ANALYSIS OF TRANSIENT G₁/S ARREST IN E1A + E1B-19kDa TRANSFORMED CELLS AFTER IONIZING RADIATION

A. I. Brichkina,¹ N. D. Aksenov, V. A. Pospelov, T. V. Pospelova

Institute of Cytology RAS, St. Petersburg, Russia;
¹ e-mail: aibri@mail.ru

Expression of human adeno virus type 5 E1A oncogene in normal rodent cells leads to disruption of the G₁/S cell cycle arrest realization in response to DNA damage. It has been shown here that rat embryo fibroblasts transformed by E1Aad5 oncogene in complementation with E1B-19kDa gene realize the irradiation-in-duced transient G₁/S arrest, which depends on selective suppression of CyclinE-Cdk2 activity despite functional inactivation of p21^Wafl inhibitor. Inhibitor p21^Wafl is not revealed in complexes with cyclins E and A in E1A + E1B-19kDa transformants, however, it is not due to p21^Wafl interaction with E1A oncoproteins, because the E1A-p21Wafl complex formation in E1A + cHa-ras transformants does not prevent the high level of CyclE, A-p21^Wafl association. In the case of p21^Wafl inactivaton, the main way of cyclin-kinase activity regulation in E1A + E1B-19kDa cells may be Cdk2 phosphorylation. However, irradiation of E1A + E1B-19kDa transformed cells induces no changes in CAK (Cdk7-associated) kinase activity and in the protein level of Cdc25A phosphatase, which are responsible for activating Thrl60 phosphoralation and Tyrl5 dephosphorylation on Cdk2. Using phospho-Tyr15-Cdk2 specific antibodies, no increase of phosphorylation at Tyrl5 position on immunoprecipitated Cdk2 was detected after irradiation. It seems likely that in the case of inactivated inhibitor p21^Wafl the transient G₁/S block after irradiation in E1A + E1B-19kDa transformants depends on suppression of Cycl-E-Cdk2 activity caused by inhibition of Thrl60 Cdk2 phosphorylation, but this occurs with the involvement of other kinases rather than CAK.

Key words:  oncogenes E1A and E1B-19kDa, G₁/S cell cycle block, CAK, Cdc25A phosphatase, inhibitor p21^Wafl

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