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WO1997010843A1 - Constructions de p53 modifiees et utilisations leur convenant - Google Patents

Constructions de p53 modifiees et utilisations leur convenant Download PDF

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WO1997010843A1
WO1997010843A1 PCT/US1996/015188 US9615188W WO9710843A1 WO 1997010843 A1 WO1997010843 A1 WO 1997010843A1 US 9615188 W US9615188 W US 9615188W WO 9710843 A1 WO9710843 A1 WO 9710843A1
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PCT/US1996/015188
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Thanos D. Halazonetis
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The Wistar Institute Of Anatomy And Biology
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Priority claimed from US08/697,221 external-priority patent/US5847083A/en
Application filed by The Wistar Institute Of Anatomy And Biology filed Critical The Wistar Institute Of Anatomy And Biology
Priority to AU72429/96A priority Critical patent/AU7242996A/en
Publication of WO1997010843A1 publication Critical patent/WO1997010843A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates generally to the field of oncoproteins, and more specifically to p53.
  • Wild-type (wt) p53 is a sequence-specific DNA binding protein found in humans and other mammals, which has tumor suppressor function [See, e.g., Harris, Science. 262: 1980-1981 (1993)].
  • the wild-type p53 protein functions to regulate cell proliferation and cell death (also known as apoptosis) . It also participates in the response of the cell to DNA damaging agents [Harris (1993), cited above].
  • DNA damaging agents such as radiation and che otherapeutics commonly used for cancer treatment.
  • Fig. 3 illustrates the effects of a number of amino acid substitutions on DNA binding of the tumor- derived p53His273 mutant. Binding was assayed using a high affinity DNA site (oligonucleotide BC) by gel retardation shift analysis on native electrophoretic gels. The amino acids are abbreviated using the single letter code: His, H; Arg, R; Lys, K.
  • Fig. 4 illustrates the effect of the Thr284 to
  • the amino acids are abbreviated using the single letter code: Arg, R; Cys, C; Gin, Q; His, H; Lys, K.
  • Fig. 5 illustrates the results of an experiment relating to rescue of the transcriptional and tumor suppressor activities of tumor-derived p53 mutants.
  • Transcriptional activities from a reporter plasmid containing a high affinity p53 DNA site are presented as means ⁇ SE.
  • the activity of wild ⁇ type p53 was adjusted to 100%. No transcription was detected from a reporter lacking a p53 site.
  • Tumor suppressor activities in Saos-2 osteosarcoma cells are presented as means ⁇ SE of the number of tumor cell colonies per plate.
  • the amino acids are abbreviated using the single letter code: Arg, R; Cys, C; Gin, Q; His, H. threonine corresponding to amino acid residue 284 of the wild-type human p53 protein is changed to arginine.
  • the invention provides a method of enhancing the DNA-binding ability of a p53 construct having a p53 DNA binding domain comprising the step of modifying the codon encoding amino acid 284 to a codon encoding arginine.
  • the present invention provides a nucleic acid sequence encoding a protein of the invention.
  • These nucleic acids may be inserted into an appropriate vector for delivery to patients for gene therapy.
  • the nucleic acids may be inserted into a vector for in vitro expression of a protein of the invention, which is then introduced into patients.
  • Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.
  • Fig. 1 illustrates activation of DNA binding of common Class I mutants by antibody PAb421. Binding was assayed using a high affinity DNA site (oligonucleotide BC) by gel retardation shift analysis on native electrophoretic gels.
  • the amino acids are abbreviated using the single letter code: Cys, C; Gin, Q; His, H; Ser, S; Trp, W.
  • Fig. 2 illustrates activation of DNA binding of common Class I mutants by deletion of the p53 C-terminal 30 amino acids (residues 364-393) .
  • Binding was assayed using a high affinity DNA site (oligonucleotide BC) by gel retardation shift analysis on native electrophoretic gels in the presence of specific (S - oligonucleotide BC) or non-specific (NS - oligonucleotide TF3) unlabeled excess competitor DNA.
