Fraction Of Cells In G1 Research Articles

The PAX-FOXO1s trigger fast trans-differentiation of chick embryonic neural cells into alveolar rhabdomyosarcoma with tissue invasive properties limited by S phase entry inhibition.

The chromosome translocations generating PAX3-FOXO1 and PAX7-FOXO1 chimeric proteins are the primary hallmarks of the paediatric fusion-positive alveolar subtype of Rhabdomyosarcoma (FP-RMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently promote malignant transformation in vivo. The reason for this is unclear. To address this, we developed an in ovo model to follow the response of spinal cord progenitors to PAX-FOXO1s. Our data demonstrate that PAX-FOXO1s, but not wild-type PAX3 or PAX7, trigger the trans-differentiation of neural cells into FP-RMS-like cells with myogenic characteristics. In parallel, PAX-FOXO1s remodel the neural pseudo-stratified epithelium into a cohesive mesenchyme capable of tissue invasion. Surprisingly, expression of PAX-FOXO1s, similar to wild-type PAX3/7, reduce the levels of CDK-CYCLIN activity and increase the fraction of cells in G1. Introduction of CYCLIN D1 or MYCN overcomes this PAX-FOXO1-mediated cell cycle inhibition and promotes tumour growth. Together, our findings reveal a mechanism that can explain the apparent limited oncogenicity of PAX-FOXO1 fusion transcription factors. They are also consistent with certain clinical reports indicative of a neural origin of FP-RMS.

Human shelterin protein POT1 prevents severe telomere instability induced by homology-directed DNA repair.

The evolutionarily conserved POT1 protein binds single-stranded G-rich telomeric DNA and has been implicated in contributing to telomeric DNA maintenance and the suppression of DNA damage checkpoint signaling. Here, we explore human POT1 function through genetics and proteomics, discovering that a complete absence of POT1 leads to severe telomere maintenance defects that had not been anticipated from previous depletion studies in human cells. Conditional deletion of POT1 in HEK293E cells gives rise to rapid telomere elongation and length heterogeneity, branched telomeric DNA structures, telomeric R-loops, and telomere fragility. We determine the telomeric proteome upon POT1-loss, implementing an improved telomeric chromatin isolation protocol. We identify a large set of proteins involved in nucleic acid metabolism that engage with telomeres upon POT1-loss. Inactivation of the homology-directed repair machinery suppresses POT1-loss-mediated telomeric DNA defects. Our results unravel as major function of human POT1 the suppression of telomere instability induced by homology-directed repair.

Growth Arrest-Specific 6 (GAS6) Promotes Prostate Cancer Survival by G1 Arrest/S Phase Delay and Inhibition of Apoptosis During Chemotherapy in Bone Marrow.

Prostate cancer (PCa) is known to develop resistance to chemotherapy. Growth arrest-specific 6 (GAS6), plays a role in tumor progression by regulating growth in many cancers. Here, we explored how GAS6 regulates the cell cycle and apoptosis of PCa cells in response to chemotherapy. We found that GAS6 is sufficient to significantly increase the fraction of cells in G1 and the duration of phase in PCa cells. Importantly, the effect of GAS6 on G1 is potentiated during docetaxel chemotherapy. GAS6 altered the levels of several key cell cycle regulators, including the downregulation of Cyclin B1 (G2 /M phase), CDC25A, Cyclin E1, and CDK2 (S phase entry), while the upregulation of cell cycle inhibitors p27 and p21, Cyclin D1, and CDK4. Importantly, these changes became further accentuated during docetaxel treatment in the presence of GAS6. Moreover, GAS6 alters the apoptotic response of PCa cells during docetaxel chemotherapy. Docetaxel induced PCa cell apoptosis is efficiently suppressed in PCa cell culture in the presence of GAS6 or GAS6 secreted from co-cultured osteoblasts. Similarly, the GAS6-expressing bone environment protects PCa cells from apoptosis within primary tumors in vivo studies. Docetaxel induced significant levels of Caspase-3 and PARP cleavage in PCa cells, while GAS6 protected PCa cells from docetaxel-induced apoptotic signaling. Together, these data suggest that GAS6, expressed by osteoblasts in the bone marrow, plays a significant role in the regulation of PCa cell survival during chemotherapy, which will have important implications for targeting metastatic disease. J. Cell. Biochem. 117: 2815-2824, 2016. © 2016 Wiley Periodicals, Inc.

