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WO2003048361A2 - P55pik - Google Patents

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Publication number
WO2003048361A2
WO2003048361A2 PCT/GB2002/005547 GB0205547W WO03048361A2 WO 2003048361 A2 WO2003048361 A2 WO 2003048361A2 GB 0205547 W GB0205547 W GB 0205547W WO 03048361 A2 WO03048361 A2 WO 03048361A2
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Prior art keywords
p55pik
expression
cells
apoptosis
cell
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WO2003048361A3 (fr
Inventor
Ian Hayes
Thomas Cotter
Finbarr Murphy
Liam Seery
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EiRx Therapeutics Ltd
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EiRx Therapeutics Ltd
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Priority claimed from GB0129377A external-priority patent/GB0129377D0/en
Priority claimed from GB0200831A external-priority patent/GB0200831D0/en
Application filed by EiRx Therapeutics Ltd filed Critical EiRx Therapeutics Ltd
Priority to AU2002347368A priority Critical patent/AU2002347368A1/en
Publication of WO2003048361A2 publication Critical patent/WO2003048361A2/fr
Publication of WO2003048361A3 publication Critical patent/WO2003048361A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01137—Phosphatidylinositol 3-kinase (2.7.1.137)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • the present invention relates to the use of the gene p55PIK in the detection and modulation of apoptosis in cells.
  • it relates to a method for identifying genes associated with p55PIK gene expression and thus their association with apoptosis.
  • Programmed cell death or apoptosis is a genetically programmed process by which cells die under both physiological and a variety of pathological conditions (Kerr et al, Br. J. Cancer, 26, 239-257, 1972). It serves as the counter-balancing force to mitosis during adult life and is a major contributor to the sculpting of physiological structures during the many processes of development (Wyllie et al, Int. Rev. Cytol, 68, 251-305, 1980). It is characterised by a number of well-defined biochemical hallmarks.
  • DNA fragmentation caused by the activation of an endogenous endonuclease enzyme (Wyllie, Nature, 284, 555-556,1980; Enari et al., Nature, 391, 43-50, 1998).
  • endogenous endonuclease enzyme Wang, Nature, 284, 555-556,1980; Enari et al., Nature, 391, 43-50, 1998.
  • the result is a DNA ladder pattern that can be readily visualised in agarose cells. Coupled with DNA fragmentation is cell shrinkage (Wesselbory et al., Cell Immunol. 148, 234-41, 1993) where water is actively extruded from the cell.
  • the apoptotic cell then undergoes fragmentation into apoptotic bodies that are engulfed by neighbouring cells or cells of the reticuloendothelial system.
  • a second well-defined characteristic is the exposure of the phospholipid phosphatidylserine to the outside surface of the plasma membrane of the cell as it undergoes apoptosis (Fadok et al., J Immunol. 148, 2207-16, 1992). Normally this lipid is located on the inner side of the membrane lipid bilayer. The underlying mechanism responsible for this lipid flipping is poorly understood at present. Its expression serves as a signal for the recognition and phagocytosis of the apoptotic cell (Fadok et al., J Immunol. 148, 2207-16, 1992) Under normal physiological conditions apoptosis is tightly regulated.
  • apoptosis is retarded or inhibited
  • ARDS acute respiratory distress syndrome
  • Inappropriate or excessive apoptosis occurs under conditions of ischaemia (stroke, myocardial infarction, etc) Linnik et al., Blood. 80, 1750-7, 1992, Gorman et al., J Neurol Sci. 139, 45-52, 1996) a series of neurodegenerative conditions, myelosuppression (Mori et al, Blood. 92, 101-7, 1998) following chemotherapy or irradiation (Lotem et al., Blood. 80, 1750-7, 1992) and a significant number of other diseases where cell death is a key feature of the pathology.
  • ROS reactive oxygen species
  • ROS molecules cause oxidative damage not only to cellular structures, but may also act to initiate the expression of apoptosis regulating genes.
  • the physiological role for such ROS molecules is far less well characterised than that of other related molecules such as nitric oxide (NO).
  • NO nitric oxide
  • the published literature is unclear, with examples of NO both driving and inhibiting apoptosis (Brune et al., Cell Death Differ. 1999 10,969-975, 1999).
  • NO nitric oxide
  • ROS may act as second messengers in signal transduction pathways in the context of cytokine/growth factor stimulation of cells.
  • Other more recent studies have indicated that they may also activate unique pathways.
  • the specific targets of ROS generated intracellularly are largely unknown at present, but it is known that the addition of hydrogen peroxide or other ROS generators to cells in culture leads to the activation of the transcription factor Nf/kB (Schreck et al., EMBO J 10, 2247-2258, 1991). This in turn controls the expression of a series of genes involved in a variety of cellular functions.
  • ROS mitogen activated protein kinase
  • a cell has the ability to produce ROS at a number of different sites. In relation to signal transduction events it is still unclear where the source of ROS is within the cell. There are a number of potential enzyme systems capable of ROS generation. Perhaps the best documented one, particularly in neutrophils and other phagocytic cells, is NADPH oxidase. Studies using inhibitors ofthis enzyme such as DPI suggest that this enzyme is also involved in the generation of ROS in non phagocytic cells (Griendling et al., Circulation, 74, 1141-1148, 1994). Mitochondria play a key role in apoptosis and are also a major site of ROS generation.
  • the loss of mitochondrial membrane potential is coupled to the release of cytochrome C and this in turn has two effects.
  • the first is the generation of ROS, since the respiratory chain is disrupted by the removal of cytochrome C.
  • the second is the cleavage of cellular DNA through a series of cytochrome C mediated caspase activation steps, which is an end point of the apoptosis process.
  • ROS ROS are involved in p53 mediated apoptosis (Johnson et al., Proc. Natl. Acad. Sci. USA, 93, 11848-11852, 1997). Cells generated to over-express p53 undergo apoptosis, accompanied by ROS production and this can be blocked by anti-oxidants (Polyak et al., Nature, 389, 300-305, 1997).
  • ROS production is closely associated with the initiation and propagation of apoptosis.
  • the mechanism of ROS activity in apoptosis has until recently been unclear.
  • a series of enzymes involved in maintaining the redox balance within a cell contribute to the ability of that cell to survive in the presence of elevated ROS levels.
  • Such enzymes include catalase and superoxide dismutase which work to reduce the oxidative stress in cells.
  • catalase and superoxide dismutase which work to reduce the oxidative stress in cells.
  • superoxide dismutase which work to reduce the oxidative stress in cells.
  • Bcl-2 proteins most notably Bcl-2 are thought to mediate their anti-apoptotic effects via an anti-oxidant process (Hockenberry et al., Cell. 1993 75 :241-51). The precise mechanism by which Bcl-2 mediates its effects are still not quite defined.
  • NF-kB redox sensitive transcription factor
  • the first of these is tyrosine phosphorylation at the plasma membrane due either to intrinsic receptor tyrosine kinase activity (e.g. the insulin growth factor 1 receptor), or indirectly coupled to tyrosine kinases or alternatively directly coupled to several transmembrane G protein-coupled receptors.
  • intrinsic receptor tyrosine kinase activity e.g. the insulin growth factor 1 receptor
  • indirectly coupled to tyrosine kinases or alternatively directly coupled to several transmembrane G protein-coupled receptors.
  • Blood neutrophils have relatively short lives with greater than 80% of them apoptosing within the first 24 hours.
  • Apoptotic neutrophils are phagocytosed by macrophages via thrombospondin and macrophage CD36/ vitronectin receptor (Savil 1992, Clinical Science 83, 649-55, Savil et al. 1993, Immunology Today 14,131-136) and thus prevent release of potentially lethal cocktail of enzymes in the host, should the neutrophil undergo necrosis.
  • certain inflammatory environments favour the survival of neutrophils.
  • cytokines including GM-CSF, IL-1, IL-2, IL- 8 and IFN ⁇ can delay neutrophil apoptosis (Brach et al, 1992, Blood 80, 2920 -2924; Calotta et al 1992, Blood 80, 2012-2020, Lee et al 1993, J Leuk Biol 54, 283 - 388, Pericle et al 1994, Eur. J. Immunol24, 440 - 444, Get ref for IL8).
  • inflammatory proteins e.g. C5A
  • bacterial products e.g. LPS
  • Granulocyte macrophage colony-stimulating factor (GM-CSF) is known to inhibit PMN apoptosis both in vitro and in vivo (Cox et al. 1992, Am. J. Respiratory Cell Mol
  • GM-CSF selectively induced tyrosine phosphorylation of Extracellular Signal-Related kinase (ERK), a member of microtubule associated protein kinase (MAPK) family
  • ERK Extracellular Signal-Related kinase
  • MAPK microtubule associated protein kinase
  • Al-Shami et al. (Blood 1997, 89(3) 1035-1044) has shown that GM-CSF induces both a time and concentration- dependent increase in the level of tyrosine phosphorylation of the PI-3- kinase regulatory subunit p85, possibly via lyn kinase.
  • Klein et al. J. Immunol.
  • MCL1 myeloid cell leukaemia 1
  • MCL1 myeloid cell leukaemia 1
  • Neutrophils also express mRNA for Al, another Bcl-2 homologue with anti-apoptotic properties (Chuang PI, Yee E, Karsan A, Winn RK, Harlan JM, Biochem Biophys Res Commun 1998; 249(2): 361-5).
  • GM-CSF inhibits death through apoptosis by the regulation of 'effector genes' that control the process of apoptosis.
  • a signal acts through a signal transduction cascade and is associated with significant changes, or patterns of changes, in gene expression in the cell.
  • the identities of such 'effector genes' and their role in the signalling pathways that lead to the biochemical events of cell death have been incompletely determined.
  • apoptosis represents a significant therapeutic target, since many diseases are due to defects in this process. Many physiological factors induce and prevent cell apoptosis. For example, cytokines or growth factors such as GM-CSF inhibit death through apoptosis. There is an acute need to identify the genes that regulate this process. In other words, if one identifies a gene that prevents apoptosis, then this gene/gene product or its function can be blocked by a drug and apoptosis allowed to occur. To-date many of the genes found have certain fundamental flaws e.g. they act late in the process, after the cell has committed to a death programme, or they are ubiquitous, that is they are not restricted to a particular cell type. The ideal target to control apoptosis act early in the process and are restricted to a particular cell type.
  • the present invention identifies that the expression of the gene p55PIK is correlated with an early stage in apoptosis.
  • p55PIK gene expression is decreased in neutrophil apoptosis and increased in neutrophil survival when apoptosis is inhibited by the presence of GM-CSF.
  • GM-CSF inhibition of apoptosis is blocked by gliotoxin, p55PIK expression is down regulated.
  • expression of recombinant p55PIK in HeLa, or growth factor dependent TF1 resulted in significant inhibition of proliferation/viability thus identifying p55PIK as a modulator of cell growth survival.
  • p55PIK (Synonyms.- PI3-Kinase P85-gamma subunit, PtdIns-3 -kinase P85-gamma, p55PIK, Phosphophatidylinositol 3 kinase, regulatory, 55KD, gamma p55-gamma; Gene name: PIK3R3), is identified in Swiss Prot, Accession number: Q92569; gi:12643788; GenBank, Accession number D88532; gi: 1661000.
  • amino acid sequence is set out in SEQ ID NO: 1.
  • bladder blood, breast, breast_normal, cervix, colon, connective tissue, eye, foreskin, gall bladder, germ cell, head_neck, lung_tumor, nose, ovary, placenta, pool, pooled brain, lung, testis, pooled lung and spleen, pooled pancreas and spleen, prostate, prostate_tumor, skin, spleen, stomach, cell line, testisjiormal, uterus, whole embryo).
  • the gene maps to chromosomal position lpter-p32.1.
  • model discovery assays are configured to target the 'early' regulatory events occurring in apoptosis induced by ROS and, in particular, in the inhibition of apoptosis by GM-CSF.
  • apoptosis by GM-CSF is itself inhibited by a drug, such as gliotoxin, then changes, or patterns of changes can be targeted by clustering those changes that are common and both increase and/or decrease depending on the treatment.
  • a change that is a 'decrease' following induction of apoptosis is a candidate target gene, however, a change that is additionally an 'increase' following inhibition of apoptosis by GM-CSF has a higher probability of being a target gene because its regulation shows increased correlation with the process. Likewise, a change that is further a 'decrease' following inhibition of GM-CSF inhibitory effect has a yet higher probability of being a target gene because its regulation shows increased correlation with the process.
  • Genes regulated in these models following modulation of apoptosis include genes that 1) are 'effector' genes involved in the cells defence mechanisms aimed at preventing apoptosis (anti-apoptotic genes) and thus represent therapeutic targets, 2) make up aspects of the apoptosis and/or GM-CSF signal cascade and thus represent therapeutic targets, 3) initiate the process of apoptosis (pro-apoptotic genes) and thus represent therapeutic targets, and 4) are associated with the processes of apoptosis and defence that will aid in the understanding of key pathways, processes and mechanisms that may subsequently lead to the identification of therapeutic targets.
  • a method for detecting apoptosis in a cell comprising detecting a decrease in any one of: i) a p55PIK polypeptide having an amino acid sequence as set out in SEQ ID NO: i) a p55PIK polypeptide having an amino acid sequence as set out in SEQ ID NO: i) a p55PIK polypeptide having an amino acid sequence as set out in SEQ ID NO: i) a p55PIK polypeptide having an amino acid sequence as set out in SEQ ID
  • Levels of gene expression may be determined in any appropriate manner. Detecting a decrease in gene expression may be achieved by measuring p55PIK gene expression in treated versus non-treated cells. Preferably, gene expression may be measured by detecting nucleic acid encoding a p55PIK polypeptide such as p55PIK mRNA transcripts, or a fragment thereof. In one embodiment, the method of measuring mRNA transcripts may use an amplification technique as described herein. In another embodiment, p55PIK expression may be measured by detecting the p55PIK polypeptide gene product, or fragment thereof, using, for example, agents that bind p55PIK. Suitable agents include anti-p55PIK antibodies.
  • a method of detecting GM-CSF-induced cell survival by detecting an increase in p55PIK gene expression in another aspect, there is provided a method of detecting GM-CSF-induced cell survival by detecting an increase in p55PIK gene expression.
  • a method of modulating apoptosis in a cell comprising the step of increasing, decreasing or otherwise altering the functional activity of p55PIK or the nucleic acid encoding it.
  • said modulation of apoptosis is inhibition.
  • said modulation of apoptosis confers survival in a cell.
  • a method of modulating cell growth in a cell comprising the step of increasing, decreasing or otherwise altering the functional activity of p55PIK or the nucleic acid encoding it.
  • the modulation of cell growth is the inhibition of proliferation.
  • the cell may be a therapeutic target for the treatment of disease.
  • a cell may be a cancer cell, a cell involved in an inflammatory disorder, a cell involved in an autoimmune disorder or in a neurodegenerative disorder.
  • the term 'altered functional activity of p55PIK or the nucleic acid encoding it' includes within its scope increased, decreased or an otherwise altered activity of p55PIK as compared with the native protein functioning in its normal environment, that is within a single cell under native conditions. In addition, it also includes within its scope an increased or decreased level of expression and/or altered intracellular distribution of the nucleic acid encoding p55PIK, and/or an altered the intracellular distribution of p55PIK_itself.
  • the method of modulating apoptosis or cell growth involves decreasing p55PIK gene expression.
  • the expression of p55PIK is reduced by greater than 50%, 60%, 70%, 80%, 90% or more of its normal level in untreated cells.
  • a decrease in p55PIK gene expression may be effected by antisense expression.
  • Other means of decreasing p55PIK gene expression will be recognised by those skilled in the art and include introducing dominant negatives, peptides or small molecules including RNA molecules such as siRNA molecules which cause a decrease in gene expression through RNA interference. Suitable siRNA molecules are described in the Examples section herein.
  • said method involves increasing p55PIK gene expression and therefore increasing cell survival.
  • the expression of p55PIK is increased by greater than 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500% or more of its normal level in untreated cells.
  • said method comprises providing an expression vector comprising a nucleic acid sequence encoding a p55PIK polypeptide; introducing the expression vector into the cell and maintaining the cell under conditions permitting expression of the encoded polypeptide in the cell.
  • a nucleic acid encoding p55PIK or a p55PIK polypeptide encompasses fragments thereof.
  • p55PIK or an agent that alters p55PIK expression in a cell, in the modulation of apoptosis.
  • apoptosis is assessed by expression of p55PIK in a test system and measuring the impact on cell growth and viability.
  • p55PIK may itself be used to identify other candidate genes or proteins which are involved in apoptosis.
  • molecules which 'interact' with p55PIK include molecules which bind to p55PIK either directly or indirectly.
  • Methods for detecting those molecules include physical methods and molecular biology techniques as herein described. Suitable standard laboratory techniques will be familiar to those skilled in the art and include immunoprecipitation, immunoblotting and fluorescence techniques. One skilled in the art, will appreciate that this list is not intended to be exhaustive. Suitable molecular biology techniques include phage display and the yeast two-hybrid system described herein. p55PIK may itself be used to identify other candidate genes or proteins which are involved in apoptosis.
  • a method for identifying a gene product whose expression is modulated by the expression of p55PIK comprising the steps of:
  • said method further comprises the step of exposing said cell to conditions which promote apoptosis or cell survival prior to measuring global gene expression.