  • S - oligonucleotide BC specific - oligonucleotide BC
  • NS - oligonucleotide TF3 non-specific
  • the invention provides modified p53 protein constructs in which the amino acid residue corresponding to residue 284 of wild-type or native human p53 is modified from the native threonine to arginine.
  • the native Thr residue at position 284 may be substituted with Lys (K284) .
  • Lys284 was introduced into a p53His273 mutant, it was found to bind oligonucleotide BC somewhat better than the original p53His273 mutant, using the assay described in detail in Example 3. It will be understood that where reference is made to R284 in the following discussion, K284 may be substituted.
  • the modified p53 constructs of the invention may be derived from full-length p53.
  • p53Arg284 [SEQ ID NO: 3] and p53Lys284 [SEQ ID NO:4] are example ⁇ of such modified constructs.
  • the modified p53 constructs of the invention may contain a C-terminal p53 deletion.
  • a preferred deletion involves truncation of amino acid residues 364 - 393.
  • One example of such a truncated construct is p53Arg284 ⁇ 364-393 [SEQ ID NO: 17].
  • suitable deletions include truncation following amino acid residue 355, and deletions internal to this region (corresponding to residues 356-393 of SEQ ID N0:2) .
  • p53 protein constructs encompasses full-length and truncated p53 proteins containing a p53 DNA binding domain. Included in this Detailed Description of the Invention
  • the present invention provides modified p53 constructs containing arginine at the amino acid residue corresponding to residue 284 of wild-type human p53 [SEQ ID NO: 2].
  • the inventor has found that such a modification results in an increase in the DNA binding avidity of the p53 and more efficient tumor suppression than the corresponding unmodified construct.
  • the R284p53 [SEQ ID NO: 3] was found to bind DNA more avidly than wild-type p53 in vitro and to suppress colony growth of tumor cells about five- to six-fold more efficiently than wild-type p53 in tissue culture experiments.
  • the inventor demonstrates herein that the tumor suppressor function of common Class I tumor-derived p53 mutants can be restored and provides the means for pharmacological rescue of p53 function in cancer patients.
  • the inventor introduced a novel p53-DNA contact between a phosphate of the DNA backbone and p53. This was done by replacing Thr284 of wild-type human p53 with Arg. This substitution, in conjunction with the conformational switch that involves the C-terminus of p53 and allosterically regulates the activity of the p53 DNA binding domain, fully restored DNA binding of the tumor-derived p53 mutants. Furthermore, the transcriptional and tumor suppressing activities of these p53 mutants were also restored. 8
  • chimeric p53 proteins include proteins containing the N-terminal portion of p53 fused, optionally via a suitable linker, to a heterologous tetramerization domain.
  • a heterologous tetramerization domain includes any sequence of amino acids heterologous to p53 which forms stable homo-tetramers.
  • One particularly desirable tetramerization domain includes the tetrameric variant of the GCN4 LZ [Harbury et al,
  • GCN4 numbering follows Hinnenbusch et al, Proc. Natl. Acad. Sci. USA, 81:6442- 6446 (1984) and Ellenberger et al, Cell, 71: 1223-1237 (1992) .
  • Wild-type GCN4 is provided in SEQ ID NOS: 5 and 6.
  • the LZ variant has Ile at positions d of the coil and Leu at positions a [SEQ ID NO: 33], in contrast to the original zipper which has Leu and Val, respectively.
  • Suitable chimera include (from N-terminus to C-terminus) :
  • the above nucleotide sequences can be included within larger DNA or RNA fragments, or may be interrupted by introns.
  • nucleic acids encoding the modified proteins of the invention are present in the context of vectors suitable for amplification in prokaryotic or eukaryotic cells or for expression in cell-free extracts or lysates or in prokaryotic or eukaryotic cells.
  • vectors suitable for amplification in prokaryotic or eukaryotic cells or for expression in cell-free extracts or lysates or in prokaryotic or eukaryotic cells are known and many of these are commercially available.
