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DePamphilis Lab: Section on Eukaryotic DNA Replication

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Research and Techniques

Genome Replication and Cancer

Cancer cells are abnormal cells that continue to proliferate under conditions where normal cells cannot. Cancer cells arise from genetic mutations and alterations that result predominantly from genomic instability. Genomic stability depends on the number of active replication origins per genome. Low origin densities produce fewer replication forks that must travel greater distances, thus increasing the probability that forks will stall or suffer damage. Conversely, high origin densities increase the possibility of DNA damage during the initiation process, and the possibility that too many forks will trigger a DNA damage response. Thus, the purpose of the mitotic cell cycle is to restrict genome duplication to once per cell division so that each daughter cell receives one complete set of chromosomes from each parent.

Initiation of DNA replication in eukarya is a highly conserved process that occurs in two steps. First, prereplication complexes (preRCs) are assembled at specific sites termed DNA replication origins that are distributed throughout the genome. This process (termed 'origin licensing') involves sequential assembly of a six subunit origin recognition complex (ORC), Cdc6 and Cdt1, which together with ORC load the six subunit DNA helicase Mcm2-7 (MCM helicase) onto chromatin. Replication origins in single cell eukarya are determined by DNA sequence, but those in multicellular eukarya are determined both by DNA sequence and epigenetic factors such as chromatin structure. PreRC assembly occurs as cells transit from anaphase to G1-phase of the mitotic cell cycle. The preRC is converted into an active replication complex by the action of two protein kinases, Dbf4 dependent kinase (DDK) and cyclin-dependent kinase (CDK).

Restricting genome duplication to once per cell division requires that cells do not reinitiate nuclear DNA replication within regions that have already replicated until cell division is completed. PreRC assembly occurs when CDK activity is suppressed during the anaphase to G1 phase transition, and preRC activation occurs when CDK and DDK activities are up-regulated during the G1 to S phase transition. PreRCs are then inactivated during S phase and preRC assembly inhibited until metaphase is completed. Mammalian cells accomplish this by multiple concerted pathways. Cdk2•Cyclin A/CcnA phosphorylates Orc1, Cdc6, and Cdt1, thereby suppressing their activities. Cdc6-P localizes to the cytoplasm. The ubiquitin ligase CRL1•Skp2 targets Orc1-P and Cdt1-P for export to the cytoplasm and degradation by the 26S proteasome. CRL4•Cdt2 ubiquitinates Cdt1, but only when Cdt1 is bound to both PCNA and DNA. Geminin binds Cdt1, thereby preventing the MCM helicase from loading onto ORC•Cdc6•chromatin sites.

Agents that interfere with the mechanisms that govern genome duplication frequently induce reinitiation of nuclear DNA replication before cells have entered mitosis (DNA re-replication). This results in the accumulation of cells containing a heterogeneous mixture of partially and fully completed chromosomes with a heterogeneous DNA content between tetraploid (4N) and octoploid (8N). These cells contain a giant aneuploid nucleus that frequently degenerates into micronuclei [termed "mitotic slippage"]. DNA re-replication produces an excess of DNA replication forks, thereby increasing the frequency of stalled forks and DNA damage, which induces apoptosis.

For a complete description of genome duplication and its relationship to cell division, human disease and evolution in the three domains of Life, see the textbook by M. L. DePamphilis and S. P. Bell, Genome Duplication, published by Garland Science.

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Selective Killing Of Cancer Cells By Induction Of DNA Re-Replication

