Imperial College London

DrAnthonyUren

Faculty of MedicineInstitute of Clinical Sciences

Lecturer
 
 
 
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Contact

 

+44 (0)20 3313 5843anthony.uren Website

 
 
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Location

 

3005CRB (Clinical Research Building)Hammersmith Campus

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Summary

 

Publications

Publication Type
Year
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25 results found

Curry E, Green I, Chapman-Rothe N, Shamsaei E, Kandil S, Cherblanc FL, Payne L, Bell E, Ganesh T, Srimongkolpithak N, Caron J, Li F, Uren AG, Snyder JP, Vedadi M, Fuchter MJ, Brown R, Curry E, Green I, Chapman-Rothe N, Shamsaei E, Kandil S, Cherblanc FL, Payne L, Bell E, Ganesh T, Srimongkolpithak N, Caron J, Li F, Uren AG, Snyder JP, Vedadi M, Fuchter MJ, Brown R, Curry E, Green I, Chapman-Rothe N, Shamsaei E, Kandil S, Cherblanc FL, Payne L, Bell E, Ganesh T, Srimongkolpithak N, Caron J, Li F, Uren AG, Snyder JP, Vedadi M, Fuchter MJ, Brown R, Curry E, Green I, Chapman-Rothe N, Shamsaei E, Kandil S, Cherblanc FL, Payne L, Bell E, Ganesh T, Srimongkolpithak N, Caron J, Li F, Uren AG, Snyder JP, Vedadi M, Fuchter MJ, Brown R, Curry E, Green I, Chapman-Rothe N, Shamsaei E, Kandil S, Cherblanc F, Payne L, Bell E, Ganesh T, Srimongkolpithak N, Caron J, Li F, Uren AG, Snyder JP, Vedadi M, Fuchter MJ, Brown Ret al., 2015, Dual EZH2 and EHMT2 histone methyltransferase inhibition increases biological efficacy in breast cancer cells, CLINICAL EPIGENETICS, Vol: 7, ISSN: 1868-7083

BACKGROUND: Many cancers show aberrant silencing of gene expression and overexpression of histone methyltransferases. The histone methyltransferases (HKMT) EZH2 and EHMT2 maintain the repressive chromatin histone methylation marks H3K27me and H3K9me, respectively, which are associated with transcriptional silencing. Although selective HKMT inhibitors reduce levels of individual repressive marks, removal of H3K27me3 by specific EZH2 inhibitors, for instance, may not be sufficient for inducing the expression of genes with multiple repressive marks. RESULTS: We report that gene expression and inhibition of triple negative breast cancer cell growth (MDA-MB-231) are markedly increased when targeting both EZH2 and EHMT2, either by siRNA knockdown or pharmacological inhibition, rather than either enzyme independently. Indeed, expression of certain genes is only induced upon dual inhibition. We sought to identify compounds which showed evidence of dual EZH2 and EHMT2 inhibition. Using a cell-based assay, based on the substrate competitive EHMT2 inhibitor BIX01294, we have identified proof-of-concept compounds that induce re-expression of a subset of genes consistent with dual HKMT inhibition. Chromatin immunoprecipitation verified a decrease in silencing marks and an increase in permissive marks at the promoter and transcription start site of re-expressed genes, while Western analysis showed reduction in global levels of H3K27me3 and H3K9me3. The compounds inhibit growth in a panel of breast cancer and lymphoma cell lines with low to sub-micromolar IC50s. Biochemically, the compounds are substrate competitive inhibitors against both EZH2 and EHMT1/2. CONCLUSIONS: We have demonstrated that dual inhibition of EZH2 and EHMT2 is more effective at eliciting biological responses of gene transcription and cancer cell growth inhibition compared to inhibition of single HKMTs, and we report the first dual EZH2-EHMT1/2 substrate competitive inhibitors that are functional in cells.

JOURNAL ARTICLE

Westerman BA, Blom M, Tanger E, van der Valk M, Song JY, van Santen M, Gadiot J, Cornelissen-Steijger P, Zevenhoven J, Prosser HM, Uren A, Aronica E, van Lohuizen M, Westerman BA, Blom M, Tanger E, van der Valk M, Song J-Y, van Santen M, Gadiot J, Cornelissen-Steijger P, Zevenhoven J, Prosser HM, Uren A, Aronica E, van Lohuizen M, Westerman BA, Blom M, Tanger E, van der Valk M, Song J-Y, van Santen M, Gadiot J, Cornelissen-Steijger P, Zevenhoven J, Prosser HM, Uren A, Aronica E, van Lohuizen M, Westerman BA, Blom M, Tanger E, van der Valk M, Song JY, van Santen M, Gadiot J, Cornelissen-Steijger P, Zevenhoven J, Prosser HM, Uren A, Aronica E, van Lohuizen Met al., 2012, GFAP-Cre-mediated transgenic activation of Bmi1 results in pituitary tumors., PLoS One, Vol: 7, Pages: e35943-e35943, ISSN: 1932-6203

