• 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)
    • An MVA-based vaccine targeting the oncofetal antigen 5T4 in patients undergoing surgical resection of colorectal cancer liver metastases.

      Elkord, Eyad; Dangoor, Adam; Drury, Noel L; Harrop, Richard; Burt, Deborah J; Drijfhout, Jan W; Hamer, Caroline; Andrews, Danielle; Naylor, Stuart; Sherlock, David J; et al. (2009-03-17)
      We investigated the use of a therapeutic vaccine, TroVax in patients undergoing surgical resection of colorectal cancer liver metastases. Systemic immunity generated by vaccination before and after resection of metastases was measured in addition to assessing safety and analyzing the function and phenotype of tumor-associated lymphocytes. Twenty patients were scheduled to receive 2 TroVax vaccinations at 2-week intervals preoperatively and 2 postoperatively; if immune responses were detected, 2 further vaccinations were offered. Blood was taken at trial entry and 2 weeks after each vaccination; tumor biopsies were collected at surgery. 5T4-specific cellular responses were assessed by lymphocyte proliferation and enzyme-linked immunosorbent spot, with antibody responses by enzyme-linked immunosorbent assay. Immunohistochemistry characterized the phenotype of tumor-infiltrating lymphocytes. Seventeen of 19 colorectal cancer patients showed 5T4 expression in the liver metastases or surrounding stroma and 18 mounted a 5T4-specific cellular and/or humoral response. In patients who received at least 4 vaccinations and potentially curative surgery (n=15), those with above median 5T4-specific proliferative responses or T-cell infiltration into the resected tumor showed significantly longer survival compared with those with below median responses. Seven of 8 patients who had preexisting proliferative responses to 5T4 were longer-term survivors; these patients showed significantly higher proliferative responses after vaccination than those who subsequently died. These data suggest that the magnitude of 5T4 proliferative responses and the density of CD3 cells in colorectal cancer liver metastases are associated with longer survival. These observations warrant more studies to identify the precise underlying mechanisms.
    • Novel therapeutic strategies by regulatory T cells in allergy.

      Elkord, Eyad; Immunology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK. eelkord@picr.man.ac.uk (2008)
      Natural CD4+CD25+FoxP3+ regulatory T cells (Treg) actively suppress physiological and pathological responses, therefore playing a critical role in controlling peripheral tolerance to self antigens and maintaining immune homeostasis. In normal individuals, natural Treg and interleukin- 10-secreting Treg are able to suppress Th2 responses to allergens, whereas lower levels of Treg or defect in their functionality have been described as potential mechanisms for inducing allergic diseases. In animal models, adoptive transfer of CD4+CD25+Treg has been shown as a promising strategy for preventing or treating allergic disorders. Recent studies show that induction of Treg activity is associated with suppression of allergic responses in allergic patients treated with specific immunotherapy. Herein, I review the potential of Treg as exciting targets for developing new immunotherapeutic strategies for treating allergic diseases.
    • O(6)-methylguanine-DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib.

      Watson, Amanda J; Middleton, Mark R; McGown, Gail; Thorncroft, Mary R; Ranson, Malcolm R; Hersey, Peter; McArthur, Grant A; Davis, Ian D; Thomson, D; Beith, Jane; et al. (2009-04-21)
      We evaluated the pharmacodynamic effects of the O(6)-methylguanine-DNA methyltransferase (MGMT) inactivator lomeguatrib (LM) on patients with melanoma in two clinical trials. Patients received temozolomide (TMZ) for 5 days either alone or with LM for 5, 10 or 14 days. Peripheral blood mononuclear cells (PBMCs) were isolated before treatment and during cycle 1. Where available, tumour biopsies were obtained after the last drug dose in cycle 1. Samples were assayed for MGMT activity, total MGMT protein, and O(6)-methylguanine (O(6)-meG) and N7-methylguanine levels in DNA. MGMT was completely inactivated in PBMC from patients receiving LM, but detectable in those on TMZ alone. Tumours biopsied on the last day of treatment showed complete inactivation of MGMT but there was recovery of activity in tumours sampled later. Significantly more O(6)-meG was present in the PBMC DNA of LM/TMZ patients than those on TMZ alone. LM/TMZ leads to greater MGMT inactivation, and higher levels of O(6)-meG than TMZ alone. Early recovery of MGMT activity in tumours suggested that more protracted dosing with LM is required. Extended dosing of LM completely inactivated PBMC MGMT, and resulted in persistent levels of O(6)-meG in PBMC DNA during treatment.
    • O6-Methylguanine-DNA methyltransferase inactivation and chemotherapy.

