Aksenov Nd

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 p21waf1(Cdkn1n gene) in cell cycle arrest, senescence markers (cell hypertrophy, SA-bGal staining and accumulation of gH2AX foci) in p21Waf1+/+ versus p21Waf1-/- 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 p21Waf1-/- cells, long-term treatment led to dramatic changes in p21Waf1+/+ cells only: cell cycle arrest became irreversible and cells become hypertrophic, SA-bGal-positive and accumulated gH2AX foci associated with mTORC1 activation. The p21Waf1+/+ 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 p21Waf1 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 p21Waf1+/+ cells. Taken together, our data indicate a new role of p21Waf1 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.

Deregulation of p53/p21Cip1/Waf1 pathway contributes to polyploidy and apoptosis of E1A+cHa-ras transformed cells after γ-irradiation

The p53/p21Cip1/Waf1-dependent checkpoint control of G1/S and G2/M phases of the cell cycle in response to DNA damage is an important mechanism of genome stability maintenance in normal cells. In many tumor cells, due to frequent point mutations and deletions of p53, the stringent control of the cell cycle and apoptosis is compromised. We have examined the cell cycle control and cell death of the rat embryo fibroblast cells (REF) transformed by E1A+cHa-ras oncogenes and expressing wild type p53. Gamma-irradiation at a dosage of 6 Gy has been used to analyse the p53-dependent trans-activation of the target p21cip1/waf1 gene and the levels of activity of cyclin-dependent kinases. Our results show that the cell cycle inhibitors p21Cip1/Waf1 and p27KIP accumulate in response to irradiation both in REF and E1A+cHa-ras cells. In contrast to normal REF cells, the accumulation of p21Cip1/Waf1 and p27KIP inhibitors, however, does not lead to inhibition of Cdk2 and cyclins E, A-associated kinase activities and to a G1/S block in E1A+cHa-ras cells. It is unlikely that the lack of inhibitory function of p21Cip1/Waf1 can be explained by its inability to bind Cdk2 and Cdk4 kinases or PCNA. Moreover, the p21Cip1/Waf1-associated kinase activity is increased upon γ-irradiation of E1A+cHa-ras cells. We suggest that inactivation of p21Cip1/Waf1 may be accounted for by its interaction with E1A oncoproducts as the inhibitor is detected in immunoprecipitates using E1A-specific antibodies. During a temporary G2/M delay induced by γ-irradiation, E1A+cHa-ras transformants continue DNA replication, which leads to accumulation of polyploid cells with lobulated nuclei and micronuclei. Thus, DNA damage of E1A+cHa-ras transformed cells, with a combination of functionally active wild type p53 and inactive p21Cip1/Waf1, contributes to formation of polyploid cells which then die due to apoptosis.

By blocking apoptosis, Bcl-2 in p38-dependent manner promotes cell cycle arrest and accelerated senescence after DNA damage and serum withdrawal.

E1A+ras-transformed rodent fibroblasts are unable to be arrested in the cell cycle and die by apoptosis in response to cytostatics, ionizing radiation (IR), or serum withdrawal. Overexpression of the human antiapoptotic gene bcl-2 suppresses apoptosis and induces reversible cell cycle arrest after IR or serum withdrawal and cell senescence after adriamycin treatment. Bcl-2-sustained adriamycin-induced cell senescence requires p38 MAPK, since the knockout of p38 MAPK abrogated anti-apoptotic and senescence-inducing effects of Bcl-2 in adriamycin-treated cells. Moreover, resistance to apoptosis and cell cycle arrest were not observed in p38 -/- E1A+ras+bcl-2-transformants following IR or serum deprivation. However, the pro-apoptotic effect of nocodazole in E1A+ras-transformed cells can not be prevented by Bcl-2 overexpression independently of the presence of p38 MAPK. These results allow us to conclude that p38 is necessary for Bcl-2-induced inhibition of apoptosis, induction of cell cycle arrest and accelerated senescence after DNA damage and serum starvation, but not after nocodazole treatment.

Evidence for the existence of satellite DNA‐containing connection between metaphase chromosomes

Physical connections between mitotic chromosomes have been reported previously. It was assumed that the interchromosome connection was based on the DNA‐protein thread. However, the data about DNA sequences and protein component in the thread is fragmentary. We demonstrated on the mouse cultured cell line and prematurely condensed chromosomes that: (a) all four mouse satellite DNA fragments (major and minor satellite, mouse satellite 3 (MS3) and mouse satellite 4 (MS4)) were involved in the thread formation; (b) MS4 was involved in the thread to the least extent among all the other fragments; (c) telomere was never a member of the thread; (d) the thread was synthesized at a late G2 phase; (e) RNA helicase p68 and CENP‐B were among the protein components of the interchromosome connection. It was shown by FACS analysis that in mouse and human cell lines: (1) the flow karyotype spectrums were never free from chromosome aggregates; (2) chromosome association did not depend on the chromosome length and each chromosome was free to associate with the other. J. Cell. Biochem. 101: 1046–1061, 2007.

Reactive Oxygen Species Are Required for Human Mesenchymal Stem Cells to Initiate Proliferation after the Quiescence Exit.

