Prevention of Methotrexate Cytotoxicity by Asparaginase Inhibition of Methotrexate Polyglutamate Formation1

[CANCER RESEARCH 45, 217-220, January 1985]

Jacques Jolivet,2 Diane E. Cole, John S. Holcenberg, and David G. Poplack

Institut du Cancer de Montreal, Montreal, Canada H2L 4M1 [J. J.]; Pediatrie Branch, National Cancer Institute, Bethesda, Maryland 20205 [D. E. C., D. G. P.]; and Children's Hospital of Los Angeles, Los Angeles, California 90027 [J. S. H.J

ABSTRACT L5178Y cells in vitro when given before the antifolate and aborts its actions when given after MTX. This effect has been ascribed Escherlchla coli asparaginase (Asnase) pretreatment of As- to nonspecific inhibition of protein synthesis and decreased MTX nase-sensitive L5178Y cells in vitro is thought to antagonize uptake by tumor cells (2, 13). We have reexamined the mecha methotrexate (MIX) cytotoxicity through nonspecific inhibition nisms underlying this interaction in L5178Y cells in view of recent of protein synthesis and MIX uptake. We have rÃ©examinÃ©e!the data describing the importance of the metabolism of MTX to mechanism of this interaction in view of recent data demonstrat polyglutamate derivatives in its mechanism of action (8). We ing the importance of MIX metabolism to polyglutamate deriva have found that Asnase antagonizes the effects of MTX by tives (MTXPGs) in the cytotoxic effects of the antifolate. After a decreasing its metabolism to polyglutamate derivatives. 3-hr exposure to 0.5 /Â¿MMTX,67% of intracellular drug was in the form of MTXPGs containing a total of 2 to 5 glutamyl residues (MTX-Glu2_5), and cloning efficiency in drug-free medium was MATERIALS AND METHODS only 7% of untreated control. After a 3-hr pretreatment with E. coli Asnase (0.1 unit/ml), [3H]thymidine incorporation dropped Chemicals. [3',5',9-3H]MTX (specific activity, 20 Ci/nmol) was pur by 29%, MTXPG formation during subsequent MTX exposure chased from Amersham Corp. (Arlington Heights, IL) and further purified by DEAE-cellulose chromatography with elution along a linear gradient decreased by more than one-half (MTX-Glu2 unchanged; MTX- of 0.1 to 0.4 M NH4HCO3 (6). Escherichia coli Asnase (Elspar) was Glu3aixi4decreased to 51.7 and 18.5% of levels achieved in cells purchased from Merck, Sharp & Dohme (West Point, PA). Asnase and not pretreated with Asnase; no MTX-Glu5 formed), and cloning L-glutaminase activities in the preparation used were determined by efficiency increased to 71 % of untreated control. This effect was direct nesslerization of the NH3 produced (15) and were found to be 73.3 not due to decreased MTX uptake into L5178Y cells or to and 2.0 units/mg, respectively. [6-3H]dThd (21.5 Ci/nmol) was purchased decreased intracellular free L-glutamate or L-glutamine levels. A from the New England Nuclear (Boston, MA). PCS from a single lot was 3-hr exposure of L5178Y cells to media lacking L-isoleucine, an used and purchased from Grand Island Biological Co. (Grand Island, NY) essential amino acid for cell growth, prior to MTX exposure and dialyzed (DFCS) as follows. PCS (100 ml) was placed in a dialysis inhibited [3H]thymidine incorporation by 37%, decreased subse membrane (27 DM; Union Carbide, Chicago, IL) and dialyzed against PBS at 4Â°until more than 95% of [3H]dThd added to the PCS before quent MTXPG formation by 62%, and increased subsequent cloning in drug-free medium to control levels. Decreased MTXPG dialysis had disappeared. All chemicals were of reagent grade and were purchased from either Fisher Scientific Co. (Pittsburgh, PA) or Sigma formation was responsible for the prevention of MTX cytotoxicity Chemical Co. (St. Louis, MO). seen after both pretreatments. Unmetabolized MTX rapidly left Propagation of Cells in Culture. Asnase-sensitive L5178Y cells were L5178Y cells after removal of extracellular MTX. Consequently, obtained from E. G. and G. Mason Research Institute (Worcester, MA). lower levels of unbound intracellular drug, a prerequisite of drug The cells were grown in continuous-suspension culture in RPMI 1640 activity, were maintained in pretreated than in control cells after (Grand Island Biological Co.) and 10% FCS under 5% CO2 at 37Â°.One passage in drug-free medium. Asnase pretreatment protects day prior to drug exposure, the cells were diluted to a density of 3 x â€¢\leukemia (3,18). Clinically, Asnase acts as a "rescue" agent and procedure repeated 2 additional times. Cells were cloned in RPM11640 permits the administration of normally toxic doses of MTX (3). and 10% DFCS with a control cloning efficiency of 20%. Colonies were Asnase antagonizes the effects of MTX in Asnase-sensitive counted visually after 10 days of incubation. Assay of Intracellular MTXPGs. The intracellular content of MTX and 1This study has been supported by the Pediatrie Branch of the National Cancer MTXPGs was determined as follows. At the end of a drug incubation, Institute and the Medical Research Council of Canada (Grant DG-302). 2 To whom requests for reprints should be addressed. the cells were added to 10 ml of ice-cold PBS, spun down at 275 x g at 3 The abbreviations used are: Asnase, u-asparagine aminohydrolase; MTX, 4Â°,and washed 3 times by resuspension in 10 ml of ice-cold PBS and methotrexate; MTXPGs or MTX-Glu2-s, methotrexate polyglutamates containing a recentrifugation. The cells were then lysed in 5 ml of 10% TCA. The total of 2 to 5 glutamyl residues; PCS, fetal calf serum; PBS, phosphate-buffered sample was processed, and MTX and MTXPGs were assayed by HPLC saline (in g/liter: NaCI, 9.0; KH2PO4, 0.21; NaaHPCv 0.726, pH 7.2); dThd, thymidine; DFCS, dialyzed fetal calf serum; TCA, trichtoroacetic acid; HPLC, high- as described previously (9). pressure liquid chromatography; DHFR, dihydrofolate reducÃase. Assay of Intracellular Enzyme-bound and Unbound MTX. DHFR- Received June 25,1984; accepted October 3,1984. bound and -unbound (free) MTX and MTXPGs were determined as

