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Effects of human interferons on human choriocarcinoma cells in vitro and in vivo
GYNECOLOGIC
ONCOLOGY
25, 115-124 (1986)
Effects of Human lnterferons on Human Choriocarcinoma Cells in Vitro and in Vivo S. SEKIYA,* *Department
T. KUWATA,? H. ISHIGE,” Y. TOMITA,~ AND H. TAKAMIZAWA*
of Obstetrics and Gynecology and )‘Department of Microbiology, School of Medicine, 1-8-l Inohana, Chiba 280, Japan
Chiba University
Received July 11, 1985 Three kinds of human choriocarcinoma cell lines (BeWo, HCCM-5, and NUC-1) were used for examining the antiviral and antiproliferative activities of human interferons (IFNs) in vitro and in vivo. All of the cell lines showed only low sensitivity to the antiviral action of every IFN-a, IFN-/3, and IFN-y against vesicular stomatitis virus infection. However, 2-5A synthetase was normally induced by IFN-a in all of the cell lines. The [‘Hlthymidine incorporation of both BeWo and HCCM-5 cells was suppressed in dose-dependent manner at 48 hr after treatment with 1 to 1000 units (U)/ml of IFN-(U or IFN-/3 and the growth of them was also slightly inhibited when treated continuously with 1000 U/ml for 6 days in vitro. Another cell line NUC-1 was the least sensitive to these IFNs among the three cell lines. IFN-7 did not show any antiproliferative effect on these cell lines. The intraperitoneal administration of 5000 or 10,000 U of IFN-/3 suppressed the growth of xenografts developed in hamster cheek pouches and subcutis of nude mice when its administration was initiated on the first day of cell inoculation. These results indicate that although some heterogeneities exist among the cell lines choriocarcinoma cells are weakly sensitive to the antiproliferative activity of human IFNs. 0 1986 Academic Press. Inc.
INTRODUCTION
Recently mass production of three types of purified human interferons (IFN-(Y, IFN-P, and IFN-y) has been established and their therapeutic application to various kinds of human malignant tumors is now being carried out [4]. However, clinical studies on human IFNs as antitumor agents are few and most of them are not completed [6,24]. Therefore, it is necessary to explore human malignant tumors against which human IFNs are effective by using many histologically different types of human malignant tumor cells. In this report, we describe the effects of three types of human IFNs on three different human choriocarcinoma cell lines in vitro and in vivo. MATERIALS
AND METHODS
Cell lines. We used the following five human cell lines: choriocarcinoma cell lines BeWo [23], HCCM-5 [22], and NUC-I [28], virus-transformed embryo fibroblast cell line RSa [ 161, and uterine adenocarcinoma cell line HEC-1 [ 151. The biologic characteristics of choriocarcinoma cell lines were described previously 115 0090-8258/86 $1.50 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
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ET AL.
[25]. Monolayer cultures were maintained in RPM1 1640 medium supplemented with 10% newborn bovine serum (Flow Laboratories, North Ryde, New South Wales, Australia) and antibiotics in a humidified 5% CO, and 95% air atmosphere at 37°C. ZFNS. Human leukocyte IFN (IFN-a) with a specific activity of 6 x lo6 U/mg protein was kindly provided by Dr. K. Cantell, Central Public Health Laboratory, Helsinki, Finland. Human fibroblast IFN (IFN-P) with a specific activity of 1 x 10’ U/mg protein was provided by Mochida Pharmaceutical Company, Ltd. (Tokyo, Japan). Human recombinant immune IFN (IFN-7) with a specific activity of 1 x 10’ U/mg protein was supplied by Torey Industries (Kamakura, Japan). The stock solutions of IFNs (10’ U/ml in Hank’s balanced salt solution) were kept at -70°C until use. Assay of antiviral activity. Antiviral activity was measured in a yield reduction assay with vasicular stomatitis virus (VSV). Cells cultivated in 16 mm Limbro multiplates were treated with 10 to 1000 U/ml of IFNs for 20 hr. Cells were washed and then infected with VSV at multiplicity of 1.0. After 1 hr of virus adsorption at 37”C, cells were washed and culture medium containing 5% newborn bovine serum was added to each culture. After 18 hr of incubation, sample medium was centrifuged and stored at - 70°C until virus titration. VSV in samples was assayed by 50% tissue culture infective dose (TCID& in microtrays, as described elsewhere [ 171. Assay of2-5A sythetase. Cells (5 to 8 x 10”) treated with 10 to 1000 U/ml IFN-(U at 37°C for 16 hr were suspended in 150 ~1 of 10 mM KCI, 1.5 mM Mg (OAc),, 20% (v/v) glycerol, and 20 mM Hepes-KOH buffer, pH 7.4. The cells were sonicated for 10 sec. The samples were centrifuged at 10,OOOgfor 20 min and 20 ~1 of the supernatant was used for enzyme assay. The reaction mixture contained 20 ~1 of the supernatant, 1 pg poly(1) * (C), 3 &I [3H]ATP ([2,8,5lHIATP, 44.5 Ci/mmole; New England Nuclear Corp., Boston, Mass.), 5 mM ATP, 20 mM Hepes/KOH, pH 7.4, 25 n&f Mg(OAc)*, 80 mM KCl, and 7 mM 2-mercaptoethanol in a final incubation volume of 50 ~1. The 2-5A synthesized from ATP was prepared as previously described [29]. The results were expressed as cpm recovered in 2-5A for 20 ~1 supernatant of cell extract at 1.0 OD,,,. [3HJThymidine incorporation in vitro. Cells (10’) in 1 ml of culture medium were seeded into a series of sterile glass scintillation vials. After 48 hr of incubation, the exponentially growing cells were exposed to IFNs at doses from 1 to 1000 U/ml for 48 hr. After collection of culture medium for radioimmunoassay (RIA) of human chorionic gonadotropin (hCG), 6-[3H]thymidine (15 Ci/mmole; New England Nuclear Corp.) was added to each vial in 1 ml of culture medium at 1 Z-Xi. After 30 min of incubation, each vial was quenched by the addition of 10 ml of ice-cold isotonic saline and the incorporation was counted with a scintillation counter, RackBeta 1215 (Wallac Oy, Turku, Finland), following the method of Ball et al. [31. Cell growth in vitro. Cells (1.5 x 105)were seeded into 60-mm plastic culture dishes in 5 ml of culture medium. After 48 hr, the culture medium was changed for that with or without IO00 U/ml of IFNs and then the culture medium was changed every other day for 6 days. The cells were detached from dishes with
