Serum Protease Inhibitor Abrogation of Newcastle Disease Virus Enhancement of Cytolysis by Recombinant Tumor Necrosis Factors Alpha and Beta Pamela A. Rood, Robert M. Lorence, Keith W. Kelley*
Newcastle disease virus (NDV) is beneficial in the treatment of paVol. 82, No. 3, February 7, 1990
Protease inhibitors are known to abrogate the enhancement of TNF cytotoxicity caused by the inhibition of RNA and protein synthesis (20,27-29). Previous data from our laboratory showed that NDV makes TNF-resistant human melanoma cells sensitive to killing by TNF-a (7). We have now extended this' finding by showing that virulent, but not inacti-
vated, NDV is required to induce transformed L929 cells to become susceptible to killing by TNF. In contrast to L929 cells, neither NDV nor dactinomycin (DACT) (7,30) could sensitize WEHI 164 clone 13 cells to the lytic effects of TNF. We explored the difference between these two cell lines that are commonly used to detect TNF activity by using a serine protease inhibitor, N-1 -tosylamide-2-phenylethylchloromethyl ketone (TPCK). This inhibitor blocked the cytotoxicity caused by TNF on NDV-treated L929 cells but had no effect on that of WEHI 164 clone 13 cells. These results suggest that L929 cells but not WEHI 164 clone 13 cells can synthesize TNFinduced protective proteins. We propose that both NDV and RNA synthesis inhibitors block the synthesis of protective proteins in L929 cells and that these proteins can also be degraded by serine proteases within the cell. Therefore, resistance or sensitivity to TNF may depend on the synthesis of these protective proteins relative to the activity of serine proteases that degrade them.
Received June 6, 1989; revised September 26, 1989; accepted October 3, 1989. Supported in part by Public Health Service grant AG-06246 from the National Institute on Aging, National Institutes of Health, Department of Health and Human Services; grant N00014-89-J-1956 from the Office of Naval Research; grant 89-37265-4536 from the U.S. Department of Agriculture; and the Moorman Manufacturing Company. R. M. Lorence is a fellow of the James Ewing Foundation (Society of Surgical Oncology). Supported in part by the Independent Study Program, University of Illinois College of Medicine, Chicago, IL. P. A. Rood, R. M. Lorence, K. W. Kelley, Department of Animal Sciences, Laboratory of Immunophysiology, University of Illinois, Urbana, IL. We thank Drs. L. Schook, H. Lewin, and S. Arkins for helpful comments on this manuscript; Dr. John Kaumeyer for providing recombinant TNF reagents and specific antibodies; Dr. S. Gillis for providing recombinant murine IL-4; Dr. Terje Espevik for the gift of WEHI 164 clone 13 cells; and Dr. D. N. Tripathy for providing a wild isolate of Newcastle disease virus. * Correspondence to: Keith W. Kelley, Ph.D., Laboratory of Immunophysiology, University of Illinois, 162 ASL, 1207 W. Gregory Dr., Urbana, IL 61801.
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Newcastle disease virus (NDV) has been used to induce regression of tumors in human cancer patients. We recently demonstrated that human malignant melanoma cells resistant to the lytic effects of tumor necrosis factor-a (TNF-a) become susceptible after treatment with NDV. We examined the effects of a serine protease inhibitor, A'-1 -tosylamide-2-phenylethyl-chloromethyl ketone (TPCK), on viral enhancement of TNF cytotoxicity. Virulent NDV (but neither heatnor UV-inactivated NDV) induced a 100-fold increase in the sensitivity of murine fibroblast L929 cells to recombinant human TNF-a (rHuTNF-a), rHuTNF-/8, and recombinant murine TNF-a (rMuTNF-a). TPCK, which is an inhibitor of chymotrypsin-like proteases, blocked between 42% and 93% of the cytolytic activity of rMuTNFa, rHuTNF-a, and rHuTNF-/? toward NDV-treated L929 cells. Similarly, TPCK abrogated 62% of the cytotoxicity of rMuTNF-a toward dactinomycin-treated L929 cells. In contrast, TPCK had no effect on WEHI 164 clone 13 cells, a murine fibrosarcoma cell line that is much more sensitive to the lytic effects of TNF and does not show enhanced sensitivity to TNF after treatment with either NDV or dactinomycin. These results suggest a role for a cellular protease in the mechanism by which some viruses sensitize tumor cells to the cytolytic activity of TNF. [J Natl Cancer Inst 82:213-217, 1990]
tients with metastatic cancer (1-4) and tumor-bearing rodents (5,6). Although the exact mechanism is unknown, we recently reported that NDV is a potent inducer of the synthesis of TNF-a in human and rat mononuclear cells (7). In addition, NDV can also induce TNF sensitivity in the TNF-resistant human malignant melanoma MEL-14 and MEL-21 cells. Other viruses are also capable of inducing TNF production (8-10) and sensitizing target cells to TNF cytotoxicity (10-15). Although the exact difference between TNF-susceptible and -resistant cells is not known, several lines of evidence suggest that TNF-a induces TNF-protective proteins in only TNFresistant cells. First, inhibitors of protein and RNA synthesis are well-known for their enhancement of TNF-a and TNF(5 cytotoxicity (16-20). Some viruses have been shown to inhibit RNA synthesis in target cells (21,22), which may explain why several viruses can increase sensitivity of target cells to TNF cytotoxicity (7,10-15). Second, pretreatment of target cells with TNF-a abrogates the enhanced sensitivity to this factor caused by a protein synthesis inhibitor (23,24). These results strongly suggest that in resistant target cells, TNF-a induces synthesis of TNF-protective proteins. Production of these proteins is blocked if the target cells are pretreated with protein or RNA synthesis inhibitors, which makes the target cells susceptible to TNF-a. Third, resistance to TNF-a is transferred to heterokaryons formed by the fusion of TNF-sensitive and -resistant cells (25). Some protective factors from the TNF-resistant cells must have been transferred to the heterokaryons to make them resistant to TNF. Fourth, TNF-a is indeed capable of inducing synthesis of proteins (16,26) in resistant cells, including manganese superoxide dismutase (26).
