Identification of novel anticancer drug candidates from Chinese medicinal herbs with DNA replication-initiation proteins as the targets
by Shen Yi
xv, 82 leaves : ill. (some col.) ; 30 cm
This thesis contains two main parts: (1) Developing a new screening strategy and identifying candidates for anticancer drugs with DNA replication-initiation proteins as the targets; (2) Identifying and characterizing Rrp12p as a novel DNA replication-initiation protein....[ Read more ]
This thesis contains two main parts: (1) Developing a new screening strategy and identifying candidates for anticancer drugs with DNA replication-initiation proteins as the targets; (2) Identifying and characterizing Rrp12p as a novel DNA replication-initiation protein.
In the first part, eukaryotic DNA replication is tightly controlled by DNA replication-initiation proteins and cell cycle check points to ensure that the genome is exactly copied once per cell cycle. Different from normal cells, most of cancer cells are deficient in the check point systems and have over-expression of DNA replication-initiation proteins. Therefore DNA replication-initiation proteins are excellent targets for cancer therapy. Inhibiting the replication-initiation proteins such as MCM (Minichromosome Maintenance) proteins effectively damages DNA replication of cancer cells and induces cancer cell apoptosis, leaving normal cell mostly unharmed. To develop novel anticancer drugs that inhibit DNA replication, a preliminary screen platform was set up to test chemicals from a traditional Chinese medicine library (TCM).
A fraction (numbered 303) of a tropical plant was identified from the screening platform. It specifically disrupts the interaction of MCM proteins in the yeast two-hybrid system, and induces cancer cells to undergo apoptosis in human cancer cell lines. In anticancer mechanism studies, fraction 303 inhibits DNA replication of cancer cells as determined by the BrdU assay, and affects the localizations of MCM-GFP proteins in cancer cells. Chromatin binding assays indicate that fraction 303 affects binding abilities of MCM proteins on chromatin. FACS results of block-and-release experiments show that fraction 303 arrests the cancer cells in G1 phase, and leads them to pro-apoptotic status. From these data, the anticancer mechanism of fraction 303 is that it disrupts the interaction of MCM protein and inhibits the formation of pre-replication complex (pre-RC) on chromatin. Consequently, incomplete DNA replication induces cancer cell apoptosis. Therefore, fraction 303 is a promising anticancer drug candidate.
In the second part, Rrp12p as a novel DNA replication-initiation protein was identified and characterized. In Saccharomyces cerevisiae, initiation of DNA replication (IDR) requires replication-initiation proteins and the replicator elements. Identification and characterization of DNA replication-initiation proteins have advanced our understanding of eukaryotic DNA replication.
A novel IDR gene, RRP12 (Ribosomal RNA Processing protein 12) was identified by screening a collection of td (temperature-sensitive degron) mutants by using the colony size assay. FACS analysis in block-and-release experiments indicates that Rrp12p is required for the initiation of DNA replication, but not for replication elongation or mitosis. Reciprocal co-immunoprecipitation experiments show that Rrp12p interacts with Mcm2p. Moreover, overexpression of RRP12 suppresses the temperature sensitive lethal phenotype of mcm3-1 mutant. These data suggest that Rrp12p is an essential DNA replication-initiation protein and plays roles in the assembly of pre-RC complex.