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0.25% trypsin solution and viable cells were counted by the trypan blue dye exclusion test using a hemocytometer. Assay of hCG. Assay of hCG in culture medium was done by using CEAIRE-SORIN hCG RIA kits (Green Cross Co., Tokyo, Japan). Znoculation into hamster cheek pouch. BeWo or HCCM-5 cells (2 x 106) suspended in 0.2 ml of culture medium were inoculated into the cheek pouches of 4-week-old female golden hamsters (Takasugi Experimental Animals, Saitama, Japan). They were conditioned with 10 mg/week of cortisone acetate (Corton, Nippon Merck-Banyu Co., Osaka, Japan) during a 14-day observation period. IFN-p at doses of 10,000 U was administered into the intraperitoneal cavity at either of the following administration schedules: (1) on every day from the first till seventh day after the cell inoculation and (2) on every day from the eighth day till the fourteenth day after the cell inoculation. Inoculation into subcutis of nude mouse. A human choriocarcinoma strain, CC-I-JCK[13], maintained by the serial subcutaneous inoculations into 6- to 8week-old female BALB/c nu/nu nude mice (Clea Japan, Tokyo, Japan) was used. IFN-/3 at a dose of 5000 U was administered by intraperitoneal route either on every 3 days from the first after the inoculation of l- to 2-mm pieces of the tumor tissues for 42 days or on every 3 days from the 28th day after the inoculation until the death of the animals. RESULTS
Antiviral Activity of ZFNs in Choriocarcinoma Cells As shown in Table 1, all of the human choriocarcinoma cell lines showed only low sensitivity to the antiviral activity of IFNs against VSV infection, as compared to RSa cells which were shown to be sensitive to the antiviral activity [16,30]. Dose-dependent inhibition of viral replication was not evident by treatment with up to 1000 U/ml and difference in the antiviral activity was not observed among the three types of IFNs (Table 1). However, 2-5A synthetase activity was normally TABLE
I
ANTIVIRAL ACTION OF HUMAN INTERFERONSIN VSV-INFECTED CHORIOCARCINOMACELL LINES Cell line Interferon IFN-a
IFN-P
IFN-y
c Yield reduction b Not tested.
IU/ml
BeWo
10 100 1000 10 100 1000
0 0.2 0.5 0.2 0.2 1.0 0 0.3 0.5
10 100 1000
of VSV (log TCIDJ0.2
ml).
HCCM-5 0.2 0.2 0.5 0.2 0.2 h
NUC-I 0 0.2 0.5 0 0.7
0
1.0 -
0.6 0.5
-
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induced in cell-free extracts of every choriocarcinoma cell line treated with IFN(Y (Fig. 1). Thus, discrepancy between low sensitivity to antiviral activity and normal activation of 2-5A synthetase seemed to be the unique property of choriocarcinoma cells. Antiproliferative
Activity of IFNs in Choriocarcinoma Cells in Vitro
We previously reported that RSa cells were highly sensitive to the antiproliferative action of IFNs [16,29], in contrast to HEC-I cells which showed complete resistance [21,32]. These cell lines were used as controls of antiproliferative activity of IFNs against choriocarcinoma cells in vitro. As shown in Fig. 2, two choriocarcinoma cell lines, BeWo and HCCM-5, were relatively sensitive to both IFNa! and IFN-/3 and their [3H]thymidine incorporations were suppressed at 48 hr after treatment with 1 to 1000 U/ml. However, these cells showed resistance to antiproliferative action of IFN-y, in contrast to RSa cells which showed sensitivity also to IFN--y. Another choriocarcinoma cell line, NUC-1, was resistant to all types of IFNs. As shown in Fig. 3, the growth of both BeWo and HCCM-5 cells was also slightly suppressed by either IFN-a or IFN-j3 at 1000U/ml in vitro. The suppression became evident when the treatment was prolonged to 2 days or more. IFN--y did not affect the growth of every choriocarcinoma cell line. NUC-1 cells showed resistance to the growth inhibitory effect of all types of IFNs. From these results, heterogeneities of both sensitivity to IFN and antiproliferative activity exist among choriocarcinoma cells and types of IFNs, respectively. Effect of IFNs on hCG Secretion of Choriocarcinoma Cells The hCG values in culture medium were suppressed in parallel with the suppression of [ 3H]thymidine incorporation or growth of choriocarcinoma cells in vitro (data not shown). The values of hCG secreted per cell were not different
IFN-a(U/m6)
FIG. 1. Induction of 2-5A synthetase activities by IFN-o( in cell-free extracts of three human choriocarcinoma cell lines.
1
10
100 1000 1
I
Concentration
0
I
I
100
I
1000
of five different
of IFN (U/me>
10
I
T
IFN-,8
FIG. 2. Effect of three types IFNs on [‘Hlthymidine incorporation into DNA RSa; (A), HCCM-5; (m), BeWo; (x), NUC-1. Bars indicate standard deviation.
0
IFN-a
I
I
1
I
IO
human tumor cell lines in vitro.
0
IFN- y
I
I
1000
CO), HEC-I;
100
(O),
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SEKIYA ET AL.
