Discussion

The MCM and Cdc6 proteins are important regulators in the process of eukaryotic DNA replication. In vitro studies have shown that these proteins are detectable throughout the proliferative phases of the cell cycle but are lost in differentiation and quiescence (Williams et a l, 1998; Stoeber et a l, 1998), making them specific indicators of cell proliferation.

One marked advantage of MCM and Cdc6 proteins over other proliferation markers is the fact that their presence throughout the cell cycle has been well characterised. The pre- replicative complex is essential for the initiation of DNA replication and represents the point of convergence of numerous signalling pathways involved in cell growth. The MCM and Cdc 6 proteins are therefore better markers of cells in cycle than other candidates such as growth factor receptors or signal transduction molecules, the functions of which are inherently redundant. Moreover, antibodies against MCM and Cdc 6 proteins do not detect cells undergoing DNA repair, which is a feature of the existing ‘proliferation’ marker PCNA. The proteins of the pre-replicative complex are readily accessible in frozen tissues and cytological preparations (Williams et a l, 1998), such that antigen retrieval methods are not necessary for their detection. The antibodies used in this study have the additional benefit that they are able to localise MCM and Cdc 6 proteins in formalin-fixed, paraffin-embedded tissues.

This study examines MCM protein expression in a wide range o f tissue systems including skin, lung, colon and bladder. It was consistently observed that antibodies against MCM proteins identify a greater number of cells in cycle than do antibodies against PCNA and Ki67. Moreover antibodies against MCM proteins are able to detect proliferating cells in paraffin and frozen tissues, unlike antibodies against PCNA, which only reliably label cells in paraffin tissues and require antigen retrieval for optimal staining.

Although only a small range of tissues could be examined for the expression o f Cdc6 and Mcm7 proteins, as only limited amounts of rabbit polyclonal antibodies against these molecules were available, the evidence obtained suggests that Cdc6 and Mcm7 are as reliable in identifying proliferating cells as Mcm5 and Mcm2. Indeed, no significant difference was observed in the expression patterns of any of the pre-replication proteins examined in this study, with all antibodies used producing essentially similar results.

Immunohistochemical detection of MCM protein expression in normal tissues shows that these molecules are restricted to the basal and parabasal compartments of stratified epithelia. Loss of MCM proteins and Cdc 6 occurs in differentiating cells that have lost the ability to proliferate. In all of the stratified squamous epithelia examined there was around 15% overall nuclear staining with Mcm2, Mcm5 and PCNA, suggesting that there is close regulation of the proliferative components of these normal epithelia. Similar findings regarding normal proliferative compartments have also been made by others in studies of the expression of PCNA (Demeter et al., 1994), Ki67 (Isacson et al., 1996) Mcm2 (Todorov et al., 1998) and Mcm7 (Hiraiwa et al., 1997) in a range of tissues.

In this study, normal transitional epithelium of the bladder differed in that occasional surface cells showing nuclear staining for Mcm2 and Mcm5 were seen, although these accounted for 5% or less of the total surface urothelial cells. This observation may be related to the fact that transitional epithelium contains relatively few layers o f cells. As a result o f this, cells may transit through the epithelium in a relatively short period of time and reach the epithelial surface before the loss of MCM proteins associated with cell differentiation can occur.

The findings in colon and endometrium confirm those made by others with markers such as PCNA (Hall et al., 1990; Yu et al., 1993). In normal colon, expression o f Mcm2 and Mcm5 occurs in the lower half of colonic crypts, with loss of these proteins from differentiated cells in the upper portion of the crypts. A cyclical change occurs in the endometrium, with the percentage of stained cells varying with the menstrual cycle, resulting in a lower frequency of Mcm2 and Mcm5 expression in the secretory phase.

The observation that epithelial inclusion cysts o f the ovary show a high frequency of expression of MCM proteins is interesting. These inclusions are thought to represent invaginations of the ovarian surface epithelium, and are usually lined by a single layer of epithelial cells. It has been suggested that the majority of epithelial neoplasms of the ovary are likely to originate from these inclusions, rather than from the ovarian surface epithelium (Blaustein et al., 1982). Indeed, ovarian carcinoma antigens are expressed far more frequently in inclusion cyst epithelium than in surface epithelium (Scully, 1995). The presence o f a high frequency of expression of MCM proteins in these inclusions is intriguing and raises the possibility that a high level of proliferative activity may contribute to the development of neoplasia in such cells.

