WO2002000241A2 - Compositions et procedes pour reguler le cycle cellulaire - Google Patents

Compositions et procedes pour reguler le cycle cellulaire Download PDF

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Publication number
WO2002000241A2
WO2002000241A2 PCT/EP2001/008096 EP0108096W WO0200241A2 WO 2002000241 A2 WO2002000241 A2 WO 2002000241A2 EP 0108096 W EP0108096 W EP 0108096W WO 0200241 A2 WO0200241 A2 WO 0200241A2
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Prior art keywords
cell
gene
cells
polypeptide
apoptosis
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PCT/EP2001/008096
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English (en)
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WO2002000241A3 (fr
Inventor
Didier Branellec
Elisabeth Lavenne
Aude Le Roux
Abderrahim Mahfoudi
Harris Perlman
Kenneth Walsh
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Aventis Pharma S.A.
Saint Elizabeth's Medical Center
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Priority to AU2001289652A priority Critical patent/AU2001289652A1/en
Publication of WO2002000241A2 publication Critical patent/WO2002000241A2/fr
Publication of WO2002000241A3 publication Critical patent/WO2002000241A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • compositions and methods for regulating the cell cycle are provided.
  • the present invention relates, generally, to compositions and methods for regulating the cell cycle, in particular apoptosis.
  • the invention relates more specifically to compositions and methods using a Ki gene or polypeptide to regulate the cell cycle, in particular apoptosis, in cells, in vitro, ex vivo or in vivo.
  • the invention also relates to compositions, vectors, cells and the like, which can be used to implement the above methods. This invention can be used to protect cells from apoptosis or to mediate or enhance apoptosis in cells, in various physio-pathological conditions.
  • Homeostasis is achieved through a balance of cell growth and cell death. Many recent studies have been conducted to determine key events which lead to cell death or cell survival (for review see (Best, Hasdai et al. 1999)). The withdrawal of growth factors in vitro (Batistatou and Greene 1993; Wang and Walsh 1996) and in vivo (Colombel, Olsson et al. 1992) from differentiated cells provokes cell cycle arrest and consequently apoptosis. Homeobox transcription factors have been shown to be implicated in cell differentiation and migration and deregulation of the expression of some of them may be associated with abnormal cell proliferation. However most of them have been associated with cell proliferation, some exhibit growth suppressive properties.
  • Gax Gax (Growth Arrest Homeobox) was isolated from an adult aorta VSMC cDNA library (Gorski, LePage et al. 1993; LePage, Altomare et al. 1994).
  • Gax expression is rapidly repressed by growth factor stimulation, and upregulated under conditions that promote quiescence (Gorski, LePage et al. 1993).
  • expression of gax is rapidly downregulated in rat carotid arteries following an injury that stimulates VSMC proliferation (Weir, Chen et al. 1995).
  • gax gene transfer leads first to cell cycle arrest and then cell apoptosis in the rat carotid model (Perlman, Luo et al. 1999).
  • gax is an important gene in SMC biology in vitro and in vivo. Owing to gax properties toward SMCs we investigated whether gax overexpression may affect human tumoral cell proliferation.
  • Ki antigen was first identified as an autoantigen in patients with systemic lupus erythematosus (SLE) (Tojo, Kaburaki et al. 1981). It is a 32 kDa protein which exhibits a nuclear localization and the expression of which has been shown to increase during proliferation and in transformed fibroblasts.
  • the present invention relates, generally, to compositions and methods for regulating the cell cycle, in particular apoptosis, more specifically using a Ki gene or polypeptide, in vitro, ex vivo or in vivo.
  • Ki antigen a component of the proteasome (also referred as to PA28 ⁇ )
  • PA28 ⁇ a component of the proteasome
  • Ki antigen is able to control cell cycle (including apoptosis), since Gax-mediated cell killing activity was reduced by Ki overexpression. Fuilhermore, this application also demonstrates that Ki protective activity is not restricted to Gax since p53-mediated cytotoxicity towards tumor cells was also reduced by Ki.
  • a particular aspect of the present invention resides in a method of inhibiting or reducing apoptosis in a cell, comprising contacting the cell with a Ki polypeptide or gene.
  • a further aspect of this invention relates to the use of a Ki polypeptide or gene to inhibit or reduce apoptosis in a cell.
  • the invention relates to methods of inhibiting or reducing p53- mediated apoptosis or GAX-mediated apoptosis.
  • a further variant of this invention relates to a method of treating a cell to protect said cell from death or degeneration, comprising contacting the cell with a Ki polypeptide or gene.
