Use of the comet assay to identify cells sensitive to tirapazamine in multicell spheroids and tumors in mice.

Tirapazamine, a bioreductive drug preferentially toxic to hypoxic cells, produces significant numbers of DNA single-strand breaks that can be detected using the alkaline comet assay. Our goal was to determine whether single-strand breaks measured using this assay could act as a surrogate end point for cell killing in multicell spheroids and solid tumors in mice. In spheroids composed of Chinese hamster V79 cells, WiDr human colon carcinoma cells, or SiHa human cervical carcinoma cells, histograms of tail moments (indicators of DNA damage in the comet assay) could be used to identify the percentage of cells that sustained sufficient DNA damage to cause cell death after treatment with tirapazamine. The proportion of comets with tail moments </= 20 (i.e., with damage comparable to that produced by about 10 Gy) correlated with cell survival irrespective of cell type, dose of tirapazamine, time of treatment, or position of cell in the spheroid. Single-cell suspensions from squamous cell carcinoma VII tumors in C3H mice or SiHa xenografts in severe combined immunodeficient mice were also analyzed for clonogenicity and DNA damage. Again, the percentage of comets with tail moments </= 20 was found to be a good predictor of cell killing for both tumor types, providing tumor samples were obtained no more than 1 h after i.p. drug administration. Because tirapazamine is currently undergoing clinical trials, application of this procedure could provide an early indicator of tumors likely to contain hypoxic, susceptible cells and also the extent of the response.