[AICAR-Dependent Activation of AMPK Kinase Is Not Accompanied by G1/S Block in Mouse Embryonic Stem Cells].

Embryonic stem cells (ESCs) have the capacity for self-renewal and pluripotency. Due to high proliferative activity, ESCs use a specific pathway of the formation of ATP molecules, which can lead to the development of the adaptive metabolic response under the conditions of energy deficiency (which is different from the response of differentiated cells). It is known that metabolic signals are integrated with the cell cycle progression; however, the signaling pathways that connect the availability of nutrients with the regulation of cell cycle in ESCs are insufficiently studied. We have studied the effect of the AICAR agent, which imitates an increase in AMP level and induces the activation of the metabolic sensor AMPK, on proliferation, cell cycle distribution, and pluripotency of mouse ESCs (mESCs). It has been demonstrated that cells treated with AICAR do not stop at the control G1/S point of the cell cycle, since they do not accumulate P21/WAF1 (G1/S checkpoint regulator), despite P53 activation. On the contrary, AICAR increases the rate of mESC proliferation, which correlates with increased expression of pluripotency marker genes (OCT3/4, NANOG, SOX2, KLF4, ESRRB, PRDM14). In addition, an increase in the transcription of the HIFlα gene (a key regulator of the cell proliferation and viability, as well as glucose metabolism under stress) was detected. An increase in the expression of glycolytic enzyme genes (LDHA, ALDOA, PCK2, GLUT4) under the effect of AICAR indicates a change in mESC metabolism towards increased glycolysis. Thus, AICAR-dependent AMPK activation as one of possible mechanisms of the mESC adaptive response to the emergence of energetic imbalance is not accompanied by a cell cycle arrest at the G1/S checkpoint, but involves the processes of increasing glycolytic activity.

Overexpression of Adenoviral E1A Sensitizes E1A+Ras-Transformed Cells to the Action of Histone Deacetylase Inhibitors.

The adenoviral E1A protein induces cell proliferation, transformation, and tumor formation in rodents, on the one hand. On the other hand, E1A expression increases cell sensitivity to a number of cytotoxic agents. Therefore, E1A is a candidate for use as a component of combination therapy for malignant tumors. The highest augmentation in the cytotoxic effect was achieved by a combined use of E1A expression and histone deacetylases (HDAC) inhibitors. However, HDAC inhibitors do not induce apoptosis in cells transformed with E1A and cHa-ras oncogenes. In this study, it was shown that HDAC inhibitors reduce the expression of adenoviral E1A. However, under unregulated E1A overexpression, these cells undergo apoptosis in the presence of HDAC inhibitors. Treatment with a HDAC inhibitor, sodium butyrate (NaBut), was shown to activate the anti-apoptotic factor NF-kB in control cells. However, NaBut was unable to modulate the NF-kB activity in E1A overexpressed cells. Therefore, it is fair to postulate that cells transformed with E1A and cHa-ras oncogenes avoid the apoptosis induced by HDAC inhibitors thanks to a NaBut-dependent decrease in E1A expression.

Leukemia inhibitory factor (LIF) withdrawal activates mTOR signaling pathway in mouse embryonic stem cells through the MEK/ERK/TSC2 pathway.

Leukemia inhibitory factor (LIF) is indispensable to maintain the pluripotent state of mouse embryonic stem cells (ESCs), but the mechanisms underlying the role of LIF/STAT3 pathway are yet poorly understood. Here we first showed that the LIF/STAT3-regulated signaling pathway contributes to the maintenance of self-renewal and pluripotency of mouse ESCs by suppressing mTOR (mammalian target of rapamycin), which is necessary for early differentiation. When LIF is withdrawn from culture medium, the mTOR activity rapidly increases as detected by phosphorylation of its targets - ribosomal protein S6 and translation factor 4EBP1. In turn, suppression of STAT3 phosphorylation on Tyr-705 by a specific small molecule WP1066 also activates phosphorylation of the mTOR target S6 ribosomal protein. LIF removal strongly activates ERK activity indicating that ERK can be involved in either direct phosphorylation of mTOR or phosphorylation of an upstream negative regulator of mTOR - TSC1/TSC2 proteins. According to western blotting data, LIF withdrawal leads to phosphorylation of TSC2 protein thereby relieving its negative effect on mTOR activity. mTOR activation is accompanied by a decrease of pluripotent gene expression Oct-4, Nanog, Sox2 and by an augmentation of fgf5 gene expression - a marker of post-implantation epiblast. Together, these data indicate that LIF-depleted mouse ESCs undergo a transition from the LIF/STAT3-supported pluripotent state to the FGFR/ERK-committed primed-like state with expression of early differentiation markers mediated through activation of mTOR signaling.