Dynamics of oscillatory phenotypes in Saccharomyces cerevisiae reveal a network of genome-wide transcriptional oscillators

Genetic and environmental factors are well-studied influences on phenotype; however, time is a variable that is rarely considered when studying changes in cellular phenotype. Time-resolved microarray data revealed genome-wide transcriptional oscillation in a yeast continuous culture system with ∼ 2 and ∼ 4 h periods. We mapped the global patterns of transcriptional oscillations into a 3D map to represent different cellular phenotypes of redox cycles. This map shows the dynamic nature of gene expression in that transcripts are ordered and coupled to each other through time and concentration space. Although cells differed in oscillation periods, transcripts involved in certain processes were conserved in a deterministic way. When oscillation period lengthened, the peak to trough ratio of transcripts increased and the fraction of cells in the unbudded (G0/G1) phase of the cell division cycle increased. Decreasing the glucose level in the culture medium was one way to increase the redox cycle, possibly from changes in metabolic flux. The period may be responding to lower glucose levels by increasing the fraction of cells in G1 and reducing S-phase gating so that cells can spend more time in catabolic processes. Our results support that gene transcripts are coordinated with metabolic functions and the cell division cycle.

Abstract 2967: Nucleolin phosphorylation by CK2 modulates its role in cell cycle checkpoint activation

Abstract Nucleolin is a multifunctional abundant nucleolar protein and its gene expression is transactivated by the proto-oncogene c-myc during exponential cellular growth. This non-ribosomal protein is a major nucleolar phosphoprotein which is involved in ribosomal RNA processing, RNA polymerase I-catalyzed transcription, and the regulation of apoptosis, demonstrating both positive and negative effects on cell proliferation. During an unperturbed cell-cycle, interphase (CK2) and mitotic (Cdc2) kinases regulate nucleolin phosphorylation, modulating its function and nucleolar localization. Following cellular stress, nucleolin has been shown to inhibit chromosomal replication and to relay nucleolar stress signals mediated by its re-localization within the nucleus. Nucleolin also interacts with prominent components of the ARF (Alternate Reading Frame)/p53 checkpoint pathway that controls growth upon oncogenic stress signals. We have previously demonstrated that nucleolin binds to the p53-antagonist Hdm2, inhibits Hdm2-mediated degradation of p53 and causes p53-mediated apoptosis in an ARF-independent manner. Here we further dissect role/s of nucleolin phosphorylation in regulating the ARF/p53 checkpoint. We have generated a novel system by engineering human osteocarcinoma (NARF6) cells to induce expression of nucleolin-wt (wild-type) or -S6A (a phospho-mutant with six consensus site serines mutated to alanine, impairing phosphorylation by CK2). This novel system can control expression of both nucleolin and ARF by different promoters, allowing us to dissect the downstream events that are solely nucleolin- or ARF- dependent, or regulated by both. We found that increased levels of nucleolin-S6A significantly decreased ARF protein levels as compared to nucleolin-wt. Nucleolin-S6A was significantly more nucleoplasmic and, interestingly, cells that express S6A lacked nucleolar ARF expression. Although a dramatic loss in both ARF and p21 protein levels is evident with nucleolin-S6A expression, there was a net increase in p53 levels. Significantly, nucleolin-S6A expression caused an increase in the fraction of G1 cells in an ARF-independent manner. Thus, reduction of nucleolin phosphorylation by CK2 acts as a molecular switch causing the sub-nuclear re-localization of both nucleolin and ARF. These events lead to p53 activation, and the control of growth in both normal and stressed cells. These results also provide validation of our unique cellular system that allows us to manipulate nucleolin wt and mutant levels, facilitating the study of its physiological functions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2967. doi:10.1158/1538-7445.AM2011-2967

