatic foci/aggregates. However, melanoma cultures also showed features of telomeric crisis (arrest because of ultrashort telomeres). Moreover, crisis markers including anaphase bridges were frequent in uncultured vertical growth-phase (VGP) melanomas. Conversely, all immortal melanoma cultures expressed telomerase reverse transcriptase and telomerase, showing aneuploidy. The findings suggest that primary melanomas are typically precrisis, with immortalization/telomere maintenance as a late event. PMID:21418545

Osteoblasts Growth Behaviour on Bio-Based Calcium Carbonate Aragonite Nanocrystal

PubMed Central

Zakaria, Zuki Abu Bakar

2014-01-01

Calcium carbonate (CaCO3) nanocrystals derived from cockle shells emerge to present a good concert in bone tissue engineering because of their potential to mimic the composition, structure, and properties of native bone. The aim of this study was to evaluate the biological response of CaCO3 nanocrystals on hFOB 1.19 and MC3T3 E-1 osteoblast cells in vitro. Cell viability and proliferation were assessed by MTT and BrdU assays, and LDH was measured to determine the effect of CaCO3 nanocrystals on cell membrane integrity. Cellular morphology was examined by SEM and fluorescence microscopy. The results showed that CaCO3 nanocrystals had no toxic effects to some extent. Cell proliferation, alkaline phosphatase activity, and protein synthesis were enhanced by the nanocrystals when compared to the control. Cellular interactions were improved, as indicated by SEM and fluorescent microscopy. The production of VEGF and TGF-1 was also affected by the CaCO3 nanocrystals. Therefore, bio-based CaCO3 nanocrystals were shown to stimulate osteoblast differentiation and improve the osteointegration process. PMID:24734228

Transdifferentiation and Proliferation in Two Distinct Hemocyte Lineages in Drosophila melanogaster Larvae after Wasp Infection

PubMed Central

Ihalainen, Teemu O.; Vanha-aho, Leena-Maija; Andó, István; Rämet, Mika

2016-01-01

Cellular immune responses require the generation and recruitment of diverse blood cell types that recognize and kill pathogens. In Drosophila melanogaster larvae, immune-inducible lamellocytes participate in recognizing and killing parasitoid wasp eggs. However, the sequence of events required for lamellocyte generation remains controversial. To study the cellular immune system, we developed a flow cytometry approach using in vivo reporters for lamellocytes as well as for plasmatocytes, the main hemocyte type in healthy larvae. We found that two different blood cell lineages, the plasmatocyte and lamellocyte lineages, contribute to the generation of lamellocytes in a demand-adapted hematopoietic process. Plasmatocytes transdifferentiate into lamellocyte-like cells in situ directly on the wasp egg. In parallel, a novel population of infection-induced cells, which we named lamelloblasts, appears in the circulation. Lamelloblasts proliferate vigorously and develop into the major class of circulating lamellocytes. Our data indicate that lamellocyte differentiation upon wasp parasitism is a plastic and dynamic process. Flow cytometry with in vivo hemocyte reporters can be used to study this phenomenon in detail. PMID:27414410

Transdifferentiation and Proliferation in Two Distinct Hemocyte Lineages in Drosophila melanogaster Larvae after Wasp Infection.

PubMed

Anderl, Ines; Vesala, Laura; Ihalainen, Teemu O; Vanha-Aho, Leena-Maija; Andó, István; Rämet, Mika; Hultmark, Dan

2016-07-01

Cellular immune responses require the generation and recruitment of diverse blood cell types that recognize and kill pathogens. In Drosophila melanogaster larvae, immune-inducible lamellocytes participate in recognizing and killing parasitoid wasp eggs. However, the sequence of events required for lamellocyte generation remains controversial. To study the cellular immune system, we developed a flow cytometry approach using in vivo reporters for lamellocytes as well as for plasmatocytes, the main hemocyte type in healthy larvae. We found that two different blood cell lineages, the plasmatocyte and lamellocyte lineages, contribute to the generation of lamellocytes in a demand-adapted hematopoietic process. Plasmatocytes transdifferentiate into lamellocyte-like cells in situ directly on the wasp egg. In parallel, a novel population of infection-induced cells, which we named lamelloblasts, appears in the circulation. Lamelloblasts proliferate vigorously and develop into the major class of circulating lamellocytes. Our data indicate that lamellocyte differentiation upon wasp parasitism is a plastic and dynamic process. Flow cytometry with in vivo hemocyte reporters can be used to study this phenomenon in detail.

Irradiation at 636 nm positively affects diabetic wounded and hypoxic cells in vitro.

PubMed

Sekhejane, Palesa R; Houreld, Nicolette N; Abrahamse, Heidi

2011-08-01

This study investigated the effect of low-intensity laser irradiation (LILI) on pro-inflammatory cytokines involved in wound healing processes in diabetes and hypoxia. Diabetes is associated with impaired wound healing and a prolonged inflammatory phase. Pro-inflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6 are elevated in diabetes. LILI has been reported to accelerate wound healing and decrease inflammatory cytokines. A human skin fibroblast cell line (WS1) was used in vitro. Cells were exposed to various insults, namely, wounding, and a diabetic or hypoxic environment. Experimental cells were exposed to an energy density of 5  J/cm(2) using a continuous wave 636-nm diode laser at an average power of 95  mW, an illuminated area of 9.05  cm(2), and an irradiance of 11 mW/cm(2) (irradiation time, 476  sec). The effect of laser irradiation on cytokine expression was examined at 1 or 24  h post-irradiation. Cellular morphology, viability, proliferation, and cytokine expression (IL-1β, IL-6, and TNF-α) were investigated. Translocation of nuclear factor-kappa B (NF-κB) was also determined. There was a higher rate of migration in irradiated wounded cultures, and irradiated hypoxic cells showed an improvement in cellular morphology. All cell models showed an increase in proliferation. Normal wounded cells showed a decrease in apoptosis, TNF-α, and IL-1β. Diabetic wounded cells showed an increase in viability and a decrease in apoptosis and IL-1β, whereas hypoxic cells showed an increase in viability and IL-6, and a decrease in apoptosis and TNF-α. NF-κB was translocated into the nucleus post-irradiation. Phototherapy resulted in hastened wound closure, increased proliferation, and normalization of cellular function. The decrease in the different pro-inflammatory cytokines and NF-κB translocation was model and time dependent. Overall, laser irradiation resulted in a reduction in inflammatory cytokines and directed cells into the cell survival pathway.

Knockdown of hTERT and concurrent treatment with interferon-gamma inhibited proliferation and invasion of human glioblastoma cell lines

PubMed Central

George, Joseph; Banik, Naren L.; Ray, Swapan K.

