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Role of Asparagine Synthetase and Asparagyl-Transfer RNA Synthetase in the Cell-Killing Activity of Asparaginase in Chinese Hamster Ovary Cell Mutants1

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Role of Asparagine Synthetase and Asparagyl-Transfer RNA Synthetase in the Cell-Killing Activity of Asparaginase in Chinese Hamster Ovary Cell Mutants1 [CANCER RESEARCH 41, 3104-3106, August 1981] 0008-5472/81 /0041-OOOOS02.00 Role of Asparagine Synthetase and Asparagyl-transfer RNA Synthetase in the Cell-killing Activity of Asparaginase in Chinese Hamster Ovary Cell Mutants1 Mary M. Y. Waye2 and Clifford P. Stanners3 Department of Medical Biophysics, University of Toronto, and the Ontario Cancer Institute, Toronto, Ontario M4X 1K9, Canada ABSTRACT was of interest to compare the sensitivity of the 2 types of mutants having the same genetic background to the cell-killing The cell-killing activity of asparaginase on three classes of activity of asparaginase. Any differences in sensitivity to as Chinese hamster ovary cell mutants was examined: a mutant paraginase between mutants with different enzymatic lesions which overproduces asparagine Synthetase (AH5); mutants could provide an explanation for some of the variability in drug defective in asparagine synthetase (N3 and N4); and mutants sensitivity of human lymphoid cell lines. conditionally defective in asparagyl-transfer RNA synthetase (Asn 3, Asn 7, and Asn 9). The overproducer was more resistant to the cell-killing activity of asparaginase than wild- MATERIALS AND METHODS type Chinese hamster ovary cells, while mutants defective in Chemicals. Asparaginase, or asparagine amidohydrolase, asparagine synthetase were more sensitive. Surprisingly, the or Kidrolase (commercial name), EC 3.5.1.1 type EC2, partially asparagyl-transfer RNA synthetase mutants were even more purified from Escherichia coli, was obtained from Poulenc, sensitive to asparaginase than the asparagine synthetase mu Ltd., Montreal, Quebec, Canada (specific activity, approxi tants. In a preliminary survey of four human lymphoid cell lines mately 200 units/mg). This relatively impure enzyme prepara (RPMI 8402, RPMI 8392, B46M, and Molt-4F) which showed tion is used clinically and hydrolyzes asparagine to aspartic dramatically different asparaginase sensitivity, however, sen acid but also contains a low level of glutaminase activity [about sitivity to the cell-killing activity of asparaginase was correlated 3% of the asparaginase activity (9)]. It should be pointed out with reduced levels of asparagine synthetase and not with that our tissue culture medium contains a relatively high con reduced levels of asparagyl-transfer RNA synthetase. centration of glutamine (292 mg/liter) which would minimize any anticellular effects of the contaminating glutaminase. Also, INTRODUCTION the cell-killing activity of the preparation was strongly reduced by the inclusion of asparagine in the treatment medium (Chart The reason for variability in clinical response to the cancer 1). chemotherapeutic agent asparaginase is unknown at present Radioactive isotopes were obtained from Amersham/Searle (4, 17). This is perhaps not surprising as the basic mechanism Corp., Oakville, Ontario, Canada; amino acids were obtained for cell killing by the drug is also unknown. Evidence has been from GIBCO, Burlington, Ontario, Canada; liver tRNA was presented in support of cell-killing mechanisms involving se obtained from A. Hampel, Northern Illinois University. vere starvation for asparagine (3, 8), cleavage of cell surface Cells and Culture Conditions. The wild-type CHO strain, protein at asparagine residues (5), and disruption of the cellular Gat", isolated by McBurney and Whitmore (10), will be referred energy-producing reactions (9). Regardless of mechanism, the to as the WT strain. This strain was also used by Thompson ef efficacy of asparaginase will obviously be affected by the al. (15, 16) and Waye and Stanners (18) to isolate asparagyl- extracellular and intracellular levels of asparagine. These levels tRNA synthetase mutants (Asn 3, Asn 7, and Asn 9) and are determined in part by the intracellular biosynthetic enzyme asparagine synthetase mutants (N3 and N4), respectively. asparagine synthetase, and some of the variability in response AH5, isolated from fsH1 (13), is a CHO cell mutant that over to asparaginase has been ascribed to variability in the activity produces asparagine synthetase (7) and was obtained from S. of this enzyme. Arfin, University of California, Irvine. Human T-cell lines (RPMI We have recently reported the isolation of mutants of CHO" 8402 and Molt-4F) and human B-cell lines (RPMI 8392 and cells with reduced or absent asparagine synthetase activity B46M) were obtained from J. Minowada (11) (Roswell Park (18). Evidence was presented in support of the hypothesis that Memorial Institute, Buffalo, N. Y.). CHO cells were maintained the mutations were in the structural