0eWo
HCCM-5
5X106-
NUC-1
IO6 m z 0 %
5x105
2 IO5 \ 5X10"
IO"
I
I
I
t
2468
2468
I
1
I
2468
Days
FIG. 3. Effect of three types of IFNs on growth of human choriocarcinoma cell lines in vitro. (O), Nontreated; (A), 1000 U/ml IFN-(Y; (m), 1000 U/ml IFN-0; (x), 1000 U/ml IFN-y.
between with and without treatments of IFNs. Thus, the effect of IFNs on hCG secretion seemed to be negligible. Antiproliferative
Activity of ZFNs in Xenografts in Vivo
As shown in Table 2, both BeWo and HCCM-5 cells were tumorigenic to hamster cheek pouches. After 7 days of cell inoculation, the tumors began to TABLE 2 ANTITUMOR
ACTION
OF HUMAN
IFN-p
ON TUMORS DEVELOPED IN HAMSTER CHEEK POUCHES
Cell line BeWo Treatment Nontreated control Schedule 1’ Schedule 2d
No. of ch.p. 5 3 2
Tumor wt’ (mg) 1101 + 201b 415 2 308 1176
HCCM-5 No. of ch.p.
Tumor wt (ms)
3 3 3
1654 +- 101 776 k 80 1562 f 128
a At 15th day after tumor cell inoculation. b Mean -r- SE. ’ IFN-j3 at dose of lo4 U/day was administered into peritoneal cavity on every day from the first till seventh day after tumor cell inoculation. d IFN-p at the same doses on every day from the seventh day till the thirteenth day after tumor cell inoculation.
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grow logarithmically. When the tumor-bearing animals were given intraperitoneal injections of 10,000 U of IFN-P on every day from the first day after inoculation, the tumor weights decreased to about one-half of nontreated controls in both BeWo and HCCM-5 cells (Table 2). In contrast, the tumor weights were almost the same as nontreated controls when IFN-/3 was given from the eighth day (Table 2). The inhibition of tumor growth was also evident in the xenografts developed in subcutis of nude mice by the intraperitoneal administration of 5000 U IFN-/3 when the administration was started on the first day of cell inoculation (Fig. 4). When the start of administration was delayed until the 28th day, IFN/3 did not show any antiproliferative activity (Fig. 4). These results indicate that IFNs may be effective in animals with a low tumor load. DISCUSSION We reported previously that a patient with refractory choriocarcinoma showed a transient stable state in growth of metastatic tumors to the lungs and urinary
Days after
transplantation
FIG. 4. Effect of intraperitoneal administration of 5000 U IFN-/3 (arrows) on the subcutaneous xenografts of a human choriocarcinoma strain CC-I-JCK in nude mice. Cross marks indicate the death of the animals.
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hCG values during and after treatment with IFN-P [ 181. This result led us to examine the effects of IFNs on choriocarcinoma cells in vitro and in vivo. In the present studies using three kinds of choriocarcinoma cell lines, two choriocarcinoma cell lines, BeWo and HCCM-5, showed weak sensitivity to the growthinhibiting action of IFN-a and IFN-P in vitro and IFN-/I in vim. Another cell line NUC-1 showed resistance to these IFNs in vitro. These heterogenous sensitivities to IFNs among the cell lines derived from the same histologic malignancy have been also pointed out in other human malignant tumor cell lines [8,10,26,27]. On the other hand, all of the three choriocarcinoma cell lines were resistant to the antiproliferative action of IFN-7 in vitro. This result indicates that various IFNs may differ in their antiproliferative activity, as also suggested by other investigators [5,12]. In the present experiment, all of the choriocarcinoma cell lines showed some resistance to the antiviral effect of IFNs, irrespective of the types. However, activities of both enzymes, 2-5A synthetase (Fig. 1) and dsRNA-dependent protein phosphokinase [31], which are considered to be indicators of IFN sensitivity were normally induced in the choriocarcinoma cells. In addition, receptor to IFN-(r was detected in NUC-1 cells which showed the least sensitivity to IFNs [ll]. Choriocarcinoma cells are characterized by their stable secretion of hCG and all of choriocarcinoma cell lines used secreted more than 200 ng/106/48 hr of hCG [25]. Therefore, the role of hCG in IFN sensitivity was examined. However, hCG did not modify the antiproliferative activity of IFN-a on these choriocarcinoma cells [19]. Other inhibitory factors may be involved in the mechanism of the weak activity of IFNs in choriocarcinoma cells. The inhibitory effect of IFNs against human malignant tumors grown in nude mice is controversial among the reporters [ 1,2,7,9,14,20]. Since IFNs are generally considered to be species specific in their action, the antiproliferative activity of them on xenografts is not due to the host-mediated action but due to direct action. Therefore, contrarities of antiproliferative action of IFNs among xenografts may be due to the following factors: the histologic types of xenografts, volume at the start of IFN administration, types of IFNs, routes of administration, and administration schedules. From the results of the present experiment, the volume of xenografts at the start of IFN administration seems to be an important factor. IFN-P at 5000 or 10,000 U showed antitumor effect when the administrations were initiated immediately after cell inoculation. This result suggested that IFN may be effective when it is used as adjuvant therapy after conventional therapies in patient with a low tumor burden. REFERENCES 1. Balkwill, F., Taylor-Papadimitriou, J., Fames, K. H., and Sebesteny, A. Human lymphoblastoid interferon can inhibit the growth of human breast cancer xenografts in athymic nude mice, Eur. J. Cancer 16, 569-573 (1980). 2. Balkwill, F., Moodie, E. M., Freedman, V., and Fantes, K. H. Human interferon inhibits the growth of established human breast tumors in the nude mouse, In?. J. Cancer 30, 231-235 (1982). 3. Ball, C. R., Poynter, R. W., and van den Berg, H. W. A novel method for measuring incorporation of radioactive precursors into nucleic acid and proteins of cells in monolayer culture, Anal. Biochem. 46, 101-107 (1972).
