- Checkpoint control and DNA damage response signaling pathways in embryonic stem cells
Embryonic stem cells (ESC), in contrast to somatic cells, fail to stop proliferation after removal of serum growth factors and in response to DNA damaging and stress factors
and subsequently die due to programmed cell death (apoptosis). Although ES cells express wild type p53, an important cell cycle regulator and "guardian of the genome", the
expression level of a p53 and its target gene p21/Waf, which codes for inhibitor of cyclin-kinase complexes p21Waf1 protein, is very low. Moreover, the p21Waf1 protein is not
accumulated in response to DNA-damaging agents and stress factors, being degraded by an ubiquitin-proteosome pathway. ES cells are tolerant to relatively high level of
uninduced single-stranded DNA breaks detected in these cells by antibody to phosphorylated histone H2AX (γH2AX). Nevertheless, the uninduced H2AX foci are not a signal
for activation of a sensor ATM kinase, which is however activated after DNA damage. The observed tolerancy can be accounted for to some extent by a dysfunction of
tumor-suppressor p53/p21Waf1/Chk2 pathway, which is indispensable for checkpoint control and subsequent DNA repair in somatic cells. In spite of the absence of G1/S and
G2/M checkpoints, ES cells maintain genome stability and the capability for differentiation due to elimination of cells with genetic defects by apoptosis. Studying the functional
state of ATM/Chk2- and ATR/Chk1-regulated DNA Damage Response (DDR) signaling as well as the role of dysfunction of p53/p21Waf1 pathway for maintenance of ESC
pluripotency is currently under investigation.
- Molecular mechanisms of mammalian cell transformation by viral and cellular oncogenes
A collection of stable, immortalized, and transformed cell lines has been established by transfection of viral (E1A, E1B) and cellular oncogenes (cHa-Ras) into
primary rodent embryonic fibroblasts as well as into cells with knockout of the key cell cycle-controlling genes: p53, p21Waf1, p16/p19, gadd45 and on stress-kinases
p38 and JNK. The process of transformation was shown to be associated with a dysfunction of the programs controlling cell cycle, apoptosis, and premature cell
senescence. Viral oncogenes display transforming activity by inactivation of negative regulators of the cell cycle - tumor-suppressor proteins p53 and pRb as well as inhibitor
of cyclin-dependent kinases p21Waf1 by forming with them inactive complexes. Cells transformed by oncogenes E1A and Ras are characterized by
serum growth factor-independent proliferation due to constitutive activation of MAP kinase cascades, constitutive activity of transcription factors AP-1 and NF-kB, and
replacement of AP-1 transcription complexes from Fos/Jun to Jun/ATF2. These transformed cells have a high and constitutive activity of cyclin-kinase complexes, therefore
they are unable to realize G1/S and G2/M arrest of the cell cycle and the mitotic checkpoint after DNA-damaging and stress factors, and subsequently
die by apoptosis. The model cell lines are very suitable for studying regulation of cell cycle, apoptosis and cellular senescence of transformed cells.
- Epigenetic mechanisms of gene regulation in transformed cells
Oncogenic transformation results to significant changes of expression of positive and negative cell cycle regulators. It has been shown that transcription of early response genes,
such as protooncogene c-fos, which contributes to AP-1 formation, is being repressed and is not activated by serum growth factors. Analysis of mechanisms of
c-fos down-regulation in transformed cells demonstrates that in the region of SRE/AP-1 element of c-fos promoter the formation of a compact inactive nucleosome
structure takes place. This is provided by recruitment of histone deacetylase (HDAC) activity that eventually leads to histone deacetylation and the nucleosome compaction.
