Abstract
Mutations in the p53 tumor suppressor gene frequently fall within the specific DNA-binding domain and prevent the molecule from transactivating normal targets. DNA-binding activity is regulated in vitro by metal ions and by redox conditions, but whether these factors also regulate p53 in vivo is unclear. To address this question, we have analyzed the effect of pyrrolidine dithiocarbamate (PDTC) on p53 DNA-binding activity in cell lines expressing wild-type p53. PDTC is commonly regarded as an antioxidant, but it can also bind and transport external copper ions into cells and thus exert either pro- or antioxidant effects in different situations. We report that PDTC, but not N-acetyl-L-cysteine, down-regulated the specific DNA-binding activity of p53. Loss of DNA binding correlated with disruption of the immunologically "wild-type" p53 conformation. Using different chelators to interfere with copper transport by PDTC, we found that bathocuproinedisulfonic acid (BCS), a non-cell-permeable chelator of Cu1+, prevented both copper import and p53 down-regulation. In contrast, 1,10-orthophenanthroline, a cell-permeable chelator of Cu2+, promoted the redox activity of copper and up-regulated p53 DNA-binding activity through a DNA damage-dependent pathway. We have previously reported that p53 protein binds copper in vitro in the form of Cu1+ (P. Hainaut, N. Rolley, M. Davies, and J. Milner, Oncogene 10:27-32, 1995). The data reported here indicate that intracellular levels and redox activity of copper are critical for p53 protein conformation and DNA-binding activity and suggest that copper ions may participate in the physiological control of p53 function.
Full Text
The Full Text of this article is available as a PDF (1.1 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Artuso M., Esteve A., Brésil H., Vuillaume M., Hall J. The role of the Ataxia telangiectasia gene in the p53, WAF1/CIP1(p21)- and GADD45-mediated response to DNA damage produced by ionising radiation. Oncogene. 1995 Oct 19;11(8):1427–1435. [PubMed] [Google Scholar]
- Becker T. W., Krieger G., Witte I. DNA single and double strand breaks induced by aliphatic and aromatic aldehydes in combination with copper (II). Free Radic Res. 1996 May;24(5):325–332. doi: 10.3109/10715769609088030. [DOI] [PubMed] [Google Scholar]
- Berg J. M., Shi Y. The galvanization of biology: a growing appreciation for the roles of zinc. Science. 1996 Feb 23;271(5252):1081–1085. doi: 10.1126/science.271.5252.1081. [DOI] [PubMed] [Google Scholar]
- Bessho R., Matsubara K., Kubota M., Kuwakado K., Hirota H., Wakazono Y., Lin Y. W., Okuda A., Kawai M., Nishikomori R. Pyrrolidine dithiocarbamate, a potent inhibitor of nuclear factor kappa B (NF-kappa B) activation, prevents apoptosis in human promyelocytic leukemia HL-60 cells and thymocytes. Biochem Pharmacol. 1994 Nov 16;48(10):1883–1889. doi: 10.1016/0006-2952(94)90586-x. [DOI] [PubMed] [Google Scholar]
- Bhunya S. P., Jena G. B. Clastogenic effects of copper sulphate in chick in vivo test system. Mutat Res. 1996 Feb;367(2):57–63. doi: 10.1016/0165-1218(95)00061-5. [DOI] [PubMed] [Google Scholar]
- Burkitt M. J., Milne L., Nicotera P., Orrenius S. 1,10-Phenanthroline stimulates internucleosomal DNA fragmentation in isolated rat-liver nuclei by promoting the redox activity of endogenous copper ions. Biochem J. 1996 Jan 1;313(Pt 1):163–169. doi: 10.1042/bj3130163. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cho Y., Gorina S., Jeffrey P. D., Pavletich N. P. Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science. 1994 Jul 15;265(5170):346–355. doi: 10.1126/science.8023157. [DOI] [PubMed] [Google Scholar]
- Delmolino L., Band H., Band V. Expression and stability of p53 protein in normal human mammary epithelial cells. Carcinogenesis. 1993 May;14(5):827–832. doi: 10.1093/carcin/14.5.827. [DOI] [PubMed] [Google Scholar]
- Ebadi M., Iversen P. L. Metallothionein in carcinogenesis and cancer chemotherapy. Gen Pharmacol. 1994 Nov;25(7):1297–1310. doi: 10.1016/0306-3623(94)90152-x. [DOI] [PubMed] [Google Scholar]
- Funk W. D., Pak D. T., Karas R. H., Wright W. E., Shay J. W. A transcriptionally active DNA-binding site for human p53 protein complexes. Mol Cell Biol. 1992 Jun;12(6):2866–2871. doi: 10.1128/mcb.12.6.2866. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gottlieb T. M., Oren M. p53 in growth control and neoplasia. Biochim Biophys Acta. 1996 Jun 7;1287(2-3):77–102. doi: 10.1016/0304-419x(95)00019-c. [DOI] [PubMed] [Google Scholar]
