Nicotine induces the fragile histidine triad methylation in human esophageal squamous epithelial cells

The fragile histidine triad (FHIT) gene has been proposed to have an important role in very early carcinogenesis. Methylation of the FHIT gene is associated with transcriptional inactivation in esophageal squamous cell carcinoma, and FHIT inactivation has been linked to smoking-related carcinogenesis. In this study, we confirmed methylation of the FHIT gene in human esophageal squamous epithelial cells (HEECs) and examined whether nicotine induced alteration of FHIT. Methylation status in the promoter region of the FHIT gene and p16(INK4A) gene was determined by methylation-specific PCR in HEECs exposed to nicotine under various conditions. Methylation status of the FHIT gene was confirmed by DNA-sequencing analysis. Protein expression of Fhit and the DNA methyltransferases (DNMTs) DNMT1 and DNMT3a were assessed by immunoblot analysis. In the absence of nicotine, methylation of the FHIT gene and attenuation of Fhit protein were not detected in HEECs. Nicotine induced the methylation of FHIT gene and attenuated Fhit protein in association with increased expression of DNMT3a. Reexpression of Fhit protein in HEECs was found after cessation of moderate- to long-term exposure to nicotine. Our results show that nicotine induces methylation of the FHIT gene followed by loss of Fhit protein expression in HEECs. Continuous smoking may thus increase the risk of esophageal cancer. (c) 2006 Wiley-Liss, Inc.