• Gene therapy of patient-derived T lymphocytes to target and eradicate colorectal hepatic metastases.

      Sheen, Aali J; Irlam, Joely J; Kirillova, Natalia; Guest, Ryan D; Sherlock, David J; Hawkins, Robert E; Gilham, David E; Cancer Research United Kingdom, Department of Medical Oncology, Paterson Institute for Cancer Research, University of Manchester, United Kingdom. (2003-06)
      PURPOSE: The overall aim of this study was to develop a novel treatment for colorectal cancer based on the use of gene therapy. Genetic modification of T lymphocytes has been used to specifically target and kill tumor cell lines directly. To test the efficacy of this method with clinically relevant materials, this study investigated the potential of T lymphocytes derived from patients with advanced colorectal disease to target autologous primary tumor material. METHODS T lymphocytes isolated preoperatively were modified genetically with recombinant retroviruses encoding CD3zeta-based chimeric immune receptors and were tested for functional activity against freshly isolated autologous tumor cells harvested from hepatic colorectal metastases. RESULTS: Patient-derived T cells were successfully transduced, and chimeric immune receptor expression was confirmed. Carcinoembryonic antigen expression on freshly isolated colorectal tumor cells was also demonstrated by molecular and immunohistochemical techniques. T cells expressing the anticarcinoembryonic antigen receptor were specifically activated by coculture with disaggregated or intact, diced tumor, whereas control non-carcinoembryonic antigen-targeted T-cell populations failed to activate. CONCLUSIONS: These results indicate that gene-targeted primary T lymphocytes depict specific functional activity against autologous colorectal tumor cells. This evidence indicates that chimeric immune receptor-expressing T cells may be able to circumvent the mechanisms used by tumor cells to avoid immune cell activity in vivo. This study emphasizes the potential of this approach as a therapy for carcinoembryonic antigen-expressing primary colorectal tumor and its metastases.
    • Gene transfer to augment the therapeutic index of anticancer chemotherapy.

      Baum, C; Margison, Geoffrey P; Eckert, H G; Fairbairn, Leslie J; Ostertag, W; Rafferty, Joseph A (1996-01)
    • Gene-specific linear trends constrain transcriptional variability of the toll-like receptor signaling

      Bagnall, J.; Rowe, W.; Alachkar, N.; Roberts, J.; England, H.; Clark, Christopher; Platt, M.; Jackson, D. A.; Muldoon, M.; Paszek, P.; et al. (2020)
      Single-cell gene expression is inherently variable, but how this variability is controlled in response to stimulation remains unclear. Here, we use single-cell RNA-seq and single-molecule mRNA counting (smFISH) to study inducible gene expression in the immune toll-like receptor system. We show that mRNA counts of tumor necrosis factor α conform to a standard stochastic switch model, while transcription of interleukin-1β involves an additional regulatory step resulting in increased heterogeneity. Despite different modes of regulation, systematic analysis of single-cell data for a range of genes demonstrates that the variability in transcript count is linearly constrained by the mean response over a range of conditions. Mathematical modeling of smFISH counts and experimental perturbation of chromatin state demonstrates that linear constraints emerge through modulation of transcriptional bursting along with gene-specific relationships. Overall, our analyses demonstrate that the variability of the inducible single-cell mRNA response is constrained by transcriptional bursting.
    • General criteria for radioassays by colloid scintillation counting under optimal conditions.

      Fox, Brian W; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1974-05)
    • A general method for the detection and mapping of submicrogram quantities of glycosaminoglycan oligosaccharides on polyacrylamide gels by sequential staining with azure A and ammoniacal silver.

      Lyon, Malcolm; Gallagher, John T; Cancer Research Campaign Department of Medical Oncology, Christie Hospital, Manchester, United Kingdom. (1990-02-15)
      A sensitive method has been developed for the visualization of nonradiolabeled glycosaminoglycan oligosaccharides resolved by polyacrylamide gel electrophoresis using fixation with azure A followed by staining with ammoniacal silver. This method, which can detect as little as 1-2 ng of a single oligosaccharide species, can be used to stain a few micrograms of a complex oligosaccharide mixture. The combination of gradient polyacrylamide gel electrophoresis and sequential azure A/silver staining can be applied to the analysis of all the complex glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate) and hyaluronate, as well as to comparisons of specificities of the glycosaminoglycan-degrading enzymes. This procedure may be particularly valuable in situations where the availability of glycosaminoglycan is very limited and/or where radiolabeling is impractical or undesirable.
    • General report on the European Union concerted action workshop on 11q23, London, UK, May 1997.

