3-(4-Maleimidylphenyl)-4-methyl-7-diethylamino coumarin (CPM), is a fluorescent thiol-binding agent. CPM is nontoxic toChinese hamster V-79 cells (2 x los cells/ milliliter) exposed to 2.5 pg/milliliter for 30 minutes. However, both toxicity and cellular binding were directly dependent on the drugxell ratio. Using flow cytometry, cellular binding of CPM correlated with inhibition of DNA synthesis. In cells pretreated with the thiol binding drugs N-ethylmaleimide, diamide and diethylmaleate or the carcinogens 4-nitroquinoline-1-oxide and AF-2 (furylfuramide), the subsequent binding of CPM was reduced by 40% or more.
Key terms: DNA damage, flow cytometry, thiolsCellular thiols, especially glutathione, act as scavenger nucleophiles and can protect against toxicity, mutagenicity or transformation by radiation and many carcinogens (5,6,8, 10, 13, 19). While biochemical techniques are available to quantitate protein and nonprotein thiol levels within cells, these assays generally require in excess of lo6 cells. In addition, only population averages can be obtained and the degree of cellular heterogeneity in thio1,content cannot be determined. Therefore, the development of a rapid single cell assay to quantitate the intracellular content of thiols could be valuable in measuring differences in thiol content of cells and in the ability of drugs to bind cellular thiols.3-(4-maleimidylphenyl)-4-methyl-7-diethylamino coumarin (CPM, Molecular Probes Inc., Junction City, OR) is a thiolbinding fluorochrome which was recently developed for use in histopathology to stain protein sulphydryls (15). We have found that viable cultured cells can be stained by CPM and, at concentrations of CPM that produce minimal toxicity, the amount of intracellular fluorescence of CPM can be easily quantified intracellularly using flow cytometry (FCM). Cells pretreated with the carcinogens 4-nitroquinoline-1-oxide
Materials and MethodsChinese hamster V-79 lung fibroblasts were grown in Eagle's minimal essential medium containing 15% fetal bovine serum. For experiments, cells were either incubated in suspension cultures in Bellco glass stirring vessels (Bellco Glass Inc., Vineland, N J ) or on Petri dishes. Incubation of cells with all drugs was carried out at 37°C in Gibco formulation phosphate-buffered saline, pH 7.4, containing Ca++ and Mg++ (Gibco, Grand Island, NY). Cells were released from plates using 0.1% trypsin (Gibco) in a citrate/saline buffer (1.5 mM sodium citrate, 0.135 M KCI, pH 7.6). Clonogenicity was determined by seeding approximately 800 surviving cells/100-mm Petri dish. Colonies were stained with malachite green and counted 8 days later.CPM was dissolved in ethanol a t a concentration of 1 mg/ml. DNA strand breakage was measured using the alkali-unwinding technique (1, 14) as previously described (11). Cells (2 X 10' cells/60 mm dish) were incubated overnight with ['4C]thymidine (80 mCi/mmole; 0.01 pCi/ml) followed by treatment with CPM. Cells were then released from plates using trypsin, and resuspended a t 5 X lob cells/ml.A...
