EP1212418A2 - P193 proteine und nuklene säure, und deren anwendung - Google Patents

P193 proteine und nuklene säure, und deren anwendung

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Publication number
EP1212418A2
EP1212418A2 EP00957721A EP00957721A EP1212418A2 EP 1212418 A2 EP1212418 A2 EP 1212418A2 EP 00957721 A EP00957721 A EP 00957721A EP 00957721 A EP00957721 A EP 00957721A EP 1212418 A2 EP1212418 A2 EP 1212418A2
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European Patent Office
Prior art keywords
cell
protein
seq
acid sequence
cells
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French (fr)
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Loren J. Field
Shih-Chong Tsai
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Indiana University Research and Technology Corp
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Indiana University Research and Technology Corp
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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
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to cell physiology, and more particularly to cell cycle regulatory proteins. Specifically, the present invention relates to a novel apoptosis associated protein designated pi 93 and modified forms thereof; to nucleotide sequences encoding pi 93 proteins; and to products and processes involved in the cloning, preparation and expression of nucleotide sequences encoding p 193.
  • apoptosis can be induced by at least two independent regulatory pathways.
  • the first pathway relies on direct activation of the death receptors (members of the tumor necrosis factor receptor superfamily, reviewed in Ashkenazi, A. et al. (1998) Science 281, 1305-1308).
  • the death receptors members of the tumor necrosis factor receptor superfamily, reviewed in Ashkenazi, A. et al. (1998) Science 281, 1305-1308.
  • FADD Fis-associated death domain
  • Apoptosis can also be regulated through the activities of Bcl-2 family members (reviewed in Adams, J.M. et al. (1998) Science 281, 1322-1326).
  • Bcl-2 The prototypical family member, Bcl-2, was originally identified as a gene activated by chromosomal translocation in some human lymphomas (Tsujimoto, Y. et al. (1984) Science 226, 1097-1099; Bakhshi, A. et al. (1985), Cell 41, 899-906; Cleary, M.L. et al. (1986) Cell 47, 19-28). Subsequent analyses have identified a family of approximately 20 proteins which share homology to Bcl-2 at one or more domains (known as Bcl-2 Homology domains BH1 through BH4). Functional analyses have shown that family members with the greatest homology to Bcl-2 tend to promote cell survival while those more distantly related tend to promote apoptosis.
  • the pro-apoptosis group is further subdivided into the Bax sub-family (which contain BH1, 2 and 3 domains, see Oltvai, Z.N. et al. (1993) Cell 74, 609-619; Chittenden, T. et al. (1995) Nature 374, 733-736; Kiefer, M.C., et al. (1995) Nature 374, 736-739; Farrow, S.N. et al. (1995) Nature 374, 731-733; Hsu, Y.T. et al. (1997) Proc. Natl. Acad. Sci.
  • the Bax sub-family which contain BH1, 2 and 3 domains, see Oltvai, Z.N. et al. (1993) Cell 74, 609-619; Chittenden, T. et al. (1995) Nature 374, 733-736; Kiefer, M.C., et al. (1995) Nature 374,
  • apoptosis Commitment to apoptosis is governed, at least in part, by the relative levels of pro-survival and pro-apoptosis Bcl-2 family members which, in turn, regulate the activity of Apaf-1 (an activator of caspase 8).
  • the caspase family of cysteine proteases are the downstream effectors of apoptosis, regardless of the initial regulatory pathway. Once activated, the caspases effect cell death by initiating a proteolytic cascade which destroys cellular organelles thereby giving rise to distinct morphologic changes which are diagnostic for apoptosis (reviewed in Thornberry, N.A. et al. (1998) Science 281, 1312-1316). These include nuclear condensation, fragmentation of DNA at nucleosomal junctions, mitochondrial disintegration and ultimately autolysis of the cell.
  • the DNA tumor virus oncoproteins have provided a more useful model system with which to dissect the molecular regulation of cell growth and death.
  • the transforming activities of these proteins reside largely in their ability to bind to, and thereby alter the activity of, endogenous cell cycle and cell death regulatory proteins (reviewed in Ludlow, J.W. et al. (1995) Virus Research 35, 113-121; and Moran, E. (1993) FASEB Journal 7, 880-885).
  • T Antigen T-Ag
  • amino acid residues 105 through 115 are required for binding to members of the Retinoblastoma family (RB and the related proteins pi 07 and pi 30, see DeCaprio, J.A. et al. (1988) Cell 54, 275-283; Ewen, M.E. et al. (1991) Cell 66, 1155-1 164; Li, Y. et al. (1993) Genes Dev. 7, 2366-2377; and Hannon, G.J. et al. (1993) Genes Dev. Dec. 7, 2378-2391).
  • T-Ag/RB binding blocks sequestration of E2F family members (which are maintained in an inactive state by binding to RB).
  • T-Ag/p53 binding prevents transcriptional activation of these genes, and concomitantly inhibits their activities (Bates, S. et al. (1999) Cell. & Mol. Life Sci. 55, 28-37; and Ko, L.J. et al. (1996) Genes Dev. 10, 1054-1072).
  • FIG. 1 Immune complex from metabolically labeled AT-2 cardiomyocytes generated with anti-T-AG or anti-p53 monoclonal antibodies. pl93 is present in anti-T-AG (lane 3) and anti-p53 (lanes 2 and 6) immune complex from 35 S -methionine labeled AT-2 cardiomyocytes, but not in immune complex prepared with IgG subtype-matched nonspecific control antibodies (lanes 1 and 5), nor in controls lacking primary antibody (lane 4). Molecular weight standards are indicated on the left, (b) PSD MALDI mass spectrum and sequence of a p 193 tryptic peptide. The b and y ions and immonium ions that were detected are shown, (c) Schematic diagram of pi 93 protein and cDNAs. The positions of several structural motifs are shown. Horizontal black lines indicate the relative position of the cDNA clones.