The present study focuses on the involvement of reactive oxygen species (ROS) in the process of mesenchymal stem cells “waking up” and entering the cell cycle after the quiescence. Using human endometrial mesenchymal stem cells (eMSCs), we showed that intracellular basal ROS level is positively correlated with the proliferative status of the cell cultures. Our experiments with the eMSCs synchronized in the G0 phase of the cell cycle revealed a transient increase in the ROS level upon the quiescence exit after stimulation of the cell proliferation. This increase was registered before the eMSC entry to the S-phase of the cell cycle, and elimination of this increase by antioxidants (N-acetyl-L-cysteine, Tempol, and Resveratrol) blocked G1–S-phase transition. Similarly, a cell cycle arrest which resulted from the antioxidant treatment was observed in the experiments with synchronized human mesenchymal stem cells derived from the adipose tissue. Thus, we showed that physiologically relevant level of ROS is required for the initiation of human mesenchymal stem cell proliferation and that low levels of ROS due to the antioxidant treatment can block the stem cell self-renewal.

Hepatitis C virus core protein transforms murine fibroblasts by promoting genomic instability

The oncogenic potential of hepatitis C virus (HCV) core protein has been demonstrated, but the precise mechanism of cell transformation triggered by HCV core is still unclear. This study shows that constitutive expression of HCV core protein (core) in NIH 3T3 murine fibroblasts triggers malignant transformation. At the preneoplastic stage, clones that expressed HCV core constitutively demonstrated genomic instability seen as disruption of the mitotic spindle cell checkpoint leading to increased ploidy. Transformation was completed by the loss of DNA and resistance to apoptosis induced by serum starvation. Simultaneously, cells acquired a capacity for anchorage independent growth and absence of contact inhibition. Inoculation of these transformed cells into severe combined immune deficiency (SCID) mice led to formation of solid core-expressing tumors. Transformation and tumorigenicity of core-expressing cell lines coincided with a 5- to 10-fold repression of endogenous p53 transactivation. Thus, long-term HCV core expression alone is sufficient for complete transformation of immortal fibroblasts that can then induce tumors in a susceptible host. This data suggests that malignant transformation by HCV core may occur through primary stress, induction of genomic instability, and further HCV core-induced rescue of surviving mutated cells.

Intracellular oxidation of hydroethidine: compartmentalization and cytotoxicity of oxidation products.

Hydroethidine (HE) is a blue fluorescent dye that is intracellularly converted into red-emitting products on two-electron oxidation. One of these products, namely 2-hydroxyethidium, is formed as the result of HE superoxide anion-specific oxidation, and so HE is widely used for the detection of superoxide in cells and tissues. In our experiments we exploited three cell lines of different origin: K562 (human leukemia cells), A431 (human epidermoid carcinoma cells), and SCE2304 (human mesenchymal stem cells derived from endometrium). Using fluorescent microscopy and flow cytometry analysis, we showed that HE intracellular oxidation products accumulate mostly in the cell mitochondria. This accumulation provokes gradual depolarization of mitochondrial membrane, affects oxygen consumption rate in HE-treated cells, and causes cellular apoptosis in the case of high HE concentrations and/or long cell incubations with HE, as well as a high rate of HE oxidation in cells exposed to some stimuli.

Different transformation pathways of murine fibroblast NIH 3T3 cells by hepatitis C virus core and NS3 proteins

The oncogenic potential of both Hepatitis C virus (HCV) core and HCV NS3 proteins has been demonstrated, but these proteins induce transformation of immortal murine fibroblasts NIH 3T3 via different pathways. As long‐term expression (50–100 passages) of HCV core triggers neoplastic transformation of NIH 3T3 through crisis of growth, HCV NS3 induces transformation shortly after transfection. We explain this distinction by different effects of core and NS3 on p53‐mediated transactivation: inhibition by NS3 and activation by core protein.

Redox environment in stem and differentiated cells: A quantitative approach

Stem cells are believed to maintain a specific intracellular redox status through a combination of enhanced removal capacity and limited production of ROS. In the present study, we challenge this assumption by developing a quantitative approach for the analysis of the pro- and antioxidant ability of human embryonic stem cells in comparison with their differentiated descendants, as well as adult stem and non-stem cells. Our measurements showed that embryonic stem cells are characterized by low ROS level, low rate of extracellular hydrogen peroxide removal and low threshold for peroxide-induced cytotoxicity. However, biochemical normalization of these parameters to cell volume/protein leads to matching of normalized values in stem and differentiated cells and shows that tested in the present study cells (human embryonic stem cells and their fibroblast-like progenies, adult mesenchymal stem cells, lymphocytes, HeLa) maintain similar intracellular redox status. Based on these observations, we propose to use ROS concentration averaged over the cell volume instead of ROS level as a measure of intracellular redox balance. We show that attempts to use ROS level for comparative analysis of redox status of morphologically different cells could lead to false conclusions. Methods for the assessment of ROS concentration based on flow cytometry analysis with the use of H2DCFDA dye and HyPer, genetically encoded probe for hydrogen peroxide, are discussed.

HDAC inhibitor sodium butyrate sensitizes E1A+Ras-transformed cells to DNA damaging agents by facilitating formation and persistence of γH2AX foci.

HDAC inhibitors (HDACi) suppress the growth of tumor cells due to induction of cell cycle arrest, senescence or apoptosis. Recent data demonstrate that HDACi can interfere with DNA Damage Response (DDR) thereby sensitizing the cells to DNA damaging agents. Here, we show that HDACi sodium butyrate (NaBut) potentiates the formation of γH2AX foci predominantly in S-phase E1A+Ras cells. Accumulation of γH2AX foci sensitizes the cells toward such DNA damaging agents as irradiation (IR) and adriamycin. In fact, 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. Indeed, NaBut treatment for 24 h 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 γH2AX foci unrepaired. However, the IR-induced γH2AX foci persist after long-term NaBut treatment (72 h) even after washing the drug. Thus, although signaling pathways regulating H2AX phosphorylation in NaBut-treated cells remain to be investigated, the obtained results show that NaBut potentiates effects of DNA damaging agents by facilitating formation and persistence of γH2AX foci.