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[CANCER RESEARCH 51, 2379-2385, May 1. 1991] Inter leu kin 1, Interleukin 6, Tumor Necrosis Factor, and Transforming Growth Factor ßIncrease Cell Resistance to Tumor Necrosis Factor Cytotoxicity by Growth Arrest in the G! Phase of the Cell Cycle1 JoséE. Belizario and Charles A. Dinarello2 Department of Medicine, Tufts University School of Medicine, and New England Medical Center, Boston, Massachusetts 02111 ABSTRACT During infection, inflammation, immune responses, and neoplastia growth, various cytokines are produced affecting both susceptibility to and protection from cellular death. We have studied the protective effect of pretreatment of the L929 fibroblast cell line with interleukin 10 (II1/3), IL-6, tumor necrosis factor a (TNF-a), or transforming growth factor 0, (TGF-/9) on subsequent TNF/actinomycin D-induced cytotoxicity. The protective effects of these cytokines on TNF cytotoxicity were time and concentration dependent. TGF-/3 was the most effective cytokine, followed by TNF, IL-1/3, and IL-6. Activators of protein kinase C also afforded protection, and TGF-/3 acted synergistically with either phorbol 12-myristate 13-acetate or the calcium ionophore A-23187. TGF-/3induced protection against TNF was observed in cells subjected to pro longed treatment with phorbol 12-myristate 13-acetate. Cells pretreated with prostaglandin E2 or cholera toxin amplified the sensitivity to TNF and inhibited TGF-/3-mediated resistance, whereas indomethacin en hanced the protective effect of TGF-/3. Cells cultured in the presence of IL-1/3, IL-6, TNF-a, or TGF-0 for 6 h inhibited DNA synthesis, and this was associated with concomitant growth arrest in the Gì phase of the cell cycle. On the other hand, prostaglandin K; or cholera toxin stimulated the progression of cells from G, toward G2 + M which was associated with increased TNF sensitivity. We conclude that these cytokines protect against death by arresting growth in the G, phase of the cell cycle. INTRODUCTION Cytokines are biologically potent polypeptides produced by a variety of cells in response to infection, microbial toxins, in flammatory agents, and neoplastic growth, as well as to them selves. Several cytokines share the ability to stimulate cell proliferation, initiate the synthesis of new proteins, and induce the production of inflammatory metabolites in a wide variety of cells. Of the various cytokines which have been identified, considerable interest has focused on IL-13 (1), IL-6 (2), TNF (3), TGF-/3 (4), epidermal growth factor, interferon, and plate let-derived growth factor as mediators of the host response to various disease states or as part of pathological processes. The ability to lyse neoplastic and virus-transformed cells in vitro and in vivo is a property of TNF (3) and is of great interest in view of its possible use in cancer therapy. On the other hand, because overproduction of this cytokine is associated with the pathogenesis of many inflammatory diseases, approaches to attenuating the action of TNF have been sought. The protective Received 8/30/90; accepted 2/22/91. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This study is supported by NIH Grant Al-15614. J. E. B. is supported by a Fellowship of the Ministry of Science and Technology of Brazil (CNPq). 2 To whom requests for reprints should be addressed, at the Department of Medicine, New England Medical Center, 750 Washington St., Boston, MA 02111. 