Lung Cancer (2007) 55, 205—213 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/lungcan Alteration of DNA methyltransferases contributes to 5 ′ CpG methylation and poor prognosis in lung cancer  Ruo-Kai Lin a , Han-Shui Hsu b , Jer-Wei Chang a , Chih-Yi Chen c , Jung-Ta Chen d , Yi-Ching Wang a,e,∗ a Department of Life Sciences, National Taiwan Normal University, Taipei, Taiwan, ROC b Division of Thoracic Surgery, Taipei Veterans General Hospital, National Yang-Ming University School of Medicine, Taipei, Taiwan, ROC c Division of Thoracic Surgery, Taichung Veterans General Hospital, Taichung, Taiwan, ROC d Department of Pathology, Taichung Veterans General Hospital, Taichung, Taiwan, ROC e Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC Received 9 April 2006; received in revised form 29 July 2006; accepted 19 October 2006 KEYWORDS DNMT; MeCP2; 5 ′ CpG hypermethylation; NSCLC; Immunohistochemistry; Tissue chromatin immunoprecipitation Summary Overexpression of DNA methyltransferases DNMT1, DNMT3a and DNMT3b has been reported in various cancers. However, physical binding of DNA methyltransferase (DNMT) to the hypermethylated promoter of tumor suppressor genes (TSGs) has never been demonstrated in tumor tissues. In addition, alteration of DNMT at the protein level has never been reported in the same series of cancer patients. By immunohistochemical analysis, we demonstrated that DNMT1, DNMT3a and DNMT3b proteins were highly expressed in a coordinate manner in lung tumors, particularly in smokers (P = 0.037, by the Fisher exact test). Patients with DNMT1 overexpression had a trend of poorer prognosis than those without such overexpression, and this prognostic significance was apparent in squamous carcinoma (SQ) patients (P = 0.041, by the log-rank test). Both DNMT1 and DNMT3b overexpressions correlated with hypermethylation in the TSG promoters, especially among smoking SQ patients (P =0.012). To further explore the molecular mechanisms between altered TSGs promoter methylation and overexpression of DNMTs protein, we performed a tissue chromatin-immunoprecipitation polymerase chain reac- tion assay for lung tumors and showed that the methylated FHIT, p16 INK4a and RARˇ promoters were bound by both DNMT protein and methyl-CpG-binding protein 2. These data suggest that overexpression and strong binding of various DNMTs may result in promoter hypermethylation of multiple TSGs and ultimately lead to lung tumorigenesis and poor prognosis. © 2006 Elsevier Ireland Ltd. All rights reserved.  Our data provide new evidences for a concurrent overexpression of DNMT1, 3a and 3b proteins, which are associated with hypermethylated tumor suppressor genes by physical binding of DNMTs to promoter of tumor suppressor genes in lung tumors. ∗ Corresponding author at: Department of Pharmacology, College of Medicine, National Cheng Kung University, No. 1, Ta-Hsueh Road, Tainan 701, Taiwan, ROC. Tel.: +886 6 2353535x5835; fax: +886 6 2749296. E-mail address: ycw5798@mail.ncku.edu.tw (Y.-C. Wang). 0169-5002/$ — see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2006.10.022