Ini J. Radimon Oncology Ed. Phys., Vol. 19. pp. 69-74 Printed in the U.S.A. All rights reserved.
0360-3016/90 $3.00 + .oO Copyright 0 1990 Pergamon Pres plc
??Original Contribution
THE INTERACTION BETWEEN RECOMBINANT HUMAN TUMOR NECROSIS FACTOR AND RADIATION IN 13 HUMAN TUMOR CELL LINES DENNIS
E. HALLAHAN,
M.D.,’
MICHAEL
AND RALPH
A. BECKETT,
R. WEICHSELBAUM,
B.S., t DONALD
KUFE,
M.D.2
M.D.’
‘Michael Reese/University of Chicago Center for Radiation Therapy, Department of Radiation and Cellular Oncology, University of Chicago, Pritzker School of Medicine; and *Laboratory of Clinical Pharmacology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA Tumor necrosis factor (TNF) was cytotoxic at concentrations of 10 to 1000 units/ml to 12 of 14 human tumor cell lines. Synergistic or additive cell killing between TNF and radiation was observed in 7 of 10 tumor cell lines, while independent tumor cell killing by each agent occurred in two tumor cell lines. The maximum synergistic effect was observed when TNF was added 4-12 hr prior to irradiation. This interaction was absent when TNF was added after irradiation. TNF also reduced potentially lethal damage repair in 3 of 5 cell lines tested. Possible mechanisms of interaction of TNF and X rays including induction of hydroxyl radicals and subsequent DNA damage by TNF and radiation are discussed. Tumor necrosis factor, Ionizing radiation, Synergy, Human tumor cell lines. INTRODUCTION
and Adriamycin ( 12). The oxidative damage produced by TNF may enhance cellular damage produced by ionizing radiation and suggests that an additive or synergistic interaction may occur between these two agents. We investigated the interaction between TNF and X rays in human tumor cell lines. Data presented here suggest a direct interaction between TNF and X rays, including the inhibition of potentially lethal damage repair (PLDR) in some human tumor cell lines.
Tumor
necrosis factor (TNF) is a polypeptide mediator of the cellular immune response which is produced by’
macrophages and lymphocytes (2, 12). The range of actions ascribed to this antitumor cytokine include increased vascular endothelial adhesiveness to neutrophils and platelets ( l), and promotion of thrombosis (10). The resulting tumor microvascular obliteration is associated with hemorrhagic necrosis of tumors in vivo (2, 12). TNF has a variety of other actions including fibroblast proliferation, activation of macrophages and NK cells, and induction of interleukin- 1 production ( 12, 18). Neutrophil peroxide radical production is also enhanced by TNF (6, 19). Finally, TNF is one of the components associated with respiratory distress syndrome and septic shock ( 12). In addition to enhancement of host immune response, TNF is also directly cytotoxic to tumor cell lines in vitro (2, 11, 18). Cell killing by TNF is associated with the production of hydroxyl radicals within the sensitive cell lines (23, 25) resulting in oxidative damage to DNA (24) and subsequently DNA fragmentation ( 14). TNF enhances the direct cytotoxic effects of a number of antitumor agents whose cytotoxic action is proposed to act through free radicals. These agents include mitomycin-C
METHODS
AND
MATERIALS
Cell
lines The establishment of human epithelial tumor and sarcoma cell lines has been previously described (20, 21). Human epithelial tumor cell culture medium consists of 72.5% Dulbecco’s Modification of Eagle’s Medium, 22.5% Ham’s Nutrient Mixture F-12, (DMEM/F12, at 3:1), 5% fetal bovine serum (FIB), 5 mcg/ml insulin, 5 mcg/ml transferrin, 2 X lo-” M 3,3’,5-triodo-L-thyronine, lo-” M cholera toxin, 1.8 X 10e4 M adenine, 0.4 mcg/ml hydrocortisone, 100 units/ml penicillin and 100 mcg/ml streptomycin (Pen/Strep). Culture medium for the human sarcoma cell lines and normal human fibroblast AG- 1522
Presented at the 3 1st Annual Scientific Meeting of the American Society for Therapeutic Radiology and Oncology San Francisco, CA, 3 October 1989. Reprint requests to: Dennis Hallahan, M.D. Dept. of Radiation Oncology, University of Chicago, 5841 S. Maryland, Chicago, IL 60637.
Supported by NC1 grant CA4 1068 and a gift from the Passis Family. Accepted for publication 24 January 1990.
69
70
I. J. Radiation
Oncology 0 Biology 0 Physics
cell line consists Pen/Strep.
of DMEM/FI
2 (3: 1) with 15% FBS and
Radiation/TNF
survival experiments
The colony forming assay and growth curve analyses were used to assess TNF sensitivity in each cell line. Human recombinant TNF* was added at concentrations ranging from 1 to 10,000 units/ml 12 hr after cells were plated. Cells were plated at densities of 200 to 10,000 in 100 mm dishes. Colonies were counted after lo- 14 days. During growth curve analysis, 1O3 cells were plated in 60 mm dishes. Cultures were trypsinized and total cell number per plate was calculated using a Coulter cell counter. Cell counts of exponentially growing cells with and without TNF were performed at days 1 through 7. X-ray survival experiments were performed on exponentially growing tumor cells and survival curves were determined as follows: Cells at the 7th to 25th passage were maintained in medium at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were trypsinized with 0.05% trypsin:0.02% EDTA from stock cultures and between 500 and 40,000 cells were plated in 100 mm diameter tissue culture dishes and allowed to enter exponential growth. Radiation was carried out 24 hr later with a 250 kV x-ray generator,+ operating at 26 mA at a dose rate of 1.07 Gy per minute. Immediately after irradiation, the cultures were returned to the incubator. After 18-24 days, cells were fixed and stained with crystal violet. Only colonies of >50 cells were scored as survivors. TNF was added to cell cultures lo-14 hr prior to irradiation. Concentrations of TNF ranged from 10 to 1000 units/ml (2.3 X lo6 units/mg). Surviving fractions were corrected for the reduced plating efficiency with TNF. Experimentally derived data points are the mean of 2-4 experiments. The multi-target model survival curves were fit to a single hit multi-target model (S = 1 - (-emD’Do)“. From th ese data points, Do and n were derived using a least square regression analysis omitting the 100 cGy data point.
