• Purification of haemopoietic stem cells--the end of the road?

      Lord, Brian I; Dexter, T Michael; Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Wilmlsow Road, Manchester M20 9BX, UK (1988-12)
    • Purification of the E. coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethylguanine and O4-methylthymine in dodecadeoxyribonucleotides.

      Wilkinson, M C; Potter, P M; Cawkwell, L; Georgiadis, P; Patel, D; Swann, P F; Margison, Geoffrey P; Department of Carcinogenesis, Christie Hospital and Holt Radium Institute, Manchester, UK. (1989-11-11)
      The E. coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme (approximately 0.2% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography. Good correlation was found between the determined and predicted amino acid compositions. The ability of the purified protein to act on O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and O4-methylthymine (O4-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein. With both proteins the rate order was O6-MeG greater than O6-EtG greater than O4-MeT, however, the ogt protein was found to repair O6-MeG, O6-EtG and O4-Met, 1.1, 173 and 84 times, respectively, faster than the ada protein.
    • Purification to apparent homogeneity and partial amino acid sequence of rat liver O6-alkylguanine-DNA-alkyltransferase.

      Wilkinson, M C; Cooper, Donald P; Southan, C; Potter, P M; Margison, Geoffrey P; Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (1990-01-11)
      O6-alkylguanine-DNA-alkyltransferase (ATase) activity was increased in rat liver from 80 to 320 fmoles/mg total protein 48 h after administration of 2-acetylaminofluorene at 60 mg/kg body weight. This tissue was used as a source of ATase which was purified by ammonium sulphate precipitation and DNA-cellulose, molecular exclusion and ion exchange chromatography (IEC). IEC purified material showed a major 24 kDa band after polyacrylamide gel electrophoresis (PAGE) with silver staining. Fluorography of purified ATase following incubation with [3H]-methylated substrate DNA and PAGE showed a single band at 24 kDa suggesting that, as with bacterial ATases, the protein itself accepts the alkyl group from O6-alkylguanine in substrate DNA during the repair reaction. Further purification of the protein using reverse phase HPLC resulted in a single peak representing approximately 125,000 fold purification. This was subjected to amino-terminal sequencing and it was found that the protein was blocked at the amino-terminal end: it was cleaved using trypsin or cyanogen bromide and the amino acid sequence of several reverse phase HPLC purified fragments was determined.
    • Purified tumour angiogenesis factor enhances proliferation of capillary, but not aortic, endothelial cells in vitro.

      Keegan, A; Hill, C; Kumar, Shant; Phillips, P; Schor, Ana M; Weiss, J; Christie Hospital, Manchester, M13 9PL, UK (1982-06)
      Purified tumour angiogenesis factor (TAF) obtained from rat Walker 256 carcinoma and found to induce neovascularization in vivo was examined for its effect on endothelial cell cultures of capillary (CBEC), cow aorta (CAEC) and pig aorta (PAEC) in vitro. Treatment with TAF increased the growth of capillary but not aortic endothelial cells, and then only when the cells were growing on a native collagen substratum. These data show an important growth difference between endothelial cells, in that the ability to proliferate in response to TAF depends not only on the substratum used but also on the vascular origin of the cells.
    • A putative human breast stem cell population is enriched for steroid receptor-positive cells.

      Clarke, Robert B; Spence, Katherine; Anderson, Elizabeth; Howell, Anthony; Okano, Hideyuki; Potten, Christopher S; Breast Biology Group, Cancer Research UK Department of Medical Oncology, University of Manchester, Christie Hospital (NHS) Trust, Wilmslow Road, Withington, Manchester M20 4BX, UK. rclarke@picr.man.ac.uk (2005-01-15)
      Breast epithelial stem cells are thought to be the primary targets in the etiology of breast cancer. Since breast cancers mostly express estrogen and progesterone receptor (ERalpha and PR), we examined the biology of these ERalpha/PR-positive cells and their relationship to stem cells in normal human breast epithelium. We employed several complementary approaches to identify putative stem cell markers, to characterise an isolated stem cell population and to relate these to cells expressing the steroid receptors ERalpha and PR. Using DNA radiolabelling in human tissue implanted into athymic nude mice, a population of label-retaining cells were shown to be enriched for the putative stem cell markers p21(CIP1) and Msi-1, the human homolog of Drosophila Musashi. Steroid receptor-positive cells were found to co-express these stem cell markers together with cytokeratin 19, another putative stem cell marker in the breast. Human breast epithelial cells with Hoechst dye-effluxing "side population" (SP) properties characteristic of mammary stem cells in mice were demonstrated to be undifferentiated "intermediate" cells by lack of expression of myoepithelial and luminal apical membrane markers. These SP cells were 6-fold enriched for ERalpha-positive cells and expressed several fold higher levels of the ERalpha, p21(CIP1) and Msi1 genes than non-SP cells. In contrast to non-SP cells, SP cells formed branching structures in matrigel which included cells of both luminal and myoepithelial lineages. The data suggest a model where scattered steroid receptor-positive cells are stem cells that self-renew through asymmetric cell division and generate patches of transit amplifying and differentiated cells.
    • Pyrrolidinedithiocarbamate increases the therapeutic index of 5-fluorouracil in a mouse model.

