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Research Articles: Therapeutics

Mode of action of the chloroethylating and carbamoylating moieties of the prodrug cloretazine

Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, Connecticut

Requests for reprints: Alan C. Sartorelli, Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Phone: 203-785-4533; Fax: 203-737-2045. E-mail: alan.sartorelli{at}yale.edu

Abstract

Cloretazine is an antitumor sulfonylhydrazine prodrug that generates both chloroethylating and carbamoylating species. The cytotoxic potency of these species was analyzed in L1210 leukemia cells using analogues with chloroethylating or carbamoylating function only. Clonogenic assays showed that the chloroethylating-only agent 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) produced marked differential cytotoxicity against wild-type and O⁶-alkylguanine-DNA alkyltransferase–transfected L1210 cells (LC₁₀, 1.4 versus 31 µmol/L), indicating that a large portion of the cytotoxicity was due to alkylation of DNA at the O-6 position of guanine. Consistent with the concept that O-6 chloroethylation of DNA guanine progresses to interstrand cross-links, the comet assay, in which DNA cross-links were measured by a reduction in DNA migration induced by strand breaks, showed that cloretazine and 90CE, but not the carbamoylating-only agent 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]hydrazine (101MDCE), produced DNA cross-links and that cloretazine caused more DNA cross-links than 90CE at equimolar concentrations. Cell cycle analyses showed that 90CE and 101MDCE at concentrations of 5 and 80 µmol/L, respectively, produced similar degrees of G₂-M arrest. 90CE produced selective inhibition of DNA synthesis after overnight incubation, whereas 101MDCE caused rapid and nonselective inhibition of RNA, DNA, and protein syntheses. Both 90CE and 101MDCE induced phosphorylation of histone H2AX, albeit with distinct kinetics. These results indicate that (a) differential expression of O⁶-alkylguanine-DNA alkyltransferase in tumor and host cells seems to be responsible for tumor selectivity exerted by cloretazine; (b) 101MDCE enhances DNA cross-linking activity; and (c) 90CE induces cell death at concentrations lower than those causing alterations in the cell cycle and macromolecular syntheses. [Mol Cancer Ther 2006;5(4):969–76]

Grant support: National Cancer Institute/U.S. Public Health Service grant CA-90671.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Penketh et al., manuscript in preparation.

Received 12/19/05; revised 2/ 1/06; accepted 2/21/06.

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