Correlation between the cytotoxicity of melphalan and DNA crosslinks as detected by the ethidium bromide fluorescence assay in the F1 variant of B16 melanoma cells.

The relationship between DNA crosslinks and cell death as a result of exposure to melphalan (MLN) was studied in the F1 variant of B16 melanoma cells. The formation of DNA crosslinks is believed to represent the lethal lesion following exposure of cells to bifunctional alkylating agents. The production of DNA crosslinks by MLN was determined by the recently described ethidium bromide fluorescence assay [De Jong et al., Int. J. Cancer 37, 557 (1986)]. A direct correlation between the percentage of DNA crosslinks (Ct) and cytotoxicity of melphalan has not been previously reported utilizing the fluorescence assay. The cytotoxicity of MLN and the production of DNA crosslinks by this drug were determined following a 1-hr incubation at 37 degrees. The concentrations of MLN necessary to reduce colony growth to 37% of control and 10% of control were 6.7 microM (EC37) and 26 microM (EC10) respectively. Utilizing the ethidium bromide fluorescence assay (EFA), the relationship between MLN concentration (x axis) and DNA crosslinks expressed as Ct (y axis) was best described by a power curve (y = 0.28 x 0.81; r = 0.985). The respective Ct values at the EC37 and EC10 of MLN were 1.3 and 3.8%. It appears that the sensitivity of the EFA is similar to the alkaline elution assay and, in addition, that the EFA is less technically difficult to employ with tumor cells obtained from patients.