Effect of Human Fibroblast Interferon on the Antiviral Activity of Mammalian Cells Treated with Bleomycin, Vincristine, Or Mitomycin C1

[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-/Ã•alone 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 triphosphate: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 triphosphate^',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-2Â«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 BiomÃ©dicalResearch 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'-Aâ€žsynthe exclusion. Twenty-four hr prior to treatment, the cells are resuspended tase, ATP:2',5'-oligoadenylic acid adenyltransferase (EC 2.2.2.-; 2' ,5'-Aâ€ž,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-2Â«, Â«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-ÃŸandAntineoplastic 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'-Aâ€žsyn- 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, 0Â°, 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 4Â°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'-Aâ€žsynthetase 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'-Aâ€žsynthetase 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 25Â°,destained (soaking the gel once in 7% acetic IFN-o alone); 1, bleomycin; 2, mitomycin C; 3, vincristine (100% of 2',5'-Aâ€ž acid:30% 2-propanol and 3 to 4 times thereafter in 7% acetic acid), dried, synthetase represents 45,000 dpm) (C). Cell density is a mean of 10 determinations. and autoradiographed. [3H]Thymidine incorporation into DNA is a mean of 4 determinations; 100% repre VSV Titration. VSV was added to 1.5 to 2 x 106 HeLa cells (0.2 ml) sents 18,500 dpm (B). Data are normalized by using the same concentration of at a multiplicity of infection of 0.1 and allowed to adsorb at 37Â°for 1 hr. protein. After 1 hr, the infected cells were diluted to 106/ml with Eagle's MEM plus 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (pH 7.3), of [3H]thymidine into acid-precipitable material did not diminish incubated 9 hr at 37Â°, and frozen at -70Â°. After 3 rounds of freezing and thawing, the cell debris was pelleted (1000 x g, 15 min, 4Â°).The to zero. supernatant was used in a plaque assay modified from that of Stanwick Interferon-induced Enzymes and Antiviral Activity. The an ef al. (35) to measure the yield of VSV as PFU/ml. Briefly, monolayers of tiviral activity of HeLa cells treated with bleomycin, mitomycin C, Vero cells in 60-mm dishes were inoculated with dilutions of supernatant, or vincristine alone or added simultaneously with IFN-ÃŸwas overlaid with 0.4% Seaplaque agarose (FMC Corp., Rockland, Maine) in determined. When HeLa cells were treated with IFN-ÃŸ(100units/ Eagle's MEM with 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic ml) for 20 hr, 2',5'-An synthetase activity was induced 11-fold acid (pH 7.3). The dishes were incubated 24 hr, fixed, and then stained (Chart 1C; 11-fold stimulation equals 100%). The simultaneous according to the method of Rager-Zisman and Merigan (30), and plaques addition of IFN-ÃŸwith increasing concentrations of bleomycin (0 were counted. to 20 Mg/ml), mitomycin C (O to 0.5 ^g/ml), or vincristine (0 to Interferon Neutralization Assay. Antiserum to IFN-.; was titrated 0.08 Â¿ig/ml)didnot affect the induction of the 2' ,5'-An synthetase according to Research Reference Reagent Note 24 (31 ). Neutralization of IFN-/3 is described in the legend to Table 2. activity (Chart 1C; Table 1), even though there was a decrease in cell density and a decrease in the incorporation of [3H]thymi-

