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A group of homologous DNA replication origins and replicators dispersed throughout the human genome & anticancer compounds targeting DNA replication initiation proteins from Chinese medicinal herbals

Anticancer compounds targeting DNA replication initiation proteins from Chinese medicinal herbals

Authors Wang, Ziyi
Issue Date 2010
Summary Initiation of DNA replication is controlled by the cis-acting replicators and the trans-acting initiation proteins and their regulators. The replicator/replication origins have been defined as specific DNA sequences in yeasts. However, although over one thousand unique metazoan replicators/origins have been identified, they seem not to share similar features at the sequence level. On the other hand, initiation proteins including ORC, Noc3, Cdc6, Cdt1 and MCM are highly conserved in all eukaryotes, from yeast to humans, and DNA replication does not initiate randomly in somatic cells, implying the conserved regulation of initiation of DNA replication in eukaryotes. We have identified a group of general human replicators / replication origins by using chromatin immunoprecipitation (ChIP) with antibodies against ORC and MCM proteins. The replicators are homologous to one another and dispersed relatively evenly throughout the human genome with an average density of one in every ~100 kb. We found that active replication origins are located near the replicators by nascent strand abundance assays and nascent strand switching assays. In addition, DNA combing assays confirm that in vivo DNA replication initiates from the area of the replicators, and chromatin combing assays indicate significant overlap of the hORC2 binding sites and the replicator/origin sequences identified. Furthermore, episomal replication assays show that the replicator/origin-containing plasmids are capable of autonomous replication in human cells and that the replication of these plasmids was semi-conservative as demonstrated by CsCl density gradient centrifugation. Proteins involved in the initiation of DNA replication represent excellent targets for cancer therapy as well as biomarkers for cancer detection. More importantly, silencing of these proteins can induce apoptosis in cancer but not normal cells. Based on these observations, we have developed a platform for anticancer drug screening by employing the reverse yeast two-hybrid system and a pair of human cell lines, L-02 (normal liver cells) and HepG2 (liver cancer cells). After screening and subsequent testing, a small compound designated as M1 from Chinese medicinal herbs has been identified that can specifically disrupt the interaction between hMcm2 and hMcm6 which is essential for the functional MCM complex. We found that M1 could kill cancer cells but not normal cells. M1 also prevents the nuclear localization and chromatin association of hMcm2 and hMcm6, blocks DNA replication, and induces apoptosis in cancer cells. Furthermore, M1 shows its profound antitumor activity with little side effects in the nude mice xenograph models.
Note Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2010
Language English
Format Thesis
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