  • FIG. 1 (a) Deduced amino acid sequence of pl93. Underlined sequences correspond to the peptides identified by PSD mass spectrometry. Bold sequence corresponds to the BH3 domain homology. (b) Comparison of the BH3 domain in pl93 and several other apoptosis regulatory proteins.
  • FIG. 3 (a) pl93 binds to T-Ag in NIH-3T3 cells. Protein prepared from cells co-transfected with CMV-pl93myc (which encodes a pi 93 protein harboring a c-terminal myc epitope tag) and CMV-T-Ag (which encodes SV40 T-Ag) was reacted with the indicated antibodies, and the resulting immune complex was analyzed by Western blotting using anti-myc and anti-T-Ag antibodies. Tfx, transfection; Tot. Pro., total protein; IP, immune precipitation, (b) In vitro translated pi 93 binds to recombinant T-Ag.
  • CMV-pl93myc which encodes a pi 93 protein harboring a c-terminal myc epitope tag
  • CMV-T-Ag which encodes SV40 T-Ag
  • Radiolabeled in vitro translated pi 93 was mixed with recombinant T-Ag, and then reacted with the indicated antibodies.
  • the resulting immune complexes were displayed on a polyacrylamide gel and transferred to nylon membranes.
  • pl93 was visualized by autoradiography, and T- Ag was visualized by Western blot, (c) Northern blot analysis of pl93 expression in adult mice.
  • Total RNA (10 micrograms) prepared from the indicated tissues was probed with a full-length pi 93 cDNA. The integrity of the RNA samples was confirmed by staining the Northern blots with methylene blue (lower panel).
  • FIG. 4 pi 93 binds to the N-terminus of T-Ag.
  • the schematic diagram depicts the T-Ag constructs used in the mapping experiments. These products were translated in vitro and mixed with in vitro translated full length pi 93. Immune complex generated with anti -T-Ag antibody PAb419 was resolved on a polyacrylamide gel and visualized by autoradiography. Construct l-92myc encoded a myc epitope-tag at the C-terminus.
  • FIG. 1 pi 93 promotes apoptosis.
  • NIH-3T3 cells were transfected as indicated; the total number of pi 93 positive cells at 68 hours post-transfection is shown. Also note that cells transfected with the pl93deltaBH (which harbors a deletion spaning the pi 93 BH3 domain) are viable.
  • FIG. 7 (A). NIH-3T3 colony growth assay with expression constructs encoding pi 93 in the sense (CMV-pl93s) and anti-sense (CMV-pl93as) orientation. Expression vector lacking insert (CMV-null) was used as a control.
  • Figure 8. (A). Structure of CMV expression vectors with nested pl93 C- terminal truncations, as described in Example 4. (B). Colony growth assay using expression constructs of Figure 8A, as described in Example 4. (C) DNA fragmentation studies confirming that pl93dn encodes dominant negative activity which blocks MMS-induced apoptosis, as described in Example 4.
  • FIG. 9 Schematic diagram of MHC-pl93dn transgene used to generate transgenic mice, as further described in Example 5.
  • FIG. 1 Heart sections showing myocardial damage in response to isoproterenol infusion in control and MCH-pl93dn transgenic mice, obtained as described in Example 6. Sections were stained with sirius red (which reacts with collagen to produce a dark signal) and counterstained with fast green (which reacts with cardiomyocytes to produce a light signal).
  • FIG 12. ES cell-derived cardiomyocyte colony growth assay showing the effects of p53dn, pl93dn, and El A gene expression, alone or in combination, as further described in Example 7.
  • Figure 13. (A). Western blot analysis of protein prepared from the ES cell- derived cardiomyocyte colony growth assay shown in Figure 12 with anti-ElA or anti-T-Ag antibodies; (B). DNA fragmentation studies showing that E1A expression in the absence of co-expression of both pl3dn and pl93dn induced apoptosis (see Example 7).
  • Figure 15 Colony growth assay demonstrating that isoproternol induces growth in cardiomyocytes which co-express 193dn and p53dn, as described in Example 9.
  • a feature of the present invention is the identification and characterization of an apoptosis associated protein, designated pi 93.
  • pi 93 is a SV40 T-Ag binding protein and appears to be a new member of the BH3 only pro-apoptosis family. This is supported by the observation that pi 93 expression promoted a prompt apoptotic response in NIH-3T3 cells. Immune cytologic analysis indicated that pi 93 is a cytoplasmic protein, and that co-expression of T-Ag resulted in the cytoplasmic localization of both proteins. pl93-induced apoptosis occurs in Gi, and pulse chase experiments revealed that T-Ag is also localized in the cytoplasm (albeit transiently) at the same point of the cell cycle. The data are consistent with the conclusion that T-Ag possesses an anti-apoptosis activity, independent of p53 sequestration, which is actuated by T-Ag/pl93 binding in the cytoplasm.
  • one aspect of the present invention concerns a method for modifying the cell cycle of a cell which involves modulating the level of pi 93 protein within the cell and/or interfering with the pi 93 protein signal transduction pathway in the cell.
