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Cadmium Induces Conformational Modifications of Wild-type p53 and Suppresses p53 Response to DNA Damage in Cultured Cells

1999; Elsevier BV; Volume: 274; Issue: 44 Linguagem: Inglês

10.1074/jbc.274.44.31663

ISSN

1083-351X

Autores

Catherine Méplan, Kris Mann, Pierre Hainaut,

Tópico(s)

Epigenetics and DNA Methylation

Resumo

The p53 tumor suppressor protein is a transcription factor that binds DNA in a sequence-specific manner through a protein domain stabilized by the coordination of zinc within a tetrahedral cluster of three cysteine residues and one histidine residue. We show that cadmium, a metal that binds thiols with high affinity and substitutes for zinc in the cysteinyl clusters of many proteins, inhibits the binding of recombinant, purified murine p53 to DNA. In human breast cancer MCF7 cells (expressing wild-type p53), exposure to cadmium (5–40 μm) disrupts native (wild-type) p53 conformation, inhibits DNA binding, and down-regulates transcriptional activation of a reporter gene. Cadmium at 10–30 μm impairs the p53 induction in response to DNA-damaging agents such as actinomycin D, methylmethane sulfonate, and hydrogen peroxide. Exposure to cadmium at 20 μm also suppresses the p53-dependent cell cycle arrest in G1 and G2/M phases induced by γ-irradiation. These observations indicate that cadmium at subtoxic levels impairs p53 function by inducing conformational changes in the wild-type protein. There is evidence that cadmium is carcinogenic to humans, in particular for lung and prostate, and cadmium is known to accumulate in several organs. This inhibition of p53 function could play a role in cadmium carcinogenicity. The p53 tumor suppressor protein is a transcription factor that binds DNA in a sequence-specific manner through a protein domain stabilized by the coordination of zinc within a tetrahedral cluster of three cysteine residues and one histidine residue. We show that cadmium, a metal that binds thiols with high affinity and substitutes for zinc in the cysteinyl clusters of many proteins, inhibits the binding of recombinant, purified murine p53 to DNA. In human breast cancer MCF7 cells (expressing wild-type p53), exposure to cadmium (5–40 μm) disrupts native (wild-type) p53 conformation, inhibits DNA binding, and down-regulates transcriptional activation of a reporter gene. Cadmium at 10–30 μm impairs the p53 induction in response to DNA-damaging agents such as actinomycin D, methylmethane sulfonate, and hydrogen peroxide. Exposure to cadmium at 20 μm also suppresses the p53-dependent cell cycle arrest in G1 and G2/M phases induced by γ-irradiation. These observations indicate that cadmium at subtoxic levels impairs p53 function by inducing conformational changes in the wild-type protein. There is evidence that cadmium is carcinogenic to humans, in particular for lung and prostate, and cadmium is known to accumulate in several organs. This inhibition of p53 function could play a role in cadmium carcinogenicity. actinomycin D gray(s) metallothionein phosphate-buffered saline ribosomal gene cluster electrophoretic mobility shift assay The p53 protein is a tumor-suppressive transcription factor activated in response to multiple signals including radiation, genotoxic chemicals, hypoxia, depletion of ribonucleotides, and poisoning of the mitotic spindle. In most normal, nonexposed cells, p53 is a latent factor. Induction in response to stress involves nuclear accumulation (as a result of escape from mdm-2-mediated degradation and nuclear export) and conversion to an active form with high affinity for specific DNA sequences. Activation requires post-translational modifications at both the N and C terminus of the protein, including changes in phosphorylation, acetylation, and binding to heterologous proteins (1Liu L. Scolnick D.M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell. Biol. 1999; 19: 1202-1209Crossref PubMed Scopus (648) Google Scholar, 2Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1013) Google Scholar, 3Waterman M.J. Stavridi E.S. Waterman J.L. Halazonetis T.D. Nat. Genet. 