213
Materials and Methods Cell Lines and Cytokines
Virus A wild isolate of NDV was obtained from Dr. Deoki N. Tripathy at the University of Illinois (Urbana, IL). Virus propagation and titer measurement were performed as described (37). The NDV was inactivated by heating to 65 °C for 1 hour or by UV irradiation at 0-5 °C in an uncovered 100- X 15-mm petri dish 4 cm from a 366-milliwatt source for either 20 minutes or 3 hours. Replicative capability of NDV was tested by our infecting chicken embryos and measuring viral titer (31).
Inhibition of TNF Activity With TPCK
The TPCK obtained from Sigma Chemical Co. (St. Louis, MO) was dissolved in 50% RPMI-1640/50% dimethyl sulfoxide to make a 5-mM stock solution. The protease inhibitor was diluted in RPMI-1640 and added at the same time as NDV in the TNF assay described above. The final concentration of TPCK/well was 4 nM. The total volume per well before the addition of TNF was 100 yL. Final concentration of dimethyl sulfoxide in control and TPCK-treated cells was 0.04%.
TNF Bioassay
The L929 or WEHI 164 clone 13 cells (4 X 104) and 1 /nCi Na251CrO4 [Amersham Corp. (Arlington Heights, IL)], in a total volume of 200 nL of complete RPMI medium, were added to each well of a Becton Dickinson (Oxnard, CA) 96-well, flat-bottom microtiter plate. After a 15- to 20-hour incubation at 38 °C and 7% CO2, the wells were washed three times with serum-free RPMI-1640 for removal of unincorporated radioisotope and serum. Cells in each well were treated by the addition of either control serum-free medium or 10 hemagglutination units 214
Results Enhancement by NDV of Cytotoxicity of TNF Toward L929 Cells
The NDV-treated L929 cells were at least 100-fold more sensitive to the cytolytic effects of TNF when compared with untreated L929 cells (fig. 1). For example, 1 U/well or 34 pg of rMuTNF-a killed 45% of the NDVtreated L929 cells, whereas 100 U/well or 3,400 pg killed only 30% of the untreated cells (fig. 1A). Similarly, treatment with NDV caused a 100-fold increase in the sensitivity of L929 cells
toward rHuTNF-a (1 U/well or 20 pg vs. 100 U/well or 2,000 pg; fig. IB) and rHuTNF-/? (1 U/well or 8 pg vs. 100 U/well or 800 pg; fig. 1C). A 2-hour preincubation with NDV before the addition of TNF gave optimum results (data not shown). We demonstrated that the cytotoxicity against NDV-treated L929 cells was a specific property of TNF-a and TNF-/3 by: (a) using a serum-free assay with recombinant human and murine TNF proteins, (b) performing TNF antibody inhibition studies, and (c) adding other cytokines at much greater concentrations than those used for TNF. The TNF antibodies gave 87%, 84%, and 134% inhibition of rHuTNF-a, rMuTNF-a, and rHuTNF/?, respectively, on NDV-treated L929 cells, whereas the appropriate murine and rabbit control antibodies had no effect. The rHuTNF-a, rHuTNF-)3, and rMuTNF-a (200, 80, and 340 pg/well, respectively) caused specific cytotoxicities ranging from 31% to 34%, whereas all of the other cytokines tested caused less than 5% specific cytotoxicity on NDV-treated L929 target cells as follows: rHu-interleukin-lj3, 1,460; rMu-interleukin-4, 1; purified Mu-interferon-a, 500; purified Mu-interferon-a+/?, 250; r-rat interferon-y, 5; and rMu-interferon-7, 100 ng/well. . Kinetics of TNF Cytotoxicity
The kinetics of TNF-a killing of NDV-treated L929 cells are shown in figure 2. The cytotoxic effects of treatment of L929 cells with the combination of 10 hemagglutination units of NDV and 10 U of rMuTNF-a were first detected at 8 hours. Cytotoxic activity against L929 cells remained at a low level with either rMuTNF-a alone or NDV alone through 14 hours « 20% cytotoxicity). In contrast, rMuTNF-a cytotoxicity was greater than 35% for NDV-treated cells after 10 hours and increased to 55% cytotoxicity by 14 hours. Comparison of Enhancement in Cytotoxicity Caused by NDV and DACT Because inhibitors of RNA synthesis such as DACT are known to enhance the sensitivity of L929 cells toward Journal of the National Cancer Institute
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The transformed murine fibroblastic L929 cell line was obtained from the American Type Culture Collection (ATCC CCL 1; Rockville, MD). The WEHI 164 clone 13 cells (30) were a gift from Dr. Terje Espevik of the Institute of Cancer Research (Trondheim, Norway). Cells were grown in complete RPMI-1640 obtained from GIBCO (Grand Island, NY) prepared by supplementation with 100 U of penicillin/mL, 100 ng of streptomycin/mL, 2 ng of sodium bicarbonate/mL, and 10% heatinactivated fetal bovine serum obtained from Flow Laboratories (McLean, VA). Recombinant murine TNF-a (rMuTNF-a; sp act = 2.9 X 107 U/mg), recombinant human TNF-a (rHuTNFa; sp act = 5.0 X 107 U/mg), and rHuTNF-/? (sp act = 1.2 X 108 U/mg) were gifts from Dr. John Kaumeyer of Genentech, Inc. (South San Francisco, CA).
of NDV in a volume of 100 yL. After a 2-hour incubation at 38 °C, TNF was added in 100 iiL of serum-free RPMI-1640, and cells were incubated for an additional 8 hours at 38 °C and 7% CO2. We determined maximum release by adding 100 yL of 0.02% Triton X-100 to the appropriate wells. After centrifugation of the plates at 400 g for 10 minutes at room temperature, 100 nL of the supernatant/well was removed, added to 3 mL of liquid scintillation cocktail [Aquasol-2, New England Nuclear Corp. (Boston, MA)], and assessed in a Beckman LS-5801 scintillation counter. Percent specific 51Cr release was calculated according to the following equation: Percent specific release = 100 X sample counts/minute (cpm) — spontaneous cpm/maximum cpm — spontaneous cpm. Spontaneous release was always less than 25% of the maximum. Treatment with NDV was omitted for the WEHI 164 clone 13 cells, because NDV had no effect on TNF cytotoxicity (data not shown).
