Low expression of asparagine synthetase in lymphoid blasts precludes its role in sensitivity to L-asparaginase.

Childhood acute lymphoblastic leukemia (ALL) is treated with combined chemotherapy, including L-asparaginase (L-asp). Recent studies question the traditional view that the level of asparagine synthetase (ASNS), an enzyme producing the intracellular asparagine, correlates with the response to L-asp treatment. However, the importance of ASNS in response to L-asp has neither been confirmed nor refuted so far. In this study, we wanted to elucidate the effect of ASNS expression level on the sensitivity of ALL cells to L-asp treatment. We used four ALL cell lines (NALM-6, RS4;11, REH, and UOCB6) and 30 diagnostic bone marrow samples of ALL patients to study the relationship between ASNS expression and sensitivity to L-asp using MTS proliferation assay. RNA interference was used to study the effect of a range of ASNS levels on the response to L-asp treatment. Using a cell line model with a gradually knocked-down ASNS gene, we defined a cutoff level below which ASNS gene expression does not correlate with sensitivity to L-asp. Importantly, ASNS gene expression in patients' ALL blasts is below this level. We confirmed that there was no correlation between ASNS gene expression and sensitivity to L-asp in ALL blasts. In addition, we show that cells with low ASNS expression level do not respond to asparagine deprivation by upregulation of ASNS gene expression. In conclusion, the ASNS expression level does not predict sensitivity to L-asp in leukemic blasts. Moreover, cell lines with high basal expression of ASNS cannot serve as a valid model for studies on the relationship between the ASNS and L-asp cytotoxic effect.

Asparagine synthetase: a new potential biomarker in ovarian cancer.

L-Asparaginase (L-ASP) is an enzyme drug that has been an asset to leukemia treatment regimens for four decades. Variability in its clinical efficacy, however, has prompted the search for biomarkers capable of distinguishing responders from non-responders. In that regard, the NCI-60 cell line panel has served as a biomarker discovery platform and has led to the identification of a correlation between L-ASP efficacy and asparagine synthetase (ASNS) expression in cultured cells. The presence of that correlation in the ovarian subpanel of the NCI-60 has made a case for repositioning L-ASP to ovarian cancer. This review presents an overview of the biomarker development process, summarizes the efforts that have been invested thus far in developing ASNS as a biomarker for ovarian cancer treatment, highlights the role of RNAi and the limitations of the NCI-60 in that process, and addresses important considerations for next steps in the development of ASNS as a predictive biomarker.

Expression levels of asparagine synthetase in blasts from children and adults with acute lymphoblastic leukaemia.

L-asparaginase is active in the treatment of acute lymphoblastic leukaemia (ALL) through the depletion of serum asparagine. Here we report that median asparagine synthetase (AS) mRNA levels were higher in acute myeloid leukaemia (AML) than ALL blasts in both children and adults, with intermediate levels in normal peripheral blood mononuclear cells (NPBMC). NPBMC versus child ALL (Tukeys multiple comparison test, P < 0.05); child ALL versus child AML (P < 0.001) and adult ALL versus adult AML (P < 0.01) were all significant and support the hypothesis that selectivity to treatment with l-asparaginase is due, at least in part, to lower AS expression.

High levels of asparagine synthetase in hypocotyls of pine seedlings suggest a role of the enzyme in re-allocation of seed-stored nitrogen.

A pine asparagine synthetase gene expressed in developing seedlings has been identified by cloning its cDNA (PsAS1) from Scots pine (Pinus sylvestris L.). Genomic DNA analysis with PsAS1 probes and a sequence-based phylogenetic tree are consistent with the possibility of more than one gene encoding asparagine synthetase in pine. However, the parallel patterns of free asparagine content and PsAS1 products indicate that the protein encoded by this gene is mainly responsible for the accumulation of this amino acid during germination and early seedling development. The temporal and spatial patterns of PsAS1 expression together with the spatial distribution of asparagine content suggest that, early after germination, part of the nitrogen mobilized from the megagametophyte is diverted toward the hypocotyl to produce high levels of asparagine as a reservoir of nitrogen to meet later specific demands of development. Furthermore, the transcript and protein analyses in seedlings germinated and growth for extended periods under continuous light or dark suggest that the spatial expression pattern of PsAS1 is largely determined by a developmental program. Therefore, our results suggest that the spatial and temporal control of PsAS1 expression determines the re-allocation of an important amount of seed-stored nitrogen during pine germination.

Asparagine synthetase expression is linked with L-asparaginase resistance in TEL-AML1-negative but not TEL-AML1-positive pediatric acute lymphoblastic leukemia.

