• Ionizing radiation induces O6-alkylguanine-DNA-alkyltransferase mRNA and activity in mouse tissues.

      Wilson, Rosemary E; Hoey, Brigid M; Margison, Geoffrey P; Cancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1993-04)
      The effect of exposure to whole-body gamma-irradiation or fast electrons on O6-alkylguanine-DNA-alkyltransferase (ATase) activity and mRNA abundance has been examined in mice. In response to gamma-radiation, hepatic ATase activity was significantly raised in BDF1 mice 24 h post-irradiation, reaching a maximum of 2- to 3-fold at 36 h and beginning to decrease by 48-60 h. A small but consistently higher level of induction was achieved when mice were exposed using a low dose rate (0.015 Gy/min) compared to a high dose rate (0.5 Gy/min). ATase activity was also induced approximately 2-fold 48 h post-irradiation in brain, kidney, lung and spleen, with a greater induction again observed in response to the lower dose rate. In response to fast electrons from a linear accelerator hepatic ATase activity was also induced 2- to 3-fold 48 h post-irradiation in BDF1, BALB/c, C57Bl and DBA2 strains. Induction of ATase activity in livers of BDF1 mice was observed 48 h after a total single dose of 5 Gy gamma-radiation (2-fold), increasing to a slightly higher level at 15 Gy, but no induction was observed at doses of 2 Gy and below. Although a maximum 2- to 3-fold induction of ATase activity was observed, mRNA levels were induced 3- to 4-fold by 48 h after a dose of 15 Gy. Furthermore, significant increases in mRNA levels were detected at low doses (1-2 Gy) at which there was no apparent increase in ATase activity. This suggests that ionizing radiation increases ATase levels by a process involving transcriptional upregulation but that strong post-transcriptional and/or translational controls operate to limit induction of enzyme activity to 2- to 3-fold. This is the first report of an in vivo induction of ATase by ionizing radiation in a species other than the rat.
    • iRFP (near-infrared fluorescent protein) imaging of subcutaneous and deep tissue tumours in mice highlights differences between imaging platforms

      Hall, Callum; von Grabowiecki, Yannick; Pearce, Simon P; Dive, Caroline; Bagley, Steven; Muller, Patricia; Tumour Suppressors Group, CRUK Manchester Institute, University of Manchester, Alderley Park, Manchester, SK10 4T (2021)
      Background: In vivo imaging using fluorescence is used in cancer biology for the detection, measurement and monitoring of tumours. This can be achieved with the expression of fluorescent proteins such as iRFP, which emits light at a wavelength less attenuated in biological tissues compared to light emitted by other fluorescent proteins such as GFP or RFP. Imaging platforms capable of detecting fluorescent tumours in small animals have been developed but studies comparing the performance of these platforms are scarce. Results: Through access to three platforms from Xenogen, Bruker and Li-Cor, we compared their ability to detect iRFP-expressing subcutaneous tumours as well as tumours localised deeper within the body of female NSG mice. Each platform was paired with proprietary software for image analyse, but the output depends on subjective decisions from the user. To more objectively compare platforms, we developed an 'in house' software-based approach which results in lower measured variability between mice. Conclusions: Our comparisons showed that all three platforms allowed for reliable detection and monitoring of subcutaneous iRFP tumour growth. The biggest differences between platforms became apparent when imaging deeper tumours with the Li-Cor platform detecting most tumours and showing the highest dynamic range.
    • Irradiated Blm-deficient mice are a highly tumor prone model for analysis of a broad spectrum of hematologic malignancies.

