• Predicting the myelotoxicity of chemotherapy: the use of pretreatment O6-methylguanine-DNA methyltransferase determination in peripheral blood mononuclear cells.

      Sabharwal, A; Waters, R; Danson, Sarah; Clamp, Andrew R; Lorigan, Paul C; Thatcher, Nick; Margison, Geoffrey P; Middleton, Mark R; Department of Medical Oncology, University of Oxford, Oxford, UK. (2011-12-21)
      To assess the value of pretreatment O-methylguanine-DNA methyltransferase (MGMT) expression in peripheral blood mononuclear cells (PBMCs) in predicting haematological toxicity with O-alkylating agent chemotherapy, we explored this relationship retrospectively in melanoma patients. Ninety-three patients treated with temozolomide or dacarbazine in four clinical trials were assessed, and a model of the interaction between MGMT expression and haematological toxicity was constructed. Nadir white-cell and platelet counts were related to, and hence could be predicted from, pretreatment MGMT. Leucopenia and thrombocytopenia were more prevalent amongst patients with low pretreatment MGMT, according to the highest grades of toxicity experienced and/or the dose intensity patients could sustain. Addition of interferon to chemotherapy or compression of the temozolomide schedule increased the toxicity. The model also predicts significant myelotoxicity where PBMC MGMT is inactivated, consistent with the experience in the clinic with lomeguatrib and O-benzylguanine. Determination of MGMT in PBMC can identify patients at greatest risk of toxicity or who are suitable for dose intensification.
    • The differential activities of Runx1 promoters define milestones during embryonic hematopoiesis.

      Sroczynska, Patrycja; Lancrin, Christophe; Kouskoff, Valerie; Lacaud, Georges; Cancer Research UK Stem Cell Biology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom. (2009-12-17)
      The transcription factor RUNX1/AML1 is a master regulator of hematopoietic development. Its spatiotemporal expression is tightly regulated during embryonic development and is under the control of 2 alternative promoters, distal and proximal. Despite the functional significance of Runx1, the relative and specific activities of these 2 promoters remain largely uncharacterized. To investigate these activities, we introduced 2 reporter genes under the control of the proximal and distal promoters in embryonic stem cell and transgenic mouse lines. Our study reveals that both in vitro and in vivo the proximal Runx1 isoform marks a hemogenic endothelium cell population, whereas the subsequent expression of distal Runx1 defines fully committed definitive hematopoietic progenitors. Interestingly, hematopoietic commitment in distal Runx1 knockout embryos appears normal. Altogether, our data demonstrate that the differential activities of the 2 Runx1 promoters define milestones of hematopoietic development and suggest that the proximal isoform plays a critical role in the generation of hematopoietic cells from hemogenic endothelium. Identification and access to the discrete stages of hematopoietic development defined by the activities of the Runx1 promoters will provide the opportunity to further explore the cellular and molecular mechanisms of hematopoietic development.
    • Detection of BRAF mutations in the tumour and serum of patients enrolled in the AZD6244 (ARRY-142886) advanced melanoma phase II study.

      Board, Ruth E; Ellison, G; Orr, M C M; Kemsley, K R; McWalter, G; Blockley, L Y; Dearden, S P; Morris, C; Ranson, Malcolm R; Cantarini, M V; et al. (2009-11-17)
      BACKGROUND: This study investigated the potential clinical utility of circulating free DNA (cfDNA) as a source of BRAF mutation detection in patients enrolled into a phase II study of AZD6244, a specific MEK1/2 inhibitor, in patients with advanced melanoma. METHODS: BRAF mutations were detected using Amplification Refractory Mutation System allele-specific PCR. BRAF mutation status was assessed in serum-derived cfDNA from 126 patients enrolled into the study and from 94 matched tumour samples. RESULTS: Of 94 tumour samples, 45 (47.9%) were found to be BRAF mutation positive (BRAF+). Serum-derived cfDNA was BRAF+ in 33 of 126 (26.2%) samples, including in five samples for which tumour data were unavailable. Of BRAF+ tumours, 25 of 45 (55.6%) were BRAF+ in cfDNA. In three cases in which the tumour was negative, cfDNA was BRAF+. Progression-free survival (PFS) of patients with BRAF+ tumour and cfDNA was not significantly different compared with tumour BRAF+ but cfDNA BRAF-negative patients, indicating that cfDNA BRAF detection is not associated with poorer prognosis on PFS in stage III/IV advanced melanoma. CONCLUSIONS: These data demonstrate the feasibility of BRAF mutation detection in cfDNA of patients with advanced melanoma. Future studies should aim to incorporate BRAF mutation testing in cfDNA to further validate this biomarker for patient selection.
    • Reactivating HIF prolyl hydroxylases under hypoxia results in metabolic catastrophe and cell death.

