[CANCER RESEARCH 46, 5646-5648, November 1986] Transplantability and Sensitivity to Natural Killer Cells of Aclarubicin-resistant Murine Lymphoma1

Yoshikazu Sugimoto,2 Yoko Hirakawa, Nobuyuki Tanaka, Makoto Tañara,Isao Sato, Toshio Nishimura, Hideo Suzuki, and Nobuo Tanaka3

Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT Cells. The parental and drug-resistant cell sublines of murine lym phoblastoma L5178Y were maintained in Fischer's medium supple DBA/2 mice implanted i.p. with an aclarubicin (A( 'R(-resistant subline mented with 10% horse serum, and YAC-1 cells were in RPMI 1640 of L5178Y cells survived 4- to S-fold longer than those with the parental medium with 10% fetal calf serum. cells; and animals with the Adriamycin- or bleomycin-resistant subline Animals. Female DBA/2 mice were propagated at Charles River displayed an intermediate survival period. The i.p. treatment of mice with Japan, Atsugi, Kanagawa-ken, Japan. cyclophosphamide markedly enhanced i.p. growth of the ACR-resistant Transplantation of I.5I7SY Cells to DBA/2 Mice. Female DBA/2 cells, suggesting that a certain host defense mechanism participates in mice (6 wk old) were implanted i.p. with 5 x 10* or 5 x IO6 L5178Y the lower transplantability. In vitro, the ACR-resistant subline showed cells. Mice were given injections i.p. of cyclophosphamide (100 mg/kg) much higher sensitivity to natural killer cells. The i.p. pretreatment with 2 days, of anti-asialo-GMl antibody (50 ^I/mouse) 4 days, or of anti-asialo-GMl antibody markedly reduced the mean survival period of carrageenan (50 mg/kg) 2 days before tumor transplantation. mice implanted i.p. with the ACR-resistant cells, suggesting that natural Assay of NK Cell Activity in Vitro. Spleen cells of female DBA/2 killer cells play an important role in the defense against transplantation mice (6 wk old) were treated with 0.83% M I4( I and passed through a of the ACR-resistant cells. Sephadex G-10 column and used as NK cells. Each subline of L5178Y cells or YAC-1 (1.5 x IO6in 0.2 ml of medium) was mixed with 0.2 ml of ["Crjsodium chromate (1 mCi/ml), incubated at 37"C for 1 h, and INTRODUCTION washed 4 times with the same medium. The cells (2 x IO4)in 100 n\ of We have isolated cell sublines of murine lymphoblastoma RPMI 1640 medium with 10% fetal calf serum were distributed into L5178Y for resistance to ADM4 (1), ACR (2), BLM (3), or each well of U-bottomed 96-well plates (Nunc). Spleen cells, prepared as described above, were aliquoted to the microplate and incubated at MCR (4) and observed that the resistance is due to a change of 37°Cfor 20 h. Released SICr in the culture fluid was counted by a plasma membrane and its transport system (5, 6). The ADM-, gamma counter (16). Results were expressed as the percentage of lysis ACR-, and MCR-resistant sublines show pleiotropic resistance, of the target cells according to the following formula but the BLM-resistant subline has selective resistance (1-4). We have also found that 5 '-nuclei>t ¡dephosphodiesterase activ % of lysis ity of plasma membrane is higher in the four drug-resistant experimental 51Cr release - spontaneous "Cr release sublines than the parental cells, although the relationship of maximal "Cr release - spontaneous "Cr release x 100 the enzyme activity to the drug resistance remains to be deter mined (7, 8). For the purpose of