Bmi1 is a member of the polycomb repressive complex 1 and plays different roles during embryonic development, depending on the developmental context. Bmi1 over expression is observed in many types of cancer, including tumors of astroglial and neural origin. Although genetic depletion of Bmi1 has been described to result in tumor inhibitory effects partly through INK4A/Arf mediated senescence and apoptosis and also through INK4A/Arf independent effects, it has not been proven that Bmi1 can be causally involved in the formation of these tumors. To see whether this is the case, we developed two conditional Bmi1 transgenic models that were crossed with GFAP-Cre mice to activate transgenic expression in neural and glial lineages. We show here that these mice generate intermediate and anterior lobe pituitary tumors that are positive for ACTH and beta-endorphin. Combined transgenic expression of Bmi1 together with conditional loss of Rb resulted in pituitary tumors but was insufficient to induce medulloblastoma therefore indicating that the oncogenic function of Bmi1 depends on regulation of p16(INK4A)/Rb rather than on regulation of p19(ARF)/p53. Human pituitary adenomas show Bmi1 overexpression in over 50% of the cases, which indicates that Bmi1 could be causally involved in formation of these tumors similarly as in our mouse model.

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March HN, Rust AG, Wright NA, ten Hoeve J, de Ridder J, Eldridge M, van der Weyden L, Berns A, Gadiot J, Uren A, Kemp R, Arends MJ, Wessels LF, Winton DJ, Adams DJ, March HN, Rust AG, Wright NA, ten Hoeve J, de Ridder J, Eldridge M, van der Weyden L, Berns A, Gadiot J, Uren A, Kemp R, Arends MJ, Wessels LFA, Winton DJ, Adams DJ, March HN, Rust AG, Wright NA, ten Hoeve J, de Ridder J, Eldridge M, van der Weyden L, Berns A, Gadiot J, Uren A, Kemp R, Arends MJ, Wessels LFA, Winton DJ, Adams DJet al., 2011, Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis., Nat Genet, Vol: 43, Pages: 1202-1209, ISSN: 1061-4036

The evolution of colorectal cancer suggests the involvement of many genes. To identify new drivers of intestinal cancer, we performed insertional mutagenesis using the Sleeping Beauty transposon system in mice carrying germline or somatic Apc mutations. By analyzing common insertion sites (CISs) isolated from 446 tumors, we identified many hundreds of candidate cancer drivers. Comparison to human data sets suggested that 234 CIS-targeted genes are also dysregulated in human colorectal cancers. In addition, we found 183 CIS-containing genes that are candidate Wnt targets and showed that 20 CISs-containing genes are newly discovered modifiers of canonical Wnt signaling. We also identified mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included those encoding members of the FGF signaling cascade. Some 70 genes had co-occurrence of CIS pairs, clustering into 38 sub-networks that may regulate tumor development.

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Vassiliou GS, Cooper JL, Rad R, Li J, Rice S, Uren A, Rad L, Ellis P, Andrews R, Banerjee R, Grove C, Wang W, Liu P, Wright P, Arends M, Bradley A, Vassiliou GS, Cooper JL, Rad R, Li J, Rice S, Uren A, Rad L, Ellis P, Andrews R, Banerjee R, Grove C, Wang W, Liu P, Wright P, Arends M, Bradley A, Vassiliou GS, Cooper JL, Rad R, Li J, Rice S, Uren A, Rad L, Ellis P, Andrews R, Banerjee R, Grove C, Wang W, Liu P, Wright P, Arends M, Bradley Aet al., 2011, Mutant nucleophosmin and cooperating pathways drive leukemia initiation and progression in mice., Nat Genet, Vol: 43, Pages: 470-475, ISSN: 1061-4036

Acute myeloid leukemia (AML) is a molecularly diverse malignancy with a poor prognosis whose largest subgroup is characterized by somatic mutations in NPM1, which encodes nucleophosmin. These mutations, termed NPM1c, result in cytoplasmic dislocation of nucleophosmin and are associated with distinctive transcriptional signatures, yet their role in leukemogenesis remains obscure. Here we report that activation of a humanized Npm1c knock-in allele in mouse hemopoietic stem cells causes Hox gene overexpression, enhanced self renewal and expanded myelopoiesis. One third of mice developed delayed-onset AML, suggesting a requirement for cooperating mutations. We identified such mutations using a Sleeping Beauty transposon, which caused rapid-onset AML in 80% of mice with Npm1c, associated with mutually exclusive integrations in Csf2, Flt3 or Rasgrp1 in 55 of 70 leukemias. We also identified recurrent integrations in known and newly discovered leukemia genes including Nf1, Bach2, Dleu2 and Nup98. Our results provide new pathogenetic insights and identify possible therapeutic targets in NPM1c+ AML.