RESULTS dine into trichloroacetic acid precipitates (Chart 1, A and B). Similarly, there was little or no effect on the ability of the HeLa Bleomycin, Mitomycin C, and Vincristine Treatment of HeLa cells to induce the synthesis of the protein kinase when IFN-/3- Cells. Before studying the combined effect of IFN-ÃŸand bleo- treated cells were compared with IFN-ÃŸ-treated HeLa cells to mycin, mitomycin C, or vincristine treatment on the antiviral state which bleomycin (10 /Â¿g/ml),mitomycin C (0.5 Ã/g/ml),or vincris of HeLa cells, the cell density and incorporation of [3H]thymidine tine (0.8 ng/m\) was added simultaneously (Table 1). The IFN-/3- into the trichloroacetic acid precipitates were measured using induced protein kinase activity was analyzed by a 2-step assay. cells treated simultaneously with bleomycin, mitomycin C, or In Step 1, to reduce the number of phosphorylated proteins, vincristine and IFN-ÃŸfor 20 hr. Addition of bleomycin, mitomycin protein kinase activation was assayed with or without dsRNA C, or vincristine (at concentrations approximating serum concen (20 /Â¿g/ml)and without ATP. In Step 2, the reactions were trations after therapeutic doses) plus IFN-/3 produced dose- supplemented with elF-2a and [a-32P]ATP. The autoradiogram dependent decreases in cell density and incorporation of after gel electrophoresis shows clearly that the phosphorylation [3H]thymidine into the trichloroacetic acid precipitates (Chart 1, of elF-2Â«was enhanced when the HeLa cells were treated with A and B). Cell densities and the decreased incorporation IFN-ÃŸin the presence of dsRNA, compared to untreated cells

NOVEMBER 1983 5463

Table 1 Effect of bleomycin, mitomycin C, and vincristine when added simultaneously wth IFN-ÃŸto HeLa cells on the induction of the dsRNA-dependent enzymes and the antiviral activity Untreated and treated cells (Chart 1) were infected with VSV, and viral yields were determined as described in "Materials and Methods." Data are the mean of duplicate determinations. 2',5'-Aâ€žsynthetase and protein kinase were assayed as described in "Materials and Methods." Total 2',5'A, synthetase activity was calculated and expressed relative to the untreated control. A value of 1.0 is equivalent to 4090 dpm [^PJATP incorporated into 2',5'-Aâ€žper >ig protein per min. The autoradiogram of the protein kinase assay gel (Chart 2) was scanned at 560 nm using a Model SD200 spectrodensitometer with a SDC 300 density computer (Scheoffel Instrument Corp.). Quantitation of the phosphorylation of elF-2Â« was done by calculating the ratio of the absorbance peak height of the elF-2n band to the peak height of a reference band (M, 54,000) which was phosphorylated to the same extent in all cases. treatmentDrug of2 titerPFUx

dose ',5 '-Aâ€žsyn ki 10e/10Â« PFU/ Experiment1234567891011121314IFN-0(units/ml)0100100100001001000010010000Cell(ng/ml)00Bleomycin Dose thetase111.011.612.11110.98.51111.010.711Proteinnase0.67.95.36.59.4VSVcells/ml161.40.20.10.60.40.50.21.10.90.30.21.20.4log,oml2.4-1.1-1.9-2.2-1.4-1.6-1.5-1.9-1.2-1.2-1.7-1.9-1.1-1.6