Resistance to L-asparaginase in leukemic cells may be caused by an elevated cellular expression of asparagine synthetase (AS). Previously, we reported that high AS expression did not correlate to L-asparaginase resistance in TEL-AML1-positive B-lineage acute lymphoblastic leukemia (ALL). In the present study we confirmed this finding in TEL-AML1-positive patients (n = 28) using microarrays. In contrast, 35 L-asparaginase-resistant TEL-AML1-negative B-lineage ALL patients had a significant 3.5-fold higher AS expression than 43 sensitive patients (P < .001). Using real-time quantitative polymerase chain reaction (RTQ-PCR), this finding was confirmed in an independent group of 39 TEL-AML1-negative B-lineage ALL patients (P = .03). High expression of AS was associated with poor prognosis (4-year probability of disease-free survival [pDFS] 58% +/- 11%) compared with low expression (4-year pDFS 83% +/- 7%; P = .009). We conclude that resistance to l-asparaginase and relapse risk are associated with high expression of AS in TEL-AML1-negative but not TEL-AML1-positive B-lineage ALL.

Establishment of real-time polymerase chain reaction method for quantitative analysis of asparagine synthetase expression.

We established a real-time quantitative PCR (RQ-PCR) with which to measure abundance of the asparagine synthetase (AS) mRNA. The level of AS mRNA paralleled AS enzyme activity, as well as the AS protein level detected by Western blotting and by in situ immunostaining. Cytotoxicity tests in vitro showed that the AS mRNA level also synchronized with cellular resistance to L-asparaginase in cell lines. Cellular levels of AS enzyme activity correlated with resistance to L-asparaginase. These results indicate that the AS mRNA level is an index of resistance to L-asparaginase. RQ-PCR is superior to enzyme assays, Western blotting, and immunostaining in the following ways: less labor and time, accurate and reproducible quantitativity, and broad dynamic range. In addition, RQ-PCR could evaluate differences in L-asparaginase sensitivity although immunostaining could not. And in clinical samples, we analyzed eight pediatric leukemia cases by this RQ-PCR to evaluate whether this method was applicable to clinical laboratories and the expression level of AS mRNA in each case were predictable for the effectiveness of L-asparaginase treatment. Consequently, this method was useful enough in defining candidates for selective therapy that targets an AS deficiency.

Childhood blastic NK cell leukemia successfully treated with L-asparagenase and allogeneic bone marrow transplantation.

Blastic NK cell lymphoma/leukemia is a rare and highly malignant neoplasia in both adults and children. It is characterized by lymphoblastoid morphology without cytoplasmic granules and immature NK cell immunophenotypes (CD56+, CD57-, CD16-). It has predilection for extranodal organ involvement, and the prognosis of affected patients is extremely poor under the current chemotherapy. We present a 14-year-old girl who was diagnosed as having blastic NK cell leukemia with mediastinal, pleural, and pericardial involvement. Immunophenotyping of her leukemic cells showed positive for CD2, CD5, CD7, CD34, CD56, HLA-DR, and cytoplasmic CD3. T cell receptor (TCR) and Immunoglobulin heavy chain genes were not rearranged. She received chemotherapy for acute lymphoblastic leukemia incorporating L-asparaginase (L-asp) which successfully induced complete remission. Bone marrow transplantation (BMT) from her HLA-identical sibling was conducted after two courses of consolidation therapy. Expression of aspargine synthetase (AS) protein in the leukemic cells at diagnosis was examined by an immunocytochemical method. She remains in hematological remission for over 36 months after BMT. The expression of AS protein was negative, suggesting that the leukemic cells were sensitive to L-asp. Induction and consolidation therapy incorporating L-asp followed by allo-BMT might be a promising treatment for child hood blastic NK cell leukemia, but more samples of the rare leukemia need to be studied before any definitive conclusions can be drawn.

Sensitivity to L-asparaginase is not associated with expression levels of asparagine synthetase in t(12;21)+ pediatric ALL.

The (12;21) translocation resulting in TEL/AML1 gene fusion is present in about 25% of childhood precursor B-lineage acute lymphoblastic leukemia (ALL) and is associated with a good prognosis and a high cellular sensitivity to L-asparaginase (L-Asp). ALL cells are thought to be sensitive to L-Asp due to lower asparagine synthetase (AS) levels. Resistance to L-Asp may be caused by an elevated cellular level of AS or by the ability of resistant cells to rapidly induce the expression of the AS gene on L-Asp exposure. AS may be a target regulated by t(12;21). We studied the relationship between t(12;21) and the mRNA level of AS to investigate a possible mechanism underlying L-Asp sensitivity. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis surprisingly revealed that 30 patients positive for t(12;21) expressed 5-fold more AS mRNA compared with 17 patients negative for t(12;21) (P =.008) and 11 samples from healthy controls (P =.016). The mRNA levels of AS between t(12;21)(-) ALL and healthy controls did not differ. No difference was found between ALL patients positive or negative for t(12;21) in the capacity to up-regulate AS after in vitro L-Asp exposure, excluding a defective capacity for t(12;21) cells in up-regulating AS on L-Asp exposure. Moreover, no correlation was observed between AS mRNA expression and sensitivity to L-Asp. We conclude that the sensitivity of t(12;21)(+) childhood ALL to L-Asp is not associated with the expression level of the AS gene. Furthermore, we contradict the general thought that leukemic cells specifically lack AS compared with normal bone marrow and blood cells.

Biochemical characterization of U937 cells resistant to L-asparaginase: the role of asparagine synthetase.