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Kool J, Uren AG, Martins CP, Sie D, de Ridder J, Turner G, van Uitert M, Matentzoglu K, Lagcher W, Krimpenfort P, Gadiot J, Pritchard C, Lenz J, Lund AH, Jonkers J, Rogers J, Adams DJ, Wessels L, Berns A, van Lohuizen M, Kool J, Uren AG, Martins CP, Sie D, de Ridder J, Turner G, van Uitert M, Matentzoglu K, Lagcher W, Krimpenfort P, Gadiot J, Pritchard C, Lenz J, Lund AH, Jonkers J, Rogers J, Adams DJ, Wessels L, Berns A, van Lohuizen M, Kool J, Uren AG, Martins CP, Sie D, de Ridder J, Turner G, van Uitert M, Matentzoglu K, Lagcher W, Krimpenfort P, Gadiot J, Pritchard C, Lenz J, Lund AH, Jonkers J, Rogers J, Adams DJ, Wessels L, Berns A, van Lohuizen M, Kool J, Uren AG, Martins CP, Sie D, de Ridder J, Turner G, van Uitert M, Matentzoglu K, Lagcher W, Krimpenfort P, Gadiot J, Pritchard C, Lenz J, Lund AH, Jonkers J, Rogers J, Adams DJ, Wessels L, Berns A, van Lohuizen Met al., 2010, Insertional Mutagenesis in Mice Deficient for p15(Ink4b), p16(Ink4a), p21(Cip1), and p27(Kip1) Reveals Cancer Gene Interactions and Correlations with Tumor Phenotypes, CANCER RESEARCH, Vol: 70, Pages: 520-531, ISSN: 0008-5472

The cyclin dependent kinase (CDK) inhibitors p15, p16, p21, and p27 are frequently deleted, silenced, or downregulated in many malignancies. Inactivation of CDK inhibitors predisposes mice to tumor development, showing that these genes function as tumor suppressors. Here, we describe high-throughput murine leukemia virus insertional mutagenesis screens in mice that are deficient for one or two CDK inhibitors. We retrieved 9,117 retroviral insertions from 476 lymphomas to define hundreds of loci that are mutated more frequently than expected by chance. Many of these loci are skewed toward a specific genetic context of predisposing germline and somatic mutations. We also found associations between these loci with gender, age of tumor onset, and lymphocyte lineage (B or T cell). Comparison of retroviral insertion sites with single nucleotide polymorphisms associated with chronic lymphocytic leukemia revealed a significant overlap between the datasets. Together, our findings highlight the importance of genetic context within large-scale mutation detection studies, and they show a novel use for insertional mutagenesis data in prioritizing disease-associated genes that emerge from genome-wide association studies.

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Mattison J, Kool J, Uren AG, de Ridder J, Wessels L, Jonkers J, Bignell GR, Butler A, Rust AG, Brosch M, Wilson CH, van der Weyden L, Largaespada DA, Stratton MR, Futreal PA, van Lohuizen M, Berns A, Collier LS, Hubbard T, Adams DJ, Mattison J, Kool J, Uren AG, de Ridder J, Wessels L, Jonkers J, Bignell GR, Butler A, Rust AG, Brosch M, Wilson CH, van der Weyden L, Largaespada DA, Stratton MR, Futreal PA, van Lohuizen M, Berns A, Collier LS, Hubbard T, Adams DJ, Mattison J, Kool J, Uren AG, de Ridder J, Wessels L, Jonkers J, Bignell GR, Butler A, Rust AG, Brosch M, Wilson CH, van der Weyden L, Largaespada DA, Stratton MR, Futreal PA, van Lohuizen M, Berns A, Collier LS, Hubbard T, Adams DJ, Mattison J, Kool J, Uren AG, de Ridder J, Wessels L, Jonkers J, Bignell GR, Butler A, Rust AG, Brosch M, Wilson CH, van der Weyden L, Largaespada DA, Stratton MR, Futreal PA, van Lohuizen M, Berns A, Collier LS, Hubbard T, Adams DJet al., 2010, Novel Candidate Cancer Genes Identified by a Large-Scale Cross-Species Comparative Oncogenomics Approach, CANCER RESEARCH, Vol: 70, Pages: 883-895, ISSN: 0008-5472

Comparative genomic hybridization (CGH) can reveal important disease genes but the large regions identified could sometimes contain hundreds of genes. Here we combine high-resolution CGH analysis of 598 human cancer cell lines with insertion sites isolated from 1,005 mouse tumors induced with the murine leukemia virus (MuLV). This cross-species oncogenomic analysis revealed candidate tumor suppressor genes and oncogenes mutated in both human and mouse tumors, making them strong candidates for novel cancer genes. A significant number of these genes contained binding sites for the stem cell transcription factors Oct4 and Nanog. Notably, mice carrying tumors with insertions in or near stem cell module genes, which are thought to participate in cell self-renewal, died significantly faster than mice without these insertions. A comparison of the profile we identified to that induced with the Sleeping Beauty (SB) transposon system revealed significant differences in the profile of recurrently mutated genes. Collectively, this work provides a rich catalogue of new candidate cancer genes for functional analysis.