510510Mitomycin

0.52.00.52.0VincristineC

0.040.080.040.08Induction

IO'3 (Fig. 1, Lanes 1 and 5). Similarly, when cells were treated with IFNM in combination with either bleomycin (Fig. 1, Lanes 7 and 2), mitomycin C (Fig. 1, Lanes 7 and 3), or vincristine (Fig. 1, Lanes 7 and 4), the phosphorylation of elF-2Â« was increased when compared to untreated cells (Fig. 1, Lane 5). The phospho rylation of elF-2Â«was quantitated by scanning the autoradiogram and comparing the absorbance peak heights of the elF-2Â«bands. Ribosomal preparations from untreated cells show little phos phorylation of elF-2Â«with or without dsRNA present (data not shown). To supplement the data on the induction of the 2',5'-An â€”30 synthetase and protein kinase activity, additional experiments were done to determine the development of the antiviral state by infecting HeLa cells with VSV. These experiments were â€” 21 performed by comparing the antiviral state of HeLa cells treated only with bleomycin, mitomycin C, or vincristine with the antiviral state of HeLa cells treated simultaneously with the drugs and IFN-/3. The antiviral activity as determined by PFU/ml of VSV- Lane 12345 Fig. 1. Effect of bleomycin, mitomycin C, and vincristine on the dsRNA-depend- infected cells was determined, and the virus titer was measured ent phosphorylation of the elF-2Â«in IFN-/j-treated cells. HeLa cells were obtained in a standard plaque assay on Vero cell monolayers following from the experiment in Chart 1. Assays were as described in "Materials and VSV infection of treated cells. IFN-(j in combination with bleo Methods." Cells were treated as follows: Lane 1. IFN-/Ã•(100 units/ml); Lane 2, IFN- ti (100 units/ml) plus bleomycin (10 Mg/ml); Lane 3, IFN-/J (100 units/ml) plus mycin, mitomycin C, or vincristine prevents the replication of mitomycin C (2 ng/ml); lane 4, IFN-J (100 units/ml) plus vincristine (0.08 wg/ml); VSV (Table 1). Bleomycin, mitomycin C, and vincristine treatment Lane 5, no treatment. Protein markers were: phosphorylase Ã¼(M,94,000); bovine of HeLa cells also decreased the PFU/ml. serum albumin (M, 68.000); ovalbumin (M, 45,000); carbonic anhydrase (M, 30,000); and chymotrypsinogen (M, 25,000). Effect of Antiinterferon Serum on the Antiviral State and the Induction of 2'.5'-A,, Synthetase Activities of IFN-/3- treated HeLa Cells. To eliminate the possibility that impurities Size Analysis of the Newly Synthesized DNA of the IFN-0- in the IFN-Â¿were responsible for the antiviral activity and the treated HeLa Cells. To determine that the incorporation of induction of 2',5'-An synthetase, specific sheep antiserum to [3H]thymidine into trichloroacetic acid precipitates represented IFN-|tf or the corresponding mock antiserum was added to IFN- incorporation into DNA, size analysis of the newly synthesized ÃÂ¡beforethe addition of the IFN-0 to HeLa cells. Antiserum to DNA was done by alkaline sucrose gradient centrifugation (Chart IFN-/i neutralized the antiviral activity of IFN-/j (Table 2). The 2). The size of the DNA strands of the IFN-/i-treated asynchro- mock \FN-ii antiserum did not neutralize IFN-ÃŸ(data not shown). nously grown HeLa cells was the same or slightly higher than The induction of 2',5'-An synthetase was also prevented by that observed for the untreated cells. Size analysis of DNA by neutralization of IFN-ji with the specific antiserum (Table 2). alkaline sucrose gradient centrifugation demonstrates that the

