Rat embryo fibroblasts transformed by complementation with oncogenes E1A+E1B-19 and E1A+cHa-ras differ in the ability to realize the G1/S block in serum free media

The capability of REF cells transformed by EA + E1B-19 kDa and EA + cHa-ras oncogenes to realize the G1/S cell cycle arrest upon serum starvation was studied. The amount of cyclin-kinase inhibitor protein p27/Kip was shown to increase in both normal and transformed cells. However, the p27/Kip-bound cyclin-kinase complexes of transformed cells were found to be active, implying the functional inactivation of p27/Kip inhibitor. Nevertheless, in contrast to E1A + cHa-ras transformants, E1A + E1B-19 kDa transformants undergo the G1 cell cycle arrest. The G1 cell cycle block correlates with the decrease in cyclinE-Cdk2 activity. Since cyclinE-Cdk2 complexes need Thr-160 phosphorylation of Cdk2 by CAK-kinase for full activity, we have analysed the Cdk-7 associated activity upon serum starvation using gst-Cdk2 as a substrate. Serum starvation did not affect CAK activity either in E1A + cHa-ras or in E1A + E1B-19 kDa transformants. Thus, selective suppression of cyclineE-Cdk2 activity in E1A + E1B-19 kDa transformants upon serum starvation does not arise from the action of cyclin-kinase inhibitors, or from change in CAK activity.     

Function of an inhibitor of cyclin-dependent kinase p27/Kip in cells transformed by E1A + E1B19 kDa + E1A + cHa-Ras, differing in their ability to realize a G1-block during serum starvation

We studied the capability of E1A + cHa-ras and E1A + E1B19kDa transformants to undergo the G1/S arrest of the cell cycle following depletion of serum growth factors. It has been shown that serum starvation induced the G1/S arrest both in normal rat embryo fibroblasts (REF) and in E1A + E1B19kDa transformants, whereas E1A + cHa-ras transformed cells lost this feature. To analyse the mechanisms underlying these differences, we studied the expression of p27/KIP, its intracellular distribution and association with E1A oncoproducts. The content of the p27/KIP inhibitor of cyclin-dependent kinases was found to change a little upon transformation by two complementary oncogene pairs. However, serum starvation for 24 h led to a significant increase in the content of p27/KIP in E1A + E1B19kDa transformants, while E1A + cHa-ras cells accumulated p27/KIP less markedly. According to the immunofluorescence study, the p27/KIP inhibitor is located in the nucleus of both normal and transformed cells. Moreover, serum starvation did not lead to its inhibition due to redistribution to the cytoplasm in both cell lines. Also, we were unable to detect association of p27/KIP with E1A oncoproducts in immunoprecipitated complexes. The obtained data indicate that, in contrast to E1A + cHa-ras transformants, in E1A + E1B19kDa cells the p27/KIP inhibitor is functional and it is capable of inducing the G1/S block after serum starvation.     

Specific regulated endonuclease activity of small RNP, containing prosomes from the A431 cell line: possible mechanisms of regulating the stability of RNA by epidermal growth factor

A comparative study was made of reactive oxygen species (ROS) in rat embryo fibroblasts and their transformants. Primary rat embryo fibroblasts (REF), REF transformed by the complementing oncogenes E1A plus cHa-ras (cell line E1A + Ras), and REF transformed by E1A plus E1B-19 kDa (cell line E1A + E1B) were studied. ROS generation was measured with microfluorometric assay using fluorescent probe 2',7'-dichlorofluorescin diacetate. It has been shown that the block of REF and E1A + 1B cells in the G1/S under serum-starved conditions (0.5% serum) for 24-48 h was paralleled by a decrease in ROS generation. Activation of serum-starved REF and E1A + 1B cells with 10% serum resulted in reactivation of cell cycle and gradual increase in ROS generation. The maximum intracellular level of ROS correlated in time with the phase of DNA synthesis. Serum-starved E1A + Ras cells were not stopped in the G1/S and ROS production of these cells was not dependent on serum growth factors. The prolonged cultivation of E1A + Ras cells in the medium with low serum content (0.5%) caused a sharp increase in ROS generation, which was accompanied by apoptotic death.     

Anti-apoptotic and antiproliferative effect of bcl-2 gene transferred to E1A+cHa-ras-transformed cells

Transformed rat embryo fibroblasts E1A + cHa-ras known to possess high proapoptotic sensitivity and not to be arrested after DNA damage or upon serum starvation, were transfected with bcl-2 gene using calcium-phosphate precipitation method. Triple transformants E1A + cHa-ras + bcl-2 appeared to be protected from damage- and serum depletion-induced apoptosis and to restore cell cycle checkpoint control. Using the method of flow cytometry we have shown that these transformants are arrested in different phases of cell cycle in response to irradiation, adriamycin treatment and serum deprivation. Overexpression of bcl-2 in E1A + cHa-ras-transformed cells entirely suppresses adriamycin-induced apoptosis and significantly reduces the level of apoptosis triggered by irradiation and growth factor withdrawal, as we have revealed by the test of clonogenic survival and electrophoretic analysis of oligonucleosomal DNA fragmentation. Our results have demonstrated, for the first time, that the oncogenic Ras co-immunoprecipitates with transfected Bcl-2 in E1A + cHa-ras + bcl-2 transformed cells after irradiation but not after adriamycin treatment. Bcl-2-Ras complexes were also observed in transformants E1A + cHa-ras + bcl-2 after serum starvation. Taken together, these data suggest that Bcl-2 and Ras interaction might play a crucial role in the cell cycle checkpoints restoration and apoptotic events regulation in transformants E1A + cHa-ras + bcl-2 exposed to DNA-damaging factors or growth factor-deprived.     

The c-fos proto-oncogene promotor is not regulated by serum, epidermal growth factor, and phorbol ester in embryonal fibroblasts transformed by E1Aad5+cHa-ras-oncogenes]

Regulation of c-fos protooncogene activity in rat embryonal fibroblasts (REF), E1Aad5-immortalized REF cells, and E1Aad5 + cHa-ras transformed REF cells has been investigated. The analysis of regulation of fos-promoter activity was done by means of transient and stable transfection of fos-CAT plasmid into immortalized and transformed cells. In parallel, the regulation of cellular c-fos as well as c-jun and c-myc genes expression has been studied. It has been found that in E1Aad5 + cHa-ras-transformed cells the expression of c-fos promoter has a constitutive, non-inducible character while in REF cells and cells immortalized by E1Aad5 the fos-promoter can be regulated by serum growth factors, EGF, and TPA.     

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.     

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.