elucidating membrane altera tion of drug-resistant neoplastic cells, we have prepared synge- Assay of Cytotoxicity of Macrophage in Vitro. Female DBA/2 mice (10 wk old) were immunized with viable M. bovis strain BCG intrader- neic monoclonal antibody specific for the ACR-resistant subline mally (IO7 plaque forming units/mouse). Peritoneal exúdatecells were of LSI 78Y cells (9). collected 10 days after immunization, washed with phosphate-buffered Several investigators have reported that drug-resistant tumor saline, and suspended in RPMI 1640 medium containing 10% fetal calf cells are often immunogenic and show lower transplantability serum and lipopolysaccharide (10 /tg/ml). Nonadherent peritoneal cells than the parental cells (10-15). We have also found that the were removed by repeated washing, and adherent monolayer cells were ACR-resistant subline of LSI 78Y cells shows lower transplant- incubated in the same medium for 2 days and used as macrophages (17). "Cr-labeled L5178Y cells, prepared as described above, were ability to DBA/2 mice, the syngeneic host, than the parental added to the macrophage monolayer and incubated at 37*C for 20 h. cells, and studied the mechanism of lower transplantability. The Released 51Cr was counted by a gamma counter, and results were results are presented in this paper. expressed as the percentage of lysis as described above. Statistical Test. Student's t test was used for statistical analysis. MATERIALS AND METHODS Rabbit anti-asialo-GMl antibody was purchased from Wako Pure RESULTS Chemical Industries, Osaka, Japan. Viable Mycobacterium bovis strain Transplantability of L5178Y Cell Sublines to Syngeneic Host. BCG was obtained from Nippon BCG Manufacture Co., Tokyo, Japan, Female DBA/2 mice were implanted i.p. with the parental, or and lipopolysaccharide of Escherichia coli 055:B5 was from Difco Lab., ACR-, ADM-, or BLM-resistant subline of LSI78Y cells. As- Detroit, MI. ["CrJSodium enrómate (590.67 mCi/mg) was a product citic tumor appeared in all the animals. The mean survival of New England Nuclear, Boston, MA. times of tumor-bearing mice and survivors on Day 60 are Received 12/10/85; revised 5/27/86; accepted 7/29/86. presented in Table 1. Mice implanted with the ACR-resistant The costs of publication of this article were defrayed in pan by the payment cells survived 4- to 5-fold longer than those with the parental of page charges. This article must therefore be hereby marked advertisement in cells, and animals with ADM- or BLM-resistant cells showed accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was partly supported by a grant-in-aid for cancer research from intermediate survival periods. The longer survival time of mice, the Ministry of Education, Science, and Culture, Japan. 1 Present address: Cancer Chemotherapy Center, Japanese Foundation for bearing ACR-resistant cells, was repeatedly confirmed by fur Cancer Research, Toshima-ku. Tokyo 170, Japan. ther experiments (data not shown). 3To whom requests for reprints should be addressed. The doubling time of LSI78Y cell sublines cultured in vitro 4The abbreviations used are: ADM, Adriamycin; ACR, aclarubicin; BLM, in Fischer's medium with 10% horse serum is shown in Table bleomycin; MCR, macromomycin; NK, natural killen BCG, Bacillus Calmette- Guerin. 2. The ACR-resistant cells grew more slowly in vitro than the 5646

