WO2005073404A2 - Element fonctionnel de siah-1b sensible a p53 - Google Patents

Element fonctionnel de siah-1b sensible a p53 Download PDF

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WO2005073404A2
WO2005073404A2 PCT/IB2005/000392 IB2005000392W WO2005073404A2 WO 2005073404 A2 WO2005073404 A2 WO 2005073404A2 IB 2005000392 W IB2005000392 W IB 2005000392W WO 2005073404 A2 WO2005073404 A2 WO 2005073404A2
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siah
cell
compound
binding
cells
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WO2005073404A3 (fr
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Robert Amson
Adam Telerman
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Molecular Engines Laboratories
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • FILED OF THE INVENTION This invention relates to the functional element in siah-lb promotor, which directly interacts with the tumor suppressor p53. Methods of screening agents useful in treating, preventing and managing cancer or a neurodegenerative disorder are also encompassed.
  • the Drosophila SL A is a protein required for neuronal differentiation of the R7 photoreceptor cells in the eye of Drosophila melanogastex (1). It acts downstream of the Sevenless tyrosine kinase receptor to degrade the transcriptional repressor Tramtra (2, 3).
  • the Drosophila SINA protein and its mammalian Siah homologues are phylogenetically conserved E3 ligases, enzymes involved in ubiquitination and proteasome-mediated degradation of protein substrates. This function is due to their N-terminal RING-finger domain, which recruits ubiquitin conjugating E2 enzymes and promotes the ligation of ubiquitin to the substrate (4).
  • siah-1 and siah-2 are responsible for degradation of Kid, BOB/OBF1, synaptophysin, synphilin, TGF Beta-induced early gene and TRAF2 (5-10).
  • Siah-1 through the binding and downmodulation of Numb, is a positive regulator of Notch activity (11).
  • siah-1 and its mouse homologous siah-lb are induced by p53 during apoptosis and tumor reversion, it is unclear whether siah genes are directly interacting with p53.
  • determining whether siah genes are direct target of interaction by p53 is important in understanding the mechanism of tumor reversion and/or cell apoptosis, thereby allowing the screening and designing of an effective anti-tumor agents.
  • an effective agents for treating, preventing or managing a neurogenerative disorder can also be screened or designed using that piece of information.
  • this invention is directed to a DNA fragment comprising 2155 to 2103 nucleotides downstream from translational start site of siah-lb.
  • this invention encompasses a method of identifying a compound that reduces or inhibits cell apoptosis comprising: (a) contacting the compound with a DNA fragment comprising 2155 to 2103 nucleotides downstream from translational start site of siah-lb and p53; (b) determining whether binding between the DNA fragment and p53 is decreased; and if the binding between the DNA fragment and p53 is decreased, then, (c) contacting the compound with a cell; and (d) determining whether cell apoptosis is reduced as compared to a cell that has not been contacted with the compound.
  • this invention encompasses a method of treating, preventing or managing a neurodegenerative disorder comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of the compound identified using the screening method above.
  • the neurodegenerative disorders that can be treated, prevented or managed using the methods of this invention include, but are not limited to, Alzheimer's disease, Huntington's disease and Parkinson's disease.
  • this invention encompasses a method of identifying a compound that promotes cell apoptosis comprising: (a) contacting the compound with a DNA fragment comprising 2155 to 2103 nucleotides downstream from translational start site of siah-lb and 53; (b) determining whether binding between the DNA fragment and p53 is decreased; and if the binding between the DNA fragment and p53 is decreased, then, (c) contacting the compound with a cell; and (d) determining whether cell apoptosis is increased as compared to a cell that has not been contacted with the compound.
  • this invention encompasses a method of treating, preventing or managing cancer comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective arriount of the compound identified using the screening method described above.
