• Fission yeast MAP kinase Sty1 is recruited to stress-induced genes.

      Reiter, Wolfgang; Watt, Stephen; Dawson, Keren; Lawrence, Clare L; Bähler, Jürg; Jones, Nic; Wilkinson, Caroline R M; Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, UK. (2008-04-11)
      The stress-induced expression of many fission yeast genes is dependent upon the Sty1 mitogen-activated protein kinase (MAPK) and Atf1 transcription factor. Atf1 is phosphorylated by Sty1 yet this phosphorylation is not required for stress-induced gene expression, suggesting another mechanism exists whereby Sty1 activates transcription. Here we show that Sty1 associates with Atf1-dependent genes and is recruited to both their promoters and coding regions. This occurs in response to various stress conditions coincident with the kinetics of the activation of Sty1. Association with promoters is not a consequence of increased nuclear accumulation of Sty1 nor does it require the phosphorylation of Atf1. However, recruitment is completely abolished in a mutant lacking Sty1 kinase activity. Both Atf1 and its binding partner Pcr1 are required for association of Sty1 with Atf1-dependent promoters, suggesting that this heterodimer must be intact for optimal recruitment of the MAPK. However, many Atf1-dependent genes are still expressed in a pcr1Delta mutant but with significantly delayed kinetics, thus providing an explanation for the relatively mild stress sensitivity displayed by pcr1Delta. Consistent with this delay, Sty1 and Atf1 cannot be detected at these promoters in this condition, suggesting that their association with chromatin is weak or transient in the absence of Pcr1.
    • 'Fit-for-purpose' validation of SearchLight multiplex ELISAs of angiogenesis for clinical trial use.

      Backen, Alison C; Cummings, Jeffrey; Mitchell, Claire L; Jayson, Gordon C; Ward, Timothy H; Dive, Caroline; CR-UK Translational Angiogenesis Group, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK. (2009-03-15)
      Validated assays of circulating biomarkers of angiogenesis to predict and determine the efficacy of vascular-targeted anticancer drugs would facilitate successful drug development. Multiple biomarker candidates exist and a multiplex approach was sought to minimise the requisite patient blood volume and to aid selection of those biomarkers with greatest potential clinical utility. Validation of the SearchLight multiplex ELISA platform comprising two multiplex assays of nine potential angiogenesis biomarkers was conducted (plex 1; VEGF R1 and R2, IL-8, KGF, PlGF; plex 2; PDGFbb, HGF, FGFb and VEGF). The study focused on instrument qualification, analyte specificity within the multiplex format, assay precision and reproducibility. No evidence was found within the multiplex that signals output from one analyte impinged on another or that antibody cross-reactivity occurred. Spike recovery for 5 between-experiment repeats was within +/-15% of input values for 7 of the 9 multiplexed analytes, with a coefficient of variation (CV) of <20% for 6 of the 9 analytes. Plasma samples from 8 ovarian cancer patients (who were not receiving therapy) were assessed using the two multiplexes on this platform to explore the likely baseline variability in this disease context. This study suggests that the platform and the multiplex approach will be useful to evaluate pharmacodynamic responses to vascular targeted therapy in early clinical trials.
    • 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.
    • 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.
    • 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.
    • The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage.

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

      Somervaille, Tim C P; Matheny, Christina J; Spencer, Gary J; Iwasaki, Masayuki; Rinn, John L; Witten, Daniela M; Chang, Howard Y; Shurtleff, Sheila A; Downing, James R; Cleary, Michael L; et al. (2009-02-06)
      The genetic programs that promote retention of self-renewing leukemia stem cells (LSCs) at the apex of cellular hierarchies in acute myeloid leukemia (AML) are not known. In a mouse model of human AML, LSCs exhibit variable frequencies that correlate with the initiating MLL oncogene and are maintained in a self-renewing state by a transcriptional subprogram more akin to that of embryonic stem cells (ESCs) than to that of adult stem cells. The transcription/chromatin regulatory factors Myb, Hmgb3, and Cbx5 are critical components of the program and suffice for Hoxa/Meis-independent immortalization of myeloid progenitors when coexpressed, establishing the cooperative and essential role of an ESC-like LSC maintenance program ancillary to the leukemia-initiating MLL/Hox/Meis program. Enriched expression of LSC maintenance and ESC-like program genes in normal myeloid progenitors and poor-prognosis human malignancies links the frequency of aberrantly self-renewing progenitor-like cancer stem cells (CSCs) to prognosis in human cancer.
    • 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.
    • Identification of gene transcript signatures predictive for estrogen receptor and lymph node status using a stepwise forward selection artificial neural network modelling approach.

