Kinetics of H2AX phosphorylation after exposure to cisplatin.
BACKGROUND
Cisplatin is a widely used cancer chemotherapeutic drug that causes DNA crosslinking and stimulates H2AX phosphorylation. Our goal was to assess the potential of gammaH2AX to help predict tumor response to cisplatin treatment.


METHODS
The kinetics of cisplatin-induced DNA interstrand crosslinks was measured using the alkaline comet assay and compared with gammaH2AX formation and clonogenic cell survival in several DNA repair proficient or deficient human and rodent cell lines.


RESULTS
The comet assay was effective in ranking cell lines according to their relative sensitivity to cisplatin based on reduced crosslink formation measured 6 h after drug exposure or by the failure of irs3 and UV41 cell lines to subsequently remove crosslinks. In comparison, the initial rate of phosphorylation of H2AX measured over the first 6 h after cisplatin treatment was unrelated to drug sensitivity or crosslinking proficiency. However, for proliferating cell cultures, the fraction of cells that retained gammaH2AX foci 24 h after cisplatin treatment was correlated with the fraction of cells that lost clonogenic potential (slope = 1.1, r(2) = 0.85).


CONCLUSIONS
H2AX phosphorylation occurs in response to replication fork damage caused by cisplatin induced DNA lesions, probably interstrand crosslinks. Although early kinetics of gammaH2AX formation was uninformative, retention of gammaH2AX foci 24 h after treatment was shown to be a useful indicator of cell response to killing by cisplatin. However, for gammaH2AX to serve as an indicator of cell viability after cisplatin treatment, cells must have the opportunity to transit S phase during the recovery period.
Olive
Peggy L
PL
Medical Biophysics Department, British Columbia Cancer Research Centre, Vancouver, BC, Canada. polive@bccrc.ca
Banáth
Judit P
JP
D014408:Biomarkers, Tumor / Q000032:analysis / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002945:Cisplatin / Q000494:pharmacology*; D020552:Comet Assay / Q000379:methods*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004249:DNA Damage / Q000187:drug effects / Q000502:physiology; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D006657:Histones / Q000032:analysis / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D020127:Recovery of Function / Q000187:drug effects / Q000235:genetics; D016196:S Phase / Q000187:drug effects / Q000235:genetics
D014408:Biomarkers, Tumor; D003432:Cross-Linking Reagents; C468783:H2AX protein, human; D006657:Histones; D002945:Cisplatin