The location and development of Replicon Cluster Domains in early replicating DNA

Background: It has been known for many years that in metazoan cells, replication origins are organised into clusters where origins within each cluster fire near-synchronously. Despite clusters being a fundamental organising principle of metazoan DNA replication, the genomic location of origin clusters has not been documented. Methods: We synchronised human U2OS by thymidine block and release followed by L-mimosine block and release to create a population of cells progressing into S phase with a high degree of synchrony. At different times after release into S phase, cells were pulsed with EdU; the EdU-labelled DNA was then pulled down, sequenced and mapped onto the human genome. Results: The early replicating DNA showed features at a range of scales. Wavelet analysis showed that the major feature of the early replicating DNA was at a size of 500 kb, consistent with clusters of replication origins. Over the first two hours of S phase, these Replicon Cluster Domains broadened in width, consistent with their being enlarged by the progression of replication forks at their outer boundaries. The total replication signal associated with each Replicon Cluster Domain varied considerably, and this variation was reproducible and conserved over time. We provide evidence that this variability in replication signal was at least in part caused by Replicon Cluster Domains being activated at different times in different cells in the population. We also provide evidence that adjacent clusters had a statistical preference for being activated in sequence across a group, consistent with the ‘domino’ model of replication focus activation order observed by microscopy. Conclusions: We show that early replicating DNA is organised into Replicon Cluster Domains that behave as expected of replicon clusters observed by DNA fibre analysis. The coordinated activation of different Replicon Cluster Domains can generate the replication timing programme by which the genome is duplicated.</p

Unreplicated DNA remaining from unperturbed S phases passes through mitosis for resolution in daughter cells

To prevent rereplication of genomic segments, the eukaryotic cell cycle is divided into two nonoverlapping phases. During late mitosis and G1 replication origins are “licensed” by loading MCM2-7 double hexamers and during S phase licensed replication origins activate to initiate bidirectional replication forks. Replication forks can stall irreversibly, and if two converging forks stall with no intervening licensed origin—a “double fork stall” (DFS)—replication cannot be completed by conventional means. We previously showed how the distribution of replication origins in yeasts promotes complete genome replication even in the presence of irreversible fork stalling. This analysis predicts that DFSs are rare in yeasts but highly likely in large mammalian genomes. Here we show that complementary strand synthesis in early mitosis, ultrafine anaphase bridges, and G1-specific p53-binding protein 1 (53BP1) nuclear bodies provide a mechanism for resolving unreplicated DNA at DFSs in human cells. When origin number was experimentally altered, the number of these structures closely agreed with theoretical predictions of DFSs. The 53BP1 is preferentially bound to larger replicons, where the probability of DFSs is higher. Loss of 53BP1 caused hypersensitivity to licensing inhibition when replication origins were removed. These results provide a striking convergence of experimental and theoretical evidence that unreplicated DNA can pass through mitosis for resolution in the following cell cycle

Combined PI3K and CDK2 inhibition induces cell death and enhances in vivo antitumour activity in colorectal cancer

Background: The phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway is commonly deregulated in human cancer, hence many PI3K and mTOR inhibitors have been developed and have now reached clinical trials. Similarly, CDKs have been investigated as cancer drug targets. Methods: We have synthesised and characterised a series of 6-aminopyrimidines identified from a kinase screen that inhibit PI3K and/or mTOR and/or CDK2. Kinase inhibition, tumour cell growth, cell cycle distribution, cytotoxicity and signalling experiments were undertaken in HCT116 and HT29 colorectal cancer cell lines, and in vivo HT29 efficacy studies. Results: 2,6-Diaminopyrimidines with an O4-cyclohexylmethyl substituent and a C-5-nitroso or cyano group (1,2,5) induced cell cycle phase alterations and were growth inhibitory (GI50<20 μM). Compound 1, but not 2 or 5, potently inhibits CDK2 (IC50=0.1 nM) as well as PI3K, and was cytotoxic at growth inhibitory concentrations. Consistent with kinase inhibition data, compound 1 reduced phospho-Rb and phospho-rS6 at GI50 concentrations. Combination of NU6102 (CDK2 inhibitor) and pictilisib (GDC-0941; pan-PI3K inhibitor) resulted in synergistic growth inhibition, and enhanced cytotoxicity in HT29 cells in vitro and HT29 tumour growth inhibition in vivo. Conclusions: These studies identified a novel series of mixed CDK2/PI3K inhibitors and demonstrate that dual targeting of CDK2 and PI3K can result in enhanced antitumour activity

Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) is a cell-cycle checkpoint effector and inducer of senescence, regulated by p53. Yet, evidence suggests that p21 could also be oncogenic, through a mechanism that has so far remained obscure. We report that a subset of atypical cancerous cells strongly expressing p21 showed proliferation features. This occurred predominantly in p53-mutant human cancers, suggesting p53-independent upregulation of p21 selectively in more aggressive tumour cells. Multifaceted phenotypic and genomic analyses of p21-inducible, p53-null, cancerous and near-normal cellular models showed that after an initial senescence-like phase, a subpopulation of p21-expressing proliferating cells emerged, featuring increased genomic instability, aggressiveness and chemoresistance. Mechanistically, sustained p21 accumulation inhibited mainly the CRL4–CDT2 ubiquitin ligase, leading to deregulated origin licensing and replication stress. Collectively, our data reveal the tumour-promoting ability of p21 through deregulation of DNA replication licensing machinery—an unorthodox role to be considered in cancer treatment, since p21 responds to various stimuli including some chemotherapy drugs

The high-affinity interaction between ORC and DNA that is required for replication licensing is inhibited by 2-arylquinolin-4-amines

In late mitosis and G1, origins of DNA replication must be "licensed" for use in the upcoming S phase by being encircled by double hexamers of the minichromosome maintenance proteins MCM2-7. A "licensing checkpoint" delays cells in G1 until sufficient origins have been licensed, but this checkpoint is lost in cancer cells. Inhibition of licensing can therefore kill cancer cells while only delaying normal cells in G1. In a high-throughput cell-based screen for licensing inhibitors we identified a family of 2-arylquinolin-4-amines, the most potent of which we call RL5a. The binding of the origin recognition complex (ORC) to origin DNA is the first step of the licensing reaction. We show that RL5a prevents ORC forming a tight complex with DNA that is required for MCM2-7 loading. Formation of this ORC-DNA complex requires ATP, and we show that RL5a inhibits ORC allosterically to mimic a lack of ATP

The location and development of Replicon Cluster Domains in early replicating DNA

Background: It has been known for many years that in metazoan cells, replication origins are organised into clusters where origins within each cluster fire near-synchronously. Despite clusters being a fundamental organising principle of metazoan DNA replication, the genomic location of origin clusters has not been documented.Methods: We synchronised human U2OS by thymidine block and release followed by L-mimosine block and release to create a population of cells progressing into S phase with a high degree of synchrony. At different times after release into S phase, cells were pulsed with EdU; the EdU-labelled DNA was then pulled down, sequenced and mapped onto the human genome.Results: The early replicating DNA showed features at a range of scales. Wavelet analysis showed that the major feature of the early replicating DNA was at a size of 500 kb, consistent with clusters of replication origins. Over the first two hours of S phase, these Replicon Cluster Domains broadened in width, consistent with their being enlarged by the progression of replication forks at their outer boundaries. The total replication signal associated with each Replicon Cluster Domain varied considerably, and this variation was reproducible and conserved over time. We provide evidence that this variability in replication signal was at least in part caused by Replicon Cluster Domains being activated at different times in different cells in the population. We also provide evidence that adjacent clusters had a statistical preference for being activated in sequence across a group, consistent with the ‘domino’ model of replication focus activation order observed by microscopy.Conclusions: We show that early replicating DNA is organised into Replicon Cluster Domains that behave as expected of replicon clusters observed by DNA fibre analysis. The coordinated activation of different Replicon Cluster Domains can generate the replication timing programme by which the genome is duplicated

