• Cytokine stimulation and the choice of promoter are critical factors for the efficient transduction of mouse T cells with HIV-1 vectors.

      Gilham, David E; Lie-A-Ling, Michael; Taylor, Naomi; Hawkins, Robert E; Cell Therapy Group, Cancer Research UK Department of Medical Oncology, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK. dgilham@picr.man.ac.uk (2010-02)
      BACKGROUND: HIV-1 fails to successfully infect mouse T cells as a result of several blocks in the viral replication cycle. We investigated whether this also impacted on the use of HIV-1 derived lentiviral vectors for stable gene transfer into mouse T cells. METHODS: Freshly isolated primary mouse T cells were immediately mixed with lentiviral vectors encoding an enhanced green fluorescent protein marker gene and transduction frequency was determined after 5 days of culture. RESULTS: Optimal transduction required both mouse T cell activation and cytokine support. Furthermore, transduction was also dependent upon the promoter chosen, with the rank order of potency being PGK > EF1 > SFFV > CMV. HIV-1 lentiviral vectors also efficiently transduced cytokine-stimulated T cells (in the absence of antibody driven T cell activation), albeit with a lower level of transgene expression compared to fully-activated T cells. CONCLUSIONS: The present study demonstrates that primary mouse T cells can be efficiently transduced with HIV-1 lentiviral vectors, opening up prospects for their use in mouse models of gene-modified adoptive cellular therapy.
    • Cytokine-mediated protein kinase C activation is a signal for lineage determination in bipotential granulocyte macrophage colony-forming cells.

      Whetton, Anthony D; Heyworth, Clare M; Nicholls, S E; Evans, C A; Lord, J M; Dexter, T Michael; Owen-Lynch, P J; Department of Biochemistry and Applied Molecular Biology, UMIST, Manchester, United Kingdom. (1994-05)
      Granulocyte macrophage colony-forming cells (GM-CFC) have the potential to develop into either macrophages and/or neutrophils. With a highly enriched population of these cells we have found that although GM-CFC are equally responsive to macrophage colony stimulating factor (M-CSF) and stem cell factor (SCF) in terms of DNA synthesis, M-CSF stimulated the development of colonies containing macrophages in soft gel assays, while SCF promoted neutrophilic colony formation. When SCF and M-CSF were combined, mainly macrophage development was stimulated both in soft agar colony-forming assays and liquid cultures. An analysis of some potential signaling mechanisms associated with cytokine-mediated developmental decisions in GM-CFC revealed that M-CSF, but not SCF, was able to chronically stimulate phosphatidylcholine breakdown and diacylglycerol production, indicating that protein kinase C (PKC) may be involved in the action of M-CSF. Furthermore, M-CSF, but not SCF, can increase the levels of PKC alpha (PKC alpha) expression and stimulate the translocation of PKC alpha to the nucleus. When the PKC inhibitor, calphostin C, was added to GM-CFC cultured in M-CSF then predominantly neutrophils were produced, conversely PKC activators added with SCF stimulated macrophage development. The data indicate a role for PKC in M-CSF-stimulated macrophage development from GM-CFC.
    • The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import.

      Walther, Tobias C; Pickersgill, Helen; Cordes, Volker C; Goldberg, Martin W; Allen, Terence D; Mattaj, Iain W; Fornerod, Maarten; Gene Expression Program, EMBL, D-69117 Heidelberg, Germany. (2002-07-08)
      The nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, CAN/Nup214 and RanBP2/Nup358. Consistent with previous data, RanBP2 was localized at the cytoplasmic filaments. In contrast, CAN was localized near the cytoplasmic coaxial ring. Unexpectedly, extensive blocking of RanBP2 with gold-conjugated antibodies failed to inhibit nuclear import. Therefore, RanBP2-deficient NPCs were generated by in vitro nuclear assembly in RanBP2-depleted Xenopus egg extracts. NPCs were formed that lacked cytoplasmic filaments, but that retained CAN. These nuclei efficiently imported nuclear localization sequence (NLS) or M9 substrates. NPCs lacking CAN retained RanBP2 and cytoplasmic filaments, and showed a minor NLS import defect. NPCs deficient in both CAN and RanBP2 displayed no cytoplasmic filaments and had a strikingly immature cytoplasmic appearance. However, they showed only a slight reduction in NLS-mediated import, no change in M9-mediated import, and were normal in growth and DNA replication. We conclude that RanBP2 is the major nucleoporin component of the cytoplasmic filaments of the NPC, and that these filaments do not have an essential role in importin alpha/beta- or transportin-dependent import.
    • Cytotoxic Michael-type amine adducts of alpha-methylene lactones alantolactone and isoalantolactone.