  • plasmids with bacterial or yeast replication origins allow amplification in bacteria or yeast, respectively.
  • Such vectors allow the production of large quantities of nucleic acids encoding the proteins of the invention, which nucleic acids can be used for gene therapy or for expression of the modified p53 proteins of the invention.
  • expression vectors are known.
  • the vector pGEM4 (Promega, Madison, WI) is suitable for expression of the p53 proteins in cell-free lysates
  • the vector pSV2 [Mulligan et al, Proc. Natl. Acad. Sci. USA. 18:2072-2076 (1981)] is suitable for expression in mammalian cells.
  • Such vectors allow the production of the modified proteins of the invention in vitro for analysis of their functional properties or for delivery to patients.
  • one of skill in the art may readily select or construct another suitable expression vector.
  • nucleic acid sequences of the invention may be inserted into a vector capable of targeting and infecting a desired cell, either in vivo or ex vivo for which is fused via an Ile linker, to aa residues 352-393 of p53wt [SEQ ID NO: 2].
  • mutants include p53 having glutamine at residue 248 (p53Q248) [SEQ ID NO: 11], p53 having histidine at residue 273 (p53H273) [SEQ ID NO: 12], and p53 having cysteine at residue 273 (p53C273) [SEQ ID NO: 13].
  • Other p53 mutants which may be susceptible to this R284 mutation are known in the art.
  • Modifying the p53 protein construct according to the method of the invention involves altering the residue corresponding to aa residue 284 of human p53wt or of a p53 mutant containing the native Thr284 to Arg. This modification can be achieved by mutating the 284 codon using conventional site-directed mutagenesis techniques [R. Higuchi et al, in M. A. Innis et al, (eds.) , PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, pp. 177-183
  • the native codon 284 (ACA) is modified by site-directed mutagenesis to CGA or preferably CGT, which encodes Arg.
  • CGA native codon 284
  • CGT preferably CGT
  • the present invention further provides nucleic acid sequences encoding the modified p53 protein constructs of this invention.
  • the nucleic acid sequences of the invention 12 a pharmaceutically acceptable carrier, such as saline, and administered parenterally (or by other suitable means) in sufficient amounts to infect the desired cells and provide sufficient levels of p53 activity to arrest abnormal cellular proliferation.
  • a pharmaceutically acceptable carrier such as saline
  • Other pharmaceutically acceptable carriers are well known to those of skill in the art.
  • a suitable amount of the vector containing the chimeric nucleic acid sequences is between about 10 6 to 10° infectious particles per mL carrier. The delivery of the vector may be repeated as needed to sustain satisfactory levels of p53 activity, as determined by monitoring clinical symptoms.
  • this therapy may be combined with other therapies for the disease or condition being treated.
  • therapy involving the administration of a vector capable of expressing a modified p53 protein construct of the invention is well suited for use in conjunction with conventional cancer therapies, including surgery, radiation and chemotherapy.
  • nucleic acid sequences driving expression of a p53 protein of the invention may also be introduced as "naked DNA" by "carriers" other than viral vectors, such as liposomes, nucleic acid-coated gold beads or can simply be suspended in saline or the like and injected in situ [Fujiwara et al (1994) , cited above; Fynan et al , Proc. Natl. Acad. Sci.
  • a suitable amount of nucleic acid is between about 10 ⁇ g to about 1 mg per mL carrier.
  • a suitable amount of nucleic acid is between about 10 ⁇ g to about 1 mg per mL carrier.
  • many such viral vectors are useful for this purpose, e.g., adenoviruses, retroviruses and adeno-associated viruses (AAV) [Schreiber et al,
  • a recombinant viral vector e.g. an adenovirus
  • a recombinant viral vector comprises DNA of at least that portion of the viral genome which is capable of infecting the target cells operatively linked to the nucleic acid sequences of the invention.
  • infection is generally meant the process by which a virus transfers genetic material to its host or target cell.