      Secker-Walker, L M; Harrison, Christine J; Department of Haematology, The Royal Free Hospital School of Medicine, London, UK. (1998-05)
      Seventeen cytogenetic laboratories in eight European countries contributed karyotypic, hematological, clinical and follow-up data from 550 patients with an acquired abnormality of 11q23. The patients had acute lymphoblastic leukemia (254), acute myeloid leukemia (250), unspecified, undifferentiated, biphenotypic acute leukemia or myeloproliferative disorder (18 cases together), or myelodysplastic syndrome (MDS) (28). The patients were classified by cytogenetic subgroup as t(4;11) (183 cases), t(6;11) (30) cases), t(9;11) (125 cases), t(10;11) (20 cases), t(11;19) (53 cases), 'other' abnormalities of 11q23 (82 cases) and del(11)(q23) (57 cases). Manuscripts were prepared on each cytogenetic subgroup, on MDS, on secondary hematological malignancies (40 cases) and on 11q23-translocation derivatives. For each subgroup the following aspects were investigated: associated clinical features, additional karyotypic change, distribution between hematological subtypes and between different age groups, prognosis at different age groups, and the impact of bone marrow transplantation on survival. The Workshop confirmed some previous findings from smaller studies, challenged others, identified new chromosomal partners and threw new light on less well documented aspects of 11q23 malignancies. The large number of cases investigated in a coordinated manner gives authoritative support to the findings. The Workshop thus demonstrates the value of collaborative European studies in the cytogenetics of malignancy.
    • Generalizability of potential biomarkers of response to CTLA-4 and PD-1 blockade therapy in cancer

      Bortone, D.; Vensko, S.; Entwistle, S.; Cogdill, A.; Monette, A.; Najjar, Y.; Sweis, R.; Tschernia, N.; Wennerberg, E.; Bommareddy, P.; et al. (2020)
      Background Multiple genomics-based biomarkers of response to immune checkpoint inhibition have been reported or proposed, including tumor mutation/neoantigen frequency, PD-L1 expression, T cell receptor repertoire clonality, interferon gene signature expression, HLA expression, and others.1 Although genomics associations of response have been reported, the primary studies have used a variety of data generation and processing techniques. There is a need for data harmonization and assessment of generalizability of potential biomarkers across multiple datasets. Methods We acquired patient-level RNA sequencing FASTQ data files from 10 data sets reported in seven pan-cancer PD-1 and CTLA-4 immune checkpoint inhibition trials with matched clinical annotations.2–7 We applied a common bioinformatics workflow for quality control, mapping to reference (STAR), generating gene expression matrices (SALMON), T cell receptor repertoire inference (MiXCR), extraction of immune gene signatures and immune subtypes,8 and differential gene expression analysis (DESeq2). We analyzed i) immunogenomics features proposed as biomarkers, and ii) gene expression signatures built from each trial for association with overall survival across the set of trials using univariable Cox proportional hazards regression. In all, we assessed 9 total immunogenomics features/signatures. P-values were adjusted for multiple testing using the Benjamini-Hochberg method. Results Of the 9 immunogenomics features assessed, cytolytic activity score and expression of the Follicular Dendritic Cell Secreted Protein gene (FDCSP) were associated with survival in two of seven studies, respectively (adjusted p < 0.05) (figure 1). No proposed biomarkers were significantly associated with survival in more than two studies. The sets of genes significantly associated with clinical benefit across the studies were highly disjoint, with only three genes significant in three studies and thirteen genes significant in two studies (figure 2). No genes were significantly associated with clinical benefit in more than three of seven studies
    • Generation of a contig comprising YACs and BACs within chromosome region 1p13.1.

