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The Role of Drug Transport in Resistance to Nitrogen Mustard and Other Alkylating Agents in L5178Y Lymphoblasts1

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The Role of Drug Transport in Resistance to Nitrogen Mustard and Other Alkylating Agents in L5178Y Lymphoblasts1 [CANCER RESEARCH 35.1687 1692, July 1975] The Role of Drug Transport in Resistance to Nitrogen Mustard and Other Alkylating Agents in L5178Y Lymphoblasts1 Gerald J. Goldenberg2 Department of Medicine. University of Manitoba, and the Manitoba Institute of Cell Biology. Winnipeg. Manitoba. R3E OV9, Canada SUMMARY (19, 20) in normal and leukemic human lymphoid cells (25) and in rat Walker 256 carcinosarcoma cells in vitro (18). An investigation was undertaken of the mechanism of Choline, a close structural analog of HN2, has been resistance to nitrogen mustard (HN2) and other alkylating identified as the native substrate for the HN2 transport agents, with particular emphasis on the interaction between system (19). Other alkylating agents, including chlorambu cross-resistance and drug transport mechanisms in LSI78Y cil, melphalan, and intact and enzyme-activated cyclophos lymphohlasts. Dose-survival curves demonstrated that the DOfor HN2-sensitive cells (L5178Y) treated with HN2 in phamide, did not inhibit HN2 transport, suggesting inde vitro was 9.79 ng/ml and the D0 for HN2-resistant cells pendent transport mechanisms for these agents (20). Unlike HN2 transport, a study of cyclophosphamide uptake by (L5178Y/HN2) was 181.11 ng/ml; thus, sensitive cells were 18.5-fold more responsive than were resistant cells and the LSI78Y lymphoblasts demonstrated biphasic kinetics and was mediated by a facilitated diffusion mechanism (15). In difference was highly significant (p < 0.001). A similar common with HN2 transport, uptake of cyclophosphamide evaluation of 5 additional alkylating agents, including chlorambucil, melphalan, l,3-bis(2-chloroethyl)-l-nitro- was not blocked by other alkylating agents such as HN2, sourea, Mitomycin C, and 2,3,5-tris(ethyleneimino)-l,4- chlorambucil, melphalan and isophosphamide, providing additional evidence that these drugs are transported by benzoquinone, revealed that L5178Y/HN2 cells were also cross-resistant, in part, to each of these compounds. Fur independent mechanisms. Accordingly, an investigation was thermore, the degree of cross-resistance was remarkably undertaken to determine the interrelationship of drug transport systems and the occurrence of cross-resistance similar; for each drug, dose-survival studies showed that HN2-resistant cells were approximately 2- to 3-fold more between HN2 and other alkylating agents in L5178Y lymphoblasts. resistant to therapy than were sensitive cells. L5I78Y/ HN2 cells were also cross-resistant to cyclophosphamide MATERIALS AND METHODS in vivo; after treatment with cyclophosphamide, DBA/2 female mice that were given inoculations of L5I78Y cells, Cross-resistance Studies. HN2-sensitive (L5178Y) and but not those given transplants of L5178Y/HN2 cells, -resistant (L5178Y/HN2) lymphoblasts were maintained in showed a significant prolongation of survival time (p < DBA/2 female mice by weekly i.p. transplantation and were 0.01). also cultured in vitro by methods described previously (13, Transport of HN2, hydroly/ed derivative of HN2, and 14, 20). Earlier studies have shown that both dose- and choline by L5178Y lymphoblasts in vitro was not competi time-survival curves of cells treated with alkylating agents tively inhibited by any of the other alkylating agents, in vitro follow an exponential relationship (2, 14, 33, 38, 39). suggesting that transport of these compounds was by an Time-dose treatment schedules were selected for each drug independent mechanism. These findings suggest that the so that maximal cell kill would approximate 3 logs; mechanism whereby L5178Y/HN2 cells are cross-resistant HN2-sensitive and -resistant cells were treated in vitro with to other alkylating agents may involve nontransport factors various drug concentrations for a fixed time as described and that these other drugs may bypass a major portion of (16, 17). The treated cells were washed once with 5 ml of HN2 resistance by using independent transport systems. Fischer medium (Grand Island Biological Co., Grand Island, N. Y.), and cell survival was determined by the INTRODUCTION cloning method of Chu and Fischer (5). Linear regression The alkylating agent HN23 is transported by an active, analysis of the dose-survival curves was performed, the carrier-mediated process in murine LSI78Y lymphoblasts regression equations being in the form log fy = mx + b, 1This work was supported by a grant from the National Cancer where y is surviving cell fraction, x is dose of alkylating Institute of Cunada. agent, m is slope of the regression line, and b is the y "Clinical Research Associate of the National Cancer Institute of intercept. Da (the dose of drug reducing survival to \/e, i.e., Canada. 