• Frequency of fibroblast growth factor receptor 3 mutations in sporadic tumours.

      Sibley, Kathryn; Stern, Peter L; Knowles, Margaret A; ICRF Clinical Centre, St. James's University Hospital, Leeds, LS9 7TF, UK. (2001-07-19)
      Mutations in FGFR3 have been identified in several tumour types including bladder carcinoma, cervical carcinoma, and multiple myeloma. In bladder carcinoma, we recently identified FGFR3 mutations in 41% of tumours, making this the most frequently mutated putative oncogene identified in bladder cancer to date. We have now investigated the frequency of FGFR3 mutation in a panel of 125 tumours and 13 cell lines from various other organs. We analysed the mutation hotspots in exons 7, 10 and 15 by direct DNA sequencing, and found one mutation in exon 7 (S249C) in 1/28 (3.5%) cervical tumours. Mutations were not detected in stomach, rectum, colon, prostate, ovarian, breast, brain, or renal tumours, nor were they found in any of the cell lines included in this study. We conclude that FGFR3 is commonly mutated in bladder carcinoma and only rarely in cervical carcinoma. Several tumour types appear not to possess any mutations in FGFR3, suggesting that these mutations are important only in the development of certain types of tumour.
    • Frequency of human T regulatory cells in peripheral blood is significantly reduced by cryopreservation.

      Elkord, Eyad; Clinical Immunotherapy Laboratory, Department of Medical Oncology, University of Manchester, Wilmslow Road, Manchester M204BX, UK. eelkord@picr.man.ac.uk (2009-08-15)
      Cryopreservation of peripheral blood mononuclear cells (PBMC) is essential for many clinical and research assays. Some studies reported consistent changes in PBMC phenotype following cryopreservation. We hypothesized that PBMC freezing may have a negative impact on estimation of the frequency of T regulatory cell (Treg). Treg levels were measured in 6 fresh PBMC samples isolated from 6 healthy donors and these levels were re-measured after freezing for three weeks. Herein, we report a significant reduction in Treg frequency in all samples following cryopreservation.
    • Frequency of Ki-ras mutations and DNA alkylation in colorectal tissue from individuals living in Manchester.

      Jackson, Peta E; Hall, C N; Badawi, Alaa F; O'Connor, Peter J; Cooper, Donald P; Povey, Andrew C; Cancer Research Campaign Department of Carcinogenesis, Paterson Institute for Cancer Research, Manchester, United Kingdom. (1996-05)
      Most human colorectal cancers arise through the accumulation of a series of genetic alterations such as point mutations within the Ki-ras and p53 genes, but the chemical carcinogens that may be implicated in these events are still unidentified. In a previous study, we showed that DNA from human colorectal tissue contained O6-methyldeoxyguanosine (O6-MedG), a promutagenic lesion arising from exposure to as yet unidentified methylating agents. To address whether such exposure may result in oncogene activation in human colorectal tumors, we examined another series of paired normal and tumor DNA samples from the lower intestinal tract for the presence of O6-MedG in DNA (as a marker of exposure) and for mutations within the Ki-ras gene. After isolation by high pressure liquid chromatography, O6-MedG was quantified by a radioimmunoassay with a limit of detection of 0.01 mumol O6-MedG/mol dG. The frequencies of methylation were 33%, 52%, and 48% for normal DNA and 58%, 32%, and 63% for tumor DNA isolated from the cecum, sigmoid colon, and rectum, respectively. Overall, 35% of the individuals had no detectable O6-MedG in the DNA from both their tumor and normal tissue. Ki-ras mutations were initially identified by a restriction site mutation assay and then sequenced to ascertain the mutations thus detected. The frequencies of mutations in tumor DNA isolated from the cecum, sigmoid colon, and rectum were 28%, 29%, and 42%, respectively. DNA isolated from macroscopically normal tissue was found to contain Ki-ras mutations in 14% of sigmoid colon samples and 12% of rectal samples. Most base mutations were in codon 12 (72%), and 64% were GC-->AT transitions: 28% and 8% were GC-->TA and CG-->CG transversions, respectively. All mutations were at the second base of either codon 12 or codon 13 except for a single GC-->TA transversion at the first base of codon 13 in a rectal tumor sample. There was no association between the presence of O6-MedG in DNA from either normal or tumor tissue or both normal and tumor tissue and the incidence of Ki-ras mutations or GC-->AT transitions in mutated Ki-ras genes. It remains to be determined, however, whether there is a relationship between methylating-agent exposure and Ki-ras mutations, as (i) the presence of O6-MedG in colorectal DNA in these samples may not represent the exposure when Ki-ras mutational activation was occurring (i.e., at some unknown time in the past), (ii) interindividual differences in repair-enzyme activity may alter susceptibility to a mutational event after exposure, (iii) the predominant mutagen in the colon and rectum may not be a methylating agent (e.g., nitric oxide), and (iv) exposure to methylating agents need not result in oncogene activation in human tissues but may perhaps promote the emergence of the mutator phenotype.
    • The frequency of osteolytic bone metastasis is determined by conditions of the soil, not the number of seeds; evidence from in vivo models of breast and prostate cancer.

