Vol. 171, No. 3, 1990 September 28, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1378-1386
STIMULATION OF Mn-SUPEROXIDE DISMUTASE EXPRESSION BY TUMOR NECROSIS FACTOR-a: QUANTITATIVE DETERMINATION OF Mn-SOD PROTEIN LEVELS IN TNF-RESISTANT AND SENSITIVE CELLS BY ELISA+ Tetsuo Udal,
Kawaguchi',
Akira
Takeyasu',
Keiji
Matsunobu',
Taizo
Minoru Ishizawa', Keiichiro Suzuki4, Tetsuo Nishiura4, 4* Mutsuo Ishikawa3 and Naoyuki Taniguchi 1
Ube Laboratory,
2 Bioscience 3 Department
Ube Industries,
Laboratory,
of Obstetrics
Ube Industries, and Gynecology, Asahikawa
4 Department
of Biochemistry,
4-3-57
Ube 755
Kita-ku
Noda 278, Asahikawa
Tokyo
Medical
School,
075
Osaka University
Nakanoshima,
Medical
School,
Osaka 530 Japan
Received August 20, 1990 Summary: Marked
increase
in protein
levels
dismutase (Mn-SOD) was found in TNF-resistant after treatment with Tumor Necrosis Factor increase Zn-SOD)
was observed protein
in
in either
Cu,Zn-superoxide TNF-resistant
of Mn-superoxide cell (TNF).
lines No such
dismutase
(Cu,
or sensitive
cells.
These results support the data that the Mn-SOD is one of the rescue proteins required for resistance to TNF cytotoxicity in these cell lines (Wong et al., Cell 58, 923-931, 1990). Mn-SOD was also responsive to TNF stimulation in KURAMOCHI, a human ovarian our
previous
in epithelial
adenocarcinoma
result ovarian 1990).
2538-2542,
that
line.
Mn-SOD protein
cancer 01990
cell
Academic
(Ishikawa Press,
This is
et --
highly al.
may explain expressed
Cancer
Inc.
'Part of this study was presented at the 49th Annual Meeting of Japanese Cancer Association at Sapporo, Japan, on July 5, 1990. * To whom correspondence should be addressed. Abbreviations: Hn-SOD, Mn-Superoxide dismutase; Cu,Zn-SOD, Cu,Zn-Superoxide dismutase; TNF, tumor necrosis factor; rhTHF, recombinant human tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay. 0006-291W90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
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Mn-Superoxide dismutase (Mn-SOD) is widely distributed in The the mitochondrial matrix of various tissues (l-3). enzyme plays an important role in the antioxidant defense system against superoxide anion generated and -in --in vitro vivo. Our current studies are directed at the physiological role of Mn-SOD during various carcinogenic processes. In, a previous study we established hybridoma cell lines producing monoclonal antibodies against human Mn-SOD. One of these antibodies binds an epitope located in the C-terminus region We also developed a sandwich enzyme-linked (3). immunoassay(ELISA) for measuring the Mn-SOD protein levels (4) and found that Mn-SOD is one of the best markers for the epithelial type of ovarian cancer (5). Two groups (6,7) have reported that tumor necrosis factor (TNF-u) or IL-l specifically induces mRNA for Mn-SOD and that this effect is blocked by actinomycin D but not cyclohexamide, indicating that the increase in Mn-SOD mRNA results from an increase in transcription of the Mn-SOD gene. Lipopolysaccharide also induces Mn-SOD mRNA in pulmonary epithelial cells by a similar mechanism (8). The importance of Mn-SOD for cellular resistance to TNF cytotoxicity has been reported However no data was available regarding the levels (9,lO). of Mn-SOD protein after TNF treatments. The development of a monoclonal antibody and ELISA made it possible to quantitatively determine Mn-SOD protein levels. This study describes the immunoreactive Mn-SOD levels in TNF-resistant and sensitive human cell lines in response to TNF. Materials
and Methods