Dejnitions Interactions between cytotoxic agents may be operationally defined. As defined by Dewey and Steele, an interaction is present when a radiation enhancing agent used concurrently with X rays results in cell killing which is greater than the expected independent killing from each agent alone (3, 17). In contrast, independent cell killing is the quantity of cell death which is expected from each agent alone (3, 17). Interactive killing may be additive or synergistic. Additive killing is defined as enhancement in x-ray induced cell killing such that the resulting survival curve is parallel to that of irradiation alone but greater than expected by the independent killing of each agent alone (3). Synergistic killing is operationally defined
* Asahi Chemical
Industries,
New York.
July 1990, Volume
19. Number
1
as a decrease in Do when both agents are used concurrently resulting in a steeper slope in the radiation survival curve (3). Potentially lethal damage repair (PLDR) is defined as damage which if unrepaired, is lethal (7). We specifically define PLDR to be the enhancement in survival as measured by increased colony-forming ability resulting from a delay in subculture of plateau phase tumor cells following radiation exposure (4). PLDR in plateau phase cultures has been reported to occur in the G1 phase of the cell cycle (4). Damage is fixed as cells cross the G&S interface and then becomes lethal (4).
Time course studies To determine the optimal time for the addition of TNF, cells were irradiated at regular intervals before and after TNF was added. Time course experiments were performed on exponentially growing tumor cells. Cell line SQ-20B was selected because of previously demonstrated synergy (5). Cells were trypsinized and plated at a density of 10,000 cells in 100 mm tissue culture dishes. TNF ( 10 units/ml) was added at 48, 24, 12, 10, 8, 6, 4, 2 hr prior to irradiation and at 0, 2, 12, and 24 hr after irradiation. Cultures were immediately returned to the incubator after irradiation. After 14 days, plates were stained and colonies ~50 cells were scored as survivors.
PLDR experiments Studies of potentially lethal damage repair (PLDR) were performed as follows. Cells were initially seeded into 35mm plastic tissue culture dishes and grown to confluency. Culture medium was renewed daily for 3 days and experiments were performed on day 4. Cells were irradiated at room temperature and then were returned to the incubator. Single dishes were removed and cells were subcultured and seeded at low density (5,000-50,000 cells per 100-mm dish) at regular intervals thereafter. Equitoxic doses were used to measure PLDR. The doses used to study PLDR were 500 cGy for cell line STSAR-13 and 700 cGy for all other cell lines tested. The enhancement in survival, as measured by the factor of increased colonyforming ability resulting from a 24 hr delay in subculture after irradiation, is interpreted as being due to the repair of potentially lethal damage. Thus, PLDR is expressed in terms of enhancement in surviving fraction as a function of the time interval between irradiation and subculture (S/So).
RESULTS Figure 1 demonstrates TNF-sensitivity in four tumor cell lines. Cell survival was reduced upon exposure to increasing TNF concentration. Cell line SCC-6 1 was killed by concentrations as low as 1 unit/ml whereas cell line
t GE Maxitron.
71
TNF and radiation interaction 0 D. E. HALLAHANet al.
0 0 -
STSAR-90 At+1522 Control STSAR-90
6
I
lb
Id0 TNF
lob0
lO,bOO
Wnitslmi)
Fig. 1. Response of human tumor cell lines to recombinant human TNF. Cell lines were grown in the presence of increasing concentrations of TNF (1 to lo4 units/ml). Plating efficiencies were compared to control plates grown without TNF (100% survival).
STSAR-5 required exposure to 1000 units/ml for detectable cell killing. Table 1 lists the related cytotoxicities of human tumor cell lines and a normal fibroblast line at concentrations of 10 and 1000 units/ml. TNF was cytotoxic to 3 of 4 soft tissue sarcoma cell lines, 3 of 3 bone sarcomas, and 4 of 5 epithelial tumor cell lines. Squamous cell carcinoma cell lines were more sensitive to lower concentrations of TNF than were the sarcoma cell lines. For example, a TNF concentration of 10 units/ml reduced the survival of epithelial tumor cells to 10% to 50% of normal, whereas 1000 units/ml was required to achieve similar cell killing in sarcoma cells (Table 1). However, at the concentrations tested, TNF was not directly cyto-
Table 1. Sensitivity
of human
1.0-7m
4
Soft tissue sarcomas STSAR-5 STSAR- 13 STSAR-48 STSAR-90 Bone sarcomas STSAR-33 STSAR-43 STSAR-75 Squamous cell carcinomas SQ-20B SCC-2 5 SCC-6 1 HNSCC-68 HNSCC- 15 1 Fibroblasts AG- 1522
Origin
toxic to all cell lines. Cell line STSAR-48 had no cytotoxicity from TNF, whereas cell lines HNSCC-68 and AGI522 responded to TNF with enhanced proliferation (Fig. 2). In contrast, cell line STSAR-90 responded to TNF with a prolonged doubling time (Fig. 2).
tumor
cell lines to TNF surviving
TNF (10 units/ml)
(1000 units/ml)
MFH Liposarcoma Neurofibrosarcoma MFH
100 50 100 100
53* 12* 100 18*
Ewings sarcoma Osteosarcoma Osteosarcoma
100 100 100
50* 30* 47
SCCA SCCA SCCA SCCA SCCA
50* 25* 12* 100 10*
3 6 0.6 100 0.3
100
100
Human
6
Fig. 2. Growth curves of human tibroblasts (AG-1522) and human sarcoma cell line @TSAR-90) with TNF (1000 units/ml) 0 and without TNF 0.
Percent Cell line
5
Days
fibroblast
Cytotoxicity of human tumor cell lines to TNF (10 to 1000 units/ml) as compared MFH = Malignant fibrous histiocytoma; SCCA = Squamous cell carcinoma. * p < 0.05.
to untreated
cell lines.
