Ribosomal 18S RNA processing by the IGF-I-responsive WDR3 protein is integrated with p53 function in cancer cell proliferation.

Insulin-like growth factor-I (IGF-I) signaling is strongly associated with cell growth and regulates the rate of synthesis of the rRNA precursor, the first and the key stage of ribosome biogenesis. In a screen for mediators of IGF-I signaling in cancer, we recently identified several ribosome-related proteins, including NEP1 (nucleolar essential protein 1) and WDR3 (WD repeat 3), whose homologues in yeast function in ribosome processing. The WDR3 gene and its locus on chromosome 1p12-13 have previously been linked with malignancy. Here we show that IGF-I induces expression of WDR3 in transformed cells. WDR3 depletion causes defects in ribosome biogenesis by affecting 18 S rRNA processing and also causes a transient down-regulation of precursor rRNA levels with moderate repression of RNA polymerase I activity. Suppression of WDR3 in cells expressing functional p53 reduced proliferation and arrested cells in the G1 phase of the cell cycle. This was associated with activation of p53 and sequestration of MDM2 by ribosomal protein L11. Cells lacking functional p53 did not undergo cell cycle arrest upon suppression of WDR3. Overall, the data indicate that WDR3 has an essential function in 40 S ribosomal subunit synthesis and in ribosomal stress signaling to p53-mediated regulation of cell cycle progression in cancer cells.

The Deubiquitinating Enzyme USP17 Is Highly Expressed in Tumor Biopsies, Is Cell Cycle Regulated, and Is Required for G1-S Progression

Ubiquitination is a reversible posttranslational modification that is essential for cell cycle control, and it is becoming increasingly clear that the removal of ubiquitin from proteins by deubiquitinating enzymes (DUB) is equally important. In this study, we have identified high levels of the DUB USP17 in several tumor-derived cell lines and primary lung, colon, esophagus, and cervix tumor biopsies. We also report that USP17 is tightly regulated during the cell cycle in all the cells examined, being abundantly evident in G1 and absent in S phase. Moreover, regulated USP17 expression was necessary for cell cycle progression because its depletion significantly impaired G1-S transition and blocked cell proliferation. Previously, we have shown that USP17 regulates the intracellular translocation and activation of the GTPase Ras by controlling Ras-converting enzyme 1 (RCE1) activation. RCE1 also regulates the processing of other proteins with a CAAX motif, including Rho family GTPases. We now show that USP17 depletion blocks Ras and RhoA localization and activation. Moreover, our results confirm that USP17-depleted cells have constitutively elevated levels of the cyclin-dependent kinase inhibitors p21cip1 and p27kip1, known downstream targets of Ras and RhoA signaling. These observations clearly show that USP17 is tightly regulated during cell division and that its expression is necessary to coordinate cell cycle progression, and thus, it may be considered a promising novel cancer therapeutic target. Cancer Res; 70(8); 3329–39. ©2010 AACR.

Predicting Cell Cycle Regulated Genes by Causal Interactions

The fundamental difference between classic and modern biology is that technological innovations allow to generate high-throughput data to get insights into molecular interactions on a genomic scale. These high-throughput data can be used to infer gene networks, e. g., the transcriptional regulatory or signaling network, representing a blue print of the current dynamical state of the cellular system. However, gene networks do not provide direct answers to biological questions, instead, they need to be analyzed to reveal functional information of molecular working mechanisms. In this paper we propose a new approach to analyze the transcriptional regulatory network of yeast to predict cell cycle regulated genes. The novelty of our approach is that, in contrast to all other approaches aiming to predict cell cycle regulated genes, we do not use time series data but base our analysis on the prior information of causal interactions among genes. The major purpose of the present paper is to predict cell cycle regulated genes in S. cerevisiae. Our analysis is based on the transcriptional regulatory network, representing causal interactions between genes, and a list of known periodic genes. No further data are used. Our approach utilizes the causal membership of genes and the hierarchical organization of the transcriptional regulatory network leading to two groups of periodic genes with a well defined direction of information flow. We predict genes as periodic if they appear on unique shortest paths connecting two periodic genes from different hierarchy levels. Our results demonstrate that a classical problem as the prediction of cell cycle regulated genes can be seen in a new light if the concept of a causal membership of a gene is applied consequently. This also shows that there is a wealth of information buried in the transcriptional regulatory network whose unraveling may require more elaborate concepts than it might seem at first.

ATM acts downstream of ATR in the DNA damage response signaling of bystander cells.

