THE EFFECT OF RADIATION ON G(2) BLOCKS, CYCLIN-B EXPRESSION AND CDC2 EXPRESSION IN HUMAN SQUAMOUS CARCINOMA CELL-LINES WITH DIFFERENT RADIOSENSITIVITIES

The purpose of the present study was to investigate the role of cyclin B and cdc2 in the G(2) delay and to test whether the magnitude of the G(2) delay correlated with sensitivity to ionizing radiation in two human cell lines. Cell cycle delays were measured by flow cytometry after pulse labeling with bromodeoxyuridine, and expression of cell cycle control genes were measured in Western blots in radiosensitive SCC61 and radioresistant SQ20B cell lines. Flow cytometry data demonstrated that the duration of the G(2) arrest was dose dependent in both cell lines, amounting to approximately 1.1 h/Gy. No difference was found between the cell lines in the length of the G(2) block. Radiation exposure did not result in a decrease of cyclin B. Cyclin B protein levels in both asynchronous and synchronized populations in fact showed a dose dependent increase, concomitant with the rise in the fraction of cells in G(2)/M. Similarly, the cdc2 protein levels did not decrease after irradiation. However, it was found that the levels of hyperphosphorylated, and therefore inactive, kinase were significantly higher in irradiated cells than in unirradiated cells. The accumulation of this hyperphosphorylated form correlated with the arrest of cells in the G(2) phase. Finally, immunocytochemical staining of cyclin B revealed an increase of this protein in the cytoplasm after irradiation and a decrease in nuclear staining. This differential localization could possibly account for the reduced nuclear phosphorylation of cdc2 kinase leading to the G(2) arrest.