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4. Billiau, A. The clinical value of interferons as antitumor agents, Eur. J. C/in. Oncol. 17, 949967 (1981). 5. Borden, E. C., Hogan, T. F., and Voelkel, J. G. Comparative antiproliferative activity in vitro of natural interferons a and p for diploid and transformed human cells, Cancer Res. 42, 49484953 (1982). 6. Borden, E. C. Progress toward therapeutic application of interferons, 1979-1983, Cancer 54, 2770-2776 (1984). 7. Clutterbuck, R. D., Millar, J. L., and Alexander, P. Failure of high doses of LYinterferon to affect the growth of human carcinoma, melanoma, and myeloid leukemia xenografts, Brif. J. Cancer 48, 445-447 (1983). 8. Creasey, A. A., Bartholomew, J. C., and Merigan, T. C. Role of C&-G, arrest in the inhibition of tumor cell growth by interferon, Proc. Nut/. Acad. Sci. USA 77, 1471-1475 (1980). 9. de Clercq, E., Georgiades, J., Edy, V. G., and Sobis, H. Effect of human and mouse interferon and polyriboinosinic acid polyribocytidylic acid on the growth of fibrosarcoma and melanoma tumors in nude mice, Eur. J. Cancer 14, 1273-1282 (1978). 10. Epstein, L. B., Shen, J-T., Abele, J. S., and Reese, C. C. Sensitivity of human ovarian carcinoma cells to interferon and other antitumor agents as assessedby an in vitro semisolid agar technique, Ann. N. Y. Acud. Sci. 350, 228-244 (1980). 11. Fuse, A., Ashino-Fuse, H., and Kuwata, T. Binding of ‘j’l-labeled human interferon to cell lines with low sensitivity to interferon, Gann 7.5, 379-384 (1984). 12. Gomi K., Morimoto, M., and Nakamizo, N. Characteristics of antiviral and anticellular activities of human recombinant interferon-y, Japan. J. Cancer Res. 76, 224-234 (1985). 13. Hayashi, H. A study of tumor growth, cell differentiation and hormone production on two transplantable human choriocarcinomas in nude mice, Keio Igaku 54, 465-476 (1978) (in Japanese). 14. Kohno, M., Ida, N., Nagao, S., Tanaka, N., Sekiguchi, F., Uenishi, N., Kajita, A., and Ogawa, H. Effects of human fibroblast interferon on human tumors transplanted into nude mice: Sensitivity of human tumors in interferon, Gann 73, 945-951 (1982). 15. Kuramoto, H., Tamura, S., and Notake, Y. Establishment of a cell line of human endometrial adenocarcinoma in vitro, Amer. J. Obstet. Gynecol. 114, 1012-1019 (1972). 16. Kuwata, T., Oda, T., Sekiya, S., and Morinaga, N. Characteristics of a human cell line successively transformed by Rous sarcoma virus and simian virus 40, J. Nut/. Cancer Inst. 56, 919-926 (1976). 17. Kuwata, T., Fuse, A., and Morinaga, N. Effects of interferon on cell and virus growth in transformed human cell lines, J. Gen. Viral. 33, 7-15 (1976). 18. Kuwata, T., Sekiya, S., Fuse, A., and Morinaga, N. Effects of interferon on the growth of human choriocarcinoma cells and hCG production in vitro and in viva, in The biology of the inferferon system (H. Kirchner and H. Schellekens, Eds.), Elsevier, Amsterdam, pp. 213-219 (1985). 19. Kuwata, T., and Sekiya, S. Effects of interferons on the biological characteristics of human choriocarcinoma cells, in The inrerferon system (F. Dianzani and G. B. Rossi, Eds.) Raven Press, New York, pp. 309-312 (1985). 20. Masuda, S., Fukuma, H., and Beppu, Y. Antitumor effect of human leukocyte interferon on humanosteosarcoma transplanted into nude mice, Eur. 1. Cancer C/in. Oncol. 19, 1521-1528 (1983). 21. Morinaga, N., Yonehara, S., Tomita, Y., and Kuwata, T. Insensitivity to interferon of two subclones of human endometrial carcinoma cell line, HEC-1, Int. J. Cancer 31, 21-28 (1983). 22. Nakamoto, 0. Properties of strain cells of the uterine invasive mole and its metastatic choriocarcinomas. Asia-Oceania J. Obstet. Gynaecol. 6, 177-189 (1980). 23. Pattillo, R. A., and Gey, G. 0. The establishment of a cell line of human hormone-synthesizing trophoblastic cells in vitro. Cancer Res. 28, 1231-1236 (1969). 24. Priestman, T. J. Interferons and cancer therapy, .I. Pathol. 141, 287-295 (1983). 25. Sekiya, S., Iwasawa, H., Sugita, M., Inaba, N., Kaiho, T., Shirotake, S., and Takamizawa, H. Characterization of human choriocarcinoma cell lines secreting high and low amounts of chorionic gonadotropin, Int. J. Gynecol. Pathol. 2, 121-133 (1983).
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26. Shibata, H., and Taylor-Papadimitriou, J. Effects of human lymphoblastoid interferon on cultured breast cancer cells. In?. 1. Cancer 28, 447-453 (1981). 27. Strander, H., and Einhorn, S. Effects of human leukocyte interferon on the growth of human osteosarcoma cells in tissue culture, Int. .Z. Cancer 19, 468-473 (1977). 28. Suzumori, K., Sugimoto, Y., Suzumori, K., Yagami, Y., and Takeda, A. The establishment of human choriocarcinoma cell line in vitro, Asia-Ocennia J. Obstet. Gynaecol. 9,309-315 (1983). 29. Tomita, Y., Cantell, K., and Kuwata, T. Effects of human y interferon on cell growth, replication of virus and induction of 2’-5’ oligoadenylate synthetase in three human lymphoblastoid cell lines and K562 cells, Znt. J. Cancer 30, 161-165 (1982). 30. Vandenbussche, P., Devizia, M., Verhaegen-Lewalle, M., Fuse, A., Kuwata, T., de Clercq, E., and Content, J. Enzymatic activities induced by interferon in human fibroblast cell lines differing in their sensitivity to the anticellular activity of interferon, Virology 111, 11-22 (1981). 31. Vandenbussche, P., Kuwata, T., Verhaegen-Lewalle, M., and Content, J. Effect of interferon on two choriocarcinoma-derived cell lines, Virology 128, 474-479 (1983). 32. Verhaegen, M., Divizia, M., Vandenbussche, P., Kuwata, T., and Content, J. Abnormal behavior of interferon-induced enzymatic activities in an interferon-resistant cell line, Proc. Natl. Acad. Sci. USA 77, 4479-4483 (1980).