Isolation and Characterization of Radiation-resistant Lung Cancer D6-R Cell Line

To isolate an isogenic radioresistant cancer cell line after fractioned X-ray radiation and characterize the resistant cells. D6 cells were exposed to repeated X-ray irradiation, and after a total dose of 5200 cGy in 8 fractions, a radioresistant monoclone D6-R was obtained. The radiosensitivity and drug sensitivity of the novel radioresistant D6-R cells, together with their parent D6 cells, were measured using clonogenic assay and MTT assay respectively. Cell cycle distribution was analyzed by flow cytometry. Fluorescence microscopy and flow cytometry were applied for apoptosis detection. Comet assay was used for the detection of DNA damage and repair. D6-R cells showed higher and broader initial shoulder (D0=2.08 Gy, Dq=1.64 Gy, N=2.20) than the parent D6 cells (D0=1.84 Gy, Dq=0.34 Gy, N=1.20). They were 1.65-fold more radioresistant than D6 cells in terms of SF2 (63% vs 38%) and were more resistant to ADM (3.15-fold) and 5-FU (3.86-fold) as compared with the latter. It was found that D6-R cells had higher fractions of cells in S phase (53.4% vs 37.8%) and lower fractions of cells in G1 (44.1% vs 57.2%) and G2-M phase (2.5% vs 5%). There was no difference in radiation-induced apoptosis between D6-R and D6 cells. D6-R cells showed less initial DNA damage and increased capacity in DNA repair after irradiation, as compared with the parent cells. D6-R cells have been isolated by exposing the parental D6 cells to repeated irradiation. The difference in cell cycle pattern together with the induction and repair of DNA damage might, at least partially, explain the mechanism of the radioresistance.

Induction of ATM Activation, Histone H2AX Phosphorylation and Apoptosis by Etoposide: Relation to Cell Cycle Phase

Etoposide (VP-16) belongs to the family of DNA topoisomerase II (topo2) inhibitors, drugs widely used in cancer chemotherapy. Their presumed mode of action is stabilization of “cleavable complexes” between topo2 and DNA; collisions of DNA replication forks with these complexes convert them into DNA double-strand breaks (DSBs), potentially lethal lesions that may trigger apoptosis. Immunocytochemical detection of activation of ATM (ATM-S1981P) and histone H2AX phosphorylation (γH2AX) provides a sensitive probe of the induction of DSBs in individual cells. Using multiparameter cytometry we measured the expression of ATM-S1981P and γH2AX as well as initiation of apoptosis (caspase-3 activation) in relation to the cell cycle phase in etoposide-treated human lymphoblastoid TK6 cells. The induction of ATM-S1981P and γH2AX was seen in all phases of the cell cycle. The G1-phase cells, however, preferentially underwent apoptosis. The extent of etoposide-induced H2AX phosphorylation was partially reduced by N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS).The maximal reduction of H2AX phosphorylation by NAC, seen in G1-phase cells, was nearly 50%. NAC also protected a fraction of G1 cells from etoposide-induced apoptosis, but had no such effect on S or G2M cells. However, no significant rise in the intracellular level of ROS upon treatment with etoposide was detected. The effects of etoposide were compared with the previously investigated effects of another topo2 inhibitor, mitoxantrone. The latter was seen to induce a maximal level of ATM-S1981P and γH2AX (partially abrogated by NAC) in G1-phase cells, but unlike etoposide, triggered apoptosis exclusively of S-phase cells. The data suggest that in addition to the generally accepted mechanism involving collisions of replication forks with the “cleavable complexes”, other mechanisms which appear to be different for etoposide vs. mitoxantrone, may contribute to formation of DSBs and to triggering of apoptosis.

Thioredoxin Is Required for Deoxyribonucleotide Pool Maintenance during S Phase

Thioredoxin was initially identified by its ability to serve as an electron donor for ribonucleotide reductase in vitro. Whether it serves a similar function in vivo is unclear. In Saccharomyces cerevisiae, it was previously shown that Deltatrx1 Deltatrx2 mutants lacking the two genes for cytosolic thioredoxin have a slower growth rate because of a longer S phase, but the basis for S phase elongation was not identified. The hypothesis that S phase protraction was due to inefficient dNTP synthesis was investigated by measuring dNTP levels in asynchronous and synchronized wild-type and Deltatrx1 Deltatrx2 yeast. In contrast to wild-type cells, Deltatrx1 Deltatrx2 cells were unable to accumulate or maintain high levels of dNTPs when alpha-factor- or cdc15-arrested cells were allowed to reenter the cell cycle. At 80 min after release, when the fraction of cells in S phase was maximal, the dNTP pools in Deltatrx1 Deltatrx2 cells were 60% that of wild-type cells. The data suggest that, in the absence of thioredoxin, cells cannot support the high rate of dNTP synthesis required for efficient DNA synthesis during S phase. The results constitute in vivo evidence for thioredoxin being a physiologically relevant electron donor for ribonucleotide reductase during DNA precursor synthesis.