      Verbeek, Barbara; Southgate, Thomas D; Gilham, David E; Margison, Geoffrey P; Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. (2008)
      INTRODUCTION: Alkylating agents are frequently used in the chemotherapy of many types of cancer. This group of drugs mediates cell death by damaging DNA and therefore, understandably, cellular DNA repair mechanisms can influence both their antitumour efficacy and their dose-limiting toxicities. SOURCES OF DATA: This review focuses on the mechanism of action of the DNA repair protein, O(6)-methylguanine-DNA methyltransferase (MGMT) and its exploitation in cancer therapy and reviews the current literature. AREAS OF AGREEMENT: MGMT can provide resistance to alkylating agents by DNA damage reversal. Inhibition of tumour MGMT by pseudosubstrates to overcome tumour resistance is under clinical evaluation. In addition, MGMT overexpression in haematopoietic stem cells has been shown in animal models to protect normal cells against the myelosuppressive effects of chemotherapy: this strategy has also entered clinical trials. AREAS OF CONTROVERSY: MGMT inhibitors enhance the myelotoxic effect of O(6)-alkylating drugs and therefore reduce the maximum-tolerated dose of these agents. Retroviral vectors used for chemoprotective gene therapy are associated with insertional mutagenesis and leukaemia development. GROWING POINTS: The results of ongoing preclinical and clinical research involving various aspects of MGMT modulation should provide new prospects for the treatment of glioma, melanoma and other cancer types. AREAS TIMELY FOR DEVELOPING RESEARCH: Tissue- and tumour-specific approaches to the modulation of MGMT together with other DNA repair functions and in combination with immuno- or radiotherapy are promising strategies to improve alkylating agent therapy.
    • Obesity and cancer: pathophysiological and biological mechanisms.

      Renehan, Andrew G; Roberts, Darren L; Dive, Caroline; Department of Surgery, School of Cancer and Imaging Sciences, University of Manchester, Christie Hospital NHS Foundation Trust, Wilmslow Road, Manchester, UK. arenehan@picr.man.ac.uk (2008-02)
      Excess body weight (overweight and obesity) is characterized by chronic hyperinsulinaemia and insulin resistance, and is implicated both in cancer risk and cancer mortality. The list of cancers at increased risk of development in an "obesogenic" environment include common adult cancers such as endometrium, post-menopausal breast, colon and kidney, but also less common malignancies such as leukaemia, multiple myeloma, and non-Hodgkin's lymphoma. The pathophysiological and biological mechanisms underpinning these associations are only starting to be understood. Insulin resistance is at the heart of many, but there are several other candidate systems including insulin-like growth factors, sex steroids, adipokines, obesity-related inflammatory markers, the nuclear factor kappa beta (NF-kappa B) system and oxidative stresses. With such as diversity of obesity-related cancers, it is unlikely that there is a "one system fits all" mechanism. While public health strategies to curb the spread of the obesity epidemic appear ineffective, there is a need to better understand the processes linking obesity and cancer as a pre-requisite to the development of new approaches to the prevention and treatment of obesity-related cancers.
    • Optimisation of circulating biomarkers of cell death for routine clinical use.