Correspondingly, inhibitors of HDAC activity induce histone acetylation, nucleosome unfolding and activation of c-fos transcription. Detailed analysis of nucleosome-bound
histones by in situ chromatin immunoprecipitation (ChIP-assay) shows that the c-fos promoter-associated nucleosome located between CRE and SRE elements within
the c-fos promoter has constitutively modified H3 histone diacetylated on Lys-9/14 and phosphorylated on Ser-10 even under conditions when cells are serum starved and
c-fos is not transcribed. Thus, in spite of relaxed nucleosome conformation, which is competent for transcription, the c-fos promoter-bound nucleosome is silenced
not only through recruitment of HDAC activity but also through a changed balance of kinase/phosphatase activities associated with the transcription factors. This follows from
data that anisomycin, a stress-inducing agent, induces c-fos transcription via activation of ERK kinase by elevating radical oxygen species (ROS) level. In vitro kinase
assay data demonstrate that anisomycin increases phosphorylation of transactivation domain of Elk-1 transcription factor - a key regulator of inducible c-fos transcription.
The activating effect of anisomycin on c-fos transcription can be abrogated by a prior treatment with ROS scavenger N-acetyl-L-cysteine. Thus, stress-factors potentiate
generation of ROS, which, in turn, can modulate the activity of MAP kinase-specific phosphatases (MKPs) thereby modulating gene transcription.
- Molecular and cellular mechanisms of histone deacetylase (HDAC) inhibitors
HDAC inhibitors induce p21Waf1-dependent and p21Waf1-independent cell cycle arrest in E1A + Ras-transformed cells. The cell cycle arrest is accompanied by decreased
expression of positive cell cycle regulators: cyclins A, D, E, c-myc, and cdc25A. Besides, e2f1 gene transcription was found to be down-regulated in part via activation of
Wnt signaling pathway. Data obtained evidence that e2f1 expression is regulated through the Wnt/Tcf pathway: e2f1 promoter activity is inhibited by sodium butyrate
(NaBut) and by overexpression of β-catenin/Tcf. The e2f1 promoter was found to contain two putative Tcf-binding elements: the proximal element competes well
with canonical Tcf element in DNA-binding assay. Being inserted into a luciferase reporter vector, the identified element provides positive transcriptional regulation in response
to β-catenin/Tcf co-transfection and NaB treatment. Thus, e2f1 belongs to a set of genes regulated through Wnt/β-catenin/Tcf pathway.
Depending on the cellular context, HDAC inhibitors can induce cell cycle arrest or apoptosis in tumor cells. The antiapoptotic effect of HDACIs in E1A + Ras is likely
to be a result of activation of NF-κB-dependent signaling pathway. 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 HDACIs-arrested cells by the chemical inhibitor CAPE or
RelA-siRNA resulted in the induction of an apoptotic program. This suggests 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 program of cell cycle arrest and cellular senescence. The results provide possibility to predict to some extent the
output of HDAC inhibitor treatment, arrest or apoptosis, depending on the level of NF-κB activity.
- HDAC inhibitors as new cancer drugs: sensibilization to DNA-damaging agents
HDAC inhibitors (HDACi) suppress the growth of tumor cells due to induction of cell cycle arrest, senescence or apoptosis. Recent data demonstrate that HDACIs can
interfere with DNA Damage Response (DDR) thereby sensitizing the cells to DNA damaging agents. Firstly, it has been shown that HDACI sodium
butyrate alone potentiates the formation of γH2AX foci predominantly in S-phase of E1A + Ras cells. Accumulation of γH2AX foci sensitizes the cells towards such
DNA damaging agents as irradiation (IR) and adriamycin. NaBut potentiates the persistence of γH2AX foci induced by genotoxic agents. The synergizing effects depend
on DNA damaging factors and on the order of NaBut treatment. Short-term NaBut treatment leads to an accumulation of G1-phase cells and a lack of S-phase cells,
therefore, adriamycin, a powerful S-phase-specific inhibitor, when added to NaBut-treated cells, is unable to substantially add γH2AX foci. In contrast, IR produces both
single- and double-strand DNA breaks at any stage of the cell cycle and was shown to increase γH2AX foci in NaBut-treated cells. Further, a lifetime of IR-induced
γH2AX foci depends on the subsequent presence of HDACi. Correspondingly, NaBut withdrawal leads to the extinction of IR-induced γH2AX foci. This necessitates
HDACi to hold the IR-induced H2AX foci unrepaired. However, the IR-induced γH2AX foci persist after long-term NaBut treatment even after washing the drug. Thus,
although signaling pathways regulating H2AX phosphorylation in NaBut-treated cells are to be investigated in more detail, combination of HDACIs and antitumor drugs increases
their effectiveness by facilitating formation and persistence of γH2AX foci.
