• H2O2 stress-specific regulation of S. pombe MAPK Sty1 by mitochondrial protein phosphatase Ptc4.

      Di, Yujun; Holmes, Emily J; Butt, Amna; Dawson, Keren; Mironov, A; Kotiadis, V N; Gourlay, C W; Jones, Nic; Wilkinson, Caroline R M; Cell Regulation Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK. (2012-02-01)
      In fission yeast, the stress-activated MAP kinase, Sty1, is activated via phosphorylation upon exposure to stress and orchestrates an appropriate response. Its activity is attenuated by either serine/threonine PP2C or tyrosine phosphatases. Here, we found that the PP2C phosphatase, Ptc4, plays an important role in inactivating Sty1 specifically upon oxidative stress. Sty1 activity remains high in a ptc4 deletion mutant upon H(2)O(2) but not under other types of stress. Surprisingly, Ptc4 localizes to the mitochondria and is targeted there by an N-terminal mitochondrial targeting sequence (MTS), which is cleaved upon import. A fraction of Sty1 also localizes to the mitochondria suggesting that Ptc4 attenuates the activity of a mitochondrial pool of this MAPK. Cleavage of the Ptc4 MTS is greatly reduced specifically upon H(2)O(2), resulting in the full-length form of the phosphatase; this displays a stronger interaction with Sty1, thus suggesting a novel mechanism by which the negative regulation of MAPK signalling is controlled and providing an explanation for the oxidative stress-specific nature of the regulation of Sty1 by Ptc4.
    • HaeIII polymorphism within 3' untranslated region of PRAD1.

      Heighway, Jim; CRC Department of Cancer Genetics, Paterson Institute for Cancer Research, Manchester, UK. (1991-10-11)
    • Haemangioblast commitment is initiated in the primitive streak of the mouse embryo.

      Huber, Tara L; Kouskoff, Valerie; Fehling, H Joerg; Palis, James; Keller, Gordon; Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York City, New York 10029, USA. (2004-12-02)
      Haematopoietic and vascular cells are thought to arise from a common progenitor called the haemangioblast. Support for this concept has been provided by embryonic stem (ES) cell differentiation studies that identified the blast colony-forming cell (BL-CFC), a progenitor with both haematopoietic and vascular potential. Using conditions that support the growth of BL-CFCs, we identify comparable progenitors that can form blast cell colonies (displaying haematopoietic and vascular potential) in gastrulating mouse embryos. Cell mixing and limiting dilution analyses provide evidence that these colonies are clonal, indicating that they develop from a progenitor with haemangioblast potential. Embryo-derived haemangioblasts are first detected at the mid-streak stage of gastrulation and peak in number during the neural plate stage. Analysis of embryos carrying complementary DNA of the green fluorescent protein targeted to the brachyury locus demonstrates that the haemangioblast is a subpopulation of mesoderm that co-expresses brachyury (also known as T) and Flk-1 (also known as Kdr). Detailed mapping studies reveal that haemangioblasts are found at highest frequency in the posterior region of the primitive streak, indicating that initial stages of haematopoietic and vascular commitment occur before blood island development in the yolk sac.
    • The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage.

      Lancrin, Christophe; Sroczynska, Patrycja; Stephenson, Catherine; Allen, Terence D; Kouskoff, Valerie; Lacaud, Georges; Cancer Research UK Stem Cell Biology Group. (2009-02-12)
      It has been proposed that during embryonic development haematopoietic cells arise from a mesodermal progenitor with both endothelial and haematopoietic potential called the haemangioblast. A conflicting theory instead associates the first haematopoietic cells with a phenotypically differentiated endothelial cell that has haematopoietic potential (that is, a haemogenic endothelium). Support for the haemangioblast concept was initially provided by the identification during mouse embryonic stem cell differentiation of a clonal precursor, the blast colony-forming cell (BL-CFC), which gives rise to blast colonies with both endothelial and haematopoietic components. Although recent studies have now provided evidence for the presence of this bipotential precursor in vivo, the precise mechanism for generation of haematopoietic cells from the haemangioblast still remains completely unknown. Here we demonstrate that the haemangioblast generates haematopoietic cells through the formation of a haemogenic endothelium intermediate, providing the first direct link between these two precursor populations. The cell population containing the haemogenic endothelium is transiently generated during BL-CFC development. This cell population is also present in gastrulating mouse embryos and generates haematopoietic cells on further culture. At the molecular level, we demonstrate that the transcription factor Tal1 (also known as Scl; ref. 10) is indispensable for the establishment of this haemogenic endothelium population whereas the core binding factor Runx1 (also known as AML1; ref. 11) is critical for generation of definitive haematopoietic cells from haemogenic endothelium. Together our results merge the two a priori conflicting theories on the origin of haematopoietic development into a single linear developmental process.
    • Haemopoiesis in long-term bone marrow cultures. A review.

