[CANCER RESEARCH 43, 5462-5466. November 1983] Effect of Human Fibroblast Interferon on the Antiviral Activity of Mammalian Cells Treated with Bleomycin, Vincristine, or Mitomycin C1 Robert J. Suhadolnik,2 Yosuke Sawada,3 Maryann B. Flick, Nancy L. Reichenbach, and Joseph D. Mosca3 Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 ABSTRACT protein kinase (2,9,28,34,36). In addition to the use of interferon in the treatment of cancer (1, 11, 12, 29, 34), combination Bleomycin, vincristine, or mitomycin C, when added to HeLa chemotherapy of interferon and methotrexate, c/s-platinum diam- cells simultaneously with human fibroblast interferon (IFN-0), minedichloride, cyclophosphamide, or 1,3-bis(/i-chloroethyl)-1- caused a decrease in cell density and inhibited DMA synthesis nitrosourea on either tumor cells in culture or in leukemic mice compared with HeLa cells treated with IFN-/3 alone. However, has been reported (5, 7, 10, 25). Furthermore, Stolfi ef al. (37) the IFN-0-induced antiviral processes were unaffected by the reported recently that the administration of mouse interferon to presence of these drugs as determined by in vitro enzyme assays mice following the administration of 5-fluorouracil protected the and the development of the antiviral state in the intact HeLa cell. mice from mortality. Because it is possible to selectively inhibit HeLa cells treated with IFN-/Õalone or with IFN-/3 in combination proliferation of tumor cells with chemotherapeutic drugs, we with bleomycin, vincristine, or mitomycin C were able to induce reasoned that the ability of the normal cell to maintain the antiviral the double-stranded RNA-dependent adenosine triphos- phate:2',5'-oligoadenylic acid adenyltransferase (EC 2.2.2.-) and state might be adversely affected by such drugs and that the antiproliferative action of interferons might affect the activity of the double-stranded RNA-dependent protein kinase. Further drugs used in the treatment of human cancers. To provide a firm more, the antiviral state as measured by the reduction of plaque- foundation for the use of interferons in combination with antineo- forming units after infection of treated cells (with IFN-0 alone or plastic drugs in the treatment of cancer, we have determined the with IFN-0 plus drugs) with vesicular stomatitis virus was not effect of combination treatments of IFN-/3 added simultaneously affected. These results indicate that, under these experimental with either bleomycin, mitomycin C, or vincristine on cell metab conditions, the double-stranded RNA-dependent adenosine tri- phosphate^',5'-oligoadenylic acid adenyltransferase and pro olism, the potentiation of interferon action, and induction of the antiviral state. tein kinase can be induced by IFN-,3 in cells treated with bleo mycin, vincristine, or mitomycin C. These cells also develop the MATERIALS AND METHODS antiviral state. These experiments could provide a basis for a careful examination of the effects of interferon on the develop Interferon and Other Chemicals. IFN-/J (8 x 105 lU/mg protein) was ment of the antiviral state when testing potentially active anti- obtained from Dr. C. Baglioni, State University of New York at Albany, neoplastic agents. The possibility that IFN-/3 potentiates the and Dr. D. S. Rabson, Chief, Cancer Biology and Drug Program, NIH; cytotoxic effects of bleomycin and mitomycin C on HeLa cells is sheep-specific antiserum to IFN-/5 and sheep mock antiserum were also discussed. obtained from Dr. S. Cunningham, National Institute of Allergy and Infectious Diseases. Bleomycin and mitomycin C were from Sigma Chemical Co. (St. Louis, Mo.); vincristine was from Eli Lilly and Co. INTRODUCTION (Indianapolis, Ind.); P. chrysogenum dsRNA was from Dr. R. J. Douthart, Interferons are a family of proteins produced in mammalian Eli Lilly and Co.; and elF-2«was from Dr. W. C. Merrick, Case Western Reserve University. Noncovalently associated polyriboinosinic-polyribo- cells in response to viral infection or other stimuli (2, 9, 28, 34, cytidylic acid, noncovalently associated polyriboinosinic:polyribocytidylic 36). There is evidence for potent, multiple effects of interferons acid:agarose, and 2',5'-oligoadenylate core dimer and core trimer were in mammalian cells. For example, interferon inhibits virus infec from P-L Biochemicals (Milwaukee, Wis.). [Me(/iy/-3H]thymidine (6.7 Ci/ tion, cell division, and cell functions; augments natural killer cell mmol) and [8-3H]ATP (22 Ci/mmol) were obtained from New England activity; modulates immune responses; and has been reported Nuclear (Boston, Mass.); [a-32P]ATP (410 Ci/mmol) and [-y-^PJATP (410 to have antitumor action (2, 3, 9,11-14, 34, 36). The anticellular Ci/mmol) were obtained from Amersham/Searle Corp. (Arlington Heights, effect of interferon was recognized as distinct from its antiviral III.). VSV and tritiated T-4 DNA were gifts from Dr. Earl E. Henderson, activity by Paucker ef al. (27) in 1962. The antiviral response of Temple University School of Medicine. interferons in mammalian cells appears to be mediated by the Cell Culture. HeLa S-3 cells (Flow Laboratories, McLean, Va.) were induction of the dsRNA4-dependent 2',5'-An synthetase and a maintained in suspension culture in Joklik's modified MEM, pH 7.3, supplemented with 5% fetal calf serum and 5% calf serum (Grand Island 1Supported in part by BiomédicalResearch Support Grant S07 RR05417 from Biological Co., Grand Island, N. Y.) (generation time, 20 to 22 hr). Vero the Division of Research Resources, NIH. cells (Flow Laboratories) were grown in monolayer culture in Eagle's 2 Recipient of NIH Research Grant GM-26134. To whom requests for reprints MEM, pH 7.0, supplemented with 6% fetal calf serum. Cells were counted should be addressed. 