2011-01-01

Human telomerase reverse transcriptase (hTERT) is the catalytic component of telomerase that facilitates tumor cell invasion and proliferation. Telomerase and hTERT are remarkably upregulated in majority of cancers including glioblastoma. Interferon-gamma (IFN-γ) modulates several cellular activities including cell cycle and multiplication through transcriptional regulation. The present investigation was designed to unravel the molecular mechanisms of the inhibition of cell proliferation, migration, and invasion of human glioblastoma SNB-19 and LN-18 cell lines after knockdown of hTERT using a plasmid vector based siRNA and concurrent treatment with IFN-γ. We observed more than 80% inhibition of cell proliferation, migration, and invasion of both cell lines after the treatment with combination of hTERT siRNA and IFN-γ. Our studies also showed accumulation of apoptotic cells in subG1 phase and an increase in cell population in G0/G1 with a reduction in G2/M phase indicating cell cycle arrest in G0/G1 phase for apoptosis. Semiquantitative and real-time RT-PCR analyses demonstrated significant downregulation of c- Myc and upregulation of p21 Waf1 and p27 Kip1. Western blotting confirmed the downregulation of the molecules involved in cell proliferation, migration, and invasion and also showed upregulation of cell cycle inhibitors. In conclusion, our study demonstrated that knockdown of hTERT siRNA and concurrent treatment with IFN-γ effectively inhibited cell proliferation, migration, and invasion in glioblastoma cells through downregulation of the molecules involved in these processes and cell cycle inhibition. Therefore, the combination of hTERT siRNA and IFN-γ offers a potential therapeutic strategy for controlling growth of human glioblastoma cells. PMID:20394835

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

Kim, Yoo-Shin; Lee, Tae Hoon; O'Neill, Brian E., E-mail: BEOneill@houstonmethodist.org

Non-lethal hyperthermia is used clinically as adjuvant treatment to radiation, with mixed results. Denaturation of protein during hyperthermia treatment is expected to synergize with radiation damage to cause cell cycle arrest and apoptosis. Alternatively, hyperthermia is known to cause tissue level changes in blood flow, increasing the oxygenation and radiosensitivity of often hypoxic tumors. In this study, we elucidate a third possibility, that hyperthermia alters cellular adhesion and mechanotransduction, with particular impact on the cancer stem cell population. We demonstrate that cell heating results in a robust but temporary loss of cancer cell aggressiveness and metastatic potential in mouse models.more » In vitro, this heating results in a temporary loss in cell mobility, adhesion, and proliferation. Our hypothesis is that the loss of cellular adhesion results in suppression of cancer stem cells and loss of tumor virulence and metastatic potential. Our study suggests that the metastatic potential of cancer is particularly reduced by the effects of heat on cellular adhesion and mechanotransduction. If true, this could help explain both the successes and failures of clinical hyperthermia, and suggest ways to target treatments to those who would most benefit. - Highlights: • Non-lethal hyperthermia treatment of cancer cells is shown to cause a reduction in rates of tumor initiation and metastasis. • Dynamic imaging of cells during heat treatment shows temporary changes in cell shape, cell migration, and cell proliferation. • Loss of adhesion may lead to the observed effect, which may disproportionately impact the tumor initiating cell fraction. • Loss or suppression of the tumor initiating cell fraction results in the observed loss of metastatic potential in vivo. • This result may lead to new approaches to synergizing hyperthermia with surgery, radiation, and chemotherapy.« less

Stochastic cellular automata model of neurosphere growth: Roles of proliferative potential, contact inhibition, cell death, and phagocytosis.

PubMed

Sipahi, Rifat; Zupanc, Günther K H

2018-05-14

Neural stem and progenitor cells isolated from the central nervous system form, under specific culture conditions, clonal cell clusters known as neurospheres. The neurosphere assay has proven to be a powerful in vitro system to study the behavior of such cells and the development of their progeny. However, the theory of neurosphere growth has remained poorly understood. To overcome this limitation, we have, in the present paper, developed a cellular automata model, with which we examined the effects of proliferative potential, contact inhibition, cell death, and clearance of dead cells on growth rate, final size, and composition of neurospheres. Simulations based on this model indicated that the proliferative potential of the founder cell and its progenitors has a major influence on neurosphere size. On the other hand, contact inhibition of proliferation limits the final size, and reduces the growth rate, of neurospheres. The effect of this inhibition is particularly dramatic when a stem cell becomes encapsulated by differentiated or other non-proliferating cells, thereby suppressing any further mitotic division - despite the existing proliferative potential of the stem cell. Conversely, clearance of dead cells through phagocytosis is predicted to accelerate growth by reducing contact inhibition. A surprising prediction derived from our model is that cell death, while resulting in a decrease in growth rate and final size of neurospheres, increases the degree of differentiation of neurosphere cells. It is likely that the cellular automata model developed as part of the present investigation is applicable to the study of tissue growth in a wide range of systems. Copyright © 2018 Elsevier Ltd. All rights reserved.

Near-infrared laser increases MDPC-23 odontoblast-like cells proliferation by activating redox sensitive pathways.

PubMed

Rizzi, Manuela; Migliario, Mario; Rocchetti, Vincenzo; Tonello, Stelvio; Renò, Filippo

2016-11-01

Near infrared laser is known to induce biostimulatory effects, resulting in cell proliferation enhancement. Although such positive effect is widely exploited in various clinical applications, molecular mechanisms involved are still poorly understood. The aim of the study was to investigate the ability of laser stimulation to increase cell proliferation through an early activation of three redox sensitive pathways, namely Nrf-2, NF-κB and ERK in a rat odontoblast-like cell line (MDPC-23 cells). MDPC-23 cells were irradiated with different energy settings (0-50J, corresponding to 0-32.47J/cm 2 ) and cell proliferation was evaluated by cell counting. Nrf-2, NF-κB and ERK signaling pathways activation was investigated through Western blot analysis. Our results show that a single 25J laser stimulation is able to increase cell proliferation and that this effect could be increased by repeating the stimulation twice with a time lapse of 24h. Western blot experiments demonstrated that laser stimulation is able to induce an early activation response in intracellular signaling, with an overlapping time pattern between the three considered pathways. Results discussed in this paper reveal a complex mechanism underlying near-infrared induced increase in pre-odontoblasts proliferation, involving three survival pathways that can act both separately or through reciprocal crosstalk. In particular, data presented suggest an important role for ERK pathway that could act directly by stimulating cell proliferation but can also induce both Nrf-2 and NF-κB activation, acting as a critical cellular checkpoint in response to imbalanced redox state generated by a laser induced increase in ROS production. Copyright © 2016 Elsevier B.V. All rights reserved.

Overexpression of human Hsp27 inhibits serum-induced proliferation in airway smooth muscle myocytes and confers resistance to hydrogen peroxide cytotoxicity.