  • Such a method may be modulated to identify compounds that modulate p55PIK protein function. Accordingly, in another aspect, there is provided a method for identifying a compound that modulates p55PIK protein function comprising:
  • expression levels are assessed by measuring gene transcription. This is preferably carried out by measuring the rate and/or amount of specific mRNA production in the cell.
  • a preferred embodiment ofthis aspect of the invention involves the use of arrayed oligonucleotide probes capable of hybridising to mRNA populations. Differences in hybridisation patterns of different mRNA populations may be used to identify genes which are differentially expressed in the two populations. Differential expression may include different expression patterns observed across a time course.
  • the arrayed oligonucleotide probes are advantageously derived from cDNA or EST libraries, and represent genes which are expressed by the cells under investigation. Levels of gene expression may be determined in any appropriate manner. Preferably, levels of gene expression may be determined by the measurement of protein production by mRNA translation to detect increases or decreases in the rate or amount of mRNA translation.
  • global gene expression is measured by assaying gene transcription using a microarray.
  • global gene expression is measured by protein array.
  • Another aspect of the invention is directed to the identification of agents capable of modulating p55PIK gene expression or protein function.
  • the invention provides assays for determining compounds that modulate the function and/or expression of p55PIK.
  • the identification of agents capable of modulating p55PIK protein function can be detected by measuring the expression of a gene whose expression is regulated byp55PIK.
  • p55PIK in an assay for identifying an agent which modulates apoptosis.
  • a method for identifying a compound that modulates p55PIK protein function comprising:
  • a method for identifying a compound that modulates p55PD protein function comprising: - taking a cell expressing p55PIK;
  • nucleic acid construct comprising the promoter sequence of a p55PIK-regulated gene operably linked to a reporter gene
  • a 'cell expressing p55PIK' is a cell which has been transfected with a nucleic acid construct encoding p55PIK preferably by providing a vector encoding p55PIK and introducing said vector into a cell under conditions to promote expression of p55PIK, as described above. Transfection may result in transient, stable or inducible expression of p55PIK using methods familiar to those skilled in the art or as described herein.
  • the p55PIK-regulated gene is selected from the group consisting of Phospholipase A2, group IVA (cytosolic), Phosphodiesterase 4A, cAMP-specific (dunce (Drosophila)-homolog phosphodiesterase E2), Phosphodiesterase IC, calmodulin-dependent (70kD), Neurogranin (protein kinase C substrate, RC3), Protein tyrosine phosphatase, non-receptor type 13 (APO-1/CD95 (Fas)-associated phosphatase) and Apolipoprotein A-II.
  • group IVA cytosolic
  • Phosphodiesterase 4A cAMP-specific (dunce (Drosophila)-homolog phosphodiesterase E2)
  • Phosphodiesterase IC calmodulin-dependent (70kD)
  • Neurogranin protein kinase C substrate, RC3
  • Protein tyrosine phosphatase non-re
  • the p55PIK-regulated gene is selected from the group consisting of lung resistance related protein, v-maf musculoaponeutoric fibrosarcoma (avian) oncogene family protein G, adenine nucleotide translocator 2 (fibroblast) and TGFB1 -induced anti-apoptotic factor 1.
  • a system for screening for compounds that modulate p55PIK protein function comprising a cell expressing p55PIK which is co-transfected with a nucleic acid construct encoding a p55PIK-regulated gene or the promoter sequence of such a gene operably linked to a reporter gene.
  • Suitably said method can be used to identify compounds that enhance cell survival.
  • Cells useful in the methods of the invention may be from any source, for example from primary cultures, from established cell lines, in organ culture or in vivo.
  • Cell lines useful in the invention include cells and cell lines of haematopoietic origin. Suitable cells include HeLa, U937 (monocyte), TF-1, HEK293 (T), primary cultures of neutrophils or cells having neutrophil characteristics, for example HL60 cells, murine FDCP-1, FDCPmix, 3T3, primary or human stem cells.
  • cells may be disease-associated cells such as cancer, inflammatory, autoimmune or neurodegeration-associated cells.
  • the modulation of apoptosis can be for therapeutic purposes. Accordingly in another aspect of the invention there is provided a method of treatment of disease comprising administering a modulator of p55PIK gene expression or functional activity to an individual.
  • a modulator of p55PIK expression or activity in the manufacture of a medicament for use in the treatment of disease.
  • said modulator is an antisense molecule or an RNA molecule which mediates RNA interference and thus causes a decrease in p55PIK expression.
  • Suitable diseases include cancer, inflammation, autoimmune disease and neurodegenerative disorders.
  • a number of inflammatory diseases such as asthma, chronic obstructive pulmonary disease (COPD), Cystic Fibrosis (CF), Rheumatoid Arthritis (RA) and Inflammatory bowel disease (IBD) are characterised by a) elevated levels and expression of cytokines and growth factors that act predominantly on myeloid cells, b) prolonged survival of myeloid cells, and c) prolonged activation of myeloid cells.
  • COPD chronic obstructive pulmonary disease
  • CF Cystic Fibrosis
  • RA Rheumatoid Arthritis
  • IBD Inflammatory bowel disease
  • p55PIK or an agent that alters p55PIK expression in a cell, in the treatment of inflammatory diseases through the modulation of myeloid cell apoptosis.
  • myeloid cell refers to terminally differentiated, non-dividing cells of the myeloid lineage. These cells include neutrophils, eosinophils and monocytes/macrophages. In one embodiment of any aspect of the present invention, the myeloid cell is a neutrophil, eosinophil or monocyte/macrophage.
  • Inflammatory diseases include, but are not limited to, diseases such as sepsis, Acute Respiratory Distress Syndrome, Pre enclampsia, Myocardial ischemia, reperfusion injury, Psoriasis, Asthma, COPD, bronchiolitis, Cystic Fibrosis, Rheumatoid Arthritis, Inflammatory Bowel Disease, Crohns Disease and Ulcerative colitis.
  • diseases such as sepsis, Acute Respiratory Distress Syndrome, Pre enclampsia, Myocardial ischemia, reperfusion injury, Psoriasis, Asthma, COPD, bronchiolitis, Cystic Fibrosis, Rheumatoid Arthritis, Inflammatory Bowel Disease, Crohns Disease and Ulcerative colitis.
  • an isolated nucleic acid molecule comprising a promoter, said nucleic acid sequence being selected from the group consisting of: i) a nucleic acid molecule having the sequence set out in SEQ ID NO:2; ii) a nucleic acid molecule having at least 60% homology with i); iii) a nucleic acid molecule hybridising under stringent conditions to i) or ii); and iv) the complement of the sequences set out in i) to iii).
  • the p55PIK promoter region has been identified to comprise a number of sites which bind specific transcription factors or enhancers. Accordingly, in one embodiment there is provided a nucleic acid sequence as set out in i), ii) or iii) above which comprises one or more of the enhancer or transcription factor binding elements selected from the group consisting of p53, C/EBP, NRE-1, AP2, CCAAT and Spl and including others known to the art but not specified herein. In another embodiment, said nucleic acid sequence comprises all of these enhancer or transcription factor binding elements.
  • GM-CSF GM-CSF
  • the promoter sequence comprises the sequence set out SEQ ID NO:2. In another embodiment, the promoter sequence comprises the sequence in the EC ACC deposit Accession no. 01120311.
  • the promoter sequence comprises the region of the genomic sequence from -2128 to + 19 of the transcription start site for p55PIK.
  • a vector comprising a nucleic acid as defined above.
  • said vector comprises a nucleic acid in accordance with the invention operably linked to a reporter gene.
  • the vector may further comprise other sequences such as sequences encoding selectable markers.
  • modulation of expression may be an increase (activation) or a decrease (inhibition) of expression from the p55PIK promoter.
  • an isolated nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule encoding a p55PIK polypeptide having the sequence as set out in SEQ ID NO: 1 but having a single nucleotide polymorphism mapping to amino acid 297; ii) a nucleic acid molecule having at least 80% homology with i); iii) a nucleic acid molecule hybridising under stringent conditions to i) or ii); and iv) the complement of the sequences set out in i) to iii).
  • Figure 1 shows the dose responsiveness of the anti-apoptotic effect of GM-CSF. Optical densities are read at 570nm using a plate reader. The results indicate a direct correlation between survival and concentrations of GM-CSF added to the culture medium.
  • Figure 2 shows that the fungal metabolite gliotoxin blocks the GM-CSF in the inhibition of neutrophil apoptosis.
  • the method is as described in Example 1.
  • Optical densities are read at 570nm using a plate reader.
  • Gliotoxin effectively blocks the GM- CSF mediated inhibition of neutrophil apoptosis.
  • the blocking effect is not seen when the inactive analogue of gliotoxin, methylgliotoxin is added with GM-CSF.
  • No increased neutrophil apoptosis is seen with the addition of gliotoxin alone to isolated neutrophils, demonstrating that the effect is specific to, and limited to, a reversal of the protective effects of GM-CSF.
  • Figure 3 shows a phosphoimage scan of a microarray.
  • Figure 4 shows the analysis of a captured image film by Array VisionTM software.
  • Figure 5 shows the results of combined code cluster analysis.
  • Figure 6 shows cluster analysis of LifeGrid filters.
  • Human purified peripheral blood neutrophils are either allowed to undergo spontaneous apoptosis (Apop), or else are treated with 5U/ml GM-CSF to inhibit apoptosis (GM-CSF).
  • Samples are isolated for RNA extraction and microarray gene analysis, 2 h (Apop2 and GMCSF2), 3 h (A ⁇ o ⁇ 3), 4 h (Apop4 and GMCSF4), 5 h (Apop5) and 6 h (Apo ⁇ 6 and GMCSF6) post- isolation.
  • Gliotoxin 0.1 ⁇ g/ml; Glio
  • Methyl Gliotoxin 0.1 ⁇ g/ml; Methyl
  • Average fold change values are compared to time zero controls (except GM4 which compares fold change of 4 h treatment of GM-CSF plus Gliotoxin with 4 h treatment of GM-CSF with Methyl Gliotoxin control), are analysed by GeneMaths using a Pearson correlation and Ward cluster algorithms. Increased expression (light) and decreased expression (dark) are represented and referenced by a color scale bar. p55PD gene is indicated.
  • Figure 7 shows p55PIK mRNA is increased in GM-CSF-induced neutrophil survival, and this increased expression is blocked by Gliotoxin. Human purified peripheral blood neutrophils are treated as described in Figure 6. The relative amounts of p55PIK transcripts are shown.
  • Figure 8 shows a dendrogram representation of cluster analysis for Figure 6. Marker genes, with known function in apoptosis and survival are indicated. p55PIK_gene is marked.
  • FIG. 9 shows P55PIK is adjacently correlated with two PI3K-signal transduction associated mRNAs.
  • Human purified peripheral blood neutrophils are either allowed to undergo spontaneous apoptosis (Apop), or else are treated with 5U/ml GM-CSF to inhibit apoptosis (GM-CSF).
  • Samples are isolated for RNA extraction and microarray gene analysis, 2 h (Apop2 and GMCSF2), 3 h (A ⁇ op3), 4 h (Apop4 and GMCSF4), 5 h (Apop5) and 6 h (Apop6 and GMCSF6) post-isolation.
  • Gliotoxin O.l ⁇ g/ml; Glio
  • Methyl Gliotoxin 0.1 ⁇ g/ml; Methyl
  • Average fold change values (from two spots on the filters) for all (>8,000) genes on the Incyte LifeGrid filters are compared to time zero controls (except GM4 which compares fold change of 4 h treatment of GM-CSF plus Gliotoxin with 4 h treatment of GM-CSF with Methyl Gliotoxin control), and are analysed by GeneMaths using a Pearson correlation and Ward cluster algorithms. Increased expression (light) and decreased expression (dark) are represented and referenced by a color scale bar. P55PIK gene is indicated.
  • Figure 10 shows P55PIK expression correlates with apoptosis, survival and associated signal transduction pathways.
  • Human purified peripheral blood neutrophils are either allowed to undergo spontaneous apoptosis (Apop), or else are treated with 5U/ml GM- CSF to inhibit apoptosis (GM-CSF).
  • Samples are isolated for RNA extraction and microarray gene analysis, 2 h (Apop2 and GMCSF2), 3 h (Apop3), 4 h (Apop4 and GMCSF4), 5 h (Apop5) and 6 h (Apop ⁇ and GMCSF6) post-isolation.
  • Gliotoxin O.l ⁇ g/ml; Glio
  • Methyl Gliotoxin O.l ⁇ g ml; Methyl
  • Gliotoxin O.l ⁇ g ml
  • Methyl Methyl
  • Average normalised sDens from two spots on the filters
  • GeneMaths using a Pearson correlation and Ward cluster algorithms.
  • Expression levels are represented and referenced by a color scale bar. p55PIK gene is indicated.
  • Figure 11 shows a time course of neutrophil differentiation, measured by NBT reduction assay.
  • HL-60 cells are seeded at 5xl0 5 /ml and incubated with lO ⁇ M Retinoic acid for 24, 48 and 72 hours, or untreated (HL60). Cells are harvested and stimulated with PMA and incubated for 15 minutes in the presence of NBT. Approximately 5x10 4 cells are transferred by cytospin onto slides and counter stained with Eosin. Slides are analysed blind and only whole cells containing formazan deposits were considered positive. Graph demonstrates differentiation towards the neutrophil lineage, as measured by percentage NBT positive cells.
  • Figure 12 shows a time course of neutrophil apoptosis, following differentiation of HL60 cells.
  • HL60 cells are seeded at 5xl0 6 cells in T25 flasks and incubated with lO ⁇ M Retinoic acid for 24, 48, 72, 96, 120 and 144hrs. Mock treated HL-60 cells were used as a control. Cellular DNA was analysed for fragmentation into oligonucleosomal-size fragments and their multiples by agarose gel electrophoresis. Lanes 1,3,5,7,9,11 contain DNA from HL-60 cells that were differentiated with lO ⁇ M Retinoic Acid over the indicated period whereas samples in lanes 2,4,6,8,10,12,14 are from control HL-60 cells treated for the same time period. Lane 15 is a 1Kb Plus Ladder Molecular Marker.
  • Figure 13 shows p55PIK is differentially regulated during neutrophil diferentiation.
  • Human purified peripheral blood neutrophils are treated as described in Figure 11.
  • cDNA is hybridised on Incte LifeGrid filters. Average fold change (from two spots on the filters) for p55PIK gene on the Incyte LifeGrid filters are compared to time zero controls.
  • Figure 14 shows p55PIK gene expression is decreased by cisplatin-induced apoptosis in HeLa cells.
  • HeLa cells are plated into 75cm 2 flasks (6xl0 6 cells/ flask) and allowed to adhere for four hours. After this period, cells are treated with Cisplatin (lug/ml) and incubated at 37°C.
  • RNA samples are isolated and analysed by microarray, using Incyte LifeGrid filters at 0 h, 2 h and 4 h following the addition of cisplatin. Average fold change of the p55PIK gene at 2 h and 4h, compared with 0 h is indicated.
  • Figure 15 shows a graphical representation of the effect of known survival and pro- apoptotic genes on the proliferation/ viability of HeLa cells, as determined by a plaque assay.
  • Figure 16 shows a graphical representation of the effect of p55PIK on the proliferation/ viability of HeLa cells, as determined by a plaque assay.
  • Figure 17 shows the nucleotide sequence for the p55PIK promoter.
  • the underlined sequence represents the beginning of the mRNA transcript.
  • Figure 18 shows enhancer and transcription factor binding elements in the p55PIK promoter. Numbers in brackets indicate positions derived from the sequence presented in Figure 17 (SEQ ID NO:2).
  • FIG 19 shows Forward and Side Scatter analysis of TF-1 population by Flow Cytometry.
  • TF-1 cells are cultured for 48h in the presence or absence of GM-CSF (2ng/ml) prior to acquisition and analysis using a FacsCalibre flow cytometer.
  • the area enclosed in the gate represents the live gate region. From this figure, it is observed that there is a decrease of approx. 50% in the number of cells with side scatter parameters of healthy cells in the factor deprived cells.
  • Figure 20 shows cell cycle analysis by flow cytometry. Apoptotic cells have less DNA, resulting in an increase in low intensity staining, measured as a sub GI peak in fluorescent histograms.
  • Figure 21 shows a composite of dot plots showing a typical result for both TF-1 cells treated under the various conditions.
  • Cells are transduced with either an empty expression vector containing no insert or BCL2 and subjected to the indicated conditions.
  • BCL2 in TF-1 cells allows more cells to survive GMCSF withdrawal, as determined by the percentage of cells in the live gate.
  • Figure 22 Overexpression of p55PIK induces apoptosis in TF1 cells.
  • TF-1 cells were transduced with either p55PIK in a retroviral expression cassette or expression cassette alone (Control) and examined 96h later for viability based on the Fsc/ Ssc parameters.
  • Apoptosis or programmed cell death is a controlled intracellular process characterised by the condensation and subsequent fragmentation of the cell nucleus during which the plasma membrane remains intact. It is an active, highly regulated process distinguished by cell shrinkage and packaging of the cell contents into apoptotic bodies that are subsequently engulfed by macrophages, thus avoiding activation of the inflammatory response (for review see Wyllie, Br. Med. Bull. 53:451- 465, 1997). Apoptotic death is distinct from other cell processes including necrotic cell death and replicative senescence.