      Dexter, T Michael; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1979)
      Bone marrow-derived adherent cell layers, containing endothelial cells, fat cells and macrophages will support prolonged haemopoiesis in vitro. Evidence suggests that the adherent layer is acting as an in vitro haemopoietic inductive environment for stem cell proliferation and differentiation into the variety of committed progenitor cells of the granulocytic, erythroid, megakaryocytic and lymphoid lineages. Using this system we have analysed the factors controlling proliferation of stem cells (CFU-S), differentiation and maturation of granulocyte/macrophage precursor cells (CFU-C), leukaemic transformation by chemicals and viruses, the role of environment versus stem cells in the aetiology of haemopoietic aplasias and the possible usefulness of long-term cultures for bone marrow transplantation.
    • Haemopoietic cell growth factor mediates cell survival via its action on glucose transport.

      Whetton, Anthony D; Bazill, G W; Dexter, T Michael; Experimental Haematology Section, Paterson Laboratories, Christie Hospital and Radium Institute, Withington, Manchester M20 9BX, UK (1984-02)
      A number of haematopoietic precursor cell lines have been established which exhibit an absolute dependence on haematopoietic cell growth factor (HCGF) which is secreted by WEHI-3 myelomonocytic leukaemia cells. In the presence of HCGF, ATP levels are maintained in these factor-dependent cells (FDC-P cells); in the absence of HCGF, intracellular ATP levels undergo a steady depletion. The cell death that follows this ATP depletion can be prevented by supplying exogenous ATP suggesting that HCGF maintains these cells via its effects on energy metabolism. We have investigated the effect of HCGF on FDC-P cells further and found that: (i) HCGF markedly and rapidly increases lactate production; (ii) high extracellular glucose or glycolytic intermediate concentrations can maintain FDC-P cell viability to some extent whilst stimulating lactate production; (iii) the uptake of 2-deoxyglucose by FDC-P2 cells is stimulated by HCGF in a dose-dependent fashion. This uptake is inhibited by cytochalasin B; (iv) HCGF does not stimulate L-glucose uptake by FDC-P cells. These results suggest that HCGF acts to maintain FDC-P cells via its action on glucose transport. The significance of these results to haemopoiesis and leukaemogenesis is discussed.
    • Haemopoietic cell kinetics in humans treated with rGM-CSF.

      Lord, Brian I; Gurney, H; Chang, James; Thatcher, Nick; Crowther, Derek; Dexter, T Michael; Cancer Research Campaign Department of Experimental Haematology, Paterson Institute for Cancer Research, Manchester, UK. (1992-01-02)
      We have investigated the kinetics of myeloid cell proliferation in the marrow of patients with small-cell lung cancer and treated with 10 daily subcutaneous injections of granulocyte/macrophage colony-stimulating factor (GM-CSF). Bone marrow, obtained before and during treatment with the growth factor, was labelled in vitro with tritiated thymidine (3H-TdR). A 3rd bone-marrow sample was obtained 1 hr following an intravenous injection of 3H-TdR. Subsequent daily blood samples were also collected, and 3H-TdR labelling was assessed on these and the marrow preparations by autoradiography. GM-CSF treatment increased the peripheral granulocytic cells nearly 5-fold, but this included significant eosinophilia, so that the neutrophilic granulocytes increased only 3.3-fold. These cells were released from the marrow over a normal time scale, but their peripheral half-life was about 6 times longer than normal and they were probably functionally defective. Furthermore, significant numbers of immature cells were released from the marrow. Neutrophil production stimulated by GM-CSF was thus overestimated by measurement of the apparent peripheral granulocytosis. Increased labelling indices and grain counts in the proliferating granulocytic cells of the marrow indicate shortened cell-cycle times, and the excess granulocyte production appears to be the result of extra amplification divisions in the proliferative compartments.
    • Haemopoietic colony stimulating factors promote cell survival bysuppressing apoptosis

