Histopathology 1992. 21, 591-594
COMMENTARY
The complexities of proliferating cell nuclear antigen D.McCORMICK 81 P.A.HALL Division of Histoputhology, United Medical and Dental School, London, UK
The last 2 years have seen a flood of reports in Histopathology and other pathology journals describing the application of antibodies that recognize proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation in histological material. This interest is a consequence of the widely held belief that such markers may prove to be useful objective indicators of biological behaviour of at least some forms of tumour. This is coupled with the resistance of some PCNA epitopes to conventional fixation and embedding. The literature relating to PCNA is already rather confused and some rather extravagant claims and suggestions have been made, sometimes without a clear understanding of the biology of the PCNA molecule and the complexitiesof the processes in which it is involved. We will briefly review the history of PCNA and attempt to clarify our current understanding of this player in the processes of DNA replication. In 1978 Miyachi et al.' described an autoimmune serum from patients with systemic lupus erythematosus which recognized a nuclear antigen distributed in proliferating cells which was consequently termed proliferating cell nuclear antigen (PCNA). PCNA is a 36 kDa acidic non-histone nuclear protein which functions as an auxiliary protein for DNA polymerase 6 and is an absolute requirement for DNA synthesis24. Immunofluorescent studies have shown the existence of two populationsof PCNA during S phase of the cell cycle, one that is nucleoplasmic as in quiescent cells and is easily extracted by detergent and another that is associated to specific nuclear structures5. Detergent resistant PCNA immunoreactivity is co-localized with bromodeoxyuridine (incorporated into DNA during S phase) in replication complexes, and their order of appearances throughout the S phase are i d e n t i ~ a l ~This - ~ . demonstrates that PCNA is tightly associated to the sites of DNA replication and probably must have a role in DNA synthesis8. This is supported by the use of antisense oligonucleotides both of which lower levels of PCNA protein and are associated with inhibition of DNA synthesis9.In the presence of PCNA and a multi-subunit complex called replication factor C, polymerase 6 catalyses elongation of Okazaki fragments to long DNA
chains representing leading strand DNA synthesis10-12. PCNA is also involved in unscheduled DNA synthesis (i.e. nucleotide excision-repair) since tightly bound PCNA can be found associated with chromatin at all phases of the cell cycle after ultra violet (UV) irradiation in v i t r ~ ' ~ .The ' ~ . explanation for this lies in an understanding of the process of DNA excision repair. In the initial phase of DNA excision repair a multi subunit endonuclease forms at the site of damage and enzymatically removes the damaged strand. This is followed by DNA synthesis to repair the defect. Consequently there may be an overlap between the proteins involved in semi-conservative DNA replication and DNA repair. Direct experimental proof of this with demonstration of involvement of PCNA has recently come from analysis of a cell free model of DNA damage15. Furthermore, a role in DNA excision repair is reflected in the expression of PCNA in non-cycling normal human keratinocytes in vivo after mild UV exposure16. The gene encoding PCNA has been cloned from a number of evolutionarily diverse species' 7-21. Although there is variation at the DNA level, there are only four amino acid differences between rat and human PCNA and about 70% of the Drosophila protein is identical to the rat and human proteins. Furthermore, while the sequence of yeast PCNA shows only 35% homology at the DNA level with man, the molecules are functionally interchangeable22. The evolutionary conservation of PCNA throughout the animal (and plant) kingdom is entirely consistent with the product of the gene having an essential role in DNA replication in eukaryotes. Regulation of PCNA expression is complex22.PCNA expression is regulated at both the transcriptional and post-transcriptional leve122-24. Induction of PCNA mRNA by growth factors is well d o c ~ r n e n t e d ' ~ * ~ ~ - ~ ~ However, it is important to realise that there appear to be differences between the regulation in quiescent cells and those which are continuously cycling22,26. In the latter there is very little variation in protein or mRNA levels during the cell cycle. While the PCNA promoter region has been the contribution of transcriptional control is still unclear22. However, it is of note that homeodomain-containing proteins bind to the PCNA 591
