DNA methyltransferase and demethylase in human prostate cancer

Abstract

Recent studies have shown that cytosine-5 methylation at CpG islands in the regulatory sequence of a gene is one of the key mechanisms of inactivation. The enzymes responsible for CpG methylation are DNA methyltransferase (DNMT) 1, DNMT3a, and DNMT3b, and the enzyme responsible for demethylation is DNA demethylase (MBD2). Studies on methylation-demethylation enzymes are lacking in human prostate cancer. We hypothesize that MBD2 enzyme activity is repressed and that DNMT1 enzyme activity is elevated in human prostate cancer. To test this hypothesis, we analyzed enzyme activities, mRNA, and protein levels of MBD2 and DNMT1, DNMT3a, and DNMT3b in human prostate cancer cell lines and tissues. The enzyme activities of DNMTs and MBD2 were analyzed by biochemical assay. The mRNA expression was analyzed by reverse transcriptase-polymerase chain reaction and by Northern blotting. The protein expression was measured by immunohistochemistry with specific antibodies. The results of these experiments demonstrated that (1) the activity of DNMTs was twofold to threefold higher in cancer cell lines and cancer tissues, as compared with a benign prostate epithelium cell line (BPH-1) and benign prostatic hyperplasia (BPH) tissues; (2) MBD2 activity was lacking in prostate cancer cell lines but present in BPH-1 cells; (3) immunohistochemical analyses exhibited higher expression of DNMT1 in all prostate cancer cell lines and cancer tissues, as compared with BPH-1 cell lines and BPH tissues; (4) MBD2 protein expression was significantly higher in BPH-1 cells and lacking in prostate cancer cell lines and, in BPH tissues, MBD2 protein expression was poorly observed, as compared with no expression in prostate cancer tissues; and (5) mRNA expression for DNMT1 was upregulated in prostate cancer, as compared with BPH-1, and mRNA expression for MBD2 was found to be significantly expressed in all cases. The results of these studies clearly demonstrate that DNMT1 activity is upregulated, whereas MBD2 is repressed at the level of translation in human prostate cancer. These results may demonstrate molecular mechanisms of CpG hypermethylation of various genes in prostate cancer.

Expression changes in EZH2, but not in BMI-1, SIRT1, DNMT1 or DNMT3B are associated with DNA methylation changes in prostate cancer

“…66 Only one group had studied DNMT expression in prostate cancer so far and reported overexpression of DNMT1 and DNMT3B. 15 Our data fit theirs with respect to DNMT3B, which we observed to be significantly overexpressed in cancers as compared to benign tissues at the mRNA level and overall at the protein level. However, DNMT3B protein expression was heterogeneous both in benign and tumor tissues and consequently differences in DNMT3B expression between tumor and benign tissues were not always clear cut.…”

“…13 So far, only one group has studied DNMTs in prostate cancers. 14,15 In various tumor types, DNMT expression changes correlated poorly with DNA methylation alterations. 4,7,8,16 Moreover, many cancers exhibit hypermethylation at specific loci concomitant with a genome-wide decrease in methylcytosine content, which is most obvious at CpG-rich satellites and retrotransposon sequences like LINE-1 interspersed in the genome.…”

“…We also investigated expression of SIRT1, to our knowledge for the first time in human prostate cancer. Furthermore, we studied expression of the DNA methyltransferases DNMT1 and DNMT3B, because only one study on prostate cancer tissues is available so far 15 in which the association with DNA methylation alterations was not addressed. Finally, we explored whether polycomb genes might be regulated by DNA methylation in prostate cancer cell lines.…”

Cigarette smoke induces demethylation of prometastatic oncogene synuclein-γ in lung cancer cells by downregulation of DNMT3B

“…De novo DNA methyltransferase activity assay The DNA methyltransferase assay was performed as described previously (Belinsky et al, 1996;Patra et al, 2006;Guo et al, 2002) with some modification. Twenty micrograms of nuclear extract was incubated at 371C with 1.5 mg of unmethylated poly(dI-dC)-poly(dI-dC) (Sigma) as the template and 2 mCi of 3 H-labeled S-adenosylmethionine (Amersham Biosciences, Piscataway, NJ, USA) in a total volume of 40 ml of reaction buffer (pH 7.4), containing 20 mM Tris-HCl, 25% glycerol (v/v), 10 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride and 20 mM 2-mercaptoethanol.…”

Expression of methylation-related genes is associated with overall survival in patients with non-small cell lung cancer

“…In contrast, DNA demethylase-methyl-CpG-binding domain 2 (MBD2) performs the reverse reaction to DNMTs (Bhattacharya et al, 1999). Compared to the adjacent normal tissue, a significant decrease in MBD2 mRNA expression has been observed in various tumour tissue types (Kanai et al, 1999;Patra et al, 2002). These findings suggest that DNA methyltransferases and DNA demethylases play pivotal roles in the initiation and progression of tumours and thus may be useful in the clinical diagnosis and prognostic assessment of cancer.…”