Characterization of Epigenetic Alterations of Tumor Suppressor Genes in Chemoresistant Head and Neck Squamous Cell Carcinoma
C-Y Fan, C Xie, H Zhang, BR Smoller, JY Suen. University of Arkansas for Medical Sciences, Little Rock, AR; John L. McClellan Memorial Veterans Hospital, Little Rock, AR
Background: Despite multimodality treatment regimens, the overall 5-year survival rate for HNSCC has not exceeded 50% for the past three decades. One major factor for this dismal survival is the development of chemoresistance, resulting in frequent recurrences. Molecular mechanisms of chemoresistance in HNSCC have been extensively studies but remain elusive. Here, we attempt to characterize the patterns of epigenetic alterations of tumor-associated genes in a laryngeal cancer cell line that develop resistant to most chemotherapeutic agents.
Design: A HNSCC cell line, HEp2, which is highly resistant to Docetaxel and Cisplatin (DR-HEp2) and its parental sensitive cell line (HEp2) were used for RNA extraction, followed by AffyMetrix microarray analysis using human U133A and U133B gene chips, which contain 33,000 unique genes. Genes that displayed reduced expression in DR-HEp2 as compared to parental HEp2 cells, and subsequently showed increased expression following treatment with 5-aza cytidine, a DNA demethylating agent were selected. Those with promoter CpG island were then subjected to PCR-based restriction fragment length polymorphism (RFLP) analysis to determine the status of promoter methylation.
Results: Among 33,000 genes, there were 129 genes in DR-HEp2 cancer cells that showed decreased expression by 2-fold or greater as compared to its parental HEp2 cells, and increased in expression by 2-fold or greater following the treatment of 5-aza-cytidine. The promoter sequences of these 129 genes were then analyzed and 23 genes were identified, which contained a CpG island in the promoter. Five of these 23 genes, CLGN, BCAT1, HDGFRP3, MAP1B and CaRF were further analyzed by PCR-based RFLP and 2 of them, namely HDGFRP3 and MAP1B demonstrated promoter hypermethylation in DR-HEp2 but not in HEp2 cancer cells.
Conclusions: Using microarray technology combined with genetic analysis for promoter CpG island in chemoresistant head and neck cancer, we have tentatively identify a subset of tumor suppressor genes that may undergo gene silencing through promoter methylation as compared to its parental sensitive cells. These data should be invaluable in designing epigenetic therapies aiming at overcoming chemoresistance commonly seen in human cancer.
Category: Head & Neck
Tuesday, March 10, 2009 1:30 PM
Platform Session: Section H, Tuesday Afternoon