592
1). McCorniick and P.A. Hall
pronioter'y~". Given that in other contexts homeodomain proteins regulate gradients of gene expression, it might be that similar mechanisms regulate the graded proliferative activity in certain tissues (e.g. in the intestinal crypt). Regulation of PCNA expression by post-transcriptional mechanisms appears to be very important, at least in vitro2?, Of particular note is the observation that PCNA m K N A is stabilized, and can thus be translated into protein with greater efficiency, in the presence of growth factors15.j1. It would alppear that many cells continuously make PCNA mRNA (at least at low levels) but rapidly degrade this without translation. Consequently cells are able, under the correct circumstances, to rapidly respond to growth factors by inducing PCNA production by stabilizing PCNA mRNA. The mechanism underlying this is intriguing and involves alteration in mRNA stability as a consequence of splicing of intron 422.3'. A further point is that oncogenes may participate in the regulation of PCNA mRNA level^^'-^^, and it is conceivable that alterations of oncogene expression in neoplasia may lead to the deregulation of PCNA expression. A wide range of antibodies thiat recognize PCNA are available. In addition to human sera from patients with SLB and related auto-immune diseases, a number of monoclonal antibodies have been generated. Commercially available reagents includle the monoclonal antibodies 19A2, 19F434*25 and PC103h. PClO and several related antibodies were generatled by immunizing mice with recombinant human PCNA expressed in E . colij6. It is apparent that the epitopes recognized by these antibodies are different37 and there are differences in the effects of fixation and processing on the detectability of these epitopes. Moreover, there appear to be differences between the immunochemical behaviour of these antibodies in comparison with the human auto-antibodie~j~. This is perhaps a partial explanation for discrepancies between reports on the relationship between PCNA immunoreactivity and other cell cycle markers. In addition, the differences observed between antibodies to PCNA may be the physical nature of the immunogens involved37and, in particular, whether they are in the native state (as seems to be the case with the autoantibodies) or are denatured (as in the case of PClO for example). Whatever the explanation, it is essential to recognize that different antibodies may have very different properties even though they recognize the same protein. This point is illustrated by the observation that one particular antibody, PC9. raised to recombinant PCNA only recognizes PCNA when in the nucleolus. This particular epitope is not seen anywhere else in the cellj6. In addition to the existence of differences between 1r)s31.
antibodies that recognize PCNA, it is clear that there are important technical considerations. The effects of different fixation conditions are of particular concern. These include duration of fixation, size of tissue block and type of fixative3x.For example, prolonged fixation can dramstically reduce PClO imrnunorea~tivity~~. PCNA detected by the 19A2 antibody after methanol fixation is closely associated with S phasejg. This is not seen in histological material with PCl 0, although under some conditions of enzyme treatment and tixation S phase distribution for PCNA can be seen by flow cytometric a n a l y ~ i s ~ " . ~ ' . Whether specific S phase PCNA immunoreactivity could be identified in conventionally fixed and processed tissue sections remains uncertain. The practice of baking sections on to glass can also be detrimental to PCNA