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The protein thiol metallothionein as an antioxidant and protectant against antineoplastic drugs
Chemico-Biological Interactions 111 – 112 (1998) 255 – 262
The protein thiol metallothionein as an antioxidant and protectant against antineoplastic drugs John S. Lazo *, Shiu-Ming Kuo 1, Elizabeth S. Woo, Bruce R. Pitt Department of Pharmacology, Uni6ersity of Pittsburgh, Pittsburgh, PA 15261, USA
Abstract Metallothioneins (MTs) are major zinc-binding protein thiols that are readily inducible and whose functions remain unclear. Recent evidence supports a role for MT as an antioxidant. Mechanisms underlying this function may include direct interception of free radicals, complexation of redox sensitive transition metals, altered zinc homeostasis or interaction with glutathione (GSH). MT overexpression after direct gene transfer in cultured cells, decreases cytotoxicity, to partially reduce reactive oxygen and nitrogen species and markedly attenuates intracellular oxidation of reporter molecules including dichlorofluorescein and cis-parinaric acid. Conversely, enhanced intracellular oxidation is seen in cells derived from mice lacking both functional MTI and MTII genes. GSH levels are unaffected in MT null cells relative to wildtype, suggesting the antioxidant function of MT is independent of GSH. In tumor cells there is at least a 400-fold range in MT levels and a 10-fold difference in the ratio of nuclear to cytoplasmic distribution. No correlation exists between MT levels and GSH levels demonstrating the autonomous regulation of intracellular thiol pools. This may be important for cancer chemotherapies since MT overexpression is seen in human tumor cells with acquired drug resistance. The authors found no evidence for altered MT isoform profiles in drug resistant cells that overexpress MT. Recent evidence suggests MT subcellular location may dictate functionality and MT may help determine the threshold for apoptosis. Thus, MT is a stress-inducible protein with antioxidant attributes that may participate independently or in conjunction with GSH to protect cells against injurious agents. © 1998 Elsevier Science Ireland Ltd. All rights reserved. * Corresponding author. 1 Present address: Nutrition Program, School of Health Related Professions, University at Buffalo, State University of New York, 323 Kimball Tower, Buffalo, NY 14214, USA. 0009-2797/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0009-2797(97)00165-8
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Keywords: Metallothionein; Antioxidant; Apoptosis; Thiols
1. Introduction Metallothioneins (MTs) are a family of ubiquitous, low molecular weight (6–7 kDa), highly conserved, thiol rich proteins. Like glutathione (GSH), one third of the 61 amino acids in the prototypic MT are cysteines. The high thiol content of MT imparts a well-characterized tertiary structure associated with the seven metalthiolate clusters. An excellent discussion of the evolution and structure of MT is located elsewhere [1]. Interest in the biological functions of MT can be attributed to the ubiquitous nature of the protein, the large number of human genes (seven) encoding MT and the induction of the protein by many endogenous and exogenous substances. Nonetheless, there remains controversy about the biological function of MT, despite a considerable number of studies. The subject of this article is the biological functions of MT, especially in relationship to cancer chemotherapy and the main nonprotein thiol GSH.
2. MT expression in tumors MT is expressed both in normal and malignant cells. In 52, human tumor cell lines from the National Cancer Institute’s tumor panel, there is almost a 400-fold difference in basal MT levels even though the cells are grown at the same site and under similar conditions [2]. Furthermore, these cells can reproducibly retain high nuclear or cytoplasmic MT phenotypes. Quantitative analyses of the relative nuclear to cytoplasmic concentration indicate almost a 10-fold difference among the cells with no relationship between the total MT intracellular content and the nuclear/cytoplasmic distribution [2]. The pioneering studies of Cherian and co-workers [3] demonstrate both nuclear and cytoplasmic MT in human tumor samples patients by immunohistochemistry. Others have used immunochemical, heavy metal binding or chromatographic methods to show MT expression in human testicular, bladder, breast and colorectal tumors [4]. In some studies, tumor cells with acquired resistance to electrophilic anticancer drugs have elevated MT levels compared with the untreated parent tumor cells [4,5]. We have compared the MT isoform composition in a human squamous cell carcinoma line with 7-fold acquired resistance to cis-diammedichloroplatinum (SCC25/CP) to the parental SCC25 cells (Fig. 1). These SCC25/CP cells have 4-fold higher basal MT levels [4]. After metabolic radiolabeling with 35S cysteine, the authors examined the MT isoform composition by fast protein liquid chromotography as previously described [6]. The major MT isoform detected in both SCC25 and SCC25/CP cells was MTIIa (Fig. 1). This HPLC method does not permit us to discriminate among the minor MTI isoforms but they
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clearly are minor components of the MT pool in these cells. Most importantly, however, was the retention of MTIIa as the primary isoform in the cis-diamminedichloroplatinum resistant cells and the lack of any obvious gross difference in MT isoform profile. These results suggest overexpression of MT in drug resistance SCC25/CP cells is due to an increase in MTIIa expression and a parallel coordinated increase in MTI expression.
