Abstract
Mutations of the p53 gene are the most common genetic changes in human malignancies; therefore their detection is of practical importance. In contrast to wild-type p53 in resting normal cells, mutant p53 proteins are easily detectable by immunocytochemical methods due to their abnormally extended half-life. Several methods of immunocytochemistry can be used to analyze the presence and localization of p53 protein in cells or tissues. The most important is immunocytochemical p53 staining of sections from paraffin embedded tissues. This method is used as a relatively reliable surrogate marker for p53 mutations and has the advantage of being easy, fast, and suitable for mass screening of large archival tissue banks. Frozen sections can also be used to detect and localize the p53 proteins. p53 can also be detected in tissue culture cells. p53 can be detected in situ through a secondary antibody coupled to a fluorescent dye or an enzymatic activity that reacts with certain chromogens.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mao, S.-Y., Javois, L. C., and Kent, U. M. (1999) Overview of antibody use in immunocytochemistry, in Methods in Molecular Medicine, vol 115: Immunocytochemical Methods and Protocols (Javois, J. C., ed), Humana Press, Totowa, pp. 3–10.
Sternberger, L. A. (1979) Immunocytochemistry, 2nd ed. Wiley, New York.
Greenblatt, M. S., Bennett, W. P., Hollstein, M., and Harris, C. C. (1994) Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 54, 4855–4878.
Lutzker, S. G. and Levine, A. J. (1996) A functionally inactive p53 protein in teratocarcinoma cells is activated by either DNA damage or cellular differentiation. Nat. Med. 2, 804–810.
Riou, G., Barrois, M., Prost, S., Terrier, M. J., Theodore, C., and Levine, A. J. (1995) The p53 and mdm-2 genes in human testicular germ-cell tumors. Mol. Carcinog. 12, 124–131
Murty, V. V., Bosl, G. J., Houldsworth, J., et al. (1994) Allelic loss and somatic differentiation in human male germ cell tumors. Oncogene 9, 2245–2251.
Peng, H. Q., Hogg, D., Malkin, D., et al. (1993) Mutations of the p53 gene do not occur in testis cancer. Cancer Res. 53, 3574–3578.
Schenkman, N. S., Sesterhenn, I. A., Washington, L., et al. (1995) Increased p53 protein does not correlate with mutations in microdissected human testicular germ cell tumors. J. Urol. 154, 617–621.
Xu, L., Chen, Y. T., Huvos, A. G., et al. (1994) Overexpression of p53 in squamous cell carcinomas of head and neck without apparent gene mutations. Diagn. Mol. Pathol. 3, 83–92.
Kennedy, S. M., MacGeogh, C., Jaffe, R., and Spurr, N. K. (1994) Overexpression of the oncoprotein p53 in primary hepatic tumor of childhood does not correlate with the gene mutations. Hum. Pathol. 25, 438–442.
Ambros, R. A., Ross, J. F., Kallakury, B. V. S., et al. (1995) p53 gene status in endometrial carcinomas showing diffuse positivity for p53 protein by immunohistochemical analysis. Mod. Pathol. 8, 441–445.
King, S. A., Adas, A. A., LiVolsi, V. A., et al. (1995) Expression and mutation analysis of the p53 gene in uterine papillary serous carcinoma. Cancer 75, 2700–2705.
Marchenko, N. D. and Moll, U. M. (1997) Nuclear overexpression of p53 protein does not correlate with gene mutation in primary peritoneal carcinoma. Hum. Pathol. 28, 1002–1006.
Zhang, W., Hu, G., Estey, E., Hester, J., and Deisseroth, A. (1992) Altered conformation of the p53 protein in myeloid leukemia cells and mitogen-stimulated normal blood cells. Oncogene 7, 1645–1647.
Moll, U. M., LaQuaglia, M., Benard, J., and Riou, G. (1995) Wild-type p53 protein undergoes cytoplasmic sequestration in undifferentiated neuroblastomas but not in differentiated tumors. Proc. Natl. Acad. Sci. USA 92, 4407–4711.
Stommel, J. M., Marchenko, N. D., Jimenez, G. S., Mol, U. M., Hope, T. J., and Wahl, G. M. (1999) A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J. 18, 1660–1672.
Haupt, Y., Maya, R., Kazaz, A., and Oren, M. (1997) Mdm2 promotes the rapid degradation of p53. Nature 387, 296–299.
Kubbutat, M. H., Jones, S. N., and Vousden, K. H. (1997) Regulation of p53 stability by Mdm2. Nature 387, 299–303.
Maki, C. G., Huibregtse, J. M., and Howley, P. M. (1996) In vivo ubiquitination and proteasome-mediated degradation of p53. Cancer Res. 56, 2649–2654.
Shirangi, T. R., Zaika, A., and Moll, U. M. (2002) Nuclear degradation of p53 occurs during down-regulation of the p53 response after DNA damage. FASEB J. 16, 420–422.
Honda, R., Tanaka, H., and Yasuda, H. (1997) Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett. 420, 25–27.
Kubbutat, M. H., Ludwig, R. L., Levine, A. J., and Vousden, K. H. (1999) Analysis of the degradation function of Mdm2. Cell Growth Differ. 10, 87–92.
Fang, S., Jensen, J. P., Ludwig, R. L., Vousden, K. H., and Weissman, A. M. (2000) Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53. J. Biol. Chem. 275, 8945–8951.
Grossman, S. R., Perez, M., Kung, A. L., et al. (1998) p300/MDM2 complexes participate in MDM2-mediated p53 degradation. Mol. Cell 2, 405–415.
Bottger, A., Bottger, V., Garcia-Echeverria, C., et al. (1997) Molecular characterization of the hdm2-p53 interaction. J. Mol. Biol. 269, 744–756.
Fritsche, M., Haessler, C., and Brandner, G. (1993) Induction of nuclear accumulation of the tumor suppressor protein p53 by DNA-damaging agents. Oncogene 8, 307–318.
Ko, L. and Prives, C. (1996) p53: puzzle and paradigm. Genes Dev. 10, 1054–1072.
Waggoner, S. E., Anderson, S. M., Luce, M. C., Takahashi, H., and Boyd, J. (1996) p53 protein expression and gene analysis in clear cell adenocarcinoma of the vagina and cervix. Gynecol. Oncol. 60, 339–344.
Yewdell, J. W., Gannon, J. V., and Lane, D. P. (1986) Monoclonal antibody analysis of p53 expression in normal and transformed cells. J. Virol. 59, 444–452.
Banks, L., Matlashewski, G., and Crawford, L. (1986) Isolation of human p53 specific monoclonal antibodies and their use in human p53 expression. Eur. J. Biochem. 159, 529–534.
Vojtesek, B., Bartek, J., Midgley, C. A., and Lane, D. P. (1992) An immunochemical analysis of the human nuclear phosphoprotein p53: new monoclonal antibodies and epitope mapping using recombinant p53. J. Immunol. Methods 151, 237–244.
Midgley, C. A., Fisher, C. J., Bartek, J., Vojtesek, B., Lane, D., and Barnes, D. M. (1992) Analysis of p53 expression in human tumours: an antibody raised against p53 expressed in Escherichia coli. J. Cell Sci. 101, 183–189.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Slade, N., Moll, U.M. (2003). Mutational Analysis of p53 in Human Tumors. In: Deb, S., Deb, S.P. (eds) p53 Protocols. Methods in Molecular Biology, vol 234. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-408-5:231
Download citation
DOI: https://doi.org/10.1385/1-59259-408-5:231
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-1-58829-106-6
Online ISBN: 978-1-59259-408-5
eBook Packages: Springer Protocols