ONCOLOGY
25, 115-124 (1986)
Effects of Human lnterferons on Human Choriocarcinoma Cells in Vitro and in Vivo S. SEKIYA,* *Department
T. KUWATA,? H. ISHIGE,” Y. TOMITA,~ AND H. TAKAMIZAWA*
of Obstetrics and Gynecology and )‘Department of Microbiology, School of Medicine, 1-8-l Inohana, Chiba 280, Japan
Chiba University
Received July 11, 1985 Three kinds of human choriocarcinoma cell lines (BeWo, HCCM-5, and NUC-1) were used for examining the antiviral and antiproliferative activities of human interferons (IFNs) in vitro and in vivo. All of the cell lines showed only low sensitivity to the antiviral action of every IFN-a, IFN-/3, and IFN-y against vesicular stomatitis virus infection. However, 2-5A synthetase was normally induced by IFN-a in all of the cell lines. The [‘Hlthymidine incorporation of both BeWo and HCCM-5 cells was suppressed in dose-dependent manner at 48 hr after treatment with 1 to 1000 units (U)/ml of IFN-(U or IFN-/3 and the growth of them was also slightly inhibited when treated continuously with 1000 U/ml for 6 days in vitro. Another cell line NUC-1 was the least sensitive to these IFNs among the three cell lines. IFN-7 did not show any antiproliferative effect on these cell lines. The intraperitoneal administration of 5000 or 10,000 U of IFN-/3 suppressed the growth of xenografts developed in hamster cheek pouches and subcutis of nude mice when its administration was initiated on the first day of cell inoculation. These results indicate that although some heterogeneities exist among the cell lines choriocarcinoma cells are weakly sensitive to the antiproliferative activity of human IFNs. 0 1986 Academic Press. Inc.
INTRODUCTION
Recently mass production of three types of purified human interferons (IFN-(Y, IFN-P, and IFN-y) has been established and their therapeutic application to various kinds of human malignant tumors is now being carried out [4]. However, clinical studies on human IFNs as antitumor agents are few and most of them are not completed [6,24]. Therefore, it is necessary to explore human malignant tumors against which human IFNs are effective by using many histologically different types of human malignant tumor cells. In this report, we describe the effects of three types of human IFNs on three different human choriocarcinoma cell lines in vitro and in vivo. MATERIALS
AND METHODS
Cell lines. We used the following five human cell lines: choriocarcinoma cell lines BeWo [23], HCCM-5 [22], and NUC-I [28], virus-transformed embryo fibroblast cell line RSa [ 161, and uterine adenocarcinoma cell line HEC-1 [ 151. The biologic characteristics of choriocarcinoma cell lines were described previously 115 0090-8258/86 $1.50 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
116
SEKIYA
ET AL.
[25]. Monolayer cultures were maintained in RPM1 1640 medium supplemented with 10% newborn bovine serum (Flow Laboratories, North Ryde, New South Wales, Australia) and antibiotics in a humidified 5% CO, and 95% air atmosphere at 37°C. ZFNS. Human leukocyte IFN (IFN-a) with a specific activity of 6 x lo6 U/mg protein was kindly provided by Dr. K. Cantell, Central Public Health Laboratory, Helsinki, Finland. Human fibroblast IFN (IFN-P) with a specific activity of 1 x 10’ U/mg protein was provided by Mochida Pharmaceutical Company, Ltd. (Tokyo, Japan). Human recombinant immune IFN (IFN-7) with a specific activity of 1 x 10’ U/mg protein was supplied by Torey Industries (Kamakura, Japan). The stock solutions of IFNs (10’ U/ml in Hank’s balanced salt solution) were kept at -70°C until use. Assay of antiviral activity. Antiviral activity was measured in a yield reduction assay with vasicular stomatitis virus (VSV). Cells cultivated in 16 mm Limbro multiplates were treated with 10 to 1000 U/ml of IFNs for 20 hr. Cells were washed and then infected with VSV at multiplicity of 1.0. After 1 hr of virus adsorption at 37”C, cells were washed and culture medium containing 5% newborn bovine serum was added to each culture. After 18 hr of incubation, sample medium was centrifuged and stored at - 70°C until virus titration. VSV in samples was assayed by 50% tissue culture infective dose (TCID& in microtrays, as described elsewhere [ 171. Assay of2-5A sythetase. Cells (5 to 8 x 10”) treated with 10 to 1000 U/ml IFN-(U at 37°C for 16 hr were suspended in 150 ~1 of 10 mM KCI, 1.5 mM Mg (OAc),, 20% (v/v) glycerol, and 20 mM Hepes-KOH buffer, pH 7.4. The cells were sonicated for 10 sec. The samples were centrifuged at 10,OOOgfor 20 min and 20 ~1 of the supernatant was used for enzyme assay. The reaction mixture contained 20 ~1 of the supernatant, 1 pg poly(1) * (C), 3 &I [3H]ATP ([2,8,5lHIATP, 44.5 Ci/mmole; New England Nuclear Corp., Boston, Mass.), 5 mM ATP, 20 mM Hepes/KOH, pH 7.4, 25 n&f Mg(OAc)*, 80 mM KCl, and 7 mM 2-mercaptoethanol in a final incubation volume of 50 ~1. The 2-5A synthesized from ATP was prepared as previously described [29]. The results were expressed as cpm recovered in 2-5A for 20 ~1 supernatant of cell extract at 1.0 OD,,,. [3HJThymidine incorporation in vitro. Cells (10’) in 1 ml of culture medium were seeded into a series of sterile glass scintillation vials. After 48 hr of incubation, the exponentially growing cells were exposed to IFNs at doses from 1 to 1000 U/ml for 48 hr. After collection of culture medium for radioimmunoassay (RIA) of human chorionic gonadotropin (hCG), 6-[3H]thymidine (15 Ci/mmole; New England Nuclear Corp.) was added to each vial in 1 ml of culture medium at 1 Z-Xi. After 30 min of incubation, each vial was quenched by the addition of 10 ml of ice-cold isotonic saline and the incorporation was counted with a scintillation counter, RackBeta 1215 (Wallac Oy, Turku, Finland), following the method of Ball et al. [31. Cell growth in vitro. Cells (1.5 x 105)were seeded into 60-mm plastic culture dishes in 5 ml of culture medium. After 48 hr, the culture medium was changed for that with or without IO00 U/ml of IFNs and then the culture medium was changed every other day for 6 days. The cells were detached from dishes with