JOURNAL ARTICLE

Uren A, Berns A, Uren A, Berns A, Uren A, Berns Aet al., 2009, Jump-starting cancer gene discovery., Nat Biotechnol, Vol: 27, Pages: 251-252, ISSN: 1087-0156

JOURNAL ARTICLE

Uren AG, Mikkers H, Kool J, van der Weyden L, Lund AH, Wilson CH, Rance R, Jonkers J, van Lohuizen M, Berns A, Adams DJ, Uren AG, Mikkers H, Kool J, van der Weyden L, Lund AH, Wilson CH, Rance R, Jonkers J, van Lohuizen M, Berns A, Adams DJ, Uren AG, Mikkers H, Kool J, van der Weyden L, Lund AH, Wilson CH, Rance R, Jonkers J, van Lohuizen M, Berns A, Adams DJ, Uren AG, Mikkers H, Kool J, van der Weyden L, Lund AH, Wilson CH, Rance R, Jonkers J, van Lohuizen M, Berns A, Adams DJet al., 2009, A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites, NATURE PROTOCOLS, Vol: 4, Pages: 789-798, ISSN: 1754-2189

Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice; however, until recently, the cost-effective isolation and sequencing of insertion sites has been a major limitation to performing screens on this scale. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA isolation, DNA digestion, linker or splinkerette ligation, primary and secondary PCR amplification, and sequencing. This method, which takes about 1 week to perform, has allowed us to isolate hundreds of thousands of insertion sites from mouse tumors and, unlike other methods, has been specifically optimized for the murine leukemia virus (MuLV), and can easily be performed in a 96-well plate format for the efficient multiplex isolation of insertion sites.

JOURNAL ARTICLE

Csikos T, Reijmers RM, Uren AG, Spaargaren M, Pals ST, Csikós T, Reijmers RM, Uren AG, Spaargaren M, Pals ST, Csikós T, Reijmers RM, Uren AG, Spaargaren M, Pals ST, Csikós T, Reijmers RM, Uren AG, Spaargaren M, Pals STet al., 2008, Instant conditional transgenesis in the mouse hematopoietic compartment, JOURNAL OF IMMUNOLOGICAL METHODS, Vol: 339, Pages: 259-263, ISSN: 0022-1759

Adoptive transfer of retrovirally transduced stem cells has recently been described for instant transgenesis in the hematopoietic compartment of mice. This method circumvents the need to manipulate the germline. However, cell type specific gene expression in this 'retrogenic' mouse model has remained tedious. Here we report a single retroviral vector-based method to rapidly generate conditional retrogenic mice. For this purpose, mutated loxP-flanked DNA segments are transduced into hematopoietic stem cells isolated from Cre recombinase transgenic mice, which are subsequently transferred into immunodeficient mice. In this way gene expression can be restricted to hematopoietic cell lineages of choice in the acquired immune system.

JOURNAL ARTICLE

Uren AG, Kool J, Matentzoglu K, de Ridder J, Mattison J, van Uitert M, Lagcher W, Sie D, Tanger E, Cox T, Reinders M, Hubbard TJ, Rogers J, Jonkers J, Wessels L, Adams DJ, van Lohuizen M, Berns A, Uren AG, Kool J, Matentzoglu K, de Ridder J, Mattison J, van Uitert M, Lagcher W, Sie D, Tanger E, Cox T, Reinders M, Hubbard TJ, Rogers J, Jonkers J, Wessels L, Adams DJ, van Lohuizen M, Berns A, Uren AG, Kool J, Matentzoglu K, de Ridder J, Mattison J, van Uitert M, Lagcher W, Sie D, Tanger E, Cox T, Reinders M, Hubbard TJ, Rogers J, Jonkers J, Wessels L, Adams DJ, van Lohuizen M, Berns A, Uren AG, Kool J, Matentzoglu K, de Ridder J, Mattison J, van Uitert M, Lagcher W, Sie D, Tanger E, Cox T, Reinders M, Hubbard TJ, Rogers J, Jonkers J, Wessels L, Adams DJ, van Lohuizen M, Berns Aet al., 2008, Large-scale mutagenesis in p19(ARF)- and p53- Deficient mice identifies cancer genes and their collaborative networks, CELL, Vol: 133, Pages: 727-741, ISSN: 0092-8674

