• P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation.

      Yague, Ernesto; Armesilla, Angel L; Harrison, Georgina; Elliott, James; Sardini, Alessandro; Higgins, Christopher F; Raguz, Selina; Medical Research Council Clinical Sciences Centre, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, United Kingdom. (2003-03-21)
      Multidrug resistance in acute myeloid leukemia is often conferred by overexpression of P-glycoprotein, encoded by the MDR1 gene. We have characterized the key regulatory steps in the development of multidrug resistance in K562 myelogenous leukemic cells. Unexpectedly, up-regulation of MDR1 levels was not due to transcriptional activation but was achieved at two distinct post-transcriptional steps, mRNA turnover and translational regulation. The short-lived (half-life 1 h) MDR1 mRNA of naive cells (not exposed to drugs) was stabilized (half-life greater than 10 h) following short-term drug exposure. However, this stabilized mRNA was not associated with translating polyribosomes and did not direct P-glycoprotein synthesis. Selection for drug resistance, by long-term exposure to drug, led to resistant lines in which the translational block was overcome such that the stabilized mRNA was translated and P-glycoprotein expressed. The absence of a correlation between steady-state MDR1 mRNA and P-glycoprotein levels was not restricted to K562 cells but was found in other lymphoid cell lines. These findings have implications for the avoidance or reversal of multidrug resistance in the clinic.
    • The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance in vitro and in vivo to temozolomide in combination with the novel MGMT inactivator O(6)-(4-bromothenyl)guanine.

      Woolford, Lorna B; Southgate, Thomas D; Margison, Geoffrey P; Milsom, Michael 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. (2006-01)
      The O(6)-methylguanine-DNA-methyltransferase (MGMT) inactivator O(6)-benzylguanine (O(6)-beG) is currently under clinical investigation as a potential tumour-sensitising agent. In clinical trials its use has been associated with increased myelotoxicity and a reduced maximum tolerated dose (MTD) for BCNU. Thus the concept of myeloprotection by gene therapy with an O(6)-beG-insensitive mutant of MGMT is soon to be tested. Recently, an alternative inactivator has been described (O(6)-(4-bromothenyl)guanine, PaTrin-2), which shows potential advantages over O(6)-beG in terms of higher activity against wild-type MGMT and oral formulation. The use of PaTrin-2 has also been associated with increased myelotoxicity in clinical trials and thus PaTrin-2 may also be a candidate for use in conjunction with mutant MGMT gene transfer in genetic chemoprotective strategies. However, its activity against mutant MGMTs has not been reported. We show here that the P(140)K mutant of MGMT is highly resistant to inactivation by PaTrin-2. Furthermore, we show that a human haemopoietic cell line (K562) transduced with a retroviral vector encoding MGMT(P140K) is highly resistant to the cytotoxic effects of PaTrin-2 in combination with the methylating agent temozolomide, and that cells expressing MGMT(P140K) can be effectively enriched in vitro following challenge with this drug combination. Finally, we show that animals reconstituted with bone marrow expressing MGMT(P140K) exhibit haemopoietic resistance to PaTrin-2/temozolomide, which results in in vivo selection of gene-modified cells. All of these effects were comparable to those also achieved using O(6)-beG/temozolomide. Thus our data show that MGMT(P140K) is a suitable candidate for chemoprotective gene therapy where PaTrin-2 is being used in conjunction with temozolomide.
    • p21(WAF1/Cip1) - PCNA: fatal attraction.

      Galanos, Panagiotis; Pappas, George; Gorgoulis, Vassilis G; Molecular Carcinogenesis Group , Department of Histology and Embryology , School of Medicine, University of Athens , Athens , Greece (2016-08-11)
    • p210 Bcr-Abl expression in a primitive multipotent haematopoietic cell line models the development of chronic myeloid leukaemia.

