Hypermethylation of the Keap1 gene in human lung cancer cell lines and lung cancer tissues

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Abstract

Low expression of the oxidative stress sensor Keap1 is thought to be involved in carcinogenesis. However, the mechanisms responsible for inactivation of the Keap1 gene remain unknown. We investigated Keap1 expression using RT-PCR and found that it was downregulated in lung cancer cell lines and tissues when compared with a normal bronchial epithelial cell line. Treatment with 5-Aza-2′-deoxycytidine restored Keap1 expression in lung cancer cell lines, indicating the silencing mechanism to be promoter methylation. Moreover, we evaluated cytosine methylation in the Keap1 promoter and demonstrated that the P1 region, including 12 CpG sites, was highly methylated in lung cancer cells and tissues, but not in normal cells. Importantly, we found evidence that three specific CpG sites (the 3rd, 6th, and 10th CpGs of P1) might be binding sites for proteins that regulate Keap1 expression. Thus, our results suggest for the first time that Keap1 expression is regulated by an epigenetic mechanism in lung cancer.

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Materials and methods

Cell lines and tissue samples. Human lung cancer cell lines were obtained from the Respiratory Research Department of the Third Military Medical University. A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, CA, USA) supplemented with 5% fetal bovine serum (FBS). SPC-A1 and NCI-H460 cells were grown in RMPI 1640 (Invitrogen) supplemented with 10% FBS. The human normal bronchial epithelial cell line (BEAS-2B) was a gift from the Academy of Military Medical Sciences

Expression of Keap1 was down-regulated in lung cancer lines and lung cancer tissues

Keap1 mRNA expression in four lung cell lines and cancer tissues was significantly decreased in three lung cancer cell lines and five cancer tissues when compared to the normal cell line BEAS-2B, where it was highly expressed (Fig. 1). To our knowledge, this is the first determination of relative Keap1 mRNA levels in lung cell lines and cancer tissues by RT-PCR. However, these results were consistent with other reports that Keap1 protein levels were dramatically decreased in lung cancer lines

Acknowledgments

We thank Dajun Deng (Peking University School of Oncology and Beijing Institute for Cancer Research, China) for valuable suggestions. We are also grateful for Zhihua Yang (Academy of Military Medical Sciences, China) and Guansong Wang (Respiratory Research Department of Xinqiao Hospital, Third Military Medical University, China) for providing cell lines. This work was supported in parts by Grants Nos. 30570983 and 30640065 from the National Science Foundation of China (NSFC).

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