  • increasing the wild-type pro-apototic pi 93 activity can be used to induce apoptosis
  • decreasing the level of pro-apoptotic pi 93 activity in the cell can be used to suppress apoptosis and/or promote cellular proliferation.
  • Increases in pro-apoptotic pi 93 activity can be achieved, for example, by expression of introduced DNA encoding a pro-apoptotic pi 93 protein.
  • Decreases in pro-apoptotic pi 93 activity can be achieved, illustratively, by decreasing the level of expression of the native pi 93 of the cell (e.g. by antisense technology), and/or by interference with the pathway through which the native pi 93 acts, for example by the introduction of a dominant negative pi 93 protein which antagonizes at least a portion of the biological function of the native pl93 protein.
  • methods for modifying the cell cycle of a cell include decreasing the level of expression of the native pi 93 protein of the cell and/or interfering with the pi 93 pathway, in conjunction with decreasing the level of expression of p53 protein in the cell or interfering with the p53 pathway, and/or in conjunction with increasing the level of expression of El A protein in the cell.
  • the present invention provides an expression vector including nucleic acid encoding a pi 93 polypeptide.
  • Such vectors can be used in inventive methods to genetically transduce host cells, and in the case of pro- apoptotic pi 93 polypeptides to induce apoptosis in the cells.
  • pi 93 polypeptides with a dominant negative character such transduction may be used to effectively suppress apoptosis or induce proliferation.
  • Another preferred embodiment of the invention provides an isolated pi 93 protein, preferably an isolated, recombinant pi 93 protein.
  • Such proteins can be combined with an appropriate pharmaceutically acceptable carrier to produce pharmaceutical compositions, also constituting a part of the present invention.
  • Such proteins can also be used in the preparation of inventive antibodies to pi 93.
  • the present invention also concerns a method for producing a pi 93 protein, comprising culturing a host cell having introduced DNA encoding a pi 93 protein under conditions suitable from expression of said introduced DNA.
  • the present invention provides a newly characterized apoptosis associated protein designated pi 93, and novel modified pi 93 proteins, including those exhibiting a dominant negative character; nucleotide sequences encoding such pi 93 proteins; products and processes involved in the cloning, preparation and expression of nucleotide sequences encoding pi 93 proteins; methods and materials for modifying the cell cycle in cells, for example regulating apoptosis and/or proliferation of cells; and methods for screening for pharmacological or other chemical agents for effect on cell cycle which involve assessing their impact on pi 93 or its signal transduction pathway in cells. Additional embodiments as well as features and advantages of the invention will be apparent from the descriptions herein. DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • the present invention provides a novel apoptosis associated protein designated pi 93 and modified forms thereof; nucleotide sequences encoding pi 93 proteins; and products and processes involved in the cloning, preparation and expression of nucleotide sequences encoding pi 93 proteins.
  • SEQ. I.D. NO. 1 shows the nucleotide sequence and deduced amino acid sequence (see also SEQ. I.D. NO. 2) for mouse pi 93 as utilized in the Examples herein.
  • SEQ. I.D. NO. 3 shows the nucleotide sequence and deduced amino acid sequence (see also SEQ. I.D. NO. 4) for human pl93.
  • nucleotide sequence is intended to refer to a natural or synthetic sequential array of nucleotides and/or nucleosides, and derivatives thereof.
  • amino acid sequence is intended to refer to a natural or synthetic sequential array of amino acids and/or derivatives thereof.
  • encoding and “coding” refer to the process by which a nucleotide sequence, through the mechanisms of transcription and translation, provides the information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce a polypeptide.
  • nucleotide sequences and amino acid sequences which differ from the specific pi 93 sequences disclosed herein, but which have substantial identity thereto and exhibit pro-apoptotic or proliferative activities as identified herein.
  • sequences will be considered to provide pi 93 nucleic acid and pi 93 proteins for use in the various aspects of the present invention.
  • nucleic acid sequences encoding variant amino acid sequences are within the scope of the invention. Modifications to a sequence, such as deletions, insertions, or substitutions in the sequence, which produce "silent" changes that do not substantially affect the functional properties of the resulting polypeptide molecule are expressly contemplated by the present invention.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine can also generally be expected to produce a biologically equivalent product.
  • modifications to the pi 93 sequence which substantially affect the functional properties of the resulting polypeptide can be made, and such changes are also expressly contemplated by the present invention.
  • modifications of the pi 93 amino acid sequence can be used to produce dominant-negative pi 93 proteins which antagonize at least a portion of the wild-type pi 93 activity, and which lead to suppression of apoptotic activity in the cells and/or an enhanced proliferative capacity of the cells.
  • nucleic acid e.g. DNA
  • nucleic acid may be used that has a coding sequence that differs from that set forth in SEQ. I.D. NO. 1 (nucleotides 62-5128) or SEQ. I.D. NO. 3 (nucleotides 87-5183), wherein the nucleic acid, or at least the coding portion thereof, will bind to nucleic acid having nucleotides 62-5128 of SEQ. I.D. NO. 1 or nucleotides 87-5183 of SEQ. I.D. NO. 3, or at least about nucleotides 62-3517 of SEQ. I.D. NO. 1 or about nucleotides 87-3615 of SEQ.
  • stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60°C.
  • nucleic acid may be used that encodes a polypeptide that has an amino acid sequence which has at least about 70% identity, more preferably at least about 80% identity, most preferably a least about 90% identity, with the amino acid sequence set forth in SEQ. I.D. NO. 2 or in SEQ. I.D. NO. 4, or with at least one significant length (i.e. at least 40 amino acid residues) segment thereof, and which polypeptide possesses a pro-apoptotic pi 93 activity or a dominant-negative pi 93 character.
