INDUCTION OF A RETINOBLASTOMA PHOSPHATASE-ACTIVITY BY ANTICANCER DRUGS ACCOMPANIES P53-INDEPENDENT G(1) ARREST AND APOPTOSIS

DNA-damaging agents induce accumulation of the tumor suppressor and G(1) checkpoint protein p53, leading cells to either growth arrest in G(1) or apoptosis (programmed cell death). The p53-dependent G(1) arrest involves induction of p21 (also called WAF1/CIP1/SDI1), which prevents cyclin kinase-mediated phosphorylation of retinoblastoma protein (RE). Recent studies suggest a p53-independent G(1) checkpoint as well; however, little is known about its molecular mechanisms. We report that induction of a protein-serine/threonine phosphatase activity by DNA damage signals is at least one of the mechanisms responsible for p53-independent, RB-mediated G(1) arrest and consequent apoptosis. When two p53-null human leukemic cell lines (HL-60 and U-937) were treated with a variety of anticancer agents, RE became hypophosphorylated, accompanied with G(1) arrest. This was followed immediately (in less than 30 min) by apoptosis, as determined by the accumulation of pre-G(1) apoptotic cells and the internucleosomal fragmentation of DNA. Addition of calyculin A or okadaic acid (specific serine/threonine phosphatase inhibitors) or zinc chloride (apoptosis inhibitor) prevented the G(1) arrest- and apoptosis-specific RE dephosphorylation. The levels of cyclin E- and cyclin A-associated kinase activities remained high during RE dephosphorylation, supporting the involvement of a chemotherapy-induced serine/threonine phosphatase(s) rather than p21. Furthermore, the induced phosphatase activity coimmunoprecipitated with the hyperphosphorylated RE and was active in a cell-free system that reproduced the growth arrest- and apoptosis-specific RE dephosphorylation, which was inhibitable by calyculin A but not zinc, We propose that the RE phosphatase(s) might be one of the p53-independent G(1) checkpoint regulators.