1998; 19: 175-178Crossref PubMed Scopus (402) Google Scholar, 4Meek D.W. Cell Signal. 1998; 10: 159-166Crossref PubMed Scopus (177) Google Scholar, 5Rainwater R. Parks D. Anderson M.E. Tegtmeyer P. Mann K. Mol. Cell. Biol. 1995; 15: 3892-3903Crossref PubMed Scopus (273) Google Scholar). Activated p53 controls several sets of genes to prevent the proliferation of cells under stress conditions. Genes transactivated by p53 include inhibitors of cell cycle progression in G1 and G2 (p21waf-1 ,14-3-3ς, GADD 45), regulators of apoptosis (APO1-Fas/CD95, Bax-1, KILLER/DR5), and genes involved in the metabolism of reactive oxygen species (such as PIG-3, PIG-6, and PIG-12) that may play a role in induction of apoptosis (6Polyak K. Waldman T. He T.C. Kinzler K.W. Vogelstein B. Genes Dev. 1996; 10: 1945-1952Crossref PubMed Scopus (473) Google Scholar, 7Yin Y. Terauchi Y. Solomon G.G. Aizawa S. Rangarajan P.N. Yazaki Y. Kadowaki T. Barrett J.C. Nature. 1998; 391: 707-710Crossref PubMed Scopus (151) Google Scholar). p53 also represses a number of promoters and modulates transcription, replication, and DNA repair through interaction with proteins such as RP-A and components of TFIID and TFIIH complexes (for recent reviews see Refs. 4Meek D.W. Cell Signal. 1998; 10: 159-166Crossref PubMed Scopus (177) Google Scholar and8Agarwal M.L. Taylor W.R. Chernov M.V. Chernova O.B. Stark G.R. J. Biol. Chem. 1998; 273: 1-4Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar, 9Giaccia A.J. Kastan M.B. Genes Dev. 1998; 12: 2973-2983Crossref PubMed Scopus (1172) Google Scholar, 10Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6698) Google Scholar, 11Hainaut P. Hollstein M. Adv. Cancer Res. 2000; 77: 81-137Crossref PubMed Scopus (843) Google Scholar).High affinity binding of p53 to specific DNA sequences is mediated by a conformation-sensitive structure in the central portion of the protein (residues 102–292) (12Pavletich N.P. Chambers K.A. Pabo C.O. Genes Dev. 1993; 7: 2556-2564Crossref PubMed Scopus (443) Google Scholar). The structure of the DNA-binding domain consists of two β-sheets supporting a loop-sheet-helix motif (that interacts with the major groove of DNA) and a loop-helix motif (L2/L3, that interacts with the minor groove). L2/L3 is stabilized by tetrahedric coordination of zinc by residues Cys176, His179, Cys238, and Cys242 (13Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2130) Google Scholar). Folded and unfolded forms of human wild-type p53 are distinguishable by their reactivity with the conformation-specific monoclonal antibodies PAb1620 (folded form, often termed "wild-type" conformation) and PAb240 (unfolded form, often termed "mutant" conformation).The folding of the DNA-binding domain is sensitive to metal substitution and to oxido-reduction in vitro and in intact cells. Removal of zinc by chelation reversibly alters p53 conformation, with loss of DNA binding capacity (5Rainwater R. Parks D. Anderson M.E. Tegtmeyer P. Mann K. Mol. Cell. Biol. 1995; 15: 3892-3903Crossref PubMed Scopus (273) Google Scholar, 14Calmels S. Hainaut P. Ohshima H. Cancer Res. 1997; 57: 3365-3369PubMed Google Scholar, 15Hainaut P. Milner J. Cancer Res. 1993; 53: 4469-4473PubMed Google Scholar, 16Verhaegh G.W. Richard M.J. Hainaut P. Mol. Cell. Biol. 1997; 17: 5699-5706Crossref PubMed Scopus (159) Google Scholar). Furthermore,metals such as copper, cadmium, or mercury induce p53 to adopt a PAb240+ phenotype in vitro (17Hainaut P. Milner J. Cancer Res. 1993; 53: 1739-1742PubMed Google Scholar;18Hainaut P. Rolley N. Davies M. Milner J. Oncogene. 