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WO2009029054A1 - Gène(s) isoforme(s) p53 et utilisations de celui-ci (ceux-ci) - Google Patents

Gène(s) isoforme(s) p53 et utilisations de celui-ci (ceux-ci) Download PDF

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Publication number
WO2009029054A1
WO2009029054A1 PCT/SG2008/000321 SG2008000321W WO2009029054A1 WO 2009029054 A1 WO2009029054 A1 WO 2009029054A1 SG 2008000321 W SG2008000321 W SG 2008000321W WO 2009029054 A1 WO2009029054 A1 WO 2009029054A1
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Prior art keywords
cancer
gene
expression
activity
nucleic acid
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Jinrong Peng
Jun Chen
David P. Lane
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Agency for Science Technology and Research Singapore
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    • G01N33/5758—
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • 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
    • 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
    • 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
    • C12Q2600/118—Prognosis of disease development
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4748—Details p53
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to p53 isoform gene(s) and uses thereof, wherein the gene(s) are capable of modulating the expression and/or activity of p53.
  • the gene(s) are capable of modulating the expression and/or activity of p53.
  • the ⁇ 133p53 and/or ⁇ 113p53 gene or transcript thereof or a portion thereof are provided.
  • p53 is a tumour suppressor gene. Activation of p53, either at the transcriptional level or protein modification level, induces expression of many genes that form a network to regulate cell cycle or cell apoptosis. This network initiated from p53 is essential to eliminate abnormal cells (especially cancer-predisposing cells) and maintain normal cells. Thus, p53 is a crucial factor in suppressing tumorigenesis. Enhancing p53 activity in tumour cells is thought to be an efficient way to kill cancer cells. However, practically, in many cases enhancing p53 activity is not correlated to cancer cell death and the reason is unknown.
  • ⁇ 133p53 might act as a dominant negative regulator of p53 since co-transfection p53 with ⁇ 133p53 impaired p53-induced cell apoptosis (Bourdon et al., 2005).
  • the present invention addresses the problems above and in particular provides new uses for p53 isoform gene(s).
  • a method of treating and/or preventing cancer comprising reducing and/or inhibiting the expression of at least one p53 isoform gene and/or transcript thereof in at least one cancer cell, wherein the isoform gene and/or transcript thereof modulates the expression and/or activity of p53.
  • a method comprising reducing and/or inhibiting the expression and/or activity of the ⁇ 133p53 or ⁇ 113p53 gene and/or transcript thereof in at least one cancer cell.
  • the cancer may be selected from the group consisting of: bone cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, gastrointestinal cancer, leukaemia, lung cancer, lymphoma, melanoma, neuroblastoma, ovarian cancer, pancreatic cancer, prostrate cancer, renal cancer, retinoblastoma, small-cell lung cancer, non- small cell lung cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, astrocytoma, glioblastoma cancer, head cancer, and hepatocarcinoma.
  • the method for treating and/or preventing cancer may further comprise administering at least one compound capable of binding to the p53 isoform gene, mRNA, promoter and/or to a portion thereof and/or a portion thereof, thereby reducing and/or inhibiting the expression and/or activity of the p53 isoform gene and/transcript thereof, wherein the at least one compound may be capable of binding to the ⁇ 133p53 or ⁇ 113p53 gene, ⁇ 133p53 or ⁇ 113p53 mRNA, ⁇ 133p53 or ⁇ 113p53 promoter and/or to a portion thereof, thereby reducing and/or inhibiting the expression and/or activity of ⁇ 133p53 or ⁇ 113p53 gene and/or transcript.
  • the compound may be at least one nucleic acid molecule comprising a nucleotide sequence complementary to and/or which hybridises to the p53 isoform gene, mRNA, promoter and/or to a portion thereof. More in particular the at least one nucleic acid molecule may comprise a nucleotide sequence complementary to and/or which hybridises to the ⁇ 133p53 or ⁇ 113p53 gene, ⁇ 133p53 or ⁇ 113p53 mRNA, ⁇ 133p53 or ⁇ 113p53 promoter and/or to a portion thereof.
  • the nucleic acid molecule may be an antisense DNA and/or RNA molecule.
  • the nucleic acid molecule may be antisense single strand RNA (sRNA), double strand RNA (dsRNA), double strand DNA (dsDNA), double strand hybrid RNA/DNA (RNA/DNA), small interfering RNA (siRNA), micro RNA (miRNA), morpholinos (MO/PMO) and/or ribozymes.
  • sRNA single strand RNA
  • dsRNA double strand RNA
  • dsDNA double strand DNA
  • RNA/DNA double strand hybrid RNA/DNA
  • small interfering RNA siRNA
  • miRNA micro RNA
  • MO/PMO morpholinos
  • ribozymes ribozymes.
  • the nucleic acid molecule is morpholino (MO/PMO).
  • the nucleic acid molecule may further comprise a nucleotide sequence which is complementary to and/or which hybridizes to a 4113bp 5'-upstream region, or portion thereof, of the transcription start site of the
  • the nucleic acid molecule may comprise a nucleotide sequence which is complementary to and/or which hybridizes to a 5'-upstream region - 1041 to -1991bp or portion thereof of the translation start site of the p53 isoform gene. More in particular, the nucleic acid molecule may comprise a nucleotide sequence which is complementary to and/or which hybridizes to a 5'-upstream region -1 to -239bp or portion thereof of the transcription start site of the p53 isoform gene.
  • nucleic acid molecule comprises a nucleotide sequence complementary to and/or which hybridizes to a p53 isoform gene, mRNA, promoter, and/or a portion thereof, wherein the p53 isoform gene modulates the expression and/or activity of p53.
  • an isolated nucleic acid molecule wherein the isoform gene may be ⁇ 133p53 or ⁇ 113p53 gene and the nucleic acid molecule may comprise a nucleotide sequence complementary to and/or which hybridises to ⁇ 133p53 or ⁇ 113p53 gene, ⁇ 133p53 or ⁇ 113p53 mRNA, ⁇ 133p53 or ⁇ 113p53 promoter and/or to a portion thereof. More in particular, the isolated nucleic acid molecule(s) may be for use in medicine for treating and/or preventing cancer.
  • the isolated nucleic acid molecule may be used in the preparation of a medicament for use in therapy for reducing and/or inhibiting the expression and/or activity of the p53 isoform gene and/or transcript thereof (in particular, for reducing and/or inhibiting the expression and/or activity of ⁇ 133p53 or ⁇ 113p53 gene and/or transcript thereof) in at least one cancer cell.
  • composition comprising at least one nucleic acid molecule described above, optionally in the presence of at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.
  • a method of screening and/or identifying at least one drug candidate capable of reducing and/or inhibiting the expression of a p53 isoform and/or transcript in at least one cancer cell comprising: (a) administering at least one drug candidate to the at least one cancer cell, and (b) determining the ability of the drug candidate to reduce and/or inhibit the expression and/or activity of the p53 isoform gene and/or transcript in the cell, wherein the p53 isoform gene is capable modulating the expression and/or activity of p53.
  • kits for reducing and/or inhibiting the expression and/or activity of a p53 isoform gene and/or transcript comprising at least one compound which binds to the p53 isoform gene, mRNA, promoter and/or to a portion thereof, thereby reducing and/or inhibiting the expression and/or activity of the p53 isoform gene and/or transcript in at least one cancer cell, and wherein the p53 isoform gene is capable modulating the expression and/or activity of p53.
  • the kit may be for reducing and/or inhibiting the expression and/or activity of a p53 isoform gene and/or transcript thereof wherein the p53 isoform gene is ⁇ 133p53 or ⁇ 113p53.
  • a method of detecting the expression and/or activity of a p53 isoform gene in at least one cancer cell wherein the detecting is carried out before and/or after treating the cancer cell with chemotherapy and/or radiotherapy, and wherein the p53 isoform gene is capable of modulating the expression and/or activity of p53,
  • a method of diagnosis and/or prognosis of cancer in a subject comprising: a) providing at least one sample from a subject; b) quantitating the expression and/or activity of a p53 isoform gene, wherein the p53 isoform gene is capable of modulating the expression and/or activity of p53; and c) comparing the expression and/or activity of the p53 isoform gene, wherein increase and/or up-regulation in the p53 isoform gene, compared to the at least one control, is indicative of presence of, predisposition to cancer.
