DNA replication-initiation proteins: Novel cancer detection markers and anticancer targets.

摘要

DNA replication is one of the fundamental processes in cell proliferation. Initiation of DNA replication is regulated by the binding of the initiation proteins to the DNA elements (replicators and origins). In this thesis, we investigated replication-initiation proteins in cancer detection and anticancer strategy and identified some potential replication origins in the human genome.;Since replication-initiation proteins are required for DNA replication in all cancer cells, we tested whether these proteins could be used as novel markers for bladder cancer diagnosis and surveillance. In voided urine cells, the mRNAs of CDC6, CDC45, MCM2 and CDT1 were examined by RT-PCR. When we combined two markers, either CDC45 with CDC6 or CDC45 with CDT1, the molecular test was 100% (46/46) sensitive and 94.5% (52/55) specific. For surveillance of previous TCC (transitional cell carcinoma) patients, 92 with clinical recurrent TCC and 321 with no recurrence, the optimal sensitivity (96.7%) and specificity (60.1%) could be obtained for the method of CDC45 combining CDC6; CDC45 combining CDT1 also gave 98.9% sensitivity and 43.6% specificity. Therefore, the molecular detection of mRNAs of the replication-initiation proteins is a promising and practical method for cancer diagnosis and surveillance.;To help to develop a novel anticancer strategy, both antisense oligodeoxynucleotides (as-ODNs) and siRNAs targeted to CDC6, CDC45 and MCM2 were used to specifically inhibit target genes expression as measured by RT-PCR, DNA replication as assayed by BrdU incorporation, and cell proliferation as determined with the WST-1 assay in HepG2 and Hep3B liver cancer cell lines. Furthermore, the as-ODNs and siRNAs could induce apoptosis in a p53-independent manner in cancer cells as examined with annexin V assay. Thus, as-ODNs and siRNAs targeted to the replication-initiation mRNAs are potential anticancer drugs, and these genes are attractive anticancer targets.;Since some of the replication-initiation proteins in human cells have been discovered and the binding of the initiation proteins should occur at or very near the replicators and replication origins, we used ChIP assay and tagged random PCR (T-PCR) followed by cloning to determine the localities of ORC2- and MCM2-binding so as to identify the positions of replicators and potential replication origins across the human genome. To date, 235 MCM2 or ORC2 putative binding sites have been identified and sequenced, and the physical interactions between some of the DNA fragments and the initiation proteins have been confirmed by independent standard ChIP assays. Whether these DNA fragments serve as origins or replicators needs further functional and biochemical studies.