1995; 10: 27-32PubMed Google Scholar). These observations raise the possibility that exposure to toxic metals and perturbation of the physiological metal supply may affect p53 functionin vivo.Metals such as cadmium, chromium, nickel, and arsenic are classified in group 1 of the International Agency for Research on Cancer categories of carcinogens (carcinogenic to humans; for reviews, see Refs. 19Beyersmann D. Hechtenberg S. Toxicol. Appl. Pharmacol. 1997; 144: 247-261Crossref PubMed Scopus (497) Google Scholar, 20Waalkes M.P. Coogan T.P. Barter R.A. Crit. Rev. Toxicol. 1992; 22: 175-201Crossref PubMed Scopus (272) Google Scholar, 21International Agency for Cancer Research IARC Monogr. Eval. Carcinog. Risks Hum. 1993; 58: 119-237PubMed Google Scholar). Cadmium is chemically close to zinc and binds with high affinity within the tetrahedral zinc-binding domains of several metalloproteinsin vitro (22Freedman L.P. Luisi B.F. Korszun Z.R. Basavappa R. Sigler P.B. Yamamoto K.R. Nature. 1988; 334: 543-546Crossref PubMed Scopus (346) Google Scholar, 23Glusker J.P. Adv. Protein Chem. 1991; 42: 1-76Crossref PubMed Google Scholar, 24Thiesen H.J. Bach C. Biochem. Biophys. Res. Commun. 1991; 176: 551-557Crossref PubMed Scopus (76) Google Scholar). Cadmium is a widespread environmental pollutant that is also present in tobacco smoke (1–3 μg/cigarette). Smoking, together with occupation, are the major sources of human exposure. Cadmium is absorbed by inhalation and ingestion and has a very long biological half-life (>25 years). Epidemiological studies have identified lung, prostate, and, to a lesser extent, kidney and stomach as primary targets for cadmium-induced tumorigenesis (21International Agency for Cancer Research IARC Monogr. Eval. Carcinog. Risks Hum. 1993; 58: 119-237PubMed Google Scholar). In exposed industrial workers, cadmium accumulates in the kidneys (100–400 μg/g, wet weight) and liver (20–100 μg/g, wet weight), at levels that are 5–9 times higher than those of unexposed workers (25Ellis K.J. Morgan W.D. Zanzi I. Yasumura S. Vartsky D. Cohn S.H. Am. J. Ind. Med. 1980; 1: 339-348Crossref PubMed Scopus (15) Google Scholar, 26Roels H.A. Lauwerys R.R. Buchet J.P. Bernard A. Chettle D.R. Harvey T.C. Al-Haddad I.K. Environ. Res. 1981; 26: 217-240Crossref PubMed Scopus (137) Google Scholar). The kidneys and liver express high levels of metallothioneins, a class of stress response proteins that bind and detoxify cadmium.The mechanisms of cadmium carcinogenesis are poorly understood.In vitro, at concentrations between 0.1 and 10 mmol, cadmium is cytotoxic and induces radical-dependent DNA damage (27Coogan T.P. Bare R.M. Waalkes M.P. Toxicol. Appl. Pharmacol. 1992; 113: 227-233Crossref PubMed Scopus (112) Google Scholar,28Tsuzuki K. Sugiyama M. Haramaki N. Environ. Health Perspect. 1994; 102 Suppl. 3: 341-342PubMed Google Scholar). However, compared with other carcinogenic metals, cadmium is a weak mutagen (29Rossman T.G. Roy N.K. Lin W.C. IARC Sci. Publ. 1992; 118: 367-375PubMed Google Scholar). At lower concentrations (1–100 μmol), cadmium binds to proteins, decreases DNA repair (30Dally H. Hartwig A. Carcinogenesis. 1997; 18: 1021-1026Crossref PubMed Scopus (243) Google Scholar, 31Nocentini S. Nucleic Acids Res. 1987; 15: 4211-4225Crossref PubMed Scopus (67) Google Scholar), activates protein degradation, up-regulates cytokines and proto-oncogenes such as c-fos, c-jun, and c-myc (32Abshire M.K. Buzard G.S. Shiraishi N. Waalkes M.P. J. Toxicol. Environ. Health. 