      Brintnell, Bill; Hey, Yvonne; Jones, David; Hoggard, Nigel; James, Louise A; Varley, Jennifer; CRC Department of Cancer Genetics, Paterson Institute for Cancer Research, Manchester, UK. (1997-03)
      Chromosome region 1p13 is known to show loss of heterozygosity (LOH) in a number of human tumor types, including breast. We have generated a contig comprising YACs and BACs spanning part of 1p13.1 which includes the smallest region of overlapping loss identified in our earlier studies. The contig is anchored to the genetic map by a number of microsatellite markers, and by the use of CEPH YACs. We have excluded a number of candidate genes from this region, and we have oriented the contig with respect to the centromere and a number of other genes and markers on 1p13. This resource will be valuable in mapping the target for LOH in breast and other tumors, and may also be useful for the genetic analysis of other genes or diseases known to map to this region.
    • Generation of a mouse SWATH-MS spectral library to quantify 10148 proteins involved in cell reprogramming

      Ulanga, U.; Russell, M.; Patassini, Stefano; Brazzatti, J.; Graham, C.; Whetton, Anthony D; Graham, R. L. J.; Clinical Proteomics Research Group, Division of Molecular and Clinical Cancer Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road Manchester, Manchester, U (2021)
      Murine models are amongst the most widely used systems to study biology and pathology. Targeted quantitative proteomic analysis is a relatively new tool to interrogate such systems. Recently the need for relative quantification on hundreds to thousands of samples has driven the development of Data Independent Acquisition methods. One such technique is SWATH-MS, which in the main requires prior acquisition of mass spectra to generate an assay reference library. In stem cell research, it has been shown pluripotency can be induced starting with a fibroblast population. In so doing major changes in expressed proteins is inevitable. Here we have created a reference library to underpin such studies. This is inclusive of an extensively documented script to enable replication of library generation from the raw data. The documented script facilitates reuse of data and adaptation of the library to novel applications. The resulting library provides deep coverage of the mouse proteome. The library covers 29519 proteins (53% of the proteome) of which 7435 (13%) are supported by a proteotypic peptide.
    • Generation of assays and antibodies to facilitate the study of human 5'-tyrosyl DNA phosphodiesterase.

      Thomson, Graeme J; Watson, Amanda J; Caldecott, K; Denneny, Olive; Depledge, Paul; Hamilton, Nicola S; Hopkins, Gemma V; Jordan, Allan M; Morrow, Christopher J; Raoof, Ali; et al. (2013-02-12)
      Topoisomerases regulate DNA topology by the transient cleavage and re-ligation of DNA during transcription and replication. Topoisomerase II (Topo II) poisons such as etoposide can induce abortive DNA strand breaks in which Topo II remains covalently bound to a 5' DNA strand terminus via a phosphotyrosyl linker. Tyrosyl DNA phosphodiesterase 2 (Tdp2) is a recently discovered human 5'-tyrosyl DNA phosphodiesterase which repairs this topoisomerase-mediated DNA damage, thus playing a central role in maintaining normal DNA topology in cells. Cellular depletion of Tdp2 has been shown to result in an increased susceptibility and sensitivity to Topo II-induced DNA double strand breaks, thereby revealing Tdp2 as a potentially attractive anti-cancer target. No drug-like inhibitors of Tdp2 have been identified to date and assays suitable for high throughput screening (HTS) have not been widely reported. Here we have identified a new and effective chromogenic substrate for Tdp2 and developed a homogenous and robust HTS assay. A second novel Tdp2 assay was also developed to cross-validate hit matter identified from an HTS. Additionally, a new and specific Tdp2 antibody is described. Together these new tools will aid in the identification of novel Tdp2 inhibitors and the investigation of the role of Tdp2 in cancer.
    • Generation of cell-free extracts of Xenopus eggs and demembranated sperm chromatin for the assembly and isolation of in vitro-formed nuclei for Western blotting and scanning electron microscopy (SEM).