This study identifies ataxia-telangiectasia mutated (ATM) as a further component of the complex signaling network of radiation-induced DNA damage in nontargeted bystander cells downstream of ataxia-telangiectasia and Rad3-related (ATR) and provides a rationale for molecular targeted modulation of these effects. In directly irradiated cells, ATR, ATM, and DNA-dependent protein kinase (DNA-PK) deficiency resulted in reduced cell survival as predicted by the known important role of these proteins in sensing DNA damage. A decrease in clonogenic survival was also observed in ATR/ATM/DNA-PK–proficient, nonirradiated bystander cells, but this effect was completely abrogated in ATR and ATM but not DNA-PK–deficient bystander cells. ATM activation in bystander cells was found to be dependent on ATR function. Furthermore, the induction and colocalization of ATR, 53BP1, ATM-S1981P, p21, and BRCA1 foci in nontargeted cells was shown, suggesting their involvement in bystander DNA damage signaling and providing additional potential targets for its modulation. 53BP1 bystander foci were induced in an ATR-dependent manner predominantly in S-phase cells, similar to ?H2AX foci induction. In conclusion, these results provide a rationale for the differential modulation of targeted and nontargeted effects of radiation.

Signaling by the Mpl receptor involves IKK and NF-kappaB.

Generated data, interpreted results and helped write and edit the manuscript.

Association of LETM1 and MRPL36 Contributes to the Regulation of Mitochondrial ATP Production and Necrotic Cell Death

Leucine zipper/EF hand-containing transmembrane-1 (LETM1) is a mitochondrial inner membrane protein that was first identified in Wolf-Hirschhorn syndrome, and was deleted in nearly all patients with the syndrome. LETM1 encodes for the human homologue of yeast Mdm38p, which is a mitochondria-shaping protein of unclear function. Here, we describe LETM1-mediated regulation of mitochondrial ATP production and biogenesis. We show that LETM1 overexpression can induce necrotic cell death in HeLa cells, in which LETM1 reduces mitochondria) biogenesis and ATP production. LETM1 acts as an anchor protein and associates with mitochondrial ribosome protein L36. Adenovirus-mediated overexpression of LETM1 reduced mitochondrial mass and expression of many mitochondrial proteins. LETM1-mediated inhibition of mitochondrial biogenesis enhanced glycolytic ATP supply and activated protein kinase B activity and cell survival signaling. The expression levels of LETM1 were significantly increased in multiple human cancer tissues compared with normals. These data suggest that LETM1 serves as an anchor protein for complex formation with the mitochondrial ribosome and regulates mitochondrial biogenesis. The increased expression of LETM1 in human cancer suggests that deregulation of LETM1 is a key feature of tumorigenesis. [Cancer Res 2009;69(8):3397-404]

RUNX3 attenuates beta-catenin/T cell factors in intestinal tumorigenesis

In intestinal epithelial cells, inactivation of APC, a key regulator of the Wnt pathway, activates beta-catenin to initiate tumorigenesis. However, other alterations may be involved in intestinal tumorigenesis. Here we found that RUNX3, a gastric tumor suppressor, forms a ternary complex with beta-catenin/7CF4 and attenuates Wnt signaling activity. A significant fraction of human sporadic colorectal adenomas and Runx3(+/-) mouse intestinal adenomas showed inactivation of RUNX3 without apparent beta-catenin accumulation, indicating that RUNX3 inactivation independently induces intestinal adenomas. In human colon cancers, RUNX3 is frequently inactivated with concomitant beta-catenin accumulation, suggesting that adenomas induced by inactivation of RUNX3 may progress to malignancy. Taken together, these data demonstrate that RUNX3 functions as a tumor suppressor by attenuating Wnt signaling.

Cell cycles and endocycles in the model brown seaweed, Ectocarpus siliculosus

The recent announcement of the first genome sequence of a brown macroalga, the filamentous Ectocarpus, has been accompanied by a number of companion papers in New Phytologist. In a paper which contributes to this special issue, we classified the core cell cycle components of Ectocarpus, comparing them to the previously studied cell cycle components of diatoms. We then carried out fluorescence microscopy experiments to show that the Ectocarpus cell cycle could be deregulated during early development to give endopolyploid adults. We discuss here how our findings complement recent studies on endopolyploidy in plant and algal systems.