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Table 1 Survivat time of DBA/2 mice, implanted i.p. with the parental, or ACR-, Table 6 Effect of anti-asialo-GM I antibody or Carrageenan on transplantability ADM-, or BLM-resistant subline of LSI 7SY cells of the parental or ACR-resistant cell line of LSI 78Y lymphoma The mean survival times of all three resistant sublines significantly differ from Each group consisted of ten mice. that of the parental cell line (P < 0.001). Each group consisted of ten mice, and each mouse was implanted i.p. with 5 x 10*cells. Pretreatment line time withAnti-asialo-GMl transplantedParental (days)13.2 Survival time ±0.8« Cell line (days) Parental 12.6 ±1.0 Parental 12.9 ±0.9° ACR resistant 65.2 ±10.6 ACR resistant 66.9 ±9.9 Anti-asialo-GM1Carrageenan ACRresistantParental 29.0 ±4.9(13.6 ADM resistant 35.9 ±3.1 BLM resistant 24.3 ±1.2 ±0.6 1Mean ±SE. Parental 12.6±0.6ÕÕÎS ACR resistant CarrageenanCell ACR resistantSurvival <'<»•<»> Table 2 Growth rate of the parental and drug-resistant sublines of LSI 78 Y cells •Mean±SE. in culture Cell line Doubling time (h) Table 7 Effect of anti-asialo-GM 1 antibody on tumoricidal activity of spleen cells Parental 9.2 ±0.2° in mice ACR resistant 15.1 ±0.3 ratioTarget ADM resistant 10.8 ±0.1 Treatment with BLM resistant 16.4 ±0.1 anti-asialo-GM1+ cellParental •Mean±SE. ±1.2** ±2.6 ±3.1 Parental 0.2 ±0.8 1.1 ±2.2 0.3 ±1.041.2 ACR resistant 11.5 ±2.9 21.8 + 2.7 + 4.3' Table 3 Effect of cyclophosphamide on i.p. transplantation of the parental or +EffectontargetACR resistant25:10.80.8 ±1.250:10.91.6 ±2.4100:12.43.1 ±2.3 ACR-resistant LSI 78Y cells * Percentage of lysis. Cyclophosphamide of 100 mg/kg was injected i.p. 2 days before tumor trans * Mean ±SE. plantation. Each group consisted often mice. c P < 0.001 compared with the control. Cyclo Mean survival Cell line phosphamide time (days) Parental 13.8 ±0.8" Effect of Cyclophosphamide on Transplantability of L5178Y (P < 0.05) 12.2 ±0.8 Cells. Female DBA/2 mice were given injections i.p. of 100 mg of cyclophosphamide per kg 2 days before transplantation of ACR resistant 60.0 ±7.7 (P< 0.001) the ACR-resistant cells. The drug treatment markedly reduced 26.6 ±4.0 1Mean ±SE. the mean survival period of the tumor-bearing mice, presumably by disrupting the host defense system (Table 3). The results suggest that a certain host defense mechanism participates in Table 4 Sensitivity of various sublines of LSI 78Y cells to NK cells in vitro the lower transplantability of ACR-resistant cells. Effector (NK):target ratio Sensitivity to NK Cells or Cytotoxic Macrophages in Vitro. Target cell 25:1 50:1 100:1 The NK sensitivity of LSI78Y cell sublines is presented in Experiment1Parental Table 4. The ACR-resistant subline displayed high sensitivity L5I78YACR ±1.2**11.5 ±2.621.8 ±3.14 to NK cells, but the parental, ADM-resistant, and BLM-resist resistantADM ±2.93.0 ±2.74.2 1.2±4.3'7.9 resistantBLM ±2.51.3 ±0.63.0 ±3.34.8 ant sublines were rather resistant to NK cells. The degree of resistantExperiment ±1.81.9 ±3.92.2 ±1.23.4 NK sensitivity of the ACR-resistant cells was similar to that of YAC-1 cells. The latter was used as a positive control. All the 2Parental L5178YACR ±1.06.3 ±2.819.8 ±2.430.9 four sublines showed a similar level of sensitivity to cytotoxic resistantYAC-10.8 ±1.912.5 ±4.821.4 ±5.2'25.1 macrophages, although the ACR-resistant cell subline exhibited ±3.6' ±3.60.9 ±2.72.4 a little higher sensitivity than the other cell lines (Table 5). * Percentage of lysis. * Mean ±SE. Effect of Anti-Asialo-GM 1 Antibody or Carrageenan on Trans ' /' - 0.001 compared with the parental L5178Y cells. plantability of LS178Y Cells. Murine NK cells are damaged by anti-asialo-GM 1 antibody more markedly than cytotoxic mac rophages (18, 19). As summarized in Table 6, the pretreatment Table 5 Sensitivity of various sublines of LS178Y cells to cytotoxic macrophages with anti-asialo-GM 1 antibody markedly enhanced growth of in vitro the ACR-resistant cells and reduced the mean survival period ratioTarget Effectontarget of the tumor-bearing mice, but it did not significantly affect cellParental that of the parental cells. On the other hand, the pretreatment ±0.3* * ±0.4 with Carrageenan, which affects macrophages more profoundly ACR resistant 6.8 ±0.5 11.5 ±0.4' ADM resistant 2.9 ±1.9 7.5 ±1.3 than NK cells, slightly reduced the mean survival time of BLM resistant5:12.8 2.9 ±1.610:14.8 4.6 ±1.3 animals bearing the ACR-resistant cells, but it did not signifi °Percentage of lysis. * Mean ±SE. cantly affect that of mice bearing the parental cells. The NK ' P < 0.01 compared with the parental cells. activity of spleen cells was markedly reduced by the treatment of mice with anti-asialo-GM 1 antibody (Table 7). The results suggest that NK cells play a more important role in the defense parental cells. The results suggest that the intrinsic growth mechanism against transplantation of the ACR-resistant cells potential is one of the mechanisms of slow growth //; vivo. than cytotoxic macrophages. Although the ACR-resistant cells grew a little more rapidly than the BLM-resistant cells in vitro, the former grew more DISCUSSION slowly than the latter in vivo. The results suggest that lower transplantability of the ACR-resistant cells is not only due to The current studies reveal that the in vivo growth of the ACR- lower intrinsic growth ability, but also to the host factors. resistant subline of LS178Y cells is suppressed by a host defense 5647