  • modulating apoptosis is meant that for a given cell, under certain environmental conditions, its normal tendency to undergo apoptosis is changed compared to an untreated cell.
  • blood neutrophils have a defined apoptotic tendency - within a population of cells, greater than 80% will apoptose within the first 24 hours. Modulating the apoptosis of blood neutrophils means changing this normal apoptotic tendency such that apoptosis is increased or decreased relative to the normal rate.
  • blood neutrophils in the presence of GM-CSF have a decreased tendency to apoptose.
  • modulating apoptosis of blood neutrophils in the presence of GM-CSF means increasing or decreasing apoptosis relative to their normal decreased tendency under these conditions.
  • a decreased tendency to apoptose may also be a measurable increase in cell survival and may be the result of an inhibition of apoptosis by inhibiting one or more components of the apoptotic pathway.
  • expression refers to the transcription of a genes DNA template to produce the corresponding mRNA and translation ofthis mRNA to produce the corresponding gene product (i.e., a peptide, polypeptide, or protein).
  • activates gene expression refers to inducing or increasing the transcription of a gene in response to a treatment where such induction or increase is compared to the amount of gene expression in the absence of said treatment.
  • decreases gene expression or “down-regulates gene expression” refers to inhibiting or blocking the transcription of a gene in response to a treatment and where such decrease or down- regulation is compared to the amount of gene expresssion in the absence of said treatment.
  • Antibodies can be whole antibodies, or antigen-binding fragments thereof.
  • the invention includes fragments such as Fv and Fab, as well as Fab' and F(ab') 2 , and antibody variants such as scFv, single domain antibodies, Dab antibodies and other antigen-binding antibody-based molecules.
  • the "functional activity" of a protein in the context of the present invention describes the function the protein performs in its native environment. Altering the functional activity of a protein includes within its scope increasing, decreasing or otherwise altering the native activity of the protein itself. In addition, it also includes within its scope increasing or decreasing the level of expression and/or altering the intracellular distribution of the nucleic acid encoding the protein, and/or altering the intracellular distribution of the protein itself.
  • variants or derivatives in relation to p55PIK polypeptide includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the polypeptide sequence of p55PIK.
  • nucleic acids encoding p55PIK are understood to comprise variants or derivatives thereof.
  • nucleic acid refers to single stranded or double stranded DNA and RNA molecules including natural nucleic acids found in nature and/or modified, artificial nucleic acids having modified backbones or bases, as are known in the art.
  • an "isolated" nucleic acid refers to material removed from its original environment (for example, the natural environment in which it occurs in nature), and thus is altered by the hand of man from its natural state.
  • an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.
  • the term "isolated” does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the nucleic acids of the present invention.
  • Vector refers to any agent such as a plasmid, cosmid, virus, autonomously replicating sequence, phage, or linear single-stranded, circular single-stranded, linear double-stranded, or circular double-stranded DNA or RNA nucleotide sequence that carries exogenous DNA into a host cell or organism.
  • the recombinant vector may be derived from any source and is capable of genomic integration or autonomous replication.
  • promoter refers to a nucleic acid sequence, usually found upstream (5') to a coding sequence, that is capable of directing transcription of a nucleic acid sequence into mRNA.
  • the promoter or promoter region typically provide a recognition site for RNA polymerase and the other factors necessary for proper initiation of transcription.
  • a promoter or promoter region includes variations of promoters derived by inserting or deleting regulatory regions, subjecting the promoter to random or site-directed mutagenesis, etc.
  • the activity or strength of a promoter may be measured in terms of the amounts of RNA it produces, or the amount of protein accumulation in a cell or tissue, relative to a promoter whose transcriptional activity has been previously assessed.
  • nucleic acid encoding the promoter sequence of p55PIK means a nucleic acid which is capable of directing transcription of p55PIK gene expression.
  • the term moreover includes those polynucleotides capable of hybridising, under stringent hybridisation conditions, to the naturally occurring nucleic acids identified above, or the complement thereof.
  • “Stringent hybridisation conditions” refers to an overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulphate, and 20 pg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65°C.
  • operably linked refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences.
  • a promoter region may be positioned relative to a nucleic acid sequence such that transcription of a nucleic acid sequence is directed by the promoter region.
  • a promoter region is “operably linked” to the nucleic acid sequence.
  • Transcription refers to the process of producing an RNA copy from a DNA template.
  • a promoter or promoter region for a gene typically provides a recognition site for RNA polymerase and for the other factors, such as transcription factors or enhancers, which are necessary for proper initiation of transcription.
  • the p55PIK promoter region has been identified to comprise a number of sites which bind specific transcription factors or enhancers.
  • reporter gene is a gene which is incorporated into an expression vector and placed under the same controls as a gene of interest to express an easily measurable phenotype.
  • myeloid cell encompasses terminally differentiated, non-dividing (i.e. non- proliferative) cells derived from the myeloid cell lineage and includes neutrophils or polymorphonuclear neutrophils (PMNs), eosinophils and mononuclear phagocytes.
  • PMNs polymorphonuclear neutrophils
  • eosinophils eosinophils
  • mononuclear phagocytes The latter cells are known as monocytes when in the blood and macrophages when they have migrated into the tissues.
  • Terminal differentiation is the normal endpoint in cellular differentiation and is usually not reversible.
  • “Inflammatory disorders” or “inflammatory diseases” are disorders characterised by chronic or acute inflammation. This, in turn, is characterised by elevated levels of cytokines and/or survival factors for myeloid cells. These disorders are characterised by the prolonged survival of myeloid cells including neutrophils, eosinophils and monocytes/macrophages which can be present as a mixture of one or more of these cell types. Accordingly, reference to treatment of inflammatory disorders or diseases includes treatment of the individual cell types or treatment of a mixture of different cell types. The resultant increased numbers of these inflammatory cells is associated with the disease pathology. In chronic inflammation a persistent inflammatory response causes damaging effects such as tissue damage.
  • Chronic Inflammatory Diseases include cystic fibrosis, acute respiratory distress syndrome, chronic obstructive pulmonary disease, inflammatory bowel disease and rheumatoid arthritis.
  • Other inflammatory diseases are known to those skilled in the art and include sepsis, Pre enclampsia, Myocardial ischemia, reperfusion injury, Psoriasis, Asthma, bronchiolitis, Crohns Disease and Ulcerative colitis.
  • PIK3R3 binds to activated (phosphorlated) protein-tyrpsine kinases through its SH2 domain and regulates their kinase activity. During insulin simulation, it also binds to IRS-1. The interaction of PIK3R3 with IGFIR and INSR provides an alternative pathway for the activation of PI 3 -kinase by these 2 receptors.
  • a number of sequence domains are found within the amino acid sequence for p55PIK (as given in SEQ ID NO: 1). These are as follows:
  • SH2 Domain 1 Amino acids 65-160
  • SH2 Domain 2 Amino acids 358-452
  • SH2 Src homology 2 domains bind phosphotyrosine-containing polypeptides via 2 surface pockets. Specificity is provided via interaction with residues that are distinct from the phosphotyrosine. Only a single occurrence of a SH2 domain has been found in S. cerevisiae.
  • the Src homology 2 (SH2) domain is a protein domain of about 100 amino-acid residues first identified as a conserved sequence region between the oncoproteins Src and Fps. Similar sequences were later found in many other intracellular signal-transducing proteins.
  • SH2 domains function as regulatory modules of intracellular signalling cascades by interacting with high affinity to phosphotyrosine-containing target peptides in a sequence-specific and strictly phosphorylation-dependent manner. They are found in a wide variety of protein contexts e.g., in association with catalytic domains of phospholipase Cy (PLCy) and the nonreceptor protein tyrosine kinases; within structural proteins such as fodrin and tensin; and in a group of small adaptor molecules, i.e Crk and Nek. In many cases, when an SH2 domain is present so too is an SH3 domain, suggesting that their functions are inter-related. The domains are frequently found as repeats in a single protein sequence.
  • PLCy phospholipase Cy
  • Nek small adaptor molecules
  • the structure of the SH2 domain belongs to the alpha+beta class, its overall shape forming a compact flattened hemisphere.
  • the core structural elements comprise a central hydrophobic anti-parallel beta-sheet, flanked by 2 short alpha- helices.
  • the loop between strands 2 and 3 provides many of the binding interactions with the phosphate group of its phosphopeptide ligand, and is hence designated the phosphate binding loop.
  • PI3 kinases are enzymes that phosphorylate phosphoinositides on the 3 -hydroxyl group of the inositol ring.
  • the precise functions of the three products of PI3 kinase (PI-3-P, PI-3,4-P and PI-3,4,5-P) are not yet known, but it is suggested that they function as second messengers in signal transduction events in organisms ranging from yeast to mammals.
  • PI3 kinase There are several forms of PI3 kinase. One of these is the mammalian enzyme, a heterodimer of a 1 lOkd catalytic subunit and an 85kd regulatory subunit, which allows it to bind to activated tyrosine protein kinases.
  • PI3 kinase P85 alpha subunits contain an N-terminal SH3 domain, and two SH2 domains in the C-terminal half of the sequence.
  • PI3KINASEP85 is a 10-element fingerprint that provides a signature for PI3 kinase P85-alpha subunits.
  • the fingerprint was derived from an initial alignment of 8 sequences: the motifs were drawn from conserved regions spanning the C-terminal half of the alignment - motifs 1 and 2 lie in the first SH2 domain; and motifs 8-10 span the second SH2 domain. Two iterations on OWL 29.1 were required to reach convergence, at which point a true set comprising 13 sequences was identified.
  • SH2DOMAIN is a 5-element fingerprint that provides a signature for SH2 domains.
  • the fingerprint was derived from an initial alignment of 21 sequences: the motifs were drawn from short conserved regions spanning the full alignment length, and largely correspond to the core structural elements (i.e., the N-terminal helix, each of the central 3 strands, and the C-terminal helix respectively) - motif 2 contains a highly conserved FLVRES sequence involved in phosphate binding (cf. PROSITE profile SH2 (PS50001)). Five iterations on OWL26.2 were required to reach convergence, at which point a true set comprising 187 sequences was identified. Thirty-nine partial matches were also found (the fingerprint does not perform perfectly because of the low level of sequence similarity between SH2 domains (the result of their disparate functions), and is further complicated by their multiple repeat nature).
  • p55PIK also includes within its scope, variants, derivitives and fragments thereof, in as far as they possess the requisite ability to modulate apoptosis.
  • Natural variants of p55PIK are likely to comprise conservative amino acid substitutions.
  • Conservative substitutions may be defined, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
  • Suitable fragments of p55PIK will be at least about 5, e.g. 10, 12, 15 or 20 amino acids in length. They may also be less than 100, 75 or 50 amino acids in length. They may contain one or more (e.g. 5, 10, 15, or 20) substitutions, deletions or insertions, including conserved substitutions.
  • a fragment of p55PIK used in the methods of the present invention must possess the requisite activity of being capable of modulating apoptosis.
  • Levels of gene expression may be determined using a number of different techniques.
  • RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), or RNeasy RNA preparation kits (Qiagen).
  • RNAzol B acid phenol/guanidine isothiocyanate extraction
  • RNeasy RNA preparation kits Qiagen.
  • Typical assay formats utilising ribonucleic acid hybridisation include nuclear run-on assays, RT-PCR and RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035. Methods for detection which can be employed include radioactive labels, enzyme labels, chemiluminescent labels, fluorescent labels and other suitable labels.
  • RT-PCR is used to amplify RNA targets.
  • the reverse transcriptase enzyme is used to convert RNA to complementary DNA (cDNA) which can then be amplified to facilitate detection.
  • DNA amplification methods are known, most of which rely on an enzymatic chain reaction (such as a polymerase chain reaction, a ligase chain reaction, or a self- sustained sequence replication) or from the replication of all or part of the vector into which it has been cloned.
  • an enzymatic chain reaction such as a polymerase chain reaction, a ligase chain reaction, or a self- sustained sequence replication
  • PCR is a nucleic acid amplification method described inter alia in U.S. Pat. Nos. 4,683,195 and 4,683,202. PCR can be used to amplify any known nucleic acid in a diagnostic context (Mok et al., (1994), Gynaecologic Oncology, 52: 247-252).
  • Self- sustained sequence replication (3SR) is a variation of TAS, which involves the isothermal amplification of a nucleic acid template via sequential rounds of reverse transcriptase (RT), polymerase and nuclease activities that are mediated by an enzyme cocktail and appropriate oligonucleotide primers (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874).
  • Ligation amplification reaction or ligation amplification system uses DNA ligase and four oligonucleotides, two per target strand. This technique is described by Wu, D. Y. and Wallace, R. B. (1989) Genomics 4:560. In the Q ⁇ Replicase technique, RNA replicase for the bacteriophage Q ⁇ , which replicates single-stranded RNA, is used to amplify the target DNA, as described by Lizardi et al. (1988) Bio/Technology 6:1197.
  • rolling circle amplification (Lizardi et al., (1998) Nat Genet 19:225) is an amplification technology available commercially (RCATTM) which is driven by DNA polymerase and can replicate circular oligonucleotide probes with either linear or geometric kinetics under isothermal conditions.
  • RCATTM rolling circle amplification
  • SDA strand displacement amplification
  • Primers suitable for use in various amplification techniques can be prepared according to methods known in the art.
  • RNA transcripts RNA transcripts present.
  • Methods for detection which can be employed include radioactive labels, enzyme labels, chemiluminescent labels, fluorescent labels and other suitable labels.
  • the detection of nucleic acids encoding p55PIK can be used, in the context of the present invention, to identify early stage apoptosis in cells - a decrease in p55PIK transcripts is associated with the onset of apoptosis. An increase is associated with cell survival and, in particular, is an early response in GM-CSF-mediated inhibition of apoptosis in neutrophils. b at the polypeptide level
  • Gene expression may also be detected by measuring the p55PIK polypeptide. This may be achieved by using molecules which bind to the p55PIK polypeptide. Suitable molecules/agents which bind either directly or indirectly to p55PIK in order to detect the presence of the protein include naturally occurring molecules such as peptides and proteins, for example antibodies, or they may be synthetic molecules.
  • Standard laboratory techniques such as immunoblotting can be used to detect altered levels of p55PIK, as compared with untreated cells in the same cell population.
  • An example of a suitable protocol is detailed below:
  • Gene expression may also be determined by detecting changes in post-translational processing of polypeptides or post-transcriptional modification of nucleic acids. For example, differential phosphorylation of polypeptides, the cleavage of polypeptides or alternative splicing of RNA, and the like may be measured. Levels of expression of gene products such as polypeptides, as well as their post-translational modification, may be detected using proprietary protein assays or techniques such as 2D polyacrylamide gel electrophoresis.
  • a number of methods are known in the art for monitoring the onset of apoptosis. These include morphological analysis, DNA ladder formation, cell cycle analysis, externalisation of membrane phospholipid phosphatidyl serine and caspase activation analysis.
  • Cell survival may be monitored by a number of techniques including cell cycle analysis and measuring cell viability. Measurements of cell proliferation may be made using a number of techniques including a plaque assay in which adherent cells are plated out in tissue culture plates and left to grow prior to fixing and staining. The number of colonies formed reflects the amount of cell proliferation.
  • the functional activity of p55PIK may be modified by suitable molecules/agents which bind either directly or indirectly to p55PIK, or to the nucleic acid encoding it.
  • Agents may be naturally occurring molecules such as peptides and proteins, for example antibodies, or they may be synthetic molecules.
  • Methods of modulating the level of expression of p55PIK include, for example, using antisense techniques.
  • Antisense constructs i.e. nucleic acid, preferably RNA, constructs complementary to the sense nucleic acid or mRNA, are described in detail in US 6,100,090 (Monia et al), and Neckers et al., 1992, Crit Rev Oncog 3(1 -2): 175-231, the teachings of which document are specifically incorporated by reference.
  • Suitable antisense molecules may be variants, based on these molecules, which have been chemically modified.
  • the antisense nucleic acids can usefully include altered, often nuclease- resistant, internucleoside bonds. See Hartmann et al.
  • modified oligonucleotide backbones are, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • modified oligonucleotide backbones for antisense use that do not include a phosphorus atom have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • RNA-mediated interference is another method for modulating gene expression based on a biological response to double-stranded RNA (dsRNA) resulting in the degradation of homologous mRNA (Demberg and Karpen, Cell 111:159-162, 2002). Since its description in 1998 (Fire and Mello, Nature 391:806-811, 1998) RNAi has rapidly become a standard experimental tool for targeted destruction of mRNAs in worms, flies, plants and mammals (for review see McManus and Sharp, Nature Genetics 3:737-747, 2002).
  • dsRNA double-stranded RNA
  • RNAi is believed to function primarily as a cellular defence mechanism against viruses and transposable elements (Ketting et al, Cell 99:133-141, 1999; Tabara et al, Cell 99:132-132, 1999; see also Elbashir et al, Genes Dev. 15:188-200, 2001).
