• A B cell specific immediate early human gene is located on chromosome band 1q31 and encodes an alpha helical basic phosphoprotein.

      Newton, J S; Deed, Richard W; Mitchell, Erika L D; Murphy, J J; Norton, John D; Immunology Section, Kings College London, UK. (1993-11-16)
      We have determined the cDNA sequence of a human B cell specific, immediate early gene, designated 1R20, which is inducible in response to several B cell activation signals. The cDNA sequence predicts a 196 amino acid open reading frame comprising numerous highly basic residues and the predicted structure contains several potential alpha helical domains together with eight consensus protein phosphorylation sites. The 1R20 gene has been localised by fluorescence in situ hybridisation to chromosome band 1q31, a region known to be implicated in the pathogenesis of haemopoietic malignancies.
    • B7 integrin-deficient mice: delayed leukocyte recruitment and attenuated protective immunity in the small intestine during enteric helminth infection

      Artis, David; Humphreys, Neil E; Potten, Christopher S; Wagner, Norbert; Muller, Werner; McDermott, Jacqueline R; Grencis, Richard K; Else, Kathryn J; Immunology Group, School of Biological Sciences, University of Manchester, Manchester, GB (2000)
    • The bacterial alkyltransferase-like (eATL) protein protects mammalian cells against methylating agent-induced toxicity.

      Tomaszowski, K; Aasland, D; Margison, Geoffrey P; Williams, Emma L; Pinder, Sarah; Modesti, M; Fuchs, R; Kaina, B; Department of Toxicology, University Medical Center, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany (2015-01-31)
      In both pro- and eukaryotes, the mutagenic and toxic DNA adduct O(6)-methylguanine (O(6)MeG) is subject to repair by alkyltransferase proteins via methyl group transfer. In addition, in prokaryotes, there are proteins with sequence homology to alkyltransferases, collectively designated as alkyltransferase-like (ATL) proteins, which bind to O(6)-alkylguanine adducts and mediate resistance to alkylating agents. Whether such proteins might enable similar protection in higher eukaryotes is unknown. Here we expressed the ATL protein of Escherichia coli (eATL) in mammalian cells and addressed the question whether it is able to protect them against the cytotoxic effects of alkylating agents. The Chinese hamster cell line CHO-9, the nucleotide excision repair (NER) deficient derivative 43-3B and the DNA mismatch repair (MMR) impaired derivative Tk22-C1 were transfected with eATL cloned in an expression plasmid and the sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was determined in reproductive survival, DNA double-strand break (DSB) and apoptosis assays. The results indicate that eATL expression is tolerated in mammalian cells and conferes protection against killing by MNNG in both wild-type and 43-3B cells, but not in the MMR-impaired cell line. The protection effect was dependent on the expression level of eATL and was completely ablated in cells co-expressing the human O(6)-methylguanine-DNA methyltransferase (MGMT). eATL did not protect against cytotoxicity induced by the chloroethylating agent lomustine, suggesting that O(6)-chloroethylguanine adducts are not target of eATL. To investigate the mechanism of protection, we determined O(6)MeG levels in DNA after MNNG treatment and found that eATL did not cause removal of the adduct. However, eATL expression resulted in a significantly lower level of DSBs in MNNG-treated cells, and this was concomitant with attenuation of G2 blockage and a lower level of apoptosis. The results suggest that eATL confers protection against methylating agents by masking O(6)MeG/thymine mispaired adducts, preventing them from becoming a substrate for mismatch repair-mediated DSB formation and cell death.
    • Bacterial and mammalian DNA alkyltransferases sensitize Escherichia coli to the lethal and mutagenic effects of dibromoalkanes.

