Loss of heterozygosity at 9q33 and hypermethylation of the DBCCR1 gene in oral squamous cell carcinoma

Abstract

The DBCCR1 gene at chromosome 9q33 has been identified as a candidate tumour suppressor, which is frequently targeted by promoter hypermethylation in bladder cancer. Here, we studied the possible involvement of DBCCR1 in the development of oral squamous cell carcinoma. DNA from 34 tumours was examined for loss of heterozygosity (LOH) at three markers surrounding DBCCR1 and for hypermethylation of the DBCCR1 promoter, using methylation-specific PCR and methylation-specific melting-curve analysis. LOH was found in 10 of 31 cases (32%), and DBCCR1 hypermethylation was present in 15 of 34 cases (44%). Hypermethylation of DBCCR1 was also present in three of seven epithelial tissues adjacent to the tumours, including two hyperplastic and one histologically normal epithelia. Furthermore, of four oral leukoplakias with dysplasia, one showed LOH at 9q33 and two showed DBCCR1 hypermethylation. These data suggest that LOH at 9q33 and hypermethylation of the DBCCR1 promoter are frequent and possibly early events in oral malignant development.

Figure 1

LOH analysis of 9q33 in oral squamous cell carcinomas. T, tumour; N, normal tissue; T1, well-differentied tumour cells adjacent to normal epithelium; T2, poor-differentied tumour cells far away from normal epithelium. Arrows indicate LOH.

Figure 2

Methylation analysis of the DBCCR1 gene promoter in oral squamous cell carcinomas. Left, MS-PCR. Genomic DNA was treated with sodium bisulphfite and PCR-amplified with primer pairs specific for methylated (M) and unmethylated (U) alleles. Right, MS-MCA. Bisulphfite-treated DNA was amplified in the presence of SYBR Green I using primers that do not discriminate between methylated and unmethylated DBCCR1 alleles. The melting characteristics of the PCR products were determined directly in the PCR tube by continuous fluorescence monitoring during a temperature transition. SssI-methylated DNA and genomic DNA from normal peripheral blood lymphocytes (PBL) provided positive controls for methylated and unmethylated DBCCR1 alleles, respectively. Tu, tumour; Ep, epithelium; Cn, connective tissue; T1, well-differentiated tumour cells adjacent to normal epithelium; T2, poor-differentiated tumour cells far away from normal epithelium.