Roscovitine up-regulates p53 protein and induces apoptosis in human HeLaS(3) cervix carcinoma cells.

Exposure of human HeLaS(3) cervix carcinoma cells to high doses of conventional cytostatic drugs, e.g. cisplatin (CP) strongly inhibits their proliferation. However, most cytostatic agents are genotoxic and may generate a secondary malignancy. Therefore, therapeutic strategy using alternative, not cytotoxic drugs would be beneficial. Inhibition of cyclin-dependent kinases (CDKs) by pharmacological inhibitors became recently a promising therapeutic option. Roscovitine (ROSC), a selective CDK inhibitor, efficiently targets human malignant cells. ROSC induces cell cycle arrest and apoptosis in human MCF-7 breast cancer cells. ROSC also activates p53 protein. Activation of p53 tumor suppressor protein is essential for induction of apoptosis in MCF-7 cells. Considering the fact that in HeLaS(3) cells wt p53 is inactivated by the action of HPV-encoded E6 oncoprotein, we addressed the question whether ROSC would be able to reactivate p53 protein in them. Their exposure to ROSC for 24 h induced cell cycle arrest at G(2)/M and reduced the number of viable cells. Unlike CP, ROSC in the used doses did not induce DNA damage and was not directly cytotoxic. Despite lack of detectable DNA lesions, ROSC activated wt p53 protein. The increase of p53 levels was attributable to the ROSC-mediated protein stabilization. Further analyses revealed that ROSC induced site-specific phosphorylation of p53 protein at Ser46. After longer exposure, ROSC induced apoptosis in HeLaS(3) cells. These results indicate that therapy of HeLaS(3) cells by ROSC could offer an advantage over that by CP due to its increased selectivity and markedly reduced risk of generation of a secondary cancer.

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.