37% of the initial cell population) was derived from the 3The abbreviations used are: HN2, nitrogen mustard; HN2-OH, negative reciprocal of the slope of the regression line (14, 16, hydrolyzed derivative of nitrogen mustard: BCNU, l,3-bis(2-chloro- ethyl)-l-nitrosourea; trenimon, 2,3,5-tris(ethyleneimino)-l.4-benzo- 17). quinone. In vivo studies of the effect of HN2 and cyclophospham Received January 13, 1975; accepted March 27, 1975. ide were performed on 6- to 8-week-old DBA/2 female JULY 1975 1687 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1975 American Association for Cancer Research. G. J. Goldenberg mice (The Jackson Laboratory, Bar Harbor, Maine) given L5178Y and L5178Y/HN2 cells treated with HN2 in vitro s.c. or i.p. inoculations of approximately 2 x IO7L5178Y or are shown in Chart 1. The £>„forsensitive cells was 9.79 L5178Y/HN2 cells. Groups of 10 mice each were treated ng/ml and that for HN2-resistant cells was 181.11 ng/ml; within 20 min of tumor transplantation with either HN2, 3.1 thus sensitive cells were 18.5-fold more responsive to HN2 mg/kg (16 /tmoles/kg), or cyclophosphamide, 88 mg/kg than were resistant cells, and the difference was highly (337 /¿moles/kg),as a single i.p. injection; control animals significant (p < 0.001). The response of sensitive cells was received an equivalent volume of 0.9% NaCl solution. The similar to that reported previously (16-18). survival data were evaluated statistically by a 2-tailed t test. The response of HN2-sensitive and -resistant cells to Transport Studies. Transport studies were performed on chlorambucil was determined. The £>„forsensitive cells was suspension cultures of L5178Y lymphoblasts incubated in 1.84 /ig/ml and that for resistant cells was 3.62 /¿g/ml; vitro at 37°asdescribed previously (19, 20). [2-chloroethyl- HN2-sensitive cells were twice as susceptible to chlorambu 1,2-14C]HN2 (specific activity, 3.1 mCi/mmole) was ob cil as were resistant cells, and the difference was highly tained from Mallinckrodt Chemical Works, St. Louis, Mo.; significant (p < 0.001). HN2-OH was prepared by alkaline hydrolysis of the parent Dose-survival curves for cells treated with melphalan compound in 0.1 N NaOH at 60°for 2 hr and provided an exhibited a D0 of 0.45 /ig/ml for sensitive cells and 1.02 advantage over HN2 in that transport could be studied /ig/ml for resistant cells; the 2.3-fold difference in sensitivity without the complication of alkylation reactions (19, 20). was highly significant (p < 0.001). [l,2-14CjCholine chloride (specific activity, 6.2 mCi/ HN2-sensitive and -resistant cells treated with the ni- mmole) was obtained from New England Nuclear, Boston, trosourea compound, BCNU, also demonstrated some Mass. Uptake of labeled substrate was determined in the degree of cross-resistance to BCNU. The D0 for sensitive absence and presence of a variety of alkylating agents. cells was 0.27 ¿ig/mland that for resistant cells was 0.77 Mitomycin C was purchased from Nutritional Biochemicals /ig/ml; thus HN2-resistant cells were 2.8-fold more refrac Corp., Cleveland, Ohio. Unlabeled HN2 hydrochloride tory to BCNU treatment and the difference was highly (Mustargen) was kindly supplied by Dr. W. Dorian (Merck, significant (p < 0.001). Sharp and Dohme, Dorval, Quebec, Canada); cyclophos Dose-survival curves of cells treated with Mitomycin C phamide (Procytox) was provided by Dr. R. Watters (Frank revealed that the D0 for HN2-sensitive cells was 0.089 W. Homer, Ltd., Montreal, Quebec, Canada); chlorambu- /ig/ml and that for resistant cells was 0.26 /¿g/ml;thus cil (Leukeran) and melphalan (Alkeran) were contributed by sensitive cells were 3-fold more responsive to Mitomycin, Dr. J. R. MacDougal (Burroughs Wellcome and Co., Ltd., and this difference was highly significant (p < 0.001). Lachine, Quebec, Canada); and BCNU, synthesized by Ben Finally, the response of cells to the alkylating agent Venue Laboratories Inc., Bedford, Ohio, was obtained from trenimon was evaluated. The D0 for HN2-sensitive cells was Dr. Stephen K. Carter (Division of Cancer Treatment, 3.14 ng/ml and that of resistant cells was 5.55 ng/ml; National Cancer Institute, Bethesda, Md.). Previous studies sensitive cells were 1.8-fold more susceptible to the cytocidal of HN2, HN2-OH, and choline transport by LSI78Y action of trenimon, and this difference was also highly lymphoblasts indicated that uptake was linear for at least 60 significant (p < 0.001). min (20); therefore, incubation times were set at that interval in order to approximate initial uptake velocity conditions. Incubations were terminated by rapid chilling to 4°and by centrifugation of the cells through a layer of 0.25 Msucrose to remove extracellular radioactivity. The washed cells were solubilized in 0.5 N NaOH, and radioactivity was determined by liquid scintillation spectrometry. The kinetic parameters Km and Vmaxwere derived from linear regression equations of Lineweaver-Burk plots in which the slope represents Km/Vmax, the y intercept is l/Vmail, and the x intercept is -\/Km (19, 20). For competitive inhibitors, K, was determined from the for mula: apparent Km (from x intercept of curve with inhibitor present) = Km (1 + [/]/£,), where Km was obtained from 0.001 the x intercept of the control curve in the absence of 0.25 0.5 0.75 1.25 inhibitor and [/] was the concentration of inhibitor (20, 26).
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