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Inhibition of p34^cdc2 Kinase Activity by Etoposide or Irradiation as a Mechanism of G₂ Arrest in Chinese Hamster Ovary Cells¹

The J. Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky 40292

The mammalian homologue of the yeast cdc2 gene product, p34^cdc2, is a cell cycle-regulated protein essential for mitosis. We have used polyclonal antisera raised against a peptide corresponding to the carboxyl terminus of the sequence of human cdc2 to study p34^cdc2 in Chinese hamster ovary (CHO) cells. Major bands are immunoprecipitated at a molecular weight of 34,000, although not in the presence of competing antigenic peptide. p34^cdc2 is coimmunoprecipitated with proteins of molecular weights of 52,000 and 57,000. Immunoprecipitates express histone H1 kinase activity which varies throughout the cell cycle, maximal activity being observed in G₂-M. The activity of the p34^cdc2 kinase varies according to its association with the M_r 52,000 and 57,000 proteins and according to their phosphorylation state. Treatment of either asynchronous CHO cells or an enriched G₂ population with the antitumor agent, etoposide, results in rapid inhibition of immunoprecipitated p34^cdc2 kinase activity, which is not due to a direct effect of drug upon the enzyme. p34^cdc2 kinase activity recovers as cells arrest in G₂ and a second etoposide treatment further inhibits p34^cdc2 kinase activity and prolongs G₂ arrest. Exposure of asynchronous CHO cells to -irradiation also inhibits p34^cdc2 kinase activity within 1 h. Again this activity recovers as cells accumulate in G₂. These results suggest that DNA damage in CHO cells elicits a response which results in inhibition of p34^cdc2 kinase activity and, consequently, G₂ arrest.

¹ This work was supported by funds from the NIH (USPHS Grant CA-24586).

² To whom requests for reprints should be addressed.

³ Present address: The College of Medicine, University of Florida, Gainesville, FL 32610.

Received 10/18/89. Revised 2/14/90.

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