Radiation-induced intercellular signaling mediated by cytochrome-c via a p53-dependent pathway in hepatoma cells

The tumor suppressor p53 has a crucial role in cellular response to DNA damage caused by ionizing radiation, but it is still unclear whether p53 can modulate radiation-induced bystander effects (RIBE). In the present work, three different hepatoma cell lines, namely HepG2 (wild p53), PLC/PRF/5 (mutation p53) and Hep3B (p53 null), were irradiated with c-rays and then co-cultured with normal Chang liver cell (wild p53) in order to elucidate the mechanisms of RIBE. Results showed that the radiosensitivity of HepG2 cells was higher than that of PLC/PRF/5 and Hep3B cells. Only irradiated HepG2 cells, rather than irradiated PLC/PRF/5 or Hep3B cells, could induce bystander effect of micronuclei (MN) formation in the neighboring Chang liver cells. When HepG2 cells were treated with 20 mu M pifithrin-alpha, an inhibitor of p53 function, or 5 lM cyclosporin A (CsA), an inhibitor of cytochrome- c release from mitochondria, the MN induction in bystander Chang liver cells was diminished. In fact, it was found that after irradiation, cytochrome- c was released from mitochondria into the cytoplasm only in HepG2 cells in a p53- dependent manner, but not in PLC/PRF/5 and Hep3B cells. Interestingly, when 50 lg/ml exogenous cytochrome- c was added into cell co- culture medium, RIBE was significantly triggered by irradiated PLC/PRF/5 and Hep3B cells, which previously failed to provoke a bystander effect. In addition, this exogenous cytochrome- c also partly recovered the RIBE induced by irradiated HepG2 cells even with CsA treatment. Our results provide new evidence that the RIBE can be modulated by the p53 status of irradiated hepatoma cells and that a p53- dependent release of cytochrome- c may be involved in the RIBE. Oncogene (2011) 30, 1947- 1955; doi: 10.1038/onc. 2010.567; published online 6 December 2010

Prognostic Significance of TRAIL Signaling Molecules in Stage II and III Colorectal Cancer

Purpose: We previously found that cellular FLICE-inhibitory protein (c-FLIP), caspase 8, and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptor 2 (DR5) are major regulators of cell viability and chemotherapy-induced apoptosis in colorectal cancer. In this study, we determined the prognostic significance of c-FLIP, caspase 8, TRAIL and DR5 expression in tissues from patients with stage II and III colorectal cancer.

Experimental Design: Tissue microarrays were constructed from matched normal and tumor tissue derived from patients (n = 253) enrolled in a phase III trial of adjuvant 5-fluorouracil–based chemotherapy versus postoperative observation alone. TRAIL, DR5, caspase 8, and c-FLIP expression levels were determined by immunohistochemistry.

Results: Colorectal tumors displayed significantly higher expression levels of c-FLIP (P < 0.001), caspase 8 (P = 0.01), and DR5 (P < 0.001), but lower levels of TRAIL (P < 0.001) compared with matched normal tissue. In univariate analysis, higher TRAIL expression in the tumor was associated with worse overall survival (P = 0.026), with a trend to decreased relapse-free survival (RFS; P = 0.06), and higher tumor c-FLIP expression was associated with a significantly decreased RFS (P = 0.015). Using multivariate predictive modeling for RFS in all patients and including all biomarkers, age, treatment, and stage, we found that the model was significant when the mean tumor c-FLIP expression score and disease stage were included (P < 0.001). As regards overall survival, the overall model was predictive when both TRAIL expression and disease stage were included (P < 0.001).

Conclusions: High c-FLIP and TRAIL expression may be independent adverse prognostic markers in stage II and III colorectal cancer and might identify patients most at risk of relapse.

Platelet 12-lipoxygenase activation via glycoprotein VI - Involvement of multiple signaling pathways in agonist control of H(P)ETE synthesis

Lipoxygenases (LOX) contribute to vascular disease and inflammation through generation of bioactive lipids, including 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE). The physiological mechanisms that acutely control LOX product generation in mammalian cells are uncharacterized. Human platelets that contain a 12-LOX isoform (p12-LOX) were used to define pathways that activate H( P) ETE synthesis in the vasculature. Collagen and collagen-related peptide (CRP) (1 to 10 mug/mL) acutely induced platelet 12-H(P)ETE synthesis. This implicated the collagen receptor glycoprotein VI ( GPVI), which signals via the immunoreceptor-based activatory motif (ITAM)-containing FcRgamma chain. Conversely, thrombin only activated at high concentrations (> 0.2 U/mL), whereas U46619 and ADP alone were ineffective. Collagen or CRP-stimulated 12-H( P) ETE generation was inhibited by staurosporine, PP2, wortmannin, BAPTA/AM, EGTA, and L-655238, implicating src-tyrosine kinases, PI3-kinase, Ca2+ mobilization, and p12-LOX translocation. In contrast, protein kinase C (PKC) inhibition potentiated 12-H( P) ETE generation. Finally, activation of the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing platelet endothelial cell adhesion molecule (PECAM-1) inhibited p12-LOX product generation. This study characterizes a receptor-dependent pathway for 12-H(P) ETE synthesis via the collagen receptor GPVI, which is negatively regulated by PECAM-1 and PKC, and demonstrates a novel link between immune receptor signaling and lipid mediator generation in the vasculature.