      Abril, N; Luque-Romero, F L; Prieto-Alamo, M J; Rafferty, Joseph A; Margison, Geoffrey P; Pueyo, C; Departamento de Bioquímica y Biología Molecular, Universidad de Córdoba, España. (1997-10)
      Here we confirm and extend our previous studies demonstrating that the mutagenic potency of 1,2-dibromoethane (DBE) and dibromomethane (DBM) is markedly enhanced (not prevented) in bacteria expressing the O6-alkylguanine-DNA alkyltransferase (ATase) encoded by the Escherichia coli ogt gene. We demonstrate that, in close parallel with mutagenesis, the Ogt ATase sensitizes the bacteria to the lethal effects of these carcinogens, suggesting that one or more of the potentially mutagenic lesions induced by DBE and DBM in the presence of Ogt has additional lethal capacity. We further demonstrate that the sensitization to both lethality and mutagenesis by DBE and DBM is a property shared by other DNA alkyltransferases. This objective was accomplished by quantifying the induction of mutations and lethal events in ogt- ada- E. coli expressing an exogenous bacterial or mammalian ATase from a multicopy plasmid. Mammalian recombinant ATases enhanced the lethal and mutagenic actions of DBE and suppressed the lack of sensitivity of the vector-transformed bacteria to DBM. In most cases the order of effectiveness of the ATases ranked: murine > human > Ogt > rat. Further comparisons included the full-length Ada ATase from E. coli and a truncated Ada version (T-ada) that retains the O6-methylguanine binding domain of the protein. The full-length Ada ATase was effective in enhancing the lethality but not the mutagenicity induced by DBE and DBM. The T-ada ATase provided less sensitization than Ada to lethality by DBE, but of the three bacterial ATases T-ada yielded the highest sensitization to mutagenesis by this compound. T-ada and Ada ATases were in general less effective than the mammalian versions, with the exception of the rat recombinant ATase. The effectiveness of the different mammalian and bacterial ATases in promoting the deleterious actions of dibromoalkanes was compared with the effectiveness of these proteins in suppressing the lethal and mutagenic effects induced by N-nitroso-N-methylurea. The ability to sensitize E. coli to the lethal and mutagenic effects of DBE and DBM seems restricted to DNA alkyltransferase, since overexpression of thioredoxin (Trx) or glutaredoxin (Grx1) in ogt- ada- cells showed no effect, in spite of the reported potential of bioactive dihaloethane-derived species to alkylate Trx.
    • Bacterial expression and functional reconstitution of human heparanase.

      Winkler, S; Schweiger, D; Wei, Zheng; Rajkovic, E; Kungl, A; Department of Pharmaceutical Sciences, University of Graz, Humboldtstrasse 46, 8010 Graz, Austria (2014-01-14)
      Human heparanase is a heparan sulfate degrading enzyme located in the extracellular matrix playing a decisive role in angiogenesis and tumor metastasis. Translated as a 65kDa inactive prae-form, the protein is processed into an 8kDa and a 50kDa subunit which form a non-covalently associated active heterodimer. We have expressed the two subunits separately in Escherichia coli which yielded active human heparanase upon reconstitution. The two purified subunits folded independently and secondary structure analysis by far-UV CD spectroscopy gave 33.1/11.1% α/β content for the 50kDa subunit and 6.9/49% α/β content for the 8kDa subunit. This heparanase expression system is easy and can be used for efficient screening for enzyme inhibitors.
    • The Basal Transcription Complex Component TAF3 Transduces Changes in Nuclear Phosphoinositides into Transcriptional Output.

      Stijf-Bultsma, Yvette; Sommer, Lilly; Tauber, M; Baalbaki, M; Giardoglou, P; Jones, D; Gelato, K; van Pelt, J; Shah, Z; Rahnamoun, H; et al. (2015-05-07)
      Phosphoinositides (PI) are important signaling molecules in the nucleus that influence gene expression. However, if and how nuclear PI directly affects the transcriptional machinery is not known. We report that the lipid kinase PIP4K2B regulates nuclear PI5P and the expression of myogenic genes during myoblast differentiation. A targeted screen for PI interactors identified the PHD finger of TAF3, a TATA box binding protein-associated factor with important roles in transcription regulation, pluripotency, and differentiation. We show that the PI interaction site is distinct from the known H3K4me3 binding region of TAF3 and that PI binding modulates association of TAF3 with H3K4me3 in vitro and with chromatin in vivo. Analysis of TAF3 mutants indicates that TAF3 transduces PIP4K2B-mediated alterations in PI into changes in specific gene transcription. Our study reveals TAF3 as a direct target of nuclear PI and further illustrates the importance of basal transcription components as signal transducers.
    • Basic fibroblast growth factor increases the multiplication and migration of a serum-free derivative of CACO-2 but does not affect differentiation.

