Most cells in the human body have two sets of 23 chromosomes, one from each parent. Chromosomes are single pieces of coiled DNA containing many genes. Chromosomes are replicated in a tightly controlled process during cell division so that each chromosome is replicated only once. DNA re-replication is an abnormal form of replication that produces a mixture of partially and fully completed chromosomes. This results in cells with repeated DNA content of between four and eight sets of chromosomes. To better understand how over-replication occurs, the investigators on this project will look for genes that prevent this process. Using small interfering RNAs (siRNAs), which selectively and systematically inhibit gene activity, the researchers will screen for genes that, when silenced, lead to over-replication of DNA in colorectal cancer cells. The study may help scientists better understand DNA re-replication and identify new treatment targets for colorectal and other cancers, since cancer cells may be particularly vulnerable to triggering re-replication.
Genome duplication in mammals normally occurs only once each time a cell divides. This restriction is circumvented on rare occasions during animal development to allow certain cells to differentiate into specialized polyploidy cells containing multiple copies of their nuclear genome. Such cells are stable, but no longer proliferate. Otherwise, agents that cause mammalian cells to re-replicate their DNA before completing mitosis cause replication forks to stall and DNA strands to break. These events trigger DNA damage response pathways and eventually, apoptosis.
Such events can also lead to the progression of human disease, especially cancer. Unfortunately, the mechanisms that prevent over-replication of DNA during proliferation of human cells are still poorly understood. Therefore, the goal of this project is to identify genes that contribute to preventing either DNA re-replication or the induction of polyploidy in human cells. To this end, we are screening for siRNAs that induce over-replication in human cancer cells. Identified genes are being screened further in a panel of both cancer and normal cells to identify cancer-selective targets. These efforts will not only illuminate genes associated with this poorly-mapped cellular process, but identify new molecular targets for clinical development.
Screen for DNA Re-Replication in Cancer Cells
Eunice Kennedy Shriver National Institute of Child Health and Human Development
Mel Depamphilis, Ph.D.
George Washington University
Wenge Zhu, Ph.D.
National Center for Advancing Translational Sciences
Madhu Lal-Nag, Ph.D.
Public Health Impact
This study will reveal determinants of DNA re-replication and identify putative strategies for the treatment of cancer.
Zhu W, Depamphilis ML. Selective killing of cancer cells by suppression of geminin activity. Cancer Res, 2009;69(11):4870-4877.
These studies have confirmed the acute importance of geminin in the replication of cancer cells and identified additional genes, which are the subject of ongoing follow-up studies.
The transfection of siRNA targeting geminin into HCT-116 colorectal cancer has previously been shown to induce a significant amount of DNA re-replication. Based on these findings, we conducted preliminary studies to determine whether these observations would translate to a high-throughput amenable RNAi assay. As shown in the figure, the transfection of an siRNA against geminin induced a significant amount of re-replication in HCT-116 cells, 72 h post-transfection. Furthermore, a comparison of negative control and geminin siRNA transfected cells indicated a good assay Z"-factor of 0.5 in 384 well plates. Pilot screens comprising two independent screens of an siRNA library directed against the human kinome also showed good correlation. We have now completed a comprehensive genome-wide screening campaign. Identified genes are the subject of intense validation and secondary assays.