p53 and p19(ARF) are tumor suppressors frequently mutated in human tumors. In a high-throughput screen in mice for mutations collaborating with either p53 or p19(ARF) deficiency, we identified 10,806 retroviral insertion sites, implicating over 300 loci in tumorigenesis. This dataset reveals 20 genes that are specifically mutated in either p19(ARF)-deficient, p53-deficient or wild-type mice (including Flt3, mmu-mir-106a-363, Smg6, and Ccnd3), as well as networks of significant collaborative and mutually exclusive interactions between cancer genes. Furthermore, we found candidate tumor suppressor genes, as well as distinct clusters of insertions within genes like Flt3 and Notch1 that induce mutants with different spectra of genetic interactions. Cross species comparative analysis with aCGH data of human cancer cell lines revealed known and candidate oncogenes (Mmp13, Slamf6, and Rreb1) and tumor suppressors (Wwox and Arfrp2). This dataset should prove to be a rich resource for the study of genetic interactions that underlie tumorigenesis.

JOURNAL ARTICLE

de Ridder J, Kool J, Uren A, Bot J, Wessels L, Reinders M, de Ridder J, Kool J, Uren A, Bot J, Wessels L, Reinders M, de Ridder J, Kool J, Uren A, Bot J, Wessels L, Reinders Met al., 2007, Co-occurrence analysis of insertional mutagenesis data reveals cooperating oncogenes., Bioinformatics, Vol: 23, Pages: i133-i141, ISSN: 1367-4803

MOTIVATION: Cancers are caused by an accumulation of multiple independent mutations that collectively deregulate cellular pathways, e.g. such as those regulating cell division and cell-death. The publicly available Retroviral Tagged Cancer Gene Database (RTCGD) contains the data of many insertional mutagenesis screens, in which the virally induced mutations result in tumor formation in mice. The insertion loci therefore indicate the location of putative cancer genes. Additionally, the presence of multiple independent insertions within one tumor hints towards a cooperation between the insertionally mutated genes. In this study we focus on the detection of statistically significant co-mutations. RESULTS: We propose a two-dimensional Gaussian Kernel Convolution method (2DGKC), a computational technique that identifies the cooperating mutations in insertional mutagenesis data. We define the Common Co-occurrence of Insertions (CCI), signifying the co-mutations that are statistically significant across all different screens in the RTCGD. Significance estimates are made on multiple scales, and the results visualized in a scale space, thereby providing valuable extra information on the putative cooperation. The multidimensional analysis of the insertion data results in the discovery of 86 statistically significant co-mutations, indicating the presence of cooperating oncogenes that play a role in tumor development. Since oncogenes may cooperate with several members of a parallel pathway, we combined the co-occurrence data with gene family information to find significant cooperations between oncogenes and families of genes. We show, for instance, the interchangeable cooperation of Myc insertions with insertions in the Pim family. AVAILABILITY: A list of the resulting CCIs is available at: http://ict.ewi.tudelft.nl/~jeroen/CCI/CCI_list.txt.

JOURNAL ARTICLE

de Ridder J, Uren A, Kool J, Reinders M, Wessels L, de Ridder J, Uren A, Kool J, Reinders M, Wessels L, de Ridder J, Uren A, Kool J, Reinders M, Wessels Let al., 2006, Detecting statistically significant common insertion sites in retroviral insertional mutagenesis screens., PLoS Comput Biol, Vol: 2, Pages: e166-e166, ISSN: 1553-734X

Retroviral insertional mutagenesis screens, which identify genes involved in tumor development in mice, have yielded a substantial number of retroviral integration sites, and this number is expected to grow substantially due to the introduction of high-throughput screening techniques. The data of various retroviral insertional mutagenesis screens are compiled in the publicly available Retroviral Tagged Cancer Gene Database (RTCGD). Integrally analyzing these screens for the presence of common insertion sites (CISs, i.e., regions in the genome that have been hit by viral insertions in multiple independent tumors significantly more than expected by chance) requires an approach that corrects for the increased probability of finding false CISs as the amount of available data increases. Moreover, significance estimates of CISs should be established taking into account both the noise, arising from the random nature of the insertion process, as well as the bias, stemming from preferential insertion sites present in the genome and the data retrieval methodology. We introduce a framework, the kernel convolution (KC) framework, to find CISs in a noisy and biased environment using a predefined significance level while controlling the family-wise error (FWE) (the probability of detecting false CISs). Where previous methods use one, two, or three predetermined fixed scales, our method is capable of operating at any biologically relevant scale. This creates the possibility to analyze the CISs in a scale space by varying the width of the CISs, providing new insights in the behavior of CISs across multiple scales. Our method also features the possibility of including models for background bias. Using simulated data, we evaluate the KC framework using three kernel functions, the Gaussian, triangular, and rectangular kernel function. We applied the Gaussian KC to the data from the combined set of screens in the RTCGD and found that 53% of the CISs do not reach the significance thresh

