• 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.
    • Genetic change in the cancer cell.

      Harnden, David G (1984-01)
    • Genetic chemoprotection with mutant O6-alkylguanine-DNA-alkyltransferases.

      Hobin, D A; Fairbairn, Leslie J; CRC Gene Therapy Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, M20 4BX, UK. (2002-02)
      One of the main barriers to more efficacious use of modern chemotherapeutic agents, is the collateral toxicity exhibited in normal, highly proliferative tissues, primarily the haemopoietic, gastrointestinal and pulmonary tissues. Drug resistance of tumours to these drugs compounds this problem. This review discusses the role of O6-alkylguanine-DNA alkyltransferase (ATase) in conferring protection against O6-alkylating agents in normal tissue, focusing mainly on the haemopoietic compartment. The development of mutant forms of ATase, which are resistant to the effects of soluble analogues of O6-alkylation such as O6-benzylguanine, is examined and the gene therapy approach of combining these two strategies to confer chemoprotection to vulnerable tissues whilst sensitising malignant tissue is reviewed.
    • Genetic content and preliminary transcriptional analysis of a representative region of murine gammaherpesvirus 68.

      Mackett, Mike; Stewart, James P; Pepper, Stuart D; Chee, M; Efstathiou, S; Nash, A A; Arrand, John R; Department of Molecular Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. mlbmm@picr.cr.man.ac.uk (1997-06)
      Murine gammaherpesvirus 68 (MHV-68) is a relatively recently discovered pathogen of wild rodents and provides a unique opportunity to explore in detail the interactions of a gammaherpesvirus with its natural host. It may also provide a much needed small animal model for human gammaherpesviruses. As a step in the detailed analysis of virus gene structure and expression we have sequenced over 20 kb of the MHV-68 genome and mapped gene transcripts by Northern blot hybridization. The region we chose to analyse contains several conserved gene blocks as well as some less well conserved genes and allowed us to estimate the relationship of this virus to other herpesvirus family members. Of particular interest is the fact that none of the characteristic Epstein-Barr virus (EBV) genes is present at this genomic locus although MHV-68 does have one gene encoding a membrane glycoprotein, 9p150, which shows similarities to the major membrane glycoprotein of EBV. Our results further confirm that MHV-68 is a gammaherpesvirus marginally more closely related to a cluster of gammaherpesviruses including herpesvirus salmiri than to EBV. Northern analysis shows that the temporal regulation of expression is broadly similar to that of other herpesviruses in this region of the genome. We also show that like other gammaherpesviruses, MHV-68 splices its homologue of the EBV transcriptional activator gene BMRF1.
    • Genetic induction of the warburg effect inhibits tumor growth.

      Sotgia, Federica; Martinez-Outschoorn, U; Lisanti, Michael P; Manchester Breast Centre and Breakthrough Breast Cancer Research Unit, Paterson Institute for Cancer Research; Institute of Cancer Sciences, Manchester Academic Health Science Centre, University of Manchester, UK. (2012-11)
      In the model of metabolic-coupling, the oxidative capacity of the tumor mitochondria and the concomitant bypassing of autophagy both act in concert to support cancer growth. Therefore, it is not unlikely that the mitochondrial citrate carrier, SLC25A1 or CIC is also involved in the positive-feedback and crosstalk provided by the stroma. It will be important to determine whether CIC impinges upon other tumor-promoting metabolic branches, particularly the catabolism of glutamine.
    • Genetic manipulation of drug sensitivity in haematopoietic cells.

      Southgate, Thomas D; Fairbairn, Leslie J; Cancer Research UK Gene Therapy Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, M20 4BX, UK. tsouthgate@picr.man.ac.uk (2004-08-06)
      The haematopoietic system can be manipulated genetically to increase either its resistance to drugs or its sensitivity to certain agents. Gene transfer and expression of specific drug-resistance factors might protect haematopoietic function during antitumour chemotherapy, or allow enrichment of gene-modified cells in vivo. By contrast, gene transfer of a prodrug activator, to confer sensitivity to otherwise nontoxic prodrugs, might allow deletion of engrafted cells in the event of an adverse effect such as graft-versus-host disease or the induction of a neoplasm. In addition, expression of a prodrug activator in tumour-infiltrating haematopoietic cells could provide a means of specifically activating a cytotoxic agent within a tumour mass.
    • Genetic predisposition to human lung cancer.

      Heighway, Jim; Thatcher, Nick; Cerny, T; Hasleton, Philip S; Department of Cell Biology, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Wilmslow Road, Manchester M20 9BX. (1986-04)
      The influence of polymorphic variants of the human c-Ha-ras gene on predisposition to lung cancer has been investigated. The human c-Ha-ras gene has been shown to reside on a polymorphic BamH1 restriction fragment. This restriction fragment length polymorphism (RFLP) results from variation in the size of a region of repetitive DNA 3' to the gene. An attempt has been made to characterise and compare the c-Ha-ras RFLP's in a normal population and in a group of cancer patients. DNA was extracted from the white blood cells of 101 normal donors and four common Ha-ras alleles identified, with occasional rare alleles of various sizes. The allele frequencies were examined in 132 lung cancer patients, comprising 66 individuals with small cell carcinoma of the lung (SCCL) and 66 with non-small cell carcinoma of the lung (non-SCCL). An abnormal allele distribution was found in individuals with non-SCCL compared to both control and SCCL values suggesting a degree of genetic pre-position to non-SCCL. In addition, analysis of the Ha-ras RFLP's in solid samples inferred a deletion of material from the short arm of chromosome 11 in two of 16 informative samples.
    • A genetic study of type 2 neurofibromatosis in the United Kingdom. I. Prevalence, mutation rate, fitness, and confirmation of maternal transmission effect on severity.