3. MT as a nucleophile Because of the nucleophilicity of MT, there has been considerable interest in its ability to protect cells against electrophilic toxins. Indeed, the authors and others [4,7 – 9] previously demonstrated increased MT levels protect against cytotoxicity from carbon-, oxygen- and nitrogen-based radicals including electrophilic mutagens, antineoplastic drugs, nitric oxide and environmental oxidants. Recently, cells isolated from mice deficient in MT by targeted disruption of MTI and MTII genes were found to be more sensitive to the toxic effects of oxidants, the heavy metal cadmium, anticancer drugs and electrophilic mutagens [10,11]. These cells do not have altered levels of GSH, catalase or GSH peroxidase [11]. MT thiolate groups clearly can interact covalently and sequester electrophilic agents in vitro [15 – 17] with some anticancer drugs, such as melphalan, showing remarkable specificity for cysteines-33 and -48 [17]. Covalent thiolate binding to cis-diamminedichloroplatinum or melphalan, in vitro, releases zinc from MT [15,17], which may cause increases in intracellular zinc that could affect other
Fig. 1. HPLC profile of MT isoforms in human SCC25 and SCC25/CP cells. Cells were preincubated with 40–50 mCi/ml (1000 Ci/mmol) 35S-cysteine for 48 h. To separate and quantify MTI and MTII, the authors prepared cell lysates and subjected them to Sephadex G-75 column chromatography followed by fast protein liquid chromatography with a HR5/5 MonoQ column as described previously [6]. The radioactivity in each fraction was then determined by liquid scintillation counting.
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processes. Nevertheless, the importance of covalent interactions between anticancer drugs and MT in vivo has not been established. Because ectopic expression of MT after transfection does not reduce DNA adduct formation seen with exposure to electrophilic mutagens, other mechanisms may be operative [4,8]. Furthermore, MT appears to protect cells against antimetabolite anticancer drugs, such as cytosine arabinoside (ara-C) and tumor necrosis factor-a [4,18,19], an effect that cannot be readily rationalized by a hypothesis of simple covalent drug sequestration.
4. MT as an antioxidant There is growing evidence that MT can function as antioxidant. Yeast and simian MT can functionally substitute for copper/zinc superoxide dismutase in a strain of S. cere6isiae lacking superoxide dismutase [20]. Both embryonic cells and hepatocytes from MT null mice are more sensitive to tert-butylhydroperoxide [4,21]. Induction of MT expression in a variety of cells leads to resistance to oxidants [4]. Overexpression of MT in NIH3T3 cells after gene transfer results in resistance to the toxic effects of both tert-butylhydroperoxide and nitric oxide [4]. The mechanism by which MT acts as an antioxidant remains unclear. In vitro experiments clearly show that MT can scavenge superoxide anions or hydroxyl radicals [22], phenoxyl radicals [23] and nitric oxide [9]. Overexpression of MT in cultured endothelial cells inhibits transition metal-independent, azo-initiator induced lipid peroxidation as detected after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid [24]. Thus, MT might function as an expendable target for oxidants due to its highly enriched cysteine residue structure. These cysteines, while involved in thiolate clusters, are quite labile and freely exchange native metals with electrophiles. This was recently verified with intact cells when Petering and co-workers [25] showed the sulfhydryl groups of MT isolated from HL-60 cells were oxidized by H2O2. It is interesting to speculate whether or not a metallothionein reductase system may exist. Alternatively, MT may function as an antioxidant indirectly by affecting two important metals: zinc and copper. Zinc and copper per se may facilitate antioxidant enzymes such as superoxide dismutase [4]. There has been some suggestion that MT and GSH cooperate within cells and that their intracellular synthetic pools are not independently regulated. Ochi et al. [12] report that GSH and MT both function to protect cells against cadmium toxicity. Ferreira et al. [13] propose intracellular movement of copper(I) from GSH to MT. Brouwer and co-workers [14] find a complex between GSH and MT in vitro and estimate a GSH/MT stoichiometry of 1.4 and a Kdiss of 14 mM. They also predict based on molecular modeling that a cleft on MT can accommodate GSH binding at cysteine-26. Interestingly, however, no relationship has been found between the basal MT and GSH levels in the National Cancer Institute’s tumor panel [2]. These results are most consistent with the two intracellular thiols pools being regulated autonomously.