1 hour or UV inactivation of the virus for 20 minutes or 3 hours resulted in a 59%, 56%, and 77% decrease (P < .01), respectively, in the ability of the virus to sensitize L929 cells to the cytotoxic effects of rMuTNF-a. Heat inactivation as well as UV irradiation was verified to be effective at inactivating over 90% of viral infectivity as assessed by viral propagation in chicken embryos.
100 90A
0 HAU NDV
t
•
10 HAU NDV
0.010
0.100
1.000
10.000
100.000
Effect of Inhibitor on TNF Cytotoxicity Toward NDV-Treated L929 Cells
Murine TNF-a (Units/well) 100-p 908070SOSO40 30 20 10-
B
A
A
0 HAU NDV
•
•
10 HAU NDV
o!! -10 -
H
0.010
0.100
h-
1.000
10.000
100.000
10.000
100.000
Human TNF-a (Units/well)
A
A
0 HAU NDV
•
•
10 HAU NDV
0.010
0.100
1.000
Human TNF-/? (Units/well)
TNF-a (20,21,24,27,28), we compared DACT with NDV. Ten units/well of rMuTNF-a killed 79% of DACTtreated L929 cells and 72% of the NDV-treated L929 cells, in contrast to
less than 10% of the untreated cells (table 1). We needed an infectious virus for inducing sensitivity to TNF in L929 cells. Heat inactivation of NDV at 65 °C for
60
Figure 2. Time course of NDV enhancement of rMuTNF-a cytotoxicity against L929 cells. Combined treatment of NDV and rMuTNF-a at 8 hr yielded a significant (P < .01) increase of cytotoxic activity from time zero. Spontaneous release was less than 20% of maximum release at every time period, and the standard deviation at each time point did not exceed 6% of the mean.
A
50
A NOV/TNF
• N0V 40-
•
TNF
3020 10
u
0
— 10-1
8
10
Time (Hours)
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Because NDV and DACT had similar abilities to sensitize L929 cells toward rMuTNF-a (table 1), we. investigated whether the protease inhibitor TPCK would have comparable effects on NDV- and DACT-treated L929 cells. TPCK abrogated 93% of the cytolytic activity of rMuTNF-a against NDV-treated L929 cells in one experiment (table 2) and 42% of the activity in another (data not shown). It was also capable of abrogating a significant percentage (62%) of the activity of rMuTNF-a against DACT-treated L929 cells (table 2). We also showed that TPCK abrogated 54% and 7 1 % of the cytolytic activities of rHuTNF-a and rHuTNF-/?, respectively, toward NDV-treated L929 cells (table 2). The addition of TPCK before, simultaneously, or after exposure to NDV did not alter inhibition of cytotoxicity caused by TPCK (data not shown). Effects of TPCK on TNF Cytotoxicity Toward WEHI 164 Clone 13 Cells At the same concentration that it inhibited TNF cytotoxicity of L929 cells (4 nM), TPCK had no such inhibitory effect on MuTNF-a cytolytic activity toward WEHI 164 clone 13 cells (table 2). This result showed that the activity of TPCK was different for L929 cells and WEHI 164 clone 13 cells. Additionally, these data demonstrated that TPCK was not blocking TNF-a cytotoxicity of L929 cells by directly inactivating this factor or by preventing it from binding to TNF receptors on murine target cells. Finally, dimethyl sulfoxide had no effect on TNF cytotoxicity at the low concentrations (0.04%) we used to solubilize TPCK, which indicated that the diluent was not inREPORTS
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Figure 1. Sensitivity of NDVtreated and control L929 cells to the lytic effects of rMuTNFa (A), rHuTNF-a (B), and rHuTNF-/3 (C). Percent cytotoxicity of NDV-treated and control L929 cells was determined after 8 hr for a range of concentrations of TNF. For each TNF species, a significant increase (P < .01) in cytotoxicity from zero was achieved at approximately 1 U/well for NDV-treated cells and 100 U/well for control cells. Standard deviation for rMuTNF-a did not exceed 13%, and standard deviation for rHuTNF-a and rHuTNF-/3 did not exceed 5%. HAU = hemagglutination units.
A
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Table 1. Comparison of ability of NDV and DACT to sensitize L929 cells toward lytic effects of rMuTNF-a
Table 2. Effects of TPCK on cytotoxic activity of TNF toward NDV- and DACT-treated L929 and WEHI 164 clone 13 cells* TNF
rMuTNF-a rMuTNF-o rMuTNF-a rMuTNF-a rMuTNF-a rMuTNF-a rHuTNF-a rHuTNF-a rHuTNF-0 rHuTNF-0
TPCK (^/W)
Target cells
0 4
NDV-treated L929 NDV-treated L929 DACT-treated L929 DACT-treated L929 WEHI 164 clone 13 WEHI 164 clone 13 NDV-treated L929 NDV-treated L929 NDV-treated L929 NDV-treated L929 '
0 4 0 4 0 4 0 4
Cytotoxicity
72 ± 2 5 ±8 79 ± 5 30 ± 6 100 ± 2 103 ± 0 51 ± 3 23 ± 2 69 ± 2 20 ± 1
Inhibition (%) 93 62 -3 54 71
*NDV, TPCK, or dimethyl sulfoxide was added 2 hr prior to aliquots of 10 U of TNF-a or 100 U of TNF-/}. Average spontaneous cpm was 10,720 and maximum release was 54,289 cpm for L929 cells; respective values for WEHI 164 clone 13 cells were 8,949 and 37,573 cpm. t Values are means ± standard deviation.