  • this invention encompasses a method of diagnosing a cause of a disease or disorder associated with reduced Siah-1 expression comprising: (a) obtaining sequence information of2155 to 2103 nucleotides downstream from translational start site of siah-lb obtained from a patient; (b) obtaining sequence information of 2155 to 2103 nucleotides downstream from translational start site of siah-lb obtained from a normal subject; and (c) comparing the sequences obtained from steps (a) and (b), wherein if t ie sequence obtained from step (a) is not identical to the sequence obtained from step (b), the cause is a defect in binding of p53 to siah-lb or activation of siah-lb by ⁇ 53.
  • FIG. 1 illustrates activation of p53 induces increased siah-lb mRNA levels.
  • B Northern blots of mouse lriRNA hybridized with the siah-1 CDS (probe CDS), the siah-la probe (probe a) or the siah-lb probe (probe b).
  • Promoter activity was measured in luciferase assays with reporter plasmids comprising the region nt -2613/- 1694 of siah-lb or its three mutants (siah-lb mutl, siah-lb mut2 and siah-lb ⁇ ), following transient transfection into HI 299 cells.
  • the different constructs are schematically represented on the left panel and their corresponding promoter activity is shown on the right panel.
  • a p21 promoter fragment containing the distal p53RE was used as positive control and empty pGL3 vector as negative control. Luciferase activity is reported as fold activation, representing the ratio of the values standardized by protein concentration measured in presence or absence of p53.
  • Fig. 3 illustrates that p53 binds to the siah-lb promoter in vitro :
  • A EMSA was performed with a probe corresponding to the p53RE of the siah-lb promoter (nt -2160/-2098). This probe was incubated with recombinant p53 protein (lanes 2, 3, 4, 6 and 7) in absence (lanes 1, 2, 4 and 6) or in presence (lanes 3, 5 and 7) of the PAb421 antibody. In lanes 6 and 7, the probe contained a double mutation in the p53 consensus site (siah-lb mut3).
  • a pool of mouse IgGs was used as control for the PAb421 antibody specificity (lane 4) ; and (B) in vitro DNase I footprinting experiment with labeled probes designed to have the p53RE placed in their center.
  • Probes containing the p53RE of siah-lb (lanes 1, 2 and 3) or its mutants (lanes 4, 5 and 6 for siah-lb mutl and lanes 7, 8 and 9 for siah-lb mut2) were incubated with either 0.5 (lanes 2, 5 and 8) or 1 ⁇ g of purified p53 (lanes 3, 6 and 9) or with an irrelevant protein (lane 10) before digestion with DNase I.
  • Fig. 4 illustrates that p53 binds to siah-lb chromatin in vivo. Chromatin immunoprecipitation (ChIP) was performed following p53 activation in vivo, using antibodies against clathrin heavy chain (ip ctrl) or p53 (ip p53). PCR was performed with gene-specific primers. Total lysate was used as a control for PCR amplification (input). p53 binding was tested using siah-lb specific primers; p21 primers and ⁇ 2 microglobulin primers were used as positive and negative controls, respectively.
  • ChIP Chromatin immunoprecipitation
  • EMSA was performed with a probe containing the p53 responsive element of the siah-lb promoter (nt -2160/-2098) (lanes 1, 2, 3, 4, 7 and 8) was incubated with the p53 produced by INT (lanes 2, 3, 4, 5 and 6) in absence (lanes 1, 2, 4, 5 and 7) or in presence (lanes 3, 6 and 8) of the PAb421 antibody.
  • the effect of the double mutation in the p53 consensus sites (siah-lb mut3) is shown in lanes 5 and 6.
  • reticulocyte lysate alone was used as control of the antibody specificity (lane 4).
  • the supershift and a non specific gel retardation observed without p53 (INT control) are indicated by an arrow.
  • siah-1 was able to reorganize MCF7 cells in structures similar to those observed with normal breast cells (14, 15).
  • Siah-1 showed an increase in apoptosis and gave rise to significantly fewer tumors than the parental cells when injected into scid/scid mice (13, 14).