      Tennant, D A; Frezza, C; MacKenzie, E D; Nguyen, Q D; Zheng, L; Selak, M A; Roberts, Darren L; Dive, Caroline; Watson, D G; Aboagye, E O; et al. (2009-11-12)
      Cells exposed to low-oxygen conditions (hypoxia) alter their metabolism to survive. This response, although vital during development and high-altitude survival, is now known to be a major factor in the selection of cells with a transformed metabolic phenotype during tumorigenesis. It is thought that hypoxia-selected cells have increased invasive capacity and resistance to both chemo- and radiotherapies, and therefore represent an attractive target for antitumor therapy. Hypoxia inducible factors (HIFs) are responsible for the majority of gene expression changes under hypoxia, and are themselves controlled by the oxygen-sensing HIF prolyl hydroxylases (PHDs). It was previously shown that mutations in succinate dehydrogenase lead to the inactivation PHDs under normoxic conditions, which can be overcome by treatment with alpha-ketoglutarate derivatives. Given that solid tumors contain large regions of hypoxia, the reactivation of PHDs in these conditions could induce metabolic catastrophe and therefore prove an effective antitumor therapy. In this report we demonstrate that derivatized alpha-ketoglutarate can be used as a strategy for maintaining PHD activity under hypoxia. By increasing intracellular alpha-ketoglutarate and activating PHDs we trigger PHD-dependent reversal of HIF1 activation, and PHD-dependent hypoxic cell death. We also show that derivatized alpha-ketoglutarate can permeate multiple layers of cells, reducing HIF1alpha levels and its target genes in vivo.
    • A small molecule inhibitor of XIAP induces apoptosis and synergises with vinorelbine and cisplatin in NSCLC.

      Dean, Emma J; Ward, Timothy H; Pinilla, C; Houghten, R; Welsh, K; Makin, Guy W J; Ranson, Malcolm R; Dive, Caroline; Department of Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, England, UK [2] Derek Crowther Unit, Christie Hospital NHS Foundation Trust, Wilmslow Road, Manchester M20 4BX, England, UK. (2009-11-10)
      Background:Evasion of apoptosis contributes to the pathogenesis of solid tumours including non-small cell lung cancer (NSCLC). Malignant cells resist apoptosis through over-expression of inhibitor of apoptosis proteins (IAPs), such as X-linked IAP (XIAP).Methods:A phenylurea-based small molecule inhibitor of XIAP, XIAP antagonist compound (XAC) 1396-11, was investigated preclincally to determine its ability to sensitise to clinically relevant cytotoxics, potentially allowing dose reduction while maintaining therapeutic efficacy.Results:XIAP protein expression was detected in six NSCLC cell lines examined. The cytotoxicity of XAC 1396-11 against cultured NSCLC cell lines in vitro was concentration- and time-dependent in both short-term and clonogenic assays. XAC 1396-11-induced apoptosis was confirmed by PARP cleavage and characteristic nuclear morphology. XAC 1396-11 synergised with vinorelbine+/-cisplatin in H460 and A549 NSCLC cells. The mechanism of synergy was enhanced apoptosis, shown by increased cleavage of caspase-3 and PARP and by the reversal of synergy by a pan-caspase inhibitor. Synergy between XAC 1396-11 and vinorelbine was augmented by optimising drug scheduling with superior effects when XAC 1396-11 was administered before vinorelbine.Conclusion:These preclinical data suggest that XIAP inhibition in combination with vinorelbine holds potential as a therapeutic strategy in NSCLC.British Journal of Cancer advance online publication, 10 November 2009; doi:10.1038/sj.bjc.6605418 www.bjcancer.com.
    • Mutant CEBPA: priming stem cells for myeloid leukemogenesis.