INFLUENCE OF HDAC INHIBITOR SODIUM BUTYRATE ON THE EXPRESSION OF DNA REPAIR GENES Rad51 AND XRCC5 IN mEras-Waf1+/+ AND mEras-Waf1­/­.

Mouse embryonal fibroblasts with knockout of CDKN1A gene encoding p21/Waf1 protein transformed by oncogenes E1A and cHa-ras (mEras-Waf1­/­ cell line) have been used to assess the level of DNA repair genes expression ­ Rad51 and XRCC5 after treatment with HDAC inhibitor sodium butyrate as compared with their control counterparts (mEras-Waf1+/+ cells). mEras-Waf1­/­ cells are characterized by the elevated amount of single-stranded DNA breaks and g-H2A.X histone foci associated with these breaks. According to immunofluorescence and immunobloting data, Rad51 and Ku80 proteins are highly expressed in the nuclei of both studied cell lines. The level of Ku80 is higher in cells with CDKN1A gene knockout. When cells were treated with DNA-damaging agent adriamycin, there was an additional accumulation of Rad51 foci in the nuclei. However, sodium butyrate reduced considerably the content of Rad51 and Ku80 proteins both in mEras-Waf1+/+ and mEras-Waf1­/­ cells as well as in the cells treated by adriamycin. RT-PCR and immunobloting data show that inhibitory effect of sodium butyrate takes place at the level of Rad51 and XRCC5 gene transcription and the content of Rad51 and Ku80 proteins. The observed suppressive effect of HDACI on DNA repair components explains in part the mechanisms of antiproliferative function of HDAC inhibitors. Surprisingly, sodium butyrate was shown to activate the pluripotent genes transcription in mEras-Waf1+/+ and mEras-Waf1­/­ cells, as exemplified by upregulation of Oct-4, Sox-2, Klf4, implying that these pluripotent genes are under negative control at the level of chromatin structure.

MEK/ERK-PATHWAY IS REQUIRED TO MAINTAIN CYTOPROTECTIVE AUTOPHAGY PROCESS IN IRRADIATED E1A+cHa-Ras TRANSFORMANTS.

Autophagy is a conservative process of misfolded protein and damaged organelle degradation that serves to support cellular viability. Autophagy is often induced in response to stress, DNA damage, retinoids, starvation and growth factor withdrawal. The aim of the present work was to study autophagic response of E1A+cHa-Ras-transformed cells to irradiation and to analyze the role of MEK/ERK pathway in regulation of autophagy induced by irradiation. MEK/ERK suppression has been found to decrease the viability of irradiated cells. Inhibition of MEK/ERK pathway leads to the changes in the autophagy induced by irradiation connected with disturbances of final stages followed by accumulation of adaptor protein p62/SQSTM1 in autophagic cavities within cytoplasm. Thus, the data obtained allow to suggest that active MEK/ERK pathway is required to support, the cytoprotective autophagy which is induced in response to irradiation of transformed E1A+cHa-ras cells.

HDAC inhibitors induce apoptosis but not cellular senescence in Gadd45α-deficient E1A+Ras cells.