immunoreactivity, although some degree of antigen retrieval using microwave methods may be possible. Of considerable importance is the observation that low levels of PCNA may be present throughout the cell As a consequence, varying the sensitivity of the immunohistological detection system may alter the number of PCNA immunoreactive cells. In the original description of PClO it was reported that cells at the lower end of small intestinal villi showed weak PCNA immunoreactivity. This was explained by the half-life of the PCNA protein being in excess of 2 0 h5. Support for this interpretation comes from the work of Scott et aL4j who reported that in tumour xenografts with a defined growth fraction and a cell cycle time of about 20 h the number of PCNA immunoreactive cells was nearly 100%.Thus, there may be situations where cells that have recently left the cell cycle express PCNA. PCNA is apparently expressed in non-cycling cells in other situations including in normal tissues adjacent to t u m o u r ~and ~ ~ possibly in some tumours where the number of PCNA immunoreactive cells does not correlate with other indices of p r ~ l i f e r a t i o n ~Hall ~ . et ~71.~' speculated that this might reflect the role of growth factors in regulating PCNA mRNA stability and protein expression. Some recent experiments have confirmed these observations and support a role for growth factors in mediating the expression of PCNA in non-cycling cells. Human carcinoma cell lines were injected into the liver or kidney of nude mice. Using tritiated thymidine a s a n objective measure of proliferation, control liver or kidney showed very few S phase cells and a similar number of PClO immunoreactive cells. In contrast, in animals with xenografts in liver or kidney, numerous PClO immunoreactive cells were seen but this was not mirrored by the number of S phase cells. In a separate set of experiments, rats fed by total parenteral nutrition were given infusions of TGFcr or EGF. In the pancreatic ductal and islet
Complexities ofproli/erating cell nuclear antigen
epithelium of both TGFa and EGF treated animals there was a more than tenfold increase in the number of PClO immunoreactive cells compared with controls, while there was no change in number of S phase cells. These experiments demonstrate that PCNA immunoreactivity (as detected by PC10) can occur without cell proliferation in association with neoplasia, and that this may be mediated by growth factors in V ~ V O In ~ ~simple . terms it would seem that expression of PCNA is a necessary but not sufficient requirement for proliferation. In summary, PCNA is a nuclear protein that is intimately involved in DNA synthesis and can be detected immunologically. There are important and poorly understood differences between the patterns of expression of some epitopes on the PCNA molecule and thus differences may exist in the data generated by different anti-PCNA antibodies. The effect of technical factors, including fixation, on patterns of PCNA staining should not be underestimated. The association of PCNA with DNA repair processes indicates that the expression of PCNA is not necessarily associated only with DNA synthesis in the context ofS phase. Finally, since PCNA is a necessary but not sufficient requirement for DNA synthesis (whether scheduled on unscheduled) PCNA may be expressed by cells that are not cycling, and this induction may be mediated by increased mRNA stability induced by growth factors. What message does this complex story have? The use of PCNA antibodies as markers of cell proliferation is not simple and straightforward but requires careful analysis and consideration of all these caveats (and perhaps others yet to be identified). As a general point, this story indicates the absolute requirement for us as pathologists to have a clear understanding of the immunological reagents we use and the biology of the antigens recognized. The PCNA story is similar to that recently put forward for the immunohistological detection of the p5 3 oncoprotein46-oversimpZ$cation will lead to false dawns! By all means employ immunological reagents but interpret with care, be cautious in drawing conclusions and always consider the biology of the molecules being investigated.
