Hypomethylation and reactivation of the asparagine synthetase gene induced by L-asparaginase and ethyl methanesulfonate.

Successful chemotherapeutic treatment of drug-responsive cancers can be compromised by the acquisition of drug resistance. Standard remission induction therapy for childhood acute lymphoblastic leukemia includes L-asparaginase, since the leukemic cells lack asparagine synthetase (AS) activity and require exogenous asparagine. We have used the Chinese hamster ovary cell line N3, which lacks AS activity, as a model to examine a novel mechanism involved in the development of drug resistance in acute lymphoblastic leukemia. Expression of AS in Chinese hamster ovary cells is associated with hypomethylation in the 5' region of the gene. Activation of AS in concert with hypomethylation occurs spontaneously at a frequency of about 10(-6); we have found that treatment with the hypomethylating drug 5-azacytidine induces a reversion frequency of 10(-2). To investigate the possibility that chemotherapeutic drugs induce similar changes, the asparagine auxotrophic cell line N3 was treated with the chemotherapeutic agents L-asparaginase, vincristine, and 1-beta-D-arabinofuranosylcytosine and with the mutagen ethyl methanesulfonate. Both L-asparaginase and ethyl methanesulfonate increased the frequency of reversion to asparagine prototrophy to about 10(-5), whereas vincristine and 1-beta-D-arabinofuranosylcytosine had no such effect. Asparagine prototrophy correlated with the demethylation of CpG sites in the 5' region of the AS gene and with the appearance of AS mRNA in revertants. In addition to the specific effect seen with the AS gene, L-asparaginase and ethyl methanesulfonate induced global reductions in methylation of up to 25 and 10%, respectively. The ability of chemotherapeutic drugs to inhibit DNA methylation and thereby activate previously silent genes may enable them to promote the aggressiveness of cancers in vivo, including the expression of drug resistance.