Enhanced Inhibition of Anticancer Drugs by Human Recombinant Gamma-Interferon for Human Renal Cell Carcinoma in Vitro

0022-534'1 /87 /1373-0539$02.00/0 Vol. 137, }/larch

THE JOURNAI, OF UnOLCGY

Printed iri U.S.A.

Copyright© 1987 by The Wiliiams & Wilkins Co.

ENHANCED INHIBITION OF ANTICANCER DRUGS BY HUMAN RECOMBINANT GAI\IIMA-INTERFERON FOR HUJ\tIAN RENAL CELL CARCINOMA IN VITRO KAZUO GOHJI,* SAKAN MAEDA, TAKETOSHI SUGIYAMA, JOJI ISHIGAMI* SADAO KAMIDONO

AND

From the Departments of Urology and Pathology, Kobe University School of Medicine, Kobe, Japan

ABSTRACT

We studied the inhibitory effect of seven anticancer drugs and Cl'-, recombinant (3-, and recombinant )'-interferons on the in vitro growth of two established human renal cell carcinomas and 16 renal cell carcinomas obtained from patients using monolayer culture and the double-layer soft agar system. Recombinant )'-interferon was the most effective of three types of interferon. Combined treatment with recombinant -y-interferon and some anticancer drugs inhibited the cell growth in both cell lines more than treatment with each drug alone. Treatment with recombinant -y-interferon and cisplatinum or 5-fluorouracil following a 24-hour incubation with the interferon was more effective than when interferon and the drug were given simultaneously. Treatment using doxorubicin, cisplatinum, or vinblastine with recombinant -y-interferon synergistically inhibited colony formation in 11 of the 16 renal cell carcinomas that showed clonal growth. Our results suggest that treatment with anticancer drugs in combination with recombinant -yinterferon is effective for renal cell carcinoma. Renal cell carcinoma is the third most common urogenital cancer in Japan. 1 Surgical excision is thought to be the only effective management for primary lesions. 2 Other treatments such as chemotherapy and radiation therapy are almost ineffective. 2 Treatment with a-interferon (a-IFN) has some efficacy against human renal cell carcinoma,3' 4 although it gives rise to side effects such as loss of appetite, fever, and bone marrow suppression. 8 ' 4 Combined treatment using a-, recombiant f3 - (rec(3-), and recombinant 'Y-interferon (rec'Y-IFN) with other anticancer drugs may strongly inhibit cell growth. Such combined therapy if used with chemotherapy and surgical treatment might prolong the survival of patients. Despite many technical problems," colony forming assay using double layer soft agar, as originally developed by Hamburger and Salmon, 6 appears to be useful for predicting the clinical response of each patient to anticancer drugs, 7 Here, we investigated the effects of these interferons (IFNs) alone and in combination with anticancer drugs on two established culture lines derived from human renal cell carcinomas and on 11 of the 16 primary renal cell carcinomas that showed clonal in double-layer soft agar, MATERIALS AND METHODS

Cells and culture conditions. Two human renal cell carcinoma lines (KO-RCC-1 8 and RCC-nu-1 9 ) were used. The cells were grown in Petri dishes in Dulbecco's modified Eagle (DME) medium with 10 per cent fetal bovine serum (FBS), 100 units/ ml. penicillin, and 100 µg./ml. streptomycin. Single cell suspensions were obtained by treating with 0.1 per cent trypsin. Renal cell carcinomas were obtained from primary tumors by nephrectomy at our hospital. Aseptically resected tumors were dissociated for two hours with 1000 units/ml. dispase in DME Accepted for publication September 3, 1986.

* Requests for reprints: Department of Urology, Kobe University School of Medicine, Chuo-ku, Kobe, Japan 650. Supported in part by Grant-in-Aid for Cancer Research No. 60570157 from the Ministry of Education, Science and Culture of ,Japan. 539

