Molecular Cancer Therapeutics 1061 Blockage of abasic site repair enhances antitumor efficacy of 1,3-bis-(2-chloroethyl)-1-nitrosourea in colon tumor xenografts Lili Liu, Ling Yan, Jon R. Donze, and human tumors to these agents are the common cause of Stanton L. Gerson treatment failure. An effort to overcome drug resistance by targeting a specific DNA repair pathway has yielded Division of Hematology and Oncology, Department of Medi- O6 cine, CaseWestern Reserve University, Cleveland, OH promising results. For instance, inactivation of -alkyl- guanine-DNA alkyltransferase (AGT) with O6-benzylgua- nine (BG) potentiates alkylating agents, such as Abstract temozolomide (TMZ) and 1,3-bis-(2-chloroethyl)-1-nitro- Methoxyamine (MX) has been shown to potentiate the sourea (BCNU). Unfortunately, this combined treatment antitumor effect of temozolomide (TMZ) in human tumor is limited by a narrow therapeutic index due to severe xenograft models. This potentiation is due to the reactivity myelosuppression. As with other targeted therapeutics, of MX with apurinic/pyrimidinic (AP) sites in DNA, which agents that block a single pathway are not completely are formed following DNA glycosylase removal of TMZ- effective because tumors maintain a complex machinery to induced methyl-purine adducts. MX-bound AP sites cannot repair DNA lesions induced by therapeutic agents. Thus, be further processed by base excision repair (BER), resulting to develop new targets to improve therapeutic efficacy is in cell death. On the basis of this finding, we investigated in particularly urgent. vivo whether MX enhanced therapeutic efficacy of other We have previously shown that methoxyamine (MX) agents, such as 1,3-bis-(2-chloroethyl)-1-nitrosourea potentiates the cytotoxic effect of methylating agent, TMZ, (BCNU) that generates AP sites during DNA repair. Nude both in vitro and in human colon tumor xenograft models mice carrying human colon tumor xenografts, HCT116 and (1, 2). We proposed that this potentiation was mediated HCT116-Ch3, were treated by a single injection (i.p) of through the known ability of MX to bind apurinic/ BCNU alone (30 mg/kg) or MX (2 mg/kg) combined with pyrimidinic (AP) sites in DNA generated by methyl-purine BCNU. The effect on tumor growth of BCNU alone was very glycosylase, the first step to remove abnormal bases moderate. Combined administration of MX and BCNU induced by alkylating agents in base excision repair produced significant inhibition of tumor growth. Tumor (BER). MX-bound AP sites are very poor substrates for growth delays were 14 F 3 days in HCT116 and 16 F AP endonuclease (APE), so that BER pathway is inter- 2 days in HCT116-Ch3 tumors, respectively (P < 0.05 rupted and cell death ensues (1, 2). Because AP sites are the versus control or BCNU alone groups). Similar results were common intermediates produced during repair of a also observed in SW480 and DLD1 tumors. Importantly, no number of DNA adducts, we hypothesized that MX- systemic toxicity was noted with BCNU and MX. In initiated interruption of BER pathway would potentiate contrast, BCNU (at dose of 25 mg/kg) combined with O6- the cytotoxic effects of another alkylating agent, BCNU. benzylguanine (BG), an inhibitor of O6-alkylguanine-DNA MX was first introduced as a tool to study BER pathway alkyltransferase (AGT) being tested in clinical trials, caused in 1985 (3). The specific action of MX in disruption of BER toxic death in all treated mice. However, a lower dose has been clearly demonstrated: (a) MX reacts with the BCNU (10 mg/kg) combined with BG and MX had tautomeric open-ring form of deoxyribose generated from significant antitumor effect without toxic death. Thus, the removal of an abnormal base by any one of the DNA targeting BER with MX is a promising strategy to improve glycosylases (4). (b) The reaction of MX with AP sites is fast, the antitumor activity of BCNU and perhaps other DNA- even faster than APE (4). (c) MX blocks repair of AP site damaging agents. (Mol Cancer Ther. 2003;2:1061–1066) due to the chemical modification of AP sites, rather than poisoning of the enzyme, APE (3). (d) MX-bound AP sites Introduction are resistant to cleavage by dRP lyase activity of DNA h Alkylating agents are a major class of chemotherapeutic polymerase by a factor of 300-fold (5). Although MX has drugs. However, intrinsic or acquired drug resistances in been a known inhibitor of BER for over 20 years, we were the first to explore MX as a modulator of alkylating agents in anticancer treatment. Received 4/3/03; revised 6/4/03; accepted 6/27/03. The impact of BER on BCNU cytotoxicity has not been The costs of publication of this article were defrayed in part by the fully evaluated. A recent study has shown that cells from payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mice