      Jayson, Gordon C; Evans, Gareth S; Pemberton, P W; Lobley, R W; Allen, Terence D; Department of Medical Oncology, Paterson Institute for Cancer Research, Withington, Manchester, United Kingdom. (1994-11-01)
      Basic fibroblast growth factor (bFGF) is found in the extracellular matrix and around the endothelial and epithelial cells of some human colon carcinomas. It is believed to play a role in angiogenesis, but in addition, recent data suggest that it can directly stimulate mitogenesis in some colon carcinoma cell lines. To clarify the role of bFGF in human colon carcinoma, we developed a model of Caco-2 which grew in serum-free conditions so that the effect of bFGF on multiplication, migration, and differentiation could be studied in defined conditions. Through morphological and biochemical studies in serum-free conditions, we demonstrated that this subline of Caco-2 differentiated spontaneously on reaching confluence. Using this model, we found that bFGF did not affect differentiation but that multiplication and migration were increased. The implication of these findings is that bFGF, released from the extracellular matrix by invading cells or produced by neovascular endothelial cells, can increase the mitogenic rate and migratory potential of colon carcinoma cells. In addition, the dual role of bFGF in stimulating colon carcinoma cells directly and promoting angiogenesis suggests that anti-bFGF strategies could form the basis of a novel approach to the treatment of colon carcinoma.
    • Bayesian inference supports a location and neighbour-dependent model of DNA methylation propagation at the MGMT gene promoter in lung tumours.

      Bonello, N; Sampson, J; Burn, J; Wilson, I; McGown, Gail; Margison, Geoffrey P; Thorncroft, Mary R; Crossbie, P; Povey, A; Santibanez-Koref, M; et al. (2013-11-07)
      We exploit model-based Bayesian inference methodologies to analyse lung tumour-derived methylation data from a CpG island in the O6-methylguanine-DNA methyltransferase (MGMT) promoter. Interest is in modelling the changes in methylation patterns in a CpG island in the first exon of the promoter during lung tumour development. We propose four competils of methylation state propagation based on two mechanisms. The first is the location-dependence mechanism in which the probability of a gain or loss of methylation at a CpG within the promoter depends upon its location in the CpG sequence. The second mechanism is that of neighbour-dependence in which gain or loss of methylation at a CpG depends upon the methylation status of the immediately preceding CpG. Our data comprises the methylation status at 12 CpGs near the 5' end of the CpG island in two lung tumour samples for both alleles of a nearby polymorphism. We use approximate Bayesian computation, a computationally intensive rejection-sampling algorithm to infer model parameters and compare models without the need to evaluate the likelihood function. We compare the four proposed models using two criteria: the approximate Bayes factors and the distribution of the Euclidean distance between the summary statistics of the observed and simulated datasets. Our model-based analysis demonstrates compelling evidence for both location and neighbour dependence in the process of aberrant DNA methylation of this MGMT promoter CpG island in lung tumours. We find equivocal evidence to support the hypothesis that the methylation patterns of the two alleles evolve independently.
    • BB-10010/MIP-1 alpha in vivo maintains haemopoietic recovery following repeated cycles of sublethal irradiation.

      Lord, Brian I; Marshall, E; Woolford, Lorna B; Hunter, M G; CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Manchester, UK. (1996-10)
      Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is an inhibitor of stem cell proliferation affording protection against damage from agents that express their cytotoxicity specifically in the DNA synthesis phase of the cell cycle. Its ability also to modify the self-renewal capacity of the regenerating cells is now shown to improve and maintain haemopoietic recovery following therapy (sublethal irradiation) whose cytotoxic damage is not limited solely to the DNA-S phase of this cycle. Such non-cell cycle-active cytotoxic agents are used clinically in repeated treatment regimens, which are often limited or terminated because of accumulating haemopoietic damage. BB-10010, a non-aggregating variant of MIP-1 alpha, was administered as a continuous dose (1600 micrograms kg-1 24 h-1) via a subcutaneously implanted pump over a period of 7 days. A dose of 4.5 Gy total-body gamma-rays was given 3-4 h after implantation. Day 8 and 12 spleen colony-forming units (CFU-S) were assayed on days 1, 7 and 14 after irradiation. This cycle of treatment was repeated four times (total 56 days), and on day 14 of the last two cycles the marrow-repopulating ability (MRA) was also measured. In the control bone marrow (no BB-10010) CFU-S fell to < 1% of normal within 1 day of irradiation and recovered to 40% at 14 days. Repeated treatments increased the level of damage, and after four cycles CFU-S recovered to only 10% of normal. BB-10010 afforded little benefit in the first treatment cycle, but by the end of the fourth cycle CFU-S still recovered to 35% of normal. MRA was reduced to 7% of normal by the irradiation protocol-about half that maintained by BB-10010 protection. We conclude that BB-10010 (MIP-1 alpha) reduces the degree of accumulated haemopoietic stem cell damage following repeated non-cell cycle-specific cytotoxic insults-a principle which should be valuable in repeated clinical cytotoxic therapy regimens.
    • bcl-2 delay of alkylating agent-induced apoptotic death in a murine hemopoietic stem cell line.