  • RNAi-like processes also appear to be involved in the post-transcriptional regulation of a variety of metazoan developmental processes (reviewed in Ruvkun, Science 294:797-799, 2001).
  • RNA interference is initiated when the dsRNA is processed to short 21- 23nt fragments (Zamore et al, Cell 101:25-33, 2000; Bernstein et al, Nature 409:363- 366, 2001).
  • siRNAs small interfering RNAs
  • siRNAs are now used routinely in mammalian cells to study the functional consequences of reducing the expression of specific genes (McManus and Sharp, Nature Genetics 3:737-747, 2002). Methods for designing effective siRNAs are described, for example, in http://www.ambion.com/hottopics/rnai.
  • changes in events immediately down-stream of p55PIK such as expression of genes whose transcription is regulated by p55PIK expression, can be used as an indication that a molecule in question affects the functional activity of p55PIK.
  • Compounds having inhibitory, activating, or modulating activity can be identified using in vitro and in vivo assays for p55PIK activity and/or expression, e.g., ligands, agonists, antagonists, and their homologs and mimetics.
  • Modulator screening may be performed by adding a putative modulator test compound to a tissue or cell sample, and monitoring the effect of the test compound on the function and/or expression of p55PIK. A parallel sample which does not receive the test compound is also monitored as a control. The treated and untreated cells are then compared by any suitable phenotypic criteria, including but not limited to microscopic analysis, viability testing, ability to replicate, histological examination, the level of a particular RNA or polypeptide associated with the cells, the level of enzymatic activity expressed by the cells or cell lysates, and the ability of the cells to interact with other cells or compounds.
  • phenotypic criteria including but not limited to microscopic analysis, viability testing, ability to replicate, histological examination, the level of a particular RNA or polypeptide associated with the cells, the level of enzymatic activity expressed by the cells or cell lysates, and the ability of the cells to interact with other cells or compounds.
  • Methods for inducing apoptosis include, without limitation, exposure to chemotherapy or radiotherapy agents and withdrawal of obligate survival factors (e.g. GM-CSF, NGF) if applicable. Differences between treated and untreated cells indicates effects attributable to the test compound.
  • obligate survival factors e.g. GM-CSF, NGF
  • Myeloid cells die spontaneously in culture although with differing time courses depending on the cell type. Neutrophils in culture apoptose within 24 hours although this can be delayed to over 48 hours in the presence of survival factors. Eosinophil apoptosis is observed over 48 hours with a delay to several days in the presence of survival factors. Macrophages are generally much longer lived. Thus, the ability of a compound to modulate myeloid cell apoptosis can be assessed by monitoring the rate of apoptosis in the presence or absence of the test compound and after the withdrawal of obligate survival factors (e.g. GM-CSF, IL-8, IL-5, G-CSF or BAL) if applicable. Differences between treated and untreated cells indicates effects attributable to the test compound.
  • obligate survival factors e.g. GM-CSF, IL-8, IL-5, G-CSF or BAL
  • p55PIK may be expressed in cells by introducing vectors encoding the p55PIK polypeptide.
  • vectors that will drive expression of polypeptides from the inserted heterologous nucleic acid
  • expression vectors will often include a variety of other genetic elements operatively linked to the protein-encoding heterologous nucleic acid insert, typically genetic elements that drive transcription, such as promoters and enhancer elements, those that facilitate RNA processing, such as transcription termination and/or polyadenylation signals, and those that facilitate translation, such as ribosomal consensus sequences.
  • expression vectors will often include a variety of other genetic elements operatively linked to the protein-encoding heterologous nucleic acid insert, typically genetic elements that drive transcription, such as promoters and enhancer elements, those that facilitate RNA processing, such as transcription termination and/or polyadenylation signals, and those that facilitate translation, such as ribosomal consensus sequences.
  • conditions that permit expression of the polypeptide from these vectors will depend on the type of vector and cell expression system chosen.
  • Vectors for expressing proteins are known for expression in prokaryotic cells, in yeast cells, typically S. cerevisiae and in mammalian cells and each include the specifc genetic elements for expression in the particular cell type.
  • Vector-drive protein expression can be constitutive or inducible.
  • Inducible vectors include either naturally inducible promoters, such as the trc promoter, which is regulated by the lac operon, and the pL promoter, which is regulated by tryptophan, the MMTV-LTR promoter, which is inducible by dexamethasone, or can contain synthetic promoters and/or additional elements that confer inducible control on adjacent promoters.
  • Plasmid vectors will typically be introduced into chemically competent or electrocompetent bacterial cells.
  • Vectors can be introduced into yeast cells by spheroplasting, treatment with lithium salts, electroporation, or protoplast fusion.
  • Mammalian and insect cells can be directly infected by packaged viral vectors, or transfected by lipid, chemical or electrical means.
  • Expression vectors can be designed to fuse the expressed polypeptide to small protein tags that facilitate purification and/or visualization.
  • proteins can be expressed with a tag that facilitates purification of the fusion protein.
  • Suitable tags and their purification means are known and include poly- his/immobilized metal affinity chromatography, glutathione-S-transferase/glutathione affinity resins, Xpress epitope/detectable by anti-Xpress antibody (Invitrogen, Carlsbad, CA, USA), myc tag/anti-myc tag antibody, V5 epitope/anti-V5 antibody (Invitrogen, Carlsbad, CA, USA) and FLAG® epitope/anti-FLAG ® antibody (Stratagene, La Jolla, CA, USA).
  • vectors can include appropriate sequences that encode secretion signals, such as leader peptides.
  • Expression vectors can also be designed to fuse proteins encoded by the heterologous nucleic acid insert to polypeptides larger than purification and/or identification tags.
  • Useful protein fusions include those that permit display of the encoded protein on the surface of a phage or cell, fusions to intrinsically fluorescent proteins, such as those that have a green fluorescent protein (GFP)-like chromophore, fusions to the IgG Fc region, and fusions for use in two hybrid systems.
  • GFP green fluorescent protein
  • p55PIK protein can be expressed and purified from systems such as these for use in methods for detecting molecules which interact with p55PIK.
  • p55PIK as an affinity ligand to identify agents which bind to it; labeling p55PIK with a detectable label and using it as a probe to detect apoptotic products in electrophoresis gels; labeling the p55PIK target and using it to probe libraries of genes and/or cDNAs; labeling the p55PIK target and using it to probe cDNA expression libraries to find clones synthesizing proteins which can bind to the target; performing UV-crosslinking studies to identify agents which can bind to the target; using the p55PIK in gel retardation assays which would detect its ability to bind to nucleic acid encoding identified agents; performing footprinting analyses to identify the regions within a nucleic acid to which the target
  • immunoprecipitation Another technique that allows the identification of protein-protein interactions is immunoprecipitation.
  • An example of a protocol for immunoprecipitation is detailed below:
  • lysates from sonicated, Triton X-100-solublized cells (60 ⁇ g protein in lOO ⁇ l PBS with protease inhibitors) are incubated for 90 min at 37°C with 500 ng affinity-purified rabbit polyclonal antibodies specific for p55PIK, followed by an addition of lO ⁇ l packed protein A/G-agarose beads (30 min, 37°C: Santa Cruz Biotechnology), vigorous washing of the pellet (10 min at lOOOOg, 3 x) in PBS, 5% SDS PAGE, and immunodetection with a p55PJX-specific mAb.
  • yeast-two hybrid system Another useful technique for identifying interacting protein is the yeast-two hybrid system described, for example in Bartel et al. (eds.), The Yeast Two-Hybrid System. Oxford University Press (1997) (ISBN: 0195109384) the disclosure of which is incorporated herein by reference.
  • Protein interactions can also be analysed using protein arrays. These may be generated by a range of different techniques which allow proteins to be deposited on a flat surface at different densities. High density protein arrays can be generated using automated approaches similar to those described for DNA arrays (see below). Proteins interacting with p55PIK may be identified by, for example, using p55PIK protein to probe an expression array. Positive interactions could then be detected by the presence of, for example, a labelled antibody or by placing a tag on p55PIK. The identity of the interacting protein can be determined by techniques such as mass spectrometry.
  • Cells useful in the method of the invention may be from any source, for example from primary cultures, from established cell lines, in organ culture or in vivo.
  • Cell lines useful in the invention include cells and cell lines of haematopoietic origin. Suitable cells include HeLa, U937 (monocyte), TF-1, HEK293 (T), primary cultures of neutrophils or cells having neutrophil characteristics, for example HL60 cells, murine FDCP-1, FDCPmix, 3T3, primary or human stem cells.
  • Expression of recombinant p55PIK in cells can be induced using an expression system including any of those described herein.
  • a number of individual gene product types whose expression or function is associated with p55PIK gene expression may be screened for in the present invention. These products include polypeptides and nucleic acids.
  • the expression levels assessed may be absolute levels of production of a particular polypeptide or nucleic acid, or the levels of production of a derivative of any polypeptide or nucleic acid.
  • the invention may be configured to measure the level of expression of a particular mRNA splice variant, or the amount present of a phosphorylated derivative of a particular polypeptide.
  • the gene product to be monitored is unknown, however, methods are employed which facilitate the identification of the gene product whose expression is to be measured.
  • the gene product is a nucleic acid
  • arrays of oligonucleotide probes may be used as a basis for screening populations of mRNA derived from cells.
  • Gene Arrays can additionally be constructed specifically, by spotting nucleotide sequences derived from cDNA clones generated from novel libraries or from cDNA clones purchased commercially. Such arrays allow the expression profiling of proprietary and/or novel nucleotide sequences.
  • Gene Arrays are additionally constructed by commercial sources (e.g. Genescreen), by spotting nucleotide sequences derived from cDNA clones generated from novel libraries or from cDNA clones purchased commercially. Such arrays allow the expression profiling of proprietary and/or novel nucleotide sequences.
  • cDNA sequences or EST (expressed sequence tag) sequences deposited in the public domain databases are derived from a restricted set of tissue types, such as liver, brain and foetal tissue.
  • tissue types such as liver, brain and foetal tissue.
  • the cloning of in-house cDNA libraries which are focused to specific cellular events, such as ROS-mediated apoptosis offers the possibility to identify, clone and characterise novel genes which are associated with this process.
  • the clomng of in-house cDNA libraries which are focused to specific tissue types, such as the neutrophil offers the possibility to identify, clone and characterise novel genes whose expression is restricted to this cell type.
  • cDNA constructed using a physical subtraction, such as the ClonTech 'Select' SSH method (suppression hybridisation) and novel modifications of such, as described, allow the selective cloning of genes whose expression is differentially regulated in the process or cell type being studied.
  • Gene Array technology is combined with SSH cDNA libraries to identify false-positives and further focus on truly differentially expressed genes.
  • Clones from each SSH library constructed are picked, cultured and archived as glycerol stocks.
  • the cDNA inserts contained within individual plasmid clone are PCR amplified and spotted onto in-house arrays. Differential expression is confirmed using hybridisation with a radiolabelled probe generated from the mRNA used for each reciprocal subtractions.
  • Arrays of nucleic acids may be prepared by direct chemical synthesis of nucleic acid molecules. Chemical synthesis involves the synthesis of arrays of nucleic acids on a surface in a manner that places each distinct nucleic acid (e.g., unique nucleic acid sequence) at a discrete, predefined location in the array. The identity of each nucleic acid is determined by its spatial location in the array. These methods may be adapted from those described in U.S. Patent No. 5,143,854; WO90/15070 and WO92/10092; Fodor et al (1991) Science, 251: 767; Dower and Fodor (1991) Ann. Rep. Med. Chem., 26: 271.
  • arrays of nucleic acids may be prepared by gridding of nucleic acid molecules.
  • Oligonucleotides may be advantageously arrayed by robotic picking, since robotic techniques allow the most precise and condensed gridding of nucleic acid molecules; however, any technique, including manual techniques, which is suitable for locating molecules at discrete locations on a support, may be used.
  • the gridding may be regular, such that each colony is at a given distance from the next, or random. If molecules are spaced randomly, their density can be adjusted to statistically reduce or eliminate the probability of overlapping on the chosen support.
  • Apparatus for producing nucleic acid microarrays is available commercially, for example from Genetix and Genetic Microsystems. Moreover, pre-prepared arrays of nucleic acid molecules are available commercially, for example from Incyte Genomics Inc. (Human LifeGrid (TM) ). Such arrays will comprise expressed sequence tags (ESTs) representative of most or all the genes expressed in a cell or organism, thus providing a platform for the screening of mRNA populations from multiple ROS-treated cells.
  • ESTs expressed sequence tags
  • Samples for mRNA population analysis may be isolated and purified by any suitable mRNA production method; for example an RNA isolation kit is available from Stratagene.
  • arrays of antibodies may be used as a basis for screening populations of polypeptides derived from cells. Examples of protein and antibody arrays are given in Proteomics: A Trends Guide, Elsevier Science Ltd., July 2000 which is incorporated by reference.
  • 2D PAGE typically involves sample preparation, electrophoresis in a first dimension on an immobilised pH gradient, SDS-PAGE electrophoresis in a second dimension, and sample detection. Protocols for 2D PAGE are widely available in the art, for example at http://www.expasy.ch /ch2d/protocols/, the contents of which as of 30.11.2001 are incorporated herein by reference.
  • Samples for 2D PAGE may be prepared by conventional techniques.
  • HeLa cells transfected with p55PIK are grown in a suitable medium, such as RPMI 1640 containing 10% foetal calf serum (FCS).
  • FCS foetal calf serum
  • the method of the present invention advantageously employs a step of establishing a reference expression level for the gene products being investigated.
  • This can be carried out by using un-transfected HeLa cells to serve as a standard for one or more subsequent assays; or it may be an integral part of every assay.
  • mRNA or polypeptide populations from HeLa transfected and untransfected cells may be assessed simultaneously on a nucleic acid array or by 2D PAGE, and changes in expression patterns identified by direct comparison. Analysis of 2D PAGE results, using appropriate software where necessary, reveals polypeptides of interest which may be isolated, sequenced and used to identify genes encoding them.
  • primers may be selected using the Primer Design facility of the GeneTool Lite software (Biotools Inc.), which have minimal internal stability and annealing temperatures of 60°C.
  • PCR templates are prepared using genomic DNA purified from HL60 cells, isolated using the Qiagen 'Blood and cell culture DNA mini Kit', (Cat. 13323), as per manufacturers instructions. PCR amplifications are performed using lOOng of genomic DNA as template. Amplimers are gel purified using the Qiaquick gel isolation kit (Qiagen, cat. 28706) and ligated to pCDNA3.1 using the Topo-TA cloning kit (Invitrogen, cat. 45-0005) according to the manufacturers instructions. Ligated DNA is transformed to E.coli (ToplO). Transformants are selected for plasmid DNA preparation and sequence analysis.
  • Plasmid DNA is prepared using either the Qiagen miniprep (cat. 27106) or midiprep (cat. 12643) kits as described by the manufacturer. Insert orientation is determined by PCR with p55PIK-specific reverse primer and vector-specific forward primer (T7 primer). Plasmid miniprep DNA (100 ng to 5 ⁇ g) is sent to MWG Biotech or Lark Technologies for contract sequencing.
  • p53 protein is a transcriptional regulator that plays an important role in cell cycle control, cellular differentiation, apoptosis, genomic stability, and response to DNA damage(Donehower and Bradley (1993) Biochim. Biophys. Acta 1155, 181-205; Friend (1994) Science 265, 334-335; Oren (1992) FASEB J. 6, 3169-3176; Vogelstein and Kinzler (1992) Cell 70, 523-526).
  • the p53 nuclear phosphoprotein can either positively or negatively regulate transcription in a gene- and tissue-specific manner.
  • the promoters of a variety of cellular genes associated with diverse p53- mediated responses are transcriptionally activated by p53 through p53-responsive elements in their promoter regions (Zhao et al (1994) Mol. Cell. Biol. 14, 8483-8492; Zambetti et al (1992) Genes & Dev. 6, 1143-1152; Shivakumar et al (1995) Mol. Cell. Biol. 15, 6785-6793; Shin et al (1995) Mol. Cell. Biol. 15, 4694-4701).
  • the C/EBP SV40 enhancer binding site represents the recognition sequence for a liver-specfic transcription factor implicated in the coordinated and tissue-specific transcriptional regulation.
  • This DNA-binding protein also binds to TTR, alpha 1-AT, and albumin regulatory sites (Costa et al (1988) Proc Natl Acad Sci U S A 85: 3840- 3844).
  • the CCAAT motif is the recognition sequence for the C/EBP family of transcription factors.
  • C/EBPs are a family of transcription factors sharing numerous structural and functional features and to date include C/EBP, ⁇ , ⁇ , ⁇ , ⁇ , ⁇ and ⁇ .
  • C/EBP was the first family member isolated and characterized, and its essential role in hepatocyte and adipocyte differentiation has been demonstrated in knockout mouse models (Lenny et al (1997) Mol.Biol.Rep 24:157-168). Subsequent analysis of the hematopoietic elements in fetal mouse liver revealed its critical role in myelopoiesis (Ward et al (2000) Leukaemia 14:973-990).
  • C/EBP ⁇ has been identified as a critical regulator of terminal granulopoiesis. Alterations in C/EBP ⁇ represent one of the causative mutations in the human disease, neutrophil-specific granule deficiency Yamanaka et al (1997) Proc Natl. Acad. Sci. (USA) 94:13189-13192).