medium with 10 per cent FBS. Single cell suspensions were obtained by passing the cells through a No. 80 stainless steel mesh. Drugs. The seven standard anticancer drugs and IFNs used in these experiments are listed in table 1 along with the abbreviations and concentrations used. Rec/3- and rec'Y-IFN 10 were gifts from Dr. K. Inoue (Kyowa Hakko Co, Tokyo, Japan). aIFN was a generous gift from the Hayashibara Biological Laboratory (Okayama, Japan). The tumor cells were initially treated for one hour with drugs at concentrations equivalent to 10 per cent of the peak plasma level obtained after standard drug administration. However, virtually no cytotoxic effects again the human renal cell carcinomas were observed with such a short exposure. Therefore, we elected to study the effects of continuous exposure. This method differs from the method of drug exposure that is commonly used. 6 ' 7 Measurement of cell number.KO-RCC-1 and RCC-nu-1 cells at the logarithmic grovvth phase were seeded in 2.5 x 104 cells/ dish and 1 x 104 cells/dish, respectivley, using 60-mm. Petri dishes, and incubated for 24 hours. The medium was then changed to DME medium containing 10 per cent FBS and anticancer drug or an IFN, or both. After 12 to 96 hours incubation, the cells were harvested, stained with 0,5 per cent trypan blue, and counted on a hemocytometer. Each assay was repeated at least three times, In vitro clonogenic assay, The in vitro chemosensitivity test using a soft agar colony forming assay was similar to that originally described by Hamburger and Salmon. 6 Viable tumor cells were adjusted to a final concentration of 3 x 103 /ml. for the established cells or 5 x 105 /ml. for the primary renal cell carcinomas per 35-mm. dish. The cells were continuously incubated for 21 days with one concentration of an anticancer drug, with one concentration of an IFN, or with both, which was added to the top agar layer with tumor cells. Established cells were treated with rec-y-IFN for 24 hours, then the cells were trypsinized, washed twice, and continuously treated with an anticancer drug (ADM, CDDP, or 5-FU) and with 10, 100, or 1000 U/ml. rec'Y-IFN. Each assay was done in three dishes at each drug concentration. The clonogenic assay using cell

540

GOHJI AND ASSOCIATES

(fig. 1). The IFNs alone slightly inhibited RCC-nu-1 cell growth, but higher doses of rec,,-IFN significantly enhanced the effects of ADM, CDDP, MMC, and AC-D (fig. 1 B). In vitro clonogenic assay. The concentration of an agent required to suppress colony formation to 50 per cent of the control was defined as the 50 per cent inhibitory dose (ID 50 ). The ID5o of the IFNs alone against KO-RCC-1 could not be evaluated, because of their relative weak effects, but their inhibition of colony formation increased dose-dependently. Rec-y-lFN had greater effects than the other two IFN (data not shown), 12 inhibiting colony formation by nine, 15, and 41 per cent at 10, 100, and 1000 U./ml., respectively (fig. 2 A). Only rec,,-IFN was used for the following experiments. The ID 50 of anticancer drugs used alone for the two established line cells is listed in table 2. When the seven drugs were given in combination with rec,,-IFN, synergistic effects were observed between the IFN and ADM, MMC, and AC-D, but higher doses ofIFN antagonized the effects of 5-FU (table 3). Rec,,-IFN had slightly inhibited the colony formation of RCC-nu-1 cells. Additive effects were noted between higher dose ofrec-y-lFN and 5-Fu, and synergistic effects with CDDP, MMC and 40487S (table 3, fig. 2 B).

lines was repeated at least three times. Plating efficiency (PE) was calculated as the number of colonies/the number of viable cells plated without drugs. Evaluation of effectiveness of combined treatment. The effectiveness of inhibition of colony formation by the combination of two agents was evaluated by the method of Valeriote and Lin. 11 RESULTS

Growth inhibition tests. The IFNs inhibited proliferation of KO-RCC-1 cells dose- and time-dependently. The order of potency was rec-y- > rec/3- > a-IFN, with rec,,-IFN being about twice as potent as either rec/3- or a-lFN. All drugs caused more than 50 per cent inhibition of KO-RCC-1 cell growth compared to the inhibition observed at concentration equivalent to 10 per cent of peak plasma concentrations after a standard drug administration protocol. The effects of ADM, CDDP and ACD on the proliferation of KO-RCC-1 cells were significantly enhanced for that of drug or rec-y-IFN alone when the drugs were combined with higher doses of rec,,-IFN (fig. 1 A). The RCC-nu-1 cells were more resistant than the KO-RCC-1 cells TABLE

1. Drugs and concentrations used Concentration**

Drugs*

Peak Plasma Level in Humans

Doxorubicin (ADM) Cis-platinum (CDDP) Vinblastine (VLB) Mitomycin C (MMC) 5-fluorouracil (5-FU) Actinomycin D (AC-D) 40487St a-interferon (CY-!FN) Recj:/-interferon (Recj:/-IFN) Rec-y-interferon (Rec-y-lFN)