      Warren, Madhuri; Chung, Yeun-Jun; Howat, William J; Harrison, Hannah; McGinnis, Ralph; Hao, Xingpei; McCafferty, John; Fredrickson, Torgny N; Bradley, Allan; Morse, Herbert C; et al. (2010-02)
      Mutations in the BLM gene cause human Bloom syndrome (BS), an autosomal recessive disorder of growth retardation, immunodeficiency and cancer predisposition. Homozygous null Blm(m3/m3) mice are cancer prone with a 5-fold increased risk of cancer compared with Blm(m3/+) and Blm(+/+) mice. Irradiation of Blm(m3/m3) mice increased the risk to 28-fold. Tumors occurred mainly in the hematopoietic system and were similar to those in BS based on detailed histologic and immunohistochemical analyses. Irradiated Blm-deficient mice thus provide a novel model for understanding accelerated malignancies in BS and a new platform for investigating the molecular basis for a wide range of hematopoietic neoplasms.
    • Irradiation induces up-regulation of E9 protein (CD105) in human vascular endothelial cells.

      Wang, Ji Min; Kumar, Shant; Van Agthoven, A; Kumar, Patricia; Pye, David A; Hunter, Robin D; Christie Hospital, Manchester, UK. (1995-09-15)
      The use of MAb E-9 raised against tissue-cultured endothelial cells (EC) has shown marked heterogeneity in vascular EC lining the blood vessels of normal and tumour tissues. MAb E-9 is human EC-specific and the protein recognized by it is a homodimer with a molecular mass of 97 kDa. The E-9 protein is resistant to treatment by 3 mM sodium periodate, but is sensitive to 10% trichloroacetic acid and 70% ethanol. E-9 protein has been assigned to a new cluster, CD105, and mapped to human chromosome 9q3. It has approximately 70% homology with type-III cell-surface receptor for transforming growth factor beta (TGF-beta). Recently CD105 has been reported to be the gene in patients with hereditary haemorrhagic telangiectasia. We have examined the effects of radiation on its expression in normal human umbilical-vein endothelial cells (HUVEC) and brain-tumour-derived endothelial cells (BTEC). Irradiation induced dose- and time-dependent up-regulation in the expression of the E-9 protein on the plasma membranes of EC, and also resulted in greater increase in the expression of the E-9 protein in semi-confluent (proliferating) as compared with confluent (non-proliferating) EC. It may well be that, following radiotherapy in cancer patients, E-9 protein is also up-regulated. The presence of increased amounts of E-9 protein in EC makes it an attractive target in the control of angiogenesis, especially after radiotherapy in cancer patients. The time scale involved in the up-regulation of E-9 protein following irradiation has led us to suggest that it may be a secondary event, the primary being the production and release of mitogenic factors (such as basic fibroblast growth factor) from irradiated EC.
    • Is immunity in cancer the key to improving clinical outcome?: Report on the International Symposium on Immunotherapy

      Stern, Peter L; Division of Molecular & Clinical Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Paterson Building, Wilmslow Road, Manchester M20 4BX, UK (2017-06)
    • Is serum or plasma more appropriate for intersubject comparisons in metabolomic studies? An assessment in patients with small-cell lung cancer.

      Wedge, D C; Allwood, J W; Dunn, W; Vaughan, A A; Simpson, Kathryn L; Brown, M; Priest, Lynsey; Blackhall, Fiona H; Whetton, Anthony D; Dive, Caroline; et al. (2011-09-01)
      In clinical analyses, the most appropriate biofluid should be analyzed for optimal assay performance. For biological fluids, the most readily accessible is blood, and metabolomic analyses can be performed either on plasma or serum. To determine the optimal agent for analysis, metabolic profiles of matched human serum and plasma were assessed by gas chromatography/time-of-flight mass spectrometry and ultrahigh-performance liquid chromatography mass spectrometry (in positive and negative electrospray ionization modes). Comparison of the two metabolomes, in terms of reproducibility, discriminative ability and coverage, indicated that they offered similar analytical opportunities. An analysis of the variation between 29 small-cell lung cancer (SCLC) patients revealed that the differences between individuals are markedly similar for the two biofluids. However, significant differences between the levels of some specific metabolites were identified, as were differences in the intersubject variability of some metabolite levels. Glycerophosphocholines, erythritol, creatinine, hexadecanoic acid, and glutamine in plasma, but not in serum, were shown to correlate with life expectancy for SCLC patients, indicating the utility of metabolomic analyses in clinical prognosis and the particular utility of plasma in relation to the clinical management of SCLC.
    • Is there a role for an 18F-fluorodeoxyglucose-derived biological boost in squamous cell anal cancer?