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5. MT and apoptosis Cellular oxidation state is almost certainly important for life and death. Apoptosis is an active form of cell death that occurs after cellular exposure to antineoplastic agents, other genotoxic substances, mutagens, oxidants and nitric oxide as well as after growth and survival factor withdrawal. Among the many putative mediators of apoptosis, reactive oxygen species are prominent [26]. Several apoptotic agonists have been shown to generate reactive oxygen species prior to overt evidence of apoptosis [26,27]. Reduced expression of antioxidant enzymes, such as superoxide dismutase or GSH peroxidase, increase apoptosis cased by many stimuli [28]. Small organic antioxidants, such as N-acetyl-L-cysteine, protect cells against apoptosis [29]. Redox sensitive protein, such as NF-kB, also appear to act in a complex manner to regulate apoptosis [30]. Previous studies indicate loss of MT expression enhances cellular sensitivity to agents that are not formally electrophilic, suggesting MT might affect signaling processes fundamental for controlling death. Thus, we and others [31,32] have investigated the role of MT in apoptosis using complementary approaches. AbdelMageed and Agrawal [32] demonstrated in MCF7 cells that transient downregulation of MT by phosphorothiolate antisense oligomer blocks proliferation and induces apoptosis. The studies revealed mouse MTI/MTII null cells are more susceptible to apoptotic death after exposure to tert-butylhydroperoxide or antineoplastic agents, including ara-C, compared with the wildtype cells [31]. Basal levels of p53 and the death effector protein Bax are higher in MT null cells compared with the wildtype, which may reflect differences in the oxidative state of the null cells. Collectively these results suggest a role for the stress- responsive factor MT in controlling the threshold for apoptotic response in cells.
6. Regional regulation of MT functionality As mentioned above, sequestration of MT in the nucleus or cytoplasm has been noted repeatedly. This is somewhat surprising for a protein of only 6–7 kDa as the nuclear pore complex generally restricts movement of proteins that are \ 50–60 kDa in mass, while smaller proteins are thought to freely diffuse between these two cellular compartments. The sequestration of small peptides or proteins is, however, certainly not without precedent: discrete pools of GSH or calmodulin (16 kDa), for example, have been noted [33,34]. The nuclear sequestration of MT appears to be independent of cell cycle but sensitive to reduced temperature; moreover, nuclear retention of MT is an energy dependent process [35]. In vivo binding partners for MT remain to be determined. That cytoplasmic location is important for protective functionality oxidants was deduced from studies with NIH3T3 cells transfected with gene encoding mouse MTI [23]. These cells, which showed a cytoplasmic MT phenotype, were protected against tert-butylhydroperoxide cytotoxicity and intracellular oxidation of reporter molecules but not against the DNA damage caused by this compound [23]. These
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results underscore the central role of cytoplasmic mechanisms in the lethal effects of oxidants. Interestingly, cytoplasmic overexpression of MT provides substantial protection against nuclear damage of the highly diffusible free radical NO [9]. Zn-pretreatment of wildtype NIH3T3 cells enhanced nuclear expression of MT and was associated with protection against an NO donor in isolated nuclei further supporting a role for nuclear MT in reducing DNA damage [9]. A system has recently been developed to address whether the subcellular location of MT affects its protective functionality [2]. A plasmid containing a regulated gene encoding b-galactosidase-MT fusion protein was introduced into MT null cells. This fusion protein was directed either to the nucleus or the cytoplasm due to the presence or absence of the SV40 large T antigen nuclear localization sequence. Both fusion proteins retain the ability to bind cadmium in intact cells. Cytoplasmic but not nuclear expression of MT decreases the formation of reactive oxygen species generated by tert-butylhydroperoxide [23]. Cytoplasmic MT also reduces the cytotoxicity of cadmium, while nuclear expression protects against the cytotoxicity of the genotoxic agent N-methyl-N%-nitro-N-nitrosoguanidine [2]. These data establish the importance of MT partitioning into the nucleus and cytoplasm for at least some of its functionality.
7. Conclusion MT are inducible protein thiols that function to control intracellular oxidation state. Elevated levels of MT can protect cells against a variety of toxic substances including reactive carbon- and nitrogen-based compounds. MT overexpression has been seen in human tumor cells with acquired drug resistance but the MT isoform profile appears not markedly altered. A detailed study of the National Cancer Institute tumor panel revealed a 400-fold range in MT levels and 10-fold difference in the nuclear to cytoplasmic distribution. There was no correlation between MT levels and GSH levels demonstrating the autonomous regulation of thiol pools. Recent evidence suggest that the subcellular location of MT may dictate its functionality and that MT could have a role in determining the threshold for apoptosis. Thus, MT is a stress-inducible, thiol rich, protein that has antioxidant attributes and may participate in conjunction with GSH to protect cells against injurious agents.
