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Maureen H. Berehi'* Barry J. Sugarman2** H. Michael Shepard2 Lois B. Epstein' 'Cancer Research Institute and Department of Pediatrics, University of California, San Francisco, CA 2Departmentof Pharmacological Sciences, Genentech, Inc., San Francisco, CA

Synergistic induction of polypeptides by tumor necrosis factor and interferon-gamma in cells sensitive or resistant to tumor necrosis factor: Assessment by computer based analysis of two-dimensional gels using the PDQUEST system Tumor necrosis factor (TNF) synergistically enhanced the antiproliferative activity of interferon-gamma (IFN-y) in both TNF-sensitive and TNF-resistant variants of the cervical carcinoma line, ME- 180. T N F alone had no apparent effect on the levels of synthesis of individual proteins in either of these variant cell lines as assessed by computerized two-dimensional gel analysis of cell lysates using the PDQUEST system. However, IFN-y enhanced the levels of 18 polypeptides and suppressed the levels of 10 polypeptides in both cell lines. When used in combination in both cell lines, T N F and IFN-y induced the synthesis of 10 polypeptides that were not induced by either agent alone. These synergistically induced polypeptides may be crucial to the mechanism of the synergistic antiproliferative action of T N F and IFN-y in ME- 180 cells.

1 Introduction Tumor necrosis factor (TNF) and interferon-gamma (IFN-y) are both cytokines which are produced as part of the immune response [ 1, 21. Both exhibit a variety of activities, some of which they share. In particular, they both demonstrate antiproliferative actions against tumor cells. The effects of T N F and IFN-y on cell growth have been widely studied. T N F was found to inhibit the growth of some cell lines, to stimulate the growth ofother cell lines, and to have no effect on the growth of yet a third group of cell lines 13-51. All of the cell lines that experienced growth enhancement in response to T N F treatment were normal lines 141. All of the cell lines undergoing an antiproliferative response were malignaint lines 13-51. Nevertheless, not all malignant cell lines are susceptible to the antiproliferative effects of TNF. IFN-y has also demonstrated broad ranging antiproliferative effects in vitro; again, cells vary in their sensitivity to IFN-y, although enhancement of cell growth has not been observed [61. Thie antitumor effects of these agents have also been demonstrated in vivo in mice [7,8], and both agents are being evaluated iri clinical trials either alone or in combination for the treatment of human malignancy L9, 101. Examination of the effects of T N F and IFN-y in combination is relevant for three reasons: it is likely that they coexist physiologically, they are known to act synergistically in the enhancement of a variety of activities, and their use in combination has potential in the development of anticancer therapies. Their synergistic antiproliferative activity has been widely examined [3-5,8, 111. Synergy in the antiproliferative effects of these two agents was seen in cell lines which were sensitive to T N F as well as in cell lines which wererellatively or completely insensitive to T N F 13-51. Furthermore, there was no correlation between sensitivity to IFN-y and synergy between T N F andIFN-y [3-51, and synergistic antiproliferative activity was not observed in normal cell lines [3,4]. Synergy between T N F Correspondence: Lois B. Epstein, M. D., Cancer Research Institute, Box 0128, University of California, San Francisco, C A 94143, USA

Abbreviations: HLA, histocompatibility antigen; IFN-y, interferon gamma;M,,pZ, molecular weightiisoelectricpoint; TPJF, tumor necrosis factor 0VCH Verlagsgesellschaft mbH, D-6940 Weinheirn. 1990