The human recombinant TNF-or Lrh-TNF) was an Ube Chemicals: Industries Ltd. product. The6specific activity of the purified rh-TNF was 2.0 X 10 unit/mg protein (11). Cells and Cell culture: A549, a human lung adenocarcinoma cell line, and KYM-1, a human myosarcoma cell line were kindly supplied by Drs. Chiba and Akiyama, respectively. KURAMOCHI, a human undifferentiated ovarian cell line was obtained from the Japan Cell Bank. WI-38, a human normal fetal lung diploid cell, T24, a human bladder transitional cell carcinoma cell line, ME-180, a human cervical epidermoid carcinoma cell line, and ZR-75-1, human breast cancer cell line were purchased from Dainippon Seiyaku. L-M, a murine transformed fibroblast cell line, was maintained in our laboratory. A549 and KURAMOCHI were grown in Iscove's modification of Dulbecco's medium (IMDM). T24 and WI-38 were grown in 1379
Vol. 171, No. 3, 1990
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minimum essential medium. ZR-75-1 was grown in RPM11640. All media were ME-180 was grown in McCoy's 5A medium. contained 10% fetal calf serum (Gibco), and 3% glutamine, IMDM 100 units/ml penicillin, and 100 ug/ml streptomycin. Each cell line was contained 0.05 mM 2-mercaptoethanol. plated in culture flasks (Falcon) and routinely maintained in a CO2 incubator at 37'C. TNF treatment: Human cell lines were cultured in 6-well When the cells had grown plates (Falcon) for several days. confluently, the medium was replaced with medium containing various concentrations of TNF. After incubation with TNF for 7 to 24 hrs, the medium removed and each well was washed with PBS. A solution of 5% trypsin, 1 mM EDTA in PBS was added, and the plates were incubated for 5 to 10 min. The enzyme reaction was stopped by adding culture medium. All cells were removed by centrifugation at 3000 rpm for 5 min. Finally to each tube was added 0.5 ml PBS, and the mixture was frozen at -80°C until used. Assay for TNF cytotoxicity: Cells lines (1 x lo4 cells per plate) were plated in 96-well plates and incubated for 72 Viability of cells was hrs with the addition of TNF. L-M cells determined by the methylene blue method (11). were used in the control experiments. Assay of Mn-SOD and Cu, Zn-SOD protein levels: A sandwich Commercially available ELISA ELISA was used in this study. kits which had been developed in our group for Mn-SOD Ube Industries) (4) and (Mn-superoxide dismutase ELISA kit, ELISA kit, Ube Industries)(12) cu, Zn-SOD (Cu, Zn-superoxide were used throughout these experiments. Each sample was thawed at room temperature and then sonicated. After centrifugation at 3,000 rpm for 15 min, the supernatant was diluted 50 to 100 fold with 10 mM phosphate buffer, pH 7.4 containing 0.5 M NaCl, 0.1% Bovine serum albumin and 0.09 % Kathon CG (w/v) (Rohm and Haas, Japan). Protein assay: Protein concentration was determined using the BioRad Protein Assay Kit using bovine serum albumin as the standard.
Results Cytotoxic The
test for TNF cytotoxic test for
TNF was carried
out
using
various
cells. L-M cells, a well-known TNF-sensitive cell line, were used as the control. Typical results for cytotoxicity by TNF are shown in Fig. 1. ME-180, KYM-1 and ZR-75-l were all found to be all TNF-sensitive cells. Among them KYM-1 was most sensitive and ZY-75-1 was least sensitive. On the other hand, TNF-resistant
A549, WI-38, T24 and KURAMOCHI were found to be cells, as shown in Fig. 2. In the case of WI-38 cells, TNF had a slightly stimulative effect on cell growth. In case of A-549 cells, TNF had a slightly inhibitory effect on cell viability. 1380
Vol.
171, No. 3, 1990
130sage
BIOCHEMICAL
of rh-TNF
AND BIOPHYSICAL
(nqlml)
RESEARCH COMMUNICATIONS
Dosage
of rh-TNF
(mglml)
Dosage
of rh-TNF
(mglml)
KYM-1 125 g100 -2 F E
50
?I a 25 '5
750 r.L 0
10-a
10-7 Dosage
166 of rh-TNF
lrJ5
16’
10-J
(mglml)
Fia. 1. Test for dose-dependent TNF cytotoxicity in TNF-sensitive cells. Each cell line was treated with the ind$ated6concentrations of human recombinant TNF(rh-TNF) was mg/ml) for 72 hrs, and the percent viability (10 -10 determined from triplicate analysis.