      Allen, Terence D; Rutherford, Sandra A; Murray, Stephen M; Sanderson, Helen S; Gardiner, Fiona; Kiseleva, Elena; Goldberg, Martin W; Drummond, Sheona P; Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Withington, Manchester M20 4BX, UK. tallen@picr.man.ac.uk (2007)
      This protocol details methods for the generation of cell-free extracts and DNA templates from the eggs and sperm chromatin, respectively, of the clawed toad Xenopus laevis. We have used this system with scanning electron microscopy (SEM), as detailed herein, to analyze the biochemical requirements and structural pathways for the biogenesis of eukaryotic nuclear envelopes (NEs) and nuclear pore complexes (NPCs). This protocol requires access to female frogs, which are induced to lay eggs, and a male frog, which is killed for preparation of the sperm chromatin. Egg extracts should be prepared in 1 d and can be stored for many months at -80 degrees C. Demembranated sperm chromatin should take only approximately 2-3 h to prepare and can be stored at -80 degrees C almost indefinitely. The time required for assembly of structurally and functionally competent nuclei in vitro depends largely on the quality of the cell-free extracts and, therefore, must be determined for each extract preparation.
    • Generation of cells expressing improved doxycycline-regulated reverse transcriptional transactivator rtTA2S-M2.

      Welman, Arkadiusz; Barraclough, Jane; Dive, Caroline; Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, United Kingdom. awelman@picr.man.ac.uk (2006)
      Tet-on cell lines engineered to stably express doxycycline (Dox)-regulated reverse transcriptional transactivator (rtTA) have many applications in biomedical research and biotechnology. Unfortunately, construction and maintenance of such cells often proves to be costly, labor intensive and ineffective. Moreover, the Tet-on clones generated using standard methodology were often unstable and frequently displayed significantly changed physiological properties compared with their parental cells. Here we describe an optimized protocol for generation of Tet-on cells. The protocol is based on the use of a recently developed pN1p beta actin-rtTA2S-M2-IRES-EGFP vector (where IRES is an internal ribosome entry site) and permits relatively inexpensive construction of many Tet-on clones with essentially 100% efficiency. The method is well suited for 'difficult' cell lines displaying genetic instability and high levels of epigenetic silencing. The constructed Tet-on cells remain stable with time in the absence of any selection agents, are easy to monitor and preserve the characteristics of parental cells. The protocol can be completed in 2 months.
    • Generation of neomucosa in vivo by transplantation of dissociated rat postnatal small intestinal epithelium.

      Tait, I S; Flint, Neil; Campbell, F C; Evans, Gareth S; Department of Surgery, Ninewells Hospital and Medical School, Dundee, UK. (1994-04)
      A novel method to study the generation of rat small intestinal mucosa, by transplantation of disaggregated postnatal rat small intestinal epithelium is described. Cellular aggregates, comprised of epithelium with attached proliferative cells and closely associated stromal tissue, were isolated from postnatal rat small intestine by enzymatic digestion, then grafted immediately to the subcutaneous plane of adult recipients. On graft retrieval after 14 days, 39% of cellular transplants to nude mice, and 84% of cellular transplants to inbred rats had developed into small intestine-like structures. These structures were comprised of a circumferential layer of epithelium surrounding a central mucin filled lumen. This neomucosal layer exhibited well formed crypts and villi, and contained all epithelial stem cell lineages i.e. absorptive enterocytes, goblet cells, Paneth's cells and entero-endocrine cells. Proliferative activity within this neomucosa was confined to crypt regions as in normal postnatal small intestine. Developmental maturation within the regenerated neomucosa was demonstrated by organotypic morphogenesis, i.e. formation of mature crypts and villi, and progressive cytodifferentiation with increased numbers of goblet cells, entero-endocrine cells and Paneth's cells. Altered patterns of brush border enzyme expression further confirmed a temporal progression of development within neomucosal enterocytes. It is concluded that after "extensive" mucosal disaggregation, postnatal small intestinal epithelial progenitor cells retain the capacity for organotypic regeneration of neomucosa when transplanted to ectopic sites in adult recipients. These small aggregates of epithelium and stroma are capable of generating the topographical signals necessary for the three dimensional regeneration of this tissue. Furthermore, the multipotent generative potential of the stem cells within these cellular aggregates is maintained with production of all progeny.
    • Generation of non MHC restricted killing in non-cytotoxic T-lymphocytes from leukaemia patients.