3The abbreviations used are: IL-1. interleukin 1; IL-6, interleukin 6; TNF-«, tumor necrosis factor a; TGF-/3, transforming growth factor ß,;PKC, protein kinase C; A-23187, calcium ionophore A-23187; PGE;. prostaglandin E2; ACT D, actinomycin D; MnSOD, manganous Superoxide dismutase; PMA. phorbol 12-myristate 13-acetate; TNF LDSO,concentration of TNF/ACT D necessary to achieve 50% cell cytotoxicity. effect afforded by preadministration of IL-1 (5-7), TNF (810), epidermal growth factor, TGF-a, or TGF-/3 ( 11-12) usually 24 h before lethal doses of TNF, bacteria, or some inflammatory agents has been demonstrated in various in vivo and in vitro models. The mechanisms which protect target cells from being killed by TNF remain unclear. Previous reports (13-15) have shown that activation of PKC (16) plays a role in the modulation of TNF sensitivity, as suggested by the effect if its inducers PMA and A-23187. However, it is uncertain whether cytokine-induced resistance is also dependent on PKC activation. Further more, the kinases associated with the majority of cytokine receptor-mediated responses remain to be defined. The present experiments were undertaken to study how IL-1/3, IL-6, TNFa, and TGF-,0 induce an increase in cell resistance to TNF cytotoxicity in murine L929 cells. Because TGF-/3 possessed marked activity to protect these cells from the action of TNF, additional experiments were carried out to evaluate the inter actions between TGF-/3 and PKC inducers. Furthermore, in view of the evidence from a previous study (17) that sensitivity of cells to the lytic effects of TNF is increased by cell arrest in mitosis, we also investigated the effect of cytokines and other pharmacological agents on the L929 cell cycle to determine whether cell resistance to TNF would be a cell cycle-linked process. Our data suggest that increased cellular resistance induced by these cytokines and PKC activators correlates with the accumulation of cells in GI, whereas the increased sensitivity induced by PGE2 and cholera toxin is associated with the transition of cells from the G, to the G2 + M phase of the cell cycle. MATERIALS AND METHODS Reagents. Recombinant human IL-1/3 was the kind gift of Dr. Alan R. Shaw (Glaxo Institute of Molecular Biology, Geneva, Switzerland). Recombinant human TNF-a, TGF-/3, and IL-6 were provided by Genentech, Inc. (South San Francisco, CA). PMA (Sigma, St. Louis, MO) and A-23187 (Calbiochem, La Jolla, CA) were dissolved in dimethyl sulfoxide and further diluted in cell culture medium. The control dilutions with dimethyl sulfoxide or alcohol had no effect in our experiments. Actinomycin D was obtained from Calbiochem, and chol era toxin was from List Biological (Campbell, CA). Prostaglandin E2 was purchased from Sigma, and a stock solution of 50 Mg/ml was prepared by adding 1.0 ml of absolute ethanol followed by 19 ml of tissue culture medium. A stock solution of 1.0 HIM indomethacin (Sigma) was prepared in 0.1 M Tris buffer (pH 8.0) and was further diluted with culture medium at the time of use. Cell Culture. L929 mouse fibrosarcoma cells (CCL1) were purchased from the American Type Culture Collection (Rockville, MD). Cells were maintained in culture using RPMI 1640 supplemented with 5% (v/v) heat-inactivated fetal calf serum (HyClone, Logan, UT) plus 2 mM L-glutamine, 10 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, 100 units/ml penicillin G, and 100 Mg/ml streptomycin sulfate at 37°Cin a 5% CO2-humidified air incubator. Cells were washed with saline, detached using 0.25% (v/v) trypsin and 20 Mg/ml EDTA, and then resuspended in complete medium at a density of 50 x 10" 2379 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research. CYTOKINE-INDUCED RESISTANCE TO TNF CYTOTOXICITY cells/ml. \lillois (100 n\) were dispensed into 96-well flat-bottomed microtiter plates, and the cells were then allowed to adhere for 3-4 h in a 5% CO2 humidified air incubator. Thereafter, 10-^1 aliquots of different concentrations of cytokines dissolved in complete medium were added to the wells. L929 Cell Cytotoxicity Assay. The assay was performed as described by Flick and Gifford (18) with modifications. Briefly, 5 x IO4 L929 cells/well were incubated in 100 ^1 of RPMI containing 5% fetal calf serum in 96-well plates overnight. Subsequently, the medium was removed and replaced with 100 p\ RPMI containing 2.5% fetal calf serum, supplemented with 5 /jg/ml actinomycin D (control) and serial dilutions of TNF. Each dilution was performed in quadruplicate. The plates were then incubated for a further 18-20 h. The plates were agitated on a plate