      Greystoke, Alastair; Cummings, Jeffrey; Ward, Timothy H; Simpson, Kathryn L; Renehan, Andrew G; Butt, Fouziah; Moore, David; Gietema, J; Blackhall, Fiona H; Ranson, Malcolm R; et al. (2008-05)
      BACKGROUND: M30 and M65 enzyme-linked immunosorbent assays detect circulating cytokeratin 18 fragments released during caspase-dependent or total cell death, respectively, and have potential as biomarkers in epithelial cancers. While these assays have been validated, their robustness for routine clinical use is unknown. PATIENTS AND METHODS: M30 and M65 were measured in matched serum and plasma samples from 31 lung cancer patients and 18 controls. RESULTS: Time allowable between sample acquisition and processing is critical for assays in clinical use. A 4-h delay in processing at room temperature increased M30 (P < 0.0001), an effect minimised by incubation on ice. M30 and M65 in serum were resistant to processing variations including delays. Serum and plasma measurements correlated well although M30 but not M65 was lower in serum (P < 0.0005). Less variation between duplicate assays was observed in serum. Prolonged storage (-80 degrees C) led to increased M30 (12%, 6 months; 34%, 1 year). Sample dilution in the supplied assay diluent proved non-linear, whereas dilution in donor serum or porcine plasma restored linearity up to a ratio of 1 : 6. CONCLUSION: We present recommendations that improve the reliability of these assays for clinical use and recommend serum as the preferred matrix with data more resistant to variations in collection.
    • Phase I trial of AEG35156 administered as a 7-day and 3-day continuous intravenous infusion in patients with advanced refractory cancer.

      Dean, Emma J; Jodrell, Duncan; Connolly, Kate; Danson, Sarah; Jolivet, Jacques; Durkin, J; Morris, Stephen; Jowle, Debra; Ward, Timothy H; Cummings, Jeffrey; et al. (2009-04-01)
      PURPOSE: To establish the maximum-tolerated dose and evaluate tolerability, pharmacokinetics, pharmacodynamic effects, and antitumor activity of AEG35156, a second-generation antisense to X-linked inhibitor of apoptosis (XIAP) protein, in patients with advanced refractory malignant tumors. PATIENTS AND METHODS: This was a first-in-man, open-label, phase I dose-escalation study. AEG35156 was administered by continuous intravenous infusion over 7 days (7DI) or 3 days (3DI) of a 21-day treatment cycle. Dose escalation started at 48 mg/m(2)/d and continued until consistent dose-limiting toxicity (DLT) was observed. RESULTS: Thirty-eight patients were entered in seven cohorts. Grade 3 to 4 adverse events were uncommon and were predominantly abnormal laboratory values: elevated ALT, thrombocytopenia, and lymphopenia. DLTs comprised elevated hepatic enzymes, hypophosphatemia, and thrombocytopenia. The maximum-tolerated doses were defined as 125 mg/m(2)/d for the 7DI regimen and < or = 213 mg/m(2)/d for the 3DI schedule. AEG35156 area under the plasma concentration curve and peak plasma concentration increased proportionally with dose. Suppression of XIAP mRNA levels was maximal at 72 hours (mean suppression, 21%), and this coincided with a dramatic decrease in circulating tumor cells in a patient with non-Hodgkin's lymphoma. Two further patients had unconfirmed partial responses. Circulating biomarkers of cell death and apoptosis altered in association with drug infusion and toxicity. CONCLUSION: In this first-in-man study, AEG35156 was well tolerated, with predictable toxicities, pharmacokinetic properties, and clinical evidence of antitumor activity in patients with refractory lymphoma, melanoma, and breast cancer. Phase I/II trials of AEG35156 chemotherapy combinations are ongoing in patients with pancreatic, breast, non-small-cell lung cancer, acute myeloid leukemia, lymphoma, and solid tumors for which docetaxel is indicated.
    • A phase II trial of lomeguatrib and temozolomide in metastatic colorectal cancer.