      Somervaille, Tim C P; Cleary, M L; Cancer Research UK Leukaemia Biology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, M20 4BX, UK. (2009-11-06)
    • Contribution of HIF-1 and drug penetrance to oxaliplatin resistance in hypoxic colorectal cancer cells.

      Roberts, Darren L; Williams, Kaye J; Cowen, Rachel L; Barathova, M; Eustace, A J; Brittain-Dissont, S; Tilby, Michael J; Pearson, D Graham; Ottley, Christopher J; Stratford, Ian J; et al. (2009-10-20)
      BACKGROUND: Hypoxia is as an indicator of poor treatment outcome. Consistently, hypoxic HCT116 colorectal cancer cells are resistant to oxaliplatin, although the mechanistic basis is unclear. This study sought to investigate the relative contribution of HIF-1 (hypoxia-inducible factor-1)-mediated gene expression and drug penetrance to oxaliplatin resistance using three-dimensional spheroids. METHODS: Hypoxia-inducible factor-1alpha function was suppressed by the stable expression of a dominant-negative form in HCT116 cells (DN). Cells were drug exposed as monolayer or multicellular spheroid cultures. Cells residing at differing oxygenation status were isolated from Hoechst 33342-treated spheroids using flow cytometry. Sub-populations were subjected to clonogenic survival assays and to Inductively-Coupled Plasma Mass Spectroscopy to determine oxaliplatin uptake. RESULTS: In spheroids, a sensitivity gradient (hypoxic
    • The transcription factor Atf1 binds and activates the APC/C ubiquitin ligase in fission yeast.

      Ors, Aslihan; Grimaldi, Margaret; Kimata, Yuu; Wilkinson, Caroline R M; Jones, Nic; Yamano, Hiroyuki; Cell Cycle Control Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey RH8 0TL, United Kingdom. (2009-09-04)
      Fission yeast Atf1 is a member of the ATF/CREB basic leucine zipper (bZIP) family of transcription factors with strong homology to mammalian ATF2. Atf1 regulates transcription in response to stress stimuli and also plays a role in controlling heterochromatin formation and recombination. However, its DNA binding independent role is poorly studied. Here, we report that Atf1 has a distinct role in regulating the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. We have identified atf1(+) as a dose-dependent suppressor of apc5-1, a mutation causing mitotic arrest. Remarkably, the suppression is not dependent upon the bZIP domain and is therefore independent of the ability of Atf1 to bind DNA. Interestingly, Atf1 physically binds the APC/C in vivo. Furthermore, we show that addition of purified Atf1 proteins into a cell-free system stimulates ubiquitylation of cyclin B and securin by the APC/C. These results reveal a novel role for Atf1 in cell cycle control through protein-protein interaction.
    • Frequency of human T regulatory cells in peripheral blood is significantly reduced by cryopreservation.

      Elkord, Eyad; Clinical Immunotherapy Laboratory, Department of Medical Oncology, University of Manchester, Wilmslow Road, Manchester M204BX, UK. eelkord@picr.man.ac.uk (2009-08-15)
      Cryopreservation of peripheral blood mononuclear cells (PBMC) is essential for many clinical and research assays. Some studies reported consistent changes in PBMC phenotype following cryopreservation. We hypothesized that PBMC freezing may have a negative impact on estimation of the frequency of T regulatory cell (Treg). Treg levels were measured in 6 fresh PBMC samples isolated from 6 healthy donors and these levels were re-measured after freezing for three weeks. Herein, we report a significant reduction in Treg frequency in all samples following cryopreservation.
    • Evaluation of circulating tumor cells and serological cell death biomarkers in small cell lung cancer patients undergoing chemotherapy.

