Sources/Clones
Antibodies to both wild-type and mutant p53 are available from Accurate, Biodesign (Pab1801, 53-12), BioSource, Bioprobe (BP53-12), Chemicon, Cymbus Bioscience, Medac (CM-1), Dako (DO-7), Gibco BRL, Immunotech, Novocastra, Oncogene (Pab1801, Pab421, Pab122), Oncor, Pharmingen (G59-12), Serotec (Pab1801, BP53-12) and Signet.
Antibodies to mutant p53
Biogenesis, Chemicon and Oncogene (Pab240)
Antibodies to Wild-Type p53
Biodesign (Pab246), Oncogene (Pab1620) and Serotec (Pab246)
Fixation/Preparation
Fresh or frozen tissues can be used. Clones Pab1801 and DO7 are effective in formalin-fixed tissue with best results following MW epitope retrieval.
Background
In the current constellation of oncogenes and recessive tumor suppressor genes, the p53 molecule represents one of the most common genetic changes associated with human cancer, being implicated in a wide range of malignancies. The p53 gene displays several unusual features, the most important of which is the ability to act as either a dominant oncogene or a recessive tumor suppressor gene. A combination of genetic events that affect both alleles of the gene results in the loss of expression of wild-type (WT) p53. This may occur as a complete loss of one allele of the gene as a result of a large chromosomal deletion combined with a point missense mutation on the other allele. Mutation leads to the loss of DNA binding and transcriptional regulatory activities of the p53 phosphoprotein with a corresponding loss of its growth suppressive activity and its role as "the guardian of the genome". The mutated protein has abnormal conformation, impaired DNA binding and a prolonged or stabilized half-life, the latter resulting in immunohistochemically stainable levels within nuclei in nearly all tumors showing p53 gene mutation. While a loss of transformation suppression activity and a gain of transforming potential often accompany mutation of p53, not all p53 mutants are equal in terms of their biological activity. Mutations at different hotspots manifest different and distinct phenotypes and there is geographic variation in the sites of mutations thought to reflect the effects of different environmental and regional carcinogens and cofactors.
The p53 gene is located on the short arm of human chromosome 17 and the majority of mutations in the gene are clustered in the most highly conserved domains spanned by 4-9 axons. An important relationship exists between DNA damage hotspots and the capacity to repair the DNA as mutation abolishes the arrest or delay seen in the normal cellular response to DNA damage. Although the WT p53 gene product is not essential for progress of cells through the cell cycle, it does negatively regulate cell growth or division. By binding to specific DNA sequences, the p53 WT product is able to inhibit adjacent gene transcription and serves to prevent uncontrolled cellular proliferation. Thus, loss of WT p53 activity induces a release from G1-S cell cycle checkpoint control following DNA damage, increasing genomic instability and promoting gene amplification.
Binding of WT p53 to a variety of viral proteins such as protein E6, a product of the human papilloma virus, simian virus 40 T-antigen and the Epstein-Barr nuclear antigen, as well as to cellular proteins such as heat shock protein 70 and MDM-2 replication protein, may result in an inactivated complex and a loss of transformation suppression activity (Chang et al, 1993; Batsakis & El-Naggar, 1995).
Applications
Immunohistochemical detection of nuclear p53 protein is based on the increase in concentration of the protein to detectable levels, secondary to an increased synthesis and a lower degradation with longer half-life. In general, there is good agreement between the frequency of positive immunostaining and the frequency of tumors with mutations detected by direct DNA sequencing. However, there are discrepancies between these findings and analysis at the protein level. There is also a danger in assuming positive staining to be an indication of an underlying mutation as p53 protein can be stabilized by other means such as sequestration of normal nuclear protein in the cytoplasm with inactivation of its tumor suppressor function or by binding with the cellular proteins previously mentioned (Hall & Lane, 1994). Also, the use of anti-p53 antibodies that do not react with all mutant forms and other events may lead to failure to detect p53 in neoplasms. The analysis of p53 in neoplastic and preneoplastic states is a powerful tool which provides molecular information on the oncogenic process and the ability to stain for abnormal forms of the protein in tissue sections, particularly those fixed in formalin, allows an important avenue of investigation. Furthermore, there is evidence to suggest that the expression of abnormal p53 may be a prognostic parameter in some neoplasms (Batsakis & El-Naggar, 1995).
Comments
Immunostaining of p53 can be affected by degradation of antigen during tissue processing and it is important to recognize the fixation conditions and the nature of the antibody employed (Fisher et al, 1994). Monoclonal antibody PabI801 (Biogenesis, Gibco BRL, Medac) recognizes most of the mutant and wild types of p53 but 1801 is not suitable for paraffin-embedded tissues. Our own experience is largely with DO7 (Medac, Biogenex, Dako) which identifies both wild-type and mutant protein in formalin-fixed, paraffin-embedded sections and best results are obtained after MW epitope retrieval. Phenol formol saline has been suggested to be a useful fixative for this antigen.
References
•Batsakis JG, El-Naggar AK 1995. p53: 15 years after discovery. Advances in Anatomic Pathology 2: 71-88.
•Chang F, Syrjanen S, Tervahauta A, Syrjanen K 1993. Tumorigenesis associated with the p53 tumour suppressor gene. British Journal of Cancer 68: 653-661.
•Fisher DJ, Gillett CE, Vojtesek B et al 1994. Problems with p53 immunohistochemical staining: the effect of fixation and variation in the methods of evaluation. British Journal of Cancer 69: 26-31.
