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Analysis of aberrant methylation of the VHL gene by transgenes, monochromosome transfer, and cell fusion

Oncogene volume 18, pages 5672–5679 (1999)Cite this article

Abstract

Several tumor suppressor genes were shown to be inactivated by a process involving aberrant de novo methylation of their GC-rich promoters which is usually associated with transcriptional repression. The mechanisms underlying this process are poorly understood. In particular this abnormal methylation may be caused and/or maintained by either deficiency of some trans-acting factor(s) or by various malfunctions acting in cis. Here we studied the nature of aberrant methylation of the von Hippel-Lindau (VHL) disease tumor suppressor gene in a human clear cell renal carcinoma cell line, UOK 121, that contains a silent hypermethylated endogenous VHL allele. First, we transfected unmethylated VHL transgenes, driven by the VHL promoter, into UOK 121 cells. Next, to exclude possible position effects that may influence methylation of the introduced VHL genes, we transferred a single chromosome 3, carrying an apparently normal hypomethylated VHL allele into the UOK 121 cells. Finally, we created somatic cell hybrids between UOK 121 and UMRC 6 cells containing a mutant VHL-expressing hypomethylated allele. In these three experiments both the methylation of the VHL promoter and the transcriptional status of the introduced and endogenous VHL alleles remained unchanged. Our results demonstrate that the putative trans-acting factors present in the UOK 121 and UMRC 6 cells are unable to induce changes in methylation pattern of the VHL alleles in all cell lines and hybrids studied. Taken together, the results indicate that cis-specific local features are pivotal in maintaining and perpetuating aberrant methylation of the VHL CpG island. Contribution of some putative trans-acting factors cannot be excluded during a period when the aberrant VHL methylation pattern was first generated.

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Acknowledgements

We wish to thank Dr MW Linehan for providing the UOK 121 cells. We acknowledge the National Cancer Institute for allocation of computing time and staff support at the Frederick Biomedical Supercomputer Center of the Frederick Cancer Research and Development Center. This project has been funded with federal funds from the National Cancer Institute, National Institutes of Health, under contract No NO1-CO-56000 and also supported, in part, by NCI grant 19401 to EJ Stanbridge.

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Authors and Affiliations

  1. Intramural Research Support Program, SAIC-Frederick, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, 21702, Maryland, MD, USA

    Igor Kuzmin, Laura Geil & Fuh-Mei Duh

  2. Department of Microbiology and Molecular Genetics, University of California, Irvine, 92697-4025, California, CA, USA

    Haiyan Ge, Ulla Bengtsson & Eric J Stanbridge

  3. Laboratory of Immunobiology, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, 21702, Maryland, MD, USA

    Michael I Lerman

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  1. Igor Kuzmin
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Kuzmin, I., Geil, L., Ge, H. et al. Analysis of aberrant methylation of the VHL gene by transgenes, monochromosome transfer, and cell fusion. Oncogene 18, 5672–5679 (1999). https://doi.org/10.1038/sj.onc.1202959

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