Abstract
The cell-cycle checkpoint protein p53 both directs terminal differentiation and protects embryos from DNA damage. To study invertebrate p53 during early development, we identified three differentially expressed p53 family members (p53, p97, p120) in the surf clam, Spisula solidissima. In these mollusks, p53 and p97 occur in both embryonic and adult tissue, whereas p120 is exclusively embryonic. We sequenced, cloned, and characterized p120 cDNA. The predicted protein, p120, resembles p53 across all evolutionarily conserved regions and contains a C-terminal extension with a sterile alpha motif (SAM) as in p63 and p73. These vertebrate forms of p53 are required for normal inflammatory, epithelial, and neuronal development. Unlike clam p53 and p97, p120 mRNA and protein levels are temporally expressed in embryos, with mRNA levels decreasing with increasing p120 protein (R(2) = 0.97). Highest surf clam p120 mRNA levels coincide with the onset of neuronal growth. In earlier work we have shown that neuronal development is altered by exposure to polychlorinated biphenyls (PCBs), a neurotoxic environmental contaminant. In this study we show that PCBs differentially affect expression of the three surf clam p53 family members. p120 mRNA and protein are reduced the most and earliest in development, p97 protein shows a smaller and later reduction, and p53 protein levels do not change. For the first time we report that unlike p53 and p97, p120 is specifically embryonic and expressed in a time-dependent manner. Furthermore, p120 responds to PCBs by 48 hr when PCB-induced suppression of the serotonergic nervous system occurs.
Full Text
The Full Text of this article is available as a PDF (1.6 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Angus W. G., Contreras M. L. Aroclor 1254 alters the binding of 125I-labeled nerve growth factor in PC12 cells. Neurosci Lett. 1995 May 19;191(1-2):23–26. doi: 10.1016/0304-3940(94)11547-x. [DOI] [PubMed] [Google Scholar]
- Barker C. M., Calvert R. J., Walker C. W., Reinisch C. L. Detection of mutant p53 in clam leukemia cells. Exp Cell Res. 1997 May 1;232(2):240–245. doi: 10.1006/excr.1997.3513. [DOI] [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]
- Choi J., Donehower L. A. p53 in embryonic development: maintaining a fine balance. Cell Mol Life Sci. 1999 Jan;55(1):38–47. doi: 10.1007/s000180050268. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dunn S. D. Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Anal Biochem. 1986 Aug 15;157(1):144–153. doi: 10.1016/0003-2697(86)90207-1. [DOI] [PubMed] [Google Scholar]
- Freedman D. A., Wu L., Levine A. J. Functions of the MDM2 oncoprotein. Cell Mol Life Sci. 1999 Jan;55(1):96–107. doi: 10.1007/s000180050273. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harlow P., Nemer M. Coordinate and selective beta-tubulin gene expression associated with cilium formation in sea urchin embryos. Genes Dev. 1987 Dec;1(10):1293–1304. doi: 10.1101/gad.1.10.1293. [DOI] [PubMed] [Google Scholar]
- Harper D. M., Flessas D. A., Reinisch C. L. Specific reactivity of leukemia cells to polyclonal anti-PCB antibodies. J Invertebr Pathol. 1994 Nov;64(3):234–237. doi: 10.1016/s0022-2011(94)90300-x. [DOI] [PubMed] [Google Scholar]
- Hay B. A., Wassarman D. A., Rubin G. M. Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell. 1995 Dec 29;83(7):1253–1262. doi: 10.1016/0092-8674(95)90150-7. [DOI] [PubMed] [Google Scholar]
- Jacobson J. L., Jacobson S. W. Intellectual impairment in children exposed to polychlorinated biphenyls in utero. N Engl J Med. 1996 Sep 12;335(11):783–789. doi: 10.1056/NEJM199609123351104. [DOI] [PubMed] [Google Scholar]
- Jessen-Eller K., Picozza E., Crivello J. F. Quantitation of metallothionein mRNA by RT-PCR and chemiluminescence. Biotechniques. 1994 Nov;17(5):962–973. [PubMed] [Google Scholar]
- Jessen-Eller K., Steele M., Reinisch C., Spitzer N. Blockade of ryanodine receptors stimulates neurite outgrowth in embryos of Spisula solidissima. Biol Bull. 1998 Oct;195(2):206–207. doi: 10.2307/1542841. [DOI] [PubMed] [Google Scholar]
