Biochunical Pharmacology, Vol. 37, No. 16, pp. 3189-3192, 1988. Printed in Great Britain. CQO6-295@3 53.00 + 0.00 @ 1988. Pergamon Press plc zyxwvu CORRELATION BETWEEN THE CYTOTOXICITY OF MELPHALAN AND DNA CROSSLINKS AS DETECTED BY THE ETHIDIUM BROMIDE FLUORESCENCE ASSAY IN THE F1 VARIANT OF B16MELANOMA CELLS SANJUANA TORRES GARCIA, ANGELAMCQUILLAN and LAWRENCE PANASCI* Lady Davis Institute for Medical Research of the Sir Mortimer B. Davis, Jewish General Hospital, Montreal, Quebec, Canada H3T lE2 (Received 7 December 1987; accepted 22 February 1988) Abstract-The relationship between DNA crosslinks and cell death as a result of exposure to melphalan (MLN) was studied in the F, 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., Znt. J. Cancer 37, 557 (1986)]. A direct correlation between the percentage of DNA crosslinks (C,) 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”. The concentrations of MLN necessary to reduce colony growth to 37% of control and 10% of control were 6.7 PM (ECJ,) and 26 PM (ECIo) respectively. Utilizing the ethidium bromide fluorescence assay (EFA), the retationship between MLN concentration (x axis) and DNA crosslinks expressed as C, (y axis) was best described by a power curve (y = 0.28 x0.*‘;r = 0.985). The respective C, values at the ECU, and ECl,, 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. To study the mechanisms through which DNA is damaged by alkylating agents, methods have been developed to detect and quantify the formation of interstrand crosslinks. There are several methods which measure the formation of DNA crosslinks in cells, such as: (a) the renaturation method [1,2], (b) the alkaline elution assay (AEA) [3,4], (c) the alkaline sucrose sedimentation [2], (d) the density labeled hybrid DNA method [S], and (e) the ethid- ium bromide fluorescence assay (EFA) [6]. The alkaline elution technique is one of the most widely employed assays for the detection of DNA crosslinks. This assay can detect protein-DNA cross- links, interstrand DNA crosslinks and also DNA strand breaks [4,7-g]. There have been several com- parisons of the drug concentrations necessary to achieve cytotoxicity compared with the dose nec- essary for detection of DNA crosslink formation using this method [lO-141. It is possible to detect DNA crosslink formation with concentrations of nitrogen mustard (NH?) and melphalan (MLN) that produce 30 and 45% cell kill in L1210 cells respectively [lo]. Similarly, con- centrations of NH2 and MLN that produce approxi- mately a 30 and 60% cell kill, respectively, in human melanoma cells result in detectable crosslinking using the AEA [14]. However, in fresh human tumor cells * Correspondence to: L. Panasci, M.D., Oncology Centre (8 East), Sir Mortimer B. Davis-Jewish General Hospital, 37.55 Cote Saint Catherine Road, Montreal, Que- bec, Canada H3T lE2. the AEA is laborious and difficult to use because the DNA cannot be easily radiolabeled, and thus it is necessary to measure DNA fluorometrically in each fraction of the AEA [15]. The EFA is based on the fact that the fluorescence of ethidium bromide increases 20 to 25”fold (depending on the ionic strength of the medium) on binding to double-stranded DNA as compared to single-stranded DNA [ 161. The production of single- strand breaks in DNA up to 750 Rad-equivalents does not interfere with the EFA [6,17]. However, it is difficult to know if the EFA has a sensitivity similar to the AEA. Results with the EFA have been compared with AEA [6,12]. This comparison can only be qualitative because the end points of both assays are quite different. A comparison of EFA results with cytotoxicity was done for cyclophos- phazenes, but the drug incubation for cytotoxicity was 1 hr whereas a 6-hr incubation was utilized for the EFA [12]. Furthermore, to our knowledge, the EFA has not been utilized to detect crosslinks pro- duced by nitrogen mustards. In this report we describe the production of DNA crosslinks by MLN and compare these results to cytotoxicity with the F1 variant of B16 melanoma cells. A good indication of the sensitivity of a cross- link assay is to compare the concentration of drug necessary to produce cytotoxicity to a given con- centration which would yield DNA crosslinks. To determine the sensitivity of EFA, we compared the cytotoxicity of MLN to the production of DNA cross- links by MLN in the EFA with the F1 variant of B16 melanoma cells. 3189