  • a diagnostic and/or prognostic kit for the diagnosis and/or prognosis of cancer in a subject comprising at least one nucleic acid molecule comprising a nucleotide sequence complementary to and/or hybridisable to a p53 isoform gene, mRNA, promoter and/or portion thereof, and wherein the p53 isoform gene is capable of modulating the expression and/or activity of p53, wherein the kit comprises at least one nucleic acid molecule comprising a nucleotide sequence complementary to and/or hybridisable to the p53 isoform gene (in particular, the nucleotide sequence is complementary to and/or hybridisable to ⁇ 133p53 or ⁇ 113p53 gene), mRNA, promoter and/or portion thereof.
  • a method of determining the susceptibility of a patient to cancer treatment comprising: a) providing at least one sample from the patient before and after chemotherapy and/or radiotherapy; b) quantitating the expression and/or activity of a p53 isoform gene before and after chemotherapy and/or radiotherapy, wherein the p53 isoform gene is capable of modulating the expression and/or activity of p53; and c) comparing the expression and/or activity of the p53 isoform gene before and after chemotherapy and/or radiotherapy, wherein decrease and/or down-regulation in the p53 isoform gene following chemotherapy and/or radiotherapy, is indicative of susceptibility of the patient to chemotherapy and/or radiotherapy.
  • the isolated cancer cell may be present in the form of cell culture.
  • the cell may further be a mammalian cell.
  • the cell may be a human cell.
  • the cell may further be implanted in a non- human animal model.
  • Figure 1 Diagram showing the genomic structure of the zebrafish p53 gene and the relative position of the 4.113 kb genomic DNA fragment cloned for the ⁇ 113p53 promoter activity analyses.
  • the zebrafish p53 gene has 10 exons (light blue or grey box) and 9 introns (black lines linking boxes).
  • the start codon ATG of p53 is located in the second exon.
  • the lengths of intron 1-4 of p53 are 637 bp, 240 bp, 92 bp and 2692, respectively, as shown in the diagram.
  • ⁇ 113p53 Transcription of ⁇ 113p53 starts in the intron 4 (dark blue/grey box ES) and the mature ⁇ 113p53 transcript contains 155 bp intron 4 sequence joined to the exon 5 of p53 after splicing an intron of length 842 bp (intron 1 for ⁇ 113p53).
  • the start codon ATG of ⁇ 113p53 is located in the exon 5 of p53.
  • the 4.113 kb genomic DNA fragment cloned is immediately upstream of the ⁇ 113p53 start codon ATG and ends in the exon 1 of p53, thus it excludes the p53 promoter sequences.
  • the 4.113 kb DNA fragment was cloned into the pEGFP vector to generate the ⁇ 113p53:gfp plasmid.
  • FIG. 1 Generation of Tg( ⁇ 113p53:gfp) transgenic fish.
  • A Plasmid DNA injection induced ⁇ 113p53 expression in the injected WT zebrafish embryos. Uninjected and buffer-injected embryos were used as the controls.
  • B Gfp fluorescence observed in the embryos injected with the ⁇ 113p53::gfp plasmid..
  • C To generate the Tg( ⁇ 113p53:gfp) transgenic fish, the ⁇ 113p53:gfp plasmid was linearized and injected into single-cell stage embryos and individual fish were screened based on gfp fluorescence as described in the 'Materials and methods' section.
  • the homozygous Tg( ⁇ 113p53:gfp) transgenic fish were mated with def 11429 heterozygous fish and their F2 progenies were examined for Gfp fluorescence and genotyped for def 1 ' 429 mutation.
  • Gfp expression was weakly expressed in WT ⁇ def+/+) and the heterozygotes (def+/-) siblings (sb) whilst the del 111429 homozygous embryos (def-/-) had strong and ubiquitous Gfp expression in the head region.
  • the start position of intron 4 of p53 is also highlighted (-1692).
  • the nucleotide position +1054 is immediately 5'- upstream of the start codon ATG of ⁇ 113p53.
  • Total nine deletion plasmids (P1 to P9) were constructed, including five plasmids with 5'-deletions (P1 to P5), two with 3'-deletions (P7 and P8) and two with internal deletions (P6 and P9).
  • FIG. 4 Promoter activity test for each ⁇ 113p53:gfp deletion plasmid.
  • A PO- P9 plasmid DNAs were injected into one-cell stage WT embryos and Gfp fluorescence in each test was visualized under a fluorescence microscope at 24 hours post-injection.
  • B The levels of gfp transcripts in each test were examined via RNA gel blot hybridization using a gfp specific probe (top panel).
  • the 18S rRNA was used as the loading control (second panel).
  • the levels of the endogenous ⁇ 113p53 expression in the same samples were examined via semi-quantitative RT-PCR.
  • the elongation factor a gene (elfa) was used as the control for RT-PCR.
  • WT uninjected wild type control.
  • FIG. 5 p53 directly regulates the ⁇ 113p53 expression.
  • A PO construct as shown in Figure 3 with addition of showing the three predicted p53-binding sites in the regulatory regions I and Il in the ⁇ 113p53 promoter. Site 1 :,
  • GGGCATGTTC SEQ ID NO: 24
  • site 2 TGACATGTTA (SEQ ID NO: 25); site
  • primer pairs for exon 10 (+1264 to +1480 in p53 cDNA with Accession Number AF365873) amplified a product only from the input sample (lower panel) but not the ChIP sample (upper panel) whilst both primer pairs for region -112 to +98 and region -1086 to -1300 yielded size-predicted products from the ChIP product.
  • ⁇ 113p53 directly interacts with p53.
  • A HA-tagged p53 (left) and MYC-tagged ⁇ 113p53 (right) cloned into the expression vector pCS2+, respectively.
  • B HA-p53 and MYC- ⁇ 113p53 mRNAs were injected alone or co- injected into single-cell stage embryos and total proteins were extracted from embryos at 5 hours post-injection.
  • Immunoprecipitation (IP) with anti-HA antibody precipitated HA-p53 and the IP products were detected for MYC- ⁇ 113p53 using an anti-MYC antibody.
  • the ⁇ fVC- ⁇ 113p53 protein was detected only in the IP products from embryos co-injected with HA-p53 and ⁇ 113p53 mRNAs (top two panels). HA-p53 and MYC- ⁇ 113p53 proteins were expressed in the injected embryos (input, bottom two panels).
  • Figure 7 The expression of ⁇ 113p53 is induced by ⁇ -ray irradiation and drug treatments.
  • A RNA gel blot hybridization using a p53 probe (top panel) or a
  • ⁇ 113p53 specific probe (middle panel) showing that ⁇ 113p53 expression was greatly induced in the embryos treated with either ⁇ -ray or carcinogenic drugs camptothecin and rostovitine. 18S rRNA was used as the loading control. (B, C)
  • lane 1 uninjected control; lane 2, ⁇ 113p53:gfp plasmid injection; lane 3, ⁇ 113p53:gfp plasmid + ⁇ 113p53-MO co-injection.
  • C Injection of ⁇ 113p53-MO sensitized the WT embryos to ⁇ -ray irradiation treatment and caused 100% mortality to the treated embryos (bottom right panel) at 5 days post-treatment.
  • the standard control morpholino injected embryos though somehow deformed, exhibited -50% surviving rate after the ⁇ -ray irradiation treatment (top right panel) at 5 days post-treatment.
  • the morpholino-injected ⁇ -ray untreated controls are shown on the left.
  • FIG. 9 Knock-down ⁇ 113p53 caused massive cell apoptosis. Embryos 18 hpf post- ⁇ -ray-treatment as in Figure 8 were used in TUNEL assay to detect cell apoptosis in each case.
  • the present invention provides to p53 isoforms, capable of being employed in diagnostic, therapeutic, and/or prognostic applications.
  • p53 isoforms refers to alternatively spliced products transcribed from alternative promoter in the p53 gene.
  • the p53 gene has a complex transcriptional expression pattern encoding different p53 variants, through the use of alternative splicing and the existence of an internal promoter in intron 4 (Bourdon et al, 2005).
  • This alternative promoter leads to the expression of an N-terminally truncated p53 protein initiated at codon 133 in humans, therefore named ⁇ 133p53.
  • FIG. 1 shows the genomic structure of the zebrafish p53 gene.
  • the inventors have shown that ⁇ 113p53 can form a complex with p53. More importantly, while most of the wild type control fish still could survive from ⁇ -ray irradiation treatment knock-down of ⁇ 113p53 in irradiation-treated fish caused -100% mortality. These results demonstrate that ⁇ 113p53 and/or the human homolog ⁇ 133p53 acts as dominant negative regulators of p53 to protect cells from p53-induced and/or other factor-induced cell death.
  • the present invention has immense implications in cancer treatment since the knock- down of ⁇ 133p53 and/or ⁇ 133p53 prevents complex formation with p53 thus facilitating p53 induced apoptosis. Therefore, cancer treatment coupled with knock-down of ⁇ 133p53 by appropriate means will become more efficient due to increased cancer cell death.