Altered localisation of MCM proteins occurs in dysplastic epithelia. All grades of dysplasia show staining for Mcm2 and Mcm5 in over 90% of basal cells. In addition, there is a strong correlation between the number of nuclei positive for Mcm2 and Mcm5 at the surface of dysplastic epithelia and the severity o f the dysplasia. Only 40% of surface epithelial cells express MCM proteins in mild dysplasia, compared to over 80% in severe dysplasia. This observation applies to most stratified epithelia studied, including those o f the skin, bladder and cervix.

Although studies of histological sections inevitably provide only ‘static’ representations of dynamic cellular processes, in vitro studies of MCM proteins have shown that these proteins are present throughout the cell cycle, but not in quiescent or differentiated cells (Stoeber et a/., 1998). Thus, in all 47 cases of epithelial dysplasia examined, the finding that over 80% of cells in high grade dysplasias, and 90% of basal cells in all grades of dysplasia, express MCM proteins indicates that the majority of dysplastic cells are in cycle at any given time point. These observations suggest that dysplastic cells may be characterised in functional terms as remaining in cell cycle, due to deregulation of normal controls over cell proliferation.

Interestingly, it has recently been shown that Mem? interacts with the E6 protein of human papillomavirus type 18 and is also a substrate of the E6-AP/E3 ubiquitin ligase (Kuhne and Banks, 1998). The functional significance o f such interactions in dysplastic epithelia remains uncertain. However, it may be speculated that bypass of normal pathways o f ubiquitination within a cell by viral or cellular oncogenes may contribute to stabilisation of proteins such as the MCMs within dysplastic and neoplastic cells.

It can be hypothesised that detection o f pre-replication proteins might enable the identification of dysplastic cells in clinical settings. The proteins may be o f value, for example, in the distinction of dysplastic cells from those showing reactive changes, which can often be a very difficult exercise using morphological criteria alone. The current data in bladder, skin and cervix suggests that pre-replication proteins show more restricted expression patterns in reactive/hyperplastic conditions than in dysplasia. Nevertheless, further work is required to rigorously test the potential clinical utility of the pre­ replication proteins, by examining their expression in a larger range of reactive conditions.

A difference was also observed in the staining pattern of well differentiated, moderately differentiated and poorly differentiated carcinomas, with a reduction in the number o f positively staining nuclei in better differentiated tumours (Table 3.5). This was

seen most clearly in well-differentiated SCC of the skin, where foci of unstained cells lacking expression of Mcm2 and Mcm5 proteins were identified in differentiated areas adjacent to keratin pearls. This observation is consistent with previous findings in normal tissues and cell lines (Williams et a l, 1998; Stoeber et al., 1998) that there is gradual loss of MCM proteins in cells undergoing differentiation. These results also agree with data from other groups showing an increase in the frequency of PCNA staining in less well differentiated TCCs of the bladder (Hattori et a l, 1995). However, the role of members of the pre-replicative complex in the process o f differentiation in epithelial cells has not been addressed experimentally and the functional significance of these observations in carcinomas and normal epithelia currently remains unclear.

Results from the immunoblotting experiments show that there is a quantitative difference in the level of Mcm5 protein between neoplastic and normal tissues. High levels of Mcm5 were observed in carcinomas of bladder and colon, although there was no detectable protein in the equivalent normal tissues (Figure 3.6). The immunofluorescence data further suggest that the overall increase in the level o f MCM proteins in neoplastic tissues is not due to overexpression of protein in individual cells. Instead the increase can be attributed to a greater number of cells showing normal levels of expression. In particular, frozen sections stained by immunofluorescence show patterns of staining similar to those observed using immunoperoxidase (Figures 3.7, 3.8) and may enable automated detection of MCM and Cdc6 proteins for future clinical applications.