Sources/Clones
Biodesign (PC10), Biogenesis (PC10), Biogenex (19A2), Boehringer Mannheim (PC10), Camon (19A2), Chemicon, Coulter (19A2), Dako (PC10), Diagnostic Biosystems (PC10), Medac (PC10), Oncogene (PC10, 19F4) 19A2), Serotec (19.A2), Signet (PC10) and Zymed (ZO49).
Fixation/Preparation
Both the main clones, PC10 and 19A2, to proliferating cell nuclear antigen (PCNA) are immunoreactive in fixed embedded tissues. However, the antigen is fixation dependent and HIER produces significant enhancement of immunostaining. HIER in 1% zinc sulfate is reported to produce the best staining (Shin et al, 1994), although we have found citrate buffer at pH 6.0 to be sufficiently effective.
Background
PCNA, formerly called cyclin (now recognized to be a much wider class of proteins ssociated with cell proliferation), represents a component of DNA polymerase-b (Bravo & Macdonald-Bravo, 1987) and is a 36 kD intranuclear proliferation-associated antigen. An antibody to this antigen was first described in the blood of selected patients with systemic lupus erythematosus. This polypeptide has since been found in both normal and transformed cells and is tightly associated to the sites of DNA replication. Its expression is highest during S-phase of the cell cycle and there is generally a good correlation between expression of PCNA and the S-phase fraction determined by flow cytometry in a given tumor cell population.
However, certain caveats apply to the use of anti-PCNA antibodies as markers of cell proliferation. In malignant cell lines such as HeLa, PCNA levels increase during S-phase but are not zero during the other phases of the cell cycle. Indeed, in this cell line the levels of PCNA increase only by a factor of 2-3 during S-phase (Morris & Matthews, 1989). There are also at least two forms of PCNA, one associated with the "replicon" structure and the other more loosely associated in the cell nucleus. Both proteins are retained by formalin fixation but only the former is retained by alcoholic fixatives such as methacarn (Bravo & MacDonald Bravo 1987). Furthermore, the antigen persists in cells that are no longer in the cycling phase and are in G0. Generally PCNA counts obtained with clone PC10 have been higher than those obtained with Ki-67 (Leong et al, 1995) or S-phase fraction measured by flow cytometry, despite PCNA being considered to be primarily an S-phase-associated protein. PCNA has a relatively long
half-life of 20 h and may be immunohistochemically detected in cells that have recently left the cell cycle and may be in G0. Discrepancies have also been demonstrated between PCNA counts obtained with PC10 and that of S-phase fraction by thymidine and bromodeoxyuridine uptake in a variety of tumors and in the experimental situation (Yu et al, 1991; Scott et al 1991). The PCNA index was found to be 2-3 times that of values obtained with DNA polymerase-a (Kawakita et al, 1992). There is ample evidence that the antigen is very fixation dependent and different antibody clones show vastly different sensitivities for the antigen (Leong et al, 1993; Coltrera et al, 1993).
Applications
PCNA immunostaining offers an alternative to the well-established but cumbersome methods of assessing tumor growth fractions, namely tritiated thymidine or bromodeoxyuridine incorporation, or flow cytometry and has been enthusiastically employed in numerous publications, despite the limitations discussed above.
Comments
PC10 is the most sensitive of the antibody clones available but bearing in mind the fixation dependency of PCNA, comparisons between laboratories are invalid unless fixation protocols are standardized. Furthermore, because of the long half-life of the antigen, only strongly stained cells should be counted and weakly stained cells show poor correlation with the S-phase fraction (Yu et al, 1995). Methanol is the fixative of choice (Burford-Mason et al, 1994).
References
•Bravo R, Macdonald-Bravo H 1987. Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. Journal of Cell Biology; 105: 1549-1554.
•Burford-Mason AP, MacKay AJ, Cummins M, Dardick I 1994. Detection of proliferating cell nuclear antigen in paraffin-embedded specimens is dependent on preembedding tissue handling and fixation. Archives of Pathology and Laboratory Medicine; 118: 1007-1013.