0.4 2.0 0.1 1.0 10.0 0.01 30.0 *** *** ***

Established Cell Lines 0.01 0.01 0.0001 0.0001 0.01 0.00001 0.01 10 10 10

0.1 0.1 0.001 0.001 0.1 0.0001 0.1 100 100 100

Tumor Cells from Resections:j:

1.0 1.0 0.01 0.01 1.0 0.001 1.0 1000 1000 1000

0.01 0.01 0.01 0.01 0.1 0.0001 0.01 10 10

0.1

0.1 0.1 0.1 0.1 1.0 0.001 0.1 100 100 100

10

1.0 1.0 1.0 1.0 10.0 0.01 1.0 1000 1000 1000

* Drug abbreviations shown in parenthesis. t In vitro-active type of cyclophosphamide. The peak plasma level of cyclophosphamide was shown. ** Concentration in µg./ml. in anticancer drugs and U./ml. in IFNs. :j: Three final concentrations were used, including 10 percent of the peak plasma level in clinical use. *** Peak plasma level in humans increased 3000 U./ml. at least in a dose-dependent manner and was not calculated.

FIG. 1 . A

B 0 f:,

D

.t.

•

C

0

1111

:;; u(II

a

'-

(1)

Recr-IFN ADM (0.01 µ9;./mR,,) COOP (0 .1 µ9;,/m!l,.) VLB (0.0001µ9;./mil.) MMC (O.OOlµg,/mR,.) 5-FU (0.1 µg./mil.) AC·D (0 .00001 µg./mR,.)

100

~ NS o ·'

Recr·IFN ADM (0.01 µg,/ml.) o: CDDP(O.lµg,/rd.) a: VLB (0.0001 mg./ld.) MMC (0 .001 µg./mt) 111: 5-FU (1.0µg,/mt) (!): AC-D (0 .0001 µg./m2.) f:,. :

,--;___...c

••

'+-

a; u (II

.

':::,


0

10

100

1000

Concentration of Rec r·IFN (U ./m2.)

0

10

100

1000

Concentration of Rec r-IFN (U./m2.)

FIG. 1. Inhibition of cell growth in monolayer cultures of A, KO-RCC-1 and B, RCC-nu-1 treated wtih rec-y-IFN and anticancer drugs after 96 hours of incubation. Values represent mean± S.E.M. Abbreviations used: ADM, doxorubicin; CDDP, cis-~latinum; VLB, vinblastine; MMC, mitomycin C; 5-FU, 5-fluorouracil; AC-D, actinomycin D. a, significantly different from control (p <0.01), , significantly different from drug alone or req-IFN alone (p <0.01). C, significantly different from rec-y-IFN alone (p <0.001). rl, significantly different from MMC alone (p <0.05). ', significantly different from drug alone or req-IFN alone (p <0.05). NS, not significant. Paired Student's t test.

641

8 ADM Req!Fil (µi/el.) (U./ol.)

0 0.01

0 0 0 0.01 0.01 0.01

COOP RecrIFN (µi/rJ.) (U./cd.)

.

0 ..i 0±4 0 10 100 1000 10 100 1000

*d

0

0

10 100

0 0 0.01 0.01 0.01

90±5

MMC Recr!FN (µ~/Ill.) (U./o!.)

0

1000 10 100 1000

56±6

,;..i

82±5

MMC Rec,!FN (µi/11.) (U./ol.)

(l 0.0-01 0.001 10 .0.001 100 0.001 1000

0.001

0 0.001 10 0.001 100 0.001 1000

*' 78±5

5-FU Rec;IFN

5-FU Recr!FN

(µ~/I'll.) (U./fll.)

0.1 0.1 0,1 0.1

0 0.01

(µ~/r!J.) (U./ol.)

0 10 100 1000

0.01 0.01 0.01 0 .01

0

10 20 30

40 50

60

0 10 100 1000

70 80 90 100

0

% Inhibition of Colony Formation

10 20 30 40 50 60 70

80 90 100

% Inhibition of Colony Formation

FIG. 2. inhibition by rec,-IFN alone or in combination with anticancer drugs of A, KO-RCC-1 and B, RCC-nu-1 cells. Values represent means S.E.M. *, synergistic effect. Abbreviations used: ADM, doxorubicin; MMC, mitomycin C; CDDP, cis-platinum; 5-FU, 5f1uorouracil. •, significantly different from control <0.01). h, significantly different from control <0.05).' significantly different from ADM only <0.01). a, significantly different from only (p <0.05), ', significantly different from alone <0.05). r, significantly different from alone (p <0.01). ", significantly different from 5-FU alone (p <0.01). ', significantly different from 5-FU alone (p <0.0,5). NS not significant. Paired Student's t test. '

TABLE

2. ID 50 ( µg./ml.) of anticancer drugs {?iven alone to established line cells using in double-layer soft agar system Drugs

KO-RCC-1 Cells

RCC-nu-1 Cells

0.08

0.005 0.2

Doxoruhicin

0.4 0.008 0.002 0.1 0.0002 >1.0

IV1itomycin C ,5-fluorouracil Actinon1ycin-D J,048,S

TABLE 3.