References [1] J.H.R. Kagi, Evolution, structure and chemical activity of class I metallothioneins: An overview, in: K.T. Suzuki, N. Imura, M. Kimura (Eds.), Metallothionein III: Biological Roles and Medical Implications, Birkha¨user, Basel, 1993, pp. 29 – 56. [2] E.S. Woo, A. Monks, S.C. Watkins, A.S. Wang, J.S. Lazo, Diversity of metallothionein content and subcellular localization in the National Cancer Institute tumor Panel, Cancer Chemother. Pharmacol. 41 (1997) 61–68.
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[3] D. Banerjee, S. Onosaka, M.G. Cherian, Immunohistochemical localization of metallothionein in cell nucleus and cytoplasm of rat liver and kidney, Toxicology 24 (1982) 95 – 105. [4] J.S. Lazo, B.R. Pitt, Metallothioneins and cell death by anticancer drugs, in: A.K. Cho, T.F. Blaschke, H.H. Loh, J.L. Way (Eds.), Annual Review of Pharmacology and Toxicology, Annual Reviews, Palo Alto, CA, 1995, pp. 635 – 653. [5] K. Kasahara, Y. Fujiwara, K. Nishio, T. Ohmori, Y. Sugimoto, K. Komiya, T. Matsuda, N. Saijo, Metallothionein content correlates with the sensitivity of human small cell lung cancer cell lines to cisplatin, Cancer Res. 51 (1991) 3237 – 3242. [6] S.-M. Kuo, Y. Kondo, J.M. DeFilippo, M.S. Ernstoff, R.R. Bahnson, J.S. Lazo, Subcellular localization of metallothionein IIA in human bladder tumor cells using a novel epitope-specific antiserum, Toxicol. Appl. Pharmacol. 125 (1994) 104 – 110. [7] S.L. Kelley, A. Basu, B.A. Teicher, M.P. Hacker, D.H. Hamer, J.S. Lazo, Overexpression of metallothionein confers resistance to anticancer drugs, Science 241 (1988) 1813 – 1815. [8] B. Kaina, H. Lohrer, M. Karin, P. Herrlich, Overexpressed human metallothionein IIA gene protects Chinese hamster ovary cells from killing by alkylating agents, Proc. Natl. Acad. Sci. USA 87 (1990) 2710–3714. [9] M.A. Schwarz, J.S. Lazo, J.C. Yalowich, W.P. Allen, M. Whitmore, H.A. Bergonia, E. Tzeng, T.R. Billiar, P.D. Robbins, J.R. Lancaster Jr., B.R. Pitt, Metallothionein protects against the cytotoxic and DNA damaging effects of nitric oxide, Proc. Natl. Acad. Sci. USA 92 (1995) 4452 – 4456. [10] Y. Kondo, E. Woo, A.E. Michalska, A.C. Choo, J.S. Lazo, Metallothionein null cells have increased sensitivity to anticancer drugs, Cancer Res. 55 (1995) 2021 – 2023. [11] J.S. Lazo, Y. Kondo, D. Dellapiazza, A.E. Michalska, K.H.A. Choo, B.R. Pitt, Enhanced sensitivity to oxidative stress in cultured embryonic cells from transgenic mice deficient in metallothionein I and II genes, J. Biol. Chem. 270 (1995) 5506 – 5510. [12] T. Ochi, F. Otsuka, K. Takahashi, M. Ohsawa, Glutathione and metallothioneins as cellular defense against cadminum toxicity in cultured Chinese hamster cells, Chem. Biol. Interact. 65 (1988) 1–14. [13] A.M. Ferreira, M.R. Ciriolo, L. Marcocci, G. Rotilio, Copper(I) transfer into metallothionein mediated by glutathione, Biochem. J. 292 (1993) 673 – 676. [14] M. Brouwer, B.T. Hoexum-Brouwer, R.E. Cashon, A putative glutathione-binding site in CdZnmetallothionein identified by equilibrium binding and molecular-modelling studies, Biochem. J. 294 (1993) 219–225. [15] D.H. Petering, A. Quesada, M. Dughish, S. Krull, T. Gan, D. Lemkuil, A. Pattanaik, R.W. Byrnes, M. Savas, H. Whelan, C.F. Shaw III, Metallothionein in tumor and host: Intersections of zinc metabolism, the stress response and tumor therapy, in: K.T. Suzuki, N. Imura, M. Kimura (Eds.), Metallothionein III: Biological Roles and Medical Implications, Birkha¨user, Basel, 1993, pp. 329–346. [16] X. Yu, Z. Wu, C. Fenselau, Covalent sequestration of melphalan by metallothionein and selective alkylation of cysteines, Biochemistry 34 (1995) 3377 – 3385. [17] J. Zaia, L. Jiang, M.S. Han, J.R. Tabb, Z. Wu, D. Fabris, C. Fenselau, A binding site for chlorambucil on metallothionein, Biochemistry 35 (1996) 2830 – 2835. [18] Y. Kondo, S.