The Role of Cyclin-dependent Kinase Inhibitor p27Kip1 in Anti-HER2 Antibody-induced G1 Cell Cycle Arrest and Tumor Growth Inhibition

Cyclin-dependent kinase (CDK) inhibitor p27Kip1 binds to the cyclin E.CDK2 complex and plays a major role in controlling cell cycle and cell growth. Our group and others have reported that anti-HER2 monoclonal antibodies exert inhibitory effects on HER2-overexpressing breast cancers through G1 cell cycle arrest associated with induction of p27Kip1 and reduction of CDK2. The role of p27Kip1 in anti-HER2 antibody-induced cell cycle arrest and growth inhibition is, however, still uncertain. Here we have provided several lines of evidence supporting a critical role for p27Kip1 in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition. Induction of p27Kip1 and G1 growth arrest by anti-HER2 antibody, murine 4D5, or humanized trastuzumab (Herceptin) are concentration-dependent, time-dependent, irreversible, and long-lasting. The magnitude of G1 cell cycle arrest induced by trastuzumab or 4D5 is well correlated with the level of p27Kip1 protein induced. Up-regulation of p27Kip1 and G1 growth arrest could no longer be removed with as little as 14 h of treatment with trastuzumab. Anti-HER2 antibody-induced p27Kip1 protein, G1 arrest, and growth inhibition persist at least 5 days after a single treatment. The magnitude of growth inhibition of breast cancer cells induced by anti-HER2 antibody closely parallels the level of p27Kip1 induced. Induced expression of exogenous p27Kip1 results in a p27Kip1 level-dependent G1 cell cycle arrest and growth inhibition similar to that obtained with anti-HER2 antibodies. Reducing p27Kip1 expression using p27Kip1 small interfering RNA blocks anti-HER2 antibody-induced p27Kip1 up-regulation and G1 arrest. Treatment with anti-HER2 antibody significantly increases the half-life of p27Kip1 protein. Inhibition of ubiquitin-proteasome pathway, but not inhibition of calpain and caspase activities, up-regulates p27Kip1 protein to a degree comparable with that obtained with anti-HER2 antibodies. We have further demonstrated that anti-HER2 antibody significantly decreases threonine phosphorylation of p27Kip1 protein at position 187 (Thr-187) and increases serine phosphorylation of p27Kip1 protein at position 10 (Ser-10). Expression of S10A and T187A mutant p27Kip1 protein increases the fraction of cells in G1 and reduces a further antibody-induced G1 arrest. Consequently, p27Kip1 plays an important role in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition through post-translational regulation. Regulation of the phosphorylation of p27Kip1 protein is one of the post-translational mechanisms by which anti-HER2 antibody upregulates the protein.

Low dose of wortmannin reduces radiosensitivity of cells

Wortmannin is an inhibitor of PI3-kinase and acts on cultured cells at doses below 1 μM. Wortmannin also inhibits the PI3-kinase family such as ATM or DNA-PK at doses above 10 μM. There are many reports on the enhancement of cell radiosensitivity by using a high dose of wortmannin inhibiting the proteins of the PI3-kinase family. However, there have been no reports of the effects on the radiosensitivity of low doses of wortmannin inhibiting PI3-kinase. We found that low doses of wortmannin reduced the radiosensitivity of human glioblastoma cells, which had wild-type p53. A low dose of wortmannin did not affect the accumulation of p53 and the phosphorylation of p53 at ser-15; however, a low dose reduced the induction of Waf-1 and enhanced the induction of GADD45. As the fraction of G2/M cells was reduced, however, the fraction of G1 cells was increased by a low dose of wortmannin after X-ray irradiation.

Diabetic LDL inhibits cell-cycle progression via STAT5B and p21waf

Modified LDL is a major cause of injury to the endothelium in diabetes. In the present study, we analyzed the effects on endothelial cells of LDL recovered from type 2 diabetic patients (dm-LDL) or from nondiabetic subjects (n-LDL). Treatment of human umbilical vein endothelial cells with dm-LDL, but not n-LDL, led to the accumulation of cells in G1. To dissect the molecular mechanisms of this effect, we analyzed the expression and function of the cyclin-dependent kinase inhibitor p21waf, a cell cycle regulator known to be a target of the signal transducers and activators of transcription (STATs). dm-LDL led to transient STAT5 phosphorylation and the formation of a STAT5-containing complex and activated p21waf expression at the transcriptional level. Expression of the dominant-negative form of STAT5B, but not of STAT5A, significantly decreased both p21waf expression and the fraction of cells in G1. Finally, immunofluorescence analysis demonstrated that activated STAT5 is expressed in newly formed intraplaque vessels and in endothelial cells lining the luminal side of the plaque. Similarly, p21waf immunoreactivity was found in the neointimal vasculature. Our results suggest a role of STAT5B as a regulator of gene expression in diabetes-associated vascular disease.