References 1. Miyachi K. Fritzler MJ,Tan EM. Autoantibody to a nuclear antigen in proliferating cells. 1. Irnmunol. 1978; 121: 2228-2234. 2. Tan CK, Castillo C, So AG, Downey KM. An auxiliary protein for DNA polymerase4 from foetal calf thymus. ]. Biol. Chem. 1986: 261; 12310-1231 6. 3. Prelich G. Tan CK. Kostura M et al. Functional identity of proliferating cell nuclear antigen and DNA polymerase 6 auxiliary protein. Nature 1987; 3 2 6 517-520. 4. Bravo R, Frank R. Blundell PA, Macdonald-Bravo H. Cyclin/PCNA
59 3
is the auxiliary protein of DNA polymerase-6. Nature 1987; 326: 5 1 5-520. 5. Bravo R. Macdonald-Bravo H. Existence of two populations of Cyclin/proliferating cell nuclear antigen during the cell cycle: associated with DNA replication sites. 1. Cell Biol. 1987; 105: 1549-1 554. 6. Bravo R. Macdonald-Bravo H. Induction of the nuclear protein ‘cyclin’ in quiescent mouse 3T3 cells stimulated by serum and growth factors. Correlation with DNA synthesis. CMBO 1. 1984; 3; 3177-3181. 7. Bravo R, Macdonald-Bravo H. Changes in the nuclear distribution of cyclin (PCNA) but not its synthesis depend on DNA replication. EMBO]. 1985; 4; 655-661. 8. Wilcock D, Lane DP. Localisation of p53, retinoblastoma and host replication proteins at sites of viral replication in herpes-infected cells. Nature 1991: 349; 429-432. 9 . Jaskulski D, Driel JK. Mercer WE, Calabretta B. Baserga R. Inhibition of cellular proliferation by antisense oligodeoxynucleotides to PCNA cyclin. Science 1988; 2 4 0 1544-1 546. 1 0 . Burgers PMJ. Succharornyces cerevisiae Replication Factor C. 11. Formation and activity of complexes with the proliferating cell nuclear antigen and with DNA polymerases 6 and E . I. Biol. Chem. 1991; 266; 22698-22706. 1 1 . Fairman MP. DNA polymerase S/PCNA: actions and interactions. ]. Cell Sci. 1990; 95; 1-4. 12. Prelich G, Stillman R. Coordinated leading and lagging strand synthesis during SV40 DNA replication in vitro requires PCNA. Cell 1988; 53; 117-126. 1 3 . Celis JE, Madsen P. Increased nuclear cyclin/PCNA antigen staining of non S-phase transformed human amnion cells engaged in nucleotide excision DNA repair. FEBS Lett. 1986; 209; 277283. 14. Toschi L. Bravo R. Changes in Cyclin/proliferating cell nuclear antigen distribution during DNA repair synthesis. I. Cell Biol. 1988; 107; 1623-1628. 1 5 . Shivji MKK, Kenny MK. Wood RD. Proliferating cell nuclear antigen (PCNA) is required for DNA excision repair. Cell 1992: 69; 1-20. 16. Hall PA, McKee PH. du P Menage H. Dover R. Lane DP. High levels of p53 protein in UV irradiated normal human skin. Oncogene. In press. 17. Almendral JM. Huebsch D. Blundell PA, Macdonald-Bravo H. Bravo R. Cloning and sequence of the human nuclear protein cyclin: homology with DNA binding proteins. Proc. Nutl. Acad. Sci. USA 1987; 84; 1575-1579. 18. Matsumoto K. Moriuchi T. Koji T. Nakane PK. Molecular cloning of cDNA coding for rat proliferating cell nuclear antigen (PCNA)/ cyclin. EMBO I. 1987; 6: 637-642. 19. Yamaguchi M, Nishida Y. Moriuchi T et al. Drosphila proliferating cell nuclear antigen (cyclin) gene: structure, expression during development, and specific binding of homeodomain proteins to its 5’-flanking region. Mol. Cell. Biol. 1990; 10; 872-879. 20. Bauer GA. Burgers PM. The yeast analogue of mammalian cyclin/ proliferating cell nuclear antigen interacts with mammalian DNA polymerase 6. Proc. Nutl. Acad. Sci. USA 1988; 85; 7506-7510. 21. Suzuka I. Daidoji €I, Matsuoka M et al. Gene for proliferating-cell nuclear antigen (DNA polyermase 6 auxiliary protein) is present in both mammalian and higher plant genomes. Proc. Nutl. Acad. Sci. USA 1989; 86; 3189-3193. 22. Baserga R. Growth regulation of the PCNA gene. 1. Cell Sci. 1991; 98: 433-436. 23. Shipman-Appasamy P. Cohen KS. Prystowsky MB. Interleukin 2induced expression of proliferating cell nuclear antigen is regu-
594
D.McCorrnick and P.A.Hal1