  • C/EBPs and specific Ets family members are important for eosinophil lineage determination (McNagny KM et al. EMBO J 1998 Jul l;17(13):3669-80).
  • GATA-1 and C/EBPbeta synergistically transactivate the promoter of an eosinophil-specific granule protein gene and FOG may act as a negative cofactor for the eosinophil lineage (Yamaguchi Y et al. Blood 1999 Aug 15;94(4):1429-39).
  • FOG is a repressor of the eosinophil lineage
  • C/EBP -mediated down-regulation of FOG is a critical step in eosinophil lineage commitment.
  • Maintenance of a multipotent state in hematopoiesis is achieved through cooperation between FOG and GATA-1 (Querfurth E et al. Genes Dev 2000 Oct l;14(19):2515-25).
  • the NRE-1 box was originally identified from the promoter of the chicken lysosomal gene, which is expressed in macrophages.
  • the NRE-1 element functions as a transcriptional silencer, exhibiting an inverse linear relationship between the transcriptional activity of the promoters containing this element: weak transcription units can be strongly repressed, whereas strong transcription units can be only weakly repressed (Baniahmad et al (1987) EMBO J. 6: 2297-2303). Such a mechanism may help to turn off completely a particular gene in situations or tissues where strong positive regulators are inactive.
  • AP-2 transcription factors represent a family of three closely related and evolutionarily conserved sequence-specific DNA-binding proteins, AP-2alpha, -beta and -gamma.
  • AP-2 proteins play a critical role in cell growth, differentiation and programmed death, where mutations or changes in precisely programmed expression patterns are likely to contribute to congenital malformations or neoplastic diseases (Hilger-Eversheim et al (2000) Gene 260(1 -2): 1-12).
  • thioredoxin is a potent stimulator of AP-2 DNA binding, probably through the oxidation state of conserved cysteine residues in the AP-2 DNA binding domain (Huang et al (1998) Biochem Biophys Res Commun 249(2):307-12).
  • the Sp/KLF family is comprised of at least twenty members which include Spl-4 and numerous kruppel-like factors. Members of this family bind with varying affinities to sequences designated as 'Spl sites' (e.g., GC-boxes, CACCC-boxes, and basic transcription elements). Family members have different transcriptional properties and can modulate each other's activity by a variety of mechanisms. Since cells can express multiple family members, Sp/KLF factors are likely to make up a transcriptional network through which gene expression can be fine-tuned. 'Spl site'-dependent transcription can be growth-regulated, and the activity, expression, and/or posttranslational modification of multiple family members is altered with cell growth.
  • 'Spl site'-dependent transcription can be growth-regulated, and the activity, expression, and/or posttranslational modification of multiple family members is altered with cell growth.
  • Sp/KLF factors are involved in many growth-related signal transduction pathways and their overexpression can have positive or negative effects on proliferation.
  • Sp/KLF factors have been implicated in apoptosis and angiogenesis; thus, the family is involved in several aspects of tumorigenesis (Black et al, (2001) J. Cell Physiol. 188(2): 143-60; Philipsen et al (1999) Nucleic Acids Res. 27(15):2991-3000).
  • reporter gene is a gene which is incorporated into an expression vector and placed under the same controls as a gene of interest to express an easily measurable phenotype.
  • reporter genes are known whose expression may be detectable by histochemical staining, liquid scintillation, spectrophotometry or luminometry.
  • reporter systems have been adapted for a broad range of assays, including colorimetric, fluorescent, bioluminescent, chemiluminescent, ELISA, and/or in situ staining. Suitable reporter systems are based on the expression of enzymes such as chloramphenicol acetyltransferase (CAT), b-galatosidase (b-gal), b-glucuronidase, alkaline phosphatase and luciferase. More recently, a number of reporter systems have been developed which are based on using Green fluorescent proteins (GFP) or various derivatives or mutant forms including EGFP. Reporter genes and detection systems are reviewed by Sussman in The Principle 15[15]:25, Jul. 23, 2001 which is incorporated by reference.
  • GFP Green fluorescent proteins
  • Primers to amplify the promoter region are selected, using the Primer Designer facility of the GeneTool Lite software (Biotools fric), which have minimal internal stability and annealing temperatures of 60°C.
  • a PCR reaction is carried out to amplify the promoter sequence from genomic DNA.
  • Amplimers are gel purified using the Qiaquick gel isolation kit (Qiagen, cat. 28706) and ligated to pCDNA3.1 using the Topo-TA cloning kit (Invitrogen, cat. 45-0005) according to the manufacturers instructions.
  • Ligated DNA is transformed to E.coli (ToplO). Transformants are selected for plasmid DNA preparation and sequence analysis.
  • Plasmid DNA is prepared using either the Qiagen miniprep (cat. 27106) or midiprep (cat. 12643) kits as described by the manufacturer. Insert orientation is determined by PCR with p55PEK-specific reverse primer and vector-specific forward primer (T7 primer). Plasmid miniprep DNA (100 ng to 5 ⁇ g) is sent to MWG Biotech or Lark Technologies for contract sequencing.
  • U937 cells are transfected with lO ⁇ g of an EGFP reporter construct (pEGFP, Clontech), containing a genomic fragment driving the expression of the EGFP gene (p55PIK -EGFP).
  • the fragment includes the putative p55PIK promoter region.
  • Cells are transfected by the calcium phosphate method. Transfection of the pEGFP vector without the p55PIK genomic fragment is used as a negative control whereas a construct containing the CMV promoter serves as a positive control.
  • U937 cells containing either pEGFP or p55PIK-EGFP are treated with GM-CSF (50 Units) either in the presence or absence of gliotoxin (O.l ⁇ g/ml).
  • GM-CSF 50 Units
  • gliotoxin O.l ⁇ g/ml
  • cells are examined for EGFP expression by flow cytometric analysis using a FacsCalibre (Becton Dickinson). Cells are considered positive for EGFP expression when the FL1 signal is greater than the background signal generated by either pEGFP or untreated p55PIK-EGFP. All values are corrected for transfection efficiency by standardization against ⁇ -gal activity, derived from the cotransfected plasmid pSV ⁇ -gal (Promega).
  • SNPs Single nucleotide polymorphisms
  • SNPs Single nucleotide polymorphisms
  • SNPs Single nucleotide polymorphisms
  • SNPs may responsible for variations between individuals, including variations which predispose an individual to a disease or cause it. Approximately half of all coding sequence SNPs result in synonymous (i.e. silent) codon changes. Even those these SNPs may have no effect on protein function, they are potentially useful for tracking other variations nearby as adjacent stretches of DNA tend to be inherited together ('linkage disequilibrium').
  • Informative SNPs accelerate the identification of disease genes by allowing researchers to look for associations between a disease and specific SNPs in a population. SNP distributions are informative for drug response and allow stratification of populations for particular therapy or drug treatment regimens.
  • DoubleTwist (www.doubletwist.com) tools were used to analyse the target sequences retrieved from Genbank.
  • the DoubleTwist suite incorporates a number of research agents to generate computational analysis outputs using algorithms that search multiple gene, protein, and patent databases for information about query sequences. These tools access the DoubleTwist annotated databases and all published information about the query sequences. For the purpose of this study the following agents were used: Perform Comprehensive Sequence Analysis; Retrieve Assembled ESTs; Retrieve and Analyse Human Genome.
  • the Comprehensive Sequence Analysis agent uses the BLAST2N, BLAST2X, TBLAST2N, and BLAST2P algorithms to search the following databases: SwissProt; NR-Nuc; NR-Pro; dbEST; PDB; PAT; PATaa; HTG; Genbank's cumulative nightly nucleotide and protein database updates; and Myriad Genetics ProNet database. Additionally the Blimps and Blkprob algorithms are used to search the Blocks ⁇ database. This agent provides information about functional protein identities and similarities, DNA identities and similarities, patented sequences, protein domains, structural identities and similarities, and genomic DNA identities and similarities.
  • the Assembled ESTs agent (Human) identifies matching EST clusters derived from the DoubleTwist Gene Indices.
  • the Gene Indices are collections of assembled EST and mRNA sequences derived by, screening out non-informative sequences (such as vector and ribosomal sequences), clustering the remaining sequences, first by matching pairs for overlapping bases, then by sub-dividing into gene variants (subclusters); aligning the sequences in each cluster, and deriving a consensus sequence for each cluster and subcluster.
  • the sequence collection is therefore checked and statistically corrected for many sequencing and cloning errors such as orientation, chimerism, and contamination.
  • DoubleTwist' s interactive data-mining tool Cluster Viewer was used to visualise the alignments.
  • the "Analyse Human Genome” agent also uses a proprietary DoubleTwist genome database derived from public data. Genomic sequences that are at least 15 kilobases in length are obtained from Genbank's Genomic Sequences Primate (GB PRI) division. Unfinished human genomic sequences are obtained from Genbank's High Throughput Genomic (HTG) Sequences division. The data is annotated by splitting the HTG sequences phase 0, 1, and 2 into component fragments while maintaining the GB PRI sequences intact.
  • Sequence contamination, from vector, bacterial, yeast or mitochrondrial sequences are masked and the Repeat Masker program (http://repeatmasker.genome.wasliington.edu/cgi-bin/RM2_req.pl) is used to mask repetitive elements and regions of low complexity.
  • the GrailEXP, FGENESH and Genscan algorithms are then employed to predict coding regions, introns and exons.
  • the Halfwise algorithm is used to match predicted coding regions with models from the Pfam database.
  • the Unigene database and the DoubleTwist Human Gene Index are further searched for DNA similarities using the BLASTN algorithm and the NR Pro database is searched, using BLASTX, for similar proteins.
  • Double Twist Genomic Viewer an interactive data mining and visualization tool was used to examine the output from the Genome Analysis agent.
  • Example 1 Gene expression and cluster analysis of neutrophil apoptosis and survival; p55PIK is identified by association, as a modulator of apoptosis and cell survival
  • the pellet is resuspended in 1 ml cell culture tested water (Sigma) for 40 sec, followed by the addition of 14ml Hanks buffer (Sigma) and centrifuged (300g, 10 min.). This lysis step is repeated to ensure removal of all erythrocytes.
  • the remaining pellet is resuspended in RPMI 1640 supplemented with 10% foetal calf serum (Sigma), L-glutamine (2mM), penicillin (100 U/ml; Sigma), streptomycin (100 ⁇ g/ml; Sigma) and amphotericin B (2.5 ⁇ g/ml; Sigma). Cell number and viability is checked using trypan blue exclusion (Boyum, (1968) ScandJClin Lab Invest Suppl; 97:77-89).
  • Isolated neutrophils are maintained at a density of 2 xlO 6 / ml in RPMI 1640 supplemented with 10% foetal calf serum (Sigma). Further additions to the medium included L-glutamine (2mM), penicillin (100 U/ml), streptomycin (100 ⁇ g/ml) and amphotericin B (2.5 ⁇ g/ml) (Sigma). Cells are incubated at 37°C in a humidified CO 2 (5%) incubator. As described in by Haslett (Clinical Science 83, pp 639-648, 1992), WO 01/46469 and WO 02/04657, upon culture in a serum-containing cell culture medium these neutrophils undergo spontaneous apoptosis.
  • GM-CSF Primary human neutrophils are isolated and purified from peripheral blood of normal healthy individuals. Neutrophils are resuspended in serum containing culture medium together with various amounts of GM-CSF at a density of 2xl0 6 /ml, with lOO ⁇ l plated into a 96 well plate and cultured for 18h at 37°C. After this time lO ⁇ l of MTT (5mg/ml) is added to the cultures and incubated for a further 4h at 37°C before solubilisation of the purple coloured formazan with acidic isopropanol. Optical densities are read at 570nm using a plate reader. Figure 1 shows there is a direct conelation between survival and concentrations of GM-CSF added to the culture medium.
  • neutrophils are resuspended in serum containing culture medium containing 5 U/ml of GM-CSF.
  • Fungal metabolite Gliotoxin blocks GM-CSF inhibition of neutrophil apoptosis.
  • This inhibitor allows us to focus in on the specific biochemical events mediating the GM-CSF survival events. In turn one is able to remove some of the noise associated GM-CSF treatment.
  • Neutrophils are isolated and purified from peripheral blood of normal healthy individuals. Neutrophils are resuspended in serum containing culture medium containing 5 U/ml of GM-CSF at a concentration of 2xl0 6 /ml. Also added to the culture mix is either O.l ⁇ g/ml of the fungal metabolite Gliotoxin or its inactive analogue bis -Dethio -bis (Methylthio) Methyl Gliotoxin, with lOO ⁇ l/well plated into a 96 well plate and culture at 37°C commenced.
  • lO ⁇ l of MTT 5mg/ml are added to the cultures and incubated for a further 4h at 37°C before solubilisation of the purple coloured formazan with acidic isopropanol.
  • Optical densities are read at 570nm using a plate reader.
  • Figure 2 demonstrates that gliotoxin effectively blocks the GM-CSF inhibition of neutrophil apoptosis. This blocking effect is not seen when the inactive analogue of gliotoxin, methylgliotoxin is added with GM-CSF. No increased neutrophil apoptosis is seen with the addition of gliotoxin alone to isolated neutrophils demonstrating that the effect is specific to and limited to a reversal of the protective effects of GM-CSF.
  • microarrays are used to measure global gene expression associated with neutrophil apoptosis, GM-CSF inhibition of neutrophil apoptosis, and the inhibition of this effect using the fungal metabolite Gliotoxin.
  • an inactive analogue of Gliotoxin Methyl Gliotoxin is used. Analysis of such microarray results identifies genes whose expression pattern changes (either up-regulation or down- regulation) in an association with a measurable apoptotic phenotype.
  • RNAzol B acid phenol/guanidine isothiocyanate extraction
  • RNeasy RNA preparation kits Qiagen. Any contaminating genomic DNA is removed by DNase treatment (DNase I, Gibco-BRL).
  • RNA is also prepared from neutrophil cells following treatment (for the time indicated in hours) with GM-CSF (50units/ml), Gliotoxin (lO ⁇ M) or MethylGliotoxin (lO ⁇ M). RNA is also prepared from neutrophils that have not been exposed to drug (i.e. as an untreated control). RNA is prepared from these cells using two sequential extractions with RNAzol B.
  • microarraying can be used to profile gene expression of thousands of genes simultaneously.
  • the microarray process is described both for the use of Human LifeGridTMmicroarray filters and can be separated into three parts: the filter, the hybridisation of radiolabelled cDNA probe, and the detection and quantitation of the microarray results.
  • microarray filter This example describes the use of the Human LifeGridTMmicroarray filters obtained from Incyte Genomics (USA). These filters contain cDNA probes representing approximately 8,400 human mRNAs.
  • Hybridisation of radiolabelled cDNA probes This example describes the synthesis of a radiolabelled cDNA from total cellular mRNA.
  • the labeled cDNA is used to 'probe' DNA fragments, which have been immobilised on to a filter membrane, by complementary hybridisation.
  • RNA is reverse transcribed to first strand cDNA in a reaction containing M-MLV reverse transcriptase (RT; alternatively Superscript II is used (Life Sciences)), RT buffer, dNTPs and [ ⁇ - 33 P] dCTP (2000- 4000 Ci/mmol) at 42°C for 1 to 5hours. Unincorporated nucleotides are removed using spin-columns and the labeled probe stored at -80°C until required.
  • RT M-MLV reverse transcriptase
  • Labeled probes may also be generated from cDNA, genomic DNA or PCR products.
  • a random primed labeling procedure can be used, for example the Ready- Prime Labeling kit (APBiotech), applied as per manufacturers instructions.
  • Radiolabelled cDNA probe is hybridised to DNA fragments immobilised onto a membrane (typically a nylon or nitrocellulase filter).
  • membrane filters are pre-hybridised in hybridisation buffer (5 to 20 ml) at 42°C for 2 to 16 h using a hybridisation oven (Hybaid). Following pre-hybridisation, the labeled cDNA probe is added to fresh hybridisation buffer (5 to 20 ml) and hybridised at 42°C for 14 to 16 h. Following hybridisation, the hybridisation mix is removed and the filters washed with 2 x SSC buffer at RT for 5 min., twice with 2 x SSC, 1% SDS buffer at 68°C for 30 min. and twice with 0.6 x SSC, 1% SDS buffer at 68°C for 30 min.
  • Hybridised filters are wrapped in plastic wrap (Saran) and exposed to a Low-Energy Phosphoimaging screen (Molecular Dynamics). The screen is then placed on the phosphoimager and the gel image captured by scanning at a resolution of 50 microns (See Figure 3).
  • the captured image file is then analysed using software such as Array Vision (Imaging Research Inc.; See Figure 4).
  • ArrayVision Imaging Research Inc.; See Figure 4
  • This program contains facilities for spot detection and quantification, and background detection and quantification.
  • This data is then exported to a text file for further analysis.
  • a variety of data fields are exported from the AnayVision analysis, including; Spot Label, Position, Density, Background, and particularly, Background subtracted density (sDens) and signal noise ratio (S/N).
  • the exported text file is up-loaded to an SQL-7.0 database, to populate a table containing anay data from all experiments. As the data is imported to the database, a Normalisation factor is calculated and the sDENs values modified accordingly.