PubMed

Salinthone, Sonemany; Ba, Mariam; Hanson, Lisa; Martin, Jody L; Halayko, Andrew J; Gerthoffer, William T

2007-11-01

Airway smooth muscle (ASM) hypertrophy and hyperplasia are characteristics of asthma that lead to thickening of the airway wall and obstruction of airflow. Very little is known about mechanisms underlying ASM remodeling, but in vascular smooth muscle, it is known that progression of atherosclerosis depends on the balance of myocyte proliferation and cell death. Small heat shock protein 27 (Hsp27) is antiapoptotic in nonmuscle cells, but its role in ASM cell survival is unknown. Our hypothesis was that phosphorylation of Hsp27 may regulate airway remodeling by modifying proliferation, cell survival, or both. To test this hypothesis, adenoviral vectors were used to overexpress human Hsp27 in ASM cells. Cells were infected with empty vector (Ad5) or wild-type Hsp27 (AdHsp27 WT), and proliferation and death were assessed. Overexpressing Hsp27 WT caused a 50% reduction in serum-induced proliferation and increased cell survival after exposure to 100 microM hydrogen peroxide (H(2)O(2)) compared with mock-infected controls. Overexpression studies utilizing an S15A, S78A, and S82A non-phosphorylation mutant (AdHsp27 3A) and an S15D, S78D, and S82D pseudo-phosphorylation mutant (AdHsp27 3D) showed phosphorylation of Hsp27 was necessary for regulation of ASM proliferation, but not survival. Hsp27 provided protection against H(2)O(2)-induced cytotoxicity by upregulating cellular glutathione levels and preventing necrotic cell death, but not apoptotic cell death. The results support the notion that ASM cells can be stimulated to undergo proliferation and death and that Hsp27 may regulate these processes, thereby contributing to airway remodeling in asthmatics.

JNK Controls the Onset of Mitosis in Planarian Stem Cells and Triggers Apoptotic Cell Death Required for Regeneration and Remodeling

PubMed Central

Almuedo-Castillo, María; Crespo, Xenia; Seebeck, Florian; Bartscherer, Kerstin; Salò, Emili; Adell, Teresa

2014-01-01

Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun–NH2–kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal. PMID:24922054

Substance P enhances proliferation and paracrine potential of adipose-derived stem cells in vitro.

PubMed

Kim, Suna; Piao, Jiyuan; Son, Youngsook; Hong, Hyun Sook

2017-03-25

Stem cells have tremendous promise to treat intractable diseases. Notably, adipose-derived stem cells (ADSCs) are actively being investigated because of ease of sampling and high repopulation capacity in vitro. ADSCs can exert a therapeutic effect through differentiation and paracrine potential, and these actions have been proven in many diseases, including cutaneous and inflammatory diseases. Transplantation of ADSCs necessitates therapeutic quantities and thus, long term ex vivo culture of ADSCs. However, this procedure can impair the activity of ADSCs and provoke cellular senescence, leading to low efficacy in vivo. Accordingly, strategies to restore cellular activity and inhibit senescence of stem cells during ex vivo culture are needed for stem cell-based therapies. This study evaluated a potential supplementary role of Substance P (SP) in ADSC ex vivo culture. After confirming that the ADSC cell cycle was damaged by passage 6 (p6), ADSCs at p6 were cultured with SP, and their proliferation rates, cumulative cell numbers, cytokine profiles, and impact on T/endothelial cells were assessed. Long-term culture weakened proliferation ability and secretion of the cytokines, transforming growth factor-beta 1 (TGF-beta1), vascular endothelial growth factor (VEGF), and stromal cell derived factor-1 alpha (SDF-1alpha) in ADSCs. However, SP treatment reduced the population doubling time (PDT), enabling gain of a sufficient number of ADSCs at early passages. In addition, SP restored cytokine secretion, enhancing the ADSC-mediated paracrine effect on T cell and human umbilical vein endothelial cells (HUVECs). Taken together, these results suggest that SP can retain the therapeutic effect of ADSCs by elevating their proliferative and paracrine potential in ex vivo culture. Copyright © 2017 Elsevier Inc. All rights reserved.

Blocking Glycolytic Metabolism Increases Memory T Cells and Antitumor Function | Center for Cancer Research

Cancer.gov

CD8+ T cells are a major component of the cellular immune response, which is necessary to control a variety of bacterial and viral infections. CD8+ T cells also play a major role in the cell-mediated antitumor immune response. After encountering antigen, naïve CD8+ T cells undergo an extensive period of proliferation and expansion, and differentiate into effector cells and distinct memory T cell subsets. Preclinical studies using adoptive transfer of purified CD8+ T cells have shown that the ability of T cells to proliferate and survive for a long time after transfer is associated with effective antitumor and antiviral responses. Understanding how the formation of long-lived memory T cell subsets is controlled may enable development of more potent immunotherapies against cancer and infectious diseases.

The nucleolus—guardian of cellular homeostasis and genome integrity.

PubMed

Grummt, Ingrid

2013-12-01

All organisms sense and respond to conditions that stress their homeostasis by downregulating the synthesis of rRNA and ribosome biogenesis, thus designating the nucleolus as the central hub in coordinating the cellular stress response. One of the most intriguing roles of the nucleolus, long regarded as a mere ribosome-producing factory, is its participation in monitoring cellular stress signals and transmitting them to the RNA polymerase I (Pol I) transcription machinery. As rRNA synthesis is a most energy-consuming process, switching off transcription of rRNA genes is an effective way of saving the energy required to maintain cellular homeostasis during acute stress. The Pol I transcription machinery is the key convergence point that collects and integrates a vast array of information from cellular signaling cascades to regulate ribosome production which, in turn, guides cell growth and proliferation. This review focuses on the mechanisms that link cell physiology to rDNA silencing, a prerequisite for nucleolar integrity and cell survival.

MiR-188 Inhibits Glioma Cell Proliferation and Cell Cycle Progression through Targeting ß-catenin.

PubMed

Li, Nan; Shi, Hangyu; Zhang, Lu; Li, Xu; Gao, Lu; Zhang, Gang; Shi, Yongqiang; Guo, Shiwen

2017-12-21

MicroRNAs (miRNAs) play important roles in several human cancers. Although miR188 has been suggested to function as a tumor repressor in cancers, its precise role in glioma and the molecular mechanism remain unknown. In the present study, we investigated the effect of miR-188 on glioma and explored its relevant mechanisms. We found that the expression of miR-188 is dramatically downregulated in glioma tissues and cell lines. Subsequent investigation revealed that miR-188 expression was inversely correlated with ß-catenin expression in glioma tissue samples. Using a luciferase reporter assay, ß-catenin was determined to be a direct target of miR-188. Overexpression of miR-188 reduced ß-catenin expression at both the mRNA and protein levels, and inhibition of miR-188 increased ß-catenin expression. Moreover, we found that overexpression of miR-188 suppressed glioma cell proliferation and cell cycle G1-S transition, whereas inhibition of miR-188 promoted glioma cell proliferation. Importantly, silencing ß-catenin recapitulated the cellular and molecular effects seen upon miR-188 overexpression, which included inhibiting glioma cell proliferation and G1-S transition. Taken together, our results demonstrated that miR188 inhibits glioma cell proliferation by targeting ß-catenin, representing an effective therapeutic strategy for glioma.

Laser direct writing of combinatorial libraries of idealized cellular constructs: Biomedical applications

NASA Astrophysics Data System (ADS)

Schiele, Nathan R.; Koppes, Ryan A.; Corr, David T.; Ellison, Karen S.; Thompson, Deanna M.; Ligon, Lee A.; Lippert, Thomas K. M.; Chrisey, Douglas B.

