Deregulation of cyclin E expression has been associated with a broad spectrum of human malignancies. Analysis of DNA replication in cells constitutively expressing cyclin E at levels similar to those observed in a subset of tumor-derived cell lines indicates that initiation of replication and possibly fork movement are severely impaired. Such cells show a specific defect in loading of initiator proteins Mcm4, Mcm7, and to a lesser degree, Mcm2 onto chromatin during telophase and early G1 when Mcm2–7 are normally recruited to license origins of replication. Because minichromosome maintenance complex proteins are thought to function as a heterohexamer, loading of Mcm2-, Mcm4-, and Mcm7-depleted complexes is likely to underlie the S phase defects observed in cyclin E–deregulated cells, consistent with a role for minichromosome maintenance complex proteins in initiation of replication and fork movement. Cyclin E–mediated impairment of DNA replication provides a potential mechanism for chromosome instability observed as a consequence of cyclin E deregulation.

Peroxisome proliferator-activated receptor ␥ (PPAR␥) coactivator-1␣ (PGC-1␣) is a highly regulated transcriptional coactivator that coordinates energy metabolism in mammals. Misregulation of PGC-1␣ has been implicated in the pathogenesis of several human diseases, including diabetes, obesity, and neurological disorders. We identified SCF Cdc4 as an E3 ubiquitin ligase that regulates PGC-1␣ through ubiquitin-mediated proteolysis. PGC-1␣ contains two Cdc4 phosphodegrons that bind Cdc4 when phosphorylated by Glycogen Synthase Kinase 3␤ (GSK3␤) and p38 MAPK, leading to SCF Mitochondrial dysfunction and alterations in PGC-1 levels have been implicated in the pathogenesis of several diseases, including diabetes, heart disease, and neurological disorders Leone et al. 2005;Cui et al. 2006;Finck and Kelly 2006;St-Pierre et al. 2006). The reduced levels of PGC-1␣ and PGC-1␤ in the muscle of type 2 diabetic patients and individuals predisposed to diabetes, have been proposed to underlie decreased mitochondrial function and the development of insulin resistance (Mootha et al. 2003;Patti et al. 2003). Loss of PGC-1␣ activity in the heart correlates with heart fail-

Overexpression of cyclin E, an activator of cyclin-dependent kinase 2, has been linked to human cancer. In cell culture models, the forced expression of cyclin E leads to aneuploidy and polyploidy, which is consistent with a direct role of cyclin E overexpression in tumorigenesis. In this study, we show that the overexpression of cyclin E has a direct effect on progression through the latter stages of mitotic prometaphase before the complete alignment of chromosomes at the metaphase plate. In some cases, such cells fail to divide chromosomes, resulting in polyploidy. In others, cells proceed to anaphase without the complete alignment of chromosomes. These phenotypes can be explained by an ability of overexpressed cyclin E to inhibit residual anaphase-promoting complex (APCCdh1) activity that persists as cells progress up to and through the early stages of mitosis, resulting in the abnormal accumulation of APCCdh1 substrates as cells enter mitosis. We further show that the accumulation of securin and cyclin B1 can account for the cyclin E–mediated mitotic phenotype.

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Summary Cell cycle progression is regulated by the cyclin-dependent kinase (Cdk) family of protein kinases, so named because their activation depends on association with regulatory subunits known as cyclins [1]. Cyclin E normally accumulates at the G1/S boundary, where it promotes S phase entry and progression by activating Cdk2. In normal cells, cyclin E/Cdk2 activity is associated with DNA replication-related functions [2]. However, deregulation of cyclin E leads to inefficient assembly of pre-replication complexes [3], replication stress [4], and chromosome instability [5]. In malignant cells, cyclin E is frequently overexpressed, correlating with decreased survival in breast cancer patients [6, 7]. Transgenic mice deregulated for cyclin E in the mammary epithelia develop carcinoma [8], confirming that cyclin E is an oncoprotein. However, it remains unknown how cyclin E-mediated replication stress promotes genomic instability during carcinogenesis. Here we show that deregulation of cyclin E causes human mammary epithelial cells to enter into mitosis with short unreplicated genomic segments at a small number of specific loci, leading to anaphase anomalies and ultimately deletions. Incompletely replicated regions are preferentially located at late-replicating domains, fragile sites and breakpoints, including the mixed-lineage leukemia breakpoint cluster region (MLL BCR). Furthermore, these regions are characterized by a paucity of replication origins or unusual DNA structures. Analysis of a large set of breast tumors shows a significant correlation between cyclin E amplification and deletions at a number of the genomic loci identified in our study. Our results demonstrate how oncogene-induced replication stress contributes to genomic instability in human cancer.