  • This Normalised data is stored in a newly created column within the table.
  • the Normalisation factor facilitates accurate comparison between datasets.
  • a number of different calculations may be used.
  • a normalization factor may be derived from Linear Regression calculated by reference to housekeeping genes.
  • the Global Mean is calculated as the average of the sDens values across all of the arrays to be compared and a normalisation factor is then derived by division of the overall spot density with the Global Mean value. Spot density values (individual sDens) are then corrected by multiplying across all values with the normalisation factor.
  • a Global Geometric Mean normalization factor may be calculated and used to adjust the dataset.
  • the data from multiple hybridisation experiments can then be stored in a suitable format, for example in an Access or SQL 7.0 database.
  • Comparison between arrays generates an output file containing the gene identifier and the fold-change in expression relative to the reference dataset.
  • Fold change, (Tx vs Ty) is calculated by dividing the normalised spot density values of Tx with Ty.
  • multiple time-course experiments are prepared and fold-change values calculated with reference to the TO time point.
  • the fold change data derived from comparison of multiple hybridisation experiments can be analysed using a variety of approaches, including hierarchical clustering, (supervised or unsupervised), k-means clustering or self-organising maps.
  • Software enabling these analyses includes the Cluster and Treeview software (M.Eisen, Stanford Uni, USA), J-Express (European Bioinformatics Institute), GeneMaths (Applied Maths, Belgium) or GeneSpring (Silicon Genetics, USA).
  • Hierarchical clustering is implemented using the GeneMaths software. Trees are generated using the WARD algorithm with distance calculated using the Pearson similarity metric. Alternatively Euclidean distance metrics are used. Simplification of Fold-change data
  • fold-change data can be difficult to interpret owing to either a very large dataset and/or a wide range in fold change values.
  • the visualization and interpretation of these datasets may be simplified using codes or combined codes.
  • each unique gene is represented by at least two identical cDNAs on the array.
  • the fold change value is calculated as described, then for each spot, a value above 5-fold change is accorded a code of 2, a fold-change value of less then 5 but greater then 2 is accorded a code of 1 and a fold-change value of less then 2 is accorded a code value of 0.
  • a combined code is then derived by adding the code values for each identical cDNA on the array.
  • the use of combined codes can greatly simplify the Cluster analysis and the subsequent visualisation (See Figure 5).
  • Comparison of coordinate patterns of gene expression, by bioinformatic data analysis, using this model system, allows the identification of cell pathways and processes associated with apoptosis and survival.
  • 'differentially regulated' genes are identified and clustered by either normalised sDens (level of expression) or by fold chance values.
  • Candidate genes, associated with apoptosis and survival are those that are reproducibly differentially regulated in multiple experiments or time courses and are additionally 'reciprocally regulated' in conditions that permit apoptosis versus survival, respectively.
  • Figure 6 shows the visual representation of a clustered selection of candidate neutrophil apoptosis/survival-associated genes identified of LifeGrid filters. Each row represents the differential regulation of an individual gene. The Fold Change colour scale is shown.
  • Apop neutrophil apoptosis time course experiments are represented (Apop), with RNA samples isolated at 2 h (Apop2), 3 h (Apop3), 4 h (Apop4), 5 h (Apop5) and 6 h (Apop ⁇ ) post-isolation of neutrophils. Fold change values are expressed relative to zero hour control samples.
  • GM-CSF Three representative GM-CSF time course experiments are represented (GM-CSF), with RNA samples isolated at 2 h (GMCSF2), 4 h (GMCSF4) and 6 h (GMCSF6) post-treatment with GM-CSF. Fold change values are expressed relative to zero hour control samples.
  • GM- CSF Blockage of GM-CSF -mediated inhibition of neutrophil apoptosis by treatment with Gliotoxin
  • Gliotoxin Gliotoxin
  • Methodyl Methyl Gliotoxin
  • Fold change values are expressed relative to zero hour control samples.
  • GM 4 RNA samples are isolated at 4 h post-treatment with GM-CSF, and fold change values are expressed relative to Methyl Gliotoxin control samples.
  • RNA sample profiled by microarray, represents the pool of multiple experiments carried out on neutrophils isolated from individual human donors.
  • the number of donor samples used for each experiment/time course is summarised in Table 1.
  • Average fold change values (from two spots on the array filters) are clustered with GeneMaths, using a Pearson correlation and Ward clustering algorithm.
  • Candidate genes represented in this selection share similar overall expression characteristics, that of an 'apoptosis/survival cluster'.
  • Candidate genes tend to be down-regulated (dark) in multiple experiments and time courses for apoptosis (Apop, GM and Glio; see legend) and up-regulated (light) in experiments and time courses for survival (Methyl and GMCSF; see legend).
  • p55PIK One of the differentially expressed genes associated with apoptosis and survival is identified as p55PIK.
  • Example 2 pSSPIK mRNA is increased in GM-CSF-induced neutrophil survival, and this increased expression is blocked by Gliotoxin
  • Figure 7 shows the relative amounts of p55PIK transcripts isolated from neutrophils treated according to Example 1. Experimental conditions and cluster analysis of average fold change comparisons are as described in Example 1.
  • p55PIK is up-regulated in multiple experiments between 2 and 6 h following addition of GM-CSF. Up-regulated genes may represent potential survival factor genes, which block or delay the apoptosis in neutrophils. Increase expression of p55PIK, following GM-CSF treatment, is blocked by the fungal inhibitor gliotoxin (Glio and GM; see legend).
  • Example 3 Cluster analysis and gene function
  • Figure 8 shows a dendrogram representation of the association of candidate genes from the cluster analysis illustrated in Figure 1 (performed using the method detailed in Example 1) of p55PIK expression compared to other known genes that have a similar pattern of gene expression across multiple experiments.
  • these are cytochrome c oxidase subunit Nllb (2060789), BH3 interacting domain death agonist (2782033), BCL2-related protein Al (2555673), CD53 antigen (3003048), interleukin 1 receptor antagonist (519653), ATP-binding cassette, sub-family B (MDR/TAP), member (2887130), GRO3 oncogene (617159) and nerve growth factor, beta polypeptide (2887215). All of these genes are known to be involved in apoptosis and survival.
  • cytochrome c oxidase, CD53 and interleukin 1 receptor antagonist are also associated with Redox regulation.
  • Cytochrome c oxidase (COX), the terminal component of the respiratory chain complex of most aerobic organisms, is composed of 13 subunits in mammals. Mitochondrial release of cytochrome c is one of the principle stepps initiating the execution of apoptosis. Mitochondrial antisense R ⁇ A for cytochrome C oxidase can induce morphologic changes and cell death in human hematopoietic cell lines (Blood 1997 Dec l;90(l l):4567-77).
  • BH3 interacting domain death agonist otherwise known as BID, is activated by the pro-apoptotic cascade. This causes BID to oligomerize BAK or BAX into pores that result in the release of cytochrome c. (for review see Cell Death Differ 2000 Dec;7(12):l 166-73).
  • BCL2-related protein Al otherwise known as Bfl-1 was first isolated by Lin et al. (1993) as a novel mouse cDNA sequence, designated BCL2 -related protein Al (BCL2al) and was identified as a member of the Bcl-2 family of apoptosis regulators by the predicted protein sequence.
  • BCL2al BCL2 -related protein Al
  • An anti-apoptotic role of Bfl-1 is described in staurosporine-treated B-lymphoblastic cells (Int J Hematol 2000 Dec;72(4):484-90).
  • CD53 is an N-glycosylated pan-leukocyte antigen of 35,000 to 42,000 MW. Increased expression of CD53 has been described on apoptotic human neutrophils (J Leukoc Biol 2000 Mar;67(3):369-73). Noehringer DW et al, described CD53 associated with resistance to ionising radiation, using microarray experiments. Expression of CD53 can lead to the increase of total cellular glutathione, which is the principle intracellular antioxidant and has been shown to inhibit many forms of apoptosis (Proc ⁇ atl Acad Sci U S A 2000 Mar 14;97(6):2680-5).
  • the Inter Leukin 1 receptor antagonist (IL1R ⁇ ) is a protein that binds to ILl receptors and inhibits the binding of ILl -alpha and ILl -beta.
  • Interleukin- 1 receptor antagonist provides cardioprotection against ischemia-reperfusion injury associated with reduction in apoptosis (Circulation 2001 Sep 18;104(12 Suppl 1):I308- 13). Hypoxia induces the expression and release of interleukin 1 receptor antagonist in mitogen-activated mononuclear cells (Cytokine 2001 Mar 21;13(6):334-41).
  • IL-lra gene up-regulates interleukin- 1 beta converting enzyme (ICE) gene expression: possible mechanism underlying IL-lbeta-resistance of cancer cells (Br J Cancer 1999 Sep;81(2):277-86).
  • ICE interleukin- 1 beta converting enzyme
  • MDR/TAP ATP-binding cassette, sub-family B
  • MDRl multiple drug resistance
  • Increased expression and amplification of MDRl sequences were also found in multidrug-resistant sublines of human leukemia and ovarian carcinoma cells.
  • Overexpression of MDRl appears to be a consistent feature of mammalian cells displaying resistance to multiple anticancer drugs and has been postulated to mediate resistance.
  • GRO3 oncogene The GRO gene, a CXC chemokine otherwise known as macrophage inflammatory protein 1 beta (MIP-1B), was initially identified by Anisowicz et al. (1987) by its constitutive overexpression in spontaneously transformed Chinese hamster fibroblasts. Neutrophils have been shown regulate their own apoptosis via preservation of CXC receptors. Gro-alpha and IL-8 (CXC chemokines) suppress neutrophil apoptosis (Neu J Surg Res 2000 May l;90(l):32-8).
  • MIP-1B macrophage inflammatory protein 1 beta
  • Nerve growth factor is a well-characterised cytokine survival factor. NGF withdrawal induces apoptosis in a range of cells in-vitro and in- vivo. Nerve growth factor suppresses apoptosis of murine monrophils (Biochem Biophys Res Commun 1992 Jul 31;186(2): 1050-6).
  • Example 4 pSSPIK is adjacently correlated with two PDK-signal transduction associated mRNAs.
  • Phosphoinositide 3-kinases phosphorylate the 3'-OH position of the inositol ring of inositol phospholipids, producing three lipid products: Ptdfr ⁇ s(3)P, PtdIns(3,4)P(2) and Ptdh ⁇ s(3,4,5)P(3). These lipids bind to the pleckstiin homology (PH) domains of proteins and control the activity and subcellular localisation of a diverse array of signal transduction molecules.
  • PH pleckstiin homology
  • guanine- nucleotide-exchange proteins for Rho family GTPases
  • the TEC family tyrosine kinases such as BTK and ITK in B and T lymphocytes, respectively
  • AGC superfamily of serine/threonine protein kinases are activated by a variety of extracellular stimuli and have been implicated in a wide range of cellular processes, including cell cycle progression, cell growth, cell motility, cell adhesion and cell survival.
  • PI3K has been implicated as a key mediator of GM-CSF-mediated inhibition of neutrophils apoptosis (J Immunol 2000 Apr 15;164(8):4286-91).
  • P55PIK The catalytic sub-unit of PI3K, pi 10 interacts with regulatory sub units, p85 and p55 (p55PIK).
  • P55PIK has been described to have multiple translation initiation start sites coding for multiple proteins (Biochem J 1999 Aug 1;341 ( Pt 3):831-7).
  • p55PIK regulatory sub unit The function of p55PIK regulatory sub unit is largely unknown. It has been shown to interacts in vitro with other non-PI3K proteins, such as the activated Insulin Growth Factor Receptor and Insulin Receptor derived from mammalian cells. p55PD interaction with the IGFIR and IR and may be involved in PI 3-kinase activation by these receptors (Gene 1998 Mar 16;209(1 -2): 175-83).
  • Thyroid stimulating hormone, beta Recombinant TSHbeta was found to significantly enhance the phagocytic activity of dendritic cells and to selectively augment IL-1 beta and IL-12 cytokine responses of dendritic cells following phagocytic activation (J Txnm ⁇ nol 2000 Jun 15; 164(12):6158-65). TSHbeta also inhibits Fas-mediated apoptosis by cytotoxic CD4+ T cells (Lab Invest 2000 Apr;80(4):471-84). The thyroid-stimulating hormone (TSH) binds to a receptor, which activates adenylate cyclase and elevates cAMP concentration. In addition, effects of TSH on intracellular calcium and inositol phosphate accumulation, through activation of PI3K, have been reported (J Endocrinol 1998 Jun; 157(3):415-24).
  • Thyroid stimulating hormone acting through cAMP, can induce DNA synthesis. This effect however also requires stimulation of the insulinlike growth factor- 1 (IGF-1) receptor by either IGF-1 or insulin, which are not themselves mitogenic agents.
  • IGF-1 insulinlike growth factor- 1
  • the stimulation of the PI 3-kinase/PKB pathway may account for the permissive action of insulin/IGF- 1 in the proliferation of these cells (Biochem J 2000 Jun 1 ;348 Pt 2:351 -8).
  • TNFRSF TNFRSF-interacting serine-threonine kinase 1
  • FAS Two cell surface cytokine receptors, FAS and the tumor necrosis factor (TNF) receptor trigger apoptosis by natural ligands. Both receptors contain a conserved intracellular death domain.
  • the death domain of TNFRl triggers distinct signaling pathways leading to apoptosis and NF-kappa-B activation through its interaction with the death domain protein TRADD.
  • TRAF protein family have been implicated in the activation of NF-kappa-B by the TNF superfamily. Hsu et al. (1996) isolated a full- length human RIP cDNA.
  • the predicted 671 -amino acid RIP protein contains an N-terminal protein kinase domain, a C-terminal death domain, and a unique internal region that they called the intermediate domain.
  • RIP and TRADD interacted via their respective death domains; RIP interacted with TRAF2 via either the kinase or the intermediate domain.
  • TRADD acted as an adaptor protein to recruit RIP to the TNFRl complex in a TNF-dependent process.
  • the death domain kinase, receptor interacting protein (RIP), is one of the major components of the tumor necrosis factor receptor 1 (TNFRl) complex and plays an essential role in tumor necrosis factor (TNF)-mediated nuclear factor kappaB (NF- kappaB) activation.
  • TNF tumor necrosis factor
  • NF- kappaB nuclear factor kappaB
  • Phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) zeta were identified as downstream signaling molecules of TNFalpha-activation of c-Rel transactivating activity.
  • PKC protein kinase C
  • dominant negative forms of PI3K inhibited PKCzeta activation and dominant negative PKCzeta inhibited PI3K-mediated activation of c-Rel transactivating activity, indicating a cross-talk between both enzymes (J Biol Chem 2001 May 11 ;276(19): 15840-9).
  • TNF can also inhibit aspects of insulin signaling through the insulin receptor, such as activation and tyrosine phosphorylation of insulin receptor substrate- 1.
  • Example 5 p55PIKis adjacently correlated with mRNAs associated with apoptosis, survival and associated signal transduction pathways, which further illustrate the cooperativity of PI3K activation.
  • NADH dehydrogenase ubiquinone 1 alpha subcomplex, 1 (NADHtubiquinone oxidoreductase (complex I)): The multisubunit NADH:ubiquinone oxidoreductase (complex I) is the first enzyme complex in the electron transport chain of mitochondria. Antiapoptotic properties of Bcl-2 are related to the reduction of mitochondrial complex I activity (Biochem Biophys Res Commun 2001 Feb 2;280(4): 1021-7).
  • the mitochondrial electron transport enzyme NADH:ubiquinone oxidoreductase (complex I) which is encoded by both mitochondrial DNA and nuclear DNA, is defective in multiple tissues in persons with Parkinson's disease (PD).
  • NQO1 may play an important role in the neurodegeneration of PD by fostering reactive oxygen species production and conferring increased neuronal susceptibility to mitochondrial toxins (Ann Neurol 1996 Oct;40(4):663-71).
  • Cells overexpressing NQO1 were resistant to dicoumarol, and this finding indicates the direct involvement of NQO1 in p53 stabilization.
  • NQO1 inhibition induced p53 degradation and blocked wild-type ⁇ 53-mediated apoptosis in gamma-irradiated normal thymocytes and in Ml myeloid leukemic cells that overexpress wild-type p53.
  • Dicoumarol also reduced the level of p53 in its mutant form in Ml cells.
  • the results indicate that NQO1 plays an important role in regulating p53 functions by inhibiting its degradation (Proc Natl Acad Sci U S A 2001 Jan 30;98(3):1188-93).
  • ING1 Inhibitor of growth family, member 1 ING1): ING1 shares many biological functions with p53. It has been reported to mediate growth arrest, senescence, apoptosis, anchorage-dependent growth, and chemosensitivity. Some of these functions, such as cell-cycle arrest and apoptosis, have been shown to be dependent on the activity of both ING1 and ⁇ 53 proteins (Exp Cell Res 2001 Aug l;268(l):l-6). ING1 competitively binds proliferating cell nuclear antigen (PCNA) through a site used by growth regulatory and DNA damage proteins, and may contribute to regulating the switch from DNA replication to DNA repair by altering the composition of the PCNA protein complex. J Cell Sci 2001 Oct;l 14(Pt 19):3455-62.