shaker the next day, and the medium was removed by suction. The remaining cells were reincubated for an additional 3-4 h in RPMI containing 500 jig/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (Sigma). The medium was aspirated, and 100 fii of 2-propanol were added to each well to dissolve the formazan crystals. The absorbance of each well was determined using a Bio-Rad model 3550 microplate reader. Reproducibility in the quadruplicate experiments was 5-15%. The percentage of cell survival was calculated as RESULTS Dose-Response and Time Course of Cytokine-mediated In creases in Resistance to TNF. Pretreatment of L929 cells with the cytokines IL-1, IL-6, TNF, or TGF-0 induced a significant (P < 0.05) increase in the dose of TNF/ACT D required to kill 50% of the cells (Fig. 1). The ability of these cytokines to reduce the TNF cytotoxic effects was time and dose dependent. The maximal effect of IL-1 was observed in cells incubated for 12 h at a dose of 10 ng/ml (Fig. \A). The percentage of increase in the TNF LD50 in cells pretreated with IL-6 was similar to that of IL-1, but the effect was most pronounced after 24 h of treatment (Fig. IB). The addition of TNF itself before chal lenging cells with the combination TNF/ACT D also signifi200 150 too Mean absorbance wells at a particular dose of TNF x 100 Mean absorbance wells in control 50 Dose-response curves were calculated from the percentage of cell sur vival versus logio dilution using the least squares method. One unit of TNF was defined as TNF LD50 (5 ¿ig/ml).The change in the cell survival due to the treatment with cytokines or other reagents is expressed as a percentage of increase in the TNF LD50 over the value in control. In most experiments, the TNF LD50 for the cells in control ranged from 0.3 to 0.6 ng/ml TNF. Comparison between experimental groups was done by Student's t test, and significance was defined as P 200 150 too 50 < 0.05. Flow Cytometric Analysis and Cell Cycle Distribution. The flow cytometric assay was performed after staining of DNA utilizing the propidium iodide method (19). Briefly, exponentially growing L929 cells were treated with the cytokines and other reagents for 6 h. The cells were washed, and then trypsin-EDTA (Irvine Scientific, Santa Ana, CA) was added for 1 min. The cells were suspended in medium and centrifuged at 500 x g for 5 min, and the pellet was resuspended in 2 ml phosphate-buffered saline (20 IHMsodium phosphate-150 mM sodium chloride, pH 7.2). The cell suspension was fixed in 5 ml 95% ethanol. The fixed cell suspension was allowed to stand for 30 min at room temperature and was subsequently stored at 4°C.Before staining, 400 300 200 100 the sample was centrifuged, and I ml trypsin-EDTA was added. After incubation for 2 min, the trypsin was inactivated by the addition of 2 ml medium. Cells were again centrifuged and stained by the addition of I ml propidium iodide staining solution (50 Mg/ml propidium iodide, 100 Mg/ml RNase A, and 0.1% Triton X-100 in phosphate-buffered saline). FACScan flow cytometric analysis was performed using a Becton Dickinson immunocytometry system. The number of cells in GI, S, and G2 + M compartments was obtained using the sum of rectangles model. The experiments were repeated at least twice, and 10,000 cells were counted per sample. |'ll| I Immillilo Incorporation Assay. Cells (5 x 104/well) were cul tured overnight in 100 .//Iof medium in 96-well microtiter plates in a humidified, 5% CO2 atmosphere at 37°C.The next day, cytokines or other experimental reagents were added, and the cells were pulsed with [3H]thymidine, 0.5 ^Ci/well (New England Nuclear, Boston, MA). Four h later, cells were harvested onto paper filters with a cell harvester (Cambridge Technology, Cambridge. MA). Incorporated radioactivity was determined by liquid scintillation, and the results were expressed as cpm ±SE of quadruplicate wells. 