- Jin S., Martinek S., Joo W. S., Wortman J. R., Mirkovic N., Sali A., Yandell M. D., Pavletich N. P., Young M. W., Levine A. J. Identification and characterization of a p53 homologue in Drosophila melanogaster. Proc Natl Acad Sci U S A. 2000 Jun 20;97(13):7301–7306. doi: 10.1073/pnas.97.13.7301. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kaelin W. G., Jr The p53 gene family. Oncogene. 1999 Dec 13;18(53):7701–7705. doi: 10.1038/sj.onc.1202955. [DOI] [PubMed] [Google Scholar]
- Kelley M. L., Winge P., Heaney J. D., Stephens R. E., Farell J. H., Van Beneden R. J., Reinisch C. L., Lesser M. P., Walker C. W. Expression of homologues for p53 and p73 in the softshell clam (Mya arenaria), a naturally-occurring model for human cancer. Oncogene. 2001 Feb 8;20(6):748–758. doi: 10.1038/sj.onc.1204144. [DOI] [PubMed] [Google Scholar]
- Komarova E. A., Chernov M. V., Franks R., Wang K., Armin G., Zelnick C. R., Chin D. M., Bacus S. S., Stark G. R., Gudkov A. V. Transgenic mice with p53-responsive lacZ: p53 activity varies dramatically during normal development and determines radiation and drug sensitivity in vivo. EMBO J. 1997 Mar 17;16(6):1391–1400. doi: 10.1093/emboj/16.6.1391. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kreiling J. A., Jessen-Eller K., Miller J., Seegal R. F., Reinisch C. L. Early development of the serotonergic and dopaminergic nervous system in Spisula solidissima (surf clam) larvae. Comp Biochem Physiol A Mol Integr Physiol. 2001 Sep;130(2):341–351. doi: 10.1016/s1095-6433(01)00394-4. [DOI] [PubMed] [Google Scholar]
- Kreiling J. A., Stephens R. E., Kuzirian A. M., Jessen-Eller K., Reinisch C. L. Polychlorinated biphenyls are selectively neurotoxic in the developing Spisula solidissima embryo. J Toxicol Environ Health A. 2000 Dec 29;61(8):657–675. doi: 10.1080/00984100050195143. [DOI] [PubMed] [Google Scholar]
- Kussie P. H., Gorina S., Marechal V., Elenbaas B., Moreau J., Levine A. J., Pavletich N. P. Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science. 1996 Nov 8;274(5289):948–953. doi: 10.1126/science.274.5289.948. [DOI] [PubMed] [Google Scholar]
- Lesser M. P., Farrell J. H., Walker C. W. Oxidative stress, DNA damage and p53 expression in the larvae of atlantic cod (Gadus morhua) exposed to ultraviolet (290-400 nm) radiation. J Exp Biol. 2001 Jan;204(Pt 1):157–164. doi: 10.1242/jeb.204.1.157. [DOI] [PubMed] [Google Scholar]
- Levine A. J. p53, the cellular gatekeeper for growth and division. Cell. 1997 Feb 7;88(3):323–331. doi: 10.1016/s0092-8674(00)81871-1. [DOI] [PubMed] [Google Scholar]
- Levrero M., De Laurenzi V., Costanzo A., Gong J., Wang J. Y., Melino G. The p53/p63/p73 family of transcription factors: overlapping and distinct functions. J Cell Sci. 2000 May;113(Pt 10):1661–1670. doi: 10.1242/jcs.113.10.1661. [DOI] [PubMed] [Google Scholar]
- Longo F. J., Mathews L., Palazzo R. E. Sperm nuclear transformations in cytoplasmic extracts from surf clam (Spisula solidissima) oocytes. Dev Biol. 1994 Mar;162(1):245–258. doi: 10.1006/dbio.1994.1082. [DOI] [PubMed] [Google Scholar]
- Moreno H., Nadal M., Leznik E., Sugimori M., Lax I., Schlessinger J., Llinás R. Nerve growth factor acutely reduces chemical transmission by means of postsynaptic TrkA-like receptors in squid giant synapse. Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14997–15002. doi: 10.1073/pnas.95.25.14997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nigro J. M., Baker S. J., Preisinger A. C., Jessup J. M., Hostetter R., Cleary K., Bigner S. H., Davidson N., Baylin S., Devilee P. Mutations in the p53 gene occur in diverse human tumour types. Nature. 1989 Dec 7;342(6250):705–708. doi: 10.1038/342705a0. [DOI] [PubMed] [Google Scholar]
- Norrander J. M., Linck R. W., Stephens R. E. Transcriptional control of tektin A mRNA correlates with cilia development and length determination during sea urchin embryogenesis. Development. 1995 Jun;121(6):1615–1623. doi: 10.1242/dev.121.6.1615. [DOI] [PubMed] [Google Scholar]
- Oakley G. G., Devanaboyina U., Robertson L. W., Gupta R. C. Oxidative DNA damage induced by activation of polychlorinated biphenyls (PCBs): implications for PCB-induced oxidative stress in breast cancer. Chem Res Toxicol. 1996 Dec;9(8):1285–1292. doi: 10.1021/tx960103o. [DOI] [PubMed] [Google Scholar]