      Pierce, A; Owen-Lynch, P J; Spooncer, Elaine; Dexter, T Michael; Whetton, Anthony D; Leukaemia Research Fund Cellular Development Unit, UMIST, Manchester, UK. (1998-08-06)
      Chronic myeloid leukaemia (CML) is a clonal disorder of the pluripotent haemopoietic stem cell, the hallmark of which is the constitutively activated Bcr-Abl protein tyrosine kinase. During the initial chronic phase of CML the primitive multipotent leukaemic progenitor cells remain growth factor dependent and are capable of producing terminally differentiated cells. Although the available evidence suggests that Bcr-Abl directly affects signalling pathways involved in controlling the development of primitive haemopoietic progenitors the identification of the specific biological consequences of Bcr-Abl activity in these progenitors has been hampered by the lack of suitable systems modelling CML. By transfecting the multipotent haemopoietic cell line FDCP-Mix with a temperature sensitive mutant of Bcr-Abl we have developed the first working model that mirrors the chronic phase of CML. FDCP-Mix cells expressing Bcr-Abl tyrosine kinase activity remain growth factor dependent and retain their ability to differentiate. Normal neutrophilic cells are formed in response to G-CSF and GM-CSF. In addition, the transfected FDCP-Mix cells grown at the permissive temperature for Bcr-Abl tyrosine kinase activity display enhanced survival and proliferation in low concentrations of growth factor. These findings are consistent with the initial subtle changes seen in CML progenitor cells during the chronic phase and confirm that Bcr-Abl effects are context specific, i.e. they depend on the origin and developmental potential of the transfected cells. This questions the significance of studies in non-haemopoietic and differentiation blocked haemopoietic cells.
    • P27(KIP1) expression indicates that steroid receptor-positive cells are a non-proliferating, differentiated subpopulation of the normal human breast epithelium.

      Clarke, Robert B; Howell, Anthony; Potten, Christopher S; Anderson, Elizabeth; Clinical Research Department, Christie Hospital, M20 4BX, Manchester, UK. (2000-09)
      To test the hypothesis that steroid receptor-expressing cells are derived from the proliferative population, we examined expression of the p27(KIP1) inhibitor of cyclin-dependent kinase activity (a differentiation marker) while tracking the fate of proliferating cells in normal human breast tissue implanted into athymic nude mice using tritiated thymidine [3H]-dT. We identified a small number of cells that appeared to have divided just once before switching on p27(KIP1) expression. p27(KIP1)+ve cells also expressed steroid receptors, but not the Ki67 proliferation-associated antigen. These data support the hypothesis that steroid receptor-expressing cells are a differentiated population within the normal human breast epithelium.
    • p27(Kip1) is a predictive factor for tamoxifen treatment response but not a prognostic marker in premenopausal breast cancer patients.

      Stendahl, M; Nilsson, Sofie; Wigerup, C; Jirström, K; Jönsson, P; Stål, O; Landberg, Göran; Center for Molecular Pathology, Lund University, Malmö University Hospital, Malmö, Sweden. (2010-03-03)
      The cell cycle regulating protein p27(Kip1) (p27) has dual roles by acting as both a cdk-inhibitor and as an assembly factor for different cdk-complexes. Loss of p27 has been linked to malignant features in tumours, however the exact role of p27 deregulation in breast cancer regarding prognostic and treatment predictive information has not been fully clarified. We have evaluated p27 expression in 328 primary, stage II breast cancers from premenopausal patients who had been randomised to either tamoxifen treatment or no adjuvant treatment after surgery. p27 was associated with the oestrogen receptor (ER) and cyclin D1, and p27 downregulation was associated with high proliferation. There was no association between recurrence-free-survival (RFS) and p27 (HR=0.800, 95%CI 0.523-1.222, p=0.300), indicating that p27 is not a prognostic marker. The predictive value of p27 was analysed by comparing RFS in tamoxifen treated and untreated patients in subgroups of low and high p27 expression (HR=0.747, 95%CI 0.335-1.664, p=0.474 and HR=0.401, 95%CI 0.240-0.670, p<0.001, respectively). Only patients with p27 high tumours benefited from tamoxifen (multivariate interaction analysis p=0.034). Our study suggests that p27 downregulation is associated with tamoxifen resistance in premenopausal breast cancer but is not linked to impaired prognosis. (c) 2010 UICC.
    • p27KIP1 phosphorylation by PKB/Akt leads to poor breast cancer prognosis.

