WO2002020834A2 - Method for the identification of genes that are involved in the modulation of a poptosis in cells - Google Patents

Method for the identification of genes that are involved in the modulation of a poptosis in cells Download PDF

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WO2002020834A2
WO2002020834A2 PCT/GB2001/003969 GB0103969W WO0220834A2 WO 2002020834 A2 WO2002020834 A2 WO 2002020834A2 GB 0103969 W GB0103969 W GB 0103969W WO 0220834 A2 WO0220834 A2 WO 0220834A2
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cell
apoptosis
cells
expression
gene
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PCT/GB2001/003969
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French (fr)
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Tom Cotter
Peter Daly
Ian Hayes
Finbarr Murphy
Liam Seery
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Eirx Therapeutics Limited
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Priority to AU2001284264A priority Critical patent/AU2001284264A1/en
Publication of WO2002020834A2 publication Critical patent/WO2002020834A2/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • the present invention relates to a method for the identification of genes that are involved in the modulation of apoptosis in cells.
  • the invention relates to a novel method for identifying and characterising those genes, and the molecular mechanisms involved in the modulation of cell death by apoptosis.
  • the present invention relates to a novel method for inducing the expression of gene products involved in the modulation of the early stages of apoptosis, and their subsequent identification and characterisation.
  • Programmed cell death or apoptosis is a genetically regulated 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).
  • apoptosis Under normal physiological conditions apoptosis is tightly regulated. However, there are a number of diseases where the process becomes deregulated, leading to a particular pathology. Examples of where apoptosis is retarded or inhibited include some types of tumour development, a number of inflammatory conditions such as adult respiratory distress syndrome (ARDS) and other related conditions (Matute-Bello et al, Am J Respir Crit Care Med. 56, 1969-77, 1997). 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.
  • ARDS adult respiratory distress syndrome
  • Apoptosis is characterised by a number of well-defined biochemical hallmarks. These include DNA fragmentation, lipid flipping and caspase activation. By way of example, DNA fragmentation is caused by the activation of an endogenous endonuclease enzyme (Wyllie, Nature, 284, 555-556,1980; Enari et al, Nature, 391, 43-50, 1998).
  • the result is a DNA ladder pattern, which 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 ofthe reticulo-enothelial system.
  • Lipid flipping is a second well-defined characteristic resulting in the exposure of the phospholipid phosphatidylserine to the outside surface of the plasma membrane of the cell as it undergoes apoptosis (Fadok et al., I Immunol. 148, 2207-16, 1992). Normally this lipid is localised to the inner surface of the membrane lipid bilayer. Although the underlying mechanism responsible for this lipid flipping is poorly understood at present, it serves as a signal for the recognition and phagocytosis of the apoptotic cell (Fadok et al., J Immunol. 148, 2207-16, 1992)
  • caspases A series of enzymes termed caspases have also been shown over the last few years to be involved in different phases of apoptosis. Although the signal transduction system of apoptosis is relatively poorly understood at present, caspase enzymes, in particular, are thought to play a role in the propagation and execution phases of the process (Samali et al., Cell Death Differ. 6, 495-6, 1999). For example, caspase 3 is involved in the activation of CAD, an endonuclease involved in the cleavage of DNA to yield the hallmark DNA ladder of apoptosis described above (Mcllroy et al., Cell Death Differ. 6, 495-6, 1999).
  • This caspase cleaves ICAD, a natural inhibitor of the CAD endonuclease.
  • Other caspases are involved in the propagation and execution phase of the process, an example of which is caspase 8.
  • Apoptosis can also occur independently of caspase activation as not all incidences of apoptosis involve these enzymes. There is evidence that under conditions of high oxidative stress these cysteine-containing proteases are inactivated. To date 14 caspases have been identified as playing a role in the process (Alnemri et al, J Cell Biochem. 64, 33-42, 1997).
  • NO nitric oxide
  • studies over the past few years have demonstrated that a variety of biological response modifiers such as cytokines, growth factors and agents that induce apoptosis can lead to the generation of NO (Brune et al., Cell Death & Differentiation, 10, 969-75, 1999).
  • cytokines cytokines
  • growth factors growth factors
  • agents that induce apoptosis can lead to the generation of NO (Brune et al., Cell Death & Differentiation, 10, 969-75, 1999).
  • a change in intracellular redox levels or an increase in NO levels is capable of triggering apoptosis.
  • the mechanisms by which these changes occur is largely unknown.
  • the specific targets of NO generated intracellularly are largely unknown at present.
  • the present invention demonstrates that NO is responsible for the modulation of early transcription and/or translation, and/or post-translational modification in cells of genes which control the progression of the cell towards apoptosis.
  • the present invention demonstrates that the genes modulated by changes in NO exposure are required for induction or inhibition of apoptosis, before the cell has made a commitment to die.
  • other genes involved in apoptosis such as caspase genes which are known to, only act at the execution stage of apoptosis and are only activated once the cell is committed to the apoptotic fate.
  • the present invention provides a mechanism for the regulation of expression of NO-modulated genes which may be used to induce or prevent the apoptotic process.
  • the present inventors hypothesised that if a model was designed such that the course of apoptosis following induction by NO was functionally characterised, then changes, or patterns of changes, in the 'early' regulatory events could be studied. Studies of changes in expression of those genes involved in these early events, would lead to the identification of potential therapeutic targets.
  • the present inventors have set out to provide a model system for the NO mediated modulation of apoptosis such that those genes involved in the early regulatory events of apoptosis, can be identified and characterised.
  • apoptosis represents a significant therapeutic target, since many diseases are due to defects in this process. Many physiological factors prevent cell apoptosis. For example cytokines or growth factors 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 targets to control apoptosis act early in the process and are restricted to a particular cell type.
  • NO induces cell 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. If model discovery assays are configured to target these 'early' regulatory events occurring in the inhibition of apoptosis it is possible to identify the key genes to control apoptosis.
  • the invention provides a method for modulating the expression of gene products that regulate the transition of a cell between a non-apoptotic state and an apoptotic state, comprising increasing or reducing the intracellular concentration of nitric oxide (NO).
  • NO nitric oxide
  • the invention provides a method for identifying one or more gene product(s) that modulate the transition of a cell between a non-apoptotic state and an apoptotic state, comprising the steps of:
  • the method ofthe invention includes the steps of: a) determining the level(s) of expression of one or more genes or gene product(s) in a cell to establish a reference expression level; and b) comparing the expression level(s) after NO exposure to the reference expression level(s).
  • the invention relates to the use of NO to alter the expression of gene products that modulate the transition of a cell between a non-apoptotic state and an apoptotic state.
  • the present invention provides a system for modelling NO mediated modulation of apoptosis in a cell comprising the steps of: (a) providing a population of cells; (b) modulating the intracellular NO. concentration in the cells; and
  • Reference levels of expression of gene products may be determined, in the absence of modulation by NO.
  • a reference expression level once established for a given cell type, may be used for repeated screens, thus facilitating the performance of repeated rounds of screening.
  • Levels of expression may be determined by assessing expression at the nucleic acid or polypeptide level. For example, mRNA levels, protein levels or the extent of posttranslational modification of proteins may be used to assess expression in accordance with the present invention.
  • NO functions to facilitate apoptotic processes in cells.
  • exposure of a cell to NO leads to induction of apoptosis in the cell more rapidly than occurs under identical conditions in the absence of NO.
  • Modulation of intracellular NO concentration may be effected by any suitable technique.
  • NO concentrations may be increased by administration of an NO donor, such as sodium nitroprusside which spontaneously releases NO on culture, or increasing the activity of the cellular enzyme NO synthase.
  • Decrease in NO concentrations may be effected by, for example, inhibition of NO synthase, the use of NO scavenging agents such as urea or NAC, or by boosting the cellular defences against NO.
  • Glutathione is a tripepetide synthesized from the amino acids glutamate, cysteine and gly cine.
  • the levels of the limiting amino acid cysteine determine its rate of synthesis. Consequently, elevation of cysteine levels in the cell increases GSH.
  • addition of cysteine to cells is not feasible since cysteine auto-oxidizes to an insoluble form and is toxic to cells.
  • cysteine is supplied to cells in an acetylated form as N- acetyl-cysteine (NAC) which when taken up by the cell is readily deacetylated intracellularly and provides the rate limiting amino acid.
  • NAC N- acetyl-cysteine
  • inhibitors of apoptosis are useful in the assays of the invention in order to extend the "window" between NO treatment and the onset of apoptosis. It is during this window that NO-induced gene expression may be observed.
  • Various inhibitors of apoptosis are known in the art and may be employed, as described below.
  • Inhibitors of NO or NO activity may be used to further investigate the involvement of NO in the induction of gene expression or other events associated with apoptosis. Suitable NO inhibitors are described in more detail below.
  • the NO inhibitors may, for example, be active against NO Synthase (NOS). Specific isoforms of NOS may be targeted in order to monitor the effect of cell-specific genes on NO-induced apoptosis.
  • NOS NO Synthase
  • the model system and assays described here may also be used to identify compounds or substances capable of promoting or inhibiting (i.e., modulating) apoptosis. Preferably, such compounds are capable of inhibiting apoptosis.
  • Candidate compounds may be identified which are capable of modulating expression of genes which are capable of modulating the transition between a non-apoptotic state and an apoptotic state of cell. Such genes may be identified by the methods as described here. Screens are usefully configured to screen libraries of candidate compounds, which libraries may be in the form of arrays as known in the art.
  • a screen may be configured by inducing apoptosis in the cell by increasing the NO concentration (e.g., by exposure of a cell to sodium nitroprusside) and gene expression observed to identify genes capable of regulating apoptosis, preferably early stages of apoptosis.
  • Candidate compounds may be exposed to cells expressing such genes, or to in vitro systems which express such genes, or to the gene products (polypeptides) themselves, whether substantially purified or partially purified.
  • Compounds identified which modulate e.g, up-regulate or down-regulate) the activity, level or expression of such genes or gene products are useful to modulate apoptosis.
  • the present invention provides numerous advantages over the prior art. In particular, it:
  • Figure 1 Sodium nitroprusside induces death in HeLa cells, which is reversed by NAC. HeLa cells are cultured ⁇ sodium nitroprusside (0.9 xlO "6 M) ⁇ NAC (20mM) for 18 h prior to detection of viability by MTT (abs 570nM). Survival corresponds positively with increases in optical densities.
  • Figure 2 H 2 O 2 induces death in HeLa cells, which is reversed by NAC. HeLa cells are culture ⁇ H 2 O 2 (1.3xlO "6 M) ⁇ NAC (20mM) for 18 h prior to detection of viability by MTT (abs 570nM). Survival corresponds positively with increases in optical densities.
  • HeLa cells are treated with sodium nitroprusside (0.9 xl0 "6 M) for 4 h (column 1) and 6 h (column 2) + NAC (20mM; 4 h column 3 and 6 h column 4).
  • Each column represents the expression of each gene (in rows) in duplicate (measured using human Incyte LifeGrid microarrays); fold change relative to a time zero control. Black indicates increased expression and white indicates decreased gene expression.
  • a greyscale bar is included to indicate respective expression levels. The same scale is used for the following figures.
  • HeLa cells are treated with sodium nitroprusside (0.9 xl0 "6 M) for 4 h (column 1) and 6 h (column 2) ⁇ NAC (20mM; 4 h column 3 and 6 h column 4).
  • Each column represents the expression of each gene (in rows) in duplicate (measured using human Incyte LifeGrid microarrays); fold change relative to a time zero control.
  • HeLa cells are treated with sodium nitroprusside (0.9 xlO '6 M) for 4 h (column 1) and 6 h (column 2) + NAC (20mM; 4 h column 3 and 6 h column 4).
  • Each column represents the expression of each gene (in rows) in duplicate (measured using human Incyte LifeGrid microarrays); fold change relative to a time zero control.
  • NAC NAC. HeLa cells are treated with sodium nitroprusside (0.9 xlO "6 M) for 4 h (SNP T4- AvgFoldChange) and 6 h (SNP T6-AvgFoldChange) ⁇ NAC (20mM) for 4 h (SNP+NAC T4-AvgFoldChange) and 6 h (SNP+NAC T6-AvgFoldChange).
  • Figure 7 Aryl hydrocarbon receptor nuclear translocator-like gene expression is decreased (expressed as fold change relative to a time zero control) associated with sodium nitroprusside induced HeLa cell apoptosis but this decrease is blocked by treatment with NAC.
  • HeLa cells are treated with sodium nitroprusside (0.9 xlO "6 M) for 4 h (SNP T4-AvgFoldChange) and 6 h (SNP T6-AvgFoldChange) ⁇ NAC (20mM) for 4 h (SNP+NAC T4-AvgFoldChange) and 6 h (SNP+NAC T6-AvgFoldChange).
  • Figure 8 Calpastatin gene expression is decreased (expressed as fold change relative to a time zero control) associated with sodium nitroprusside induced HeLa cell apoptosis but this decrease is blocked by treatment with NAC.
  • HeLa cells are treated with sodium nitroprusside (0.9 xlO "6 M) for 4 h (SNP T4-AvgFoldChange) and 6 h (SNP T6- AvgFoldChange) ⁇ NAC (20mM) for 4 h (SNP+NAC T4- AvgFoldChange) and 6 h (SNP+NAC T6-AvgFoldChange).
  • FIG. 9 Thioredoxin reductase 1 gene expression is decreased (expressed as fold change relative to a time zero control) associated with sodium nitroprusside induced HeLa cell apoptosis but this decrease is blocked by treatment with NAC.
  • HeLa cells are treated with sodium nitroprusside (0.9 xlO "6 M) for 4 h (SNP T4-AvgFoldChange) and 6 h (SNP T6- AvgFoldChange) + NAC (20mM) for 4 h (SNP+NAC T4-AvgFoldChange) and 6 h (SNP+NAC T6-AvgFoldChange).
  • Figure 10 Shows that - matrix matalloproteinase 2 gene expression is decreased (expressed as fold change relative to a time zero control) associated with sodium nitroprusside induced HeLa cell apoptosis but this decrease is blocked by treatment with NAC.
  • HeLa cells are treated with sodium nitroprusside (0.9 xl0 "6 M) for 4 h (SNP T4- AvgFoldChange) and 6 h (SNP T6-AvgFoldChange) ⁇ NAC (20mM) for 4 h (SNP+NAC T4-AvgFoldChange) and 6 h (SNP+NAC T6-AvgFoldChange).
  • Figure 11 Shows that Ubiquitin C-terminal hydrolase Ll is increased (expressed as fold change relative to a time zero control) associated with sodium nitroprusside induced HeLa cell apoptosis but this increase is blocked by treatment with NAC.
  • HeLa cells are treated with sodium nitroprusside (0.9 xlO "6 M) for 4 h (SNP T4-AvgFoldChange) and 6 h (SNP T6- AvgFoldChange) + NAC (20mM) for 4 h (SNP+NAC T4-AvgFoldChange) and 6 h (SNP+NAC T6-AvgFoldChange).
  • Fig. 12 Photomicrograph of HeLa cells exposed to Sodium Nitroprusside either in the presence or absence of NAC.
  • Cells were treated with SNP in the absence of NAC for Oh (A), 6h (B) or 24h (D) or in the presence of 20mM NAC for 6h (C) or 24h (E).
  • HeLa cells were then trypsinized and slides were made by cytospinning cells onto glass slides and staining using the Diff Quik II staining kit. Apoptosis was assessed by nuclear condensation and membrane blebbing.
  • Magnification X 400 Magnification X 400.
  • apoptosis refers to genetically programmed cell death which is regulated throughout the lifetime of an organism.
  • a triggering agent from either outside or inside the cell causes "cell-suicide” genes to produce enzymes that damage the cell in several ways, including disrupting its cytoskeleton and nucleus.
  • the cell shrinks and pulls away from neighbouring cells.
  • the DNA within the nucleus fragments, and the cytoplasm shrinks, although the plasma membrane remains intact. Phagocytes in the vicinity then ingest the dying cell.
  • Apoptosis may be regarded as a normal type of cell death and contrasts with necrosis which is a pathological type of cell death that results from tissue injury.
  • Apoptosis removes unneeded cells during development before birth. It continues to occur after birth to regulate the number of cells in a tissue and eliminate potentially dangerous cells such as cancer cells.
  • NO is formed from the amino acid arginine by the enzyme nitric oxide synthase (NOS) which is widely distributed in cells and tissues. NO is different from all previously known neurotransmitters because it is not synthesised in advance and packaged into synaptic vesicles. In contrast, NO is formed on demand and acts immediately. Its action is brief because NO is a highly reactive free radical that lasts less than 10 seconds before it combines with oxygen and water to form nitrates and nitrites. The precise functions of NO released by neurons are still unclear. Because NO is lipid soluble, it diffuses out of cells that produce it and into neighbouring cells, where it activates an enzyme for production of a second messenger called cyclic GMP. Some research suggests that NO may play a role in memory and learning.
  • NOS nitric oxide synthase
  • NO endothelial cells in blood vessel walls release NO, which diffuses into neighbouring smooth muscle cells and causes relaxation. The result is vasodilation, an increase in blood vessel diameter. The effects of such vasodilation range from a lowering of blood pressure to erection of the penis in males.
  • NO is highly toxic. Phagocytic cells, such as macrophages and certain white blood cells, produce NO to kill microbes and tumour cells. Based on the presence of NOS, it is estimated that more than 2% of the neurons in the brain produce NO. NOS is also highly concentrated in autonomic neurons that cause either relaxation of smooth muscle in the gut or release of epinephrine and norepinephrine from the adrenal medulla.
  • Nitric oxide has been shown to play a key signalling role in a range of physiological and pathophysiological states. It is involved in the cytotoxic effects of immune cells, neurotransmission, smooth muscle cell contractility, platelet reactivity and it is implicated in atherosclerosis, inflammatory conditions, septic shock, neurodegenerative diseases, retinal diseases and cancer metastasis. In acute lung injury, neutrophil apoptosis may be important in regulating the inflammatory process by controlling neutrophil numbers and thus activity. Exogenous inhaled NO is used therapeutically in patients with acute lung injury and its effects on apoptosis of these cells may be important. NO has been shown to induce apoptosis in renal cells (Amore et al. Kidney Int.
  • NO synthesis is controlled by the action of Nitric Oxide Synthase (NOS).
  • NOS Nitric Oxide Synthase
  • NOS consists of a family of related isoforms which have different physiological roles and tissue specificity. Three distinct isoforms have been identified ; nNOS whose activity is regulated by Ca++ and calmodulin, is found in neural tissue. A second Ca++/calmodulin -requiring constitutive enzyme (eNOS) is present in vascular endothelial cells. A third Ca++ independent isoform (iNOS) can be isolated from a variety of cells following induction with inflammatory mediators and bacterial products. Hobbs et al., Annu. Rev. Pharmacol. Toxicol. 1999, 39,:191-220)
  • Nitric oxide can cause oxidative damage to cellular structures.
  • NO can react with reactive oxygen species (ROS) to produce reactive nitrogen species/substances such as peroxynitrite which is a powerful inducer of apoptosis.
  • ROS reactive oxygen species
  • Peroxynitrite itself can also generate ROS in at least some systems and ROS are also pro- apoptotic.
  • pre-treatment with NAC, a ROS scavenger fully scavenged peroxynitrite induced ROS generation and thus effectively inhibited peroxynitrite induced apoptosis.
  • NO may be involved in p53 mediated apoptosis (Brune et al., Cell Death & Differentiation, 10, 969-75, 1999).
  • NO induces alterations in the expression patterns of the Bcl-2 family of proteins known to be involved in the regulation of apoptosis.
  • Nf-k ⁇ the transcription factor Nf-k ⁇
  • Peroxynitrite induces apoptosis in epithelial and macrophage cells lines and this may be attenuated by pre-incubation of cells with L-ascorbic acid. Apoptosis is induced at peroxynitrite levels of 5-75 ⁇ M, concentrations greater than this inducing necrosis (Free Radic Biol Med 3, 489-95, 1997). Such results suggest that peroxynitrite may contribute to the pathology of gut inflammation by promoting apoptosis and this can be blocked the antioxidant ascorbic acid.