5464 CANCER RESEARCH VOL. 43

Table 2 Neutralizationof the antiviral activity and the induction of2',5'-Aâ€žsynthetase bleomycin, mitomycin C, and vincristine) and the antiviral activity activity of IFN-ÃŸbyanti-IFN-ÃŸserum of interferon. IFN-0 was incubated for 1 hr at 37Â°with either antisemm to IFN-tf (effective The work reported here correlates the effects of IFN-ÃŸand IFN-tf neutralizingtiter = 51.2 units/ml), an equal volume of mock antiserum, or an bleomycin, mitomycin C, and vincristine alone or in combination equal volume of sterile phosphate-buffered 0.9% NaCI solution. The pretreated IFN-0was then added to cells (5 x ICP/ml,50 ml). After 20 hr, cells were counted, on the maintenance of the antiviral state, cell density, incorpo and 2',5'-Aâ€žsynthetasewas assayed as described in "Materials and Methods." ration of [3H]thymidine into DNA, and the induction of the dsRNA- For viral titer, cells (2 x 10") from each culture were infected with VSV. Total 2',5'- dependent 2',5'-An synthetase and the dsRNA-dependent pro Aâ€žsynthetaseactivity was calculated and expressed relative to the untreated control. A value of 1.0 is equivalent to 0.74 pmol of ATP incorporated into 2',5'-Aâ€ž tein kinase. There are several interesting aspects of the findings synthetase per ^g protein per min. Data reported are the result of duplicate reported here, (a) The development of the antiviral state of the determinations. cell as determined by inhibition of virus replication, the induction syn of the dsRNA-dependent 2',5'-An synthetase, and induction of thetase activ protein kinase is not affected when IFN-ÃŸis added simultane TreatmentNone ity1.0 yield(PFU/ml)4.2 x 10Â« ously with either bleomycin, mitomycin C, or vincristine or when IFN-/ÃŒ(100units/ml) 24.6 1.1 x 10s HeLa cells are treated with IFN-ÃŸalone. The inhibition of cell IFN-0(100 units/ml) + anti-IFN-/3serum2',5'-Aâ€ž 1.9VSV 4.8 x 10Â« proliferation and the inhibition of incorporation of [3H]thymidine into DNA by bleomycin, mitomycin C, or vincristine at the con centrations used did not interfere with the ability of the cell to o respond to IFN-ÃŸandto develop the antiviral state, as determined A 10 by the inhibition of VSV replication, the induction of 2',5'-An w T4 dcDNA O a synthetase, and the induction of the protein kinase. o (b) Preliminary experiments were done to determine if IFN-ÃŸ o could function as a positive or negative modulator of bleomycin and mitomycin C activity. Similar types of experiments were a> x reported by Stolfi ef al. (37); they demonstrated that the toxicity II of 5-fluorouracil yielded significant protection from mortality when mouse interferon was administered following the administration of 5-fluorouracil. However, protection against 5-fluorouracil tox icity was observed when polyinosinic-polycytidylic acid was ad ministered simultaneously with 5-fluorouracil. In our experiments, exposure of HeLa cells to bleomycin (10 Â¿tg/ml)showed a 49% O 10 20 30 inhibition of incorporation of [3H]thymidine into DNA. However, Fraction Number the simultaneous addition of IFN-ÃŸ(100 units/ml) to HeLa cell BOTTOM TOP cultures containing bleomycin (10 u.g/m\) showed a 76% inhibition of incorporation of [3H]thymidine into DNA. Similarly, the simul Chart 2. Alkalinesucrose gradientcentrifugationof IFN-0-treatedand-untreated cells. IFN-Â£)-treatedand-untreated HeLa cells (2x10* cells, 5 ml) were harvested taneous addition of mitomycin C (10 Mg/ml) and IFN-ÃŸ(100units/ 20 hr after treatment and incubated with [3H]thymidine(10 Â»iCi/ml)asdescribed in ml) inhibited the incorporation of [3H]thymidine into DNA by 58%.5 "Materials and Methods." Cells were washed twice with phosphate-buffered0.9% NaCI solution at 4Â°.The DNA from nuclei was subjected to alkaline sucrose To our knowledge, this is the first report on the influence of gradient centrifugation (34).â€¢,untreated;A, IFN-0treated. J, position of T-4 DNA. interferon on the increased cytotoxic activity of bleomycin and dcDNA, demethylated cytosine containing T-4 bacteriophage DNA (16). mitomycin C when compared with the maintenance of the anti viral state of the cell plus the induction of the dsRNA-dependent [3H]thymidine incorporated into the trichloroacetic acid precipi 2',5'-An synthetase and protein kinase. Therefore, it appears tates of IFN-ÃŸ-treated HeLa cells was incorporated into newly that, under proper experimental conditions, IFN-ÃŸcan function synthesized DNA. CsC1 buoyant density ultracentrifugation of the [3H]DNA following the incorporation of [3H]thymidine of 5-bro- as a positive modulator of bleomycin and mitomycin C toxicity. These preliminary findings support our 2 earlier reports on the modeoxyuridine-treated cells showed a density of 1.75, which antiproliferative properties of IFN-ÃŸin HeLa cells, in that there is represents replicative DNA (data not shown). an accumulation of low-molecular-weight DNA in IFN-ÃŸ-treated HeLa cells (32, 38), which could potentiate the cytotoxicity of DISCUSSION bleomycin and mitomycin C. These preliminary observations are also in agreement with the report of Miyoshi ef al. (22) that Many agents have been developed that have increased our interferon increases the cytotoxic effects of 5-fluorouracil in understanding of cancer biology and the pharmacology of anti- human cell lines originating from neoplastic tissues. However, it cancer drugs. However, among the difficulties encountered in is important to note that BrostrÃ¶m (5) did not observe an en the treatment of human cancers with antineoplastic agents is the hanced cytotoxic effect in human osteosarcoma and lymphoma inhibition of the immune response system and interference with cell lines treated with methotrexate and interferon. The potentia the antiviral state (1, 19, 26). In addition to the use of antineo tion of interferon and drug-treated human neoplastic cell lines plastic agents in the treatment of human cancers, recent studies might be explained by combining the above observations with have reported the successful use of interferon in the treatment the recent observation by deFerra and Baglioni (8). They showed of human cancers (1, 11, 12, 29, 34). If antineoplastic drugs are an increase in the intracellular concentration of S-adenosylhom- to be used successfully in combination with interferon, it is ocysteine in interferon-treated HeLa cells. The accumulation of essential to determine the potentiation of the cytotoxic activity of drugs used in the treatment of human cancers (such as 'â€¢'Manuscriptin preparation.