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0.25% trypsin solution and viable cells were counted by the trypan blue dye exclusion test using a hemocytometer. Assay of hCG. Assay of hCG in culture medium was done by using CEAIRE-SORIN hCG RIA kits (Green Cross Co., Tokyo, Japan). Znoculation into hamster cheek pouch. BeWo or HCCM-5 cells (2 x 106) suspended in 0.2 ml of culture medium were inoculated into the cheek pouches of 4-week-old female golden hamsters (Takasugi Experimental Animals, Saitama, Japan). They were conditioned with 10 mg/week of cortisone acetate (Corton, Nippon Merck-Banyu Co., Osaka, Japan) during a 14-day observation period. IFN-p at doses of 10,000 U was administered into the intraperitoneal cavity at either of the following administration schedules: (1) on every day from the first till seventh day after the cell inoculation and (2) on every day from the eighth day till the fourteenth day after the cell inoculation. Inoculation into subcutis of nude mouse. A human choriocarcinoma strain, CC-I-JCK[13], maintained by the serial subcutaneous inoculations into 6- to 8week-old female BALB/c nu/nu nude mice (Clea Japan, Tokyo, Japan) was used. IFN-/3 at a dose of 5000 U was administered by intraperitoneal route either on every 3 days from the first after the inoculation of l- to 2-mm pieces of the tumor tissues for 42 days or on every 3 days from the 28th day after the inoculation until the death of the animals. RESULTS
Antiviral Activity of ZFNs in Choriocarcinoma Cells As shown in Table 1, all of the human choriocarcinoma cell lines showed only low sensitivity to the antiviral activity of IFNs against VSV infection, as compared to RSa cells which were shown to be sensitive to the antiviral activity [16,30]. Dose-dependent inhibition of viral replication was not evident by treatment with up to 1000 U/ml and difference in the antiviral activity was not observed among the three types of IFNs (Table 1). However, 2-5A synthetase activity was normally TABLE
I
ANTIVIRAL ACTION OF HUMAN INTERFERONSIN VSV-INFECTED CHORIOCARCINOMACELL LINES Cell line Interferon IFN-a
IFN-P
IFN-y
c Yield reduction b Not tested.
IU/ml
BeWo
10 100 1000 10 100 1000
0 0.2 0.5 0.2 0.2 1.0 0 0.3 0.5
10 100 1000
of VSV (log TCIDJ0.2
ml).
HCCM-5 0.2 0.2 0.5 0.2 0.2 h
NUC-I 0 0.2 0.5 0 0.7
0
1.0 -
0.6 0.5
-
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induced in cell-free extracts of every choriocarcinoma cell line treated with IFN(Y (Fig. 1). Thus, discrepancy between low sensitivity to antiviral activity and normal activation of 2-5A synthetase seemed to be the unique property of choriocarcinoma cells. Antiproliferative
Activity of IFNs in Choriocarcinoma Cells in Vitro
We previously reported that RSa cells were highly sensitive to the antiproliferative action of IFNs [16,29], in contrast to HEC-I cells which showed complete resistance [21,32]. These cell lines were used as controls of antiproliferative activity of IFNs against choriocarcinoma cells in vitro. As shown in Fig. 2, two choriocarcinoma cell lines, BeWo and HCCM-5, were relatively sensitive to both IFNa! and IFN-/3 and their [3H]thymidine incorporations were suppressed at 48 hr after treatment with 1 to 1000 U/ml. However, these cells showed resistance to antiproliferative action of IFN-y, in contrast to RSa cells which showed sensitivity also to IFN--y. Another choriocarcinoma cell line, NUC-1, was resistant to all types of IFNs. As shown in Fig. 3, the growth of both BeWo and HCCM-5 cells was also slightly suppressed by either IFN-a or IFN-j3 at 1000U/ml in vitro. The suppression became evident when the treatment was prolonged to 2 days or more. IFN--y did not affect the growth of every choriocarcinoma cell line. NUC-1 cells showed resistance to the growth inhibitory effect of all types of IFNs. From these results, heterogeneities of both sensitivity to IFN and antiproliferative activity exist among choriocarcinoma cells and types of IFNs, respectively. Effect of IFNs on hCG Secretion of Choriocarcinoma Cells The hCG values in culture medium were suppressed in parallel with the suppression of [ 3H]thymidine incorporation or growth of choriocarcinoma cells in vitro (data not shown). The values of hCG secreted per cell were not different
IFN-a(U/m6)
FIG. 1. Induction of 2-5A synthetase activities by IFN-o( in cell-free extracts of three human choriocarcinoma cell lines.
1
10
100 1000 1
I
Concentration
0
I
I
100
I
1000
of five different
of IFN (U/me>
10
I
T
IFN-,8
FIG. 2. Effect of three types IFNs on [‘Hlthymidine incorporation into DNA RSa; (A), HCCM-5; (m), BeWo; (x), NUC-1. Bars indicate standard deviation.
0
IFN-a
I
I
1
I
IO
human tumor cell lines in vitro.
0
IFN- y
I
I
1000
CO), HEC-I;
100
(O),
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SEKIYA ET AL.
0eWo
HCCM-5
5X106-
NUC-1
IO6 m z 0 %
5x105
2 IO5 \ 5X10"
IO"
I
I
I
t
2468
2468
I
1
I
2468
Days
FIG. 3. Effect of three types of IFNs on growth of human choriocarcinoma cell lines in vitro. (O), Nontreated; (A), 1000 U/ml IFN-(Y; (m), 1000 U/ml IFN-0; (x), 1000 U/ml IFN-y.
between with and without treatments of IFNs. Thus, the effect of IFNs on hCG secretion seemed to be negligible. Antiproliferative
Activity of ZFNs in Xenografts in Vivo
As shown in Table 2, both BeWo and HCCM-5 cells were tumorigenic to hamster cheek pouches. After 7 days of cell inoculation, the tumors began to TABLE 2 ANTITUMOR
ACTION
OF HUMAN
IFN-p
ON TUMORS DEVELOPED IN HAMSTER CHEEK POUCHES
Cell line BeWo Treatment Nontreated control Schedule 1’ Schedule 2d
No. of ch.p. 5 3 2
Tumor wt’ (mg) 1101 + 201b 415 2 308 1176
HCCM-5 No. of ch.p.