      Khan, O A; Ranson, Malcolm R; Michael, M; Olver, I; Levitt, N C; Mortimer, Peter; Watson, Amanda J; Margison, Geoffrey P; Midgley, R; Middleton, Mark R; et al. (2008-05-20)
      To evaluate the tumour response to lomeguatrib and temozolomide (TMZ) administered for 5 consecutive days every 4 weeks in patients with metastatic colorectal carcinoma. Patients with stage IV metastatic colorectal carcinoma received lomeguatrib (40 mg) and TMZ (50-200 mg m(-2)) orally for 5 consecutive days every 4 weeks. Response was determined every two cycles. Pharmacokinetics of lomeguatrib and TMZ as well as their pharmacodynamic effects in peripheral blood mononuclear cells (PBMC) were determined. Nineteen patients received 49 cycles of treatments. Despite consistent depletion of O(6)-methylguanine-DNA methyltransferase in PBMC, none of the patients responded to treatment. Three patients had stable disease, one for the duration of the study, and no fall in carcinoembryonic antigen was observed in any patient. Median time to progression was 50 days. The commonest adverse effects were gastrointestinal and haematological and these were comparable to those of TMZ when given alone. This combination of lomeguatrib and TMZ is not efficacious in metastatic colorectal cancer. If further studies are to be performed, emerging data suggest that higher daily doses of lomeguatrib and a dosing period beyond that of TMZ should be evaluated.
    • 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.
    • Preclinical evaluation of M30 and M65 ELISAs as biomarkers of drug induced tumor cell death and antitumor activity.

      Cummings, Jeffrey; Hodgkinson, Cassandra L; Odedra, Rajesh; Sini, Patrizia; Heaton, Simon P; Mundt, Kirsten E; Ward, Timothy H; Wilkinson, Robert W; Growcott, Jim; Hughes, Andrew; et al. (2008-03)
      M30 and M65 are ELISAs that detect different circulating forms of cytokeratin 18. Using the aurora kinase inhibitor AZD1152 and the SW620 human colon cancer xenograft, experiments were conducted to qualify preclinically both assays as serologic biomarkers of cell death. Using two different apoptotic markers, the kinetics of cell death induced by AZD1152 was first characterized in vitro in three different cell lines and shown to peak 5 to 7 days after drug addition. Treatment of non-tumor-bearing rats with AZD1152 (25 mg/kg) produced no alterations in circulating baseline values of M30 and M65 antigens. In treated, tumor-bearing animals, M30 detected a 2- to 3-fold (P < 0.05) increase in plasma antigen levels by day 5 compared with controls. This correlated to a 3-fold increase in the number of apoptotic cells detected on day 5 in SW620 xenografts using immunohistochemistry. By contrast, M65 did not detect a drug-induced increase in circulating antigen levels at day 5. However, M65 plasma levels correlated to changes in tumor growth in control animals (r(2) = 0.93; P < 0.01) and also followed the magnitude of the temporal effect of AZD1152 on tumor growth. An intermediate but active dose of AZD1152 (12.5 mg/kg) produced a less significant increase in M30 plasma levels at day 5. It was also confirmed that the plasma profiles of M30 and M65 mirrored closely those measured in whole tumor lysates. We conclude that M30 is a pharmacodynamic biomarker of AZD1152-induced apoptosis in the SW620 xenograft model, whereas M65 is a biomarker of therapeutic response.
    • 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.
    • 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.
    • Quantitative multiplexed quantum dot immunohistochemistry.

      Sweeney, Elizabeth; Ward, Timothy H; Gray, N; Womack, C; Jayson, Gordon C; Hughes, Andrew; Dive, Caroline; Byers, Richard J; Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, Wilmslow Road, Manchester, 420 4BX, UK. (2008-09-19)
      Quantum dots are photostable fluorescent semiconductor nanocrystals possessing wide excitation and bright narrow, symmetrical, emission spectra. These characteristics have engendered considerable interest in their application in multiplex immunohistochemistry for biomarker quantification and co-localisation in clinical samples. Robust quantitation allows biomarker validation, and there is growing need for multiplex staining due to limited quantity of clinical samples. Most reported multiplexed quantum dot staining used sequential methods that are laborious and impractical in a high-throughput setting. Problems associated with sequential multiplex staining have been investigated and a method developed using QDs conjugated to biotinylated primary antibodies, enabling simultaneous multiplex staining with three antibodies. CD34, Cytokeratin 18 and cleaved Caspase 3 were triplexed in tonsillar tissue using an 8h protocol, each localised to separate cellular compartments. This demonstrates utility of the method for biomarker measurement enabling rapid measurement of multiple co-localised biomarkers on single paraffin tissue sections, of importance for clinical trial studies.
    • Quantitative proteomics analysis demonstrates post-transcriptional regulation of embryonic stem cell differentiation to hematopoiesis.

