G1 block of the cell cycle during differentiation of F9 cells correlates with accumulation of inhibitors of the activity of cyclin-kinase complexes of proteins p21/Waf1 and p27/Kip

F9 teratocarcinoma cells have a very short duration of the cell cycle with a short G1-period typical for early embryonic cells. The cells are capable of differentiating towards parietal endoderm cells after the treatment with retinoic acid (RA) and dibutyryl-cAMP (db-cAMP). This leads to changes in the cell cycle; in particular, G1-period becomes longer, and then differentiated F9 cells leave the cycle to stay in G0-phase. It was previously reported that undifferentiated F9 cells undergo no G1 arrest of the cell cycle after DNA damage (Malashicheva et al., 2000). In the present work mechanisms of accumulation of G1-phase cells during differentiation induced by retinoic acid and db-cAMP were studied. Kinase activity of cyclin-Cdk complexes regulating the G1/S transition was analyzed. In differentiated F9 cells, the activity of cyclin-Cdk complexes, comprising Cdk4 and Cdk2 kinases and cyclins A and E, was significantly decreased. A decrease of Cdk4 kinase activity correlates with a drop of the cyclin D1 content. The amount of p21/Waf1 and p27/Kip inhibitors of the cyclin-kinase complexes increased in differentiated F9 cells. p21/Waf1 protein, which undergoes proteasomal degradation in undifferentiated F9 cells, was shown to be stable in their differentiated derivatives. Besides, in differentiated F9 cells p21/Waf1 and p27/Kip proteins can be detected with Cdk4/Cdk2-cyclin E complexes, in contrast to undifferentiated cells. Thus, we suggest that a G1/G0 block of the cell cycle taking place upon differentiation of F9 cells is likely to be caused by a decrease in cyclin-kinase activity due to stabilization and accumulation of p21/Waf1 and p27/Kip inhibitors and to their ability to associate with Cdk-cyclin complexes.     

Inhibition of the ATR/Chk1 Pathway Induces a p38-Dependent S-phase Delay in Mouse ES Cells

Ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR)kinases, family members of the PI-3 kinase related proteins, play a key role in checkpointactivation and maintenance of genomic stability following DNA damage. We have usedwild type (WT) and p38?-deficient mouse embryonic stem (ES) cells to investigate therole of ATR and ATM kinases during embryonic cell cycle. We have found thatinhibition of ATR and ATM kinases with caffeine or Chk1 with UCN-01, results inactivation of a p38-dependent intra-S-phase checkpoint and activation of apoptosis in EScells. However, wortmannin at a concentration, that inhibits ATM kinase but not ATRkinase, did not affect cell cycle progression. Furthermore, the presence of caffeine resultsin activation of p38 kinase, accumulation of p21/Waf1 in a complex with Cdk2 anddecrease of Cdk2 kinase activity. In contrast, caffeine-treated p38?-/- ES cells show lessapoptosis, and fail to trigger an effective S-phase checkpoint and accumulation ofp21/Waf1. We conclude that ATR kinase activity is essential for normal cell cycleprogression of exponentially proliferating mouse ES cells even in the absence ofexogenous DNA damage, and ATR deregulation triggers p38?-dependent cell-cyclecheckpoint and apoptotic responses.     

Activation of DNA damage response signaling in mouse embryonic stem cells

Mouse embryonic stem cells (mESC) are characterized by high proliferation activity. mESC are highly sensitive to genotoxic stresses and do not undergo G1/S checkpoint upon DNA-damage. mESC are supposed to develop sensitive mechanisms to maintain genomic integrity provided by either DNA damage repair or elimination of defected cells by apoptosis. The issue of how mESC recognize the damages and execute DNA repair remains to be studied. We analyzed the kinetics of DNA repair foci marked by antibodies to phosphorylated ATM kinase and histone H2AX (γH2AX). We showed that mESC display non-induced DNA single-strand breaks (SSBs), as revealed by comet-assay, and a noticeable background of γH2AX staining. Exposure of mESC to γ-irradiation induced the accumulation of phosphorylated ATM-kinase in the nucleus as well as the formation of additional γH2AX foci, which disappeared thereafter. To decrease the background of γH2AX staining in control non-irradiated cells, we pre-synchronized mESC at the G2/M by low concentration of nocodazol for a short time (6 h). The cells were then irradiated and stained for γH2AX. Irradiation induced the formation of γH2AX foci both in G2-phase and mitotic cells, which evidenced for the active state of DNA-damage signaling at these stages of the cell cycle in mESC. Due to the G1/S checkpoint is compromised in mESCs, we checked, whether wild-type p53, a target for ATM kinase, was phosphorylated in response to γ-irradiation. The p53 was barely phosphorylated in response to irradiation, which correlated with a very low expression of p53-target p21/Waf1 gene. Thus, in spite of the dysfunction of the p53/Waf1 pathway and the lack of cell cycle checkpoints, the mESC are capable of activating ATM and inducing γH2AX foci formation, which are necessary for the activation of DNA damage response.     

