• IAPs as a target for anticancer therapy.

      Danson, Sarah; Dean, Emma J; Dive, Caroline; Ranson, Malcolm R; Christie Hospital NHS Trust and Paterson Institute for Cancer Research, Wilmslow Road, Manchester, M20 4BX, United Kingdom. s.danson@sheffield.ac.uk (2007-12)
      The avoidance of apoptosis is one of the hallmarks of cancer cells. In addition, failure to induce apoptosis by anticancer agents, either due to limitations of the drug or the tumour cell evading apoptosis, is a reason for chemotherapeutic failure. Two general pathways for apoptotic cell death have been characterised, the extrinsic and intrinsic pathways which merge in the final common pathway. X-linked inhibitor of apoptosis protein (XIAP) is an anti-apoptotic protein in the final common pathway that inhibits caspases and suppresses apoptosis. XIAP is over-expressed in many cancer cell lines and cancer tissues. High XIAP expression has been correlated with resistance to chemotherapy and radiotherapy and to poor clinical outcome by some investigators. Manipulation of apoptosis is an attractive therapeutic concept. Much effort has been spent on inhibiting the anti-apoptotic protein, B cell lymphoma gene 2 (Bcl-2) which is part of the intrinsic pathway. Now attention is turning to inhibition of XIAP as a cancer drug target. It has been argued that it is more effective to block the final common pathway rather than just the intrinsic arm. Inhibition of XIAP can be with either antisense oligonucleotides (ASO) or small molecule inhibitors. In vitro, XIAP antagonists produce XIAP knockdown and apoptosis which is associated with sensitisation of tumour cells to radiotherapy and cytotoxic drugs. In vivo, XIAP antagonists have antitumour effects and sensitise tumours to the effects of chemotherapy. This review will summarise the preclinical data for both ASO and small molecule inhibition of XIAP and discuss emerging Phase I data. Future strategies for manipulation of XIAP and the clinical development of XIAP inhibitors will be discussed.
    • Id helix-loop-helix proteins antagonize pax transcription factor activity by inhibiting DNA binding.

      Roberts, E Claire; Deed, Richard W; Inoue, Toshiaki; Norton, John D; Sharrocks, Andrew D; Department of Biochemistry and Genetics, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom. (2001-01)
      The Id subfamily of helix-loop-helix (HLH) proteins plays a fundamental role in the regulation of cellular proliferation and differentiation. The major mechanism by which Id proteins are thought to inhibit differentiation is through interaction with other HLH proteins and inhibition of their DNA-binding activity. However, Id proteins have also been shown to interact with other proteins involved in regulating cellular proliferation and differentiation, suggesting a more widespread regulatory function. In this study we demonstrate functional interactions between Id proteins and members of the Pax-2/-5/-8 subfamily of paired-domain transcription factors. Members of the Pax transcription factor family have key functions in regulating several developmental processes exemplified by B lymphopoiesis, in which Pax-5 plays an essential role. Id proteins bind to Pax proteins in vitro and in vivo. Binding occurs through the paired DNA-binding domain of the Pax proteins and results in the disruption of DNA-bound complexes containing Pax-2, Pax-5, and Pax-8. In vivo, Id proteins modulate the transcriptional activity mediated by Pax-5 complexes on the B-cell-specific mb-1 promoter. Our results therefore demonstrate a novel facet of Id function in regulating cellular differentiation by functionally antagonizing the action of members of the Pax transcription factor family.
    • Id helix-loop-helix proteins in cell growth and differentiation.

      Norton, John D; Deed, Richard W; Craggs, Graham; Sablitzky, F; Cancer Research Campaign Dept of Gene Regulation, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. JNorton@picr.man.ac.uk (1998-02)
      Id helix-loop-helix proteins function at a general level as positive regulators of cell growth and as negative regulators of cell differentiation. They act as dominant-negative antagonists of other helix-loop-helix transcription factors, which drive cell lineage commitment and differentiation in diverse cell types of higher eukaryotes. In addition, the functions of Id proteins are integrated with cell-cycle-regulatory pathways orchestrated by cyclin-dependent kinases and the retinoblastoma protein. Here, some of the recent advances that highlight the importance of Id proteins as regulatory intermediates for coordinating differentiation-linked gene expression with cell-cycle control in response to extracellular signalling are reviewed.
    • Id helix-loop-helix proteins inhibit nucleoprotein complex formation by the TCF ETS-domain transcription factors.