1996; 48: 359-377Crossref PubMed Scopus (60) Google Scholar, 33Abshire M.K. Devor D.E. Diwan B.A. Shaughnessy J.D.J. Waalkes M.P. J. Biol. Chem. 1996; 17: 1349-1356Google Scholar), and induces the expression of metallothioneins (34Durnam D.M. Palmiter R.D. J. Biol. Chem. 1981; 256: 5712-5716Abstract Full Text PDF PubMed Google Scholar). Thus, cadmium carcinogenicity may involve multiple factors, including up-regulation of mitogenic signals and interference with DNA repair (for a review, see Ref. 19Beyersmann D. Hechtenberg S. Toxicol. Appl. Pharmacol. 1997; 144: 247-261Crossref PubMed Scopus (497) Google Scholar).In this study, we have examined the effects of cadmium on p53 protein conformation, DNA binding, and transcriptional activity. Using the breast carcinoma MCF7 cell line, which expresses high levels of wild-type p53, we show that cadmium at subtoxic concentrations (10–30 μm) perturbs the folding of p53, disrupts DNA binding, impairs p53 induction by DNA-damaging agents, inhibits transactivation of a reporter gene and of target genes such as p21waf-1 , and prevents cell cycle arrest in response to γ-irradiation. Based on these results, we propose that cadmium may inactivate wild-type p53 by altering metal-dependent folding and that this effect may contribute to cadmium carcinogenesis.DISCUSSIONZinc is essential for correct folding of wild-type p53. The DNA-binding domain contains a tetrahedrally coordinated zinc that stabilizes two loops at the DNA-binding surface of the protein (13Cho Y. Gorina S. Jeffrey P.D. Pavletich N.P. Science. 1994; 265: 346-355Crossref PubMed Scopus (2130) Google Scholar). Several in vitro studies have shown that metal chelation abolishes binding of p53 to specific DNA (12Pavletich N.P. Chambers K.A. Pabo C.O. Genes Dev. 1993; 7: 2556-2564Crossref PubMed Scopus (443) Google Scholar, 17Hainaut P. Milner J. Cancer Res. 1993; 53: 1739-1742PubMed Google Scholar, 37Verhaegh G.W. Parat M.O. Richard M.J. Hainaut P. Mol. Carcinog. 1998; 21: 205-214Crossref PubMed Scopus (99) Google Scholar, 48Srinivasan R. Roth J.A. Maxwell S.A. Cancer Res. 1993; 53: 5361-5364PubMed Google Scholar). In addition, metal chelation increases oxidation of cysteines in p53, indicating that zinc binding is not purely structural but also controls the sensitivity of p53 to oxidation-reduction (17Hainaut P. Milner J. Cancer Res. 1993; 53: 1739-1742PubMed Google Scholar, 49Delphin C. Cahen P. Lawrence J.J. Baudier J. Eur. J. Biochem. 1994; 223: 683-692Crossref PubMed Scopus (71) Google Scholar). Reduction of cysteines stimulates p53 DNA-binding (5Rainwater R. Parks D. Anderson M.E. Tegtmeyer P. Mann K. Mol. Cell. Biol. 1995; 15: 3892-3903Crossref PubMed Scopus (273) Google Scholar, 15Hainaut P. Milner J. Cancer Res. 1993; 53: 4469-4473PubMed Google Scholar, 50Hupp T.R. Meek D.W. Midgley C.A. Lane D.P. Nucleic Acids Res. 1993; 21: 3167-3174Crossref PubMed Scopus (185) Google Scholar), and Ref-1, a protein that regulates the redox state of several transcription factors, is a potent activator of p53 (51Jayaraman L. Murthy K.G. Zhu C. Curran T. Xanthoudakis S. Prives C. Genes Dev. 1997; 11: 558-570Crossref PubMed Scopus (442) Google Scholar). In vitro, the conformation and DNA-binding capacity of p53 are altered by incubation with metals chemically close to zinc, such as cadmium and copper, but not with cobalt, magnesium, manganese, or iron (17Hainaut P. Milner J. Cancer Res. 1993; 53: 1739-1742PubMed Google Scholar, 52Coffer A.I. Knowles P.P. Biochim. Biophys. Acta. 1994; 1209: 279-285Crossref PubMed Scopus (19) Google Scholar). These observations have led to the suggestion that specific metals and redox factors may affect the fine tuning of p53 and participate in the physiological control of p53 functions (5Rainwater