  • the polypeptide may, for example, have an amino acid sequence which has at least about 70% , 80%, or 90% identity with at least about amino acid residues 1-1 152 of SEQ. I.D. NO.
  • polypeptides especially when a functional pro-apoptotic protein is desired, will preferably include the characteristic pi 93 BH3 domain occurring at residues 1566 to 1572 of SEQ. I.D. NO. 2 or at residues 1575 to 1581 of SEQ. I.D. NO. 4:
  • Percent identity is intended to mean percent identity as determined by comparing sequence information using the advanced BLAST computer program, version 2.0.8, available from the National Institutes of Health, USA.
  • the BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68 (1990) and as discussed in Altschul, et al., J. Mol. Biol 215:403-10 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-7 (1993); and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.
  • the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences.
  • the program may be used to determine percent identity over the entire length of the proteins being compared.
  • the program also uses an SEG filter to mask-off segments of the query sequence as determined by the SEG program of Wootton and Federhen Computers and Chemistry 17: 149-163, (1993).
  • nucleic acid may be used that includes a coding sequence that has at least about 70% identity with the coding portion of the nucleotide sequence set forth in SEQ. I.D. NO. 1 (nucleotides 62 to 5128) or in SEQ. I.D. NO. 3 (nucleotides 87 to 5183), or with at least one significant length (i.e. at least 100 nucleotides) segment thereof, and which nucleic acid encodes a polypeptide possessing pro-apoptotic pi 93 activity or dominant-negative 193 activity as identified herein.
  • the nucleic acid may, for example, have a coding sequence which has at least about 70% at least about 80%, or at least about 90%, identity with nucleotides 62 to 5128 of SEQ. I.D. NO. 1 or with nucleotides 87 to 5183 of SEQ. ID. NO. 3, or at least with about nucleotides 62 to 3517 of SEQ. I.D. NO. 1 or about nucleotides 87 to 3615 of SEQ. I.D. NO. 3.
  • the pi 93 nucleotide sequence may be operably linked to a promoter sequence as known in the art to provide recombinant nucleic acid useful in a variety of applications including, for example, in the provision of vehicles such as vectors for functionally introducing the nucleic acid in to mammalian or other eukaryotic cells, such as cardiomyocytes, hepatocytes, smooth muscle cells, hemotpoietic stem cells, tumorogenic cells, and the like.
  • a nucleotide sequence is "operably linked" to another nucleotide sequence (e.g. a regulatory element such as a promoter) when it is placed into a functional relationship with the other nucleotide sequence.
  • a nucleotide sequence is operably linked to a promoter sequence
  • this generally means that the nucleotide sequence is contiguous with the promoter and the promoter exhibits the capacity to promote transcription of the gene.
  • promoters are known in the art, including cell-specific promoters, inducible promoters and constitutive promoters.
  • the promoters may be selected so that the desired product produced from the nucleotide sequence template is produced constitutively in the target cells.
  • promoters such as inducible promoters, may be selected that require activation by activating elements known in the art, so that production of the desired product may be regulated as desired.
  • promoters may be chosen that promote transcription of the gene in one or more selected cell types, e.g. the so-called cell-specific promoters.
  • Expression vectors in accordance with the present invention can be designed to effectively increase wild-type pi 93 activity in a cell thus inducing apoptosis, or to interfere with wild-type pi 93 activity in a cell thus suppressing apoptosis and/or inducing proliferation.
  • expression vectors incorporating nucleic acid encoding a pro-apoptotic pi 93 polypeptide can be employed to increase apoptotic activity in a cell.
  • vectors incorporating nucleic acid encoding a modified pi 93 polypeptide for example truncation mutants of pi 93 exhibiting activity consistent with dominant negative (pl93dn), can be used to interfere with wild-type pi 93 activity and thereby suppress apoptosis in the cell and/or induce proliferation of the cell.
  • Genetic transduction of cells with vectors incorporating antisense (as) pi 93 nucleotide sequences can also be used to effectively suppress apoptotic activity and/or induce proliferation in the cells.
  • pi 93 antisense RNA may be administered to cells so as to decrease pi 93 and apoptotic activity and/or induce proliferation in the cells.
  • the pi 93 nucleotide sequence is operably linked to a cell-specific promoter, for example, providing for constitutive expression of the nucleotide sequence in a selected cell type.
  • a cell-specific promoter for example, providing for constitutive expression of the nucleotide sequence in a selected cell type.
  • Illustrative candidates for such promoters include cardiomyocyte-specific promoters such as the ⁇ -myosin heavy chain ( ⁇ -MHC) promoter, the ⁇ -myosin heavy chain ( ⁇ - MHC) promoter, the myosin light chain-2V (MLC-2V) promoter, the atrial natriuretic factor (ANF) promoter, and the like.
  • Additional cell-specific promoters include liver-specific cells such as PePCK, albumin, transthyretin, and major urinary protein (MUP). Any cell type expressing endogenous gene, and its exressed ubiquitous, lung, heart, liver, eyes. Such constructs enable the expression of the pi 93 nu
  • Another aspect of the invention provides recombinant nucleic acid that includes a pi 93 nucleotide sequence encoding a pi 93 polypeptide operably linked to an inducible promoter.
  • the pi 93 nucleotide sequence may, for instance, encode a pro-apoptosis polypeptide, such that expression and induces of apoptosis in cells, or an apoptosis-suppressing and/or proliferation-inducing polypeptide, such that expression suppresses apoptosis and/or promotes cellular proliferation.
