Chromosome 16q Located Genes CDH1, CDH13 and ADAMTS18 Are Correlated and Frequently Methylated But Not Associated With DNMTs levels In Human Lymphoma

Hypermethylation of promoter contributes to the transcriptional repression of a number of cancer associated genes. In lymphoma, the promoter hypermethylation of many tumor suppressor genes (TSGs), such as p16, has been already known. Using methylation-specific PCR (MSP) and quantitative real-time PCR (qRT-PCR), we examined promoter methylation status and mRNA expression levels of E-cadherin (CDH1), H-cadherin (CDH13) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS18) which are putative TSGs located on chromosome 16q, and also examined the mRNA expression levels of DNA methyltransferases (DNMT1, 3A and 3B) and studied the correlation between these different tested parameters in 36 of lymphomas [included 29 diffuse large B cell (DLBCL) and seven mantle cell lymphoma (MCL)] and 16 non-malignant lymphoid tissues after obtaining informed consent. The expression of DNMTs mRNA were significantly higher in lymphomas compared to non-malignant tissues (p<0.05). Promoter hypermethylation of CDH1, CDH13, and ADAMTS18 was detected in 31/36 (86%), 33/36 (91.6%) and 28/36 (77.7%) of lymphoma, respectively. The expression of CDH1 and ADAMTS18 was significantly reduced in the patients with hypermethylated promoter when compared to unmethylated (p<0.01 and p<0.05, respectively), while no significant difference was found in CDH13. CDH1 and ADAMTS18 expressions were significantly reduced in lymphomas compared to non-malignant tissues (p<0.01), while CDH13 showed no significant difference. Notably, there was significant positive correlation between the expression levels of CDH1 and CDH13 (r = 0.735, p<0.01) (Fig. 1A). Moreover, ADAMTS18 expression showed significant positive correlation with both CDH1 and CDH13 expression levels (r = 0.625, p<0.01; r = 0.720, p<0.01, respectively) (Fig. 1B, C). Also there was significant negative correlation between the expression levels of DNMT3A and 3B with ADAMTS18 (r = -0.396, p<0.01; r = -0.364, p<0.01, respectively), but not with CDH1 and CDH13 (Fig. 2A and B). We could not find any correlation between the levels of DNMTs mRNA and the methylation status of CDH1, CDH13 and ADAMTS18. We examined the effect of 5-Aza-2-deoxycytidine (5-aza-dC) treatment on CDH1, CDH13 and ADAMTS18 expression levels and their methylated promoters in 3 of lymphoma cell lines (Raji, CTB-1 and SLVL) and one patient primary DLBCL cell line. We found that 5-aza-dC treatment of CDH1, CDH13 and ADAMTS18-methylated cell lines led to restoration of their expression levels (Fig. 3A, B, and C). Our results showed that CDH1, CDH13 and ADAMTS18, tumor suppressor genes adjacently located at chromosome 16q, are remarkably correlated and frequently methylated in human lymphoma and their methylation could not be explained solely by the expression level of DNMTs mRNA.