      Hou, Jian-Mei; Greystoke, Alastair; Lancashire, Lee J; Cummings, Jeffrey; Ward, Timothy H; Board, Ruth E; Amir, Eitan; Hughes, Sarah; Krebs, Matthew G; Hughes, Andrew; et al. (2009-08)
      Serological cell death biomarkers and circulating tumor cells (CTCs) have potential uses as tools for pharmacodynamic blood-based assays and their subsequent application to early clinical trials. In this study, we evaluated both the expression and clinical significance of CTCs and serological cell death biomarkers in patients with small cell lung cancer. Blood samples from 88 patients were assayed using enzyme-linked immunosorbent assays for various cytokeratin 18 products (eg, M65, cell death, M30, and apoptosis) as well as nucleosomal DNA. CTCs (per 7.5 ml of blood) were quantified using Veridex CellSearch technology. Before therapeutic treatment, cell death biomarkers were elevated in patients compared with controls. CTCs were detected in 86% of patients; additionally, CD56 was detectable in CTCs, confirming their neoplastic origin. M30 levels correlated with the percentage of apoptotic CTCs. M30, M65, lactate dehydrogenase, and CTC number were prognostic for patient survival as determined by univariate analysis. Using multivariate analysis, both lactate dehydrogenase and M65 levels remained significant. CTC number fell following chemotherapy, whereas levels of serological cell death biomarkers peaked at 48 hours and fell by day 22, mirroring the tumor response. A 48-hour rise in nucleosomal DNA and M30 levels was associated with early response and severe toxicity, respectively. Our results provide a rationale to include the use of serological biomarkers and CTCs in early clinical trials of new agents for small cell lung cancer.
    • Early chromatin unfolding by RUNX1: a molecular explanation for differential requirements during specification versus maintenance of the hematopoietic gene expression program.

      Hoogenkamp, Maarten; Lichtinger, Monika; Krysinska, Hanna; Lancrin, Christophe; Clarke, Deborah; Williamson, Andrew J K; Mazzarella, Luca; Ingram, Richard; Jorgensen, Helle; Fisher, Amanda; et al. (2009-07-09)
      At the cellular level, development progresses through successive regulatory states, each characterized by their specific gene expression profile. However, the molecular mechanisms regulating first the priming and then maintenance of gene expression within one developmental pathway are essentially unknown. The hematopoietic system represents a powerful experimental model to address these questions and here we have focused on a regulatory circuit playing a central role in myelopoiesis: the transcription factor PU.1, its target gene colony-stimulating-factor 1 receptor (Csf1r), and key upstream regulators such as RUNX1. We find that during ontogeny, chromatin unfolding precedes the establishment of active histone marks and the formation of stable transcription factor complexes at the Pu.1 locus and we show that chromatin remodeling is mediated by the transient binding of RUNX1 to Pu.1 cis-elements. By contrast, chromatin reorganization of Csf1r requires prior expression of PU.1 together with RUNX1 binding. Once the full hematopoietic program is established, stable transcription factor complexes and active chromatin can be maintained without RUNX1. Our experiments therefore demonstrate how individual transcription factors function in a differentiation stage-specific manner to differentially affect the initiation versus maintenance of a developmental program.
    • Preclinical efficacy of the bioreductive alkylating agent RH1 against paediatric tumours.

      Hussein, Deema; Holt, Sarah V; Brookes, K E; Klymenko, T; Adamski, J K; Hogg, Alison; Estlin, E J; Ward, Timothy H; Dive, Caroline; Makin, Guy W J; et al. (2009-07-07)
      BACKGROUND: Despite substantial improvements in childhood cancer survival, drug resistance remains problematic for several paediatric tumour types. The urgent need to access novel agents to treat drug-resistant disease should be expedited by pre-clinical evaluation of paediatric tumour models during the early stages of drug development in adult cancer patients. METHODS/RESULTS: The novel cytotoxic RH1 (2,5-diaziridinyl-3-[hydroxymethyl]-6-methyl-1,4-benzoquinone) is activated by the obligate two-electron reductase DT-diaphorase (DTD, widely expressed in adult tumour cells) to a potent DNA interstrand cross-linker. In acute viability assays against neuroblastoma, osteosarcoma, and Ewing's sarcoma cell lines RH1 IC(50) values ranged from 1-200 nM and drug potency correlated both with DTD levels and drug-induced apoptosis. However, synergy between RH1 and cisplatin or doxorubicin was only seen in low DTD expressing cell lines. In clonogenic assays RH1 IC(50) values ranged from 1.5-7.5 nM and drug potency did not correlate with DTD level. In A673 Ewing's sarcoma and 791T osteosarcoma tumour xenografts in mice RH1 induced apoptosis 24 h after a single bolus injection (0.4 mg/kg) and daily dosing for 5 days delayed tumour growth relative to control. CONCLUSION: The demonstration of RH1 efficacy against paediatric tumour cell lines, which was performed concurrently with the adult Phase 1 Trial, suggests that this agent may have clinical usefulness in childhood cancer.
    • To determine the cytotoxicity of chlorambucil and one of its nitro-derivatives, conjugated to prasterone and pregnenolone, towards eight human cancer cell-lines.