R. Parks D. Anderson M.E. Tegtmeyer P. Mann K. Mol. Cell. Biol. 1995; 15: 3892-3903Crossref PubMed Scopus (273) Google Scholar, 16Verhaegh G.W. Richard M.J. Hainaut P. Mol. Cell. Biol. 1997; 17: 5699-5706Crossref PubMed Scopus (159) Google Scholar, 51Jayaraman L. Murthy K.G. Zhu C. Curran T. Xanthoudakis S. Prives C. Genes Dev. 1997; 11: 558-570Crossref PubMed Scopus (442) Google Scholar, 53Parks D. Bolinger R. Mann K. Nucleic Acids Res. 1997; 25: 1289-1295Crossref PubMed Scopus (80) Google Scholar).We show here that sequence-specific DNA binding of p53 in vitro is decreased by cadmium in a dose-dependent manner. In MCF7 cells, transient exposure (4 h) to cadmium at 20 μm and above induced a change in p53 conformation (to the unfolded, PAb240+ form) with loss of DNA binding and transcriptional activity. These results are consistent with this idea that cadmium perturbs the folding of p53 in a direct or indirect manner.In the neuroblastoma cell line HT4, cadmium at concentrations up to 100 μm, equal to or greater than those used here, has been shown to induce disruption of intracellular sulfhydryl homostasis and depletion of pools of GSH. These effects were accompanied by an increase in protein thiolation and ubiquitination (40Figueiredo-Pereira M.E. Yakushin S. Cohen G. J. Biol. Chem. 1998; 273: 12703-12709Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Extensive oxidative stress induced by cadmium may perhaps inactivate p53 by oxidation, a phenomenon that has been observed in cultured cells exposed to nitric oxide (14Calmels S. Hainaut P. Ohshima H. Cancer Res. 1997; 57: 3365-3369PubMed Google Scholar), to hydrogen peroxide (53Parks D. Bolinger R. Mann K. Nucleic Acids Res. 1997; 25: 1289-1295Crossref PubMed Scopus (80) Google Scholar), and to glutathione-depleting agents (54Russo T. Zambrano N. Esposito F. Ammendola R. Cimino F. Fiscella M. Jackman J. O'Connor P.M. Anderson C.W. Appella E. J. Biol. Chem. 1995; 270: 29386-29391Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar).On the other hand, cadmium easily substitutes for zinc in several zinc-dependent DNA-binding proteins and inhibits many enzymes containing essential dithiols (55Simons Jr., S.S. Chakraborti P.K. Cavanaugh A.H. J. Biol. Chem. 1990; 265: 1938-1945Abstract Full Text PDF PubMed Google Scholar). For example, with the zinc-inducible transcription factor MTF-1, cadmium at 6 μm partially abrogates activation of DNA binding by the addition of 30 μm zinc and totally inactivates it at 60 μm (56Bittel D. Dalton T. Samson S.L.-A. Gedamu L. Andrews G.K. J. Biol. Chem. 1998; 273: 7127-7133Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). MTF-1 contains six Cys2-His2 zinc fingers, which are thought to bind cadmium with high affinity (56Bittel D. Dalton T. Samson S.L.-A. Gedamu L. Andrews G.K. J. Biol. Chem. 1998; 273: 7127-7133Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Cadmium differs from zinc in that it has a higher affinity for thiols and has a larger atomic radius. The binding affinity of cadmium to cysteine thiolate clusters in zinc finger proteins is 2–3 orders of magnitude higher than that of zinc (22Freedman L.P. Luisi B.F. Korszun Z.R. Basavappa R. Sigler P.B. Yamamoto K.R. Nature. 1988; 334: 543-546Crossref PubMed Scopus (346) Google Scholar, 24Thiesen H.J. Bach C. Biochem. Biophys. Res. Commun. 