  • expression of the polypeptide encoded by the cells incorporating the nucleic acid can be upregulated in response to an inducing agent.
  • Illustrative candidate inducible promoter systems include, for example, the metallothionein (MT) promoter system, wherein the MT promoter is induced by heavy metals such as copper sulfate; the tetracycline regulatable system, which is a binary system wherein expression is dependent upon the presence or absence of tetracycline; a glucocorticoid responsive promoter, which uses a synthetic sequence derived from the glucocorticoid response element and is inducible in vivo by administering dexamethasome (cells having the appropriate receptor); a muristerone-responsive promoter, which uses the ganodotropin-releasing hormone promoter and is inducible with muristerone (cells having the appropriate receptor); and TNF responsive promoters.
  • MT metallothionein
  • tetracycline regulatable system which is a binary system wherein expression is dependent upon the presence or absence of tetracycline
  • Additional inducible promoters which may be used, and which are more preferred, include the ecdysone promoter system, which is inducible using an insect hormone (ecdysone) and provides complete ligand- dependent expression in mammals; the ⁇ -GAL system, which is a binary system utilizing an E. coli lac operon operator and the I gene product in trans, and a gratuitous inducer (IPTG) is used to regulate expression; and, the RU486 inducible system, which uses the CYP3A5 promoter and is inducible by RU486, a well defined pharmaceutical.
  • ecdysone promoter system which is inducible using an insect hormone (ecdysone) and provides complete ligand- dependent expression in mammals
  • the ⁇ -GAL system which is a binary system utilizing an E. coli lac operon operator and the I gene product in trans, and a gratuitous inducer (IPTG) is used to regulate expression
  • IPTG gratuitous inducer
  • One aspect of the present invention concerns the discovery that blocking pl93 and p53 activity (by expression of dominant negative cDNA variants) protects against proliferation-induced apoptotic signals. This in turns renders cardiac myocytes responsive to the pro-proliferation signals, such signals encoded for example by El A.
  • Therapeutic approaches may be adopted which promote controlled regeneration of cardiac tissue, or alternatively controlled proliferation of engrafted cardiomyocytes, which rely upon the use of regulatable promoters to drive expression of the dominant negative cDNAs in addition to the growth promoting gene.
  • An alternative approach may rely on pharmaceutical blockade of the p53 and/or the pi 93 pathways, in conjunction with expression of growth- promoting genes in combination with a regulatable promoter.
  • HSV-TK Herpes simplex virus thymidine kinase
  • the HSV-TK gene can incorporate normal nucleotides as well as the nucleotide analog gancyclovir at a high efficiency whereas mammalian thymidine kinase does not incorporate gancyclovir into cells at high efficiency. Incorporation of gancyclovir is cytotoxic.
  • illegitimate activation of the regulatable promoter would result in expression of the anti-apoptosis (e.g. p53dn and pl93dn) and pro-growth (e.g. E1A) genes, as well as the HSV-TK gene.
  • Inappropriately growing cells e.g. those where illegitimate promoter activity has occurred) can be eliminated by simple treatment with gancyclovir.
  • the present invention also concerns vectors which incorporate a pi 93 nucleotide sequence and which are useful in the genetic transduction of cells in vitro or in vivo.
  • vector systems are suitable for these purposes. These include, for example, viral vectors such as adenovirus vectors as disclosed for example in Franz et al., Cardiovasc. Res. 35(3):560-566 (1997); Inesi et al., Am. J. Physiol 274 (3 Pt. l):C645-653 (1998); Kohout et al., Circ. Res. 78(6):971-977 (1996); Leor et al., J. Mol Cell Cardiol. 28(10):2057-2067 (1996); March et al., Clin.
  • Adeno-Associated Virus (AAV) vectors are also suitable, and are illustratively disclosed in Kaptlitt et al., Ann. Thora. Surg. 62(6): 1669- 1676 (1996); and Svensson et al., Circulation 99(2):201-205 (1999).
  • Additional viral vectors which may be used include retroviral vectors (see e.g. Prentice et al., J. Mol. Cell Cardiol. 28(1): 133-140 (1996); and Petropoulos et al., J. Virol.
  • a preferred class of expression vectors will incorporate the pi 93 nucleic acid operably linked to a cardiomyocyte-specific promoter, such as one of those identified above. Still further, AAV vectors are highly compatible for use in transfection of myocardial and other cells and tissue, and are preferred from among those identified above.
  • cells can also be genetically transduced with pi 93 nucleic acid in vitro or in vivo using liposome -based transduction systems.
  • liposomal transduction systems are known, and have been reported to successfully deliver recombinant expression vectors to a variety of cells. Illustrative teachings may be found for example in R.W. Zajdel, et al., Developmental Dynamics. 213(4):412-20 (1998); Y. Sawa, et al., Gene Therapy.5(11): 1472-80 (1998); Y. Kawahira, et al., Circulation 98(19 Suppl):II262-7; discussion II267-8 (1998); G.
  • liposomal recombinant expression vectors including pi 93 DNA can also be utilized to tranduce cells in vitro and in vivo for the purposes described herein.
  • Nucleic acid constructs can be used for example to introduce nucleotide sequences encoding a pi 93 protein into cells in vivo or in vitro, to achieve a level of intracellular pi 93 activity that is increased relative to the native level of the cells. Such increased activity can induce apoptosis in the cells. Induction of apoptotic activity can be evidenced, for example, by cell death and other characteristic morphological changes such as cell shrinkage and nuclear condensation and fragmentation.
