DNA Damage-Processing Pathways Involved in the Eukaryotic Cellular Response to Anticancer DNA Cross-Linking Drugs Vladimir Beljanski, Luigi G. Marzilli, and Paul W. Doetsch Departments of Chemistry (V.B., L.G.M.) and Biochemistry (P.W.D.) and Division of Cancer Biology and Department of Radiation Oncology (P.W.D.), Emory University, Atlanta, Georgia; and Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana (L.G.M.) Received November 12, 2003; accepted March 5, 2004 This article is available online at http://molpharm.aspetjournals.org ABSTRACT We used a panel of isogenic Saccharomyces cerevisiae strains compromised in several different DNA damage-processing pathways to assess in vivo processing of DNA adducts induced by four cross-linking anticancer drugs. By examining cytotox- icity profiles, cell cycle arrest patterns, and determining recom- bination and mutation frequencies, we found that cisplatin-, nitrogen mustard-, mitomycin-, and carmustine-induced DNA adducts in S. cerevisiae are processed by components of the nucleotide excision repair (NER), recombination repair (RR), and translesion synthesis (TLS) pathways, with substantially different contributions of each pathway for the drugs studied here. In contrast to previous studies that used single pathway- compromised strains to identify genes that mediate sensitivity to DNA cross-linking drugs, we used strains that were compro- mised in multiple pathways. By doing so, we were able to establish several functions that were previously unknown and interconnections between different DNA damage-processing pathways. To our surprise, we found that for cisplatin-induced cytotoxicity, TLS and RR contribute to survival to a significant extent. In the case of nitrogen mustard DNA adduct processing, equal involvement of two major pathways was established: one that requires functional RR and NER components and one that requires functional TLS and NER components. These data re- veal the complexity of DNA cross-link processing that, in many cases, requires interactions of components from several differ- ent DNA damage-processing systems. We demonstrate the usefulness of yeast strains with multiple simultaneous defects in DNA damage-processing pathways for studying the modes of action of anticancer drugs. Compounds that form chemical bonds with DNA molecules represent several important classes of anticancer drugs. A large number of such compounds have been synthesized and tested for anticancer activity, and many of them are in clin- ical use. These drugs include nitrogen mustards, the aziri- dines, the alkane sulfonates, the nitrosoureas, and platinum compounds (Pratt, 1994; Lawley and Phillips, 1996; Tannock and Goldenberg, 1998). All of these drugs interact with a variety of biomolecules in cells, but the most important as- pect responsible for their cytotoxicity seems to be inhibition of cell division or stimulation of apoptosis caused by the formation of DNA adducts (Tannock and Goldenberg, 1998). Important examples of such drugs include cisplatin, nitrogen mustard, mitomycin, and carmustine (Fig. 1) (Dronkert and Kanaar, 2001). Once DNA adducts are formed, to carry out normal DNA transactions, such as replication and transcription, cells must be able to remove or to tolerate the presence of such DNA damage. Generally, base excision repair (BER) removes base damage that causes relatively minor distortions in DNA (Memi- soglu and Samson, 2000). Nucleotide excision repair (NER) is a major pathway by which cells remove bulky, DNA helix-distort- ing adducts (Friedberg et al., 1995). Mismatch repair (MMR) corrects errors (insertions, deletions, and/or base substitutions) created during DNA replication (Harfe and Jinks-Robertson, 2000). Homologous recombination repair (RR) and translesion synthesis (TLS) also provide routes by which cells can continue replication despite the presence of replication fork-blocking ad- ducts (Doetsch et al., 2001). RR and TLS are often regarded as DNA damage-tolerance pathways because they allow cells to complete replication and mitosis at the expense of increasing mutation and recombination frequencies. These pathways are summarized in Fig. 2. Resistance of cancer cells to chemotherapeutic drugs is a major problem encountered in the treatment of tumors (Tan- nock and Goldenberg, 1998). The basis for resistance has been studied extensively and can be drug-specific, or nonspe- cific as in the case of alkylating and platinum drugs (Pratt, This research was supported by National Institutes of Health Grants CA073041 and ES11163 (to P.W.D.) and GM29222 (to L.G.M.). ABBREVIATIONS: BER, base excision repair; NER, nucleotide excision repair; MMR, mismatch repair; RR, recombination repair; TLS, translesion synthesis; YEPD, medium composed of 1% yeast extract, 2% Bacto-peptone, 2% dextrose, 2% agar, and 0.005% adenine sulfate. 0026-895X/04/6506-1496 –1506$20.00 MOLECULAR PHARMACOLOGY Vol. 65, No. 6 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 3093/1153867 Mol Pharmacol 65:1496–1506, 2004 Printed in U.S.A. 1496 at ASPET Journals on June 17, 2017 molpharm.aspetjournals.org Downloaded from