NOVEMBER 1983 5465

S-adenosylmethionine and S-adenosylhomocysteine would de 10. Gresser, I., Maury, C., and Tovey, M. Efficacy of combined interferon cydo- phosphamidetherapy after diagnosisof lymphomain AKR mice.Eur. J. Cancer, crease in the formation of homocysteine, which would accumu 14: 97-99, 1978. late methyl Bi2 coenzyme and accumulate methyltetrahydrofol- 11. Gresser, I., and Tovey, M. G. Antitumor effects of interferon. Biochim. Biophys. Acta, 576:231-247,1978. ate. The accumulation of methyltetrahydrofolate would deplete 12. Gutterman, J. U., Blumanschein, G. R., Alexian, R., Yap, H., Buzdar, A. V., the cell of available tetrahydrofolate, such that there would be Cabanillas, F., Hortobagyi, G. N., Hersh, E. M.. Rasmussen, S. L., Harmon, insufficient A/5,/V10-methylenetetrahydrofolate to convert dUMP to M., Kramer,M., and Pestka, S. Leukocyte interferon inducedtumor regression in human metastatic breast cancer, multiple myeloma, and malignant lym dTMP by thymidylate synthetase and eventually to form dTTP. phoma. Ann. Intern. Med., 93: 399-406, 1980. The decreased de novo synthesis of dTTP would then inhibit 13. Harfast, B., Huddlestone, J. R., Casali, P., Merigan, T. C., and Oldstone, M. B. A. Interferon acts directly on human B lymphocytes to modulate immuno- DNA synthesis. This is also in agreement with the report of globulin synthesis. J. Immunol., 727: 2146-2150,1981. Moore ef al. (23, 24) on the accumulation of low-molecular- 14. Herberman, R. B., Ortaldo, J. R., Mantovani, A., Hobbs, D. S., Kung, H., and weight DNA and the rapid turnover of newly replicated DNA of Pestka, S. Effect of human recombinant Â¡nterferononcytotoxic activity of interferon-treated Daudi cells.6 natural killer (NK)cells and monocytes. Cell. Immunol.,67: 160, 1982. 15. Krokan, H.. Cooke, L., and Prydz, H. DNA synthesis in isolated HeLa cell (c) Treatment of HeLa cells with IFN-/3 and either bleomycin, nuclei. Evidence for in vitro initiation of synthesis of small pieces of DNA and mitomycin C, or vincristine for 20 hr before VSV infection shows their subsequent ligation. Biochemistry, 74: 4233-4237, 1975. 16. Laemmli,U. K. Cleavageof structural protein during the assembly of the head that IFN-0 is still an effective inhibitor of virus replication (Table bacteriophageT4. Nature (Lond.), 227: 680-685,1970. 1, Experiments 3, 4, 7, 8, 11, and 12). At first, it appears that 17. Levin, D. H., Petryshyn, R., and London, I. M. Characterization of purified double stranded RNA-activated elF-2 kinase from rabbit reticulocytes. J. Biol. bleomycin, mitomycin C, and vincristine had antiviral activity as Chem., 256: 7638-7641,1981. determined by VSV titer in the absence of IFN-ÃŸ(Table 1, 18. Lowry, O. H., Rosebrough, N. J.. Fair, A. L.. and Randall, R. J. Protein Experiments 5, 6, 9,10,13, and 14). However, when the antiviral measurement with the Polin phenol reagent. J. Biol. Chem., 793: 265-275, 1951. state as determined by VSV titer was compared to the activities 19. Merigan, T. C., Rand, K. H., Pollard, R. B., Abdallah, P. S., Jordan, G. W., and of the 2 dsRNA-dependent enzymes induced by interferon (;.e., Fried, R. P. Human leukocyte interferon for the treatment of herpes zoster in 2',5'-An synthetase and protein kinase), there was no decrease patients with cancer. N. Engl. J. Med., 298: 981-987, 1978. 