  • the ⁇ 113p53 and/or the human homolog ⁇ 133p53 can be used interchangeably and hereafter referred to as ⁇ 113p53 or ⁇ 133p53.
  • the present invention provides to method of treating and/or preventing cancer comprising reducing and/or inhibiting the expression and/or activity of at least one p53 isoform gene and/or transcript thereof in at least one cancer cell, wherein the isoform gene and/or transcript thereof modulates the expression and/or activity of p53.
  • the isoform gene may be ⁇ 133p53 or ⁇ 113p53 gene and accordingly the method comprises reducing and/or inhibiting the expression and/or activity of the ⁇ 133p53 or ⁇ 113p53 gene and/or transcript thereof in at least one cancer cell.
  • the present invention is not limited to ⁇ 133p53 or ⁇ 113p53 but encompasses any homolog or ortholog of ⁇ 133p53 and/or ⁇ 113p53.
  • the method of treating and/or preventing cancer may further comprise reduction and/or inhibition of the p53 isoform gene and/or transcript preventing the down-regulation of p53 in at least one cancer cell.
  • the method of reducing and/or inhibiting the expression and/or activity p53 isoform gene and/or transcript may induce and/or sensitize at least one cancer cell to apoptosis.
  • up-regulation means that the expression and/or activity of particular gene and/or mRNA transcript or the expression of a particular protein is higher or increased compared to the basal level of expression constitutively present in a cell in the absence of any stimulation.
  • down-regulation would refer to the lower or decreased expression and/or activity of particular gene and/or mRNA transcript or of a particular protein compared to the basal level of expression constitutively present in a cell in the absence of any stimulation. Using established techniques known in the art this up- or down- regulation may be quantitated.
  • the method, comprising treating and/or preventing cancer in at least one cell may be in the presence of chemotherapy and/or radiotherapy.
  • “Chemotherapy” refers to the use of chemical substances primarily to cytotoxic drugs used to treat cancer.
  • Radiotherapy refers to the medical use of ionizing radiation as part of cancer treatment to control malignant cells.
  • Chemotherapy and radiotherapy are known in the art as the conventional or mainstream therapies. However the method of the current invention can also be used along with alternative and/or complementary therapies used to relieve the pain and side effects of the conventional therapies.
  • the method of treating and/or preventing cancer in at least one cell may be in vitro or in vivo.
  • the cancer may mammal cancer, in particular human cancer.
  • the cancer may be selected from the group consisting of: bone cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, gastrointestinal cancer, leukaemia, lung cancer, lymphoma, melanoma, neuroblastoma, ovarian cancer, pancreatic cancer, prostrate cancer, renal cancer, retinoblastoma, small-cell lung cancer, non-small cell lung cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, astrocytoma, glioblastoma cancer, head cancer, and hepatocarcinoma.
  • cancer refers to the uncontrolled growth of the unhealthy cells that crowds out and destroys healthy cells.
  • the method is for the treating and/or preventing cancer in a subject in need of the treatment.
  • a "subject” may be a patient suffering from cancer.
  • a person skilled in the art will know how to select subjects based on their amenability to a particular treatment, or their susceptibility to disease.
  • the method therefore comprises administering in vivo or in vitro to cancer cells administering at least one compound capable of binding to the p53 isoform gene, mRNA, promoter and/or to a portion thereof and/or a portion thereof, thereby reducing and/or inhibiting the expression and/or activity of the p53 isoform gene and/transcript thereof.
  • inhibitor and/or reducing is herein understood to mean any drug, compound, substance or nucleic acid capable of binding, interacting or hybridizing with the p53 isoform and inhibiting, reducing or substantially inhibiting or reducing the expression and/or activity of the p53 isoform in the cells. For example, by blocking or degrading the p53 isoform gene and/or transcript.
  • Drugs, compounds or substances capable of inhibiting or reducing the activity of p53 isoform can be selected among the drugs, compounds and substances known in the art or can be newly discovered or synthesised.
  • the inhibition and/or reduction of the expression and/or activity of p53 isoform gene and/or transcript can be carried out according to any suitable technology known in the art. The screening, selection of these types of drugs, compounds or substances will be described later.
  • the method comprises administering at least one compound which hybridizes to the p53 isoform, thereby inhibiting and/or reducing the expression and/or activity of the p53 isoform.
  • the method comprises administering a compound that is at least one nucleic acid molecule complementary to or which hybridises to the p53 isoform, thereby inhibiting and/or reducing the expression and/or activity of p53 isoform.
  • the method comprises administering at least one compound capable of binding to the ⁇ 133p53 or ⁇ 113p53 gene, ⁇ 133p53 or ⁇ 113p53 mRNA, ⁇ 133p53 or ⁇ 113p53 promoter and/or to a portion thereof, thereby reducing and/or inhibiting the expression and/or activity of ⁇ 133p53 or ⁇ 113p53 gene and/or transcript.
  • the nucleic acid molecule is an anti-sense RNA molecule.
  • the nucleic acid molecule may be an antisense DNA and/or RNA molecule.
  • the antisense molecule may be an anti-miRNA oligonucleotide (AMO), antagomir, locked-nucleic-acid anti-sense oligonucleotide, single strand RNA (sRNA), double strand RNA (dsRNA), double strand DNA (dsDNA), double strand hybrid RNA/DNA (RNA/DNA), small interfering RNA (siRNA), morpholinos (MO/PMO) and/or ribozymes.
  • AMO anti-miRNA oligonucleotide
  • dsRNA double strand RNA
  • dsDNA double strand DNA
  • RNA/DNA double strand hybrid RNA/DNA
  • siRNA small interfering RNA
  • MO/PMO morpholinos
  • ribozymes ribozymes.
  • the nucleic acid molecule may be morpholinos (MO/
  • the compound is at least one nucleic acid molecule comprising a nucleotide sequence complementary to and/or which hybridises to the p53 isoform gene, mRNA, promoter and/or to a portion thereof.
  • the at least one nucleic acid molecule may comprise a nucleotide sequence complementary to and/or which hybridises to the ⁇ 133p53 or ⁇ 113p53 gene, ⁇ 133p53 or ⁇ 113p53 mRNA, ⁇ 133p53 or ⁇ 113p53 promoter and/or to a portion thereof.
  • the nucleic acid molecule may further comprise a nucleotide sequence which is complementary to and/or which hybridizes to a 4113bp 5'-upstream region, or portion thereof, of the translation start site of the p53 isoform gene.
  • the nucleic acid molecule may comprise a nucleotide sequence which is complementary to and/or which hybridizes to a 5'-upstream region -1041 to - 1991 bp or portion thereof of the transcription start site of the p53 isoform gene.
  • nucleic acid molecule may comprise a nucleotide sequence which is complementary to and/or which hybridizes to a 5'-upstream region -1 to -239bp or portion thereof of the transcription start site of the p53 isoform gene.
  • nucleic acid molecule comprises a nucleotide sequence complementary to and/or which hybridizes to a p53 isoform gene, mRNA, promoter, and/or a portion thereof, wherein the p53 isoform gene modulates the expression and/or activity of p53.
  • the isolated nucleic acid molecule may be ⁇ 133p53 or ⁇ 113p53 gene and the nucleic acid molecule may comprise a nucleotide sequence complementary to and/or which hybridises to ⁇ 133p53 or ⁇ 113p53 gene, ⁇ 133p53 or ⁇ 113p53 mRNA, ⁇ 133p53 or ⁇ 113p53 promoter and/or to a portion thereof.
  • the isolated nucleic acid molecule may comprise a nucleotide sequence which is complementary to and/or which hybridizes to a 5'-upstream region -1041 to - 1991 bp or portion thereof of the transcription start site of the p53 isoform gene.
  • the isolated nucleic acid molecule may further comprise a nucleotide sequence which is complementary to and/or which hybridizes to a 5'- upstream region -1 to -239bp or portion thereof of the transcription start site of the p53 isoform gene.
  • the isolated nucleic acid may be for use in medicine for treating and/or preventing cancer. Further the at least one nucleic acid may be used in the preparation of a medicament for use in therapy, wherein the preparation of a medicament is for reducing and/or inhibiting the expression and/or activity of p53 isoform gene and/or transcript, in particular ⁇ 133p53 or ⁇ 113p53 gene and/or transcript, in at least one cancer cell. The use may be for treating and/or preventing cancer.
  • the term "nucleic acid" is well known in the art and is used to generally refer to a molecule (one or more strands) of DNA, RNA or a derivative or analog thereof comprising nucleobases.