      Yates, Paula R; Atherton, Graham T; Deed, Richard W; Norton, John D; Sharrocks, Andrew D; Department of Biochemistry and Genetics, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH. (1999-02-15)
      The Id subfamily of helix-loop-helix (HLH) proteins plays a fundamental role in the regulation of cellular proliferation and differentiation. Id proteins are thought to inhibit differentiation mainly through interaction with other HLH proteins and by blocking their DNA-binding activity. Members of the ternary complex factor (TCF) subfamily of ETS-domain proteins have key functions in regulating immediate-early gene expression in response to mitogenic stimulation. TCFs form DNA-bound complexes with the serum response factor (SRF) and are direct targets of MAP kinase (MAPK) signal transduction cascades. In this study we demonstrate functional interactions between Id proteins and TCFs. Ids bind to the ETS DNA-binding domain and disrupt the formation of DNA-bound complexes between TCFs and SRF on the c-fos serum response element (SRE). Inhibition occurs by disrupting protein-DNA interactions with the TCF component of this complex. In vivo, the Id proteins cause down-regulation of the transcriptional activity mediated by the TCFs and thereby block MAPK signalling to SREs. Therefore, our results demonstrate a novel facet of Id function in the coordination of mitogenic signalling and cell cycle entry.
    • Id2 expression increases with differentiation of human myeloid cells.

      Ishiguro, A; Spirin, K S; Shiohara, M; Tobler, A; Gombart, A F; Israel, M A; Norton, John D; Koeffler, H P; Department of Medicine, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, CA 90048, USA. (1996-06-15)
      Id proteins are helix-loop-helix (HLH) transcriptional factors that lack the basic DNA binding domain. The Id proteins have been reported generally to function as inhibitors of cell differentiation, and their gene expression is often downregulated during cell differentiation. We examined the expression of human Id mRNAs by Northern hybridization in 11 human myeloid cell lines, several myeloid cell lines induced to differentiate, fresh myeloid leukemia samples, and normal human myeloid cells. Id2 mRNA was expressed in myelomonoblastic and monoblastic leukemic cells (PLB-985, THP-1, and U-937) but was weakly expressed in myeloblastic leukemic cells (KG-1 and HL-60). Id2 mRNA levels markedly increased with induction of differentiation of myeloid blasts (HL-60, PLB-985, THP-1, and U-937) toward either granulocytes or macrophages. Examination of fresh acute myeloid leukemic samples from 22 individuals also showed prominent Id2 mRNA expression in those samples having more differentiated blasts. Using the French-American-British classification, only 2 of 8 M0/M1 samples expressed Id2 mRNA; however, 10 of 13 M2/M3/M4 samples expressed it. In normal human myeloid cells, Id2 mRNA was expressed in cultured macrophages from bone marrow and in mature granulocytes and monocytes from peripheral blood. The half-life of Id2 mRNA was short (1 hour), and its expression was inducible by cessation of protein synthesis. Id3 mRNA was moderately expressed in monoblastic cell lines (THP-1 and U-937), and levels decreased with their differentiation. Almost no Id3 expression was detectable in either other myeloid leukemia lines, fresh leukemic samples, or normal human myeloid cells by Northern analyses. Id1 mRNA was not detected by polymerase chain reaction in either leukemic or normal myeloid cells except in K562 myeloid/erythroid cells. These results showed that Id2 mRNA was constitutively expressed in more mature myeloid blast cells and level markedly increased with terminal myeloid differentiation, suggesting that Id2 protein may inhibit an HLH transcriptional complex that normally represses myeloid differentiation.
    • Identification and characterisation of the Drosophila melanogaster O6-alkylguanine-DNA alkyltransferase cDNA.