and IFN-y has been demonstrated in vivo in the antitumor effects of these agents against human tumor xenografts in nude mice [ 81. In addition, T N F and IFN-y act synergistically in reducing c-myc encogene expression in HeLa cells [ 121, causing damage to liposome membranes 1131, inducing histocompatibility (HLA) class I1 antigens [ 14- 161, inducing differentiation of human myeloid cell lines [15], inhibiting viral replication [ 171, reorganizing vascular endothelial cell monolayers in culture [ 181, and inhibiting hematopoietic colonyformation and growth [ 19,201. Lymphotoxin (or TNF-P) which possesses 30 ?h sequence homology with T N F and which binds to the same cellular receptor [2 11, also exhibits synergy with IFN-y in its antiproliferative activity as well as in other activities [ 15, 22-25 I. Cells of the cervical carcinoma line ME-180 were shown to be sensitive to the antiproliferative effects of both T N F and IFNy alone and in combination [3,41. Together these agents acted synergistically to inhibit the growth of ME- 180 cells. In this work, we examined the antiproliferative effects of T N F and I F N y alone and in combination on TNF-sensitive and TNFresistant, neomycin-resistant variants of the ME- 180 cell line. In an attempt to uncover the molecular basis for the observed synergy in their actions, we used two-dimensional gel analysis to examine the levels of synthesis of individual proteins during treatment of these cells with T N F and IFN-y.

2 Materials and methods 2.1 Cell lines ME- 180 cervical carcinoma cells were purchased from the American Type Culture Collection (Rockville, MD). Clones ofME-180 cells, ME-18ONEo(NEO;T N F sensitive) and ME18ONE0R3'(NEO R3 1;T N F resistant), werederivedfromthe parent line by transfection with a plasmidpSVENE%al6 [261, conferring resistance to the aminoglycoside Geneticin G-4 18

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Current address: Syva Company, Box2-102-1,900Arastradero Road, Palo Alto, CA 94304, USA Current address: Amgen, Inc., Amgen Center, Thousand Oaks, CA 9 1320, USA 01 73-0835/90/0303-0232 %02.50/0

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(GIBCO). Such transfection facilitates the isolation of TNFsensitive and TNF-resistant variants. These lines are derived from a clonal isolate and are not the result of transfection en masse. Both cell lines retain their sensitivity to the antiproliferative effects of IFN-y, and each retain their characteristic positive or negative response to TNF, indicating that the presence of the plasmid did not alter the response of the cell variants to these cytokines. The cells were maintained in Mc-Coy’s 5a medium supplemented with 10 % heat inactivated fetal calf serum, 100 units/mL penicillin, and 100pg/mL streptomycin. NEO R3 1 cells were carried in the presence of 5000 units/mL TNF. Cells were split twice weekly 1:lO or 1:12. Once every three weeks, 400 pg/mL Geneticin G-4 18 was added to the medium.

fluorography and four exposures of each gel were scanned with an Optronics P-1000 scanner interfaced to a PDP-I 1/60 computer. Computerized analysis of the gels employed the PDQUEST System which is based upon modifications of the system described by Garrels et aZ. 1291. Proteins in each gel were identified with a number and quantitated in terms of ppm. Next, they were matched from sample to sample within 4 subsets(basedon twocelllines and twoperiodsoflabe1ing)ofeach experiment. This was followed by matching across subsets and finally matching across the two experiments. Proteins were assigned molecular weights and isoelectric points within 15 % error. These parameters were used to identify the proteins (molecular weight/isoelectric point; M,/p/). The data was analyzed for the induction or suppression of protein levels by TNF, IFN-y, or the combination ofTNF and IFN-yin each of the two cell lines over each of the two labeling periods. The 2.2 Agents criteria for induction or suppression is a two-fold increase or decrease in the levels of the protein over those seen in the conRecombinant human TNF-a and IFN-y were products of trols in both experiments. The quantitative data obtained from Genentech (San Francisco, CA). Their specific activities were Protein Databases, Inc. was confirmed or revised in our labologunits/mL and 3.4 x lo7I.U./mL, respectively. ratory both visually and with the PDQUEST workstation. Because ofthelargevolume ofinformation generated, the data on polypeptide induction will be presented for only one of two 2.3 Antiproliferative assay experiments. Nevertheless, all polypeptides for which inducCells were seeded at lo4cells in 0.2 mL medium in the wells of tion was confirmed met the criteriain both experiments. In adthree microtiter plates. The plates were incubated for 24 h. At dition, to further simplify the data, theresults for only one conthis time, the medium was aspirated from 6 replicate wells centration of T N F (1000 units/mL) will be shown graphicalon each plate and replaced with 100 units/mL TNF, 1000 ly. units/mL TNF, 100 I.U./mL IFN-y, 100 units/mL T N F + 100 I.U./mL IFN-y, or 1000 units/mL T N F + 100 I.U./mL IFN-y. Eighteen wells per plate to which fresh medium alone 3 Results was added served as controls. The plates were incubated for 48, 72 or 96 h. They were subsequently stained with Crystal T N F at 1000 units/mL inhibited the growth of the NEO cells Violet, and the dye was eluted and quantitated spectrophoto- but not the NEO R3 1 cells; the inhibition was most evident metrically as described previously [271. after 96 h of continuous exposure (Fig. 1). T N F at 100 units/ mL also inhibited the growth of NEO cells; it reached a max2.4 Radiolabelingof cellularproteinsand preparationof cell lysates Cells were radiolabeled as described previously [271. Two 96well microtiter plates were seeded at 2.5 x lo4cells per well in 0.2 mL medium. The plates were incubated for 24 h. The medium was aspirated and replaced with 25 pCi/wellof a 14Camino acid mixture (Amersham, Arlington Heights, IL). The following reagents were added to duplicate wells on each ofthe two plates to the following final concentrations: 100units/mL TNF, 1000 units/mL TNF, 100 I.U./mL IFN-y, 100 units/ mL T N F + 100 I.U./mL IFN-y, and 1000 units/mL T N F + 100 I.U./mL IFN-y. Four wells per plate to which no cytokines were added served as controls. The plates were incubated for 24 or 32 h. The cells were harvested in lysis buffer and stored at -70 OC as describedpreviously [271. Celllysates were prepared in duplicate experiments, experiments 1 and 2.