- Cellular senescence as a powerful antitumor mechanism
Most types of tumor cells do not execute G1/S and/or G2/M checkpoints due to dysfunction of p53-signaling pathway. There is a large body of data
showing that HDAC inhibitors are able to stop proliferation and induce cell cycle arrest or apoptosis in tumor cells with various defects of p53 pathway. Currently, HDAC
inhibitors are considered as promising drugs for antitumor therapy. HDAC inhibitors induce chromatin remodeling in those loci where HDAC-dependent repression of genes took
place, in particular, of tumor suppressor genes, which are negative cell cycle regulators. Among others, HDAC inhibitors activate transcription of p21Waf1 gene by a
p53-indpendent manner. Long-term treatment of transformed cells causes reactivation of cellular senescence program, which is often compromised in tumor cells. Cellular
senescence is accompanied by cell hypertrophy, irreversible cell cycle arrest and development of various senescence markers such as SA-βGal staining. In addition,
HDACI-induced senescent cells reveal accumulation of γH2AX foci, a marker of DNA repair, in spite of HDACIs by themselves do not cause double strand DNA breaks.
There are two important findings: (1) p21Waf1 is indispensable for realization of irreversible cell cycle arrest and cellular senescence, (2) HDAC inhibitors activate mTOR
signaling. As rapamycin, an inhibitor of mTORC1 activity, abrogates both irreversible cell cycle arrest and cellular senescence, this implies that HDACI-induced senescence
can be mediated through mTORC1 signaling pathway. The ongoing researches are directed towards elucidation of mTOR-dependent mechanisms of HDAC-induced cellular
senescence in various tumor cell lines. In turn, use of non-toxic mTOR inhibitors (rapamycin and its rapalogs) opens a very promising approach to prevent cellular senescence
and development of age-related diseases, including cancer.
Traditional methods of molecular and cell biology including cultivation of normal, immortalized and cancer cells, establishment of new cell lines after transformation with
oncogenes, analysis of gene expression by RT PCR, immunoblotting, confocal immunoflurescence, in vitro kinase assay, luciferase assay, analysis of apoptotic cell death,
two-parametric flow cytometry.
The work of the Laboratory was supported by grants of the Russian Foundation for Basic Research RFBR), grant of the Russian Academy of Sciences "Molecular and
Cell Biology", International grants of the European Scientific Foundation (ESF), National Cancer Institute (NCI NIH), INTAS, NATO, CRDF, the Program of St.Petersburg
State University "Biomedicine and Human Health", grants of St. Petersburg's Government for young scientists.
- Laboratory of Prof. Peter Herrlich, Institute of Age Research, Jena, Germany;
- Laboratory of Prof. Albert J. Fornace, Georgetown University, Wasington D.C., USA;
- Laboratory of Prof. Michael Blagosklonny, Department of Cell Stress Biology; Roswell Park Cancer Institute; BLSC; Buffalo, NY USA.
- Faculty of Medicine and Pharmacy, University of Burgundy, 21078 Dijon, France (Dr. Oleg Demidov and Dr. Anastasia Goloudina).
- Department of Histology and Cytology (Biological Faculty St.Peterburg State University) in the frame of the Program "Biomedicine and Human Health".
- N.N. Petrov Institute of Oncology (Laboratory headed by prof. V.N. Anisimov).
Pospelov V.A., Pospelova T.V., Julien J.P. (1994) AP-1 and Krox-24 transcription factors activate the NF-L gene promoter in P19 embryonal
teratocarcinoma cells. Cell Growth and Differ. 5: 187-196.
