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Inhibition of the Ligase Step of Excision Repair by 2-Chloroethyl Isocyanate, a Decomposition Product of 1,3-Bis(2-chloroethyl)-1-nitrosourea¹

Laboratory of Molecular Pharmacology, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20014 [A. J. F., K. W. K.], and Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30303 [H. E. K.]

The compound 2-chloroethyl isocyanate, a decomposition product of 1,3-bis(2-chloroethyl)-1-nitrosourea, was studied for its effects on excision repair of DNA in normal human fibroblasts exposed to ultraviolet (UV) radiation. For examination of the initial step in repair (UV endonuclease), the frequency of UV-induced strand breaks was analyzed by alkaline elution. Repair polymerase activity was assessed from the rate of UV-stimulated incorporation of radioactive thymidine and from the amount of repair replication (isopycnic gradient analysis for newly synthesized patches of DNA within strands of prevlously synthesized parental DNA). The final step in repair (strand rejoining) was monitored by following the disappearance of strand breaks with time after UV, with the use of the alkaline elution technique. Chloroethyl isocyanate, 75 µM, did not inhibit production of strand breaks after UV; however, rejoining of breaks was inhibited. Since neither UV-stimulated incorporation of radioactive thymidine nor repair replication was affected by chloroethyl isocyanate, inhibition of strand rejoining is attributed to an effect on the ligase step. The biological significance of this effect with respect to the mechanism of action of chloroethyl nitrosoureas is uncertain; in theory this effect could amplify cytotoxicity, since an excision-type repair mechanism is involved in repair of DNA damaged by alkyiation, and alkylation is considered to be fundamentally responsible for the antitumor activity of chloroethyl nitrosoureas.

¹ Supported in part by Grant CA 16620, awarded by the National Cancer Institute, Department of Health, Education and Welfare.

² Present address: Department of Pathology, Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, Mass. 02115.

³ To whom requests for reprints should be addressed, at Department of Medicine, Emory University School of Medicine, 69 Butler Street, S.E., Atlanta, Ga. 30303.

Received 9/23/77. Accepted 1/ 9/78.