      Williamson, Andrew J K; Smith, Duncan L; Blinco, David; Unwin, Richard D; Pearson, Stella; Wilson, Claire L; Miller, Crispin J; Lancashire, Lee J; Lacaud, Georges; Kouskoff, Valerie; et al. (2008-03)
      Embryonic stem (ES) cells can differentiate in vitro to produce the endothelial and hematopoietic precursor, the hemangioblasts, which are derived from the mesoderm germ layer. Differentiation of Bry(GFP/+) ES cell to hemangioblasts can be followed by the expression of the Bry(GFP/+) and Flk1 genes. Proteomic and transcriptomic changes during this differentiation process were analyzed to identify mechanisms for phenotypic change during early differentiation. Three populations of differentiating Bry(GFP) ES cells were obtained by flow cytometric sorting, GFP-Flk1- (epiblast), GFP+Flk1- (mesoderm), and GFP+Flk1+ (hemangioblast). Microarray analyses and relative quantification two-dimensional LCLC-MS/MS on nuclear extracts were performed. We identified and quantified 2389 proteins, 1057 of which were associated to their microarray probe set. These included a variety of low abundance transcription factors, e.g. UTF1, Sox2, Oct4, and E2F4, demonstrating a high level of proteomic penetrance. When paired comparisons of changes in the mRNA and protein expression levels were performed low levels of correlation were found. A strong correlation between isobaric tag-derived relative quantification and Western blot analysis was found for a number of nuclear proteins. Pathway and ontology analysis identified proteins known to be involved in the regulation of stem cell differentiation, and proteins with no described function in early ES cell development were also shown to change markedly at the proteome level only. ES cell development is regulated at the mRNA and protein level.
    • Quantum dots light up pathology.

      Tholouli, E; Sweeney, Elizabeth; Barrow, E; Clay, V; Hoyland, Judith A; Byers, Richard J; Department of Clinical Haematology, Manchester Royal Infirmary, Oxford Road, Manchester, UK. (2008-11)
      Quantum dots (QDs) are novel nanocrystal fluorophores with extremely high fluorescence efficiency and minimal photobleaching. They also possess a constant excitation wavelength together with sharp and symmetrical tunable emission spectra. These unique optical properties make them near-perfect fluorescent markers and there has recently been rapid development of their use for bioimaging. QDs can be conjugated to a wide range of biological targets, including proteins, antibodies, and nucleic acid probes, rendering them of particular interest to pathology researchers. They have been used in multiplex immunohistochemistry and in situ hybridization, which when combined with multispectral imaging, has enabled quantitative measurement of gene expression in situ. QDs have also been used for live in vivo animal imaging and are now being applied to an ever-increasing range of biological problems. These are detailed in this review, which also acts to outline the important advances that have been made in their range of applications. The relative novelty of QDs can present problems in their practical use and guidelines for their application are given.
    • 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.
    • Reciprocal relationship between expression of hypoxia inducible factor 1alpha (HIF-1alpha) and the pro-apoptotic protein bid in ex vivo colorectal cancer.

      Seenath, M M; Roberts, Darren L; Cawthorne, Christopher; Saunders, Mark P; Armstrong, G; O'Dwyer, Sarah T; Stratford, Ian J; Dive, Caroline; Renehan, Andrew G; Clinical and Experimental Pharmacology, Paterson Institute of Cancer Research, Manchester, UK. (2008-08-05)
      Hypoxia inducible factor 1 (HIF-1) represses the transcription of pro-apoptotic bid in colorectal cancer cells in vitro. To assess the clinical relevance of this observation, HIF-1alpha and Bid were assessed in serial sections of 39 human colorectal adenocarcinomas by immunohistochemistry. In high HIF-1alpha nuclear-positive cell subpopulations, there was a significant reduction in Bid expression (ANOVA, P=0.04). Given the role of Bid in drug-induced apoptosis, these data add impetus to strategies targeting HIF-1 for therapeutic gain.
    • Reciprocal relationship between O6-methylguanine-DNA methyltransferase P140K expression level and chemoprotection of hematopoietic stem cells.

