The complexities of proliferating cell nuclear antigen

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 complexities of 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 populations of 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 iden 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