      Fairbairn, Leslie J; Cowling, Graham J; Dexter, T Michael; Rafferty, Joseph A; Margison, Geoffrey P; Reipert, Brigit M; CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital National Health Service Trust, Manchester, United Kingdom. (1994-09)
      Many cytotoxic agents kill cells by invoking a specific death pathway termed physiological cell death, or apoptosis. Treatment of a murine hemopoietic stem cell line, FDCP-mix, with methylmethanesulfonate (MMS) or N'-methyl-N'-nitrosourea (MNU) leads to death by apoptosis. Retroviral gene transfer was used to overexpress the bcl-2 oncogene in FDCP-mix cells, and this was associated with a delay in apoptosis in these cells after treatment with MNU and MMS and decreased sensitivity of colony formation to the cytotoxic effects of MMS. These data suggest an explanation for the refractory nature of bcl-2-expressing follicular lymphoma to cytotoxic chemotherapy and furthermore suggest that DNA-damaging antitumor therapy may contribute to the progression of disease.
    • Bcl-w expression in colorectal adenocarcinoma.

      Wilson, James W; Nostro, M C; Balzi, Manuela; Faraoni, P; Cianchi, F; Becciolini, Aldo; Potten, Christopher S; CRC Epithelial Biology Laboratory, Paterson Institute for Cancer Research, Manchester, UK. (2000-01)
      We have found that the anti-apoptotic Bcl-2 family protein, Bcl-w, was frequently expressed in colorectal adenocarcinomas, with 69/75 showing positive staining with anti-Bcl-w IgG. Adenomas demonstrated a much lower frequency of Bcl-w expression (only 1 of 17), as did adenocarcinomas from other epithelial tissues such as breast (0/8), stomach (1112) and cervix (0/12). Bcl-w status could be related to the histopathological classification of the tumours, with TNM stage III tumours showing significantly higher levels of expression than tumours of better prognostic grade (at P = 0.009). Those patients with node involvement also had tumours with significantly elevated levels of Bcl-w (at P = 0.02), compared to those which were node-negative. The results suggest that Bcl-w could play a general role in the progression from adenoma to adenocarcinoma in the colorectal epithelium. Currently, more data are being collected to allow us to assess the importance of Bcl-w for disease progression and patient survival.
    • Bcl-w is an important determinant of damage-induced apoptosis in epithelia of small and large intestine.

      Pritchard, D Mark; Print, C; O'Reilly, L; Adams, J M; Potten, Christopher S; Hickman, John A; CRC Department of Epithelial Biology, Paterson Institute, Christie Hospital NHS Trust, Manchester, UK. (2000-08-10)
      The potential role of the bcl-2 relative bcl-w as a physiological regulator of apoptosis in intestinal epithelia has been investigated. Immunoblots for bcl-w with new monoclonal antibodies revealed that it was expressed in the small intestine and colon, among other murine tissues, as well as in six human tumour cell lines of epithelial origin, including two colon carcinoma lines. To assess whether bcl-w regulates either spontaneous or damage-induced apoptosis in the small intestine or colon, apoptosis in intestinal crypts of bcl-w -/- and wild-type mice was quantified microscopically on a cell positional basis. Spontaneous apoptosis within crypt epithelia was not significantly increased by loss of bcl-w, in either the small intestine or midcolon. However, after treatment with the cytotoxic drug 5-fluorouracil or with gamma-radiation, the bcl-w-null animals exhibited substantially more apoptosis than their wild-type counterparts in both tissues. The greatest enhancement of apoptosis attributable to the absence of bcl-w (up to sixfold) occurred in the small intestine. Hence, bcl-w is an important determinant of damage-induced apoptosis in intestinal epithelia, and unlike bcl-2, which regulates only colonic apoptosis, plays a major role in small intestinal epithelium.
    • Bcr-Abl protein tyrosine kinase activity induces a loss of p53 protein that mediates a delay in myeloid differentiation.