- Guillot C., Falette N., Paperin M. P., Courtois S., Gentil-Perret A., Treilleux I., Ozturk M., Puisieux A. p21(WAF1/CIP1) response to genotoxic agents in wild-type TP53 expressing breast primary tumours. Oncogene. 1997 Jan 9;14(1):45–52. doi: 10.1038/sj.onc.1200803. [DOI] [PubMed] [Google Scholar]
- Hainaut P., Milner J. A structural role for metal ions in the "wild-type" conformation of the tumor suppressor protein p53. Cancer Res. 1993 Apr 15;53(8):1739–1742. [PubMed] [Google Scholar]
- Hainaut P., Milner J. Interaction of heat-shock protein 70 with p53 translated in vitro: evidence for interaction with dimeric p53 and for a role in the regulation of p53 conformation. EMBO J. 1992 Oct;11(10):3513–3520. doi: 10.1002/j.1460-2075.1992.tb05434.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hainaut P., Milner J. Redox modulation of p53 conformation and sequence-specific DNA binding in vitro. Cancer Res. 1993 Oct 1;53(19):4469–4473. [PubMed] [Google Scholar]
- Hainaut P., Rolley N., Davies M., Milner J. Modulation by copper of p53 conformation and sequence-specific DNA binding: role for Cu(II)/Cu(I) redox mechanism. Oncogene. 1995 Jan 5;10(1):27–32. [PubMed] [Google Scholar]
- Halazonetis T. D., Davis L. J., Kandil A. N. Wild-type p53 adopts a 'mutant'-like conformation when bound to DNA. EMBO J. 1993 Mar;12(3):1021–1028. doi: 10.1002/j.1460-2075.1993.tb05743.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hansen S., Hupp T. R., Lane D. P. Allosteric regulation of the thermostability and DNA binding activity of human p53 by specific interacting proteins. CRC Cell Transformation Group. J Biol Chem. 1996 Feb 16;271(7):3917–3924. doi: 10.1074/jbc.271.7.3917. [DOI] [PubMed] [Google Scholar]
- Hupp T. R., Lane D. P. Two distinct signaling pathways activate the latent DNA binding function of p53 in a casein kinase II-independent manner. J Biol Chem. 1995 Jul 28;270(30):18165–18174. doi: 10.1074/jbc.270.30.18165. [DOI] [PubMed] [Google Scholar]
- Hupp T. R., Meek D. W., Midgley C. A., Lane D. P. Activation of the cryptic DNA binding function of mutant forms of p53. Nucleic Acids Res. 1993 Jul 11;21(14):3167–3174. doi: 10.1093/nar/21.14.3167. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hupp T. R., Meek D. W., Midgley C. A., Lane D. P. Regulation of the specific DNA binding function of p53. Cell. 1992 Nov 27;71(5):875–886. doi: 10.1016/0092-8674(92)90562-q. [DOI] [PubMed] [Google Scholar]
- Juven T., Barak Y., Zauberman A., George D. L., Oren M. Wild type p53 can mediate sequence-specific transactivation of an internal promoter within the mdm2 gene. Oncogene. 1993 Dec;8(12):3411–3416. [PubMed] [Google Scholar]
- Kastan M. B., Zhan Q., el-Deiry W. S., Carrier F., Jacks T., Walsh W. V., Plunkett B. S., Vogelstein B., Fornace A. J., Jr A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell. 1992 Nov 13;71(4):587–597. doi: 10.1016/0092-8674(92)90593-2. [DOI] [PubMed] [Google Scholar]
- Li Y., Trush M. A. DNA damage resulting from the oxidation of hydroquinone by copper: role for a Cu(II)/Cu(I) redox cycle and reactive oxygen generation. Carcinogenesis. 1993 Jul;14(7):1303–1311. doi: 10.1093/carcin/14.7.1303. [DOI] [PubMed] [Google Scholar]
- Margalioth E. J., Schenker J. G., Chevion M. Copper and zinc levels in normal and malignant tissues. Cancer. 1983 Sep 1;52(5):868–872. doi: 10.1002/1097-0142(19830901)52:5<868::aid-cncr2820520521>3.0.co;2-k. [DOI] [PubMed] [Google Scholar]
- Meyer M., Schreck R., Baeuerle P. A. H2O2 and antioxidants have opposite effects on activation of NF-kappa B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J. 1993 May;12(5):2005–2015. doi: 10.1002/j.1460-2075.1993.tb05850.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Milner J. Flexibility: the key to p53 function? Trends Biochem Sci. 1995 Feb;20(2):49–51. doi: 10.1016/s0968-0004(00)88954-9. [DOI] [PubMed] [Google Scholar]
- Miyashita T., Krajewski S., Krajewska M., Wang H. G., Lin H. K., Liebermann D. A., Hoffman B., Reed J. C. Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene. 1994 Jun;9(6):1799–1805. [PubMed] [Google Scholar]
- Monaco A. P., Chelly J. Menkes and Wilson diseases. Adv Genet. 1995;33:233–253. doi: 10.1016/s0065-2660(08)60335-1. [DOI] [PubMed] [Google Scholar]