  • the method of this invention comprises contacting the cell(s), in vivo or ex vivo, with a vector encoding a Ki polypeptide, such as a viral vector or a plasmid.
  • Ki gene or polypeptide may be used alone or in combination(s) with other therapeutic or prophylactic treatment(s) or agent(s).
  • the above methods/uses can be applied to various cell types, either isolated or in tissues, organs or whole organisms, in healthy or pathological condition, such as more particularly smooth muscle cells (in particular cardiomyocytes), muscle cells, fibroblasts, hepatic cells, endothelial cells, nervous cells, etc.
  • smooth muscle cells in particular cardiomyocytes
  • muscle cells in particular fibroblasts
  • hepatic cells in particular hepatic cells
  • endothelial cells hepatic cells
  • nervous cells etc.
  • Another aspect of this invention resides in a method of causing or stimulating (or enhancing) apoptosis in a cell, comprising contacting the cell with a compound that inhibits Ki gene expression or activity.
  • This invention also concerns the use of a compound that inhibits Ki gene expression or activity to cause or stimulate (or enhance) apoptosis in a cell.
  • the compound is an antisense nucleic acid molecule that inhibits Ki gene expression, an anti-Ki antibody, or a chemical inhibitor or competitor, including peptides (or nucleic acids encoding the same).
  • the compound can be used either alone or in combination(s) with additional cytotoxic agent(s) or treatment(s), such as tumor suppressor genes or polypeptides, anti-tumor chemical agents or treatments, etc.
  • cytotoxic agent such as tumor suppressor genes or polypeptides, anti-tumor chemical agents or treatments, etc.
  • the cytotoxic agent may be used before, after or together with the compound that inhibits Ki gene expression or activity.
  • compositions comprising a compound that inhibits Ki gene expression or activity and a cytotoxic agent, for simultaneous, separate or sequential use.
  • the invention also relates to compositions comprising a Ki gene or polypeptide and a cyto- protective agent, for simultaneous, separate or sequential use.
  • a further aspect of this invention comprises a viral vector comprising a Ki gene, more preferably an adeno viral vector or an AAV-derived vector.
  • compositions such as pharmaceutical compositions, nucleic acid constructs, recombinant cells as well as methods of treating or preventing various pathological conditions where abnormal cell cycle regulation occurs, such as tumors, neuro-degeneration, stenosis, stroke, myocardial disorders (e.g., myocardial ischemia, heart failure), hepatitis, cyrrhosis, etc.
  • FIG. 1 Transfection assay in H1299 tumor cells. 72hrs after transfection using
  • FIG. 1 Transfection assay in MEF fibroblast cells. 48hrs after transfection using Lipofectamine, MEF cells viability was assessed.
  • the present invention relates, generally, to compositions and methods for regulating the cell cycle, in particular apoptosis, more specifically using a Ki gene or polypeptide, in vitro, ex vivo or m vivo.
  • the regulation of cell cycle comprises, within the context of the present invention, a regulation of growth stimulating and/or growth inhibiting genes or polypeptides or pathways in a cell.
  • the invention is more particularly suited to regulate apoptosis (i.e. programmed cell death) in a cell, or related gene pathways.
  • This invention also includes methods of protecting cell from death or degeneration, i.e., methods of inhibiting or reducing cell death or degeneration.
  • the invention can also be used to prolong cell survival.
  • inhibiting or reducing means either partial or complete suppression, more generally partial suppression.
  • inhibiting cell death designates an inhibition of at least 20%, preferably at least 30% of the cellular death, or a prolonged cell survival time of at least 20%, more preferably at least 30%.
  • an inhibition of apoptosis designates an inhibition of at least 20%, preferably at least 30% of the cellular death, or a prolonged cell survival time of at least 20%), more preferably at least 30%.
  • Ki polypeptide designates the human Ki polypeptide comprising the amino acid sequence SEQ ID NO:2.
  • a Ki polypeptide also includes any variants or homologs of the above sequence, more particularly naturally-occurring variants or homologs, such as polymorphisms, splicing variants, homologs from other species, etc., that have the ability to interact with a GAX polypeptide in vitro, or to reduce Gax-mediated or p53 -mediated apoptosis in a cell.
  • Ki polypeptides also include derivatives of the above sequence, comprising one or several amino acid modifications, such as a mutation, a deletion, a substitution and/or an addition of one or several amino acid residues, and having the above-described property(ies).