  • An additional example is the use of epigallocatechin gallate which protects U937 cells from nitric oxide induced cells cycle arrest and apoptosis (J Cell Biochem 81, 647-58, 2001).
  • the expression of gene products may regulate the transition of a cell between a non-apoptotic state and an apoptotic state after exposure to increasing levels of NO (5-100 ⁇ M).
  • a change in intracellular redox levels or an increase in NO levels may be capable of triggering apoptosis.
  • redox refers to a coupled reaction referred to as an oxidation- reduction (redox) reaction.
  • oxidation and reduction reactions are always coupled - whenever one substance is oxidised, another is almost simultaneously reduced.
  • oxidation refers to inter alia: (i) the action or process of reacting with oxygen, especially the addition of oxygen to a substance; (ii) the loss or removal of hydrogen from a substance; and (iii) the loss or removal of one or more electrons from a molecular entity, with or without concomitant loss or removal of a proton or protons. In this sense, oxidation is the opposite of, and is always coupled to, reduction.
  • reduction refers to the chemical process by which oxygen is withdrawn from, hydrogen is added to, or (more generally) an electron is added to (with or without addition of a proton) a molecular entity.
  • nitric oxide may be generated in the cell under a number of condition including, but not limited to, exposure of cells to ultra violet irradiation, cytokines (inc. IFN ⁇ , TNF ⁇ , ILl ⁇ , ILl ⁇ , IL-2, etc.), lipopolysaccharide, arachidonic acid, 17 ⁇ -Estradiol, L-glutamic acid, histamine, bradykinin, acetycholine hydroxyguanidine, A23187 and tetrahydro-L-biopterin.
  • cytokines inc. IFN ⁇ , TNF ⁇ , ILl ⁇ , ILl ⁇ , IL-2, etc.
  • lipopolysaccharide arachidonic acid
  • 17 ⁇ -Estradiol lipopolysaccharide
  • L-glutamic acid histamine
  • bradykinin bradykinin
  • acetycholine hydroxyguanidine A23187 and tetrahydro-L-b
  • an inhibitor of apoptosis may be added to the cells.
  • Nitric Oxide may be applied to the cell by exposure of the cell to a number of nitric oxide donors.
  • nitric oxide donors include, but are not limited to, sodium Nitroprusside, isosorbide-5-mononitrate, molsidomine, S-Nitrosocaptopril, S- Nitrosoglutathione, S-Nitrosoglutathione Monoethyl ester, + -S- Nitroso-N- acetylpenicillimine, Glyco-SNAP-1, Glyco-SNAP-2, S,S'-Dinitrosodithiol, Diethylamine NONOate, BNN3, BNN5Na, BNN5 Methyl Ester, NOC-5, NOC-7, NOC-9, NOC-12, NOC-18, NOR-1, NOR-3, NOR-4, 4-phenyl-3-fi_roxancarbonitrile, PROLI/NO, SIN-1, SNVP, Spermine NONOate,
  • an inhibitor of apoptosis may be added to the cells.
  • Nitric Oxide may be inhibited by treatment of cells with nitric oxide synthase inhibitors.
  • inhibitors may include, but are not limited to, Actinomycin D, cycloheximide, AET, ALLM, ALLN, N G -Allyl-L-arginine, aminoguanidine, l-Amino-2- hydroxyguanidine, p-Toluenesulfonate, 2-Amino-4-methylpyridine, AMITU, AMT, S- Benzykisothiourea, urea, trimethylphenylfluoroimidazole, Bromocriptine Mesylate, L- Canavanine sulfate, chlorpromazine, curcumin, cyclosporine, dexamethasone, 2,4- Diamino-6-hydroxypyrimidine, N G , N G - Dimethyl-L-arginine, Diphenyleneiodonium chloride, (-)- Epigallocatechin gallate, S-
  • the present invention includes the use of such selective NOS inhibitors to selectively modulate NO levels in cells (where the endpoint is apoptosis) and to identify novel genes whose expression is thereby modulated.
  • reactive Nitric Oxide species may be scavenged by the addition of reagents including, but not limited to Carboxy-PTIO, Hemoglobin, DTCS, NOX 100, MGD, PTIO, (+)-Rutin Hydrate, Quercetin pentaacetate.
  • reagents including, but not limited to Carboxy-PTIO, Hemoglobin, DTCS, NOX 100, MGD, PTIO, (+)-Rutin Hydrate, Quercetin pentaacetate.
  • Spin Traps include, but not limited to, N-tert-Butyl- ⁇ -phenylnitrone, DEMPO, DTCS, TEMPOL or POBN.
  • antioxidants may include, but are not limited to, N-acetyl-L-cysteine, AG 1714, Ferritin, CAPE, Caffeic acid, Bilirubin, Deferoxamine Mesylate, R-(-)- Deprenyl, DMNQ, DTP A, Dianhydride, Ebselen, Ellagic Acid, (-)-Epigallocatechin, L- Ergothioneine, EUK-8, reduced Glutathione, Glutathione Monoethyl ester, Glutathione Diethyl ester, ⁇ -Lipoic acid, Luteolin, Manoalide, MCI-186, MnTBAP, MnTNPyP, Morin Hydrate, NDGA, (+)- Taxifolin, Tetrandrine, Thioredoxin, Thioredoxin II, DL- ⁇ - Tocopherol, Trolox, U-74389G
  • the method of the invention is applicable to the discovery of gene products, and the genes encoding them, which are involved in apoptosis.
  • the term "gene product” includes but is not limited to polypeptide gene products and/or RNA gene products.
  • the gene products ofthe present invention may be natural gene products, which are encoded by naturally-occurring genes in the cell being investigated and are assembled in the cell using natural components such as amino acids or nucleotides.
  • the present invention also encompasses screening for gene products encoded by genes that are not endogenous to the cell being investigated.
  • genes may be, for example, heterologous genes from other cells or organisms, artificial genes encoding polypeptides comprising domains from different sources or composite RNA molecules, and wholly or partially randomised genes encoding repertoires of polypeptide or nucleic acid gene products.
  • polypeptide refers to any peptide comprising two or more amino acids, whether comprising a single domain or multiple domains, and includes multi-subunit proteins, which are cellular gene products.
  • RNA GENE PRODUCTS As used herein, the term "RNA gene products” includes but is not limited to ribozymes, antisense RNA molecules and/or mRNA molecules.
  • Levels of gene expression may be determined in any appropriate manner.
  • the invention comprises the measurement of protein production by mRNA translation, and is configured to detect increases or decreases in the rate or amount of mRNA translation.
  • the invention may also be configured to detect changes in post-translational processing of polypeptides or post-transcriptional modification of nucleic acids.
  • the invention may be configured to detect the phosphorylation of polypeptides, the cleavage of polypeptides or alternative splicing of RNA, and the like.
  • Levels of expression of gene products such as polypeptides, as well as their post-translational modification, may be detected using protein assays or techniques such as 2D polyacrylamide gel electrophoresis. Polypeptide or nucleic acid populations may be assessed individually, or together, in order to identify candidate gene products.
  • expression levels are assessed by measuring gene transcription. This is preferably carried out by measuring the rate and/or amount of specific mRNA produced in the cell.
  • a preferred embodiment of this 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 that are differentially expressed in the two populations.
  • the arrayed oligonucleotide probes are advantageously derived from cDNA or EST libraries, and represent genes that are expressed by the cells under investigation.
  • oligonucleotide and “polynucleotide” are equivalent, and imply no limitation as to maximum or minimum length.
  • 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. Examples of such cells include but- are not limited to, epithelial cells, cardiomyocytes, peripheral blood leukocytes and neurons.
  • Cell lines useful in the present invention include but are not limited to HeLa cells, fibroblast cell lines, myeloid cell lines such as HL-60, carcinoma cell lines and neuroblastoma cell lines.
  • Cells may be primary cultures of neurons or cells having neuronal characteristics. These cells include but are not limited to Ntera-II (human embryonal carcinoma cells which can be induced to differentiate towards neurons), PC- 12 (rat pheochromocytoma cells) and SH-SY5Y cells (neuroblastoma cell which can be induced to differentiate towards neurons when cultured in the presence of retinoic acid) or cells of haematopoietic origin such as HL-60 (human myeloid cell line which can be induced to differentiate towards neutrophils and monocytes and readily undergoes apoptosis).
  • Ntera-II human embryonal carcinoma cells which can be induced to differentiate towards neurons
  • PC- 12 rat pheochromocytoma cells
  • SH-SY5Y cells neuroroblastoma cell which can be induced to differentiate towards neurons when cultured in the presence of retinoic acid
  • haematopoietic origin such as HL-60 (human myeloid cell line which can
  • HeLa cells in the methods of the invention is effective and efficient, HeLa cells being easy to culture and well-known to those skilled in the art.
  • Cells and cell lines having documented pro- and anti-apoptotic reactions following NO treatment are listed in Brune et al, Apoptotic cell death and nitric oxide: activating and antagonistic transducing pathways; Biochemistry (Moscow) Vol 63, No 7 1998, p817-825.
  • Any suitable inhibitor of apoptosis may be employed, including physiological growth factors, for example, NGF or inhibitors of caspase enzymes such as Z-VAD.
  • Apoptosis may be inhibited through expression of genes, e.g., BCL-2.
  • An example of a suitable inhibitor is Neuronal Growth Factor (NGF).
  • NGF Neuronal Growth Factor
  • NGF has been shown to inhibit apoptosis and prolongs survival of neurons in culture Culturing neurons in NGF provides a very useful model system with which to study neuronal apoptosis. NO induced apoptosis can be directly stimulated in this system though the exogenous addition of NO generating agents such as sodium nitroprusside.
  • human peripheral blood neutrophils can also be induced to undergo apoptosis and this can be blocked by culturing cells in the presence of GM-CSF a survival factor similar to NGF.
  • a number of methods are known in the art for monitoring the onset of apoptosis. These include but are not limited to morphological analysis, DNA ladder formation, externalisation of membrane phospholipid phosphatidyl serine (lipid flipping) and caspase activation analysis.
  • the ability of NO to induce apoptosis is preferably confinned by monitoring the onset thereof according to one or more ofthe above methods.
  • the ability of NO to induce apoptosis is preferably confirmed by monitoring the onset of lipid flipping.
  • lipid flipping refers to 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 localised to the inner surface 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) Preferably, the ability of NO to induce apoptosis is preferably confirmed by monitoring the onset of caspase activation.
  • caspases The signal transduction system of apoptosis is relatively poorly understood at present, but a series of enzymes termed caspases have been shown over the last few years to be involved in different phases of apoptosis.
  • caspase 3 is involved in the activation of CAD, an endonuclease involved in the cleavage of DNA to yield the hallmark DNA ladder of apoptosis described above (Mcllroy et al, Cell Death Differ. 6, 495-6, 1999).
  • This caspase cleaves ICAD, a natural inhibitor ofthe CAD endonuclease.
  • caspase 8 Other caspases are involved in the propagation and execution phase ofthe process, an example of which is caspase 8. Not all incidences of apoptosis involve these enzymes and there is evidence that under conditions of high oxidative stress these cysteine-containing proteases are inactivated. To date 14 caspases have been identified as playing a role in the process (Alnemri et al., J Cell Biochem. 64, 33-42, 1997).
  • the ability of NO to induce apoptosis is preferably confirmed by monitoring the onset of a caspase independent activation mechanism.
  • a number of individual gene product types 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 assay ofthe invention may be configured to identify gene products which accelerate or retard the induction of apoptosis.
  • the assay detects gene products, which accelerate the induction of apoptosis.
  • the gene product to be monitored is unknown, however, methods are employed which facilitate the identification ofthe 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 of oligonucleotides specific to gene sequences archived in public domain databases, such as GenBank, are available commercially from a number of suppliers (such as GenomeSystems). Examples of such commercial arrays are in the form of either nucleotides spotted onto a membrane filter (such a nitrocellulose), or a solid support (such as glass). Commercial Gene Arrays may be used to profile the patterns of gene expression which are associated with the process of apoptosis in cells such as but not limited to neuronal cells.
  • Gene Arrays may be constructed in-house, by spotting nucleotide sequences derived from cDNA clones generated from in-house libraries or from cDNA clones purchased commercially. Such arrays allow the expression profiling of proprietary novel nucleotide sequences.
  • Candidate compounds capable of inhibiting or promoting apoptosis, preferably by interacting with gene products of genes identified by the methods described here, may be arranged in the form of arrays for screening purposes, as known in the art.
  • 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 cDNA libraries that are focused to specific cellular events, such as NO mediated apoptosis, offers the possibility to identify, clone and characterise novel genes that are associated with this process.
  • tissue types such as the neuron
  • cDNA constructed using a physical subtraction, such as the ClonTech 'Select' SSH (suppression hybridisation) method, allow the selective cloning of genes whose expression is differentially regulated in the process or cell type being studied.
  • Gene Array technology may be 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.
  • Libraries of candidate compounds which are capable of inhibiting or promoting (preferably inhibiting) apoptosis, preferably by interacting with gene products of genes identified by the methods described here, may be generated by methods known in the art. Such libraries may take the form of combinatorial libraries. Libraries of such compounds are also available commercially.
  • 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.
  • a ⁇ rays of nucleic acids may be prepared by spotting 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 are 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 Genome Sciences 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 commercial mRNA production method.
  • 2D PAGE 2-dimensional polyacrylamide gel electrophoresis
  • 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.
  • Samples for 2D PAGE may be prepared by conventional techniques.
  • Ntera-II is grown in a suitable medium, such as Dulbecco's modified Eagle medium (DMEM) containing 10% foetal calf serum (FCS), and treated with NO inducers as necessary.
  • DMEM Dulbecco's modified Eagle medium
  • FCS foetal calf serum
  • Cells are then rinsed, for example with DMEM without FCS and removed from the flask, for example by incubating them with a solution containing trypsin (0.5 g/1) and EDTA (0.2 g/1).
  • DMEM containing FCS is added into the flask to stop the action of the trypsin.
  • the cells are detached from the surface of the flask by squirting the solution onto the cells.
  • the suspension is transferred into a tube and the cells are centrifuged at 1000 g for 5 minutes. Supernatant is discarded and the cells are washed with DMEM without FCS. After centrifugation and removal of DMEM, 0.8 x 10 6 cells are mixed and solubilised with 60 ⁇ l of a solution containing urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM) and a trace of bromophenol blue. The whole final diluted NTERA-II sample is loaded on the first dimensional separation.
  • 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 before addition of NO to the cells, and serve as a standard for one or more subsequent assays; or it may be an integral part of every assay. For example mRNA or polypeptide populations from NO-induced and un-induced cells may be assessed simultaneously on a nucleic acid array or by 2D PAGE, and changes in expression patterns identified by direct comparison.
  • Polypeptides of interest may be isolated, sequenced and used to identify genes encoding them in the cell under investigation.
  • the invention may moreover be configured to screen libraries for genes capable of modulating the response of a cell to apoptosis.
  • apoptosis may be induced in cells by production/administration of NO, as described above, and libraries, such as cDNA libraries, of genes, screened in such cells.
  • Cells which demonstrate resistance to apoptosis, for example by increased survival times, are transformed with a nucleic acid . encoding a gene product which interferes with NO-induced apoptosis.
  • the genes may thus be cloned on the basis of function in such a screen.
  • pro- or anti-apoptotic compounds may also be determined in as assay as described herein. For instance, cells may be exposed to NO and then treated with the compound(s) to be tested. Variation in the survival of the cells is indicative of an effect ofthe compound(s) on NO-induced apoptosis.
  • apoptosis Many diseases are known to involve apoptosis and are thus targets for apoptotic therapies developed in accordance with the present invention.
  • the control of apoptosis in neurons may be useful in the treatment of a number of diseases, including but not limited to atherosclerosis, inflammatory conditions, systemic inflammatory response syndrome (SIRS), neurodegenerative diseases, retinal diseases, cancer metastasis, Alzheimer's and Parkinson's disease, adult respiratory distress syndrome (ARDS) and other related conditions, stroke, myocardial infarction, myelosuppression following chemotherapy or irradiation and a significant number of other diseases where cell death is a key feature ofthe pathology.
  • SIRS systemic inflammatory response syndrome
  • ARDS adult respiratory distress syndrome
  • Example 1 The invention will now be further described for the purpose of non-limiting illustration by way of examples.
  • Example 1 The invention will now be further described for the purpose of non-limiting illustration by way of examples.
  • This example describes the cellular and biochemical characterisation of the apoptosis process in the model systems described in the following examples.
  • a range of assays are established and used to measure the magnitude and temporal induction of apoptosis.
  • the earliest biochemical measurement of the apoptosis phenotype by these assays is considered to be the point beyond which the cells are 'committed' to the process of apoptosis.
  • these measurements determine the reproducibility of induction of apoptosis in the model systems.
  • these measurements determine the cellular mechanisms of apoptosis in these systems (such as whether apoptosis is caspase- dependent or caspase-independent).
  • Caspase activity (Caspase-3) is measured using a commercial kit (CaspACETM Assay System, Promega). The methodology is essentially as described by the manufacturer. Cells are removed from culture and centrifuged (300g/ 10 mi ⁇ ) at 4°C. The pellet is kept on ice, washed in ice-cold Hanks buffer and then resuspended in Cell Lysis Buffer at 10 / ml. Cells are lysed by freeze-thawed once, incubated on ice for 15 min, followed by centrifugation (15,000g /20 min) at 4°C. The caspase 3 activity present in the supernatant fraction is measured using the absorbance at 405nm.
  • binding buffer 400 ⁇ l is added to each tube and cells were analysed by flow cytometry using a Becto ⁇ Dickenson FACScan equipped with CellQuest software with excitation at 488nm and emission collected through a 530/30 bandpass filter for FITC (FL-1) and a 585/42 band pass filter for propidium iodide.
  • Excess stain is drained from the slide prior to immersion in solution C containing; methylene blue (0.4%w/v), Azure A (0.04% w/v), sodium phosphate dibasic (0.4% w/v), potassium phosphate monobasic (0.05% w/v) and potassium phosphate monobasic (0.4% w/v), to counterstain the cytoplasm.
  • Excess dye is rinsed; the slides air-dried and mounted in DPX aqueous mountant (BDH Laboratory Supplies, U.K.). Morphological examination is then carried out by light microscopy for the presence of apoptotic cells as determined by the loss of membrane asymmetry and condensation of cytoplasm and nuclei (Cotter and Martin, 1996).
  • HeLa cells are trypsinized, and made up to a concentration of 6 x 10 4 cells/ml.
  • One ml of cells are plated/well of a 24 well plate and allowed to adhere overnight. The following day cells are incubated with Sodium Nitroprusside (made up in DMEM; final cone 0.9 xlO "6 M) in the presence or absence of 20mM N-acetyl-L-cysteine (NAC). Cells are incubated for the indicated period after which cells removed and examined for signs of apoptosis by microscopy. Slides of the cells are made by placing 3x 10 4 cells on a slide using a cytospin (Shandon II cytocentrifuge).
  • This example describes the biochemical characterisation of intracellular NO status in the model systems described below, as cells undergo apoptosis.
  • Primary neurons or neuronal cell lines are cultured in medium deficient in the survival factor NGF.
  • the earliest biochemical measurement of production or inhibition of NO generation is considered to be the point beyond which cells are undergoing the process of "Induction of Commitment" to apoptosis.
  • the measurements determine the reproducibility of NO generation in the model systems.
  • Nitric oxide can be generated in cells under a number of conditions including the addition of sodium nitroprusside to the culture medium.
  • sodium nitroprusside is added at concentrations 0.1-100 ⁇ M to induce NO production. The effects of this on the induction of apoptosis are monitored using the assays described previously.
  • Nitric oxide is measured in cells undergoing apoptosis using a flow cytometry based assay with the NO sensitive probe DAF-2 DA (Kojima et al., Neuroreport, 9, 3345-8, 1998).