•Coltrera MD, Skelly M, Gown AM 1993. Anti-PCNA antibody PC10 yields unreliable proliferation indexes in routinely processed, deparaffinized, formalin-fixed tissue. Applied Immunohistochemistry; 1: 193-200.
•Kawakita N, Seki S, Yanani A et al 1992. Immunocytochemical identification of proliferating hepatocytes using mononuclear antibody to proliferating nuclear cell antigen (PCNA/cyclin). Comparison with immunocytochemical staining for DNA polymerase-alpha. American Journal of Clinical Pathology; 97 (suppl 1): S14-20.
•Leong AS-Y, Milios J, Tang SK 1993. Is immunolocalization of proliferating cell nuclear antigen (PCNA) in paraffin sections a valid index of cell proliferation? Applied Immunohistochemistry; 1: 127-135.
•Leong AS-Y, Vinyuvat S, Suthipintawong C 1995. A comparative study of cell proliferation markers in breast carcinomas. Journal of Clinical Pathology: Molecular Pathology; 48: M83-M87.
•Morris GF, Matthews MB 1989. Regulation of proliferating cell nuclear antigen during the cell cycle. Journal of Biological Chemistry; 264: 3856-3864.
•Scott RJ, Hall PA, Haldane JS 1991. A comparison of immunohistochemical markers of cell proliferation with experimentally determined growth fraction. Journal of Pathology; 165: 173-178.
•Shin HJ, Shin DM, Shah T, Ro JY 1994. Optimization of proliferating cell nuclear antigen immunohistochemical staining by microwave heating in zinc sulfate solution. Modern Pathology; 7: 242-248.
•Yu CC-W, Hall PA, Fletcher CDM et al 1991. Hemangiopericytomas: the prognostic value of immunohistochemical staining with a monoclonal antibody to proliferating cell nuclear antigen (PCNA). Histopathology; 1929-1933.
•Yu CC-W, Dublin EA, Camplejohn RS, Levison DA 1995. Optimization of immunohistochemical staining of proliferating cells in paraffin sections of breast carcinoma using antibodies to proliferating cell nuclear antigen and the Ki-67 antigen. Annals of Cell Pathology; 9: 45-52.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
Biodesign (PC10), Biogenesis (PC10), Biogenex (19A2), Boehringer Mannheim (PC10), Camon (19A2), Chemicon, Coulter (19A2), Dako (PC10), Diagnostic Biosystems (PC10), Medac (PC10), Oncogene (PC10, 19F4) 19A2), Serotec (19.A2), Signet (PC10) and Zymed (ZO49).
Fixation/Preparation
Both the main clones, PC10 and 19A2, to proliferating cell nuclear antigen (PCNA) are immunoreactive in fixed embedded tissues. However, the antigen is fixation dependent and HIER produces significant enhancement of immunostaining. HIER in 1% zinc sulfate is reported to produce the best staining (Shin et al, 1994), although we have found citrate buffer at pH 6.0 to be sufficiently effective.
Background
PCNA, formerly called cyclin (now recognized to be a much wider class of proteins ssociated with cell proliferation), represents a component of DNA polymerase-b (Bravo & Macdonald-Bravo, 1987) and is a 36 kD intranuclear proliferation-associated antigen. An antibody to this antigen was first described in the blood of selected patients with systemic lupus erythematosus. This polypeptide has since been found in both normal and transformed cells and is tightly associated to the sites of DNA replication. Its expression is highest during S-phase of the cell cycle and there is generally a good correlation between expression of PCNA and the S-phase fraction determined by flow cytometry in a given tumor cell population.