lated by transcriptional and post-transcriptional mechanisms. J. Biol. Chem. 1990: 265; 19180-19184. 24. Koniecki J, Nugent P. Kordowska 1. Bakrga R.Effect of the SV40 T antigen on the post-transcriptional regulation of the proliferating cell nuclear antigen and DNA polyerase-a genes. Cancer Res. 1991; 51: 1465-1471. 25. JaskulskiD, Gatti C, Travali S,Calabretta B, Baserga R. Regulation of the proliferating cell nuclear antigen/cyclin and thymidine kinase mRNA levels by growth factors. I. Biol. Chem. 1988: 263; 101 75-101 79. 26. Morris GF, Matthews MB.Regulation of proliferating cell nuclear antigen during the cell cycle. J. Biol. Chem. 1989: 264; 1385613864. 27. Travali S. Ku DH. Rim0 MG,Ottavio L, Baserga R, Calabretta B. Structure of the human gene for the proliferating cell nuclear antigen. I. Bid. Chem. 1989: 264; 7466-7472. 28. Mtavio L. Chang CD. Rizzo MG,Petralia S,Travali S. Baserga R. The promoter of the proliferating cell nuclear antigen (PCNA) is active in serum deprived cells. Biochern. Biophus. Res. Commun. 1990; 169; 509-516. 29. Yamaguchi M, Hirose P, Nishlda Y. Matsukage A. Repression of the Drosophila proliferatingcell nuclear antigen gene promoter by zerknullt protein. Mol. Cell. Blol. 1991: 11; 4 9 0 9 4 9 1 7 . 30. Chang C-D, Ottavio L. Travali S,Lipson KE. Baserga R. Transcrip tional and posttranscriptionalregulation of the proliferating cell nuclear antigen gene. Mol. Cell. Bfol. 1 9 9 0 10; 3289-3296. 3 1. Ottavio L. Chang C-D, R i m M-G. Travah S,Casadevall C, Baserga R. Importance of introns In the growth regulation ofmRNA levels of the proliferatingcell nuclear antigen gene. Mol. Cell. Bfol. 1 9 9 0 1 0 303-309. 32. Travali S,Perber A. Reis K et al. Effect of the mub gene product on expression of the PCNA gene in Ebroblasts. Oncogene 1991: 6; 887-894. 33. Mercer WE, Shields MT. Ling D, Appella E, Ullrich SJ. Growth suppression Induced by wild-type p53 protein Is accompanied by selective down-regulation of proliferating-cell nuclear antigen expression. Proc. Natl. Acad. Sci. USA 1991: 88; 1958-1962. 34. Ogata K. Kwki P, Celis JE, Nakamura RM. Tan EM. Monoclonal antibodies to a nuclear protein (PCNA/Cyclin) associated with DNA replication. Exp. Cell. Res. 1987: 168; 475-486. 35. Ogata K, Ogata Y. Takasaki Y,Tan EM. Epitopes on proliferating
cell nuclear antigen recognised by human lupus autoantibody and murine monoclonal antibody. 1.hnmunol. 1987: 139; 29422946. 36. Waseem NH. Lane DP. Monoclonal antibody analysis of the proliferatingcell nuclear antigen (PCNA)Structural conservation and the detection of a nucleolar form. I. Cell. Sci. 1990: 96; 121129. 37. Huff JP,Rms G, Peebles CL. Houghten R, Sullivan KE. Tan EM. Insights Into native epitopes of proliferating cell nuclear antigen uslng recombinant DNA protein products. 1.Exp. h4ed. 1 9 9 0 172; 419-429. 38. Hall PA. Levison DA, Woods AL et al. Proliferating cell nuclear antigen (PCNA)immunolocalizationin par& sections:an index of cell proliferation with evidence of deregulated expression in some neoplasms. J. Pathol. 1 9 9 0 162; 285-294. 39. Galand P, Degraef C. Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells In para& sections from animal and human tissues. Cell Tissue Kinet. 1989: 22; 383-392. 40. Landberg G, Roos G. Antibodies to proliferating cell nuclear antigen as S-phase probes in flow cytometric cell cycle analysis. Cancer Res. 1991: 51; 4570-4574. 41. Wilson GD, Camplejohn RS. Martindale CA. Brock A, Lane DP. Barnes DM. Flow cytometric characterisation of proliferating cell nuclear antigen using the monoclonal antibody PC10. Eur. I. Cancer. In press. 42. Celis JE, Celis A. Cell cycle-dependent variations in the distributions of the nuclear protein cyclin proliferating cell nuclear antigen in cultured cells: Subdivision of S phase. Proc. Natl. Acad. Sci. USA 1985: 82; 3262-3266. 43. Scott RJ, Hall PA, Haldane JSet al. A comparison of immunohisbchemical markers of cell proliferating with experimentally determined growth fraction. ]. Pathol. 1991: 165; 173-178. 44. Yu CCW, Hall PA, Fletcher CDM et al. Haemangiopericytomas: the prognostic value of immunohistochemical stafning with a monoclonal antibody to proliferatingcell nuclear antigen (PCNA). Histo@hologu 1991: 19; 29-34. 45. Hall PA, Hart I, Goodlad R. Coates PJ, Lane DP. Expression of proliferating cell nuclear antigen in noncycling cells. In press. 46. Wynford-Thomas D. p53 in tumour pathology: can we trust immunocytochemistry?I. Pathol. 1992: 166; 329-330.