      Kooistra, Rolf; Zonneveld, Jose B M; Watson, Amanda J; Margison, Geoffrey P; Lohman, Paul H; Pastink, Albert; MGC Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. (1999-04-15)
      The protein O 6-alkylguanine-DNA alkyltransferase(alkyltransferase) is involved in the repair of O 6-alkylguanine and O 4-alkylthymine in DNA and plays an important role in most organisms in attenuating the cytotoxic and mutagenic effects of certain classes of alkylating agents. A genomic clone encompassing the Drosophila melanogaster alkyltransferase gene ( DmAGT ) was identified on the basis of sequence homology with corresponding genes in Saccharomyces cerevisiae and man. The DmAGT gene is located at position 84A on the third chromosome. The nucleotide sequence of DmAGT cDNA revealed an open reading frame encoding 194 amino acids. The MNNG-hypersensitive phenotype of alkyltransferase-deficient bacteria was rescued by expression of the DmAGT cDNA. Furthermore, alkyltransferase activity was identified in crude extracts of Escherichia coli harbouring DmAGT cDNA and this activity was inhibited by preincubation of the extract with an oligonucleotide containing a single O6-methylguanine lesion. Similar to E.coli Ogt and yeast alkyltransferase but in contrast to the human alkyltransferase, the Drosophila alkyltransferase is resistant to inactivation by O 6-benzylguanine. In an E.coli lac Z reversion assay, expression of DmAGT efficiently suppressed MNNG-induced G:C-->A:T as well as A:T-->G:C transition mutations in vivo. These results demonstrate the presence of an alkyltransferase specific for the repair of O 6-methylguanine and O 4-methylthymine in Drosophila.
    • Identification and characterization of a novel transcriptional target of RUNX1/AML1 at the onset of hematopoietic development.

      Ferreras, Cristina; Lancrin, Christophe; Lie-A-Ling, Michael; Kouskoff, Valerie; Lacaud, Georges; Cancer Research UK Stem Cell Biology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom. (2011-07-21)
      Although the critical requirement for the transcription factor RUNX1/AML1 at the onset of hematopoietic development is well established, little is known about its transcriptional targets at this pivotal stage of blood development. Using microarrays, we identified the uncharacterized gene AI467606 as a gene whose expression level is dramatically reduced in the absence of RUNX1. We further demonstrated by chromatin immunoprecipitation and promoter assay a direct regulation of its transcription by RUNX1. Using a bacterial artificial chromosome transgenic approach, we established that AI467606 is expressed during the development of the hematopoietic system in vivo and in vitro and that its expression is detected within the CD41(+) population and marks definitive hematopoietic potential. Similarly, in the adult mouse, all hematopoietic cell lineages, except mature erythrocytes, express AI467606. Taken together, these findings indicate that AI467606 is a novel transcriptional target of RUNX1/AML1 at the onset of hematopoietic development that is extensively expressed within the hematopoietic system.
    • Identification and characterization of murine gammaherpesvirus 68 gp150: a virion membrane glycoprotein.

