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.
Increased expression and activity of proteins driving cell cycle progression as well as inactivation of endogenous inhibitors of cyclin-dependent kinases (CDKs) enhance the proliferative potential of cells. Escape of cells during malignant transformation from the proper cell cycle control rendering them independent from growth factors provides rationale for therapeutic targeting of CDKs. Exposure of rapidly growing human MCF-7 breast cancer and HeLa cervix cancer cells to roscovitine (ROSC), a selective inhibitor of CDKs, inhibits their proliferation by induction of cell cycle arrest and/or apoptosis. The outcome strongly depends on the intrinsic traits of the tumor cells, on their cell cycle status prior to the onset of treatment and also on ROSC concentration. At lower dose ROSC primarily inhibits the cell cycle-related CDKs resulting in a strong cell cycle arrest. Interestingly, ROSC arrests asynchronously growing cells at the G(2)/M transition irrespective of the status of their restriction checkpoint. However, the exposure of cancer cells synchronized after serum starvation in the late G(1) phase results in a transient G(1) arrest only in cells displaying the intact G(1)/S checkpoint. At higher dosage ROSC triggers apoptosis. In HeLa cells inhibition of the activity of CDK7 and, in consequence, that of RNA polymerase II is a major event that facilitates the initiation of caspase-dependent apoptosis. In contrast, in the caspase-3-deficient MCF-7 breast cancer cells ROSC induces apoptosis by a p53-dependent pathway. HIPK2-mediated activation of the p53 transcription factor by phosphorylation at Ser46 results in upregulation of p53AIP1 protein. This protein after de novo synthesis and translocation into the mitochondria promotes depolarization of the mitochondrial membrane.
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