1991; 176: 551-557Crossref PubMed Scopus (76) Google Scholar, 57Predki P.F. Sarkar B. J. Biol. Chem. 1992; 267: 5842-5846Abstract Full Text PDF PubMed Google Scholar). Consistent with this notion, we found that the effect of cadmium on p53 was not reversed by the addition of excess ZnCl2 (up to 25-fold) to cadmium-treated MCF7 cells (data not shown). Therefore, although our data do not provide a formal proof that cadmium can displace zinc from native p53, our results are consistent with the idea that cadmium perturbs the metal-dependent folding of the DNA-binding domain.In MCF7 cells, cadmium exerts complex, biphasic effects on p53 protein levels and DNA binding activity. At low concentrations (up to 10 μm), cadmium alone induces a small (2–3-fold) but reproducible accumulation of p53 protein, correlated with slightly enhanced DNA binding activity (2.28 ± 0.26-fold). This effect may be due to p53 protein stabilization by low levels of oxidative DNA-damage induced by cadmium. However, increasing the concentration of cadmium does not result in higher levels of p53 protein activation. In contrast, it significantly decreases p53 DNA binding activity (at 20 μm) and protein levels (at 40 μm). Furthermore, inhibition of p53 activity correlates with a change in protein conformation, with loss of PAb1620 reactivity (wild type-specific) and acquisition of the PAb240-positive phenotype (Fig.3 C). Along with the observation that cadmium does not prevent p53 localization in the nucleus (Fig. 3 B), these data indicate that cadmium inhibits p53 by turning it into an inactive, "mutant-like," form.Cadmium at 30 μm induces total inhibition of p53 protein activation in response to DNA-damaging agents such as Act D, methylmethane sulfonate, or H2O2. This inhibition resulted in a loss of transcriptional activation of several p53 target genes including p21waf-1 . Moreover, cadmium at noncytotoxic concentrations (10 μm) is sufficient to significantly reduce (by about 40%) the extent of p53 induction by DNA-damaging agents and therefore to perturb the response of p53 to DNA damage.The apparent contradiction between the effects of cadmium at 10 and 30 μm may be resolved by considering that cadmium has two opposite effects on p53, with first protein stabilization as a result of generation of DNA damage by low doses of cadmium and, second, direct inhibition of p53 protein by metal substitution and conformational modifications at higher doses of cadmium. The level of p53 DNA binding activity detected in the presence of cadmium would thus depend upon a subtle balance between these two mechanisms.Inhibition of p53 DNA binding activity by cadmium has important functional consequences in cultured cells. First, cadmium reduces p53-dependent transactivation of reporter or endogenous target genes. Second, cadmium prevents the cell cycle arrest induced by low doses of γ-irradiation in MCF7 cells, suggesting that cadmium can effectively suppress p53 protein function. Cells exposed to cadmium thus behave in a manner analogous to p53-deficient cells that retain the capacity to proliferate after exposure to DNA-damaging agents. A similar hypothesis has been proposed in the case of excess production of nitric oxide, which also induces conformational and functional changes in wild-type p53 (14Calmels S. Hainaut P. Ohshima H. Cancer Res. 1997; 57: 3365-3369PubMed Google Scholar). Impairment of p53 function by cadmium may contribute to decrease the cell capacity to respond to the DNA damage induced by other carcinogens, thereby increasing the likelihood of acquiring mutations leading to cancer.Cadmium is highly toxic in most biological systems and has a very long biological half-life (about 25 years in humans (20Waalkes M.P. Coogan T.P. Barter R.A. Crit. Rev. Toxicol. 