  • L-NAME a synthetic inhibitor of NO synthase (5-50 ⁇ M) is incubated with cells prior to the induction of apoptosis by survival factor withdrawal. The level of apoptosis is monitored by caspase activation, phosphatidylserine exposure and morphological assays. NO Scavenging
  • ⁇ X 100 a nitric oxide scavenger is used to neutralise the effects of NO produced following the withdrawal of survival factors such as NGF from cells in culture. At indicated time points NOX 100 at 30 ⁇ M is added to cell cultures prior to NGF withdrawal. Cells are monitored for the induction of apoptosis using assays already described in this document.
  • This example describes the establishment of two model cell systems to study NO mediated apoptosis in cell lines and in an in vivo model system.
  • neuronal cell lines such as Ntera-II, SH-SY5Y, HL-60 or PC- 12 are induced to undergo apoptosis by treatment with either sodium nitroprusside or removal of survival factors to induce NO production and subsequent apoptosis.
  • NO scavengers such as NOX 100 to block the effects of NO.
  • mice In the second model system that is an in vivo one, adult male Balb-c mice are maintained in the dark for 18 hours before being exposed to constant light. Immediately prior to light exposure their pupils are dilated with 5% cyclopentolate. The mice are then exposed to 2 hours of cool white fluorescent light at an illuminesence level of 5000 lux. The mice are sacrificed after treatment by cervical dislocation at the following time points: 30min and lhr after light onset, immediately after light exposure (Ohr) and after 6hr 14hr and 24hr of darkness, followed by 2hr light exposure. Under these condition extensive apoptosis occurs in neuronal derived retinal cells and this is a particularly good in vivo model for the study of apoptosis.
  • DNA strand breaks in photoreceptor nuclei are detected by in situ end-labelling (TUNEL).
  • TUNEL in situ end-labelling
  • enucleated eyes were fixed in 10% buffered formalin for 24 hours, dehydrated, processed and embedded in paraffin. Sections (5 ⁇ m) are incubated in 50 ⁇ l of reaction buffer containing 2.5mM CoCl 2 , O.lU/ml terminal deoxynucleotidyl transferase (TdT) in a 0.1M Na cacodylate (pH 7.0) buffer and 0.75nM fluorescein- 12-dUTP (Boehringer Mannheim, Germany). These sections are incubated at 37°C for 1 hour in a humidified chamber.
  • reaction buffer containing 2.5mM CoCl 2 , O.lU/ml terminal deoxynucleotidyl transferase (TdT) in a 0.1M Na cacodylate (pH 7.0) buffer and 0.75nM
  • the cells are then washed in PBS and fixed in 1% paraformahdehyde at 4°C for 30 min.
  • Cells are washed in permeablization buffer (PB: lOmM HEPES, 150mM NaCl, 4% FCS, 0.1% sodium azide and 0.1% Triton-X-100) and resuspended in PBS containing 0.125 ⁇ g anti-active caspase-3 antibody (PharMingen International, San Diego, CA, USA), or the same concentration of an isotype control (goat IgG, Sigma, UK) and incubated for one hour at 4°C.
  • PB permeablization buffer
  • PBS 0.125 ⁇ g anti-active caspase-3 antibody
  • an isotype control goat IgG, Sigma, UK
  • HeLa cells are obtained from the ATCC (Manassas, Virginia, USA) and maintained in DMEM medium with 10% FCS at 37°C in a 5% CO2 atmosphere.
  • Sodium Nitroprusside, N-acety-L-cysteine, MTT, and DMSO are all purchased from Sigma (Fancy Road, Poole, Dorset, UK).
  • Treatment of HeLa cells with the NO donor Sodium Nitroprusside in presence or absence of N-acetyl-L-cysteine
  • HeLa cells are trypsinized, and made up to a concentration of 5 x 10 4 cells/ml. One hundred microlitres of cells are plated/well of a 96 well plate and allowed to adhere overnight. The following day cells are incubated with Sodium Nitroprusside (made up in DMEM; final cone 0.9 xlO " M) in the presence or absence of 20mM N-acetyl-L-cysteine (NAC). Cells are incubated for a further 18h.
  • HeLa cells are trypsinized, and made up to a concentration of 5 x 10 4 cells/ml. One hundred microlitres of cells are plated/well of a 96 well plate and allowed to adhere overnight. The following day cells are incubated with Hydrogen Peroxide soln (final cone 1.3xlO "6 M) in the presence or absence of 20mM N-acetyl-L-cysteine (NAC). Cells are incubated for a further 18h.
  • Hydrogen Peroxide soln final cone 1.3xlO "6 M
  • NAC N-acetyl-L-cysteine
  • HeLa cells (3x106/ 35mls) are cultured overnight in a T175 flask to allow adherence. Following morning cells are treated with Sodium Nitroprusside (0.9 xl0 "6 M) + NAC (20mM) and further incubated for 4 or 6 hours. Cells are harvested for RNA by adding 4mls RNAzol to the cells at the indicated times. Sodium Nitroprusside (an NO donor) induces HeLa cell death, which is reversed by NAC Treatment
  • HeLa cells obtained from the ATCC are maintained in DMEM medium with 10% FCS at 37oC in a 5% CO 2 atmosphere. HeLa cells are trypsinized, and made up to a concentration of 5 x 10 4 cells/ml. One hundred microlitres of cells are plated/well of a 96 well plate and allowed to adhere overnight. The following day cells are incubated with sodium nitroprusside (made up in DMEM; final cone 0.9 xlO "6 M) in the presence or absence of 20mM N-acetyl-L-cysteine (NAC; Sigma, UK). Cells are incubated for a further 18h.
  • sodium nitroprusside made up in DMEM; final cone 0.9 xlO "6 M
  • NAC N-acetyl-L-cysteine
  • Cell viability is measured by MTT assay. At 18 hours post-treatment the supernatant is removed and cells are washed in 2X serum free DMEM medium prior to adding lOO ⁇ l fresh medium containing 0.5mg/ml MTT (Sigma, UK) and allowed to incubate for a further 4 hours before developing the insoluble formazan products with DMSO. Viability is positively correlated with the optical density at 570nm.
  • H 2 O 2 (a ROS) induces HeLa cell death, which is reversed by NAC Treatment
  • HeLa cells obtained from the ATCC are maintained in DMEM medium with 10% FCS at 37°C in a 5% CO 2 atmosphere. HeLa cells are trypsinized, and made up to a concentration of 5 x 10 4 cells/ml. One hundred microlitres of cells are plated/well of a 96 well plate and allowed to adhere overnight. The following day cells are incubated with H 2 O (made up in DMEM; final cone 1.3xl0 "6 M) in the presence or absence of 20mM N-acetyl-L-cysteine (NAC; Sigma, UK). Cells are incubated for a further 18h. Cell viability is measured by MTT assay.
  • This example describes the characterisation, cloning and analysis of oligonucleotide/polynucleotide sequences whose expression changes are associated with apoptosis.
  • This example describes the use of transcription inhibitors such as, Actinomycin D (actD) to determine the dependence of apoptosis on gene transcription.
  • transcription inhibitors such as, Actinomycin D (actD) to determine the dependence of apoptosis on gene transcription.
  • Genomics techniques such as, microarray and subtraction cDNA cloning.
  • microarrays are used to measure global gene expression associated with apoptosis. 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.
  • Suppression Subtractive Hybridisation is used to identify and clone cDNA sequences derived from differentially expressed genes. Such differential gene expression is associated with a measurable apoptotic phenotype. Such cDNA sequences are extended to encompass the full length coding sequence for the mRNA gene product.
  • This example describes the process of microarraying (in the context of a filter based microarraying) and its use to profile gene expression of thousands of genes simultaneously.
  • the microarray process can be separated into three parts: the preparation ofthe microarray filter, the hybridisation of radiolabelled cDNA probes, and the detection and quantitation of the microarray results .
  • DNA 'probe' sequences are obtained for genes to be represented on a particular microarray. These sequences would typically be EST cDNA sequences cloned in a plasmid vector, either from in-house libraries or obtained from commercial sources (e.g.
  • the DNA sequences used to construct the microarray are amplified by PCR using common primer sequences found flanking the multiple cloning sites of most commercial cloning vector plasmids as follows:
  • T7 Primer 5' gta ata cga etc act ata ggg c 3' PCR reactions are carried out as follows: l-2 ⁇ l of glycerol stock, miniprep DNA or overnight culture is added to a 20 ⁇ l reaction containing, 0.4 ⁇ l lOmM dNTP mix, 1 x reaction buffer, 0.4 ⁇ l each 20 ⁇ M primer and 0.5U Taq polymerase (Qiagen). PCR amplification is earned out with a MWG HT Primus 4 x 96 well thermocycler as follows: 94°C 60 s, 35 cycles of 94°C 40 s, 55°C 30 s, 72°C 120 s, followed by 72°C 120 s.
  • PCR reaction products are purified using 96-Well PCR Multiscreen (Millipore) as described by the manufacturer.
  • PCR products are resuspended at lOOng/ ⁇ l TE in V bottom 96-well microtitre plates.
  • Filter microarrays are spotted as a custom service by GeneScreen Ltd (UK).
  • commercial filter arrays 'Human LifeGridTM' are purchased from Genome Systems Inc. (USA).
  • This example describes the synthesis of a radiolabelled cDNA from total cellular mRNA.
  • the labelled 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 stand 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 5 h. Unincorporated nucleotides are removed using spin-columns and the labelled probe stored at -80°C until required.
  • RT M-MLV reverse transcriptase
  • This example describes the complementary hybridisation of radiolabelled cDNA probe to DNA fragments immobilised onto a membrane.
  • Methodology is as described by manufacturer, for Human LifeGridTM arrays.
  • 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).
  • Hybaid hybridisation oven
  • the labelled cDNA probe is added to fresh hybridisation buffer (5 to 20 ml) and hybridised at 42°C for 14 to 16 h.
  • 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.
  • This example describes the use of a Phosphoimager to quantitatively image positive signals across the filter arrays.
  • Hybridised filters are wrapped in plastic wrap and imaged on a Storm Phosphoimager (Amersham Pharmacia Biotech), with a 5 to 48 h exposure and a resolution of 50 to 100 microns.
  • the *.tif image generated by the Phosphoimager is analysed, quantitated and background subtracted using Array Vision software (Imaging Research Inc.).
  • SSH Suppression Subtr active Hybridisation
  • This example describes a method to identify and clone differentially expressed genes.
  • RNAzol B guanidine isothiocyanate lysis followed by caesium chloride gradient centrifugation, or acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), or RNeasy RNA preparation kits (Qiagen). Any contaminating genomic DNA is removed by DNase treatment (DNase I, Gibco-BRL).
  • RNA is then used to prepare mRNA, using the Oligotex mRNA purification kit (Qiagen), or a similar system (Clontech or Strategene). Briefly, mRNA is purified by passing the total RNA over an oligo-dT column.
  • the oligo-dT may be attached to cellulose or glass beads or biotin, and the column may be either spin or gravity format.
  • the bound mRNA is subsequently washed and eluted, ready for use in subtractive hybridisation.
  • SSH is performed on mRNA purified from treatment or control cells essentially as described (Diatchenko et al 1996), using a PCR-select cDNA subtraction kit (Clontech, K1804). Briefly, cDNA is synthesised from the two pools of mRNA (control and treatment, driver and tester respectively). The resulting cDNA is digested with a restriction enzyme generating a blunt end product (typically Rsal). The tester cDNA is divided into two subsets and distinct adaptor molecules are ligated to each of the cDNA pools.
  • tester cDNA samples are then separately combined with driver cDNA in a solution containing 50mM HEPES, pH 8.3; 0.5M NaCl; 0.02mM EDTA, pH 8.0.
  • driver cDNA samples are then separately combined with driver cDNA in a solution containing 50mM HEPES, pH 8.3; 0.5M NaCl; 0.02mM EDTA, pH 8.0.
  • the tester and driver cDNAs are allowed to anneal for lOhrs at 68C.
  • the two samples are combined and a fresh portion of heat denatured driver cDNA is added. The samples are allowed to anneal for a further 10 hours at 68°C.
  • the hybridised cDNA is then diluted in a solution containing 20mM HEPES, pH 8.3; 50mM NaCl; 0.2mM EDTA) and heated at 72C for 7minutes, prior to PCR amplification.
  • PCR is performed under standard conditions using the Advantage cDNA PCR kit (Clontech). Only cDNAs that have the correct primer combination, the differentially expressed cDNAs, will amplify exponentially.
  • PCR products from the subtractive hybridisation are inserted into a TA cloning vector, for example pCRII T/A cloning kit (Invitrogen).
  • TA cloning vector for example pCRII T/A cloning kit (Invitrogen).
  • This library of differentially expressed cDNAs is then transformed to E.coli and the transformants selected for miniprep and sequence analysis. Plasmid miniprep
  • This example describes the extraction of double stranded plasmid DNA from cDNA clones.
  • This example describes the di-deoxy sequencing of cloned cDNA inserts within the vector pT-Adv (Clontech).
  • Plasmid miniprep DNA (100 ng to 5 ⁇ g) is sent to MWG Biotech for contract sequencing. Sequencing reactions are primed using one of the following universal primer sequences:
  • the SSH procedure for the cloning of differentially expressed gene products also generates a portion of artifactual cDNAs 'false positives' which are not differentially expressed.
  • This example describes the use of microarray to confirm the identity of truly differentially expressed clones prior to the laborious task of sequencing.
  • cDNA insert sequences are amplified by PCR and spotted onto microarray filters (GeneScreen Ltd).
  • Duplicate filters are hybridised with radiolabelled cDNA probes generated from the reciprocal RNA material used to generate each SSH library (i.e. if an SSH library was made from a subtraction between diseased vs. normal cells, then one filter is hybridised with probe synthesised from RNA isolated from diseased cells and the other from normal cells.
  • An analysis of the filters identifies with cDNA clone is differentially expressed.
  • Full length clones are isolated from poly A+ RNA using SmartTM RACE Technology (Clontech) according to the manufacturers protocol. Briefly, double stranded cDNA is generated using the Smart II oligonucleotide and the CDS Primer (both supplied in the kit). Specific PCR products are then generated using the PCR primer (supplied in the kit) and gene specific primers (sense and antisense primers generated from the DNA sequence to be extended). To increase specificity, nested sense and antisense primers are used in secondary PCR amplification. PCR products are then ligated into plasmids, such as the
  • Plasmids are purified using mini-prep isolation system (Qiagen) and plasmids are submitted to MWG for sequencing. Specific 5' and 3' sequences are identified using sense and antisense gene specific primers. Products are to be sequenced approximately 10 bases 5' of the initiation codon for 5' PCR products and 10 bases 3' of stop codons for 3' PCR products. Using sequence data, 5' and 3' primers are made and full length cDNA is amplified.
  • RNA is isolated and examined for gene expression changes using microarray. Significant transcription of mRNA is observed.
  • 1169 genes are detected using Incyte human 'LifeGrid' filters; these genes are increased or decreased by greater than 2-fold over a 6hour time course as compared with the time zero control HeLa cells.
  • 943 genes are increased or decreased as early as 4 hours post-treatment, and prior to the 'commitment to die' point identified as described.
  • 469 genes are increased or decreased by 6 hours post-treatment.
  • Figures 3, 4 and 5 show cluster analysis of genes regulated during sodium nitroprusside-induced HeLa cell apoptosis time course. Genes fall into primary clusters of up (white) and down- (black) regulated genes. By 6 host post treatment - 184 genes are up-regulated and -285 down-regulated.
  • Inhibition of apoptosis by the redox modulator NAC blocks changes in gene expression associated with, sodium nitroprusside -induced HeLa cell apoptosis.
  • NAC Sodium nitroprusside-induced HeLa cell apoptosis is inhibited by the addition of NAC (20mM). Gene expression change most closely and reciprocally correlated with HeLa cell survival (with NAC), or death (without NAC), is most likely to be causally involved in the apoptosis regulatory process. Furthermore, the use of NAC is directed at removing or modulating the route cause of apoptosis-induction i.e. the production and action of reactive nitrogen species.
  • Changes in 'early' gene expression include classes of genes regulated by NO (RNS) and/or by hypoxic stress and include classes of genes known to be associated with cell defence mechanisms to apoptosis and ROS/RNS induced stress.
  • RNS NO
  • hypoxic stress include classes of genes known to be associated with cell defence mechanisms to apoptosis and ROS/RNS induced stress.
  • genes regulated as early as 4 and 6 hours post-treatment with sodium nitroprusside some include genes that are known to be transcriptionally regulated by NO and/or RNS. Also included are genes that are known to be transcriptionally regulated by hypoxic stress which is well characterised as involving ROS and RNS.
  • Scleroderma is a disease characterised by hypoxia and reactive oxygen species, where antioxidant therapy has been proposed as a treatment (Toxicology 2000 Nov 30;155(1- 3): 1-15 Emerging potentials for an antioxidant therapy as a new approach to the treatment of systemic sclerosis. Simonini G et al).
  • polymyositis/scleroderma autoantigen 2 (lOOkD) is a prominent autoantigen associated with the disease.
  • Figure 6 shows that polymyositis/scleroderma autoantigen 2 (lOOkD) is decreased associated with sodium nitroprusside induced HeLa cell apoptosis but blocked by treatment with NAC.
  • aryl hydrocarbon receptor nuclear translocator and hypoxia-inducible factor (HIF)-l alpha mediate cellular responses to hypoxia.
  • Rht hypoxia-inducible factor
  • HIF hypoxia-inducible factor
  • Figure 7 shows that aryl hydrocarbon receptor nuclear translocator- like gene is decreased associated with sodium nitroprusside induced HeLa cell apoptosis but blocked by treatment with NAC.
  • Calpain is a noncaspase protease mediator of apoptosis (Leukemia 2000, 14(9) 1695-703 Noncaspase proteases in apoptosis Johnson DE).
  • Calpastatin (calpain inhibitor 1) is a physiological inhibitor of calpstatin. Calpain inhibitorl reduces the renal dysfunction and injury associated with ischemia/reperfiision of the kidney, a disease associated with hypoxic stress and apoptosis (Kidney Int 2001 Jun;59(6):2073-83 Calpain inhibitor-1 reduces renal injury in the rat. Chatterjee PK et al).
  • NO nitric oxide
  • Nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin.
  • the nitric oxide donor sodium nitroprusside prevented many ofthe effects of calcium ionophore on C(2)C(12) muscle cells, indicating that nitric oxide inhibition of calpain protected the cells from ionophore-induced proteolysis.
  • Calpain inhibitor I and a cell-permeable calpastatin peptide also protected the cells from proteolysis, confirming that ionophore- induced proteolysis was primarily calpain mediated (Am J Physiol Cell Physiol 2000 Sep;279(3):C806-12 Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells. Koh TJ and Tidball JG.
  • Figure 8 shows that Calpastatin is decreased associated with sodium nitroprusside induced HeLa cell apoptosis but blocked by treatment with NAC.
  • a network of radical acceptors and enzymes is thought to play an important redox- buffering role to protect cells against NO-mediated injury.
  • Thioredoxin reductase is one of the key cellular antioxidant defense enzymes.
  • An inverse correlation between cell susceptibility to NO damaging effects and Trx expression has been described, suggesting that the Trx system may have important preventative capacities towards NO-mediated cellular injury in monocytic macrophage cells (Biochem J 2000 Mar 15;346 Pt 3:759-65 Protective effect of thioredoxin upon NO-mediated cell injury in THP1 monocytic human cells. Ferret PJ et al).
  • Figure 9 shows that thioredoxin reductase 1 is decreased associated with sodium nitroprusside induced HeLa cell apoptosis but blocked by treatment with NAC. These results suggest that thioredoxin reductase 1 may have a protective role in cells challenged with NO/RNS.
  • Matrix metalloproteinases play an integral part in tumor spread and the metastatic cascade.
  • NO donors such as S-nitroso-n-acetylpenicillamine and S- nitrosoglutathione (both at 0.01-100 microM) inhibit MMP-2 release from platelets and tumor cells (Cancer Res 2001 Jan l;61(l):376-82
  • Jurasz P et al Figure 10 shows that matrix matalloproteinase 2 is decreased associated with sodium nitroprusside induced HeLa cell apoptosis but blocked by treatment with NAC.