  • Siah-1 acts in a complex with Skpl, Ebl, SIP (Siah interacting protein), and adenomatous polyposis coli protein (pAPC) to facilitate, in a p53 -dependent manner, the degradation of beta-catenin thus inducing apoptosis and inhibiting cell proliferation and transformation (17, 18).
  • Pwl/Peg3 is another p53-inducible gene product that cooperates with Siah-1 in promoting cell death (19) whereas BAG-1, an anti-apoptotic protein, antagonizes ' the effect of Siah-1 on apoptosis (20).
  • the human siah-1 gene has two murine homologues, siah-la and siah-lb, which are widely expressed in various tissues of the embryo and adult (21).
  • the RNA sequences of siah-la and siah-lb are 95% homologues with each other, 90% with their human counterpart and 72% with Drosophila SINA.
  • Siah-la drives the degradation of c-myb, a proto-oncogene involved in cellular proliferation and apoptosis (22), and is necessary for progression past metaphase during meiosis I of spermatogenesis (23).
  • siah-lb by cDNA differential display as a gene induced by p53 in murine Ml myeloid leukemia cells (24).
  • p53 is a homotetrameric transcription factor that can activate or repress the transcription of a series of genes controlling cell cycle progression, apoptosis, DNA repair, and other types of stress response. These genes include p21, MDM2, cyclin G, BAX, noxa, puma, TSAP6 (26-32) and many others.
  • the p53 protein is normally short-lived and present at low levels, but in response to stress it accumulates in the nucleus where it binds to specific DNA sequences within chromatin (33).
  • the consensus p53 binding site is composed of two 10 bases half sites, each conforming to the sequence 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3' (where Pu and Py represent purines and pyrimidines, respectively) and separated by a spacer of 0-13 bp (34-37).
  • a computer algorithm developed to identify p53 binding sites in the human genome disclosed siah-1 as a possible downstream target of p53 (38).
  • siah-lb promoter contains a functional p53 responsive element (RE), which is able to bind p53 in vitro and in vivo.
  • RE functional p53 responsive element
  • siah-lb gene is a direct transcriptional target of p53.
  • the mouse siah-lb gene shown previously to be upregulated during p53-mediated apoptosis (24), contains a functional p53RE within its second intron. This p53RE enables the transactivation of the siah-lb gene by p53 in response to anti-cancer agents such as ActD.
  • siah-lb mRNA is increased as a, result of p53 activation, thus resolving previous ambiguities stemming from the use of non-specific probes (12, 16, 21, 24, 40). Basal levels of siah-lb mRNA are also present before p53 activation, suggesting that the protein has a role also in non-stressed cells. Moreover, siah-lb expression can also be induced by p53-independent mechanisms (13), although expression is maximal when p53 is activated.
  • Siah-lb is not involved in the p53 pathway (40). However, it is possible that in such cells p53 activates multiple downstream effectors. The role of Siah-lb could thus be substituted by proteins encoded by one or more of the other p53 -responsive genes that are transactivated simultaneously with siah-lb. Moreover, the apparent discrepancy between our data and those of Bowtell and co-workers (40) may also be due to different experimental conditions; it is conceivable that the contribution of Siah-lb to the p53 response may vary as a function of the cellular context.
  • this invention encompasses methods of treating, preventing and/or managing cancer comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of a compound identified using certain methods of this invention.
  • the terms “treat,” “treating” and “treatment” refer to the eradication or amelioration of a disease or condition, or of one or more symptoms associated with the disease or condition. In certain embodiments, the terms refer to minimizing the spread or worsening of the disease or condition resulting from the administration of one or more prophylactic or therapeutic agents to a subject with such a disease or condition.
  • the terms “prevent,” “preventing” and “prevention” refer to the prevention of the onset, recurrence or spread of a disease or condition, or of a symptom thereof.