2009-03-01

The ability to control cell placement and to produce idealized cellular constructs is essential for understanding and controlling intercellular processes and ultimately for producing engineered tissue replacements. We have utilized a novel intra-cavity variable aperture excimer laser operated at 193 nm to reproducibly direct write mammalian cells with micrometer resolution to form a combinatorial array of idealized cellular constructs. We deposited patterns of human dermal fibroblasts, mouse myoblasts, rat neural stem cells, human breast cancer cells, and bovine pulmonary artery endothelial cells to study aspects of collagen network formation, breast cancer progression, and neural stem cell proliferation, respectively. Mammalian cells were deposited by matrix assisted pulsed laser evaporation direct write from ribbons comprised of a UV transparent quartz coated with either a thin layer of extracellular matrix or triazene as a dynamic release layer using CAD/CAM control. We demonstrate that through optical imaging and incorporation of a machine vision algorithm, specific cells on the ribbon can be laser deposited in spatial coherence with respect to geometrical arrays and existing cells on the receiving substrate. Having the ability to direct write cells into idealized cellular constructs can help to answer many biomedical questions and advance tissue engineering and cancer research.

Non-monotonic changes in clonogenic cell survival induced by disulphonated aluminum phthalocyanine photodynamic treatment in a human glioma cell line

PubMed Central

2010-01-01

Background Photodynamic therapy (PDT) involves excitation of sensitizer molecules by visible light in the presence of molecular oxygen, thereby generating reactive oxygen species (ROS) through electron/energy transfer processes. The ROS, thus produced can cause damage to both the structure and the function of the cellular constituents resulting in cell death. Our preliminary investigations of dose-response relationships in a human glioma cell line (BMG-1) showed that disulphonated aluminum phthalocyanine (AlPcS2) photodynamically induced loss of cell survival in a concentration dependent manner up to 1 μM, further increases in AlPcS2concentration (>1 μM) were, however, observed to decrease the photodynamic toxicity. Considering the fact that for most photosensitizers only monotonic dose-response (survival) relationships have been reported, this result was unexpected. The present studies were, therefore, undertaken to further investigate the concentration dependent photodynamic effects of AlPcS2. Methods Concentration-dependent cellular uptake, sub-cellular localization, proliferation and photodynamic effects of AlPcS2 were investigated in BMG-1 cells by absorbance and fluorescence measurements, image analysis, cell counting and colony forming assays, flow cytometry and micronuclei formation respectively. Results The cellular uptake as a function of extra-cellular AlPcS2 concentrations was observed to be biphasic. AlPcS2 was distributed throughout the cytoplasm with intense fluorescence in the perinuclear regions at a concentration of 1 μM, while a weak diffuse fluorescence was observed at higher concentrations. A concentration-dependent decrease in cell proliferation with accumulation of cells in G2+M phase was observed after PDT. The response of clonogenic survival after AlPcS2-PDT was non-monotonic with respect to AlPcS2 concentration. Conclusions Based on the results we conclude that concentration-dependent changes in physico-chemical properties of sensitizer such as aggregation may influence intracellular transport and localization of photosensitizer. Consequent modifications in the photodynamic induction of lesions and their repair leading to different modes of cell death may contribute to the observed non-linear effects. PMID:20433757

Cold inducible RNA binding protein upregulation in pituitary corticotroph adenoma induces corticotroph cell proliferation via Erk signaling pathway

PubMed Central

Fu, Wei; Tang, Hao; Chen, Xiao; Zhao, Yao; Zheng, Lili; Pan, Sijian; Wang, Weiqing; Bian, Liuguan; Sun, Qingfang

2016-01-01

Cushing's disease is caused by pituitary corticotroph adenoma, and the pathogenesis of it has remained obscure. Here, we showed that cold inducible RNA binding protein (CIRP) was markedly elevated in corticotroph tumors. Forced overexpression of CIRP in murine AtT20 pituitary corticotroph cell line increased corticotroph precursor hormone proopiomelanocortin (POMC) transcription, ACTH secretion and cellular proliferation. In vivo, CIRP overexpression promotes murine corticotroph tumor growth and enhances ACTH production. Mechanistically, we show that CIRP could promote AtT20 cells proliferation by inducing cyclinD1 and decreasing p27 expression via Erk1/2 signaling pathway. Clinically, CIRP overexpression is significantly correlated with Cushing's disease recurrence. CIRP appears to play a critical tumorigenesis function in Cushing's disease and its expression might be a useful biomarker for tumor recurrence. PMID:26824322

Modulation of paracrine signaling by CD9 positive small extracellular vesicles mediates cellular growth of androgen deprived prostate cancer

PubMed Central

Soekmadji, Carolina; Riches, James D.; Russell, Pamela J.; Ruelcke, Jayde E.; McPherson, Stephen; Wang, Chenwei; Hovens, Chris M.; Corcoran, Niall M.; Hill, Michelle M.; Nelson, Colleen C.

2017-01-01

Proliferation and maintenance of both normal and prostate cancer (PCa) cells is highly regulated by steroid hormones, particularly androgens, and the extracellular environment. Herein, we identify the secretion of CD9 positive extracellular vesicles (EV) by LNCaP and DUCaP PCa cells in response to dihydrotestosterone (DHT) and use nano-LC–MS/MS to identify the proteins present in these EV. Subsequent bioinformatic and pathway analyses of the mass spectrometry data identified pathologically relevant pathways that may be altered by EV contents. Western blot and CD9 EV TR-FIA assay confirmed a specific increase in the amount of CD9 positive EV in DHT-treated LNCaP and DUCaP cells and treatment of cells with EV enriched with CD9 after DHT exposure can induce proliferation in androgen-deprived conditions. siRNA knockdown of endogenous CD9 in LNCaPs reduced cellular proliferation and expression of AR and prostate specific antigen (PSA) however knockdown of AR did not alter CD9 expression, also implicating CD9 as an upstream regulator of AR. Moreover CD9 positive EV were also found to be significantly higher in plasma from prostate cancer patients in comparison with benign prostatic hyperplasia patients. We conclude that CD9 positive EV are involved in mediating paracrine signalling and contributing toward prostate cancer progression. PMID:28881726

miR-192 suppresses leptomeningeal dissemination of medulloblastoma by modulating cell proliferation and anchoring through the regulation of DHFR, integrins, and CD47.

PubMed

Yang, Seung Yeob; Choi, Seung Ah; Lee, Ji Yeoun; Park, Ae-Kyung; Wang, Kyu-Chang; Phi, Ji Hoon; Koh, Eun Jung; Park, Woong-Yang; Park, Sung-Hye; Hwang, Do Won; Jung, Hee Won; Kim, Seung-Ki

2015-12-22

The main cause of death in medulloblastoma is recurrence associated with leptomeningeal dissemination. During this process, the role of microRNAs (miRs) in the acquisition of metastatic phenotype remains poorly understood. This study aimed to identify the miR involved in leptomeningeal dissemination and to elucidate its biological functional mechanisms. We analyzed the miR expression profiles of 29 medulloblastomas according to the presence of cerebrospinal fluid (CSF) seeding. Differentially expressed miRs (DEmiRs) were validated in 29 medulloblastoma tissues and three medulloblastoma cell lines. The biological functions of the selected miRs were evaluated using in vitro and in vivo studies. A total of 12 DEmiRs were identified in medulloblastoma with seeding, including miR-192. The reduced expression of miR-192 was confirmed in the tumor seeding group and in the medulloblastoma cells. Overexpression of miR-192 inhibited cellular proliferation by binding DHFR. miR-192 decreased cellular anchoring via the repression of ITGAV, ITGB1, ITGB3, and CD47. Animals in the miR-192-treated group demonstrated a reduction of spinal seeding (P < 0.05) and a significant survival benefit (P < 0.05). Medulloblastoma with seeding showed specific DEmiRs compared with those without. miR-192 suppresses leptomeningeal dissemination of medulloblastoma by modulating cell proliferation and anchoring ability.