  • Ubiquitin C-terminal hydrolase Ll represents 1 to 2% of total soluble brain protein. Its occurrence in Lewy bodies and its function in the proteasome pathway make it a compelling candidate gene in Parkinson disease, a disease characterized by reactive oxygen stress and apoptosis. A marked increase of ubiquitin C-terminal hydrolase Ll immunoreactivity in neurons 1 hr after ischemia/hypoxia in a rat model. These data suggest that ubiquitin C-terminal hydrolase Ll is involved in the neuronal stress response (Neurochem Res 1997 Jan;22(l):93-100 Ubiquitin-mediated stress response in a rat model of brain transient ischemia/hypoxia. Gubellini P et al).
  • FIG. 11 shows that ubiquitin C-terminal hydrolase Ll is increased associated with sodium nitroprusside induced HeLa cell apoptosis but blocked by treatment with NAC.
  • Changes in transcription are not necessarily paralleled by similar changes in translation.
  • protein expression may also be dependent upon post-translational modification such as phosphorylation, proteolytic cleavage or changes in intracellular location.
  • This example describes proteomic studies, which complement the use of genomic studies.
  • Translation inhibitors such as cyclohexamide, are used to establish the dependence of apoptosis/NO-mediated apoptosis on protein synthesis.
  • changes in expression/translation of candidate genes are measured using FACS, or 20- PAGE to measure individual proteins or hundreds or thousands of proteins simultaneously.
  • Proteomics may be applied to the identification of novel apoptosis-associated proteins in a number of ways.
  • the proteins may be labelled metabolically using 35 -S or 33 -P radioactive sources and then detected through phosphoimaging or by conventional autoradiography.
  • Proteins from different treatment groups may be differentially labelled with either P or a Cy5 derivative (Amersham Pharmacia Biotech) and then analysed simultaneously on the same gel, thereby facilitating identification of differentially expressed products.
  • post-staining, approaches may be used, including silver staining, anti-phosphotryosine antibody staining, anti-phosphoserine antibody staining, carbohydrate-specific staining procedures (for example based on hydrazine modification) or similar post-translational modification detection techniques known to the art.
  • Gel images derived from post-stained gels are captured using CCD camera, phosphoimaging or by densitometry and analysed digitally to determine differences in protein expression patterns using software such as Melanie (BioRad) or Phoretix 2D (Phoretix). Measurement of individual or global protein expression and/or post-translational modification by 2D-PAGE
  • Neurons (1.5xl0 7 cells/ml) are incubated in RPMI-1640 supplemented with 60 ⁇ Ci/ml [ 35 S]-methionine. Cells are challenged with agents influencing the process of apoptosis and are harvested at appropriate time points following treatment, from 0 to 22 hours.
  • Total cell protein is prepared by lysing the cells in a solution comprising 8M Urea (Ultrapure); 2% CHAPS (Sigma); 40mM Tris(HCl) (Sigma). In some cases addition or omission of DTT may be desirable. RNA and DNA contamination may be removed by treatment with RNases and DNases. A sequential extraction procedure may be used with which more hydrophobic proteins can be solubilised. Polypetides synthesised following the addition of the radioactive tracer may then be detected by 2D-PAGE analysis of the lysates.
  • TrisHCI Tris base, glycine, (Sigma); trichloracetic acid (TCA), NaOH, glycerol, n-butanol, bromophenol blue (Merck); [ 35 S]-methionine (SJ 204, specific activity: > 1.000 Ci/mmol, containing 0.1% 2mercaptoethanol), Amplify (Amersham International, Amersham, UK); filters (HAWP 0.25 m pore size) (Millipore, Boston, USA); RNase A, DNase I; urea (ultra pure); acrylamide, bisacrylamide, TEMED, ammonium persulphate (BioRad, Richmond, CA,USA); ampholytes: pH 5-7, pH 3.5-10, pH 7-9, pH 8-9.5 (Pharmacia, Uppsala, Sweden); Nonidet P-40 (BDH, Poole, UK); ampholytes: pH 5-7 and sodium dodecyl sulphate
  • first dimension gels contain 4% acrylamide, 0.25% bisacrylamide and ampholytes, and are 18 cm long and 0.4 cm in diameter. Equal numbers of counts of each cell lysates sample are applied to the gels. In the case of lower amounts of radioactivity it may be necessary to regulate the exposure time ofthe gel so that comparable total optical densities are obtained.
  • the samples are first resolved on isoelectric focusing (IEF; pH 3.5-7) gels. IEF gels may be pre-focused for approximately 30mins at 140 ⁇ A/gel (limiting current), the sample is then applied, by either cup-loading (Multiphor) or in-gel rehydration (IPG-phor), and focused for varying times and voltages appropriate to short or long IEF strips.
  • IEF isoelectric focusing
  • Second dimension gels 1 mm thick, 200 mm long and 185 mm wide contain acrylamide and bisacrylamide, prepared as gradient (9-16%) or single percentage (12%) gels. Focused IEF strips are loaded to the second dimension gels through a 1.0% LMP-agarose top layer. After electrophoresis in Tris-glycine buffer, the gels are fixed in 45% methanol and 7.5% acetic acid for 30 min and treated for fluorography with Amplify for 30 min before being dried. The gels are placed in contact with X-ray films and exposed at -70 C for 1 to 40 days. Each gel is exposed for at least 3 time periods to compensate for the lack of dynamic range of X-ray films.
  • Proteins from 2D gels may be identified by immuno-blotting, N-terminal sequencing, internal peptide sequencing, co-migration of unknown with known proteins, or over- expression in a model organism. Additionally proteins will be prepared for analysis by either Electrospray Ionisation (ESI)-MS or Matrix Assisted Laser Desorption Ionization- time of flight (MALDI-TOF)-MS. Using these techniques it is possible to measure the masses of peptides derived from protease or chemical digestion. The set of masses produced by such a digestion is unique to a protein, and so constitutes a peptide mass fingerprint, which can form the basis of a database search.
  • ESI Electrospray Ionisation
  • MALDI-TOF Matrix Assisted Laser Desorption Ionization- time of flight
  • Triple quadrupole or ion trap mass spectrometers can be used to generate fragmentation spectra from peptides (MS/MS), from which sequence data can be obtained.
  • Peptides are fragmented by isolating the 'parent' peptide ion and then accelerating it into a region filled with an inert gas, typically argon.
  • the peptide ion undergoes multiple collisions with the gas and fragments in a random manner along the amide backbone.
  • the resulting 'daughter' ions are detected and a fragmentation pattern, which contains sequence specific information, is derived. This information can be used to extend database searches, or to derive degenerate oligonucleotides for library screening.
  • Additional analysis techniques include amino-acid analysis through HPLC, or direct HPLC of peptides derived through protease cleavage of the target protein.
  • the genomics strategy described in the present invention provides the means to characterise the mechanism of apoptosis.
  • the strategy provides the means to identify, characterise, clone and validate molecules, including oligonucleotides and polypeptides, associated, both causally and consequentially, to apoptosis.
  • the model described enables the identification of genes which are involved in, or cause, early stages of apoptosis.
  • the model also provides a screen for substances which interact with such genes to inhibit apoptosis. Such compounds and substances identified may be used in treating diseases involving apoptosis.
  • cells such as HeLa cells are cultured, and apoptosis induced by administration of a suitable substance, for example, Sodium nitroprusside (SNP).
  • suitable substances include nitric acid donors which are capable of generating reactive nitric oxide (NO) species.
  • gene expression may be studied at times which precede the earliest morphological manifestation of apoptosis (for example, gene expression may be studied at 1, 2, 3, 4, or 5 hours, whereas the earliest morphological signs of apoptosis generally appear at 6 hours).
  • the model system described here therefore is capable of blocking the root cause of apoptosis by modulating the action of nitric oxide.
  • it is capable of identifying genes which are involved in the earliest stages of apoptosis, before morphological signs of apoptosis appear.
  • Other cells may also be used in the model system and assays described here.
  • Cells such as the human Ntera-II neuronal cell line or primary rodent neurons may be cultured in a serum-free cell culture medium these cells undergo apoptosis following withdrawal of serum (Forgie et al, (2000) Eur J Neurosci, 2, 610-616).
  • the onset of apoptosis is characterised using markers, such as caspase activation and externalisation of the membrane phospholipid phosphatidylserine. These assays serve to identify the earliest measurable onset of the cells commitment to apoptosis. Intracellular events that drive commitment of a cell to apoptosis, occur before this earliest measurement of the commitment.
  • the intracellular initiator of the commitment to apoptosis is a change in REDOX manifested by an increase in level of NO. Intracellular NO is measured to confirm this increase preceding the measurement of apoptosis.
  • Suitable inhibitors of NO production include NOX 100 and L-NAME are used to confirm the causal role of NO in mediating apoptosis in this model system.
  • the characterisation of the apoptosis process and NO involvement in this model system serve to establish a temporal window between the onset of NO (which is the initial trigger for the apoptosis process) and the measurement of cellular apoptosis (at which point the cell is committed to undergoing apoptosis).
  • the key early intracellular events that induce the commitment of cells to undergoing apoptosis (and those which may represent the most attractive therapeutic targets) occur within this window.
  • NGF has been shown to inhibit apoptosis and prolongs survival of neurons in culture Culturing neurons in NGF provides a very useful model system with which to study neuronal apoptosis.
  • NO induced apoptosis can be directly stimulated in this system though the exogenous addition of NO generating agents such as sodium nitroprusside.
  • NO generating agents such as sodium nitroprusside.
  • apoptosis may be induced by administration of sodium nitroprusside or any other source of NO to the cell.
  • Apoptosis may be inhibited in various ways, for example, by administration of NAC.
  • Signal transduction events are associated with induction of commitment of cells to undergoing apoptosis. Signal transduction would typically involve events such as protein phosphorylation and/or de-phosphorylation. Transcription events are associated with induction of commitment of cells to undergoing apoptosis. Transcription inhibitors, such as Actinomycin D (actD) are used to measure the role of transcription events in the induction process. In addition, the use of actD in pulse-chase experiments is used to establish the precise time point at which transcription events exert an effect.
  • actD Actinomycin D
  • the invention provides the means to allow the study of the transcription of individual mRNAs in the early 'induction of commitment' phase of apoptosis, and in particular following NO.
  • One candidate gene that may be associated with this process is FasL.
  • the expression of individual candidate genes is measured by RT-PCR or hybridisation with a gene specific radiolabelled probe.

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Abstract

The present invention describes a method for modulating the expression of gene products that regulate the transition of a cell from a non-apoptotic state to an apoptotic state, comprising modulating the intracellular concentration of Nitric Oxide (NO). The present invention provides a mechanism for the regulation of expression of NO-induced genes which may be used to induce or prevent the apoptotic process.

Description

SCREENING METHOD 2
FIELD OF THE INVENTION
The present invention relates to a method for the identification of genes that are involved in the modulation of apoptosis in cells.
In particular, the invention relates to a novel method for identifying and characterising those genes, and the molecular mechanisms involved in the modulation of cell death by apoptosis. Moreover, the present invention relates to a novel method for inducing the expression of gene products involved in the modulation of the early stages of apoptosis, and their subsequent identification and characterisation.
BACKGROUND TO THE INVENTION
Programmed cell death or apoptosis is a genetically regulated 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).
Under normal physiological conditions apoptosis is tightly regulated. However, there are a number of diseases where the process becomes deregulated, leading to a particular pathology. Examples of where apoptosis is retarded or inhibited include some types of tumour development, a number of inflammatory conditions such as adult respiratory distress syndrome (ARDS) and other related conditions (Matute-Bello et al, Am J Respir Crit Care Med. 56, 1969-77, 1997). 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 ofthe pathology. Apoptosis is characterised by a number of well-defined biochemical hallmarks. These include DNA fragmentation, lipid flipping and caspase activation. By way of example, DNA fragmentation is caused by the activation of an endogenous endonuclease enzyme (Wyllie, Nature, 284, 555-556,1980; Enari et al, Nature, 391, 43-50, 1998). The result is a DNA ladder pattern, which 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 ofthe reticulo-enothelial system.
Lipid flipping is a second well-defined characteristic resulting in the exposure of the phospholipid phosphatidylserine to the outside surface of the plasma membrane of the cell as it undergoes apoptosis (Fadok et al., I Immunol. 148, 2207-16, 1992). Normally this lipid is localised to the inner surface of the membrane lipid bilayer. Although the underlying mechanism responsible for this lipid flipping is poorly understood at present, it serves as a signal for the recognition and phagocytosis of the apoptotic cell (Fadok et al., J Immunol. 148, 2207-16, 1992)
A series of enzymes termed caspases have also been shown over the last few years to be involved in different phases of apoptosis. Although the signal transduction system of apoptosis is relatively poorly understood at present, caspase enzymes, in particular, are thought to play a role in the propagation and execution phases of the process (Samali et al., Cell Death Differ. 6, 495-6, 1999). For example, caspase 3 is involved in the activation of CAD, an endonuclease involved in the cleavage of DNA to yield the hallmark DNA ladder of apoptosis described above (Mcllroy et al., Cell Death Differ. 6, 495-6, 1999). This caspase cleaves ICAD, a natural inhibitor of the CAD endonuclease. Other caspases are involved in the propagation and execution phase of the process, an example of which is caspase 8. Apoptosis can also occur independently of caspase activation as not all incidences of apoptosis involve these enzymes. There is evidence that under conditions of high oxidative stress these cysteine-containing proteases are inactivated. To date 14 caspases have been identified as playing a role in the process (Alnemri et al, J Cell Biochem. 64, 33-42, 1997).
It has also been suggested that nitric oxide (NO) may play a key role in the regulation of apoptosis by acting as a second messenger in signal transduction pathways. In this regard, studies over the past few years have demonstrated that a variety of biological response modifiers such as cytokines, growth factors and agents that induce apoptosis can lead to the generation of NO (Brune et al., Cell Death & Differentiation, 10, 969-75, 1999). It is also known that a change in intracellular redox levels or an increase in NO levels is capable of triggering apoptosis. However, the mechanisms by which these changes occur is largely unknown. Moreover, the specific targets of NO generated intracellularly are largely unknown at present.
It would be desirable to identify the mechanisms by which NO modulation is capable of triggering apoptotic processes or inhibiting them In this way, treatments could then be developed to modulate and/or control apoptotic processes.
It would also be desirable to identify the specific targets for NO which has been generated intracellularly.
SUMMARY OF THE INVENTION
The present invention demonstrates that NO is responsible for the modulation of early transcription and/or translation, and/or post-translational modification in cells of genes which control the progression of the cell towards apoptosis. The present invention demonstrates that the genes modulated by changes in NO exposure are required for induction or inhibition of apoptosis, before the cell has made a commitment to die. In contrast, other genes involved in apoptosis (such as caspase genes) which are known to, only act at the execution stage of apoptosis and are only activated once the cell is committed to the apoptotic fate. Accordingly, the present invention provides a mechanism for the regulation of expression of NO-modulated genes which may be used to induce or prevent the apoptotic process. Based on the findings ofthe present invention, the present inventors hypothesised that if a model was designed such that the course of apoptosis following induction by NO was functionally characterised, then changes, or patterns of changes, in the 'early' regulatory events could be studied. Studies of changes in expression of those genes involved in these early events, would lead to the identification of potential therapeutic targets. Thus, the present inventors have set out to provide a model system for the NO mediated modulation of apoptosis such that those genes involved in the early regulatory events of apoptosis, can be identified and characterised.
The control of apoptosis represents a significant therapeutic target, since many diseases are due to defects in this process. Many physiological factors prevent cell apoptosis. For example cytokines or growth factors 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 targets to control apoptosis act early in the process and are restricted to a particular cell type.
The inventors have discovered that NO induces cell 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. If model discovery assays are configured to target these 'early' regulatory events occurring in the inhibition of apoptosis it is possible to identify the key genes to control apoptosis.
The present inventors have further discovered that if a model is designed such that the modulation of apoptosis by NO is itself inhibited by a drug, 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. For example, a change that is a 'decrease' following induction of apoptosis is a candidate target gene. DETAILED ASPECTS OF THE INVENTION
In a first aspect, therefore, the invention provides a method for modulating the expression of gene products that regulate the transition of a cell between a non-apoptotic state and an apoptotic state, comprising increasing or reducing the intracellular concentration of nitric oxide (NO).
In a second aspect, the invention provides a method for identifying one or more gene product(s) that modulate the transition of a cell between a non-apoptotic state and an apoptotic state, comprising the steps of:
a) exposing the cell to NO or to an agent which induces the production of NO in the cell or which inhibits the production of NO in the cell; b) determining the level(s) of expression of one or more gene product(s) in a cell to establish a reference expression level; c) monitoring the ievel(s) of expression of said one or more gene product(s) in the cell; and d) identifying one or more of the gene product(s) whose expression has been increased, decreased or modified as a result ofthe NO exposure.
Preferably, the method ofthe invention includes the steps of: a) determining the level(s) of expression of one or more genes or gene product(s) in a cell to establish a reference expression level; and b) comparing the expression level(s) after NO exposure to the reference expression level(s).
In a further aspect, the invention relates to the use of NO to alter the expression of gene products that modulate the transition of a cell between a non-apoptotic state and an apoptotic state.
In a still further aspect, the present invention provides a system for modelling NO mediated modulation of apoptosis in a cell comprising the steps of: (a) providing a population of cells; (b) modulating the intracellular NO. concentration in the cells; and
(c) identifying genes whose expression is modulated as a result of changes in intracellular NO concentration.
Reference levels of expression of gene products may be determined, in the absence of modulation by NO.
A reference expression level, once established for a given cell type, may be used for repeated screens, thus facilitating the performance of repeated rounds of screening.
Levels of expression may be determined by assessing expression at the nucleic acid or polypeptide level. For example, mRNA levels, protein levels or the extent of posttranslational modification of proteins may be used to assess expression in accordance with the present invention.
In general, NO functions to facilitate apoptotic processes in cells. Thus, exposure of a cell to NO leads to induction of apoptosis in the cell more rapidly than occurs under identical conditions in the absence of NO.
Modulation of intracellular NO concentration may be effected by any suitable technique. For example, NO concentrations may be increased by administration of an NO donor, such as sodium nitroprusside which spontaneously releases NO on culture, or increasing the activity of the cellular enzyme NO synthase. Decrease in NO concentrations may be effected by, for example, inhibition of NO synthase, the use of NO scavenging agents such as urea or NAC, or by boosting the cellular defences against NO.
Glutathione is a tripepetide synthesized from the amino acids glutamate, cysteine and gly cine. The levels of the limiting amino acid cysteine determine its rate of synthesis. Consequently, elevation of cysteine levels in the cell increases GSH. However, addition of cysteine to cells is not feasible since cysteine auto-oxidizes to an insoluble form and is toxic to cells. To overcome this, cysteine is supplied to cells in an acetylated form as N- acetyl-cysteine (NAC) which when taken up by the cell is readily deacetylated intracellularly and provides the rate limiting amino acid. The use of inhibitors of apoptosis is useful in the assays of the invention in order to extend the "window" between NO treatment and the onset of apoptosis. It is during this window that NO-induced gene expression may be observed. Various inhibitors of apoptosis are known in the art and may be employed, as described below.
Inhibitors of NO or NO activity may be used to further investigate the involvement of NO in the induction of gene expression or other events associated with apoptosis. Suitable NO inhibitors are described in more detail below. The NO inhibitors may, for example, be active against NO Synthase (NOS). Specific isoforms of NOS may be targeted in order to monitor the effect of cell-specific genes on NO-induced apoptosis.
The model system and assays described here may also be used to identify compounds or substances capable of promoting or inhibiting (i.e., modulating) apoptosis. Preferably, such compounds are capable of inhibiting apoptosis. Candidate compounds may be identified which are capable of modulating expression of genes which are capable of modulating the transition between a non-apoptotic state and an apoptotic state of cell. Such genes may be identified by the methods as described here. Screens are usefully configured to screen libraries of candidate compounds, which libraries may be in the form of arrays as known in the art. Thus, a screen may be configured by inducing apoptosis in the cell by increasing the NO concentration (e.g., by exposure of a cell to sodium nitroprusside) and gene expression observed to identify genes capable of regulating apoptosis, preferably early stages of apoptosis. Candidate compounds may be exposed to cells expressing such genes, or to in vitro systems which express such genes, or to the gene products (polypeptides) themselves, whether substantially purified or partially purified. Compounds identified which modulate (e.g, up-regulate or down-regulate) the activity, level or expression of such genes or gene products are useful to modulate apoptosis.
relevant gene expression, i..e, expression of genes identified by the model system as described here. Other aspects of the present invention are presented in the accompanying claims and in the following description and drawings. These aspects are presented under separate section headings. However, it is to be understood that the teachings under each section are not necessarily limited to that particular section heading.