      Wang, N; Reeves, Kimberley J; Brown, H; Fowles, A; Docherty, F; Ottewell, P; Croucher, P; Holen, I; Eaton, C; The Mellanby Centre for Bone Research, Department of Human Metabolism, Medical School, University of Sheffield, Beech Hill Road, Sheffield, S10 2RX (2015)
      While both preclinical and clinical studies suggest that the frequency of growing skeletal metastases is elevated in individuals with higher bone turnover, it is unclear whether this is a result of increased numbers of tumour cells arriving in active sites or of higher numbers of tumour cells being induced to divide by the bone micro-environment. Here we have investigated how the differences in bone turnover affect seeding of tumour cells and/or development of overt osteolytic bone metastasis using in vivo models of hormone-independent breast and prostate cancer.
    • Frequency of regulatory T cells in renal cell carcinoma patients and investigation of correlation with survival.

      Griffiths, Richard W; Elkord, Eyad; Gilham, David E; Ramani, Vijay A C; Clarke, Noel W; Stern, Peter L; Hawkins, Robert E; Department of Medical Oncology, Christie Research Centre, Paterson Institute for Cancer Research, Manchester, UK. (2007-11)
      BACKGROUND: Regulatory T cells are important in maintaining immune homeostasis, mediating peripheral tolerance and preventing autoimmunity. Increased frequencies of CD4(+)CD25(high )T regulatory (T(Reg)) cells have been documented in the peripheral blood of patients with several types of cancer consistent with a role in tumour escape from immunological control. We have investigated the presence of T(Reg) cells systemically and in situ in previously untreated patients with renal cell carcinoma (RCC). RESULTS: We have shown that there is a significant increased frequency of CD4(+)CD25(high) T cells in RCC patients (n = 49) compared to normal donors (n = 38), respectively, 2.47% versus 1.50%; P < 0.0001. We confirmed these data using the FOXP3 marker of T(Reg) cells in a subset of these patients and normal donors. The population of T(Reg) cells identified showed the expected phenotype with CD4(+)CD25(high) population in both RCC patients and normal donors contained higher proportions of CD45RO and GITR than CD4(+)CD25(-/low) populations and exhibiting suppressive activity in an anti-CD3 and anti-CD28 induced proliferation assay. CD4(+)FOXP3(+) T cells were detected in the tumour microenvironment by immunofluorescence and the numbers enumerated in lymphocytes recovered following enzymatic disaggregations of biopsies; their frequency was higher in the tumour than the peripheral blood of the same patients. The early follow up data show an association between higher peripheral blood regulatory T-cell count and adverse overall survival. CONCLUSION: These data confirm the increase of T(Reg) cells in RCC patients and provide impetus to further investigate modulation of T(Reg) activity in RCC patients as part of therapy.
    • Frequent alterations of cell cycle regulators in early-stage breast lesions as detected by immunohistochemistry.