      Shervington, Leroy A; Smith, Nigel K; Norman, Emma; Ward, Timothy H; Phillips, Roger M; Shervington, Amal; School of Pharmacy and Pharmaceutical Sciences, University of Central Lancashire, Preston, UK. lashervington@uclan.ac.uk (2009-07)
      Four ester prodrugs derived from the bifunctional alkylating agent chlorambucil, and one of its nitro-derivatives, 3-nitrochlorambucil conjugated to prasterone and pregnenolone, were synthesized and tested for their cytotoxic activity against eight human cell lines, using the standard MTT assay. A comparison between the esters and the controls, namely chlorambucil and 3-nitrochlorambucil would suggest that all four esters possess to varying degrees, specificity towards the breast adenocarcinoma cell line (MDA-mb468) than the other seven cells' lines tested. The overall findings are encouraging since it infers that these lipophilic esters not only have the ability to traverse specific cell membranes but also exhibit cytotoxicity towards most of the cell lines tested.
    • Methods comparison for high-resolution transcriptional analysis of archival material on Affymetrix Plus 2.0 and Exon 1.0 microarrays.

      Linton, Kim M; Hey, Yvonne; Dibben, Sian; Miller, Crispin J; Freemont, Anthony J; Radford, John A; Pepper, Stuart D; Cancer Research UK Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester, UK. kim.linton@christie.nhs.uk (2009-07)
      Microarray gene expression profiling of formalin-fixed paraffin-embedded (FFPE) tissues is a new and evolving technique. This report compares transcript detection rates on Affymetrix U133 Plus 2.0 and Human Exon 1.0 ST GeneChips across several RNA extraction and target labeling protocols, using routinely collected archival FFPE samples. All RNA extraction protocols tested (Ambion-Optimum, Ambion-RecoverAll, and Qiagen-RNeasy FFPE) provided extracts suitable for microarray hybridization. Compared with Affymetrix One-Cycle labeled extracts, NuGEN system protocols utilizing oligo(dT) and random hexamer primers, and cDNA target preparations instead of cRNA, achieved percent present rates up to 55% on Plus 2.0 arrays. Based on two paired-sample analyses, at 90% specificity this equalled an average 30 percentage-point increase (from 50% to 80%) in FFPE transcript sensitivity relative to fresh frozen tissues, which we have assumed to have 100% sensitivity and specificity. The high content of Exon arrays, with multiple probe sets per exon, improved FFPE sensitivity to 92% at 96% specificity, corresponding to an absolute increase of ~600 genes over Plus 2.0 arrays. While larger series are needed to confirm high correspondence between fresh-frozen and FFPE expression patterns, these data suggest that both Plus 2.0 and Exon arrays are suitable platforms for FFPE microarray expression analyses.
    • Flipping of alkylated DNA damage bridges base and nucleotide excision repair.

