Published OnlineFirst May 26, 2009; DOI: 10.1158/1541-7786.MCR-08-0519 Impairment of APE1 Function Enhances Cellular Sensitivity to Clinically Relevant Alkylators and Antimetabolites Daniel R. McNeill,1 Wing Lam,3 Theodore L. DeWeese,2 Yung-Chi Cheng,3 and David M. Wilson III1 1Laboratory of Molecular Gerontology, Biomedical Research Center, National Institute on Aging, NIH; 2Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University, Baltimore, Maryland; and 3Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut Abstract Introduction Base excision repair (BER) is the major pathway for To cope with the deleterious consequences of endogenous removing mutagenic and cytotoxic oxidative and alkylation and exogenous DNA-damaging agents, cells have evolved re- DNA modifications. Using a catalytically inactive, dominant pair systems that maintain genome integrity (1).Defects in negative protein form of human APE1, termed ED, which DNA repair processes are linked to genomic instability syn- binds with high affinity to substrate DNA and blocks dromes and cancer predisposition.A significant yet evolution- subsequent repair steps, we assessed the role of BER in arily unintended role for DNA repair is its involvement in mediating cellular resistance to clinically relevant alkylating influencing cellular resistance to anticancer agents (2, 3).In par- drugs and antimetabolites. Colony formation assays ticular, most drugs used to eradicate neoplastic disease operate revealed that ED expression enhanced cellular sensitivity by inducing the formation of complex DNA lesions that ulti- to melphalan not at all; to decarbazine, thiotepa, busulfan mately prevent replication and activate cell death responses. and carmustine moderately (1.2- to 2.4-fold); and to The best established demonstration of a role of DNA repair in 6 streptozotocin and temozolomide significantly (2.0- to mitigating therapeutic agent responsiveness is with O -methyl- 5.3-fold). The effectiveness of ED to promote enhanced guanine DNA methyltransferase (MGMT), which plays a prom- cytotoxicity generally correlated with the agent's inent part in adduct repair that limits the cytotoxic effect of (a) monofunctional nature, (b) capacity to induce clinical alkylating (methylating or chloroethylating) agents (4). N7-guanine and N3-adenine modifications, and (c) inability to Most agents used to treat cancer fall into the following major generate O6-guanine adducts or DNA cross-links. ED also categories: antimetabolites, DNA-interactive drugs (e.g., alky- enhanced the cell killing potency of the antimetabolite lators, cross-linking agents, intercalating agents, topoisomerase troxacitabine, apparently by blocking the processing of DNA inhibitors, and DNA cleaving agents), antitubulin agents, mo- strand breaks, yet had no effect on the cytotoxicity of lecularly targeted drugs, hormonal therapies, tumor-targeting gemcitabine, results that agree well with the known strategies, and biological agents (5).Relevant to the studies efficiency of APE1 to excise these nucleoside analogues herein are the alkylating compounds and the antimetabolites. from DNA. Most impressively, ED expression produced an Alkylating agents represent the earliest of anticancer therapies ∼5- and 25-fold augmentation of the cell killing effect of and have great utility in both hematologic and solid tumor ma- 5-fluorouracil and 5-fluorodeoxyuridine, respectively, lignancies.The most common of the alkylating agents used in implicating BER in the cellular response to such clinical practice include nitrogen mustards, nitrosoureas, plati- antimetabolites; the increased 5-fluorouracil sensitivity was num complexes, methanesulfonate esters, and aziridines.These associated with an accumulation of abasic sites and active compounds, or their active metabolites, react with a range of caspase–positive staining. Our data suggest that APE1, nucleophilic targets, particularly in DNA, to form covalent and BER more broadly, is a potential target for inactivation intermediates that induce cell death (6, 7). in anticancer treatment paradigms that involve Antimetabolites account for nearly one fifth of all drugs cur- select alkylating agents or antimetabolites. rently approved by the Food and Drug Administration for the (Mol Cancer Res 2009;7(6):897–906) treatment of cancer.These compounds, which are structural analogues of natural compounds, are used primarily in the treat- ment of hematologic malignancies, although some of the more recently developed agents have shown activity against solid tu- Received 11/6/08; revised 1/28/09; accepted 2/11/09; published OnlineFirst mors.The majority of antimetabolites are analogues of purines 5/26/09. or pyrimidines and must be