800 600 400 200 -6h -12 h -24 h Time of treatment Fig. 1. Dose response and time course of the increase in cell resistance to TNF/ACT D cytotoxicity mediated by the cytokines IL-10 (A), IL-6 (A), TNF-o (C), and TGF-fi (D). Exponentially growing cultures of L929 cells were pretreated with a selected cytokine at the indicated concentrations and times followed by the TNF killing assay. The percentage of increase in TNF LD50 was calculated by comparing the values obtained from untreated control cells to those from cytokine-treated cells. Columns, means from three independent experiments; bars, SE. Each value was significantly different (/' < 0.05) as compared with the noncytokine-treated control response. 2380 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research. CYTOKINE-INDUCED RESISTANCE TO TNF CYTOTOXICITY cantly protected L929 cells. The percentage of increase in TNF LD50 in cells pretreated with TNF alone for 6 h ranged from 83 to 279%. However, in cells incubated for 24 h, the increases ranged from 79 to 193% (Fig. 1C), indicating a moderate decrease in the acquired protection induced by TNF. TGF-ß treatment induced a dramatic and rapid augmentation in de fense against TNF cytotoxicity (Fig. 10). Cells incubated with 5 ng/ml for only 6 h increased the TNF LD50 by 620%. The profiles of protection were similar in cells exposed to TGF-0 for 12 or 24 h. Additional experiments showed that exposure of cells to TGF-/3 plus TNF had an additive effect on the cell resistance, but not IL-1 plus IL-6, IL-1 plus TGF-0, IL-1 plus TNF, IL-6 plus TNF, or IL-6 plus TGF-/3 (data not shown). On the other hand, the incubation of a single cytokine simultaneously with the combination TNF/ACT D did not increase the LD50 in the assay, suggesting that the synthesis or suppression of interme diate molecules is required to increase cell resistance. The autocrine production of TNF, as has been described (20), was not an explanation for the increase in resistance, since TNF activity was not found in the conditioned medium of cells culture in the presence of the cytokines used in this study. TGF-ßActs Synergistically with Activators of PKC. As re ported previously, activators of protein kinase C increase cell survival through down-regulation of TNF surface receptors (13-15). In view of these findings, we investigated the interac tions of PKC activators on the TGF-0-mediated cellular resist ance to TNF in cells exposed to either PMA or A-23187 (Fig. 2). The TNF LD50 in cells pretreated with PMA (Fig. 2A) for 7 h increased by only 193%, whereas in cells treated with the combination of PMA plus TGF-/3 it increased to 1910%. Since TGF-ßalone caused an increase of 932%, the data suggest a synergy between TGF-ßand PMA. Similar synergistic effects were also observed in cells treated with the combination of TGF-ßplus A-23187 (Fig. 2B). In cells exposed to this combi nation, the LD50 for TNF ranged from 3700 to 5500% (Fig. 2B). A previous study demonstrated that exposure of cells to high concentrations of PMA for 3 days depletes them of protein kinase C activity (22). We examined the effect of treatment of L929 cells with PMA (100 ng/ml) for 3 days on the subsequent effect of TGF-/3-mediated protection (Fig. 3). As expected, short-term addition of PMA did not increase the TNF LD50 in cells pretreated for 3 days with PMA, as compared to control cells. The protective effect of TGF-ßwas more pronounced in cells treated with PMA for 3 days. However, the synergy between TGF-ßand short-term PMA was significantly reduced, as compared to control cells. Thus, the protective effects in duced by TGF-ßplus PMA may be regulated through distinct pathways or by isoenzymes of PKC resistant to depletion by prolonged PMA treatment (16). Cholera Toxin and PGE2 Block, whereas Indomethacin En hances, TGF-0-mediated Increase in Cellular Resistance. Six h of pretreatment of L929 cells with various concentrations of PGE2 (Fig. 4A) resulted in a marked decrease in the ability of the cells to respond to TGF-ß.In addition, cells pretreated with PGE2 alone were more sensitive to TNF as compared to control cells. This effect was significant (P < 0.05) at 100, 1000, and 2500 ng/ml (Fig. 