      Lancashire, Lee J; Rees, Robert C; Ball, Graham R; Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom. LLancashire@picr.man.ac.uk (2008-06)
      OBJECTIVE: The advent of microarrays has attracted considerable interest from biologists due to the potential for high throughput analysis of hundreds of thousands of gene transcripts. Subsequent analysis of the data may identify specific features which correspond to characteristics of interest within the population, for example, analysis of gene expression profiles in cancer patients to identify molecular signatures corresponding with prognostic outcome. These high throughput technologies have resulted in an unprecedented rate of data generation, often of high complexity, highlighting the need for novel data analysis methodologies that will cope with data of this nature. METHODS: Stepwise methods using artificial neural networks (ANNs) have been developed to identify an optimal subset of predictive gene transcripts from highly dimensional microarray data. Here these methods have been applied to a gene microarray dataset to identify and validate gene signatures corresponding with estrogen receptor and lymph node status in breast cancer. RESULTS: Many gene transcripts were identified whose expression could differentiate patients to very high accuracies based upon firstly whether they were positive or negative for estrogen receptor, and secondly whether metastasis to the axillary lymph node had occurred. A number of these genes had been previously reported to have a role in cancer. Significantly fewer genes were used compared to other previous studies. The models using the optimal gene subsets were internally validated using an extensive random sample cross-validation procedure and externally validated using a follow up dataset from a different cohort of patients on a newer array chip containing the same and additional probe sets. Here, the models retained high accuracies, emphasising the potential power of this approach in analysing complex systems. These findings show how the proposed method allows for the rapid analysis and subsequent detailed interrogation of gene expression signatures to provide a further understanding of the underlying molecular mechanisms that could be important in determining novel prognostic markers associated with cancer.
    • Immune evasion mechanisms in colorectal cancer liver metastasis patients vaccinated with TroVax (MVA-5T4).

      Elkord, Eyad; Dangoor, Adam; Burt, Deborah J; Southgate, Thomas D; Daayana, Sai; Harrop, Richard; Drijfhout, Jan W; Sherlock, David J; Hawkins, Robert E; Stern, Peter L; et al. (2009-02-17)
      We have recently reported the results of a phase II trial in which two TroVax [modified vaccinia ankara (MVA) encoding the tumour antigen 5T4] vaccinations were given to patients both pre- and post-surgical resection of liver metastases secondary to colorectal cancer (CRC). 5T4-specific cellular responses were assessed at the entry and 2 weeks after each vaccination by proliferation of fresh lymphocytes and ELISA for antibody responses; 18 from the 19 CRC patients mounted a 5T4-specific cellular and/or humoral response. Here, we present a comparison of individual and between patient responses over the course of the treatments using cryopreserved peripheral blood mononuclear cells (PBMC) samples from the baseline until after the fourth vaccination at 14 weeks. Assays used were proliferation assay with 5T4-Fc fusion protein, overlapping 32mer 5T4 peptides, MVA-LacZ and MVA-5T4 infected autologous monocytes. Responses to 5T4 protein or one or more peptide pools were pre-existing in 12/20 patients and subsequently 10 and 12 patients showed boosted and/or de novo responses, respectively. Cumulatively, 13/20 patients showed proliferative responses by week 14. We also assessed the levels of systemic T regulatory cells, plasma cytokine levels, phenotype of tumour-infiltrating lymphocytes including T regulatory cells and tumour HLA class I loss of expression. More than half of the patients showed phenotypes consistent with relative immune suppression and/or escape highlighting the complexity of positive and negative factors challenging any simple correlation with clinical outcome.
    • In silico screening and biological evaluation of inhibitors of Src-SH3 domain interaction with a proline-rich ligand.

      Atatreh, Noor; Stojkoski, Cvetan; Smith, Phillippa; Booker, Grant W; Dive, Caroline; Frenkel, A David; Freeman, Sally; Bryce, Richard A; School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PT, UK. (2008-02-01)
      Src signalling and transduction are directly involved in cell growth, cell cycle, malignant transformation and cell migration, providing therapeutic opportunities through inhibition of Src. Here we report virtual screening for novel compounds that inhibit the Src-SH3 protein-protein interaction with a proline-rich peptide ligand. Computational docking of the ZINC compound database was performed using GOLD. Top-scoring compounds were assayed using a fluorescence polarization-based assay. A benzoquinoline derivative showed micromolar inhibition of binding between Src-SH3 and the proline-rich peptide. Several analogues were subsequently assayed showing the requirement of a linker between the benzoquinoline and phenyl rings, and electron donating substituents on the phenyl ring.
    • Inn1 couples contraction of the actomyosin ring to membrane ingression during cytokinesis in budding yeast.