Cell survival responses after exposure to modulated radiation fields.

In the present study survival responses were determined in cells with differing radiosensitivity, specifically primary fibroblast (AG0-1522B), human breast cancer (MDA-MB-231), human prostate cancer (DU-145) and human glioma (T98G) cells, after exposure to modulated radiation fields delivered by shielding 50% of the tissue culture flask. A significant decrease (P < 0.05) in cell survival was observed in the shielded area, outside the primary treatment field (out-of-field), that was lower than predicted when compared to uniform exposures fitted to the linear-quadratic model. Cellular radiosensitivity was demonstrated to be an important factor in the level of response for both the in- and out-of-field regions. These responses were shown to be dependent on secretion-mediated intercellular communication, because inhibition of cellular secreted factors between the in- and out-of-field regions abrogated the response. Out-of-field cell survival was shown to increase after pretreatment of cells with agents known to inhibit factors involved in mediating radiation-induced bystander signaling (aminoguanidine, DMSO or cPTIO). These data illustrate a significant decrease in survival out-of-field, dependent upon intercellular communication, in several cell lines with varying radiosensitivity after exposure to a modulated radiation field. This study provides further evidence for the importance of intercellular signaling in modulated exposures, where dose gradients are present, and may inform the refinement of established radiobiological models to facilitate the optimization of advanced radiotherapy treatment plans.

Organizational structure and the periphery of the gene regulatory network in B-cell lymphoma

Background:
The physical periphery of a biological cell is mainly described by signaling pathways which are triggered by transmembrane proteins and receptors that are sentinels to control the whole gene regulatory network of a cell. However, our current knowledge about the gene regulatory mechanisms that are governed by extracellular signals is severely limited.Results: The purpose of this paper is three fold. First, we infer a gene regulatory network from a large-scale B-cell lymphoma expression data set using the C3NET algorithm. Second, we provide a functional and structural analysis of the largest connected component of this network, revealing that this network component corresponds to the peripheral region of a cell. Third, we analyze the hierarchical organization of network components of the whole inferred B-cell gene regulatory network by introducing a new approach which exploits the variability within the data as well as the inferential characteristics of C3NET. As a result, we find a functional bisection of the network corresponding to different cellular components.

Conclusions:
Overall, our study allows to highlight the peripheral gene regulatory network of B-cells and shows that it is centered around hub transmembrane proteins located at the physical periphery of the cell. In addition, we identify a variety of novel pathological transmembrane proteins such as ion channel complexes and signaling receptors in B-cell lymphoma. © 2012 Simoes et al.; licensee BioMed Central Ltd.

IHG-1 amplifies TGF-beta1 signaling and is increased in renal fibrosis

Induced in high glucose-1 (IHG-1) is an evolutionarily conserved gene transcript upregulated by high extracellular glucose concentrations, but its function is unknown. Here, it is reported that the abundance of IHG-1 mRNA is nearly 10-fold higher in microdissected, tubule-rich renal biopsies from patients with diabetic nephropathy compared with control subjects. In the diabetic nephropathy specimens, in situ hybridization localized IHG-1 to tubular epithelial cells along with TGF-beta1 and activated Smad3, suggesting a possible role in the development of tubulointerstitial fibrosis. Supporting this possibility, IHG-1 mRNA and protein expression also increased with unilateral ureteral obstruction. In the HK-2 proximal tubule cell line, overexpression of IHG-1 increased TGF-beta1-stimulated expression of connective tissue growth factor and fibronectin. IHG-1 was found to amplify TGF-beta1-mediated transcriptional activity by increasing and prolonging phosphorylation of Smad3. Conversely, inhibition of endogenous IHG-1 with small interference RNA suppressed transcriptional responses to TGF-beta1. In summary, IHG-1, which increases in diabetic nephropathy, may enhance the actions of TGF-beta1 and contribute to the development of tubulointerstitial fibrosis.