Glutaminase activity determines cytotoxicity of L-asparaginases on most leukemia cell lines - PubMed (original) (raw)
Glutaminase activity determines cytotoxicity of L-asparaginases on most leukemia cell lines
Jean Hugues Parmentier et al. Leuk Res. 2015 Jul.
Abstract
L-Asparaginase (ASNase) is a front-line chemotherapy for acute lymphoblastic leukemia (ALL), which acts by deaminating asparagine and glutamine. To evaluate the importance of glutaminase activity, we exploited a recently developed mutant of Helicobacter pylori ASNase (dm HpA), with amino acid substitutions M121C/T169M. The mutant form has the same asparaginase activity as wild-type but lacks glutaminase activity. Wild-type and dm HpA were compared with the clinically used ASNases from Escherichia coli (l-ASP) and Erwinia chrysanthemi (ERWase). Asparaginase activity was similar for all isoforms, while glutaminase activity followed the rank order: ERWase>l-ASP>wild-type HpA>dm HpA. Cytotoxic efficacy of ASNases was tested on 11 human leukemia cell lines and two patient-derived ALL samples. Two cell lines which we had previously shown to be asparagine-dependent were equally sensitive to the asparaginase isoforms. The other nine lines and the two patient-derived samples were more sensitive to isoforms with higher glutaminase activities. ERWase was overall the most effective ASNase on all cell lines tested whereas dm HpA, having the lowest glutaminase activity, was the least effective. These data demonstrate that asparaginase activity alone may not be sufficient for ASNase cytotoxicity, and that glutaminase activity may be required for full anti-leukemic efficacy.
Keywords: ALL; Asparaginase; Glutaminase; Leukemia.
Copyright © 2015 Elsevier Ltd. All rights reserved.
Conflict of interest statement
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Figures
Figure 1
Dose response fit of asparaginase activity (A) and glutaminase activity (B) of four ASNases (n =2). The X-axis represents the Log IU for each isoforms calculated from 10 to 12 different concentrations ranging from 0.001 to 20 IU. Graphs shown are a non-linear regression 5-parameter model.
Figure 2
Cytotoxic efficacy of ASNases on human leukemia cells. A, Cells were cultured for 72 hours with a range of 0.03 IU/ml to 3 IU/ml ASNase isoforms. After 72 hours, cell viability was determined in triplicate by trypan blue exclusion (n = 4 or 5 per cell line). Percentage of live cells at 5 concentrations compared to untreated control (100%) is plotted in each graph. Statistical analysis was performed with two way ANOVA with Bonferroni post-tests for each ASNase isoforms vs. control and also dm HpA vs. other isoforms however for clarity, symbols are not indicated on the graphs. B, RS4;11 and BV173 cells were cultured for 48 hours with 3 IU/ml ASNase isoforms (n = 3). Cell death was quantified with DAPI-positive cells using flow cytometry, and expressed as a percentage of total measured cells. dm: double-mutant H. pylori, wt: wild-type H. pylori, L-Asp: E. coli, ERWase: Erwinia chrysanthemi. *p<0.05 vs. control was determined by t-test. C, Primary human leukemia cells in direct co-culture with OP9 feeder layer were incubated for 72 hours with 3 IU/ml ASNase isoforms (n = 4). Percentage of live cells compared to untreated control (100%) is indicated;. *p<0.05 vs. dm HpAse was determined by two way ANOVA with Bonferroni post-tests.
Figure 2
Cytotoxic efficacy of ASNases on human leukemia cells. A, Cells were cultured for 72 hours with a range of 0.03 IU/ml to 3 IU/ml ASNase isoforms. After 72 hours, cell viability was determined in triplicate by trypan blue exclusion (n = 4 or 5 per cell line). Percentage of live cells at 5 concentrations compared to untreated control (100%) is plotted in each graph. Statistical analysis was performed with two way ANOVA with Bonferroni post-tests for each ASNase isoforms vs. control and also dm HpA vs. other isoforms however for clarity, symbols are not indicated on the graphs. B, RS4;11 and BV173 cells were cultured for 48 hours with 3 IU/ml ASNase isoforms (n = 3). Cell death was quantified with DAPI-positive cells using flow cytometry, and expressed as a percentage of total measured cells. dm: double-mutant H. pylori, wt: wild-type H. pylori, L-Asp: E. coli, ERWase: Erwinia chrysanthemi. *p<0.05 vs. control was determined by t-test. C, Primary human leukemia cells in direct co-culture with OP9 feeder layer were incubated for 72 hours with 3 IU/ml ASNase isoforms (n = 4). Percentage of live cells compared to untreated control (100%) is indicated;. *p<0.05 vs. dm HpAse was determined by two way ANOVA with Bonferroni post-tests.
Figure 3
Glutaminase activity vs. cytotoxic efficacy. A, Each cell line data for percentage inhibition at 3 IU/ml was plotted and mean ± SD was calculated for the four ASNase isoforms (n=11 cell lines per ASNase). B, Data for 3 asparaginase-sensitive cell lines (Sup-B15, RS4;11 and MOLT-4) without other 8 cell lines are shown for % inhibition at 3 IU/ml. C, Data for 3 asparaginase-sensitive cell lines (Sup-B15, RS4;11 and MOLT-4) were excluded and the remaining 8 cell lines are shown for percentage inhibition at 3 IU/ml. Relative glutaminase activity for each isoform is indicated below graphs.
References
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