[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.

[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.

[PI 3-kinase activity is necessary for F9 mouse embryonic carcinoma cell proliferation]. / Neobkhodimost' aktivatsii PI 3-kinazy dlia proliferatsii kletok émbrional'noi kartsinomy myshi linii F9.

Embryonic carcinoma (EC) cells and embryonic stem (ES) cells have short cell cycles and, accordingly, proliferate very fast. Serum starvation does not suppress proliferation of EC and ES cells that allows to assume independence of their proliferation from the activity of cascades induced by serum. In the present work, we used flow cytometry to investigate how specific MAP-kinase and PI3-kinase inhibitors may influence proliferation and cell cycle of EC F9 cells. It is established that inhibitors of ERK-, JNK- and p38-kinases do not suppress EC F9 cell proliferation. It is possible to assume that proliferation of EC cells is supported by constitutive activity of down-stream cell cycle regulators, for example, E2F1 transcription factor. Since PI3-kinase inhibitor LY294002 causes reduction of S-phase and accumulation of G1-phase F9 cells, PI3-kinase mediated cascades seem to be constantly activated and involved in phosphorylation of important cell cycle regulators. The analysis of transcription of immediate-early genes in undifferentiated cells has shown that c-fos and c-jun genes are strongly activated by serum, and that ERK-kinase plays the main role in activation of c-fos transcription, while activation of c-jun transcription depends predominantly on p38-kinase. It is necessary to note that PI3-kinase inhibitor increases effect of serum stimulation of c-fos promoter. It means that the PI3-kinase dependent cascade negatively influences the cascade, which activates c-fos transcription. Thus, the transcription of c-fos and c-jun is not connected with of EC F9 cell proliferation. The proliferation of these cells depends on PI3-kinase activity.

[Transcription of c-fos gene and DNA binding activity of transcription factor AP-1 increase upon differentiation of mouse F9 teratocarcinoma cells]. / Transkriptsiia gena c-fos i DNK-sviazyvaiushchaia aktivnost' transkriptsionnogo faktora AP-1 uvelichivaiutsia v rezul'tate differentsirovki kletok émbrional'noi kartsinomy myshi linii F9.

Retinoic acid (RA) causes differentiation of mouse F9 embryonic carcinoma cell line into primitive and parietal (with dibutiril-cAMP) endoderm. The role of AP-1 transcription factor during RA-induced differentiation was studied in F9 cell line. It was shown that differentiated cells acquired protein complexes, which are specifically bound to well characterized AP-1 32P-labeled binding sites from collagenase (Col-AP-1) and c-jun (Jun2-AP-1) promoters. These complexes contain c-Fos/c-Jun with Col-AP-1 site and c-Jun/ATF-2 with Jun2-AP-1 site as revealed by supershift analysis. DNA-binding activity of these complexes is high in parietal endoderm but low-detectable in undifferentiated cells. DNA-binding activity of AP-1 transcription factor correlates with increased expression of c-fos and c-jun genes. RT-PCR analysis showed an increase in steady-state level of c-fos and c-jun gene transcription at the stage of parietal endoderm (terminally differentiated F9 cells). Transcription of immediate early c-fos and c-jun genes and DNA-binding activity of c-Fos/c-Jun complex are serum dependent. The rate of c-fos and c-jun gene transcription and DNA-binding activity of c-Fos/c-Jun complex decreased in serum-starved cells, but was rapidly induced upon stimulation with serum. Undifferentiated F9 cells contain a very low level of c-fos mRNA, with may be a consequence of repressive chromatin structure in promoter region. Histone deacetylase (HDAC) activity is necessary to restrict expression of specific number of genes, also HDAC inhibitors are well known inductors of differentiation and anticancer agents. Frow cytometry analysis showed a decreased rate of proliferation of F9 cells after their incubation with HDAC inhibitors, sodium butirate and trichostatin A. Also, these ihibitors induced the transcription of c-fos gene. So, we conclude that HDAC activity may be necessary to sustain a high proliferative rate of undifferentiated F9 cells.

[Noninduced single-stranded breaks in DNA in murine F9 teratocarcinoma cells]. / Neindutsirovannye odnonitevye razryvy v DNK kletok linii F9 teratokartsinomy myshi.

Our previous study demonstrated the high incidence of non-induced DNA single strand breaks (SSB) in preimplantation mouse embryo genom (Patkin et al., 1994). F9 mouse teratocarcinoma cell line is an in vitro model for early embryonal differentiation, since F9 cells remind in many respects the inner cell mass cells of mouse blastocyst and are capable of differentiation under retinoic acid (RA) and dibutyryl cAMP (db-cAMP) treatment. Using gap filling reaction of F9 metaphase chromosomes and single-cell DNA electrophoresis, we have observed multiple SSB in undifferentiated F9 cells as well as in F9 cells at the early steps of RA-induced differentiation (days of RA treatment), but not in terminally differentiated F9 cells and in mouse embryonal fibroblasts. Rad51 nuclear protein that binds specifically single stranded DNA is highly expressed in all cells of undifferentiated F9 population and is not expressed in terminally differentiated F9 population. Multiple SSB could lead to enhanced rate of sister chromatid exchanges (SCE) in F9 cells. In undifferentiated F9 population the level of SCE was 9.6 +/- 0.44 per metaphase, that was not higher than in NIH 3T3 cell line. However, RA treatment for 48 h led to rising the SCE level up to 16.68 +/- 0.72 followed by its decrease to the initial rate by 72 h of RA treatment. Since the enhanced level of SSB in undifferentiated F9 cells and in mouse blastocyst does not normally lead to chromosomal instability, we consider SSB to be a natural consequence of fast-going DNA replication in these cells.

[Differentiated murine F9 teratocarcinoma cells are susceptible to apoptosis upon contact with substrate]. / Differentsirovannye kletki teratokartsinomy myshi linii F9 vstupaiut v apoptoz pri razryve kontakta s substratom.

Epithelial and endothelial cells are susceptible to a subset of apoptosis known as anoikis. This type of programmed cell death is activated upon disruption of cell-substrate contacts. Here we demonstrate that mouse F9 embryonal carcinoma cell line acquires susceptibility to anoikis upon retinoic acid-induced differentiation towards non-malignant pariental endoderm-like cells. F9 cells survival becomes dependent on the substrate by the 4th day of retinoic acid treatment, when cells assume epithelial phenotype as revealed by actin, alpha-actinin and vinculin expression and distribution, and when focal adhesion contacts are formed. Differentiated F9 cells die in suspension by apoptosis as revealed by oligonucleosomal DNA laddering, DAPI staining and DNA flow cytometry analysis. On the contrary, undifferentiated F9 cells form large multicellular aggregates in suspension and survive. Thus, F9 cell line provides a new model to study pathways involved in both anoikis induction and inhibition.