Orlovskaia EI, Kisliakova TV, Osipov KA, Malashicheva AB, Pospelov VA. (1997) Functional status of the p53 gene in murine F9 embryonal
teratocarcinoma cells. Mol Biol (Mosk). 31(2): 224-31.
Pospelova T.V., Medvedev A.V., Kukushkin A.N., Svetlikova S.B., van der Eb, A.J. Dorsman J.C., Pospelov V.A. (1999) E1A+cHa-ras transformed
rat embryo fibroblast cells are characterized by high and constitutive DNA binding activities of AP-1 dimers with significantly altered composition. Gene Expression 8: 19-32.
Bulavin D.V., Tararova N.D., Aksenov N.D., Pospelov V.A., Pospelova T.V. (1999) Deregulation of p53/Rb pathway does contribute to polyploidy and
apoptosis of E1A+cHa-ras transformed cells after γ-irradiation. Oncogene. 18: 5611-5619.
Malashicheva A.B., Kislyakova T.V., Aksenov N.D., Osipov K.A., Pospelov V.A. (2000) F9 embryonal carcinoma cells fail to stop at G1/S after
γ-irradiation due to p21waf1/cip1 degradation. Oncogene. 19: 3858-3865.
Kisliakova TV, Kustova ME, Lianguzova M.S., Malashicheva A.B., Strunnikova M.A., Suchkova I.O., Pospelov V.A., Patkin E.L.(2000)
Noninduced single-stranded breaks in DNA in murine F9 teratocarcinoma cells. Tsitologiya. 42 (11): 1060-1068 (in Russian).
Are A.F., Galkin V.E., Pospelova T.V., Pinaev G.P. (2000) The p65/RelA subunit of NF-kappaB interacts with actin-containing structures. Exp Cell Res.
256 (2): 533-544.
Smirnova I.S., Yakovleva T.K., Rosanov Y.M., Aksenov N.D., Pospelova T.V. (2001) Analysis of p53-dependent mechanisms in the maintenance of genetic
stability in diploid tumourigenic line SK-UT-1B of human uterine leiomyosarcoma. Cell Biol Int. 25 (11): 1101-1115.
Malashicheva A.B., Kisliakova T.V., Savatier., Pospelov V.A. (2002) Embryonic stem cells do not undergo cell cycle arrest upon exposure to
damaging factors. Tsitologiya. 44 (7): 643-648.
Kukushkin A.N., Abramova M.V., Svetlikova S.B., Darieva Z.A., Pospelova T.V., Pospelov V.A. (2002) Downregulation of c-fos gene in
E1A+cHa-ras-transformed cells is mediated through constitutive activation of MAP kinase cascades and recruitment of histone de-acetylase activity. Oncogene. 21: 719-730.
Goloudina A.R., Yamaguchi H., Chervyakova D.B., Appela E., Fornace Jr. A.J., Bulavin D.V. (2003) Regulation of human Cdc25A stability by
Serine 75 phosphorylation is not sufficient to activate S-phase checkpoint. Cell Cycle. 2 (5): 473-478.
Abramova M.V., Kukushkin A.N., Svetlikova S.B., Pospelov V.A. (2003). Selective repression of c-fos gene transcription in rat embryo fibroblasts
transformed by oncogenes E1A and cHa-ras. Biochem Biophys Res Comm. 306: 483-487.
Usenko T.N., Kukushkin A.N., Pospelova T.V., Pospelov V.A. (2003) Downregulation of c-fos gene revealed in the established E1A+cHa-ras-
transformed cells can be reproduced upon transient transfection of E1A and Ras oncogenes into non-transformed REF52 cells. Oncogene. 22 (48): 7661-7667.
Nelioudova A.M., Tararova N.D., Aksenov N.D., Pospelov V.A., Pospelova T.V. (2004). Restoration of G1/S arrest in E1A+cHa-ras-transformed
cells by Bcl-2 overexpression. —ell Cycle. 3 (11): 1427-1432.