  • the virus used in the construction of a vector of the invention is rendered replication-defective to remove the effects of viral replication on the target cells.
  • the replication-defective viral genome can be packaged by a helper virus in association with conventional techniques.
  • the vector(s) containing the nucleic acids encoding a protein of the invention is suspended in 14 effective to treat the conditions referred to below.
  • a preferred dose of a pharmaceutical composition containing a protein of this invention is generally effective above about 0.1 mg modified p53 protein, and preferably from about 1 mg to about 100 mg. Dosage units of such pharmaceutical compositions containing the proteins of this invention preferably contain about 1 mg to 5 g of the protein. These doses may be administered with a frequency necessary to achieve and maintain satisfactory p53 DNA binding and tumor suppressor activity levels. Although a preferred range has been described above, alternative doses for treatment of each type of tumor or other condition may be determined by those of skill in the art.
  • nucleic acids and proteins of the invention can be introduced into human patients for therapeutic benefits in conditions characterized by insufficient wild-type p53 activity.
  • the nucleic acids of the invention may be introduced into the patient in the form of a suitable viral vector (or by direct DNA delivery) to harness the patient's cellular machinery to express the proteins of the invention in vivo.
  • the proteins of the invention may be introduced into the patient in appropriate pharmaceutical formulations as described above.
  • compositions of thi ⁇ invention containing a protein of the invention or a nucleic acid or a viral vector which express a protein of the invention in vivo, may be employed to induce the cellular defense to DNA damaging agents.
  • DNA damaging agents include sunlight, UV irradiation, as well as radiation and chemotherapeutics used for cancer treatment.
  • UV irradiation examples include sunlight, UV irradiation, as well as radiation and chemotherapeutics used for cancer treatment.
  • modified p53 protein constructs of this invention may also be formulated into pharmaceutical compositions and administered using a therapeutic regimen compatible with the particular formulation.
  • compositions within the scope of the present invention include compositions containing a protein of the invention in an effective amount to have the desired physiological effect, e.g. to arrest the growth of cancer cells without causing unacceptable toxicity for the patient.
  • Suitable carriers for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form, e.g. saline.
  • suspensions of the active compounds may be administered in suitable conventional lipophilic carriers or in liposomes.
  • compositions may be supplemented by active pharmaceutical ingredients, where desired.
  • Optional antibacterial, antiseptic, and antioxidant agents in the compositions can perform their ordinary functions.
  • the pharmaceutical compositions of the invention may further contain any of a number of suitable viscosity enhancers, stabilizers, excipients and auxiliaries which facilitate processing of the active compounds into preparations that can be used pharmaceutically.
  • these preparations, as well as those preparations discussed below, are designed for parenteral administration.
  • compositions designed for oral or rectal administration are also considered to fall within the scope of the present invention.
  • suitable amount or “effective amount” means an amount which is 16
  • Plasmids of the pGEM series were used to generate in vitro translated p53 proteins, as previously described [T. Halazonetis and A. Kandil, EMBO J.. 12:5057-5064 (1993a); T. Halazonetis and A. Kandil, EMBO J. , 12:1021-1028 (1993b) ; J. L. Waterman et al, EMBO J. , 14:512-519 (1995)].
  • plasmid pGEMhump53wt (also termed pGEMhp53wtB) encodes full-length human wild-type p53.
  • This plasmid was prepared by PCR using a human p53 cDNA, which is readily available to those practicing the art. The PCR procedure was designed to incorporate unique restriction sites within the coding sequence of human p53: Kpn I at codon 218, Sst I at codon 299, Sst II at codon 333, Bst BI at codon 338 and Sal I immediately following the termination codon. An Msc I site at codon 138 was eliminated.
  • the proteins were derived from pGEMhump53wt by site-directed mutagenesis [Higuchi, in Innis et al, PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, pp. 177-183 (1990) ] of the codons indicated below.
  • In vitro amount of a composition of this invention patients may tolerate higher doses of such DNA damaging agents.