  • “managing” and “management” refer to preventing or slowing the progression, spread or worsening of a disease or condition. Often, the beneficial effects that a subject derives from a prophylactic or therapeutic agent do not result in a cure of the disease or condition.
  • the term "managing” encompasses preventing the recurrence of cancer in a patient who had suffered from cancer, lengthening the time a patient who had suffered from cancer remains in remission, preventing the occurrence of cancer in patients at risk of suffering from cancer (e.g., patients who had been exposed to high amounts of radiation or carcinogenic materials, such as asbestos; patients infected with viruses associated with the occurrence of cancer, such as, but not limited to, HIN and Kaposi's sarcoma-associated herpes virus; and patients with genetic predispositions to cancer, such as those suffering from Downs syndrome), and preventing the occurrence of malignant cancer in patients suffering from pre-malignant or non- malignant cancers.
  • cancer e.g., patients who had been exposed to high amounts of radiation or carcinogenic materials, such as asbestos; patients infected with viruses associated with the occurrence of cancer, such as, but not limited to, HIN and Kaposi's sarcoma-associated herpes virus; and patients with genetic predis
  • a “therapeutically effective amount” of a compound is an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease or condition, or to delay or minimize one or more symptoms associated with the disease or condition.
  • a therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment or management of the disease or condition.
  • the term "therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.
  • a “prophylactically effective amount” of a compound is an amount sufficient to prevent a disease or condition, or prevent its recurrence.
  • a prophylactically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the disease.
  • prophylactically effective amount can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
  • cancer includes, but is not limited to, solid tumors and blood born tumors.
  • cancer refers to disease of skin tissues, organs, blood, and vessels, including, but not limited to, cancers of the bladder, bone or blood, brain, breast, cervix, chest, colon, endrometrium, esophagus, eye, head, kidney, liver, lymph nodes, lung, mouth, neck, ovaries, pancreas, prostate, rectum, stomach, testis, throat, and uterus.
  • Specific cancers include, but are not limited to, advanced malignancy, amyloidosis, neuroblastoma, meningioma, hemangiopericytoma, multiple brain metastases, glioblastoma multiforms, glioblastoma, brain stem glioma, poor prognosis malignant brain tumor, malignant glioma, recurrent malignant giolma, anaplastic astrocytoma, anaplastic oligodendroglioma, neuroendocrine tumor, rectal adenocarcinoma, Dukes C & D colorectal cancer, unresectable colorectal carcinoma, metastatic hepatocellular carcinoma, Kaposi's sarcoma, karotype acute myeloblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-Cell lymphoma, cutaneous B- Cell lymphoma, diffuse large B-Cell lymph
  • the cancer is metastatic.
  • the cancer is refractory or resistance to chemotherapy or radiation.
  • compounds of this invention can be administered, sequentially or simultaneously, with another anticancer agent. The administration may be via same route or different routes.
  • anti- cancer agents include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; celecoxib; chlorambucil; ciro
  • anti-cancer drugs include, but are not limited to: 20-epi-l,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; arnrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-
  • CaRest M3 CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cyto statin; dacliximab; decitabine; dehydrodidemnin B; deslore
  • N-substituted benzamides N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; oblimersen
  • a prophylactic or therapeutic dose of each active ingredient in the treatment, prevention and/or management of cancer will typically vary with the specific active ingredients, the severity and type of cancer, and the route of administration.
  • the dose, and perhaps the dose frequency may also vary according to age, body weight, response, and the past medical history of the patient. Suitable dosing regimens can be readily selected by those skilled in the art with due consideration of such factors by following, for example, dosages reported in the literature and recommended in the Physician 's Desk Reference® (54th ed., 2000).
  • the patient is an animal.
  • the patient is a mammal, specifically, a human.
  • this invention encompasses methods of treating, preventing and managing a neurodegenerative disorder comprising administering to a patient in need of such treatment, prevention and/or management a therapeutically or prophylactically effective amount of a compound identified certain methods of this invention.