deficient in 3-methyladenine DNA glycosylase have indicate this fact. increased sensitivity to BCNU (6). Reduced DNA damage Grant support: Public Health Service Grants RO1CA86357, and cytotoxicity induced by BCNU was observed in human RO1CA82292, and CA43703. lung cells transfected with genes of Escherichia coli forma- Requests for Reprints: Lili Liu, Division of Hematology and Oncology, midopyrimidine-DNA glycosylase or human 8-oxoguanine- Department of Medicine, Case Western Reserve University, 10900 Euclid h Avenue, BRB-3, Cleveland, OH 44106. Phone: (216) 368-5194; DNA glycosylase (7). Overexpression of polymerase , the Fax: (216) 368-1166. E-mail: [email protected] major polymerase involved in short patch BER, increased Downloaded from mct.aacrjournals.org on September 23, 2021. © 2003 American Association for Cancer Research. 1062 Blockage of Abasic Site Repair Enhances BCNU Efficacy cell resistance to both mono- and bifunctional-alkylating react specifically with an aldehyde group, an open-ring agents (8). It has also been suggested that BCNU resistance is form of the AP sites. The AP site assay was essentially associated with enhanced capacity for repairing AP sites, performed as described by Nakamura et al. (12). Briefly, because the predominant DNA adducts involve alkylation at DNA (15 Ag) extracted from cells with different treatments N7 position of guanine, and these lesions appear to be was incubated with 1 mM ARP (Dojindo Molecular substrates for BER (9–11). Technologies Inc., Gaithersburg, MD) at 37jC for 10 min. In this study, we used colon tumor xenograft models to After precipitation and wash with ethanol, DNA was investigate the potential of MX-mediated BER blockage to resuspended in TE buffer [10 mM Tris-HCl, 1 mM EDTA enhance antitumor effect of BCNU. We chose colon tumor (pH 7.2)]. DNA was heat-denatured at 100jC for 5 min, cells because these cells are well characterized for genetic quickly chilled on ice, and mixed with an equal amount of defects/status in DNA repair, such as mismatch repair ammonium acetate (2 M). The single-stranded DNA was (MMR), p53, and AGT, each of which may confer selective then immobilized on a BAS-85 NC membrane (Schleicher resistance to different DNA damages induced by BCNU. and Schuell, Keene, NH), using a vacuum filter device Thus, they may serve to link specific genetic defect with (Schleicher and Schuell). The NC membrane was incubated drug target. We initially failed to observe significant with streptavidin-conjugated horseradish peroxidase (Bio- potentiation of BCNU by MX in vitro (1), presumably due Genex, San Ramon, CA) at room temperature for 30 min. to different microenvironments in vitro versus in vivo that After NC membrane was rinsed with washing buffer influence drug sensitivities. For instance, in vivo, angio- containing NaCl (0.26 M), EDTA (1 mM), Tris-HCl (20 genesis affects drug pharmacokinetics that may affect the mM), and Tween 20 (1%), ARP-AP sites were visualized duration of interaction between drug and molecular with ECL reagents (Amersham Corp., Piscataway, NJ). targets; or signal transduction cascades and cell to cell Comet Assay interaction differ from in vitro may affect sensitivity of cells The principle of Comet assay is based on the ability of to the apoptotic signaling. In this report, we provide evi- denatured, cleaved DNA fragments to migrate out of the dence that MX potentiates BCNU antitumor effect without cell under the influence of an electric field. Undamaged additive systemic toxicity in in vivo studies. When BG, DNA migrates slower and remains within the confines of BCNU, and MX were used together, lower doses of BCNU the nuclei when a current is applied. DNA damage was are comparable or more effective than higher doses of assessed in a cell based on evaluation of the DNA ‘‘comet’’ BCNU and either agent alone. Thus, blocking both repair tail shape and migration distance (13). Cells were harvested pathways, BER and AGT with MX and BG, improved and washed with PBS after exposure to BCNU (25 AM)or BCNU therapeutic efficacy. MX (25 mM) plus BCNU (25 AM) for 2 h. Cell suspension (1  105/ml cold PBS) was mixed with 1% low gelling Materials and Methods temperature agarose at 42jC at a ratio of 1:10 (v/v) and immediately pipetted 75 Al onto CometSlide (Trevigen, Inc., Tu m o r s in Nu d e Mi c e Gaithersburg, MD). When the low gelling temperature  6 Tumor cells (5 10 ) were injected into the bilateral agarose had set, the slides were submerged in prechilled flanks of female athymic nude mice, at 6–8 weeks of age. lysis buffer [2.5 M NaCl, 100 mM EDTA, and 10 mM Tris- The tumors were measured with calipers using the HCl (pH 10.5–11.5), containing 1% Triton X-100, added just  2 formula: V = L (mm) W (mm)/2, where L is the largest before use] at 4jC for 1 h.