      Lawrence, Nicholas J; McGown, Alan T; Nduka, Jane; Hadfield, John A; Pritchard, Robin G; Department of Chemistry, UMIST, Manchester, UK. lawrencenj1@cardiff.ac.uk (2001-02-12)
      Two series of cytotoxic (IC50, K562 cell line, 1-24 microM) alpha-aminomethyl substituted lactones 3 and 4 were prepared by stereoselective Michael-type addition of amines to alantolactone (1) and isoalantolactone (2). The lactones 1 and 2 and their amine adducts induce apoptosis and act as alkylating agents.
    • Cytotoxicity of adherent cells associated with some human tumours and lung tissues.

      Vose, Brent M; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1978)
    • Cytotoxicity of the bioreductive agent RH1 and its lack of interaction with radiation.

      Kim, Joo-Young; West, Catharine M L; Valentine, Helen R; Ward, Timothy H; Patterson, Adam V; Stratford, Ian J; Roberts, Stephen A; Hendry, Jolyon H; Cancer Research UK Groups of Experimental Radiation Oncology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK. (2004-03)
      BACKGROUND AND PURPOSE: RH1 is a new bioreductive agent that was developed as a cytotoxic agent with selectivity for tumour cells expressing high levels of the enzyme DT-diaphorase (DTD). The aim of the present study was to investigate the cytotoxicity of RH1 in relation to cellular levels of reducing enzymes and any interaction of RH1 with ionizing radiation under oxic and hypoxic conditions. PATIENTS AND METHODS: The MB-MDA231 human breast cancer cell line (WT) and WT cells transfected with the NQO1 gene encoding DTD (the D7 cell line) were used to examine the dependency of RH1's cytotoxicity on cellular DTD activity. The role of the 1-electron reducing enzyme P450 reductase was also studied using a P450 reductase-transfected isogenic cell line (R4). A clonogenic assay was used to investigate the cytotoxicity of RH1 with and without irradiation in air and in nitrogen. In all cases drug exposure was for 3 h. RESULTS: DTD levels were around 300-fold higher in D7 compared to WT and R4 cells. RH1 was cytotoxic at nanomolar concentrations to all the cell lines, and was 2-3 times more toxic in the D7 cells with high DTD than in the other two cell lines. Doses of RH1 was around 2-fold more effective in hypoxic than in oxic WT cells, but not by as much in D7 cells. RH1 did not radiosensitise the cells but showed an additive effect when combined with irradiation under oxic and hypoxic conditions. CONCLUSIONS: RH1 shows high clonogenic cytotoxicity to MDA231 cells with high DTD activity but its selectivity based on the presence of DTD is much less than as shown in previous reports. RH1 showed an additive cell killing effect when combined with irradiation under both oxic and hypoxic conditions.
    • D-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition

      de Chiara C; Homsak M; Prosser, GA; Purkiss AG; Tate EW; de Carvalho LPS; Douglas, HL; Garza-garcia, A; Kelly, G; Mycobacterial Metabolism and Antibiotic Research Laboratory, The Francis Crick Institute, London, UK. (2020)
    • Damage-induced apoptosis in intestinal epithelia from bcl-2-null and bax-null mice: investigations of the mechanistic determinants of epithelial apoptosis in vivo.

