Quantification of DNA adducts in individual cells by immunofluorescence: effects of variation in DNA conformation

Abstract

Previously reported detection of melphalan-DNA adducts by immunofluorescent staining indicated considerable intercell variation in fluorescence levels. Investigations were undertaken to determine whether this variation reflected actual intercell differences in adduct levels. Melphalan-treated CCRF-CEM leukaemia cells were analysed by the trapped-in-agarose DNA immunostaining (TARDIS) method using fluorescein immunofluorescence and Hoechst dye-DNA fluorescence. Increasing the time of DNA denaturation in alkali affected the staining intensity, in agreement with known adduct properties, but failed to reduce intercell heterogeneity. To test the hypothesis that heterogeneity resulted from variation in levels of DNA strand breaks, drug-treated cells were exposed to ionising radiation. An increase in level and reduction in heterogeneity of immunofluorescence were observed, optimal at 10 Gy. When samples were irradiated after lysis, 1 Gy was optimal. At the optimal doses, irradiation before or after lysis resulted in similar levels of DNA strand breaks. Our conclusions are as follows: (a) There was no major intercell variation in the number of adducts other than from variation in DNA content. (b) Detection of melphalan, and possibly other adducts, by immunofluorescence can be markedly influenced by the level of strand breaks present in the DNA. (c) Samples analysed for melphalan adducts by immunofluorescence should be irradiated to minimise errors due to this factor.