      Clarke, Robert B; Breast Biology Group, Clinical Research Department, Christie Hospital NHS Trust, Wilmslow Road, Withington, Manchester, UK. RClarke@PICR.man.ac.uk (2003)
    • The p38 alpha stress kinase suppresses aneuploidy tolerance by inhibiting Hif-1 alpha.

      Simoes-Sousa, Susana; Littler, Samantha; Thompson, Sarah L; Minshall, Paul; Whalley, Helen J; Bakker, B; Belkot, Klaudyna; Moralli, D; Bronder, Daniel; Tighe, Anthony; et al. (2018)
      Deviating from the normal karyotype dramatically changes gene dosage, in turn decreasing the robustness of biological networks. Consequently, aneuploidy is poorly tolerated by normal somatic cells and acts as a barrier to transformation. Paradoxically, however, karyotype heterogeneity drives tumor evolution and the emergence of therapeutic drug resistance. To better understand how cancer cells tolerate aneuploidy, we focused on the p38 stress response kinase. We show here that p38-deficient cells upregulate glycolysis and avoid post-mitotic apoptosis, leading to the emergence of aneuploid subclones. We also show that p38 deficiency upregulates the hypoxia-inducible transcription factor Hif-1? and that inhibiting Hif-1? restores apoptosis in p38-deficent cells. Because hypoxia and aneuploidy are both barriers to tumor progression, the ability of Hif-1? to promote cell survival following chromosome missegregation raises the possibility that aneuploidy tolerance coevolves with adaptation to hypoxia.
    • p53 and related proteins in epithelial ovarian cancer.

      Sengupta, P S; McGown, Alan T; Bajaj, V; Blackhall, Fiona H; Swindell, Ric; Bromley, Michael; Shanks, Jonathan H; Ward, Timothy H; Buckley, C H; Reynolds, K; et al. (2000-12)
      We conducted a retrospective immunohistochemical evaluation of the prognostic significance of the expression of p53 and the related proteins Bax, Bcl-2, growth arrest and DNA damage (Gadd45), murine double minute 2 (Mdm2) and p21(WAF1/CIP1) in chemonaive tumours taken from 66 patients with ovarian cancer. Ki-67 expression (a marker of cell proliferation) was also evaluated immunohistochemically, while apoptosis within malignant cells was determined with the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) assay. The expression of each of the following proteins was significantly associated in the tumours (P < 0.05 unless otherwise stated): Bax with Bcl-2 (P < 0.01); Bax with Mdm2; p21(WAF1/CIP1) with Gadd45 (P < 0.01); p21(WAF1/CIP1) with p53; p53 with Mdm2. Univariate analysis showed that expression of p53, Bax, bulk residual disease and International Federation of Gynecology and Obstetricians (FIGO) stage were all strongly correlated with response to chemotherapy (P < 0.01). Similarly, the FIGO stage and Ki-67 expression (P < 0.01), as well as pathological subtype and bulk residual disease (P < 0.05), were prognostic factors for disease progression. The FIGO stage and Ki-67 expression were significant prognostic factors for overall survival (P < 0.01), with Gadd45 expression and pathological subtype also significant (P < 0.05) in a univariate analysis. Multivariate analysis for response to chemotherapy showed that expression of p53, Bax and FIGO stage were all independent prognostic factors (P < 0.01). The FIGO stage was the most important independent prognostic factor for progression and survival on multivariate analysis (P < 0.01). However, Ki-67 expression was also an independent prognostic factor for disease progression (P < 0.05) and approached significance for survival (P = 0.055). Taken together, these data suggest that determination of Ki-67 expression could supplement established prognostic factors.
    • p53 deficiency sensitizes clonogenic cells to irradiation in the large but not the small intestine.

