Identifying specific genes involved in altered DNA methylation in carcinogenesis.
Lee, Peter Ju-Heung
1995
Abstract
Alterations of DNA methylation in carcinogenesis have attracted much attention because such changes have been found in all human cancers and pre-malignant lesions examined. However, the precise role of altered DNA methylation in cancer is unclear because neither the specific gene targets nor causes of the pathogenic alteration of DNA methylation are known. I have sought to identify those gene targets using an in vitro cell culture system and examined the DNA methyltransferase (MTase), a candidate gene which might cause altered DNA methylation in human colon cancers. The DNA hypomethylating drug 5-aza-2$\sp\prime$-deoxycytidine (5-aza-dCyd) transforms mouse embryo fibroblast cells at a high frequency. Although the mechanism of transformation by 5-aza-dCyd is unknown but it has been suggested that 5-aza-dCyd either causes a generalized disturbance in DNA methylation which predisposes toward transformation, or it hypomethylates and activates specific transforming genes. To investigate the latter possibility, I designed a novel approach using a classical method of DNA transfection to detect and clone such genes. I found that transfection of DNA from 5-aza-CdR-transformed cells indeed transformed recipient cells at a frequency of 0.7 $\times$ 10$\sp{-3}$, suggesting 5-aza-CdR activated at least one specific transforming gene. Although the process of cloning is still underway, I have validated the cloning strategy and generated valuable resources for future studies. Recently, DNA MTase has been suggested as a candidate gene responsible for alteration in DNA methylation because levels of DNA MTase mRNA were significantly elevated in colon cancers as determined by polymerase chain reaction. I developed a sensitive and quantitative method using RNase protection assay to measure DNA MTase mRNA levels in colon cancer samples. Unlike the previously reported results, I found the MTase mRNA levels to be only moderately increased in colon cancers when compared to adjacent normal mucosa. Furthermore, when normalized to histone H4 mRNA, no increase in MTase mRNA was observed, indicating that the increase in MTase mRNA could be explained by increased mitotic activities in these cancers. Thus, no aberrant regulation of DNA MTase mRNA expression is present in colon cancer. In summary, I have established by genomic transfection experiments that 5-aza-dCyd transforms mouse cells by activating one or more transforming genes suggesting the existence of specific gene targets for pathologic alterations of DNA methylation in cancer. In addition, I have found that DNA MTase is not likely to be responsible for alteration of DNA methylation since no aberrant changes in its expression are found in colon cancers.Subjects
Altered Carcinogenesis Dna Genes Identifying Involved Methylation Methyltransferase Specific
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