HDAC inhibitors (HDIs) induce irreversible cell cycle arrest and senescence in E1A+Ras expressing cells. Furthermore, HDIs activate Gadd45α/NF-κB signaling pathway to suppress apoptosis thereby promoting the cell survival. Here, to clarify the role of Gadd45α in realization of the antiapoptotic program, we compared wild-type E1A+Ras cells and the cells with knockout of gadd45α gene (Gadd45α-/- cells). As in Gadd45α-expressing E1A+Ras cells, HDIs induce irreversible cell cycle arrest in Gadd45α-/- cells, but the arrested cells do not senesce and eventually die due to activation of the apoptotic death program. These data suggest that the expression of Gadd45α is involved in maintaining the balance of pro- and anti-apoptotic stimuli, while lack or loss of Gadd45 directs the cells to apoptosis after HDIs treatment. Appropriately Gadd45α-deficient cells demonstrate a higher level of pro-apoptotic signals, whereas the anti-apoptotic program is suppressed. The elevated apoptotic background of Gadd45α-/- cells is accompanied by higher levels of Ser15-phosphorylated p53 and p21/Waf1 proteins that additionally commit the cells to HDIs-induced apoptosis. Additionally, loss of Gadd45α protein activates the DDR signaling pathway as demonstrated by nuclear pATM staining, accumulation of γH2AX foci and an increase of single-strand DNA breaks. Thus, in wild-type E1A+Ras cells the p53-dependent expression of Gadd45α is necessary not only for DNA repair and HDI-induced cellular senescence, but also to withstand to apoptosis after DNA damage and stress. Therefore the use of HDIs in combination with agents that block Gadd45α function may have promise for cancer therapy.

[Analysis of irradiation-induced repair foci in mouse embryonic stem cells].

Somatic cells in response to DNA damage activate two important protective mechanisms: G1 checkpoint control and a program for recognizing and repairing DNA defects (DDR signaling). Both mechanisms are triggered by the activation of common sensor kinases ATM and ATR, which in turn phosphorylate downstream targets. Mouse embryonic stem cells (mESCs) lack of G1 checkpoint and undergo only temporary G2 delay after DNA damage. We have analyzed the ability of mESCs to detect DNA damage and to form repair foci after irradiation. We showed irradiation-induced activation of ATM and ATR is followed by formation of γH2AX foci co-localized with DNA repair proteins Rad51, DNA-PK and adapter protein 53BP1. Furthermore, we checked contribution of ATM/Chk2 and ATR/Chk1 cascades to cell cycle control and viability of mESCs after DNA damage. Inhibition of ATR/Chk1 cascade leads to accumulation of G1 phase cells, whereas perturbation of ATM/Chk2 activity causes no such effect. Moreover, inhibition of ATR/Chk1 activity, but not ATM/Chk2, substantially augments the killing effect of ionizing radiation on mESCs. In summary, our results indicate that mESCs are capable of recognizing DNA damage and forming repair foci, but their DDR signaling it seems to be distinct from somatic cells and tightly connected with maintaining of pluripotency and self-renewal.

[ATM/ATR signaling pathway activation in human embryonic stem cells after DNA damage].

Embryonic stem cells (ESCs) are the progenitors of all adult cells so any disruption in their genome can have disastrous consequences for the developing organism. ESCs are characterized by a high proliferation activity and do not undergo checkpoints upon DNA-damage executing only G2/M delay after DNA damage. ATM and ATR kinase are key sensors of DNA double strands breaks and activate downstream signaling pathways involving checkpoints, DNA repair and apoptosis. We estimated ATM/ATR signaling pathway activation in human ESCs and have revealed that irradiation induced ATM, ATR Chk2 phosphorylation, γH2AX foci formation and their co-localization with 53BP1 and Rad51 proteins. Interestingly, human ESCs display non-induced yH2AX foci co-localized with Rad51 and marking DNA single-strand breaks. Next we have revealed the substantial contribution of ATM, Chk1 and Chk2 kinases to G2/M block after irradiation of human ESCs and ATM-dependent activation (phosphorylation) of p53. However p53 activation and subsequent induction of p21 gene expression after DNA damage do not result in p21 protein accumulation due to proteasomal degradation.