0.005 0.1 >1.0 0.0008 1.0

Colony formation in cultured line cells treated with various combinations of req-IFN and anticancer drugs KO-RCC-1

Drugs (µg./ml.) 0 100 Doxorubicin (0.01) Cis-platinum (0.1) Vinblastine (0.0001) Mitomycin C (0.001) 5-fluorouracil (0.1) Actinomycin D (0.0001) 40487S (LO)

92

65 88 74 50 58 78

rec-y-lFN (U./ml.) 10 100 91 68'a 55+h SONS 52·b 62$b 42"b 73NS

85 45'b 5z+b 65+b 32·b 62'b 6"b 72"'

RCC-nu-1 rec-y-!FN (U./ml.) 100

1000

0

10

59 lO'b 33+a 55NS 22'a 62#b 2•a 53NS

100 36 60 100 100 84 54 88

98 32+NS 48"b lOONS 73•a 82NS 5o+b so··

98 25··

45•a 94NS 82'" SONS 38'' 77·,

1000 96 g•a 42·· s2•a 7o·b so+a 30'b 72"b

Numbers are the percentage of surviving colonies, calculated as the number of colonies in a treated culture divided by the number of colonies in the control, times 100. • Synergistic effect; + additive effect; £ subadditive effect; 'interference effect; # antagonistic effect. Evaluation of effectiveness of the combined treatment was done by tbe method of Valeriote and Lin.11 "Significantly different from drug alone or rec-y-IFN alone (p <0.05). b Significantly different from drug alone or rec-yIFN alone (p <0.01). NS Not significant. Paired Student's t test.

542

GOHJI AND ASSOCIATES

The inhibition by CDDP or 5-FU was significantly greater when cells were pre-treated with rec-y-IFN than when the agents were given simultaneously with IFN; inhibition by ADM was similar with either treatment to rec-y-lFN (fig. 3). Inhibition of colony formation in primary renal cell carcinomas. In 11 of 16 patients (69 per cent) who underwent nephrectomy for renal cell carcinoma, the colony-forming ability of the malignant cells was sufficient to evaluate the efficacy of the anticancer agents. With the other five cases, the number of colonies in the control dish was less than 30. The PE values of the cells from these 11 patients ranged from 0.13 X 10-3 to 9.20 X 10-:i, and were not correlated with sensitivity to the drugs. The ID 50 was calculated for the specimen from only one patient (case 3) treated wtih 1000 U./ml. rec"(-IFN alone. The IDso of the other drugs could not be calculated in any case. When rec"(IFN was also given, however, calculation of IDso was possible for all of the drugs except 5-FU in cells obtained from about one third or half of the patients. Synergistic effects were often observed between the IFN and ADM, CDDP, or VLB (table 4).

DISCUSSION

The antitumor effects of "(-IFN are reported to be 100 times more potent than those of a- or /3-IFN.13 The effects of rec"(IFN were the strongest among the three IFNs in our study, but the differences were much smaller than in the above report. The effects of anticancer drugs used in combination with a- or /3-IFN have been studied in vitro 14- 16 and in vivo; 17 they appear to vary widely with the method of administration and concentration of the drugs as well as the tumor cells studied. Kataoka et al. 14 suggested that IFN together with vincristine and ADM modifies microtubules of the cells, and Inoue et al. 18 found that IFN may enhance the binding of AC-D and CDDP to cellular DNA, thereby increasing the metabolic activity of the cells. IFN may also inhibit the metabolism of 5-FU, since IFN inhibited the release of uridine from cells, 19 or increase its uptake by cells. 10 Greater antitumor effects were observed by treating the cells first with IFN and then with anticancer drugs than by treating them with both agents simultaneously. 14• 18 Our

FIG. 3. A 100

s'1. C

0

·.;; Ill

...0E ....