  • neurodegenerative disorders that can be treated using methods of this invention include, but are not limited to, amylotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease and Parkinson's disease.
  • the neurodegenerative disorder that can be treated, prevented and/or managed using methods of this invention is Alzheimer's disease.
  • the patient is a mammal, in particular, a human.
  • compound of this invention may be administered, sequentially or simultaneously, with another agent useful in treating a neurodegenerative disorder.
  • the administration may be via same route or different routes.
  • other agents include, but are not limited to: acridine derivatives such as tacrine; amantadine; dopamine depleting agents such as tetrabenazine and reserpine; dopamine receptor antagonists such as bromocriptine and pergolide; metabolic precursors of dopamine such as levodopa; inhibitors of acetylcholinesterase (AChE) such as physostigmine; inhibitors of L-amino acid decarboxylase such as benserazide and carbidopa; muscarinic receptor antagonists such as benzotropine mesylate, diphenhydramine hydorchloride and trihexyphenidyl; precursors of acetylcholine synthesis such as choline chloride and phosphatidy
  • a prophylactic or therapeutic dose of each active ingredient in the treatment, prevention and/or management of a neurodegenerative disorder will typically vary with the specific active ingredients, the severity and type of disorder, and the route of administration.
  • the dose, and perhaps the dose frequency may also vary according to age, body weight, response, and the past medical history of the patient. Suitable dosing regimens can be readily selected by those skilled in the art with due consideration of such factors by following, for example, dosages reported in the literature and recommended in the Physician's
  • siah-lb promoter region (nt -2613/-1694) was cloned into the pGL3- enhancer vector (Promega).
  • the siah-lb p53 binding element was mutated (CATG to TATA) either within the first (siah-lb mutl) or within the second half site (siah-lb mut2), using the QuikChange Multi SiteDirected Mutagenesis kit (Stratagene).
  • the p53RE (nt -2160/-2098) was deleted in the siah-lb ⁇ with the following primers: Forward 5'- CTAAAATGGGTCTCAAGACCTCCCCTGAGA-3' and Reverse 5'- TCTCAGGGGAGGTCTTGAGACCCATTTTAG-3'.
  • the distal region of the p21 promoter (nt -7615/-7354 from ATG) containing the p53RE was cloned into the pGL3 enhancer vector using the primers Forward 5'- CTAGGTACCCCAGAGGATACCTTGCAAGGCTGCA-3' and Reverse 5'- TATAGATCTTCTCTGTCTCCATTCATGCTCCTCC-3'.
  • the mouse p53 coding sequence (CDS) was cloned in a pCMN vector. 6.1.3 Proteins and Antibodies The human p53 protein was purified from insect cells infected with an appropriate recombinant baculovirus. The following antibodies were used: anti- p53 FL-393 (Santa Cruz Biotechnologies) and PAb421 (Oncogene); anti-clathrin heavy chain H-300 (Santa Cruz Biotechnologies); anti alpha-tubulin B-5-1-2
  • siah-1 Forward 5'-CCCCTTGTGAGTCAACACATAGTGCTGC-3'
  • siah-1 Reverse 5'- TGGGGCGACAGTTGCTACAAACAAG-3'.
  • Luciferase Assays 120,000 cells were transfected with 300 ng of the different siah-lb promoter constructs (see Cloning and Mutagenesis) and 300 ng of pCMN vector, either empty or expressing mouse p53. Transfection was performed using LipofectAMI ⁇ E PLUS reagent (Life Technologies Inc.) according to the manufacturer's recommendations. 24 hr after transfection the cells were washed and cell extracts prepared using a reporter lysis reagent (25 mM Tris, 8 mM MgCl 2 , 1 mM DTT, 1% Triton X-100 and 15% glycerol).