JOURNAL ARTICLE

Uren AG, Kool J, Berns A, van Lohuizen M, Uren AG, Kool J, Berns A, van Lohuizen M, Uren AG, Kool J, Berns A, van Lohuizen M, Uren AG, Kool J, Berns A, van Lohuizen Met al., 2005, Retroviral insertional mutagenesis: past, present and future, ONCOGENE, Vol: 24, Pages: 7656-7672, ISSN: 0950-9232

Retroviral insertion mutagenesis screens in mice are powerful tools for efficient identification of oncogenic mutations in an in vivo setting. Many oncogenes identified in these screens have also been shown to play a causal role in the development of human cancers. Sequencing and annotation of the mouse genome, along with recent improvements in insertion site cloning has greatly facilitated identification of oncogenic events in retrovirus-induced tumours. In this review, we discuss the features of retroviral insertion mutagenesis screens, covering the mechanisms by which retroviral insertions mutate cellular genes, the practical aspects of insertion site cloning, the identification and analysis of common insertion sites, and finally we address the potential for use of somatic insertional mutagens in the study of nonhaematopoietic and nonmammary tumour types.

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Speliotes EK, Uren A, Vaux D, Horvitz HR, Speliotes EK, Uren A, Vaux D, Horvitz HR, Speliotes EK, Uren A, Vaux D, Horvitz HRet al., 2000, The survivin-like C. elegans BIR-1 protein acts with the Aurora-like kinase AIR-2 to affect chromosomes and the spindle midzone., Mol Cell, Vol: 6, Pages: 211-223, ISSN: 1097-2765

Baculoviral IAP repeat proteins (BIRPs) may affect cell death, cell division, and tumorigenesis. The C. elegans BIRP BIR-1 was localized to chromosomes and to the spindle midzone. Embryos and fertilized oocytes lacking BIR-1 had defects in chromosome behavior, spindle midzone formation, and cytokinesis. We observed indistinguishable defects in fertilized oocytes and embryos lacking the Aurora-like kinase AIR-2. AIR-2 was not present on chromosomes in the absence of BIR-1. Histone H3 phosphorylation and HCP-1 staining, which marks kinetochores, were reduced in the absence of either BIR-1 or AIR-2. We propose that BIR-1 localizes AIR-2 to chromosomes and perhaps to the spindle midzone, where AIR-2 phosphorylates proteins that affect chromosome behavior and spindle midzone organization. The human BIRP survivin, which is upregulated in tumors, could partially substitute for BIR-1 in C. elegans. Deregulation of bir-1 promotes changes in ploidy, suggesting that similar deregulation of mammalian BIRPs may contribute to tumorigenesis.

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Uren AG, O'Rourke K, Aravind L, Pisabarro MT, Seshagiri S, Koonin EV, Dixit VM, Uren AG, O'Rourke K, Aravind LA, Pisabarro MT, Seshagiri S, Koonin EV, Dixit VM, Uren AG, O'Rourke K, Aravind LA, Pisabarro MT, Seshagiri S, Koonin EV, Dixit VM, Uren Aet al., 2000, Identification of paracaspases and metacaspases: Two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma, MOLECULAR CELL, Vol: 6, Pages: 961-967, ISSN: 1097-2765

Caspases are cysteine proteases essential to apoptosis. We have identified two families of caspase-like proteins, Paracaspases (found in metazoans and Dictyostelium) and metacaspases (found in plants, fungi, and protozoa). Metazoan paracaspase prodomains contain a death domain and immunoglobulin domains. Several plant metacaspase prodomains contain zinc finger motifs resembling those in the plant hypersensitive response/cell death protein Isd-1. The human paracaspase prodomain binds Bcl10, a protein involved in the t(1;14)(p22;q32) translocation of mucosa-associated lymphoid tissue (MALT) lymphoma. Another MALT lymphoma translocation, t(11;18)(q21;q21), fuses the IAP-2 gene to the MLT1/MALT1 locus, which encodes the human paracaspase. We find that this fusion activates NF-kappaB and that the caspase domain is required for this function, since mutation of the conserved catalytic cysteine attenuates NF-kappaB activation.

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Uren AG, Wong L, Pakusch M, Fowler KJ, Burrows FJ, Vaux DL, Choo KHA, Uren AG, Wong L, Pakusch M, Fowler KJ, Burrows FJ, Vaux DL, Choo KH, Uren AG, Wong L, Pakusch M, Fowler KJ, Burrows FJ, Vaux DL, Choo KH, Uren AG, Wong L, Pakusch M, Fowler KJ, Burrows FJ, Vaux DL, Choo KHAet al., 2000, Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype, CURRENT BIOLOGY, Vol: 10, Pages: 1319-1328, ISSN: 0960-9822