      Stewart, J P; Janjua, N J; Pepper, Stuart D; Bennion, Gordon; Mackett, Mike; Allen, Terence D; Nash, A A; Arrand, John R; Department of Veterinary Pathology, The University of Edinburgh, United Kingdom. (1996-06)
      Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring virus of murid rodents which displays pathobiological characteristics similar to those of other gammaherpesviruses, including Epstein-Barr virus (EBV). However, unlike EBV and many other gammaherpesviruses, MHV-68 replicates in epithelial cells in vitro and infects laboratory strains of mice and therefore provides a good model for the study of gammaherpesviruses. Studies of sequences around the center of the MHV-68 genome identified a gene (designated BPRF1 for BamHI P fragment rightward open reading frame 1) whose putative product had motifs reminiscent of a transmembrane glycoprotein. All other gammaherpesviruses have a glycoprotein in this genomic position, but the BPRF1 gene showed sequence homology with only the EBV membrane antigen gp340/220. Biochemical analysis showed that the product of BPRF1 was a glycoprotein present on the surface of infected cells, and immunoelectron microscopy showed that it was present in the virus particle. In addition, antibodies to the BPRF1 product raised by using a bacterial fusion protein neutralized the virus in the absence of complement. The predominant molecular weights of the protein were 150,000 and 130,000. Pulse-chase analysis and endoglycosidase-H digestion showed that the 130,000-molecular-weight form was a precursor of the 150,000-molecular-weight form, and cell surface labelling showed that the 150,000-molecular-weight form alone was on the cell surface. We therefore named the protein gp150. Since gp150 is the first virion-associated glycoprotein and neutralizing determinant of MHV-68 to be characterized, it provides a valuable tool for the future study of virus-host interactions.
    • Identification and characterization of novel functional markers during the hematopoietic stem cell specification process

      Buchler-Schaff, M.; Kaschutnig, P.; Thambyrajah, Roshana; Nadler, W.; Hanke, S.; Pfaffenholz, S.; Block, M.; Grasel, J.; Kremer, J.; Bayindir-Buchhalter, I.; et al. (2020)
      Endothelial to hematopoietic transition (EHT) is a crucial step in the formation of definitive hematopoietic stem cells during embryonic development. Holding such a critical developmental role not many details regarding molecular changes and cell surface marker (CSM) expression on these transitional cells are known. We think that sub-segregating the process of EHT holds great therapeutic potential by giving new indications to understand the hematopoietic stem cell maturation. We generated embryonic stem (ES) cell lines from Hoxb4-YFP reporter mice (Hills, 2011), in which YFP expression marks functional HSCs in adult and embryonic mice. When these cell lines were subject to embryoid body differentiation assays, we observed a transient Hoxb4/YFP+ cell population, which corresponded to the emergence of hemogenic endothelial cells in culture. We then analyzed the gene expression profile of the Hoxb4+ cells and compared it to immediate precursor (Hoxb4-Flk1+) and daughter (Hoxb4-CD41+) cell populations. As well as documenting the expression of numerous molecular markers previously associated with EHT, we also observed an elevated inflammatory gene expression signature that has previously been characterized as a mediator of hematopoietic specification in vivo. We were additionally able to identify 45 novel cell surface markers that could potentially be used to prospectively isolate and sub segregate cells undergoing EHT. 26 of these targets were subsequently verified using MRM mass spectrometry. To establish if any of these markers were functionally relevant, we generated knockout (KO) cell lines using CRISPR/Cas9. Upon EB differentiation, we observed a profound block in hematopoietic differentiation for ES cells that were KO for the membrane proteins Evi2a and Lyve1. This block was manifest at the EHT stage, as verified by time-lapse video imaging, and was equivalent in magnitude to Runx1 KO, suggesting a crucial role of Evi2a and Lyve1 during the EHT stage of hematopoietic development. Notably, this defect could be rescued by genetic replacement of the deleted gene. When comparing our data to human fetal liver data sets Evi2a is highly expressed in fetal liver, as well as in HSCs isolated from murine AGM and fetal liver.
    • Identification and cloning in yeast artificial chromosomes of a region of elevated loss of heterozygosity on chromosome 1p31.1 in human breast cancer.

      Hoggard, Nigel; Hey, Yvonne; Brintnell, Bill; James, Louise A; Jones, David; Mitchell, Erika L D; Weissenbach, J; Varley, Jennifer; CRC Department of Cancer Genetics, Paterson Institute for Cancer Research, Manchester, United Kingdom. (1995-11-20)
      We have mapped a region of high loss of heterozygosity in breast cancer to a 2-cM interval between the loci D1S430 and D1S465 on chromosome 1p31.1. This region shows allelic imbalance in around 60% of breast tumors. As part of a strategy to clone the target gene(s) within this interval, we have generated a yeast artificial chromosome contig spanning over 7 Mb. YACs from the CEPH and Zeneca (formerly ICI) libraries have been obtained by screening with PCR-based STSs from the region for both previously identified loci and newly isolated STSs. The YACs have been assembled into a contig by a combination of approaches, including analysis of their STS content, generation of new STSs from the ends of key YACs, and long-range restriction mapping. These YAC clones provide the basis for complete characterization of the region of high loss in breast cancer and for the ultimate identification of the target gene(s).
    • Identification and partial characterisation of a low Mr collagen synthesised by bovine retinal pericytes. Apparent relationship to type X collagen.

