DNA replication licensing and human cell proliferation

The convergence point of mitogenic and transforming growth regulatory pathways is the initiation of chromosomal replication, the core of which is assembly of pre-replicative complexes (pre-RCs) resulting in chromatin being "licensed" for replication in the subsequent S phase (figure 1). This evolutionary conserved final and important step in growth control is therefore an attractive target for therapeutic and diagnostic intervention.

Control of chromosomal replication

The fundamental growth regulatory mechanisms that allow cells to withdraw from the proliferative cell cycle into quiescent or differentiated out-of-cycle states operate stringently in untransformed cells but are defective in transformed cells. Using a combination of tissue-culture model systems and novel cell-free systems that initiate semi-conservative DNA replication in vitro (figure 2) (1) we are investigating the causal molecular events that establish replication competence as cells re-enter the proliferative cycle and how these mechanisms become deregulated in early tumorigenesis. These studies focus on the key replication licensing factors origin-recognition complex (ORC), Cdc6, Cdt1, and the minichromosome maintenance (MCM) family of proteins (Mcm2-7) and on cellular regulators of pre-RC function, including the Dbf4-dependent Cdc7 kinase, S-phase promoting cyclin/CDK complexes, and the inhibitor geminin. Using direct biochemical and functional studies in the reductionist cell-free DNA replication assay, we have shown that a powerful ubiquitous mechanism by which cells suppress growth in quiescent, differentiated, and replicative senescent out-of-cycle states is by downregulation of the Cdc6 and MCM replication licensing factors (1,2). Moreover, we have shown that expression of Cdc6 is rate limiting in late G1 for entry into S phase, a potential target for tumorigenesis (1) and that failure to downregulate the Cdc6 and MCM proteins characterises the block to differentiation found in dysplasia, a precursor neoplastic lesion of epithelia (figure 3) (3,4).

Growth-regulating mechanisms in human tissues and their deregulation in neoplasia

The constituents of the pre-RC can be regarded as relay stations coupling growth-regulatory pathways with initiation of DNA replication. Regulation of pre-RC function is thus a powerful mechanism for control of cell proliferation in tissues of multicellular organisms. We are therefore systematically studying expression profiles of replication licensing factors and cellular and viral modulators of pre-RC function (figure 4) in permanent, stable, and self-renewing human tissues, and we are determining how these key replication proteins become deregulated in the different tumour types arising from these tissues. Results of our studies with antibodies raised against the MCM family of proteins have shown that these proteins represent novel biomarkers of growth that identify not only proliferating cells but also licensed cells with proliferative potential (2). We have shown that the MCM proteins, which swivel along the DNA molecule unwinding the two daughter strands (DNA helicase) are deregulated at an early stage in epithelial carcinogenesis and therefore are of potential value in cancer diagnosis (3,4).

Replication licensing proteins as diagnostic cancer markers and therapeutic targets

We have shown in several self-renewing stem-cell tissues, including oesophagus, cervix, bladder, and prostate, that MCM proteins can be potentially exploited as sensitive markers to facilitate early cancer detection. Using a two site time-resolved immunofluorometric assay for biochemical detection of MCM replication licensing factors in body fluids we have shown - in the first blinded clinical trial using this approach - that Mcm5 represents a powerful diagnostic agent for non-invasive detection and surveillance of patients with genitourinary tract malignancies (figure 5) (5). Supported by Cancer Research UK and industrial sources, we are about to start a multicentre trial in the North of England to validate this simple non-invasive test for front-line clinical applications in the management of patients with prostate and bladder cancer.

Exploiting the multidisciplinary environment at the Wolfson Institute for Biomedical Research, we are using a combination of structural biology and medicinal chemistry to dissect pre-RC function at the molecular level, with the aim of designing small molecule inhibitors to selectively target the replication licensing machinery in cancer cells. The cell-free DNA replication systems that we have developed are being exploited as high-throughput screens to identify candidate inhibitors.