50

>, C

C

0

0

u a C

a

0

d

B 1001

~

ADM

CDDP a

s'1. C

.Q

..., Ill

50

E L

....0

>, C

0

0

0

a NS -Ns

0 0

0.01

0.1

1.0

0

0.01

0.1

1.0

0

0 .01

0 .1

1 .0

Drug Concentration (µg,/m/),,)

FIG. 3. Effects of doxorubicin (ADM), cis-platinum (CDDP) and 5-fluorouracil (5-FU) used in combination with rec-y-lFN in different ways on colony formation of A, KO-RCC-1 and B, RCC-nu-1 cells. 0-0 Drugs alone. 8-9 Drug and 100 units/ml. rec,,-IFN used simultaneously. X-X Drug and 100 U./ml. rec,,-IFN given after 24 hours of pretreatment with 100 U./ml. rec,,-IFN. Values represent means ± S.E.M. •, significantly different from control (p <0.01). h, significantly different from control (p <0.05). C, significantly different from drug alone (p <0.01). d, significantly different from drug alone (p <0.05). e, significantly different from drug and rec,,-IFN used simultaneously (p <0.01). r, significantly different from drug and rec,,-IFN used simultaneously (p <0.05). NS, not significant. Paired Student's t test.

543 TABLE

4. Colony fonnation in 11 primary renal cell carcinonia.s with colony growth treated with various cornbinatzons of rec-y-LPN- and anticancer drugs rec-y-IFN (1000 U./ml.)

Case No. 1

2 3

4 5 6 7 8

9 10 11

PE (x10-')

9.20 0.24 0.62 0.50 0.28 0.15 0.13 0.34 0.59 0.45 0.13

recy-!FN (1000 U/ml.) 72 52 38 58 58 92 78 90

79 92 62

ADM (0.1 µg./ml.)

CDDP (0.1 µg./ml.)

0

rec-y-IFN

0

rec,,-IFN

95 90 95 60 100 100 100 100 100 95 60

90' 32' 80' 46' 52· 82' 72'

95 100 100 75 100 95 100 95

35+ 85' 48' 30" 53+ 50" 90' 75·

so· 65' 42' 18"

98 80

75' 25"

+ Drugs

VBL (0.1 µg./ml.) 0

recy-IFN

5-Fu (1.0 µg./mL) 0

rec-y-!FN

92

85"'

92 82 100 75 55 80 55

58' 95"

so·

100

95•

33• 62' 32'

~ Not tested. Values are the percentage of surviving colonies.· Synergistic effect;+ additive effect;' subadditive effect;' interference effect;# antagonistic effect. The statistical analysis was not done since the number of each experiment was too small.

findings support the speculation by Inoue et al. 18 that pretreatment with IFN enhances the cytotoxic effect of CDDP. Furthermore, the time-dependency of 5-FU suggests that a larger amount of active 5-FU may be taken up by the cells as more cells enter the S phase during the pre-treatment with IFN and undergo changes in their membrane. In our study, higher doses of rec')'-IFN enhanced the effects of some anticancer drugs. Particularly, the growth of RCC-nu1 cells, which was negligibly affected by rec')'-IFN alone, was inhibited by these combinations, suggesting their clinical usefulness for therapy. IFN also had very different effects on the two established cell lines of the same histological type, suggesting ths.t IFN may act by a mechanism different from those of other anticancer drugs. This study employed not only established cultured cells but also cells collected from resected carcinomas, so it reflected the clinical stiuation better than studies in which only established cell lines were used. 14· 16· 18 The ID 50 against renal cell carcinoma could not be determined for any of the drugs examined except for req-IFN; there are no clinically effective drugs for this tumor yet. 2 Synergistic effects were observed between IFN and ADM, CDDP, or VLB when used against cells obtained from some patients, as had been observed with established cell lines. However the extent of the effect varied considerably with the individual. To use the same protocol for all patients as in conventional chemotherapy for cancer is probably to be avoided. Stolfi et al. 20 succeeded in reducing the toxicity of 5-FU by administering it with IFN or an IFN inducer in mice. With selection of an optimum combination of drugs for individual patients m vitro clonogenic some combinations of rec-1-IFN an anticancer drug be effective for advanced renal cell carcinomas. REFERENCES L Ueno, A.: Malignant renal tumors. Jap. J. Clin. Med., 4l(Suppl.): 1392, 1983. 2< Dekernion, J. B.: Treatment of advanced renal cell carcinoma: traditional methods and innovative approaches. J. Urol., 130: 2, 1983. 3. Dekernion, J.B., Sarna, G., Figlin, R., Lindner, A. and Smith, R. B.: The treatment of renal cell carcinoma with human leukocyte alpha-interferon. J. Urol., 130: 1063, 1983. 4. Kirkwood, J.M., Harris, J.E., Vera, R., Sandler, S., Fischer, D.S., Khandekar, J., Ernstoff, M. S., Gordon, L., Lutes, R., Bonomi, P., Lytton, B., Cobleigh, M. and Taylor IV, S. J.: A randomized study of low and high doses of leukocyte a-interferon in metastatic renal cell carcinoma: the American Cancer Society collaborative trial. Cancer Res., 45: 863, 1985. 5. Lieber, M. M.: Soft agar colony formation assay for in vitro chemosensitivity testing of human renal cell carcinoma: Mayo