  • a Ki polypeptide has at least 75% amino acid identity, even more preferably at least 90% amino acid identity with sequence SEQ ID NO:2.
  • a Ki polypeptide may also be modified by addition of a foreign moiety, such as a tag, marker, linker, etc.
  • Ki polypeptides include any polypeptide encoded by a nucleic acid molecule that hybridises, under stringent conditions, with a nucleic acid encoding the Ki polypeptide of SEQ ID NO:2 and having the above-mentioned property(ies).
  • a Ki gene designates any nucleic acid molecule encoding a Ki polypeptide as described above, more preferably a Ki polypeptide having the sequence of SEQ ID NO:2.
  • the Ki gene may be a DNA (cDNA, gDNA, synthetic or semi-synthetic DNA, etc.) or a RNA (mRNA, premRNA, synthetic or semi-synthetic RNA, etc.).
  • a Ki gene comprises the nucleic acid sequence SEQ ID NO:l, and is, more preferably, a cDNA.
  • a particular object of the present invention resides in a method of inhibiting or reducing apoptosis in a cell, comprising contacting the cell with a Ki polypeptide or gene.
  • the invention relates to methods of inhibiting or reducing p53- mediated apoptosis or GAX-mediated apoptosis.
  • Apoptosis has been associated with various pathological conditions and occurs in response to various stimuli.
  • the invention now offers novel and efficient strategies to counteract the effect of apoptosis, such as Gax- orp53-mediated apoptosis.
  • the method of this invention comprises contacting the cell(s), in vivo or ex vivo (or in vitro), with a vector encoding a Ki polypeptide, such as a viral vector or a plasmid.
  • a vector encoding a Ki polypeptide such as a viral vector or a plasmid.
  • a Ki gene is preferred to deliver to the cells (or tissues, organs, organisms), allowing the gene to be expressed in the cells and to produce its cyto- protective effect.
  • Preferred vectors are viral vectors such as adenoviral vectors, AAV-based vectors or lentiviral vectors, more specifically replication-defective adenoviral vectors, lentiviral vectors or AAV-based vectors.
  • Adenoviral vectors may be prepared from various serotypes, such as Ad2 or Ad5, by deleting one or several viral genes (such as El, E4, E2, E3 and/or Iva2, etc.), and by inserting the Ki gene in said defective vector.
  • the viruses may be produced in any suitable packaging cell, according to techniques known in the art.
  • AAV-based vectors may be prepared from various serotypes, by deleting one or several viral genes (such as Rep and/or Cap), and by inserting the Ki gene in said defective vector.
  • the viruses may be produced in any suitable packaging cell, according to techniques known in the art. Specific Adenoviral vectors have been described for instance in WO00/12738.
  • Preferred viral vectors are: - replication-defective adenoviral vectors having at least a non-functional El region and comprising a Ki gene, under the control of a transcriptional promoter,
  • adenoviral vectors lacking all endogenous viral genes (gutless or minimal vectors) and comprising a Ki gene, under the control of a transcriptional promoter
  • Plasmids such as pCOR plasmids comprising a conditional origin of replication (WO97/10343) or any other plasmid preferably lacking an antibiotic- resistance gene or a fully functional origin of replication.
  • plasmids may be obtained or prepared according to conventional methods.
  • the Ki gene may be placed under the control of any suitable transcriptional promoter, preferably a promoter functional in mammalian cells, more preferably in human cells.
  • the promoter may be constitutive, ubiquitous, regulated, inducible, tissue-specific, etc. The choice of a particular promoter can be made by the skilled person, depending on the cell to be treated.
  • Promoters could also be of synthetic origin such as inducible or regulated promoters (modulated by tetracycline, rapamycin, PPAR activator, hormone analogs, physiopathological situations etc. or any promoters derived from fragments of the above mentioned using techniques known by the skilled person (deletion mutants, chimeric promoter, ).
  • any cell containing a vector as denied above is the object of this invention, particularly cells infected with a viral vector as defined above.
  • the invention also encompasses methods of producing viral vectors as defined above, comprising introducing a viral vector comprising a Ki gene in a packaging cell, and isolating the viruses prepared.
  • the present invention can be used for the treatment or prevention of disorders resulting from excessive cell death that affect muscle cells, or cardiovascular vessels including but not limited to myocardial, cerebral or peripheral ischemia, infarct, heart failure, aneurisms and also non cardiovascular disorders such as inflammatory diseases, neurodegenrative diseases, stroke, ulcers, infertility, scleroderma, renal diseases, eye diseases, hepatic diseases and pulmonary disesases .