The present invention provides numerous advantages over the prior art. In particular, it:
(i) provides a method by which genes involved in the early stages of apoptosis, whose expression is modulated by reactive nitrogen species in the cell, can be identified and isolated.
(ii) provides a means by which therapeutics may be developed to modulate and/or control apoptotic processes.
(iii) overcomes a flaw associated with the methods currently being used to manipulate the apoptotic process. In this regard, some of the methods currently being used are based on the regulation of caspases, enzymes which only become involved in apoptosis after the cell has made a commitment to the apoptotic pathway. Accordingly, the regulation of these down-stream enzymes can only serve to delay the onset of apoptosis and not to prevent it. The present invention avoids this fundamental drawback, (iv) It provides a method by which cell specific genes modulated by NO and involved in apoptosis signalling may be identified.
Other advantages are discussed and are made apparent in the following description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 : Sodium nitroprusside induces death in HeLa cells, which is reversed by NAC. HeLa cells are cultured ± sodium nitroprusside (0.9 xlO"6M) ± NAC (20mM) for 18 h prior to detection of viability by MTT (abs 570nM). Survival corresponds positively with increases in optical densities. Figure 2: H2O2 induces death in HeLa cells, which is reversed by NAC. HeLa cells are culture ± H2O2 (1.3xlO"6M) ± NAC (20mM) for 18 h prior to detection of viability by MTT (abs 570nM). Survival corresponds positively with increases in optical densities.
Figure 3: Representative excerpt of cluster analysis of genes increased (>=2 fold) or decreased (<= 2 fold) by HeLa cells following treatment with sodium nitroprusside. HeLa cells are treated with sodium nitroprusside (0.9 xl0"6M) for 4 h (column 1) and 6 h (column 2) + NAC (20mM; 4 h column 3 and 6 h column 4). Each column represents the expression of each gene (in rows) in duplicate (measured using human Incyte LifeGrid microarrays); fold change relative to a time zero control. Black indicates increased expression and white indicates decreased gene expression. A greyscale bar is included to indicate respective expression levels. The same scale is used for the following figures.
Figure 4: Cluster analysis of genes increased (>=2 fold) or decreased (<= 2 fold) by HeLa cells at both 4 h and 6 h following treatment with sodium nitroprusside, which are blocked by the addition of NAC. HeLa cells are treated with sodium nitroprusside (0.9 xl0"6M) for 4 h (column 1) and 6 h (column 2) ± NAC (20mM; 4 h column 3 and 6 h column 4). Each column represents the expression of each gene (in rows) in duplicate (measured using human Incyte LifeGrid microarrays); fold change relative to a time zero control.
Figure 5: Cluster analysis of genes increased (>=2 fold) or decreased (<= 2 fold) by HeLa cells at 6 h following treatment with sodium nitroprusside, which are blocked by the addition of NAC. HeLa cells are treated with sodium nitroprusside (0.9 xlO'6M) for 4 h (column 1) and 6 h (column 2) + NAC (20mM; 4 h column 3 and 6 h column 4). Each column represents the expression of each gene (in rows) in duplicate (measured using human Incyte LifeGrid microarrays); fold change relative to a time zero control.
Figure 6: polymyositis/scleroderma autoantigen 2 (lOOkD) gene expression is decreased (expressed as fold change relative to a time zero control) associated with sodium nitroprusside induced HeLa cell apoptosis but this decrease is blocked by treatment with
NAC. HeLa cells are treated with sodium nitroprusside (0.9 xlO"6M) for 4 h (SNP T4- AvgFoldChange) and 6 h (SNP T6-AvgFoldChange) ± NAC (20mM) for 4 h (SNP+NAC T4-AvgFoldChange) and 6 h (SNP+NAC T6-AvgFoldChange).
Figure 7: Aryl hydrocarbon receptor nuclear translocator-like gene expression is decreased (expressed as fold change relative to a time zero control) associated with sodium nitroprusside induced HeLa cell apoptosis but this decrease is blocked by treatment with NAC. HeLa cells are treated with sodium nitroprusside (0.9 xlO"6M) for 4 h (SNP T4-AvgFoldChange) and 6 h (SNP T6-AvgFoldChange) ± NAC (20mM) for 4 h (SNP+NAC T4-AvgFoldChange) and 6 h (SNP+NAC T6-AvgFoldChange).
Figure 8: Calpastatin gene expression is decreased (expressed as fold change relative to a time zero control) associated with sodium nitroprusside induced HeLa cell apoptosis but this decrease is blocked by treatment with NAC. HeLa cells are treated with sodium nitroprusside (0.9 xlO"6M) for 4 h (SNP T4-AvgFoldChange) and 6 h (SNP T6- AvgFoldChange) ± NAC (20mM) for 4 h (SNP+NAC T4- AvgFoldChange) and 6 h (SNP+NAC T6-AvgFoldChange).
Figure 9: Thioredoxin reductase 1 gene expression is decreased (expressed as fold change relative to a time zero control) associated with sodium nitroprusside induced HeLa cell apoptosis but this decrease is blocked by treatment with NAC. HeLa cells are treated with sodium nitroprusside (0.9 xlO"6M) for 4 h (SNP T4-AvgFoldChange) and 6 h (SNP T6- AvgFoldChange) + NAC (20mM) for 4 h (SNP+NAC T4-AvgFoldChange) and 6 h (SNP+NAC T6-AvgFoldChange).
Figure 10: Shows that - matrix matalloproteinase 2 gene expression is decreased (expressed as fold change relative to a time zero control) associated with sodium nitroprusside induced HeLa cell apoptosis but this decrease is blocked by treatment with NAC. HeLa cells are treated with sodium nitroprusside (0.9 xl0"6M) for 4 h (SNP T4- AvgFoldChange) and 6 h (SNP T6-AvgFoldChange) ± NAC (20mM) for 4 h (SNP+NAC T4-AvgFoldChange) and 6 h (SNP+NAC T6-AvgFoldChange).
Figure 11: Shows that Ubiquitin C-terminal hydrolase Ll is increased (expressed as fold change relative to a time zero control) associated with sodium nitroprusside induced HeLa cell apoptosis but this increase is blocked by treatment with NAC. HeLa cells are treated with sodium nitroprusside (0.9 xlO"6M) for 4 h (SNP T4-AvgFoldChange) and 6 h (SNP T6- AvgFoldChange) + NAC (20mM) for 4 h (SNP+NAC T4-AvgFoldChange) and 6 h (SNP+NAC T6-AvgFoldChange).
Fig. 12 Photomicrograph of HeLa cells exposed to Sodium Nitroprusside either in the presence or absence of NAC. Cells were treated with SNP in the absence of NAC for Oh (A), 6h (B) or 24h (D) or in the presence of 20mM NAC for 6h (C) or 24h (E). HeLa cells were then trypsinized and slides were made by cytospinning cells onto glass slides and staining using the Diff Quik II staining kit. Apoptosis was assessed by nuclear condensation and membrane blebbing. Magnification X 400.
DETAILED DESCRIPTION OF THE INVENTION
APOPTOSIS
As used herein, the term "apoptosis" refers to genetically programmed cell death which is regulated throughout the lifetime of an organism. In apoptosis, a triggering agent from either outside or inside the cell causes "cell-suicide" genes to produce enzymes that damage the cell in several ways, including disrupting its cytoskeleton and nucleus. As a result, the cell shrinks and pulls away from neighbouring cells. The DNA within the nucleus fragments, and the cytoplasm shrinks, although the plasma membrane remains intact. Phagocytes in the vicinity then ingest the dying cell. Apoptosis may be regarded as a normal type of cell death and contrasts with necrosis which is a pathological type of cell death that results from tissue injury. Apoptosis removes unneeded cells during development before birth. It continues to occur after birth to regulate the number of cells in a tissue and eliminate potentially dangerous cells such as cancer cells.
NITRIC OXIDE (NO)
As used herein, the term "nitric oxide" means the gas nitric oxide (NO) depicted as: N= O
which is produced by endothelial cells lining blood vessels and is both a hormone and a neurotransmitter.
NO is formed from the amino acid arginine by the enzyme nitric oxide synthase (NOS) which is widely distributed in cells and tissues. NO is different from all previously known neurotransmitters because it is not synthesised in advance and packaged into synaptic vesicles. In contrast, NO is formed on demand and acts immediately. Its action is brief because NO is a highly reactive free radical that lasts less than 10 seconds before it combines with oxygen and water to form nitrates and nitrites. The precise functions of NO released by neurons are still unclear. Because NO is lipid soluble, it diffuses out of cells that produce it and into neighbouring cells, where it activates an enzyme for production of a second messenger called cyclic GMP. Some research suggests that NO may play a role in memory and learning.
Endothelial cells in blood vessel walls release NO, which diffuses into neighbouring smooth muscle cells and causes relaxation. The result is vasodilation, an increase in blood vessel diameter. The effects of such vasodilation range from a lowering of blood pressure to erection of the penis in males. In larger quantities, NO is highly toxic. Phagocytic cells, such as macrophages and certain white blood cells, produce NO to kill microbes and tumour cells. Based on the presence of NOS, it is estimated that more than 2% of the neurons in the brain produce NO. NOS is also highly concentrated in autonomic neurons that cause either relaxation of smooth muscle in the gut or release of epinephrine and norepinephrine from the adrenal medulla.
Nitric oxide (NO) has been shown to play a key signalling role in a range of physiological and pathophysiological states. It is involved in the cytotoxic effects of immune cells, neurotransmission, smooth muscle cell contractility, platelet reactivity and it is implicated in atherosclerosis, inflammatory conditions, septic shock, neurodegenerative diseases, retinal diseases and cancer metastasis. In acute lung injury, neutrophil apoptosis may be important in regulating the inflammatory process by controlling neutrophil numbers and thus activity. Exogenous inhaled NO is used therapeutically in patients with acute lung injury and its effects on apoptosis of these cells may be important. NO has been shown to induce apoptosis in renal cells (Amore et al. Kidney Int. 2000, 57,1549-59), gastric epithelial cells (Watanabe et al, J Gastroenterol Hepatol. 2000, 15, 168-74, cardiomyocytes (Taimor et al.,. Cardiovasc Res. 2000 45, 588-94 and in neurons (Estevez at al, Science. 1999, 286, :2498-500).
NO synthesis is controlled by the action of Nitric Oxide Synthase (NOS). NOS consists of a family of related isoforms which have different physiological roles and tissue specificity. Three distinct isoforms have been identified ; nNOS whose activity is regulated by Ca++ and calmodulin, is found in neural tissue. A second Ca++/calmodulin -requiring constitutive enzyme (eNOS) is present in vascular endothelial cells. A third Ca++ independent isoform (iNOS) can be isolated from a variety of cells following induction with inflammatory mediators and bacterial products. Hobbs et al., Annu. Rev. Pharmacol. Toxicol. 1999, 39,:191-220)
REACTIVE NITROGEN SPECIES
Nitric oxide can cause oxidative damage to cellular structures. By way of example, NO can react with reactive oxygen species (ROS) to produce reactive nitrogen species/substances such as peroxynitrite which is a powerful inducer of apoptosis. Peroxynitrite itself can also generate ROS in at least some systems and ROS are also pro- apoptotic. For instance in HL-60 cells, pre-treatment with NAC, a ROS scavenger, fully scavenged peroxynitrite induced ROS generation and thus effectively inhibited peroxynitrite induced apoptosis. (Reactive oxygen species participate in peroxynitrite induced apoptosis in HL-60 cells, Lin KT et al Biochem Biophys Res Commun 1997, 230:1 115-9). NO scavengers, also enhances organ graft acceptance probably by its ability to ameliorate the apoptotic effects of NO.
It has been suggested that NO may be involved in p53 mediated apoptosis (Brune et al., Cell Death & Differentiation, 10, 969-75, 1999). In addition NO induces alterations in the expression patterns of the Bcl-2 family of proteins known to be involved in the regulation of apoptosis. Moreover, it is known that the addition of NO generators to cells in culture lead to the activation of the transcription factor Nf-kβ (Yan et al., Arterioscler Thromb Vase Biol. 12:2854-62, 1999). This factor, may, in turn may controls the expression of a series of genes involved in a variety of cellular functions.
Peroxynitrite induces apoptosis in epithelial and macrophage cells lines and this may be attenuated by pre-incubation of cells with L-ascorbic acid. Apoptosis is induced at peroxynitrite levels of 5-75 μM, concentrations greater than this inducing necrosis (Free Radic Biol Med 3, 489-95, 1997). Such results suggest that peroxynitrite may contribute to the pathology of gut inflammation by promoting apoptosis and this can be blocked the antioxidant ascorbic acid. An additional example is the use of epigallocatechin gallate which protects U937 cells from nitric oxide induced cells cycle arrest and apoptosis (J Cell Biochem 81, 647-58, 2001).
In one embodiment ofthe present invention, it has been found that exposure ofthe cell to NO leads to induction or inhibition of apoptosis in the cell at a different rate than occurs under identical conditions, but in the absence of NO.
In another embodiment of the present invention, the expression of gene products may regulate the transition of a cell between a non-apoptotic state and an apoptotic state after exposure to increasing levels of NO (5-100μM).
REDOX
In one embodiment of the present invention, a change in intracellular redox levels or an increase in NO levels may be capable of triggering apoptosis.
As used herein, the term "redox" refers to a coupled reaction referred to as an oxidation- reduction (redox) reaction. In this regard, within a cell, oxidation and reduction reactions are always coupled - whenever one substance is oxidised, another is almost simultaneously reduced. As used herein, the term "oxidation" refers to inter alia: (i) the action or process of reacting with oxygen, especially the addition of oxygen to a substance; (ii) the loss or removal of hydrogen from a substance; and (iii) the loss or removal of one or more electrons from a molecular entity, with or without concomitant loss or removal of a proton or protons. In this sense, oxidation is the opposite of, and is always coupled to, reduction.
As used herein, the term "reduction" refers to the chemical process by which oxygen is withdrawn from, hydrogen is added to, or (more generally) an electron is added to (with or without addition of a proton) a molecular entity.
NITRIC OXIDE GENERATION
In one embodiment of the present invention, nitric oxide may be generated in the cell under a number of condition including, but not limited to, exposure of cells to ultra violet irradiation, cytokines (inc. IFNγ, TNFα, ILlα, ILlβ, IL-2, etc.), lipopolysaccharide, arachidonic acid, 17β-Estradiol, L-glutamic acid, histamine, bradykinin, acetycholine hydroxyguanidine, A23187 and tetrahydro-L-biopterin. In order to increase the length of the window between NO addition and the onset of apoptosis, which is the time period during which modulation of the gene expression of genes involved in apoptosis induction is detectable, an inhibitor of apoptosis may be added to the cells.
In a second embodiment, Nitric Oxide may be applied to the cell by exposure of the cell to a number of nitric oxide donors. These include, but are not limited to, sodium Nitroprusside, isosorbide-5-mononitrate, molsidomine, S-Nitrosocaptopril, S- Nitrosoglutathione, S-Nitrosoglutathione Monoethyl ester, + -S- Nitroso-N- acetylpenicillimine, Glyco-SNAP-1, Glyco-SNAP-2, S,S'-Dinitrosodithiol, Diethylamine NONOate, BNN3, BNN5Na, BNN5 Methyl Ester, NOC-5, NOC-7, NOC-9, NOC-12, NOC-18, NOR-1, NOR-3, NOR-4, 4-phenyl-3-fi_roxancarbonitrile, PROLI/NO, SIN-1, SNVP, Spermine NONOate, and streptocin. In order to increase the length ofthe window between NO addition and the onset of apoptosis, which is the time period during which modulation of the gene expression of genes involved in apoptosis induction is detectable, an inhibitor of apoptosis may be added to the cells. INHIBITION OF NITRIC OXIDE
Generation of Nitric Oxide may be inhibited by treatment of cells with nitric oxide synthase inhibitors. Such inhibitors may include, but are not limited to, Actinomycin D, cycloheximide, AET, ALLM, ALLN, NG-Allyl-L-arginine, aminoguanidine, l-Amino-2- hydroxyguanidine, p-Toluenesulfonate, 2-Amino-4-methylpyridine, AMITU, AMT, S- Benzykisothiourea, urea, trimethylphenylfluoroimidazole, Bromocriptine Mesylate, L- Canavanine sulfate, chlorpromazine, curcumin, cyclosporine, dexamethasone, 2,4- Diamino-6-hydroxypyrimidine, NG, NG- Dimethyl-L-arginine, Diphenyleneiodonium chloride, (-)- Epigallocatechin gallate, S-Ethyl-N-phenylisothiourea, Hydrogen iodide, 2- Ethyl-2-thioseudourea, hydrobromide, ETPI, GED, GW 274150, Haloperidol, L-N-(l- Iminoethyl)ornithine, LY 83583, MEG, Melatonin, S-Methylisothiourea sulfate, S- Methyl-L-thiocitrulline, NG-Monoethyl-L-arginine, NG-Monomethyl-D-arginine, NG- Monomethyl-L-arginine, Mycophenolic acid, L-NIL, NG-Nitro-D-arginine, NG-Nitro-D- arginine Methyl ester, NG-Nitro-L-arginine, NG-Nitro-L-arginine methyl ester, ^Nitroblue Tetrazolium Chloride, 7-Nitroindazole, Osteopontin, 1,3-PBITU, Pentamidine Isethionate, PPM- 18, NG-Propyl-L-arginine, 1-Pyrrolidinecarbodithiioic acid, SKF- 525A, SKF-96365, Sodium Salicylate, Spermidine, Spermine, L-Thiocitrulline, N - Tosyl-Lys Chloromethyl ketone, Nα-Tosyl-Phe Chloromethyl ketone, TRIM, and Zinc (II) Protoporphyrin IX. Since certain inhibitors exhibit varying degrees of specificity for specific isoforms of NOS, the present invention includes the use of such selective NOS inhibitors to selectively modulate NO levels in cells (where the endpoint is apoptosis) and to identify novel genes whose expression is thereby modulated.
In a further embodiment, reactive Nitric Oxide species may be scavenged by the addition of reagents including, but not limited to Carboxy-PTIO, Hemoglobin, DTCS, NOX 100, MGD, PTIO, (+)-Rutin Hydrate, Quercetin pentaacetate. Furthermore, the effects of Nitric oxide may be inhibited by addition of Spin Traps, which include, but not limited to, N-tert-Butyl-α-phenylnitrone, DEMPO, DTCS, TEMPOL or POBN.
Furthermore, the effects of nitric oxide may be ameliorated by treatment of cells with antioxidants. Such antioxidants may include, but are not limited to, N-acetyl-L-cysteine, AG 1714, Ferritin, CAPE, Caffeic acid, Bilirubin, Deferoxamine Mesylate, R-(-)- Deprenyl, DMNQ, DTP A, Dianhydride, Ebselen, Ellagic Acid, (-)-Epigallocatechin, L- Ergothioneine, EUK-8, reduced Glutathione, Glutathione Monoethyl ester, Glutathione Diethyl ester, α-Lipoic acid, Luteolin, Manoalide, MCI-186, MnTBAP, MnTNPyP, Morin Hydrate, NDGA, (+)- Taxifolin, Tetrandrine, Thioredoxin, Thioredoxin II, DL-α- Tocopherol, Trolox, U-74389G, U-83836E , Uric Acid or Vitamin E Succinate.
GENE PRODUCTS
The method of the invention is applicable to the discovery of gene products, and the genes encoding them, which are involved in apoptosis.