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mycin resistance in a subline of mouse lymphoblastoma LS178Y cells. J. mechanism and that the cell subline is highly sensitive to NK Antibiot. (Tokyo), 32: 518-522, 1979. cells. 6. Sugimoto, Y., Nishimura, T., Suzuki, H., and Tanaka, N. Evidence of altered NK cells participate in an anticancer immune defense mech influx of Adriamycin into anthracycline-resistant cells. J. Antibiot. (Tokyo), 34:1064-1066, 1981. anism (20). It is of interest how NK cells recognize and kill 7. Sugimoto, Y., Nishimura, T., Suzuki, H., and Tanaka, N. Alteration of tumor cells. The parental cell line of LSI78Y is resistant to, membrane-associated enzymes in drug-resistant sublines of mouse lympho blastoma L5178Y cells. J. Antibiot. (Tokyo), 34: 1200-1205, 1981. but the ACR-resistant subline is sensitive to, NK cells. There 8. Fukazawa, H., Suzuki, H., and Tanaka, N. A novel tumor-associated molec fore, comparative studies of both cell lines may be useful for ular species of 5'-nucleotide phosphodiesterase. Biochem. Biophys. Res. elucidating the mechanism of NK sensitivity. Commun., 130:1072-1077, 1985. 9. Sugimoto, Y,. Suzuki, H., and Tanaka, N. Alteration of plasma membrane Several investigators reported that drug-resistant tumor cells of drug-resistant tumor cells: 230-kilodalton protein identified by monoclonal are highly immunogenic and are not easily transplanted to the antibody. Biochem. Biophys. Res. Commun., 7/4:969-975, 1983. syngeneic host (10-15). The multiple drug resistance of tumor 10. Fuji!, H., and Miliirti, E. Selection of high immunogenicity in drug-resistant sublines of murine lymphomas demonstrated by plaque assay. Cancer Res., cells is due to membrane alteration (2, 6, 21). Since tumor- 35:946-952, 1975. 11. Fujii, H., Mihich, I .. and Pressman, D. Differential tumor immunogenicity associated transplantation antigens are related to plasma mem of LI 210 and its sublines. I. Effect of an increased antigen density on tumor brane, the antigens may become manifest simultaneously with cell surfaces on primary B cell responses in vitro. J. Immunol., 119: 983- membrane change of drug resistance. Therefore, it remains to 12. Strzadala,986, 1977. I... Opplski, A., Radzikowski, ('.. and Mihich, E. Differential be determined whether the lower transplantability of the drug- expression of murine leukemia antigen on LI 210 parental and drug-resistant resistant sublines of L5178Y is partly due to immunogenicity. sublines. Cancer Res., 41:4934-4937, 1981. 13. Rapp, L., and Fujii, H. Differential antigenic expression of the DBA/2 lymphoma LI210 and its sublines: cross-reactivity with C3H mammary tumors as defined by some syngeneic monoclonal antibodies. Cancer Res., ACKNOWLEDGMENTS 43: 2592-2599, 1983. 14. Ilium. D. B. S., /¡ola.B., and Ramshaw, I. 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Drug sensitivity of an Adriamycin- 2042, 1978. resistant mutant subline of mouse lymphoblastoma LSI 78Y cells. J. Antibiot. 18. Habu, S., Fukui, H.. Shimamura, K., Kasai, M., Nagai, Y., Okumura, K., (Tokyo), 31:493-495, 1978. and Tamaoki, N. in vivo effects of anti-asialo GM1. I. Reduction of NK 2. Nishimura, T., Suzuki, II. Muto, K., Tanaka, Y., and Tanaka, N. Studies activity and enhancement of transplanted tumor growth in nude mice. J. on aclacinomycin A resistance in mouse lymphoblastoma. J. Antibiot. (To Immunol., 127: 34-38, 1981. kyo), 33:737-743,1980. 19. Akigawa, K. S., and Tokunaga, T. Appearance of a cell surface antigen 3. Suzuki, II.. Nishimura, T., and Tanaka, N. Drug sensitivity and some associated with the activation of peritoneal macrophages in mice. Microbiol. characteristics or a bleomycin-resistant subline of mouse lymphoblastoma Immunol., 26:831-842,1982. LSI78Y cells. J. Antibiot. (Tokyo), 34:1210-1212, 1981. 20. Haller, O., Hansson, M., KJessling, K , and Wigzell, H. Role of non- 4. Suzuki, H., Sugimoto, Y., Tanaka, K., Nishimura, T., and Tanaka, N. conventional natural killer cells in resistance against syngeneic tumour cells Isolation, drug sensitivity, and some biochemical and genetica! properties of in vivo. Nature (Lond.), 270:609-611. 1977. macromomycin-resistant mouse lymphoblastoma L5178Y cells. J. Antibiot. 21. Inaba, M., and Johnson, R. K. Decreased retention of actinomycin D as the (Tokyo), 37: 602-609, 1984. basis for cross-resistance in anthracycline-resistant sublines of P388 leuke 5. Nishimura, T., Suzuki, H., Muto, K., and Tanaka, N. Mechanism of Adria- mia. Cancer Res., 37:4629-4634, 1977.

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Yoshikazu Sugimoto, Yoko Hirakawa, Nobuyuki Tanaka, et al.

Cancer Res 1986;46:5646-5648.

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