  • PCNA proliferating cell nuclear antigen
  • Leukemia-associated phosphoprotein pl8 (stathmin): Increased level of Stathmin (an 18-kD cytosolic phosphoprotein (pi 8)) is found in the cells of various types of human acute leukemia. Its expression and phosphorylation are regulated throughout development in response to extracellular signals regulating cell proliferation, differentiation, and function. The overall pattern of its molecular forms reflects the activation of corresponding second messenger pathways. This phosphoprotein is therefore a good candidate for a general relay in signal transduction, possibly integrating diverse signals from the cell's environment
  • Stathmin is an abundant cytosolic phosphoprotein that plays an important role in the regulation of cellular proliferation. A major function is to promote depolymerization of the microtubules that make up the mitotic spindle. There is growing evidence that the p53 tumor suppressor protein not only can function to activate gene transcription but also to repress the expression of specific genes, including stathmin (Genes Dev 1999 Oct 1;13(19):2490-501).
  • GNGll Guanine nucleotide binding protein 11
  • GNG11 encodes a 73-amino acid protein, the sequence of which is only 33 to 44% homologous to the sequence of most known gamma subunits but 76% homologous to that of gamma- 1.
  • GNGll was found to associate with G protein beta-1.
  • Integrin, alpha M complement component receptor 3, alpha; also known as CDl lb
  • a major surface antigen family on human leukocytes includes complement receptor type 3 (CR3A; also called integrin alpha-M, Macl or Mol).
  • CD1 lb promotes adhesion of granulocytes to each other and to endothelial cell monolayers.
  • TNFalpha elicits association of PI 3-kinase with the p ⁇ OTNFR and activation of PI 3-kinase in adherent neutrophils (Biochem Biophys Res Commun 2001 Mar 2;281(3):651-6).
  • Tumor necrosis factor requires the engagement of beta integrins to trigger secretion of superoxide anion (O(-)(2)).
  • the p60 TNF receptor (p ⁇ OTNFR) is responsible for signal transduction for activation of O(-)(2) generation.
  • Activation of TNFalpha-triggered O(-)(2) generation in neutrophils adherent to fibrinogen-coated surfaces involves the CDllb/CD18 receptor.
  • Phosphoinositide 3-kinase PI 3-kinase is essential for activation of O(-)(2) generation.
  • PI3K also synergises with SHIP and CDllb in mediating outside-in signalling of beta (2) integrin to regulate phagocytosis (J Exp Med 2001 Jan 1;193(1):61-71).
  • Complement component 4-binding protein, beta also regulated.
  • Example 6 P55PIK expression in neutrophil differentiation.
  • P55PIK is differentially regulated during neutrophil differentiation.
  • HL60 cells are plated in 75cm 2 flasks at a concentration of 0.5 xl0 6 /ml in RPMI+10% FCS (20 ml/ flask) and incubated for the indicated period of time with lO ⁇ M Retinoic acid after which time 1.5xl0 6 trypan blue negative cells are resuspended in 1 ml of RPMI medium and stimulated with 50ng ml Phorbal Myristate Acetate (PMA, Sigma) for 2 minutes. Nitroblue Tetrazolium salt is added to a final concentration of 50 ⁇ g/ml. Following incubation for 15 minutes at 37°C the samples are placed on ice to terminate the reaction.
  • PMA Phorbal Myristate Acetate
  • Cells are then centrifuged at 300xg for 5 minutes and the supernatant removed. Cells are washed once in PBS and resuspended in 1ml PBS. Cells are then cytocentrifuged onto glass slides using a cytospin (Shandon II). . Slides are allowed to air dry and cells are then fixed in methanol (Rapi Diff Kit; Diachem Int, UK). A counter stain is applied by immersing the slides in an Eosin stain (Solution B; Rapi Diff Kit; Diachem Int, UK) for 10 minutes. Excess stain is removed by gentle washing with water. The slide is then air-dried and a cover slip applied. Positive and negative cells are enumerated at 40x magnification. Slides are assessed blind at 3 different fields and the mean calculated. NBT positive cells are determined as those that contained blue intracellular deposits. NBT negative cells are stained pink and are free of blue particles.
  • Figures 11 and 12 show time courses of neutrophil differentiation and apoptosis, respectively.
  • HL60 cells Upon treatment of HL60 cells with retinoic acid, HL60 cells arrest their cell cycle and differentiate into neutrophils across a 5-day time course. Markers of differentiated neutrophils are increasingly detected at day 2 and day 3, as measured by reduction of NBT (see Figure 11 and also Martin SJ et al, Clin. Exp. Immunol. (1990)).
  • Apoptosis begins to occur around day 4 (96 h) as shown in Figure 12.
  • RNA samples are isolated by lysing cells and adding RNA zol (5xl0 6 cells/ml RNAzol), purifying RNA as described previously and analysed by microarray using Incyte LifeGrid filters as described previously.
  • Figure 13 shows P55PIK gene expression fold change in retinoic acid treated HL60 cells.
  • Example 7 Effect of cisplatin treatment on pSSPIK expression.
  • HeLa cells are obtained from the ATCC (Manassas, Virginia, USA), maintained in DMEM medium with 10% FCS at 37°C in a 5% CO2 atmosphere and treated with cisplatin (1 ⁇ g/ml). At the time points indicated RNA samples are isolated and analysed for gene expression changes by microarray using Incyte LifeGrid filters as described previously.
  • ⁇ 55PIK is decreased by cisplatin-induced apoptosis in HeLa cells.
  • Figure 14 shows p55PIK gene expression fold change in Cisplatin treated HeLa cells.
  • Example 8 Expression of recombinant p55PIK is associated with changes in gene expression, associated with apoptosis and survival
  • This example describes the analysis of oligonucleotide/polynucleotide sequences whose expression changes are associated with expression of p55PIK.
  • microarrays are used to measure global gene expression associated with p55PIK expression in HeLa cells. Analysis of such microarray results identifies genes whose expression pattern changes (either up-regulation or down-regulation) in a functional association with expression of recombinant p55PIK. We demonstrate that the genes identified using this approach include many genes whose products have been associated with apoptosis and survival. This identification further establishes a functional cellular role of p55PIK in the modulation of growth and survival.
  • HeLa cells are transiently transfected with pcDNA3.1 containing full-length cDNA for p55PIK using the CalPhos Mammalian Transfection Kit (Clontech) according to the manufacturers instructions. HeLa cells are plated in 75cm flasks at a concentration of 1.5x10° cells I flask. The following day, when cells are 70% to 80% confluent cells are transfected and the Cal/Phos soln left on the cells for a further 18 hours at 37°C in a 5% CO 2 humidified incubator. Typically, this transfection procedure yield 60 - 70 % transfection efficiency, as judged by FacsCalibre analysis of control EGFP transfected cells.
  • RNAzol RNAzol
  • lOO ⁇ l of chloroform lOO ⁇ l of chloroform
  • the aqueous layer is removed and the RNA is precipitated following addition of an equal volume of ice-cold isopropanol and centrifugation for 20 mins at 12000g at 4°C.
  • the RNA is further cleaned by addition to an RNeasy minispin column (Qiagen) according to the manufacturers instructions. Any contaminating DNA remaining in the elutant is removed by DNAase treatment of samples.
  • cDNA is hybridised to Human Life GridTM arrays and subjected to quantitative imaging and analysis using a STORM phosphoimager.
  • Example 9 Expression of recombinant pSSPIK is associated with changes in gene expression, associated with signal transduction and apoptosis
  • neutrophils Exposure of neutrophils to agents such as lipopolysaccharide, tumor necrosis factor- alpha (TNF-alpha), and the granulocyte-macrophage colony-stimulating factor causes a major activation. Activation is associated with the production of intracellular second messengers such as Ca++, inositol phosphates, cyclic AMP (cAMP) and aracidonic acids. Following the removal of the insult, the immune system must be resolved in order to prevent damage to the host. Resolution of neutrophil mediated inflammation is achieved, in part, through induction of neutrophil apoptosis.
  • agents such as lipopolysaccharide, tumor necrosis factor- alpha (TNF-alpha), and the granulocyte-macrophage colony-stimulating factor causes a major activation. Activation is associated with the production of intracellular second messengers such as Ca++, inositol phosphates, cyclic AMP (cAMP) and ara
  • Phosphodiesterase 4A cAMP-specific (dunce (Drosophila)-homolog + 3 phosphodiesterase E2)
  • Phosphodiesterase IC cahnodulin-dependent (70kD +3 Neurogranin (protein kinase C substrate, RC3) + >7
  • Phospholipase A2 group IVA (cytosolic): One of the key modulators of inflammation produced by the monrophil is arachidonic acid.
  • Phospholipase A2 (PLA2) enzyme cleave esterified fatty acids from membrane glycerophospholipids. The 20-carbon polyunsaturated fatty acid, arachidonic acid, is used as substrate by intermediate enzymes for the generation of eicosanoids, including leukotrienes and prostanoid products.
  • levels of phospholipase A2 serve a rate-limiting role in arachidonic release and consequently protanoid systhesis.
  • Phosphodiesterase 4A cAMP-specific (dunce (Drosophila)-homolog phosphodiesterase E2) and Phosphodiesterase IC, cahnodulin-dependent (70kD):
  • the second messenger molecule cyclic AMP dramatically modulates the apoptotic program in a wide variety of cells, accelerating apoptosis in some and delaying the rate of apoptosis in others.
  • Human neutrophil apoptosis is profoundly delayed by the cell- permeable analog of cyclic AMP, dibutyryl-cAMP.
  • Martin et al have investigated the mechanisms underlying cyclic AMP-mediated delay of neutrophil apoptosis, and showed that cyclic AMP inhibits loss of mitochondrial potential occurring during constitutive damrophil apoptosis. Furthermore, they demonstrated that cyclic AMP also suppresses caspase activation in these inflammatory cells (J Biol Chem 2001 276(48): 45041 - 50).
  • Intracellular levels of cyclic nucleotide second messengers are regulated predominantly by the complex superfamily of cyclic nucleotide phosphodiesterase (PDE) enzymes (Biochem Pharmacol 1999 May l;57(9):965-73).
  • PDE cyclic nucleotide phosphodiesterase
  • phophodiesterases will decrease the levels of cAMP, and hence reduce the survival of the cell mediated by cAMP.
  • Neurogranin protein kinase C substrate, RC3
  • calmodulin dependent Phosphodiesterase IC One member of the phosphodiesterase family we see elevated due to expression of recombinant p55PIK is calmodulin dependent Phosphodiesterase IC.
  • neurogranin protein kinase C substrate
  • neurogranin has been shown to be a regulator of calmodulin and that elevated levels of calcium increase levels of calmodulin (Mol Neurolbiol 1997, 15(20): 131- 63).
  • expression of neurogranin is elevated greater than seven fold when p55pik is expressed. This would presumably alleviate any limiting concentrations of calmodulin required for full activation of Phosphodiesterase IC.
  • Apolipoprotein A-II An understanding of apolipoprotein A-II (apoA-II) physiology is much more limited than that of apoA-I. However, important and surprising advances have been produced, mainly through analysis of genetically modified mice. These results reveal a positive association of apoA-II with FFA and VLDL triglyceride plasma concentrations; however, whether this is due to increased VLDL synthesis or to decreased VLDL catabolism remains a matter of controversy.
  • Example 10 Expression of recombinant pSSPIK is associated with changes in gene expression, associated with modulation of apoptosis.
  • LRP Multidrug-resistant (MDR) cancer cells frequently overexpress the 110-kD lung resistance-related protein (LRP). Overexpression of LRP often predicts a poor response to chemotherapy.
  • MDR Multidrug-resistant
  • LRP-specific monoclonal antibody By screening a multidrug-resistant non-P-glycoprotein fibrosarcoma cell line with an LRP-specific monoclonal antibody, Scheffer et al. (1995) isolated a cDNA encoding LRP. Lung resistance-related protein/major vault protein and vaults in multidrug-resistant cancer. Curr Opin Oncol 2000 Nov;12(6):550-6. Thus are finding presented here suggest that the up regulation ofthis protein may confer an anti-apoptotic advantage to these cells.
  • v-Maf can bind as a homodimer to a variant Maf recognition element located between -66 and -54 upstream in the mouse p53 promoter.
  • V-Maf and its cellular counterparts are shown to activate p53 expression through this site.
  • the ability of v-Maf to activate p53 expression is modulated by API family members.
  • overexpression of v- Maf in primary cells leads to a p53 -dependent cell death.
  • Maf and members of the API family are able to regulate p53 expression through this site in the p53 promoter. J Biol Chem 2000 Jun 16;275(24): 17991-9. Consequently, down regulation of this gene would restrict the activation of p53 and presumably confer an anti- apoptotic advantage to cells expressing this gene.
  • Adenine nucleotide translocator 2 (ANT-2; fibroblast): It is assumed that ANT-1 and ANT-2 co-exist in every single mitochondrion and might be differently distributed within the membrane structures that constitute the peripheral inner membrane or the crista membrane. To discriminate between ANT originating from peripheral or from cristal inner membranes, two ANT-isotype-specific antibodies were used and by performing sequence analysis of the N-terminal end , it was discovered that the peripheral inner membrane contained ANT-1 and ANT-2, whereas the cristal membrane contained exclusively ANT-2 (Biochem J 2001 358 (Pt 2): 349 - 59).
  • ANT has two opposite functions. On the one hand, ANT is a vital, specific antiporter which accounts for the exchange of ATP and ADP on IM. On the other hand, ANT can form a non-specific pore, as this has been shown by electrophysiological characterization of purified ANT reconstituted into synthetic lipid bilayers or by measuring the permeabilization of proteoliposomes containing ANT. Pore formation by ANT is induced by a variety of different agents (e.g. Ca(2+), atractyloside, thiol oxidation, the pro-apoptotic HIV-1 protein Vpr, etc.) and is enhanced by Bax and inhibited by Bcl-2, as well as by ADP.
  • agents e.g. Ca(2+), atractyloside, thiol oxidation, the pro-apoptotic HIV-1 protein Vpr, etc.
  • ANT In isolated mitochondria, pore formation by ANT leads to an increase in IM permeability to solutes up to 1500 Da, swelling of the mitochondrial matrix, and OM permeabilization, presumably due to physical rupture of OM. Although alternative mechanisms of mitochondrial membrane permeabilization may exist, ANT emerges as a major player in the regulation of cell death. (Cell Death and Differentiation (2000) 7, 1146 - 1154). Consequently, our results presented here show decreased expression of ANT-2 which would presumably result in reduced apoptosis in cells, particularly those that are subjected to ROS-induced stress and thiol oxidation etc. TGFB1 -induced anti-apoptotic factor 1:
  • TGF-betal protects L929 fibroblasts against TNF-alpha cytotoxicity
  • GFP- tagged TIAFl protein is present mostly in perinuclear and nuclear locations.
  • TIAFl inhibits the cytotoxic effects of TNF-alpha and overexpressed TNF receptor adaptors TRADD, FADD, and RIP.
  • L929 stable transfectants expressing TIAFl do not have significant changes in the expression of TNF receptors and effector or regulatory proteins in apoptosis, which may account for the acquired TNF resistance in these cells.
  • these cells have a significantly suppressed IkappaB-alpha protein expression, and IkappaB-alpha degradation is blocked when exposing these cells to TNF-alpha.
  • stimulation of untransfected L929 cells with TGF-betal results in suppression of IkappaB-alpha expression and retarded IkappaB-alpha degradation in response to TNF-alpha.
  • TIAFl is apparently involved in TGF-betal inhibition of IkappaB-alpha expression and suppression of TNF-mediated IkappaB-alpha degradation (Biochem Biophys Res Commun 1998 Dec 30;253(3):743-9).
  • gene function associated with proliferation, survival and death can be ascertained by the expression of the recombinant gene (mRNA) in a test (model) system by measurement of impact on cell growth and viability.
  • mRNA recombinant gene
  • the 'readout' in this assay identifies a gene function as a 'modulator of cell growth/survival'.
  • HeLa cells are plated into a 24 well tissue culture plate at a concentration of 1.5 xlO 4 /ml.
  • Cells are left to adhere overnight, before transfecting the cells with a pcDNA3.1 plasmid containing the gene of interest (full-length coding mRNA sequence) using a Calcium Phosphate transfection kit (Clontech). Cells are left in the transfection medium for 24h, prior to replacing it with fresh culture medium. Following 24 h, transfected cells are treated with G418 (an antibiotic to select for cells containing an integrated copy of the plasmid and gene of interest, by virtue of the plasmid containing and expressing a gene for neomycin resistance) at a concentration of 500 ⁇ g/ ml and culture maintained for a further 7 days or until cells in test or confrol wells become confluent. Cells are then fixed and stained with Crystal Violet (1% in ethanol) for five minutes. To quantify, cells are solubilized by adding 33% Acetic Acid and the absorbance measured by reading the plate at 570nm using a colorimetric plate reader.
  • G418 an antibiotic to select for cells
  • This assay is validated by a number of control genes, which are known to affect cell growth/survival, including superoxide dismutase (SOD), glutathione peroxidase, p53 and p73.
  • SOD superoxide dismutase
  • glutathione peroxidase glutathione peroxidase
  • p53 p73
  • p73 and p53 are known tumor suppressor genes, which induce cell apoptosis.
