Supplementary Material for: An Epigenetic Screen Unmasks Metallothioneins as Putative Contributors to Renal Cell Carcinogenesis

Objective: Functional epigenetic studies aimed to re-express transcriptionally silenced genes in renal cell carcinoma (RCC) may facilitate the ongoing search for appropriate markers supporting clinical decision-making. Methods: The RCC cell line A-498 was treated with the DNA methyltransferase inhibitor zebularine under low-cytotoxicity conditions. RNA chip analyses revealed several upregulated transcripts that were further validated by qPCR on 49 matched pairs of human kidney tissues to identify suitable marker candidates. Results: Members of the metallothionein (MT) group were remarkably downregulated in tumor tissues. MT1G and MT1H expression was decreased in 98% of cases, whereas MT2A expression was downregulated in 73% of all cases. Comparison of 308 reactivated transcripts upregulated more than 1.5-fold to published data revealed a high number of shared candidates, which supports the consistency of this experimental approach. Conclusion: MTs were found to be transcriptionally inactivated in human RCC. Our observations support the hypothesis of a possible involvement of these metalloproteins in renal cell carcinogenesis. Additional functional studies of these genes may provide clues for understanding renal cancers as essentially metabolic diseases.