Growth fraction measured using the comet assay

Growth fraction, an important determinant of tumour response to therapy, was measured using a novel assay in WiDr human colon carcinoma cells grown as monolayers, spheroids, or xenografts. The assay is based on the fact that the anticancer agent etoposide produces DNA strand breaks in proliferating but not non-proliferating cells. Strand breaks were detected in individual cells using the alkaline 'comet' assay, and growth fraction was defined as the fraction of cells containing elevated numbers of DNA strand breaks. The specificity of the method for detecting proliferating cells was verified directly by allowing cells to incorporate bromodeoxyuridine (BrdUrd) into DNA, followed by exposure to etoposide and treatment of the comets with anti-BrdUrd antibodies. All cells stained with anti-BrdUrd antibodies were also damaged by etoposide. Similarly, growth fraction measured using Ki-67 correlated well with the new assay. The accuracy, speed and convenience of the comet assay for measuring growth fraction suggest that it may be useful for predicting response of human cancers to therapy.