Cyclin-dependent kinases (CDKs) play a central role in the orderly transition from one phase of the eukaryotic mitotic cell division cycle to the next. In this context, p27 Kip1 (one of the CIP/KIP family of CDK specific inhibitors in mammals) or its functional analogue in other eukarya prevents a premature transition from G1 to S-phase. Recent studies have revealed that expression of a second member of this family, p57 Kip2 , is induced as trophoblast stem (TS) cells differentiate into trophoblast giant (TG) cells. p57 then inhibits CDK1 activity, an enzyme essential for initiating mitosis, thereby triggering genome endoreduplication (multiple S-phases without an intervening mitosis). Expression of p21 Cip1 , the third member of this family, is also induced in during differentiation of TS cells into TG cells where it appears to play a role in suppressing the DNA damage response pathway. Given the fact that p21 and p57 are unique to mammals, the question arises as to whether one or both of these proteins are responsible for the induction and maintenance of polyploidy during mammalian development. The road to polyploidyWhen metazoan cells proliferate, they employ the mitotic cell cycle in which separation of sibling chromosomes during mitosis (M-phase) and DNA synthesis during genome duplication (S-phase) are separated by two intervening gaps of time called the G1 and G2-phases to generate a repeating series of events: M→G1→S→G2→M. Cell division (cytokinesis) occurs immediately after mitosis. Cell growth occurs primarily during G1-phase. In addition, metazoan cells can exit their mitotic cell cycle and enter a quiescent state termed G0 in which the living state is maintained in the absence of either cell growth or proliferation. Mitotic cell cycles restrict genome duplication to once and only once per cell division. Therefore, G1-phase somatic cells contain two copies of their genome (2N or diploid), whereas somatic cells in G2 or M-phases are tetraploid (4N DNA). Cells with greater than 4N DNA content are referred to as polyploid.Polyploidy can result from aberrant DNA re-replication during S-phase. DNA re-replication occurs when newly assembled replication forks re-replicate parts of the genome that have already been replicated, resulting in replication bubbles within replication bubbles [1]. This occurs when one or more of the normal controls that prevent reutilization of replication origins during S-phase is circumvented. For example, DNA replication can be induced in some metazoan cells either by over-expression of Cdt1, a protein essential for loading the replicative MCM DNA helicase, or by suppression of the Cdt1 specific inhibitor geminin. Both changes promote loading of the MCM helicase at replication origins [2]. As DNA re-

Development of a fertilized egg into an adult human requires trillions of cell divisions, the vast majority of which duplicate their genome once and only once. Nevertheless, trophoblast giant cells and megakaryocytes in mammals circumvent this rule by duplicating their genome multiple times without undergoing cell division, a process generally referred to as 'endoreduplication'. In contrast, arthropods such as Drosophila endoreduplicate their genome in most larval tissues, as well as in many adult tissues. Endoreduplication requires that cells prevent entrance into or completion of mitosis and cytokinesis under conditions that permit assembly of prereplication complexes. In addition, cells must prevent induction of apoptosis in response to incomplete DNA replication or DNA damage that may occur during the ensuing sequence of 'endocycles'. Thus, developmentally regulated endoreduplication results in terminal cell differentiation. Recent progress has revealed both differences and similarities in the mechanisms employed by flies and mammals to change from mitotic cell cycles to 'endocycles'. The critical step, however, appears to be switching from a CDK-dependent form of the anaphase promoting complex (APC) to one that functions only in the absence of CDK activity.

Previous studies have shown DNA re-replication can be induced in cells derived from human cancers under conditions in which it is not possible for cells derived from normal tissues. Because DNA re-replication induces cell death, this strategy could be applied to the discovery of potential anticancer therapeutics. Therefore, an imaging assay amenable to high-throughput screening was developed that measures DNA replication in excess of four genomic equivalents in the nuclei of intact cells and indexes cell proliferation. This assay was validated by screening a library of 1,280 bioactive molecules on both normal and tumor-derived cells where it proved more sensitive than current methods for detecting excess DNA replication. This screen identified known inducers of excess DNA replication, such as inhibitors of microtubule dynamics, and novel compounds that induced excess DNA replication in both normal and cancer cells. In addition, two compounds were identified that induced excess DNA replication selectively in cancer cells and one that induced endocycles selectively in cancer cells. Thus, this assay provides a new approach to the discovery of compounds useful for investigating the regulation of genome duplication and for the treatment of cancer. Mol Cancer Res; 9(3); 294-310. Ó2011 AACR.

Nuclear genome duplication is normally restricted to once per cell division, but aberrant events that allow excess DNA replication (EDR) promote genomic instability and aneuploidy, both of which are characteristics of cancer development. Here we provide the first comprehensive identification of genes that are essential to restrict genome duplication to once per cell division. An siRNA library of 21,584 human genes was screened for those that prevent EDR in cancer cells with undetectable chromosomal instability. Candidates were validated by testing multiple siRNAs and chemical inhibitors on both TP53+ and TP53- cells to reveal the relevance of this ubiquitous tumor suppressor to preventing EDR, and in the presence of an apoptosis inhibitor to reveal the full extent of EDR. The results revealed 42 genes that prevented either DNA re-replication or unscheduled endoreplication. All of them participate in one or more of eight cell cycle events. Seventeen of them have not been identified previously in this capacity. Remarkably, 14 of the 42 genes have been shown to prevent aneuploidy in mice. Moreover, suppressing a gene that prevents EDR increased the ability of the chemotherapeutic drug Paclitaxel to induce EDR, suggesting new opportunities for synthetic lethalities in the treatment of human cancers.