4A). Prostaglandin modulation of TGF-ß activity was also confirmed by treating cells with the cyclooxygenase inhibitor indomethacin (Fig. 4Ä).Concentrations rang ing from 0.01 to 10 UM indomethacin caused an enhancement of the ability of TGF-ßto increase cell resistance in a bimodal, 2000 O 1500 - If 1000 500 - 7000 6000 5000 4000 K 3000 - 0.5 1.0 TGF-ß (ng/ml) 2.5 Fig. 2. TGF-(3-mediated increase in resistance to TNF cytotoxicity in cells pretreated for 1 h with either 20 ng/ml PMA (A) or 0.2 »MA-23187 (B) and then with TGI -.i at the indicated concentrations for an additional 6 h. A: •cells treated with PMA for 7 h; D, untreated cell control with TGF-ßonly for 6 h; Q, cells treated with PMA for 1 h and T(,I ,i for an additional 6 h. B: •.cells treated with A-23187 for 7 h; D, untreated cell control with TGF-ßonly for 6 h; D. cells treated with A-23187 for 1 h and TGF-ßfor an additional 6 h. The percentage of increase in TNF 1.1),,, was calculated by comparing the values obtained from untreated control cells to those from cytokine-treated cells. Col umns, means from three independent experiments; bars, SE. Each value shown is significant at P < 0.05. 1600 1200 800 - «too- Control PMA treated Fig. 3. TGF-ß-and PMA-mediated increase in cell resistance to TNF cytotox icity in cells exposed to prolonged PMA treatment. L929 cells were first cultured for 3 days in regular medium containing 100 ng/ml PMA (I'M.l treated) or 0.25% dimethyl sulfoxide vehicle (Control) and then seeded into 96-well microtiler plates. The next day, cells were pretreated for 1 h with 10 ng/ml PMA and then with 1 ng/ml TGF-0 for an additional 6 h. •cells treated with PMA for 7 h; D, untreated cell control with TGF-/3 only for 6 h; Q, cells treated with PMA for 1 h and TGF-/3 for an additional 6 h. The percentage of increase in TNF 1.1),„ was calculated by comparing the values obtained from untreated control cells to those from cytokine/reagent-treated cells. Columns, means from three experi ments; bars, SE. a, P < 0.005; ft, P < 0.05; c, P < 0.01 versus similar conditions in control cells. dose-dependent manner. However, 100 MMindomethacin re duced the TGF-ßactivity. Indomethacin per re had no effect on TNF cytotoxicity at these concentrations. Interestingly, when L929 cells were treated with different doses of cholera toxin, 2381 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research. CYTOKINE-INDUCED RESISTANCE TO TNF CYTOTOXICITY 800 PGE2 (Fig. 5, A and C) or cholera toxin (Fig. 5B). As shown in Fig. 5, the increase in TNF LD50 induced by either TGF-0 or TGF-ßplus A-23187 was nearly abolished 6 h after the treatment with PGE2 or cholera toxin. These data suggest that the effects of A-23187 or TGF-/3 on protection against TNF cytotoxicity can be down-modulated by cyclic AMP-stimulatory factors. Increased Proportion of Cells in G2 + M Phase of Cell Cycle following PGE2 or Cholera Toxin Treatment. Fig. 6 shows the cell cycle phase distributions of asynchronized growing cells after 6 h of exposure to cytokines and/or other reagents. Dif ferences no larger than 2% in the relative proportion of cells in the G i and S phases were found in cells treated with IL-1, IL6, TNF, TGF-0, or TGF-0 plus the PKC activators, as com- 600 400 200 0 Ti -200 o n PGEj + - + TGF - + + 1 10 100 1000 2500 Pros tag Iand in E2 <ng/ml ) 3000 I) O) e e w o n 0.01 0.1 1 10 Indome thac in <uM> +•f +-+ TGFPOE,1200900£00300-Tnn•-+_Bj.-+ 100 + -++ +rr-r-iI +- + 800 M 600 400 200 -200 CT TGF -J + - + TGF + CT 2500 1 10 25 Cholera Toxin 50 100 <ng/ml> Fig. 4. Changes in TGF-Ã-i-mediated increase in resistance to TNF cytotoxicity in cells pretreated for 6 h with PGE2 (A), for l h with indomethacin (B), or for 6 h with cholera toxin (C) at the indicated concentrations. These treatments were then followed by the addition of 1.0 ng/ml I (.1 .; for an additional 6 h. •(.• D, cells treated with PGE2 for 12 h; G, untreated cell control with TGF-/3 only for 6 h; •.cells treated with P(. I for 6 h and with !