  • purified (e.g. purified recombinant) pi 93 protein may be introduced into cells to increase pi 93 activity (e.g.
  • Nucleic acid constructs can also be used to introduce modified pi 93 nucleotide sequences into cells in vivo or in vitro, wherein the sequences provide characteristics of a dominant negative gene and effectively antagonize wild-type pi 93 activity, resulting in as for example a suppression of apoptosis and/or an increase in the proliferative capacity of the cells.
  • a dominant negative pi 93 protein or another molecule can be introduced into the cells which interferes with or antagonizes wild-type pi 93 activity, and thereby suppresses apoptosis and/or induces proliferation in the cells.
  • vectors incorporating antisense (as) pi 93 nucleotide sequences can be used, and/or small synthetic organic molecules serving as pharmacologic agents can be used, to effectively interfere with the expression of or the activity of wild-type pi 93 protein.
  • the present invention makes available methods which can be applied in vitro or in vivo for research, therapeutic, screening or other purposes.
  • Methods for the in vitro culture of cells expressing introduced pi 93 DNA can be used, for example, in the study and understanding of the cell cycle, in screening for chemical or physical agents which modulate pi 93 activity or other aspects of the cell cycle, or in the culture of cells having suppressed apoptotic activity and/or increased proliferative potential for subsequent engraftment into mammals, including humans.
  • Cells to be cultured in accordance with the invention can be derived from a variety of sources. For example, they may be harvested from a mammal for culture and subsequent engraftment into that mammal (autografts) or another mammal of the same species (allografts) or a different species (xenografts). Cardiomyocyte or other cells may also be derived from the differentiation of stem cells such as embryonic stem cells, somatic stem cells or other similar pluripotent cells. General methodology for such derivations is disclosed in U.S. Patent Nos. 5,602,301 and 5,733,727 to Field et al.
  • the genetic modification to incorporate the i 93 nucleic acid may take place at the stem cell level, for instance utilizing one or more vectors to introduce the pi 93 nucleic acid operably linked to a tissue-specific promoter, and nucleic acid enabling the selection of a target cell type from other cells differentiating from the stem cell and/or at a differentiated level e.g., including a selectable marker gene operably linked to a tissue-specific promoter. Nucleic acid enabling selection of transduced from non-transduced stem cells may also be used in such strategies. Such selection of the stem and/or differentiated cell types may be achieved, illustratively, utilizing a gene conferring resistance to an antibiotic (e.g. neomycin or hygromycin) or other chemical agent operably linked to an appropriate promoter.
  • an antibiotic e.g. neomycin or hygromycin
  • the genetic modification to incorporate the pi 93 and potentially other nucleic acid may also occur after differentiation of the stem cells.
  • a differentiated cell population enriched in cardiomyocytes or another target cell type for instance containing 90% or more of the target cell type, may be transformed with a vector having pl93 nucleic acid (especially antisense or including a dominant negative mutation) operably linked to a promoter (optionally tissue specific), as described above.
  • the same or a different vector may also be used to introduce other functional nucleic acid to the cells, for example providing a reporter gene and/or selectable marker, or providing for the expression of a growth factor and/or another cell cycle regulatory protein.
  • decreasing the level of pi 93 protein or interfering with the pi 93 signal transduction pathway can be used in conjunction with other means of effecting the cell cycle.
  • modifications of p 193 and/or its pathway effected e.g. by an introduced antisense pi 93 nucleic acid or a nucleic acid having a dominant negative mutation
  • a p53 nucleic acid especially antisense or a dominant negative mutation
  • an El A nucleic acid or a combination of the two.
  • pl93 and/or its pathway may be used in conjunction with other methods of relaxing or facilitating the GJS transit, for example by manipulating key regulators at the restriction point of the cell cycle such as inhibiting RB family members, overexpressing D-type cyclin or cyclin-dependent kinase activities, inhibiting cyclin-dependent kinase inhibitors, overexpressing downstream targets, and the like.
  • left ventricular, right ventricular, left atrial, or right atrial cardiomyocytes may be genetically modified in vitro to incorporate anti-apoptotic and/or proliferative pl93 nucleic acid using a suitable vector as disclosed above.
  • Cells to be genetically transduced in such protocols may be obtained for instance from animals at different developmental stages, for example fetal, neonatal and adult stages. Suitable animal sources include mammals such as bovine, porcine, equine, ovine and murine animals. Human cells may be obtained from human donors or from a patient to be treated.
  • the modified cardiomyocytes may thereafter be implanted into a mammal, for example into the left or right atrium or left or right ventricle, to establish a cellular graft in the mammal.
  • Implantation of the cells may be achieved by any suitable means, including for instance by injection or catheterization.
  • the cells may also be modified in vitro to contain other functional nucleic acid sequences which can be expressed to provide other proteins, for example or one or more additional cell cycle regulatory proteins.
  • cells are modified with nucleic acid encoding pi 93 and with nucleic acid encoding at least one other cell cycle regulatory protein, for example combining forced expression of pl93 and p53 dominant (Mowat, M., Nature Vol. 314, p. 633-636 (1985); Munroe, D.G. Mol. Cell. Biol., Vol. 10, 3307-3313 (1990) so as to suppress apoptosis in the cells.
  • Cells for culture, and potential implantation may also be obtained from a transgenic animal (especially mammal) expressing introduced pi 93 nucleic acid.