Diabetic LDL inhibits cell-cycle progression via STAT5B and p21(waf).

Modified LDL is a major cause of injury to the endothelium in diabetes. In the present study, we analyzed the effects on endothelial cells of LDL recovered from type 2 diabetic patients (dm-LDL) or from nondiabetic subjects (n-LDL). Treatment of human umbilical vein endothelial cells with dm-LDL, but not n-LDL, led to the accumulation of cells in G1. To dissect the molecular mechanisms of this effect, we analyzed the expression and function of the cyclin-dependent kinase inhibitor p21(waf), a cell cycle regulator known to be a target of the signal transducers and activators of transcription (STATs). dm-LDL led to transient STAT5 phosphorylation and the formation of a STAT5-containing complex and activated p21(waf) expression at the transcriptional level. Expression of the dominant-negative form of STAT5B, but not of STAT5A, significantly decreased both p21(waf) expression and the fraction of cells in G1. Finally, immunofluorescence analysis demonstrated that activated STAT5 is expressed in newly formed intraplaque vessels and in endothelial cells lining the luminal side of the plaque. Similarly, p21(waf) immunoreactivity was found in the neointimal vasculature. Our results suggest a role of STAT5B as a regulator of gene expression in diabetes-associated vascular disease.

Friend virus-induced erythroblastosis in STAT5a/b mutant mice: Properties of isolated erythroblasts in vitro

This study examined the roles of the Stat5a and Stat5b gene products in growth and differentiation of mouse erythroid cells. BALB/c mice bearing inactivating mutations in both Stat5a and Stat5b were infected with the anemia-inducing strain of Friend virus. The infected mice developed erythroblastosis in the spleen typical of the early phase of Friend disease. Early erythroblasts purified from the spleens of infected mice (Stat5a/b mutants and BALB/c wild type controls) were cultured in medium containing 2 U/mL of erythropoietin (EPO). During the culture period of 60 h, a significant fraction of the Stat5a/b(−/−) erythroblasts differentiated into reticulocytes. However, the population exhibited several properties that were different from controls. Control cell numbers increased 6-fold during the 60 h culture period and about 90% of the resultant population formed reticulocytes. The Stat5a/b(−/−) cells increased in number only 3.5-fold. About 30% of the cells never initiated hemoglobin synthesis, and about one-half of the remainder did not mature into reticulocytes. After 36 h of culture, the Stat5a/b(−/−) erythroblast population contained a higher fraction than normal of cells which were positive for DNA degradation by a flow cytometric TUNEL assay although morphological apoptosis was not pronounced. This pleotropic phenotype does not resemble the type or timing of apoptosis induced by EPO deprivation. The combined result of the above factors was inefficient erythropoiesis by Stat5a/b(−/−) cells in vitro. Several molecular regulators of apoptosis and of cell cycle were examined. Normal levels of Bcl-x production were observed and no activation of caspase 3 or cleavage of PARP was detected. p27 and p21 were significantly elevated in Stat5a/b(−/−) cells. The fractions of cells in G1 and S phases of the cell cycle were different in Stat5a/b (−/−) and control cells throughout the culture period.

Hepatocyte growth factor and vitamin D cooperatively inhibit androgen-unresponsive prostate cancer cell lines.