[Protein phosphatase MKP-1 participates in c-fos gene derepression under the action of stress factors on fibroblasts transformed with E1A and cHA-ras oncogenes].

Immediate-early response gene c-fos expression is repressed and not activated after serum stimulation of serum-starved fibroblasts transformed with E1A and cHa-ras oncogenes. We have previously shown that such stress factors as an anisomycin are able to activate c-fos gene transcription in E1A + cHa-ras transformants, wherein MEK/ERK signal pathway plays a major role in the activation. In the present paper, we investigated the role of MKP-1-dependent regulation of c-fos gene by dephosphorylation of ERK kinases. It has been shown that MKP-1 gene transcription in E1A + ras transformants is activated by anisomycin for a maximum of 1 h, and then a reduction in the level of transcription occurs. Use of inhibitors of MAP-kinase has revealed that MKP-1 gene transcription depends on MEK/ERK and JNK kinase cascades, but not om p38 cascade. The anisomycin-induced c-fos gene transcription intensified after transfection of siRNA MKP-1 into the cells. Thus, protein phosphatase MKP-1 carries a negative regulation of c-fos gene transcription by dephosphorylation of ERK kinases that are a key signal component under the action of such stress reagent as anisomycin on the E1A + ras-transformed cells.

Cyclin-dependent kinase inhibitor p21(Waf1): contemporary view on its role in senescence and oncogenesis.

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.

[Cell senescence induced by histone deacetylase inhibitor sodium butyrate in rodent transformed cells resistant to apoptosis].

The capacity of HDAC inhibitor sodium butyrate to induce senescence in cells derived from rat embryonic fibroblasts transformed by E1A+E1B19 kDa oncogenes has been studied. These transformants are resistant to apoptosis in response to gamma-irradiation and growth factor deprivation. The process of cell senescence was investigated by the analysis of cell growth curves, G1/S and G2/M cell cycle arrest, and senescent associated beta-galactosidase expression. The irreversibility of sodium butyrate antiproliferative activity was analyzed by clonogenic assay. We show that sodium butyrate suppresses proliferation and induces senescence in the E1A+E1B19 kDa transformed cells. Interestingly, NaB induces growth arrest due to accumulation of cells in G2/M phase, these cells are not tetraploid but mainly binuclear. Thus, in case of NaB induced senescence in E1A+E1B19 kDa transformed fibroblasts, the observed suppression of cell proliferation may be the result of cytokinesis failure leading to formation of binuclear and multinuclear cells incapable to proliferate.

p21(Waf1) 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-ßGal staining and accumulation of γH2AX 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 G(1) 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-ßGal-positive and accumulated γH2AX 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, γH2AX foci accumulation and SA-ßGal 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 only with execution of cell cycle arrest, but also with the development of mTOR-dependent markers of cellular senescence.

HDAC inhibitor-induced activation of NF-κB prevents apoptotic response of E1A+Ras-transformed cells to proapoptotic stimuli.

HDAC inhibitors (HDACIs) are capable of suppressing the cell growth of tumour cells due to the induction of apoptosis and/or cell cycle arrest. This allows of considering HDACIs as promising agents for tumour therapy. The final outcome - apoptotic cell death or cell cycle arrest - depends on the type of tumour and cellular context. In this report, we addressed the issue by analysing effects produced in E1A+Ras-transformed MEF cells by HDAC inhibitors sodium butyrate (NaB), Trichostatin A (TSA) and some others. It has been shown that the HDACIs induced cell cycle arrest in E1A+Ras-transformed cells but not apoptosis. The antiapoptotic effect of HDACIs is likely to be a result of NF-κB-dependent signaling pathway activation. HDACI-induced activation of NF-κB takes place in spite of a deregulated PI3K/Akt pathway in E1A+Ras cells, suggesting an alternative mechanism for the activation of NF-κB based on acetylation. HDACI-dependent activation of NF-κB prevents the induction of apoptosis by cytostatic agent adriamycin and serum deprivation. Accordingly, suppression of NF-κB activity in HDACI-arrested cells by the chemical inhibitor CAPE or RelA-siRNA resulted in the induction of an apoptotic programme. Thus, our findings suggest that the activation of the NF-κB pathway in HDACI-treated E1A+Ras-transformed cells blocks apoptosis and may thereby play a role in triggering the programme of cell cycle arrest and cellular senescence.