A human histiocytic lymphoma cell line, U937, is highly sensitive to L-asparaginase with an ID50 of about 0.0001 U/ml after 72 hr of culture. When U937 cells were made resistant to either L-asparaginase (1 U/ml) or asparagine deprivation, the activity of asparagine synthetase increased to 80- or 7-fold of the wild type, respectively. The phenotype of the resistance to L-asparaginase turned out to be stable under nonselective conditions for over several months. The hybrids between L-asparaginase sensitive (Molt4) and resistant (HL-60) cell lines revealed the latter phenotype in terms of L-asparaginase sensitivity and the activity of asparagine synthetase. Furthermore, U937 cells resistant to L-asparaginase could survive in glutamine-free media with 1.5-fold elevation of glutamine synthetase activity. These results altogether clarify the role of asparagine synthetase in L-asparaginase toxicity and have a good implication for the clinical use of L-asparaginase.

AsnC: an autogenously regulated activator of asparagine synthetase A transcription in Escherichia coli.

The regulation of the asparagine synthetase A gene of Escherichia coli was studied in vitro with a coupled transcription-translation system. It was shown that the 17-kilodalton gene, which is transcribed divergently from the adjacent asnA gene, codes for an activator of asnA transcription. The synthesis of the 17-kilodalton protein, which we now call AsnC, is autogenously regulated. The stimulating effect of AsnC on asnA transcription is abolished by asparagine, while the autoregulation of asnC is not affected by asparagine. The N-terminal part of the asnC protein, inferred from the DNA sequence, is homologous to the DNA-binding domain of regulatory proteins like catabolite gene activator, cro, and cI. This homology and direct repeats found in the region of the two asn promoters suggest that the asnC protein regulates transcription by binding to DNA. The asn promoters were defined by mapping of the mRNA start sites of in vitro-generated transcripts.

A high-performance liquid chromatography assay for asparagine synthetase.

A highly sensitive method for assaying asparagine synthetase and its glutaminase activity is presented. The amino acids L-asparagine, L-aspartate, L-glutamate, and L-glutamine, are separated by derivatization with o-phthaldialdehyde followed by reversed-phase high-performance liquid chromatography on an Altex ultrasphere-ODS C18 column. The elution is isocratic and the mobile phase used is 50 mM sodium acetate buffer (pH 5.9) with 30% methanol. This assay can easily detect picomoles of asparagine, which may be difficult to do with the other assays that have been described.

Assignment of structural gene for asparagine synthetase to human chromosome 7.

Somatic cell hybrids obtained from the fusion of human B lymphocytes and an asparagine synthetase-deficient Chinese hamster ovary cell line were isolated after growth in asparagine-free medium. The human and hamster forms of asparagine synthetase differ significantly in their rate of inactivation at 47.5 degrees C. The asparagine synthetase activity expressed in the hybrids was inactivated at 47.5 degrees C at the same rate as the human form of the enzyme. Karyotypic analysis and analysis for chromosome-specific enzyme markers showed that the structural gene for asparagine synthetase is located on chromosome 7 in humans. The heat-inactivation profile for asparagine synthetase in extracts of hybrids formed between human peripheral leukocytes and a hamster cell line expressing asparagine synthetase activity was intermediate between the two parental types when human chromosome 7 was present, but was identical to the hamster parent when chromosome 7 was absent.

Methotrexate stimulation of asparagine synthetase activity in rat liver.

Methotrexate was found to stimulate asparagine synthetase activity in vivo by approximately six-fold in rat liver. The maximum effect of methotrexate on hepatic asparagine synthetase activity was observed sixteen hours after intraperitoneal injection of the drug. Cycloheximide, like methotrexate, is a protein synthesis inhibitor and was used to determine that asparagine synthetase activity was not preferentially stimulated under stress. As expected, hepatic asparagine synthetase activity falls markedly with the decreased protein synthesis caused by injection of cycloheximide. It is proposed that methotrexate inhibits serine-dependent glycine biosyn-thesis by decreasing the concentration of tetrahydrofolate for serine hydroxymethyltransferase. This leads to a stimulation of asparagine synthetase to provide nitrogen for asparagine-dependent glycine synthesis. This may provide an explanation of the observed chemotherapeutic synergism between asparaginase and methotrexate treatment.

A new assay for L-asparagine synthetase.

A fast, relatively inexpensive method of measuring the enzymatic formation of L-asparagine from L-aspartate is presented. This radiochemical assay requires simple manipulations making it ideal for working with large numbers of samples, while maintaining high sensitivity and reproducibility. A mechanism similar to the enzymatic beta-decarboxylation of aspartate is utilized but in a nonenzymatic reaction. In the presence of pyridoxal and A13+ ions, the 14C of L-[4-14C]aspartate is decarboxylated while L-[4-14C]asparagine remains intact. This assay is shown to be suitable for measuring mammalian L-asparagine synthetase activity, while not requiring the isolation of assay enzymes.

Effect of ploidy on spontaneous mutation rate to asparagine non-requirement in cultured cells.

Variations in ploidy do not affect the spontaneous mutation rate to asparagine non-requirement in Jensen rat sarcoma cells cultivated in vitro.