      Hendry, Jolyon H; Cai, W B; Roberts, Stephen A; Potten, Christopher S; CRC Department of Experimental Radiation Oncology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom. (1997-09)
      The role of p53 in the survival of irradiated crypts in the small intestine and three regions of the large intestine (cecum, mid-colon and rectum) was assessed by comparing the responses in p53 null, p53 heterozygous and wild-type mice. There was no difference in the levels of crypt survival in the small intestine between the three genotypes, although the rate of cell depletion and regeneration in the null mice appeared slower. In the large intestine, crypt survival was lowest in the null mice compared to the other genotypes, in particular after high doses. The levels of crypt survival in the heterozygotes were not significantly different from those in the wild-type mice. Hence the greater radioresistance of crypts in the colon than in the small intestine, reported previously by us and others using various other mouse strains, may be partly attributable to the presence of p53. This effect is not readily explained by current knowledge concerning the decreased p53 expression and the greater expression of the survival gene Bcl2 in the stem cell zone of crypts in the colon compared to those in the small intestine. Reduced repair associated with the lack of a G2-phase checkpoint delay, the predominant arrest point for intestinal cells, is a possible explanation for the decrease in survival.
    • p53 overexpression as a marker of malignancy in gastric biopsies.

      Starzynska, T; Marsh, P J; Stern, Peter L; Department of Gastroenterology, Medical Pomeranian Academy, Szczecin, Poland. (1993-12)
      Inactivation of the p53 tumour-suppressor gene is the commonest genetic abnormality in human cancers. This results in a conformational change in the p53 protein, and a consequent prolongation in its half-life; thereby permitting the identification of p53 immunoreactivity in malignant cells. Such reactivity is observed in up to 57% of gastric carcinomas, and is a proven indicator of poor prognosis. We have investigated the use of p53 immunohistochemistry in the diagnosis of malignancy in pre-operative gastric biopsy specimens. Using a three-stage immunoperoxidase technique, p53 expression was examined in 117 gastric biopsies obtained during flexible upper gastrointestinal endoscopy: 80 of these biopsies were from known gastric carcinomas, 20 from benign gastric disorders and 17 from normal gastric mucosa. Of the gastric cancers 40% (n = 32) exhibited overexpression of p53. No reactivity was observed in any of the biopsies of gastric ulcers, polyps or normal mucosa. The expression of p53 by gastric carcinomas improved the diagnostic accuracy of conventional histopathology from 86% to 92.5%; with 5% of biopsies incorrectly diagnosed and 2.5% of an equivocal appearance. These results demonstrate that the detection of p53 is a highly specific marker of gastric malignancy, and that such a technique can easily be performed on biopsies obtained at endoscopy.
    • Pan-cancer analysis of whole genomes

      Campbell, P. J.; Getz, G.; Korbel, J. O.; Stuart, J. M.; Jennings, J. L.; Stein, L. D.; Perry, M. D.; Nahal-Bose, H. K.; Ouellette, B. F. F.; Li, C. H.; et al. (2020)
      Fibrosis and fat replacement in skeletal muscle are major complications that lead to a loss of mobility in chronic muscle disorders, such as muscular dystrophy. However, the in vivo properties of adipogenic stem and precursor cells remain unclear, mainly due to the high cell heterogeneity in skeletal muscles. Here, we use single-cell RNA sequencing to decomplexify interstitial cell populations in healthy and dystrophic skeletal muscles. We identify an interstitial CD142-positive cell population in mice and humans that is responsible for the inhibition of adipogenesis through GDF10 secretion. Furthermore, we show that the interstitial cell composition is completely altered in muscular dystrophy, with a near absence of CD142-positive cells. The identification of these adipo-regulatory cells in the skeletal muscle aids our understanding of the aberrant fat deposition in muscular dystrophy, paving the way for treatments that could counteract degeneration in patients with muscular dystrophy.
    • Papillomavirus vaccines.