2.5 Two-dimensionalgel electrophoresis and computerized analysis Electrophoresis of the cell lysates and computerized analysis of the gels were performed at Protein Databases, Inc. (Huntington Station, NY). Cell lysates were electrophoresed in two dimensions as described previously [271, based on the method of Garrels (281. The loads ofradioactivity per gel ranged from 107 744 to 315 348 cpm in experiment 1 and from 193 275 to 305 706 cpm in experiment 2. The gels were treated for

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LENGTH OF EXPOSURE (hours) Figure I. Antiproliferative activity of T N F and IFN-y in TNF-sensitive (NEO) and TNF-resistant (NEO R3 1)ME-I80 derived cell variants. NEO (A) and NEO R3 1 (B) cells were incubated for 48,72 or 96 h in the presence of medium alone, 1000 units/mLTNF, 100 I.U./mLIFN-y, or l000units/ m L T N F + 100 I.U./mLIFN-y. Cell growth was quantitated as absorbance at 550nmofthevitaldye,Crystal Violet. Theresultsarepresentedasthecell growth as a percent of the control; the data represent the mean +/- the standard error of three experiments.

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Figure 2. Fluorograms of two-dimensional gels of untreated and cytokine treated R3 1 cell lysates. Fluorograms of gels of R3 1 cell lysates illustrate the changes occurring in the levels of individual proteins upon treatment of the cells for 32 h. (A) control; (B) 1000 units/mLTNF;(C) 1001.U./mL IFN-y; and (D) 1000 units/mL T N F + 100 I.U./mL IFN-y. The large arrows in (C) and (D) indicate proteins induced in each of these samples; the small arrows in (C) indicate proteins suppressed by IFN-y. All of the altered proteins are represented by arrows in the untreated sample (A). Similar changes in the levels of specific proteins were seen in both cell lines for both labeling periods. No arrows are present in (B) as T N F alone did not induce or suppress any polypeptide greater than twofold. In panel (C) peptides 1- 18 are induced by IFN-y; peptides 5 1-60 are suppressed by IFN-y. Quantitative data for each numbered polypeptide in panel (C) are depicted in Fig. 3 (IFN-y induced peptides) and in Fig. 5 (IFN-y suppressed peptides). In panel (D) peptides 3 1-40 are synergistically induced by T N F and IFN-y. Quantitative data for each numbered polypeptide in panel (D) are depicted in Fig. 4. The molecular weights and isoelectric points for each numbered peptide are depicted in the legend of the respective figures. Panel (B) p. 235, (C) p. 236 and (D) p. 237.