p21Waf1 is required for cellular senescence but not for cell cycle arrest induced by the HDAC inhibitor sodium butyrate

Cell senescence is characterized by senescent morphology and permanent loss of proliferative potential. HDAC inhibitors (HDACI) induce senescence and/or apoptosis in many types of tumor cells. Here, we studied the role of cyclin-kinase inhibitor p21^waf1(Cdkn1n gene) in cell cycle arrest, senescence markers (cell hypertrophy, SA-bGal staining and accumulation of gH2AX foci) in p21^Waf1+/+ versus p21^Waf1-/- mouse embryonic fibroblast cells transformed with E1A and cHa-Ras oncogenes (mERas). While short treatment with the HDACI sodium butyrate (NaB) induced a reversible G1 cell cycle arrest in both parental and p21^Waf1-/- cells, long-term treatment led to dramatic changes in p21^Waf1+/+ cells only: cell cycle arrest became irreversible and cells become hypertrophic, SA-bGal-positive and accumulated gH2AX foci associated with mTORC1 activation. The p21^Waf1+/+ cells lost their ability to migrate into the wound and through a porous membrane. Suppression of migration was accompanied by accumulation of vinculin-staining focal adhesions and Ser3-phosphorylation of cofilin, incapable for F-actin depolymerization. In contrast, the knockout of the p21^Waf1 abolished most of the features of NaB-induced senescence, including irreversibility of cell cycle arrest, hypertrophy, additional focal adhesions and block of migration, gH2AX foci accumulation and SA-bGal staining. Rapamycin, a specific inhibitor of mTORC1 kinase, decreased cellular hypertrophy, canceled coffilin phosphorylation and partially restored cell migration in p21^Waf1+/+ cells. Taken together, our data indicate a new role of p21^Waf1 in cell senescence, which may be connected not with execution of cell cycle arrest, but also with the development of mTOR-dependent markers of cellular senescence.     

Histone deacetylase inhibitor blocks proliferation of cells transformed with oncogenes E1A and cHa-ras

Rat embryonic fibroblasts, transformed with E1A and cHa-ras oncogenes, are unable to stop in the cell cycle checkpoints under growth factor withdrawal and genotoxic stresses (Bulavin et al., 1999). In the present paper, we showed that sodium butyrate, an inhibitor of histone deacetyase activity, decreased the share of cells being in S-phase, and caused G1/S and G2/M blocks of the cell cycle in the transformants. By means of RT-PCR and immunoblotting, we found that NaB significantly changed the expression of genes involved in proliferation: cyclins D1, A, E and cyclin-dependent kinases Cdk2 and Cdk4, whereas the amount of p21Waf1 and p27Kip1 inhibitors greatly increased. Along with accumulation of p21Waf1 protein content, that of Cdk2-bound p21 increases. Taken together, these data allow to suggest that NaB treatment does evidently restore the capability of p21Waf1 to inhibit cyclin-kinase activity. One may suppose that inhibition of HDAC activity by sodium butyrate leads to activation of yet unknown HDAC-dependent genes, which is followed by restoration of p21Waf1 function in spite of the E1A oncogene expression.     

The molecular mechanisms of vitamin C on cell cycle regulation in B16F10 murine melanoma

Vitamin C has inconsistent effects on malignant tumor cells, which vary from growth stimulation to apoptosis induction. It is well known that melanoma cells are more susceptible to vitamin C than any other tumor cells, but the precise mechanism remains to be elucidated. In the present study, the proliferation of B16F10 melanoma cells was suppressed by vitamin C, which induced growth arrest in a dose-dependent manner without cytotoxic effects. Therefore, we investigated the changes in cell cycle distribution of B16F10 melanoma cells by staining DNAs with propidium iodide (PI). The growth inhibition of B16F10 melanoma by vitamin C was associated with an arrest of cell cycle distribution at G1 stage. In addition, the levels of p53-p21Waf1/Cip1 increased during G1 arrest, which were essential for vitamin C-induced cell cycle arrest. The increased p21Waf1/Cip1 inhibited CDK2. Moreover, the activity of p53-p21Waf1/Cip1 pathway was closely related with the activation of checkpoint kinase 2 (Chk2). Inhibitor of the PI3K-family, LY294002 and the ATM/ATR inhibitor, caffeine, blocked vitamin C-induced growth arrest in B16F10 melanoma cells. These results suggest that vitamin C might be a potent agent to inhibit proliferative activity of melanoma cells via the regulation of Chk2-p53-p21Waf1/Cip1 pathway.     