20. Merlin, G., Revel, M.. and Wallach, D. The interferon-inducedenzyme oligoad- in the induction of these 2 enzymes by either bleomycin (0 to 10 enylate synthetase: rapid determination of its in vitro product. Anal. Biochem., Mg/ml), mitomycin C (O to 0.5 nQ/m\), or vincristine (0 to 0.08 /Â¿g/ 770: 190-196, 1981. 21. Minks, M. A., Benvin, S., Maroney, P. A., and Baglioni, C. Synthesis of ml) (Chart 1; Table 1). Therefore, these 3 antineoplastic drugs 2',5'oligo (A) in extracts of interferon treated HeLa cells. J. Bid. Chem., 254: did not interfere with the antiviral activity of interferon as meas 5058-5064,1979. ured by VSV titer and 2',5'-Aâ€žsynthetase and protein kinase 22. Miyoshi, T., Ogawa, S., Kanamori.T., Nobuhara,M., and Namba, M. Interferon potentiates cytotoxic effects of 5-fluorouracilon cell proliferationof established activities. human cell lines originating from neoplastic tissues. Cancer Lett., 77: 239- Under the conditions of the experiments described here, IFN- 247, 1983. ÃŸinhibits virus replication in the presence of the 3 antineoplastic 23. Moore, G., andClemens,M. J. Inhibitionof cell proliferationby humaninterferon associated with rapid turnover of newly replicated DNA. Biochem. Soc. Trans. drugs. It is important to note that Cesario and Slater (6) showed 11: 44, 1983. that vincristine inhibited the antiviral activity of interferon in 24. Moore, G., Gewert, D. R., and Clemens, M. J. Changes in deoxynucleoside human fibroblast cells when interferon was added either simul metabolism and DNA synthesis in interferon-treated human lymphoblastoid cells. Biochem. Soc. Trans. 70: 95, 1982. taneously with vincristine or by pretreatment of cells with vincris 25. Mowshowitz, S. L., Chin-Bow, S. T., and Smith, G. D. Interferon and cis-DDP: tine followed by the addition of interferon. combination chemotherapy for P388 leukemia in CDFi mice. J. Int. Res., 2: 587-591,1982. The data reported here indicate that the combination therapy 26. Parker, L. M., Upton, J. M., Binder, N., Crawford, E. L., Kudisch, M., and of interferon plus selected antineoplastic drugs may find appli Levin, M. J. Effect of acycyclovir and interferon on human hematopoietic cation in the treatment of certain types of cancer. Although progenitor cells. Antimicrob. Agnts Chemother., 27: 146-150, 1982. 27. Paucker,K., Cantell, K., and Henle,W. Quantitative studieson viral interference normal cells were not included in this study, Miyoshi ef al. (22) in suspended L cells. III. Effect of interfering viruses and interferon on the showed that 5-fluorouracil in combination with interferon did not growth rate of cells. Virology, 77: 324-334,1962. have additive lethal effects on WI-38 normal human cells. 28. Pestka, S. (ed.).The Interferons. Methods Enzymol. 78A, 79B, 1981. 29. Priestman, T. J. Interferon: an anticancer agent? Cancer Treat. Rev., 6: 223- 237,1979. REFERENCES 30. Rager-Zisman,B., and Merigan, T. C. A useful quantitative semi-micromethod for viral plaque assay. Proc. Soc. Exp. Biol. Med., 742: 1174-1179,1973. 