Quantitative analysis of associations between DNA hypermethylation, hypomethylation, and DNMT RNA levels in ovarian tumors

Abstract

How hypermethylation and hypomethylation of different parts of the genome in cancer are related to each other and to DNA methyltransferase (DNMT) gene expression is ill defined. We used ovarian epithelial tumors of different malignant potential to look for associations between 5'-gene region or promoter hypermethylation, satellite, or global DNA hypomethylation, and RNA levels for ten DNMT isoforms. In the quantitative MethyLight assay, six of the 55 examined gene loci (LTB4R, MTHFR, CDH13, PGR, CDH1, and IGSF4) were significantly hypermethylated relative to the degree of malignancy (after adjustment for multiple comparisons; P < 0.001). Importantly, hypermethylation of these genes was associated with degree of malignancy independently of the association of satellite or global DNA hypomethylation with degree of malignancy. Cancer-related increases in methylation of only two studied genes, LTB4R and MTHFR, which were appreciably methylated even in control tissues, were associated with DNMT1 RNA levels. Cancer-linked satellite DNA hypomethylation was independent of RNA levels for all DNMT3B isoforms, despite the ICF syndrome-linked DNMT3B deficiency causing juxtacentromeric satellite DNA hypomethylation. Our results suggest that there is not a simple association of gene hypermethylation in cancer with altered DNMT RNA levels, and that this hypermethylation is neither the result nor the cause of satellite and global DNA hypomethylation.

Fig. 1. Southern blot analysis of satellite DNA hypomethylation in ovarian epithelial tumors

Examples of satellite DNA hypomethylation in BstBI digests of ovarian carcinomas in a single blot hybridized three times. Normal postnatal somatic DNAs and sperm DNA are the hypermethylated and hypomethylated standards, respectively. (A) juxtacentromeric Chr1 Sat2 probe and (B) centromeric Chr1 Satα probe under high-stringency hybridization conditions; (C), centromeric Chr1 Satα probe under low-stringency conditions that allow hybridization to DNA from most of the centromeres. Scoring of hypomethylation was by phosphorimager quantitation of <4-kb vs. >4-kb signal and visual comparison of bands b, c, d, e, and f to region a.

Fig. 2. Comparison of satellite DNA hypomethylation and global DNA hypomethylation in ovarian epithelial tumors of different malignant potential (A)

The levels of satellite DNA hypomethylation (score 0–4) and (B) the percent of total DNA cytosine residues that were methylated are plotted for individual tumors. Not shown are three LMP tumors and six cystadenomas analyzed only for satellite DNA methylation. The horizontal line is the global DNA hypomethylation threshold, i.e., the minimum percentage of methylated cytosine residues found in diverse normal postnatal somatic tissues (3.43; range for normal tissue types, 3.43–4.1). As a conservative estimate of an overall deficiency of m⁵C in tumor DNAs, global methylation levels of <3.43 were considered hypomethylated. (C) The percentage of tumors displaying global DNA hypomethylation or strong-to-moderate satellite DNA hypomethylation (score 2, 3, or 4) relative to all the somatic controls is shown. P-values for hypomethylation scores vs. degree of malignancy are given. For all of these DNA hypomethylation parameters, except the all-Satα hypomethylation, there was a significant association with the degree of malignancy after adjustment for multiple comparisons.