clinic experience. J. Urol., 131: 391, 1984. 6. Hamburger, A. W. and Salmon, S. E.: Primary bioassay of human tumor stem cells. Science (Wash. DC), 197: 461, 1977. 7. Von Hoff, D. D., Clark, G. M., Stogdil!, B. J., Sarosdy, M. F., O'Brien, M. T., Cascer, J. T., Mattox, D. E., Page, C. P., Cruz, A. B. and Sandbach, J. F.: Prospective clinical trial of a human tumor cloning system. Cancer Res., 43: 1926, 1983. 8. Okada, Y., Gohji, K., Kamidono, S. and Tatewaki, 0.: Establishment and characterization of a cell line (KO-RCC-1) from a human renal cell carcinoma. Jpn. J. Urol., 77: 780, 1986. 9. Sawaguchi, K., Takahashi, Y., Akimoto, R., Mai, M., Hatano, M., Tanaka, J. and Ohno, S.: New established human cell line divided from the clear cell carcinoma of the kidney and the chemosensitivity of this cell line. Proc. Jpn. Cancer Assoc. (abst.), 43: 214, 1984. 10. Taniguchi, T., Pang, R.H. L., Yip, Y. K., Henriksen, D. and Vilcek, J.: Partial characterization of 'Y (immune) interferon mRNA extracted from human lymphocytes. Proc. Natl. Acad. Sci. U. S. A., 7§: 3469, 1981. 11. Valeriote, F. and Lin, H.: Synergistic interaction of anticancer agents: a cellular perspective. Cancer Chemother. Rep., 59: 895, 1975. 12. Gohji, K., Murao, S., Maeda, S., Sugiyama, T. and Kamidono, S.: Enhanced Inhibition of colony formation in soft agar in human renal cell carcinoma by the combination with alpha-difluoromethy!ornithine and recombinant gamma interf~ron. Cancer Res., 46: 6264, 1986. 13. Crane, J. L., Glasgow, L. A., Kem, E. R. and Youngner, J. S.: Inhibition of murine osteogenic sarcomas by treatment with type I or type H interferon. J. Natl. Cancer Inst., 61: 871, 1978. 14. Kataoka, T., Oh-hashi, F. and Sakurai, Y.: Enhancement of antiproliferation activity of vincristine and adriamycin by interferon. Jpn. J. Cancer Res. 75: 548, 1984. 15. Oku, T., Imanishi, J. and T.: Assessment of anti-tumor cell effect of human leukoycte interferon in combination with anticancer agents a convenient assay system in monolayer cell culture. Jpn. J. Res<, 73: 667, 1982. 16. Namba, M., 1vuvu,>1u. T., Kanamori, T., Nobuhara, Kimoto, T. and Ogawa, · Combined effects of 5-fluoromacil interferon on proliferation of human neoplastic cells in culture. Jpn. J< Cancer Res. (Gann), 73: 819, 1982. 17. Marquet, R. L., Schellekens, H., Westbroek, D. L. and Jeekel, J.: Effect of treatment with interferon and cyclophosphamide on the growth of a spontaneous liposarcoma in rats. Int. J. CanceL, 31: 223, 1983. 18. Inoue, M. and Tan, Y. H.: Enhancement of actinomycin D- and cis-diamminedichloroplatinum(H)-induced killing of human fibroblasts by human f:l-interferon. Cancer Res., 43: 5484, 1983. 19. Degre, M. and Hovig, T.: Functional and ultrastructural studies of the effects of human interferon on cell membranes of in vitro cultured cells. Acta Pathol., Microbiol. Scand. (Sect. B.), 84: 347, 1976. 20. Stolfi, R. L., Martin, D. S., Sawyer, R. C. and Spiegelman, S.: Modulation of 5-fluorouracil-induced toxicity in mice with interferon or with the interferon inducer, polyinosinic-polycytidylic acid. Cancer Res., 43: 561, 1983.