      Bach, Simon P; Chinery, Rebecca; O'Dwyer, Sarah T; Potten, Christopher S; Coffey, Robert J; Watson, Alastair; Cancer Research Campaign, Department of Epithelial Biology, The Paterson Institute, Manchester, England. (2000-01)
      BACKGROUND & AIMS: The thiol-containing antioxidant pyrrolidinedithiocarbamate (PDTC) enhances the cytotoxic efficacy of 5-fluorouracil (5-FU) against human colorectal cancer cell lines in vitro and in vivo. This process appears to be mediated by a sustained increase in p21 expression, independent of p53 function, resulting in growth arrest and apoptosis. We determined whether PDTC augmented 5-FU intestinal toxicity in non-tumor-bearing mice. METHODS: Apoptotic and mitotic indices were measured in the small and large intestine on a cell positional basis at intervals throughout the 72-hour period after administration of 5-FU (40 mg/kg) and PDTC (250 mg/kg). The proportion of crypts regenerating after 5-FU (600-1200 mg/kg) and PDTC (500 mg/kg) was also measured. RESULTS: 5-FU therapy induces substantial apoptotic cell death with simultaneous inhibition of mitotic activity within the small and large intestinal epithelium. PDTC reduces 5-FU-induced apoptotic events in the colon by 49%, predominantly among clonogenic stem and transit cells while promoting the early recovery of mitotic activity. As a consequence, PDTC increased the proportion of regenerating colonic crypts after 5-FU therapy. PDTC did not, however, significantly modulate 5-FU toxicity in the small intestine. CONCLUSIONS: PDTC does not augment the intestinal toxicity of 5-FU and actually protects the colonic mucosa. These results support further investigation of PDTC and related compounds as treatments for colorectal cancer.
    • Qualification of M30 and M65 ELISAs as surrogate biomarkers of cell death: long term antigen stability in cancer patient plasma

      Cummings, Jeffrey; Ranson, Malcolm R; Butt, Fouziah; Moore, David; Dive, Caroline; Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, England. jcummings@picr.man.ac.uk (2007-11)
      PURPOSE: M30 and M65 ELISAs are proposed as surrogate biomarkers of tumour cell death in patients and are being applied increasingly in the pharmacodynamic (PD) evaluation of anticancer drugs during clinical trials. In the absence of such data, we have studied the long-term stability of the antigens of both assays in plasma of cancer patients stored at -80 degrees C over 2 years. RESULTS: No evidence was detected of degradation in the M65 antigen. However, in a proportion of patients significant increases in levels of M30 antigen were detected CONCLUSION: Plasma samples for M65 analysis can be stored at -80 degrees C for 2 years; however, caution is recommended when considering long-term storage of samples for the M30 assay.
    • Quality of life (QoL) outcomes with futibatinib treatment in FOENIX-CCA2 - A phase II study in patients (pts) with intrahepatic cholangiocarcinoma (iCCA) harboring FGFR2 gene fusions/rearrangements