      Williams, Gwyn T; Smith, Christopher A; Spooncer, Elaine; Dexter, T Michael; Taylor, Dale R; Department of Anatomy, University of Birmingham Medical School, Vincent Drive, Birmingham, B15 2TJ, UK (1990)
    • Haemopoietic growth factors

      Whetton, Anthony D; Dexter, T Michael; Department of Biochemistry and Applied Molecular Biology, University of Manchester, Institute of Science and Technology, PO Box 88, Manchester M60 1QD, UK. (1986)
    • Haemopoietic growth factors: their relevance in osteoclast formation and function.

      Testa, Nydia G; Allen, Terence D; Molineux, Graham; Lord, Brian I; Onions, D E; Paterson Institute for Cancer Research, Christie Hospital & Holt Radium Institute, Withington, Manchester, UK. (1988)
      The major recent advance in our knowledge of the haemopoietic system has been the purification and characterization of a family of haemopoietic growth factors, and their availability in recombinant form. In the bone marrow the sequences of differentiation and proliferation leading to the production of mature cells that these factors regulate may be determined by the relative availability of the factors in microenvironmental domains. The observation that growth factor-producing cells and haemopoietic progenitor cells are not evenly distributed in the bone marrow leads us to expect that the overall effect of growth factors (and other regulatory molecules) on the production and function of macrophages and osteoclasts may differ when in vivo or in vitro assays are used as end-points and, in the latter case, when whole marrow or purified cell populations are tested. The availability of an in vitro assay in which osteoclast-like cells are generated will allow these concepts to be tested.
    • Haemopoietic injury after irradiation: analysis of dose responses and repair using a target-cell model.

      Millar, W T; Hendry, Jolyon H; CRC Department of Radiation Oncology, Glasgow University, UK. (1997-11)
      Published dose-incidence data for haemopoietic lethality in mice have been analysed using a mathematical model based on target-cell survival. The analysis of three comprehensive data sets produced an initial D(o) value of about 1.4 Gy, decreasing to about 1.1 Gy at 3 Gy, 0.9 Gy at 5 Gy, and about 0.7 Gy at a dose of 10 Gy. The alpha/beta ratio was about 14 Gy, and the repair half-time was about 0.3 h. The level of target-cell depletion at LD37 was at about 6 x 10(-4). The D(o) values are compatible with those measured directly for several stages of early haemopoietic progenitor cells in the marrow. The additional use of 13 or alternatively 24 other less-comprehensive data sets increased the overall degree of heterogeneity, so flattening dose-response curves and increasing the deduced overall D(o) values by a factor of about 2. However, when these data sets were stratified with respect to ln(N(o)) where N(o) is the number of tissue rescuing units (TRU), the results were comparable to those obtained when the three comprehensive data sets were analysed individually. Also, the repair halftime was higher at about 1 h. Further, the implied radiosensitivity of the projected target-cell population comprising the TRU was similar to the survival curves obtained for CFU-S and other closely-related haemopoietic progenitor cell types. It has been shown that the number of critical TRU at risk in the marrow is the main feature modulating heterogeneity even when it is assumed that the cellular radiosensitivity does not vary between strains. The number of stem cells comprising a TRU may vary between strains and this may also be influenced by environmental and/or immunological factors. However, it is certainly the case that the initial complement of TRU plays a major role in the incidence of whole body radiation induced mortality.
    • Haemopoietic progenitor and myeloid cell kinetics in humans treated with interleukin-3 and granulocyte/macrophage colony-stimulating factor in combination.

