Enhanced H2AX phosphorylation, DNA replication fork arrest, and cell death in the absence of Chk1.
H2AX phosphorylation at serine 139 (gammaH2AX) is a sensitive indicator of both DNA damage and DNA replication stress. Here we show that gammaH2AX formation is greatly enhanced in response to replication inhibitors but not ionizing radiation in HCT116 or SW480 cells depleted of Chk1. Although H2AX phosphorylation precedes the induction of apoptosis in such cells, our results suggest that cells containing gammaH2AX are not committed to death. gammaH2AX foci in these cells largely colocalize with RPA foci and their formation is dependent upon the essential replication helicase cofactor Cdc45, suggesting that H2AX phosphorylation occurs at sites of stalled forks. However Chk1-depleted cells released from replication inhibitors retain gammaH2AX foci and do not appear to resume replicative DNA synthesis. BrdU incorporation only occurs in a minority of Chk1-depleted cells containing gammaH2AX foci after release from thymidine arrest and, in cells incorporating BrdU, DNA synthesis does not occur at sites of gammaH2AX foci. Furthermore activated ATM and Chk2 persist in these cells. We propose that the gammaH2AX foci in Chk1-depleted cells may represent sites of persistent replication fork damage or abandonment that are unable to resume DNA synthesis but do not play a direct role in the Chk1 suppressed death pathway.
Gagou
Mary E
ME
Institute for Cancer Studies, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom.
Zuazua-Villar
Pedro
P
Meuth
Mark
M
D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000737:chemistry; D016923:Cell Death; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D004261:DNA Replication; D005434:Flow Cytometry / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D006657:Histones / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D012694:Serine / Q000737:chemistry
C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; C468783:H2AX protein, human; D006657:Histones; D034741:RNA, Small Interfering; D012694:Serine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D053148:Caspase 3