      Evans, D Gareth R; Huson, S M; Donnai, D; Neary, W; Blair, Val; Teare, M Dawn; Newton, V; Strachan, T; Ramsden, R; Harris, R; et al. (1992-12)
      A clinical and genetic study of type 2 neurofibromatosis (NF2) has been carried out in the United Kingdom. Virtually complete ascertainment of cases in the north-west of England was achieved and suggests a population incidence of 1 in 33,000 to 40,000. In the UK as a whole, 150 cases have been identified and been used to study the clinical and genetic features of NF2. The autosomal dominant inheritance of NF2 was confirmed, 49% of cases were assessed as representing new mutations, and the mutation rate was estimated to be 6.5 x 10(-6). Evidence to support a maternal gene effect was found in that age at onset was 18.17 years in 36 maternally inherited cases and 24.5 in 20 paternally inherited cases (p = 0.027). The preponderance of maternally inherited cases was also significant (p = 0.03). Data are presented which suggest that there are two types of NF2, one with later onset and bilateral vestibular schwannomas as the only usual feature, and the other with earlier onset and multiple other tumours. A considerable number of cases did not fall easily into one or other group and other factors such as maternal effect on severity and anticipation need to be considered.
    • A genetic study of type 2 neurofibromatosis in the United Kingdom. II. Guidelines for genetic counselling.

      Evans, D Gareth R; Huson, S M; Donnai, D; Neary, W; Blair, Val; Newton, V; Strachan, T; Harris, R; Department of Medical Genetics, St Mary's Hospital, Manchester. (1992-12)
      The major defining features, age at onset of symptoms, and survival in 150 patients with type 2 neurofibromatosis (NF2) have been studied. The mean age at onset was 21.57 years (n = 110) and no cases presented after 55 years of age. Patients presented with symptoms attributable to vestibular schwannomas (acoustic neuroma), cranial meningiomas, and spinal tumours. In 97 cases studied personally by the authors, skin and eye examination were found to be useful to detect early signs of the condition. Examination of the skin is likely to assist in early diagnosis in at least 10% of cases and examination of the eye for a lens opacity or cataract in at least as many again. There are marked interfamilial differences in disease severity and tumour susceptibility. Vestibular schwannomas are not fully penetrant, but the condition is usually expressed in another way. Alteration to the current diagnostic criteria is advocated to cover the lack of provision for new mutations. A screening protocol is proposed and the effect of disease heterogeneity on management is discussed.
    • Genetic toxicology of nitrogen and sulfur mustard.

      Fox, Margaret; Scott, David; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX Great Britain (1980)
    • Genetically abnormal circulating cells in lung cancer patients: an antigen-independent fluorescence in situ hybridization-based case-control study.

      Katz, R L; He, W; Khanna, A; Fernandez, R L; Zaidi, T M; Krebs, Matthew G; Caraway, N P; Zhang, H Z; Jiang, F; Spitz, M; et al. (2010-08-01)
      PURPOSE: We performed a study to determine if a fluorescence in situ hybridization (FISH)-based assay using isolated peripheral blood mononuclear cells (PBMCs) with DNA probes targeting specific sites on chromosomes known to have abnormalities in non-small cell lung cancer (NSCLC) cases could detect circulating genetically abnormal cells (CACs). EXPERIMENTAL DESIGN: We evaluated 59 NSCLC cases with stage I through IV disease and 24 controls. PBMCs and matched tumors were hybridized with 2 two-color [3p22.1/CEP3 and 10q22.3 (SP-A)/CEP10) and 2 four-color [CEP3, CEP7, CEP17, and 9p21.3 (URO); and EGFR, c-MYC, 6p11-q11, and 5p15.2 (LAV)] FISH probes. Percentages of cytogenetically abnormal cells (CACs) in peripheral blood and in matched tumor specimens were quantified by using an automated fluorescent scanner. Numbers of CACs were calculated based on the percentage of CACs (defined as PBMCs with genetic abnormalities) per milliliter of blood and expressed per microliter of blood. RESULTS: Patients with NSCLC had significantly higher numbers of CACs than controls. Mean number of CACs ranged from 7.23 +/- 1.32/microL for deletions of 10q22.3/CEP10 to 45.52 +/- 7.49/microL for deletions of 3p22.1/CEP3. Numbers of CACs with deletions of 3p22.1, 10q22.3, and 9p21.3, and gains of URO, increased significantly from early to advanced stage of disease. CONCLUSIONS: We have developed a sensitive and quantitative antigen-independent FISH-based test for detecting CACs in peripheral blood of patients with NSCLC, which showed a significant correlation with the presence of cancer. If this pilot study can be validated in a larger study, CACs may have a role in the management of patients with NSCLC.
    • Genetics and genomics of radiotherapy toxicity: towards prediction.

      West, Catharine M L; Barnett, Gillian C; School of Cancer and Enabling Sciences, The University of Manchester, Manchester Academic Health Science Centre, The Christie, Wilmslow Road, Manchester M20 4BX, UK. catharine.west@manchester.ac.uk. (2011)
      Radiotherapy is involved in many curative treatments of cancer; millions of survivors live with the consequences of treatment, and toxicity in a minority limits the radiation doses that can be safely prescribed to the majority. Radiogenomics is the whole genome application of radiogenetics, which studies the influence of genetic variation on radiation response. Work in the area focuses on uncovering the underlying genetic causes of individual variation in sensitivity to radiation, which is important for effective, safe treatment. In this review, we highlight recent advances in radiotherapy and discuss results from four genome-wide studies of radiotoxicity.