  • compositions of this invention are in inducing apoptosis of specific cells, such as proliferating lymphocytes.
  • a suitable amount of an appropriate pharmaceutical composition of this invention is administered to a subject to enhance the development of immune tolerance.
  • This method may employ both in vivo and ex vivo modes of administration.
  • this therapy is useful as the sole treatment or as an accessory treatment to prevent transplant rejection, or to treat autoimmune diseases, e.g. , systemic lupus erythrematosis, rheumatoid arthritis and the like.
  • the pharmaceutical compositions of this invention may also be employed to restore p53 function in tumor cells.
  • a suitable amount of the composition of this invention is administered systemically, or locally to the site of the tumor with or without concurrent administration of conventional cancer therapy (i.e. DNA damaging agents) .
  • compositions of this invention may be administered in methods to suppress cell proliferation in diseases other than cancers, which are characterized by aberrant cell proliferation.
  • diseases include psoriasis, atherosclerosis and arterial restenosis.
  • This method is conducted by administering a suitable amount of the selected composition systemically or locally to the patient.
  • These examples illustrate the preferred method for preparing exemplary modified p53 constructs of the invention and the biological activity of the modified p53 constructs. These examples are illustrative only and do not limit the scope of the invention. 18
  • the DNA binding activity of wild-type p53 is allosterically regulated by a basic region within the C- ter inal 30 amino acids of p53.
  • Monoclonal antibodies that mask this regulatory region, such as PAb421, or deletion of this region stimulate binding to DNA [T. Halanonetis et al, EMBJO J. , 12:1021-1028 (1993) ; T.R. Hupp et al, Cell. 21:875-886 (1992) ; J.L.F. Waterman et al, EMBO J. , ⁇ :512-519 (1995)].
  • some tumor-derived mutants have also been reported to bind DNA when allosterically activated by antibody PAb421 [T. Halazonetis and A.
  • Proteins corresponding to a) to h) each containing a deletion of the C-terminal 30 amino acid of human p53 ( ⁇ 364-393) , were also generated [SEQ ID NOS: 17-24]. These deletions permit in vitro DNA binding.
  • Plasmid pBC/TKseap has one copy of oligonucleotide BC [Halazonetis, EMBO J. , 12:1021-1028 (1993) cloned in the Eco RV site of pTKseap [Waterman, 1996] and expresses secreted alkaline phosphatase in a p53-responsive manner.
  • an Arg side chain introduced at position 284 could form electrostatic interactions with the phosphate oxygen atoms of DNA closest to its ⁇ -carbon and without violating bond lengths and angles. Modeling was performed with Quanta 4.1 (Molecular Simulations Inc., Burlington, MA) .
  • Example 1 All the proteins of Example 1 containing the 30 amino acid C-terminal deletion were expressed by in vitro translation and assayed for DNA binding using 0.2 ng 32 P- labeled DNA and, where indicated below, 100 ng unlabeled competitor DNA [J. L. F. Waterman et al, EMBO J. , 14: 512- 519 (1995)], The analysis was restricted to the C- terminally truncated proteins because full-length p53 translated in vitro is in a latent state and cannot bind DNA unless activated by a C-terminal truncation or by a monoclonal antibody (PAb421) that binds to the p53 C- terminus [Waterman et al, cited above].
  • PAb421 monoclonal antibody
  • Oligonucleotide BC which has the following sequence (top strand) is: [SEQ ID NO: 29] CC-GGGCA-TGTCC- GGGCA-TGTCC-GGGCATGT, and oligonucleotide structure of their DNA binding domain, have latent sequence-specific DNA binding activity, whereas Clas ⁇ II mutants, which have unfolded DNA binding domains [C. A. Finlay et al, Mol. Cell. Biol.
  • residues of the DNA binding domain of p53His273 were replaced with basic amino acids.
  • the substitutions targeted essentially all the residues close to the DNA backbone, except for those that already contact DNA or those that unequivocally stabilize the three-dimensional structure of p53 [Cho, cited above].