[Effect of histone deacetylases inhibitor sodium butyrate (NaB) on transformants E1A + cHa-Ras expressing wild type p53 with supressed transactivation function].

Induction of cellular senescence by various antitumour agents is a promising strategy of cancer treatment. We assessed the ability of sodium butyrate (NaB), a histone deacetylase inhibitor (HDACi), to reactivate the cellular senescence program in either E1A + cHa-Ras-transformed rat embryo fibroblasts with wild-type p53 (ERas(WT)) and in the isogenic cell line where p53 was inactivated due to expression of the potent genetic suppressor element GSE56 (ERas(GSE56)). NaB treatment increased p53 transcriptional activity and induced an irreversible G1/S cell cycle arrest in ERas(WT), but not in ERas(GSE56) cells. By the transient transfections method using reporter luciferase (p53-LUC) constructions, it was shown that p53-LUC activity as a marker of p53 transactivation function did not increase after X-rays exposure of transformants ERas(GSE56). p53 activity in transformants ERas(WT) increased both after irradiation or upon NaB treatment. Interestingly, the expression of senescence-associated beta-galactosidase (SA-beta-Gal), widely used as a marker of senescence, as well as loss of clonogenic ability, were observed in both cell lines following NaB treatment. Thus, our results suggest that induction of p53 transcription activity could be the key determinant of HDACi-induced cell cycle arrest and senescence in transformed cells and provide an additional evidence of SA-beta-Gal invalidity as a sufficient senescence marker.

[Sodium butyrate do not induce the program of premature senescence in transformants with JNK1,2 knockout].

We studied the role of JNK1,2 stress-kinases in the regulation of premature senescence program, stimulated by the inhibitor of histone deacetylase, sodium butyrate (NaB). It was found, that the transformants EIA + cHa-ras selected from embryonic mouse fibroblasts with knockout jnk1,2 stress-kinase genes did not block the cell cycle after sodium butyrate treatment. The data on the cell cycle distribution and cell growth curves showed that even long term (during five days) NaB influence did not suppress proliferation. We did not also reveal any cellular hypertrophy and increase in SA-beta-galactosidase activity after NaB treatment. The data presented suggest that JNK stress-kinases are involved in sodium butyrate-induced senescence in E1A + cHa-Ras mouse transformants, and they are indicative of that JNK1,2 have tumor suppressor properties.

[Signaling pathways regulating proliferation of murine embryonic stem cells].

Murine embryonic stem cells (mESC) are capable of unlimiting proliferation with maintenance of pluripotency during long-term cultivation. Signaling pathways regulating the cell cycle of mESC are of the great interest for further investigation. This review concerns to the cell cycle regulation of mESC through different signaling pathways (LIF-STAT3, PI3K-Akt, Wnt-beta-catenin) and to the mechanisms of unlimited proliferation of mESC and their inability to undergo long-term block of proliferation in response to DNA-damaging and stress factors. The functioning of negative cell cycle regulators (cyclin-kinase inhibitors and Rb) and positive cell cycle regulators (cyclin-kinase complexes and E2F factors) are also topics of this review. It is considered that, permanent mitogenic stimuli are needed to prevent induction of apoptosis. Therefore, the agents which cause prolonged halt of proliferation without ongoing onset of differentiation or induction of apoptosis are currently unknown. The main focus is given to the role of the Wnt signaling pathway in sustaining the pluripotent state of mESC. The cell cycle regulation by downstream targets of LIF-STAT3, PI3-kinase and Wnt-beta-catenin pathways is discussed in light of cooperative action of these pathways for maintenance of undifferentiated state of mESC.

