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Chemotherapy-induced O⁶-Benzylguanine-resistant Alkyltransferase Mutations in Mismatch-deficient Colon Cancer¹

Division of Hematology and Oncology, Department of Medicine and Comprehensive Cancer Center, Case Western Reserve University and University Hospital Cleveland, Cleveland, Ohio 44106-4937

The ability of O⁶-benzylguanine (BG) to inactivate alkyltransferase (AGT) to potentiate the antitumor efficacy of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is being tested in clinical trials. As of now, there are no examples of acquired resistance to BG+BCNU in the clinical setting. However, we hypothesized that genetically unstable tumors might develop resistance to the combination after repeated drug-exposures to achieve therapeutic efficacy. To evaluate this possibility, we treated three colon cancer cell lines that are either proficient in mismatch repair (MMR) [SW480 (MMR wild type)] or deficient in MMR [HCT116 (hMLH1 mutant) and HCT15 (hMSH6 mutant)] with three cycles of BG+BCNU. After drug-treatments, HCT116 and HCT15 were completely resistant to BG-potentiated cytotoxicity of BCNU. In these two cell lines, the acquired BG resistance resulted from two de novo and different mutations at amino acid 165 in AGT: 165-lysine (K) to glutamic acid (E) (K165E in HCT116), and 165-lysine to asparagine (N) (K165N in HCT15). Both K165-mutated AGTs had markedly decreased enzymatic activity because of unstable AGT protein but were remarkably resistant to BG inactivation. FISH analysis showed that only one copy of MGMT gene exists in HCT116 cells, and the status of promoter methylation of MGMT in HCT15 showed that one allele of the MGMT promoter has an aberrant methylation. Thus, the MGMT gene expressing AGT either from one copy (HCT116) or from unmethylated allele (HCT15) was mutated because of the exposure to BG+BCNU in these two MMR-deficient cell lines. Conversely, MMR-proficient SW480 cells, treated with three cycles of BG+BCNU, maintained wt AGT and the sensitivity to BG-potentiated BCNU-cytotoxicity. To confirm that K165-mutated AGT proteins were responsible for resistance to BG+BCNU, we transfected K165E and K165N MGMT cDNAs into Chinese hampster ovary (CHO) cells. Transfected CHO cells had low AGT activity but increased IC₅₀ for either BCNU or temozolomide (TMZ), compared with parental CHO cells. BG did not potentiate the cytotoxicity of these two alkylating agents at concentrations up to 200 µM; in contrast, BG, at 25 µM, sensitized CHO-AGT (transfected with wt MGMT cDNA) cells to BCNU or TMZ-cytotoxicity by 3–4 fold. These results suggest that K165 AGT mutants arising in MMR-deficient tumor cells after treatment with chemotherapeutic agents are both resistant to BG-inactivation and are active in the repair of alkylated DNA adducts.

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