      Marsh, K L; Varley, Jennifer; CRC Department of Cancer Genetics, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (1998-05)
      Progression through G1 phase of the eukaryotic cell cycle is tightly controlled by cyclin-dependent kinases (CDK). These proteins form part of a regulatory pathway including the cyclin-dependent kinase inhibitor (CKI) p16, D-type cyclins and the product of the retinoblastoma gene pRb. Aberration of any one of these components may lead to uncontrolled proliferation contributing to neoplasia. Three of these proteins, cyclin D1, pRb and p16, were analysed by immunohistochemistry on archival paraffin sections to determine whether expression patterns were different in preinvasive ductal carcinoma in situ (DCIS) and invasive breast tumours relative to normal. Genetic analysis of the gene encoding cyclin D1 (CCND1) was also carried out, using an intragenic restriction fragment-length polymorphism (RFLP) to assess possible allelic imbalance. A majority of the tumours studied (approximately 90%) showed abnormalities in expression of at least one of these proteins. Overexpression of cyclin D1 was found in approximately 49% cases, reduced expression of p16 in approximately 46% and reduced expression of pRb in approximately 37%. Allelic imbalance of cyclin D1 was found in approximately 57% cases.
    • Frequent alterations of chromosome 1 in ductal carcinoma in situ of the breast.

      Munn, K E; Walker, R A; Varley, Jennifer; CRC Department of Cancer Genetics, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (1995-04-20)
      Ductal carcinoma in situ (DCIS) of the breast is commonly described as a premalignant lesion. Using PCR to amplify DNA from areas of tumour cells which have been microdissected from fixed material, we have studied the involvement of chromosome 1 in 19 cases of DCIS. A series of microsatellite repeat polymorphisms has been used to define regions of allelic imbalance and this has confirmed the involvement in DCIS of six of the regions previously implicated in studies of invasive breast tumours. This suggests that these regions may harbour tumour suppressor genes, the inactivation of which is important for the early stages of breast tumour development. Analysis of separate ducts from within the same tumour has revealed that the same genetic alterations are not necessarily present throughout the lesion. In addition we have found that in three cases where frank invasive carcinoma is also present, similar alterations can be detected in the in situ and invasive component.
    • Frequent loss of function mutations in TGF beta R1 and TGF beta R2 identify hair follicle bulge stem cells as the cell of origin for cutaneous squamous cell carcinoma

      Cammareri, P; Rose, A; Vincent, D; Libertini, S; Ridgway, R; Athineos, D; Coates, P; McHugh, A; Pourreyron, C; Larsson, J; et al. (2015)
    • Frequent loss of function mutations in TGF beta R1 and TGF beta R2 implicate hair follicle bulge stem cells as a cell of origin of cutaneous squamous cell carcinoma.

      Cammareri, P; Rose, A; Vincent, D; Wang, J; Nagano, A; Libertini, S; Ridgway, R; McHugh, A; Pourreyron, C; Spender, L; et al. (2016)
    • Friend disease in vitro.

      Dexter, T Michael; Allen, Terence D; Testa, Nydia G; Scolnick, E (1981-09-01)
      In long-term marrow cultures, hemopoiesis can be maintained for several months, although erythropoiesis is normally suppressed at the most primitive level of development (the erythroid colony-forming cells). Infection of these cultures with a viral complex combining helper-independent murine leukemia virus (F-MuLV) and a spleen focus-forming virus (SFFVp) results in a productive infection of both the replication defective SFFVp and the F-MuLV. After infection, the cultures show a dramatic elevation in the numbers of late erythroid progenitor cells (CFU-E), many of which will grow in the absence of added erythropoietin, and a transient erythropoietin, independent erythropoiesis, including the production of mature, enucleated erythrocytes. Hemopoiesis eventually declines, with no evidence for the generation of Friend tumor cells. When erythropoiesis is induced in the long-term cultures by addition of anemic mouse serum before infection by polycythemia-inducing Friend virus, the generation of erythropoietin-independent CFU-E and erythrocyte formation is followed by the sustained production (greater than 40 wk) of primitive erythroid cells with low spontaneous levels (less than 5%) of hemoglobinization. Although these cells will produce spleen colonies in irradiated mice and can be cloned in soft-gel media, they do not produce autonomous, permanently growing cell lines in vitro, i.e., they retain a dependency upon the marrow-adherent layer for their continued growth. However, following a further passage on a "virgin" marrow environment, permanent cell lines can be established that are able to grow independently of environmental influences. Thus, this system is the first description of a complete in vitro system for the reproducible production and isolation of Friend virus-induced erythroid cell lines.
    • From live-cell imaging to scanning electron microscopy (SEM): the use of green fluorescent protein (GFP) as a common label.