  • transgenic animals which harbor introduced pi 93 nucleic acid in essentially all of their cells can be raised, and used as sources for harvesting culturable cells (e.g. cardiomyocytes), tissues or organs, or may be used as animal models for research or screening purposes.
  • transgenic bovine, porcine, equine, ovine or murine animals may be used as sources for cells, tissues or organs, or as animal models for study.
  • transgenic animals having reduced levels of wild-type pi 93 protein and/or expressing an introduced dominant negative pi 93 protein can be used as a source for apoptotically-suppressed and/or proliferatively enhanced cells, tissue or organs, which will be protected against fibrosis or other similar damage. Such materials will thus possess significant advantages for use in transplantation into other animals, such as humans.
  • the present invention also provides for the genetic modification of cells in vivo to increase pi 93 activity (using pro-apoptotic protein) or decrease pi 93 activity (using pl93dn) in the cells (impacting transduction pathway).
  • An expression vector containing the pi 93 nucleic acid may be delivered to tissue of a recipient mammal, to achieve transduction of cells in the tissue.
  • the pi 93 nucleic acid in such vectors will be operably linked to a tissue-specific promoter, for instance a cardiomyocyte- specif ⁇ c promoter.
  • the delivery of the vector can be suitably achieved, for instance, by injection, catheterization, or infusion into the blood stream, or by other known means.
  • transduced cells any mode of delivery which enables the establishment of transduced cells within the recipient mammal is contemplated as being within the present invention.
  • a single delivery of the vector may be used, or multiple deliveries nearly simultaneous or over time may be used, in order to establish a substantial population of transduced cells within the recipient.
  • the transduced cells will then express the encoded pi 93 polypeptide, for instance under the control of a constitutive, inducible or tissue-specific promoter, and thereby exhibit a suppressed or induced level of apoptosis.
  • the implantation of cells cultured in vitro or the delivery of the vector for in vivo genetic transduction may be directed to a selected site or sites within the recipient.
  • site or sites may be in the left or right atrium or left or right ventricle of the recipient, or any combination of these.
  • the implantation or delivery site or sites will occur in the left or right ventricle of the recipient.
  • the site(s) may, for instance, be one(s) in which there is a need for additional viable cells, for example in a damaged or diseased area of the heart such as in cases of myocardial infarcts and cardiomyopathies.
  • the site(s) may also be targets for the delivery of other proteins such as growth factors, e.g. nerve growth or angiogenic factors, via expression in the grafted or in vivo transduced cells.
  • Cellular engraftment and/or in vivo genetic modification in accordance with the invention can be used, for example, to deliver therapy to mammals, including humans.
  • a variety of ex vivo cellular transplantation and implantation techniques and gene therapy techniques are thus contemplated as forming a part of the invention.
  • these techniques may be used to provide cells in the mammal having a reduced level of wild-type pi 93 protein and/or having a disrupted or partially disrupted pi 93 signal transduction pathway, the cells thereby exhibiting decreased apoptotic activity and/or an enhanced proliferative capacity.
  • such an approach may be used to target an improvement or protection of the contractile function of the heart of the patient, for example in the treatment of contractile losses due to infarcts or cardiomyopathies. They may also be used to target an improvement and/or protection of the function of other tissue or organs in the patient, for example the liver or lungs of the patient.
  • the use of a pi 93 protein having a dominant negative mutation will be especially advantageous for such purposes.
  • pro-apoptotic pi 93 protein delivered to cells, for example by in vivo genetic transduction with an appropriate pi 93 nucleic acid and consequent expression of the pro-apoptotic protein, can be used to promote apoptosis in cells in which apoptosis is desired, for example in the case of inappropriately proliferative cells.
  • the present invention also provides access to antibodies having specificity to one or more epitopes present on the pi 93 peptide, or an idiotype on the pi 93 (see e.g. Figure 14 and accompanying discussion in Examples).
  • Such antibodies can be polyclonal or monoclonal, and can be made with the pi 93 polypeptide or fragment thereof as the immunogen.
  • the term "antibody” (Ab) or “monoclonal antibody” (Mab) as used herein is meant to include intact molecules as well as fragments thereof capable of binding an antigen.
  • Antibodies to pi 93 can be used, for example, to detect the presence of the pi 93 protein in a human or other mammalian tissue sample.
  • This may involve contacting the sample with a detectably labeled antibody and detecting the label, thereby establishing the presence of the pi 93 protein in the sample. Detection can be carried out by imaging in vivo.
  • the pi 93 protein can also be detected by known immunoassay techniques, including, for example, RIA, ELISA, etc., using appropriate antibodies according to the invention.
  • Antibodies of the invention can be prepared by any of a variety of known methods.
  • cells expressing the pl93 protein can be administered to an animal in order to induce the production of serum containing polyclonal antibodies that are capable of binding the pi 93 protein.
  • the pi 93 protein or fragment thereof is chemically synthesized and purified by HPLC to render it substantially free of contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of high specific activity.
  • Polyclonal antibodies can be generated in any suitable animal including, for example, mice, rabbits or goats.
  • the pl93 immunogenic peptide or fragment thereof can be injected by itself or linked to an appropriate immunoactivating carrier.
  • Monoclonal antibodies can be prepared in various ways using techniques well understood by those having ordinary skill in the art.
  • monoclonal antibodies can be prepared using hybridoma technology (Kohler, et al., Nature 256:495 (1975); Kohler, et al., Eur. J. imninol. 6:511 (1976); Kohler, et al., Eur. J. Immunol. 6:292 (1976); Hammerling, et al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)); Roger H.
