Transcriptional regulation of the mismatch repair gene hMLH1 under hypoxic conditions

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Inactivation of the DNA mismatch repair (MMR) system induces genome instability, which is a cause of carcinogenesis and cancer progression. Hypoxia is known to induce genome instability, and has recently been shown to inactivate the MMR system via alteration of hMLH1 expression. Hypoxia may induce genome instability through alteration of hMLH1 expression, and HIF-1α, a key factor in cellular response to hypoxia, might play a part, so we attempted to clarify the role of hypoxia and HIF-1α in transcriptional regulation of hMLH1. First, a 1649 bp length of the 5’ region of hMLH1 was subcloned. Transient transfection of the reporter (pGL3-MLH1 Pro) into MCF-7, HepG2, and HSC4 cells showed strong promoter activities, and activities increased to over 2-fold when cells were incubated in hypoxic conditions (1% O₂ for 36 h). To clarify the role of HIF-1α in these hypoxic activations of hMLH1 transcription, co-transfection assays with a wild- or dominant negative-form of HIF-1α (HIF-1α or HIF-1αDN) were performed in MCF-7. We found that hypoxia-activated promoter activity was suppressed to 30% of the control by HIF-1αDN overexpression, while HIF-1α overexpression remarkably increased the promoter activity (6.5-fold). To identify the hypoxia response element (HRE) in the promoter region of hMLH1, a series of deletion mutants was constructed: Co-transfection with HIF-1α showed that the region from -273 to -4 played an important role in hypoxia- and HIF-1α-mediated transcriptional activation of hMLH1. We also investigated hMLH1 expression in clinical samples to determine whether hMLH1 was actually upregulated in vivo. Total RNAs were prepared from endometrial carcinomas and the adjacent normal tissue samples of 16 patients, and expression levels were analyzed by real-time RT-PCR method: As expected, the expression levels of hMLH1 in cancer tissues were significantly higher than those in normal tissues (P = 0.015), and we found overexpression of carbonic anhydrase IX gene (CAIX), a tumor-related hypoxic marker gene, in cancer tissues (P < 0.001), with 10 of 16 cancer tissues showing overexpression of both hMLH1 and CAIX. In conclusion, we demonstrated here that hypoxia can affect the transcriptional regulation of hMLH1 via HIF-1α in cancer cell lines and that hMLH1 is up-regulated in endometrial carcinomas.The clinical significance of hMLH1 and hypoxia in endometrial carcinoma is now under investigation.