      Lord, Brian I; Testa, Nydia G; Bretti, Sergio; Chang, James; Demuynck, Hilde; Coutinho, Lucia H; De Campos, E S; Fitzsimmons, Lesley; Scarffe, J Howard; Department of Experimental Haematology, PICR, Manchester, UK. (1994-11-15)
      Patients with advanced adenocarcinoma of the colon, rectum or pancreas were entered into trials for evaluation of treatment with sequential doses of IL-3 and GM-CSF. They received 0.25 to 5 micrograms IL-3/kg/d for up to 7 days, followed by 1 microgram GM-CSF/kg/day for a maximum of 10 further days. We assessed the kinetics of bone-marrow cell proliferation and of blood production using tritiated thymidine labelling in vitro and in vivo. Megakaryocytic-CFC were unaffected but proliferation rates of GM-CFC and BFU-E were increased. Progenitor cells were mobilized (12-fold over baseline) into the peripheral blood. The proliferative activity of maturing cells in the marrow was increased (cell-cycle times were reduced by at least 30%). This translated into amplified blood cell production (WCC approximately 30 x 10(9)/l), a 2.2-fold increase in platelet counts and significant eosinophilia. Newly generated neutrophils appeared in the circulation at the normal time and their peripheral half-life was also normal. The calculated 3.2-fold amplification in neutrophil production required nearly 2 extra divisions in the marrow, shared between the progenitors and the proliferating granulocytic cells. The results were compared with those of a previous trial using GM-CSF only, although at a 10-fold higher dose level. Comparable levels of peripheral neutrophils were obtained in both trials but significant ineffective granulopoiesis developed in the earlier study. This was overcome in the present study, the priming dose of IL-3 apparently giving the latitude to utilize lower doses of GM-CSF with less risk of complications.
    • Haemopoietic stem cell development to neutrophils is associated with subcellular redistribution and differential expression of protein kinase C subspecies.

      Shearman, M S; Heyworth, Clare M; Dexter, T Michael; Haefner, B; Owen, P J; Whetton, Anthony D; Department of Biochemistry, Kobe University School of Medicine, Japan. (1993-01)
      Multipotential FDCP-Mix A4 (A4) cells can be induced either to self-renew or to differentiate and develop into mature neutrophils in liquid culture, depending on the haemopoietic growth factors with which they are cultured. When cultured in low concentrations of interleukin 3 (IL-3, 1 unit/ml)) plus Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Granulocyte-CSF (G-CSF), A4 cells proliferate with accompanying development to form cells which resemble mature, postmitotic neutrophils. The presence of high concentrations of IL-3 (100 units/ml) blocks the development of A4 cells even in the presence of GM-CSF plus G-CSF. A4 cell development to neutrophils is accompanied by major changes in the expression of protein kinase C (PKC) subspecies in these cells. The predominant subspecies present in multipotent A4 cells, as judged by direct chromatographic analysis, was the type III enzyme (alpha) subspecies, whereas in mature A4 cell neutrophils, the type II (beta I + beta II) enzymes were predominant. Phorbol esters added to immature A4 cells resulted in a proliferative response, but when added to postmitotic A4 cells resembling neutrophils they elicited a large increase in reactive oxygen intermediate production. This suggests that the type III (alpha) subspecies may mediate proliferative responses in stem cells, whilst the type II (beta I + beta II) enzymes are more important for the mature cell functions of postmitotic neutrophils. In cultures containing IL-3 (100 units/ml) both the type III, and also the type II subspecies were predominantly membrane-associated for prolonged periods (> 24 hours).(ABSTRACT TRUNCATED AT 250 WORDS)
    • Haemopoietic stem cell proliferation: spatial and temporal considerations.

      Wright, E; Lord, Brian I; Department of Anatomy and Experimental Pathology, University of St Andrews, Bute Medical Buildings, Westburn Lane, St Andrews, Fife KY16 9TS. (1986)
    • Haemopoietic stem cells

      Lajtha, L G; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX (1975)
    • Haemopoietic stem cells and the problem of self-renewal.

      Dexter, T Michael; Spooncer, Elaine; Schofield, Raymond; Lord, Brian I; Simmons, P J (1984)
      Haemopoiesis occurs in association with a complex stromal cell network in which all levels of haemopoietic cell development can be found. In order to understand the interaction between stromal cells and growth factors with the processes of self-renewal and differentiation, we have carried out a series of experiments attempting to define the circumstances in which self-renewal occurs in long-term marrow cultures. We have found that highly purified (FACS sorted) CFU-S do not undergo significant self-renewal in vitro when inoculated onto marrow stromal cells that can support self-renewal of unfractionated CFU-S. We have examined the effects of expression of the src oncogene on self-renewal of CFU-S. We have found that, following infection of long-term cultures with a retrovirus carrying the src oncogene, there is expression of src in certain of the stromal cells. There is also a selection for CFU-S that have an extended self-renewal capacity in vivo and in vitro. These CFU-S are non-leukaemic and can reconstitute haempoiesis in irradiated mice. Cells from src-infected cultures can also be induced to proliferate and form cell lines in vitro in the presence of interleukin 3 (IL-3). The cell lines produced are multipotential and non-leukaemic. From such data we conclude that expression of the src oncogene has (directly or indirectly) permanently altered the stem cells in such a way that they can undergo extensive self-renewal in situations that are unfavourable for growth and self-renewal of normal stem cells.
    • Haemopoietic Stem-cell Kinetics during Continuous Irradiation.