[Role of p38alpha kinase in activation of premature senescence program in transformed mouse fibroblasts].

We investigated the role of p38alpha stress-kinase in regulation of premature senescence program, stimulated by histone deacetylase inhibitor--sodium butyrate (NaB)--after application to rodent transformed cell lines. Investigation was performed on the E1A + cHa-ras transformants selected from mice embryonic fibroblasts null at the p38alpha kinase gene or null fibroblasts at the PPM1D gene, which encoded phosphatase Wip1. Absence of Wip1 led to constitutive activation of p38alpha kinase. It was revealed that after NaB treatment both cell lines completely stopped proliferation due to irreversible cell cycle arrest in G1/S phase. In both cell lines sodium butyrate induced sustained block of prolifaration due to irreversible cell cycle arrest in G1/S phase. Following sodium butyrate treatment cells expressed marker of senescence--beta-galactosidase activity (SA-beta-Gal). Long-term (during several days) NaB treatment of cells led to partial restoration of actin cytoskeleton, focal adhesion contacts and heterochromatin focus formation (SAHF) in the nucleus of senescent cells. Obtained data allow us to suppose that irreversible process of cellular senescence activated by sodium butyrate can occur in the absence of functionally active p38 kinase by means of other ways of cell cycle suppression.

[Anti-proliferative effect of histone deacetylase inhibitors on murine embryonic stem cells].

The effect of histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butirate (NaBut) on the proliferation of murine embryonic stem cells (MESC) was studied. Both agents suppressed the population growth and clonability of MESC. Flow cytometry analysis showed a decrease in the amount of S-phase cells upon treatment with HDAC inhibitors. TSA treatment caused a decrease in mRNA level of such positive cell cycle regulators as cyclins D1, A, c-myc, cdc25A, and induced transcription of negative regulators of the cell cycle--p21(Wafl) and p57(kip2). Also, HDAC inhibitors decreased the level of e2f-dependent transcription, with the concominant reduction of mRNA level of e2fl gene. HDAC inhibitors also affected the survival of MESC. A 2 day TSA treatment resulted in massive detachment and cell death, as confirmed by DNA laddering and MTT assay. Treatment with TSA for 2 and 5 days did not induce SAPbetaGAL, activity and p16(ink4a) transcription, i.e., characteristic features of senescent fibroblasts. In summary, HDAC inhibitors decrease the rate of proliferation affecting cell cycle and viability of MESC. We conclude that MESC are unable to realize a sustainable block of the cell cycle upon treatment with HDAC inhibitors.

[PI3-kinase inhibitors LY294002 and wortmannin have different effects on proliferation of murine embryonic stem cells].

Murine embryonic stem (mES) cells can proliferate independently of the presence of growth factors in the medium. It is yet unknown what intrinsic activity triggers cell cycle events in mES cells. Here we investigated the contribution of the PI3-kinase cascade to autonomous proliferation of mES cell using PI3-kinase inhibitors wortmannin and LY294002. Wortmannin displays a weaker inhibitory effect on phosphorylation of PI3-kinase pathway target PKB as compared with LY294002, and does not downregulate mES cells proliferation, while LY294002 causes a strong decrease in the share of cells in S-phase and accumulation of cells in G1 phase. Both inhibitors cause significant decrease in cyclin D1 amount. The treatment with LY294002, rather than with wortmannin results in a decrease of cyclin E amount and cyclin E-assossiated kinase activity. In mES cells, inactivation of PI3-kinase-dependent pathway and G1 arrest are not accompanied by induction of p27kip 1 transcription and accumulation of this inhibitor of cyclin-cdk complexes at the protein level, implying that these events accomplished by some p27kip 1-independent mechanism. Both LY294002 and wortmannin cause apoptotic death of mES cells and downregulate the growth of population. Thus, inactivation of PI3-kinase in mES cells may lead to apoptosis rather than to cell cycle arrest.