      Valle, Juan W; Hollebecque, A.; Furuse, J.; Goyal, L.; Meric-Bernstam, F.; Morlock, R.; He, Y.; Benhadji, K.; Bridgewater, J.; Division of Cancer Sciences, University of Manchester/The Christie NHS Foundation Trust, Manchester, (2020)
      Background: Cancer treatment can produce AEs that result in a reduced QoL. Futibatinib, a highly selective irreversible FGFR1e4 inhibitor, demonstrated an objective response rate (ORR) of 37.3% and median 8.3-mo duration of response in the interim analysis of FOENIX-CCA2, a mutlicenter phase II trial of pts with advanced, refractory iCCA harboring an FGFR2 fusion/rearrangement; grade 3 treatment-related AEs (TRAEs) occurred in 57% of pts (most commonly, hyperphosphatemia [26.9%]). Change in pt-reported outcomes (PROs) from baseline (BL) for the interim data of the phase II trial are presented here. Methods: Pts enrolled into FOENIX-CCA2 (NCT02052778), had locally advanced/ metastatic unresectable iCCA and received oral futibatinib 20 mg once daily (QD) until disease progression/intolerance. PRO measures included EORTC-QLQ-C30 (5 functional and 9 physical measures) and EQ-5D-3L (utility index and 5 dimensions: anxiety/ depression, mobility, pain/discomfort, self-care, and usual activity). PROs were collected at screening, cycles 2 and 4, every 3 cycles after cycle 4 and at the end of treatment. Change in mean score from BL was assessed using predefined clinically meaningful thresholds for each time point with 19 observations (through cycle 13). Results: Sixty-seven of 103 enrolled pts had 6 months of follow-up and 57 (85.1%) had PRO completion data at BL and 1 assessment. EORTC mean global health status score was high at BL (68.7) and maintained through cycle 13 (70.8), a trend observed across all EORTC measures. The only clinically meaningful changes ( 10-point changes) in this timeframe were for constipation symptoms at cycles 2 and 4 (worsened +12.4 and +10.7, respectively) and dyspnea at cycle 10 (improved -12.2). Mean EQ-5D-3L index scores improved from 70.9 at BL to 79.1 at cycle 13 (approximately 273 days on treatment). Conclusions: Overall, the interim PROs from FOENIX-CCA2 were encouraging. These data suggest that despite the occurrence of TRAEs, a 20-mg-QD dose of futibatinib in pts with iCCA provides a promising clinical response without adversely impacting QoL.
    • Quantifying antivascular effects of monoclonal antibodies to vascular endothelial growth factor: insights from imaging.

      O'Connor, James P B; Carano, Richard A D; Clamp, Andrew R; Ross, Jed; Ho, Calvin C K; Jackson, Alan; Parker, Geoff J M; Rose, Chris J; Peale, Franklin V; Friesenhahn, Michel; et al. (2009)
    • The quantitation and kinetics of unscheduled (repair) DNA synthesis in ultraviolet-irradiated human skin by automated image analysis.

      Chadwick, Caroline A; Diggle, S P; Young, A R; Potten, Christopher S; CRC Department of Epithelial Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, U.K. (1996-10)
      Grain counting by eye is a tedious and time-consuming technique but one with great potential in cell kinetics and for the study of DNA excision repair activity (unscheduled DNA synthesis or UDS). We have been investigating the levels of UDS in human skin sections exposed in situ to ultraviolet radiation using a short-term incubation in tritiated thymidine and autoradiography and the decline in UDS levels with time (repair kinetics). We have adapted an automated image analysis system automatically to assess the number of grains over epidermal cell nuclei in autoradiographs of sections of epidermis. An excellent correlation was observed between visual counting and machine measurement of the area (in pixels) occupied by silver grains. The levels of UDS declined with time as lesions are progressively repaired. The half time (+/- standard deviation) for the reduction in UDS is 7.25 +/- 0.18 h. The grain counts can be significantly increased by increasing the autoradiographic exposure, by increasing the concentration of tritiated thymidine and by increasing the incubation time.
    • Quantitation of extracts containing tumour angiogenesis factor (TAF) by radioimmunometric and radioimmunoassays.

      Schor, Ana M; Kumar, S; Phillips, P; Clinical Research Laboratories, Christie Hospital and Holt Radium Institute, Manchester, M20 9BX, England. (1980-06-15)
      An antiserum which is able to inhibit TAF-induced neovascularization in vivo (TAF antiserum) was used to develop two quantitative assays for TAF-containing tumour extracts (tumor TAF). 1) Radioimmunometric assay (RIMA): the IgG of the TAF antiserum was labelled with 125I. An excess of 125I-IgG was incubated with increasing concentrations of tumour TAF and the antigen-bound fraction was precipitated by addition of Clq. 2) Radioimmunoassay (RIA): an excess of iodinated antigen (tumour TAF) was incubated with TAF antiserum diluted so that binding in the absence of unlabelled antigen represented 70-80% of the maximum binding. When tumour TAF was added, competition between labelled and unlabelled antigen for the TAF antibody binding sites resulted in displacement of the former by increasing concentrations of the latter. A second antibody was used to precipitate the bound labelled antigen. Of the two assays, the RIMA was the more sensitive and, due to the lack of a purified antigen, allowed standardization of the results in a more accurate manner. Our data show that there was a good correlation between the ability of tumour TAF to induce angiogenesis in vivo and the degree of antiserum binding detected in vitro by both assays. Preparations containing TAF, whatever the source (i.e. human or animal tumours or tissue culture) shared common antigenic determinants. It is suggested that the quantitative assays should prove valuable in determining the clinical relevance of TAF as a tumour marker.
    • Quantitation of proliferative and cytotoxic precursor cells directed against human tumours: limiting dilution analysis in peripheral blood and at the tumour site.