      Sabbagh, A; Jacobs, C; Cooke, R; Chu, KY; Ng, SM; Strauss, VY; Virdee, PS; Hawkins, MA; Aznar, Marianne Camille; Muirhead, R; et al. (2018)
      AIMS: To investigate the potential role for a biological boost in anal cancer by assessing whether subvolumes of high 18F-fluorodeoxyglucose (FDG) avidity, identified at outset, are spatially consistent during a course of chemoradiotherapy (CRT). MATERIALS AND METHODS: FDG-positron emission tomography (FDG-PET) scans from 21 patients enrolled into the ART study (NCT02145416) were retrospectively analysed. In total, 29 volumes including both primary tumours and involved nodes >2 cm were identified. FDG-PET scans were carried out before treatment and on day 8 or 9 of CRT. FDG subvolumes were created using a percentage of maximum FDG avidity at thresholds of 34%, 40%, 50%, on the pre-treatment scans, and 70% and 80% on the subsequent scans. Both FDG-PET scans were deformably registered to the planning computed tomography scan. The overlap fraction and the vector distance were calculated to assess spatial consistency. FDG subvolumes for further investigation had an overlap fraction >0.7, as this has been defined in previous publications as a 'good' correlation. RESULTS: The median overlap fractions between the diagnostic FDG-PET subvolumes 34%, 40% and 50% of maximum standardised uptake value (SUVmax) and subsequent FDG-PET subvolumes of 70% of SUVmax were 0.97, 0.92 and 0.81. The median overlap fraction between the diagnostic FDG-PET subvolumes 34%, 40% and 50% and subsequent FDG-PET subvolumes of 80% were 1.00, 1.00 and 0.92. The median (range) vector distance values between diagnostic FDG-PET subvolumes 34%, 40% and 50% and subsequent FDG-PET subvolumes of 80% were 0.74 mm (0.19-2.94) 0.74 mm (0.19-3.39) and 0.71 mm (0.2-3.29), respectively. Twenty of 29 volumes (69.0%) achieved a threshold > 0.7 between the FDG 50% subvolume on the diagnostic scan and the FDG 80% subvolume on the subsequent scan. CONCLUSION: FDG-avid subvolumes identified at baseline were spatially consistent during a course of CRT treatment. The subvolume of 50% of SUVmax on the pre-treatment scan could be considered as a potential target for dose escalation.
    • Is tumor cell radiation resistance correlated with metastatic ability?

      Suit, H; Allam, A; Allalunis-Turner, J; Brock, W; Girinsky, T; Hill, S; Hunter, N; Milas, L; Pearcey, R; Peters, L; et al. (1994-04-01)
      Patients who experience local failure following radiation treatment of epithelial malignancies exhibit a substantially higher rate of distant metastasis than those patients who achieve permanent local control. This fact has raised concern that the local failure to control the primary/regional tumor may serve as a marker of a particularly malignant neoplasm, i.e., high metastatic activity and radiation resistance. If this were true, there would be no gains in survival by increasing the efficacy of treating the primary/regional disease because the new local controls would develop distant metastasis. To investigate this concept, the relationship between distant metastasis probability and tumor cell radiation resistance has been studied by examining laboratory and clinical data (in vitro and in vivo assays) from six collaborating centers. TCD50s (radiation dose which inactivates half of the irradiated tumors) and incidence of distant metastasis in mice with local control have been evaluated for 24 murine tumor systems. SF2s (surviving fraction after 2 Gy) were determined in vitro for cell lines from 8 human, 13 mouse, and 15 rat tumors/tumor sublines and the metastatic activity assessed after injection of the cells into syngeneic murine hosts and xenogenic hosts for the human tumors. SF2s of cells from carcinomas of the head/neck, cervix, and endometrium which were controlled locally by radiation +/- surgery from four centers were compared for those which did and those which did not metastasize. The total number of patients studied was 222. The cumulative distributions of SF2s of locally controlled tumors which did and did not metastasize were not different in each of the data sets. Similarly, there was no demonstrable relationship between TCD50s and metastatic frequency in local control mice. Furthermore, the SF2s of murine and human tumor cell lines did not track with metastatic activity. Radiation sensitivity of clinical and laboratory tumors did not correlate with metastatic activity in studies of data from six centers.
    • Isoflavones inhibit intestinal epithelial cell proliferation and induce apoptosis in vitro.