  • This invention also finds utility for the treatment of general disorders due to excessive cell proliferation such as atheroma, retinopathy, cancers, metastasis, AIDS complications, abnormal skin proliferation, psoriasis or rhumatoid arthritis.
  • Particular objects of this invention reside in methods of treating a subject with heart disease, comprising administering to the subject, preferably through direct myocard or heart injection, an amount of a Ki gene or polypeptide, preferably a Ki gene, efficient to ensure protective activity of myocardial cells exposed to said gene or polypeptide.
  • the invention is also suitable to treat various nervous cells, to protect said cells from death or degeneration.
  • this invention can be used to prevent or treat or reduce the burden of subjects with neurodegenerative disorders (ALS, SMA, Parkinson's disease, Alzheimer's disease, etc.).
  • ALS neurodegenerative disorders
  • SMA neurodegenerative disorders
  • Parkinson's disease Alzheimer's disease, etc.
  • the invention relates to a method of preventing or treating or reducing the burden of a subjects with neurodegenerative disorder, comprising administrering to the subject an amount of a Ki gene or polypeptide, preferably a Ki gene, efficient to ensure protective activity of nervous cells exposed to said gene or polypeptide.
  • a Ki gene or polypeptide preferably a Ki gene
  • the Ki gene or polypeptide may be used either alone or in combination with additional agents or treatments, in particular agents or treatments used for cardiovascular or neurological indications.
  • Ki gene or polypeptide may be associated with other treatments as known in the state of the art for the indication, or known to enhance the desired effect of the Ki-mediated treatment by the skilled person.
  • a particular object of this invention resides in a composition
  • a composition comprising a Ki gene or polypeptide and a second therapeutic or prophylactic agent, for simultaneous, separate or sequential use.
  • the composition may comprise any pharmaceutically acceptable carrier or vehicle or excipient, such as saline buffer, isotonic solution, additives, stabilizers, etc.
  • the second agent may be a cyto-protective agent, an (anti-)coagulation factor, an (anti- )platelet agent, etc.
  • polypeptides, genes or compositions may be used in different ways.
  • they may be contacted directly with the cells, in any appropriate device (tube, plate, dish, pouch, etc.).
  • they may be administered to a subject in need thereof, by various conventional routes such as local injection, intracardiac injection, pericardia! injection, intra-tumoral injection, stereotaxic injection, intra-muscular injection, percutaneous injection, etc. It is believed that the specific administration protocol, including route, dose and frequency, may be adjusted by the clinician, depending on the situation.
  • 10 4 -10 13 viral particles may be injected per dose, more generally between 10 ⁇ -10 12 .
  • Another object of this invention resides in a method of causing or stimulating (or enhancing) apoptosis in a cell, more particularly in a tumor cell or a smooth muscle cell, comprising contacting the cell with a compound that inhibits Ki gene expression or activity.
  • the invention also provides novel strategies to cause cytotoxicity or apoptosis, or to enhance cytotoxicity or apoptosis in a cell.
  • the compound is an antisense nucleic acid molecule specific for Ki gene (as defined above, including any transcription product thereof).
  • these antisense molecules can be targeted against the genomic DNA with which they can form a triple helice, or against any transcription product of said genomic DNA, including messenger RNAs, with which they form double helices.
  • Preferred antisense molecules comprise all or part of SEQ ID NO:l (or its complementary sequence) and are directed against Ki mRNA.
  • a particular type of antisense molecules comprises synthetic single-strand oligonucleotides comprising between 5 and about 100 bases, more preferably between 5 and 40 bases and having a sequence region that is complementary to at least a region of a Ki RNA.
  • the antisense molecules may be produced by any conventional technique, including artificial nucleic acid synthesis and/or recombinant DNA techniques (isolation from DNA libraries, cleavage, ligation etc.).
  • the antisense molecules may contain non-natural bases and/or may be chemically modified to increase their stability (or the stability of the hybrid) and/or to induce a degradation of the hybrid formed.
  • the antisense molecule may also be a Peptide Nucleic Acid molecule (PNA), i.e., an antisense nucleic acid comprising one or several amino acids.
  • PNA Peptide Nucleic Acid molecule
  • the antisense molecules of this invention may also comprise double stranded DNAs encoding antisense oligonucleotides as described above, as well as ribozymes.
  • the present invention thus resides in a method of causing or stimulating apoptosis in a cell, comprising contacting said cell with an antisense nucleic acid molecule specific for Ki gene or any transcription product derived therefrom.