- Müller M. M., Ruppert S., Schaffner W., Matthias P. A cloned octamer transcription factor stimulates transcription from lymphoid-specific promoters in non-B cells. Nature. 1988 Dec 8;336(6199):544–551. doi: 10.1038/336544a0. [DOI] [PubMed] [Google Scholar]
- Nagel W. W., Vallee B. L. Cell cycle regulation of metallothionein in human colonic cancer cells. Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):579–583. doi: 10.1073/pnas.92.2.579. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nobel C. I., Kimland M., Lind B., Orrenius S., Slater A. F. Dithiocarbamates induce apoptosis in thymocytes by raising the intracellular level of redox-active copper. J Biol Chem. 1995 Nov 3;270(44):26202–26208. doi: 10.1074/jbc.270.44.26202. [DOI] [PubMed] [Google Scholar]
- O'Halloran T. V. Transition metals in control of gene expression. Science. 1993 Aug 6;261(5122):715–725. doi: 10.1126/science.8342038. [DOI] [PubMed] [Google Scholar]
- Okamoto K., Beach D. Cyclin G is a transcriptional target of the p53 tumor suppressor protein. EMBO J. 1994 Oct 17;13(20):4816–4822. doi: 10.1002/j.1460-2075.1994.tb06807.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Parat M. O., Richard M. J., Pollet S., Hadjur C., Favier A., Béani J. C. Zinc and DNA fragmentation in keratinocyte apoptosis: its inhibitory effect in UVB irradiated cells. J Photochem Photobiol B. 1997 Jan;37(1-2):101–106. doi: 10.1016/s1011-1344(96)07334-4. [DOI] [PubMed] [Google Scholar]
- Rainwater R., Parks D., Anderson M. E., Tegtmeyer P., Mann K. Role of cysteine residues in regulation of p53 function. Mol Cell Biol. 1995 Jul;15(7):3892–3903. doi: 10.1128/mcb.15.7.3892. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Remacle J., Raes M., Toussaint O., Renard P., Rao G. Low levels of reactive oxygen species as modulators of cell function. Mutat Res. 1995 Feb;316(3):103–122. doi: 10.1016/0921-8734(95)90004-7. [DOI] [PubMed] [Google Scholar]
- Schenk H., Klein M., Erdbrügger W., Dröge W., Schulze-Osthoff K. Distinct effects of thioredoxin and antioxidants on the activation of transcription factors NF-kappa B and AP-1. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1672–1676. doi: 10.1073/pnas.91.5.1672. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schulze-Osthoff K., Los M., Baeuerle P. A. Redox signalling by transcription factors NF-kappa B and AP-1 in lymphocytes. Biochem Pharmacol. 1995 Sep 7;50(6):735–741. doi: 10.1016/0006-2952(95)02011-z. [DOI] [PubMed] [Google Scholar]
- Stephen C. W., Lane D. P. Mutant conformation of p53. Precise epitope mapping using a filamentous phage epitope library. J Mol Biol. 1992 Jun 5;225(3):577–583. doi: 10.1016/0022-2836(92)90386-x. [DOI] [PubMed] [Google Scholar]
- Toledano M. B., Leonard W. J. Modulation of transcription factor NF-kappa B binding activity by oxidation-reduction in vitro. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4328–4332. doi: 10.1073/pnas.88.10.4328. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Verhaegen S., McGowan A. J., Brophy A. R., Fernandes R. S., Cotter T. G. Inhibition of apoptosis by antioxidants in the human HL-60 leukemia cell line. Biochem Pharmacol. 1995 Sep 28;50(7):1021–1029. doi: 10.1016/0006-2952(95)00233-p. [DOI] [PubMed] [Google Scholar]
- Wu J., Forbes J. R., Chen H. S., Cox D. W. The LEC rat has a deletion in the copper transporting ATPase gene homologous to the Wilson disease gene. Nat Genet. 1994 Aug;7(4):541–545. doi: 10.1038/ng0894-541. [DOI] [PubMed] [Google Scholar]
- Xanthoudakis S., Miao G., Wang F., Pan Y. C., Curran T. Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme. EMBO J. 1992 Sep;11(9):3323–3335. doi: 10.1002/j.1460-2075.1992.tb05411.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamamoto K., Kawanishi S. Site-specific DNA damage induced by hydrazine in the presence of manganese and copper ions. The role of hydroxyl radical and hydrogen atom. J Biol Chem. 1991 Jan 25;266(3):1509–1515. [PubMed] [Google Scholar]
- el-Deiry W. S., Harper J. W., O'Connor P. M., Velculescu V. E., Canman C. E., Jackman J., Pietenpol J. A., Burrell M., Hill D. E., Wang Y. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res. 1994 Mar 1;54(5):1169–1174. [PubMed] [Google Scholar]
- el-Deiry W. S., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817–825. doi: 10.1016/0092-8674(93)90500-p. [DOI] [PubMed] [Google Scholar]