Local exposure of 849 MHz and 1763 MHz radiofrequency radiation to mouse heads does not induce cell death or cell proliferation in brain

PubMed Central

Kim, Tae-Hyoung; Huang, Tai-Qin; Jang, Ja-June; Kim, Man Ho; Kim, Hyun-Jeong; Lee, Jae-Seon; Pack, Jeong Ki; Seo, Jeong-Sun

2008-01-01

Even though there is no direct evidence to prove the cellular and molecular changes induced by radiofrequency (RF) radiation itself, we cannot completely exclude the possibility of any biological effect of mobile phone frequency radiation. We established a carousel-type exposure chamber for 849 MHz or 1763 MHz of mobile phone RF radiation to expose RF to the heads of C57BL mice. In this chamber, animals were irradiated intermittently at 7.8 W/kg for a maximum of 12 months. During this period, the body weights of 3 groups-sham, 849 MHz RF, and 1763 MHz RF-did not show any differences between groups. The brain tissues were obtained from 3 groups at 6 months and 12 months to examine the differences in histology and cell proliferation between control and RF exposure groups, but we could not find any change upon RF radiation. Likewise, we could not find changes in the expression and distribution of NeuN and GFAP in hippocampus and cerebellum, or in cell death by TUNEL assay in RF exposure groups. From these data, we conclude that the chronic exposure to 849 MHz and 1763 MHz RF radiation at a 7.8 W/kg specific absorption rate (SAR) could not induce cellular alterations such as proliferation, death, and reactive gliosis. PMID:18587267

Analysis of cellular signal transduction from an information theoretic approach.

PubMed

Uda, Shinsuke; Kuroda, Shinya

2016-03-01

Signal transduction processes the information of various cellular functions, including cell proliferation, differentiation, and death. The information for controlling cell fate is transmitted by concentrations of cellular signaling molecules. However, how much information is transmitted in signaling pathways has thus far not been investigated. Shannon's information theory paves the way to quantitatively analyze information transmission in signaling pathways. The theory has recently been applied to signal transduction, and mutual information of signal transduction has been determined to be a measure of information transmission. We review this work and provide an overview of how signal transduction transmits informational input and exerts biological output. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cathepsin D non-proteolytically induces proliferation and migration in human omental microvascular endothelial cells via activation of the ERK1/2 and PI3K/AKT pathways.

PubMed

Pranjol, Md Zahidul I; Gutowski, Nicholas J; Hannemann, Michael; Whatmore, Jacqueline L

2018-01-01

Epithelial ovarian cancer (EOC) frequently metastasises to the omentum, a process that requires pro-angiogenic activation of human omental microvascular endothelial cells (HOMECs) by tumour-secreted factors. We have previously shown that ovarian cancer cells secrete a range of factors that induce pro-angiogenic responses e.g. migration, in HOMECs including the lysosomal protease cathepsin D (CathD). However, the cellular mechanism by which CathD induces these cellular responses is not understood. The aim of this study was to further examine the pro-angiogenic effects of CathD in HOMECs i.e. proliferation and migration, to investigate whether these effects are dependent on CathD catalytic activity and to delineate the intracellular signalling kinases activated by CathD. We report, for the first time, that CathD significantly increases HOMEC proliferation and migration via a non-proteolytic mechanism resulting in activation of ERK1/2 and AKT. These data suggest that EOC cancer secreted CathD acts as an extracellular ligand and may play an important pro-angiogenic, and thus pro-metastatic, role by activating the omental microvasculature during EOC metastasis to the omentum. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Penrose, Harrison; Heller, Sandra; Cable, Chloe

The proliferation of colon cancer cells is mediated in part by epidermal growth factor receptor (EGFR) signaling and requires sustained levels of cellular energy to meet its high metabolic needs. Intracellular lipid droplets (LDs) are a source of energy used for various cellular functions and they are elevated in density in human cancer, yet their regulation and function are not well understood. Here, in human colon cancer cells, EGF stimulates increases in LD density, which depends on EGFR expression and activation as well as the individual cellular capacity for lipid synthesis. Increases in LDs are blockaded by inhibition of PI3K/mTORmore » and PGE2 synthesis, supporting their dependency on select upstream pathways. In colon cancer cells, silencing of the FOXO3 transcription factor leads to down regulation of SIRT6, a negative regulator of lipid synthesis, and consequent increases in the LD coat protein PLIN2, revealing that increases in LDs depend on loss of FOXO3/SIRT6. Moreover, EGF stimulates loss of FOXO3/SIRT6, which is blockaded by the inhibition of upstream pathways as well as lipid synthesis, revealing existence of a negative regulatory loop between LDs and FOXO3/SIRT6. Elevated LDs are utilized by EGF treatment and their depletion through the inhibition of lipid synthesis or silencing of PLIN2 significantly attenuates proliferation. This novel mechanism of proliferative EGFR signaling leading to elevated LD density in colon cancer cells could potentially be therapeutically targeted for the treatment of tumor progression. - Highlights: • In colon cancer cells, EGFR activation leads to increases in LD density. • EGFR signaling includes PI3K/mTOR and PGE2 leading to lipid synthesis. • Increases in LDs are controlled by a negative regulatory loop with FOXO3/SIRT6. • EGFR mediated colon cancer cell proliferation depends on increased LD density.« less

Evaluation of PBS Treatment and PEI Coating Effects on Surface Morphology and Cellular Response of 3D-Printed Alginate Scaffolds.

PubMed

Mendoza García, María A; Izadifar, Mohammad; Chen, Xiongbiao

2017-11-01

Three-dimensional (3D) printing is an emerging technology for the fabrication of scaffolds to repair/replace damaged tissue/organs in tissue engineering. This paper presents our study on 3D printed alginate scaffolds treated with phosphate buffered saline (PBS) and polyethyleneimine (PEI) coating and their impacts on the surface morphology and cellular response of the printed scaffolds. In our study, sterile alginate was prepared by means of the freeze-drying method and then, used to prepare the hydrogel for 3D printing into calcium chloride, forming 3D scaffolds. Scaffolds were treated with PBS for a time period of two days and seven days, respectively, and PEI coating; then they were seeded with Schwann cells (RSC96) for the examination of cellular response (proliferation and differentiation). In addition, swelling and stiffness (Young's modulus) of the treated scaffolds was evaluated, while their surface morphology was assessed using scanning electron microscopy (SEM). SEM images revealed significant changes in scaffold surface morphology due to degradation caused by the PBS treatment over time. Our cell proliferation assessment over seven days showed that a two-day PBS treatment could be more effective than seven-day PBS treatment for improving cell attachment and elongation. While PEI coating of alginate scaffolds seemed to contribute to cell growth, Schwann cells stayed round on the surface of alginate over the period of cell culture. In conclusion, PBS-treatment may offer the potential to induce surface physical cues due to degradation of alginate, which could improve cell attachment post cell-seeding of 3D-printed alginate scaffolds.