As used herein, the term "gene product" includes but is not limited to polypeptide gene products and/or RNA gene products. The gene products ofthe present invention may be natural gene products, which are encoded by naturally-occurring genes in the cell being investigated and are assembled in the cell using natural components such as amino acids or nucleotides. The present invention also encompasses screening for gene products encoded by genes that are not endogenous to the cell being investigated. Such genes may be, for example, heterologous genes from other cells or organisms, artificial genes encoding polypeptides comprising domains from different sources or composite RNA molecules, and wholly or partially randomised genes encoding repertoires of polypeptide or nucleic acid gene products.
POLYPEPTIDE
As used herein, the term "polypeptide" refers to any peptide comprising two or more amino acids, whether comprising a single domain or multiple domains, and includes multi-subunit proteins, which are cellular gene products.
RNA GENE PRODUCTS As used herein, the term "RNA gene products" includes but is not limited to ribozymes, antisense RNA molecules and/or mRNA molecules.
GENE EXPRESSION LEVELS
Levels of gene expression may be determined in any appropriate manner.
Preferably, the invention comprises the measurement of protein production by mRNA translation, and is configured to detect increases or decreases in the rate or amount of mRNA translation.
POST TRANSLATIONAL MODIFICATIONS
The invention may also be configured to detect changes in post-translational processing of polypeptides or post-transcriptional modification of nucleic acids. For example, the invention may be configured to detect the phosphorylation of polypeptides, the cleavage of polypeptides or alternative splicing of RNA, and the like. Levels of expression of gene products such as polypeptides, as well as their post-translational modification, may be detected using protein assays or techniques such as 2D polyacrylamide gel electrophoresis. Polypeptide or nucleic acid populations may be assessed individually, or together, in order to identify candidate gene products.
GENE TRANSCRIPTION
Advantageously, expression levels are assessed by measuring gene transcription. This is preferably carried out by measuring the rate and/or amount of specific mRNA produced in the cell. A preferred embodiment of this 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 that are differentially expressed in the two populations. The arrayed oligonucleotide probes are advantageously derived from cDNA or EST libraries, and represent genes that are expressed by the cells under investigation. OLIGONUCLEOTIDE/POLYNUCLEOTIDE
As used herein, the terms "oligonucleotide" and "polynucleotide" are equivalent, and imply no limitation as to maximum or minimum length.
CELLS
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. Examples of such cells include but- are not limited to, epithelial cells, cardiomyocytes, peripheral blood leukocytes and neurons. Cell lines useful in the present invention include but are not limited to HeLa cells, fibroblast cell lines, myeloid cell lines such as HL-60, carcinoma cell lines and neuroblastoma cell lines.
Cells may be primary cultures of neurons or cells having neuronal characteristics. These cells include but are not limited to Ntera-II (human embryonal carcinoma cells which can be induced to differentiate towards neurons), PC- 12 (rat pheochromocytoma cells) and SH-SY5Y cells (neuroblastoma cell which can be induced to differentiate towards neurons when cultured in the presence of retinoic acid) or cells of haematopoietic origin such as HL-60 (human myeloid cell line which can be induced to differentiate towards neutrophils and monocytes and readily undergoes apoptosis).
The use of HeLa cells in the methods of the invention is effective and efficient, HeLa cells being easy to culture and well-known to those skilled in the art. Cells and cell lines having documented pro- and anti-apoptotic reactions following NO treatment are listed in Brune et al, Apoptotic cell death and nitric oxide: activating and antagonistic transducing pathways; Biochemistry (Moscow) Vol 63, No 7 1998, p817-825.
APOTOSIS INHIBITORS
Any suitable inhibitor of apoptosis may be employed, including physiological growth factors, for example, NGF or inhibitors of caspase enzymes such as Z-VAD. Apoptosis may be inhibited through expression of genes, e.g., BCL-2. An example of a suitable inhibitor is Neuronal Growth Factor (NGF). NGF has been shown to inhibit apoptosis and prolongs survival of neurons in culture Culturing neurons in NGF provides a very useful model system with which to study neuronal apoptosis. NO induced apoptosis can be directly stimulated in this system though the exogenous addition of NO generating agents such as sodium nitroprusside. Similarly human peripheral blood neutrophils can also be induced to undergo apoptosis and this can be blocked by culturing cells in the presence of GM-CSF a survival factor similar to NGF.
MONITORING APOPTOSIS ONSET
A number of methods are known in the art for monitoring the onset of apoptosis. These include but are not limited to morphological analysis, DNA ladder formation, externalisation of membrane phospholipid phosphatidyl serine (lipid flipping) and caspase activation analysis.
Preferably, the ability of NO to induce apoptosis is preferably confinned by monitoring the onset thereof according to one or more ofthe above methods.
Preferably, the ability of NO to induce apoptosis is preferably confirmed by monitoring the onset of lipid flipping.
LIPID FLIPPING
As used herein, the term "lipid flipping" refers to 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 localised to the inner surface 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) Preferably, the ability of NO to induce apoptosis is preferably confirmed by monitoring the onset of caspase activation.
CASPASE ACTIVATION
The signal transduction system of apoptosis is relatively poorly understood at present, but a series of enzymes termed caspases have been shown over the last few years to be involved in different phases of apoptosis. In particular, in the propagation and execution phases of the process (Samali et al, Cell Death Differ. 6, 495-6, 1999). For example, caspase 3 is involved in the activation of CAD, an endonuclease involved in the cleavage of DNA to yield the hallmark DNA ladder of apoptosis described above (Mcllroy et al, Cell Death Differ. 6, 495-6, 1999). This caspase cleaves ICAD, a natural inhibitor ofthe CAD endonuclease. Other caspases are involved in the propagation and execution phase ofthe process, an example of which is caspase 8. Not all incidences of apoptosis involve these enzymes and there is evidence that under conditions of high oxidative stress these cysteine-containing proteases are inactivated. To date 14 caspases have been identified as playing a role in the process (Alnemri et al., J Cell Biochem. 64, 33-42, 1997).
Preferably, the ability of NO to induce apoptosis is preferably confirmed by monitoring the onset of a caspase independent activation mechanism.
DETERMINATION OF EXPRESSION LEVELS
As indicated above, a number of individual gene product types 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. For example, 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.
ASSAYS In one embodiment ofthe present invention, the assay ofthe invention may be configured to identify gene products which accelerate or retard the induction of apoptosis. Advantageously, the assay detects gene products, which accelerate the induction of apoptosis.
Where it is desired to monitor the levels of expression of a known gene product, conventional assay techniques may be employed, including nucleic acid hybridisation studies and activity-based protein assays. Kits for the quantitation of nucleic acids and polypeptides are available commercially.
Where the gene product to be monitored is unknown, however, methods are employed which facilitate the identification ofthe gene product whose expression is to be measured. For example, where 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.
ARRAYS
Gene Arrays of oligonucleotides specific to gene sequences archived in public domain databases, such as GenBank, are available commercially from a number of suppliers (such as GenomeSystems). Examples of such commercial arrays are in the form of either nucleotides spotted onto a membrane filter (such a nitrocellulose), or a solid support (such as glass). Commercial Gene Arrays may be used to profile the patterns of gene expression which are associated with the process of apoptosis in cells such as but not limited to neuronal cells.
Gene Arrays may be constructed in-house, by spotting nucleotide sequences derived from cDNA clones generated from in-house libraries or from cDNA clones purchased commercially. Such arrays allow the expression profiling of proprietary novel nucleotide sequences.
Candidate compounds capable of inhibiting or promoting apoptosis, preferably by interacting with gene products of genes identified by the methods described here, may be arranged in the form of arrays for screening purposes, as known in the art. LIBRARIES
Many of the 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. The cloning of cDNA libraries that are focused to specific cellular events, such as NO mediated apoptosis, offers the possibility to identify, clone and characterise novel genes that are associated with this process. Similarly, the cloning of cDNA libraries that are focused to specific tissue types, such as the neuron, offers the possibility to identify, clone and characterise novel genes whose expression is restricted to this cell type. Libraries (cDNA) constructed using a physical subtraction, such as the ClonTech 'Select' SSH (suppression hybridisation) method, allow the selective cloning of genes whose expression is differentially regulated in the process or cell type being studied. Gene Array technology may be 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.
Libraries of candidate compounds which are capable of inhibiting or promoting (preferably inhibiting) apoptosis, preferably by interacting with gene products of genes identified by the methods described here, may be generated by methods known in the art. Such libraries may take the form of combinatorial libraries. Libraries of such compounds are also available commercially.
CHEMICAL SYNTHESIS OF ARRAYS
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 PREPARED BY GRIDDING
In a preferred aspect ofthe invention, aπrays of nucleic acids may be prepared by spotting 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 are 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 ARRAYS
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 Genome Sciences 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.
TEST SAMPLES
Samples for mRNA population analysis may be isolated and purified by any suitable commercial mRNA production method.
CHARACTERISATION OF GENE PRODUCTS USING 2D PAGE
For the monitoring of unknown polypeptide gene products, separation techniques such as 2-dimensional polyacrylamide gel electrophoresis (2D PAGE) are employed. 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.
Samples for 2D PAGE may be prepared by conventional techniques. In the case of one of the preferred cells for use in the invention, Ntera-II is grown in a suitable medium, such as Dulbecco's modified Eagle medium (DMEM) containing 10% foetal calf serum (FCS), and treated with NO inducers as necessary. Cells are then rinsed, for example with DMEM without FCS and removed from the flask, for example by incubating them with a solution containing trypsin (0.5 g/1) and EDTA (0.2 g/1). DMEM containing FCS is added into the flask to stop the action of the trypsin. The cells are detached from the surface of the flask by squirting the solution onto the cells. The suspension is transferred into a tube and the cells are centrifuged at 1000 g for 5 minutes. Supernatant is discarded and the cells are washed with DMEM without FCS. After centrifugation and removal of DMEM, 0.8 x 106 cells are mixed and solubilised with 60 μl of a solution containing urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM) and a trace of bromophenol blue. The whole final diluted NTERA-II sample is loaded on the first dimensional separation.
REFERENCE MEANS
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 before addition of NO to the cells, and serve as a standard for one or more subsequent assays; or it may be an integral part of every assay. For example mRNA or polypeptide populations from NO-induced and un-induced 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 changes in the expression of polypeptides or their derivatives. Polypeptides of interest may be isolated, sequenced and used to identify genes encoding them in the cell under investigation.
FUNCTIONAL CLONING The invention may moreover be configured to screen libraries for genes capable of modulating the response of a cell to apoptosis. For example, apoptosis may be induced in cells by production/administration of NO, as described above, and libraries, such as cDNA libraries, of genes, screened in such cells. Cells which demonstrate resistance to apoptosis, for example by increased survival times, are transformed with a nucleic acid . encoding a gene product which interferes with NO-induced apoptosis. The genes may thus be cloned on the basis of function in such a screen.
The effectiveness of pro- or anti-apoptotic compounds may also be determined in as assay as described herein. For instance, cells may be exposed to NO and then treated with the compound(s) to be tested. Variation in the survival of the cells is indicative of an effect ofthe compound(s) on NO-induced apoptosis.
DISEASES
Many diseases are known to involve apoptosis and are thus targets for apoptotic therapies developed in accordance with the present invention. For example, the control of apoptosis in neurons may be useful in the treatment of a number of diseases, including but not limited to atherosclerosis, inflammatory conditions, systemic inflammatory response syndrome (SIRS), neurodegenerative diseases, retinal diseases, cancer metastasis, Alzheimer's and Parkinson's disease, adult respiratory distress syndrome (ARDS) and other related conditions, stroke, myocardial infarction, myelosuppression following chemotherapy or irradiation and a significant number of other diseases where cell death is a key feature ofthe pathology.
EXAMPLES
The invention will now be further described for the purpose of non-limiting illustration by way of examples. Example 1
Cellular/biochemical characterisation of apoptosis
This example describes the cellular and biochemical characterisation of the apoptosis process in the model systems described in the following examples. A range of assays are established and used to measure the magnitude and temporal induction of apoptosis. The earliest biochemical measurement of the apoptosis phenotype by these assays is considered to be the point beyond which the cells are 'committed' to the process of apoptosis. In addition, these measurements determine the reproducibility of induction of apoptosis in the model systems. Furthermore, these measurements determine the cellular mechanisms of apoptosis in these systems (such as whether apoptosis is caspase- dependent or caspase-independent).
Caspase activation assay
Caspase activity (Caspase-3) is measured using a commercial kit (CaspACE™ Assay System, Promega). The methodology is essentially as described by the manufacturer. Cells are removed from culture and centrifuged (300g/ 10 miή) at 4°C. The pellet is kept on ice, washed in ice-cold Hanks buffer and then resuspended in Cell Lysis Buffer at 10 / ml. Cells are lysed by freeze-thawed once, incubated on ice for 15 min, followed by centrifugation (15,000g /20 min) at 4°C. The caspase 3 activity present in the supernatant fraction is measured using the absorbance at 405nm.
Assay for externalisation of membrane phospholipid phosphatidylserine
Externalisation of the membrane phospholipid is detected using a commercial kit (Annexin V-FITC, Pharmingen Europe). The methodology is essentially as described by the manufacturer. Cells are removed from culture, washed twice with ice-cold Hanks buffer and resuspended in binding buffer (10 mM Hepes, pH7.4, 140 mM NaCl, 2.5mM CaCl2; stored at 4°C) at a concentration of 1 x 106 cells/ml. Annexin- V (5μl) and propidium iodide (5ul) are added to 100 μl of cells (10s cells), gently vortexed and incubated for 15 min at 25°C. A further 400μl of binding buffer is added to each tube and cells were analysed by flow cytometry using a Bectoή Dickenson FACScan equipped with CellQuest software with excitation at 488nm and emission collected through a 530/30 bandpass filter for FITC (FL-1) and a 585/42 band pass filter for propidium iodide.
Morphological Determination of Apoptosis
An aliquot (lOOμl) of cell suspension is removed from culture and using an IEC Centra-7 centrifuge equipped with a Cytobucket™ adapter, and a monolayer of cells is concentrated onto standard microscopic slides. Preparations are allowed to air dry prior to fixing in Rapi-Diff (Diagnostic Developments, UK) solution A (reactive ingredient 100% methanol). Slides are air dried prior to immersion in solution B containing; eosin Y (0.1% w/v), formaldehyde (0.1% w/v), sodium phosphate dibasic (0.4% w/v) and potassium phosphate monobasic (0.5% w/v). Excess stain is drained from the slide prior to immersion in solution C containing; methylene blue (0.4%w/v), Azure A (0.04% w/v), sodium phosphate dibasic (0.4% w/v), potassium phosphate monobasic (0.05% w/v) and potassium phosphate monobasic (0.4% w/v), to counterstain the cytoplasm. Excess dye is rinsed; the slides air-dried and mounted in DPX aqueous mountant (BDH Laboratory Supplies, U.K.). Morphological examination is then carried out by light microscopy for the presence of apoptotic cells as determined by the loss of membrane asymmetry and condensation of cytoplasm and nuclei (Cotter and Martin, 1996).
Example 2
Morphological examination of cells exposed to Sodium Nitroprusside
HeLa cells are trypsinized, and made up to a concentration of 6 x 104 cells/ml. One ml of cells are plated/well of a 24 well plate and allowed to adhere overnight. The following day cells are incubated with Sodium Nitroprusside (made up in DMEM; final cone 0.9 xlO"6M) in the presence or absence of 20mM N-acetyl-L-cysteine (NAC). Cells are incubated for the indicated period after which cells removed and examined for signs of apoptosis by microscopy. Slides of the cells are made by placing 3x 104 cells on a slide using a cytospin (Shandon II cytocentrifuge). Cells are allowed to air dry for 5 minutes prior to fixing them in Rappi-Diff (Diachem International, Lancashire, U.K.) soln A (methanol). Excess fixative was drained from the slide and the preparation was then immersed in soln B (eosin Y, 0.1%) to achieve nuclear staining. After washing the slide in water and allowing to dry briefly, the slide was immersed in soln C (methylene blue) to counterstain the cytoplasm. Finally, excess stain is washed away and after air drying, preparations were mounted in DPX aqueous mountant (BDH Laboratory Supplies, Poole, U.K.). Morphological examination was then carried out by light microscopy.
Sodium Nitroprusside kills cells by apoptosis as determined by microscopy
Morphological examination of slides made at 0,2, 4 ,6 and 24 hours post insult either with Sodium Nitroprusside alone or with 20 mM NAC reveals that the initiation of apoptosis occurs approximately 6 hours post insult. However, the majority of apoptosis occurs between 6 and 24 hours post insult majority apoptosis (See Table 1). The presence of Nac protects the cells against apoptosis as determined by morphological examination (See Table 1 and Figure 12 A-F). It will be seen that early morphological signs of apoptosis are seen without NAC at 6 hours (Figure 12B), which become most pronounced after 24 hours (Figure 12D). These morphological signs of apoptosis are prevented by addition of NAC (Figures C and E at 6 hours and 24 hours respectively).
Table 1 : Percentage of HeLa cells undergoing apoptosis following exposure to Sodium Nitroprusside
Figure imgf000030_0001
Example 3
Biochemical characterisation of cellular NO generation
This example describes the biochemical characterisation of intracellular NO status in the model systems described below, as cells undergo apoptosis. Primary neurons or neuronal cell lines are cultured in medium deficient in the survival factor NGF. The earliest biochemical measurement of production or inhibition of NO generation is considered to be the point beyond which cells are undergoing the process of "Induction of Commitment" to apoptosis. In addition the measurements determine the reproducibility of NO generation in the model systems.
Generation of NO
Nitric oxide can be generated in cells under a number of conditions including the addition of sodium nitroprusside to the culture medium. In this instance sodium nitroprusside is added at concentrations 0.1-100μM to induce NO production. The effects of this on the induction of apoptosis are monitored using the assays described previously.
Measurement of NO
Nitric oxide is measured in cells undergoing apoptosis using a flow cytometry based assay with the NO sensitive probe DAF-2 DA (Kojima et al., Neuroreport, 9, 3345-8, 1998).
Inhibition of NO production
L-NAME a synthetic inhibitor of NO synthase (5-50 μM) is incubated with cells prior to the induction of apoptosis by survival factor withdrawal. The level of apoptosis is monitored by caspase activation, phosphatidylserine exposure and morphological assays. NO Scavenging
ΝΟX 100 a nitric oxide scavenger is used to neutralise the effects of NO produced following the withdrawal of survival factors such as NGF from cells in culture. At indicated time points NOX 100 at 30 μM is added to cell cultures prior to NGF withdrawal. Cells are monitored for the induction of apoptosis using assays already described in this document.
Example 4
Establishment and characterisation of 'Model Cell Systems' to study apoptosis
This example describes the establishment of two model cell systems to study NO mediated apoptosis in cell lines and in an in vivo model system. In the first model neuronal cell lines such as Ntera-II, SH-SY5Y, HL-60 or PC- 12 are induced to undergo apoptosis by treatment with either sodium nitroprusside or removal of survival factors to induce NO production and subsequent apoptosis. This is complemented by the use of NO scavengers such as NOX 100 to block the effects of NO.
In the second model system that is an in vivo one, adult male Balb-c mice are maintained in the dark for 18 hours before being exposed to constant light. Immediately prior to light exposure their pupils are dilated with 5% cyclopentolate. The mice are then exposed to 2 hours of cool white fluorescent light at an illuminesence level of 5000 lux. The mice are sacrificed after treatment by cervical dislocation at the following time points: 30min and lhr after light onset, immediately after light exposure (Ohr) and after 6hr 14hr and 24hr of darkness, followed by 2hr light exposure. Under these condition extensive apoptosis occurs in neuronal derived retinal cells and this is a particularly good in vivo model for the study of apoptosis.
Tissue staining for DNA fragmentation
DNA strand breaks in photoreceptor nuclei are detected by in situ end-labelling (TUNEL). Briefly, enucleated eyes were fixed in 10% buffered formalin for 24 hours, dehydrated, processed and embedded in paraffin. Sections (5μm) are incubated in 50μl of reaction buffer containing 2.5mM CoCl2, O.lU/ml terminal deoxynucleotidyl transferase (TdT) in a 0.1M Na cacodylate (pH 7.0) buffer and 0.75nM fluorescein- 12-dUTP (Boehringer Mannheim, Germany). These sections are incubated at 37°C for 1 hour in a humidified chamber. Following several washes in PBS, the sections are mounted and viewed under a fluorescence microscope (Nikon Eclipse E600) using a fluorescein isothiocyanate (FITC) filter. 3 animals are used for each of the time points; Ohr, 6hr, 14hr and 24hr after light exposure.