CANCER RESEARCH VOL. 45 JANUARY 1985 217

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1985 American Association for Cancer Research. EFFECT OF ASNASE ON MIX POLYGLUTAMATE FORMATION follows. At the end of drug incubation with [3H]MTX, the cells were ments. washed 3 times in ice-cold PBS as described above, resuspended in 1.5 Effects of Asnase on MTX Uptake. We next examined if the ml 0.15 M KH2PO4, pH 6.2, and sonicated in a Branson 350 sonicator decreased MTXPG formation following Asnase exposure could (Branson Sonic Power Co., Danbury, CT) for 3 sec at the lowest setting. be due to decreased MTX uptake across the cell membrane One-third of the cell extract was immediately added to 10% TCA for HPLC determination of MTXPGs, while the enzyme-bound and free drug resulting in decreased amounts of substrate for polyglutamate synthesis. We measured drug uptake during the first 30 min of in the remaining extract was separated by gel chrc matography as exposure to 0.5 MM[3H]MTX Â¡nAsnase-pretreated (0.1 units/ml) described previously (7). MTX Uptake Experiments. L5178Y cells (1 x 10" cells/ml) were and control L5178Y cells. As can be seen in Chart 1, no signifi incubated in 50-ml plastic flasks (Costar, Inc.) with 0.5 UM [3H]MTX in cant differences in drug uptake could be seen under the 2 RPM11640 containing 10% DFCS for various time intervals. At the end conditions examined. of incubation, 3 ml of the cell suspension were transferred into 10 ml of Effect of Asnase on Intracellular Free L-Asparagine, L- ice-cold PBS. The cells were washed 3 times in ice-cold PBS as described Glutamine, and i-Glutamate Levels. We next determined if above. The washed cell pellet was then dissolved in 1 ml of 1 N NaOH decreased MTX polyglutamate formation could be due to de overnight at room temperature. One half of this final sample was assayed creased levels of the other substrate required for polyglutamate for radioactivity using Diminscint liquid scintillation fluid (National Diag synthesis, L-glutamate (12). Intracellular levels of this compound nostics, Somerville, NJ) while a Lowry (11) protein assay was performed could have been decreased by the L-glutaminase activity in Â£. on the other half. Results were expressed in nmol of drug per g of coli Asnase. As shown on Table 2, L5178Y intracellular i_- protein. Assay for dThd Incorporation. L5178Y cell [3H]dThd incorporation glutamate and L-glutamine levels were relatively unaffected by studies were performed in 50-ml flasks (Costar, Inc.) at 37Â°under 5% exposure to E. coli Asnase (0.1 unit/ml) for up to 6 hr. L- COu in various media containing 10% DFCS and 1 UM [3H]dThd (0.2 /iCi/ Asparagine was depleted to less than 0.06 /