216
MuTNF-a caused by NDV and DACT treatment of L929 cells. Furthermore, these data extend earlier work with TNF (20,27) to include inhibition of recombinant TNF-/3 cytolytic activity on NDV-treated target cells by a protease inhibitor as well (table 2). It is believed that the proteases involved in TNF cytotoxicity are not lysosomal (20), nor do they react at the surface of the plasma membrane (28). Our results with the highly TNF-sensitive murine cell line, WEHI 164 clone 13, may provide a clue as to how proteases are involved in TNF cytotoxicity. We (7) and others (30) found that the RNA synthesis inhibitor DACT did not significantly increase sensitivity of WEHI 164 clone 13 cells to TNF cytotoxicity. Treatment with NDV also had no effect on TNF cytotoxicity of WEHI 164 clone 13 cells (data not shown). Therefore, the effect of DACT and NDV on WEHI 164 clone 13 cells was different from that on L929 cells. Because of this difference, we compared the effect of TPCK on both of these cell lines. We found that TNF-a killing of WEHI 164 clone 13 cells was unaffected by 4 fiM TPCK, the same concentration that blocked 93% of MuTNF-a cytotoxicity toward NDV-treated L929 cells (table 2). These results suggest that WEHI 164 clone 13 cells lack putative TNFprotective proteins such as manganese superoxide dismutase, which recently has been shown to protect cells against cytotoxic effects of TNF-a (33). This defect would make WEHI 164 clone 13 cells highly sensitive to TNF-a and TNF-/J and therefore unaffected by any sensitizing effects of DACT or NDV. We propose that the TPCK-reactive proteases interfere with functioning of the TNF-protective proteins, possibly by proteolyzing TNF-protective proteins and thus increasing their steadystate turnover. This hypothesis would explain why a lack of TNF-protective proteins in WEHI 164 clone 13 cells results in the absence of an effect on TNF-a cytotoxicity by TPCK, as well as by DACT and NDV. Testing of this hypothesis awaits verification and identification of the alleged TNF-protective proteins. Nevertheless, these results expand the importance of proteases in mediating cell death and demonstrate Journal of the National Cancer Institute
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by showing that virulent, but not inactivated, NDV is capable of enhancing TNF-a and TNF-/3 sensitivity in L929 Cytotoxicity toward cells, a widely used cell line for TNF L929 cells (%)t studies. Treatment* The molecular mechanism responsiMedium rMuTNF-a ble for the cytolytic activities of TNF-a None 0±2 5±1 and TNF-/3 has not been elucidated, alNDV 5±3 72 ± 1 though stimulation of arachidonic acid DACT 6±2 79 ± 5 metabolism by TNF, with subsequent *Ten hemagglutination units of NDV and 1 ng formation of toxic oxygen radicals, of DACT/mL were used. • t Average percent cytotoxicity was determined has been suggested (32). TNF-resistant after the addition of medium or 10 U of cells have also been proposed to synrMuTNF-a for the different treatments. Values thesize TNF-protective proteins, such are means ± standard deviation. as manganese superoxide dismutase, in response to TNF-a or TNF-0 (16,26). The ability of inhibitors of protein and volved in the protective effects of 4 \iM RNA synthesis to sensitize target cells TPCK (data not shown). to the lytic effects of TNF has been well documented (16-20). It is also Discussion known that many viruses can potently NDV has been used to treat pa- inhibit host RNA and protein synthetients with malignant melanoma with sis and that UV inactivation decreases reported beneficial results (3,4). In ro- the ability of viruses to inhibit prodents, NDV can also induce regression tein synthesis in L929 cells (21). Exof tumors and prevent the formation of periments in this report demonstrate metastases (5,6). Although the mecha- that treatment of L929 cells with either nism by which NDV reduces tumor bur- DACT or virulent NDV (but not UVden is unknown, it appears to have a or heat-inactivated NDV) augmented twofold effect. the cytolytic activity of TNF-a toward First, we recently reported that this L929 cells to a similar degree (table 1). virus induced the synthesis of TNF-a If both DACT and NDV sensiby human mononuclear cells (7). Sec- tize L929 cells to TNF-a by a simiond, we showed that two human ma- lar mechanism, we hypothesized from lignant melanoma cell lines, MEL-14 previous observations (20,27,28) that and MEL-21, which are normally re- serine protease inhibitors might abrosistant to the cytolytic effect of TNF, gate enhancement to TNF-a caused by become sensitive to TNF cytotoxicity both agents. The data presented in tawhen pretreated with NDV. Data in ble 2 show that this prediction is true; the present report extend this finding TPCK inhibited the cytotoxic effects of
the active role cells play in determining their fate when encountering TNF.
