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

Abstract

Childhood acute lymphoblastic leukaemia (ALL) is treated by combination chemotherapy with a number of drugs, always including the enzyme L-asparaginase (ASNase). Although the initial remission rate is quite high, relapse and associated drug resistance are a significant problem. In vitro studies have demonstrated increased asparagine synthetase (AS) expression in ASNase-resistant cells, which has led to the hypothesis that elevated AS activity permits drug-resistant survival. The data presented show that not only is elevated AS expression a property of ASNase-resistant MOLT-4 human leukaemia cells, but that short-term (12 h) treatment of the cells with ASNase causes a relatively rapid induction of AS expression. The results also document that the elevated expression of AS in ASNase-resistant cells is not fully reversible, even 6 weeks after ASNase removal from the culture medium. Furthermore, ASNase resistance, assessed as both drug-insensitive cell growth rates and decreased drug-induced apoptosis, parallels this irreversible AS expression. Mimicking the elevated AS activity in ASNase-resistant cells by overexpression of the human AS protein by stable retroviral transformation of parental MOLT4 cells is sufficient to induce the ASNase-resistance phenotype. These data document that ASNase resistance in ALL cells is a consequence of elevated AS expression and that although other drug-induced metabolic changes occur, they are secondary to the increased asparagine biosynthetic rate.

Role of Sp1 and Sp3 in the Nutrient-regulated Expression of the Human Asparagine Synthetase Gene

“…In the absence of an available insect cell culture medium lacking individual amino acids, transfected SL2 cells were incubated in Schneider's complete media without (control) or with the enzyme asparaginase (1 unit/ml) for 18 h. Asparaginase will cause complete depletion of medium asparagine and glutamine within minutes of addition, inducing amino acid limitation conditions (20). Induction of AS expression is the same regardless of whether histidine is removed from the MEM culture medium or asparaginase is added to complete medium (26).…”

ATF4 Is a Mediator of the Nutrient-sensing Response Pathway That Activates the Human Asparagine Synthetase Gene

“…32 P-Radiolabeled cDNA probe synthesis for growth hormone, glutamate dehydrogenase, and LacZ, as well as the Northern analyses, were performed as described (18). The cDNA probe for human ATF4 (nt 853-1933) was generated by reverse transcript-PCR and then subcloned into the pcDNA3.1 vector.…”

The downregulation of asparagine synthetase expression can increase the sensitivity of cells resistant to L-asparaginase

“…However, suppression of protein synthesis is an obvious potential target and it had been documented that L-asparaginase exposure could initiate apoptosis of the cells. 3 Aslanian et al 4 overexpressed the AsnS protein in the Molt-4 cells using a Moloney mouse retrovirus system and induced the L-asparaginase resistance phenotype in the parental cells. Hutson et al 5 demonstrated that the AsnS messsnger RNA (mRNA) level was in parallel to the AsnS protein level.…”