p27Kip1 and cyclin D1 are necessary for focal adhesion kinase regulation of cell cycle progression in glioblastoma cells propagated in vitro and in vivo in the scid mouse brain

We have reported previously that the expression of focal adhesion kinase (FAK) is elevated in glioblastomas and that expression of FAK promotes the proliferation of glioblastoma cells propagated in either soft agar or in the C.B.17 severe combined immunodeficiency (scid) mouse brain. We therefore determined the effect of FAK on cell cycle progression in these cells. We found that overexpression of wild-type FAK promoted exit from G(1) in monolayer cultures of glioblastoma cells, enhanced the expression of cyclins D1 and E while reducing the expression of p27(Kip1) and p21(Waf1), and enhanced the kinase activity of the cyclin D1-cyclin-dependent kinase-4 (cdk4) complex. Transfection of the monolayers with a FAK molecule in which the autophosphorylation site is mutated (FAK397F) inhibited exit from G(1) and reduced the expression of cyclins D1 and E while enhancing the expression of p27(Kip1) and p21(Waf1). Small interfering RNA (siRNA)-mediated down-regulation of cyclin D1 inhibited the enhancement of cell cycle progression observed on expression of wild-type FAK, whereas siRNA-mediated down-regulation of cyclin E had no effect. siRNA-mediated down-regulation of p27(Kip1) overcame the inhibition of cell cycle progression observed on expression of FAK397F, whereas down-regulation of p21(Waf1) had no effect. These results were confirmed in vivo in the scid mouse brain xenograft model in which propagation of glioblastoma cells expressing FAK397F resulted in a 50% inhibition of tumor growth and inhibited exit from G(1). Taken together, our results indicate that FAK promotes proliferation of glioblastoma cells by enhancing exit from G(1) through a mechanism that involves cyclin D1 and p27(Kip1).     

Overexpression of the signaling adapter FRS2 reconstitutes the cell cycle deficit of a nerve growth factor non-responsive TrkA receptor mutant

We have characterized the cell cycle deficit of a novel TrkA receptor mutant (TrkAS3) that fails to support nerve growth factor (NGF)-dependent cell cycle arrest and neurite outgrowth. TrkAS3 receptors fail to support an NGF-dependent increase in the expression of cyclin D1 and the cell cycle inhibitor, p21(Waf1/Cip1), two important regulators of G(1) /S transition, and do not down-regulate expression of the G(2) /M phase marker, cdc2/cdk1, or the S phase marker, proliferating cell nuclear antigen. Moreover, NGF-activated TrkAS3 receptors do not down-regulate cyclin-dependent kinase 4 phosphorylation of the retinoblastoma protein, essential for G(1) arrest, in comparison to NGF-activated wild-type TrkA. Collectively these data indicate that TrkAS3 receptors fail to support NGF-dependent G(1) arrest. Interestingly, ectopic expression of regulators of G(1) /S arrest, such as cyclin D1 or inhibitors of cell cycle (p21(Waf1/Cip1), p16(INK4A) ), or the fibroblast growth factor (FGF) receptor substrate-2 (FRS2) in cells expressing TrkAS3 reconstitutes NGF-dependent neurite outgrowth. Collectively, these data suggest a model in which NGF-stimulated TrkA-dependent activation of FRS2 supports neurite outgrowth through a mechanism that likely involves the induction of p21(Waf1/Cip1) expression and the arrest of cells at G(1) /S.     

The lack of G1/S arrest of teratocarcinoma F9 cells is determinated by degradation of cyclin-dependent kinases inhibitor p21waf1/cip1

We have studied the ability of F9 teratocarcinoma cells to arrest in G1/S and G2/M checkpoints following gamma-irradiation. Wild-type p53 protein is rapidly accumulated in F9 cells after gamma-irradiation, however this is not followed by G1/S arrest; there is just a reversible delay of the cell cycle in G2/M. In order to elucidate the reasons of the lack of G1/S arrest in F9 cells we investigated the levels of regulatory cell cycle proteins: G1-cyclins, cyclin dependent kinases and kinase inhibitor p21WAF1/CIP1. We have shown that in spite of p53-dependent activation of p21WAF1/CIP1 promoter, p21WAF1/CIP1 protein is not revealed by different polyclonal and monoclonal antibodies, either by immunoblotting or by immunofluorescent staining. However, when cells are treated with specific proteasome inhibitor lactacystin, p21WAF1/CIP1 protein is revealed. We therefore suggest that p21WAF1/CIP1 protein is subjected to proteasome degradation in F9 cells and probably the lack of G1/S arrest after gamma-irradiation is due to this degradation. Thus, it is the combination of functionally active p53 with low level expression of p21WAF1/CIP1 that causes a short delay of the cell cycle progression in G2/M, rather than the G1-arrest after gamma-irradiation of F9 cells.     