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We have recently shown that exogenous expression of the mouse interferon-7 (IFN-7) gene augmented the cellkilling potential of a line of cytotoxic T lymphocytes (CTLs) specific against a murine glioma line (203-glioma). In the present work, we further investigated the in vivo antitumor effects of the E7-6 and E 7 - 9 sublines of this CTL line transfected with the IFN-7gene. Using the Winn assay to test the neutralization of subcutaneous gliomas, we determined that these CTL sublines were more effective than the E-4 parent CTL line and that suppression of the tumor growth was dependent on the number of effector cells (CTLs). Moreover, intravenous injection of E7-9 cells was more effective in suppressing the tumor growth than intravenous injection of E-4 cells. These results suggest that transfection of antitumor effector cells with the IFN-7 gene could improve the efficacy of adoptive immunotherapy against cancer. [J Natl Cancer lnst 82:217-220, 1990]
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Efficient Tumor Suppression by Glioma-Specific Murine Cytotoxic T Lymphocytes Transfected With Interferon-7 Gene
Newcastle disease virus (NDV) is beneficial in the treatment of paVol. 82, No. 3, February 7, 1990
Protease inhibitors are known to abrogate the enhancement of TNF cytotoxicity caused by the inhibition of RNA and protein synthesis (20,27-29). Previous data from our laboratory showed that NDV makes TNF-resistant human melanoma cells sensitive to killing by TNF-a (7). We have now extended this' finding by showing that virulent, but not inacti-
vated, NDV is required to induce transformed L929 cells to become susceptible to killing by TNF. In contrast to L929 cells, neither NDV nor dactinomycin (DACT) (7,30) could sensitize WEHI 164 clone 13 cells to the lytic effects of TNF. We explored the difference between these two cell lines that are commonly used to detect TNF activity by using a serine protease inhibitor, N-1 -tosylamide-2-phenylethylchloromethyl ketone (TPCK). This inhibitor blocked the cytotoxicity caused by TNF on NDV-treated L929 cells but had no effect on that of WEHI 164 clone 13 cells. These results suggest that L929 cells but not WEHI 164 clone 13 cells can synthesize TNFinduced protective proteins. We propose that both NDV and RNA synthesis inhibitors block the synthesis of protective proteins in L929 cells and that these proteins can also be degraded by serine proteases within the cell. Therefore, resistance or sensitivity to TNF may depend on the synthesis of these protective proteins relative to the activity of serine proteases that degrade them.
Received June 6, 1989; revised September 26, 1989; accepted October 3, 1989. Supported in part by Public Health Service grant AG-06246 from the National Institute on Aging, National Institutes of Health, Department of Health and Human Services; grant N00014-89-J-1956 from the Office of Naval Research; grant 89-37265-4536 from the U.S. Department of Agriculture; and the Moorman Manufacturing Company. R. M. Lorence is a fellow of the James Ewing Foundation (Society of Surgical Oncology). Supported in part by the Independent Study Program, University of Illinois College of Medicine, Chicago, IL. P. A. Rood, R. M. Lorence, K. W. Kelley, Department of Animal Sciences, Laboratory of Immunophysiology, University of Illinois, Urbana, IL. We thank Drs. L. Schook, H. Lewin, and S. Arkins for helpful comments on this manuscript; Dr. John Kaumeyer for providing recombinant TNF reagents and specific antibodies; Dr. S. Gillis for providing recombinant murine IL-4; Dr. Terje Espevik for the gift of WEHI 164 clone 13 cells; and Dr. D. N. Tripathy for providing a wild isolate of Newcastle disease virus. * Correspondence to: Keith W. Kelley, Ph.D., Laboratory of Immunophysiology, University of Illinois, 162 ASL, 1207 W. Gregory Dr., Urbana, IL 61801.
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Newcastle disease virus (NDV) has been used to induce regression of tumors in human cancer patients. We recently demonstrated that human malignant melanoma cells resistant to the lytic effects of tumor necrosis factor-a (TNF-a) become susceptible after treatment with NDV. We examined the effects of a serine protease inhibitor, A'-1 -tosylamide-2-phenylethyl-chloromethyl ketone (TPCK), on viral enhancement of TNF cytotoxicity. Virulent NDV (but neither heatnor UV-inactivated NDV) induced a 100-fold increase in the sensitivity of murine fibroblast L929 cells to recombinant human TNF-a (rHuTNF-a), rHuTNF-/8, and recombinant murine TNF-a (rMuTNF-a). TPCK, which is an inhibitor of chymotrypsin-like proteases, blocked between 42% and 93% of the cytolytic activity of rMuTNFa, rHuTNF-a, and rHuTNF-/? toward NDV-treated L929 cells. Similarly, TPCK abrogated 62% of the cytotoxicity of rMuTNF-a toward dactinomycin-treated L929 cells. In contrast, TPCK had no effect on WEHI 164 clone 13 cells, a murine fibrosarcoma cell line that is much more sensitive to the lytic effects of TNF and does not show enhanced sensitivity to TNF after treatment with either NDV or dactinomycin. These results suggest a role for a cellular protease in the mechanism by which some viruses sensitize tumor cells to the cytolytic activity of TNF. [J Natl Cancer Inst 82:213-217, 1990]
tients with metastatic cancer (1-4) and tumor-bearing rodents (5,6). Although the exact mechanism is unknown, we recently reported that NDV is a potent inducer of the synthesis of TNF-a in human and rat mononuclear cells (7). In addition, NDV can also induce TNF sensitivity in the TNF-resistant human malignant melanoma MEL-14 and MEL-21 cells. Other viruses are also capable of inducing TNF production (8-10) and sensitizing target cells to TNF cytotoxicity (10-15). Although the exact difference between TNF-susceptible and -resistant cells is not known, several lines of evidence suggest that TNF-a induces TNF-protective proteins in only TNFresistant cells. First, inhibitors of protein and RNA synthesis are well-known for their enhancement of TNF-a and TNF(5 cytotoxicity (16-20). Some viruses have been shown to inhibit RNA synthesis in target cells (21,22), which may explain why several viruses can increase sensitivity of target cells to TNF cytotoxicity (7,10-15). Second, pretreatment of target cells with TNF-a abrogates the enhanced sensitivity to this factor caused by a protein synthesis inhibitor (23,24). These results strongly suggest that in resistant target cells, TNF-a induces synthesis of TNF-protective proteins. Production of these proteins is blocked if the target cells are pretreated with protein or RNA synthesis inhibitors, which makes the target cells susceptible to TNF-a. Third, resistance to TNF-a is transferred to heterokaryons formed by the fusion of TNF-sensitive and -resistant cells (25). Some protective factors from the TNF-resistant cells must have been transferred to the heterokaryons to make them resistant to TNF. Fourth, TNF-a is indeed capable of inducing synthesis of proteins (16,26) in resistant cells, including manganese superoxide dismutase (26).