COMMENTARY
The complexities of proliferating cell nuclear antigen D.McCORMICK 81 P.A.HALL Division of Histoputhology, United Medical and Dental School, London, UK
The last 2 years have seen a flood of reports in Histopathology and other pathology journals describing the application of antibodies that recognize proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation in histological material. This interest is a consequence of the widely held belief that such markers may prove to be useful objective indicators of biological behaviour of at least some forms of tumour. This is coupled with the resistance of some PCNA epitopes to conventional fixation and embedding. The literature relating to PCNA is already rather confused and some rather extravagant claims and suggestions have been made, sometimes without a clear understanding of the biology of the PCNA molecule and the complexitiesof the processes in which it is involved. We will briefly review the history of PCNA and attempt to clarify our current understanding of this player in the processes of DNA replication. In 1978 Miyachi et al.' described an autoimmune serum from patients with systemic lupus erythematosus which recognized a nuclear antigen distributed in proliferating cells which was consequently termed proliferating cell nuclear antigen (PCNA). PCNA is a 36 kDa acidic non-histone nuclear protein which functions as an auxiliary protein for DNA polymerase 6 and is an absolute requirement for DNA synthesis24. Immunofluorescent studies have shown the existence of two populationsof PCNA during S phase of the cell cycle, one that is nucleoplasmic as in quiescent cells and is easily extracted by detergent and another that is associated to specific nuclear structures5. Detergent resistant PCNA immunoreactivity is co-localized with bromodeoxyuridine (incorporated into DNA during S phase) in replication complexes, and their order of appearances throughout the S phase are i d e n t i ~ a l ~This - ~ . demonstrates that PCNA is tightly associated to the sites of DNA replication and probably must have a role in DNA synthesis8. This is supported by the use of antisense oligonucleotides both of which lower levels of PCNA protein and are associated with inhibition of DNA synthesis9.In the presence of PCNA and a multi-subunit complex called replication factor C, polymerase 6 catalyses elongation of Okazaki fragments to long DNA
chains representing leading strand DNA synthesis10-12. PCNA is also involved in unscheduled DNA synthesis (i.e. nucleotide excision-repair) since tightly bound PCNA can be found associated with chromatin at all phases of the cell cycle after ultra violet (UV) irradiation in v i t r ~ ' ~ .The ' ~ . explanation for this lies in an understanding of the process of DNA excision repair. In the initial phase of DNA excision repair a multi subunit endonuclease forms at the site of damage and enzymatically removes the damaged strand. This is followed by DNA synthesis to repair the defect. Consequently there may be an overlap between the proteins involved in semi-conservative DNA replication and DNA repair. Direct experimental proof of this with demonstration of involvement of PCNA has recently come from analysis of a cell free model of DNA damage15. Furthermore, a role in DNA excision repair is reflected in the expression of PCNA in non-cycling normal human keratinocytes in vivo after mild UV exposure16. The gene encoding PCNA has been cloned from a number of evolutionarily diverse species' 7-21. Although there is variation at the DNA level, there are only four amino acid differences between rat and human PCNA and about 70% of the Drosophila protein is identical to the rat and human proteins. Furthermore, while the sequence of yeast PCNA shows