Protecting genomic integrity in somatic cells and embryonic stem cells

Mutation frequencies at some loci in mammalian somatic cells in vivo approach 10(-4). The majority of these events occur as a consequence of loss of heterozygosity (LOH) due to mitotic recombination. Such high levels of DNA damage in somatic cells, which can accumulate with age, will cause injury and, after a latency period, may lead to somatic disease and ultimately death. This high level of DNA damage is untenable for germ cells, and by extrapolation for embryonic stem (ES) cells, that must recreate the organism. ES cells cannot tolerate such a high frequency of damage since mutations will immediately impact the altered cell, and subsequently the entire organism. Most importantly, the mutations may be passed on to future generations. ES cells, therefore, must have robust mechanisms to protect the integrity of their genomes. We have examined two such mechanisms. Firstly, we have shown that mutation frequencies and frequencies of mitotic recombination in ES cells are about 100-fold lower than in adult somatic cells or in isogenic mouse embryonic fibroblasts (MEFs). A second complementary protective mechanism eliminates those ES cells that have acquired a mutational burden, thereby maintaining a pristine population. Consistent with this hypothesis, ES cells lack a G1 checkpoint, and the two known signaling pathways that mediate the checkpoint are compromised. The checkpoint kinase, Chk2, which participates in both pathways is sequestered at centrosomes in ES cells and does not phosphorylate its substrates (i.e. p53 and Cdc25A) that must be modified to produce a G1 arrest. Ectopic expression of Chk2 does not rescue the p53-mediated pathway, but does restore the pathway mediated by Cdc25A. Wild type ES cells exposed to ionizing radiation do not accumulate in G1 but do so in S-phase and in G2. ES cells that ectopically express Chk2 undergo cell cycle arrest in G1 as well as G2, and appear to be protected from apoptosis.     

An Opposite Effect of the CDK Inhibitor,p18INK4c on Embryonic Stem Cells Compared with Tumor and Adult Stem Cells

Self-renewal is a feature common to both adult and embryonic stem (ES) cells, as well as tumor stem cells (TSCs). The cyclin-dependent kinase inhibitor, p18^INK4c, is a known tumor suppressor that can inhibit self-renewal of tumor cells or adult stem cells. Here, we demonstrate an opposite effect of p18 on ES cells in comparison with teratoma cells. Our results unexpectedly showed that overexpression of p18 accelerated the growth of mouse ES cells and embryonic bodies (EB); on the contrary, inhibited the growth of late stage teratoma. Up-regulation of ES cell markers (i.e., Oct4, Nanog, Sox2, and Rex1) were detected in both ES and EB cells, while concomitant down-regulation of various differentiation markers was observed in EB cells. These results demonstrate that p18 has an opposite effect on ES cells as compared with tumor cells and adult stem cells. Mechanistically, expression of CDK4 was significantly increased with overexpression of p18 in ES cells, likely leading to a release of CDK2 from the inhibition by p21 and p27. As a result, self-renewal of ES cells was enhanced. Our current study suggests that targeting p18 in different cell types may yield different outcomes, thereby having implications for therapeutic manipulations of cell cycle machinery in stem cells.     

Cell cycle dependent phosphorylation and subnuclear organization of the histone gene regulator p220(NPAT) in human embryonic stem cells

Human embryonic stem (ES) cells have an expedited cell cycle ( approximately 15 h) due to an abbreviated G1 phase ( approximately 2.5 h) relative to somatic cells. One principal regulatory event during cell cycle progression is the G1/S phase induction of histone biosynthesis to package newly replicated DNA. In somatic cells, histone H4 gene expression is controlled by CDK2 phosphorylation of p220(NPAT) and localization of HiNF-P/p220(NPAT) complexes with histone genes at Cajal body related subnuclear foci. Here we show that this 'S point' pathway is operative in situ in human ES cells (H9 cells; NIH-designated WA09). Immunofluorescence microscopy shows an increase in p220(NPAT) foci in G1 reflecting the assembly of histone gene regulatory complexes in situ. In contrast to somatic cells where duplication of p220(NPAT) foci is evident in S phase, the increase in the number of p220(NPAT) foci in ES cells appears to precede the onset of DNA synthesis as measured by BrdU incorporation. Phosphorylation of p220(NPAT) at CDK dependent epitopes is most pronounced in S phase when cells exhibit elevated levels of cyclins E and A. Our data indicate that subnuclear organization of the HiNF-P/p220(NPAT) pathway is rapidly established as ES cells emerge from mitosis and that p220(NPAT) is subsequently phosphorylated in situ. Our findings establish that the HiNF-P/p220(NPAT) gene regulatory pathway operates in a cell cycle dependent microenvironment that supports expression of DNA replication-linked histone genes and chromatin assembly to accommodate human stem cell self-renewal.