      Vose, Brent M; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester, M20 9BX, UK (1982-08-15)
      Blood and tumour-infiltrating lymphocytes (TIL) from 16 cancer patients have been examined under limiting dilution conditions to determine the frequency of cells responding in mixed tumour-lymphocyte cultures (MLTC) to autologous tumour and Interleukin-2 (IL-2). Tumour-derived lymphocytes showed a high spontaneous response to IL-2 alone 1/1,900 in TIL; 1/6,000 in PBL suggesting the presence of "activated" T cells in situ. Proliferative frequencies were increased in MLTC in both blood (1/3,779) and TIL (1/1,084). Phenotypic analyses showed that total T-cell contents of the responder populations were comparable but TIL were enriched for the OKT8+ subset with a corresponding reduction in OKT4+. TIL showed increased numbers of OKMI+ and Tac+ lymphocytes. The major cytotoxic precursor expanding under these conditions was reactive against autologous tumour. K562 (NK) were present at a lesser frequency--particularly in TIL. The data show a concentration and activation of reactive lymphocytes at the tumour site and establish conditions for the clonal expansion of specifically cytotoxic T cells.
    • Quantitation of the radiotherapeutic importance of naturally-hypoxic normal tissues from collated experiments with rodents using single doses.

      Hendry, Jolyon H; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1979-07)
    • Quantitative analysis of biomarkers by LC-MS/MS.

      Cummings, Jeffrey; Unwin, Richard D; Veenstra, Timothy D; University of Manchester, Manchester, UK. jcummings@picr.man.ac.uk (2009-05-01)
    • Quantitative and qualitative alterations of heparan sulfate in fibrogenic liver diseases and hepatocellular cancer.

      Tátrai, Péter; Egedi, Krisztina; Somorácz, Aron; Van Kuppevelt, Toin H; Ten Dam, Gerdy; Lyon, Malcolm; Deakin, Jon A; Kiss, András; Schaff, Zsuzsa; Kovalszky, Ilona; et al. (2010-05)
      Heparan sulfate (HS), due to its ability to interact with a multitude of HS-binding factors, is involved in a variety of physiological and pathological processes. Remarkably diverse fine structure of HS, shaped by non-exhaustive enzymatic modifications, influences the interaction of HS with its partners. Here we characterized the HS profile of normal human and rat liver, as well as alterations of HS related to liver fibrogenesis and carcinogenesis, by using sulfation-specific antibodies. The HS immunopattern was compared with the immunolocalization of selected HS proteoglycans. HS samples from normal liver and hepatocellular carcinoma (HCC) were subjected to disaccharide analysis. Expression changes of nine HS-modifying enzymes in human fibrogenic diseases and HCC were measured by quantitative RT-PCR. Increased abundance and altered immunolocalization of HS was paralleled by elevated mRNA levels of HS-modifying enzymes in the diseased liver. The strong immunoreactivity of the normal liver for 3-O-sulfated epitope further increased with disease, along with upregulation of 3-OST-1. Modest 6-O-undersulfation of HCC HS is probably explained by Sulf overexpression. Our results may prompt further investigation of the role of highly 3-O-sulfated and partially 6-O-desulfated HS in pathological processes such as hepatitis virus entry and aberrant growth factor signaling in fibrogenic liver diseases and HCC.
    • A quantitative comparison of cytogenetic effects of anti-tumor agents.

      Parkes, D J; Scott, David; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester, M20 9BX, UK (1982)
      The relative potency of different anti-tumor agents in inducing structural and numerical chromosome abnormalities and SCEs was assessed by making comparisons at equitoxic doses, measured in terms of colony forming ability, in cultured diploid human fibroblasts. At approximately 20% survival the relative potency of X-rays, daunorubicin, nitrogen mustard, adriamycin, and actinomycin D in inducing structural aberrations was 1.0, 0.85, 0.26, 0.22, and zero, respectively. SCE induction was quantitatively unrelated to the induction of chromosome aberrations. No numerical changes were observed. Accurate assessment of the yields of chromosome aberrations requires the use of multiple sampling times in asynchronous populations.
    • A quantitative description of the action of high-dose pulses of radiation on aerated acid solutions containing ferrous and chloride ions

      Bjergbakke, E; Navaratnam, S; Parsons, B; Swallow, A John; Riso National Laboratory, Post Box 49, DK-4000, Roskilde, Denmark (1987)
    • Quantitative development of adherent cell colonies in bone marrow cell culture in vitro.

