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[CANCER RESEARCH 41, 3104-3106, 0008-5472/81 /0041-OOOOS02.00 August 1981] 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 The cell-killing activity of asparaginase on three classes of Chinese hamster ovary cell mutants was examined: a mutant which overproduces asparagine Synthetase (AH5); mutants defective in asparagine synthetase (N3 and N4); and mutants 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 wildtype Chinese hamster ovary cells, while mutants defective in asparagine synthetase were more sensitive. Surprisingly, the asparagyl-transfer RNA synthetase mutants were even more sensitive to asparaginase than the asparagine synthetase mu tants. In a preliminary survey of four human lymphoid cell lines (RPMI 8402, RPMI 8392, B46M, and Molt-4F) which showed dramatically different asparaginase sensitivity, however, sen sitivity to the cell-killing activity of asparaginase was correlated with reduced levels of asparagine synthetase and not with reduced levels of asparagyl-transfer RNA synthetase. INTRODUCTION The reason for variability in clinical response to the cancer chemotherapeutic agent asparaginase is unknown at present (4, 17). This is perhaps not surprising as the basic mechanism for cell killing by the drug is also unknown. Evidence has been presented in support of cell-killing mechanisms involving se vere starvation for asparagine (3, 8), cleavage of cell surface protein at asparagine residues (5), and disruption of the cellular energy-producing reactions (9). Regardless of mechanism, the efficacy of asparaginase will obviously be affected by the extracellular and intracellular levels of asparagine. These levels are determined in part by the intracellular biosynthetic enzyme asparagine synthetase, and some of the variability in response to asparaginase has been ascribed to variability in the activity of this enzyme. We have recently reported the isolation of mutants of CHO" cells with reduced or absent asparagine synthetase activity (18). Evidence was presented in support of the hypothesis that the mutations were in the structural gene for the enzyme (18). We have also isolated CHO mutants with conditional defects in asparagyl-tRNA synthetase (15, 16), an essential enzyme for cellular protein synthesis. As the latter mutants are both tem perature sensitive and auxotrophic for asparagine (15, 16), it ' Supported by grants from the National Cancer Institute of Canada and Grant MT 1877 from the Medical Research Council of Canada. 2 Research student of the National Cancer Institute of Canada. 3 To whom requests for reprints should be addressed. ' The abbreviations used are: CHO, Chinese hamster ovary; WT, wild type. Received September 30, 1980; accepted May 7, 1981. 3104 was of interest to compare the sensitivity of the 2 types of mutants having the same genetic background to the cell-killing activity of asparaginase. Any differences in sensitivity to as paraginase between mutants with different enzymatic lesions could provide an explanation for some of the variability in drug sensitivity of human lymphoid cell lines. MATERIALS AND METHODS Chemicals. Asparaginase, or asparagine amidohydrolase, or Kidrolase (commercial name), EC 3.5.1.1 type EC2, partially purified from Escherichia coli, was obtained from Poulenc, Ltd., Montreal, Quebec, Canada (specific activity, approxi mately 200 units/mg). This relatively impure enzyme prepara tion is used clinically and hydrolyzes asparagine to aspartic acid but also contains a low level of glutaminase activity [about 3% of the asparaginase activity (9)]. It should be pointed out that our tissue culture medium contains a relatively high con centration of glutamine (292 mg/liter) which would minimize any anticellular effects of the contaminating glutaminase. Also, the cell-killing activity of the preparation was strongly reduced by the inclusion of asparagine in the treatment medium (Chart 1). Radioactive isotopes were obtained from Amersham/Searle Corp., Oakville, Ontario, Canada; amino acids were obtained from GIBCO, Burlington, Ontario, Canada; liver tRNA was obtained from A. Hampel, Northern Illinois University. Cells and Culture Conditions. The wild-type CHO strain, Gat", isolated by McBurney and Whitmore (10), will be referred to as the WT strain. This strain was also used by Thompson ef al. (15, 16) and Waye and Stanners (18) to isolate asparagyltRNA synthetase mutants (Asn 3, Asn 7, and Asn 9) and asparagine synthetase mutants (N3 and N4), respectively. AH5, isolated from fsH1 (13), is a CHO cell mutant that over produces asparagine synthetase (7) and was obtained from S. Arfin, University of California, Irvine. Human T-cell lines (RPMI 8402 and Molt-4F) and human B-cell lines (RPMI 8392 and B46M) were obtained from J. Minowada (11) (Roswell Park Memorial Institute, Buffalo, N. Y.). CHO cells were maintained in suspension culture at 34°using «-modified minimal essential medium (12) with asparagine-H2O at 50 /¿g/ml and supple mented with 10% fetal calf serum. Human lymphoid cells were maintained in static suspension cultures at 37° in the same growth medium. Cell concentrations were determined using an electronic