213
Materials and Methods Cell Lines and Cytokines
Virus A wild isolate of NDV was obtained from Dr. Deoki N. Tripathy at the University of Illinois (Urbana, IL). Virus propagation and titer measurement were performed as described (37). The NDV was inactivated by heating to 65 °C for 1 hour or by UV irradiation at 0-5 °C in an uncovered 100- X 15-mm petri dish 4 cm from a 366-milliwatt source for either 20 minutes or 3 hours. Replicative capability of NDV was tested by our infecting chicken embryos and measuring viral titer (31).
Inhibition of TNF Activity With TPCK
The TPCK obtained from Sigma Chemical Co. (St. Louis, MO) was dissolved in 50% RPMI-1640/50% dimethyl sulfoxide to make a 5-mM stock solution. The protease inhibitor was diluted in RPMI-1640 and added at the same time as NDV in the TNF assay described above. The final concentration of TPCK/well was 4 nM. The total volume per well before the addition of TNF was 100 yL. Final concentration of dimethyl sulfoxide in control and TPCK-treated cells was 0.04%.
TNF Bioassay
The L929 or WEHI 164 clone 13 cells (4 X 104) and 1 /nCi Na251CrO4 [Amersham Corp. (Arlington Heights, IL)], in a total volume of 200 nL of complete RPMI medium, were added to each well of a Becton Dickinson (Oxnard, CA) 96-well, flat-bottom microtiter plate. After a 15- to 20-hour incubation at 38 °C and 7% CO2, the wells were washed three times with serum-free RPMI-1640 for removal of unincorporated radioisotope and serum. Cells in each well were treated by the addition of either control serum-free medium or 10 hemagglutination units 214
Results Enhancement by NDV of Cytotoxicity of TNF Toward L929 Cells
The NDV-treated L929 cells were at least 100-fold more sensitive to the cytolytic effects of TNF when compared with untreated L929 cells (fig. 1). For example, 1 U/well or 34 pg of rMuTNF-a killed 45% of the NDVtreated L929 cells, whereas 100 U/well or 3,400 pg killed only 30% of the untreated cells (fig. 1A). Similarly, treatment with NDV caused a 100-fold increase in the sensitivity of L929 cells
toward rHuTNF-a (1 U/well or 20 pg vs. 100 U/well or 2,000 pg; fig. IB) and rHuTNF-/? (1 U/well or 8 pg vs. 100 U/well or 800 pg; fig. 1C). A 2-hour preincubation with NDV before the addition of TNF gave optimum results (data not shown). We demonstrated that the cytotoxicity against NDV-treated L929 cells was a specific property of TNF-a and TNF-/3 by: (a) using a serum-free assay with recombinant human and murine TNF proteins, (b) performing TNF antibody inhibition studies, and (c) adding other cytokines at much greater concentrations than those used for TNF. The TNF antibodies gave 87%, 84%, and 134% inhibition of rHuTNF-a, rMuTNF-a, and rHuTNF/?, respectively, on NDV-treated L929 cells, whereas the appropriate murine and rabbit control antibodies had no effect. The rHuTNF-a, rHuTNF-)3, and rMuTNF-a (200, 80, and 340 pg/well, respectively) caused specific cytotoxicities ranging from 31% to 34%, whereas all of the other cytokines tested caused less than 5% specific cytotoxicity on NDV-treated L929 target cells as follows: rHu-interleukin-lj3, 1,460; rMu-interleukin-4, 1; purified Mu-interferon-a, 500; purified Mu-interferon-a+/?, 250; r-rat interferon-y, 5; and rMu-interferon-7, 100 ng/well. . Kinetics of TNF Cytotoxicity
The kinetics of TNF-a killing of NDV-treated L929 cells are shown in figure 2. The cytotoxic effects of treatment of L929 cells with the combination of 10 hemagglutination units of NDV and 10 U of rMuTNF-a were first detected at 8 hours. Cytotoxic activity against L929 cells remained at a low level with either rMuTNF-a alone or NDV alone through 14 hours « 20% cytotoxicity). In contrast, rMuTNF-a cytotoxicity was greater than 35% for NDV-treated cells after 10 hours and increased to 55% cytotoxicity by 14 hours. Comparison of Enhancement in Cytotoxicity Caused by NDV and DACT Because inhibitors of RNA synthesis such as DACT are known to enhance the sensitivity of L929 cells toward Journal of the National Cancer Institute
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The transformed murine fibroblastic L929 cell line was obtained from the American Type Culture Collection (ATCC CCL 1; Rockville, MD). The WEHI 164 clone 13 cells (30) were a gift from Dr. Terje Espevik of the Institute of Cancer Research (Trondheim, Norway). Cells were grown in complete RPMI-1640 obtained from GIBCO (Grand Island, NY) prepared by supplementation with 100 U of penicillin/mL, 100 ng of streptomycin/mL, 2 ng of sodium bicarbonate/mL, and 10% heatinactivated fetal bovine serum obtained from Flow Laboratories (McLean, VA). Recombinant murine TNF-a (rMuTNF-a; sp act = 2.9 X 107 U/mg), recombinant human TNF-a (rHuTNFa; sp act = 5.0 X 107 U/mg), and rHuTNF-/? (sp act = 1.2 X 108 U/mg) were gifts from Dr. John Kaumeyer of Genentech, Inc. (South San Francisco, CA).
of NDV in a volume of 100 yL. After a 2-hour incubation at 38 °C, TNF was added in 100 iiL of serum-free RPMI-1640, and cells were incubated for an additional 8 hours at 38 °C and 7% CO2. We determined maximum release by adding 100 yL of 0.02% Triton X-100 to the appropriate wells. After centrifugation of the plates at 400 g for 10 minutes at room temperature, 100 nL of the supernatant/well was removed, added to 3 mL of liquid scintillation cocktail [Aquasol-2, New England Nuclear Corp. (Boston, MA)], and assessed in a Beckman LS-5801 scintillation counter. Percent specific 51Cr release was calculated according to the following equation: Percent specific release = 100 X sample counts/minute (cpm) — spontaneous cpm/maximum cpm — spontaneous cpm. Spontaneous release was always less than 25% of the maximum. Treatment with NDV was omitted for the WEHI 164 clone 13 cells, because NDV had no effect on TNF cytotoxicity (data not shown).
