Laura Barkley, Kathryn Leigh Eward, Hye-Kyung Hong, Sarah Kingsbury,
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.
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).
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).
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.
1 Stoeber K, Mills AD, Kubota Y, et al.
Cdc6 protein causes premature entry into S phase in a mammalian
cell-free system. EMBO J 1998; 17: 7219-29.
2 Stoeber K, Tlsty TD, Happerfield L, et al. DNA replication licensing
and human cell proliferation. J Cell Sci 2001; 114: 2027-41.
3 Williams GH, Romanowski P, Morris L, et al. Improved cervical
smear assessment using antibodies against proteins that regulate
DNA replication. Proc Natl Acad Sci USA 1998; 95: 14932-37.
4 Stoeber K, Halsall I, Freeman A, et al. Immunoassay for urothelial
cancers that detects DNA-replication protein Mcm5 in urine. Lancet
1999; 354: 1524-25.
5 Stoeber K, Swinn R, Prevost AT, et al. Diagnosis of genitourinary
tract cancer by detection of Mcm5 in urine sediments. J Natl Cancer
Inst 2002; 94: 1071-79.