      Pierce, Andrew; Spooncer, Elaine; Wooley, Sarah; Dive, Caroline; Francis, Julia M; Miyan, Jaleel; Owen-Lynch, P Jane; Dexter, T Michael; Whetton, Anthony D; Leukaemia Research Fund Cellular Development Unit, UMIST, Manchester, UK. (2000-11-16)
      Chronic myeloid leukaemia is a haemopoietic stem cell disorder, the hallmark of which is the expression of the Bcr-Abl Protein Tyrosine Kinase (PTK). We have previously reported that activation of a temperature sensitive Bcr-Abl PTK in the multipotent haemopoietic cell line FDCP-Mix for short periods resulted in subtle changes including, a transient suppression of apoptosis and no inhibition of differentiation. In contrast, activation of the Bcr-Abl PTK for 12 weeks results in cells that display a delay in differentiation at the early granulocyte stage. Flow cytometric analysis also indicates that the expression of cell surface differentiation markers and nuclear morphology are uncoupled. Furthermore, a significant number of the mature neutrophils display abnormal morphological features. Prolonged exposure to Bcr-Abl PTK results in interleukin-3 independent growth and decreased p53 protein levels. FDCP-Mix cells expressing a dominant negative p53 and p53null FDCP-Mix cells demonstrate that the reduction in p53 is causally related to the delay in development. Returning the cells to the restrictive temperature restores the p53 protein levels, the growth factor dependence and largely relieves the effects on development. We conclude that prolonged Bcr-Abl PTK activity within multipotent cells results in a reduction of p53 that drives a delayed and abnormal differentiation.
    • Bedaquiline, an FDA-approved antibiotic, inhibits mitochondrial function and potently blocks the proliferative expansion of stem-like cancer cells (CSCs)

      Fiorillo, Marco; Lamb, Rebecca; Tanowitz, H; Cappello, A; Martinez-Outschoorn, U; Sotgia, F; Lisanti, Michael P; The Breast Cancer Now Research Unit, Institute of Cancer Sciences, Cancer Research UK Manchester Institute, University of Manchester, Manchester (2016)
      Bedaquiline (a.k.a., Sirturo) is an anti-microbial agent, which is approved by the FDA for the treatment of multi-drug resistant pulmonary tuberculosis (TB). Bedaquiline is a first-in-class diaryl-quinoline compound, that mechanistically inhibits the bacterial ATP-synthase, and shows potent activity against both drug-sensitive and drug-resistant TB. Interestingly, eukaryotic mitochondria originally evolved from engulfed aerobic bacteria. Thus, we hypothesized that, in mammalian cells, bedaquiline might also target the mitochondrial ATP-synthase, leading to mitochondrial dysfunction and ATP depletion. Here, we show that bedaquiline has anti-cancer activity, directed against Cancer Stem-like Cells (CSCs). More specifically, we demonstrate that bedaquiline treatment of MCF7 breast cancer cells inhibits mitochondrial oxygen-consumption, as well as glycolysis, but induces oxidative stress. Importantly, bedaquiline significantly blocks the propagation and expansion of MCF7-derived CSCs, with an IC-50 of approx. 1-μM, as determined using the mammosphere assay. Similarly, bedaquiline also reduces both the CD44+/CD24low/- CSC and ALDH+ CSC populations, under anchorage-independent growth conditions. In striking contrast, bedaquiline significantly increases oxygen consumption in normal human fibroblasts, consistent with the fact that it is well-tolerated in patients treated for TB infections. As such, future pre-clinical studies and human clinical trials in cancer patients may be warranted. Interestingly, we also highlight that bedaquiline shares certain structural similarities with trans-piceatannol and trans-resveratrol, which are known natural flavonoid inhibitors of the mitochondrial ATP-synthase (complex V) and show anti-aging properties.
    • Bedside filtration of blood products in the prevention of HLA alloimmunization--a prospective randomized study. Alloimmunisation Study Group.