3 Recipient of partial support from USPHS Training Grant 5-T32 AM-07162. in a hemacytometer, and cell viability was determined by trypan blue 4The abbreviations used are: dsRNA, double-stranded RNA; 2',5'-A„synthe exclusion. Twenty-four hr prior to treatment, the cells are resuspended tase, ATP:2',5'-oligoadenylic acid adenyltransferase (EC 2.2.2.-; 2' ,5'-A„,oligomer in fresh medium. Immediately before treatment, the cells are diluted for of adenylic acid with 2',5'-phosphodiester linkages and a triphosphate at the 5'- 2 x 106 cells/ml in the same medium and aliquoted to separate flasks end); IFN-(i, human fibroblast interferon; elF-2«, «subunit of eukaryotic initiation for treatment. Concentrations are given in the tables and figures. factor 2; VSV, vesicular stomatitis virus; MEM, minimal essential medium; SDS, 2',5'-An Synthetase Assay. 2'.5'-A, synthetase was assayed by 2 sodium dodecyl sulfate; PFU, plaque-forming units. Received May 6. 1983; accepted July 29, 1983. methods. Treated HeLa cells were lysed, and the synthetase was as- 5462 CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1983 American Association for Cancer Research. IFN-ßandAntineoplastic Agents in HeLa Cells sayed as described (21). Protein in these lysates was determined ac cording to the method of Lowry ef al. (18). HeLa cells treated as described under interferon neutralization assay were lysed, and the 2',5'-A„syn- thetase was assayed as described (20, 33). Protein in these lysates was IO determined according to the method of Bradford (4). Incorporation of [3H]Thymidine into Acid-insoluble Material. Two- O mi aliquots of cell culture were centrifuged (1400 x g, 1 min), and the cell pellets were washed once with 1 ml of Spinner's buffer. Acid-insoluble radioactivity was collected on Whatman GF/A filters and washed with 3 ml of 5% trichloroacetic acid and 5 ml of 95% ethanol. Filters were dried under an infrared lamp, and radioactivity was determined in a Beckman A Model LS-100C liquid scintillation spectrometer using Scintillation Fluid I Z 949 (New England Nuclear). Alkaline Sucrose Gradient Centrif ugation. Lysis of cells and alkaline sucrose gradients of DNA were performed as described by Krokan ef al. (15). Gradients were fractionated dropwise from the bottom (20 drops/ fraction); 3 ml of 10% trichloroacetic acid, 0°, plus 200 /ig of bovine serum albumin were added to each fraction. The trichloroacetic acid precipitate was collected on Whatman GF/A filters and washed with 3 ml of 5% trichloroacetic acid and 5 ml of 95% ethanol. The filters were < s dried under an IR lamp, and the radioactivity was determined. Protein Kinase Assay. The interferon-induced dsRNA-dependent pro O 20 40 60 246 004 0.12 tein kinase was assayed in ribosomal fractions prepared by centrifugation Bleomycin Mitomycin C Vincristine of homogenized lysates at 225,000 x g for 1 hr at 4°as described (17). The reaction was terminated by the addition of 10 n\ of H2O and 5 M!of buffer containing 50 mM Tris-HCI (pH 7.4), 0.8% SDS, and 2.5% 2- Chart 1. Effect of increasing concentrations of bleomycin, mitomycin C, and vincristine on cell density, incorporation of [3H]thymidine into DNA, and the induction mercaptoethanol. Each sample was heated 5 min in a steam bath of 2',5'-A„synthetase in IFN-tf-treated cells. HeLa cells in the log phase of growth followed by the addition of 5 n\ of 2% bromophenol blue in glycerol. The (2 x 10s cells/ml, 50 ml) were treated simultaneously with IFN-fi (100 units/ml) and samples were applied to SDS slab gels (0.1 % SDS: 10% acrylamide:0.3% either bleomycin, mitomycin C. or vincristine at the concentrations indicated. Twenty bisacrylamide, 1 mm thick) and electrophoresed as described (17). Gels hr later, cell density, [3H]thymidine incorporation into DNA, and 2' ,5'-A„synthetase activity were determined. A, cell density; B, [3H]thymidine incorporated into DNA; were stained with 0.05% Coomassie blue in 10% acetic acid:33% 2- C, 2',5'Ai synthetase activity (100% represents the values from cells treated with propanol overnight at 25°,destained (soaking the gel once in 7% acetic IFN-o alone); 1, bleomycin; 2, mitomycin C; 3, vincristine (100% of 2',5'-A„ acid:30% 2-propanol and 3 to 4 times thereafter in 7% acetic acid), dried, synthetase represents 45,000 dpm) (C).