  • a nucleobase includes, for example, a purine or pyrimidine base found in DNA (e.g., an adenine "A”, a guanine “G", a thymine “T” or a cytosine "C”) or RNA (e.g., an A, a G, an Uracil "U” or a C).
  • the term nucleic acid encompasses the terms “oligonucleotide” and “polynucleotide” each as subgenus of the term “nucleic acid”.
  • complementar in the context of nucleic acids refers to a strand of nucleic acid non-covalently attached to another strand, wherein the complementarity of the two strands is defined by the complementarity of the bases.
  • the base A on one strand pairs with the base T or U on the other, and the base G on one strand pairs with the base C on the other.
  • An oligonucleotide or analog is of "substantial complementarity" when there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide or analog to non-target sequences under conditions in which specific binding is desired
  • a nucleic acid molecule is "hybridisable" to another nucleic acid molecule (in the present case, the p53 isoform, for example ⁇ 133p53 or ⁇ 113p53 ), when a single-stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (Sambrook and Russell, 2001 ). The conditions of temperature and ionic strength determine the "stringency" of the hybridisation. Hybridisation requires the two nucleic acids to contain complementary sequences. Depending on the stringency of the hybridisation, mismatches between bases are possible.
  • DNA RNA
  • DNA DNA
  • the nucleic acid molecule(s) may be in any suitable construct, for example in vector, phage, plasmid, or nucleic acid fragment comprising the nucleic acid molecule.
  • the nucleic acid molecule in the construct may be at least 15-30 nucleotides in length.
  • the nucleic acid molecule is introduced into a construct for example, in vector, phage or plasmid.
  • the nucleic acid molecule may be complementary and/or hybridise to a nucleic acid molecule ⁇ 133p53 or ⁇ 113p53 gene, mRNA, promoter and/or to a portion thereof.
  • the invention provides nucleic acid molecules which are complementary to the p53 isoform gene, mRNA, promoter and/or to a portion thereof. More in particular the invention provides nucleic acid molecules which are complementary to ⁇ 133p53 or ⁇ 113p53 gene, mRNA, promoter and/or to a portion thereof.
  • the vector may be administered in vitro or in vivo to the cell(s).
  • the method of the invention comprises in vivo or in vitro administering to the cells a compound reducing and/or inhibiting the expression and/or activity of p53 isoform gene and/or transcript, in particular reducing and/or inhibiting the expression and/or activity of ⁇ 133p53 or ⁇ 113p53 gene and/or transcript.
  • the method comprises administering to or transfecting in vivo or in vitro the cells with a nucleic acid construct comprising a nucleic acid molecule complementary to and/or hybridising to the p53 isoform gene, mRNA, promoter and/or a portion thereof.
  • the method comprises administering to or transfecting in vivo or in vitro the cells with a nucleic acid construct comprising a nucleic acid molecule complementary to and/or hybridising to the ⁇ 133p53 or ⁇ 113p53 gene, mRNA, promoter and/or a portion thereof.
  • “Inhibiting and/or reducing the expression” and/or “activity” of a gene, mRNA and/or promoter used herein refers to the ability of the nucleic acid molecules described in the previous section, to measurably reduce and/or inhibit the expression and/or activity of a gene, mRNA and/or promoter. In the present invention it contemplates reduction and/or inhibition of the expression and/or function of a particular gene and/or transcript. It is understood that the phrase is relative, and does not require absolute suppression of the transcript.
  • reducing and/or inhibiting the expression and/or activity of p53 isoform gene and/or transcript requires that, following application of the nucleic acid molecules mentioned in the previous section, p53 isoform gene and/or transcript, in particular ⁇ 133p53 or ⁇ 113p53 gene and/or transcript, is expressed at least 5 % less than prior to application these compounds and/or molecules, such as at least 10 % less, at least 15 % less, at least 20 % less, at least 25 % less, or even more reduced.
  • application of the nucleic acid molecules reduces and/or inhibits expression and/or activity of the the p53 isoform, in particular ⁇ 133p53 or ⁇ 113p53, by about 30 %, about 40 %, about 50 %, about 60 %, or more.
  • expression and/or activity is inhibited and/or reduced by 70 %, 85 %, 85 %, 90 %, 95 %, or even more.
  • the present invention therefore extends to the preparation of anti-sense nucleotides and ribozymes that may be used to interfere with the expression of p53 isoform gene(s) and/or transcript, in particular ⁇ 133p53 or ⁇ 113p53 gene and/or transcript.
  • This approach involves anti-sense nucleic acid molecules and ribozymes to block p53 isoform gene(s) and/or transcript, in particular ⁇ 133p53 or ⁇ 113p53 gene and/or transcript, either by masking it with an anti-sense nucleic acid or cleaving it with a ribozyme.
  • Anti-sense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule. In the cell, they hybridise to mRNA, forming an untranslatable double-stranded molecule. Therefore, antisense nucleic acids interfere with the expression of mRNA into protein. Oligomers of about fifteen nucleotides and molecules that hybridise to the AUG initiation codon will be particularly efficient, since they are easy to synthesise and are likely to pose fewer problems than larger molecules. Anti-sense methods have been used to inhibit the expression of many genes in vitro.
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns. By modifying the nucleotide sequence of these RNAs, researchers have been able to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it. Because they are sequence-specific, only mRNAs with particular sequences are inactivated. For example, the nucleic acid molecule is an antisense DNA and/or RNA molecule.
  • the nucleic acid molecule may be an antisense single strand RNA (sRNA), double strand RNA (dsRNA), double strand DNA (dsDNA), double strand hybrid RNA/DNA (RNA/DNA), small interfering RNA (siRNA) and/or ribozymes.
  • the nucleic acid construct can be any suitable vector, phage, plasmid, a nucleic acid fragment or the like comprising the nucleic acid molecule.
  • RNA interference technology is well known and consists of a process in which a double stranded RNA (dsRNA) induces the postranscriptional degradation of homologous transcripts.
  • dsRNA double stranded RNA
  • RNAi can be initiated by exposing cells to dsRNA either via transfection or endogenous expression.
  • DNA targeting sequences specific for the reduction and/or inhibition of the p53 isoform gene(s) and/or transcript, in particular reduction and/or inhibition of ⁇ 133p53 or ⁇ 113p53 gene and/or transcript, are selected and prepared according to standard technology, for example, the DNA targeting sequence are generated using Ambion siRNA target finder (http://www.ambion.com/techlib/misc/siRNA_finder.html).
  • the DNA targeting sequences may be inserted into a construct and/or vector and used to transfect the cell or cell lines in vitro or in vivo.
  • the RNA polymerase of the cell transcribes the siRNAs complementary to the p53 isoform transcript, in particular complementary to ⁇ 133p53 or ⁇ 113p53 transcript, or to a portion thereof.
  • siRNAs form a complex known as the RNA-induced silencing complex or RISC which functions in homologous target RNA destruction.
  • RISC RNA-induced silencing complex
  • the sequence-specific RNAi effect has been observed by the introduction of siRNAs either via transfection or endogenous expression of 19-23 base transcripts capable of forming duplexes, or via expression of short hairpin RNAs.
  • siRNA expression constructs and/or vectors may be constructed according to any method known in the art, for example by chemical synthesis, in vitro transcription, by digestion of long dsRNA by an RNase III family enzyme (e.g. Dicer, RNase III), by expression in cells from an siRNA expression plasmid or viral vector, and expression in cells from a PCR-derived siRNA expression cassette.
  • RNase III family enzyme e.g. Dicer, RNase III
  • the construct is directly transfected into mammalian cells resulting in functional expression of siRNAs.
  • the present invention provides a pharmaceutical composition comprising at least one nucleic acid molecule described in the preceding sections.
  • the pharmaceutical composition may be formulated treating and/or preventing cancer, wherein the pharmaceutical composition comprises a therapeutically effective amount of at least one nucleic acid molecule capable of reducing and/or inhibiting the expression of p53 isoform gene and/or transcript, in particular capable of reducing and/or inhibiting the expression of ⁇ 133p53 or ⁇ 113p53 gene and/or transcript.
  • the pharmaceutical composition may be in the presence of at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.
  • excipients examples include water, saline, dextrose, glycerol, ethanol and the like as well as combinations thereof.
  • a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or alternatively the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carrier, excipient and/or diluent.
  • Excipients normally employed for such formulations includes mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • the pharmaceutical composition is useful for treating and/or preventing cancer.
  • any pharmaceutical composition comprising a drug, compound, or substance capable of reducing and/or inhibiting the expression of p53 isoform gene and/or transcript, in particular capable of reducing and/or inhibiting the expression of ⁇ 133p53 or ⁇ 113p53 gene and/or transcript is within the scope of the present invention.
