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

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 de®ciency 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 e ects that may in¯uence 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-speci®c 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 ®rst generated.

Hypermethylation trigger of the glutathione-S-transferase gene (GSTP1) in prostate cancer cells

“…(2) The GSTP1 CpG island sequence itself is not a target for abnormal methylation in the prostate cancer cell lines because the transfected sequence remained unmethylated. Similar ®ndings were also found for the VHL gene that was not methylated when transfected into a renal colorectal cell line that methylated its endogenous VHL gene (Kuzmin et al, 1999); suggesting that it is cis-speci®c local features and not transacting factors that are pivotal in maintaining and perpetuating the hypermethylation of the VHL CpG island. (3) Sp1 binding or binding of other protein factors in the essential promoter region are not responsible for protecting the GSTP1 CpG island from de novo methylation, because both speci®c mutation or removal of the essential promoter region does not trigger hypermethylation of the transfected DNA.…”

The candidate tumor suppressor gene, RASSF1A , from human chromosome 3p21.3 is involved in kidney tumorigenesis

“…In RFX393, UOK108, UOK121, UOK127, and UOK143 cell lines, the VHL gene was silenced by hypermethylation (10,22), but the RASSF1A was hypermethylated only in RFX393, UOK108, and UOK127. RASSF1A wild-type mRNA was expressed in the remaining ''VHLmethylated'' lines UOK121 and UOK143.…”

“…We studied RASSF1A transcription and methylation in RCC somatic-cell hybrid clones that were created earlier for VHL studies (22). Both parental lines (UOK121 and UMRC6) contained multiple copies of chromosome 3 (5-6 copies; data not shown).…”

Epigenetic gene silencing in cancer

“…Similar experiments will be of interest to probe the role of transcription factor deficiency or repressor overexpression in the silencing of other TS genes that are hypermethylated in cancer cells. A recent study used somatic cell genetics and chromosome transfers (31) to test for deficiency of trans-acting factors or overexpression of transcriptional repressors in a renal cell carcinoma line with VHL silencing and hypermethylation, and the findings did not support either of these mechanisms.…”