Induction of a retinoblastoma phosphatase activity by anticancer drugs accompanies p53-independent G1 arrest and apoptosis.
DNA-damaging agents induce accumulation of the tumor suppressor and G1 checkpoint protein p53, leading cells to either growth arrest in G1 or apoptosis (programmed cell death). The p53-dependent G1 arrest involves induction of p21 (also called WAF1/CIP1/SDI1), which prevents cyclin kinase-mediated phosphorylation of retinoblastoma protein (RB). Recent studies suggest a p53-independent G1 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 G1 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, RB became hypophosphorylated, accompanied with G1 arrest. This was followed immediately (in less than 30 min) by apoptosis, as determined by the accumulation of pre-G1 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 G1 arrest- and apoptosis-specific RB dephosphorylation. The levels of cyclin E- and cyclin A-associated kinase activities remained high during RB dephosphorylation, supporting the involvement of a chemotherapy-induced serine/threonine phosphatase(s) rather than p21. Furthermore, the induced phosphatase activity coimmunoprecipitated with the hyperphosphorylated RB and was active in a cell-free system that reproduced the growth arrest- and apoptosis-specific RB dephosphorylation, which was inhibitable by calyculin A but not zinc. We propose that the RB phosphatase(s) might be one of the p53-independent G1 checkpoint regulators.
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Department of Pharmacology, University of Pittsburgh School of Medicine, PA, USA.
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D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects*; D002460:Cell Line; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D003561:Cytarabine / Q000494:pharmacology*; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000187:drug effects / Q000378:metabolism; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016193:G1 Phase; D006801:Humans; D007948:Leukemia, Monocytic, Acute; D015473:Leukemia, Promyelocytic, Acute; D008387:Marine Toxins; D010080:Oxazoles / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D016160:Retinoblastoma Protein / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D000970:Antineoplastic Agents; D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D016160:Retinoblastoma Protein; D016159:Tumor Suppressor Protein p53; D003561:Cytarabine; C059041:calyculin A; D018844:Cyclin-Dependent Kinases; D017027:Protein Tyrosine Phosphatases; D015032:Zinc