      Booth, Catherine; Hargreaves, Danielle F; Hadfield, John A; McGown, Alan T; Potten, Christopher S; Epithelial Biology Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1999-07)
      There have been many reports that high soya-based diets reduce the risk of certain types of cancer. This effect may be due to the presence of high levels of isoflavones derived from the soya bean, particularly genistein which has been shown to be a protein tyrosine kinase (PTK) inhibitor and have both oestrogenic and anti-oestrogenic properties. We have examined the effect of genistein and a number of novel synthetic analogues on both normal (IEC6, IEC18) and transformed (SW620, HT29) intestinal epithelial cell lines. Responses were compared to those elicited by oestradiol, the anti-oestrogen tamoxifen, and the tyrosine kinase inhibitor tyrphostin. Genistein and tamoxifen were potent inhibitors of cell proliferation. Of seven novel isoflavones tested, none were more potent inhibitors than genistein, and all displayed similar relative activities across the different cell lines. In addition to inhibiting cell proliferation, cell death via apoptosis was observed when the cells were exposed to the isoflavones and all but one exhibited PTK inhibitory activity. These data suggest that by reducing proliferation and inducing apoptosis, possibly due in part to PTK inhibition, isoflavones may have a role in protecting normal intestinal epithelium from tumour development (reducing the risk) and may reduce colonic tumour growth.
    • Isolation and cDNA cloning of a rat O6-alkylguanine-DNA-alkyltransferase gene, molecular analysis of expression in rat liver.

      Potter, P M; Rafferty, Joseph A; Cawkwell, L; Wilkinson, M C; Cooper, Donald P; O'Connor, Peter J; Margison, Geoffrey P; CRC Department of Chemical Carcinogenesis, Paterson Institute for Cancer Research, Manchester, UK. (1991-04)
      A rat O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA has been isolated from a rat liver cDNA library by hybridization with the human homologue. The candidate 806 bp cDNA was sequenced and shown to contain a 630 bp open reading frame that could encode a protein of 22.2 kd. Fluorography of labelled ATase indicates a 24 kd protein which is consistent with several previous reports. The derived amino acid sequence demonstrated 81% similarity with the human ATase and 94% identity over a 67 residue region encompassing the putative alkyl acceptor site. Peptide sequences derived from cleaved homogeneous rat ATase have been located in the predicted protein providing additional confirmation of the identity of the cDNA. A 1.05 kb mRNA has been detected in rat liver by Northern analysis; treatment of adult rats with 2-acetylaminofluorene causes an approximately 10-fold induction of this message in liver. Following site directed mutagenesis of the 806 bp cDNA, the 630 bp protein coding sequence has been ligated into an Escherichia coli expression vector to achieve ATase levels of greater than 3% of total protein in bacterial extracts.
    • Isolation and characterisation of a bipotential haematopoietic cell line.

      Dexter, T Michael; Allen, Terence D; Scott, David; Teich, N; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1979-02-08)
    • Isolation and characterization of 5T4, a tumour-associated antigen

      Hole, Nicholas; Stern, Peter L; Department of Cancer Biology, Salk Institute, San Diego, CA, USA (1990)
    • Isolation and characterization of a human homologue of the latrophilin gene from a region of 1p31.1 implicated in breast cancer.

