Quantitative Proteomics Reveals a "Poised Quiescence" Cellular State after Triggering the DNA Replication Origin Activation Checkpoint

An origin activation checkpoint has recently been discovered in the G1 phase of the mitotic cell cycle, which can be triggered by loss of DNA replication initiation factors such as the Cdc7 kinase. Insufficient levels of Cdc7 activate cell cycle arrest in normal cells, whereas cancer cells appear to lack this checkpoint response, do not arrest, and proceed with an abortive S phase, leading to cell death. The differential response between normal and tumor cells at this checkpoint has led to widespread interest in the development of pharmacological Cdc7 inhibitors as novel anticancer agents. We have used RNAi against Cdc7 in combination with SILAC-based high resolution MS proteomics to investigate the cellular mechanisms underlying the maintenance of the origin activation checkpoint in normal human diploid fibroblasts. Bioinformatics analysis identified clear changes in wide-ranging biological processes including altered cellular energetic flux, moderate stress response, reduced proliferative capacity, and a spatially distributed response across the mitochondria, lysosomes, and the cell surface. These results provide a quantitative overview of the processes involved in maintenance of the arrested state, show that this phenotype involves active rather than passive cellular adaptation, and highlight a diverse set of proteins responsible for cell cycle arrest and ultimately for promotion of cellular survival. We propose that the Cdc7-depleted proteome maintains cellular arrest by initiating a dynamic quiescence-like response and that the complexities of this phenotype will have important implications for the continued development of promising Cdc7-targeted cancer therapies

Cdc7 is a potent anti-cancer target in pancreatic cancer due to abrogation of the DNA origin activation checkpoint.

PURPOSE: Cdc7 is a serine/threonine kinase which is responsible for the 'firing' of replication origins leading to initiation of DNA replication. Inhibition or depletion of Cdc7 in normal cells triggers a DNA origin activation checkpoint causing a reversible G1 arrest. Here we investigate Cdc7 as a novel therapeutic target in pancreatic cancer. EXPERIMENTAL DESIGN: Cdc7 target validation was performed by immunoexpression profiling in a cohort of 73 patients with pancreatic adenocarcinoma including 24 controls. Secondly Cdc7 kinase was targeted in Capan-1 and PANC-1 pancreatic cancer cell line models using either an siRNA against Cdc7 or alternatively a small molecule inhibitor (SMI) of Cdc7 (PHA-767491). RESULTS: Cdc7 was significantly overexpressed in pancreatic adenocarcinoma compared to benign pancreatic tissue (median LI 34.3% vs. 1.3%; P<0.0001). Cdc7 knockdown using siRNA in Capan-1 and PANC-1 cells resulted in marked apoptotic cell death when compared with control cells. A prominent sub-G1 peak was seen on flow cytometry (sub-G1 51% vs. 3% and 45% vs. 0.7% in Capan-1 and PANC-1 cells, respectively). Annexin V labelling confirmed apoptosis in 64% vs. 11% and 75% vs. 8%, respectively. Western blotting showed cleavage of PARP-1 and caspase-3 and presence of γH2A.X. TUNEL assay showed strong staining in treated cells. These results were mirrored following Cdc7 kinase inhibition with PHA-767491. CONCLUSIONS: Our findings show that Cdc7 is a potent anti-cancer target in pancreatic adenocarcinoma and that Cdc7 immunoexpression levels might be used as a companion diagnostic to predict response to therapeutic siRNAs or SMIs directed against this kinase

GPS-ARM: Computational Analysis of the APC/C Recognition Motif by Predicting D-Boxes and KEN-Boxes

Anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase incorporated with Cdh1 and/or Cdc20 recognizes and interacts with specific substrates, and faithfully orchestrates the proper cell cycle events by targeting proteins for proteasomal degradation. Experimental identification of APC/C substrates is largely dependent on the discovery of APC/C recognition motifs, e.g., the D-box and KEN-box. Although a number of either stringent or loosely defined motifs proposed, these motif patterns are only of limited use due to their insufficient powers of prediction. We report the development of a novel GPS-ARM software package which is useful for the prediction of D-boxes and KEN-boxes in proteins. Using experimentally identified D-boxes and KEN-boxes as the training data sets, a previously developed GPS (Group-based Prediction System) algorithm was adopted. By extensive evaluation and comparison, the GPS-ARM performance was found to be much better than the one using simple motifs. With this powerful tool, we predicted 4,841 potential D-boxes in 3,832 proteins and 1,632 potential KEN-boxes in 1,403 proteins from H. sapiens, while further statistical analysis suggested that both the D-box and KEN-box proteins are involved in a broad spectrum of biological processes beyond the cell cycle. In addition, with the co-localization information, we predicted hundreds of mitosis-specific APC/C substrates with high confidence. As the first computational tool for the prediction of APC/C-mediated degradation, GPS-ARM is a useful tool for information to be used in further experimental investigations. The GPS-ARM is freely accessible for academic researchers at: http://arm.biocuckoo.org

Mechanism of Cancer Cell Death Induced by Depletion of an Essential Replication Regulator