Expression of MET, the receptor for hepatocyte growth factor (HGF), has been associated with androgen-insensitive prostate cancer. In this study we evaluated MET activation by HGF and HGF action in prostate cancer cell lines. HGF causes phosphorylation (activation) of the MET receptor in three androgen-unresponsive cell lines (DU 145, PC-3, and ALVA-31) together with morphological change. Although HGF is known to stimulate the growth of normal epithelial cells, including those from prostate, we found that HGF inhibited ALVA-31 and DU 145 (hormone-refractory) cell lines. Moreover, HGF and vitamin D additively inhibited growth in each androgen-unresponsive cell line, with the greatest growth inhibition in ALVA-31 cells. Further studies in ALVA-31 cells revealed distinct cooperative actions of HGF and vitamin D. In contrast to the accumulation of cells in G1 seen during vitamin D inhibition of androgen-responsive cells (LNCaP), growth inhibition of the androgen-unresponsive ALVA-31 cell line with the HGF and vitamin D combination decreased, rather than increased, the fraction of cells in G1, with a corresponding increase in the later cell cycle phases. This cell cycle redistribution suggests that in androgen-unresponsive prostate cancer cells, HGF and vitamin D act together to slow cell cycle progression via control at sites beyond the G1/S checkpoint, the major regulatory locus of growth control in androgen-sensitive prostate cells.

Strain-dependent myeloid hyperplasia, growth deficiency, and accelerated cell cycle in mice lacking the Rb-related p107 gene.

To investigate the function of the Rb-related p107 gene, a null mutation in p107 was introduced into the germ line of mice and bred into a BALB/cJ genetic background. Mice lacking p107 were viable and fertile but displayed impaired growth, reaching about 50% of normal weight by 21 days of age. Mutant mice exhibited a diathetic myeloproliferative disorder characterized by ectopic myeloid hyperplasia in the spleen and liver. Embryonic p107(-/-) fibroblasts and primary myoblasts isolated from adult p107(-/-) mice displayed a striking twofold acceleration in doubling time. However, cell sort analysis indicated that the fraction of cells in G1, S, and G2 was unaltered, suggesting that the different phases of the cell cycle in p107(-/-) cells was uniformly reduced by a factor of 2. Western analysis of cyclin expression in synchronized p107(-/-) fibroblasts revealed that expression of cyclins E and A preceded that of D1. Mutant embryos expressed approximately twice the normal level of Rb, whereas p130 levels were unaltered. Lastly, mutant mice reverted to a wild-type phenotype following a single backcross with C57BL/6J mice, suggesting the existence of modifier genes that have potentially epistatic relationships with p107. Therefore, we conclude that p107 is an important player in negatively regulating the rate of progression of the cell cycle, but in a strain-dependent manner.

Effect of phosphorus starvation on the cell cycle of the photosynthetic prokaryote Prochlorococcus spp.

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 132:265-274 (1996) - doi:10.3354/meps132265 Effect of phosphorus starvation on the cell cycle of the photosynthetic prokaryote Prochlorococcus spp. Parpais J, Marie D, Partensky F, Morin P, Vaulot D The influence of phosphorus (P) starvation on the cell cycle of Prochlorococcus was studied for several strains from the Mediterranean Sea, the Atlantic and Pacific Oceans. P starvation could only be induced at high N/P ratio (&GT=150). In P-starved stationary phase, the fraction of cells in G1 dropped markedly, while cells accumulated not only in G2, but also in the DNA synthesis phase S, indicating that cells were not arrested at a precise cell cycle location but in all cell cycle phases. The percentage of P-starved cells in each phase varied slightly between strains. For the Mediterranean Sea strain CCMP 1378, cell cycle arrest in G1 and G2 could be reversed by addition of phosphates, while cells arrested in S could not resume their cycle and died. Such irreversible arrest in S had never been reported previously in phytoplankton. This suggests that cell cycle controls are not very tight in Prochlorococcus, since cells are not prevented from entering the critical S phase in the absence of phosphorus. Prochlorococcus . Phosphate starvation . Cell cycle . Flow cytometry . Growth rate Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 132. Publication date: February 29, 1996 Print ISSN:0171-8630; Online ISSN:1616-1599 Copyright © 1996 Inter-Research.

The fraction of cells in G1 with bound retinoblastoma protein increases with the duration of the cell cycle.