Chuykin I.A., Lianguzova M.S., Pospelov V.A. (2006) Beta-catenin does not show nuclear location in undifferentiated murine embryonic stem cells.
Dokl Acad Sci. 411: 524-526 (in Russian).
Lianguzova M.S., Chuykin I.A., Nordheim A., Pospelov V.A. (2007) Phosphoinositide 3-kinase inhibitor LY294002 but not serum withdrawal
suppresses proliferation of murine embryonic stem cells. Cell Biol Int. 31 (4): 330-337.
Nelyudova A.M., Aksenov N.D., Pospelov V.A., Pospelova T.V. (2007) By blocking apoptosis, Bcl-2 in p38-dependent manner promotes cell
cycle arrest and accelerated senescence after DNA damage and serum withdrawal. Cell Cycle. 6 (17): 2171-2177.
Chuykin I.A., Lianguzova M.S., Pospelova T.V., Pospelov V.A. (2008) Activation of DNA damage response signaling in mouse embryonic stem cells.
Cell Cycle. 7 (18): 2922-2928.
Kukushkin A.N., Svetlikova S.B., Amanzholov R.A., Pospelov V.A. (2008) Anisomycin abrogates repression of protooncogene c-fos transcription in
E1A+cHa-ras-transformed cells through activation of MEK/ERK kinase cascade. J Cell Biochem. 103: 1005-1012.
Demidenko Z.N., Zubova S.G., Bukreeva E.I., Pospelov V.A., Pospelova T.V., ¬lagosklonny M.V. (2009) Rapamycin decelerates cellular senescence.
Cell Cycle. 8 (12): 1888-1895.
Pospelova T.V., Demidenko Z.N., Bukreeva E.I., Pospelov V.A., Gudkov A.V., Blagosklonny M.V. (2009) Pseudo-DNA damage response in
senescent cells. Cell Cycle. 8 (24): 4112-4118.
Abramova M.V., Zatulovskiy E.A., Svetlikova S.B., Kukushkin A.N., Pospelov V.A. (2010) E2f1 gene is a new member of Wnt/beta-catenin/Tcf-regulated
genes. Biochem Biophys Res Comm. 391: 142-146.
Abramova M.V., Zatulovskiy E.A., Svetlikova S.B., Pospelov V.A. (2010) HDAC inhibitor-induced activation of NF-kappaB prevents apoptotic response of
E1A+Ras-transformed cells to proapoptotic stimuli. Int J Biochem Cell Biol. 42: 1847-1855.
Sineva G.S., Pospelov V.A. (2010) Inhibition of GSK3beta enhances both adhesive and signaling activities of beta-catenin in mouse embryonic stem cells.
Biol Cell. 102: 549-560.
Romanov V.S., Abramova M.V., Svetlikova S.B., Bykova T.V., Zubova S.G., Fornace Jr. A.J., Pospelova T.V., Pospelov V.A. (2010).
p21Waf1 is required for cellular hypertrophy and cytoskeleton reorganization in the course of cellular senescence induced by HDAC inhibitor sodium butyrate.
Cell Cycle. 9: 3945-3955.
Romanov V.S., Bardin A.A., Zubova S.G., Bykova T.V., Pospelov V.A., Pospelova T.V. (2011) p21(Waf1) is required for complete oncogenic
transformation of mouse embryo fibroblasts by E1Aad5 and c-Ha-ras oncogenes. Biochimie. 93: 1408-1414.
Abramova M..V, Svetlikova S.B., Kukushkin A.N., Aksenov N.D., Pospelova T.V., Pospelov V.A. (2011) HDAC inhibitor sodium butyrate
sensitizes E1A+Ras-transformed cells to DNA damaging agents by facilitating formation and persistence of H2AX foci. Cancer Biology & Therapy (accepted).
Pospelova T.V., Shitikova J.V., Pospelov V.A. (2011) Assessement of morphological features of senescence. In: Methods in Molecular Biology,
Humana Press, USA.