      Canfield, Ann E; Schor, Ana M; Department of Medical Oncology, Christie Hospital and Holt Radium Institute, Manchester, UK. (1991-07-29)
      Bovine retinal pericytes (BRP) in culture synthesise a low Mr collagenous polypeptide which appears similar, but not identical, to bovine type X collagen and which we have called 'BRP collagen'. This polypeptide displays the following characteristics: (i) it is sensitive to digestion by bacterial collagenase and is resistant to pepsin digestion; (ii) it has an apparent Mr of 45 kDa (pepsinised form); (iii) it is recognised by specific antibodies to type X collagen using immunoblotting; (iv) it is present in the cell layer/matrix but not in the medium of pericyte cultures; and (v) it is not disulphide-bonded into higher Mr multimers. The latter two properties distinguish BRP collagen from bovine type X collagen. We have recently shown that pericytes calcify in vitro. We now report that this calcification is associated with an increased synthesis of BRP collagen.
    • Identification and partial characterization of three low-molecular-weight collagenous polypeptides synthesized by chondrocytes cultured within collagen gels in the absence and in the presence of fibronectin.

      Gibson, G J; Kielty, C M; Garner, C; Schor, Seth L; Grant, M E; Department of Biochemistry, University of Manchester Medical School, Manchester, M13 9PT (1983-05-01)
      Culture of chick-embryo sternal-cartilage chondrocytes within three-dimensional collagen gels promotes the synthesis of three low-molecular-weight collagenous polypeptides. The proportions of these novel collagens synthesized and released into the medium are markedly influenced by the presence or the absence of fibronectin in the serum supplement. Chondrocytes cultured on plastic dishes appear to synthesize only small amounts of these low-molecular-weight species. The three species (designated G, H and J) were characterized with respect to the proportion of [14C]proline incorporated into each polypeptide occurring as hydroxy[14C]proline and with respect to their susceptibilities to bacterial collagenase. On the basis of their electrophoretic mobilities under reducing conditions, the G, H and J polypeptides were calculated to have Mr 59 000, 69 000 and 84 000 respectively. Chymotrypsin digestion converted the G collagen into a species containing polypeptides of Mr 45 000, whereas the H and J polypeptides yielded a single band of Mr 53 000. The H and J polypeptides were found to occur as disulphide-linked aggregates, as was the chymotrypsin-digestion product. Peptide 'mapping' has shown that G, H and J polypeptides show no common identity and are distinct from the known interstitial collagens. Native G collagen was digested by human collagenase to discrete products, whereas H and J chains were not cleaved under identical conditions.
    • Identification and partial characterization of two major proteins of Mr 47,000 synthesized by bovine retinal endothelial cells in culture.

      Canfield, Ann E; Schor, Ana M; West, David C; Schor, Seth L; Grant, M E; Department of Biochemistry and Molecular Biology, School of Biological Sciences, University of Manchester, U.K. (1987-08-15)
      Biosynthetic experiments with cultured bovine retinal endothelial cells have identified a glycoprotein of Mr 47,000 (Gp47) as a major component secreted into the medium. Gp47 is a non-collagenous glycoprotein with a pI of 4.6-5.5, which does not bind to either gelatin-Sepharose or heparin-Sepharose but is retained by concanavalin A-Sepharose. The Mr of this species decreases to approx. 42,000 in the presence of tunicamycin, indicating that it contains asparagine-linked oligosaccharides. A second protein of Mr 47,000 (P47) is present in the cell layer/matrix of these cultured cells. The electrophoretic mobility of P47 remains unaltered when synthesized in the presence of tunicamycin. Peptide-mapping experiments using N-chlorosuccinimide and Staphylococcus aureus V8 proteinase demonstrate that Gp47 and P47 are distinct proteins, and are not related to colligin, a membrane-bound collagen-receptor protein of similar size, or to SPARC, a major secreted product of parietal endodermal cells and sparse cultures of aortic endothelial cells.
    • Identification in rat stomach mucosae of a cell population characterized by a deficiency for the repair of O6-methyldeoxyguanosine from DNA.