  • a reporter lysis reagent 25 mM Tris, 8 mM MgCl 2 , 1 mM DTT, 1% Triton X-100 and 15% glycerol.
  • luciferase activity was measured using the Victor Luminometer (Perkin Elmer) after addition of 20 nmol Luciferin (Roche) and 100 nmol ATP (Sigma). 6.1.7 Electrophoretic Mobility Shift Assay (EMSA Synthetic oligonucleotides containing the p53RE of siah-lb, either wild type
  • TCTCAAGATATATCCAGACCTCCCCTGATCACATTCAAAAGGGTCTCA AGATATATCCAGACC-3 ') were radiolabeled at their 5' end using T4
  • the 410 bp labeled PCR product containing the p53RE in its center was used in DNase I footprinting experiments following the Sure Core Footprinting protocol (Promega) with minor modifications.
  • 500 ng or 1 ⁇ g of p53 or 1 ⁇ g of irrelevant protein were mixed with 500 ng of PAb421 antibody.
  • 10 ng (30000 c.p.m.) of 5 '-end-labeled DNA was added and incubation carried on for another 30 min on ice.
  • concentration 5 mM CaCl 2 and 10 mM MgCl 2; samples were digested with DNase I (0.15 U) for 1 min at RT and resolved on a sequencing gel.
  • DNA sequencing reactions were done using the Sequenase Version 2.0 DNA Sequencing Kit (USB). 6.1.9 Chromatin immunoprecipitation and Western blot analysis Experiments were performed with the Chromatin Immunoprecipitation
  • TCTCTGTCTCCATTCATGCTCCTCC-3' Specificity of the primers was checked by Blast analysis using the Ensembl mouse genomic database. PCR reactions with the different primers gave rise to a single specific product of the expected size. The linear range for each primer pair was determined empirically, using increasing amounts of LTR6 genomic DNA. PCR products were resolved on 2% agarose gels. Whole cell extracts were generated using standard conditions. Extracts containing 20 ⁇ g total protein were subjected to Western blot analysis using the FL-393 and B 5-1-2 antibodies.
  • this probe hybridizes to two transcripts, corresponding to sizes of 2.3 and 1.9 kb, respectively (Fig. IB), i order to study more specifically siah-lb transcription, we designed a probe in the 5' UTR (ace. n. Z19580) of its mRNA (nt 1/230) (Fig. 1A). the same manner we designed a probe specific for siah-la (ace. n. Z19579) in the 3'UTR of its mRNA (nt 1676/1934) (Fig. 1A).
  • the siah-la probe (probe a) recognizes the 2.3 kb band
  • the siah-lb probe (probe b) recognizes only the 1.9 kb band (Fig. IB).
  • the band at 2.3 kb includes both siah-la and siah-lb transcripts.
  • the expected size of the siah-lb transcript is 1.7 kb.
  • siah-lb specific probe recognizes a band of bigger size
  • the 3' RACE PCR confirmed the published sequences, but the preliminary 5' RACE analysis revealed a possible alternative splicing of the siah-lb first exon.
  • the two different first exons described by Delia et al. (21) or sequenced by the I.M.A.G.E consortium (ace. n. BC052887) each represent 1/7 of the clones analyzed by us. The remaining 5/7 of the clones were found to contain a first exon of 231 bp, which so far has not been described (ace. n. AY495086).
  • siah-lb is ap53-inducible gene.
  • Activated p53 can trigger different effector pathways, depending on cell type and mode of activation.
  • NIH3T3 another cell line, NIH3T3, to treatment with the transcription inhibitor Actinomycin D, a well-documented activator of the p53 pathway (41, 42) .
  • Actinomycin D a well-documented activator of the p53 pathway (41, 42) .
  • Fig. ID this also resulted in a marked increase in siah-lb mRNA.
  • This DNA element consists of two identical half sites: 5'-AGACATGTCC-3'.