imum of 18 % inhibition at 72 h. IFN-y at 100 I.U./mL inhibited the growth of both cell lines. Suppression of growth was evident at 48 h, but the percent suppression increased further with time. Together, T N F and IFN-y inhibited the growth of the NEO cells to a greater extent than would be expected on the basis of the action of either agent alone. Moreover, the actions of these two agents in this cell line meet

the criteria for synergism as described by Drewinko [30].The synergism in the antiproliferative effects of TNF and IFN-y is most apparent at 48 h. Although T N F alone had no effect on the growth of the NEO R3 1, it enhanced the antiproliferative effect of IFN-y on these cells. Again, this was most evident at 48 h. Synergism in both cell lines was observed with 100unitsl mL T N F as well as with 1000 units/mL TNF.

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Two-dimensional gel analysis revealed numerous alterations in the levels of specific proteins upon treatment ofthe cells with IFN-y, alone or in combination with T N F (Fig. 2). However, comparison of polypeptide levels in T N F (both 100 and 1000 units/mL) treated cells with those in controls failed to reveal any polypeptides which met our criteriafor induction by T N F in either of the cell lines labeled for either period of time. Nevertheless, the levels of some proteins were increased by T N F but not to twofold or greater levels in both experiments. Treatment of each of the cell lines with IFN-y led to the induction of 18 polypeptides as evidenced for each of the labeling periods (Fig. 3). The differences in the patterns of IFN-y mediated polypeptide induction between the TNF-sensitive and TNF-resistant cell lines were minimal. The same proteins were found to beinduced over both the 24 and 32 h periods of continuous labeling; however, the levels with respect to the total level of labeled protein are variable. Thirteen of the 18

235

polypeptides induced in the ME- 180 cells by IFN-y have been described previously as being induced in fibroblasts treated with IFN-y [271, and their Mr/pI designated in Table 1. Protein designations vary between experiments on different cell lines because of the 15 %error inherent in the determination of the MJpI. We also observed that 10 polypeptides which were not induced by either T N F or IFN-y alone were induced by the combination of these two agents in both cell lines over both labeling periods (Fig. 4). Several of these proteins were observed only in cells that had been treated with both agents. Only 2 of these polypeptides were synthesized constitutively at detectable levels in both cell lines over both labeling periods. The synthesis of 10 proteins was suppressed fifty percent or more by IFN-y treatment of both cell lines over both periods of treatment (Fig. 5). T N F did not inhibit the synthesis of any poly-

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peptides in accordance with our criteria. Similarly, T N F did not enhance the IFN-y mediated suppres,sion of any polypeptides.

4 Discussion Synergism in the antiproliferative activities o f T N F and IFN-y was observed in both TNF-sensitive (NEO) and TNF-resistant (NEO R3 1) variants of the ME-180 cervical carcinoma line (Fig. 1). These twocytokines were previously shown to act synergistically in the TNF-sensitive parent ME- 180 cell line 13, 41. Although synergistic antiproliferative effects were observed in bothNEO and NEO R31 cells, the percent growth inhibition was greater in the T N F sensitive NEO cells. Nevertheless, the phenomenon of synergy as observed in NEO R 3 1 cells is independent of the sensitivity of the cells to T N F alone. This was demonstrated previously in other cell

Electrophoresis 1990, 11,232-241

lines resistant to T N F 13-51. It is likely that cytolysisoccurred in the presenceofboth T N F andIFN-y, becausethenumber of cells decreased over the period from 48 to 96 h. Two-dimensional gel analysis of the levels of synthesis of individual polypeptides did not reveal the induction of any polypeptides by T N F in either N E O or NEO R 3 1 cells. This is not to say that the levels of any peptides did not increase in response to T N F treatment. Rather, it indicates that no polypeptides met our criteria for twofold or greater enhancement of synthesis in both experiments. It may be important to mention that the NEO cells were only moderately sensitive to T N F ; a greater susceptibility of the parent ME-180 line to the cytostatic ef fects of T N F was previously observed [3,4l. T N F is known to lead to the induction of polypeptides in other cell lines. It enhances the synthesis of 2',5'-oligoisoadenylate synthetase 13 11, cfos I I 1 I, epidermal growth factor receptor 1321,H4/18 antigen [33 I,IFN-P2(IL-6)[31],HLA-A,Bantigens[34l,and an endothelial cell surface factor which promotes neutrophil