  • the targeted residues were: Glyll7, Thrll ⁇ , Alall9, Asn247, Thr284, Glu285 and Glu287.
  • Substitution of Thr284 with Arg enhanced binding of p53His273 to the high affinity DNA ⁇ ite, although binding wa ⁇ still dependent on allosteric activation by antibody PAb421 (Fig. 3) .
  • Substitution of Thr284 with Lys also enhanced binding of p53His273 to the high affinity DNA ⁇ ite, but less than sub ⁇ titution of 22
  • the Class I p53 mutants had either weak (p53Hi ⁇ 273) or no (p53Gln248 and p53Cys273) transcriptional activity. However, their transcriptional activity was enhanced to wild-type levels by the Thr284 to Arg substitution or, for p53Gln248, by combining the Thr284 to Arg substitution with C-terminal allosteric activation (Fig. 1) .
  • Tumor suppre ⁇ ing activity was tested in a colony formation assay, by cotransfecting Saos-2 osteosarcoma cells with 5 ⁇ g of pSV2hp53 expres ⁇ ion pla ⁇ mid directing p53 expre ⁇ ion, 0.5 ⁇ g of pSV7neo, a plasmid that confers neomycin/G418 resistance [K. Zhang et al, Proc. Natl. Acad. Sci. USA. 82:6281-6285 (1990)] and 24 ⁇ g of pBC12/PLseap [T. D. Halazonetis, Anticancer Res. , 11:285-292 (1992)], a carrier plasmid.
  • the transfected cell ⁇ were ⁇ elected for G418 re ⁇ i ⁇ tance, a neomycin relative. Two weeks later the colonies were stained with cry ⁇ tal violet and counted. High tumor suppressor activity corresponds to low colony formation.
  • the proteins containing the Arg284 modification ⁇ uppre ⁇ ed tumor colony formation more efficiently than the corresponding proteins without the Arg284 modification (Table 1) .
  • the magnitude of the effect is greater for the tumor-derived p53 mutants; however, even Ep21, which has the following sequence: [SEQ ID NO: 30] CCC-GAACA-TGTCC-CAACA-TGTTG-GGG, each contain a p53 binding site, which is underlined.
  • the BC oligonucleotide has a high affinity p53-binding site, while oligonucleotide Ep21 contains a lower affinity site, which is present in the regulatory sequences of the p21 gene [W. S.
  • Oligonucleotide Egadd45 has the sequence [SEQ ID NO: 31] ACA-GAACA-TGTCT-AAGCA-TGCTG-GGGA.
  • Oligonucleotide TF3 which contains three tandem repeats of [SEQ ID NO: 32] ATCACGTGAT, is a non-specific DNA [Halazonetis et al, EMBO J. , 11:1021-1028 (1993)].
  • Thr284 to Arg substitution enhanced binding of all p53 proteins examined (Fig. 4) .
  • oligonucleotides BC and Ep21 for wild-type p53 the effect is evident with oligonucleotides BC and Ep21, for p53Gln248 it is evident with oligonucleotide BC, for p53His273 and p53Cys273 it is evident with all oligonucleotides tested (Fig. 4) .
  • Example 3 Transcription and Tumor Suppres ⁇ ion A ⁇ ay ⁇
  • the proteins of Example 1 were examined for their transcriptional activity and tumor suppres ⁇ or activity. Wild-type p53 activate ⁇ transcription of target genes and suppresses tumor growth, whereas tumor- derived mutants lack both these activities [S.E. Kern et al, Science. 256: 827-830 (1992) ; C. A. Finlay et al, Cell, 5_7:1083-1093 (1989)].
  • the transcriptional activities of wild-type p53 and various p53 mutants were as ⁇ ayed with a p53-responsive reporter pla ⁇ mid in Saos-2 human o ⁇ teo ⁇ arcoma cells, which lack endogenous p53 [M.J.F.
