USE OF THE COMET ASSAY TO IDENTIFY CELLS SENSITIVE TO TIRAPAZAMINE INMULTICELL SPHEROIDS AND TUMORS IN MICE

Tirapazamine, a bioreductive drug preferentially toxic to hypoxic cell s, produces significant numbers of DNA single-strand breaks that can b e detected using the alkaline comet assay. Our goal was to determine w hether single-strand breaks measured using this assay could act as a s urrogate end point for cell. killing in multicell spheroids and solid tumors in mice. In spheroids composed of Chinese hamster V79 cells, Wi Dr human colon carcinoma cells, or SiHa human cervical carcinoma cells , histograms of tail moments (indicators of DNA damage in the comet as say) could be used to identify the percentage of cells that sustained sufficient DNA damage to cause cell death after treatment with tirapaz amine. The proportion of comets with tail moments less than or equal t o 20 (i.e, with damage comparable to that produced by about 10 Gy) cor related with cell survival irrespective of cell type, dose of tirapaza mine, time of treatment, or position of cell in the spheroid. Single-c ell suspensions from squamous cell carcinoma VII tumors in C3H mice or SiHa xenografts in severe combined immunodeficient mire were also ana lyzed for clonogenicity and DNA damage. Again, the percentage of comet s with tail moments less than or equal to 20 was found to be a good pr edictor 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 co ntain hypoxic, susceptible cells and also the extent of the response.