activated by cellular enzymes to Grant support: Intramural Research Program of the NIH, National Institute on nucleotide metabolites, which are incorporated into DNA Aging, and NIH grant CA63477 (Y-C.Cheng). The costs of publication of this article were defrayed in part by the payment of and/or are direct inhibitors of enzymes required for DNA syn- page charges.This article must therefore be hereby marked advertisement in thesis, such as DNA polymerases or thymidylate synthase (8). accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Regardless, nucleoside analogues interfere with normal chro- Requests for reprints: David M.Wilson III, Biomedical Research Center, National Institute on Aging, NIH, 251 Bayview Boulevard, Suite 100, Room mosome replication and thus inhibit cell growth. #06B117, Baltimore, MD 21224.Phone: 410-558-8153; Fax: 410-558-8157. Whereas DNA-interactive drugs typically exploit the high E-mail: [email protected] Copyright © 2009 American Association for Cancer Research. replicative capacity of cancerous cells, actively dividing normal doi:10.1158/1541-7786.MCR-08-0519 cells (e.g., bone marrow) are also susceptible to the toxic effects Mol Cancer Res 2009;7(6). June 2009 897 Downloaded from mcr.aacrjournals.org on September 26, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst May 26, 2009; DOI: 10.1158/1541-7786.MCR-08-0519 898 McNeill et al. of these compounds.Thus, a primary goal of current investiga- plored the role of APE1 and BER in the survival response to a tions is to devise combinatorial treatment methods that (a) pro- wide range of clinical alkylating agents and antimetabolites. tect normal cells from and (b) enhance the sensitivity of tumor cells to the toxicity of anticancer agents.As noted above, DNA Results repair systems represent a major protective mechanism against Effect of ED on Cellular Sensitivity to SN1andSN2 the cytotoxic effects of clinical DNA-interactive drugs (3). Alkylating Agents Besides MGMT, base excision repair (BER) is another promi- Alkylating agents are generally divided into two types based nent system that eliminates potentially lethal base damage in- on their reaction mechanism: S 1orS 2 (6, 7).S 2 type, be- ′ ′ N N N troduced by alkylating agents (6, 7).In addition, 3 to 5 DNA cause they have direct biomolecular reaction with DNA, exhibit exonucleases, which have the capacity to excise chain-terminat- high nucleophilic selectivity, and alkylate almost exclusively ing nucleoside analogues that have been incorporated into the highly nucleophilic nitrogen centers in DNA, producing pri- DNA, can determine the efficacy of antimetabolites (9).Strate- marily N7-alkylguanine, less amounts of N3-alkyladenine, and gic regulation of these repair mechanisms would therefore im- only small amounts of O-adducted lesions, such as O6-alkyl- prove the selectivity and effectiveness of specific anticancer guanine.S N1-type alkylating agents modify DNA via the inter- treatment paradigms. mediate methyldiazonium ion.Because of its high electrophilic Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) reactivity, this intermediate has relatively low selectivity, and is the major enzyme responsible for the repair of abasic sites therefore modifies not only the highly nucleophilic nitrogen in DNA (10).AP sites are common intermediates of alkyl- atoms but also the less nucleophilic oxygen atoms to generate ation damage to DNA, arising either via spontaneous base significant, albeit less, amounts of O-alkylated nucleotides, loss or through base release by a DNA repair glycosylase. such as O6-alkylguanine, O4-alkylthymine, and O2-alkylcyto- APE1 initiates repair of AP sites by incising the phosphodie- sine, as well as alkylphosphates. ′ ster backbone immediately 5 to the lesion, creating a single- We had previously shown that ED expression increases by strand break intermediate that is further processed by proteins 4.8- to 6.3-fold cellular sensitivity to the SN2-type alkylating of the BER pathway.In addition to its AP site incision activ- agent MMS, and concurrently leads to a hyperaccumulation ′ ′ ity, APE1 also possesses a 3 to 5 exonucleolytic function of chromosomal AP sites (25).MMS has been routinely used ′ that operates on 3 -obstructive termini, such as mismatched as a classic BER-type DNA-damaging agent because it creates nucleotides, tyrosyl groups, phosphate or phosphoglycolate re- primarily the base lesions N7-methylguanine and N3-methyl- sidues, and certain chain-terminating nucleoside analogues guanine, which are either lost spontaneously due to the in- (11-15).Indeed, past studies using antisense, RNAi, or small creased instability of the N-glycosidic bond or removed as molecule inhibitor strategies have revealed