Tumor wt (ms)
3 3 3
1654 +- 101 776 k 80 1562 f 128
a At 15th day after tumor cell inoculation. b Mean -r- SE. ’ IFN-j3 at dose of lo4 U/day was administered into peritoneal cavity on every day from the first till seventh day after tumor cell inoculation. d IFN-p at the same doses on every day from the seventh day till the thirteenth day after tumor cell inoculation.
INTERFERON AND CHORIOCARCINOMA
121
grow logarithmically. When the tumor-bearing animals were given intraperitoneal injections of 10,000 U of IFN-P on every day from the first day after inoculation, the tumor weights decreased to about one-half of nontreated controls in both BeWo and HCCM-5 cells (Table 2). In contrast, the tumor weights were almost the same as nontreated controls when IFN-/3 was given from the eighth day (Table 2). The inhibition of tumor growth was also evident in the xenografts developed in subcutis of nude mice by the intraperitoneal administration of 5000 U IFN-/3 when the administration was started on the first day of cell inoculation (Fig. 4). When the start of administration was delayed until the 28th day, IFN/3 did not show any antiproliferative activity (Fig. 4). These results indicate that IFNs may be effective in animals with a low tumor load. DISCUSSION We reported previously that a patient with refractory choriocarcinoma showed a transient stable state in growth of metastatic tumors to the lungs and urinary
Days after
transplantation
FIG. 4. Effect of intraperitoneal administration of 5000 U IFN-/3 (arrows) on the subcutaneous xenografts of a human choriocarcinoma strain CC-I-JCK in nude mice. Cross marks indicate the death of the animals.
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SEKIYA ET AL.
hCG values during and after treatment with IFN-P [ 181. This result led us to examine the effects of IFNs on choriocarcinoma cells in vitro and in vivo. In the present studies using three kinds of choriocarcinoma cell lines, two choriocarcinoma cell lines, BeWo and HCCM-5, showed weak sensitivity to the growthinhibiting action of IFN-a and IFN-P in vitro and IFN-/I in vim. Another cell line NUC-1 showed resistance to these IFNs in vitro. These heterogenous sensitivities to IFNs among the cell lines derived from the same histologic malignancy have been also pointed out in other human malignant tumor cell lines [8,10,26,27]. On the other hand, all of the three choriocarcinoma cell lines were resistant to the antiproliferative action of IFN-7 in vitro. This result indicates that various IFNs may differ in their antiproliferative activity, as also suggested by other investigators [5,12]. In the present experiment, all of the choriocarcinoma cell lines showed some resistance to the antiviral effect of IFNs, irrespective of the types. However, activities of both enzymes, 2-5A synthetase (Fig. 1) and dsRNA-dependent protein phosphokinase [31], which are considered to be indicators of IFN sensitivity were normally induced in the choriocarcinoma cells. In addition, receptor to IFN-(r was detected in NUC-1 cells which showed the least sensitivity to IFNs [ll]. Choriocarcinoma cells are characterized by their stable secretion of hCG and all of choriocarcinoma cell lines used secreted more than 200 ng/106/48 hr of hCG [25]. Therefore, the role of hCG in IFN sensitivity was examined. However, hCG did not modify the antiproliferative activity of IFN-a on these choriocarcinoma cells [19]. Other inhibitory factors may be involved in the mechanism of the weak activity of IFNs in choriocarcinoma cells. The inhibitory effect of IFNs against human malignant tumors grown in nude mice is controversial among the reporters [ 1,2,7,9,14,20]. Since IFNs are generally considered to be species specific in their action, the antiproliferative activity of them on xenografts is not due to the host-mediated action but due to direct action. Therefore, contrarities of antiproliferative action of IFNs among xenografts may be due to the following factors: the histologic types of xenografts, volume at the start of IFN administration, types of IFNs, routes of administration, and administration schedules. From the results of the present experiment, the volume of xenografts at the start of IFN administration seems to be an important factor. IFN-P at 5000 or 10,000 U showed antitumor effect when the administrations were initiated immediately after cell inoculation. This result suggested that IFN may be effective when it is used as adjuvant therapy after conventional therapies in patient with a low tumor burden. REFERENCES 1. Balkwill, F., Taylor-Papadimitriou, J., Fames, K. H., and Sebesteny, A. Human lymphoblastoid interferon can inhibit the growth of human breast cancer xenografts in athymic nude mice, Eur. J. Cancer 16, 569-573 (1980). 2. Balkwill, F., Moodie, E. M., Freedman, V., and Fantes, K. H. Human interferon inhibits the growth of established human breast tumors in the nude mouse, In?. J. Cancer 30, 231-235 (1982). 3. Ball, C. R., Poynter, R. W., and van den Berg, H. W. A novel method for measuring incorporation of radioactive precursors into nucleic acid and proteins of cells in monolayer culture, Anal. Biochem. 46, 101-107 (1972).