-M. Kuo, S.C. Watkins, J.S. Lazo, Metallothionein localization and cisplatin resistance in human hormone-independent prostatic tumor cell lines, Cancer Res. 55 (1995) 474–477. [19] K. Leyshon-Sorland, L. Morkrid, H.E. Rugstrad, Metallothionein: a protein conferring resistance in vitro to tumor necrosis factor, Cancer Res. 53 (1993) 4874 – 4880. [20] K.T. Tamai, E.B. Gralla, L.M. Ellerby, V. J.S. Valentine, D.J. Thiele, Yeast and mammalian metallothioneins functionally substitute for yeast copper – zinc superoxide dismutase, Proc. Natl. Acad. Sci. USA 90 (1993) 8013–8017. [21] H. Zheng, J. Liu, K.H.A. Choo, A.E. Michalska, C.D. Klaassen, Metallothionein-I and -II knock-out mice are sensitive to cadmium-induced liver mRNA expression of c-jun and p53, Toxicol. Appl. Pharmacol. 136 (1996) 229 – 235. [22] M. Sato, I. Bremner, Oxygen free radicals and metallothionein, Free Radic. Biol. Med. 14 (1993) 325–337.
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[23] M.A. Schwarz, J.S. Lazo, J.C. Yalowich, I. Reynolds, V.E. Kagan, V. Tyurin, T.-M. Kim, S.C. Watkins, B.R. Pitt, Cytoplasmic metallothionein overexpression protects NIH 3T3 cells from tert-butyl hydroperoxide toxicity, J. Biol. Chem. 269 (1994) 15238 – 15243. [24] B.R. Pitt, M. Schwarz, E.S. Woo, E. Yee, K. Wasserloos, S. Tran, W. Weng, R.J. Mannix, S.A. Watkins, Y.Y. Tyurina, V.A. Tyurin, V.E. Kagan, J.S. Lazo, Overexpression of metallothionein decreases the sensivitiy of pulmonary endothelial cells to oxidant injury, Am. J. Physiol. Cell Mol. Physiol. (1997) in press. [25] A.R. Quesada, R.W. Byrnes, S.O. Krezoski, D.H. Petering, Direct reaction of H2O2 with sulfhydryl groups in HL-60 cells, Arch. Biochem. Biophys. 334 (1996) 241 – 250. [26] T.M. Buttke, P.A. Sandstrom, Oxidative stress as a mediator of apoptosis, Immunol. Today 15 (1994) 7–10. [27] T.M. Johnson, Z.-X. Yu, V.J. Ferrans, R.A. Lowenstein, T. Finkel, Reactive oxygen species are downstream mediators of p53-dependent apoptosis, Proc. Natl. Acad. Sci. USA 93 (1996) 11848 – 11852. [28] L.J. Greenlund, T.L. Deckwerth, E.M. Johnson Jr., Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death, Neuron 14 (1995) 303 – 315. [29] M. Mayer, M. Noble, N-acetyl-L-cysteine is a pluripotent protector against cell death and enhancer of trophic factor-mediated cell survival in vitro, Proc. Natl. Acad. Sci. USA 91 (1994) 7496 – 7500. [30] K.I. Lin, S.H. Lee, R. Narayanan, J.M. Baraban, J.M. Hardwick, R.R. Ratan, Thiol agents and Bcl-2 identify an alphavirus-induced apoptotic pathway that requires activation of the transcription factor NF-k-B, J. Cell Biol. 131 (1995) 1149 – 1161. [31] Y. Kondo, J.M. Rusnak, D.G. Hoyt, C.E. Settineri, B.R. Pitt, J.S. Lazo, Enhanced apoptosis in metallothionein null cells, Mol. Pharmacol. (in press). [32] A.B. Abdel-Mageed, K.C. Agrawal, Antisense down-regulation of metallothionein induces growth arrest and apoptosis in human breast carcinoma cells, Cancer Gene Ther. 4 (1997) 199 – 207. [33] G. Bellomo, M. Vairetti, L. Stivala, F. Mirabelli, P. Richelmi, S. Orenius, Demonstration of nuclear compartmentalization of glutathione in hepatocytes, Proc. Natl. Acad. Sci. USA 89 (1992) 4412–4416. [34] N. Michaud, D.S. Goldfarb, Most nuclear proteins are imported by a single pathway, Exp. Cell Res. 208 (1993) 128–136. [35] E.S. Woo, Y. Kondo, S.C. Watkins, D.G. Hoyt, J.S. Lazo, Nucleophilic distribution of metallothionein tumor cells, Exp. Cell Res. 224 (1996) 365 – 371.