July 1990, Volume 19, Number I
I. J. Radiation Oncology 0 Biology ??Physics
12
Time
Course
point represents the surviving fraction after treatment with 700 cGy and 10 units/ml of TNF added at the indicated time interval before or after irradiation. Surviving fractions were corrected for the reduced plating efficiency due to TNF. The extent of cell killing when TNF was added after irradiation is equal to that of both agents added separately. In contrast, cell killing was greater than expected by either agent alone when TNF was added 4- 12 hr prior to irradiation. TNF was therefore added 12 hr prior to irradiation when studying the effects of concurrent TNF and X rays on survival curves. There was independent cell killing by TNF and X rays in human tumor cell lines STSAR 13 and STSAR-43. After correcting for the reduced plating efficiencies, there was no change in ii or Do after treating these two cell lines with TNF and radiation (Fig. 4A). The interaction between TNF and radiation was additive (3) in cell lines STSAR-5, HNSCC-68, and SCC-25 and ii was reduced (2.6 to 1.5 for STSAR-5) while Do remained unchanged (Fig. 4B). The interaction between TNF and X rays was synergistic (3) in cell lines STSAR 33, SQ-20B, HNSCC15 1. and SCC-6 1 (Do was reduced from 80, 240, 179, and 133 to 60, 130, 126 and 109, respectively). The x-ray survival data for cell line SCC-6 1 in these experiments is slightly different from that previously reported (21). Survival data for cell line SCC-61 treated with TNF and X rays (Fig. 4C) is representative of cell lines demonstrating synergistic killing between TNF and X rays. Note that
SO-208
0.6
24 TNF added
hours
I
I
I
12
6
0
I
+12
+24
prior to irradiation
Fig. 3. Time course experiment showing surviving fraction after treatment with X rays and TNF (10 units/ml) added 48 to 2 hr before irradiation and immediately after 2, 12, and 24 hr after irradiation (+). Surviving fractions are corrected for plating efficiencies with TNF added. Cells were irradiated with 700 cGy.
Time course studies demonstrate that the interaction between TNF and X rays is observed only when TNF is added several hours prior to irradiation (Fig. 3). Each data STSAR
13
10,
STSAR
10:
5
200
400 DOSE
(COY)
600
, 600
1
0
(W
(A)
.ooo 1
SCC-6
103
.oo 1
1 200
.oo 1 400 DOSE
I 600
I 600
I 200
1
I
400
600
1 600
(COY) DOSE
kGy)
Fig. 4. Radiation survival curves for human tumor cell lines with TNF 0, and without TNF 0. Surviving fractions are corrected for plating efficiencies with TNF. (A) Cell line STSAR- 13 treated with TNF (1000 units/ml) added 12 hr prior to irradiation showing no interaction between X rays and TNF. (B) Cell line STSAR-5 treated with TNF (1000 units/ml) added 12 hr prior to X ray showing reduced n but no change in Do. (C) Cell line SCC-6 1 treated with TNF (10 units/ml) prior to irradiation showing a reduced n and Do.
TNF and radiation interaction 0 D. SQ-208
0
1.0
PLDR
STSAR-90
Control
73
E. HALLAHAN et a/.
GM- 1522
PLDR
PLDR
0 controt 0
0 TNF
0 TNF
TNF
0.1 E ._ 2 I; P ._ .2 2 cz
i
I
d,
6
(4
/
12
I
18
Hours
,b-
,
0.01
I
I
24
6
W
I
I2
Hours
I
18
I
24
)
(Cl
6
I
12
I
I
I8
24
Hours
Fig. 5. Delayed plating experiments of confluent human tumor cell lines SQ-20B (A), STSAR-90 (B) and human fibroblasts AG-1522 (C). Cells were irradiated with 0 or without ??TNF added. Surviving fractions are corrected for reduced plating efficiency with TNF.
enhanced radiation killing in cell line HNSCC-68 despite promoting growth in that cell line. Finally, there was no interaction between TNF and X rays in normal human fibroblast cell line AG-1522 or tumor cell line STSAR-48. PLDR for cell line SQ20-B was reduced in the presence of TNF (Fig. 5A) at relatively equitoxic survival levels. The recovery ratio (S/So), defined as the ratio of surviving fractions at 24 and 0 hr, was reduced from 2 to 1.2 when TNF was added. TNF had no effect on PLDR in cell line STSAR-13 when surviving fractions were corrected for the reduced plating efficiency in the presence of TNF. TNF produced growth inhibition in cell line STSAR 90 and enhanced PLDR in this cell line (Fig. 5B). The 0 hr survival was reduced from 0.033 to 0.014, the recovery ratio was increased from 1.4 to 7.1 in the presence of TNF. Growth promotion was observed during treatment of cell lines AG-1522 and HNSCC-68 with TNF. The recovery ratio for cell line AG- 1522 was reduced from 12 to 3 by TNF (Fig. 5C).
TNF
DISCUSSION TNF was cytotoxic to 12 of 14 human tumor cell lines, although the sensitivity of TNF varied for each of the tested cell lines. The mechanism of direct cytotoxicity by TNF may involve hydroxyl radical production within the tumor cell (24). This possibility is supported by the findings that cell killing by TNF is reduced by anaerobic conditions, induction of superoxide dismutase, and free rad-
ical scavengers (8, 9, 22). In addition, hydroxyl radicals are not produced in cell lines which are resistant to TNF killing (23). Free radical species are also intermediates of DNA damage induced by ionizing radiation (13). Thus, pretreatment of cells with TNF may increase the level of hydroxyl radical or other radical products and thereby potentiate the oxidative damage induced by radiation. This proposed interaction is supported by the observation that 7 of 10 tumor cell lines examined in this study respond with an additive or synergistic interaction between TNF and X rays. Time course studies demonstrated that the addition of TNF 4 to 12 hr prior to irradiation maximally increases cell killing. TNF and radiation induced cell killing acted independently when TNF was added after irradiation. These findings may be explained by the observation that hydroxyl radical production begins after 1 to 4 hr (23) and oxidative damage reaches a maximum at 3-7 hr after exposure to TNF (24). This interaction between these agents is not observed when TNF is added after irradiation. These results indicated that hydroxyl or other radical species produced in response to TNF must be present prior to the time of irradiation for an interaction to exist. The effect of TNF on PLDR is dependent upon the response of the cell line to TNF. Growth promotion by TNF resulted in diminished PLDR. Maximum PLDR occurs during the G, phase of the cell cycle (4). Repair of potentially lethal damage may be decreased by agents which cause cell proliferation (7). Lethal DNA damage may be fixed as the cell crosses the G,/S interface of the
74
I. J. Radiation Oncology 0 Biology 0 Physics
cell cycle (4, 7). This may explain the diminished PLDR in cell line AG-1522 despite the lack of an interaction between TNF and X rays in these cells. Some agents which impede progression through the cell cycle may enhance PLDR (7). Thus, TNF augments PLDR in cell line STSAR 90 in which it is a growth inhibitor, whereas PLDR is diminished in cell line SQ-20B in which TNF is cytotoxic. In addition to the direct cell killing produced by TNF
July 1990, Volume 19. Number 1
in vitro. the immunologic effects of TNF enhance tumor cure rates in irradiated mice while decreasing bone marrow toxicity ( 15). Although no direct cytotoxicity of TNF on murine tumor cells was demonstrated in this study, data reported herein suggest that TNF is cytotoxic and interacts with radiation killing in some human tumor cells in vitro. Therefore, a clinical investigation of TNF and xirradiation may be warranted despite disappointing results with TNF as a sole antitumor agent ( 16).