  • transcriptional activity was determined by transfecting Saos-2 cell ⁇ with 2.5 ⁇ g pSV2hp53 expre ⁇ ion plasmid and 27.5 ⁇ g pBC/TKseap or pTKseap reporter pla ⁇ ids [Waterman et al, Cancer Res. , 24
  • MOLECULE TYPE DNA (genomic)
  • GGT TCT AAA TCA ACC AAC GAA AAT GTA TCT GCT TCC ACT TCT 879 Gly Ser Lys Ser Thr Asn Glu Asn Val Ser Ala Ser Thr Ser
  • MOLECULE TYPE DNA (genomic)
  • AAAAATTTCC GACTTTAAAT ACGGAAGATA AATACTCCAA CCTTTTTTTC 100
  • GAAAACTGTC AGTTTTTTGA AGAGTTATTT GTTTTGTTAC CAATTGCTAT 600
  • Lys Gin Arg Ser lie Pro Leu Ser Pro Ile Val Pro Glu Ser Ser
  • AAA CGT GCT AGA AAC ACT GAA GCC GCC AGG CGT TCT CGT GCG 1509 Lys Arg Ala Arg Asn Thr Glu Ala Ala Arg Arg Ser Arg Ala 235 240 245
  • MOLECULE TYPE protein Glu Ala Leu Glu Leu Lys Asp Ala Gin Ala Gly Lys Glu Pro Gly

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Abstract

La présente invention concerne une protéine p53 modifiée ou un peptide comportant une liaison ADN dans laquelle le groupe 284 d'acide aminé d'une protéine p53 ou d'un fragment de protéine p53 se change en arginine ou lysine. L'invention concerne également des séquences nucléotides codant la protéine modifiée et des vecteurs capables de l'exprimer.
PCT/US1996/015188 1995-09-22 1996-09-20 Constructions de p53 modifiees et utilisations leur convenant WO1997010843A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2000022115A3 (fr) * 1998-10-13 2000-09-21 Univ Texas Dosages permettant d'identifier des alterations fonctionnelles dans le gene suppresseur de tumeur p53

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US5362623A (en) * 1991-06-14 1994-11-08 The John Hopkins University Sequence specific DNA binding by p53
WO1995017213A1 (fr) * 1993-12-21 1995-06-29 Sloan-Kettering Institute For Cancer Research Fragments polypeptidiques a base de p53, molecules d'acides nucleiques codant ces derniers et utilisations pertinentes
US5573925A (en) * 1994-11-28 1996-11-12 The Wistar Institute Of Anatomy And Biology P53 proteins with altered tetramerization domains

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US5362623A (en) * 1991-06-14 1994-11-08 The John Hopkins University Sequence specific DNA binding by p53
WO1995017213A1 (fr) * 1993-12-21 1995-06-29 Sloan-Kettering Institute For Cancer Research Fragments polypeptidiques a base de p53, molecules d'acides nucleiques codant ces derniers et utilisations pertinentes
US5573925A (en) * 1994-11-28 1996-11-12 The Wistar Institute Of Anatomy And Biology P53 proteins with altered tetramerization domains

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CELL, 27 November 1992, Vol. 71, HUPP et al., "Regulation of the Specific DNA Binding Function of p53", pages 875-886. *
SCIENCE, 05 July 1991, Vol. 253, HOLLSTEIN et al., "p53 Mutations in Human Cancers", pages 49-53. *
THE EMBO JOURNAL, 1993, Vol. 12, No. 13, HALAZONETIS et al., "Conformational Shifts Propagate from the Oligomerization Domain of p53 to its Tetrameric DNA Binding Domain and Restore DNA Binding to Select p53 Mutants", pages 5057-5064. *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000022115A3 (fr) * 1998-10-13 2000-09-21 Univ Texas Dosages permettant d'identifier des alterations fonctionnelles dans le gene suppresseur de tumeur p53
US6429298B1 (en) 1998-10-13 2002-08-06 Board Of Regents, The University Of Texas System Assays for identifying functional alterations in the p53 tumor suppressor

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