Sensitivity to Chemotherapeutic and Immunomodulating Agents of Two Mouse Lymphomas and of a Macrophage Tumor1

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(CANCER RESEARCH 39. 3964-3967, October 1979) 0008-54 72/797 0039-OOOOS02.00 Sensitivity to Chemotherapeutic and Immunomodulating Agents of Two Mouse Lymphomas and of a Macrophage Tumor1

George S. Tarnowski, Peter Ralph,2 and C. Chester Stock

Walker Laboratory, Memorial Sloan-Kettering Cancer Center, Rye, New York 10580

ABSTRACT Table 1 Acceptance values for the MST of groups of 5 to 6 control tumor-bearing mice The effect of treatment with 15 Chemotherapeutic and 10 Ratios of MST for treated mice versus control mice from the corresponding immunomodulating agents on the growth of T-cell lymphoma therapy tests, which are greater than the acceptance value, indicate a significant prolongation of survival time; p < 0.05. EL-4, macrophage tumor J774, and B-cell lymphoma 70Z/2 Mice inoculated on Day 0 with of the mouse has been studied using the prolongation of median survival time of tumor-bearing hosts as an index of p.TumorEL-41 x 10s viable tumor cells, i. i.No. x 106 viable cells therapeutic effectiveness. The survival time of mice bearing of MST of MST 70Z/2 was prolonged more than 100% by single-agent therapy groups15 (days)13.33 groups16 (days)11.97 with actinomycin D, cyclophosphamide, 6-mercaptopurine, mitomycin C, and vinblastine; a similar response of J774 was J774 1713Av. 19.91 6.09 1.3 25 18.12 6.94 1.4 produced by therapy with Adriamycin, cyclophosphamide, or 70Z/2No. 10.31UCLa3.771.94AV1.31.21 15Av. 10.67UCL1.833.74p.AV1.21.4 6-mercaptopurine. No Chemotherapeutic agent prolonged the * UCL, upper 99.5% confidence limit of MST = 2.58 x S.D.; AV, acceptance median survival time of mice bearing EL-4 by 100% or more. value = (x + UCLJ/x, in which x is MST (days). Of the immunomodulating agents, mycobacterial preparations rophage tumor was grown in female BALB/cCrl mice from (Bacillus Calmette-GuÃ©rin or interphase material), Corynebac- Charles River Breeding Laboratories, Inc., Wilmington, Mass.; terium parvum, and polyinosinic-polycytidylic acid moderately and the 70Z/2 lymphoma was grown in B6D2F, male mice prolonged the survival time of mice bearing J774 or 70Z/2; from ARS/Sprague-Dawley, Madison, Wis. the EL-4 lymphoma was refractory to all 10 immunomodulating Tumors were maintained and tested in ascites form. Mice agents. weighed 18 to 22 g at the time of tumor implantation and were given food and water ad libitum. MST5 of mice varied from 19 INTRODUCTION to 11 days when given injections of 103 to 106 EL-4 cells, 24 We have reported recently (8) the reversal of tumor-induced to 16 days with 103 to 106 J774, and 14 to 10 days with 105 suppression of cytotoxic T-cell activity during chemotherapy of to10670Z/2. a mouse T-cell lymphoma and a macrophage tumor. Subse Chemicals. ara-C, azaserine, 1,3-bis(2-chloroethyl)-1-nitro- quently,3 this investigation has been extended to include the sourea, cyclophosphamide, 6-mercaptopurine, mitomycin C, effects of therapy on the reversal of tumor-induced suppression nitrogen mustard, and vinblastine were obtained from the Can of the humoral immune response of spleen cells of tumor- cer Chemotherapy National Service Center, Bethesda, Md.; bearing mice and on the hematopoietic progenitor cells and actinomycin D and hydrocortisone acetate from Merck, Sharp, infiltrating tumor cells in the spleens of such mice. In addition and Dohme Research Laboratories, Rahway, N. J.; Adriamycin to the T-cell lymphoma EL-4 (2) and macrophage tumor J774 from Farmitalia, Milan, Italy; bleomycin from the Bristol Labo (6), a B-cell lymphoma 70Z/2" of C57BL/6J x DBA/2 F, ratories, Inc., Syracuse, N. Y.; DON from Parke, Davis and Co. (hereafter called B6D2F,) mice has been included in this study. Research Laboratories, Ann Arbor, Mich.; 5-fluorouracil from The results of a survey of antitumor activity of a group of Hoffmann-LaRoche Inc., Nutley, N. J., and methotrexate from Chemotherapeutic and immunomodulating agents against the Lederle Laboratory Division, American Cyanamide Co., these tumors are presented in this publication. Pearl River, N. Y. Of the immunomodulating agents, Bacillus Calmette-GuÃ©rin MATERIALS AND METHODS vaccine was purchased from the BCG Laboratory, Medical Center, University of Illinois, Chicago, III.; concanavalin A was Tumors. EL-4 lymphoma was grown in male C57BL/6J mice from Sigma Chemical Co., St. Louis, Mo.; and phytohemagglu- from The Jackson Laboratory, Bar Harbor, Maine; J774 mac- tinin was from Difco Laboratories, Detroit, Mich. Corynebacte- rium parvum vaccine was provided by the Burroughs Wellcome 1 Supported in part by National Cancer Institute Grants CA-08748, CA-18856- Co., Research Triangle Park, N. C.; the mycobacterial inter- 02, and CA-24300 phase material was from Professor E. Lederer, Institut de 2 To whom requests for reprints should be addressed, at the Donald S. Walker Laboratory, Sloan-Kettering Institute for Cancer Research, 145 Boston Post Chimie des Substances Naturelles, Gif sur Yvette, France; Road. Rye. N. Y. 10580 lentinan was from Ajinomoto Co., Kawasaki, Japan; levamisole 1R. Faanes. V. J. Merluzzi. N. Williams, G. S. Tarnowski. and P. Ralph. was from Ortho Research Foundation, Raritan, N. J.; tilorone Matching of chemotherapy to mouse strain and lymphoid tumor type to prevent tumor-induced suppression of specific T- and B-cell functions. Cancer Res. (in hydrochloride from William S. Merrell Co. Research Laborato- press). * 70Z/2 was induced with methylnitrosourea in a thymectomized C57BL/6J x DBA/2 F, mouse by Dr. P. Baines (1). Its cytoplasmic and surface immuno- 5 The abbreviations used are: MST, median survival time; ara-C, 1-/y-o-arabi- globulins were the same as those described for line 70Z/3 by Paige et al. (3). nofuranosylcytosine; DON, 6-diazo-5-oxo-L-norleucine; T/C, treated/control; Received July 2. 1979; accepted July 9, 1979. id. intradermal.