      Pritchard, D Mark; Potten, Christopher S; Korsmeyer, Stanley J; Roberts, Stephen A; Hickman, John A; CRC Molecular and Cellular Pharmacology Group, School of Biological Sciences, Stopford Building (G38), University of Manchester, Manchester M13 9PT, UK. (1999-12-02)
      The influence of bcl-2 and bax expression on apoptotic cell death in mouse intestinal epithelia was assessed using homozygously null mice. Apoptosis was induced in vivo by the enterotoxin 5-fluorouracil (5FU) or by gamma-irradiation and its cell positional incidence was assessed. 5FU and gamma-radiation treated bax-null mice surprisingly showed no reductions in apoptotic yield in the small intestine or midcolon at 4.5 h at cell positions in which both agents had previously been shown to strongly induce p53 protein expression. The colonic epithelia of 5FU treated bcl-2-null mice showed elevated levels of apoptosis at 4.5 h: from 48 apoptotic events in wild-type mice to 273 in the nulls, scoring 200 half crypts. The increase occurred specifically in the cell positions considered to harbour colonic stem cells, at the base of crypts, where there is selective expression of bcl-2. There was a modest but significant increase in apoptosis in the small intestine of the bcl-2-null mice although the epithelia of wild-type mice here are not immunohistochemically positive for bcl-2 protein. These findings show that bcl-2 plays a key role in determining the sensitivity of colonic stem cells to damage-induced death but that bax is not responsible for the p53-dependent induction of apoptosis in this context.
    • DCE-MRI biomarkers in the clinical evaluation of antiangiogenic and vascular disrupting agents.

      O'Connor, James P B; Jackson, Alan; Parker, Geoff J M; Jayson, Gordon C; Imaging Science and Biomedical Engineering, University of Manchester, Oxford Road, Manchester M13 9PT, UK.james.o'connor@manchester.ac.uk (2007-01-29)
      Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is now frequently used in early clinical trial assessment of antiangiogenic and vascular disrupting compounds. Evidence of drug efficacy and dose-dependent response has been demonstrated with some angiogenesis inhibitors. This review highlights the critical issues that influence T(1)-weighted DCE-MRI data acquisition and analysis, identifies important areas for future development and reviews the clinical trial findings to date.
    • Death of intestinal crypts and of their constituent cells after treatment by chemotherapeutic drugs.

      Moore, James V; Paterson Laboratories, CHristie Hospital and Holt Radium Institute, Manchester M20 9BX (1984-01)
      The number and spatial distribution of necrotic cells in the jejunal crypts of mice, has been measured after treatment by each of 6 cytotoxic drugs. At the LD10/8 day dose of each drug, the majority of necrotic cells were found below position 9 and numbers per crypt were similar for all drugs (approximately 8). These findings resemble those for radiation. However, major differences between agents were found in the calculated numbers of the microcolony-forming units (MFU) that determine overall crypt survival or ablation after high doses of cytotoxic agent. Numbers of MFU as assayed by radiation were approximately 80 per crypt, but only 2 when assayed by mechlorethamine hydrochloride, adriamycin and 5-fluorouracil, and 7 using BCNU. No crypts were destroyed by either cyclophosphamide or actinomycin D, despite the appearance of numerous necrotic cells in the lower part of the crypt. We conclude that in drug-treated intestine, necrotic cells may arise from a non-MFU compartment and the incidence and distributions of such cells are likely to be poor indicators of the response of the MFU.
    • The decay of autoradiographic grain number over crypt base columnar cells in murine ileum as a measure of their generation time.

      Chwalinski, S; Potten, Christopher S; Department of Pathophysiology, Institute of Rheumatology, Warsaw, Poland. (1991-01)
      The decay in the number of grains over [3H]-thymidine labelled crypt base columnar cells (BCC) in autoradiographs of the ileum of BDF1 mice has been studied. The results revealed that using the conventional grain count halving (GCH) method it is possible to obtain an estimation of the generation time (Tc) of the proliferative BCC cells in the Paneth cell zone (PC-zone) of 18.8 +/- 0.74 h. This lies within the range obtained by the percent labelled mitoses (PLM) method, but is shorter than most values obtained by stathmokinetic methods. The present data show no evidence for a shortening of the cell cycle 3 days after irradiation (8 Gy) which is contrary to some earlier observations. Some reasons for this discrepancy are discussed. The comparatively high labelling index of the BCC allows a larger amount of data to be easily collected, compared with the PLM technique, and correction factors which take into account the complicated shape of the bottom of the crypt are not required.
    • Decoding the interdependence of multiparametric magnetic resonance imaging to reveal patient subgroups correlated with survivals