particle counter. Assay for Asparagine Synthetase Activity. Asparagine syn thetase activity was measured by determining the ability of sonicated whole-cell extracts to synthesize [14C]asparagine from [14C]aspartic acid as a function of time as described by Arfin ef al. (1). Reactions were carried out taking determina- CANCER RESEARCH VOL. 41 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. AsparagyltRNA 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 from centrifugation of detergent (Nonidet P-40)-lysed cells for 30 min at 30,000 x g. Details of this procedure have been reported by Thompson ef al. (15, 16). RESULTS The measured activities of asparagine synthetase and asparagyl-tRNA synthetase for the cell lines used in this study are presented in Table 1. Considering the CHO cell mutants, asparagine synthetase activities ranged from less than 2% of the activity of WT cells for the N3 mutant to 2 to 3 times WT activity for the asparagyl-tRNA synthetase mutants to 11 times WT activity for the superproducer line, AH5. Asparagyl-tRNA synthetase activities, measured at a permissive temperature for the fs mutants, showed very low levels relative to WT cells for the Asn 3 and Asn 7 mutants as reported previously (15, 16). These low levels are presumably due to inactivation of the more labile defective enzyme in the mutants during preparation of the cellular extracts. The asparagine synthetase mutant (N3), on the other hand, showed higher than WT asparagyltRNA synthetase activity. The cell-killing activity of different doses of asparaginase on Table 1 Asparagine synthetase and asparagyl-tRNA synthetase activities of various cell lines lineAH5WTN3N4Asn Cell 3Asn 7Asn 9RPMI 8402MOLT-4FRPMI 8392B46MAsparagine syn thetase*11 synthetase0ND"1 ±21 0.1<0.020.4 ± 0.22.50.3<0.010.09ND3.3 ± 0.12.0 ± 0.12.3 ± 0.22.7 ± 0.4<0.1<0.11.6 ± sensitivity'_+++ ++ +++ ++ ++ ++ +_- 0.63.1 ± 0.30.3 ± 0.21.3 ± 0.030.3 ± ±0.4Asparagyl-tRNA ±0.2Asparaginase The values shown represent the activity relative to WT cells, with the 90% confidence intervals for repeat determinations. The activity of WT cells was 2.2 nmol asparagine per mg protein per hr. The values shown represent the activity relative to WT cells, with the 90% confidence intervals for repeat determinations. The activity of WT cells was 35 pmol [14C]asparagine incorporated per mg protein per min. Asparagyl-tRNA synthetase activities of all cell lines were first normalized to the overall aminoacyltRNA synthetase activity measured with a mixture of 4 amino acids (phenylalanine, tyrosine, threonine, and valine) to correct for differences in extraction efficiency from preparation to preparation. The normalization procedure did not affect the qualitative conclusions drawn. Semiquantitative estimates of sensitivity for cell survival to asparaginase obtained from Chart 1a for CHO cell lines and from Ohnuma ef al. (11) for the human lymphoid cell lines; —,relatively insensitive; +, relatively sensitive. d ND, not determined. AUGUST 1981 ,Asn9 IO " O IO* Asporogìnose concenlration(IU/ml) IO'5 io- KI' io'! MfJ 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 for treatment with asparaginase and for outgrowth of surviving colonies. After the cells were attached, the medium was changed to growth medium lacking aspar agine and supplemented with 10% dialyzed fetal calf serum. Various concentra tions of asparaginase were added to the cells, and the cultures were incubated at either 34°(for AH5 and Asn 3) or 37°(for Asn 7, Asn 9, WT, N3, and N4) for 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 3, Asn 7, and Asn 9) and at 37° (for N3, N4. and WT) for 10 days. After incubation, the number of visible colonies was scored and expressed as a fraction of that obtained with asparagine-supplemented medium without asparaginase. b. dose-response curve of cellular colony-forming ability to asparaginase in aspar agine-supplemented medium. Different serial concentrations of cells were plated in complete growth medium containing asparagine -H2O at 50 /ig/ml. After the 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 one rinse of a-modified minimal essential medium lacking asparagine, the cells were incubated in a-modified mimimal essential medium containing asparagineH2O at 200 /ig/ml and 10% fetal calf serum at 34°for 10 days. Colonies were scored as in a. In both a and b, the plating efficiency at any particular survival level was found to be constant over a range of cell numbers plated of about 103. these CHO cell lines maintained in asparagine-free medium for a fixed exposure time of 65 hr, as assessed by colony-forming ability, is shown in Chart 1a. The viability of the asparagine synthetase superproducer, AH5, was unaffected by even the highest dose of asparaginase used, whereas WT cells showed some loss of viability at very high drug doses. The asparagine synthetase mutants, N3 and N4, were predictably much more sensitive to asparaginase than WT cells. The asparagyl-tRNA synthetase mutants, Asn 3, Asn 7, and Asn 9, when maintained under semipermissive conditions (37°; asparagine-free me dium), were found to be even more sensitive to the cell-killing activity of asparaginase than the asparagine