      Tubbs, Julie L; Latypov, Vitaly F; Kanugula, Sreenivas; Butt, Amna; Melikishvili, Manana; Kraehenbuehl, Rolf; Fleck, Oliver; Marriott, Andrew S; Watson, Amanda J; Verbeek, Barbara; et al. (2009-06-11)
      Alkyltransferase-like proteins (ATLs) share functional motifs with the cancer chemotherapy target O(6)-alkylguanine-DNA alkyltransferase (AGT) and paradoxically protect cells from the biological effects of DNA alkylation damage, despite lacking the reactive cysteine and alkyltransferase activity of AGT. Here we determine Schizosaccharomyces pombe ATL structures without and with damaged DNA containing the endogenous lesion O(6)-methylguanine or cigarette-smoke-derived O(6)-4-(3-pyridyl)-4-oxobutylguanine. These results reveal non-enzymatic DNA nucleotide flipping plus increased DNA distortion and binding pocket size compared to AGT. Our analysis of lesion-binding site conservation identifies new ATLs in sea anemone and ancestral archaea, indicating that ATL interactions are ancestral to present-day repair pathways in all domains of life. Genetic connections to mammalian XPG (also known as ERCC5) and ERCC1 in S. pombe homologues Rad13 and Swi10 and biochemical interactions with Escherichia coli UvrA and UvrC combined with structural results reveal that ATLs sculpt alkylated DNA to create a genetic and structural intersection of base damage processing with nucleotide excision repair.
    • Expression of the leukemia oncogene Lmo2 is controlled by an array of tissue-specific elements dispersed over 100 kb and bound by Tal1/Lmo2, Ets, and Gata factors.

      Landry, Josette-Renee; Bonadies, Nicolas; Kinston, Sarah; Knezevic, Kathy; Wilson, Nicola K; Oram, S Helen; Janes, Mary E; Piltz, Sandie; Hammett, Michelle; Carter, Jacinta; et al. (2009-06-04)
      The Lmo2 gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Ectopic LMO2 expression causes leukemia in T-cell acute lymphoblastic leukemia (T-ALL) patients and severe combined immunodeficiency patients undergoing retroviral gene therapy. Tightly controlled Lmo2 expression is therefore essential, yet no comprehensive analysis of Lmo2 regulation has been published so far. By comparative genomics, we identified 17 highly conserved noncoding elements, 9 of which revealed specific acetylation marks in chromatin-immunoprecipitation and microarray (ChIP-chip) assays performed across 250 kb of the Lmo2 locus in 11 cell types covering different stages of hematopoietic differentiation. All candidate regulatory regions were tested in transgenic mice. An extended LMO2 proximal promoter fragment displayed strong endothelial activity, while the distal promoter showed weak forebrain activity. Eight of the 15 distal candidate elements functioned as enhancers, which together recapitulated the full expression pattern of Lmo2, directing expression to endothelium, hematopoietic cells, tail, and forebrain. Interestingly, distinct combinations of specific distal regulatory elements were required to extend endothelial activity of the LMO2 promoter to yolk sac or fetal liver hematopoietic cells. Finally, Sfpi1/Pu.1, Fli1, Gata2, Tal1/Scl, and Lmo2 were shown to bind to and transactivate Lmo2 hematopoietic enhancers, thus identifying key upstream regulators and positioning Lmo2 within hematopoietic regulatory networks.
    • The S. pombe mitotic regulator Cut12 promotes spindle pole body activation and integration into the nuclear envelope.

      Tallada, Victor A; Tanaka, Kenji; Yanagida, Mitsuhiro; Hagan, Iain M; Cancer Research UK Cell Division Group, Paterson Institute for Cancer Research, University of Manchester, Manchester M204BX, England, UK. (2009-06-01)
      The fission yeast spindle pole body (SPB) comprises a cytoplasmic structure that is separated from an ill-defined nuclear component by the nuclear envelope. Upon mitotic commitment, the nuclear envelope separating these domains disperses as the two SPBs integrate into a hole that forms in the nuclear envelope. The SPB component Cut12 is linked to cell cycle control, as dominant cut12.s11 mutations suppress the mitotic commitment defect of cdc25.22 cells and elevated Cdc25 levels suppress the monopolar spindle phenotype of cut12.1 loss of function mutations. We show that the cut12.1 monopolar phenotype arises from a failure to activate and integrate the new SPB into the nuclear envelope. The activation of the old SPB was frequently delayed, and its integration into the nuclear envelope was defective, resulting in leakage of the nucleoplasm into the cytoplasm through large gaps in the nuclear envelope. We propose that these activation/integration defects arise from a local deficiency in mitosis-promoting factor activation at the new SPB.
    • The histone acetyl transferase activity of monocytic leukemia zinc finger is critical for the proliferation of hematopoietic precursors.