      Li, C; Wang, S; Liu, P; Torheim, Turid; Boonzaier, NR; van Dijken, BR; Schonlieb, CB; Markowetz, F; Price, SJ; Cambridge Brain Tumor Imaging Laboratory, Division of Neurosurgery, Department of Clinical Neurosciences, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK (2019)
      Glioblastoma is highly heterogeneous in microstructure and vasculature, creating various tumor microenvironments among patients, which may lead to different phenotypes. The purpose was to interrogate the interdependence of microstructure and vasculature using perfusion and diffusion imaging and to investigate the utility of this approach in tumor invasiveness assessment. A total of 115 primary glioblastoma patients were prospectively recruited for preoperative magnetic resonance imaging (MRI) and surgery. Apparent diffusion coefficient (ADC) was calculated from diffusion imaging, and relative cerebral blood volume (rCBV) was calculated from perfusion imaging. The empirical copula transform was applied to ADC and rCBV voxels in the contrast-enhancing tumor region to obtain their joint distribution, which was discretized to extract second-order features for an unsupervised hierarchical clustering. The lactate levels of patient subgroups, measured by MR spectroscopy, were compared. Survivals were analyzed using Kaplan-Meier and multivariate Cox regression analyses. The results showed that three patient subgroups were identified by the unsupervised clustering. These subtypes showed no significant differences in clinical characteristics but were significantly different in lactate level and patient survivals. Specifically, the subtype demonstrating high interdependence of ADC and rCBV displayed a higher lactate level than the other two subtypes (P?=?.016 and P?=?.044, respectively). Both subtypes of low and high interdependence showed worse progression-free survival than the intermediate (P?=?.046 and P?=?.009 respectively). Our results suggest that the interdependence between perfusion and diffusion imaging may be useful in stratifying patients and evaluating tumor invasiveness, providing overall measure of tumor microenvironment using multiparametric MRI.
    • Decoding the regulatory network of early blood development from single-cell gene expression measurements.

      Moignard, V; Woodhouse, S; Haghverdi, L; Lilly, A J; Tanaka, Y; Wilkinson, A; Buettner, F; Macaulay, I; Jawaid, W; Diamanti, E; et al. (2015-02-09)
      Reconstruction of the molecular pathways controlling organ development has been hampered by a lack of methods to resolve embryonic progenitor cells. Here we describe a strategy to address this problem that combines gene expression profiling of large numbers of single cells with data analysis based on diffusion maps for dimensionality reduction and network synthesis from state transition graphs. Applying the approach to hematopoietic development in the mouse embryo, we map the progression of mesoderm toward blood using single-cell gene expression analysis of 3,934 cells with blood-forming potential captured at four time points between E7.0 and E8.5. Transitions between individual cellular states are then used as input to develop a single-cell network synthesis toolkit to generate a computationally executable transcriptional regulatory network model of blood development. Several model predictions concerning the roles of Sox and Hox factors are validated experimentally. Our results demonstrate that single-cell analysis of a developing organ coupled with computational approaches can reveal the transcriptional programs that underpin organogenesis.
    • Decrease of pro-angiogenic monocytes predicts clinical response to anti-angiogenic treatment in patients with metastatic renal cell carcinoma (mRCC)

      Oudard, S; Benhamouda, N; Escudier, B; Ravel, P; Kothari, D; Mehmud, F; Levionnois, E; Sevin, E; Negrier, S; Barthelemy, P; et al. (2015)
    • Decreased expression of Yes-associated protein is associated with outcome in the luminal A breast cancer subgroup and with an impaired tamoxifen response.

