FreeRadical Biology& Medicine, Vol. 12, pp. 151-159, 1992 Printed in the USA. All rights reserved.
0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press plc
Original Contribution REDUCED EXPRESSION OF MANGANESE SUPEROXIDE DISMUTASE IN CELLS RESISTANT TO CYTOLYSIS BY TUMOR NECROSIS FACTOR
J U A N A. MELENDEZ and CORRADO BAGLIONI The Center for Molecular Genetics, Department of Biological Sciences, University at Albany, State University of New York, Albany, NY 12222, U.S.A.
(Received 23 July 1991; Revised 30 September 1991; Accepted 22 October 1991) Abstract--Tumor necrosis factor (TNF) induces synthesis of manganese superoxide dismutase (MnSOD). It was previously shown that overexpression of MnSOD protected some mammalian cells from TNF cytotoxicity. The purpose of this study was to establish whether MnSOD was increased in cells selected for resistance to cytolysis by TNF in combination with cycloheximide. Melanoma SK-MEL-109 and HeLa cell-resistant variants were selected by repeated treatments with TNF and cycloheximide. The SK-MEL-109 variants had relatively low levels of MnSOD that were inducible by TNF. Surprisingly, the HeLa variants had very low levels of MnSOD that were poorly inducible by either TNF or interleukin- 1a. Therefore, an elevated level of MnSOD was not required to protect these cells from TNF-mediated cytolysis. The HeLa variants were more sensitive than parental cells to superoxide radical (02-) generating compounds, such as paraquat or xanthine/xanthine oxidase. Pretreatment of these variants with TNF did not provide protection against damage by superoxide radicals. Keywords--Superoxide dismutase, Tumor necrosis factor, Cytolysis, Superoxide radicals, Free radicals
donic acid metabolism, ls,16 and lipid peroxidation. 17,1sHowever, proteins that confer complete protection from TNF-mediated cytolysis have not yet been identified. Production of hydroxyl radical species induced by TNF has been implicated in the killing of tumor cells i n v i t r o . 19 A partial protection obtained with compounds that inhibit their production provides support for this role of radical species.15,2° Superoxide radicals (O2-) are also generated in response to TNF or interleukin- 1 (IL- 1) in human diploid fibroblasts and endothelial cells. 21'22 Recent studies have shown that sensitivity to TNF is inversely related to the radical scavenging activity of different cells) 7:8 This activity is maintained by high levels of reduced glutathione and other sulfhydryl compounds that provide the major mechanism of detoxification of oxygen/lipid radical species. 23 TNF and IL-1 induce the mRNA for manganese superoxide dismutase (MnSOD), but not for copperzinc SOD (CuZnSOD) or other antioxidant enzymes. 24'25 MnSOD is a mitochondrial protein that scavenges 02- by the dismutation reaction of two superoxide radicals to H202 and water. Human embryonic kidney cells transfected with a MnSOD expression vector produce high levels of this enzyme and are
INTRODUCTION
Tumor necrosis factor (TNF) is cytotoxic for some tumor cells but is not growth inhibitory for most other cells.l'2 On the contrary, TNF stimulates the growth of normal diploid fibroblasts and of some tumor cells) -5 These opposite responses to TNF cannot be attributed to differences in the number of receptors or binding affinity between TNF-sensitive and -resistant cells) '6'7 However, TNF induces cytolysis of all cells, even normal diploid fibroblasts, when used in combination with inhibitors of RNA or protein synthesis, s Therefore, cytotoxicity or growth stimulation are celltype-specific responses to TNF, but cytolysis in the presence of inhibitors such as cycloheximide or actinomycin D is a general response to TNF. Treatment of human diploid and transformed fibroblasts with TNF prior to the addition of such inhibitors protects these cells from its cytolytic activity? This observation suggests that TNF induces the synthesis of protective proteins that inhibit cytolysis. Proteins induced by TNF are involved in a broad spectrum of biological activities, such as regulation of fibrinolysis, 1o,11transcriptional activation, 12-14arachiAddress correspondence to Dr. Corrado Baglioni, Biology-126, SUNYA, 1400 Washington Avenue, Albany, NY 12222. 151
152
J.A. MELENDEZ and C. BAGLION1
A
B
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~
~
A
80
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x
~
~
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• R4
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g " 60 ,.~
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//
I 0.3
1 I
I 3
I I0
/! TNF
•
•
I C).3
1 I
SK-MEL-I09
-.-...........~, I 3
I IO
(ng/ml)
Fig. 1. Cytolytic activity of TNF on HeLa, SK-MEL- 109, and variant cells selected for TNF resistance. The selection techniques and the cloning of variants are described in Materials and Methods. HeLa (A), SK-MEL-109 (B), and variant cells selected for TNF resistance were treated with the TNF concentration indicated in the abscissa and 10 ~tg/ml ofcycloheximide. After 18 h, live cells were stained with crystal violet. The A54orelative to control cells treated with cycloheximide alone is indicated. (Fig. 1A from Gessani et al. 3~ Reprinted by permission of Wichtig Editore.)