  • the cancer may be selected from the group consisting of: bone cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, gastrointestinal cancer, leukaemia, lung cancer, lymphoma, melanoma, neuroblastoma, ovarian cancer, pancreatic cancer, prostrate cancer, renal cancer, retinoblastoma, small-cell lung cancer, non-small cell lung cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, astrocytoma, glioblastoma cancer, head cancer, and hepatocarcinoma.
  • AMO anti-miRNA oligonucleotides
  • antagomirs locked-nucleic-acid antisense oligonucleotides
  • sRNA single strand RNA
  • dsRNA double strand RNA
  • dsDNA double strand DNA
  • RNA/DNA double strand hybrid RNA/DNA
  • siRNA small interfering RNA
  • compositions are preferably prepared and administered in dose units.
  • a subject such as but not limited to a human subject, and depending on activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the patient, different daily doses are necessary. Under certain circumstances, however, higher or lower daily doses may be appropriate.
  • the administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administrations of subdivided doses at specific intervals.
  • the pharmaceutical compositions can be administered system ica I Iy or locally, such as, but not limited to, by injection directly into a tumour.
  • the compositions are in general administered topically, intravenously, intramuscularly, orally, parenterally, or as implants, but even rectal use is possible in principle.
  • Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (micro) capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solutions in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • compositions are suitable for use in a variety of drug delivery systems.
  • a “therapeutically effective dose” used herein is the quantity of a compound and/or pharmaceutical composition necessary to inhibit and/or reduce, to cure or at least partially arrest the symptoms of cancer. Amounts effective for this use will, of course, depend on the severity of the disease and the weight and general state of the patient. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the compound and/or pharmaceutical composition, and animal models may be used to determine effective dosages.
  • systems for the introduction of a compound and/or a pharmaceutical composition of the invention designed to reduce and/or inhibit the expression and/or activity of p53 isoform gene(s) and/or transcript, in particular designed to reduce and/or inhibit the expression and/or activity of ⁇ 133p53 or ⁇ 113p53 gene and/or transcript, can be introduced into a subject by any of a number of methods.
  • a pharmaceutical preparation of the nucleic acid delivery system can be introduced systemically, e. g. by intravenous injection, and specific transduction of the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, the cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the gene, or a combination thereof.
  • initial delivery is more limited with introduction into the animal being quite localized.
  • the pharmaceutical preparation can consist essentially of the nucleic acid system in an acceptable diluent, or can be a slow release matrix in which the nucleic acid delivery vehicle is imbedded.
  • the complete delivery system can be produced from recombinant cells, e.g. retroviral packages
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • methods of introducing the viral packaging cells may be provided by, for example, rechargeable or biodegradable devices.
  • Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals, and can be adapted for release of viral particles through the manipulation of the polymer composition and form.
  • biocompatible polymers including hydro gels
  • biodegradable and non-degradable polymers can be used to form an implant for the sustained release of viral particles by cells implanted at a particular target site.
  • Such aspects can be used for the delivery of an exogenously purified virus, which has been incorporated in the polymeric device, or for the delivery of viral particles produced by a cell encapsulated in the polymeric device.
  • monomer composition or polymerization technique By choice of monomer composition or polymerization technique, the amount of water, porosity and consequent permeability characteristics can be controlled.
  • the selection of the shape, size, polymer, and method for implantation can be determined on an individual basis according to the disorder to be treated and the individual patient response.
  • the generation of such implants is generally known in the art.
  • manipulation of the polymer can be carried out to provide for optimal release of viral particles.
  • the invention provides a method for screening and/or identifying at least one drug candidate capable of reducing and/or inhibiting the expression and/or activity of a p53 isoform and/or transcript in at least one cancer cell, comprising: (a) administering at least one drug candidate to the at least one cancer cell, and (b) determining the ability of the drug candidate to reduce and/or inhibit the expression and/or activity of the p53 isoform gene and/or transcript in the cell, wherein the p53 isoform gene is capable modulating the expression and/or activity of p53.
  • the method of screening and/or identifying may be for screening and/or identifying at least one drug candidate capable of reducing and/or inhibiting the expression and/or activity of a p53 isoform ⁇ 133p53 or ⁇ 113p53 gene and/or transcript in at least one cancer cell.
  • the drug candidate may be capable of preventing the down-regulation of p53 or capable of reducing and/or inhibiting p53 activity in at least one cancer cell.
  • the drug candidate may be further capable of inducing and/or sensitizing at least one cancer cells to apoptosis.
  • Drug candidates may be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity.
  • the compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
  • the biological library approach is typically used for polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds.
  • the present invention also provides a kit for reducing and/or inhibiting the expression and/or activity of a p53 isoform gene and/or transcript, comprising at least one compound which binds to the p53 isoform gene, mRNA, promoter and/or to a portion thereof, thereby reducing and/or inhibiting the expression of the p53 isoform gene and/or transcript in at least one cancer cell, and wherein the p53 isoform gene is capable modulating the expression and/or activity of p53.
  • the kit may be for reducing and/or inhibiting the expression and/or activity of ⁇ 133p53 or ⁇ 113p53 gene and/or transcript, comprising at least one compound which binds to ⁇ 133p53 or ⁇ 113p53 gene, ⁇ 133p53 or ⁇ 113p53 mRNA, ⁇ 133p53 or ⁇ 113p53 promoter and/or to a portion thereof.
  • the at least one compound may be at least one nucleic acid molecule comprising a nucleotide sequence complementary to and/or which hybridises to the p53 isoform gene, mRNA, promoter and/or to a portion thereof, thereby inhibiting and/or reducing the expression and/or activity of the p53 isoform gene and/or transcript thereof.
  • the compound may be at least one nucleic acid molecule comprising a nucleotide sequence complementary to and/or which hybridises to the ⁇ 133p53 or ⁇ 113p53 gene, ⁇ 133p53 or ⁇ 113p53 mRNA, ⁇ 133p53 or ⁇ 113p53 promoter and/or to a portion thereof.
  • the compound may be capable of preventing the down-regulation of p53 expression and/or activity in at least one cancer cell.
  • the compound may further be capable of inducing and/or sensitizing at least one cancer cells to apoptosis.
  • the current invention provides a method of detecting the expression of a p53 isoform gene in at least one cancer cell, wherein the detecting is carried out before and/or after treating the cancer cell with chemotherapy and/or radiotherapy, and wherein the p53 isoform gene is capable of modulating the expression and/or activity of p53.
  • the p53 isoform gene may be ⁇ 133p53 or ⁇ 113p53 gene.
  • the method may further comprise means for detecting the expression and/or activity of the p53 isoform gene, wherein the p53 isoform gene may be ⁇ 133p53 or ⁇ 113p53 gene.
  • the invention provides a method for diagnosis and/or prognosis of cancer in a subject, comprising: a) providing at least one sample from a subject; b) quantitating the expression and/or activity of a p53 isoform gene, wherein the p53 isoform gene is capable of modulating the expression and/or activity of p53; and c) comparing the expression of the p53 isoform gene, wherein increase and/or up-regulation in the p53 isoform gene, compared to the at least one control, is indicative of presence of, predisposition to cancer.
  • the method may be for the diagnosis and/or prognosis of cancer in a subject, comprising: a) providing at least one sample from a subject; b) quantitating the expression and/or activity of ⁇ 133p53 or ⁇ 113p53 gene; and c) comparing the expression and/or activity of the ⁇ 133p53 or ⁇ 113p53 gene, wherein increase and/or up-regulation in the ⁇ 133p53 or
  • ⁇ 113p53 gene compared to the at least one control, is indicative of presence of, predisposition to cancer.
  • the invention further provides a diagnostic and/or prognostic kit for the diagnosis and/or prognosis of cancer in a subject comprising at least one nucleic acid molecule comprising a nucleotide sequence complementary to and/or hybridisable to a p53 isoform gene, mRNA, promoter and/or portion thereof, and wherein the p53 isoform gene is capable of modulating the expression and/or activity of p53.
  • the diagnostic and/or prognostic kit may be for the diagnosis and/or prognosis of cancer in a subject comprising at least one nucleic acid molecule comprising a nucleotide sequence complementary to and/or hybridisable to ⁇ 133p53 or ⁇ 113p53 gene, mRNA, promoter and/or portion thereof.
  • a "subject” may be a patient suffering from cancer. A person skilled in the art will know how to select subjects based on their amenability to a particular treatment, or their susceptibility to a particular disease.
  • control for example, may not be suffering cancer.