3964 CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1979 American Association for Cancer Research. Sensitivity to Chemotherapy of Mouse Lymphoid Tumors ries, Cincinnati, Ohio, and sterile zymosan was from Farben Therapy. For the tests of chemotherapeutic agents, 1 x 106 fabriken Bayer A.-G., Wuppertal-Elberfeld, West Germany. Po- viable (trypan blue-excluding) tumor cells were implanted i.p. lyinosinic-polycytidylic acid was prepared by Dr. Leonard D. on Day 0; immunomodulating agents were tested against a Hamilton, Brookhaven National Laboratory, Upton, N. Y. 6- load of 1 x 10s viable tumor cells implanted i.p. on Day 0. The Mercaptopurine was suspended in a 0.5% solution of carbox- route and schedule of administration of test materials differed ymethylceliulose in the 0.85% NaCI solution, and other chem according to the nature of materials; they are listed in Tables icals were dissolved in pyrogen-free 0.85% NaCI solution. 2 and 3. The effect of therapy was evaluated by the prolonga Solutions of azaserine, 1,3-bis(2-chloroethyl)-1-nitrosourea, tion of MST of tumor-bearing hosts which were observed for DON, and nitrogen mustard were prepared fresh daily. 56 days. The ratios of the MST of treated mice to that of the

Table 2 Effect of treatment of mouse lymphomas and of a macrophage tumor with chemotherapeutic agents T/C (% of increase of MST after therapy with a multiple of basic dose) dose img/ AgentActinomycin schedule0.85% route, and dose kg/day)0.0010.0010.0010.10.10.10.30.30.30.10.10.11010100.010.010.013331010101111110.10.10.10.010.010.010.010.010.011X-8-1822933-17019017663"187110-240-1503601950*81527-813338351807271719025"1136025183x0-1133933-1781318172563b27"13330-29-1781855"11650b38"3073b-15-1256b03073"040b64"25"1973"50"1182b0132710X0-1189b27bS30025"132742b19100b45"40b56b0-24031X25b9144b55*>273b42b42"2536-81988"-18-40118-14023b112b182b172575"-46-55-18-8125Â°172b-2525075"63"82"1519127b DAdriamycinara-CAzaserineBCNUCBleomycinCyclophosphamideDON5-FluorouracilHydrocortisoneDay1AsNaCI solution, 6x, Â¡.p.,everyday from

