Phosphorylation of chk1 at serine-345 affected by topoisomerase I poison SN-38.
Human head and neck squamous carcinoma cell lines, A253 and FaDu, were utilized to identify mediators associated with response to topoisomerase I poison, SN-38, a metabolite of irinotecan. The drug sensitivity of FaDu cells to SN-38 was significantly higher than that of the A253 cells. In A253 cells, G2/M arrest following drug treatment (0.35 microM SN-38, 2-h exposure) was accompanied by DNA fragmentation in the 50-300 kb range, but FaDu cells accumulated in S-phase concurrently with induction of smaller DNA fragmentation in the 4-80 kb range. Because the critical regulatory step in activating cdc2 during progression into mitosis appears to be dephosphorylation of Tyrosine 15 (Tyr15), we examined the Tyr15 phosphorylation status of cdc2 in both cell lines. Slightly increased levels of cdc2 phosphorylation was observed in the A253 cells, while reduced levels of cdc2 phosphorylation was noted in the FaDu cells, corresponding to the abrogation of the G2-phase arrest. Increased chk1 phosphorylation at Ser345 induced by SN-38 was accompanied by the observed G2 phase arrest in the A253 cell line, while significant downregulation of chk1 and cdc25C phosphorylation, which resulted in the abrogation of G2/M checkpoint arrest, was noted in FaDu cells at this timepoint. These results suggest that alterations of chk1 signaling are associated with the response to topoisomerase I poison SN-38. Furthermore, A253 cells possess higher levels of endogenous hMLH1, compared to FaDu cells. A deficiency in G2 arrest was observed in FaDu cells, suggesting endogenous hMLH1 protein expression is associated with the abrogation of G2/M arrest, subsequently with the response to topoisomerase I poison SN-38.
Hapke
Gunnar
G
Department of Pharmacology and Therapeutics, Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Yin
Ming-Biao
MB
Wu
Jiaxi
J
Frank
Cheryl
C
Rustum
Youcef M
YM
D048948:14-3-3 Proteins; D048868:Adaptor Proteins, Signal Transducing; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002352:Carrier Proteins; D018797:Cell Cycle Proteins / Q000032:analysis; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D053938:DNA Fragmentation / Q000187:drug effects; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016195:G2 Phase / Q000187:drug effects; D006258:Head and Neck Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D000077146:Irinotecan; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D009363:Neoplasm Proteins / Q000032:analysis; D009687:Nuclear Proteins; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000032:analysis; D012694:Serine / Q000378:metabolism*; D059004:Topoisomerase I Inhibitors; D014407:Tumor Cells, Cultured; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis; D020687:cdc25 Phosphatases / Q000032:analysis
D048948:14-3-3 Proteins; D048868:Adaptor Proteins, Signal Transducing; D000972:Antineoplastic Agents, Phytogenic; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C501486:MLH1 protein, human; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D059004:Topoisomerase I Inhibitors; D012694:Serine; D000077146:Irinotecan; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D002166:Camptothecin