      White, Gavin R M; Varley, Jennifer; Heighway, Jim; CRC Section of Molecular Genetics, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (1998-12-31)
      We have identified a region of chromosome 1p31.1 that shows high frequency loss of heterozygosity (LOH) in human breast cancer. This region forms part of a 7 Mb YAC/BAC contig. In order to identify candidate sequences, mutation of which might contribute to the development of disease, we have carried out mapping studies of ESTs localized to 1p31.1. This analysis, coupled with library screening and a modified 5' RACE-PCR strategy, resulted in the identification and characterization of a novel gene (LPHH1) which is located adjacent to the smallest region of overlapping loss (SRO) seen in tumours. The 4209 bp open reading frame of the 7 kb LPHH1 transcript encodes a peptide which shows approximately 65% identity to rat latrophilin, a G-coupled, seven span transmembrane protein, which binds alpha-latrotoxin. In the human sequence, whilst conservation of the transmembrane domain is high, the intra- and extracellular domains show two regions of variable structure, which are presumably generated by alternative splicing. Surprisingly, while expression of the rat gene is tightly restricted to neurological and perhaps some endocrine cells, the human sequence appears to be expressed very widely in all normal tissues tested. Northern and RT-PCR analysis of a panel of tumour cell lines showed that LPHH1 expression was variable, apparently elevated in some lines and absent or markedly reduced in others. Furthermore, characterization of the range of transcripts encoded in a breast tumour cell line, compared to normal breast, suggested that gene product variability was higher in the tumour.
    • Isolation and characterization of human mammary stem cells.

      Clarke, Robert B; Breast Biology Group, Division of Cancer Studies, University of Manchester, Christie Hospital, Wilmslow Road, Withington, Manchester, M20 4BX, UK. robert.clarke@manchester.ac.uk (2005-12)
      Since stem cells are present throughout the lifetime of an organism, it is thought that they may accumulate mutations, eventually leading to cancer. In the breast, tumours are predominantly oestrogen and progesterone receptor-positive (ERalpha/PR+). We therefore studied the biology of ERalpha/PR-positive cells and their relationship to stem cells in normal human mammary epithelium. We demonstrated that ERalpha/PR-positive cells co-express the putative stem cell markers p21(CIP1/WAF1), cytokeratin (CK) 19 and Musashi-1 when examined using dual label immunofluorescence on tissue sections. Next, we isolated a Hoechst dye-effluxing 'side population' (SP) from the epithelium using flow cytometry and demonstrated them to be undifferentiated cells by lack of expression of myoepithelial and luminal cell-specific antigens such as CALLA and MUC1. Epithelial SP cells were shown to be enriched for the putative stem cell markers p21(CIP1/WAF1), Musashi-1 and ERalpha/PR-positive cells. Lastly, SP cells, compared to non-SP, were highly enriched for the capacity to produce colonies containing multiple lineages in 3D basement membrane (Matrigel) culture. We conclude that breast stem cells include two populations: a primitive ERalpha/PR-negative stem cell necessary for development and a shorter term ERalpha/PR-positive stem cell necessary for adult tissue homeostasis during menstrual cycling. We speculate these two basic stem cell types may therefore be the cells of origin for ERalpha-positive and -negative breast tumours.
    • Isolation and characterization of progenitor-like cells from human renal proximal tubules.