gene for the enzyme (18). in suspension culture at 34°using «-modified minimal essential We have also isolated CHO mutants with conditional defects in medium (12) with asparagine-H2O at 50 /¿g/ml and supple asparagyl-tRNA synthetase (15, 16), an essential enzyme for mented with 10% fetal calf serum. Human lymphoid cells were cellular protein synthesis. As the latter mutants are both tem maintained in static suspension cultures at 37° in the same perature sensitive and auxotrophic for asparagine (15, 16), it growth medium. Cell concentrations were determined using an electronic particle counter. ' Supported by grants from the National Cancer Institute of Canada and Grant Assay for Asparagine Synthetase Activity. Asparagine syn MT 1877 from the Medical Research Council of Canada. thetase activity was measured by determining the ability of 2 Research student of the National Cancer Institute of Canada. sonicated whole-cell extracts to synthesize [14C]asparagine 3 To whom requests for reprints should be addressed. from [14C]aspartic acid as a function of time as described by ' The abbreviations used are: CHO, Chinese hamster ovary; WT, wild type. Received September 30, 1980; accepted May 7, 1981. Arfin ef al. (1). Reactions were carried out taking determina- 3104 CANCER RESEARCH VOL. 41 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1981 American Association for Cancer Research. Asparagine Synthetase and Asparagyl-tRNA Synthetase in Asparaginase Cell Killing tions at 0, 20, 40, and 60 min and were linear up to 40 min. Separation of the asparagine formed from aspartic acid was carried out by the electrophoretic method of Horowitz et al. (8) or by a Dowex 1-acetate column (7). The authenticity of the asparagine spot was tested by its susceptibility to hydrolysis with asparaginase. Protein determination was performed using the dye-binding protein assay of Bradford (2). The percentage of conversion of aspartic acid to asparagine per mg of extract protein per hr was determined from the slopes of straight lines relating the percentage of conversion with time. Assay for Asparagyl-tRNA Synthetase Activity. Asparagyl- tRNA synthetase activity was measured by determining the amount of [14C]asparagine accepted by rat liver tRNA at 34° as a function of time, using the supernatant fractions obtained ,Asn9 from centrifugation of detergent (Nonidet P-40)-lysed cells for IO " O IO* IO'5 io- KI' io'! 30 min at 30,000 x g. Details of this procedure have been Asporogìnose concenlration(IU/ml) MfJ reported by Thompson ef al. (15, 16). Chart 1. a, dose-response curve of cellular colony-forming ability to asparag inase in asparagine-deficient medium. Different serial concentrations of cells were plated in plastic tissue culture dishes containing complete growth medium RESULTS for treatment with asparaginase and for outgrowth of surviving colonies. After the cells were attached, the medium was changed to growth medium lacking aspar The measured activities of asparagine synthetase and as- agine and supplemented with 10% dialyzed fetal calf serum. Various concentra tions of asparaginase were added to the cells, and the cultures were incubated paragyl-tRNA synthetase for the cell lines used in this study at either 34°(for AH5 and Asn 3) or 37°(for Asn 7, Asn 9, WT, N3, and N4) for are presented in Table 1. Considering the CHO cell mutants, 65 hr. After washing with 2 rinses of phosphate-buffered saline (6), the cells were incubated in complete «-modified minimal essential medium at 34°(for AH5, Asn asparagine synthetase activities ranged from less than 2% of 3, Asn 7, and Asn 9) and at 37° (for N3, N4. and WT) for 10 days. After the activity of WT cells for the N3 mutant to 2 to 3 times WT incubation, the number of visible colonies was scored and expressed as a fraction activity for the asparagyl-tRNA synthetase mutants to 11 times of that obtained with asparagine-supplemented medium without asparaginase. b. WT activity for the superproducer line, AH5. Asparagyl-tRNA dose-response curve of cellular colony-forming ability to asparaginase in aspar agine-supplemented medium. Different serial concentrations of cells were plated synthetase activities, measured at a permissive temperature in complete growth medium containing asparagine -H2O at 50 /ig/ml. After the for the fs mutants, showed very low levels relative to WT cells cells were attached, various concentrations of asparaginase were added to the medium, and the cultures were incubated at 37°for 54 hr. After washing with for the Asn 3 and Asn 7 mutants as reported previously (15, one rinse of a-modified minimal essential medium lacking asparagine, the cells 16). These low levels are presumably due to inactivation of the were incubated in a-modified mimimal essential medium containing asparagine- H2O at 200 /ig/ml and 10% fetal calf serum at 34°for 10 days. Colonies were more labile defective enzyme in the mutants during preparation scored as in
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