      Lehn, S; Tobin, N; Sims, A; Stål, O; Jirström, K; Axelson, H; Landberg, Göran; Center for Molecular Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, 205 02 Malmö, Sweden (2014)
      Yes-associated protein (YAP1) is frequently reported to function as an oncogene in many types of cancer, but in breast cancer results remain controversial. We set out to clarify the role of YAP1 in breast cancer by examining gene and protein expression in subgroups of patient material and by downregulating YAP1 in vitro and studying its role in response to the widely used anti-estrogen tamoxifen.
    • Decreasing sensitivity to cytotoxic agents parallels increasing tumorigenicity in human fibroblasts.

      Kinsella, Anne R; Haran, M Sally; Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, United Kingdom. (1991-04-01)
      Human embryo fibroblasts of common genetic origin but exhibiting a range of phenotypes from normal to aggressively tumorigenic have been used to study resistance to the cytotoxic drugs methotrexate and N-(phosphonacetyl)-L-aspartate. Measurement of the intrinsic sensitivities of these cells to the two drugs in standard survival assays, in normal fetal bovine serum, showed increasing resistance to parallel increasing tumor-igenicity. Tumor cells were totally resistant to 10 mM N-(phosphonacetyl)-L-aspartate whereas the 50% lethal dose for methotrexate for the tumor cells was 500 nM compared with 50 nM for the normal diploid parent cell line. The difference in resistance between the immortal and tumorigenic cell lines was eliminated for both methotrexate and N-(phosphonacetyl)-L-aspartate, when the experiments were repeated in the presence of dialyzed fetal bovine serum, but could be restored by the addition of either hypoxanthine (100 microM) or uridine (10 microM). This suggested an important role for the salvage pathways of purine and pyrimidine biosynthesis in the increased resistance of the more tumorigenic cell lines. The implications of these data in relation to cancer chemotherapy will be discussed.
    • Deduction of the clonogen content of intestinal crypts: a direct comparison of two-dose and multiple-dose methodologies.

      Roberts, Stephen A; Hendry, Jolyon H; Potten, Christopher S; Cancer Research Campaign, Biomathematics and Computing Unit, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester, United Kingdom. (1995-03)
      A microcolony assay was used in conjunction with fractionated gamma irradiation to determine the number of clonogens in murine intestinal crypts with varying doses of irradiation used in the determination. The experimental design allows direct comparison between two-dose methodologies, employing one and two (or two or four) equal dose fractions, and multiple-dose methodologies involving determination of the crypt survival curves for a number of fractionation regimens using equal doses per fraction. The two-dose methodology yielded estimates of clonogen number of between 3 and 4 at low delivered dose (single and double fractions each of 6.5-7.5 Gy), rising to around 40 at high biological doses (two and four fractions each of 5.75 or 6.5 Gy). The multifraction methodology yielded estimates of clonogen number which increased from 13 after a single fraction to values of 26 and 22 after three and four fractions. However, the latter values were reduced to 11 and 9, and showed little evidence of any dependence on fraction number, when data pertaining to high biologically effective doses were excluded. Hence it is concluded that the high values for clonogen number typically deduced from such multiple-dose protocols, compared with the generally lower (but dose-dependent) values obtained from two-dose protocols, may be explained at least partially by the higher biological doses generally employed in the multiple-dose protocols.
    • A deep learning framework for predicting response to therapy in cancer

      Sakellaropoulos, T; Vougas, K; Narang, S; Koinis, F; Kotsinas, A; Polyzos, A; Moss, TJ; Piha-Paul, S; Zhou, H; Kardala, E; et al. (2019)
      A major challenge in cancer treatment is predicting clinical response to anti-cancer drugs on a personalized basis. Using a pharmacogenomics database of 1,001 cancer cell lines, we trained deep neural networks for prediction of drug response and assessed their performance on multiple clinical cohorts. We demonstrate that deep neural networks outperform the current state in machine learning frameworks. We provide a proof of concept for the use of deep neural network-based frameworks to aid precision oncology strategies.