      Chu-Tse, Wu; Lajtha, L G; Paterson Laboratories, CHristie Hospital and Holt Radium Institute, Manchester (1975)
    • Hair cortical cell counts (HCCC), a new sensitive in vivo assay with possible applications for biological dosimetry.

      Potten, Christopher S; Department of Epithelial Biology, Paterson Institute for Cancer Research, Christie Hospital, (NHS) Trust, Manchester, UK. (1993-01)
      In the region of a growing hair follicle where terminal keratinization takes place, the nuclei of the medulla and cortex are gradually degraded. In the zone between rapid cell proliferation in the matrix and the complete loss of the nuclei in the formed hair, the number of medullary cells in the hair proved to be a sensitive-radiation-dose-dependent endpoint (Potten et al. 1990). Nuclei in the cortex of the hair are similarly degraded but are more difficult to quantitate since they form a circumferential ring of cells round the medulla. However, using the confocal microscope to obtain discrete optical sections just above and below the medulla, the number of cortical cells was reproducibly counted. The maximum reduction in cortical cell count occurred on the 3rd day after irradiation. Here, the dose-response curve (0.5-4.0 Gy) is presented, for the population of cortical cells in awl guard hairs of the mouse for 137Cs gamma-rays at 4 Gy/min, which was exponential with no significant shoulder, (the D0 was 2.4 +/- 0.2 Gy and the extrapolation number 1.2 +/- 0.1). The cortical cells exhibited a slightly higher D0 value than the medullary cells but differed in that there was no evidence of a shoulder. The effects of 0.5 Gy could be detected. The degree of sensitivity, and the fact that cortical cells are readily detected in human hair follicles, unlike the medullary cells, make this a potentially valuable human biological dosimeter.
    • Hair medullary cell counts following low-dose-rate gamma- and high-energy neutron irradiation.

      Potten, Christopher S; Cancer Research Campaign Department of Epithelial Biology, Paterson Institute for Cancer Research, Christie Hospital, (NHS) Trust, Manchester, UK. (1993-01)
      Young adult Balb/c mice with hair follicles synchronously in the middle of the hair growth cycle received whole-body or partial-body doses of gamma-radiation or neutron radiation. The hair follicles were analysed either 3 days after irradiation in the dose-response experiments, or at various times after a constant dose in the time-course experiments, for changes in the number of cells in the forming medulla of the hair in the region just above the germinal matrix of the growing (anagen) hair follicle. Time-course experiments showed that 3 days after irradiating growing follicles (2 or 4 Gy of gamma-rays or 1 or 2 Gy of neutrons), the maximum reduction in the hair medullary cell count (HMCC) was observed. Survival curves were obtained for gamma-rays over a range of dose-rates (4.0-0.0023 Gy/min) using total doses between 0.5 and 5.0 Gy. A survival curve was also obtained for 62 MeV neutrons at a dose-rate of 0.31 Gy/min and doses of 0.1-2.0 Gy. The D0 for the HMCC dose-response curve following caesium gamma-irradiation at 4.0 Gy/min was 2.1 +/- 0.2 Gy with n = 1.7 +/- 0.2. The dose-response curve for low-dose-rate gamma-rays was best fitted by a simple exponential function with no evidence of a shoulder. The D0 was 3.0 +/- 0.1 Gy at a dose-rate of 0.04 Gy/min. The data for 62 MeV neutrons (at 0.35 Gy/min) had a small but significant shoulder n = 1.5 +/- 0.1 and a D0 of 1.0 +/- 0.1 Gy. These data further illustrate the sensitivity of this assay and its potential application as a biological dosimeter.