partially protected from TNF cytotoxicity, z6This finding suggests that MnSOD is one of the protective proteins induced by TNF and that generation of 02- in the mitochondria is a component of this cytotoxic activity. However, inhibitors of phospholipase A 2 can also reduce TNF cytotoxicity, 27-29 and proteinase inhibitors protect some cells from TNF-mediated cytolysis. 3°
TNF-resistant cell variants were previously selected by repeated treatments with TNF and cycloheximide. 3z In the present study, we examined whether these variants had an elevated MnSOD activity. However, analysis of enzymatic activity and mRNA levels demonstrated that HeLa cell variants had reduced amounts of MnSOD and were poorly inducible by TNF or IL-1. These TNF-resistant vail-
A
B TNF IL-!
TNF
-
+
-
+
-
+
-
--
+
--
+
+
+
-
+
-
+
+
+
+
MN-SOD-
I.leLo
R5
CUZN-SOO-
SK
R4
R6
RI2
R7
RIO
Fig. 2. Analysis of MnSOD activity in SK-MEL-109, HeLa, and TNF-resistant variants. SK-MEL-109 (A), HeLa (B), and TNF-resistant cells were treated for 18 h with 10 ng/ml TNF, 10 ng/ml IL- I a, or both cytokines, as indicated. Cell extracts were prepared as described in Materials and Methods. Superoxide dismutase activity zymograms contained 25 #g of cell protein per lane. The upper and lower bands correspond to MnSOD and CuZnSOD, respectively.
153
MnSOD in TNF-resistant variants
B
A TNF 11_-I TNF-
+
-
--
+
-
+
+
+
-
+
+
+
-I-
+
MnSOD
0891-5849/92 $5.00 + .00 Copyright © 1992 Pergamon Press plc
Original Contribution REDUCED EXPRESSION OF MANGANESE SUPEROXIDE DISMUTASE IN CELLS RESISTANT TO CYTOLYSIS BY TUMOR NECROSIS FACTOR
J U A N A. MELENDEZ and CORRADO BAGLIONI The Center for Molecular Genetics, Department of Biological Sciences, University at Albany, State University of New York, Albany, NY 12222, U.S.A.
(Received 23 July 1991; Revised 30 September 1991; Accepted 22 October 1991) Abstract--Tumor necrosis factor (TNF) induces synthesis of manganese superoxide dismutase (MnSOD). It was previously shown that overexpression of MnSOD protected some mammalian cells from TNF cytotoxicity. The purpose of this study was to establish whether MnSOD was increased in cells selected for resistance to cytolysis by TNF in combination with cycloheximide. Melanoma SK-MEL-109 and HeLa cell-resistant variants were selected by repeated treatments with TNF and cycloheximide. The SK-MEL-109 variants had relatively low levels of MnSOD that were inducible by TNF. Surprisingly, the HeLa variants had very low levels of MnSOD that were poorly inducible by either TNF or interleukin- 1a. Therefore, an elevated level of MnSOD was not required to protect these cells from TNF-mediated cytolysis. The HeLa variants were more sensitive than parental cells to superoxide radical (02-) generating compounds, such as paraquat or xanthine/xanthine oxidase. Pretreatment of these variants with TNF did not provide protection against damage by superoxide radicals. Keywords--Superoxide dismutase, Tumor necrosis factor, Cytolysis, Superoxide radicals, Free radicals
donic acid metabolism, ls,16 and lipid peroxidation. 17,1sHowever, proteins that confer complete protection from TNF-mediated cytolysis have not yet been identified. Production of hydroxyl radical species induced by TNF has been implicated in the killing of tumor cells i n v i t r o . 19 A partial protection obtained with compounds that inhibit their production provides support for this role of radical species.15,2° Superoxide radicals (O2-) are also generated in response to TNF or interleukin- 1 (IL- 1) in human diploid fibroblasts and endothelial cells. 21'22 Recent studies have shown that sensitivity to TNF is inversely related to the radical scavenging activity of different cells) 7:8 This activity is maintained by high levels of reduced glutathione and other sulfhydryl compounds that provide the major mechanism of detoxification of oxygen/lipid radical species. 23 TNF and IL-1 induce the mRNA for manganese superoxide dismutase (MnSOD), but not for copperzinc SOD (CuZnSOD) or other antioxidant enzymes. 24'25 MnSOD is a mitochondrial protein that scavenges 02- by the dismutation reaction of two superoxide radicals to H202 and water. Human embryonic kidney cells transfected with a MnSOD expression vector produce high levels of this enzyme and are