REFERENCES I. Bevilacqua, M. P.; Stengelin, S.; Gimbrone, M. A.; Seed, B. Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science 243: I 160- 1 165; 1989. 2. Carswell, E. A.; Old, L. J.; Russel, R. L.; Green, M.; Williamson, B. An endotoxin induced serum factor that causes necrosis of tumors. Proc. Natl. Acad. Sci. USA 72:36003670; 1975. 3. Dewey, W. C. In vitro systems: standardization of endpoints. Int. J. Radiat. Oncol. Biol. Phys. 5:l 163; 1979. 4. Hahn, G. M.; Little, J. B. Plateau phase cultures of mammalian cells: an in vitro model from human cancer. Curr. Top. Rad. Res. 8:39-7 1; 1972. 5. Hallahan, D. E.; Spriggs, D. R.; Beckett, M. A.; Kufe. D. W.; Weichselbaum, R. R. Increased tumor necrosis factor mRNA following ionizing radiation exposure. Proc. Natl. Acad. Sci. USA 86:10104-10107; 1989. 6. Hoffman, M.; Weinberg, J. B. Tumor necrosis factor-induced increased hydrogen peroxide production and Fc receptor expression, but not increased Ia antigen expression by peritoneal macrophages. J. Leukocyte Biol. 42:704-707; 1987. potentially lethal damage: 7. Iliakis, G. Radiation-induced DNA lesions susceptible to fixation. Int. J. Radiat. Biol. 53: 541-584; 1988. 8. Matthews, N.; Neale, M. L.; Fiera, R. A.; Jackson, S. K.; Stark, S. M. Are free radicals involved in tumor cell cytolysis by TNF? In: Bonavida, B., Gifford, G. E., Kirchner, H., Old, L. J., eds. Tumor necrosis factor/cachectin and related cytokinesis. Basel, Switzerland: S. Karger; 1988:20-25. 9. Matthews, N.; Neale, M. L.; Jackson, S. K., Stark, J. M. Tumor cell killing by tumor necrosis factor: inhibition by anaerobic conditions, free radical scavengers, and inhibitors of arachidonate metabolism. Immunology 62: 153-l 55; 1987. 10. Nawroth, P. P.; Stern, D. M. Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J. Exp. Med. 163:740-745; 1986. 11. Old, L. J. Tumor necrosis factor. Science 230:630-634; 1985. 12. Old, L. J. Antitumor activity of microbial products and tumor necrosis factor. In: Bonavida, B., Gifford, G. E., Kirchner, H., Old, L. J., eds. Tumor necrosis factor/cachectin and related cytokinesis. Basel, Switzerland: S. Karger; 1988:7-19. 13. Repine, J. B.; Pfenninger, 0. W.; Talmage, D. W.; Berger, E. M.; Pettijohn, D. E. Dimethyl sulfoxide prevents DNA nicking mediated by ionizing radiation or iron/hydrogen peroxide-generated hydroxyl radical. Proc. Natl. Acad. Sci USA 78:1001-1003; 1981.
14. Rubin, B. Y.; Smith, L. J.; Hellerman, G. R.; Lunn, R. M.; Richardson, N. K.; Anderson, S. L. Correlation between the anticellular and DNA fragmenting activities of TNF. Cancer Res. 48:6006-60 10; 1988. 15. Sersa, G.; Willingham, V.; Milas, L. Anti-tumor effects of TNF alone or combined with radiotherapy. Int. J. Cancer 42:129-134: 1988. 16. Springs, D. R.; Sherman, M. L.; Frei, E., III; Kufe, D. W. Clinical studies with tumor necrosis factor. Ciba Found. Symp. 1 (131):206-227; 1987. 17. Steele, G. G. Terminology in the description of drug-radiation interactions. Int. J. Radiat. Oncol. Biol. Phys. 5: 11451150; 1979. 18. Sugarman, H. J.; Aggarwal, B. B.; Hass, P. E.; Figari, I. S.; Palladino, M. A., Jr.; Shepard, H. M. Recombinant human tumor necrosis factor: effects on proliferation of normal and transformed cells in vitro. Science 230:943-945; 1985. 19. Taujimoto, M.; Yokota, S.; Vilcek, J.; Weissmann, G. Tumor necrosis factor provokes superoxide anion generation from neutrophils. Biochem. Biophys. Res. Commun. 137: 1094-l 100; 1986. R. R.; Beckett, M. A.; Simon, M. A.: 20. Weichselbaum. McCowley, C.; Haraf, D.; Awan, A.; Samuels, B.; Nachman, J.; Dritschilo, A. In vitro radiobiological parameters of human sarcoma ceil lines. Int. J. Radiat. Oncol. Biol. Phys. 15:937-942.20; 1988. R. R.; Dahlberg, W.; Beckett, M. A.; Kar21. Weichselbaum, rison, T.; Miller, D.; Clark, J.; Ervin, T. J. Radioresistant and repair proficient human tumor cells may be associated with radiotherapy failure in head and neck cancer patients. Proc. Natl. Acad. Sci. USA 83:2684-2688; 1986. 22. Wong, G. H. W.; Elwell, J. H.; Oberley, L. H.; Goeddel, D. V. Manganous superoxide dismutase is essential for resistance to cytotoxicity of tumor necrosis factor. Cell 58: 923-931; 1989. 23. Yamauchi, N.; Karizana, H.; Watanabe, H.; Neda, H.; Maeda, M.; Nutsu, Y. Intracellular hydroxyl radical production induced by recombinant human tumor necrosis factor. Cancer Res. 49: 167 l- 1675; 1989. 24. Zimmerman, R. J.; Chan, A.; Leadon, S. A. Oxidative damage in murine tumor cells treated in vitro by recombinant human TNF. Cancer Res. 49: 1644- 1648; 1989. 25. Zimmerman, R. J.; Marafino, B. J.; Chan, A.; Landre, P.; Winkelhake, J. L. The role of free radicals in tumor cell sensitivity to recombinant human tumor necrosis factor in viva: implications for mechanisms of action. J. Immunol. (In press) 1990.