aboveAs

dailyAsabove, fresh

above0.85%

1Saline.NaCI solution, 1 x, i.p., on Day "59b118b171963"62"3582b-15-1256"-865"218646"-333658b50"127b50"3282"81973b30

dailySaline,6x . i.p., every day from Day 1, fresh

1Saline, 6x, Â¡.p.,every day from Day

acetate6-MercaptopurineMethotrexateMitomycin1CMC0.6x, s.c., every day from Day

1Saline,6x, Â¡.p.,every day from Day

1As 6x, Â¡.p.,every day from Day

CNitrogen aboveAs

mustardVinblastineDiluent, dailyAsabove, fresh

aboveTumorEaJ7EJ7EJ7Ã•J7EJ7EJ7EJ7EJ7EJ7EJ7EJ7EJ7EJ7EJ7EJ7Basic

E. EL4; J, J774; 7, 70Z/2; 1 x 106 cells injected i.p. on Day 0. 6 Significant prolongation of MST, p < 0.05. c BCNU, 1,3-bis(2-chloroethylM-nitrosourea; CMC, 0.5% carboxymethylcellulose in 0.85% NaCI solution.

OCTOBER 1979 3965

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1979 American Association for Cancer Research. G. S. Tarnowski et al. corresponding test controls, the T/C values, for the graded bearing mice past the observation period of 56 days was doses of each test material were compared with the T/C observed only in some mice bearing J774 treated with Adria acceptance values for MST of the control mice. These values mycin. were based on the S.D. of the pooled averaged MST for groups Among the 3 alkylating agents tested, cyclophosphamide of 65 to 100 diluent-treated control mice, 5 to 6 mice per group was the most effective one, especially against 70Z/2 and (4). The higher 99% confidence limit of the distribution of the J774. Of the antimetabolities, 6-mercaptopurine was quite MST values, divided by the pooled grand average itself, yielded effective against J774 and 702/2, but not against EL-4. On the T/C acceptance values at the 99.5% level of confidence the other hand, EL-4 was susceptible to therapy with ara-C or for the inocula of 1 x 105and1 x 106 tumor cells, respectively, 5-fluorouracil, but J774 was not. In preliminary tests, ara-C for each tumor (Table 1). The T/C values greater than the retarded growth of the solid form of EL-4; J774 does not grow acceptance values were then attributed to the effect of a given as a solid tumor following i.d. or s.c. inoculation. 702/2 re dose of test material. sponded to therapy with 5-fluorouracil but was not very sensi tive to ara-C. Methotrexate prolonged MST of mice bearing RESULTS AND DISCUSSION each of the 3 tumors by 40 to 60%. The glutamine analogs, azaserine, and DON, have prolonged the survival time of 70Z/ Of the 3 test tumors, 702/2 responded with a prolongation 2-bearing mice, but not of those with J774; only azaserine was of MST by more than 100% to therapy with optimal doses of 5 effective against EL-4. chemotherapeutic agents (actinomycin D, cyclophosphamide, Of the 4 antitumor antibiotics tested, actinomycin D pro 6-mercaptopurine, mitomycin C, and vinblastine), J774 to ther longed MST of 702/2, Adriamycin and mitomycin C showed apy with 3 agents (Adriamycin, cyclophosphamide, and 6- activity, and bleomycin was inactive against all 3 tumors. mercaptopurine), and EL-4 to none (Table 2). Survival of tumor- Vinblastine and hydrocortisone acetate prolonged MST only

Table 3 Effect of treatment of mouse lymphomas and of a macrophage tumor with immunomodu/ating agents On Day 0,1 x 105 cells of EL4 (E). J774 (J). or 702/2 (7) were injected i.p. into groups of mice per dose level; diluent, pyrogen-free 0.85% NaCI solution in water.

T/C (% of increase of MST after therapy with a multiple of basic dose) scheduleBacillus Test agent Route and dose doseCells/mouse1 x100x-46-1430a100X0-808-120100X15-530a100X-14170 â€”14ConcanavalinCalmette-GuÃ©rin Â¡.p..1 x. Day x10'mg/kg/day1mg/kg10.5ng

1CorynebactenumA Â¡.p..6x, every day from Day

0Interphase parvum i.p.. 1 x , Day

-14smegmatisLentinanmaterial from Mycobacterium i.p., Ix.Day /mouse100mg/kg/day0.1mg/kg/day1mg/kg/day1mg/kg/day1mg/kg/day10mg/kg/day1IX040"60'019-10733a50"740"60a0001760800767a801300003x02780"033a70a040a60a0130744a33a850a1010X81410028150a0-80860151410-5061'50a17311000030

1Levamisole i.p . 6x. every day from Day

11Phytohemagglutinin Â¡.p.,4x, Days 1. 4, 7, and

1Polyinosinic-polycytidylic i.p., 6x, every day from Day

1Tilorone acid i.p.. 6x, every day from Day

1Zymosan i.p.. 4 x, Days 1. 4, 7. and 1

i.p., 6x. every day from Day 1TumorEJ7EJ7EJ7EJ7EJ7EJ7EJ7EJ7EJ7EJ7Basic

a Significant prolongation of MST, p < 0.05.

