Asparagine synthetase (ASNS) is a housekeeping gene responsible for the biosynthesis of L-asparagine (ASN). The gene is expressed constitutively in most mammalian cells and regulates its level of activity in response to the concentration of ASN in the plasma or medium, and the extent of tRNA aminoacylation (Andrulis et al, 1979). In contrast to normal cells, the cells of a number of animal malignancies, especially lymphoid, are ASN auxotrophs that fail to express ASNS. In these, deprivation of exogenous ASN by L-asparaginase (Asnase) induces cell death (Broome, 1963;Boyse et al, 1967). This forms the basis for the clinical use of the enzyme as a therapeutic agent for acute lymphocytic leukaemia (Tallal et al, 1970). In the prototypic mouse system, 6C3HED, an Asnasesensitive lymphoma cell line originating in the C3H strain (here designated 6C3HED-Ade), lacked ASNS activity, as did various similar lymphoma and other cell lines (Patterson and Orr, 1967;Broome, 1968; Horowitz et al, 1968). However, ASNS activity was present in Asnase-resistant variants of 6C3HED (6C3HED-Ind), which grew out during culture in the absence of ASN or after treatment with subcurative doses of enzyme in vivo. The mechanism responsible for the lack of ASNS in the Asnase-sensitive lymphoma cells as opposed to the Asnase-resistant variants has not been studied in recent years, but in addition to theoretical interest, it may be of clinical significance in understanding the responsiveness of acute lymphocytic leukemia to enzyme treatment both initially and during relapse.The regulation of ASNS mRNA by amino acid concentration has transcriptional and post-transcriptional components involving both cis-and trans-acting factors (Guerrini et al, 1993). Within the human ASNS promoter, the amino acid response element is important for both basal activity and regulation of ASNS expression under amino acid starvation. Evidence that indirectly suggested that methylation might play a role in regulating ASNS gene expression came from studies made in conjunction with cloning the human gene. It was shown that transfection with plasmids containing human ASNS cDNA into cells of the ASNdependent Jensen rat sarcoma was capable of conferring protrophy (Andrulis et al, 1987). However, several transfectants with numerous copies of the cDNA exhibited only basal levels of enzyme activity. Treatment of these transfectant cell lines with 5-azacytidine, an inhibitor of DNA methylation, greatly increased the expression of ASNS mRNA, protein and enzyme activity. This was consistent with the finding that 5-azacytidine caused a marked increase in the number of ASN protrophs in cultures of Jensen cells (Sugiyama et al, 1983). However, no full and direct examination of the methylation status of ASNS has been made until now. Our present experiments have characterized the CpG island in the promoter of the mouse ASNS gene and correlated its hypermethylation with inactivation of gene expression in ASN-dependent lymphoma cells. To our surprise, we found that partial methylation in...