0360-3016/90 $3.00 + .oO Copyright 0 1990 Pergamon Pres plc
??Original Contribution
THE INTERACTION BETWEEN RECOMBINANT HUMAN TUMOR NECROSIS FACTOR AND RADIATION IN 13 HUMAN TUMOR CELL LINES DENNIS
E. HALLAHAN,
M.D.,’
MICHAEL
AND RALPH
A. BECKETT,
R. WEICHSELBAUM,
B.S., t DONALD
KUFE,
M.D.2
M.D.’
‘Michael Reese/University of Chicago Center for Radiation Therapy, Department of Radiation and Cellular Oncology, University of Chicago, Pritzker School of Medicine; and *Laboratory of Clinical Pharmacology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA Tumor necrosis factor (TNF) was cytotoxic at concentrations of 10 to 1000 units/ml to 12 of 14 human tumor cell lines. Synergistic or additive cell killing between TNF and radiation was observed in 7 of 10 tumor cell lines, while independent tumor cell killing by each agent occurred in two tumor cell lines. The maximum synergistic effect was observed when TNF was added 4-12 hr prior to irradiation. This interaction was absent when TNF was added after irradiation. TNF also reduced potentially lethal damage repair in 3 of 5 cell lines tested. Possible mechanisms of interaction of TNF and X rays including induction of hydroxyl radicals and subsequent DNA damage by TNF and radiation are discussed. Tumor necrosis factor, Ionizing radiation, Synergy, Human tumor cell lines. INTRODUCTION
and Adriamycin ( 12). The oxidative damage produced by TNF may enhance cellular damage produced by ionizing radiation and suggests that an additive or synergistic interaction may occur between these two agents. We investigated the interaction between TNF and X rays in human tumor cell lines. Data presented here suggest a direct interaction between TNF and X rays, including the inhibition of potentially lethal damage repair (PLDR) in some human tumor cell lines.
Tumor
necrosis factor (TNF) is a polypeptide mediator of the cellular immune response which is produced by’
macrophages and lymphocytes (2, 12). The range of actions ascribed to this antitumor cytokine include increased vascular endothelial adhesiveness to neutrophils and platelets ( l), and promotion of thrombosis (10). The resulting tumor microvascular obliteration is associated with hemorrhagic necrosis of tumors in vivo (2, 12). TNF has a variety of other actions including fibroblast proliferation, activation of macrophages and NK cells, and induction of interleukin- 1 production ( 12, 18). Neutrophil peroxide radical production is also enhanced by TNF (6, 19). Finally, TNF is one of the components associated with respiratory distress syndrome and septic shock ( 12). In addition to enhancement of host immune response, TNF is also directly cytotoxic to tumor cell lines in vitro (2, 11, 18). Cell killing by TNF is associated with the production of hydroxyl radicals within the sensitive cell lines (23, 25) resulting in oxidative damage to DNA (24) and subsequently DNA fragmentation ( 14). TNF enhances the direct cytotoxic effects of a number of antitumor agents whose cytotoxic action is proposed to act through free radicals. These agents include mitomycin-C
METHODS
AND
MATERIALS
Cell
lines The establishment of human epithelial tumor and sarcoma cell lines has been previously described (20, 21). Human epithelial tumor cell culture medium consists of 72.5% Dulbecco’s Modification of Eagle’s Medium, 22.5% Ham’s Nutrient Mixture F-12, (DMEM/F12, at 3:1), 5% fetal bovine serum (FIB), 5 mcg/ml insulin, 5 mcg/ml transferrin, 2 X lo-” M 3,3’,5-triodo-L-thyronine, lo-” M cholera toxin, 1.8 X 10e4 M adenine, 0.4 mcg/ml hydrocortisone, 100 units/ml penicillin and 100 mcg/ml streptomycin (Pen/Strep). Culture medium for the human sarcoma cell lines and normal human fibroblast AG- 1522
Presented at the 3 1st Annual Scientific Meeting of the American Society for Therapeutic Radiology and Oncology San Francisco, CA, 3 October 1989. Reprint requests to: Dennis Hallahan, M.D. Dept. of Radiation Oncology, University of Chicago, 5841 S. Maryland, Chicago, IL 60637.
Supported by NC1 grant CA4 1068 and a gift from the Passis Family. Accepted for publication 24 January 1990.
69
70
I. J. Radiation
Oncology 0 Biology 0 Physics
cell line consists Pen/Strep.
of DMEM/FI
2 (3: 1) with 15% FBS and
Radiation/TNF
survival experiments
The colony forming assay and growth curve analyses were used to assess TNF sensitivity in each cell line. Human recombinant TNF* was added at concentrations ranging from 1 to 10,000 units/ml 12 hr after cells were plated. Cells were plated at densities of 200 to 10,000 in 100 mm dishes. Colonies were counted after lo- 14 days. During growth curve analysis, 1O3 cells were plated in 60 mm dishes. Cultures were trypsinized and total cell number per plate was calculated using a Coulter cell counter. Cell counts of exponentially growing cells with and without TNF were performed at days 1 through 7. X-ray survival experiments were performed on exponentially growing tumor cells and survival curves were determined as follows: Cells at the 7th to 25th passage were maintained in medium at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were trypsinized with 0.05% trypsin:0.02% EDTA from stock cultures and between 500 and 40,000 cells were plated in 100 mm diameter tissue culture dishes and allowed to enter exponential growth. Radiation was carried out 24 hr later with a 250 kV x-ray generator,+ operating at 26 mA at a dose rate of 1.07 Gy per minute. Immediately after irradiation, the cultures were returned to the incubator. After 18-24 days, cells were fixed and stained with crystal violet. Only colonies of >50 cells were scored as survivors. TNF was added to cell cultures lo-14 hr prior to irradiation. Concentrations of TNF ranged from 10 to 1000 units/ml (2.3 X lo6 units/mg). Surviving fractions were corrected for the reduced plating efficiency with TNF. Experimentally derived data points are the mean of 2-4 experiments. The multi-target model survival curves were fit to a single hit multi-target model (S = 1 - (-emD’Do)“. From th ese data points, Do and n were derived using a least square regression analysis omitting the 100 cGy data point.