  • the compounds that inhibit Ki gene expression or activity can also be a peptide, preferably a peptides of 5 to 50 amino acid resides, even more preferably 5 to 25, or a nucleic acid encoding the same.
  • the peptide preferably comprises a portion of Ki polypeptide and is able to interfere with the activity of Ki, in particular its binding to Gax and/or p53.
  • Antibodies may be polyclonal, monoclonal, recombinant, , etc.
  • Antibody derivatives or fragments include any portion of an antibody (e.g., Fab, Fab'2, CDR, etc.) that retains at least a potion of the epitope or antigen- specificity thereof.
  • Derivatives also include ScFv, which can be synthesized directly into a target cell.
  • the compounds that inhibit Ki gene expression or activity include any natural or artificial product that is able to inhibit Ki gene expression or Ki polypeptide activity. These include any product interfering with the interaction between a Ki polypeptide and a Gax or p53 protein.
  • Such product may be a peptide, nucleic acid, lipid, sugar, inorganic or organic substance, whether natural, synthetic or semi-synthetic.
  • the compound is used either alone or in combination(s) with additional cytotoxic agents, such as tumor suppressor genes or polypeptides, anti-tumor chemical agents or treatments, etc.
  • tumor suppressor genes include any nucleic acid or polypeptide selected from the p53 tumor suppressor product, the Rb tumor suppressor product, etc.
  • Cytotoxic agents include any anti-tumor agent such as, for instance, Taxol, taxotere, DNA damaging agents (cis- platinum, etc), cytotoxic gene therapy and the like.
  • the cytotoxic treatment also includes radiotherapy.
  • the cytotoxic agent or treatment may be used following conventional protocols, before, after or together with the compound that inhibits Ki gene expression or activity.
  • compositions comprising a compound that inhibits Ki gene expression or activity and a cytotoxic agent, for simultaneous, separate or sequential use.
  • the composition may contain any pharmaceutically acceptable carrier or vehicle or excipient, such as saline buffer, isotonic solution, additives, stabilizers, etc.
  • polypeptides, genes or compositions may be used in different ways.
  • they may be contacted directly with the cells, in any appropriate device (tube, plate, dish, pouch, etc.).
  • they may be administered to a subject in need thereof, by various conventional routes such as local injection, systemic injection, intra-tumoral injection, intracardiac or pericardial (stereotaxic) injection, intra-muscular injection, percutaneous catheter-mediated delivery (in particular for arterial smooth muscle cells to block restenosis), etc.
  • routes, including route, dose and frequency may be adjusted by the clinician, depending on the situation.
  • 10 4 -10 13 viral particles may be injected per dose, generally between 10 6 -10 12 .
  • compositions such as pharmaceutical compositions, nucleic acid constructs, recombinant cells as well as methods of treating or preventing various pathological conditions where abnormal cell cycle regulation occurs, such as tumors, neuro-degeneration, muscular hyperproliferative disorders, fibrosis, arterial, stenosis, myocardial disorders, hepatitis, inflammatory diseases, etc.
  • pathological conditions where abnormal cell cycle regulation occurs, such as tumors, neuro-degeneration, muscular hyperproliferative disorders, fibrosis, arterial, stenosis, myocardial disorders, hepatitis, inflammatory diseases, etc.
  • COS-1 cells were obtained from ATCC (American Type Culture Collection) and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS).
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • COS-1 cell is a fibroblast-like cell line established from CV-1 simian cells which were transformed by an origin-defective mutant of SV40 which codes for wild-type antigen.
  • Tumoral H1299 and H460 cells derived from non small cell lung cancer were obtained from ATCC (American Type Culture Collection) and were grown in RPMI 1640 medium (Gibco BRL, Cergy Pontoise, France) supplemented with 10% fetal bovine serum and penicillin (lOOmM), streptomycin (100 ⁇ g/ ml), L-glutamin (2 mM) (Gibco BRL, Cergy Pontoise, France). Cells were grown at 37°C with 95% humidity and 5% C02.
  • the GST-Gax protein production was realized from plasmid pGEX-2T-hGax. Plasmids were transformed into BL21 (Amersham Pharmacia Biotech, England) for expression. The cells were grown at 37°C in LB medium with ampicillin. The cultures were grown to an A600 between 0.6 and 0.8 and then induced with 0.1 mM isop pyl-b-thiogalactoside (IPTG). Cells were sonicated and the purification of GST-GAX protein was realized on agarose-beads coupled to glutathion.