1. Arvin, A. M.. Kushner, J. H., Feldman, S.. Baehner, R. L, Hammond, D., and 31. Research Reference Reagent Note No. 24. Sheep antiserum to human fibro Merigan, T. C. Human leukocyte Â¡nterferonforthe treatment of varicella in blast interferon. Bethesda, Md.: NIH, 1981. children with cancer. N. Engl. J. Med., 306: 761-765, 1982. 32. Sawada, Y., Wu, J., Reichenbach, N. L., and Suhadolnik, R. J. Effect of 2. Baglioni, C. Interferon-inducedenzymatic activities and their role in the antiviral interferon on DNA synthesis, poly-ADPR polymerase, and 2,5-A polymerase state. Cell, 77: 255-264, 1979. in asynchronized and synchronized HeLa S3 cells (abstract). Fed. Proc., 39: 3. Black, P. L., Suhadolnik, R. J., and Henderson, E. E. Augmentation of natural 1857,1980. killing by interferon-inducibleoligonucleotides (abstract). Fed. Proc., 42: 678, 33. Schattner, A., Merlin,G., Levin, S., Wallach, D., Hahn,T., and Revel,M. Assay 1983. of an interferon-inducedenzyme in white blood cells as a diagnostic aid in viral 4. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram diseases. Lancet, 2: 497-500,1981. quantities of protein utilizing the principle of protein dye binding. Anal. 34. Sehgal, P. B., Pfeffer, L. M., and Tamm, I. Interferon and its inducers.In: P. E. Biochem., 72: 248-254, 1976. Came and L. A. Caliguiri(eds.),Chemotherapy of Viral Infections, p. 205-311. 5. BrostrÃ¶m,L. The combined effect of interferon and methotrexate on human Springer-Verlag.1982. osteosarcoma and lymphoma cell lines. Cancer Lett., 10: 83-90,1980. 35. Stanwick, T. L., Schinazi, R. F., Campbell, D. E., and Nahmias,J. Combined 6. Cesario, T. C., and Slater, L. M. Diminishedantiviraleffect of human interferon antiviral effect of interferon and acyclovir on herpes simplex virus types 1 and in the presence of therapeutic concentrations of anti neoplastic agents. Infect. 2. Antimicrob. Agents Chemother., 79: 672-674, 1981. Immun., 27: 842-845, 1980. 36. Stewart, W. E., II. The Interferon System. New York: Springer-Verlag Ine 7. Chingos, M., and Pearson,J. Cure of murine leukemiawith drug and interferon 1979. treatment. J. Nati. Cancer Inst., 57: 1367-1368, 1973. 37. Stolfi, R. L., Martin, D. S., Sawyer, R. C., and Spiegelman, S. Modulation of 8. deFerra, F., and Baglioni, C. Increase in S-adenosylhomocysteineconcentra 5-fluorouracil-induced toxicity in mice with interferon or with the interferon tion in interferon-treated HeLa cells and inhibition of methylation of vesicular inducer, polyinosinicacid-polycytidylic acid. Cancer Res., 43: 561-566,1983. stomatitis virus mRNA. J. Biol. Chem., 258. 2118-2121,1983. 38. Suhadolnik,R. J., Sawada, Y., Gabriel,J., Henderson,E. E., and Reichenbach, 9. Friedman, R. M. Interferons, A Primer. New York: Academic Press, Inc., 1981. N. L. Effect of human fibroblast interferon on thymidine metabolism, DNA processing, and chromatin structure in HeLa cells. J. Biol. Chem., 258: in â€¢Personalcommunication. press. 1983.

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1983 American Association for Cancer Research. Effect of Human Fibroblast Interferon on the Antiviral Activity of Mammalian Cells Treated with Bleomycin, Vincristine, or Mitomycin C

Robert J. Suhadolnik, Yosuke Sawada, Maryann B. Flick, et al.

Cancer Res 1983;43:5462-5466.

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