Dejnitions Interactions between cytotoxic agents may be operationally defined. As defined by Dewey and Steele, an interaction is present when a radiation enhancing agent used concurrently with X rays results in cell killing which is greater than the expected independent killing from each agent alone (3, 17). In contrast, independent cell killing is the quantity of cell death which is expected from each agent alone (3, 17). Interactive killing may be additive or synergistic. Additive killing is defined as enhancement in x-ray induced cell killing such that the resulting survival curve is parallel to that of irradiation alone but greater than expected by the independent killing of each agent alone (3). Synergistic killing is operationally defined
* Asahi Chemical
Industries,
New York.
July 1990, Volume
19. Number
1
as a decrease in Do when both agents are used concurrently resulting in a steeper slope in the radiation survival curve (3). Potentially lethal damage repair (PLDR) is defined as damage which if unrepaired, is lethal (7). We specifically define PLDR to be the enhancement in survival as measured by increased colony-forming ability resulting from a delay in subculture of plateau phase tumor cells following radiation exposure (4). PLDR in plateau phase cultures has been reported to occur in the G1 phase of the cell cycle (4). Damage is fixed as cells cross the G&S interface and then becomes lethal (4).
Time course studies To determine the optimal time for the addition of TNF, cells were irradiated at regular intervals before and after TNF was added. Time course experiments were performed on exponentially growing tumor cells. Cell line SQ-20B was selected because of previously demonstrated synergy (5). Cells were trypsinized and plated at a density of 10,000 cells in 100 mm tissue culture dishes. TNF ( 10 units/ml) was added at 48, 24, 12, 10, 8, 6, 4, 2 hr prior to irradiation and at 0, 2, 12, and 24 hr after irradiation. Cultures were immediately returned to the incubator after irradiation. After 14 days, plates were stained and colonies ~50 cells were scored as survivors.
PLDR experiments Studies of potentially lethal damage repair (PLDR) were performed as follows. Cells were initially seeded into 35mm plastic tissue culture dishes and grown to confluency. Culture medium was renewed daily for 3 days and experiments were performed on day 4. Cells were irradiated at room temperature and then were returned to the incubator. Single dishes were removed and cells were subcultured and seeded at low density (5,000-50,000 cells per 100-mm dish) at regular intervals thereafter. Equitoxic doses were used to measure PLDR. The doses used to study PLDR were 500 cGy for cell line STSAR-13 and 700 cGy for all other cell lines tested. The enhancement in survival, as measured by the factor of increased colonyforming ability resulting from a 24 hr delay in subculture after irradiation, is interpreted as being due to the repair of potentially lethal damage. Thus, PLDR is expressed in terms of enhancement in surviving fraction as a function of the time interval between irradiation and subculture (S/So).
RESULTS Figure 1 demonstrates TNF-sensitivity in four tumor cell lines. Cell survival was reduced upon exposure to increasing TNF concentration. Cell line SCC-6 1 was killed by concentrations as low as 1 unit/ml whereas cell line
t GE Maxitron.
71
TNF and radiation interaction 0 D. E. HALLAHANet al.
0 0 -
STSAR-90 At+1522 Control STSAR-90
6
I
lb
Id0 TNF
lob0
lO,bOO
Wnitslmi)
Fig. 1. Response of human tumor cell lines to recombinant human TNF. Cell lines were grown in the presence of increasing concentrations of TNF (1 to lo4 units/ml). Plating efficiencies were compared to control plates grown without TNF (100% survival).
STSAR-5 required exposure to 1000 units/ml for detectable cell killing. Table 1 lists the related cytotoxicities of human tumor cell lines and a normal fibroblast line at concentrations of 10 and 1000 units/ml. TNF was cytotoxic to 3 of 4 soft tissue sarcoma cell lines, 3 of 3 bone sarcomas, and 4 of 5 epithelial tumor cell lines. Squamous cell carcinoma cell lines were more sensitive to lower concentrations of TNF than were the sarcoma cell lines. For example, a TNF concentration of 10 units/ml reduced the survival of epithelial tumor cells to 10% to 50% of normal, whereas 1000 units/ml was required to achieve similar cell killing in sarcoma cells (Table 1). However, at the concentrations tested, TNF was not directly cyto-
Table 1. Sensitivity
of human
1.0-7m
4
Soft tissue sarcomas STSAR-5 STSAR- 13 STSAR-48 STSAR-90 Bone sarcomas STSAR-33 STSAR-43 STSAR-75 Squamous cell carcinomas SQ-20B SCC-2 5 SCC-6 1 HNSCC-68 HNSCC- 15 1 Fibroblasts AG- 1522
Origin
toxic to all cell lines. Cell line STSAR-48 had no cytotoxicity from TNF, whereas cell lines HNSCC-68 and AGI522 responded to TNF with enhanced proliferation (Fig. 2). In contrast, cell line STSAR-90 responded to TNF with a prolonged doubling time (Fig. 2).
tumor
cell lines to TNF surviving
TNF (10 units/ml)
(1000 units/ml)
MFH Liposarcoma Neurofibrosarcoma MFH
100 50 100 100
53* 12* 100 18*
Ewings sarcoma Osteosarcoma Osteosarcoma
100 100 100
50* 30* 47
SCCA SCCA SCCA SCCA SCCA
50* 25* 12* 100 10*
3 6 0.6 100 0.3
100
100
Human
6
Fig. 2. Growth curves of human tibroblasts (AG-1522) and human sarcoma cell line @TSAR-90) with TNF (1000 units/ml) 0 and without TNF 0.
Percent Cell line
5
Days
fibroblast
Cytotoxicity of human tumor cell lines to TNF (10 to 1000 units/ml) as compared MFH = Malignant fibrous histiocytoma; SCCA = Squamous cell carcinoma. * p < 0.05.
to untreated
cell lines.