INTRODUCTION
Tumor necrosis factor (TNF) is cytotoxic for some tumor cells but is not growth inhibitory for most other cells.l'2 On the contrary, TNF stimulates the growth of normal diploid fibroblasts and of some tumor cells) -5 These opposite responses to TNF cannot be attributed to differences in the number of receptors or binding affinity between TNF-sensitive and -resistant cells) '6'7 However, TNF induces cytolysis of all cells, even normal diploid fibroblasts, when used in combination with inhibitors of RNA or protein synthesis, s Therefore, cytotoxicity or growth stimulation are celltype-specific responses to TNF, but cytolysis in the presence of inhibitors such as cycloheximide or actinomycin D is a general response to TNF. Treatment of human diploid and transformed fibroblasts with TNF prior to the addition of such inhibitors protects these cells from its cytolytic activity? This observation suggests that TNF induces the synthesis of protective proteins that inhibit cytolysis. Proteins induced by TNF are involved in a broad spectrum of biological activities, such as regulation of fibrinolysis, 1o,11transcriptional activation, 12-14arachiAddress correspondence to Dr. Corrado Baglioni, Biology-126, SUNYA, 1400 Washington Avenue, Albany, NY 12222. 151
152
J.A. MELENDEZ and C. BAGLION1
A
B
I00
~
~
~
A
80
--
o
-
x
~
x
~
x
~
~
R6 RI2
A--
• R4
R5
g " 60 ,.~
40
HeLo -e
//
I 0.3
1 I
I 3
I I0
/! TNF
•
•
I C).3
1 I
SK-MEL-I09
-.-...........~, I 3
I IO
(ng/ml)
Fig. 1. Cytolytic activity of TNF on HeLa, SK-MEL- 109, and variant cells selected for TNF resistance. The selection techniques and the cloning of variants are described in Materials and Methods. HeLa (A), SK-MEL-109 (B), and variant cells selected for TNF resistance were treated with the TNF concentration indicated in the abscissa and 10 ~tg/ml ofcycloheximide. After 18 h, live cells were stained with crystal violet. The A54orelative to control cells treated with cycloheximide alone is indicated. (Fig. 1A from Gessani et al. 3~ Reprinted by permission of Wichtig Editore.)
partially protected from TNF cytotoxicity, z6This finding suggests that MnSOD is one of the protective proteins induced by TNF and that generation of 02- in the mitochondria is a component of this cytotoxic activity. However, inhibitors of phospholipase A 2 can also reduce TNF cytotoxicity, 27-29 and proteinase inhibitors protect some cells from TNF-mediated cytolysis. 3°
TNF-resistant cell variants were previously selected by repeated treatments with TNF and cycloheximide. 3z In the present study, we examined whether these variants had an elevated MnSOD activity. However, analysis of enzymatic activity and mRNA levels demonstrated that HeLa cell variants had reduced amounts of MnSOD and were poorly inducible by TNF or IL-1. These TNF-resistant vail-
A
B TNF IL-!
TNF
-
+
-
+
-
+
-
--
+
--
+
+
+
-
+
-
+
+
+
+
MN-SOD-
I.leLo
R5
CUZN-SOO-
SK
R4
R6
RI2
R7
RIO
Fig. 2. Analysis of MnSOD activity in SK-MEL-109, HeLa, and TNF-resistant variants. SK-MEL-109 (A), HeLa (B), and TNF-resistant cells were treated for 18 h with 10 ng/ml TNF, 10 ng/ml IL- I a, or both cytokines, as indicated. Cell extracts were prepared as described in Materials and Methods. Superoxide dismutase activity zymograms contained 25 #g of cell protein per lane. The upper and lower bands correspond to MnSOD and CuZnSOD, respectively.
153
MnSOD in TNF-resistant variants
B
A TNF 11_-I TNF-
+
-
--
+
-
+
+
+
-
+
+
+
-I-
+
MnSOD