      Sanchez-Diaz, Alberto; Marchesi, Vanessa; Murray, Stephen M; Jones, Richard C; Pereira, Gislene; Edmondson, Ricky D; Allen, Terence D; Labib, Karim; Cancer Research U.K., Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK. (2008-04)
      By rapidly depleting each of the essential budding yeast proteins of unknown function, we identified a novel factor that we call Inn1, which associates with the contractile actomyosin ring at the end of mitosis and is needed for cytokinesis. We show that Inn1 has a C2 domain at the amino terminus of the protein that is required for ingression of the plasma membrane, whereas the remainder of the protein recruits Inn1 to the actomyosin ring. The lethal effects of deleting the INN1 gene can be suppressed by artificial fusion of the C2 domain to other components of the actomyosin ring, restoring membrane ingression on contraction of the actomyosin ring. Our data indicate that recruitment of the C2 domain of Inn1 to the contractile actomyosin ring is crucial for ingression of the plasma membrane during cytokinesis in budding yeast.
    • Int6/eIF3e promotes general translation and Atf1 abundance to modulate Sty1 MAPK-dependent stress response in fission yeast.

      Udagawa, T; Nemoto, N; Wilkinson, Caroline R M; Narashimhan, J; Jiang, L; Watt, S; Zook, A; Jones, Nic; Wek, R; Bähler, J; et al. (2008-08-08)
      int-6 is one of the frequent integration sites for mouse mammary tumor viruses. Although its product is the e-subunit of translation initiation factor eIF3, other evidence indicates that it interacts with proteasomes or other proteins to regulate protein stability. Here we report that the fission yeast int6(+) is required for overcoming stress imposed by histidine starvation, using the drug 3-aminotriazole (3AT). Microarray and complementary Northern studies using wild-type, int6Delta or gcn2Delta mutants indicate that 3AT-treated wild-type yeast induces core environmental stress response (CESR) genes in addition to typical general amino acid control (GAAC) genes whose transcription depends on the eIF2 kinase, Gcn2. In agreement with this, Sty1 MAPK and its target transcription factor Atf1, which signal the CESR, are required for overcoming 3AT-induced starvation. We find that Int6 is required for maintaining the basal level of Atf1 and for rapid transcriptional activation of the CESR on 3AT-insult. Pulse labeling experiments indicate that int6Delta significantly slows down de novo protein synthesis. Moreover, Atf1 protein half-life was reduced in int6Delta cells. These effects would account for the compromised Atf1 activity on 3AT-induced stress. Thus, the robust protein synthesis promoted by intact eIF3 appears to be a part of the requisites for sound Sty1 MAPK-dependent signaling governed by the activity of the Atf1 transcription factor.
    • An introduction to artificial neural networks in bioinformatics--application to complex microarray and mass spectrometry datasets in cancer studies.

      Lancashire, Lee J; Lemetre, Christophe; Ball, Graham R; Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, UK. llancashire@picr.man.ac.uk (2009-05)
      Applications of genomic and proteomic technologies have seen a major increase, resulting in an explosion in the amount of highly dimensional and complex data being generated. Subsequently this has increased the effort by the bioinformatics community to develop novel computational approaches that allow for meaningful information to be extracted. This information must be of biological relevance and thus correlate to disease phenotypes of interest. Artificial neural networks are a form of machine learning from the field of artificial intelligence with proven pattern recognition capabilities and have been utilized in many areas of bioinformatics. This is due to their ability to cope with highly dimensional complex datasets such as those developed by protein mass spectrometry and DNA microarray experiments. As such, neural networks have been applied to problems such as disease classification and identification of biomarkers. This review introduces and describes the concepts related to neural networks, the advantages and caveats to their use, examples of their applications in mass spectrometry and microarray research (with a particular focus on cancer studies), and illustrations from recent literature showing where neural networks have performed well in comparison to other machine learning methods. This should form the necessary background knowledge and information enabling researchers with an interest in these methodologies, but not necessarily from a machine learning background, to apply the concepts to their own datasets, thus maximizing the information gain from these complex biological systems.
    • Loss of regulators of vacuolar ATPase function and ceramide synthesis results in multidrug sensitivity in schizosaccharomyces pombe.

      Dawson, Keren; Toone, W Mark; Jones, Nic; Wilkinson, Caroline R M; Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, United Kingdom. (2008-06)
      We undertook a screen to isolate determinants of drug resistance in fission yeast and identified two genes that, when mutated, result in sensitivity to a range of structurally unrelated compounds, some of them commonly used in the clinic. One gene, rav1, encodes the homologue of a budding yeast protein which regulates the assembly of the vacuolar ATPase. The second gene, lac1, encodes a homologue of genes that are required for ceramide synthesis. Both mutants are sensitive to the chemotherapeutic agent doxorubicin, and using the naturally fluorescent properties of this compound, we found that both rav1 and lac1 mutations result in an increased accumulation of the drug in cells. The multidrug-sensitive phenotype of rav1 mutants can be rescued by up-regulation of the lag1 gene which encodes a homologue of lac1, whereas overexpression of either lac1 or lag1 confers multidrug resistance on wild-type cells. These data suggest that changing the amount of ceramide synthase activity in cells can influence innate drug resistance. The function of Rav1 appears to be conserved, as we show that SpRav1 is part of a RAVE-like complex in fission yeast and that loss of rav1 results in defects in vacuolar (H(+))-ATPase activity. Thus, we conclude that loss of normal V-ATPase function results in an increased sensitivity of Schizosaccharomyces pombe cells to drugs. The rav1 and lac1 genes are conserved in both higher eukaryotes and various pathogenic fungi. Thus, our data could provide the basis for strategies to sensitize tumor cells or drug-resistant pathogenic fungi to drugs.
    • Making connections at DNA replication forks: Mrc1 takes the lead.