      Williamson, L M; Wimperis, J Z; Williamson, P; Copplestone, J A; Gooi, H C; Morgenstern, Godfrey R; Norfolk, D R; Division of Transfusion Medicine, University of Cambridge, UK. (1994-05-15)
      To test the efficacy of poststorage bedside leucodepletion of blood products in the prevention of primary HLA alloimmunization and its clinical sequelae, 172 patients with hematologic malignancy requiring intensive red blood cell and platelet support were randomized to receive either standard or filtered red blood cells and platelets. Quality control of bedside filtration was explored by sequential sampling downstream of the filter, but this did not predict the total number of leucocytes transfused. After exclusions, 123 evaluable patients were assessed every two weeks until the end of therapy. HLA antibodies developed in 21 of 56 (37.5%) nonfilter (NF) and 15 of 67 (22%) filter (F) patients (risk ratio estimate, 0.60 [95% confidence interval, 0.34 to 1.05]; P = .07). Patients with acute myeloid leukemia (AML; n = 53) had higher alloimmunization rates in both arms of the study, with a greater effect of filtration (62.5% NF and 31.0% F; P = .025). Bedside filtration did not affect the overall incidence of febrile transfusion reactions (FTRs; 37% NF and 34% F; P = .71) or of platelet refractoriness assessed in 50 patients (30% NF and 26% F), despite an association between broad HLA reactivity and both FTRs and refractoriness. However, FTRs were also seen in 28 patients without HLA antibodies. Five alloimmunized refractory patients (2 F and 3 NF) required HLA-selected platelets. This report, the first prospective study of bedside filtration, has failed to show clear clinical benefit. Methodological limitations may account in part for this failure, notably the difficulties in accurately assessing the number of leucocytes transfused.
    • Benzamide potentiation of the cytotoxicity of bifunctional galactitol [correction of galacticol] in resistant P388 leukemia correlates with inhibition of DNA ligase II.

      Institoris, E; Fox, Brian W; Pályi, I; National Institute of Oncology, Budapest, Hungary. (1992)
      Benzamide (BA) enhances the cytotoxicity of 1,2:5,6-dianhydrogalactitol (DAG) in resistant P388 leukemia cell lines but not in the sensitive parent line. To examine the reason for this difference in response, we carried out an alkaline elution assay using proteinase K to study DNA interstrand cross-linking. At early time points, equal concentrations of DAG produced the same level of interstrand cross-linking (ICL) in the resistant and sensitive P388 leukemic cells, although marked differences were observed in their cytotoxicity toward the two cell lines. In the sensitive cells, neither the amount of DNA cross-linking nor the cytotoxicity changed during the observation period (38 h) in either the presence or the absence of BA. In contrast, the elution rate of the DNA of DAG-treated resistant cells increased with time and had reached the control levels by 38 h. However, when these cells were postincubated with BA for 38 h, the elution rate of DNA was much faster than that observed for the untreated resistant cells, indicating an accumulation of DNA single-strand breaks (SSB). The SSB accumulation caused by BA was associated with an inhibition of the activity of ligase II enzyme, which was stimulated when resistant cells were treated with DAG alone. The potentiating effect of BA on the resistant cells can thus be related to the inhibiting action of BA on the DNA-rejoining enzyme, ligase II. The lack of sensitization by BA of the DAG-treated parent cell line may be attributable to the absence of DNA-SSB formation, which is necessary for ligase II activation through the stimulation of poly(ADP-ribose) synthesis.
    • Bergamot natural products eradicate cancer stem cells (CSCs) by targeting mevalonate, Rho-GDI-signalling and mitochondrial metabolism.

