Gulp1, an Adaptor Protein Required for Clearance of Apoptotic Cells, Is Frequently Inactivated by Promoter Methylation in HNSCC
OF Ikpatt, CH Xie, BR Smoller, JY Suen, CY Fan. University of Arkansas for Medical Sciences, Little Rock, AR; John L. McClellan Memorial Veterans Hospital, Little Rock, AR
Background: Despite multimodality treatment, the overall survival for patients with HNSCC remains dismal. Therefore, it is of ulmost importance to identify new molecular targets that can be used for early tumor development, prediction of behavior and development of personalized, more effective antitumor therapies. Using gene expression microarray in combination of DNA demethylation by 5-azacytidine, we tentatively identified some novel genes that are epigenetically regulated in HNSCC. One such candidate gene is Gulp1, an evolutionarily conserved adaptor protein required for efficient engulfment of apoptotic cells by phagocytes. In this study, we further analyze epigenetic regulation of Gulp1 expression in HNSCC.
Design: The promoter hypermethylation in the Gulp1 gene was identified by PCR-based restriction fragment length polymorphism (RFLP) using primers specific for the Gulp1 promoter CpG island, methylation specific PCR (MSP) and further verified by direct bisulfite DNA sequencing in 11 HNSCC lines and one normal keratinocyte cell line. HNSCC and normal keratinocyte lines treated with or without DNA demethylating agents (gossypol and 5-azacytidine) were analyzed for Gulp1 mRNA expression by real-time RT-PCR. Gulp1 promoter methylation was also analysed in 154 archival HNSCC cases using MSP.
Results: 6 of 11 (54%) HNSCC lines showed promoter hypermethylation for the Gulp1 gene by both RFLP and MSP methods. Gulp1 promoter methylation was significantly reduced by the treatment of a DNA demethylating agent (5-azacytidine or gossypol) as detected by both Direct bisulfite DNA sequencing and MSP. Correspondingly, the expression of Gulp1 was significantly increased following the treatment with DNA demethylating agent. By MSP, 76 of 154 (49%) archival primary HNSCC displayed promoter hypermethylation of the Gulp1 gene.
Conclusions: Gulp1 may represent a newly identified candidate tumor suppressor gene that show frequent epigenetic inactivation in HNSCC.
Category: Head & Neck
Tuesday, March 23, 2010 1:00 PM
Poster Session IV # 154, Tuesday Afternoon