1 hour or UV inactivation of the virus for 20 minutes or 3 hours resulted in a 59%, 56%, and 77% decrease (P < .01), respectively, in the ability of the virus to sensitize L929 cells to the cytotoxic effects of rMuTNF-a. Heat inactivation as well as UV irradiation was verified to be effective at inactivating over 90% of viral infectivity as assessed by viral propagation in chicken embryos.
100 90A
0 HAU NDV
t
•
10 HAU NDV
0.010
0.100
1.000
10.000
100.000
Effect of Inhibitor on TNF Cytotoxicity Toward NDV-Treated L929 Cells
Murine TNF-a (Units/well) 100-p 908070SOSO40 30 20 10-
B
A
A
0 HAU NDV
•
•
10 HAU NDV
o!! -10 -
H
0.010
0.100
h-
1.000
10.000
100.000
10.000
100.000
Human TNF-a (Units/well)
A
A
0 HAU NDV
•
•
10 HAU NDV
0.010
0.100
1.000
Human TNF-/? (Units/well)
TNF-a (20,21,24,27,28), we compared DACT with NDV. Ten units/well of rMuTNF-a killed 79% of DACTtreated L929 cells and 72% of the NDV-treated L929 cells, in contrast to
less than 10% of the untreated cells (table 1). We needed an infectious virus for inducing sensitivity to TNF in L929 cells. Heat inactivation of NDV at 65 °C for
60
Figure 2. Time course of NDV enhancement of rMuTNF-a cytotoxicity against L929 cells. Combined treatment of NDV and rMuTNF-a at 8 hr yielded a significant (P < .01) increase of cytotoxic activity from time zero. Spontaneous release was less than 20% of maximum release at every time period, and the standard deviation at each time point did not exceed 6% of the mean.
A
50
A NOV/TNF
• N0V 40-
•
TNF
3020 10
u
0
— 10-1
8
10
Time (Hours)
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Because NDV and DACT had similar abilities to sensitize L929 cells toward rMuTNF-a (table 1), we. investigated whether the protease inhibitor TPCK would have comparable effects on NDV- and DACT-treated L929 cells. TPCK abrogated 93% of the cytolytic activity of rMuTNF-a against NDV-treated L929 cells in one experiment (table 2) and 42% of the activity in another (data not shown). It was also capable of abrogating a significant percentage (62%) of the activity of rMuTNF-a against DACT-treated L929 cells (table 2). We also showed that TPCK abrogated 54% and 7 1 % of the cytolytic activities of rHuTNF-a and rHuTNF-/?, respectively, toward NDV-treated L929 cells (table 2). The addition of TPCK before, simultaneously, or after exposure to NDV did not alter inhibition of cytotoxicity caused by TPCK (data not shown). Effects of TPCK on TNF Cytotoxicity Toward WEHI 164 Clone 13 Cells At the same concentration that it inhibited TNF cytotoxicity of L929 cells (4 nM), TPCK had no such inhibitory effect on MuTNF-a cytolytic activity toward WEHI 164 clone 13 cells (table 2). This result showed that the activity of TPCK was different for L929 cells and WEHI 164 clone 13 cells. Additionally, these data demonstrated that TPCK was not blocking TNF-a cytotoxicity of L929 cells by directly inactivating this factor or by preventing it from binding to TNF receptors on murine target cells. Finally, dimethyl sulfoxide had no effect on TNF cytotoxicity at the low concentrations (0.04%) we used to solubilize TPCK, which indicated that the diluent was not inREPORTS
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Figure 1. Sensitivity of NDVtreated and control L929 cells to the lytic effects of rMuTNFa (A), rHuTNF-a (B), and rHuTNF-/3 (C). Percent cytotoxicity of NDV-treated and control L929 cells was determined after 8 hr for a range of concentrations of TNF. For each TNF species, a significant increase (P < .01) in cytotoxicity from zero was achieved at approximately 1 U/well for NDV-treated cells and 100 U/well for control cells. Standard deviation for rMuTNF-a did not exceed 13%, and standard deviation for rHuTNF-a and rHuTNF-/3 did not exceed 5%. HAU = hemagglutination units.
A
215
Table 1. Comparison of ability of NDV and DACT to sensitize L929 cells toward lytic effects of rMuTNF-a
Table 2. Effects of TPCK on cytotoxic activity of TNF toward NDV- and DACT-treated L929 and WEHI 164 clone 13 cells* TNF
rMuTNF-a rMuTNF-o rMuTNF-a rMuTNF-a rMuTNF-a rMuTNF-a rHuTNF-a rHuTNF-a rHuTNF-0 rHuTNF-0
TPCK (^/W)
Target cells
0 4
NDV-treated L929 NDV-treated L929 DACT-treated L929 DACT-treated L929 WEHI 164 clone 13 WEHI 164 clone 13 NDV-treated L929 NDV-treated L929 NDV-treated L929 NDV-treated L929 '
0 4 0 4 0 4 0 4
Cytotoxicity
72 ± 2 5 ±8 79 ± 5 30 ± 6 100 ± 2 103 ± 0 51 ± 3 23 ± 2 69 ± 2 20 ± 1
Inhibition (%) 93 62 -3 54 71
*NDV, TPCK, or dimethyl sulfoxide was added 2 hr prior to aliquots of 10 U of TNF-a or 100 U of TNF-/}. Average spontaneous cpm was 10,720 and maximum release was 54,289 cpm for L929 cells; respective values for WEHI 164 clone 13 cells were 8,949 and 37,573 cpm. t Values are means ± standard deviation.