  • the control may exhibit decreased expression of p53 isoform gene and/or transcript, in particular the control may exhibit decreased expression of ⁇ 133p53 or ⁇ 113p53 gene and/or transcript.
  • control value may also be an average value in expression obtained from a selected population.
  • Diagnose or “diagnosis” used herein, refers to determining the nature or the identity of a condition (disease). A diagnosis may be accompanied by a determination as to the severity of the disease.
  • Prognostic or “prognosis” used herein refers to predicting the outcome or prognosis of a disease, such as to give a chance of survival based on observations and results of clinical tests.
  • Predisposition refers to the likelihood of being diagnosed with, or susceptibility to a particular disease.
  • up-regulation means that the expression of particular gene and/or mRNA transcript or the expression of a particular protein is higher or increased compared to the basal level of expression constitutively present in a cell in the absence of any stimulation.
  • down-regulation would refer to the lower or decreased expression of particular gene and/or mRNA transcript or of a particular protein compared to the basal level of expression constitutively present in a cell in the absence of any stimulation. Using established techniques known in the art this up- or down- regulation can be quantitated.
  • a method of determining the susceptibility of a patient to cancer treatment comprising: a) providing at least one sample from the patient before and after chemotherapy and/or radiotherapy; b) quantitating the expression and/or activity of a p53 isoform gene before and after chemotherapy and/or radiotherapy, wherein the p53 isoform gene is capable of modulating the expression and/or activity of p53; and c) comparing the expression and/or activity of the p53 isoform gene before and after chemotherapy and/or radiotherapy, wherein decrease and/or down-regulation in the p53 isoform gene following chemotherapy and/or radiotherapy, is indicative of susceptibility of the patient to chemotherapy and/or radiotherapy.
  • the method may be for determining the susceptibility of a patient to cancer treatment, comprising: a) providing at least one sample from the patient before and after chemotherapy and/or radiotherapy; b) quantitating the expression and/or activity of ⁇ 133p53 before and after chemotherapy and/or radiotherapy; and c) comparing the expression and/or activity of ⁇ 133p53 before and after chemotherapy and/or radiotherapy, wherein decrease and/or down- regulation in the ⁇ 133p53 following chemotherapy and/or radiotherapy, is indicative of susceptibility of the patient to chemotherapy and/or radiotherapy.
  • the invention provides an isolated ⁇ 133p53 or ⁇ 133p53 knock-out cancer cell, wherein the knock-out cell shows suppression and/or reversion of malignant phenotype.
  • the knock-out cells may be prepared according to any standard technology, for example, by knocking out the p53 isoform gene, in particular ⁇ 133p53 or ⁇ 133p53, by homologous recombination.
  • Knock-down cells may also be prepared according to any standard technology, for example by inhibiting the expression and/or activity of the p53 isoform mRNA, in particular ⁇ 133p53 or ⁇ 133p53 mRNA by RNAi as described in the earlier sections.
  • the isolated cancer cell may be present in the form of cell culture, wherein the cell may be a mammal cell. In particular the cell may be a human cell.
  • the knockout and/or knock-down cell of the invention shows an increased sensitivity to some drugs, compounds and substances. Accordingly, the invention also relates to one or more knockout and/or knockdown cells.
  • the invention provides a use for the knockout cells made according to the method of the present invention, wherein the knockout cells are implanted in a non-human animal model, for the purposes of drug testing and/or drug screening.
  • a non-human animal model for the purposes of drug testing and/or drug screening.
  • Any knockout cells prepared according to the invention may be implanted on the animal.
  • the knockout cells to be implanted on the animal are not limited to the exemplified knockout cell. Any cell showing the characteristics of any carcinoma cell or cell line according to the invention may be implanted.
  • the invention provides a non-human animal model which is implanted with the knock-out cells made according to any embodiment of the present invention for the purpose of drug screening and/or drug testing.
  • the animal model of the invention may be used as negative control in order to screen, test or discover drugs which may show suppression of malignant phenotype, reversion of malignancy, increased sensitivity of cancer cells to apoptosis, and/or a reduction in the growth rate.
  • the animal model of the invention may be a mammal, for example a mouse or rat.
  • the animal model may be a zebrafish model.
  • the non-human animal model may also be implanted with a knock-down cell made according to any embodiment of the present invention.
  • ⁇ 113p53:gfp plasmid A 4.113 kb DNA fragment immediately upstream of the start codon ATG of ⁇ 113p53 was amplified from genomic DNA (AB strain wild type zebrafish) with primer pair PO-F and PO-R (SEQ ID NO: 1 and SEQ ID NO: 2) (Table 1 ) using the Expand Long Template Kit (Roche). Both the primers were derived from the p53 mRNA (accession number AF365873).
  • PCR product was first cloned into pGEMTeasy vector (Promega), then digested with BamHI and EcoRI enzymes before being subcloned into the pEGFP-1 vector (Clontech) (T4 ligase used was from Promega) to generate the ⁇ 113p53:gfp plasmid.
  • PO-F EcoRI-zf-tp53long-u26, S'-CCGGAATTCTAACCTCCTTGTTTTGGTCAT
  • the p53 probe I that can detect both p53 and A113p53 transcripts (SEQ ID NO: 35) atggcgcaaaacgacagccaagagttcgcggagctctgggagaagaatttgattattcagcccccaggtggtggctcttg ctgggacatcattaatgatgaggagtacttgccgggatcgtttgaccccaattttttttgaaaatgtgcttgaagaacagc ctcagccatccactctcccaccaacatccactgttccggagacaagcgactatcccggcgatcatggatttaggctcagg ttcccgcagtctggcacaaaatctgtggcacaaaatctgtaacttgcacttattcaccggacctgaat
  • the A113p53 specific probe derived the 5' UTR of A113p53 (SEQ ID NO:36) cgcatttttaaaatatcctggcgaacatttggagggagatgttggtcttttatgcattttttaggatggagtgtaatacattttagg attgttaatagtgctggacagtcaagctggtgcttcacattctgtgacattacaagaccaggagg
  • the ⁇ 113p53:gfp plasmid used for injection was linearized with EcoRI.
  • the ⁇ 113p53:gfp plasmid was linearized and injected into single-cell stage embryos.
  • Approximately 25 pg of linearized ⁇ 113p53:gfp plasmid DNA was injected into the one-cell stage embryos thereby generating TO stage fish.
  • the TO fish were raised to adulthood for screening for individual fish that produced Gfp positive offspring. Total three independent TO lines were found to produce gfp positive transgenic fish (Tg( ⁇ 113p53:gfp) transgenic fish). These TO fish were then mated with the wild type (WT) fish to generate the T1 progenies.
  • WT wild type
  • the progenies T1 from each individual TO x WT were raised to adulthood and then used in screening for gfp positive fish using gfp specific primers EGFP-U322 and EGFP-L742 (SEQ ID NO: 14 and SEQ ID NO: 15)(Table 1 ).
  • Homozygous Tg( ⁇ 113p53:gfp fish were obtained by mating a male and female gfp positive T1 fish in the same family.
  • Each truncated promoter was amplified from ⁇ 113p53:gfp (PO) using iProof enzyme (Biorad) and cloned into pEGFP-1 vector using the Infusion kit (Clontech) according to manufacturer's protocol.
  • Corresponding primer pairs used are listed in Table 1.
  • the PO construct was made using primers comprising the sequence of SEQ ID NO: 1 and SEQ ID NO: 2.
  • the P1 , P2, P3, P4 and P5 constructs were made using the sequences comprising the SEQ ID NOs: 3, 4, 5, 6 and 7 respectively as forward primers.
  • the primer comprising the sequence of SEQ ID NO: 2 was used as reverse primer in the constructs P1 to P5.
  • the internal deletion constructs without the nucleotides (- 1059) to (-506) designated construct P6 or the deletion construct without the nucleotides (-239) to (-1) designated construct P9 were also derived from ⁇ 113p53:gfp using iProof enzyme (Biorad) ( Figure 3).
  • primer pairs (-U3059)-(-L1060) SEQ ID NO: 1 and SEQ ID NO: 9) and (-U505ML1054) (SEQ ID NO: 8 and SEQ ID NO: 2) were used to amplify the parts flanking the left and right side of the deletion, respectively ( Figure 3).
  • PCR products from these two pairs of primers were denatured and mixed together to allow annealing of the sticky ends and this mixture was then used as the templates for the second round PCR using primers (-U3059) and (L1054) (SEQ ID NO: 1 and SEQ ID NO: 2) to get the product P6 with the internal deletion of the nucleotides -1059 to -506.