      Fiorillo, Marco; Peiris-Pagès, Maria; Sanchez-Alvarez, Rosa; Bartella, L; Di Donna, L; Dolce, V; Sindona, G; Sotgia, Federica; Cappello, A; Lisanti, Michael P; et al. (2018-04-04)
      Here, we show that a 2:1 mixture of Brutieridin and Melitidin, termed "BMF", has a statin-like properties, which blocks the action of the rate-limiting enzyme for mevalonate biosynthesis, namely HMGR (3-hydroxy-3-methylglutaryl-CoA-reductase). Moreover, our results indicate that BMF functionally inhibits several key characteristics of CSCs. More specifically, BMF effectively i) reduced ALDH activity, ii) blocked mammosphere formation and iii) inhibited the activation of CSC-associated signalling pathways (STAT1/3, Notch and Wnt/beta-catenin) targeting Rho-GDI-signalling. In addition, BMF metabolically inhibited mitochondrial respiration (OXPHOS) and fatty acid oxidation (FAO). Importantly, BMF did not show the same toxic side-effects in normal fibroblasts that were observed with statins. Lastly, we show that high expression of the mRNA species encoding HMGR is associated with poor clinical outcome in breast cancer patients, providing a potential companion diagnostic for BMF-directed personalized therapy.
    • The best guess approach to phase I trial design.

      Rosa, Daniela D; Harris, John; Jayson, Gordon C; Cancer Research United Kingdom, Department of Medical Oncology, Christie Hospital NHS Trust, Manchester, United Kingdom. dornellesrosa@hotmail.com (2006-01-01)
    • The bi-specific CD3 x NCAM antibody: a model to preactivate T cells prior to tumour cell lysis.

      Jensen, M; Ernestus, K; Kemshead, John T; Klehr, M; Von Bergwelt-Baildon, M S; Schinköthe, T; Schultze, J L; Berthold, F; Department of Pediatric Oncology and Hematology, University of Cologne, Germany. jensen@uni-koeln.de (2003-11)
      To target the neural cell adhesion molecule (NCAM, CD56) on neuroblastoma by T cell-based immunotherapy we have generated a bi-specific CD3 x NCAM antibody (OE-1). This antibody can be used to redirect T cells to NCAM+ cells. Expectedly, the antibody binds specifically to NCAM+ neuroblastoma cells and CD3+ T cells. OE-1 induces T cell activation, expansion and effector function in peripheral blood mononuclear cell (PBMC)-derived CD4+ and CD8+ T cells. T cell activation was shown to depend on the presence of normal natural killer (NK) cells in the culture. Interestingly, while PBMC- derived T cells were activated by OE-1, NK cells were almost completely depleted, suggesting that T cells activated by OE-1 deleted the NK cells. Activated CD4+ and CD8+ T cells differentiate into a larger CCR7+ central memory and a smaller CCR7- effector memory cell population. Most importantly, preactivated T cells were highly cytotoxic for neuroblastoma cells. In eight of 11 experiments tumour-directed cytotoxicity was enhanced when NK cells were present during preactivation with OE-1. These data strongly support a bi-phasic therapeutic concept of primarily stimulating T cells with the bi-specific antibody in the presence of normal NCAM+ cells to induce T cell activation, migratory capacity and finally tumour cell lysis.
    • Biallelic mutations in p16(INK4a) confer resistance to Ras- and Ets-induced senescence in human diploid fibroblasts.

      Huot, Thomas J G; Rowe, Janice; Harland, Mark; Drayton, Sarah; Brookes, Sharon; Gooptu, Chandra; Purkis, Patricia; Fried, Mike; Bataille, Veronique; Hara, Eiji; et al. (2002-12)
      The INK4a/ARF tumor suppressor locus is implicated in the senescence-like growth arrest provoked by oncogenic Ras in primary cells. INK4a and ARF are distinct proteins encoded by transcripts in which a shared exon is decoded in alternative reading frames. Here we analyze dermal fibroblasts (designated Q34) from an individual carrying independent missense mutations in each copy of the common exon. Both mutations alter the amino acid sequence of INK4a and functionally impair the protein, although they do so to different degrees. Only one of the mutations affects the sequence of ARF, causing an apparently innocuous change near its carboxy terminus. Unlike normal human fibroblasts, Q34 cells are not permanently arrested by Ras or its downstream effectors Ets1 and Ets2. Moreover, ectopic Ras enables the cells to grow as anchorage-independent colonies, and in relatively young Q34 cells anchorage independence can be achieved without addition of telomerase or perturbation of the p53 pathway. Whereas ARF plays the principal role in Ras-induced arrest of mouse fibroblasts, our data imply that INK4a assumes this role in human fibroblasts.