  • IPTG isop pyl-b-thiogalactoside
  • Ki-myc cloning was realized in pET-29 plasmid (Novagen) from PGAD (Clontech, Palo Alto, USA) contained in the yeast. PET-29 plasmid produces the protein fused to the S-tag epitope). Plasmids were transformed into BL2, the cultures were grown to an A600 between 0.6 and 0.8 and then induced with 0.1 mM isopropyl-b-thiogalactoside (IPTG) and T7 RNA polymerase induced by BL-21. Cells were sonicated and the purification of SmycKi protein was realized on agarose-beads coupled to S-protein.
  • IPTG isopropyl-b-thiogalactoside
  • Transfection Cos-1 cell transfections were performed in a 4 wells culture dish at a density of 10 000 cells in in DMEM supplemented with 10% fetal calf serum (FCS) and transfected.
  • HI 299 cell transfections were performed in 24 well (Falcon) at a density of 100 000 cells in DMEM supplemented with 10%> fetal bovine serum by using lipofectamine (Gibco BRL Cergy Pontoise, France) at a ratio 4 / 1 (lipofectamine/ADN). After a 4 h incubation in serum free medium, medium was changed to complete medium which contained 10% serum and cells were maintained in culture for 24 h to 72 h depending on the assay.
  • cell viability was measured using crystal violet: cells are fixed by formaldehyde 4%o (diluted in H2O) for 30 minutes at room temperature, Cristal Violet at .0.1% was then added for 20 minutes at room temperature. Two washes with H2O were necessary to remove extracellular Cristal Violet. Cells made soluble in sodium deoxycholate 2% preheated at 37°C. An incubation for 30 minutes was done under agitation before transferring 100 ⁇ L of soluble fraction in a 96 wells plate in order to measure D.O at 550 nm.
  • Ki counteracts gax and p53 mediated tumor cell death ( Figure 1)
  • Figure 1 This example demonstrates the effects of Ki antigen on cell viability, in the presence or absence of Gax.
  • Ki alone does not exhibit any toxic activity or growth- stimulatory activity in our experimental conditions, like the control plasmid.
  • transfection of a plasmid encoding Gax decreased cell viability by 35% to 40%.
  • gax and ki were simultaneously expressed, we did not observe cell death. Ki is thus able to counteract gax mediated cell death.
  • the p53 tumor suppressor gene induces apoptosis in a number of different cell types (Elledge and Lee 1995; White 1996).
  • Ki could affect p53-induced cell death.
  • Using the same conditions of transfection we observed that the transfection of p53 encoding plasmid leads to up to 50% decrease in cell viability.
  • p53 induced cell death was not observed in the presence of ki.
  • Ki counteracts gax-mediated fibroblast cell death (Figure 2)
  • test plasmid (Gax or Ki) were co-transfected with 1 ⁇ g of CMV-b-Gal expression plasmid using the Lipofectamine procedure (GobcoBrl) in a 1:6 ratio, for 2-4 hours. Cultures were serum stimulated for 48 hours and fixed in 4% neutral buffered formalin for 10 minutes. Plasmids pCDNA-bcl-2, pCDNA-bcl-x and pCDNA-bax were previously described. Plasmid pCMV-HA-KI comprises the HA-tagged Ki gene under the control of the CMV promoter. Values represent the mean and standard eror, and were compared for statistical significance by Anova analysis and student t-test (p ⁇ 0.05).

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Abstract

L'invention concerne en règle générale des compositions et des procédés pour réguler le cycle cellulaire, notamment l'apoptose. La présente invention concerne plus particulièrement des compositions et des procédés faisant appel à un polypeptide Ki ou à un gène Ki pour réguler le cycle cellulaire, notamment l'apoptose, dans des cellules, in vitro, ex vivo ou in vivo. Ladite invention porte également sur des compositions, des vecteurs, des cellules et des éléments similaires, qui peuvent être utilisés pour mettre en oeuvre les procédés susmentionnés. Cette invention peut être appliquée pour protéger des cellules de l'apoptose, ou bien pour induire ou promouvoir l'apoptose dans des cellules, dans différentes conditions physio-pathologiques.
PCT/EP2001/008096 2000-06-28 2001-06-22 Compositions et procedes pour reguler le cycle cellulaire WO2002000241A2 (fr)

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US9560984B2 (en) 2009-10-29 2017-02-07 The Nielsen Company (Us), Llc Analysis of controlled and automatic attention for introduction of stimulus material

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