  • the distance between the two half sites is unusual: while the consensus specifies a spacer of 0-13 bp (33), the two half sites found in siah-lb are 33 bp apart.
  • the majority of the physiologically relevant ones have no spacer at all (34, 38). It was therefore of particular interest to test the interaction of p53 with this putative binding site.
  • Luciferase activity in the presence of p53 was normalized for the activity in the absence of p53, to obtain a fold of activation. While the empty luciferase vector (negative control) was not affected by the presence of p53, inclusion of siah-lb sequences led to an 8 fold increase in luciferase activity when p53 was present (Fig. 2).
  • Fig. 2 To verify that the positive effect of p53 on the transcriptional activity of the reporter plasmid is mediated by the consensus site identified within the siah-lb gene, we mutated nucleotides within this site that are known to be necessary for direct binding of p53. Mutation of CATG to TATA in the first half site (siah-lb mutl, Fig.
  • siah-lb and p21 chromatin were specifically immunoprecipitated with anti-p53 antibodies only from LTR6 cells maintained at 32°C; hence, like the corresponding elements in the p21 gene promoter, the p53RE within the siah-lb intron is indeed occupied by wild type p53 within living cells.
  • the in vivo binding of p53 to the siah-lb genomic DNA was due to a p53 overexpression artefact
  • we induced activation of endogenous p53 in NIH3T3 cells by treatment with Actinomycin D which led to a substantial increase in the steady state levels of p53 (Fig. 4B).

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Abstract

L'invention concerne un élément fonctionnel de siah-1b qui interagit directement avec p53, ainsi que des procédés de criblage de composés, utiles pour traiter, prévenir ou gérer le cancer ou une maladie neurodégénérative, sur la base de l'interaction entre p53 et l'élément fonctionnel. La présente invention porte également sur des méthodes pour traiter, prévenir ou gérer un cancer ou une maladie neurodégénérative au moyen de ces composés.
PCT/IB2005/000392 2004-01-29 2005-01-28 Element fonctionnel de siah-1b sensible a p53 WO2005073404A2 (fr)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2002002819A1 (fr) * 2000-06-30 2002-01-10 University Of Utah Research Foundation Procede de selection de traitements chimiotherapeutiques du cancer

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002002819A1 (fr) * 2000-06-30 2002-01-10 University Of Utah Research Foundation Procede de selection de traitements chimiotherapeutiques du cancer

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
AMSON R B ET AL: "ISOLATION OF 10 DIFFERENTIALLY EXPRESSED CNDAS IN P53-INDUCED APOPTOSIS: ACTIVATION OF THE VERTEBRATE HOMOLOGUE OF THE DROSOPHILA SEVEN IN ABSENTIA GENE" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 93, no. 9, 30 April 1996 (1996-04-30), pages 3953-3957, XP002032914 ISSN: 0027-8424 *
DATABASE ENSEMBLE [Online] XP002335757 retrieved from ENSEMBLE.ORG Database accession no. ENSMUSG00000040749 *
DELLA N G ET AL: "ISOLATION AND CHARACTERISATION OF MURINE HOMOLOGUES OF THE DROSOPHILA SEVEN IN ABSENTIA GENE (SINA)" DEVELOPMENT, COMPANY OF BIOLOGISTS, CAMBRIDGE,, GB, vol. 117, 1993, pages 1333-1343, XP000601972 ISSN: 0950-1991 *
FIUCCI GIUSY ET AL: "Siah-1b is a direct transcriptional target of p53: Identification of the functional p53 responsive element in the siah-1b promoter." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 101, no. 10, 9 March 2004 (2004-03-09), pages 3510-3515, XP002335752 ISSN: 0027-8424 *
MAEDA A ET AL: "The characterization of the human Siah-1 promoter<1>" FEBS LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 512, no. 1-3, 13 February 2002 (2002-02-13), pages 223-226, XP004341382 ISSN: 0014-5793 cited in the application *

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