CANCER RESEARCH VOL. 45 JANUARY 1985 218

Table 1 Effects of Asnaseand L-isoleucinedepletion on [3H]dThd incorporation,MTX cytotoxicity, and MTXPGformation L5178Y cells growing in log phase (1 x 10ecell/ml) in RPMI 1640 with 10% DFCS were exposed to either complete RPM11640(control cells),mediawith E. coli Asnase(0.1 unit/ml), or mediawithout L-isoleucine3 hr prior to MTX exposure. [3H]dThd was added at time zero to parallelflasks, and [3H)dThdincorporation was measuredjust prior to adding MTX. Control [3H]dThd incorporation was linear over the 3-hr period. After the 3-hr cell exposure to various media, [3H]MTX (0.5 UM)was added for an additional 3 hr to the same media, following which cloning efficiency was determined as a percentage of untreated control, and an aliquot of cells was analyzed for intracellular MTX and MTXPGs. Cloning efficiencies of cells transferred for 6 hr in E. coli Asnase (0.1 unit/ml) or in L-isoleucine-freemedia were 97 and 80% of control, respectively. protein)MTX4.69 (nmol/g Pre-MTX media incorporation efficiency formulationControl (% ofcontrol)100 (% of control)7

E. coli Asnase 71.266.3Cloning 71113MTXPGs3.814.43Glu,1.140.911.04GIÃ™:,5.032.602.14Glu43.020.560.35Glu50.18 (0.1 unit/ml) L-lsoleucmedeleted[3H]dThd

and free intracellular drug at the end of MTX exposure due to greater MTXPG formation. During the drug-free efflux period, total intracellular drug decreased in control cells mostly during the first hr due to the efflux of parent drug and MTX-Glu2. MTX- Glu3 and MTX-Glu4 levels actually increased in the cells, leading to continued presence of intracellular free drug above the DFHR- binding capacity. On the other hand, Asnase-pretreated cells had much less drug metabolism and lower total and free drug accumulation. The cells lost almost all unbound drug in the 6 hr â€¢NoL-Asparaginase due to initially insufficient amounts of the retainable MTXPGs o L-Asparaginase (MTX-Glus-s) needed to ensure maintenance of large amounts of intracellular free drug after removal of extracellular drug.

DISCUSSION 10 15 20 25 30 TIME (minutes) The present study has confirmed that E. coli Asnase pretreat Chart 1. Uptakeof MTX in control and Asnase-pretreatedL5178Ycells. L5178Y ment protects Asnase-sensitive L5178Y cells from the cytotoxic cells growing in log phase (1x10Â« cells/ml) in RPM11640 with 10% DFCS were transferred to either complete RPMI 1640 (control cells) or media with E. coli effects of MTX (2). We have, however, gained new insight into Asnase(0.1 unit/ml) for 3 hr prior to MTX exposure. The cells were then incubated the mechanisms underlying this interaction. Asnase exposure at 37Â°with 0.5 Â»u[3H]MTXin their pre-MTX exposure medium, and total MTX led to decreased MTXPG formation and, as described in other uptake was measuredover 30 min. Results are expressed in nmol MTX/g protein, and the mean results of duplicate experiments are shown. cell lines (1, 5, 7, 9,16), unmetabolized MTX quickly left the cells after transfer to drug-free medium resulting in lower levels of