      Zaidi, N H; O'Connor, Peter J; Cancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital (NHS Trust), Manchester, UK. (1995-03)
      Immunohistochemical studies have been used to show the time course for the cell-specific methylation of DNA in the upper gastrointestinal tract of rats treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The doses used were 1, 5, 25 and 50 mg MNNG/kg (i.g.) and tissue samples were analysed at intervals of from 1 to 192 h after treatment. Relatively little reaction with nuclear DNA was observed in the forestomach and still less in the oesophagus. Reaction with DNA was most extensive in the corpus, pylorus and the duodenum, reaching a peak of staining intensity between 2 and 4 h and then declining progressively there-after. Staining for the presence of O6-methyldeoxyguanosine (O6-MedG) in DNA was highly selective and tended to occur in the nuclei of the basal cell of the oesophagus and forestomach and in the cells of the lumenal border of the glands and villi of the corpus, pylorus and duodenum. There were also areas, 5-15, glands apart where staining for O6-MedG in the corpus and pylorus extended as far down as the basal mucosa. From 12 h after MNNG treatment, in the corpus and pylorus, a band of strongly methylated cells became apparent about 3-6 cells deep from the lumen and remained identifiable up to 168 h after treatment with the higher doses. These cells, which apparently have a very low O6-MedG repair capacity, are stationary (i.e. not part of the escalator) and are located in the mesenchymal tissue elements as demonstrated by staining of serial sections with cytokeratin or vementin. The significance of this population of cells is unknown.
    • Identification of a biomarker panel for early detection of lung cancer patients

      Geary, B; Walker, MJ; Snow, JT; Lee, DCH; Pernemalm, M; Maleki-Dizaji, S; Azadbakht, N; Apostolidou, S; Barnes, J; Krysiak, P; et al. (2019)
      Lung cancer is the most common cause of cancer-related mortality worldwide, characterized by late clinical presentation (49-53% of patients are diagnosed at stage IV) and consequently poor outcomes. One challenge in identifying biomarkers of early disease is the collection of samples from patients prior to symptomatic presentation. We used blood collected during surgical resection of lung tumors in an iTRAQ isobaric tagging experiment to identify proteins effluxing from tumors into pulmonary veins. Forty proteins were identified as having an increased abundance in the vein draining from the tumor compared to "healthy" pulmonary veins. These protein markers were then assessed in a second cohort that utilized the mass spectrometry (MS) technique: Sequential window acquisition of all theoretical fragment ion spectra (SWATH) MS. SWATH-MS was used to measure proteins in serum samples taken from 25 patients <50 months prior to and at lung cancer diagnosis and 25 matched controls. The SWATH-MS analysis alone produced an 11 protein marker panel. A machine learning classification model was generated that could discriminate patient samples from patients within 12 months of lung cancer diagnosis and control samples. The model was evaluated as having a mean AUC of 0.89, with an accuracy of 0.89. This panel was combined with the SWATH-MS data from one of the markers from the first cohort to create a 12 protein panel. The proteome signature developed for lung cancer risk can now be developed on further cohorts.
    • Identification of a candidate tumor-suppressor gene specifically activated during Ras-induced senescence.