  • primer pairs (-U3059M-L240) (SEQ ID NO: 1 and SEQ ID NO: 13) and (U1 )-(L1054) (SEQ ID NO: 12 and SEQ ID NO: 2) were used to amplify the two parts flanking each side of the deletion and the PCR products were mixed together and used as the template for the second round PCR using primers (-U3059) and (L1054) (SEQ ID NO: 1 and SEQ ID NO: 2) to get the internal deletion product for P9.
  • the final PCR products were cloned into pEGFP-1 to get P6 and P9 plasmids, respectively, using the Infusion kit (Clontech) according to manufacturer's protocol. All fragments cloned were sequenced and confirmed to be identical to ⁇ 113p53:gfp except the deleted regions. Ten pg of each plasmid DNA was injected into AB fish embryos at one-cell stage.
  • Chromatin immunoprecipitation (ChIP)
  • HA taggedp53 mRNA was injected into one-cell stage embryos. At 5 hours post fertilization (hpf), -500 embryos were deyolked in PBS with 1x protease inhibitor cocktail (Complete, Roche). The supernatant was removed after centrifugation at 300 g. The pellet was homogenized in 1 ml NIM buffer of 0.25mM sucrose, 25 mM KCI, 10 mM Tris. Cl (pH 7.4), 5mM MgCI2 and ixComplete and treated with formaldehyde (final concentration 1 %) at room temperature for 15 minutes. The reaction was quenched with glycine (final concentration 125mM).
  • the suspension was pelleted at 80Og in 4 0 C.
  • the pellet was washed with NIM buffer three times, and resuspended in SDS lysis buffer (provided with ChIP assay kit; Upstate Biotechnology).
  • the embryo lysate was sonicated to shear the chromatin by subjecting the lysate to 40 sets of 5- second pulses using Misonix 3000 equipped with a 2-mm tip and the energy output was set to 2.
  • the lysate was incubated on ice for 2 min between each pulse.
  • the chromatin was sheared into 200-1000 bp fragments. After sonication, 50 ul lysate was taken out as the template for positive control PCRs and 25 ul lysate for western blot.
  • the rest of lysate was spun at 14,000 rpm and the pellet was resuspended in ChIP dilution buffer. 100 ul HA antibody-matrix (Roche) was added to the suspension and incubated overnight at 4 0 C. Following the incubation the suspension was washed and the HA antibody- matrix was eluted as described in the protocol (given by the ChIP assay kit). The histone-DNA crosslinks was reversed according to manufacture's protocol. The DNA was recovered by phenol/chloroform extraction and precipitated by ethanol. The pellet was resuspended in distilled water and used as the template for PCR reactions.
  • Co-IP Co-immunoprecipitation
  • p53 was fused with a HA-tag at its N-terminal in one construct (pCS2+-HA-p53) and ⁇ 113p53 fused to a MYC-tag at its N-terminal in the other construct (pCS2+-MYC- ⁇ 113p53) ⁇ F ⁇ gure 6A).
  • the pCS2+-HA-p53 and pCS2+-MYC- ⁇ 113p53 plasmids were linearized with Not1 for synthesizing the HA-p53 and MYC- ⁇ 113p53 mRNA with the mMESSAGE mMACHINE ® SP6 kit (Ambion), respectively.
  • the pellet was homogenized in Tris.CI Lysis Buffer of 50 mM Tris.CI pH 8.0, 150 mM NaCI, 0.1 % NP40, 0.1 mM DTT and 1x Complete.
  • the lysate was centrifuged at 14,000 rpm for 5 min at 4 0 C. Before being loaded onto the CO-IP column (PIERCE), 20 ul supernatant was taken out as the input control. The rest was mixed with 6 ul Anti-HA agarose. The subsequent procedures were according to the manufacture's instructions (PIERCE). All samples were equally loaded into two PAGE gels for western blot analysis, one was probed with an anti-HA antibody and the other with an anti-MYC antibody.
  • morpholinos were purchased from Gene Tools. Two p53 antisense morpholinos, one targeting against the start codon ATG comprising the sequence of SEQ ID NO: 16 ( ⁇ '-GCGCCATTGCTTTGCAAGAATTG-S') (Langheinrich et al., 2002) (p53-MO ATG ) and the other against the splice junction between exon 5 and intron 5 (5'-AAAATGTCTGTACTATCTCCATCCG- (p53-MO spl ) comprising the sequence of SEQ ID NO: 17 (Chen et al., 2005), were designed.
  • def antisense morpholino corresponding to the splice junction between exon 2 and intron 2 and comprising the sequence of SEQ ID NO: 18 ( ⁇ '-ATGAATATAATGACTTACCAAGCGC-S 1 ) (def-MO) was also designed (Chen et al., 2005) The morpholinos were injected at a concentration was 1.0 mM.
  • An antisense morpholino comprising the sequence of SEQ ID NO: 19 (5'-GCAAGTTTTTGCCTGACAGAAG-3') ( ⁇ 113p53-MO) that specifically targets against the 5'-UTR region of ⁇ 113p53 to block its translation was designed.
  • the human beta-globin antisense morpholino comprising the sequence of SEQ ID NO: 20 (5'-CCTCTTACCTCAGTTACAATTT-S') was used as the standard control (st-MO).
  • the two later morpholinos were used in injection at a concentration of 0.4 mM.
  • Embryos at 1 day(s) post fertilization were ⁇ -ray irradiated (with a dosage of 24 Gray) or treated with 500 nM campthecin or 50 urn roscovitine, respectively.
  • the embryos were harvested for RNA extraction and the extracted RNA was used for Northern blot analysis of p53 and ⁇ 113p53 expression.
  • heterozygous Tg( ⁇ 113p53:gfp) transgenic embryos were treated with ⁇ -ray and the two drugs, respectively.
  • RNA was extracted from the treated embryos and used for the analysis of gfp transcripts.
  • Tg( ⁇ 113p53:gfp) homozygous fish was crossed with the def hl429 heterozygotes and the F2 progeny was checked for Gfp fluorescence in digestive organs.
  • F2 fish at 1 dpf and 2 dpf, only weak gfp expression was detected in the wild type (WT) and in the def 1 ' 429 heterozygous siblings (sb) but Gfp was strongly and ubiquitously expressed in the head region but relatively weakly expressed in the trunk region in the def 1 ' 429 homozygous mutant (def-/-) (Figure 2C).
  • Gfp continued to be weakly expressed in the WT and def" 429 heterozygous siblings (sb) but is highly enriched in the head region and digestive organs in the def 11429 homozygous mutants (def-/-), displaying a pattern similar to the endogenous ⁇ 113p53 expression as reported previously ( Figure 2D and 2E) (Chen et al., 2005). Therefore, these results indicate that 4.113 kb DNA fragment contained necessary regulatory elements for ⁇ 113p53 expression in response both to stress signals (e.g plasmid DNA injection) and developmental cues (e.g the def 1 ' 429 mutation).
  • stress signals e.g plasmid DNA injection
  • developmental cues e.g the def 1 ' 429 mutation
  • deletion -278 to +1054 bp from the 3'-end completely abolished the promoter activity ( Figure 4A and 4B) most likely because the -278 to +1054 bp region contains (a) crucial regulatory element(s) together with the transcription initiation start site (TSS, position +1 , Figure 3).
  • TSS transcription initiation start site
  • deletion analyses have identified two regions, namely -1041 to -1991 bp (region I) and -1 to -239 bp (region II) to contain crucial c/s-elements for the A113p53 expression.
  • RT- PCR results showed that, in the all above cases, the endogenous A113p53 expression was increased due to stress signals (plasmid DNA injection) ( Figure 4B), demonstrating that the two regions identified in our promoter analysis are likely genuinely used to regulate A113p53 expression in vivo.
  • A113p53 expression is directly regulated by p53
  • Uninjected embryos were used as the negative control and def-MO along injected embryos were used as the positive control. It has been previously shown that injection of the def-MO caused an upregulation of the A113p53 expression as that observed in the def 11429 mutant. As expected, injection of def-MO alone caused obvious upregulation of gfp expression in the Tg( ⁇ 113p53:gfp) embryos.
  • HA- tagged p53 mRNA was injected into single-cell-stage embryos. The embryos at 5 hours post-injection were harvested and used for the chromatin co- immunoprecipitation (ChIP) assay using anti-HA antibody to pull down the HA- p53-DNA complex.
  • Two pairs of primers one pair CHIP-(-U1300) and CHIP- (L1086) (SEQ ID NOs: 29 and SEQ ID NO: 30) to amply the -1086 to -1300 fragment containing the putative p53-binding site in region I and the other pair CHIP-(-U112) and CHIP-(L98) (SEQ ID NO:31 and SEQ ID NO: 32) to amplify the -112 to +98 fragment containing one putative p53-binding site in region II, were designed and used to perform PCR using the HA-p53-DNA complex as the template.