216
MuTNF-a caused by NDV and DACT treatment of L929 cells. Furthermore, these data extend earlier work with TNF (20,27) to include inhibition of recombinant TNF-/3 cytolytic activity on NDV-treated target cells by a protease inhibitor as well (table 2). It is believed that the proteases involved in TNF cytotoxicity are not lysosomal (20), nor do they react at the surface of the plasma membrane (28). Our results with the highly TNF-sensitive murine cell line, WEHI 164 clone 13, may provide a clue as to how proteases are involved in TNF cytotoxicity. We (7) and others (30) found that the RNA synthesis inhibitor DACT did not significantly increase sensitivity of WEHI 164 clone 13 cells to TNF cytotoxicity. Treatment with NDV also had no effect on TNF cytotoxicity of WEHI 164 clone 13 cells (data not shown). Therefore, the effect of DACT and NDV on WEHI 164 clone 13 cells was different from that on L929 cells. Because of this difference, we compared the effect of TPCK on both of these cell lines. We found that TNF-a killing of WEHI 164 clone 13 cells was unaffected by 4 fiM TPCK, the same concentration that blocked 93% of MuTNF-a cytotoxicity toward NDV-treated L929 cells (table 2). These results suggest that WEHI 164 clone 13 cells lack putative TNFprotective proteins such as manganese superoxide dismutase, which recently has been shown to protect cells against cytotoxic effects of TNF-a (33). This defect would make WEHI 164 clone 13 cells highly sensitive to TNF-a and TNF-/J and therefore unaffected by any sensitizing effects of DACT or NDV. We propose that the TPCK-reactive proteases interfere with functioning of the TNF-protective proteins, possibly by proteolyzing TNF-protective proteins and thus increasing their steadystate turnover. This hypothesis would explain why a lack of TNF-protective proteins in WEHI 164 clone 13 cells results in the absence of an effect on TNF-a cytotoxicity by TPCK, as well as by DACT and NDV. Testing of this hypothesis awaits verification and identification of the alleged TNF-protective proteins. Nevertheless, these results expand the importance of proteases in mediating cell death and demonstrate Journal of the National Cancer Institute
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by showing that virulent, but not inactivated, NDV is capable of enhancing TNF-a and TNF-/3 sensitivity in L929 Cytotoxicity toward cells, a widely used cell line for TNF L929 cells (%)t studies. Treatment* The molecular mechanism responsiMedium rMuTNF-a ble for the cytolytic activities of TNF-a None 0±2 5±1 and TNF-/3 has not been elucidated, alNDV 5±3 72 ± 1 though stimulation of arachidonic acid DACT 6±2 79 ± 5 metabolism by TNF, with subsequent *Ten hemagglutination units of NDV and 1 ng formation of toxic oxygen radicals, of DACT/mL were used. • t Average percent cytotoxicity was determined has been suggested (32). TNF-resistant after the addition of medium or 10 U of cells have also been proposed to synrMuTNF-a for the different treatments. Values thesize TNF-protective proteins, such are means ± standard deviation. as manganese superoxide dismutase, in response to TNF-a or TNF-0 (16,26). The ability of inhibitors of protein and volved in the protective effects of 4 \iM RNA synthesis to sensitize target cells TPCK (data not shown). to the lytic effects of TNF has been well documented (16-20). It is also Discussion known that many viruses can potently NDV has been used to treat pa- inhibit host RNA and protein synthetients with malignant melanoma with sis and that UV inactivation decreases reported beneficial results (3,4). In ro- the ability of viruses to inhibit prodents, NDV can also induce regression tein synthesis in L929 cells (21). Exof tumors and prevent the formation of periments in this report demonstrate metastases (5,6). Although the mecha- that treatment of L929 cells with either nism by which NDV reduces tumor bur- DACT or virulent NDV (but not UVden is unknown, it appears to have a or heat-inactivated NDV) augmented twofold effect. the cytolytic activity of TNF-a toward First, we recently reported that this L929 cells to a similar degree (table 1). virus induced the synthesis of TNF-a If both DACT and NDV sensiby human mononuclear cells (7). Sec- tize L929 cells to TNF-a by a simiond, we showed that two human ma- lar mechanism, we hypothesized from lignant melanoma cell lines, MEL-14 previous observations (20,27,28) that and MEL-21, which are normally re- serine protease inhibitors might abrosistant to the cytolytic effect of TNF, gate enhancement to TNF-a caused by become sensitive to TNF cytotoxicity both agents. The data presented in tawhen pretreated with NDV. Data in ble 2 show that this prediction is true; the present report extend this finding TPCK inhibited the cytotoxic effects of
the active role cells play in determining their fate when encountering TNF.
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We have recently shown that exogenous expression of the mouse interferon-7 (IFN-7) gene augmented the cellkilling potential of a line of cytotoxic T lymphocytes (CTLs) specific against a murine glioma line (203-glioma). In the present work, we further investigated the in vivo antitumor effects of the E7-6 and E 7 - 9 sublines of this CTL line transfected with the IFN-7gene. Using the Winn assay to test the neutralization of subcutaneous gliomas, we determined that these CTL sublines were more effective than the E-4 parent CTL line and that suppression of the tumor growth was dependent on the number of effector cells (CTLs). Moreover, intravenous injection of E7-9 cells was more effective in suppressing the tumor growth than intravenous injection of E-4 cells. These results suggest that transfection of antitumor effector cells with the IFN-7 gene could improve the efficacy of adoptive immunotherapy against cancer. [J Natl Cancer lnst 82:217-220, 1990]
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Efficient Tumor Suppression by Glioma-Specific Murine Cytotoxic T Lymphocytes Transfected With Interferon-7 Gene