      Barradas, Marta; Gonos, Efstathios S; Zebedee, Zoe; Kolettas, Evangelos; Petropoulou, Charikleia; Delgado, M Dolores; León, Javier; Hara, Eiji; Serrano, Manuel; Department of Immunology and Oncology, Spanish National Center of Biotechnology (CSIC), Campus de Cantoblanco, Madrid E-28049, Spain. (2002-02-15)
      Normal cells display protective responses against oncogenes. Notably, oncogenic Ras triggers an irreversible proliferation arrest that is reminiscent of replicative senescence and that is considered a relevant tumor-suppressor mechanism. Here, we have used microarrayed filters to identify genes specifically upregulated in Ras-senescent human fibroblasts. Among the initial set of genes selected from the microarrays, we found the cell-cycle inhibitor p21(Cip1/Waf1), thus validating the potency of the screening to identify markers and mediators of Ras-senescence. A group of six genes, formed by those more highly upregulated during Ras-senescence, was analyzed in further detail to evaluate their specificity. In particular, we examined their expression in cells overexpressing Ras but rendered resistant to Ras-senescence by the viral oncoprotein E1a; also, we have studied their expression during replicative senescence, organismal aging, H(2)O(2)-induced senescence, and DNA damage. In this manner, we have identified a novel gene, RIS1 (for Ras-induced senescence 1), which is not upregulated in association to any of the above-mentioned processes, but exclusively during Ras-senescence. Furthermore, RIS1 is also upregulated by the transcriptional factor Ets2, which is a known mediator of Ras-induced senescence. Interestingly, RIS1 is located at chromosomal position 3p21.3 and, more specifically, it is included in a short segment of just 1 Mb previously defined by other investigators for its tumor-suppressor activity. In summary, we report the identification of a novel gene, RIS1, as a highly specific marker of Ras-induced senescence and a candidate tumor-suppressor gene.
    • Identification of a functional prostanoid-like receptor in the protozoan parasite, trypanosoma cruzi.

      Mukherjee, S; Sadekar, N; Ashton, A; Huang, H; Spray, D; Lisanti, Michael P; Machado, F; Weiss, L; Tanowitz, H; Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, USA. sankar.mukhopadhyay@einstein.yu.edu (2013-04)
      Trypanosoma cruzi infection in humans and experimental animals causes Chagas disease which is often accompanied by myocarditis, cardiomyopathy, and vasculopathy. T. cruzi-derived thromboxane A2 (TXA2) modulates vasculopathy and other pathophysiological features of Chagasic cardiomyopathy. Here, we provide evidence that epimastigotes, trypomastigotes, and amastigotes of T. cruzi (Brazil and Tulahuen strains) express a biologically active prostanoid receptor (PR) that is responsive to TXA2 mimetics, e.g. IBOP. This putative receptor, TcPR, is mainly localized in the flagellar membrane of the parasites and shows a similar glycosylation pattern to that of bona fide thromboxane prostanoid (TP) receptors obtained from human platelets. Furthermore, TXA2-PR signal transduction activates T. cruzi-specific MAPK pathways. While mammalian TP is a G-protein coupled receptor (GPCR); T. cruzi genome sequencing has not demonstrated any confirmed GPCRs in these parasites. Based on this genome sequencing it is likely that TcPR is unique in these protists with no counterpart in mammals. TXA2 is a potent vasoconstrictor which contributes to the pathogenesis of Chagasic cardiovascular disease. It may, however, also control parasite differentiation and proliferation in the infected host allowing the infection to progress to a chronic state.
    • Identification of a major histocompatibility complex class I-restricted T-cell epitope in the tumour-associated antigen, 5T4.