      Drummond, Sheona P; Allen, Terence D; Department of Structural Cell Biology, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom. (2008)
      The identification and characterization of many biological substructures at high resolution requires the use of electron microscopy (EM) technologies. Scanning electron microscopy (SEM) allows the resolution of cellular structures to approximately 3 nm and has facilitated the direct visualization of macromolecular structures, such as nuclear pore complexes (NPCs), which are essential for nucleo-cytoplasmic molecular trafficking. However, SEM generates only static images of fixed samples and therefore cannot give unambiguous information about protein dynamics. The investigation of active processes and analysis of protein dynamics has greatly benefited from the development of molecular biology techniques whereby vectors can be generated and transfected into tissue culture cells for the expression of specific proteins tagged with a fluorescent moiety for real-time light microscopy visualization. As light microscopy is limited in its powers of resolution relative to electron microscopy, it has been important to adapt a protocol for the processing of samples for real-time imaging by conventional light microscopy with protein labels that can also be identified by SEM. This allows correlation of dynamic events with high resolution molecular and structural identification. This method describes the use of GFP for tracking the dynamic distribution of NPC components in real-time throughout the cell cycle and for high resolution immuno-SEM labeling to determine localization at the nanometer level.
    • From meiosis to mitosis: the sperm centrosome defines the kinetics of spindle assembly after fertilization.

      Cavazza, Tommaso; Peset, Martin; Vernos, I; Cell and Developmental Biology Programme, Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology, Doctor Aiguader, 88, 08003 Barcelona (2016-05-13)
      Bipolar spindle assembly in the vertebrate oocyte relies on a self-organization chromosome-dependent pathway. Upon fertilization the male gamete provides a centrosome and the first and subsequent embryonic divisions occur in the presence of duplicated centrosomes that act as dominant microtubule organizing centres (MTOCs).The transition from meiosis to embryonic mitosis involves a necessary adaptation to integrate the dominant chromosome-dependent pathway with the centrosomes to form the bipolar spindle.Here we took advantage of the Xenopus laevis egg extract system to mimic in vitro the assembly of the first embryonic spindle and investigate the respective contribution of the centrosome and the chromosome-dependent pathway to the kinetics of the spindle bipolarization. We found that centrosomes control the transition from the meiotic to the mitotic spindle assembly mechanism. By defining the kinetics of spindle bipolarization, the centrosomes ensure their own positioning to each spindle pole and thereby their essential correct inheritance to the two first daughter cells of the embryo for the development of a healthy organism.
    • Frozen surface replicas of the underside and surface of cultured-cells.

      Britch, M; Allen, Terence D; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1979)
    • A FTIR microspectroscopic study of the uptake and metabolism of isotopically labelled fatty acids by metastatic prostate cancer.

      Gazi, Ehsan; Harvey, Tim J; Brown, Michael D; Lockyer, Nicholas P; Gardner, Peter; Clarke, Noel W; Genito Urinary Cancer research Group, School of Cancer and Imaging Sciences, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK (2009)
    • FTIR-based spectroscopic analysis in the identification of clinically aggressive prostate cancer.