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adherence [351. Kirstein andBaglioni [361 observedenhancement in the synthesis of two polypeptides of molecular masses 36 and 42 kDa in response to T N F treatment of human fibroblasts. Thesefibroblastswereinsensitivetotheantiproliferative effects of T N F in the absence of cycloheximide. It was proposed that these two proteins may confer a protective effect against TNF on these cells. In human strain 153 fibroblasts, we previously observed enhanced levels of nine polypeptides (M,lpI = 21/>1, 2516.5, 33/>7,42/5.6, 7916.4, 80/6.0,961 6.7, and 9616.8) in response to treatment with 1000 unitslml T N F [271. However, T N F stirnulatedratherthan inhibited the growth of these cells. The levels of eighteen polypeptides were enhanced by treatment with IFN-y (Fig. 3). IFN-y has been reported to induce the synthesis of a wide variety of proteins including enzymes,

23 7

cell surface antigens, and a number of proteins of unknown identity that have been observed upon one- or two-dimensional gel electrophoresis (see [271 for review). Our laboratory has previously demonstrated the induction of polypeptides by IFN-y in fibroblasts and ovarian carcinoma cells [27,35-4 11. We found that thirteen of the polypeptides induced in fibroblasts are also induced in NEO and NEO R3 1 cells (Table 1). In both the fibroblasts and NEO and NEO R3 1 cells, IFN-y inhibited the growth of the cells. Thus it is intriguing to speculate that some or all of the thirteen peptides induced in common in fibroblasts and cervical carcinoma cells are involved in the antiproliferative effects of IFN-y. IFN-y was also found to suppress the levels of ten proteins (Fig. 5). T N F was not observed to suppress the levels of any proteins by 50 % or more in these cells. We previously showed that T N F at 1000 units/mL suppressed the synthesis of one polypeptide in fibro-

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POLYPEPTIDES(molecularweight/isoelectric point) Figure 3. Interferon-y induced polypeptides. NEO and NEO R3 1 cells were labeled for 24 or 32 h with a 14C-aminoacid mixture in the presence or absence of 100 I.U./mL IFN-y. Whole cell lysates were wbjected to computerized two-dimensional gel analysis. Eighteen polypeptides (identified by molecular weight/isoelectric point) were enhanced by IFN-y treatment to levels two-fold or greater above those in untreated cells in two experiments. The data represents the results of experiment 2. The asterisks (*)I indicate proteins for which accurate quantitative data was not obtained with the PDQUEST system but which were clearly visible; these proteins were determined to be induced in experiment 1 and visually appear to be induced in experiment 2. The table below permits the localization in Fig. 2, Panel (C) of each polypeptide for which quantitative data are given in this figure. 9 5315.9b Numerical designation 10 5 116.7 in Fig. 2. Panel (C) MhZ 11 4616.0 8915.9 1 12 8015.7 4516.1 2 13 44/62 7616.4 3 14 3815.4 4 54/53 15 3716.4 5316.1a 5 16 3516.3 53/6.1b 6 17 3015.6 5316.0 7 18 2915.8 8 5315.9a

Table 1. Correspondence between IFN-y induced polypeptides in ME- 180 variants and in fibroblasts Protein designation for ME-180 variantsa)

Protein designation for fibroblasts 1271

2915.8 3015.6 3516.3 3716.4 44/63 4516.1 5116.7 5315.9a 5315.9b 5316.0 5 316.1 a 8015.7 8915.9

2115.9 2815.5 3416.5 3616.6 441>7 4516.4 5016.9 5116.la 5 116.lb 5 116.2a 5 116.2b 8016.0 8916.2

a) Location of each peptide in their corresponding autoradiograms confirmed that each pair represented identical peptides. Protein designations vary between experiments on different cell lines because of t.he 15 % error inherent in the determination of the M,/pZ.