      Redchenko, Irina; Harrop, Richard; Ryan, Matthew G; Hawkins, Robert E; Carroll, Miles W; Oxford BioMedica (UK) Ltd, Medawar Centre, Oxford Science Park, Oxford, UK. i.redchenko@oxfordbiomedica.co.uk (2006-05)
      5T4 is a surface glycoprotein expressed on placental trophoblasts and also on a wide range of human carcinomas. Its highly restricted expression on normal tissues and broad distribution on many carcinomas make 5T4 a promising target for cancer immunotherapy. In the current study, we set out to investigate whether a 5T4-specific cytotoxic T lymphocyte (CTL) repertoire exists in healthy individuals. CD4-depleted peripheral blood mononuclear cells (PBMCs) from blood donors were screened using an ex vivo interferon-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay. A panel of overlapping peptides, spanning the full length of the 5T4 protein, was used as a source of antigen. In the process of screening, one out of 30 blood donors demonstrated a positive ex vivo IFN-gamma ELISPOT response to a single 5T4 peptide. A polyclonal T-cell line was derived from this donor by culturing PBMCs with autologous peptide-pulsed dendritic cells (DCs). The resulting polyclonal T-cell line and clones were tested in a 51Cr-release assay and by ELISPOT and were shown to be peptide specific. Furthermore, antigen-presenting cells (APCs), infected with a viral vector expressing 5T4, were able to stimulate IFN-gamma production by the peptide-specific T-cell clones. A minimal CD8 epitope, PLADLSPFA, has been identified and found to be restricted through human leucocyte antigen (HLA) Cw7. Subsequently, we have demonstrated that HLA-Cw7-positive colorectal cancer patients vaccinated with a recombinant vaccinia viral vector encoding 5T4 (TroVax) are capable of mounting a strong IFN-gamma ELISPOT response to this novel CTL epitope. These findings have potential application in cancer immunotherapy in terms of subunit vaccine design and the monitoring of immune responses induced in patients by 5T4-based therapies.
    • Identification of a naturally processed HLA A0201-restricted viral peptide from cells expressing human papillomavirus type 16 E6 oncoprotein.

      Bartholomew, Jennifer S; Stacey, Simon N; Coles, B; Burt, Deborah J; Arrand, John R; Stern, Peter L; Department of Immunology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, GB. (1994-12)
      Human papillomavirus (HPV) DNA encoding the oncogenic proteins E6 and E7 is usually retained in cervical carcinomas, implicating these proteins as potential target antigens for immune recognition in this virally associated tumor. We have characterized endogenously processed peptides eluted from major histocompatibility complex class I molecules in cells infected with a recombinant vaccinia expressing the HPV-16 E6 oncoprotein. The reverse-phase chromatography profile of peptides eluted from isolated HLA-A0201 molecules in cells expressing the E6 oncoprotein differs from that of cells not expressing E6. Sequential Edman degradation of novel peaks found in the peptide profiles from cells expressing HPV-16 E6 led to the identification of a naturally processed HLA-A0201-restricted E6 peptide of sequence KLPQLCTEL. This approach has allowed the identification of a viral peptide which is processed and presented by cells expressing the E6 oncoprotein and is a likely target for cytotoxic T lymphocyte recognition in HLA-A0201-positive patients.
    • Identification of a novel chalcone derivative that inhibits Notch signaling in T-cell acute lymphoblastic leukemia.

      Mori, M; Tottone, L; Quaglio, D; Zhdanovskaya, N; Ingallina, C; Fusto, M; Ghirga, F; Peruzzi, G; Crestoni, M; Simeoni, Fabrizio; et al. (2017-05-19)
      Notch signaling is considered a rational target in the therapy of several cancers, particularly those harbouring Notch gain of function mutations, including T-cell acute lymphoblastic leukemia (T-ALL). Although currently available Notch-blocking agents are showing anti-tumor activity in preclinical studies, they are not effective in all the patients and often cause severe side-effects, limiting their widespread therapeutic use. Here, by functional and biological analysis of the most representative molecules of an in house library of natural products, we have designed and synthetized the chalcone-derivative 8 possessing Notch inhibitory activity at low micro molar concentration in T-ALL cell lines. Structure-activity relationships were afforded for the chalcone scaffold. Short term treatments with compound 8 resulted in a dose-dependent decrease of Notch signaling activity, halted cell cycle progression and induced apoptosis, thus affecting leukemia cell growth. Taken together, our data indicate that 8 is a novel Notch inhibitor, candidate for further investigation and development as an additional therapeutic option against Notch-dependent cancers.