      Milsom, Michael D; Jerabek-Willemsen, Moran; Harris, Chad E; Schambach, Axel; Broun, Emily; Bailey, J; Jansen, Michael; Schleimer, David; Nattamai, Kalpana; Wilhelm, Jamie; et al. (2008-08-01)
      Retroviral-mediated delivery of the P140K mutant O(6)-methylguanine-DNA methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSC) has been proposed as a means to protect against dose-limiting myelosuppressive toxicity ensuing from chemotherapy combining O(6)-alkylating agents (e.g., temozolomide) with pseudosubstrate inhibitors (such as O(6)-benzylguanine) of endogenous MGMT. Because detoxification of O(6)-alkylguanine adducts by MGMT is stoichiometric, it has been suggested that higher levels of MGMT will afford better protection to gene-modified HSC. However, accomplishing this goal would potentially be in conflict with current efforts in the gene therapy field, which aim to incorporate weaker enhancer elements to avoid insertional mutagenesis. Using a panel of self-inactivating gamma-retroviral vectors that express a range of MGMT(P140K) activity, we show that MGMT(P140K) expression by weaker cellular promoter/enhancers is sufficient for in vivo protection/selection following treatment with O(6)-benzylguanine/temozolomide. Conversely, the highest level of MGMT(P140K) activity did not promote efficient in vivo protection despite mediating detoxification of O(6)-alkylguanine adducts. Moreover, very high expression of MGMT(P140K) was associated with a competitive repopulation defect in HSC. Mechanistically, we show a defect in cellular proliferation associated with elevated expression of MGMT(P140K), but not wild-type MGMT. This proliferation defect correlated with increased localization of MGMT(P140K) to the nucleus/chromatin. These data show that very high expression of MGMT(P140K) has a deleterious effect on cellular proliferation, engraftment, and chemoprotection. These studies have direct translational relevance to ongoing clinical gene therapy studies using MGMT(P140K), whereas the novel mechanistic findings are relevant to the basic understanding of DNA repair by MGMT.
    • Recommendations for cervical cancer prevention in Asia Pacific.

      Garland, Suzanne M; Cuzick, Jack; Domingo, Efren J; Goldie, Sue J; Kim, Young-Tak; Konno, Ryo; Parkin, D Maxwell; Qiao, You-Lin; Sankaranarayanan, Rengaswamy; Stern, Peter L; et al. (2008-08-19)
      Asia Oceania includes countries from both the Asia Pacific region and Australasia, which cover very diverse geographical areas and populations as well as bearing 52% of the cervical cancer burden in the world. Human papillomavirus (HPV) genotype distribution in women with normal cytology varies between countries in this region, as well as with the distribution typically observed in worldwide estimates or in Western countries. HPV-16 remains the predominant oncogenic type for high-grade cervical dysplasia and cervical cancer across the region, and HPV-18 is generally among the five most common types. HPV-58 is commonly found in cervical cancer as well as in women with normal cytology, and HPV-31, 33 and 35 are relatively less frequent in these regions compared to the West. While screening programmes have been proposed and implemented in several populations, successful programmes are rather limited and the majority of countries still have no or minimal screening services. Prophylactic HPV vaccination will only be feasible when it becomes affordable, thus the current priority and the short-term goal for cervical cancer control is to identify feasible and effective screening measures, and to find the most effective way to combine vaccination with sustainable screening programmes. This Regional Report has carefully described the disease burden of HPV and cervical cancer and the current situations in cervical cancer prevention for many countries in the Asia Oceania region. These data identify the many challenges and opportunities to be considered for policy decisions for cervical cancer control. Furthermore, this report presents the results of advanced decision analytic models calibrated to countries in the region that provide early insight into what strategies are most promising and those likely to be cost-effective and affordable. It thus provides a synthesis of the available evidence-based scientific information, in the context of a significant and systematic international review, that is likely to be useful to governments and public health providers.