synthetase mu tants. This difference is most apparent for Asn 3, which suffered relatively little damage from the incubation at semipermissive conditions in the absence of asparaginase. To confirm these results using incubation conditions which themselves had no toxic effect on any of the asparagyl-tRNA synthetase mutants, cultures were exposed to asparaginase in medium containing normal levels of asparagine. The results shown in Chart 1b again show that the mutants deficient in asparagyl-tRNA synthetase were far more sensitive to the cellkilling activity of asparaginase than the mutants deficient in asparagine synthetase. These results raised the possibility that the variability in asparaginase sensitivity of human leukemic cells could be due to defects in both asparagine synthetase and asparagyl-tRNA 3105 M. M. Y. Waye and C. P. Stanners synthetase. Two human T-cell lines, RPMI8402 and MOLT-4F, which were much more sensitive to asparaginase than 2 human B-cell lines, RPMI 8392 and B46M (11), however, showed low levels of asparagine synthetase only (Table 1). DISCUSSION The recent isolation of both asparagine synthetase (18)- and asparagyl-tRNA synthetase (15, 16)-deficient mutants in the same cell line, CHO, has enabled us to examine a possible role of both these enzymes in determining asparaginase sensitivity of mammalian cells. We find that the conditionally defective fs asparagyl-tRNA synthetase mutants were far more sensitive to asparaginase than the defective asparagine synthetase mu tants, even when maintained under fully permissive conditions. The cell-killing action of the drug on the former mutants is probably due to the progressive permanent inactivation of this essential protein biosynthetic enzyme by extreme asparagine starvation as reported recently in detail for several aminoacyltRNA synthetase CHO cell mutants under nonpermissive con ditions (14). What are the possible clinical ramifications of this observa tion? It would seem unlikely that human neoplastic cells would have strong mutations leading to inactive aminoacyl-tRNA synthetases, as these would be lethal. Weak mutations, producing no defects in these enzymes under normal conditions, however, are possible and would, as we have shown, lead to greatly increased sensitivity to drugs such as asparaginase. We found no evidence for asparagyl-tRNA synthetase mutations in 2 human T-lymphoid leukemic cell lines which were highly sen sitive to asparaginase but feel that more sensitive tests for cryptic mutations in these enzymes and results with a much wider spectrum of human neoplastic cells are required before any conclusions can be drawn. We are currently developing such tests using our CHO cell mutants as a guide. ACKNOWLEDGMENTS We thank Dr. S. Arfin for providing AH5 and Dr. J. Minowada for providing the human lymphoid cell lines. We thank W. A. Mehring for technical assistance. 3106 REFERENCES 1. Arfin. S. M., Simpson, D. R., Chiang, C. S., Andrulis. I. L., and Hatfield, G. W. A role for asparaginyl-tRNA in the regulation of asparagine synthetase in a mammalian cell line. Proc. Nati. Acad. Sei. U. S. A.. 74: 2367-2369, 1977. 2. Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. 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Asparagine synthetase activity of mouse leukemias. Science (Wash. D. C.), reo. 533-535. 1968. 9. Lavietes. B. B., Regan, D. H., and Demopoulos, H. B. Glutamate oxidation in 6C3HED lymphoma: effects of L-asparaginase on sensitive and resistant lines. Proc. Nati. Acad. Sei. U. S. A., 71: 3993-3997, 1974. 10. McBurney, M. W., and Whitmore. G. F. Isolation and biochemical character ization of folate deficient mutants of Chinese hamster cells. Cell, 2: 173182, 1974. 11. Ohnuma, T.. Holland, J. F., Arkin, H., and Minowada, J. L-Asparagine requirements of human T-lymphocytes and B-lymphocytes in culture. J. Nati. Cancer Inst., 59. 1061-1063, 1977. 12. Stanners, C. P., Eliceiri, G. L., and Green. H. Two types of ribosomes in mouse hamster hybrid cells. Nat. New Biol.. 230. 52-54, 1971. 13. Stanners. C. P., and Thompson, L. H. Studies on a mammalian cell mutant with a temperature-sensitive leucyl-tRNA synthetase. Cold Spring Harbor Conf. Cell Proliferation. 7: 191-203, 1974. 14. Stanners, C. P.. Wightman, T. M., and Harkins, J. L. Effect of extreme amino acid starvation on the protein synthetic machinery of CHO cells. J. Cell. Physiol., 95: 125-138, 1978. 15. Thompson, L. H., Lofgren, D. J., and Adair, G. M. CHO cell mutants for arginyl-, asparagyl-, glutaminyl-, histidyl- and methionyl-transfer RNA synthetases: identification and initial characterization. Cell, ) 1:157-168. 1977. 16. Thompson, L. H., Stanners, C. P., and Siminovitch, L. Selection by [3H]amino acids of CHO-cell mutants with altered leucyl- and asparagyltRNA synthetases. Somatic Cell Genet., Õ:187-208, 1975. 17. Uren, J. R.. and Handschumacher, R. E. Enzyme therapy. In: f. F. Becker (ed.). Cancer. A Comprehensive Treatise. Vol. 5. pp. 457-487. New York: Plenum Publishing Corp., 1977. 18. Waye. M. M. Y., and Stanners, C. P. Isolation and characterization of CHO cell mutants with altered asparagine synthetase. Somatic Cell Genet., 5: 625-639. 1979. CANCER RESEARCH VOL. 41