•Hall PA, Lane DP 1994. p53 in tumour pathology: can we trust immunohistochemistry? - Revisited. Journal of Pathology 172:1.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
Antibodies to both wild-type and mutant p53 are available from Accurate, Biodesign (Pab1801, 53-12), BioSource, Bioprobe (BP53-12), Chemicon, Cymbus Bioscience, Medac (CM-1), Dako (DO-7), Gibco BRL, Immunotech, Novocastra, Oncogene (Pab1801, Pab421, Pab122), Oncor, Pharmingen (G59-12), Serotec (Pab1801, BP53-12) and Signet.
Antibodies to mutant p53
Biogenesis, Chemicon and Oncogene (Pab240)
Antibodies to Wild-Type p53
Biodesign (Pab246), Oncogene (Pab1620) and Serotec (Pab246)
Fixation/Preparation
Fresh or frozen tissues can be used. Clones Pab1801 and DO7 are effective in formalin-fixed tissue with best results following MW epitope retrieval.
Background
In the current constellation of oncogenes and recessive tumor suppressor genes, the p53 molecule represents one of the most common genetic changes associated with human cancer, being implicated in a wide range of malignancies. The p53 gene displays several unusual features, the most important of which is the ability to act as either a dominant oncogene or a recessive tumor suppressor gene. A combination of genetic events that affect both alleles of the gene results in the loss of expression of wild-type (WT) p53. This may occur as a complete loss of one allele of the gene as a result of a large chromosomal deletion combined with a point missense mutation on the other allele. Mutation leads to the loss of DNA binding and transcriptional regulatory activities of the p53 phosphoprotein with a corresponding loss of its growth suppressive activity and its role as "the guardian of the genome". The mutated protein has abnormal conformation, impaired DNA binding and a prolonged or stabilized half-life, the latter resulting in immunohistochemically stainable levels within nuclei in nearly all tumors showing p53 gene mutation. While a loss of transformation suppression activity and a gain of transforming potential often accompany mutation of p53, not all p53 mutants are equal in terms of their biological activity. Mutations at different hotspots manifest different and distinct phenotypes and there is geographic variation in the sites of mutations thought to reflect the effects of different environmental and regional carcinogens and cofactors.
The p53 gene is located on the short arm of human chromosome 17 and the majority of mutations in the gene are clustered in the most highly conserved domains spanned by 4-9 axons. An important relationship exists between DNA damage hotspots and the capacity to repair the DNA as mutation abolishes the arrest or delay seen in the normal cellular response to DNA damage. Although the WT p53 gene product is not essential for progress of cells through the cell cycle, it does negatively regulate cell growth or division. By binding to specific DNA sequences, the p53 WT product is able to inhibit adjacent gene transcription and serves to prevent uncontrolled cellular proliferation. Thus, loss of WT p53 activity induces a release from G1-S cell cycle checkpoint control following DNA damage, increasing genomic instability and promoting gene amplification.
Binding of WT p53 to a variety of viral proteins such as protein E6, a product of the human papilloma virus, simian virus 40 T-antigen and the Epstein-Barr nuclear antigen, as well as to cellular proteins such as heat shock protein 70 and MDM-2 replication protein, may result in an inactivated complex and a loss of transformation suppression activity (Chang et al, 1993; Batsakis & El-Naggar, 1995).
Applications
Immunohistochemical detection of nuclear p53 protein is based on the increase in concentration of the protein to detectable levels, secondary to an increased synthesis and a lower degradation with longer half-life. In general, there is good agreement between the frequency of positive immunostaining and the frequency of tumors with mutations detected by direct DNA sequencing. However, there are discrepancies between these findings and analysis at the protein level. There is also a danger in assuming positive staining to be an indication of an underlying mutation as p53 protein can be stabilized by other means such as sequestration of normal nuclear protein in the cytoplasm with inactivation of its tumor suppressor function or by binding with the cellular proteins previously mentioned (Hall & Lane, 1994). Also, the use of anti-p53 antibodies that do not react with all mutant forms and other events may lead to failure to detect p53 in neoplasms. The analysis of p53 in neoplastic and preneoplastic states is a powerful tool which provides molecular information on the oncogenic process and the ability to stain for abnormal forms of the protein in tissue sections, particularly those fixed in formalin, allows an important avenue of investigation. Furthermore, there is evidence to suggest that the expression of abnormal p53 may be a prognostic parameter in some neoplasms (Batsakis & El-Naggar, 1995).
Comments
Immunostaining of p53 can be affected by degradation of antigen during tissue processing and it is important to recognize the fixation conditions and the nature of the antibody employed (Fisher et al, 1994). Monoclonal antibody PabI801 (Biogenesis, Gibco BRL, Medac) recognizes most of the mutant and wild types of p53 but 1801 is not suitable for paraffin-embedded tissues. Our own experience is largely with DO7 (Medac, Biogenex, Dako) which identifies both wild-type and mutant protein in formalin-fixed, paraffin-embedded sections and best results are obtained after MW epitope retrieval. Phenol formol saline has been suggested to be a useful fixative for this antigen.
References
•Batsakis JG, El-Naggar AK 1995. p53: 15 years after discovery. Advances in Anatomic Pathology 2: 71-88.
•Chang F, Syrjanen S, Tervahauta A, Syrjanen K 1993. Tumorigenesis associated with the p53 tumour suppressor gene. British Journal of Cancer 68: 653-661.
•Fisher DJ, Gillett CE, Vojtesek B et al 1994. Problems with p53 immunohistochemical staining: the effect of fixation and variation in the methods of evaluation. British Journal of Cancer 69: 26-31.
•Hall PA, Lane DP 1994. p53 in tumour pathology: can we trust immunohistochemistry? - Revisited. Journal of Pathology 172:1.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