(,!., for an additional 6 h. B: • cells treated with indomethacin (Indo) for 7 h; D, untreated cell control with 1111 ; only for 6 h; •.cells treated with indomethacin for 1 h and 1( ; I ; for an additional 6 h. ( '.-•cells treated with cholera toxin (('/') for 12 h; D, untreated 1500 500 cell control with TGF-/3 only for 6 h; G, cells treated with cholera toxin for 6 and TGF-tf for an additional 6 h. The percentage of change in TNF LD50 was calculated by comparing the values obtained from untreated control cells to those from cytokine/reagent-treated cells. Positive scale, increases in LDM (increased resistance); negative scale, decreases in 1.1><,>(increased sensitivity). Data of one representative experiment from four independent assays. which shares with PGE2 the ability to induce cyclic AMP, there was also inhibition of TGF-|3-mediated reduction of TNF cy totoxicity (Fig. 4C). We also observed that the cells exposed to cholera toxin are more sensitive to TNF/ACT D than are control cells. Cholera toxin and PGE2 themselves were not toxic to L929 at the concentrations used in this study. Additional experiments were undertaken to determine the sensitivity of cells pretreated for 6 h with TGF-ßor TGF-/3 plus A-23187 and subsequently incubated for an additional 6 h with n TCF/fl-23187 PGE2 0.5 TGF-p 1.0 <ng/ml> 2.5 Fig. 5. Changes in resistance to TNF cytotoxicity in cells pretreated for 6 h with either TGF-Ö or TGF-/3 plus 0.1 JIM A-23187 at the indicated concentrations and then with 1.0 Mg/ml PGE2 (A, C) or 100 ng/ml cholera toxin (B) for an additional 6 h. A: Q, cells treated with TGF-,8 for 21 h; Q. untreated cell control with PGE2 only for 6 h; •,cells treated with TGF-ß for 6 h and PGE¡ for an additional 6 h. B: G, cells treated with TGF-0 for 12 h;H, untreated cell control with cholera toxin (C7") for 6-h; H, cells with TGF-fi for 6 h and cholera toxin for an additional 6 h. C: G, cells treated with TGF-ii and A-23187 for 12 h; G, untreated cell control with PGE2 only for 6 h; O. cells treated with TGF-0/A23187 for 6 h and PGE2 for an additional 6 h. The percentage of change in TNF LDso was calculated by comparing the values obtained from untreated control cells to those from cytokine/reagent-treated cells. Positive columns, increases in LD50 (increased resistance); negative columns, decreases in LD50 (increased sen sitivity). Columns, means from three independent experiments; bars. SE. 2382 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research. CYTOKINE-INDUCED RESISTANCE TO TNF CYTOTOXICITY 800 600 400 ft § -200 THF Gì 62 8 30 G2+M 8 CT GÃŒ 41 8 33 G2+M 26 CHX E 3 Z PGE2 Gì 43 8 34 G2 + M 23 flCT D HIT C Fig. 8. Changes in the cell resistance to TNF cytotoxicity in cells pretreated for 6 h with 10 ng/ml cycloheximide (CHX), 1 nM puromycin (PURO), 1 Mg/ml actinomycin D (ACT D), or 5 ^g/ml mitomycin (MIT C). The percentage of change in TNF 1.))..,, was calculated by comparing the values obtained from untreated control cells to those from drug-treated cells. Positive columns, increases in LDM (increased resistance); negative columns, decreases in LD50 (increased sensitivity). Each value is significantly different as compared with untreated controls (P< 0.05). I« O> JO TGF + PMA GÃŒ 57 PURO pared to the control experiment cultured in medium containing 5% fetal calf serum. In cells treated with PGE2 or cholera toxin, there was a higher proportion of cells in G2 + M and a pronounced decrease in the proportion of cells in Gt. These findings suggest that agents and cytokines leading to an increase in cell resistance are likely to be associated with the arrest of cells in d; on the contrary, agents leading to an increase in cell sensitivity are associated with a marked enhancement of cells in the G2 + M phase of the cell cycle. To confirm this hypoth esis, cells were stimulated with the cytokines or other com pounds in the presence of ['11|th\ midine during the initial 4 h 0> OÃ- Relative PI Fluorescence Fig. 6. Cell cycle phase distribution (after 6 h of treatment) and [3H)thymidine incorporation (within the initial 4 h of treatment) in asynchronous growing cultures of L929 cells incubated with medium alone (.