Table 2 unbound intracellular drug, an essential prerequisite for MTX Intracellular levels of free L-asparagine, L-g/utamate, and L-glutamine during cytotoxicity (17). In addition to being preferentially retained intra- Asnase treatment in Asnase-sensitive L5178Y cells cellularly, MTXPGs also dissociate less readily from DHFR (7) L5178Y cells growing in log phase (1x10* cells/ml) in RPMI 1640 with 10% and possibly affect other folate-requiring enzymes (8), factors DFCS were transferred at t = 0 to either complete RPMI 1640 (control) or media with E. coli Asnase (0.1 unit/ml) for up to 6 hr (Asnase). Free t-asparagine, L- which might also contribute to the toxic effects of MTX. Thus, glutamate, and L-glutaminelevels were measured in sulfosalicylicacid extracts in the decrease in polyglutamate formation caused by Asnase control and treated cells after 3 and 6 hr of exposure. Results representa meanof probably accounts for the observed Asnase "protection" from 3 experiments. Increased L-glutamateand L-glutaminelevels at t = 3 hr and ( = 6 hr are secondary to the cells being refed at f = 0 with new media. MTX. Nanas and Capizzi (13) described that the Asnase-induced >imol/gprotein protection from MTX cytotoxicity in L5178Y cells was related to t = 3hr <6hr decreased drug uptake which was seen as early as 4 hr after Asnase administration but became marked by 24 hr. Although L-Asparagine we did not see any significant effect of Asnase on the uptake of L-GlutamateL-Glutamme(control)1.07 4.81 11.65 12.23 10.16 11.35 unmetabolized MTX across the cell membrane after 3 hr of 0.20Control1.771.20Asnase<0.061.97Control2.010.93Asnase<0.052.12 Asnase exposure, it is possible that decreased MTX uptake could have been observed with longer Asnase incubation or then supplemented with 10 /Â¿Mhypoxanthine and 10 U.MdThd, higher doses of the enzyme. However, since intracellular MTX resuspended in drug-free RPMI 1640 and 10% DFCS, and levels were very similar in pretreated and nonpretreated cells, incubated at 37Â°.Intracellular MTXPG and enzyme-bound and we doubt that decreased transport played an important part in free drug were then again assayed after 1,2,3, and 6 hr in drug- the observed Asnase-induced protection from MTX. free medium. The results of a representative experiment are In addition to providing confirmatory evidence of the impor illustrated in Table 3. Control cells had greater drug accumulation tance of MTXPG metabolism in MTX cytotoxicity, the present

CANCER RESEARCH VOL. 45 JANUARY 1985 219

Table3 Efflux of MTX and MTXPGsfrom control and Asnase-pretreatedcells L5178Y cells growing in log phase (1 x 10* cells/ml) in RPMI 1640 with 10% DFCS were transferred to either complete RPM11640(controlcells)or media with E. coli Asnase(0.1 unit/ml) 3 hr prior to MTX exposure. The cells were then incubated with 0.5 ^M [3H]MTXfor 3 hr in their pre-MTXexposure mediumfollowing which an aliquot was removed and assayedfor enzyme-boundand free drug and MTXPGs.The remainingcells were washed 3 times with ice-cold PBS and transferred to drug-free complete RPM11640with 10 JIMhypoxanthine, 10 ^M dThd, and 10% DFCS at 37Â°(efflux period). Enzyme-boundand free drug and MTXPGs were again assayed after 1,2,3. and 6 hr in drug-free medium. Total drug protein)%free54.646.441.033.729.3% (nmol/g Efflux pe- (nmol/g pro- bound45.453.659.066.370.7MTX3.620.680.380.220.36Glu21.570.600.410.280.13Glu,5.865.284.433.672.93GluÂ«2.113.073.903.853.78Glu,,0.100.260.450.620.77Asnase-pretreatedriod (hr)tein)Control cells0123613.269.899.588.647.97MTXPGs

cells012369.716.866.075.594.3741.123.117.110.25.958.976.982.889.894.13.671.180.790.790.591.321.000.480.650.473.883.523.502.922.150.841.161.311.221.16