Several recent papers indicate that apoptosis may proceed in a caspase independent pathway. Droerfler et al., J Immunol. 2000 164, 4071-9, Dasano and Sato, J Biol Chem. 2000 Apr 28;275(18):13967-13973. The following experiments examine whether NO induces retinal cell apoptosis in a caspase dependent or independent manner. Caspase 3 activity is assessed in retinal cells isolated from the eyes of Balb-c mice following exposure to light as described above. Tissue dissociation is achieved in 0.25% trypsin solution (Gibco-BRL, Paisley, UK). The cells are then washed in PBS and fixed in 1% paraformahdehyde at 4°C for 30 min. Cells are washed in permeablization buffer (PB: lOmM HEPES, 150mM NaCl, 4% FCS, 0.1% sodium azide and 0.1% Triton-X-100) and resuspended in PBS containing 0.125μg anti-active caspase-3 antibody (PharMingen International, San Diego, CA, USA), or the same concentration of an isotype control (goat IgG, Sigma, UK) and incubated for one hour at 4°C. Following washes in PBS, cells are re-suspended in 20μg/ml FITC conjugated secondary antibody (goat anti-rabbit, Sigma, UK) and incubated for one hour at 4°C. After a further two washes in PBS, cells are re-suspended in 0.5mls PBS for flow cytometry analysis.
Example 5
HeLa based model cell system
HeLa cells are obtained from the ATCC (Manassas, Virginia, USA) and maintained in DMEM medium with 10% FCS at 37°C in a 5% CO2 atmosphere. Sodium Nitroprusside, N-acety-L-cysteine, MTT, and DMSO are all purchased from Sigma (Fancy Road, Poole, Dorset, UK). Treatment of HeLa cells with the NO donor Sodium Nitroprusside in presence or absence of N-acetyl-L-cysteine
HeLa cells are trypsinized, and made up to a concentration of 5 x 104 cells/ml. One hundred microlitres of cells are plated/well of a 96 well plate and allowed to adhere overnight. The following day cells are incubated with Sodium Nitroprusside (made up in DMEM; final cone 0.9 xlO" M) in the presence or absence of 20mM N-acetyl-L-cysteine (NAC). Cells are incubated for a further 18h. The following morning, the supernatant is removed and cells are washed x2 in serum free DMEM medium prior to adding lOOμl fresh medium containing 0.5mg/ml MTT and allowed to incubate for a further 4 hours before developing the insoluble formazan products with DMSO. Viability is positively correlated with the optical density at 570nm.
Treatment of HeLa cells with the Hydrogen Peroxide in presence or absence of N- acetyl-L-cysteine
HeLa cells are trypsinized, and made up to a concentration of 5 x 104 cells/ml. One hundred microlitres of cells are plated/well of a 96 well plate and allowed to adhere overnight. The following day cells are incubated with Hydrogen Peroxide soln (final cone 1.3xlO"6M) in the presence or absence of 20mM N-acetyl-L-cysteine (NAC). Cells are incubated for a further 18h. The following morning, the supernatant is removed and cells are washed x2 in serum free DMEM medium prior to adding lOOμl fresh medium containing 0.5mg/ml MTT and allowed to incubate for a further 4 hours before developing the insoluble formazan products with DMSO. Viability is positively correlated with the optical density at 570nm.
Treatment of HeLa cells with Sodium Nitroprusside in presence or absence of NAC For isolation of RNA
HeLa cells (3x106/ 35mls) are cultured overnight in a T175 flask to allow adherence. Following morning cells are treated with Sodium Nitroprusside (0.9 xl0"6M) + NAC (20mM) and further incubated for 4 or 6 hours. Cells are harvested for RNA by adding 4mls RNAzol to the cells at the indicated times. Sodium Nitroprusside (an NO donor) induces HeLa cell death, which is reversed by NAC Treatment
HeLa cells obtained from the ATCC (Manassas, Virginia, USA) are maintained in DMEM medium with 10% FCS at 37oC in a 5% CO2 atmosphere. HeLa cells are trypsinized, and made up to a concentration of 5 x 104 cells/ml. One hundred microlitres of cells are plated/well of a 96 well plate and allowed to adhere overnight. The following day cells are incubated with sodium nitroprusside (made up in DMEM; final cone 0.9 xlO"6M) in the presence or absence of 20mM N-acetyl-L-cysteine (NAC; Sigma, UK). Cells are incubated for a further 18h.
Cell viability is measured by MTT assay. At 18 hours post-treatment the supernatant is removed and cells are washed in 2X serum free DMEM medium prior to adding lOOμl fresh medium containing 0.5mg/ml MTT (Sigma, UK) and allowed to incubate for a further 4 hours before developing the insoluble formazan products with DMSO. Viability is positively correlated with the optical density at 570nm.
Treatment of HeLa cells with sodium nitroprusside induces cell death, as determined by a decrease in the ability of cultures treated with sodium nitroprusside to convert the soluble MTT to the insoluble purple formazan. (Figure 1). Addition of NAC simultaneously with sodium nitroprusside significantly decreased the cytotoxicity of the NO donor, as determined by an increase in conversion in the conversion rates at 570nm.
H2O2 (a ROS) induces HeLa cell death, which is reversed by NAC Treatment
HeLa cells obtained from the ATCC (Manassas, Virginia, USA) are maintained in DMEM medium with 10% FCS at 37°C in a 5% CO2 atmosphere. HeLa cells are trypsinized, and made up to a concentration of 5 x 104 cells/ml. One hundred microlitres of cells are plated/well of a 96 well plate and allowed to adhere overnight. The following day cells are incubated with H2O (made up in DMEM; final cone 1.3xl0"6M) in the presence or absence of 20mM N-acetyl-L-cysteine (NAC; Sigma, UK). Cells are incubated for a further 18h. Cell viability is measured by MTT assay. At 18 hours post-treatment the supernatant is removed and cells are washed in 2X serum free DMEM medium prior to adding lOOμl fresh medium containing 0.5mg/ml MTT (Sigma, UK) and allowed to incubate for a further 4 hours before developing the insoluble formazan products with DMSO. Viability is positively correlated with the optical density at 570nm.
Treatment of HeLa cells with H2O induces cell death, as determined by a decrease in the ability of cultures treated with H2O2 to convert the soluble MTT to the insoluble purple formazan (Figure 2). Addition of NAC simultaneously with H2O significantly decreased the cytotoxicity of the NO donor, as determined by an increase in conversion in the conversion rates at 570nm.
Example 6
Characterisation and discovery of oligo/polynucleotides 'Genomics' associated with apoptosis
This example describes the characterisation, cloning and analysis of oligonucleotide/polynucleotide sequences whose expression changes are associated with apoptosis. This example describes the use of transcription inhibitors such as, Actinomycin D (actD) to determine the dependence of apoptosis on gene transcription. Also described are the establishment and use of Genomics techniques such as, microarray and subtraction cDNA cloning.
In one example, commercial microarrays are used to measure global gene expression associated with apoptosis. 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.
In another example., Suppression Subtractive Hybridisation is used to identify and clone cDNA sequences derived from differentially expressed genes. Such differential gene expression is associated with a measurable apoptotic phenotype. Such cDNA sequences are extended to encompass the full length coding sequence for the mRNA gene product.
Measurement of global gene expression by Microarray '
This example describes the process of microarraying (in the context of a filter based microarraying) and its use to profile gene expression of thousands of genes simultaneously. The microarray process can be separated into three parts: the preparation ofthe microarray filter, the hybridisation of radiolabelled cDNA probes, and the detection and quantitation of the microarray results .
Construction and spotting ofcDNA microarray
DNA 'probe' sequences are obtained for genes to be represented on a particular microarray. These sequences would typically be EST cDNA sequences cloned in a plasmid vector, either from in-house libraries or obtained from commercial sources (e.g.
IMAGE consortium clones). The DNA sequences used to construct the microarray are amplified by PCR using common primer sequences found flanking the multiple cloning sites of most commercial cloning vector plasmids as follows:
Ml 3 (-24) Reverse Primer: 5' aac age tat gac cat g 3'
M13 (-48) Reverse Primer: 5' age gga taa caa ttt cac aca gga 3'
Ml 3 (-20) Forward Primer: 5 ' gta aaa cga egg cca gt 3 '
Ml 3 (-40) Forward Primer: 5' gtt ttc cca gtc acg ac 3'
T3 Primer: 5' aat taa ccc tea eta aag gg 3'
T7 Primer: 5' gta ata cga etc act ata ggg c 3' PCR reactions are carried out as follows: l-2μl of glycerol stock, miniprep DNA or overnight culture is added to a 20μl reaction containing, 0.4μl lOmM dNTP mix, 1 x reaction buffer, 0.4μl each 20μM primer and 0.5U Taq polymerase (Qiagen). PCR amplification is earned out with a MWG HT Primus 4 x 96 well thermocycler as follows: 94°C 60 s, 35 cycles of 94°C 40 s, 55°C 30 s, 72°C 120 s, followed by 72°C 120 s.
PCR reaction products are purified using 96-Well PCR Multiscreen (Millipore) as described by the manufacturer.
Purified PCR products are resuspended at lOOng/μl TE in V bottom 96-well microtitre plates. Filter microarrays are spotted as a custom service by GeneScreen Ltd (UK). Alternatively, commercial filter arrays 'Human LifeGrid™' are purchased from Genome Systems Inc. (USA).
Synthesis of labelled probes
This example describes the synthesis of a radiolabelled cDNA from total cellular mRNA. The labelled cDNA is used to 'probe' DNA fragments, which have been immobilised on to a filter membrane, by complementary hybridisation.
Methodology is as described by manufacturer, for Human LifeGrid™ arrays. Essentially, total cellular RNA (1 μg to 20 μg) or polyA+ mRNA (100 ng to 5 μg) is incubated with an oligo (dT) primer. Primed RNA is reverse transcribed to first stand cDNA in a reaction containing M-MLV reverse transcriptase (RT; alternatively Superscript II is used (Life Sciences)), RT buffer, dNTPs and [α-33P] dCTP (2000-4000 Ci/mmol) at 42°C for 1 to 5 h. Unincorporated nucleotides are removed using spin-columns and the labelled probe stored at -80°C until required.
Hybridisation of filter based microarrays
This example describes the complementary hybridisation of radiolabelled cDNA probe to DNA fragments immobilised onto a membrane. Methodology is as described by manufacturer, for Human LifeGrid™ arrays. Essentially, 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 labelled 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.
Quantitative imaging and analysis of microarray filters
This example describes the use of a Phosphoimager to quantitatively image positive signals across the filter arrays.
Hybridised filters are wrapped in plastic wrap and imaged on a Storm Phosphoimager (Amersham Pharmacia Biotech), with a 5 to 48 h exposure and a resolution of 50 to 100 microns. The *.tif image generated by the Phosphoimager is analysed, quantitated and background subtracted using Array Vision software (Imaging Research Inc.).
Suppression Subtr active Hybridisation (SSH) cloning of differentially expressed genes
This example describes a method to identify and clone differentially expressed genes.
Total RNA isolation
Primary or cultured cells are split into control and treatment groups and the treatment group is then challenged with apoptotic stimuli or inhibitors as appropriate. Total RNA is then prepared from both groups using guanidine isothiocyanate lysis followed by caesium chloride gradient centrifugation, or acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), or RNeasy RNA preparation kits (Qiagen). Any contaminating genomic DNA is removed by DNase treatment (DNase I, Gibco-BRL).
Extraction of mRNA The total RNA is then used to prepare mRNA, using the Oligotex mRNA purification kit (Qiagen), or a similar system (Clontech or Strategene). Briefly, mRNA is purified by passing the total RNA over an oligo-dT column. The oligo-dT may be attached to cellulose or glass beads or biotin, and the column may be either spin or gravity format. The bound mRNA is subsequently washed and eluted, ready for use in subtractive hybridisation.
SSH is performed on mRNA purified from treatment or control cells essentially as described (Diatchenko et al 1996), using a PCR-select cDNA subtraction kit (Clontech, K1804). Briefly, cDNA is synthesised from the two pools of mRNA (control and treatment, driver and tester respectively). The resulting cDNA is digested with a restriction enzyme generating a blunt end product (typically Rsal). The tester cDNA is divided into two subsets and distinct adaptor molecules are ligated to each of the cDNA pools. These two samples of tester cDNA are then separately combined with driver cDNA in a solution containing 50mM HEPES, pH 8.3; 0.5M NaCl; 0.02mM EDTA, pH 8.0. Following denaturation, (1.5min, 98°C), the tester and driver cDNAs are allowed to anneal for lOhrs at 68C. After this first hybridisation, the two samples are combined and a fresh portion of heat denatured driver cDNA is added. The samples are allowed to anneal for a further 10 hours at 68°C. The hybridised cDNA is then diluted in a solution containing 20mM HEPES, pH 8.3; 50mM NaCl; 0.2mM EDTA) and heated at 72C for 7minutes, prior to PCR amplification. PCR is performed under standard conditions using the Advantage cDNA PCR kit (Clontech). Only cDNAs that have the correct primer combination, the differentially expressed cDNAs, will amplify exponentially.
Construction of SSH cDNA libraries
PCR products from the subtractive hybridisation are inserted into a TA cloning vector, for example pCRII T/A cloning kit (Invitrogen). This library of differentially expressed cDNAs is then transformed to E.coli and the transformants selected for miniprep and sequence analysis. Plasmid miniprep
This example describes the extraction of double stranded plasmid DNA from cDNA clones.
Individual colonies (E. coli DH5α or TOPI OF') are picked into 96-deep well culture blocks containing 2.3 ml LB + ampicillin. The culture blocks are shaken at 300 rpm 37°C for 24 h. Plasmid DNA is isolated using MultiScreen-FB and MultiScreen-NA 96-well plates (Millipore). The methodology is as described by the manufacturer. DNA sequencing
This example describes the di-deoxy sequencing of cloned cDNA inserts within the vector pT-Adv (Clontech).
Plasmid miniprep DNA (100 ng to 5 μg) is sent to MWG Biotech for contract sequencing. Sequencing reactions are primed using one of the following universal primer sequences:
Ml 3 (-24) Reverse Primer: 5' aac age tat gac cat g 3'
Ml 3 (-48) Reverse Primer: 5' age gga taa caa ttt cac aca gga 3'
Ml 3 (-20) Forward Primer: 5' gta aaa cga egg cca gt 3'
Ml 3 (-40) Forward Primer: 5' gtt ttc cca gtc acg ac 3'
T3 Primer: 5' aat taa ccc tea eta aag gg 3'
T7 Primer: 5' gta ata cga etc act ata ggg c 3' Use of microarray to confirm differential expression of SSH clones
The SSH procedure for the cloning of differentially expressed gene products also generates a portion of artifactual cDNAs 'false positives' which are not differentially expressed. This example describes the use of microarray to confirm the identity of truly differentially expressed clones prior to the laborious task of sequencing.
Approximately 1000 colonies are isolated from each reciprocal SSH cDNA library. cDNA insert sequences are amplified by PCR and spotted onto microarray filters (GeneScreen Ltd). Duplicate filters are hybridised with radiolabelled cDNA probes generated from the reciprocal RNA material used to generate each SSH library (i.e. if an SSH library was made from a subtraction between diseased vs. normal cells, then one filter is hybridised with probe synthesised from RNA isolated from diseased cells and the other from normal cells. An analysis of the filters identifies with cDNA clone is differentially expressed.
Isolation of Full Length Clones for Functional Cloning
Full length clones are isolated from poly A+ RNA using Smart™ RACE Technology (Clontech) according to the manufacturers protocol. Briefly, double stranded cDNA is generated using the Smart II oligonucleotide and the CDS Primer (both supplied in the kit). Specific PCR products are then generated using the PCR primer (supplied in the kit) and gene specific primers (sense and antisense primers generated from the DNA sequence to be extended). To increase specificity, nested sense and antisense primers are used in secondary PCR amplification. PCR products are then ligated into plasmids, such as the
TA cloning system (Invitrogen), transformed into competent cells and expanded.
Plasmids are purified using mini-prep isolation system (Qiagen) and plasmids are submitted to MWG for sequencing. Specific 5' and 3' sequences are identified using sense and antisense gene specific primers. Products are to be sequenced approximately 10 bases 5' of the initiation codon for 5' PCR products and 10 bases 3' of stop codons for 3' PCR products. Using sequence data, 5' and 3' primers are made and full length cDNA is amplified. Example 7
Sodium nitroprusside-induced HeLa cell apoptosis is associated with significant changes in gene expression.
Following treatment of cultured HeLa cells with sodium nitroprusside, at a dose that induces apoptosis (0.9 xlO" M), total cellular RNA is isolated and examined for gene expression changes using microarray. Significant transcription of mRNA is observed. In one experiment, as many as 1169 genes are detected using Incyte human 'LifeGrid' filters; these genes are increased or decreased by greater than 2-fold over a 6hour time course as compared with the time zero control HeLa cells. As many as 943 genes are increased or decreased as early as 4 hours post-treatment, and prior to the 'commitment to die' point identified as described. As many as 469 genes are increased or decreased by 6 hours post-treatment. Figures 3, 4 and 5 show cluster analysis of genes regulated during sodium nitroprusside-induced HeLa cell apoptosis time course. Genes fall into primary clusters of up (white) and down- (black) regulated genes. By 6 host post treatment - 184 genes are up-regulated and -285 down-regulated.
Many of these regulated genes are ESTs (e.g. 124 at 6h post-treatment) with little or no known biological function annotation.
Inhibition of apoptosis by the redox modulator NAC blocks changes in gene expression associated with, sodium nitroprusside -induced HeLa cell apoptosis.
Sodium nitroprusside-induced HeLa cell apoptosis is inhibited by the addition of NAC (20mM). Gene expression change most closely and reciprocally correlated with HeLa cell survival (with NAC), or death (without NAC), is most likely to be causally involved in the apoptosis regulatory process. Furthermore, the use of NAC is directed at removing or modulating the route cause of apoptosis-induction i.e. the production and action of reactive nitrogen species.
Following treatment of cultured HeLa cells with sodium nitroprusside in the presence of NAC, at a dose that blocks apoptosis (20mM), total cellular RNA is isolated and examined for gene expression changes using microarray. Significant transcription of mRNA is observed. In one experiment, using Incyte human 'LifeGrid' filters, as many as 43 genes increased or decreased by greater than 2-fold at both 4 and 6 h following treatment with sodium nitroprusside are blocked by the addition of NAC. As many as 209 genes are increased or decreased by greater than 2-fold at 6 h following treatment with sodium nitroprusside are blocked by the addition of NAC. Figures 4 and 5 show cluster analysis of genes regulated during sodium nitroprusside-induced HeLa cell apoptosis time course, which are blocked by NAC. Genes fall into primary clusters of up (white) and down- (black) regulated genes.
Many of these regulated genes are ESTs (e.g. 51 at 6h post-treatment) with little or no known biological function annotation.
Changes in 'early' gene expression include classes of genes regulated by NO (RNS) and/or by hypoxic stress and include classes of genes known to be associated with cell defence mechanisms to apoptosis and ROS/RNS induced stress.
Of those genes regulated as early as 4 and 6 hours post-treatment with sodium nitroprusside, some include genes that are known to be transcriptionally regulated by NO and/or RNS. Also included are genes that are known to be transcriptionally regulated by hypoxic stress which is well characterised as involving ROS and RNS.
Scleroderma is a disease characterised by hypoxia and reactive oxygen species, where antioxidant therapy has been proposed as a treatment (Toxicology 2000 Nov 30;155(1- 3): 1-15 Emerging potentials for an antioxidant therapy as a new approach to the treatment of systemic sclerosis. Simonini G et al). polymyositis/scleroderma autoantigen 2 (lOOkD) is a prominent autoantigen associated with the disease. Figure 6 shows that polymyositis/scleroderma autoantigen 2 (lOOkD) is decreased associated with sodium nitroprusside induced HeLa cell apoptosis but blocked by treatment with NAC. These results suggest that polymyositis/scleroderma autoantigen 2 (lOOkD) may have a protective role in cells challenged with NO/RNS.