-I) or supplemented with 10 ng/ml IL-1 (B), 10 ng/ml IL-6 (C), 5 ng/ml TGF-,3 (D), 10 ng/ml TNF (£),5 ng/ml TGF-0 plus 10 ng/ml PMA (F). 5 ng/ml TGF-0 plus 0.2 «MA-23187 (C), 100 ng/ml cholera toxin (//) or 1.0 fig/ml PGE2 (/). DNA histograms were generated on a FACScan using propidium iodide (19). The percentage of cells in the GI, S, or GI + M compartments was obtained using the sum of rectangles model. Columns, cpm of quadruplicate determinations; bars, SE. *, P < 0.05 venus control. CT, cholera toxin; PI, propidium iodide; TGF, TGF-/3; A-23, A23187. of incubation (Fig. 6, columns). The treatment of cells with the cytokines IL-1, IL-6, TNF, or TGF-0 (Fig. 6B-E) caused a significant decrease in the incorporation of thymidine. The combination of TGF-/3 plus PMA (Fig. 6F) did not change thymidine incorporation, whereas the combination TGF-,0 plus A-23187, cholera toxin, or PGE2 significantly increased the uptake. Interestingly, the rates of thymidine incorporation within 4-8 h of incubation were significantly (P< 0.05) reduced in cells incubated in the presence of cytokines and/or other reagents (Fig. 7), as compared with the control experiment incubated with medium. Inhibitors of Cell Cycle Progression Increase Cellular Resist ance to TNF. To further study the relationship between cell cycle progression and protection, we carried out experiments to ascertain whether prior exposure of cells to cycloheximide, puromycin, or actinomycin would inhibit cell growth predomi nantly during the GI and S phases of the cell cycle (23, 24) and thus interfere with the resistance of cells to TNF. As depicted in Fig. 8, pretreatment of cells with these metabolic inhibitors resulted in a significant increment in the TNF LD50, whereas treatment with mitomycin, a DNA-intercalating agent that causes cells to arrest in mitosis, reduced resistance. DISCUSSION Fig. 7. [3H]Thymidine incorporation during the 4-8-h time period in cultures of L929 cells incubated with culture medium alone (control) or supplemented with cytokines and/or other agents as described in Fig. 6. Columns, cpm of quadruplicate determinations: bars, SE. All treatments resulted in significant inhibition of thymidine uptake as compared to control (P < 0.05). CT, cholera toxin. Most cells are inherently resistant to the cytotoxic effects of TNF (3, 11, 12). The molecular mechanism leading from a resistant to a sensitive state remains unknown. The mechanism by which a short exposure to some factors induces a transient increase in the relative resistance of susceptible target cells to the cytotoxicity of TNF is not fully understood. The results of the current study provide evidence for a possible mechanism by which prior administration of the cytokines IL-1, IL-6, TNF, 2383 Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research. CYTOKINE-INDUCED RESISTANCE TO TNF CYTOTOXICITY or TGF-ßor various pharmacological agents for 12-24 h before challenging murine L929 cells with a lethal dose of TNF/ACT D can attenuate cellular death. Several mechanisms of cellular regulation of the action of TNF have been described. The activation of PKC leads to the desensitization to TNF (13-15). We observed that treatment of L929 cells with PMA or A-23187, as well as with the PKC inhibitor sphingosine (21), enhances cell survival (25). Addi tional experiments showed that either PMA or A-23187 to gether with TGF-/3 acts in a synergistic manner to augment the cell resistance. Despite these observations, it was shown that TGF-/J continued to exert its protective effect on cells made deficient in protein kinase C by prolonged incubation with PMA, suggesting that molecular events associated with either PKC activation or a non-PKC pathway may also protect cells from death. The treatment of cells with either TNF (26) or IL-1 (26, 27) induces MnSOD, a mitochondrial enzyme involved in the scav enging of Superoxide anions. Such activated oxygen species have been proposed to be