Inhibition of tumorigenesis by a cytosine–DNA, methyltransferase, antisense oligodeoxynucleotide

Abstract

This paper tests the hypothesis that cytosine DNA methyltransferase (DNA MeTase) is a candidate target for anticancer therapy. Several observations have suggested recently that hyperactivation of DNA MeTase plays a critical role in initiation and progression of cancer and that its up-regulation is a component of the Ras oncogenic signaling pathway. We show that a phosphorothioate-modified, antisense oligodeoxynucleotide directed against the DNA MeTase mRNA reduces the level of DNA MeTase mRNA, inhibits DNA MeTase activity, and inhibits anchorage independent growth of Y1 adrenocortical carcinoma cells ex vivo in a dosedependent manner. Injection of DNA MeTase antisense oligodeoxynucleotides i.p. inhibits the growth of Y1 tumors in syngeneic LAF1 mice, reduces the level of DNA MeTase, and induces demethylation of the adrenocortical-specific gene C21 and its expression in tumors in vivo. These results support the hypothesis that an increase in DNA MeTase activity is critical for tumorigenesis and is reversible by pharmacological inhibition of DNA MeTase.Modification of DNA by methylation is now recognized as an important mechanism of epigenetic regulation of genomic functions (1-3). Methylation of DNA is a postreplication event catalyzed by the DNA methyltransferase (DNA MeTase) enzyme using S-adenosyl methionine as a methyl donor (4). Approximately 80% of cytosines located in the CpG dinucleotide sequence are methylated in the genome of most vertebrate cells, but the distribution of methylated sites is cell-and tissue-specific (5). Patterns of methylation are generated during development by enzymatic de novo methylation and demethylation processes (1-7) and are maintained in somatic cells.A number of observations have suggested that the pattern of DNA methylation is disrupted in cancer cells (8,9). Both hypomethylation (9) and hypermethylation (10-12) of different CpG sites in cancer cells and tissues relative to the cognate normal tissue have been documented. Some of the sites that are hypermethylated in tumors are located in tumor-suppressor loci such as p16 (13), retinoblastoma (14), von Hippel-Lindau (15), and Wilms tumor (16), and, recently, a new candidate tumorsuppressor gene was cloned by molecular analysis of the hypermethylated region in chromosome 17p13.3 (17). One possible explanation that has been proposed to explain the changes in DNA methylation observed in cancer cells is that they are the end result of a change in the enzymatic machinery controlling DNA methylation in the cell (7,12,(18)(19)(20). In accordance with this hypothesis, cancer cell lines (21) and human tumors (22) have been shown to express elevated levels of DNA MeTase. Recently, Belinsky et al. (23) showed that increased DNA MeTase activity is an early event in carcinogen-initiated lung cancer in the mouse. Forced expression of DNA MeTase cDNA in murine NIH 3T3 cells leads to genomic hypermethylation and neoplastic transformation (24), and expression of an antisense mRNA to the DNA MeTase leads to loss of tumorigenicity of t...

Inhibition of p53 represses E-cadherin expression by increasing DNA methyltransferase-1 and promoter methylation in serous borderline ovarian tumor cells

“…DNMT1 knockouts are resistant to colorectal tumorigenesis, and antisense knockdowns of DNMT1 reverse tumorigenesis in vitro and in vivo (Laird et al, 1995;MacLeod and Szyf, 1995;Ramchandani et al, 1997). In ovarian cancer, differential DNMT gene expression has been examined, and it has been suggested that alterations in DNMT expression might contribute to the CpG island methylation phenotype (Ahluwalia et al, 2001).…”

RFTS-deleted DNMT1 enhances tumorigenicity with focal hypermethylation and global hypomethylation

“…57,58 The current approach is to inhibit expression or hyperactivity of DNMT1. [59][60][61] However, demethylating agents lead to unavoidable non-specific genomic demethylation causing genomic instability, DNA damage, oncogene reactivation, and promote opportunities for cancer progression. [62][63][64][65] Because the RFTS domain functions as a key regulator of DNMT1 function, targeting RFTS interactions may revert euchromatin-associated DNMT1 activation while also normalizing pericentromeric DNA methylation.…”

Epigenomic Stress Response

“…1A, there is a 6-base pair mismatch between MG88 and the mouse dnmt1 mRNA. We therefore determined whether MG88 would inhibit DNA synthesis in a mouse adrenal carcinoma cell line, Y1, which was previously shown by us to be responsive to a mouse dnmt1 antisense oligonucleotide (23). The results shown in Fig.…”

“…For Western blot analysis of DNMT1, 50 g of nuclear protein was fractionated on a 5% SDSpolyacrylamide gel, transferred to polyvinylidene difluoride membrane, and reacted with the polyclonal anti-DNMT1 antibody (New England Biolabs) at a dilution of 1:2000 in the presence of 0.05% Tween and 5% milk, and it was then reacted with anti-rabbit IgG (Sigma) at a dilution of 1:5000. The amount of total protein per lane was determined by Amido Black staining (23). The intensity of DNMT1 and total protein signal was measured by scanning densitometry, and the ratio of DNMT1/total nuclear protein was calculated.…”