study also sheds some light into its regulatory mechanisms. asertestumor cell in vitro. J. Biol. Chem., 257:1890-1896,1982. Nimec and Galivan (14) have recently shown in cultured hepa- 6. Goldman, l. D., Lichtenstein, N. S., and Oliverio, V. T. Carrier-mediated transport of the folie acid analogue,methotrexate, in the L1210 leukemiacell. toma cells that MTXPG formation is decreased in fully grown, J. Bid. Chem., 243: 5007-5017,1968. quiescent cultures and by high-concentration folate exposure 7. Jolivet, J., and Chabner, B. A. Intracellularpharmacokineticsof methotrexate polyglutamates in human breast cancer cells. J. Clin. Invest., 72: 773-778, prior to MTX administration. Conversely, metabolism can be 1983. increased by folate depletion and nonspecifically by insulin 8. Jolivet, J., Cowan, K. H., Curt, C. A., Ctendeninn,N. J., and Chabner, B. A. The pharmacology and clinical use of methotrexate. N. Engl. J. Med., 309: administration (14). Asnase can now be added to the list of 1094-1104,1983. factors which can decrease MTXPG formation. Since the same 9. Jolivet, J., Schilsky, R. L., Bailey, B. D., Drake, J. C., and Chabner, B. A. effect was seen after cell growth inhibition by L-isoleucine dele Synthesis, retention and biological activity of methotrexate polyglutamates in cultured human breast cancer cells. J. Clin. Invest., 70: 351-360,1982. tion, the enzyme possibly caused a nonspecific decrease in MTX 10. Ley, K. D., and Tobey, R. A. Regulation of initiation of DMA synthesis in metabolism through its inhibitory effects on cell growth. The Chinese hamster cells. II. Induction of DNA synthesis and cell division by isoleucine and glutamine in G,-arrested cells in suspension culture. J. Cell mechanisms underlying this phenomenon are unknown and are Biol., 47: 453-459,1970. being investigated. Further knowledge of the regulation of MTX 11. Lowry, O. H., Rosebrough, N. J., Farr, A. J., and Randall, R. J. Protein metabolism may be of significant importance for the optimal measurement with the Folin phenol reagent. J. Bid. Chem., 193: 265-275, 1951. clinical use of MTX. 12. McGuire,J. J., Hsieh,P., Coward, J. K., and Berlino, J. R. Enzymaticsynthesis of fdylpdyglutamates. J. Bid. Chem., 255: 5776-5788,1980. 13. Nanas. A., and Capizzi, R. L. Effect of in vivo treatment with L-asparaginase on the in vitro uptake and phosphorylation of some antileukemic agents. REFERENCES Cancer Res., 34: 2689-2693,1974. 14. Nimec, Z., and Galivan,J. Regulatory aspects of the giutamyiationof metho 1. Balinska, M., Galivan, J., and Coward, J. K. Efflux of methotrexate and its trexate in cultured hepatoma cells. Arch. Biochem. Biophys., 226: 671-680, polyglutamate derivatives from hepatic cells in vitro. Cancer Res., 41: 2751- 1983. 2756,1981. 15. Roberts, J., Hdcenberg, J. S., and Dolowy, W. Isolation, crystalization and 2. Capizzi, R. L. Schedule-dependentsynergismand antagonismbetween meth- properties of Achromobacteriaceaeglutaminase-asparaginasewith antitumor otrexate and t-asparaginase. Biochem. Pharmacd., 23 (Suppl.).-151-161, activity. J. Bid. Chem., 247: 84-90,1972. 1974. 16. Rosenblatt. D. S.. Whitehead, V. M., Vera, N., Pottier, A., Dupont, M., and 3. Capizzi, R. L. Asparaginase-methotrexate in combination chemotherapy: Vuchich, M. J. Prolonged inhibition of DNA synthesis associated with the schedule-dependent differential effects on normal versus neoplastia cells. accumulation of methotrexate pdyglutamates by cultured human cells. Md. Cancer Treat Rep., 65: 115-121, 1981. Pharmacd., 14: 1143-1147,1978. 4. Chu, M. Y., and Fischer, G. A. The incorporation of 3H-cytosinearabinoside 17. White, J. C. Reversal of methotrexate binding to dihydrofdate reductase by and its effect on murine teukemic cells (L5178Y). Biochem. Pharmacol., 17: dihydrofdate: studieswith pureenzymeand computer modelingusingnetwork 753-767,1968. thermodynamics.J. Bid. Chem., 254:10889-10894,1979. 5. Fry, D. W., Yatowich, J. C., and Goldman, I. D. Rapid formation of pdy-y- 18. Yap, B.-S., McCredie, K. B., Keating, M. J., Bodey, G. P., and Freireich,E. J. glutamyl derivatives of methotrexate and their association with dihydrofolate Asparaginaseand methotrexate combinationchemotherapyin relapsed acute reductase as assessed by high-pressure liquid chromatography in the Ehrlich lymphoblasticleukemia in adults. Cancer Treat Rep., 65: 83-87,1981.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1985 American Association for Cancer Research. Prevention of Methotrexate Cytotoxicity by Asparaginase Inhibition of Methotrexate Polyglutamate Formation

Jacques Jolivet, Diane E. Cole, John S. Holcenberg, et al.

Cancer Res 1985;45:217-220.

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