part of the mechanism of TNF cytotoxicity since the overexpression of the MnSOD gene pro tects cells from lethal injury by TNF (28). Hence, the protective effects evoked by the TNF and IL-1 treatment on L929 cells might be explained by the synthesis and scavenger activity of MnSOD. On the other hand, the protective effects evoked by IL-6 and TGF-/Õare likely to be independent of antioxidant defenses because these cytokines are not inducers of MnSOD (26). Previous studies have shown that following exposure of target cells to TNF, most of them die at late stages of mitosis or soon after cytokinesis (17, 29). Consistent with these observations, it was also noted that drugs which cause cells to arrest in mitosis, such as vinblastine, Colcemid (17), or mitomycin (29), potentiate sensitivity to TNF. We have shown in the present study that both PGE2 and cholera toxin are also capable of augmenting sensitivity to TNF. Most importantly, it was ob served that soon after the addition of these compounds, an increased accumulation of cells in G2 + M occurs. DNA syn thesis is stimulated during the initial 4 h of the incubation and thereafter is inhibited. It has been established in other studies (30) that both PGE2 and cholera toxin promote cells to prolif erate through the activation of adenylate cyclase. A sustained cAMP elevation, on the other hand, inhibits the proliferation and induces the differentiation of many mammalian cells (30). It is also interesting that TNF increases the synthesis of prostaglandins in a wide variety of cells (3). Our findings confirm and further extend the conclusion of earlier studies (17, 29) that TNF cytotoxicity is associated with DNA replication and cell division and that in the M-phase cells are most sensitive to TNF action. Since the arrest of cells in G2 + M makes them more vulnerable to TNF, the arrest of cells in G, of the cell cycle in response to IL-1, IL-6, TNF, or TGF-/J makes them more resistant. In fact, analysis of viability and of the relative pro portions of cells at various cell cycle phases in cultures of Lcells (17) or L929 cells4 treated with TNF has provided evidence early and decisive biochemical events in G, that prepare them to enter into the cycle (23, 24). Indeed, several studies have shown that IL-1 (1, 31), IL-6 (2, 32), TNF (3, 11), and TGF-/ÃŽ (4, 33) exert a strong inhibition on growth of a variety of cell lines. Furthermore, the reduction of c-myc transcription by TGF-/3 (33) and TNF (34), the suppression of ornithine decarboxylase activity by IL-1 and TNF (31), the induction of inter ferons and the enzyme 2',5'-oligoadenylate synthetase by IL-1, IL-6, and TNF (32, 35), and the down-regulation of mitogen receptors by TGF-ß(36) have been described as mechanisms by which these cytokines mediate growth inhibition on a variety of tumor cell lines and normal tissue cells. More direct evidence that protection is associated with a GI phase growth arrest comes from results in this study which show that prior exposure of cells to cycloheximide, puromycin, or actinomycin D also prevents cell death by TNF/ACT D. These results further support our hypothesis that a cell popu lation resistant to TNF is generated in response to either the cytokines or other agents capable of blocking the early events in G, phase (23, 24). According to this concept, exponentially growing cells, which are not affected by a cytokine-induced protection, enter into S phase as soon as they are stimulated by the growth factors present in fetal calf serum, which is added simultaneously with the combination TNF/ACT D. Thereafter, ACT D renders these cells more sensitive to TNF cytolytic effects by blocking the synthesis of TNF-induced repair proteins (3, 18) and by arresting and synchronizing cells in the G2 + M compartment. 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Dinarello Cancer Res 1991;51:2379-2385. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/51/9/2379 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1991 American Association for Cancer Research.