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The protein thiol metallothionein as an antioxidant and protectant against antineoplastic drugs John S. Lazo *, Shiu-Ming Kuo 1, Elizabeth S. Woo, Bruce R. Pitt Department of Pharmacology, Uni6ersity of Pittsburgh, Pittsburgh, PA 15261, USA
Abstract Metallothioneins (MTs) are major zinc-binding protein thiols that are readily inducible and whose functions remain unclear. Recent evidence supports a role for MT as an antioxidant. Mechanisms underlying this function may include direct interception of free radicals, complexation of redox sensitive transition metals, altered zinc homeostasis or interaction with glutathione (GSH). MT overexpression after direct gene transfer in cultured cells, decreases cytotoxicity, to partially reduce reactive oxygen and nitrogen species and markedly attenuates intracellular oxidation of reporter molecules including dichlorofluorescein and cis-parinaric acid. Conversely, enhanced intracellular oxidation is seen in cells derived from mice lacking both functional MTI and MTII genes. GSH levels are unaffected in MT null cells relative to wildtype, suggesting the antioxidant function of MT is independent of GSH. In tumor cells there is at least a 400-fold range in MT levels and a 10-fold difference in the ratio of nuclear to cytoplasmic distribution. No correlation exists between MT levels and GSH levels demonstrating the autonomous regulation of intracellular thiol pools. This may be important for cancer chemotherapies since MT overexpression is seen in human tumor cells with acquired drug resistance. The authors found no evidence for altered MT isoform profiles in drug resistant cells that overexpress MT. Recent evidence suggests MT subcellular location may dictate functionality and MT may help determine the threshold for apoptosis. Thus, MT is a stress-inducible protein with antioxidant attributes that may participate independently or in conjunction with GSH to protect cells against injurious agents. © 1998 Elsevier Science Ireland Ltd. All rights reserved. * Corresponding author. 1 Present address: Nutrition Program, School of Health Related Professions, University at Buffalo, State University of New York, 323 Kimball Tower, Buffalo, NY 14214, USA. 0009-2797/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0009-2797(97)00165-8
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Keywords: Metallothionein; Antioxidant; Apoptosis; Thiols
1. Introduction Metallothioneins (MTs) are a family of ubiquitous, low molecular weight (6–7 kDa), highly conserved, thiol rich proteins. Like glutathione (GSH), one third of the 61 amino acids in the prototypic MT are cysteines. The high thiol content of MT imparts a well-characterized tertiary structure associated with the seven metalthiolate clusters. An excellent discussion of the evolution and structure of MT is located elsewhere [1]. Interest in the biological functions of MT can be attributed to the ubiquitous nature of the protein, the large number of human genes (seven) encoding MT and the induction of the protein by many endogenous and exogenous substances. Nonetheless, there remains controversy about the biological function of MT, despite a considerable number of studies. The subject of this article is the biological functions of MT, especially in relationship to cancer chemotherapy and the main nonprotein thiol GSH.
2. MT expression in tumors MT is expressed both in normal and malignant cells. In 52, human tumor cell lines from the National Cancer Institute’s tumor panel, there is almost a 400-fold difference in basal MT levels even though the cells are grown at the same site and under similar conditions [2]. Furthermore, these cells can reproducibly retain high nuclear or cytoplasmic MT phenotypes. Quantitative analyses of the relative nuclear to cytoplasmic concentration indicate almost a 10-fold difference among the cells with no relationship between the total MT intracellular content and the nuclear/cytoplasmic distribution [2]. The pioneering studies of Cherian and co-workers [3] demonstrate both nuclear and cytoplasmic MT in human tumor samples patients by immunohistochemistry. Others have used immunochemical, heavy metal binding or chromatographic methods to show MT expression in human testicular, bladder, breast and colorectal tumors [4]. In some studies, tumor cells with acquired resistance to electrophilic anticancer drugs have elevated MT levels compared with the untreated parent tumor cells [4,5]. We have compared the MT isoform composition in a human squamous cell carcinoma line with 7-fold acquired resistance to cis-diammedichloroplatinum (SCC25/CP) to the parental SCC25 cells (Fig. 1). These SCC25/CP cells have 4-fold higher basal MT levels [4]. After metabolic radiolabeling with 35S cysteine, the authors examined the MT isoform composition by fast protein liquid chromotography as previously described [6]. The major MT isoform detected in both SCC25 and SCC25/CP cells was MTIIa (Fig. 1). This HPLC method does not permit us to discriminate among the minor MTI isoforms but they
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clearly are minor components of the MT pool in these cells. Most importantly, however, was the retention of MTIIa as the primary isoform in the cis-diamminedichloroplatinum resistant cells and the lack of any obvious gross difference in MT isoform profile. These results suggest overexpression of MT in drug resistance SCC25/CP cells is due to an increase in MTIIa expression and a parallel coordinated increase in MTI expression.