Unreplicated DNA remaining from unperturbed S phases passes through mitosis for resolution in daughter cells

To prevent re-replication of genomic segments, the eukaryotic cell cycle is divided into two non-overlapping phases. During late mitosis and G1 replication origins are ‘licensed’ by loading MCM2-7 double hexamers and during S phase licensed replication origins activate to initiate bidirectional replication forks. Replication forks can stall irreversibly, and if two converging forks stall with no intervening licensed origin - a ‘double fork stall’ (DFS) - replication cannot be completed by conventional means. We previously showed how the distribution of replication origins in yeasts promotes complete genome replication even in the presence of irreversible fork stalling. This analysis predicts that DFSs are rare in yeasts but highly likely in large mammalian genomes. Here we show that complementary strand synthesis in early mitosis, ultrafine anaphase bridges and G1-specific 53BP1 nuclear bodies provide a mechanism for resolving unreplicated DNA at DFSs in human cells. When origin number was experimentally altered, the number of these structures closely agrees with theoretical predictions of DFSs. 53BP1 is preferentially bound to larger replicons, where the probability of DFSs is higher. Loss of 53BP1 caused hypersensitivity to licensing inhibition when replication origins are removed. These results provide a striking convergence of experimental and theoretical evidence that unreplicated DNA can pass through mitosis for resolution in the following cell cycle

Efficacy of GDC-0941 and PD 0325901 in mice bearing human colorectal tumour xenografts.

<p>± standard deviation (SD) and the median RTV3 or RTV4 for each group (± interquartile range (IR)). Time taken in days for HCT116 and HT29 tumour xenografts to reach three or four times their initial volume (time to RTV3 or RTV4) when treated with either vehicle control, 1 mg/kg PD 0325901 and 100 mg/kg GDC-0941 alone, or 1 mg/kg PD 0325901 and 100 mg/kg GDC-0941 in combination, p.o. once daily for 14 days. Data are presented as the mean time to RTV3 or RTV4 for the mice in each group </p

Plasma and tumour concentrations of GDC-0941 and PD 0325901 from mice bearing human tumour xenografts.

<p>Plasma and tumour concentrations of the PI3K inhibitor GDC-0941 (GDC) (<b>A</b>) and the MEK inhibitor PD 0325901 (PD) (<b>B</b>) measured by HPLC in samples from HCT116 tumour xenograft-bearing mice at the indicated time points after a single p.o. dose of either 100 mg/kg GDC-0941 alone, 1 mg/kg PD 0325901 alone or the combination of 1 mg/kg PD 0325901 and 100 mg/kg GDC-0941. Data are presented as the mean concentration from 3 mice in each group ± standard error. The horizontal dashed line indicates the <i>in vitro</i> GI₅₀ concentration (previously determined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081763#pone.0081763-Haagensen1" target="_blank">[38]</a>).</p

Plasma and tumour tissue concentration AUC values following GDC-0941 and PD 0325901 administration.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081763#pone-0081763-g001" target="_blank">Figure 1</a>). The area under the curve (AUC) was calculated and data are presented are the mean AUC ± standard deviation. Significant differences between groups are denoted by superscript letters; ^a,c,d,f p<0.01, ^b,g,j p = 0.04, ^e,h p = 0.01, ⁱ p = 0.02. Plasma and tumour tissue concentrations of the PI3K inhibitor GDC-0941 and the MEK inhibitor PD 0325901 were measured by HPLC in samples from HCT116 tumour xenograft-bearing mice over 24 hours after a single p.o. dose of 10 or 100 mg/kg GDC-0941 or 1 or 10 mg/kg PD 0325901, alone and in combination (</p