Non-Thermal Atmospheric Pressure Plasma Efficiently Promotes the Proliferation of Adipose Tissue-Derived Stem Cells by Activating NO-Response Pathways

PubMed Central

Park, Jeongyeon; Lee, Hyunyoung; Lee, Hae June; Kim, Gyoo Cheon; Kim, Do Young; Han, Sungbum; Song, Kiwon

2016-01-01

Non-thermal atmospheric pressure plasma (NTAPP) is defined as a partially ionized gas with electrically charged particles at atmospheric pressure. Our study showed that exposure to NTAPP generated in a helium-based dielectric barrier discharge (DBD) device increased the proliferation of adipose tissue-derived stem cells (ASCs) by 1.57-fold on an average, compared with untreated cells at 72 h after initial NTAPP exposure. NTAPP-exposed ASCs maintained their stemness, capability to differentiate into adipocytes but did not show cellular senescence. Therefore, we suggested that NTAPP can be used to increase the proliferation of ASCs without affecting their stem cell properties. When ASCs were exposed to NTAPP in the presence of a nitric oxide (NO) scavenger, the proliferation-enhancing effect of NTAPP was not obvious. Meanwhile, the proliferation of NTAPP-exposed ASCs was not much changed in the presence of scavengers for reactive oxygen species (ROS). Also, Akt, ERK1/2, and NF-κB were activated in ASCs after NTAPP exposure. These results demonstrated that NO rather than ROS is responsible for the enhanced proliferation of ASCs following NTAPP exposure. Taken together, this study suggests that NTAPP would be an efficient tool for use in the medical application of ASCs both in vitro and in vivo. PMID:27991548

GM-CSF produced by non-hematopoietic cells is required for early epithelial cell proliferation and repair of injured colonic mucosa1,2

PubMed Central

Egea, Laia; McAllister, Christopher S.; Lakhdari, Omar; Minev, Ivelina; Shenouda, Steve; Kagnoff, Martin F.

2012-01-01

GM-CSF is a growth factor that promotes the survival and activation of macrophages and granulocytes, and dendritic cell (DC) differentiation and survival in vitro. The mechanism by which exogenous GM-CSF ameliorates the severity of Crohn’s disease in humans and colitis in murine models has been considered mainly to reflect its activity on myeloid cells. We used GM-CSF deficient (GM-CSF−/−) mice to probe the functional role of endogenous host-produced GM-CSF in a colitis model induced after injury to the colon epithelium. Dextran sodium sulfate (DSS) at doses that resulted in little epithelial damage and mucosal ulceration in wild type (WT) mice resulted in marked colon ulceration and delayed ulcer healing in GM-CSF−/− mice. Colon crypt epithelial cell proliferation in vivo was significantly decreased in GM-CSF−/− mice at early times after DSS injury. This was paralleled by decreased expression of crypt epithelial cell genes involved in cell cycle, proliferation, and wound healing. Decreased crypt cell proliferation and delayed ulcer healing in GM-CSF−/− mice were rescued by exogenous GM-CSF, indicating the lack of a developmental abnormality in the epithelial cell proliferative response in those mice. Non-hematopoietic cells and not myeloid cells produced the GM-CSF important for colon epithelial proliferation after DSS-induced injury as revealed by bone marrow chimera and DC depletion experiments, with colon epithelial cells being the cellular source of GM-CSF. Endogenous epithelial cell produced GM-CSF has a novel non-redundant role in facilitating epithelial cell proliferation and ulcer healing in response to injury of the colon crypt epithelium. PMID:23325885

A cyclized peptide derived from alpha fetoprotein inhibits the proliferation of ER-positive canine mammary cancer cells.

PubMed

Torres, Cristian Gabriel; Pino, Ana María; Sierralta, Walter Daniel

2009-06-01

The effects of estradiol (E2) and of an AFP-derived cyclized peptide (cP) on the proliferation of primary cultures of cancer cells isolated from spontaneous canine mammary tumors were studied. The cellular response to E2 and cP was related to the expression of estradiol receptor (isoforms alpha and beta). In ER-positive cells, 2 nM estradiol increased cell proliferation and the phosphorylation of ERK1/2; 2 microg/ml cP inhibited all these effects. Estradiol also increased HER2 immunoreactivity in ER-positive cells, an effect that was reverted to its basal values by cP. Estradiol stimulated in these cells the release of MMP2 and MMP9 and the shedding of HB-EGF, effects that the cP did not affect. ER-negative cells were refractory to estradiol or cP. All canine mammary tumor cells in culture responded to treatments analogously to human mammary cancer cells. Our results support the proposal of cP as a new, potentially effective therapeutic agent for the management of mammary cancer.

Voltage-Gated Ion Channels in Cancer Cell Proliferation

PubMed Central

Rao, Vidhya R.; Perez-Neut, Mathew; Kaja, Simon; Gentile, Saverio

2015-01-01

Changes of the electrical charges across the surface cell membrane are absolutely necessary to maintain cellular homeostasis in physiological as well as in pathological conditions. The opening of ion channels alter the charge distribution across the surface membrane as they allow the diffusion of ions such as K+, Ca++, Cl−, Na+. Traditionally, voltage-gated ion channels (VGIC) are known to play fundamental roles in controlling rapid bioelectrical signaling including action potential and/or contraction. However, several investigations have revealed that these classes of proteins can also contribute significantly to cell mitotic biochemical signaling, cell cycle progression, as well as cell volume regulation. All these functions are critically important for cancer cell proliferation. Interestingly, a variety of distinct VGICs are expressed in different cancer cell types, including metastasis but not in the tissues from which these tumors were generated. Given the increasing evidence suggesting that VGIC play a major role in cancer cell biology, in this review we discuss the role of distinct VGIC in cancer cell proliferation and possible therapeutic potential of VIGC pharmacological manipulation. PMID:26010603

Human mesenchymal stromal cells transiently increase cytokine production by activated T cells before suppressing T-cell proliferation: effect of interferon-γ and tumor necrosis factor-α stimulation.