3. MT as a nucleophile Because of the nucleophilicity of MT, there has been considerable interest in its ability to protect cells against electrophilic toxins. Indeed, the authors and others [4,7 – 9] previously demonstrated increased MT levels protect against cytotoxicity from carbon-, oxygen- and nitrogen-based radicals including electrophilic mutagens, antineoplastic drugs, nitric oxide and environmental oxidants. Recently, cells isolated from mice deficient in MT by targeted disruption of MTI and MTII genes were found to be more sensitive to the toxic effects of oxidants, the heavy metal cadmium, anticancer drugs and electrophilic mutagens [10,11]. These cells do not have altered levels of GSH, catalase or GSH peroxidase [11]. MT thiolate groups clearly can interact covalently and sequester electrophilic agents in vitro [15 – 17] with some anticancer drugs, such as melphalan, showing remarkable specificity for cysteines-33 and -48 [17]. Covalent thiolate binding to cis-diamminedichloroplatinum or melphalan, in vitro, releases zinc from MT [15,17], which may cause increases in intracellular zinc that could affect other
Fig. 1. HPLC profile of MT isoforms in human SCC25 and SCC25/CP cells. Cells were preincubated with 40–50 mCi/ml (1000 Ci/mmol) 35S-cysteine for 48 h. To separate and quantify MTI and MTII, the authors prepared cell lysates and subjected them to Sephadex G-75 column chromatography followed by fast protein liquid chromatography with a HR5/5 MonoQ column as described previously [6]. The radioactivity in each fraction was then determined by liquid scintillation counting.
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processes. Nevertheless, the importance of covalent interactions between anticancer drugs and MT in vivo has not been established. Because ectopic expression of MT after transfection does not reduce DNA adduct formation seen with exposure to electrophilic mutagens, other mechanisms may be operative [4,8]. Furthermore, MT appears to protect cells against antimetabolite anticancer drugs, such as cytosine arabinoside (ara-C) and tumor necrosis factor-a [4,18,19], an effect that cannot be readily rationalized by a hypothesis of simple covalent drug sequestration.
4. MT as an antioxidant There is growing evidence that MT can function as antioxidant. Yeast and simian MT can functionally substitute for copper/zinc superoxide dismutase in a strain of S. cere6isiae lacking superoxide dismutase [20]. Both embryonic cells and hepatocytes from MT null mice are more sensitive to tert-butylhydroperoxide [4,21]. Induction of MT expression in a variety of cells leads to resistance to oxidants [4]. Overexpression of MT in NIH3T3 cells after gene transfer results in resistance to the toxic effects of both tert-butylhydroperoxide and nitric oxide [4]. The mechanism by which MT acts as an antioxidant remains unclear. In vitro experiments clearly show that MT can scavenge superoxide anions or hydroxyl radicals [22], phenoxyl radicals [23] and nitric oxide [9]. Overexpression of MT in cultured endothelial cells inhibits transition metal-independent, azo-initiator induced lipid peroxidation as detected after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid [24]. Thus, MT might function as an expendable target for oxidants due to its highly enriched cysteine residue structure. These cysteines, while involved in thiolate clusters, are quite labile and freely exchange native metals with electrophiles. This was recently verified with intact cells when Petering and co-workers [25] showed the sulfhydryl groups of MT isolated from HL-60 cells were oxidized by H2O2. It is interesting to speculate whether or not a metallothionein reductase system may exist. Alternatively, MT may function as an antioxidant indirectly by affecting two important metals: zinc and copper. Zinc and copper per se may facilitate antioxidant enzymes such as superoxide dismutase [4]. There has been some suggestion that MT and GSH cooperate within cells and that their intracellular synthetic pools are not independently regulated. Ochi et al. [12] report that GSH and MT both function to protect cells against cadmium toxicity. Ferreira et al. [13] propose intracellular movement of copper(I) from GSH to MT. Brouwer and co-workers [14] find a complex between GSH and MT in vitro and estimate a GSH/MT stoichiometry of 1.4 and a Kdiss of 14 mM. They also predict based on molecular modeling that a cleft on MT can accommodate GSH binding at cysteine-26. Interestingly, however, no relationship has been found between the basal MT and GSH levels in the National Cancer Institute’s tumor panel [2]. These results are most consistent with the two intracellular thiols pools being regulated autonomously.
