Polymyxin permeabilization as a tool to investigate cytotoxicity of therapeutic aromatic alkylators in DNA repair-deficient Escherichia coli strains

Chlorambucil (CLB, N.N-bis(2-chloroethyl)-p-aminophenylbutyric acid) and it s biologically active beta-oxidation product phenylacetic acid mustard (PAM ; N,N-bis(2-chloroethyl)-p-aminophenylacetic acid) are bifunctional aromati c alkylators. CLB is in wide clinical use as an anticancer drug and also as an immunosuppressant. The chemical structures indicate that CLB and PAM ar e mutagenic, teratogenic and carcinogenic, but the mode of action has remai ned obscure. We have investigated the biological effects of CLB and PAM wit h DNA repair-deficient Escherichia coli strains. In contrast to MNNG (N-met hyl-N'-nitro-N-nitrosoguanine), CLB and PAM were not toxic to E. coli, but permeabilization of the outer membrane of the cells through use of polymyxi n B nonapeptide (PMBN) rendered them susceptible to these compounds. The im portance of DNA repair, shown by reversal of damage and attenuation of the toxicity of CLB and PAM, was indicated by the susceptibility of cells lacki ng O-6-methylguanine-DNA methyltransferase I and II (ada ogt). Similarly, t he protective role of base excision repair (BER) was substantiated by demon stration of an even more increased susceptibility to CLB and PAM of cells l acking 3-methyladenine-DNA glycosylase I and II (alkA1 tag-1). Cells defici ent in mismatch repair (mutS) appeared to be slightly more sensitive than n ormal cells to CLB and PAM, although no such sensitivity to MNNG was observ ed. This implicates the role of mismatches in CLB- and PAM-related cytotoxi city. It is generally believed that bifunctional alkylating agents, like CL B and PAM, exert their cytotoxic action via DNA cross-linking. Our results with O-6-methyltransferase- and 3-methyladenine-DNA glycosylase-deficient c ells indicate that removal of the adducts prior to the formation of cross-l inks is an important mechanism maintaining cell viability. We conclude that PMBN permeabilization provides a valuable tool to investigate genetically engineered E. coli cells, whose outer membrane is not naturally permeable t o mutagens or other interesting compounds. (C) 2000 Elsevier Science B.V. A ll rights reserved.