      Baker, Matthew J; Gazi, Ehsan; Brown, Michael D; Shanks, Jonathan H; Gardner, Peter; Clarke, Noel W; Manchester Interdisciplinary Biocentre, Centre for Instrumentation and Analytical Science, School of Chemical Engineering and Analytical Science, The University of Manchester, Manchester, M1 7DN, UK. M.J.Baker@manchester.ac.uk (2008-12-02)
      Fourier transform infrared (FTIR) spectroscopy is a vibrational spectroscopic technique that uses infrared radiation to vibrate molecular bonds within the sample that absorbs it. As different samples contain different molecular bonds or different configurations of molecular bonds, FTIR allows us to obtain chemical information on molecules within the sample. Fourier transform infrared microspectroscopy in conjunction with a principal component-discriminant function analysis (PC-DFA) algorithm was applied to the grading of prostate cancer (CaP) tissue specimens. The PC-DFA algorithm is used alongside the established diagnostic measures of Gleason grading and the tumour/node/metastasis system. Principal component-discriminant function analysis improved the sensitivity and specificity of a three-band Gleason score criterion diagnosis previously reported by attaining an overall sensitivity of 92.3% and specificity of 99.4%. For the first time, we present the use of a two-band criterion showing an association of FTIR-based spectral characteristics with clinically aggressive behaviour in CaP manifest as local and/or distal spread. This paper shows the potential for the use of spectroscopic analysis for the evaluation of the biopotential of CaP in an accurate and reproducible manner.
    • Fulvestrant, an estrogen receptor downregulator, reduces cell turnover index more effectively than tamoxifen.

      Bundred, Nigel J; Anderson, Elizabeth; Nicholson, Robert I; Dowsett, Mitch; Dixon, J Michael; Robertson, John F R; Department of Surgery, South Manchester University Hospital Education and Research Centre, UK. bundredn@man.ac.uk (2002)
      BACKGROUND: This study was performed to determine whether drug action on breast tumours could also be analysed using a cell turnover index (CTI), a composite measurement of both proliferation and apoptosis. MATERIALS AND METHODS: Data were obtained from a randomized, placebo-controlled trial comparing three single doses (50, 125 and 250 mg) of intramuscular fulvestrant (Faslodex, previously known as ICI 182,780) with oral tamoxifen 20 mg/day for 14-21 days in women with early, operable, ER-positive breast cancer. CTI was calculated as the ratio of the proliferation index to the apoptotic index. RESULTS: Fulvestrant 250 mg significantly reduced CTI compared with both placebo (p = 0.0003) and tamoxifen (p = 0.026). The effect on CTI with tamoxifen was not significantly different from that with placebo. CONCLUSION: This study suggests that CTI may be a useful indicator of drug action on breast tumour growth. A significant reduction in growth was found with fulvestrant 250 mg compared with tamoxifen.
    • Functional comparison of annexin V analogues labeled indirectly and directly with iodine-124.

      Dekker, Bronwen A; Keen, Heather G; Shaw, David M; Disley, Lynn; Hastings, David L; Hadfield, John A; Reader, Andrew J; Allan, Donald; Julyan, Peter J; Watson, Alastair; et al. (2005-05)
      We are interested in imaging cell death in vivo using annexin V radiolabeled with (124)I. In this study, [(124)I]4IB-annexin V and [(124)I]4IB-ovalbumin were made using [(124)I]N-hydroxysuccinimidyl-4-iodobenzoate prepared by iododestannylation of N-hydroxysuccinimidyl-4-(tributylstannyl)benzoate. [(124)I]4IB-annexin V binds to phosphatidylserine-coated microtiter plates and apoptotic Jurkat cells and accumulates in hepatic apoptotic lesions in mice treated with anti-Fas antibody, while [(124)I]4IB-ovalbumin does not. In comparison with (124)I-annexin V, [(124)I]4IB-annexin V has a higher rate of binding to phosphatidylserine in vitro, a higher kidney and urine uptake, a lower thyroid and stomach content uptake, greater plasma stability and a lower rate of plasma clearance. Binding of radioactivity to apoptotic cells relative to normal cells in vitro and in vivo appears to be lower for [(124)I]4IB-annexin V than for (124)I-annexin V.
    • Functional expression of secreted proteins from a bicistronic retroviral cassette based on foot-and-mouth disease virus 2A can be position dependent.

