Abstract
Tirapazamine (SR 4233), a bioreductive drug selectively toxic towards hypoxic cells, is presently in phase II clinical trials. Since it would not be expected that all tumours would respond equally to the drug, we are exploring ways of predicting the response of individual tumours. In this study we have tested whether the comet assay, which measures DNA damage in individual cells, can provide a simple, surrogate end point for cell killing by tirapazamine. We examined the relationship between the cytotoxicity of tirapazamine under hypoxic conditions and tirapazamine-induced DNA strand breaks in murine (SCCVII, EMT6, RIF-1) and human (HT1080, A549, HT29) tumour cell lines. These results were compared with the relationship between tirapazamine cytotoxicity and another measure of the ability of cells to metabolise tirapazamine; high-performance liquid chromatography (HPLC) analysis of tirapazamine loss or formation of the two electron reduction product SR 4317. The correlation between the hypoxic cytotoxic potency of tirapazamine and DNA damage was highly significant (r = 0.905, P = 0.013). A similar correlation was observed for hypoxic potency and tirapazamine loss (r = 0.812, P = 0.050), while the correlation between hypoxic potency and SR 4317 formation was not significant (r = 0.634, P = 0.171). The hypoxic cytotoxicity of tirapazamine in vitro can therefore be predicted by measuring tirapazamine-induced DNA damage using the comet assay. This approach holds promise for predicting the response of individual tumours to tirapazamine in the clinic.
Highlights
Cells were cultured in Alpha MEM (HT1080, A549), McCoys 5A (HT29) or Waymouth's (SCCVII, EMT6, RIF-1) media supplemented with 10% (HT1080, A549, HT29) or 15% fetal bovine serum (FBS) (SCCVII, EMT6, RIF-1) plus penicillin (100 IU ml-') and streptomycin (100 ig ml-')
The cytotoxicity of tirapazamine towards three human (HT1080, A549, HT29) and three murine (SCCVII, RIF-1, EMT6) tumour cell lines was assessed under hypoxic conditions and correlated with three measures of drug metabolism determined in the same experiments: production of tirapazamine-induced DNA strand breaks, loss of tirapazamine and formation of SR 4317
Tirapazamine solutions were equilibrated with nitrogen-5% carbon dioxide for 60 min before initiation of drug exposure by addition of cells equilibrated under identical conditions
Summary
Details of the derivation of the SCCVII (Hirst et al, 1983), EMT6 (Rockwell et al, 1972) and RIF-1 (Twentyman et al, 1980) cell lines have been described previously. HT1080 and A549 cells were obtained from the American Type Culture Collection. HT29 cells were obtained from Dr RM Sutherland (SRI International, Menlo Park, CA, USA). Cells were cultured in Alpha MEM (HT1080, A549), McCoys 5A (HT29) or Waymouth's (SCCVII, EMT6, RIF-1) media supplemented with 10% (HT1080, A549, HT29) or 15% fetal bovine serum (FBS) (SCCVII, EMT6, RIF-1) plus penicillin (100 IU ml-') and streptomycin (100 ig ml-'). Tirapazamine was kindly supplied by Sterling Winthrop and SR 4317 and SR 4430 by SRI International
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