DNA methylation patterns associated with asparagine synthetase expression in asparagine-overproducing and -auxotrophic cells.

Abstract

In Chinese hamster ovary cells, the gene for asparagine synthetase, which spans 20 kilobase pairs, was found to contain a cluster of potential sites for CpG methylation in a 1-kilobase-pair region surrounding the first exon. Fourteen of the sites that could be assayed for methylation by MspI-HpaII digestions were found in this region, with an additional nine MspI sites spread throughout the remainder of the gene. The methylation status of the gene was analyzed in a series of cell lines that differed in the amount of asparagine synthetase activity. The level of expression showed a direct correlation with the extent of methylation of a subset of the MspI sites found in the 5' region of the gene. The rest of the gene was completely methylated in most cell lines. Wild.type cells, which expressed a basal level of asparagine synthetase activity, were partially demethylated in the 5' region. In contrast, asparagine-requiring N3 cells, which lacked detectable mRNA for asparagine synthetase, were methylated throughout the entire gene. Spontaneous revertants of strain N3, selected for growth in asparagine-free medium, exhibited extensive hypomethylation of the asparagine synthetase gene. The methylation pattern of the gene in cell lines that overproduced the enzyme was also examined. Albizziinresistant cell lines, which had amplified copies of the gene, were extensively demethylated in the 5' region. Overexpression of asparagine synthetase in j-aspartyl hydroxamate-resistant lines without amplified copies of the gene was also correlated with DNA hypomethylation.

Asparagine synthetase expression alone is sufficient to induce l-asparaginase resistance in MOLT-4 human leukaemia cells

“…With regard to mechanism, it has been documented that gene amplification is not the cause of the ASNase-induced AS expression in resistant cells [18,29]. However, the expression of AS has been shown to be inversely proportional to the degree of cytosine methylation in the region surrounding the transcriptional start site [16,30]. Indeed, Worton et al [16] obtained data documenting an ASNase-dependent 25 % reduction in global DNA methylation.…”

DNA methylation and gene expression.

“…These changes also presumably occur over a long time scale under growth-selective conditions (69,92). The same type of methylation may also be responsible for the observed modification of exogenously introduced viral sequences several generations after their transfection and of genes which have undergone repression or extinction in certain cell lines (1,27,54). Changes in the methylation patterns of specific genes are also observed in tumor cells (64).…”

Pharmacological and clinical evaluation of l-asparaginase in the treatment of leukemia

“…Asparagine synthetase is the enzyme responsible for the synthesis of l-asparagine in normal cells. Andrulis and Barrett [98], and Greco et al in the 1989 attributed the regulation of the expression of asparagine synthetase gene to the degree of methylation of cytosine residues in DNA [99].…”