Anti-Geminin siRNA selectively kills cancer cells in vitro

We proposed that Geminin might well be the "Achilles' heel" of the cancer cell, because cancer cells are far more dependent than normal cells on Geminin to regulate origin licensing (Zhu & DePamphilis, 2009). Geminin prevents DNA re-replication during S, G2 and early M phases by binding to Cdt1, thereby preventing further loading of replicative DNA helicases (MCM helicase) onto DNA. We found that siRNA suppression of Geminin expression in cells derived from a variety of cancers induces DNA re-replication, DNA damage, a DNA damage response, and finally apoptosis under conditions that cells derived from normal human tissues do not. These cancer cells were derived from carcinomas, adenocarcinomas, glioblastomas, and osteosarcomas. Under the same conditions, cells derived from normal tissues or immortalized cells that gave rise to these cancers continued to proliferate. In fact, cells derived from melanoma or cervical cancers also were resistant, suggesting that most human cells are insensitive to Geminin depletion. This distinction was not an experimental artifact, because DNA re-replication could be induced in normal cells if both Geminin and Cyclin A/CcnA expression were suppressed, either directly with individual siRNAs, or indirectly by suppressing expression of Emi1 (Lee et al., 2012). Emi1 prevents premature activation of the anaphase-promoting complex, a ubiquitin ligase that targets CcnA, CcnB and Geminin for degradation as cells exit metaphase. Therefore, cancer cells rely solely on Geminin to prevent DNA re-replication, whereas normal cells rely on additional mechanisms as well. In fact, human cells contain three cyclin A-dependent pathways that would prevent premature assembly of preRCs at replication origins.

Based on these results, we anticipate that suppressing Geminin activity in humans could selectively kill a variety of different cancer cells with little or no effect on non-cancerous tissues, because most of our cells reside in a quiescent state, and when they do proliferate, they are far less sensitive to Geminin depletion than are cancer cells. Therefore, drugs that prevent Geminin from relicensing replication origins should be excellent candidates for cancer chemotherapy.

Small Molecules That Mimic Anti-Geminin siRNA

We designed a quantitative high-throughput screen (qHTS) for small molecules that mimic the effects of siRNA against Geminin on human cells (Zhu et al., 2011). This assay, developed in collaboration with the NIH Center for Translational Therapeutics, quantifies the number of cells adhering to the bottom of a multi-well plate and the amount of nuclear DNA in each cell after culturing cells for two days in the presence of seven different concentrations of each drug. We identified 68 compounds that induce excess DNA replication in cancer cells, 13 of which had been reported previously, such as inhibitors of microtubule dynamics. Of these, only three were cancer cell selective. Tetraethylthiuram disulfide and 3 phenylpropargylamine induced DNA re-replication selectively in cancer cells, and SU6656 induced endocycles selectively in cancer cells. Their effective concentration at 50% maximum response (EC50) was at least 10-fold greater on MCF10A cells than on SW480 cells. Moreover, although tetraethylthiuram disulfide and SU6656 rapidly arrested normal cell proliferation, 3 phenylpropargylamine did not, making 3 phenylpropargylamine our first candidate for a small molecule that mimics anti-Geminin siRNA. We then applied this qHTS to the 343,078 compounds in the NIH Molecular Libraries Small Molecule Repository. From the primary screen, 2,215 compounds were chosen that appeared to selectively target SW480 cancer cells relative to MCF10A normal cells. Based on the results of three successive screens, we identified 127 compounds that appear to be cancer cell-selective. These compounds will now be validated and characterized both in vitro and in vivo.

Genes That Prevent Excess DNA Replication In Mammals

We have now screened the entire human genome for genes that mimic the Geminin phenotype as well as for all genes that are essential for preventing DNA re-replication in cancer cells as well as normal cells. The results revealed over 20 genes that are essential for preventing excess DNA replication in human cells and confirmed our hypothesis that Geminin is unique in its ability to kill cancer cells without killing normal cells. Our goal now is to use this information to identify the gene targets for the small molecules selected in our qHTS for their ability to trigger excess DNA replication in human cells. We anticipate that one or more of these molecules will prove to be effective in selectively killing cancer cells without preventing proliferation of normal cells.

Last Updated Date: 11/30/2012
Last Reviewed Date: 11/30/2012

Contact Information

Name: Prof Melvin L Depamphilis
Senior Investigator
Section on Eukaryotic DNA Replication
Phone: 301-402-8234
Fax: 301-480-9354
Email: depamphm@mail.nih.gov

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