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AND
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4. Billiau, A. The clinical value of interferons as antitumor agents, Eur. J. C/in. Oncol. 17, 949967 (1981). 5. Borden, E. C., Hogan, T. F., and Voelkel, J. G. Comparative antiproliferative activity in vitro of natural interferons a and p for diploid and transformed human cells, Cancer Res. 42, 49484953 (1982). 6. Borden, E. C. Progress toward therapeutic application of interferons, 1979-1983, Cancer 54, 2770-2776 (1984). 7. Clutterbuck, R. D., Millar, J. L., and Alexander, P. Failure of high doses of LYinterferon to affect the growth of human carcinoma, melanoma, and myeloid leukemia xenografts, Brif. J. Cancer 48, 445-447 (1983). 8. Creasey, A. A., Bartholomew, J. C., and Merigan, T. C. Role of C&-G, arrest in the inhibition of tumor cell growth by interferon, Proc. Nut/. Acad. Sci. USA 77, 1471-1475 (1980). 9. de Clercq, E., Georgiades, J., Edy, V. G., and Sobis, H. Effect of human and mouse interferon and polyriboinosinic acid polyribocytidylic acid on the growth of fibrosarcoma and melanoma tumors in nude mice, Eur. J. Cancer 14, 1273-1282 (1978). 10. Epstein, L. B., Shen, J-T., Abele, J. S., and Reese, C. C. Sensitivity of human ovarian carcinoma cells to interferon and other antitumor agents as assessedby an in vitro semisolid agar technique, Ann. N. Y. Acud. Sci. 350, 228-244 (1980). 11. Fuse, A., Ashino-Fuse, H., and Kuwata, T. Binding of ‘j’l-labeled human interferon to cell lines with low sensitivity to interferon, Gann 7.5, 379-384 (1984). 12. Gomi K., Morimoto, M., and Nakamizo, N. Characteristics of antiviral and anticellular activities of human recombinant interferon-y, Japan. J. Cancer Res. 76, 224-234 (1985). 13. Hayashi, H. A study of tumor growth, cell differentiation and hormone production on two transplantable human choriocarcinomas in nude mice, Keio Igaku 54, 465-476 (1978) (in Japanese). 14. Kohno, M., Ida, N., Nagao, S., Tanaka, N., Sekiguchi, F., Uenishi, N., Kajita, A., and Ogawa, H. Effects of human fibroblast interferon on human tumors transplanted into nude mice: Sensitivity of human tumors in interferon, Gann 73, 945-951 (1982). 15. Kuramoto, H., Tamura, S., and Notake, Y. Establishment of a cell line of human endometrial adenocarcinoma in vitro, Amer. J. Obstet. Gynecol. 114, 1012-1019 (1972). 16. Kuwata, T., Oda, T., Sekiya, S., and Morinaga, N. Characteristics of a human cell line successively transformed by Rous sarcoma virus and simian virus 40, J. Nut/. Cancer Inst. 56, 919-926 (1976). 17. Kuwata, T., Fuse, A., and Morinaga, N. Effects of interferon on cell and virus growth in transformed human cell lines, J. Gen. Viral. 33, 7-15 (1976). 18. Kuwata, T., Sekiya, S., Fuse, A., and Morinaga, N. Effects of interferon on the growth of human choriocarcinoma cells and hCG production in vitro and in viva, in The biology of the inferferon system (H. Kirchner and H. Schellekens, Eds.), Elsevier, Amsterdam, pp. 213-219 (1985). 19. Kuwata, T., and Sekiya, S. Effects of interferons on the biological characteristics of human choriocarcinoma cells, in The inrerferon system (F. Dianzani and G. B. Rossi, Eds.) Raven Press, New York, pp. 309-312 (1985). 20. Masuda, S., Fukuma, H., and Beppu, Y. Antitumor effect of human leukocyte interferon on humanosteosarcoma transplanted into nude mice, Eur. 1. Cancer C/in. Oncol. 19, 1521-1528 (1983). 21. Morinaga, N., Yonehara, S., Tomita, Y., and Kuwata, T. Insensitivity to interferon of two subclones of human endometrial carcinoma cell line, HEC-1, Int. J. Cancer 31, 21-28 (1983). 22. Nakamoto, 0. Properties of strain cells of the uterine invasive mole and its metastatic choriocarcinomas. Asia-Oceania J. Obstet. Gynaecol. 6, 177-189 (1980). 23. Pattillo, R. A., and Gey, G. 0. The establishment of a cell line of human hormone-synthesizing trophoblastic cells in vitro. Cancer Res. 28, 1231-1236 (1969). 24. Priestman, T. J. Interferons and cancer therapy, .I. Pathol. 141, 287-295 (1983). 25. Sekiya, S., Iwasawa, H., Sugita, M., Inaba, N., Kaiho, T., Shirotake, S., and Takamizawa, H. Characterization of human choriocarcinoma cell lines secreting high and low amounts of chorionic gonadotropin, Int. J. Gynecol. Pathol. 2, 121-133 (1983).
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26. Shibata, H., and Taylor-Papadimitriou, J. Effects of human lymphoblastoid interferon on cultured breast cancer cells. In?. 1. Cancer 28, 447-453 (1981). 27. Strander, H., and Einhorn, S. Effects of human leukocyte interferon on the growth of human osteosarcoma cells in tissue culture, Int. .Z. Cancer 19, 468-473 (1977). 28. Suzumori, K., Sugimoto, Y., Suzumori, K., Yagami, Y., and Takeda, A. The establishment of human choriocarcinoma cell line in vitro, Asia-Ocennia J. Obstet. Gynaecol. 9,309-315 (1983). 29. Tomita, Y., Cantell, K., and Kuwata, T. Effects of human y interferon on cell growth, replication of virus and induction of 2’-5’ oligoadenylate synthetase in three human lymphoblastoid cell lines and K562 cells, Znt. J. Cancer 30, 161-165 (1982). 30. Vandenbussche, P., Devizia, M., Verhaegen-Lewalle, M., Fuse, A., Kuwata, T., de Clercq, E., and Content, J. Enzymatic activities induced by interferon in human fibroblast cell lines differing in their sensitivity to the anticellular activity of interferon, Virology 111, 11-22 (1981). 31. Vandenbussche, P., Kuwata, T., Verhaegen-Lewalle, M., and Content, J. Effect of interferon on two choriocarcinoma-derived cell lines, Virology 128, 474-479 (1983). 32. Verhaegen, M., Divizia, M., Vandenbussche, P., Kuwata, T., and Content, J. Abnormal behavior of interferon-induced enzymatic activities in an interferon-resistant cell line, Proc. Natl. Acad. Sci. USA 77, 4479-4483 (1980).