1992; 22: 175-201Crossref PubMed Scopus (272) Google Scholar). Therefore, it is essential to consider whether the concentrations of cadmium used in our experiments are compatible with those that occur in target cells of exposed organisms. After exposure to cadmium, most of the intracellular pool of cadmium is bound to MTs, a class of inducible, metal-binding proteins that sequester cadmium and protect cells from its toxic effect. However, experiments with MT-I and -II knockout mice showed that cadmium also accumulates to high levels in the absence of MT. In MT-deficient mice, CdCl2 injected subcutaneously at 30 μg/kg accumulates in liver cells within 3–6 h at up to 20–25 μg/g of fresh tissue (58Zheng H. Liu J. Choo K.H. Michalska A.E. Klaassen C.D. Toxicol. Appl. Pharmacol. 1996; 136: 229-235Crossref PubMed Scopus (79) Google Scholar). These levels may correspond to intracellular concentrations 3–10-fold higher than those used in the present study. Although this dose of cadmium was toxic in MT-deficient mice, it produced only mild hepatotoxicity in control mice. Concentrations of cadmium of up to 25 μm are well tolerated in many cultured cell lines (30Dally H. Hartwig A. Carcinogenesis. 1997; 18: 1021-1026Crossref PubMed Scopus (243) Google Scholar). Therefore, the effects reported here are compatible with concentrations of cadmium that are not lethal and can occur in biological systems after acute or chronic exposure.Alteration of p53 protein conformation by cadmium was described previously, using in vitro translated p53, with concentrations of CdCl2 of 50–100 μm (17Hainaut P. Milner J. Cancer Res. 1993; 53: 1739-1742PubMed Google Scholar). In our study, we show that much lower concentrations of cadmium (10–30 μm) are able to alter p53 conformation and function in intact cells. This is the first report that a metal compound can inactivate p53 at doses compatible with biological effects.In 1995, Zheng et al. (58Zheng H. Liu J. Choo K.H. Michalska A.E. Klaassen C.D. Toxicol. Appl. Pharmacol. 1996; 136: 229-235Crossref PubMed Scopus (79) Google Scholar) reported that cadmium could increase p53 mRNA levels in liver cells of mice injected with CdCl2. We did not observe such an effect in cultured MCF7 cells. It is important to note that induction of p53 mRNA was observed as a late event (after 6–12 h) in mice receiving a high, hepatotoxic dose of cadmium. Therefore, it is possible that elevated p53 mRNA may represent a response to cell damage rather than a direct effect of cadmium on p53 gene expression.The mechanism of p53 inactivation described here may account for some of the unexplained properties of cadmium as a carcinogen. Indeed, cadmium is a weak genotoxic agent compared with metals such as copper, iron, nickel, and chromium. Therefore, mechanisms other than direct genotoxicity have been proposed to explain cadmium carcinogenesis (59Reid T.M. Feig D.I. Loeb L.A. Environ. Health Perspect. 1994; 102 Suppl. 3: 57-61PubMed Google Scholar). Exposure to cadmium enhances the persistence of DNA lesions induced by mutagens such as benzo(a)pyrene and methylmethane sulfonate in human cells, suggesting that cadmium may inhibit DNA repair. Recently, Dally and Hartwig (30Dally H. Hartwig A. Carcinogenesis. 1997; 18: 1021-1026Crossref PubMed Scopus (243) Google Scholar) have shown that cadmium, as well as nickel, inhibits the repair of DNA damage after irradiation. These authors propose that cadmium may either inactivate repair enzymes directly, for example by reaction with a histidine or cysteine residue, or compete with and displace essential metal ions, a hypothesis compatible with the results presented here. Inhibition of p53 function may explain the persistence of DNA lesions in cells exposed to both carcinogens and cadmium. According to this model, cadmium would not act as a conventional mutagen but rather as an indirect carcinogen that sensitizes cells to the genotoxic effects of other carcinogens by switching off essential components of cell cycle control and DNA repair pathways involving p53.Cadmium exerts complex effects on the growth and survival of normal and cancer cells. The metal was shown to induce apoptosis or necrosis in some cells and tissues and to reduce the growth and metastasis of human lung carcinoma xenograft in nude mice (60Prise K.M. Gillies N.E. Whelan A. Newton G.L. Fahey R.C. Michael B.D. Int. J. Radiat. Biol. 1995; 67: 393-401Crossref PubMed Scopus (22) Google Scholar). In contrast, cadmium was shown to inhibit apoptosis induced by DNA-damaging metals such as chromium (61Shimada H. Shiao Y.H. Shibata M. Waalkes M.P. J. Toxicol. Environ. Health. 