3966 CANCER RESEARCH VOL. 39

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1979 American Association for Cancer Research. Sensitivity to Chemotherapy of Mouse Lymphoid Tumors of mice bearing 70Z/2. In culture, J774 (5) and EL-4 (4) are ent hematopoietic progenitor cells of tumor-free mice to several resistant to corticosteroids, whereas 70Z/2 is killed by 1CT6 chemotherapeutic and immunomodulating agents (8).3 M hydrocortisone.6 Among the immunomodulating agents (Table 3), antitumor ACKNOWLEDGMENTS activity was observed only against J774 and 70Z/2; EL-4 was The authors thank Dr. C. J. Paige for the 70Z/2 tumor and advice. insensitive to all 10 tested agents. Moderate prolongation of MST in sensitive tumors was elicited by mycobacterial prepa REFERENCES rations, ÃŸ.Calmette-GuÃ©rin and interphase material, C. par- 1. Baines, P., Dexter. T. M.. and Schofield. R. Characterization of malignant vum, and polyinosinic-polycytidylic acid. Preventive treatment cell populations in MNU-induced leukemia of mice. Leukemia Res., 3. 23- with mycobacterial preparations was administered 14 days 28. 1979. 2. Gorer, P. Studies in Antibody Response of Mice to Tumor Inoculation. Br. J. prior to tumor inoculation, and C. parvum was injected on the Cancer, 4. 372-379, 1950. day of inoculation following the schedule previously used for 3. Paige, C. J., Kincade, P. W., and Ralph, P. Murine B-cell leukemia line with treatment of mouse sarcomas (8). inducible surface immunoglobulin expression. J. Immunol.. 121: 641-647, 1978. The overall results of this survey have revealed: (a) the 4. Ralph, P. Retention of lymphocyte characteristics by myelomas and 9*- greater sensitivity of the macrophage tumor J774 and B-cell lymphomas: sensitivity to Cortisol and phytohemagglutinin. J. Immunol., / 70. tumor 702/2 compared with that of the T-cell lymphoma EL-4 1470-1475, 1973. 5. Ralph, P., Ito, M., Broxmeyer, H. E., and Nakoinz. I. Corticosteroids block to several chemicals; and (b) in the case of actinomycin D, newly induced but not constitutive functions of macrophage cell lines: hydrocortisone, and vinblastine, the unique responsiveness of myeloid colony-stimulating activity production, latex phagocytosis, and an 70Z/2. Further comparative studies are needed to establish tibody-dependent lysis of RBC and tumor targets. J. Immunol., 121: 300- 303, 1978. whether differences represent properties of the 3 individual 6. Ralph. P., Prichard. J., and Cohn, M. Reticulum cell sarcoma: an effector tumors tested or would be shared by other tumors of B- or T- cell in antibody-dependent cell-mediated immunity. J. Immunol.. /14: 898- 905, 1975. lymphocytes or macrophages, respectively. For this purpose, 7. Tarnowski. G. S. Approaches to the immunotherapy of experimental tumors. it would be preferable to use tumors which have originated in In: D. Mizuno, G. Chihara, F. Fukuoka, T. Yamamoto, and Y. Yamamura the same inbred strain of mice, since we have shown mouse (eds.). Host Defense against Cancer and Its Potentiation, pp. 389-396. strain differences in sensitivity of immune response and differ- Tokyo: University of Tokyo Press. 1975. 8. Tarnowski, G. S., Faanes, R. B., Ralph, P., and Williams, N. Suppression and restoration of Cytotoxic T-cell activity during chemotherapy of a mouse T-cell lymphoma and a macrophage tumor. Cancer Res.. 38. 4540-4545. 6 P. Ralph, unpublished observation. 1978.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1979 American Association for Cancer Research. Sensitivity to Chemotherapeutic and Immunomodulating Agents of Two Mouse Lymphomas and of a Macrophage Tumor

George S. Tarnowski, Peter Ralph and C. Chester Stock

Cancer Res 1979;39:3964-3967.

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