      Labib, Karim; Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. (2008-10-24)
      In a recent issue of Molecular Cell, Lou et al. (2008) demonstrate that the Mrc1 protein associates with the DNA polymerase that acts on the leading strand at replication forks, suggesting a potential mechanism that could help to preserve genome stability.
    • 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.
    • Mitochondrial dynamics and apoptosis: a painful separation.

      James, Dominic I; Martinou, Jean-Claude; Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. (2008-09)
      Reporting in Molecular Cell, Sheridan et al. (2008) and Breckenridge et al. (2008) show that mitochondrial fragmentation is not required to induce cell death. Meanwhile, Yamaguchi et al. show that proapoptotic Bcl-2 family members promote cytochrome c mobilization through Opa1-mediated cristae remodeling. Therefore, the connection between mitochondrial structure and apoptosis is more complex than previously imagined.
    • Multiple pathways differentially regulate global oxidative stress responses in fission yeast.

      Chen, Dongrong; Wilkinson, Caroline R M; Watt, Stephen; Penkett, Christopher J; Toone, W Mark; Jones, Nic; Bähler, Jürg; Cancer Research UK Fission Yeast Functional Genomics Group, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, United Kingdom. (2008-01)
      Cellular protection against oxidative damage is relevant to ageing and numerous diseases. We analyzed the diversity of genome-wide gene expression programs and their regulation in response to various types and doses of oxidants in Schizosaccharomyces pombe. A small core gene set, regulated by the AP-1-like factor Pap1p and the two-component regulator Prr1p, was universally induced irrespective of oxidant and dose. Strong oxidative stresses led to a much larger transcriptional response. The mitogen-activated protein kinase (MAPK) Sty1p and the bZIP factor Atf1p were critical for the response to hydrogen peroxide. A newly identified zinc-finger protein, Hsr1p, is uniquely regulated by all three major regulatory systems (Sty1p-Atf1p, Pap1p, and Prr1p) and in turn globally supports gene expression in response to hydrogen peroxide. Although the overall transcriptional responses to hydrogen peroxide and t-butylhydroperoxide were similar, to our surprise, Sty1p and Atf1p were less critical for the response to the latter. Instead, another MAPK, Pmk1p, was involved in surviving this stress, although Pmk1p played only a minor role in regulating the transcriptional response. These data reveal a considerable plasticity and differential control of regulatory pathways in distinct oxidative stress conditions, providing both specificity and backup for protection from oxidative damage.
    • Multiplexed assays for detection of mutations in PIK3CA.

      Board, Ruth E; Thelwell, Nicola J; Ravetto, Paul F; Little, Stephen; Ranson, Malcolm R; Dive, Caroline; Hughes, Andrew; Whitcombe, David; Discovery Medicine, AstraZeneca Pharmaceuticals, Macclesfield, UK. ruth.board@astrazeneca.com (2008-04)
      BACKGROUND: Mutations in the PIK3CA gene (phosphoinositide-3-kinase, catalytic, alpha polypeptide) have recently been described in a number of cancers, and their detection is currently limited because of the low sensitivity of conventional sequencing techniques. METHODS: We combined Amplification Refractory Mutation System (ARMS; AstraZeneca) allele-specific PCR and Scorpions (DxS) to develop assays for tumor-borne PIK3CA mutations and used real-time PCR to develop high-throughput multiplexed assays for the most commonly reported PIK3CA mutants (H1047L, H1047R, E542K, E545K). RESULTS: These assays were more sensitive than sequencing and could detect 5 copies of mutant DNA in proportions as low as 0.1% of the total DNA. We assayed DNA extracted from human tumors and detected PIK3CA mutation frequencies of 10.2% in colorectal cancer, 38.7% in breast cancer, 1.9% in lung cancer, and 2.9% in melanoma. In contrast, sequencing detected only 53% of the mutations detected by our assay. CONCLUSIONS: Multiplexed assays, which can easily be applied to clinical samples, have been developed for the detection of PIK3CA mutations.