      Duggan-Keen, Margaret F; Brown, Michael D; Stacey, Simon N; Stern, Peter L; Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, United Kingdom. (1998-12-01)
      The considerable morbidity and mortality associated with certain human papillomaviruses (HPV) has provided the impetus for HPV vaccine development. The design of such vaccines has evolved from an understanding of the nature of HPV infections and their consequences, together with evaluation of the efficacy of different approaches to vaccination in animal models. These studies have culminated in the production of several different vaccine preparations which are currently undergoing Phase I and II clinical trials. The justification for the widespread implementation of prophylactic HPV vaccines will depend on the outcome of larger scale studies of vaccine efficacy that take into account the epidemiology of HPV infections and associated disease. The usefulness of therapeutic HPV vaccines will require evidence that they can substantially augment or substitute for the effectiveness of currently available treatments.
    • Paradoxical elevations in serum IGF-II and IGF binding protein-2 in acromegaly: insights into the regulation of these peptides.

      Renehan, Andrew G; Toogood, Andy; Ryder, W David J; Jones, Jenny; Potten, Christopher S; O'Dwyer, Sarah T; Shalet, Stephen M; Department of Surgery, Christie Hospital NHS Trust, Manchester, UK. arenehan@picr.man.ac.uk (2001-10)
      OBJECTIVE: Circulating insulin-like growth factor (IGF)-II and IGF binding protein-2 (IGFBP-2) are frequently altered, often in parallel, in numerous pathologies including neoplastic disease but little is known about their normal regulation. This study compared serum IGF-II and IGFBP-2 distributions between acromegalics and a large normal adult population to explore possible determinants. PATIENTS: Sixty acromegalic patients undergoing screening colonoscopy (age range 25-81 years); normative data from 306 healthy adults (age range 20-89 years). MEASUREMENTS: Serum IGF-I, IGF-II, IGFBP-2 and IGFBP-3 were measured in healthy adults and acromegalics. Mean growth hormone (GH) levels were obtained for acromegalic patients. Differences were compared using t-tests (unadjusted) and multiple regression models (adjusted for age and gender). Correlations were expressed as Pearson's coefficient (r). RESULTS: For acromegalic patients, GH was significantly correlated with IGF-I (r = 0.50; P < 0.001) and IGFBP-3 (r = 0.29; P = 0.03) but not IGF-II or IGFBP-2. Contrary to expectations, mean IGF-II and IGFBP-2 levels were significantly raised in the acromegalics compared with normals [adjusted mean difference (95% CI) = 226 (181, 271) microg/l and 305 (200, 410) microg/l, respectively]. Ten acromegalic patients had colorectal neoplasia but their presence did not contribute to the elevations in serum IGF-II and IGFBP-2. The (IGF-I + IGF-II)/IGFBP-3 molar ratios were remarkably constant in both healthy adults and acromegalics, but the relationships of the ligands individually with IGFBP-3 were not linear: as IGFBP-3 increased, IGF-I also increased whereas IGF-II initially increased but then decreased. IGFBP-2 did not correlate with IGF-II, but molar concentration significantly correlated with the IGF-II/IGFBP-3 molar ratio (r = 0.40; P = 0.001). CONCLUSIONS: Serum IGF-II and IGFBP-2 levels were paradoxically elevated in acromegalics, independent of the presence of colorectal neoplasia. The (IGF-I + IGF-II)/IGFBP-3 molar ratio appears to be pivotal in determining IGF-II values, which, in turn, expressed as a ratio of IGFBP-3, is related to IGFBP-2. These observations offer new insights into the regulation of these peptides.
    • Parallel evaluation of circulating tumor DNA and circulating tumor cells in metastatic colorectal cancer.

      Germano, G; Mauri, G; Siravegna, G; Dive, Caroline; Pierce, Jackie; Di Nicolantonio, F; D'Incalci, M; Bardelli, A; Siena, S; Sartore-Bianchi, A; et al. (2017-11-01)
      Tissue biopsy is the gold standard for tumor genotyping, but it is an invasive procedure providing a single snapshot into tumor heterogeneity. Liquid biopsy approaches, encompassing the analysis of circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs), have been proposed as an alternative, with the potential of providing a comprehensive portrait of the tumor molecular landscape. In metastatic colorectal cancer (mCRC), both CTCs and ctDNA analysis have been investigated, but comparative analyses are limited.
    • A parallel proteomic and metabolomic analysis of the hydrogen peroxide- and Sty1p-dependent stress response in Schizosaccharomyces pombe.

