Phosphorylation of p53 on Ser15 during cell cycle caused by Topo I and Topo II inhibitors in relation to ATM and Chk2 activation.
The DNA topoisomerase I (topo1) inhibitor topotecan (TPT) and topo2 inhibitor mitoxantrone (MXT) damage DNA inducing formation of DNA double-strand breaks (DSBs). We have recently examined the kinetics of ATM and Chk2 activation as well as histone H2AX phosphorylation, the reporters of DNA damage, in individual human lung adenocarcinoma A549 cells treated with these drugs. Using a phospho-specific Ab to tumor suppressor protein p53 phosphorylated on Ser15 (p53-Ser15(P)) combined with an Ab that detects p53 regardless of the phosphorylation status and multiparameter cytometry we correlated the TPT- and MXT-induced p53-Ser15(P) with ATM and Chk2 activation as well as with H2AX phosphorylation in relation to the cell cycle phase. In untreated interphase cells, p53-Ser15(P) had "patchy" localization throughout the nucleoplasm while mitotic cells showed strong p53-Ser15(P) cytoplasmic immunofluorescence (IF). The intense phosphorylation of p53-Ser15, combined with activation of ATM and Chk2 (involving centrioles) as well as phosphorylation of H2AX seen in the untreated mitotic cells, suggest mobilization of the DNA damage detection/repair machinery in controlling cytokinesis. In the nuclei of cells treated with TPT or MXT, the expression of p53-Ser15(P) appeared as closely packed foci of intense IF. Following TPT treatment, the induction of p53-Ser15(P) was most pronounced in S-phase cells while no significant cell cycle phase differences were seen in cells treated with MXT. The maximal increase in p53-Ser15(P) expression, rising up to 2.5-fold above the level of its constitutive expression, was observed in cells treated with TPT or MXT for 4-6 h. This maximum expression of p53-Ser15(P) coincided in time with the peak of Chk2 activation but not with ATM activation and H2AX phosphorylation, both of which crested 1-2 h after the treatment with TPT or MXT. The respective kinetics of p53-Ser15 phosphorylation versus ATM and Chk2 activation suggest that in response to DNA damage by TPT or MXT, Chk2 rather than ATM mediates p53 phosphorylation.
Zhao
Hong
H
Brander Cancer Research Institute and Department of Pathology, New York Medical College, Valhalla, New York 10595, USA.
Traganos
Frank
F
Darzynkiewicz
Zbigniew
Z
D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008942:Mitoxantrone / Q000494:pharmacology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012694:Serine / Q000235:genetics* / Q000378:metabolism; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D019772:Topotecan / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D012694:Serine; D019772:Topotecan; D008942:Mitoxantrone; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II