July 1990, Volume 19, Number I
I. J. Radiation Oncology 0 Biology ??Physics
12
Time
Course
point represents the surviving fraction after treatment with 700 cGy and 10 units/ml of TNF added at the indicated time interval before or after irradiation. Surviving fractions were corrected for the reduced plating efficiency due to TNF. The extent of cell killing when TNF was added after irradiation is equal to that of both agents added separately. In contrast, cell killing was greater than expected by either agent alone when TNF was added 4- 12 hr prior to irradiation. TNF was therefore added 12 hr prior to irradiation when studying the effects of concurrent TNF and X rays on survival curves. There was independent cell killing by TNF and X rays in human tumor cell lines STSAR 13 and STSAR-43. After correcting for the reduced plating efficiencies, there was no change in ii or Do after treating these two cell lines with TNF and radiation (Fig. 4A). The interaction between TNF and radiation was additive (3) in cell lines STSAR-5, HNSCC-68, and SCC-25 and ii was reduced (2.6 to 1.5 for STSAR-5) while Do remained unchanged (Fig. 4B). The interaction between TNF and X rays was synergistic (3) in cell lines STSAR 33, SQ-20B, HNSCC15 1. and SCC-6 1 (Do was reduced from 80, 240, 179, and 133 to 60, 130, 126 and 109, respectively). The x-ray survival data for cell line SCC-6 1 in these experiments is slightly different from that previously reported (21). Survival data for cell line SCC-61 treated with TNF and X rays (Fig. 4C) is representative of cell lines demonstrating synergistic killing between TNF and X rays. Note that
SO-208
0.6
24 TNF added
hours
I
I
I
12
6
0
I
+12
+24
prior to irradiation
Fig. 3. Time course experiment showing surviving fraction after treatment with X rays and TNF (10 units/ml) added 48 to 2 hr before irradiation and immediately after 2, 12, and 24 hr after irradiation (+). Surviving fractions are corrected for plating efficiencies with TNF added. Cells were irradiated with 700 cGy.
Time course studies demonstrate that the interaction between TNF and X rays is observed only when TNF is added several hours prior to irradiation (Fig. 3). Each data STSAR
13
10,
STSAR
10:
5
200
400 DOSE
(COY)
600
, 600
1
0
(W
(A)
.ooo 1
SCC-6
103
.oo 1
1 200
.oo 1 400 DOSE
I 600
I 600
I 200
1
I
400
600
1 600
(COY) DOSE
kGy)
Fig. 4. Radiation survival curves for human tumor cell lines with TNF 0, and without TNF 0. Surviving fractions are corrected for plating efficiencies with TNF. (A) Cell line STSAR- 13 treated with TNF (1000 units/ml) added 12 hr prior to irradiation showing no interaction between X rays and TNF. (B) Cell line STSAR-5 treated with TNF (1000 units/ml) added 12 hr prior to X ray showing reduced n but no change in Do. (C) Cell line SCC-6 1 treated with TNF (10 units/ml) prior to irradiation showing a reduced n and Do.
TNF and radiation interaction 0 D. SQ-208
0
1.0
PLDR
STSAR-90
Control
73
E. HALLAHAN et a/.
GM- 1522
PLDR
PLDR
0 controt 0
0 TNF
0 TNF
TNF
0.1 E ._ 2 I; P ._ .2 2 cz
i
I
d,
6
(4
/
12
I
18
Hours
,b-
,
0.01
I
I
24
6
W
I
I2
Hours
I
18
I
24
)
(Cl
6
I
12
I
I
I8
24
Hours
Fig. 5. Delayed plating experiments of confluent human tumor cell lines SQ-20B (A), STSAR-90 (B) and human fibroblasts AG-1522 (C). Cells were irradiated with 0 or without ??TNF added. Surviving fractions are corrected for reduced plating efficiency with TNF.
enhanced radiation killing in cell line HNSCC-68 despite promoting growth in that cell line. Finally, there was no interaction between TNF and X rays in normal human fibroblast cell line AG-1522 or tumor cell line STSAR-48. PLDR for cell line SQ20-B was reduced in the presence of TNF (Fig. 5A) at relatively equitoxic survival levels. The recovery ratio (S/So), defined as the ratio of surviving fractions at 24 and 0 hr, was reduced from 2 to 1.2 when TNF was added. TNF had no effect on PLDR in cell line STSAR-13 when surviving fractions were corrected for the reduced plating efficiency in the presence of TNF. TNF produced growth inhibition in cell line STSAR 90 and enhanced PLDR in this cell line (Fig. 5B). The 0 hr survival was reduced from 0.033 to 0.014, the recovery ratio was increased from 1.4 to 7.1 in the presence of TNF. Growth promotion was observed during treatment of cell lines AG-1522 and HNSCC-68 with TNF. The recovery ratio for cell line AG- 1522 was reduced from 12 to 3 by TNF (Fig. 5C).
TNF
DISCUSSION TNF was cytotoxic to 12 of 14 human tumor cell lines, although the sensitivity of TNF varied for each of the tested cell lines. The mechanism of direct cytotoxicity by TNF may involve hydroxyl radical production within the tumor cell (24). This possibility is supported by the findings that cell killing by TNF is reduced by anaerobic conditions, induction of superoxide dismutase, and free rad-
ical scavengers (8, 9, 22). In addition, hydroxyl radicals are not produced in cell lines which are resistant to TNF killing (23). Free radical species are also intermediates of DNA damage induced by ionizing radiation (13). Thus, pretreatment of cells with TNF may increase the level of hydroxyl radical or other radical products and thereby potentiate the oxidative damage induced by radiation. This proposed interaction is supported by the observation that 7 of 10 tumor cell lines examined in this study respond with an additive or synergistic interaction between TNF and X rays. Time course studies demonstrated that the addition of TNF 4 to 12 hr prior to irradiation maximally increases cell killing. TNF and radiation induced cell killing acted independently when TNF was added after irradiation. These findings may be explained by the observation that hydroxyl radical production begins after 1 to 4 hr (23) and oxidative damage reaches a maximum at 3-7 hr after exposure to TNF (24). This interaction between these agents is not observed when TNF is added after irradiation. These results indicated that hydroxyl or other radical species produced in response to TNF must be present prior to the time of irradiation for an interaction to exist. The effect of TNF on PLDR is dependent upon the response of the cell line to TNF. Growth promotion by TNF resulted in diminished PLDR. Maximum PLDR occurs during the G, phase of the cell cycle (4). Repair of potentially lethal damage may be decreased by agents which cause cell proliferation (7). Lethal DNA damage may be fixed as the cell crosses the G,/S interface of the
74
I. J. Radiation Oncology 0 Biology 0 Physics
cell cycle (4, 7). This may explain the diminished PLDR in cell line AG-1522 despite the lack of an interaction between TNF and X rays in these cells. Some agents which impede progression through the cell cycle may enhance PLDR (7). Thus, TNF augments PLDR in cell line STSAR 90 in which it is a growth inhibitor, whereas PLDR is diminished in cell line SQ-20B in which TNF is cytotoxic. In addition to the direct cell killing produced by TNF
July 1990, Volume 19. Number 1
in vitro. the immunologic effects of TNF enhance tumor cure rates in irradiated mice while decreasing bone marrow toxicity ( 15). Although no direct cytotoxicity of TNF on murine tumor cells was demonstrated in this study, data reported herein suggest that TNF is cytotoxic and interacts with radiation killing in some human tumor cells in vitro. Therefore, a clinical investigation of TNF and xirradiation may be warranted despite disappointing results with TNF as a sole antitumor agent ( 16).
