Is DNA Hypomethylation Like DNA Hypermethylation Sometimes Associated with Tumor Progression

Hypermethylation of a subset of CpG islands is progressive in some types of cancer although sometimes this regional hypermethylation occurs very early in tumorigenesis, and other times it can serve as a significant indicator of survival.16,82, 3 Also, DNA hypomethylation is sometimes associated with tumor progression as seen in studies of repeated DNA sequences. In a study of 31 hepatocellular carcinomas by Itano and coworkers,84 the degree ofhypomethylation of either of two repetitive sequences was significantly correlated with postoperative recurrence of hepatocellular carcinoma and was a better predictor than conventional factors. These repeats were the above-mentioned 1.4-kb Y10752/NBL2 sequence that is found in the pericentromeric regions of chromosomes 13, 14, 21, and 950 and a 13-kb repeat present in tandem about 200 times on 8q21. Hypomethylation was determined by quantitating the corresponding radioactive spots relative to reference spots by restriction landmark genomic scanning (RLGS) involving two-dimensional electrophoresis of DNA doubly digested with the CpG methylation-sensitive NotI and the CpG methylation-insensitive PvuII. The 1.4-kb Y10752/ NBL2 repeat (CNIC) and the 13-kb repeat (HTRS) were fully methylated at the examined sites in normal liver. Hypomethylation of Y10752/NBL2 and HTRS was also significantly correlated with the presence of hepatitis B or C antibodies in the serum. Itano and coworkers suggest that viral infection could influence the tumors' malignant potential and tumor recurrence indirectly by predisposing to DNA hypomethylation. In an earlier study, this group showed that the number of spots that appear in RLGS profiles of hepatocellular carcinomas but not in the profiles of normal liver was also a significant and independent prognostic indicator of postoperative occurrence of the disease.85 Their appearance specifically in the hepatocarcinoma RLGS profile is presumably because they are hypomethylated only in the tumors.

In a prostate cancer study, LINE-1 hypomethylation had a highly significant relationship with lymph node involvement for prostate adenocarcinomas.41 Recently, we have shown that hypomethylation of both Sata centromeric and Sat2 juxtacentromeric repeats is significantly associated with tumor grade and decreased survival in primary ovarian carcinomas (M. Ehrlich and M. Widschwendter, unpub. results). In collaboration with Louis Dubeau, we also demonstrated that there is a significant association of malignant potential and hypomethylation of Sat2 DNA in the juxtacentromeric heterochromatin of chromosomes 1 and 16 in a comparison of benign ovarian cystadenomas, low malignant potential tumors, and carcinomas.46 Moreover, there was also a significant association of Sat2 hypomethylation with global hypomethylation of the genome in these neoplasms, as determined by Southern blot analysis for satellite hypomethylation and high-performance liquid chromatography of DNA digested to deoxynucleosides for global hypomethylation. These studies suggest that one of the carcinogenesis-promoting advantages of global hypomethylation is that it is often linked to hypomethylation of satellite DNA sequences. Global hypomethylation might also be related to hypomethylation of interspersed DNA repeats, including retroelements.

Not only is the degree of global genomic hypomethylation in ovarian epithelial neoplasms associated with the degree of malignancy,24 but also it is significantly associated with the grade of cervical neoplasia,2 with multifocal vs. unifocal hepatocellular carcinomas,86 and the disease stage, tumor size, and histological grade for breast tumors.87 However, the latter three studies were done by incorporation of methyl groups in vitro, and it is desirable to see confirmation with a direct analysis of m5C levels. A study of global levels of DNA methylation at Hpall sites in breast cancer, as determined by the extent of smearing of DNA fragments in Hpall digests upon electrophoresis, revealed no significant association of hypomethylation with tumor grade although almost all of the tumors were hypomethylated compared to normal breast tissue.88 The quantitation of DNA methylation by this method might have been complicated by different amounts of degradation of the DNA during isolation.

As for CpG island hypermethylation in cancers, cancer-associated hypomethylation of certain DNA sequences in some types of cancers occurs early in tumorigenesis and, in others, only later. Previously, we found that 21 benign tumors from the breast, ovary, uterus, thyroid, or brain had an average genomic m5C content that was the same as that from all 15 of the various normal human tissues (0.89 mol%; percentage of the bases as m5C) examined.22 The analogous values for the 20 metastases and 62 primary tumors were 0.78 and 0.83 mol%, respectively.

In contrast, there is evidence for the early appearance of DNA hypomethylation during some types of tumorigenesis. Goelz et al71 found hypomethylation of 3-4 of10 examined genes by Southern blot analysis with CpG methylation-sensitive restriction endonucleases in adenomatous colon polyps from seven patients. Five of those patients had colon cancers also displaying hypomethylation of the same genes. In collaboration with Andy Feinberg and Charles Gehrke, we compared colon tumors and adjacent apparently normal tissue. We found that there was a reproducible decrease in global DNA methylation levels of about 8% in eight colonic polyps from colon cancer patients.74 Ribieras and coworkers observed hypomethylation of the A-y globin gene in two samples of lobular carcinoma in situ, an early stage of breast cancer.72 However, we found that only one of 19 samples from abnormal but nonmalignant breast tissue showed moderate-to-strong hypomethylation of Sat2 compared with almost half of 25 examined breast adenocarcinomas displaying moderate-to-strong hypomethylation.45 The benign samples were tissues displaying mild or moderate fibrocystic changes, fibroadenoma, gynecomastia, or benign phylloides tumor. How early DNA hypomethylation can be detected during tumorigenesis probably depends on the DNA sequence being examined, the type of tumor, and the individual tumor sample, as is often the case for CpG island hypermethylation.

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