Limited up-regulation of DNA methyltransferase in human colon cancer reflecting increased cell proliferation.

Abstract

Epigenetic alterations in the genome of tumor cells have attracted considerable attention since the discovery of widespread alterations in DNA methylation of colorectal cancers over 10 years ago. However, the mechanism of these changes has remained obscure. el-Deiry and coworkers [el-Deiry, W. S., Nelkin, B. D., Celano, P., Yen, R. C., Falco, J. P., Hamilton, S. R. & Baylin, S. B. (1991) Proc. Nati. Acad. Sci. USA 88, 3470-3474], using a quantitative reverse transcription-PCR assay, reported 15-fold increased expression of DNA methyltransferase (MTase) in colon cancer, compared with matched normal colon mucosa, and a 200-fold increase in MTase mRNA levels compared with mucosa of unaffected patients. These authors suggested that increases in MTase mRNA levels play a direct pathogenetic role in colon carcinogenesis. To test this hypothesis, we developed a sensitive quantitative RNase protection assay of MTase, linear over three orders of magnitude. Using this assay on 12 colorectal carcinomas and matched normal mucosal specimens, we observed a 1.8-to 2.5-fold increase in MTase mRNA levels in colon carcinoma compared with levels in normal mucosa from the same patients. There was no significant difference between the normal mucosa of affected and unaffected patients. Furthermore, when the assay was normalized to histone H4 expression, a measure of S-phase-specific expression, the moderate increase in tumor MTase mRNA levels was no longer observed. These data are in contrast to the previously reported results, and they indicate that changes in MTase mRNA levels in colon cancer are nonspecific and compatible with other markers of cell proliferation.

Increased DNA methyltransferase expression in leukaemia

“…21 Our results of elevated DNA methyltransferase levels in leukaemia are similar to studies in colon cancer that have used quantitative RT-PCR assays, where an average of 3.7-fold 23 and 2.6-fold 24 increases in DNA MTase transcripts were reported when normalised with GAPDH. Lee et al 24 also found that the level of DNA MTase expression in colon cancer was 1.8-fold higher when normalised to actin, however, no significant elevation was detected when normalised to histone H4. Therefore these authors argue that the observed increased in DNA MTase expression was simply a reflection of the increase in cell proliferation in colon tumour cells.…”

“…DNA MTase expression has been assayed previously in lung, liver and colon tumour samples using a wide range of methods, including protein-based assays 21,22 and RNA-based assays 20,[22][23][24]37 and the results obtained have been variable. However, in this study we developed a standardised competitive RT-PCR assay which enabled accurate measurement of DNA MTase mRNA expression within and between samples.…”

“…21,22 Elevated levels of DNA MTase mRNA transcripts have been reported in colon cancer when compared to normal colon mucosa. Using non-quantitative reverse transcription RT-PCR assay, a 200-fold increase in DNA MTase mRNA levels was reported comparing colon cancers to normal mucosa, 20 whereas increases of 3.7-and 2.5-fold were found in later studies using quantitative RT-PCR 23 and RNAase protection assays, 24 respectively. DNA methylation patterns are known to be altered in a large number of genes in haematological malignancies.…”

Expression of DNA methyl-transferase (DMT) and the cell cycle in human breast cancer cells

“…However, a recent study of colon tumors and matched normal colonic mucosa indicated that DMT expression in tumors is only modestly increased above that seen in normal colon tissue, and the increase in DMT expression was correlated with increased histone H4 expression, a measure of S phase-speci®c gene expression (Lee et al, 1996). Those results were perhaps not surprising in light of the fact that DMT expression and activity are known to vary with the cell cycle (Szyf et al, 1985(Szyf et al, , 1991.…”

Detailed methylation analysis of the glutathione S-transferase π (GSTP1) gene in prostate cancer

“…Evidence is accumulating that a loss of normal regulation of DNA methylation (both de novo and maintenance activities) is common in cancer Baylin et al, 1998). Gross changes include extensive changes in regional levels of methylation (both increased and decreased) (Laird and Jaenisch, 1994) as well as elevated levels of DNA methyltransferase (Melki et al, 1998;Lee et al, 1996). It has also recently been reported that colon cancer cell lines can be grouped into two classes which di er in their capacity to methylate introduced retroviral genomes (Lengauer et al, 1997) this has been interpreted to indicate that while defects in mismatch repair systems are critical early events leading to cancer development, alterations to the regulation of DNA methylation may provide an alternate route.…”