[CANCER RESEARCH 61, 7603–7607, October 15, 2001] Hypoxia-induced Enrichment and Mutagenesis of Cells That Have Lost DNA Mismatch Repair 1 Akira Kondo, Roohangiz Safaei, Misako Mishima, Hannes Niedner, Xinjian Lin, and Stephen B. Howell 2 Department of Medicine and the Cancer Center, University of California San Diego, La Jolla, California 92093-0058 ABSTRACT Loss of DNA mismatch repair (MMR) increases the risk of spontaneous mutations. We sought to determine whether there was an interaction between hypoxia and MMR deficiency that might contribute to the phenomenon of tumor progression. Human colon carcinoma HCT116ch2 (MMR-deficient) and HCT116ch3 (MMR-proficient) sublines were exposed for varying pe- riods of time to an environment of <0.1% O 2 and pH as low as 6.1. When a population containing 5% MMR-deficient cells and 95% MMR-proficient cells was subjected to hypoxia for 72 h, the MMR-deficient cells were en- riched by a factor of 2-fold in the surviving population, whereas no enrich- ment was detected in cells maintained under aerobic conditions. The potential of hypoxia to destabilize the genome was determined by measuring the frequency of clones in the surviving population resistant to very high con- centrations of 6-thioguanine or cisplatin. A 72-h exposure to hypoxia did not increase the frequency of resistant clones in the MMR-proficient cells but produced a 7.8-fold increase in 6-thioguanine-resistant clones and a 2.5-fold increase in cisplatin-resistant clones in the MMR-deficient cells. Loss of MMR increased the frequency of mutations in a reporter vector sensitive to frameshift mutations in a microsatellite sequence. Exposure to hypoxia for a time period as short as 48 h further increased the number of mutations in both cell types, but the absolute number of mutants was higher in the MMR-deficient cells. These results indicate that hypoxia and its accompany- ing low pH enrich for MMR-deficient cells and that loss of MMR renders human colon carcinoma cells hypersensitive to the ability of hypoxia to induce microsatellite instability and generate highly drug-resistant clones in the surviving population. INTRODUCTION As cancers grow, they often exhibit increasingly malignant behav- ior, presumably as the result of the acquisition of additional genetic alterations. The tumor microenvironment is characterized by regions of both fluctuating and chronic hypoxia, low pH, and nutrient depri- vation (1, 2). The extent of hypoxia has been reported to affect tumor cell invasiveness, metastasis, and the risk of recurrence (3). Hypoxia and its associated low pH have also been observed to augment the emergence of drug resistance (4). One hypothesis advanced to explain these observations is that the microenvironment of a solid tumor is itself mutagenic and may be an important source of genetic instability. Reynolds et al. (5) reported that the frequency of supF reporter gene mutations arising in cells within in vivo tumors was 5-fold higher than that in otherwise identical cells grown in culture and that exposure of cultured cells to hypoxia produced an elevated mutation frequency and a pattern of mutation similar to that observed in the in vivo tumors. The mutation frequency in the HGPRT (hypoxanthine gua- nine phosphoribosyl transferase) gene in tumors was also reported to be higher than that found in cultured cells (6). Several groups have observed an increased frequency of specific types of drug resistance among cells exposed to hypoxia or low pH, effects attributed to amplification of genes encoding the targets of these drugs (7–9). However, the mechanisms by which the tumor microenvironment induces genetic instability and increased mutagenesis are not well understood. It is known that hypoxic cells have numerous alterations in metabolic activity (10). These metabolic changes are assumed to lead to conditions that either cause increased damage to DNA (10) or compromise DNA repair processes (11). Cells subjected to repeated cycles of hypoxia and reoxygenation undergo transiently increased levels of intracellular reac- tive oxygen species that can damage DNA bases and may be a cause of mutation (12). In addition to generating mutations, the hypoxic environ- ment may favor the outgrowth of clones with specific types of genetic defects. Graeber et al. (13) reported that hypoxia favors the expansion of p53-deficient variants that demonstrate reduced apoptosis in response to injury that is normally cytotoxic. The MMR 3 system is particularly important to the maintenance of genomic integrity. Loss of MMR function results in microsatellite instability and underlies hereditary nonpolyposis colon cancer (14 – 18). We and others have demonstrated that loss of MMR causes resistance to the cytotoxic effects of many platinum-containing drugs (19 –21) and also renders cells hypersensitive to the mutagenic effects of cisplatin (22). We recently showed that loss of MMR renders cells both resistant to the cytotoxicity of H 2 O 2 and hypersensitive to the mutagenic effect of this oxidative stress (23). We report here that hypoxia, along with its associated low pH, enriches for MMR-defi- cient cells in the surviving population and makes cells sensitive to some forms of hypoxia-induced genomic instability. MATERIALS AND METHODS Cell Lines and Materials. Clones of the human colorectal adenocarcinoma hMLH1-deficient cell line HCT116 that had undergone chromosome 3 transfer (HCT116/3-6, identified here as HCT116+ch3) and chromosome 2 transfer (HCT116/2-1, identified here as HCT116+ch2) were obtained from Drs. C. R. Boland, M. Koi, and T. A. Kunkel (24). These cell lines were maintained in a 5% CO 2 atmosphere at 37°C in Iscove’s modified Dulbecco’s medium (Irvine Scientific, Irvine, CA) supplemented with L-glutamine, 10% heat-inactivated fetal bovine serum, and 400 g/ml Geneticin (Life Technologies, Inc., Gaith- ersburg, MD). All cell counts were performed in the presence of 0.1% trypan blue. The absence and presence of hMLH1 expression in these cell lines was verified by immunoblot analysis (data not shown). All cell lines tested negative for contamination with Mycoplasma species. Cisplatin was kindly provided by Bristol-Myers Squibb Co. (Princeton, NJ). A stock solution of 1 mM cisplatin in 0.9% NaCl was stored in the dark at room temperature. Hypoxia. Hypoxic conditions were created using plastic chambers (AnaeroPac jar; Mitsubishi Gas Chemical Co., Inc.) modified to permit gas flushing (25). Deoxygenation was accomplished by flushing the chamber for 15 min with a mixture of 95% N 2 and 5% CO 2 . To further deplete oxygen, an ascorbic acid sachet (Mitsubishi Gas Chemical Co., Inc.) was added to each chamber just before it was flushed with nitrogen/CO 2 . Anaerobic conditions were confirmed using Anaero-Indicator strips that documented the oxygen content in the chamber to be between 0 and 0.1% in all experiments. To prevent evaporation of medium from the dishes, dishes of distilled water were positioned in the chamber. Received 3/22/01; accepted 8/15/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part byNIH Grant CA78648. This work was conducted in part by the Clayton Foundation for Research-California Division. X. L. and S. B. H. are Clayton Foundation investigators. 2 To whom requests for reprints should be addressed, at Department of Medicine 0058, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0058. Phone: (858) 822-1110; Fax: (858) 822-1111; E-mail: showell@ucsd.edu. 3 The abbreviations used are: MMR, DNA mismatch repair; GFP, green fluorescence protein; 6TG, 6-thioguanine. 7603 Research. on December 14, 2021. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from