1998; 54: 159-168Crossref PubMed Scopus (73) Google Scholar). These observations suggest that the sensitivity to cadmium may vary from one cell type to another and that some cancer cells may be hypersensitive to cadmium. The cytotoxic impact of cadmium may be related to the cellular level of metallothioneins, which is frequently deregulated in cancer cells (62Waalkes M.P. Diwan B.A. Carcinogenesis. 1999; 20: 65-70Crossref PubMed Scopus (36) Google Scholar).It would be naive to suggest that effects on p53 alone can explain all of the cadmium carcinogenicity. Indeed, it is likely that cadmium substitutes for zinc and alters the function of a number of other cellular proteins. For example, Cd2+ (as well as a number of other metal ions) has been shown to alter the nucleotide selectivity of human DNA polymerase β in vitro (63Pelletier H. Sawaya M.R. Wolfle W. Wilson S.H. Kraut J. Biochemistry. 1996; 35: 12762-12777Crossref PubMed Scopus (176) Google Scholar). In addition, factors such as competition between metals and interactions with metallothioneins should also be considered. However, we believe that our observations represent a important step in the understanding of the carcinogenic potential of cadmium. Moreover, these observations also provide a model system for determining how essential metals such as zinc or metal chelators may be used in preventive approaches to reduce cadmium carcinogenesis. The p53 protein is a tumor-suppressive transcription factor activated in response to multiple signals including radiation, genotoxic chemicals, hypoxia, depletion of ribonucleotides, and poisoning of the mitotic spindle. In most normal, nonexposed cells, p53 is a latent factor. Induction in response to stress involves nuclear accumulation (as a result of escape from mdm-2-mediated degradation and nuclear export) and conversion to an active form with high affinity for specific DNA sequences. Activation requires post-translational modifications at both the N and C terminus of the protein, including changes in phosphorylation, acetylation, and binding to heterologous proteins (1Liu L. Scolnick D.M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell. Biol. 1999; 19: 1202-1209Crossref PubMed Scopus (648) Google Scholar, 2Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1013) Google Scholar, 3Waterman M.J. Stavridi E.S. Waterman J.L. Halazonetis T.D. Nat. Genet. 1998; 19: 175-178Crossref PubMed Scopus (402) Google Scholar, 4Meek D.W. Cell Signal. 1998; 10: 159-166Crossref PubMed Scopus (177) Google Scholar, 5Rainwater R. Parks D. Anderson M.E. Tegtmeyer P. Mann K. Mol. Cell. Biol. 1995; 15: 3892-3903Crossref PubMed Scopus (273) Google Scholar). Activated p53 controls several sets of genes to prevent the proliferation of cells under stress conditions. Genes transactivated by p53 include inhibitors of cell cycle progression in G1 and G2 (p21waf-1 ,14-3-3ς, GADD 45), regulators of apoptosis (APO1-Fas/CD95, Bax-1, KILLER/DR5), and genes involved in the metabolism of reactive oxygen species (such as PIG-3, PIG-6, and PIG-12) that may play a role in induction of apoptosis (6Polyak K. Waldman T. He T.C. Kinzler K.W. Vogelstein B. Genes Dev.

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