REFERENCES I. Bevilacqua, M. P.; Stengelin, S.; Gimbrone, M. A.; Seed, B. Endothelial leukocyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science 243: I 160- 1 165; 1989. 2. Carswell, E. A.; Old, L. J.; Russel, R. L.; Green, M.; Williamson, B. An endotoxin induced serum factor that causes necrosis of tumors. Proc. Natl. Acad. Sci. USA 72:36003670; 1975. 3. Dewey, W. C. In vitro systems: standardization of endpoints. Int. J. Radiat. Oncol. Biol. Phys. 5:l 163; 1979. 4. Hahn, G. M.; Little, J. B. Plateau phase cultures of mammalian cells: an in vitro model from human cancer. Curr. Top. Rad. Res. 8:39-7 1; 1972. 5. Hallahan, D. E.; Spriggs, D. R.; Beckett, M. A.; Kufe. D. W.; Weichselbaum, R. R. Increased tumor necrosis factor mRNA following ionizing radiation exposure. Proc. Natl. Acad. Sci. USA 86:10104-10107; 1989. 6. Hoffman, M.; Weinberg, J. B. Tumor necrosis factor-induced increased hydrogen peroxide production and Fc receptor expression, but not increased Ia antigen expression by peritoneal macrophages. J. Leukocyte Biol. 42:704-707; 1987. potentially lethal damage: 7. Iliakis, G. Radiation-induced DNA lesions susceptible to fixation. Int. J. Radiat. Biol. 53: 541-584; 1988. 8. Matthews, N.; Neale, M. L.; Fiera, R. A.; Jackson, S. K.; Stark, S. M. Are free radicals involved in tumor cell cytolysis by TNF? In: Bonavida, B., Gifford, G. E., Kirchner, H., Old, L. J., eds. Tumor necrosis factor/cachectin and related cytokinesis. Basel, Switzerland: S. Karger; 1988:20-25. 9. Matthews, N.; Neale, M. L.; Jackson, S. K., Stark, J. M. Tumor cell killing by tumor necrosis factor: inhibition by anaerobic conditions, free radical scavengers, and inhibitors of arachidonate metabolism. Immunology 62: 153-l 55; 1987. 10. Nawroth, P. P.; Stern, D. M. Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J. Exp. Med. 163:740-745; 1986. 11. Old, L. J. Tumor necrosis factor. Science 230:630-634; 1985. 12. Old, L. J. Antitumor activity of microbial products and tumor necrosis factor. In: Bonavida, B., Gifford, G. E., Kirchner, H., Old, L. J., eds. Tumor necrosis factor/cachectin and related cytokinesis. Basel, Switzerland: S. Karger; 1988:7-19. 13. Repine, J. B.; Pfenninger, 0. W.; Talmage, D. W.; Berger, E. M.; Pettijohn, D. E. Dimethyl sulfoxide prevents DNA nicking mediated by ionizing radiation or iron/hydrogen peroxide-generated hydroxyl radical. Proc. Natl. Acad. Sci USA 78:1001-1003; 1981.
14. Rubin, B. Y.; Smith, L. J.; Hellerman, G. R.; Lunn, R. M.; Richardson, N. K.; Anderson, S. L. Correlation between the anticellular and DNA fragmenting activities of TNF. Cancer Res. 48:6006-60 10; 1988. 15. Sersa, G.; Willingham, V.; Milas, L. Anti-tumor effects of TNF alone or combined with radiotherapy. Int. J. Cancer 42:129-134: 1988. 16. Springs, D. R.; Sherman, M. L.; Frei, E., III; Kufe, D. W. Clinical studies with tumor necrosis factor. Ciba Found. Symp. 1 (131):206-227; 1987. 17. Steele, G. G. Terminology in the description of drug-radiation interactions. Int. J. Radiat. Oncol. Biol. Phys. 5: 11451150; 1979. 18. Sugarman, H. J.; Aggarwal, B. B.; Hass, P. E.; Figari, I. S.; Palladino, M. A., Jr.; Shepard, H. M. Recombinant human tumor necrosis factor: effects on proliferation of normal and transformed cells in vitro. Science 230:943-945; 1985. 19. Taujimoto, M.; Yokota, S.; Vilcek, J.; Weissmann, G. Tumor necrosis factor provokes superoxide anion generation from neutrophils. Biochem. Biophys. Res. Commun. 137: 1094-l 100; 1986. R. R.; Beckett, M. A.; Simon, M. A.: 20. Weichselbaum. McCowley, C.; Haraf, D.; Awan, A.; Samuels, B.; Nachman, J.; Dritschilo, A. In vitro radiobiological parameters of human sarcoma ceil lines. Int. J. Radiat. Oncol. Biol. Phys. 15:937-942.20; 1988. R. R.; Dahlberg, W.; Beckett, M. A.; Kar21. Weichselbaum, rison, T.; Miller, D.; Clark, J.; Ervin, T. J. Radioresistant and repair proficient human tumor cells may be associated with radiotherapy failure in head and neck cancer patients. Proc. Natl. Acad. Sci. USA 83:2684-2688; 1986. 22. Wong, G. H. W.; Elwell, J. H.; Oberley, L. H.; Goeddel, D. V. Manganous superoxide dismutase is essential for resistance to cytotoxicity of tumor necrosis factor. Cell 58: 923-931; 1989. 23. Yamauchi, N.; Karizana, H.; Watanabe, H.; Neda, H.; Maeda, M.; Nutsu, Y. Intracellular hydroxyl radical production induced by recombinant human tumor necrosis factor. Cancer Res. 49: 167 l- 1675; 1989. 24. Zimmerman, R. J.; Chan, A.; Leadon, S. A. Oxidative damage in murine tumor cells treated in vitro by recombinant human TNF. Cancer Res. 49: 1644- 1648; 1989. 25. Zimmerman, R. J.; Marafino, B. J.; Chan, A.; Landre, P.; Winkelhake, J. L. The role of free radicals in tumor cell sensitivity to recombinant human tumor necrosis factor in viva: implications for mechanisms of action. J. Immunol. (In press) 1990.
