Establishment of Real-Time Polymerase Chain Reaction Method for Quantitative Analysis of Asparagine Synthetase Expression

Abstract

We established a real-time quantitative PCR (RQ-PCRAsparagine is not an essential amino acid obtained from outside the body because it is synthesized by using the hydrolysis energy of ATP from aspartic acid and glutamine via asparagine synthetase (AS). Even when the asparagine supply is reduced, normal cells can compensate by synthesizing L-asparagine. However, lymphoblastic cells require external asparagine for growth as they lack sufficient AS activity. 1-3 Thus, L-asparaginase is effective against childhood acute lymphoblastic leukemia (ALL) during the induction of remission or the intensification phases of treatment. 4,5 Asparagine in the blood, cerebrospinal fluid and bone marrow is depleted by L-asparaginase. A reduction of asparagine leads to cell death, since exposure to L-asparaginase in vitro induces the fragmentation of DNA and morphological changes typical of apoptosis in a mouse lymphoma cell line 6 and in NIH3T3 cells. 7 An asparagine deficiency can be evoked by the intracellular depletion of glutamate and glutamine. 7 The apoptosis of leukemia cells induced by L-asparaginase may be associated with cell cycle arrest in the G1 phase because DNA strand breaks can be seen in G1 phase cells as early as 8 hours after L-asparaginase exposure. 8 L-asparaginase activates AS expression and the overexpression of human AS protein can induce the L-asparaginase-resistance phenotype. 9 The expression of AS and sensitivity to L-asparaginase were correlated not only in leukemic, but also in ovarian cancer cells. 10 These observations indicate the importance of monitoring AS activity as a marker for clinical decisions regarding L-asparaginase therapy. The present study established real-time quantitative PCR (RQ-PCR) as a method of measuring AS expression. We discuss the clinical application of this method compared with immunohistochemistry, Western blotting, and enzyme activity. Materials and Methods Cell Lines and Culture ConditionsK562, HL60, U937, and MOLT4 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). All cells were suspension-cultured in RPMI 1640 medium (Invitrogen Corp., Carlsbad, CA) containing 10% fetal bovine serum (FBS; Sigma, St. Louis, MO) and penicillin and streptomycin under a 5% CO 2 atmosphere. MOLT4/R 11 and U937/R 1 were established by sequential

Up-regulation of asparagine synthetase expression is not linked to the clinical response l-asparaginase in pediatric acute lymphoblastic leukemia

“…21 A rapid transcriptional control of the AS gene occurs following deprivation of any single essential amino acid. 6,22 In 1997, Hutson et al 6 demonstrated that depletion of the intracellular asparagine pool by L-Asp was sufficient to activate in vitro AS expression in human leukemic cell lines. The increase in AS mRNA expression also resulted in a simultaneous up-regulation of AS protein levels and AS enzyme activity.…”

Asparagine Depletion Potentiates the Cytotoxic Effect of Chemotherapy against Brain Tumors

“…ASNS expression was measured by RT-PCR in cell lines before and after treatment with ASNase ± temozolomide (21, 29). mRNA from a housekeeping gene (18S) was used as an internal standard for the total amount of cDNA.…”

Inhibition of Glutamine Synthetase Triggers Apoptosis in Asparaginase-Resistant Cells

“…The cytotoxic effects of ASNase were attributed to asparagine starvation [3,4] and, consistently, low activity of asparagine synthetase (AS), the enzyme that obtains asparagine from aspartate and glutamine, has been considered the most important determinant of sensitivity to ASNase [2,5]. In agreement with this hypothesis, methods have been developed to measure AS expression conveniently, so as to foresee clinical sensitivity to ASNase [6], and several ASNase-resistant cell lines have been characterized that exhibit an increased expression and/ or activity of AS [5,[7][8][9].…”