BACKGROUND: Survivin is a mammalian protein that carries a motif typical of the inhibitor of apoptosis (IAP)proteins, first identified in baculoviruses. Although baculoviral IAP proteins regulate cell death, the yeast Survivin homolog Bir1 is involved in cell division. To determine the function of Survivin in mammals, we analyzed the pattern of localization of Survivin protein during the cell cycle, and deleted its gene by homologous recombination in mice. RESULTS: In human cells, Survivin appeared first on centromeres bound to a novel para-polar axis during prophase/metaphase, relocated to the spindle midzone during anaphase/telophase, and disappeared at the end of telophase. In the mouse, Survivin was required for mitosis during development. Null embryos showed disrupted microtubule formation, became polyploid, and failed to survive beyond 4.5days post coitum. This phenotype, and the cell-cycle localization of Survivin, resembled closely those of INCENP. Because the yeast homolog of INCENP, Sli15, regulates the Aurora kinase homolog Ipl1p, and the yeast Survivin homolog Bir1 binds to Ndc10p, a substrate of Ipl1p, yeast Survivin, INCENP and Aurora homologs function in concert during cell division. CONCLUSIONS: In vertebrates, Survivin and INCENP have related roles in mitosis, coordinating events such as microtubule organization, cleavage-furrow formation and cytokinesis. Like their yeast homologs Bir1 and Sli15, they may also act together with the Aurora kinase.

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Uren AG, Beilharz T, O'Connell MJ, Bugg SJ, van Driel R, Vaux DL, Lithgow T, Uren AG, Beilharz T, O'Connell MJ, Bugg SJ, van Driel R, Vaux DL, Lithgow T, Uren AG, Beilharz T, O'Connell MJ, Bugg SJ, van Driel R, Vaux DL, Lithgow T, Uren AG, Beilharz T, O'Connell MJ, Bugg SJ, van Driel R, Vaux DL, Lithgow Tet al., 1999, Role for yeast inhibitor of apoptosis (IAP)-like proteins in cell division, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 96, Pages: 10170-10175, ISSN: 0027-8424

Inhibitors of apoptosis (IAPs) are a family of proteins that bear baculoviral IAP repeats (BIRs) and regulate apoptosis in vertebrates and Drosophila melanogaster. The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe both encode a single IAP, designated BIR1 and bir1, respectively, each of which bears two BIRs. In rich medium, BIR1 mutant S. cerevisiae underwent normal vegetative growth and mitosis. Under starvation conditions, however, BIR1 mutant diploids formed spores inefficiently, instead undergoing pseudohyphal differentiation. Most spores that did form failed to survive beyond two divisions after germination. bir1 mutant S. pombe spores also died in the early divisions after spore germination and became blocked at the metaphase/anaphase transition because of an inability to elongate their mitotic spindle. Rather than inhibiting caspase-mediated cell death, yeast IAP proteins have roles in cell division and appear to act in a similar way to the IAPs from Caenorhabditis elegans and the mammalian IAP Survivin.

JOURNAL ARTICLE

Hawkins CJ, Ekert PG, Uren AG, Holmgreen SP, Vaux DL, Hawkins CJ, Ekert PG, Uren AG, Holmgreen SP, Vaux DL, Hawkins CJ, Ekert PG, Uren AG, Holmgreen SP, Vaux DL, Hawkins CJ, Ekert PG, Uren AG, Holmgreen SP, Vaux DLet al., 1998, Anti-apoptotic potential of insect cellular and viral IAPs in mammalian cells, CELL DEATH AND DIFFERENTIATION, Vol: 5, Pages: 569-576, ISSN: 1350-9047

IAPs were identified as baculoviral proteins that could inhibit the apoptotic response of insect cells to infection. Of the viral IAPs, OpIAP and CpIAP can inhibit apoptosis, whereas AcIAP cannot. OpIAP and some mammalian homologues can inhibit mammalian cell death. Two mammalian IAPs bind to TNFRII associated factors (TRAFs), but the significance of this is unclear. Here we show that Drosophila cellular IAPs and two baculoviral IAPs (OpIAP and CpIAP) can inhibit mammalian cell death induced by overexpression of Caspases 1 and 2. IAPs must act on conserved components of the apoptotic mechanism, but as none of these IAPs could bind TRAF proteins, TRAFs are not likely to be important for IAP mediated apoptosis inhibition. As OpIAP protected against death induced by ligation of TNF receptor family members, but not by factor nor serum withdrawal from dependent cells, it can inhibit certain apoptotic pathways without affecting others.