- Pozniak C. D., Radinovic S., Yang A., McKeon F., Kaplan D. R., Miller F. D. An anti-apoptotic role for the p53 family member, p73, during developmental neuron death. Science. 2000 Jul 14;289(5477):304–306. doi: 10.1126/science.289.5477.304. [DOI] [PubMed] [Google Scholar]
- Rosenthal E. T., Hunt T., Ruderman J. V. Selective translation of mRNA controls the pattern of protein synthesis during early development of the surf clam, Spisula solidissima. Cell. 1980 Jun;20(2):487–494. doi: 10.1016/0092-8674(80)90635-2. [DOI] [PubMed] [Google Scholar]
- Rosenthal E. T., Ruderman J. V. Widespread changes in the translation and adenylation of maternal messenger RNAs following fertilization of Spisula oocytes. Dev Biol. 1987 May;121(1):237–246. doi: 10.1016/0012-1606(87)90155-2. [DOI] [PubMed] [Google Scholar]
- Safe S. H. Polychlorinated biphenyls (PCBs): environmental impact, biochemical and toxic responses, and implications for risk assessment. Crit Rev Toxicol. 1994;24(2):87–149. doi: 10.3109/10408449409049308. [DOI] [PubMed] [Google Scholar]
- Schultz J., Ponting C. P., Hofmann K., Bork P. SAM as a protein interaction domain involved in developmental regulation. Protein Sci. 1997 Jan;6(1):249–253. doi: 10.1002/pro.5560060128. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Seegal R. F. Epidemiological and laboratory evidence of PCB-induced neurotoxicity. Crit Rev Toxicol. 1996 Nov;26(6):709–737. doi: 10.3109/10408449609037481. [DOI] [PubMed] [Google Scholar]
- Smith C. R., Barker C. M., Barker L. F., Jessen-Eller K., Reinisch C. L. Polychlorinated biphenyls (PCBs) selectively disrupt serotonergic cell growth in the developing Spisula embryo. Toxicol Sci. 1999 Jul;50(1):54–63. doi: 10.1093/toxsci/50.1.54. [DOI] [PubMed] [Google Scholar]
- Stephens R. E., Walker C. W., Reinisch C. L. Multiple protein differences distinguish clam leukemia cells from normal hemocytes: evidence for the involvement of p53 homologues. Comp Biochem Physiol C Toxicol Pharmacol. 2001 Aug;129(4):329–338. doi: 10.1016/s1532-0456(01)00208-3. [DOI] [PubMed] [Google Scholar]
- Strano S., Rossi M., Fontemaggi G., Munarriz E., Soddu S., Sacchi A., Blandino G. From p63 to p53 across p73. FEBS Lett. 2001 Feb 16;490(3):163–170. doi: 10.1016/s0014-5793(01)02119-6. [DOI] [PubMed] [Google Scholar]
- Tchang F., Gusse M., Soussi T., Méchali M. Stabilization and expression of high levels of p53 during early development in Xenopus laevis. Dev Biol. 1993 Sep;159(1):163–172. doi: 10.1006/dbio.1993.1230. [DOI] [PubMed] [Google Scholar]
- Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tilson H. A., Kodavanti P. R. The neurotoxicity of polychlorinated biphenyls. Neurotoxicology. 1998 Aug-Oct;19(4-5):517–525. [PubMed] [Google Scholar]
- Tschan M. P., Grob T. J., Peters U. R., Laurenzi V. D., Huegli B., Kreuzer K. A., Schmidt C. A., Melino G., Fey M. F., Tobler A. Enhanced p73 expression during differentiation and complex p73 isoforms in myeloid leukemia. Biochem Biophys Res Commun. 2000 Oct 14;277(1):62–65. doi: 10.1006/bbrc.2000.3627. [DOI] [PubMed] [Google Scholar]
- Van Beneden R. J., Walker C. W., Laughner E. S. Characterization of gene expression of a p53 homologue in the soft-shell clam (Mya arenaria). Mol Mar Biol Biotechnol. 1997 Jun;6(2):116–122. [PubMed] [Google Scholar]
- Vanden Heuvel J. P., Tyson F. L., Bell D. A. Construction of recombinant RNA templates for use as internal standards in quantitative RT-PCR. Biotechniques. 1993 Mar;14(3):395–398. [PubMed] [Google Scholar]
- Vodicnik M. J., Peterson R. E. The enhancing effect of spawning on elimination of a persistent polychlorinated biphenyl from female yellow perch. Fundam Appl Toxicol. 1985 Aug;5(4):770–776. doi: 10.1016/0272-0590(85)90201-5. [DOI] [PubMed] [Google Scholar]
- Wallingford J. B., Seufert D. W., Virta V. C., Vize P. D. p53 activity is essential for normal development in Xenopus. Curr Biol. 1997 Oct 1;7(10):747–757. doi: 10.1016/s0960-9822(06)00333-2. [DOI] [PubMed] [Google Scholar]
- Wang P., Reed M., Wang Y., Mayr G., Stenger J. E., Anderson M. E., Schwedes J. F., Tegtmeyer P. p53 domains: structure, oligomerization, and transformation. Mol Cell Biol. 1994 Aug;14(8):5182–5191. doi: 10.1128/mcb.14.8.5182. [DOI] [PMC free article] [PubMed] [Google Scholar]