only 35% homology at the DNA level with man, the molecules are functionally interchangeable22. The evolutionary conservation of PCNA throughout the animal (and plant) kingdom is entirely consistent with the product of the gene having an essential role in DNA replication in eukaryotes. Regulation of PCNA expression is complex22.PCNA expression is regulated at both the transcriptional and post-transcriptional leve122-24. Induction of PCNA mRNA by growth factors is well d o c ~ r n e n t e d ' ~ * ~ ~ - ~ ~ However, it is important to realise that there appear to be differences between the regulation in quiescent cells and those which are continuously cycling22,26. In the latter there is very little variation in protein or mRNA levels during the cell cycle. While the PCNA promoter region has been the contribution of transcriptional control is still unclear22. However, it is of note that homeodomain-containing proteins bind to the PCNA 591
592
1). McCorniick and P.A. Hall
pronioter'y~". Given that in other contexts homeodomain proteins regulate gradients of gene expression, it might be that similar mechanisms regulate the graded proliferative activity in certain tissues (e.g. in the intestinal crypt). Regulation of PCNA expression by post-transcriptional mechanisms appears to be very important, at least in vitro2?, Of particular note is the observation that PCNA m K N A is stabilized, and can thus be translated into protein with greater efficiency, in the presence of growth factors15.j1. It would alppear that many cells continuously make PCNA mRNA (at least at low levels) but rapidly degrade this without translation. Consequently cells are able, under the correct circumstances, to rapidly respond to growth factors by inducing PCNA production by stabilizing PCNA mRNA. The mechanism underlying this is intriguing and involves alteration in mRNA stability as a consequence of splicing of intron 422.3'. A further point is that oncogenes may participate in the regulation of PCNA mRNA level^^'-^^, and it is conceivable that alterations of oncogene expression in neoplasia may lead to the deregulation of PCNA expression. A wide range of antibodies thiat recognize PCNA are available. In addition to human sera from patients with SLB and related auto-immune diseases, a number of monoclonal antibodies have been generated. Commercially available reagents includle the monoclonal antibodies 19A2, 19F434*25 and PC103h. PClO and several related antibodies were generatled by immunizing mice with recombinant human PCNA expressed in E . colij6. It is apparent that the epitopes recognized by these antibodies are different37 and there are differences in the effects of fixation and processing on the detectability of these epitopes. Moreover, there appear to be differences between the immunochemical behaviour of these antibodies in comparison with the human auto-antibodie~j~. This is perhaps a partial explanation for discrepancies between reports on the relationship between PCNA immunoreactivity and other cell cycle markers. In addition, the differences observed between antibodies to PCNA may be the physical nature of the immunogens involved37and, in particular, whether they are in the native state (as seems to be the case with the autoantibodies) or are denatured (as in the case of PClO for example). Whatever the explanation, it is essential to recognize that different antibodies may have very different properties even though they recognize the same protein. This point is illustrated by the observation that one particular antibody, PC9. raised to recombinant PCNA only recognizes PCNA when in the nucleolus. This particular epitope is not seen anywhere else in the cellj6. In addition to the existence of differences between 1r)s31.