      Perez-Campo, Flor-Maria; Borrow, Julian; Kouskoff, Valerie; Lacaud, Georges; Paterson Institute for Cancer Research, University of Manchester, USA. (2009-05-14)
      The monocytic leukemia zinc finger (MOZ) gene encodes a large multidomain protein that contains, besides other domains, 2 coactivation domains for the transcription factor Runx1/acute myeloid leukemia 1 and a histone acetyl transferase (HAT) catalytic domain. Recent studies have demonstrated the critical requirement for the complete MOZ protein in hematopoietic stem cell development and maintenance. However, the specific function of the HAT activity of MOZ remains unknown, as it has been shown that MOZ HAT activity is not required either for its role as Runx1 coactivator or for the leukemic transformation induced by MOZ transcriptional intermediary factor 2 (TIF2). To assess the specific requirement for this HAT activity during hematopoietic development, we have generated embryonic stem cells and mouse lines carrying a point mutation that renders the protein catalytically inactive. We report in this study that mice exclusively lacking the HAT activity of MOZ exhibit significant defects in the number of hematopoietic stem cells and hematopoietic committed precursors as well as a defect in B-cell development. Furthermore, we demonstrate that the failure to maintain a normal number of hematopoietic precursors is caused by the inability of HAT(-/-) cells to expand. These results indicate a specific role of MOZ-driven acetylation in controlling a desirable balance between proliferation and differentiation during hematopoiesis.
    • GSTM1 copy number and lung cancer risk.

      Crosbie, Philip A J; Barber, Philip V; Harrison, Kathryn L; Gibbs, Alan R; Agius, Raymond M; Margison, Geoffrey P; Povey, Andrew C; Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom. (2009-05-12)
      The GSTM1 null genotype is associated with a small increased lung cancer risk when compared to controls with at least one copy of the GSTM1 gene. As two copies of the GSTM1 gene might provide more protection than a single copy, we have determined GSTM1 copy number in a lung cancer case-control study. Cases with incident lung cancer were identified through a Bronchoscopy Unit and two separate hospital based control groups with non-malignant disease were selected with one from the same Bronchoscopy Unit and the other from a chest clinic at the same hospital. Subjects with at least one GSTM1 copy had a decreased lung cancer risk whatever the control group: the odds ratio (95% CI), after adjustment for age, gender and smoking duration, was 0.64 (0.41-0.98) and 0.54 (0.32-0.91) with bronchoscopy and chest clinic controls, respectively. Lung cancer risk varied with GSTM1 copy number with chest clinic controls only: the OR was 0.56 (0.32-0.97) for one copy of the GSTM1 gene and with two copies 0.43 (0.15-1.22), a trend that was significant (p=0.02): with bronchoscopy controls the trend was not significant (p=0.07). Results then confirm that the presence of GSTM1 provides protection against the risk of lung cancer. In addition there is equivocal evidence that this protection varies with the number of gene copies.
    • Quantitative mass spectrometry-based techniques for clinical use: biomarker identification and quantification.

      Simpson, Kathryn L; Whetton, Anthony D; Dive, Caroline; Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, United Kingdom. KSimpson@PICR.man.ac.uk (2009-05-01)
      The potential for development of personalised medicine through the characterisation of novel biomarkers is an exciting prospect for improved patient care. Recent advances in mass spectrometric (MS) techniques, liquid phase analyte separation and bioinformatic tools for high throughput now mean that this goal may soon become a reality. However, there are challenges to be overcome for the identification and validation of robust biomarkers. Bio-fluids such as plasma and serum are a rich source of protein, many of which may reflect disease status, and due to the ease of sampling and handling, novel blood borne biomarkers are very much sought after. MS-based methods for high throughput protein identification and quantification are now available such that the issues arising from the huge dynamic range of proteins present in plasma may be overcome, allowing deep mining of the blood proteome to reveal novel biomarker signatures for clinical use. In addition, the development of sensitive MS-based methods for biomarker validation may bypass the bottleneck created by the need for generation and usage of reliable antibodies prior to large scale screening. In this review, we discuss the MS-based methods that are available for clinical proteomic analysis and highlight the progress made and future challenges faced in this cutting edge area of research.