      Mori, K; Fujitake, H; Okubo, H; Dexter, T Michael; Ito, Y (1979-04)
      Quantification of the formation of adherent cell colonies in bone marrow cell culture was attempted. By secondary transfer of the bone marrow cells as a single cell suspension after 4 days' culture of fine marrow fragments, a linear relationship was obtained between the number of adherent cell colonies developing and the number of cells secondarily inoculated into the culture bottle. This suggests that 4 days' culture of the bone marrow cells with close intercellular interactions is sufficient for the 'conditioning' of the cells to develop adherent cell colonies. Activity of such colonies to support haemopoietic stem cell proliferation was also shown.
    • A quantitative histometric murine in vivo model of radiation-induced oral mucositis.

      Wardley, Andrew M; Booth, Dawn; Roberts, Stephen A; Scarffe, J Howard; Potten, Christopher S; CRC Epithelial Biology Group, UK. (1998-07)
      Gastrointestinal toxicity is a limiting factor in the effectiveness of cancer therapy. This toxicity is most visible in the mouth. There is considerable interest in developing strategies involving growth-factor manipulation of the epithelial stem cells to afford protection to these cells during treatment and/or to speed up the regenerative process following treatment. In order for this to be achieved, studies have to be undertaken in animal systems to demonstrate the proof of principle and determine optimal protocols. Here, a murine model for oral mucositis based on measurements of tissue cellularity at various times after exposure to radiation was used to investigate cytotoxicity. Several sites in the mouth were analysed and the pronounced circadian rhythm in these various epithelial sites determined. The circadian rhythm is important in that it would determine the timing of administration of growth factors. A microscope with an interactive computer was used to define areas of epithelium and lengths of basal layer, within which, and along which, the total number of cell nuclei was determined over a range of times following exposure to 10, 20 and 30 Gy of X-rays. For various practical reasons, the ventral surface of the tongue was identified as the most appropriate tissue to analyse. Here, measurements of cellularity reached minimum values between 6 and 8 days following 20 Gy. Labelling of S-phase cells demonstrated foci of regeneration and a burst of proliferative regeneration that commenced at about 5 days and reached peak values at 8 days after irradiation. This burst of regenerative proliferation was coincident with the minimum in tissue cellularity on about day 8. The lower dose of radiation (10 Gy) had minimal effects on cellularity: after the higher dose (30 Gy), there was clearly a more severe level of cellular depletion. This quantitative model of oral mucositis could be used to study the effects of other cytotoxics, including combinations of agents, and the potential role of growth factors to reduce the severity of the cellular depletion and to speed up the kinetics of regeneration.
    • Quantitative imaging biomarkers in the clinical development of targeted therapeutics: current and future perspectives.

      O'Connor, James P B; Jackson, Alan; Asselin, Marie-Claude; Buckley, David L; Parker, Geoff J M; Jayson, Gordon C; Imaging Science and Biomedical Engineering, University of Manchester, Manchester, UK. james.o'connor@manchester.ac.uk (2008-08)
      Targeted therapeutics have challenged how imaging techniques assess tumour response to treatment because many new agents are thought to cause cytostasis rather than cytotoxicity. Advanced tracer development, image acquisition, and image analysis have been used to produce quantitative biomarkers of pathophysiology, with particular focus on measurement of tumour vascular characteristics. Here, we critically appraise strategies available to generate imaging biomarkers for use in development of targeted therapeutics. We consider important practical and technical features of data acquisition and analysis because these factors determine the precise physiological meaning of every biomarker. We discuss the merits of volume-based and other size-based metrics for assessment of targeted therapeutics, and we examine the strengths and weaknesses of CT, MRI, and PET biomarkers derived from conventional clinical data. We review imaging biomarkers of tumour microvasculature and discuss imaging strategies that probe other physiological processes including cell proliferation, apoptosis, and tumour invasion. We conclude on the need to develop comprehensive compound-specific imaging biomarkers that are appropriate for every class of targeted therapeutics, and to investigate the complementary information given in multimodality imaging studies of targeted therapeutics.