      Matera, L; Cardoso, E; Caudana, L; Moore, Michael (1987-02)
      Experiments were undertaken to determine whether the depression of natural killer (NK) activity previously observed in the peripheral blood lymphocytes (PBL) of leukaemia patients in remission extended to NK-like precursors in the blood. T-lymphocytes (E+) from leukaemia patients and normal subjects were depleted of IgG Fc-receptor-bearing (gamma FcR) fresh NK cells by passage over immune complex-coated monolayers. gamma FcR - E + PBL were cultured alone or with DAUDI cells. On day 5 of culture, cytotoxicity toward the NK-sensitive cell lines K562 and MOLT-4 was evaluated in the responder lymphocytes of leukaemia patients and controls. Negligible NK-like cytotoxicity was found in both FcR - E + PBL responder populations cultured alone. By contrast, stimulation with DAUDI induced high levels of K562 and MOLT-4 cytotoxicity in leukaemia as well as in normal responder cells. Complement-mediated cytotoxicity experiments using various McAb demonstrated that in both normal and leukaemia cultures NK-like effectors react with the pan-T OKT3 McAb and with the OKT11 McAB directed to the SRBC receptor, but not with Leu 1 1b and OKM1 McAbs, directed against antigens expressed on peripheral blood NK cells. Fractionation of the responder cells on discontinuous Percoll gradients showed that most of this activity was present in the highly dividing blast cell fraction.
    • Generation of osteoclasts in vitro.

      Testa, Nydia G; Allen, Terence D; Lajtha, L G; Onions, D; Jarret, O; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 4BX, U.K. (1981-02)
    • Generational shift in melanoma incidence and mortality in Queensland, Australia, 1995-2014.

      Aitken, Joanne F; Youlden, Danny R; Baade, Peter D; Soyer, H; Green, Adèle C; Smithers, B; Cancer Council Queensland, Brisbane, QLD, AustraliaInstitute for Resilient Regions, University of Southern Queensland, Brisbane, QLD, Australia (2017-11-06)
      Public campaigns encouraging sun protection for skin cancer prevention began in Queensland, Australia, in the early 1980s. We examined recent trends to assess whether earlier evidence of stabilizing melanoma incidence in young people has persisted. Anonymized incidence and mortality data for in situ and invasive melanoma for the 20 years 1995-2014 were obtained from the Queensland Cancer Registry. Time trends were analyzed using JoinPoint regression. Birth cohort patterns were assessed using age-period-cohort models. Melanoma incidence in Queensland remains the highest recorded in the world (age-standardized incidence of invasive melanoma (2010-2014) = 72/100,000/annum). Over the 20-year period, incidence of in situ melanoma increased in all age groups. Incidence of both thin (≤1 mm) and thick (>1 mm) invasive melanoma was either stable or decreased in people under 60, while it increased in those aged 60 and above, particularly in men. Age-period-cohort analysis revealed decreasing age-specific incidence of invasive melanoma under 40 years of age, beginning with the birth cohort born around the mid-1960s, with steepest falls for those born around 1980 and later. Age-specific incidence was stable between 40 and 59 years of age from the 1945 birth cohort onwards. Melanoma mortality over the period was stable or decreased in all groups except in men aged 60 or over. These findings are evidence of real advances in the prevention and early detection of invasive melanoma in this very high-risk population. They make a compelling case for continued public health efforts to reduce the burden of melanoma in susceptible populations.
    • Genetic ablation of caveolin-2 sensitizes mice to bleomycin-induced injury.

