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DNA Damage and Repair in Mouse Leukemia L1210 Cells Treated with Nitrogen Mustard, 1,3-Bis(2-chloroethyl)-1-nitrosourea, and Other Nitrosoureas

Laboratory of Molecular Pharmacology, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014

The technique of alkaline elution was applied to studies of DNA damage and repair in mouse leukemia L1210 cells treated with nitrogen mustard (HN2) and nitrosoureas. DNA cross-linking was measured in terms of the reduction in the effect of X-ray on the kinetics of DNA elution and was observed in cells treated with HN2 and three chloroethylnitrosoureas: 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, and chlorozotocin. Evidence was obtained that cross-links are of two types, DNA interstrand cross-links and DNA-protein cross-links, distinguishable on the basis of sensitivity to proteinase-K. HN2 and 1,3-bis(2-chloroethyl)-1-nitrosourea each produced both types of cross-links. In most of the studies reported, the combined effect of the two types of cross-links was measured. With HN2, cross-linking was observed at subtoxic doses, and the cross-links were fully repaired by 24 hr. At higher HN2 doses, which reduced colony-forming ability, cross-links were not fully repaired by 24 hr. Cross-linking by chloroethylnitrosoureas differed from that by HN2 in that the effect increased for about 6 hr after removal of the drug (treatment times were 0.5 hr for HN2 and 1 hr for nitrosoureas). The chloroethylnitrosoureas, but not HN2, also produce DNA single-strand breaks or lesions, which were converted to single-strand breaks in alkali. Methylnitrosourea produced little or no cross-linking for at least 6 hr after treatment but did produce extensive DNA damage of the alkali-labile type; this damage was repairable and had little efect on colony-forming ability. Fluoroethylnitrosourea, the fluoro analog of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, was much less effective than 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in producing cross-links. The findings on the kinetics and structure dependence of cross-linking are similar to the previously reported findings on interstrand cross-linking of purified DNA and support a two-step reaction mechanism involving a chloroethylated intermediate.

Received 12/16/76. Accepted 4/14/77.