PubMed

Cuerquis, Jessica; Romieu-Mourez, Raphaëlle; François, Moïra; Routy, Jean-Pierre; Young, Yoon Kow; Zhao, Jing; Eliopoulos, Nicoletta

2014-02-01

Mesenchymal stromal cells (MSCs) suppress T-cell proliferation, especially after activation with inflammatory cytokines. We compared the dynamic action of unprimed and interferon (IFN)-γ plus tumor necrosis factor (TNF)-α-pretreated human bone marrow-derived MSCs on resting or activated T cells. MSCs were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) at high MSC-to-PBMC ratios in the absence or presence of concomitant CD3/CD28-induced T-cell activation. The kinetic effects of MSCs on cytokine production and T-cell proliferation, cell cycle and apoptosis were assessed. Unprimed MSCs increased the early production of IFN-γ and interleukin (IL)-2 by CD3/CD28-activated PBMCs before suppressing T-cell proliferation. In non-activated PBMC co-cultures, low levels of IL-2 and IL-10 synthesis were observed with MSCs in addition to low levels of CD69 expression by T cells and no T-cell proliferation. MSCs also decreased apoptosis in resting and activated T cells and inhibited the transition of these cells into the sub-G0/G1 and the S phases. With inhibition of indoleamine 2,3 dioxygenase, MSCs increased CD3/CD28-induced T-cell proliferation. After priming with IFN-γ plus TNF-α, MSCs were less potent at increasing cytokine production by CD3/CD28-activated PBMCs and more effective at inhibiting T-cell proliferation but had preserved anti-apoptotic functions. Unprimed MSCs induce a transient increase in IFN-γ and IL-2 synthesis by activated T cells. Pre-treatment of MSCs with IFN-γ plus TNF-α may increase their effectiveness and safety in vivo. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks

PubMed Central

2012-01-01

Background In vitro fabricated tissue engineered vascular constructs could provide an alternative to conventional substitutes. A crucial factor for tissue engineering of vascular constructs is an appropriate cell source. Vascular cells from the human umbilical cord can be directly isolated and cryopreserved until needed. Currently no cell bank for human vascular cells is available. Therefore, the establishment of a future human vascular cell bank conforming to good manufacturing practice (GMP) conditions is desirable for therapeutic applications such as tissue engineered cardiovascular constructs. Materials and methods A fundamental step was the adaption of conventional research and development starting materials to GMP compliant starting materials. Human umbilical cord artery derived cells (HUCAC) and human umbilical vein endothelial cells (HUVEC) were isolated, cultivated, cryopreserved (short- and long-term) directly after primary culture and recultivated subsequently. Cell viability, expression of cellular markers and proliferation potential of fresh and cryopreserved cells were studied using trypan blue staining, flow cytometry analysis, immunofluorescence staining and proliferation assays. Statistical analyses were performed using Student’s t-test. Results Sufficient numbers of isolated cells with acceptable viabilities and homogenous expression of cellular markers confirmed that the isolation procedure was successful using GMP compliant starting materials. The influence of cryopreservation was marginal, because cryopreserved cells mostly maintain phenotypic and functional characteristics similar to those of fresh cells. Phenotypic studies revealed that fresh cultivated and cryopreserved HUCAC were positive for alpha smooth muscle actin, CD90, CD105, CD73, CD29, CD44, CD166 and negative for smoothelin. HUVEC expressed CD31, CD146, CD105 and CD144 but not alpha smooth muscle actin. Functional analysis demonstrated acceptable viability and sufficient proliferation properties of cryopreserved HUCAC and HUVEC. Conclusion Adaptation of cell isolation, cultivation and cryopreservation to GMP compliant starting materials was successful. Cryopreservation did not influence cell properties with lasting impact, confirming that the application of vascular cells from the human umbilical cord is feasible for cell banking. A specific cellular marker expression profile was established for HUCAC and HUVEC using flow cytometry analysis, applicable as a GMP compliant quality control. Use of these cells for the future fabrication of advanced therapy medicinal products GMP conditions are required by the regulatory authority. PMID:22591741

Identification of chemicals inducing cardiomyocyte proliferation in developmental stage-specific manner with pluripotent stem cells.

PubMed

Uosaki, Hideki; Magadum, Ajit; Seo, Kinya; Fukushima, Hiroyuki; Takeuchi, Ayako; Nakagawa, Yasuaki; Moyes, Kara White; Narazaki, Genta; Kuwahara, Koichiro; Laflamme, Michael; Matsuoka, Satoshi; Nakatsuji, Norio; Nakao, Kazuwa; Kwon, Chulan; Kass, David A; Engel, Felix B; Yamashita, Jun K

2013-12-01

The proliferation of cardiomyocytes is highly restricted after postnatal maturation, limiting heart regeneration. Elucidation of the regulatory machineries for the proliferation and growth arrest of cardiomyocytes is imperative. Chemical biology is efficient to dissect molecular mechanisms of various cellular events and often provides therapeutic potentials. We have been investigating cardiovascular differentiation with pluripotent stem cells. The combination of stem cell and chemical biology can provide novel approaches to investigate the molecular mechanisms and manipulation of cardiomyocyte proliferation. To identify chemicals that regulate cardiomyocyte proliferation, we performed a screening of a defined chemical library based on proliferation of mouse pluripotent stem cell-derived cardiomyocytes and identified 4 chemical compound groups: inhibitors of glycogen synthase kinase-3, p38 mitogen-activated protein kinase, and Ca(2+)/calmodulin-dependent protein kinase II, and activators of extracellular signal-regulated kinase. Several appropriate combinations of chemicals synergistically enhanced proliferation of cardiomyocytes derived from both mouse and human pluripotent stem cells, notably up to a 14-fold increase in mouse cardiomyocytes. We also examined the effects of identified chemicals on cardiomyocytes in various developmental stages and species. Whereas extracellular signal-regulated kinase activators and Ca(2+)/calmodulin-dependent protein kinase II inhibitors showed proliferative effects only on cardiomyocytes in early developmental stages, glycogen synthase kinase-3 and p38 mitogen-activated protein kinase inhibitors substantially and synergistically induced re-entry and progression of cell cycle in neonatal but also as well as adult cardiomyocytes. Our approach successfully uncovered novel molecular targets and mechanisms controlling cardiomyocyte proliferation in distinct developmental stages and offered pluripotent stem cell-derived cardiomyocytes as a potent tool to explore chemical-based cardiac regenerative strategies.

Behavior of Human Bone Marrow-Derived Mesenchymal Stem Cells on Various Titanium-Based Coatings

PubMed Central

Qu, Chengjuan; Kaitainen, Salla; Kröger, Heikki; Lappalainen, Reijo; Lammi, Mikko J.

2016-01-01

The chemical composition and texture of titanium coatings can influence the growth characteristics of the adhered cells. An enhanced proliferation of the human mesenchymal stem cells (hMSCs) would be beneficial. The present study was aimed to investigate whether titanium deposited at different atmospheres would affect the cell growth properties, cellular morphology, and expression of surface markers of hMSCs. Titanium-based coatings were deposited on silicon wafers under oxygen, nitrogen, or argon atmospheres by ultra-short pulsed laser deposition using two different gas pressures followed by heating at 400 °C for 2 h. The characteristics of the coated surfaces were determined via contact angle, zeta potential, and scanning electron microscopy (SEM) techniques. Human MSCs were cultivated on differently coated silicon wafers for 48 h. Subsequently, the cell proliferation rates were analyzed with an MTT assay. The phenotype of hMSCs was checked via immunocytochemical stainings of MSC-associated markers CD73, CD90, and CD105, and the adhesion, spreading, and morphology of hMSCs on coated materials via SEM. The cell proliferation rates of the hMSCs were similar on all coated silicon wafers. The hMSCs retained the MSC phenotype by expressing MSC-associated markers and fibroblast-like morphology with cellular projections. Furthermore, no significant differences could be found in the size of the cells when cultured on all various coated surfaces. In conclusion, despite certain differences in the contact angles and the zeta potentials of various titanium-based coatings, no single coating markedly improved the growth characteristics of hMSCs. PMID:28773947

Effects of methotrexate on the viscoelastic properties of single cells probed by atomic force microscopy.