  • ⁇ 113p53 forms a complex with p53
  • ⁇ Np63 and ⁇ Np73 are found to function as dominant negative regulators of p63 and p73, respectively, although they alone also have some biological functions (Grab et al., 2001 ; Yang et al., 1998).
  • ⁇ 133p53 has been implicated to act as a dominant negative regulator of p53 since co-transfection p53 with ⁇ 133p53 impaired p53-induced cell apoptosis (Bourdon et al., 2005).
  • the ⁇ 113p53 protein is an N-terminal truncated form of p53 with deletion of the activation domain and Mdm2-interacting motif and with partial deletion of the DNA-binding domain but retains the dimerization domain.
  • ⁇ 113p53 acts as a dominant negative regulator of p53 and that ⁇ 113p53 competes with p53 to form a heterodimer with p53.
  • p53 and ⁇ 113p53 interacted with each other to facilitate ⁇ 113p53's dominant negative role on p53.
  • the HA-tagged-p53 and MYC-tagged-A113p53 were cloned into pCS2+ expression plasmid, respectively ( Figure 6A).
  • the HA-tagged-p53 and MYC-tagged-A113p53 mRNA was obtained from in vitro transcription from corresponding plasmids and was co-injected into the single-cell stage zebrafish embryos. Injected embryos at 5 hpf were harvested and total proteins were extracted from the injected experimental and control embryos, respectively.
  • HA- p53 was immunoprecipitated with anti-HA antibody and the protein precipitates were assayed for ⁇ 113p53 using anti-MYC antibody. The results showed that ⁇ 113p53 was efficiently co-precipitated with p53 (Figure 6B), demonstrating that p53 and ⁇ 113p53 can form a complex.
  • the WT fish embryos were treated with ⁇ -ray, camptothecin and rostovitine, respectively, and embryos 18 hours post- treatment were harvested and assayed for A113p53 expression using either a p53 probe (Table 2, Chen et al., 2005) (SEQ ID NO: 35) (which can detect both p53 and A113p53) or a A113p53 specific probe (Table 3, Cheng et al., 2005) (SEQ ID NO: 36). The results showed that all treatments dramatically induced the Ai13p53 expression in the treated embryos (Figure 7A).
  • ⁇ 113p53 indeed functions as a dominant negative regulator of p53 as proposed, the fact that the A113p53 expression is greatly induced by stress signals such as ⁇ -ray irradiation, injection of plasmid DNA and drug treatment suggests that ⁇ 113p53 might be essential for cell survival by helping cells escaping from p53-induced cell apoptosis under these stress conditions.
  • stress signals such as ⁇ -ray irradiation
  • ⁇ 113p53 might be essential for cell survival by helping cells escaping from p53-induced cell apoptosis under these stress conditions.
  • ⁇ 113p53-MO morpholino derived from the 5'-UTR of Ai13p53 (SEQ ID NO: 19) (this 5'-UTR region is included in the 4.113 kb pA113p53 fragment and is transcribed together with gfp in A113p53:gfp) and then co-injected ⁇ 113p53-MO with A113p53:gfp plasmid into the single-cell stage embryos.
  • ⁇ 113p53-MO can effectively knock-down the expression of Gfp protein ( Figure 8A and 8B).

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Abstract

L'invention concerne un procédé de traitement et/ou de prévention du cancer qui consiste à réduire et/ou inhiber l'expression et/ou l'activité d'au moins un gène isoforme p53 et/ou d'un transcrit de celui-ci dans au moins une cellule cancéreuse, ce gène isoforme et/ou ce transcrit de ce gène module l'expression et/ou l'activité de p53. En particulier, le gène isoforme p53 est le gène Δ133p53 et/ou Δ113p53. L'invention concerne aussi des utilisations de ce ou de ces gènes isoformes p53.
PCT/SG2008/000321 2007-08-31 2008-09-01 Gène(s) isoforme(s) p53 et utilisations de celui-ci (ceux-ci) Ceased WO2009029054A1 (fr)

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WO2009064590A3 (fr) * 2007-11-12 2009-09-03 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Applications thérapeutiques des isoformes p53 en médecine régénératrice, pour lutter contre le vieillissement et le cancer
WO2011000891A1 (fr) * 2009-06-30 2011-01-06 Centre National De La Recherche Scientifique (Cnrs) Méthode permettant de tester un sujet qu’on pense prédisposé à souffrir d’un cancer métastatique, au moyen du delta133p53 bêta
WO2014085534A1 (fr) 2012-11-30 2014-06-05 3M Innovative Properties Company Feuille de note transparente et procédé permettant de fabriquer cette dernière
WO2016120495A1 (fr) * 2015-01-30 2016-08-04 Centre National De La Recherche Scientifique (Cnrs) Isoformes de delta133p53bêta et delta133p53gamma, biomarqueurs de cellules souches cancéreuses
EP2622065B1 (fr) * 2010-10-01 2016-09-07 The Government of the United States of America as represented by the Secretary of the Department of Health and Human Services Manipulation de la fonction de cellules souches par des isoformes p53
CN108728441A (zh) * 2018-04-18 2018-11-02 深圳市第二人民医院 特异性识别p53突变的基因系统

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US8575121B2 (en) 2007-11-12 2013-11-05 The United States of America as represented by the Secetary of the Department of Health and Human Services Therapeutic applications of p53 isoforms in regenerative medicine, aging and cancer
US9944927B2 (en) 2007-11-12 2018-04-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Therapeutic applications of p53 isoforms in regenerative medicine, aging and cancer
US9068165B2 (en) 2007-11-12 2015-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Therapeutic applications of P53 isoforms in regenerative medicine, aging and cancer
AU2008321253B2 (en) * 2007-11-12 2014-01-16 Masaryk Memorial Cancer Institute Therapeutic applications of p53 isoforms in regenerative medicine, aging and cancer
WO2009064590A3 (fr) * 2007-11-12 2009-09-03 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Applications thérapeutiques des isoformes p53 en médecine régénératrice, pour lutter contre le vieillissement et le cancer
JP2012531606A (ja) * 2009-06-30 2012-12-10 サントル、ナショナール、ド、ラ、ルシェルシュ、シアンティフィク、（セーエヌエルエス） Δ１３３ｐ５３βを用いた転移癌を有する素因があると思われる被験体の検査方法
CN102471801A (zh) * 2009-06-30 2012-05-23 国家科学研究中心 用Δ133p53β检验被认为倾向于具有转移性癌症的受试者的方法
EP2272979A1 (fr) * 2009-06-30 2011-01-12 Centre National de la Recherche Scientifique (CNRS) Procédé de test d'un sujet dont on pense qu'il est prédisposé à un cancer
CN102471801B (zh) * 2009-06-30 2016-05-11 国家科学研究中心 用Δ133p53β检验被认为倾向于具有转移性癌症的受试者的方法
WO2011000891A1 (fr) * 2009-06-30 2011-01-06 Centre National De La Recherche Scientifique (Cnrs) Méthode permettant de tester un sujet qu’on pense prédisposé à souffrir d’un cancer métastatique, au moyen du delta133p53 bêta
EP2622065B1 (fr) * 2010-10-01 2016-09-07 The Government of the United States of America as represented by the Secretary of the Department of Health and Human Services Manipulation de la fonction de cellules souches par des isoformes p53
WO2014085534A1 (fr) 2012-11-30 2014-06-05 3M Innovative Properties Company Feuille de note transparente et procédé permettant de fabriquer cette dernière
WO2016120495A1 (fr) * 2015-01-30 2016-08-04 Centre National De La Recherche Scientifique (Cnrs) Isoformes de delta133p53bêta et delta133p53gamma, biomarqueurs de cellules souches cancéreuses
CN107208066A (zh) * 2015-01-30 2017-09-26 国家科学研究中心 Δ133P53β和Δ133P53γ同种型是癌症干细胞的生物标记物
US10920198B2 (en) 2015-01-30 2021-02-16 Centre National De La Recherche Scientifique (Cnrs) Delta133p53beta and delta133p53gamma isoforms are biomarkers of cancer stem cells
US12227767B2 (en) 2015-01-30 2025-02-18 Centre National De La Recherche Scientifique (Cnrs) Delta133P53Beta and Delta133P53Gamma isoforms are biomarkers of cancer stem cells
CN108728441A (zh) * 2018-04-18 2018-11-02 深圳市第二人民医院 特异性识别p53突变的基因系统

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