Perturbation of DNA replication initiation arrests human cells in G1, pointing towards an origin activation checkpoint. We used RNAi against Cdc7 kinase to inhibit replication initiation and dissect this checkpoint in fibroblasts. We show that the checkpoint response is dependent on three axes coordinated through the transcription factor FoxO3a. In arrested cells, FoxO3a activates the ARF-|Hdm2-|p53-p21 pathway and mediates p15 INK4B upregulation; p53 in turn activates expression of the Wnt/b-catenin signalling antagonist Dkk3, leading to Myc and cyclin D1 downregulation. The resulting loss of CDK activity inactivates the Rb-E2F pathway and overrides the G1-S transcriptional programme. Fibroblasts concomitantly depleted of Cdc7/FoxO3a, Cdc7/p15, Cdc7/p53 or Cdc7/Dkk3 can bypass the arrest and proceed into an abortive S phase followed by apoptosis. The lack of redundancy between the checkpoint axes and reliance on several tumour suppressor proteins commonly inactivated in human tumours provides a mechanistic basis for the cancer-cell-specific killing observed with emerging Cdc7 inhibitors.

Locus control regions (LCRs) alleviate chromatinmediated transcriptional repression. Incomplete LCRs partially lose this property when integrated in transcriptionally restrictive genomic regions such as centromeres. This frequently results in position effect variegation (PEV), i.e. the suppression of expression in a proportion of the cells. Here we show that this PEV is in¯uenced by the heterochromatic protein SUV39H1 and by the Polycomb group proteins M33 and BMI-1. A concentration variation of these proteins modulates the proportion of cells expressing human globins in a locus-dependent manner. Similarly, the transcription factors Sp1 or erythroid Kru È ppel-like factor (EKLF) also in¯uence PEV, characterized by a change in the number of expressing cells and the chromatin structure of the locus. However, in contrast to results obtained in a euchromatic locus, EKLF in¯uences the expression of the g-more than the b-globin genes, suggesting that the relief of silencing is caused by the binding of EKLF to the LCR and that genes at an LCR proximal position are more likely to be in an open chromatin state than genes at a distal position. Keywords: gene activation/position effects/transcription IntroductionThe observation that the presence of the b globin locus control region (LCR) results in copy number-dependent expression of a globin transgene, independent of its position of integration in the host genome, led to the proposal that LCR function includes the establishment and/or maintenance of an open chromatin structure (Grosveld et al., 1987;Festenstein et al., 1996;Milot et al., 1996). Partial LCRs have lost this property and are very sensitive to position effects, particularly when integrated into transcriptionally repressive regions in the genome of transgenic mice (reviewed in Kioussis and Festenstein, 1997). Such integration leads to expression in only a proportion of the cells, which is similar to classical position effect variegation (PEV) (reviewed in Karpen, 1994) or to a novel cell timing position effect, in which all cells express but for only part of the time (Milot et al., 1996). The PEV expression was found in mice containing a deletion of the hypersensitive site 2 (HS2) of the b globin LCR, but only when the transgene was integrated in transcriptionally repressive regions such as found in centromeres (Milot et al., 1996). The subpopulation of erythroid cells that did not transcribe the human b globin gene was associated with a loss of DNase I hypersensitivity within the human b globin locus. Very similar results were obtained in other studies involving partial LCRs (Robertson et al., 1995;Festenstein et al., 1996;Boyer et al., 1997;Guy et al., 1997).Many transcription factors bind to the human b globin locus, but their role in transcriptional activation is not yet clear. An exception is the erythroid-speci®c erythroid Kru Èppel-like factor (EKLF) , which is essential for adult b globin gene expression (Nuez et al., 1995;Perkins et al., 1996;Wijgerde et al., 1996). It binds in vivo to a GT motif with...

Purpose: The DNA replication licensing machinery is integral to the control of proliferation, differentiation, and maintenance of genomic stability in human cells. We have analyzed replication licensing factors (RLF), together with DNA ploidy status, to investigate their role in progression of penile squamous cell carcinoma and to assess their utility as novel prognostic tools. Experimental Design: In a cohort of 141 patients, we linked protein expression profiles of the standard proliferation marker Ki67 and the RLFs Mcm2 and geminin to clinicopathologic variables, ploidy status, and clinical outcome. Results: Increased Ki67, Mcm2, and geminin levels were each significantly associated with arrested tumor differentiation (P < 0.0001) and aneuploidy (P ≤ 0.01). Accelerated cell cycle progression was linked to increasing tumor size, stage, and depth of invasion. Aneuploid tumors significantly correlated with tumor grade (P < 0.0001). Biomarker expression and DNA ploidy status were significant predictors of locoregional disease progression [Mcm2 (P = 0.02), geminin (P = 0.02), Ki67 (P = 0.03), and aneuploidy (P = 0.03)] in univariate analysis. Importantly, aneuploidy was a strong independent prognosticator for overall survival (hazard ratio, 4.19; 95% confidence interval, 1.17-14.95; P = 0.03). Used in conjunction with conventional pathologic information, multiparameter analysis of these variables can stratify patients into low-or high-risk groups for disease progression (Harrell's c-index = 0.88). Conclusions: Our findings suggest that RLFs and tumor aneuploidy may be used as an adjunct to conventional prognostic indicators, identifying men at high risk of disease progression. Our results also identify the DNA replication initiation pathway as a potentially attractive therapeutic target in penile squamous cell carcinoma. (Clin Cancer Res 2009;15(23):7335-44)