However, certain caveats apply to the use of anti-PCNA antibodies as markers of cell proliferation. In malignant cell lines such as HeLa, PCNA levels increase during S-phase but are not zero during the other phases of the cell cycle. Indeed, in this cell line the levels of PCNA increase only by a factor of 2-3 during S-phase (Morris & Matthews, 1989). There are also at least two forms of PCNA, one associated with the "replicon" structure and the other more loosely associated in the cell nucleus. Both proteins are retained by formalin fixation but only the former is retained by alcoholic fixatives such as methacarn (Bravo & MacDonald Bravo 1987). Furthermore, the antigen persists in cells that are no longer in the cycling phase and are in G0. Generally PCNA counts obtained with clone PC10 have been higher than those obtained with Ki-67 (Leong et al, 1995) or S-phase fraction measured by flow cytometry, despite PCNA being considered to be primarily an S-phase-associated protein. PCNA has a relatively long
half-life of 20 h and may be immunohistochemically detected in cells that have recently left the cell cycle and may be in G0. Discrepancies have also been demonstrated between PCNA counts obtained with PC10 and that of S-phase fraction by thymidine and bromodeoxyuridine uptake in a variety of tumors and in the experimental situation (Yu et al, 1991; Scott et al 1991). The PCNA index was found to be 2-3 times that of values obtained with DNA polymerase-a (Kawakita et al, 1992). There is ample evidence that the antigen is very fixation dependent and different antibody clones show vastly different sensitivities for the antigen (Leong et al, 1993; Coltrera et al, 1993).
Applications
PCNA immunostaining offers an alternative to the well-established but cumbersome methods of assessing tumor growth fractions, namely tritiated thymidine or bromodeoxyuridine incorporation, or flow cytometry and has been enthusiastically employed in numerous publications, despite the limitations discussed above.
Comments
PC10 is the most sensitive of the antibody clones available but bearing in mind the fixation dependency of PCNA, comparisons between laboratories are invalid unless fixation protocols are standardized. Furthermore, because of the long half-life of the antigen, only strongly stained cells should be counted and weakly stained cells show poor correlation with the S-phase fraction (Yu et al, 1995). Methanol is the fixative of choice (Burford-Mason et al, 1994).
References
•Bravo R, Macdonald-Bravo H 1987. Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. Journal of Cell Biology; 105: 1549-1554.
•Burford-Mason AP, MacKay AJ, Cummins M, Dardick I 1994. Detection of proliferating cell nuclear antigen in paraffin-embedded specimens is dependent on preembedding tissue handling and fixation. Archives of Pathology and Laboratory Medicine; 118: 1007-1013.
•Coltrera MD, Skelly M, Gown AM 1993. Anti-PCNA antibody PC10 yields unreliable proliferation indexes in routinely processed, deparaffinized, formalin-fixed tissue. Applied Immunohistochemistry; 1: 193-200.
•Kawakita N, Seki S, Yanani A et al 1992. Immunocytochemical identification of proliferating hepatocytes using mononuclear antibody to proliferating nuclear cell antigen (PCNA/cyclin). Comparison with immunocytochemical staining for DNA polymerase-alpha. American Journal of Clinical Pathology; 97 (suppl 1): S14-20.
•Leong AS-Y, Milios J, Tang SK 1993. Is immunolocalization of proliferating cell nuclear antigen (PCNA) in paraffin sections a valid index of cell proliferation? Applied Immunohistochemistry; 1: 127-135.
•Leong AS-Y, Vinyuvat S, Suthipintawong C 1995. A comparative study of cell proliferation markers in breast carcinomas. Journal of Clinical Pathology: Molecular Pathology; 48: M83-M87.
•Morris GF, Matthews MB 1989. Regulation of proliferating cell nuclear antigen during the cell cycle. Journal of Biological Chemistry; 264: 3856-3864.
•Scott RJ, Hall PA, Haldane JS 1991. A comparison of immunohistochemical markers of cell proliferation with experimentally determined growth fraction. Journal of Pathology; 165: 173-178.
•Shin HJ, Shin DM, Shah T, Ro JY 1994. Optimization of proliferating cell nuclear antigen immunohistochemical staining by microwave heating in zinc sulfate solution. Modern Pathology; 7: 242-248.
•Yu CC-W, Hall PA, Fletcher CDM et al 1991. Hemangiopericytomas: the prognostic value of immunohistochemical staining with a monoclonal antibody to proliferating cell nuclear antigen (PCNA). Histopathology; 1929-1933.
•Yu CC-W, Dublin EA, Camplejohn RS, Levison DA 1995. Optimization of immunohistochemical staining of proliferating cells in paraffin sections of breast carcinoma using antibodies to proliferating cell nuclear antigen and the Ki-67 antigen. Annals of Cell Pathology; 9: 45-52.
Bibliografia
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