      Lindgren, D; Boström, A; Nilsson, K; Hansson, J; Sjölund, J; Möller, C; Jirström, K; Nilsson, E; Landberg, Göran; Axelson, H; et al. (2011-02)
      The tubules of the kidney display a remarkable capacity for self-renewal on damage. Whether this regeneration is mediated by dedifferentiating surviving cells or, as recently suggested, by stem cells has not been unequivocally settled. Herein, we demonstrate that aldehyde dehydrogenase (ALDH) activity may be used for isolation of cells with progenitor characteristics from adult human renal cortical tissue. Gene expression profiling of the isolated ALDH(high) and ALDH(low) cell fractions followed by immunohistochemical interrogation of renal tissues enabled us to delineate a tentative progenitor cell population scattered through the proximal tubules (PTs). These cells expressed CD24 and CD133, previously described markers for renal progenitors of Bowman's capsule. Furthermore, we show that the PT cells, and the glomerular progenitors, are positive for KRT7, KRT19, BCL2, and vimentin. In addition, tubular epithelium regenerating on acute tubular necrosis displayed long stretches of CD133(+)/VIM(+) cells, further substantiating that these cells may represent a progenitor cell population. Furthermore, a potential association of these progenitor cells with papillary renal cell carcinoma was discovered. Taken together, our data demonstrate the presence of a previously unappreciated subset of the PT cells that may be endowed with a more robust phenotype, allowing increased resistance to acute renal injury, enabling rapid repopulation of the tubules.
    • Isolation and characterization of the integral glycosaminoglycan constituents of human amyloid A and monoclonal light-chain amyloid fibrils.

      Nelson, S R; Lyon, Malcolm; Gallagher, John T; Johnson, E A; Pepys, M B; Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, U.K. (1991-04-01)
      Amyloid fibrils were isolated by extraction in water from the livers and spleens of four patients who had died of monoclonal, light-chain (AL)-type, systemic amyloidosis and one with reactive systemic, amyloid A protein (AA)-type amyloidosis. Each fibril preparation contained 1-2% by weight of glycosaminoglycan (GAG) which was tightly associated with the fibrils and not just co-isolated from the tissues with them. After exhaustive digestion of the fibrils with papain and Pronase, the GAGs were specifically precipitated with cetylpyridinium chloride and were identified by cellulose acetate electrophoresis and selective susceptibility to specific glycosidases. All the preparations contained approximately equal amounts of heparan sulphate and dermatan sulphate. There was no evidence for the presence of chondroitin sulphate or other GAGs. Fine structural analysis by oligosaccharide mapping in gradient polyacrylamide gels, following partial digestion with specific glycosidases, showed very similar structures among the heparan sulphates and the dermatan sulphates, respectively. GAGs were also extracted by solubilizing amyloid fibrils in 4 M-guanidinium chloride followed by CsCl density-gradient ultracentrifugation. Although a minor proportion of the GAG material obtained in this way was apparently in the form of proteoglycan molecules, most of it was free GAG chains. The presence in amyloid fibrils of different types, in different organs and from different patients of particular GAG classes with similar structures supports the view that these molecules may be of pathogenic significance.
    • Isolation and partial characterisation of a Chinese hamster O6-alkylguanine-DNA alkyltransferase cDNA.

      Rafferty, Joseph A; Elder, Rhoderick H; Watson, Amanda J; Cawkwell, L; Potter, P M; Margison, Geoffrey P; CRC Department of Chemical Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (1992-04-25)
      The cDNA encoding Chinese hamster O6-alkylguanine-DNA-alkyltransferase (ATase) has been isolated from a library prepared from RNA isolated from V79 lung fibroblasts which had an upregulated level of this repair activity following stepwise selection with a chloroethylating agent (1, 2). Expression of the cDNA in E. coli produced functionally active ATase at levels of 2.5% of total cellular protein as determined by in vitro assay. The recombinant hamster protein has a molecular weight of 28 kDa as estimated by SDS-PAGE and fluorography and this was identical to that in the upregulated cells. The characteristic PCHRV pentapeptide of the alkyl acceptor site has been identified and there is a 68 amino acid residue region which is 90% conserved across all the mammalian proteins so far analysed: in contrast, the N- and C-terminal domains diverge by as much as 50% between species. Polyclonal antibodies to the human and rat ATases hybridised to the hamster protein on western analysis suggesting at least one common epitope shared across species. However, in antibody inhibition experiments neither of the antisera cross reacted with the hamster ATase in a way which interfered with functional activity whereas the anti-human antibodies inhibited the human ATase and the anti-rat antibodies inhibited the rat and mouse ATases. There may therefore be significant tertiary structural differences between the hamster protein and the other mammalian ATases.
    • Isolation and partial characterization of murine O6-alkylguanine-DNA-alkyltransferase: comparative sequence and structural properties.

