Characterization of adriamycin-induced G2 arrest and its abrogation by caffeine in FL-amnion cells with or without p53

We investigated the effect of Adriamycin on FL-amnion (FL) cells, After tre atment with the drug, the cells arrested at G2, but we did not detect an in crease in the p21 levels. We established a p53-deficient derivative of thes e cells, in which G2 arrest also occurred after treatment with Adriamycin, suggesting that the arrest we observed in these cells is independent of the p53 pathway. Low doses of Adriamycin (100-200 ng/ml) induced G2 arrest, wh ile late S-phase arrest was observed at high doses (500-1000 ng/ml) in both FL and p53-deficient FL cells, Accumulation of cyclin B1 was detected only in cells arrested at G2, and not in those arrested at S phase, suggesting that the S-phase checkpoint functioned efficiently even in p53-deficient FL cells. In both cell lines, caffeine-induced activation of CDCS kinase was detected only in cells arrested at G2 and CDC2 kinase-activated cells died exhibiting features of apoptosis, CDCS kinase activation was inhibited by c ycloheximide. Furthermore, cycloheximide inhibited activation of CDK2:cycli n A, which normally precedes CDC2 kinase activation in caffeine-treated cel ls. These results suggest that p53 and p21 do not have special roles in the S- and G2-phase checkpoints and that CDK2:cyclin A could be the target of the G2-phase DNA damage checkpoint. (C) 2001 Academic Press.