Background: Depletion of replication factors often causes cell death in cancer cells. Depletion of Cdc7, a kinase essential for initiation of DNA replication, induces cancer cell death regardless of its p53 status, but the precise pathways of cell death induction have not been characterized. Methodology/Principal Findings: We have used the recently-developed cell cycle indicator, Fucci, to precisely characterize the cell death process induced by Cdc7 depletion. We have also generated and utilized similar fluorescent cell cycle indicators using fusion with other cell cycle regulators to analyze modes of cell death in live cells in both p53-positive and-negative backgrounds. We show that distinct cell-cycle responses are induced in p53-positive and-negative cells by Cdc7 depletion. p53-negative cells predominantly arrest temporally in G2-phase, accumulating CyclinB1 and other mitotic regulators. Prolonged arrest at G2-phase and abrupt entry into aberrant M-phase in the presence of accumulated CyclinB1 are followed by cell death at the post-mitotic state. Abrogation of cytoplasmic CyclinB1 accumulation partially decreases cell death. The ATR-MK2 pathway is responsible for sequestration of CyclinB1 with 14-3-3s protein. In contrast, p53-positive cancer cells do not accumulate CyclinB1, but appear to die mostly through entry into aberrant S-phase after Cdc7 depletion. The combination of Cdc7 inhibition with known anti-cancer agents significantly stimulates cell death effects in cancer cells in a genotype-dependent manner, providing a strategic basis for future combination therapies

Metabolic alterations during the growth of tumour spheroids

Solid tumours undergo considerable alterations in their metabolism of nutrients in order to generate sufficient energy and biomass for sustained growth and proliferation. During growth, the tumour microenvironment exerts a number of influences (e.g. hypoxia and acidity) that affect cellular biology and the flux or utilisation of fuels including glucose. The tumour spheroid model was used to characterise the utilisation of glucose and describe alterations to the activity and expression of key glycolytic enzymes during the tissue growth curve. Glucose was avidly consumed and associated with the production of lactate and an acidified medium, confirming the reliance on glycolytic pathways and a diminution of oxidative phosphorylation. The expression levels and activities of hexokinase, phosphofructokinase-1, pyruvate kinase and lactate dehydrogenase in the glycolytic pathway were measured to assess glycolytic capacity. Similar measurements were made for glucose-6-phosphate dehydrogenase, the entry point and regulatory step of the pentose-phosphate pathway (PPP) and for cytosolic malate dehydrogenase, a key link to TCA cycle intermediates. The parameters for these key enzymes were shown to undergo considerable variation during the growth curve of tumour spheroids. In addition, they revealed that the dynamic alterations were influenced by both transcriptional and posttranslational mechanisms

Metabolic alterations during the growth of tumour spheroids

Solid tumours undergo considerable alterations in their metabolism of nutrients in order to generate sufficient energy and biomass for sustained growth and proliferation. During growth, the tumour microenvironment exerts a number of influences (e.g. hypoxia and acidity) that affect cellular biology and the flux or utilisation of fuels including glucose. The tumour spheroid model was used to characterise the utilisation of glucose and describe alterations to the activity and expression of key glycolytic enzymes during the tissue growth curve. Glucose was avidly consumed and associated with the production of lactate and an acidified medium, confirming the reliance on glycolytic pathways and a diminution of oxidative phosphorylation. The expression levels and activities of hexokinase, phosphofructokinase-1, pyruvate kinase and lactate dehydrogenase in the glycolytic pathway were measured to assess glycolytic capacity. Similar measurements were made for glucose-6-phosphate dehydrogenase, the entry point and regulatory step of the pentose-phosphate pathway (PPP) and for cytosolic malate dehydrogenase, a key link to TCA cycle intermediates. The parameters for these key enzymes were shown to undergo considerable variation during the growth curve of tumour spheroids. In addition, they revealed that the dynamic alterations were influenced by both transcriptional and posttranslational mechanisms

Влијанието на ХБО терапијата врз брзината на заздравувањето на раните кај пациенти со дијабетично стапало

Хипербарната оксигенотерапија претставува адјувантен третман кој се користи во лекувањето на голем број заболувања. Особено добри резултати, со скратување на времето на заздравувањето на раните,се постигнува по третманот со ХБО кај пациентите со дијабетични улцерации. Нашето двегодишно искуство покажа редуцирање на потребата од хируршки интерванции, како и намалување на процентот на ампутации на екстремитетите

Two ubiquitin ligases, APC/C-Cdh1 and SKP1-CUL1-F (SCF)-β-TrCP, regulate glycolysis during the cell cycle

During cell proliferation, the abundance of the glycolysis-promoting enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is controlled by the ubiquitin ligase APC/C-Cdh1 via a KEN box. We now demonstrate in synchronized HeLa cells that PFKFB3, which appears in mid-to-late G1, is essential for cell division because its silencing prevents progression into S phase. In cells arrested by glucose deprivation, progression into S phase after replacement of glucose occurs only when PFKFB3 is present or is substituted by the downstream glycolytic enzyme 6-phosphofructo-1-kinase. PFKFB3 ceases to be detectable during late G1/S despite the absence of Cdh1; this disappearance is prevented by proteasomal inhibition. PFKFB3 contains a DSG box and is therefore a potential substrate for SCF-beta-TrCP, a ubiquitin ligase active during S phase. In synchronized HeLa cells transfected with PFKFB3 mutated in the KEN box, the DSG box, or both, we established the breakdown routes of the enzyme at different stages of the cell cycle and the point at which glycolysis is enhanced. Thus, the presence of PFKFB3 is tightly controlled to ensure the up-regulation of glycolysis at a specific point in G1. We suggest that this up-regulation of glycolysis and its associated events represent the nutrient-sensitive restriction point in mammalian cells