      Santibanez-Koref, Mauro F; Elder, Rhoderick H; Fan, Chun-Yang; Cawkwell, Lynn; McKie, J H; Douglas, K T; Margison, Geoffrey P; Rafferty, Joseph A; Department of Cancer Genetics, University of Manchester, United Kingdom. (1992)
      A cDNA encoding murine O6-alkylguanine-DNA-alkyltransferase (ATase) has been sequenced after isolation from total liver RNA by the polymerase chain reaction using oligonucleotide primers derived from the rat ATase cDNA sequence. Functionally active murine ATase protein has been expressed in Escherichia coli at high levels (about 2% of total protein) and purified to apparent homogeneity (molecular mass 26 kDa). In liquid hybridization experiments, anti-human ATase polyclonal antibodies inhibited human but not rat or mouse ATase, whereas anti-rat polyclonal antibodies inhibited rat and mouse but not human ATase. Both antibodies detected all mammalian ATases tested by western analysis so far. These results indicate some common epitopes and at least one unique human epitope. We compared the amino-acid sequence of the murine ATase with those of other mammalian and bacterial ATases. The proteins of this family all have a large domain (approximately 70 amino acids) of highly conserved residues flanking the sequence PCHRV, which contains the alkyl-accepting cysteine residue of the active site. No evidence was found in the sequences for helix-turn-helix, leucine-zipper, or zinc-finger motifs for DNA recognition and binding. Nuclear localization signals (basic-residue-rich regions) could not be uniquely identified in the mammalian members of the family. Outside of the conserved PCHRV region, there were major differences between prokaryotic and eukaryotic proteins at the primary structure level: there was a series of proline-rich motifs, but these also varied between sequences.
    • Isolation of a cDNA encoding 5T4 oncofetal trophoblast glycoprotein. An antigen associated with metastasis contains leucine-rich repeats.

      Myers, Kevin A; Rahi-Saund, Veena; Davison, M D; Young, J A; Cheater, Amanada J; Stern, Peter L; Cancer Research Campaign Department of Immunology, Paterson Institute for Cancer Research, Christie Hospital, Manchester, United Kingdom. (1994-03-25)
      The monoclonal antibody 5T4 defines a human oncotrophoblast antigen expressed by a variety of carcinomas but with a restricted pattern of expression in normal adult tissues. The 5T4 antigen has been isolated from term placenta as a 72-kDa glycoprotein consisting of a 42-kDa core protein with extensive N-linked glycosylation. A cDNA has been isolated from a human placental library using pools of oligonucleotides based on amino acid sequence obtained from purified 5T4 molecules. The predicted open reading frame encodes a protein of 420 amino acids with a molecular mass of 46 kDa and 8 potential N-glycosylation sites. There are N- and C-terminal hydrophobic segments corresponding to putative signal and membrane anchorage sequences, respectively. Northern analysis has demonstrated a major 2.5-kilobase mRNA present in cell lines serologically reactive with the monoclonal antibody 5T4. Comparison of the 5T4 protein sequence with current sequence data bases has identified the presence of leucine-rich repeats, which are found in a variety of proteins from yeast, insects, and mammals. The 5T4 antigen expression is strongly associated with metastasis in colorectal and gastric cancer, and, hence, the possible functions of the gene product and its relationship to tumor growth and progression are discussed.
    • Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4.

      Shaw, David M; Embleton, Jim; Westwater, C; Ryan, Matthew G; Myers, Kevin A; Kingsman, Susan M; Carroll, M W; Stern, Peter L; CRC Immunology Group, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (2000-12-15)
      The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.