Oncogenes do not Fully Override Cell-intrinsic Traits: Pronounced Impact of the Cellular Programme.

Overexpression of p53 tumor suppressor protein in malignant cells induces cell cycle arrest, or alternatively, apoptosis thereby indicating that additional factors may contribute to the p53-mediated outcome. Comparison of the experimental protocols revealed that the construct encoding wild-type (wt) p53 was expressed in cells of different origin. Therefore, we decided to determine whether the intrinsic cellular program of primary cells of the same genetic background could have any effect on the oncogenic potential of mutated c-Ha-RAS and TP53. Primary rat cells (RECs) isolated from rat embryos of different age: at 13.5 gd (y) and 15.5 gd (o), were used for transfection. Immortalized rat cell clones overexpressing temperature-sensitive (ts) p53(135val) mutant and transformed cell clones after co-transfection with oncogenic c-Ha-Ras, were generated. The ts p53(135Val) mutant, switching between wt and mutant conformation, offers the possibility to study the role of p53 in cell cycle control in a model of malignant transformation in cells with the same genetic background. Surprisingly, the kinetics of cell proliferation at non-permissive temperature and that of cell cycle arrest at 32°C strongly differed between cell clones established from yRECs and oRECs. Furthermore, the kinetics of the re-enter of G1-arrested cells in the active cell cycle strongly differed between distinct cell clones. Finally, the susceptibility of immortalized and transformed cells to the pharmacological inhibitors of cyclin-dependent kinases (CDKs) considerably differed. Our results clearly show that overexpression of genes such as mutated TP53 and oncogenic c-Ha-RAS is not able to fully override the intrinsic cellular programme.

Outcome of treatment of human HeLa cervical cancer cells with roscovitine strongly depends on the dosage and cell cycle status prior to the treatment.

Exposure of asynchronously growing human HeLa cervical carcinoma cells to roscovitine (ROSC), a selective cyclin-dependent kinases (CDKs) inhibitor, arrests their progression at the transition between G(2)/M and/or induces apoptosis. The outcome depends on the ROSC concentration. At higher dose ROSC represses HPV-encoded E7 oncoprotein and initiates caspase-dependent apoptosis. Inhibition of the site-specific phosphorylation of survivin and Bad, occurring at high-dose ROSC treatment, precedes the onset of apoptosis and seems to be a prerequisite for cell death. Considering the fact that in HeLa cells the G(1)/S restriction checkpoint is abolished by E7, we addressed the question whether the inhibition of CDKs by pharmacological inhibitors in synchronized cells would be able to block the cell-cycle in G(1) phase. For this purpose, we attempted to synchronize cells by serum withdrawal or by blocking of the mitotic apparatus using nocodazole. Unlike human MCF-7 cells, HeLa cells do not undergo G(1) block after serum starvation, but respond with a slight increase of the ratio of G(1) population. Exposure of G(1)-enriched HeLa cells to ROSC after re-feeding does not block their cell-cycle progression at G(1)-phase, but increases the ratio of S- and G(2)-phase, thereby mimicking the effect on asynchronously growing cells. A quite different impact is observed after treatment of HeLa cells released from mitotic block. ROSC prevents their cell cycle progression and cells transiently accumulate in G(1)-phase. These results show that inhibition of CDKs by ROSC in cells lacking the G(1)/S restriction checkpoint has different outcomes depending on the cell-cycle status prior to the onset of treatment.

Pleiotropic effects of selective CDK inhibitors on human normal and cancer cells.

Escape from the proper control of the cell cycle by up-regulation of cyclins or aberrant activation of cyclin-dependent kinases (CDKs) as well as by inactivation of cellular inhibitors of CDKs (CKI) leads to malignant transformation. Loss of cellular CKIs in cancers provided a rationale for development of pharmacological inhibitors of CDKs. Recently synthesized CKIs, e.g., purine derivatives such as olomoucine (OLO) and roscovitine (ROSC) are non-genotoxic and exhibit increased selectivity towards CDK2 and CDK7/9. Interestingly, both drugs induce additional effects. Recently, a new, unexpected action of OLO on normal human cells was observed. OLO strongly up-regulates CLIMP-63, a 65 kD protein that mediates the anchoring of the ER to microtubules. Moreover, ROSC induces in human MCF-7 cells phosphorylation of p53 protein at Ser-46 which in turn initiates caspase-independent apoptosis. In the present contribution we raised the question whether both CKIs would be able to block cell cycle progression and to reactivate p53 protein in human HPV-positive HeLa cervix carcinoma cells. We also addressed the question whether exponentially growing cancer cells are more susceptible to the inhibitory action of CKIs than normal cells. Our results show that HeLa cells are much more sensitive to ROSC than normal fibroblasts. ROSC induces G(2) arrest and apoptosis in HeLa cells. It also reactivates and stabilizes wt p53 protein. The increase of p53 protein coincides with down-regulation of E6 oncoprotein. Thus, the biological action of substituted purines is not restricted to the inhibition of CDKs and open new perspectives for their therapeutic applications.