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5. MT and apoptosis Cellular oxidation state is almost certainly important for life and death. Apoptosis is an active form of cell death that occurs after cellular exposure to antineoplastic agents, other genotoxic substances, mutagens, oxidants and nitric oxide as well as after growth and survival factor withdrawal. Among the many putative mediators of apoptosis, reactive oxygen species are prominent [26]. Several apoptotic agonists have been shown to generate reactive oxygen species prior to overt evidence of apoptosis [26,27]. Reduced expression of antioxidant enzymes, such as superoxide dismutase or GSH peroxidase, increase apoptosis cased by many stimuli [28]. Small organic antioxidants, such as N-acetyl-L-cysteine, protect cells against apoptosis [29]. Redox sensitive protein, such as NF-kB, also appear to act in a complex manner to regulate apoptosis [30]. Previous studies indicate loss of MT expression enhances cellular sensitivity to agents that are not formally electrophilic, suggesting MT might affect signaling processes fundamental for controlling death. Thus, we and others [31,32] have investigated the role of MT in apoptosis using complementary approaches. AbdelMageed and Agrawal [32] demonstrated in MCF7 cells that transient downregulation of MT by phosphorothiolate antisense oligomer blocks proliferation and induces apoptosis. The studies revealed mouse MTI/MTII null cells are more susceptible to apoptotic death after exposure to tert-butylhydroperoxide or antineoplastic agents, including ara-C, compared with the wildtype cells [31]. Basal levels of p53 and the death effector protein Bax are higher in MT null cells compared with the wildtype, which may reflect differences in the oxidative state of the null cells. Collectively these results suggest a role for the stress- responsive factor MT in controlling the threshold for apoptotic response in cells.
6. Regional regulation of MT functionality As mentioned above, sequestration of MT in the nucleus or cytoplasm has been noted repeatedly. This is somewhat surprising for a protein of only 6–7 kDa as the nuclear pore complex generally restricts movement of proteins that are \ 50–60 kDa in mass, while smaller proteins are thought to freely diffuse between these two cellular compartments. The sequestration of small peptides or proteins is, however, certainly not without precedent: discrete pools of GSH or calmodulin (16 kDa), for example, have been noted [33,34]. The nuclear sequestration of MT appears to be independent of cell cycle but sensitive to reduced temperature; moreover, nuclear retention of MT is an energy dependent process [35]. In vivo binding partners for MT remain to be determined. That cytoplasmic location is important for protective functionality oxidants was deduced from studies with NIH3T3 cells transfected with gene encoding mouse MTI [23]. These cells, which showed a cytoplasmic MT phenotype, were protected against tert-butylhydroperoxide cytotoxicity and intracellular oxidation of reporter molecules but not against the DNA damage caused by this compound [23]. These
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results underscore the central role of cytoplasmic mechanisms in the lethal effects of oxidants. Interestingly, cytoplasmic overexpression of MT provides substantial protection against nuclear damage of the highly diffusible free radical NO [9]. Zn-pretreatment of wildtype NIH3T3 cells enhanced nuclear expression of MT and was associated with protection against an NO donor in isolated nuclei further supporting a role for nuclear MT in reducing DNA damage [9]. A system has recently been developed to address whether the subcellular location of MT affects its protective functionality [2]. A plasmid containing a regulated gene encoding b-galactosidase-MT fusion protein was introduced into MT null cells. This fusion protein was directed either to the nucleus or the cytoplasm due to the presence or absence of the SV40 large T antigen nuclear localization sequence. Both fusion proteins retain the ability to bind cadmium in intact cells. Cytoplasmic but not nuclear expression of MT decreases the formation of reactive oxygen species generated by tert-butylhydroperoxide [23]. Cytoplasmic MT also reduces the cytotoxicity of cadmium, while nuclear expression protects against the cytotoxicity of the genotoxic agent N-methyl-N%-nitro-N-nitrosoguanidine [2]. These data establish the importance of MT partitioning into the nucleus and cytoplasm for at least some of its functionality.
7. Conclusion MT are inducible protein thiols that function to control intracellular oxidation state. Elevated levels of MT can protect cells against a variety of toxic substances including reactive carbon- and nitrogen-based compounds. MT overexpression has been seen in human tumor cells with acquired drug resistance but the MT isoform profile appears not markedly altered. A detailed study of the National Cancer Institute tumor panel revealed a 400-fold range in MT levels and 10-fold difference in the nuclear to cytoplasmic distribution. There was no correlation between MT levels and GSH levels demonstrating the autonomous regulation of thiol pools. Recent evidence suggest that the subcellular location of MT may dictate its functionality and that MT could have a role in determining the threshold for apoptosis. Thus, MT is a stress-inducible, thiol rich, protein that has antioxidant attributes and may participate in conjunction with GSH to protect cells against injurious agents.
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