The retinoblastoma gene product (pRB) is a nuclear phosphoprotein with growth-suppressing effects. During early G1 phase, pRB is underphosphorylated and bound in the nucleus. The association between the duration of the cell cycle/G1 phase and the fraction of cells in G1 with bound pRB was studied in the human pre-B cell line Reh. The cell-cycle duration was varied by growing cells at different concentrations (25, 10, 2, 0.5 and 0%) of fetal calf serum (FCS); pRB binding was studied by flow cytometry. The culture doubling time increased from 21 h in 25% FCS to 54 h in 0.5% FCS. Cell death occurred in the absence of FCS, and the culture doubling time therefore could not be defined. The fraction of cells in G1 did not change significantly with decreasing FCS concentration (0.47 in 25% FCS, 0.52 in 0% FCS). In contrast, the fraction of G1 cells with bound pRB increased from 0.12 in 25% FCS to 0.65 in 0% FCS. Continuous labelling with bromodeoxyuridine demonstrated that the growth fraction was close to unity at all FCS concentrations down to 0.5%, hence, the duration of the cell cycle was equal to the culture doubling time under these conditions. The duration of early G1 phase (where pRB is underphosphorylated and bound) increased 10-fold, while the duration of late G1 phase increased twofold, for Reh cells grown in 0.5% FCS compared with cells grown in 25% FCS. The increase in the duration of late G1, and the increased S and G2+M phase transit times, indicate that other factors, in addition to pRB kinase activity, regulate the duration of G1 and the cell cycle of serum-deprived Reh cells.

Effects of melatonin on the cell cycle kinetics and “estrogen‐rescue” of MCF‐7 human breast cancer cells in culture

Melatonin has been shown to have a direct inhibitory action on the proliferation of estrogen-responsive MCF-7 human breast cancer cells in culture. In the present study, we examined by flow cytometry whether this inhibitory effect might be exerted on the G1 phase of the cell cycle, thus causing a transition delay into the S phase. In order to further verify this hypothesis we tested the ability of estradiol to "rescue" MCF-7 cells from melatonin inhibition, and the potential of this indoleamine to block the ability of estradiol to rescue the cells from tamoxifen inhibition. Following five days of incubation, melatonin (10(-9)M) increased the fraction of cells in G1 of the cell cycle while simultaneously causing a 50% reduction in the proportion of cells in S phase. The antiproliferative effect of melatonin (10(-5)M) was prevented by the simultaneous treatment of the cells with estradiol (10(-8)M) in clonogenic soft agar culture, or reversed by the addition of estradiol to cells previously incubated with and inhibited by melatonin (10(-9)M) in monolayer culture. Additionally, melatonin blocked the estrogen-rescue of tamoxifen-inhibited cells in both types of culture systems. These results support the hypothesis that the antiproliferative effect of melatonin, like tamoxifen, is cell cycle specific by causing a G1-S transition delay. These results also indicate an important interaction of melatonin with estrogen-mediated mechanisms of MCF-7 cell proliferation.

Different quiescence states of three culture cell lines detected by acridine orange staining of cellular RNA

Three cell lines (CHO, L-929, and R1H) were investigated for their growth kinetics and the difference of exponential and quiescent state of monolayers in medium with and without serum (L-929). The noncycling populations of L-929 and R1H in medium with serum contained increased G1-phase percentages but also considerable proportions of SQ and G2Q cells. Although about 90% of the cells excluded trypan blue, the viability tested by colony assay was clearly lower than for exponentially growing cultures. CHO cells showed similar fractions of cells in G1-, S-, and G2-Q compartments but in addition considerable cell loss. The RNA content of these cells was reduced in plateau phase by 7-48% depending on cell type and residence time in the noncycling state. The data suggest that the cells suffered from nutrition depletion and were arrested in all phases of the cycle. In contrast, L-929 cells in medium without serum reduced their RNA content down to one-third that of proliferating cells and still retained the full viability as shown by the same plating efficiency in a colony assay. Since about 90% of the cells had G1 DNA content, these cells resemble true G1Q or G0 cells controlled by growth factors rather than nutritional depletion.

B-cell acute lymphoblastic leukaemia: clinical and biological aspects.

Acute lymphoblastic leukaemia of B-cell phenotype (B-ALL) is uncommon. We studied eight cases of B-ALL, investigating the clinical characteristics as well as the biological features. Cytology revealed a typical L3 profile in most cases, but in one case the morphological diagnosis was L2 and morphometric analysis indicated that the blasts in B-ALL were larger than in other ALLs. Cytogenetic study detected the typical translocations (8; 14) and (8; 22) in most of the cases. Abnormalities of the long arm of the chromosome 1 were found in four cases and a major aneuploidy was observed in one case. Cell-cycle analysis showed a high degree of proliferation with, in all cases, a small fraction of cells in G1 with low protein content (corresponding to early G1). All these biological characteristics must be related to the poor prognosis of this disease.