blasts (M,/pI = 26/6.4) [271. Synergistic suppression of polypeptide synthesis by T N F plus IFN-y was not evident in either NEO or NEO R3 1 cells. The levels of ten polypeptides were synergistically enhanced by T N F and IFN-y (Fig. 4). Our criteria for assessing synergistic induction required that either the fold-increase due to the combination of agents be greater than the product of the fold-increases due to either agent alone or that if the protein was absent upon treatment with one agent its level be greater than two-fold above that due to the other agent. These ten polypeptides failed to meet our criteria for induction by either agent alone. Furthermore, we did not observe these polypeptides to be induced by single agents in fibroblasts. Some or all of these ten polypeptides may be central to the molecular mechanisms underlying the synertistic interactions between T N F and IFN-y in antiproliferative activity. The enhanced synthesis of these proteins is dependent upon interactions between the cellular effects of T N F and IFN-y. It may be that the induction of certain proteins by IFN-y is required before T N F treatment can lead to subsequent induction of these ten

Synergistic induction of polypeptides by tumor necrosis factor and interferon-y

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POLYPEPTIDES(molecularweight/isoelectric point) Figure4. Synergistic induction ofpolypeptides by T N F and IFN-y. NEO and NEO R3 1 cells were labeled with a I4C-aminoacid mixture for 24 or 32 h in the presenceofmedium alone, lOOOunits/mLTNF, 100 I.U./mLIFN-y,or lOOOunits/mLTNF + IOOI.U./mL IFN-?.Ten polypeptides weresynergistically enhanced by the combination o f T N F and IFN-y. These proteins werenot always present in untreated, T N F treated, or IFN-ytreated cells. Thisisindicated by the absence of a bar. The asterisks (*) indicate proteins for which accurate quantitative data were unavailable with the PDQUEST system; they were visually determined to be present in the respective samples in both experiments. The table below permits the localization in Fig. 2, Panel (D) of each polypeptide for which quantitative data are given in this figure.

MIPI 4715.5 4614.7 4516.2 4415.0

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35/61 2115.2 2616.1 2615.2 2515.1 2415.2

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proteins. The time course of induction of these proteins has not been studied; therefore, it is not known whether their synthesis is a primary or secondary response to treatment with these cytokines. Furthermore, the temporal relationships in administration of T N F and IFN-y required for synergy between these two agents have yet to be examined.

IFN-y mediated increase in T N F receptor number in HT-29 cells, whereas they do not have any effect on the synergistic antiproliferative activities of T N F and IFN-y in these cells [451. It therefore appears unlikely that induction of T N F receptors by IFN-y is a major mechanism for the synergy in the antiproliferative effects of these two agents.

A possible basis for the synergy in the antiproliferative effects of T N F and IFN-y lies in the up-regulation of T N F receptors by IFN-y. Treatment with IFN-y was found to increase the number of T N F receptors on the surfaces of a variety of cell lines 142-461. T N F and IFN-yexhibit marked synergy in their antiproliferative effects on ME- 180, HT-29, and HeLa cells [ 3,4 1; IFN-y was also found to induce T N F receptors in these lines [42-441. In HT-29 cells, IFN-y treatment prior to T N F treatment resulted in synergy, whereas the reverse did not [441. This is consistent with IFN-y mediated enhancement of T N F receptor number followed by enhanced binding of TNF. However, several lines of evidence argue against the involvement of up-regulation of T N F receptors in the synergy between T N F and IFN-y. First, the number of T N F receptors in a range of cell lines does not appear to be related to their sensitivity to the cytotoxic or cytostatic effects of T N F [41. Moreover, T N F and IFN-y exhibit synergy in the SK-MEL-I09 cell line, although IFN-y does not increase T N F receptor number in these cells [431. Furthermore, IFN-a and IFN-P inhibit the