Effect of TNF on the levels of Mn-SOD and Cu,Zn-SOD in TNFresistant cells. The effect of TNF on the expression of Mn-SOD protein in TNF-resistant cells was examined. In the case of WI-38, which is fetal lung cells, Mn-SOD protein levels increased dramatically, approximately 80-fold, after TNF treatment (Fig. 3). Increases in Mn-SOD protein were also observed in although the amount of increase A-549 and KUHAMOCHI cells, was not so marked. As shown in Fig. 1, A549 cells are slightly sensitive to TNF at high concentrations. On the other hand, TNF treatment did not cause any changes in Cu,Zn-SOD expression in most of the TNFresistant cells, with the exception of WI-38. Effect of TNF on the expression of Mn-SOD in KUHAMOCHI. Our previous study indicated that Mn-SOD is highly expressed in the epithelial type of ovarian cancer (5) and that Mn-SOD is one of the best marker proteins for 1381
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3, 1990
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A549 125
,
I
0 ‘, 0
I 10-a
1 lo-’
I 10-e
Dosage
I IIT5
of rh-TNF
I 16”
01
I’ 10-J
I IO-'
(mglml)
I IO-~
I 10-6
Dosage
I IO-'
of rh-TNF
I 16~
I lo-*
(mglml) KURAMOWi
125 ,
OJ, 0
1
I
lo-6
I@
Dosage
I 163
lb-4
of rh-TNF
1
I 10-z
0
(mg/ml)
10-s
io-7
Dosage
id-5
ia6
of rh-TNF
Fiq. 2. Test for dose-dependent TNF cytotoxicity in TNF-resistant cells by TNF. Each cell line2was-greated the indicated concentrations of rh-TNF (10 -10 mg/ml) 72 hrs and the percent viability was determined from triplicate analysis.
Ib-J
(mglml)
with for
epithelial ovarian cancer. Since the origin of KURAMOCHI is about the adenocarcinoma of the ovary , we were curious effect of TNF on the induction of Mn-SOD in this cell line. In the case of KURAMOCHI, the Mn-SOD level prior to treatment with TNF was 110 ng/mg protein. Addition of TNF -4 -6 to concentrations of 10 * 1o-5 and 10 mg/ml resulted in dose-dependent increases in Mn-SOD levels to 180, 270 and 360 ng/mg protein, respectively. On the other hand even at 1o-4 mg/ml, did not induce Cu,Zn-SOD protein in the KURAMOCHI cells (Fig. 3). These results again indicate that TNF specifically induces Mn-SOD protein, but not Cu,Zn-SOD protein. Effect of TNF sensitive cells. In ME-180 Mn-SOD levels TNF-resistant
on the
level
of Mn-SOD
and Cu,Zn-SOD
and KYM-1 cells, which are TNF-sensitive, are one order of magnitude lower than cells (Fig. 4). However, the ZR-75-1 1382
in
TNF
cell
io-3
I
Vol. 171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
T24
KURAMOCHI
F 6 z 6,000k = 0’ !F -ti
4,000
c i! i
2,000
-
0 0
1o‘5
1o'4
4,000 A549
2 P z ‘0 $
T24
WI-38
KURAMOCXI
T
3,000
Dosage
of rh-TNF
(mg/ml)
Fiq. 3. Effect of TNF on Mn-SOD and Cu,Zn-SOD levels in TNFresistant cells. Each cell line was treated with the indicated concentrations of TNF for 24 hrs before Mn-SOD and Cu,Zn-SOD protein levels were determined. Each value was obtained from triplicate samples as described in Materials and Methods. Bar indicates the standard deviation.
which is also TNF-sensitive, contains a relatively line, high level of Mn-SOD, even though its level is not as high The basal Mn-SOD protein as those of TNF-resistant cells. levels in KYM-1 and Me-180 cells are very low, and even in Mn-SOD was after treatment with TNF, no increase observed. In ZR-75-1 cells, however, a tendency toward increased Mn-SOD levels was observed after TNF treatment. 1383
Vol.
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BIOCHEMICAL
ZR-7% 800
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
ME-180
KYM-1
Ti
I
0
10’510-4
0
10'~10-'10-~
Dosage
3,ooor
ZR-75-l
0
10'510'4
of rh-TNF
0
lo-'10-610-S
KYM-1
0
Dosage
IO-4
(mg/ml)
ME- 180
10'8
10-7
of rh-TNF
10'6
0
10-710-6
10-S
10.6
(mg/ml)
Fig. 4. Effect of TNF on Mn-SOD and Cu,Zn-SOD levels in TNFsensitive cells. Each cell line was treated with the indicated concentrations of TNF for 24 hrs before Mn-SOD and Cu,Zn-SOD protein levels were determined. Each value was obtained from triplicate samples as described in Materials and Methods. Bar indicates the standard deviation.