      de Almeida, C; Jasmin, J; Del Galdo, F; Lisanti, Michael P; Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA USA (2013-07-15)
      Caveolar domains act as platforms for the organization of molecular complexes involved in signal transduction. Caveolin proteins, the principal structural components of caveolae, have been involved in many cellular processes. Caveolin-1 (Cav-1) and caveolin-2 (Cav-2) are highly expressed in the lung. Cav-1-deficient mice (Cav-1(-/-)) and Cav-2-deficient mice (Cav-2(-/-)) exhibit severe lung dysfunction attributed to a lack of Cav-2 expression. Recently, Cav-1 has been shown to regulate lung fibrosis in different models. Here, we show that Cav-2 is also involved in modulation of the fibrotic response, but through distinct mechanisms. Treatment of wild-type mice with the pulmonary fibrosis-inducer bleomycin reduced the expression of Cav-2 and its phosphorylation at tyrosine 19. Importantly, Cav-2(-/-) mice, but not Cav-1(-/-) mice, were more sensitive to bleomycin-induced lung injury in comparison to wild-type mice. Bleomycin-induced lung injury was characterized by alveolar thickening, increase in cell density, and extracellular matrix deposition. The lung injury observed in bleomycin-treated Cav-2(-/-) mice was not associated with alterations in the TGF-β signaling pathway and/or in the ability to produce collagen. However, apoptosis and proliferation were more prominent in lungs of bleomycin-treated Cav-2(-/-) mice. Since Cav-1(-/-) mice also lack Cav-2 expression and show a different outcome after bleomycin treatment, we conclude that Cav-1 and Cav-2 have distinct roles in bleomycin induced-lung fibrosis, and that the balance of both proteins determines the development of the fibrotic process.
    • Genetic analysis of melanoma from an albino patient

      Bracalente, Candelaria; Mundra, Piyushkumar A; Rinflerch, Adriana; Garcia-Martinez, Pablo; Volonteri, Victoria; Trucco, Lucas D; Galimberti, Gaston; Dhomen, Nathalie; Pavet, Valeria R; Marais, Richard; et al. (2018)
    • Genetic and functional studies of a germline TP53 splicing mutation in a Li-Fraumeni-like family.

      Varley, Jennifer; Chapman, P; McGown, Gail; Thorncroft, Mary R; White, Gavin R M; Greaves, M J; Scott, David; Spreadborough, Anne R; Tricker, K J; Birch, Jillian M; et al. (1998-06-25)
      We report an extensive Li-Fraumeni-like family in which there is an unusual spectrum of tumours at relatively late onset. A germline TP53 splice donor mutation in exon 4 is present in all affected family members available for testing. The mutation abolishes correct splicing of intron 4 and techniques of RT-PCR have identified three different aberrant transcripts from the mutant TP53 allele. Using the yeast functional assay to analyse transcripts in cells from a number of family members with the mutant allele, TP53 appears wild-type. Functional studies have been carried out on cells from patients with and without cancer who carry the germline mutation, and on cells from unaffected individuals from the same family who do not carry the mutation. Using a number of functional endpoints known to distinguish between cells carrying mutant or wild-type TP53 alleles, we were unable to discriminate normal (wt/wt) from heterozygous (wt/mut) cells by lymphocyte apoptosis and fibroblast survival following low dose rate ionising radiation exposure. However germline mutation carriers show increased sensitivity to radiation-induced chromosome damage in the G2 phase of the cell cycle, and decreased transient and permanent G1 arrest. These studies demonstrate the importance of fully characterising the effects of TP53 germline mutations, and may explain some of the phenotypic features of this family.
    • Genetic and non-genetic mechanisms underlying cancer evolution

      Shlyakhtina, Yelyzaveta; Moran, Katherine L; Portal, Maximiliano M; Cell Plasticity and Epigenetics Lab. Cancer Research UK-Manchester Institute, The University of Manchester, Alderley Park, SK10 4TG, UK (2021)
      Cancer development can be defined as a process of cellular and tissular microevolution ultimately leading to malignancy. Strikingly, though this concept has prevailed in the field for more than a century, the precise mechanisms underlying evolutionary processes occurring within tumours remain largely uncharacterized and rather cryptic. Nevertheless, although our current knowledge is fragmentary, data collected to date suggest that most tumours display features compatible with a diverse array of evolutionary paths, suggesting that most of the existing macro-evolutionary models find their avatar in cancer biology. Herein, we discuss an up-to-date view of the fundamental genetic and non-genetic mechanisms underlying tumour evolution with the aim of concurring into an integrated view of the evolutionary forces at play throughout the emergence and progression of the disease and into the acquisition of resistance to diverse therapeutic paradigms. Our ultimate goal is to delve into the intricacies of genetic and non-genetic networks underlying tumour evolution to build a framework where both core concepts are considered non-negligible and equally fundamental.