Nevertheless, up-regulation of T N F receptors by IFN-y may represent one possible mechanism for synergy in cell lines that constitutively express low numbers of T N F receptors. ME180 cells possess approximately 2000 T N F receptors per cell [42]. This is a relatively low number, since the number o f T N F receptors was found to range from 1000 to 20 000 per cell 143,441 in other cell types. A role for T N F receptors in the synergy between T N F and IFN-y in ME-180 cells has not been ruled out. It is also possible that the synergism in antiproliferative action observed with T N F and IFN-y may be mediated through their effects on the cell membrane. IFN has long been known to alter cell membranes 1471. Recently, the effects of T N F and IFN-y, alone andin combination, onliposome membranes have been studied 1131. T N F was effective in causing leakiness in liposomes, while IFN-y was only slightly effective. More importantly, the abilities of T N F and IFN-y to cause leakiness in liposome membranes were synergistic. This finding may be relevant to the elucidation of the mechanism of synergism in their antiproliferative effects. Confirmation of

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POLYPEPTIDES(molecularweight/isoelectric point) Figure 5. IFN-y suppressed polypeptides. Cells were treated as described for Fig. 4. IFN-y treatment suppressed the synthesis often polypeptides by 50 3' 6 or more in both experiments. The data represent the results of experiment 2. The asterisk (*) represents a protein for which valid data was not obtained through the PDQUEST system, because it was located on a streak in the control gel; this protein appeared visually to be suppressed by IFN-y treatment. A dagger (7)represents proteins which were faintly detectable by eye, but which were undetected with the PDQUEST system. The table below permits the localization in Fig. 2, Panel (C) of each polypeptide for which quantitative data are given in this figure. Numerical designation 4815.6 55 MPI in Fig. 2. Panel (C) 4716.9 56 4415.9 57 10915.7 51 4215.1 58 52 8815.9 5216.7 53 3216.3 59 60 2715.3 5215.9 54

this possibility awaits further studies with cellular membranes. Changes in biological membranes in response to T N F and IFN-y are likely to be much more complex than those occurring in liposomes. It is possible that the:se agents may have a direct effect on the cell membrane and/or that their effects on membranes may be mediated through cdlular proteins, either preexisting or induced. Synergism requires that the mechanisms of the antiproliferative action of T N F and IFN-y converge when they are used in combination. It could be that there is overlap in their mechanisms of action as single agents and that their use in combination optimizes the elements in the mechanism. Alternatively, when used alone, they may act through distinct mechanisms, but when used together an additional mechanism requiring input from both agents may be brought into play. The observed synergy in the induction of unique polypeptides by the combination of these agents suggests that the latter may be the case. Further characterization of the synergistically induced polypeptides with regard to function may be the key to understanding the basis ofthe synergistic interactions between T N F and IFN-y. Another possible mechanism for the functional synergy between T N F and IFN-y is that the latter inhibits the synthesis of a protein or proteins which ordinarily counteract the direct, non-protein synthesis-related cytotoxicity of TNF.

For this reason, consideration of identity and function of the ten polypeptides suppressed by IFN-y is also of importance in future studies.

These studies were supported by NIH grants CA27903, CA44446 and HDI 7001,M.H.B. was supported by American Cancer Society Grant PF-2879. The authors wish to thank Esther Honda and Mary Anne Fordfor their editorialassistance. Received October 16, 1989

5 References [11 Beutler, B. and Cerami, A., N. Engl. J. Med. 1987,316, 379-385. [21 Epstein, L. B., in: Vilcek, J . and DeMaeyer, E. (Eds.),InterJerons and the Immune System, Volume I1 of Finter, N. B. (Ed.), Interferon, Elsevier Science Publishers, Amsterdam 1984, pp. 185-220. 131 Fransen,L.,VanDerHeyden, J.,Ruysschaert,R. andFiers, W.,Eur. J. Cancer Clin. Oncol. 1986,22,419-426. [41 Sugarman, B. J . , Aggarwal, B. B., Hass, P. E., Figari, I. S., Palladino Jr., M. A. and Shepard, H. M., Science 1985,230,943-945. [51 Shepard, H. M. andLewis, G. D.J. Clin.Immunol. 1988,8,333-34 1. [61 Kirchner, H., Progress in Clinical Biochemistry and Medicine Springer Verlag, Berlin 1984, pp. 169-203.

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