Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.Asparagine synthetase (AS) is a housekeeping enzyme responsible for the biosynthesis of asparagine from glutamine and aspartic acid. The regulation of AS has been studied in cultured mammalian cells (for a review, see reference 1), and overproducing cell lines, some with amplified copies of the gene, have been isolated in response to drug selection (2, 3, 6, 11). Most mammalian cells express AS and regulate the level of activity in response to the concentration of asparagine in the medium and the extent of tRNA aminoacylation (4, 7). In contrast to normal cells, some tumor cells exhibit little or no AS activity and rely on the surrounding medium as a source of exogenous asparagine. This property has been exploited in the treatment of certain cancers, since the tumor cells can be selectively killed by the chemotherapeutic drug asparaginase which hydrolyzes circulating asparagine (9, 24). One of the most sensitive human malignancies was found to be childhood acute lymphoblastic leukemia, and asparaginase is now routinely used in induction and consolidation protocols for patients with this disease (23). In mouse model systems, it was found that asparaginase-sensitive tumor cells could be made asparaginase-resistant by repeated subculturing of these cells in sublethal concentrations of the drug. The resistant cells were found to have regained AS activity and in some cases had elevated levels of enzyme activity up to 60-fold higher than that in normal cells (13,24). The mechanism responsible for the lack of activity of AS in the sensitive leukemic cells and for the overproduction in the asparaginase-resistant variants is of clinical importance for the treatment of acute lymphoblastic leukemia patients both initially and during relapse.To investigate the molecular basis for the sensitivity to asparaginase in human acute...

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.