      Weeks, Mark E; Sinclair, John; Butt, Amna; Chung, Yuen-Li; Worthington, Jessica L; Wilkinson, Caroline R M; Griffiths, John R; Jones, Nic; Waterfield, Michael D; Timms, John F; et al. (2006-05)
      Using an integrated approach incorporating proteomics, metabolomics and published mRNA data, we have investigated the effects of hydrogen peroxide on wild type and a Sty1p-deletion mutant of the fission yeast Schizosaccharomyces pombe. Differential protein expression analysis based on the modification of proteins with matched fluorescent labelling reagents (2-D-DIGE) is the foundation of the quantitative proteomics approach. This study identifies 260 differentially expressed protein isoforms from 2-D-DIGE gels using MALDI MS and reveals the complexity of the cellular response to oxidative stress and the dependency on the Sty1p stress-activated protein kinase. We show the relationship between these protein changes and mRNA expression levels identified in a parallel whole genome study, and discuss the regulatory mechanisms involved in protecting cells against hydrogen peroxide and the involvement of Sty1p-dependent stress-activated protein kinase signalling. Metabolomic profiling of 29 intermediates using 1H NMR was also conducted alongside the protein analysis using the same sample sets, allowing examination of how the protein changes might affect the metabolic pathways and biological processes involved in the oxidative stress response. This combined analysis identifies a number of interlinked metabolic pathways that exhibit stress- and Sty1-dependent patterns of regulation.
    • The paralogous hematopoietic regulators Lyl1 and Scl are coregulated by Ets and GATA factors, but Lyl1 cannot rescue the early Scl-/- phenotype.

      Chan, Wan Y I; Follows, George A; Lacaud, Georges; Pimanda, John E; Landry, Josette-Renee; Kinston, Sarah; Knezevic, Kathy; Piltz, Sandie; Donaldson, Ian J; Gambardella, Laure; et al. (2007-03-01)
      Transcription factors are key regulators of hematopoietic stem cells (HSCs), yet the molecular mechanisms that control their expression are largely unknown. Previously, we demonstrated that expression of Scl/Tal1, a transcription factor required for the specification of HSCs, is controlled by Ets and GATA factors. Here we characterize the molecular mechanisms controlling expression of Lyl1, a paralog of Scl also required for HSC function. Two closely spaced promoters directed expression to hematopoietic progenitor, megakaryocytic, and endothelial cells in transgenic mice. Conserved binding sites required for promoter activity were bound in vivo by GATA-2 and the Ets factors Fli1, Elf1, Erg, and PU.1. However, despite coregulation of Scl and Lyl1 by the same Ets and GATA factors, Scl expression was initiated prior to Lyl1 in embryonic stem (ES) cell differentiation assays. Moreover, ectopic expression of Scl but not Lyl1 rescued hematopoietic differentiation in Scl-/- ES cells, thus providing a molecular explanation for the vastly different phenotypes of Scl-/- and Lyl1-/- mouse embryos. Furthermore, coregulation of Scl and Lyl1 later during development may explain the mild phenotype of Scl-/- adult HSCs.
    • PARG inhibition: development of novel compounds and a biomarker strategy to determine cell line sensitivity in breast cancer

      Waddell, Ian D; James, Dominic I; Smith, Kate M; Holt, Sarah V; Acton, Ben; Fairweather, Emma E; Hamilton, Niall M; Hamilton, Nicola S; Hitchin, James R; Huttom, Colin; et al. (2015)
    • PARG inhibitors exhibit synthetic lethality with XRCC1 deficiency and a cellular mechanism of action that is distinct from PARP inhibition

      Martin, Leenus; Cheng, Tzuling; James, Dominic I; Begum, Habiba; Smith, Kate M; Jordan, Allan M; Waddell, Ian D; Vaidya, Kedar; Fischer, Marcus; Yao, Bing; et al. (2018)
    • Partial trisomy 14q-- and pseudoxanthoma elasticum.

      Muldal, S; Enoch, B A; Ahmed, A; Harris, R; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1973-06)