JOURNAL ARTICLE

Uren AG, Coulson EJ, Vaux DL, Uren AG, Coulson EJ, Vaux DL, Uren AG, Coulson EJ, Vaux DL, Uren AG, Coulson EJ, Vaux DLet al., 1998, Conservation of baculovirus inhibitor of apoptosis repeat proteins (BIRPs) in viruses, nematodes, vertebrates and yeasts, TRENDS IN BIOCHEMICAL SCIENCES, Vol: 23, Pages: 159-162, ISSN: 0968-0004

JOURNAL ARTICLE

Uren AG, Vaux DL, Uren AG, Vaux DL, Uren AG, Vaux DLet al., 1997, Viral inhibitors of apoptosis, VITAMINS AND HORMONES - ADVANCES IN RESEARCH AND APPLICATIONS, VOL 53, Vol: 53, Pages: 175-193, ISSN: 0083-6729

JOURNAL ARTICLE

Vaux DL, Hawkins CJ, Uren AG, Ekert PG, Vaux DL, Hawkins CJ, Uren AG, Ekert PG, Vaux DL, Hawkins CJ, Uren AG, Ekert PGet al., 1997, Motor neurone disease and the life of motor neurones, MEDICAL JOURNAL OF AUSTRALIA, Vol: 166, Pages: 109-109, ISSN: 0025-729X

JOURNAL ARTICLE

Hawkins CJ, Uren AG, Hacker G, Medcalf RL, Vaux DL, Hawkins CJ, Uren AG, Häcker G, Medcalf RL, Vaux DL, Hawkins CJ, Uren AG, Häcker G, Medcalf RL, Vaux DL, Hawkins CJ, Uren AG, Hacker G, Medcalf RL, Vaux DLet al., 1996, Inhibition of interleukin 1 beta-converting enzyme-mediated apoptosis of mammalian cells by baculovirus IAP, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 93, Pages: 13786-13790, ISSN: 0027-8424

Apoptosis can be a potent weapon against viral infection and consequently has selected for viruses carrying antiapoptosis genes. Two baculovirus proteins, IAP and p35, can prevent insect cells from dying in response to infection. p35, which interferes with members of the Ced-3 family of cysteine proteases, can also function in mammalian cells. We investigated the ability of IAP from Orgyia pseudotsugata nuclear polyhedrosis virus to prevent death of mammalian cells. IAP was transiently expressed in mammalian cells and its ability to block cell death caused by expression of interleukin-1 beta converting enzyme (ICE), FADD, or the ICE homologues ICH-1 and ICE-Lap3, was investigated. IAP strongly inhibited ICE- and ICH-1-induced cell death but protected only partially against death by overexpression of FADD and not at all against death due to enforced ICE-Lap3 expression. These results demonstrate that a baculoviral IAP protein can functionally interact with conserved components of the apoptosis machinery in mammalian cells.

JOURNAL ARTICLE

Uren AG, Pakusch M, Hawkins CJ, Puls KL, Vaux DL, Uren AG, Pakusch M, Hawkins CJ, Puls KL, Vaux DL, Uren AG, Pakusch M, Hawkins CJ, Puls KL, Vaux DL, Uren AG, Pakusch M, Hawkins CJ, Puls KL, Vaux DLet al., 1996, Cloning and expression of apoptosis inhibitory protein homologs that function to inhibit apoptosis and/or bind tumor necrosis factor receptor-associated factors, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 93, Pages: 4974-4978, ISSN: 0027-8424

Baculovirus inhibitors of apoptosis (IAPs) act in insect cells to prevent cell death. Here we describe three mammalian homologs of IAP, MIHA, MIHB, and MIHC, and a Drosophila IAP homolog, DIHA. Each protein bears three baculovirus IAP repeats and an N-terminal ring finger motif. Apoptosis mediated by interleukin 1beta converting enzyme (ICE), which can be inhibited by Orgyia pseudotsugata nuclear polyhedrosis virus IAP (OpIAP) and cowpox virus crmA, was also inhibited by MIHA and MIHB. As MIHB and MIHC were able to bind to the tumor necrosis factor receptor-associated factors TRAF1 and TRAF2 in yeast two-hybrid assays, these results suggest that IAP proteins that inhibit apoptosis may do so by regulating signals required for activation of ICE-like proteases.

JOURNAL ARTICLE

Uren AG, Vaux DL, Uren AG, Vaux DL, Uren AG, Vaux DLet al., 1996, TRAF proteins and meprins share a conserved domain, TRENDS IN BIOCHEMICAL SCIENCES, Vol: 21, Pages: 244-245, ISSN: 0968-0004

JOURNAL ARTICLE

Uren AG, Vaux DL, Uren AG, Vaux DL, Uren AG, Vaux DL, Uren AG, Vaux DLet al., 1996, Molecular and clinical aspects of apoptosis, PHARMACOLOGY & THERAPEUTICS, Vol: 72, Pages: 37-50, ISSN: 0163-7258

Unwanted cells are removed by physiological cell death processes that are highly conserved throughout the animal kingdom. Physiological cell death plays an important role in development, tissue homeostasis and defence against viral infection and mutation. This review describes the molecular components that implement this process, the relevance of these to a variety of human diseases, and discusses the potential for novel therapies based on our understanding of them.

JOURNAL ARTICLE

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