  • Figure 15 shows graphical representation of the effect of these known survival and pro-apoptotic genes on the proliferation viability of HeLa cells, as determined by a plaque assay.
  • Figure 16 shows graphical representation of the effect of p55PIK (AF417321) on the proliferation/viability of HeLa cells, as determined by a plaque assay. Expression of recombinant p55PIK in HeLa cells resulted in significant increase of 5 proliferation/viability. These results identify p55PIK as a 'modulator of cell growth/survival'.
  • NCBI National Center for Bioinformatics
  • Table 4 shows a single SNP is identified mapping to amino acid 297.
  • the SNP is a 15 synonomous substitution, C-T fransition, at the third position of the Ser-297 codon.
  • the accession number of the chromosomal contig containing the p55PIK gene, and the position of the SNP within the contigs are indicated.
  • the unique SNP identification numbers are also included, as is the unique protein identifier
  • Primers are selected using the Primer Designer facility of the GeneTool Lite software (Biotools Inc). Primers are selected to have minimal internal stability and annealing temperatures of approximately 60°C.
  • An Nhel restriction site (underlined) is incorporated to the 5 'end of the forward primer, to allow for orientation of the insert.
  • a Not/ restriction site (underlined) is incorporated to the 5 'end of the Reverse primer, to allow for orientation of the insert.
  • Templates for PCRs are prepared by reverse franscription of total R ⁇ A isolated from Hela or HL60 cell lines, or Brain and kidney tissue samples (Stratagene). Briefly, total cellular R ⁇ A (1 ⁇ g to 20 ⁇ g) or polyA + mR ⁇ A (100 ng to 5 mg) is incubated with an oligo (dT) primer. Primed R ⁇ A is reverse franscribed to first stand cD ⁇ A in a reaction containing M-MLV reverse transcriptase (RT; alternatively Superscript II is used (Life Sciences)), RT buffer, and d ⁇ TPs at 42°C for 1 to 2 hours.
  • RT M-MLV reverse transcriptase
  • PCRs are prepared with primers (500nMol), appropriate templates (1/100 dilution of the reverse transcription reaction), buffer and Taq polymerase (lunit/reaction)
  • Full-length amplimers are gel purified using the Qiaquick gel isolation kit (Qiagen, cat. 28706) and ligated to pCD ⁇ A3.1 using the Topo-TA cloning kit (Invitrogen, cat. 45-0005) according to the manufacturers instructions. Ligated DNA is transformed to E.coli (ToplO). Transformants are selected for plasmid DNA preparation and sequence analysis.
  • Plasmid DNA is prepared using either the Qiagen miniprep (cat. 27106) or midiprep
  • Insert orientation is determined by restriction digestion with Nhel or Notl. Plasmid miniprep DNA (100 ng to 5 ⁇ g) is sent to MWG Biotech or Lark Technologies for contract sequencing. Sequencing reactions are primed using one of the following universal primer sequences:
  • Example 14 Amplification and cloning of pSSPIK promoter.
  • Primers are selected using the Primer Designer facility of the GeneTool Lite software (Biotools Inc). Primers are selected to have minimal internal stability and annealing temperatures of 60°C.
  • Templates for PCR are prepared using genomic DNA purified from HL60 cells using the Qiagen 'Blood and cell culture DNA mini Kit', (Cat. 13323), as per manufacturers instructions. PCR amplifications are performed as described previously using lOOng of genomic DNA as template.
  • Amplimers are gel purified using the Qiaquick gel isolation kit (Qiagen, cat. 28706) and ligated to pCDNA3.1 using the Topo-TA cloning kit (Invitrogen, cat. 45-0005) according to the manufacturers instructions. Ligated DNA is transformed to E.coli (Top 10). Transformants are selected for plasmid DNA preparation and sequence analysis.
  • Plasmid DNA is prepared using either the Qiagen miniprep (cat. 27106) or midiprep (cat. 12643) kits as described by the manufacturer.
  • Insert orientation is determined by PCR with p55PIK-specific reverse primer and vector-specific forward primer (T7 primer). Plasmid miniprep DNA (100 ng to 5 ⁇ g) is sent to MWG Biotech or Lark Technologies for contract sequencing.
  • the promoter sequence of p55pik is given in Figure 17 as SEQ ID NO:2 and has been deposited with EC ACC in accordance with the terms of the Budapest treaty, Accession Number 01120311.
  • p53 Mammalian p53 recognition site ([AG][AG][AG]C[AT][AT]G[CT][CT][CT]) el-Deiry, W.S. et al., Nature Genet 1: 45-49 (1992)
  • C/EBP SV40 C enhancer binding protein recognition site (TC*TACTC)
  • NRE-1 Vertebrate NRE Boxl (A*CCTCTC[CT]) Baniahmad A. et al., EMBO J. 6: 2297-2303 (1987)
  • AP2 Mammalian AP-2 recognition site (G[CG][CG][AT]G[CG]CC) Mitchell, P.J. et al., Cell 50: 847-861 (1987)
  • CCAAT Eukaryotic promoter CCAAT region, (T[tg]T[AG]GCCAAT[GC]A) Bucher, P. J. Mol. Biol. 212:563-578 (1990).
  • Example 15 Transient transfection in U937 cells and analysis of GM-CSF responsiveness.
  • GM-CSF enhances p55PIK promoter activity in U937 cells.
  • U937 cells are transfected with lO ⁇ g of an EGFP reporter construct (pEGFP, Clontech), containing a l,996bp human genomic fragment driving the expression of the EGFP gene (p55-EGFP).
  • the fragment represents the region from -2128 to +19 of the franscription start site, including the putative p55 promoter region.
  • the 2139bp fragment is cloned to the Topo-TA vector (described above) and subcloned to pEGFP using BamHI and Xbal restriction sites.
  • U937 macrophage cell line was cultured at 37°C under 5% CO2 in RPMI supplemented with 10% foetal calf serum. Cells were transfected by the calcium phosphate method. Transfection of the pEGFP vector without the p55 genomic fragment is used as a negative control whereas a construct containing the CMV promoter serves as a positive control.
  • U937 cells containing either pEGFP or p55-EGFP are treated with GM-CSF (50 Units) either in the presence or absence of gliotoxin (O.l ⁇ g/ml).
  • GM-CSF 50 Units
  • gliotoxin O.l ⁇ g/ml
  • flow cytometric analysis using a FacsCalibre (Becton Dickinson).
  • FacsCalibre Becton Dickinson
  • Cells are considered positive for EGFP expression when the FL1 signal is greater than the background signal generated by either pEGFP or untreated p55-EGFP. All values were corrected for transfection efficiency by standardization against ⁇ -gal activity, derived from the cotransfected plasmid pSV ⁇ - gal (Promega).
  • the erythroleukaemic TF1 cell line is growth factor dependent, requiring GM-CSF for survival in culture. Withdrawal of GM-CSF causes TF1 cells to undergo spontaneous apoptosis.
  • candidate regulators of the apoptotic process such as p55PIK, can be determined in these cells by measuring the extent of apoptosis following introduction of the candidate gene to the cells.
  • Light Scatter Analysis takes advantage of the fact that by using the laser beam of a flow cytometer one can determine the size (Forward Scatter) and granularity (Side Scatter) of a cell.
  • the morphological changes associated with apoptosis, such as decreased size (shrinkage) and granularity affect these parameters.
  • cells undergoing apoptosis will move to the left and slightly down, from the parameters of a healthy population.
  • TF-1 cells are plated into 24 well plates (2xl0 5 /ml) and are cultured for 48h in the presence or absence of GM-CSF (2ng/ml). Cells are then harvested by centrifugation (lOOOrpm, for 10 min) and washed in PBS. The pellet is resuspended in PBS (2x10 5 cells/ml) and acquired by a FacsCalibre. Forward and Side scatter parameters are assessed using Cell Quest software.
  • TF-1 cells are cultured for 48h in the presence or absence of GM-CSF (2ng/ml). Cells are then harvested by centrifugation (lOOOrpm, for 10 min) and washed in PBS. The pellet is resuspended in PBS (2x10 5 cells/ml) and 1ml is then added in a dropwise fashion to ice cold 70% EtoH whilst continuously vortexing. Cells are then permeabilized by incubating at -20°C for 24 h. To stain DNA, cells are centrifuged and washed in PBS.
  • RNAse A 50 ⁇ g/ml
  • GM-CSF withdrawal decreases the percentage of cells in live gate
  • Culturing TF-1 cells in the absence of GM-CSF induces cell death. This death induces morphological changes in the Forward and Side Scatter parameters that can be detected by FacsCalibre analysis. Consequently, by observing the percentage of cells in the live gate (which is an arbitrary region pre-set on a healthy population of TF-1 cells grown in the presence of GM-CSF (2ng/ml)), one observes that there is a decrease in the percentage of cells with these light scatter parameters, when cytokine is withdrawn.
  • Figure 19 shows the percentage of cells in the live gate decreases from 77% to 35% upon factor withdrawal for 48h, representing a decrease of approx. 50% over the time period.
  • DNA degradation due to endonucleases activated during the apoptotic process, and subsequent release during the fixation and washing process results in a population of cells with reduced fluorescence upon propidium iodide staining as analysed by a fluorescence histogram (sub GI).
  • Figure 20 shows TF-1 cells grown in the absence of GMCSF for 48 h have an increase of cells with sub-Gl profiles from 5% to approximately 55%, indicative of the cells undergoing apoptosis.
  • cells are transduced with either BCL2 plasmid or control plasmid.
  • the cells are then induced to undergo apoptosis by withdrawal of GMCSF for 48h.
  • the percentage of transduced cells remaimng in the live gate (an arbitrary region pre-set on a healthy population of TF-1 cells grown in the presence of GM-CSF (2ng/ml)) is recorded.
  • An index of antiapoptotic activity is calculated by computing the difference in samples +/- GMCSF.
  • TF1 cells are transduced with either a plasmid expressing the p55PIK coding sequence (p55PIK plasmid) or a control plasmid.
  • the p55PIK coding sequence encoding the amino acid sequence set out in SEQ ID NO: 1 is cloned into the retroviral expression vector pMSCV (Clontech). Plasmids are transfected into the retroviral packaging cell line Phoenix 293 (Standford) using the CalPhos Mammalian Transfection Kit (Clontech) according to the manufacturer's instructions. Refrovirus is harvested 72 hours later. Retrovirus is transduced to TF1 cells by incubation for 18 hours. Media is changed and cells are incubated for an additional 96 hours.
  • RNAi interfering RNA
  • the ⁇ 55PIK CDS is screened for AAN19TT siRNA target sequences with a GC content of 40-55%.
  • Candidate targets are subject to a BLAST search against the Genbank database to ensure that the selected sequences share no significant homology with any other human genes.
  • oligonucleotides selected are:
  • the p55PIK siRNA is initially tested for efficacy in Hela cells.
  • Cells were plated at a density of 7.2 x 10 4 in each well of a 6 well plate and incubated at 37°C overnight in DMEM culture medium (IX Dulbeccos modified Eagles medium (Sigma D2554), 0.004% folic acid, 4 mM L-glutamine, 0.37% sodium bicarbonate, 0.1 mM sodium pyruvate) containing 10% foetal calf serum (FCS).
  • DMEM culture medium IX Dulbeccos modified Eagles medium (Sigma D2554), 0.004% folic acid, 4 mM L-glutamine, 0.37% sodium bicarbonate, 0.1 mM sodium pyruvate
  • FCS foetal calf serum
  • 2 ⁇ g of duplex siRNA is fransfected into Hela cells using DMRIE-C reagent (Invitrogen 10459-014) according to the manufacturers recommendations.
  • RNA is isolated using Qiagen' s RNeasy Miniprep columns (Qiagen 74104) after lysis on QIAshredder columns (Qiagen 79654). The RNA is quantified by spectrophotometric analysis and 1 ⁇ g was reverse franscribed into cDNA using SuperScriptll RNAseH- Reverse Transcriptase (Invitrogen 18064-014).
  • Q-PCR primers are designed to amplify a PCR product of 149 nucleotides in length from the CDS of p55PIK, following the guidelines outlined in the Quantitect SYBR Green PCR handbook from Qiagen.
  • a control RPS13 (NM_001017) amplified PCR product diluted to [1000,100,10,1 and 0.1 fg] respectively, used to quantify the level of transcript; 2. RPS13 amplified from sample templates as separate PCR reactions for normalisation purposes.
  • the cDNA templates from Hela cells fransfected with either p55PIK or Lamin A/C siRNA, and a mock-transfected control, are amplified with p55PIK Q-PCR primers using QuantiTectSYBR Green PCR kit (Qiagen 204143) on a DNA Engine Opticon System (MJ Research).
  • the amplification conditions include a 95°C step for 15 min for initial activation of HotStarTaq DNA polymerase, followed by 35 cycles of (15s at 94°C, 30s at 60°C, 30s at 72°).
  • the fluorescence of the samples at 521 nm is read between the annealing and extension steps of the protocol.
  • the melting curves are calculated at the end of the 35 cycles and confirm product homogeneity.
  • a standard curve is plotted using the log [template quantity] of the RPS13 confrol template dilutions (as above) versus cycle number at which the fluorescence intensity measured in the well exceeds the level specified in the cycle threshold parameters (the C(T) value).
  • An estimate of the quantity of initial template in treatment samples is determined from this plot and the amount of p55PIK present in each sample is normalized across samples by calculating the ratio of p55PIK to RPS13 for each sample.
  • the normalised expression values are used to estimate percentage knockdown of p55PD with reference to the control transfected cells, (in this case cells transfected with Lamin A/C RNAi).
  • the siRNA duplex is introduced to the cell line, TF1.
  • TF1 cells 4 x 10 5 cells/ well, are fransfected with 2 ⁇ g of siRNA in each of 7 wells of a 24 well plate using 8 ⁇ l DMRIE-C reagent (InVitrogen). Cells are simultaneously fransfected in parallel with a confrol siRNA directed to Lamin A C.
  • Recombinant GMCSF is added to the OPTI-MEM media during transfection. The transfection media is replaced after four hours with fresh RPMI 1640 containing GMSCF and 10% FCS and incubated at 37°C.
  • Cell counts are determined using a haemocytometer. Cell viability is determined using the MTT assay. Briefly, 5mg/ml MTT in PBS was added to lOO ⁇ l aliquots of cells, mixed thoroughly and incubated for 4 hours at 37°C. Mitochondrial succinate dehydrogenase of viable cells can convert the soluble MTT salt to an insoluble blue formazan crystal. Addition of lOO ⁇ l of 0.1N HCl/Isopropanol allows the samples to be read at 570nm on a Molecular Devices Emax precision micro plate reader.
  • FSC/SSC Forward Scatter/Side Scatter
  • TF-1 cells are plated into 24 well plates (2xl05/ml) and are cultured for 48h in the presence or absence of GM-CSF (2ng/ml). Cells are then harvested by centrifugation
  • SubGl parameters are obtained by resuspending cells in a buffer, (0.1% Sodium Citrate, 0.1% TritonX-100, 200 ⁇ l of Propidium Iodide at 5mg/ml made up to 20mls in PBS) and staining for 7hours at 4°C in the dark. The PI stained cells are then acquired by the flow cytometer. Analysis of FL2 fluorescence is performed on Cell Quest software to allow quantification of the Sub-Gl phase of the cell.
  • Example 18 Differential expression of pSSPIK is associated with inflammatory diseases
  • CF Cystic Fibrosis
  • COPD Chronic Obstructive Pulmonary Disease
  • Sepsis Neufrophils are prepared from these samples as described (Example 1).
  • the isolated neutrophils are lysed in RNAZol (Biogenesis) and RNA is prepared as described (Example 1).
  • cDNA from these samples is used for anay hybridization and quantitative PCR.
  • Differential gene expression is determined by reference to a pooled set of samples from normal controls. Up regulation of p55PIK is confirmed by quantitative PCR.

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Abstract

L'invention concerne un procédé permettant de détecter l'apoptose dans une cellule et consistant entre autres à détecter une altération d'un quelconque élément suivant : i) un polypeptide de p55PIK présentant une séquence d'acides aminés telle que représentée par SEQ ID NO:1 ; ii) un polypeptide présentant au moins 80 % d'homologie avec i) ; iii) un acide nucléique codant pour un polypeptide présentant la séquence établie en i) ou en ii) ; iv) un acide nucléique s'hydridant avec la séquence établie en iii) lorsqu'il est soumis à des conditions rigoureuses ; ou v) le complément de iii) ou de iv). En conséquence, l'invention concerne un procédé permettant de moduler l'apoptose, par modulation de l'expression du gène p55PIK, et un procédé permettant d'identifier des gènes associés à l'expression du gène p55PIK et ainsi d'identifier d'autres gènes associés à l'apoptose. L'invention se rapporte en outre à une nouvelle séquence d'acides nucléiques codant pour la région promoteur de p55PIK.
PCT/GB2002/005547 2001-12-07 2002-12-06 P55pik Ceased WO2003048361A2 (fr)

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GB0129377A GB0129377D0 (en) 2001-12-07 2001-12-07 p55PIK
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US6100090A (en) * 1999-06-25 2000-08-08 Isis Pharmaceuticals Inc. Antisense inhibition of PI3K p85 expression
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