The aryl hydrocarbon receptor nuclear translocator (Arnt) and hypoxia-inducible factor (HIF)-l alpha mediate cellular responses to hypoxia. We have previously shown that expression of these genes, and aryl hydrocarbon receptor nuclear translocator-like gene is associated with ROS-induced apoptosis in HeLa cells and neutrophils (see PCT/IB00/02054). Figure 7 shows that aryl hydrocarbon receptor nuclear translocator- like gene is decreased associated with sodium nitroprusside induced HeLa cell apoptosis but blocked by treatment with NAC. These results suggest that aryl hydrocarbon receptor nuclear translocator-like may have a protective role in cells challenged with NO/RNS.
Calpain is a noncaspase protease mediator of apoptosis (Leukemia 2000, 14(9) 1695-703 Noncaspase proteases in apoptosis Johnson DE). Calpastatin (calpain inhibitor 1) is a physiological inhibitor of calpstatin. Calpain inhibitorl reduces the renal dysfunction and injury associated with ischemia/reperfiision of the kidney, a disease associated with hypoxic stress and apoptosis (Kidney Int 2001 Jun;59(6):2073-83 Calpain inhibitor-1 reduces renal injury in the rat. Chatterjee PK et al). An excessive production of nitric oxide (NO) in response to cytokines has been shown to be the major cause of the destruction of islet beta-cells associated with type 1 (insulin-dependent) diabetes mellitus. The NO-induced beta-cell death is the typical apoptosis. SNAP -induced apoptosis was blocked by an inhibitor of a Ca2+-dependent protease, calpain (Cell Struct Funct 1999 Dec;24(6):451-5 Nitric oxide induces apoptosis via Ca2+-dependent processes in the pancreatic beta-cell line MIN6. Nakata M et al). Nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many ofthe effects of calcium ionophore on C(2)C(12) muscle cells, indicating that nitric oxide inhibition of calpain protected the cells from ionophore-induced proteolysis. Calpain inhibitor I and a cell-permeable calpastatin peptide also protected the cells from proteolysis, confirming that ionophore- induced proteolysis was primarily calpain mediated (Am J Physiol Cell Physiol 2000 Sep;279(3):C806-12 Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells. Koh TJ and Tidball JG. Figure 8 shows that Calpastatin is decreased associated with sodium nitroprusside induced HeLa cell apoptosis but blocked by treatment with NAC. These results suggest that calpastatin may have a protective role in cells challenged with NO/RNS.
A network of radical acceptors and enzymes is thought to play an important redox- buffering role to protect cells against NO-mediated injury. Thioredoxin reductase is one of the key cellular antioxidant defense enzymes. An inverse correlation between cell susceptibility to NO damaging effects and Trx expression has been described, suggesting that the Trx system may have important preventative capacities towards NO-mediated cellular injury in monocytic macrophage cells (Biochem J 2000 Mar 15;346 Pt 3:759-65 Protective effect of thioredoxin upon NO-mediated cell injury in THP1 monocytic human cells. Ferret PJ et al). Figure 9 shows that thioredoxin reductase 1 is decreased associated with sodium nitroprusside induced HeLa cell apoptosis but blocked by treatment with NAC. These results suggest that thioredoxin reductase 1 may have a protective role in cells challenged with NO/RNS.
Matrix metalloproteinases (MMPs) play an integral part in tumor spread and the metastatic cascade. NO donors such as S-nitroso-n-acetylpenicillamine and S- nitrosoglutathione (both at 0.01-100 microM) inhibit MMP-2 release from platelets and tumor cells (Cancer Res 2001 Jan l;61(l):376-82 Matrix metalloproteinase 2 in tumor cell-induced platelet aggregation: regulation by nitric oxide. Jurasz P et al). Figure 10 shows that matrix matalloproteinase 2 is decreased associated with sodium nitroprusside induced HeLa cell apoptosis but blocked by treatment with NAC. These results suggest that thioredoxin reductase 1 may have a protective role in cells challenged with NO/RNS.
Ubiquitin C-terminal hydrolase Ll represents 1 to 2% of total soluble brain protein. Its occurrence in Lewy bodies and its function in the proteasome pathway make it a compelling candidate gene in Parkinson disease, a disease characterized by reactive oxygen stress and apoptosis. A marked increase of ubiquitin C-terminal hydrolase Ll immunoreactivity in neurons 1 hr after ischemia/hypoxia in a rat model. These data suggest that ubiquitin C-terminal hydrolase Ll is involved in the neuronal stress response (Neurochem Res 1997 Jan;22(l):93-100 Ubiquitin-mediated stress response in a rat model of brain transient ischemia/hypoxia. Gubellini P et al). Figure 11 shows that ubiquitin C-terminal hydrolase Ll is increased associated with sodium nitroprusside induced HeLa cell apoptosis but blocked by treatment with NAC. These results suggest that ubiquitin C-terminal hydrolase Ll may mediate the pro-apoptotic actions of sodium nitroprusside in NO/RNS . Example 8
Characterisation and discovery of polypeptides/proteins 'Proteomics' associated with apoptosis
Changes in transcription are not necessarily paralleled by similar changes in translation. Furthermore, protein expression may also be dependent upon post-translational modification such as phosphorylation, proteolytic cleavage or changes in intracellular location. This example describes proteomic studies, which complement the use of genomic studies. Translation inhibitors, such as cyclohexamide, are used to establish the dependence of apoptosis/NO-mediated apoptosis on protein synthesis. In addition, changes in expression/translation of candidate genes are measured using FACS, or 20- PAGE to measure individual proteins or hundreds or thousands of proteins simultaneously.
Proteomics may be applied to the identification of novel apoptosis-associated proteins in a number of ways. The proteins may be labelled metabolically using 35-S or 33-P radioactive sources and then detected through phosphoimaging or by conventional autoradiography. Proteins from different treatment groups may be differentially labelled with either P or a Cy5 derivative (Amersham Pharmacia Biotech) and then analysed simultaneously on the same gel, thereby facilitating identification of differentially expressed products. Alternative, post-staining, approaches may be used, including silver staining, anti-phosphotryosine antibody staining, anti-phosphoserine antibody staining, carbohydrate-specific staining procedures (for example based on hydrazine modification) or similar post-translational modification detection techniques known to the art. Gel images derived from post-stained gels are captured using CCD camera, phosphoimaging or by densitometry and analysed digitally to determine differences in protein expression patterns using software such as Melanie (BioRad) or Phoretix 2D (Phoretix). Measurement of individual or global protein expression and/or post-translational modification by 2D-PAGE
Characterisation of phosphorylation by metabolic labelling
Neurons (1.5xl07 cells/ml) are incubated in RPMI-1640 supplemented with 60μCi/ml [35S]-methionine. Cells are challenged with agents influencing the process of apoptosis and are harvested at appropriate time points following treatment, from 0 to 22 hours.
Preparation of cell lysates
Total cell protein is prepared by lysing the cells in a solution comprising 8M Urea (Ultrapure); 2% CHAPS (Sigma); 40mM Tris(HCl) (Sigma). In some cases addition or omission of DTT may be desirable. RNA and DNA contamination may be removed by treatment with RNases and DNases. A sequential extraction procedure may be used with which more hydrophobic proteins can be solubilised. Polypetides synthesised following the addition of the radioactive tracer may then be detected by 2D-PAGE analysis of the lysates.
2D-PAGE
Isoelectric focusing and polyacrylamide gel electrophoresis
The following reagents are used: TrisHCI, Tris base, glycine, (Sigma); trichloracetic acid (TCA), NaOH, glycerol, n-butanol, bromophenol blue (Merck); [35S]-methionine (SJ 204, specific activity: > 1.000 Ci/mmol, containing 0.1% 2mercaptoethanol), Amplify (Amersham International, Amersham, UK); filters (HAWP 0.25 m pore size) (Millipore, Boston, USA); RNase A, DNase I; urea (ultra pure); acrylamide, bisacrylamide, TEMED, ammonium persulphate (BioRad, Richmond, CA,USA); ampholytes: pH 5-7, pH 3.5-10, pH 7-9, pH 8-9.5 (Pharmacia, Uppsala, Sweden); Nonidet P-40 (BDH, Poole, UK); ampholytes: pH 5-7 and sodium dodecyl sulphate; agarose (GibcoBRL); ethanol (absolute 96%) (BDH); methanol (BDH); acetic acid (99% glacial) (BDH) and X-ray film (Curix RP-2) (AGFA). Briefly, first dimension gels contain 4% acrylamide, 0.25% bisacrylamide and ampholytes, and are 18 cm long and 0.4 cm in diameter. Equal numbers of counts of each cell lysates sample are applied to the gels. In the case of lower amounts of radioactivity it may be necessary to regulate the exposure time ofthe gel so that comparable total optical densities are obtained. The samples are first resolved on isoelectric focusing (IEF; pH 3.5-7) gels. IEF gels may be pre-focused for approximately 30mins at 140μA/gel (limiting current), the sample is then applied, by either cup-loading (Multiphor) or in-gel rehydration (IPG-phor), and focused for varying times and voltages appropriate to short or long IEF strips.
Second dimension gels, 1 mm thick, 200 mm long and 185 mm wide contain acrylamide and bisacrylamide, prepared as gradient (9-16%) or single percentage (12%) gels. Focused IEF strips are loaded to the second dimension gels through a 1.0% LMP-agarose top layer. After electrophoresis in Tris-glycine buffer, the gels are fixed in 45% methanol and 7.5% acetic acid for 30 min and treated for fluorography with Amplify for 30 min before being dried. The gels are placed in contact with X-ray films and exposed at -70 C for 1 to 40 days. Each gel is exposed for at least 3 time periods to compensate for the lack of dynamic range of X-ray films.
Identification of proteins associated with apoptosis
Proteins from 2D gels may be identified by immuno-blotting, N-terminal sequencing, internal peptide sequencing, co-migration of unknown with known proteins, or over- expression in a model organism. Additionally proteins will be prepared for analysis by either Electrospray Ionisation (ESI)-MS or Matrix Assisted Laser Desorption Ionization- time of flight (MALDI-TOF)-MS. Using these techniques it is possible to measure the masses of peptides derived from protease or chemical digestion. The set of masses produced by such a digestion is unique to a protein, and so constitutes a peptide mass fingerprint, which can form the basis of a database search. Triple quadrupole or ion trap mass spectrometers can be used to generate fragmentation spectra from peptides (MS/MS), from which sequence data can be obtained. Peptides are fragmented by isolating the 'parent' peptide ion and then accelerating it into a region filled with an inert gas, typically argon. The peptide ion undergoes multiple collisions with the gas and fragments in a random manner along the amide backbone. The resulting 'daughter' ions are detected and a fragmentation pattern, which contains sequence specific information, is derived. This information can be used to extend database searches, or to derive degenerate oligonucleotides for library screening. Additional analysis techniques include amino-acid analysis through HPLC, or direct HPLC of peptides derived through protease cleavage of the target protein.
SUMMARY
The genomics strategy described in the present invention provides the means to characterise the mechanism of apoptosis. In addition, the strategy provides the means to identify, characterise, clone and validate molecules, including oligonucleotides and polypeptides, associated, both causally and consequentially, to apoptosis. The model described enables the identification of genes which are involved in, or cause, early stages of apoptosis. The model also provides a screen for substances which interact with such genes to inhibit apoptosis. Such compounds and substances identified may be used in treating diseases involving apoptosis.
In a preferred configuration ofthe present invention, cells such as HeLa cells are cultured, and apoptosis induced by administration of a suitable substance, for example, Sodium nitroprusside (SNP). Suitable substances include nitric acid donors which are capable of generating reactive nitric oxide (NO) species.
In the model described here, gene expression may be studied at times which precede the earliest morphological manifestation of apoptosis (for example, gene expression may be studied at 1, 2, 3, 4, or 5 hours, whereas the earliest morphological signs of apoptosis generally appear at 6 hours). The model system described here therefore is capable of blocking the root cause of apoptosis by modulating the action of nitric oxide. Thus, it is capable of identifying genes which are involved in the earliest stages of apoptosis, before morphological signs of apoptosis appear. Other cells may also be used in the model system and assays described here. Cells such as the human Ntera-II neuronal cell line or primary rodent neurons may be cultured in a serum-free cell culture medium these cells undergo apoptosis following withdrawal of serum (Forgie et al, (2000) Eur J Neurosci, 2, 610-616). The onset of apoptosis is characterised using markers, such as caspase activation and externalisation of the membrane phospholipid phosphatidylserine. These assays serve to identify the earliest measurable onset of the cells commitment to apoptosis. Intracellular events that drive commitment of a cell to apoptosis, occur before this earliest measurement of the commitment.
The intracellular initiator of the commitment to apoptosis is a change in REDOX manifested by an increase in level of NO. Intracellular NO is measured to confirm this increase preceding the measurement of apoptosis. Suitable inhibitors of NO production include NOX 100 and L-NAME are used to confirm the causal role of NO in mediating apoptosis in this model system.
The characterisation of the apoptosis process and NO involvement in this model system serve to establish a temporal window between the onset of NO (which is the initial trigger for the apoptosis process) and the measurement of cellular apoptosis (at which point the cell is committed to undergoing apoptosis). The key early intracellular events that induce the commitment of cells to undergoing apoptosis (and those which may represent the most attractive therapeutic targets) occur within this window.
NGF has been shown to inhibit apoptosis and prolongs survival of neurons in culture Culturing neurons in NGF provides a very useful model system with which to study neuronal apoptosis. NO induced apoptosis can be directly stimulated in this system though the exogenous addition of NO generating agents such as sodium nitroprusside. Where HeLa cells are used, apoptosis may be induced by administration of sodium nitroprusside or any other source of NO to the cell. Apoptosis may be inhibited in various ways, for example, by administration of NAC.
Signal transduction events are associated with induction of commitment of cells to undergoing apoptosis. Signal transduction would typically involve events such as protein phosphorylation and/or de-phosphorylation. Transcription events are associated with induction of commitment of cells to undergoing apoptosis. Transcription inhibitors, such as Actinomycin D (actD) are used to measure the role of transcription events in the induction process. In addition, the use of actD in pulse-chase experiments is used to establish the precise time point at which transcription events exert an effect.
The invention provides the means to allow the study of the transcription of individual mRNAs in the early 'induction of commitment' phase of apoptosis, and in particular following NO. One candidate gene that may be associated with this process is FasL. The expression of individual candidate genes is measured by RT-PCR or hybridisation with a gene specific radiolabelled probe.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridisation techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al, Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc. which are incorporated herein by reference), chemical methods, pharmaceutical formulations and delivery and treatment of patients.
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be covered by the present invention.

Claims

1. A method for modulating the expression of gene products that regulate the transition of a cell between a non-apoptotic state and an apoptotic state, comprising modulating the intracellular concentration of nitric oxide (NO).
2. A method for identifying one or more gene product(s) that modulate the transition of a cell between a non-apoptotic state and an apoptotic state, comprising the steps of:
a) exposing the cell to NO or to an agent which induces the production of NO in the cell or inhibits the production of NO in the cell; b) determining the level(s) of expression of one or more gene product(s) in a cell to establish a reference expression level; c) monitoring the level(s) of expression of said one or more gene product(s) in the cell; and d) identifying one or more of the gene product(s) whose expression has been increased, decreased or modified as a result ofthe NO modulation.
3. A method according to claim 2, further comprising the steps of: a) determining the level(s) of expression of one or more gene product(s) in a cell to establish a reference expression level; and b) comparing the expression level(s) after NO modulation to the reference expression level(s).
4. A method according to any preceding claim, wherein exposure of the cell to NO leads to induction of apoptosis in the cell more rapidly than occurs under identical conditions but in the absence of NO.
5. A method according to any one of claims 2 to 4, wherein expression levels are determined by assessing polypeptide production.
6. A method according to any one of claims 2 to 4, wherein expression levels are determined by assessing polypeptide post-translational modification.
7. A method according to any one of claims 2 to 4, wherein expression levels are determined by assaying gene transcription.
8. A method according to any preceding claim, wherein the cell is a HeLa cell.
9. A method according to any of claims 1 to 7, wherein the cell is a neuron or a cell with neuronal characteristics (e.g. PC-12).
10. A method according to any preceding claim, in which the cell is exposed to aa chemical inducer of apoptosis, preferably sodium nitroprusside.
11. A method according to any preceding claim, wherein the cell is cultured in the presence of an inhibitor of apoptosis, which inhibitor acts to delay the onset of apoptosis in the cell.
12. A method according to claim 11 , wherein the inhibitor is N-acetyl-cysteine (NAC) or nerve growth factor (NGF).
13. A method according to any preceding claim, wherein the onset of apoptosis is monitored by morphological analysis, externalisation of membrane phospholipid phosphatidyl serine or caspase activation analysis.
14. A method according to claim 13, wherein the involvement of NO in the induction of the onset of apoptosis is measured by further exposing the cells to one or more NO inhibitors.
15. A method wherein the NO inhibitors are selected from the group consisting of NOX 100, or L-NAME or other L-arginine analogues, L-Citrulline analogues, aminoguanidine, S-substituted isothioureas or bis-isothioureas, N (3aminomethyl) benzyl) acetamidine, 7-nitroindazole, trimethylphenylfluoroimidazole, mercaptoethyl and mercaptopropyl-guanidinnes
16. A method according to any preceding claim wherein the NO inhibitors are selective against specific isoforms of Nitric Oxide Synthase (NOS) thereby providing a means of identifying cell-specific genes involved in NO-mediated apoptosis signalling.
17. A method according to claim 16 wherein the NO inhibitor is 7-nitroindazole such that neuronal specific genes involved NO-mediated apoptotic signalling may be identified.
18. A method according to claim 17 wherein the NO inhibitor is a mercaptoethyl- guanidine or a mercaptopropyl -guanidine such that inflammatory cell specific genes involved in NO-mediated apoptotic signalling may be identified.
19. A method according to any one of claims 9 to 16, wherein the expression levels of a plurality of gene product(s) are determined by hybridisation of one or more mRNA populations to a set of polynucleotides arrayed on to a substrate.
20. A method according to claim 5 or claim 6, wherein the expression levels of a plurality of gene product(s) are determined by 2D-polyacrylamide gel electrophoresis of one or more polypeptide populations.
21. A method according to any preceding claim whereby gene targets for therapeutic intervention may be identified where the disease is selcted from the group consiting of: septic shock, cerebral ischaemia, neurodegenerative diseases, inflammatory diseases, inflammatory pain, migraine, diabetes and meningitis.
22. A model system for modelling NO mediated modulation of apoptosis in a cell comprising the steps of:
(a) providing a population of cells;
(b) modulating the intracellular NO concentration in the cells; and (c) identifying genes whose expression is modulated as a result of changes in intracellular NO concentration.
23. The use of NO to induce the expression of gene products that modulate the transition of a cell from a non-apoptotic state to an apoptotic state.
24. A method of identifying an inhibitor of apoptosis, the method comprising the identifying a compound which interacts with a gene identified in a method according to claim 22.
25. A method of identifying an inhibitor of apoptosis, the method comprising the steps of:
(a) providing cell;
(b) modulating the intracellular NO concentration in the cell; (c) identifying a gene whose expression is modulated as a result of changes in intracellular NO concentration;
(d) exposing the cell to a candidate compound; and
(e) identifying candidate compounds which modulate the expression of a gene identified in (c).
26. An inhibitor of apoptosis identified by a method according to claim 24 or 25.
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US8598150B1 (en) 2008-04-02 2013-12-03 Jonathan R. Brestoff Composition and method for affecting obesity and related conditions
US8809312B2 (en) 2008-04-02 2014-08-19 Jonathan R. Brestoff Composition and method for affecting obesity and related conditions
US8987245B2 (en) 2008-04-02 2015-03-24 Jonathan R. Brestoff Parker Composition and method for affecting obesity and related conditions

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