      Rothwell, Dominic G; Crossley, Rachel; Bridgeman, John S; Sheard, Victoria; Zhang, Y; Sharp, T; Hawkins, Robert E; Gilham, David E; McKay, Tristan R; Cancer Research UK Department of Medical Oncology, School of Cancer and Imaging Sciences, University of Manchester, Manchester Academic Health Science Centre, Christie NHS Trust, Manchester M20 4BX, United Kingdom. drothwell@picr.man.ac.uk (2010-11)
      The expression of two or more genes from a single viral vector has been widely used to label or select for cells containing the transgenic element. Identification of the foot-and-mouth disease virus (FMDV) 2A cleavage peptide as a polycistronic linker capable of producing equivalent levels of transgene expression has greatly improved this approach in the field of gene therapy. However, as a consequence of 2A posttranslational cleavage the upstream protein is left with a residual 19 amino acids from the 2A sequence on its carboxy terminus, and the downstream protein is left with an additional 2 to 5 amino acids on its amino terminus. Here we have assessed the functional consequences of the FMDV 2A cleavage motif on two secreted proteins (interleukin [IL]-2 and transforming growth factor [TGF]-β) when expressed from a retroviral bicistronic vector. Whereas IL-2 expression and function were found to be unaffected by the 2A motif in either orientation, functional expression of secreted TGF-β was significantly abrogated when the transgene was expressed upstream of the 2A sequence. We believe this is a consequence of aberrant cleavage and intracellular trafficking of the TGF-β polyprotein. These results highlight that to achieve functional expression of secreted proteins consideration must be taken of the transgenic protein's posttranslational modification and trafficking when using 2A-based bicistronic cassettes.
    • Functional heterogeneity among cytotoxic clones derived from natural killer cells.

      Christmas, Stephen E; Moore, Michael; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester, UK. (1987)
      Clones were obtained from highly purified populations of human peripheral blood natural killer (NK) cells propagated in the presence of interleukin-2 and phytohaemagglutinin. Almost all clones were cytotoxic against standard NK targets and many were also able to kill the B lymphoblastoid cell line BSM. This latter property was not necessarily a result of the incorporation of this cell line into the feeder mixture used to derive the clones. In most cloning experiments there was a high degree of concordance between the killing of the NK targets K562 and Molt 4 by panels of clones. In some cases this extended to the killing of BSM targets but in other instances there was no relationship or even an inverse correlation between killing of BSM and other targets. In a single cloning experiment there was no relationship between killing of BSM and Raji targets. In some cases a panel of clones could be divided into two or more distinct groups based on their differential activity towards BSM and K562. Such differences were not solely due to inter-donor variation. These findings were extended by cold target inhibition experiments in which at least three types of clone were identified. In one group of clones, which was nonreactive towards BSM, cold BSM significantly enhanced the killing of K562 in a dose-dependent fashion. These experiments provide evidence for a limited degree of functional heterogeneity among clones derived from human peripheral blood NK cells.
    • Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo.

      Kanemaki, Masato; Sanchez-Diaz, Alberto; Gambus, Agnieszka; Labib, Karim; Cancer Research UK, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, M20 4BX, UK. (2003-06-12)
      Evolutionarily diverse eukaryotic cells share many conserved proteins of unknown function. Some are essential for cell viability, emphasising their importance for fundamental processes of cell biology but complicating their analysis. We have developed an approach to the large-scale characterization of such proteins, based on conditional and rapid degradation of the target protein in vivo, so that the immediate consequences of bulk protein depletion can be examined. Budding yeast strains have been constructed in which essential proteins of unknown function have been fused to a 'heat-inducible-degron' cassette that targets the protein for proteolysis at 37 degrees C (ref. 4). By screening the collection for defects in cell-cycle progression, here we identify three DNA replication factors that interact with each other and that have uncharacterized homologues in human cells. We have used the degron strains to show that these proteins are required for the establishment and normal progression of DNA replication forks. The degron collection could also be used to identify other, essential, proteins with roles in many other processes of eukaryotic cell biology.