antibodies that recognize PCNA, it is clear that there are important technical considerations. The effects of different fixation conditions are of particular concern. These include duration of fixation, size of tissue block and type of fixative3x.For example, prolonged fixation can dramstically reduce PClO imrnunorea~tivity~~. PCNA detected by the 19A2 antibody after methanol fixation is closely associated with S phasejg. This is not seen in histological material with PCl 0, although under some conditions of enzyme treatment and tixation S phase distribution for PCNA can be seen by flow cytometric a n a l y ~ i s ~ " . ~ ' . Whether specific S phase PCNA immunoreactivity could be identified in conventionally fixed and processed tissue sections remains uncertain. The practice of baking sections on to glass can also be detrimental to PCNA immunoreactivity, although some degree of antigen retrieval using microwave methods may be possible. Of considerable importance is the observation that low levels of PCNA may be present throughout the cell As a consequence, varying the sensitivity of the immunohistological detection system may alter the number of PCNA immunoreactive cells. In the original description of PClO it was reported that cells at the lower end of small intestinal villi showed weak PCNA immunoreactivity. This was explained by the half-life of the PCNA protein being in excess of 2 0 h5. Support for this interpretation comes from the work of Scott et aL4j who reported that in tumour xenografts with a defined growth fraction and a cell cycle time of about 20 h the number of PCNA immunoreactive cells was nearly 100%.Thus, there may be situations where cells that have recently left the cell cycle express PCNA. PCNA is apparently expressed in non-cycling cells in other situations including in normal tissues adjacent to t u m o u r ~and ~ ~ possibly in some tumours where the number of PCNA immunoreactive cells does not correlate with other indices of p r ~ l i f e r a t i o n ~Hall ~ . et ~71.~' speculated that this might reflect the role of growth factors in regulating PCNA mRNA stability and protein expression. Some recent experiments have confirmed these observations and support a role for growth factors in mediating the expression of PCNA in non-cycling cells. Human carcinoma cell lines were injected into the liver or kidney of nude mice. Using tritiated thymidine a s a n objective measure of proliferation, control liver or kidney showed very few S phase cells and a similar number of PClO immunoreactive cells. In contrast, in animals with xenografts in liver or kidney, numerous PClO immunoreactive cells were seen but this was not mirrored by the number of S phase cells. In a separate set of experiments, rats fed by total parenteral nutrition were given infusions of TGFcr or EGF. In the pancreatic ductal and islet
Complexities ofproli/erating cell nuclear antigen
epithelium of both TGFa and EGF treated animals there was a more than tenfold increase in the number of PClO immunoreactive cells compared with controls, while there was no change in number of S phase cells. These experiments demonstrate that PCNA immunoreactivity (as detected by PC10) can occur without cell proliferation in association with neoplasia, and that this may be mediated by growth factors in V ~ V O In ~ ~simple . terms it would seem that expression of PCNA is a necessary but not sufficient requirement for proliferation. In summary, PCNA is a nuclear protein that is intimately involved in DNA synthesis and can be detected immunologically. There are important and poorly understood differences between the patterns of expression of some epitopes on the PCNA molecule and thus differences may exist in the data generated by different anti-PCNA antibodies. The effect of technical factors, including fixation, on patterns of PCNA staining should not be underestimated. The association of PCNA with DNA repair processes indicates that the expression of PCNA is not necessarily associated only with DNA synthesis in the context ofS phase. Finally, since PCNA is a necessary but not sufficient requirement for DNA synthesis (whether scheduled on unscheduled) PCNA may be expressed by cells that are not cycling, and this induction may be mediated by increased mRNA stability induced by growth factors. What message does this complex story have? The use of PCNA antibodies as markers of cell proliferation is not simple and straightforward but requires careful analysis and consideration of all these caveats (and perhaps others yet to be identified). As a general point, this story indicates the absolute requirement for us as pathologists to have a clear understanding of the immunological reagents we use and the biology of the antigens recognized. The PCNA story is similar to that recently put forward for the immunohistological detection of the p5 3 oncoprotein46-oversimpZ$cation will lead to false dawns! By all means employ immunological reagents but interpret with care, be cautious in drawing conclusions and always consider the biology of the molecules being investigated.
References 1. Miyachi K. Fritzler MJ,Tan EM. Autoantibody to a nuclear antigen in proliferating cells. 1. Irnmunol. 1978; 121: 2228-2234. 2. Tan CK, Castillo C, So AG, Downey KM. An auxiliary protein for DNA polymerase4 from foetal calf thymus. ]. Biol. Chem. 1986: 261; 12310-1231 6. 3. Prelich G. Tan CK. Kostura M et al. Functional identity of proliferating cell nuclear antigen and DNA polymerase 6 auxiliary protein. Nature 1987; 3 2 6 517-520. 4. Bravo R, Frank R. Blundell PA, Macdonald-Bravo H. Cyclin/PCNA
59 3
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