Therefore, may control
in this cell line Mn-SOD expression.
it
seems a different
mechanism
Discussion In the present study we found that most of the TNFresistant cells responded to TNF treatment with increases the Mn-SOD protein levels. Inducibility of Mn-SOD in the TNF-resistant cell lines was abolished by actinomycin D (data not shown), which indicates that the effect is the 1384
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Vol. 171, No. 3, 1990
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AND BIOPHYSICAL RESEARCH COMMUNICATIONS
result of --de novo biosynthesis of the Mn-SOD protein. The differences in the levels of Mn-SOD expression among the TNF-resistant cells suggest that the amount of superoxide anion produced by TNF is different in each cell environment. There is also evidence of another possible mechanism for which Mn-SOD expression. In contrast, the levels of Cu,Zn-SOD were essentially unchanged by addition of TNF to the various cell lines, irrespective of whether the cells were sensitive or resistant to TNF. In our previous studies, we demonstrated that Mn-SOD is expressed in the epithelial type of ovarian cancer and is one of the best markers for diagnosing and monitoring ovarian cancer (5). Immunochemical measurement of Mn-SOD showed high levels of patients with ovarian cancer, and immunohistochemical studies also suggested that the cancer tissues produced Mn-SOD protein. Two groups (6,7) reported that treatment with TNF or IL-l induces the expression of mRNA for Mn-SOD but not for other antioxidant enzymes such as Cu,Zn-SOD, catalase, glutathione peroxidase or glutathione transferase. In the present study we conclude that TNF induces Mn-SOD protein but not Cu,Zn-SOD, as judged by quantitating the SOD protein levels by ELISA. The induction of Mn-SOD protein observed in A549, a human adenocarcinoma cell line, and KURAMOCHI, a human ovarian adenocarcinoma, both of which have epithelial-like characteristics, could explain the mechanism by which Mn-SOD expression was elevated in the epithelial ovarian cancers as described above. One possible explanation is that TNF or IL-l is produced as an autocrine factor in the cancer Regarding the tissues or in cancer-associated macrophages. TNF-sensitivity of the cell lines, induction of Mn-SOD was generally marked in the TNF-resistant cells but not in the Exceptions to this pattern, TNF-sensitive cell lines. suggest that additional factors may be involved in however, the regulation of Mn-SOD expression.
Acknowledgments This Cancer Culture,
work was in part supported by Grants-in-Aid for Research by the Ministry of Education, Science and Japan. 1385
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AND BIOPHYSICAL RESEARCH COMMUNICATIONS
References 1. McCord, J. M., Boyle, J. A., Day, E. D., Rizzolo, L, J., and Salin, M. L. (1977) In Superoxide and Superoxide Dismutase, A. M., Michelson, J. M. MacCord, and I. Fridovich, eds. (London, Academic Press), pp129-138. J. B., Bannister, W. H., and Rotellio, G. 2. Bannister, (1987). Crit. Rev. Biochem, 22, 111-180, 1980 3. Kawaguchi,T., Noji, S., Uda, T., Nakashima, Y., Takeyasu, A., Kawai Y., Takagi, H., Tohyama, M., and N-(1989) J. Biol. Chem., 264, 5762-5767, 1989 Taniguchi, 4. Kawaguchi, T., Suzuki, K., Matsuda, Y., Nishura, T., Uda, T., Ono, M., Sekiya, C., Ishikawa, M, Iino, S., Endo, Y., and Taniguchi, N. (1990) J. Immunol. Methods, 127, 249-254 5. Ishikawa, M., Yaginuma, Y., Hayashi, H., Shimizu, t., Endo, Y., Taniguchi, N., (1990) Cancer Res., 50, 538-2542 6. wowI G. H. W. and Goeddel, D. V. (1988) Science 242, 941-944 7. Masuda, A., Longo, D. L., Kobayashi, Y., Appella, E., Oppenheim, J. J. and Matsushima, K. (1989) FASEB J. 2;-3087-3086 8. Visner, G. A., Dougall, W. C., Wilson, J. M., Burr, I. A. and Nick, H. S., (1990) J. Biol. Chem. 265, 2856-2864 J. H., Oberley, L. W., and 9. wowI G. H. W., Elwell, Goeddel, D. V., (1989) Cell 58, 923-931 10. Asoh, K., Watanabe, Y., Mizoguchi, H., Nawatari, M., Ono, Y., Kohno, K. and Kuwano, M., (1989) Biochem. Biophys. Res. Commun. 162, 794-801 11. Yamaszaki, S., Onishi, E., Enami, K., Natori, K., Kohase, M., Skamoto, H., Tanouchi, M. and Hayashi, H. (1986) Japan. J. Med. Sci. Biol. 39, 105-118 K., Matsuua, S., Yosimura, S., Yamamoto, K., 12. Ogino, Okawaki Y., Takemoto T., Kato, N., and Uda, T. (1989) Clin. Chim. Acta 182, 209-220
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