PubMed

Li, Mi; Liu, Lianqing; Xiao, Xiubin; Xi, Ning; Wang, Yuechao

2016-10-01

Methotrexate is a commonly used anti-cancer chemotherapy drug. Cellular mechanical properties are fundamental parameters that reflect the physiological state of a cell. However, so far the role of cellular mechanical properties in the actions of methotrexate is still unclear. In recent years, probing the behaviors of single cells with the use of atomic force microscopy (AFM) has contributed much to the field of cell biomechanics. In this work, with the use of AFM, the effects of methotrexate on the viscoelastic properties of four types of cells were quantitatively investigated. The inhibitory and cytotoxic effects of methotrexate on the proliferation of cells were observed by optical and fluorescence microscopy. AFM indenting was used to measure the changes of cellular viscoelastic properties (Young's modulus and relaxation time) by using both conical tip and spherical tip, quantitatively showing that the stimulation of methotrexate resulted in a significant decrease of both cellular Young's modulus and relaxation times. The morphological changes of cells induced by methotrexate were visualized by AFM imaging. The study improves our understanding of methotrexate action and offers a novel way to quantify drug actions at the single-cell level by measuring cellular viscoelastic properties, which may have potential impacts on developing label-free methods for drug evaluation.

Thiazolidinediones abrogate cervical cancer growth

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

Wuertz, Beverly R., E-mail: knier003@umn.edu; Darrah, Lindsay, E-mail: ldarrah@obgynmn.com; Wudel, Justin, E-mail: drwudel@drwudel.com

Peroxisome proliferator-activated receptor gamma (PPAR γ) is activated by thiazolidinedione drugs (TZDs) and can promote anti-cancer properties. We used three TZDs (pioglitazone, rosiglitazone, and ciglitazone) to target cervical cancer cell lines and a nude mouse animal model. Each agent increased activation of PPAR γ, as judged by a luciferase reporter gene assay in three HPV-associated cell lines (CaSki, SiHa, and HeLa cells) while decreasing cellular proliferation in a dose-dependent manner. They also promoted Oil Red O accumulation in treated cell lines and upregulated the lipid differentiation marker adipsin. Interestingly, xenograft HeLa tumors in nude mice treated with 100 mg/kg/day pioglitazonemore » exhibited decreased growth compared to control mice or mice treated with standard cervical chemotherapy. In conclusion, TZDs slow tumor cell growth in vitro and in vivo with decreases in cell proliferation and increases in PPAR γ and adipsin. These agents may be interesting treatments or treatment adjuncts for HPV-associated cancers or perhaps even precancerous conditions. - Highlights: • Thiazolidinediones decreases cervical cancer proliferation. • Pioglitazone increases cervical cancer differentiation. • Pioglitazone decreases tumor growth in mice. • Pioglitazone may be a useful treatment adjunct.« less

Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells

PubMed Central

Nieborowska-Skorska, Margaret; Sullivan, Katherine; Dasgupta, Yashodhara; Podszywalow-Bartnicka, Paulina; Maifrede, Silvia; Di Marcantonio, Daniela; Bolton-Gillespie, Elisabeth; Cramer-Morales, Kimberly; Lee, Jaewong; Li, Min; Slupianek, Artur; Gritsyuk, Daniel; Cerny-Reiterer, Sabine; Seferynska, Ilona; Bullinger, Lars; Gorbunova, Vera; Piwocka, Katarzyna; Valent, Peter; Civin, Curt I.; Muschen, Markus; Dick, John E.; Wang, Jean C.Y.; Bhatia, Smita; Bhatia, Ravi; Eppert, Kolja; Minden, Mark D.; Sykes, Stephen M.

2017-01-01

Quiescent and proliferating leukemia cells accumulate highly lethal DNA double-strand breaks that are repaired by 2 major mechanisms: BRCA-dependent homologous recombination and DNA-dependent protein kinase–mediated (DNA-PK–mediated) nonhomologous end-joining, whereas DNA repair pathways mediated by poly(ADP)ribose polymerase 1 (PARP1) serve as backups. Here we have designed a personalized medicine approach called gene expression and mutation analysis (GEMA) to identify BRCA- and DNA-PK–deficient leukemias either directly, using reverse transcription-quantitative PCR, microarrays, and flow cytometry, or indirectly, by the presence of oncogenes such as BCR-ABL1. DNA-PK–deficient quiescent leukemia cells and BRCA/DNA-PK–deficient proliferating leukemia cells were sensitive to PARP1 inhibitors that were administered alone or in combination with current antileukemic drugs. In conclusion, GEMA-guided targeting of PARP1 resulted in dual cellular synthetic lethality in quiescent and proliferating immature leukemia cells, and is thus a potential approach to eradicate leukemia stem and progenitor cells that are responsible for initiation and manifestation of the disease. Further, an analysis of The Cancer Genome Atlas database indicated that this personalized medicine approach could also be applied to treat numerous solid tumors from individual patients. PMID:28481221

Neonatal Heart-Enriched miR-708 Promotes Proliferation and Stress Resistance of Cardiomyocytes in Rodents

PubMed Central

Deng, Shengqiong; Zhao, Qian; Zhen, Lixiao; Zhang, Chuyi; Liu, Cuicui; Wang, Guangxue; Zhang, Lin; Bao, Luer; Lu, Ying; Meng, Lingyu; Lü, Jinhui; Yu, Ping; Lin, Xin; Zhang, Yuzhen; Chen, Yi-Han; Fan, Huimin; Cho, William C.; Liu, Zhongmin; Yu, Zuoren

2017-01-01

Adult heart has limited potential for regeneration after pathological injury due to the limited cell proliferation of cardiomyocytes and the quiescent status of progenitor cells. As such, induction of cell-cycle reentry of cardiomyocytes is one of the key strategies for regeneration of damaged heart. In this study, a subset of miRNAs including miR-708 were identified to be much more abundant in the embryonic and neonatal cardiomyocytes than that in adult rodents. Overexpression of miR-708 promoted cellular proliferation of H9C2 cells or primary cardiomyocytes from neonatal rats or mice in vitro. Lipid nanoparticle delivery of miR-708 promoted myocardial regeneration and heart function recovery in vivo. In addition, miR-708 protected cardiomyocytes against stress-induced apoptosis under hypoxia or isoproterenol treatments. miR-708 inhibited the expression of MAPK14, which has been demonstrated arresting the cell cycle in cardiomyocytes. The cell proliferation-promoting function of miR-708 was dependent at least partly on the expression of MAPK14. These findings strengthen the potential of applying miRNAs to reconstitute lost cardiomyocytes in injured hearts, and may provide a novel miRNA candidate for promoting heart regeneration. PMID:28638481