  • AT-2 cardiomyocytes were homogenized in 20 ml of NET, pre-cleared with protein A sepharose beads, and mixed with anti-T-Ag monoclonal antibody PAb419 (90 min., 4°C). Immune complexes were collected with Protein A- sepharose, displayed on polyacrylamide gels and visualized by staining with Coomassie Brilliant Blue. The region of the gel containing pi 93 was excised, alkylated with isopropylacetamide, and digested with F-trypsin (0.2 ⁇ g trypsin, 37°C, 17 hrs) as described ( Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Anal. Chem.
  • the peptides were then extracted with 5% formic acid/50% acetonitrile and separated on a C18 0.32 x 100 mm capillary column (LC Packing, Inc.). An aliquot of each of the isolated HPLC fractions was applied to a pre-made spot of matrix (0.5 ml of 20 mg/ml ⁇ -cyano-4- hydroxycinammic acid + 5 mg/ml nitrocellulose in 50% acetone/50% 2-propanol) on the target plate. Ions were formed by matrix-assisted laser desorption/ionization with a nitrogen laser, 337 nm.
  • Spectra were acquired with a PerSeptive Biosystems Voyager Elite time-of-flight mass spectrometer, operated in linear delayed extraction mode. Subsequently, fragment ions for selected precursor masses were obtained from post-source decay (PSD) experiments (Kaufman, R., Kirsch, D. and Spengler, B. (1994) International J. Mass Spec, and Ion Proc. 131, 355-385). Automated protein sequencing was performed on a model 470A Applied Biosystems sequencer equipped with an on-line PTH analyzer using modified cycles as described (Henzel, W.J., Grimley, C, Bourell, J.H., Billed, T.M., Wong, S.C. and Stults, J.T.
  • pi 93 cDNAs were isolated from an adult heart cDNA library generated from C3HeB/FeJ inbred mice (Kim, K.K., Daud, A.I., Wong, S.C, Pajak, L., Tsai, S.C, Wang, H., Henzel, W.J., and Field, L.J. (1996) J. Biol. Chem. 271, 29255- 29264). Plaque hybridizations, phage DNA isolation and subcloning were performed using standard methodologies (Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Sequence was determined for both strands of the cDNA clone examined using the dideoxy chain terminating approach (Sequenase, United States Biochemicals, Cleveland OH).
  • a full length pi 93 cDNA was subcloned into the pcDNA3.1/A yc-His expression vector (Invitrogen, Carlsbad CA) such that the epitope tag was incorporated into the C-terminus of the molecule (construct designated CMV-pl93myc).
  • a T-Ag cDNA was subcloned into pcDNA3.1 expression vector (which lacks the epitope tag; construct designated CMV-T-Ag).
  • NIH-3T3 cells (ATCC, Rockville MD) were co-transfected with CVM-5-Ag and CMV-pl93myc using the calcium phosphate approach (Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Protein (1 mg) prepared from the transfected cells was reacted with an anti- T-Ag (PAb419), an anti-myc (9E10, Santa Cruz Biotech.), or an IgG subtype- matched non-specific antibody (anti-GST, Pharmacia), and the resulting immune complexes were subjected to Western blot analysis.
  • TNT kit 35 S-methionine labeled in vitro transcription/translation (TNT kit, Promega) product obtained from a full length pi 93 cDNA subcloned into pBluescript IISK (Stratagene, LaJolla CA) was mixed with 1.2 ⁇ g of recombinant SV40 T-Ag (Molecular Biology Resource), and reacted with anti-T-Ag (PAb419) or an IgG subtype-matched nonspecific control antibody (anti-MAP kinase #D2, Santa Cruz Biotech.). Immune complex was then visualized via autoradiography (pi 93) or Western blotting (T-Ag) as described above.
  • RNA was denatured with glyoxal, displayed on agarose gels, transferred to Genescreen (NEN) and reacted with a nick-translated full-length pi 93 cDNA as described (Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
  • N-Ag N-terminal specific anti-T-Ag monoclonal antibody
  • NIH-3T3 cells transfected with either CMV- ⁇ - GALmyc or CMV-pl93myc were labeled with Hoechst, trypsinized and fixed in 5% acetic acid in ethanol, rehydrated in PBS and reacted with FITC-conjugated anti-myc antibody (9E10, Oncogene Sciences) as described (Esser, C, Gottlinger, C, Kremer, J., Hundeiker, C, and Radbruch, A. (1995) Cytometry 21, 382-386; Brown, D.R., Thomas, C.A., and Deb, S.P. (1998) EMBO , 17, 2513-2525).
  • FITC-positive cells were then analyzed on a Becton Dickinson FACS-PLUS instrument. Immune cytologic analyses were as described (44), and images were captured using a BioRad laser scanning confocal microscope or photographed directly using conventional light or fluorescent microscopy.
  • NIH-3T3 cells synchronized by two rounds of serum depletion (starvation media contained 0.1 % FBS in DMEM) were transfected with either CMV-pl93myc or CMV- ⁇ GALmyc using Lipofectin (Gibco-Life Sciences, Grand Island NY) for 24 hrs. The cultures were then rinsed with PBS, and cultured for an additional 6 hrs in starvation media.
  • immune complexes were generated using protein prepared form 35 S -methionine labeled AT- 2 cells, a cell line derived form the transgenic heart tumors (Daud, A. I., Lanson, N.A., Jr., Claycomb, W.C, and Field, L.J. (1993) Am. J. Physiol 264, H1693- 700). Proteins with apparent molecular weights of 380, 193 and 120 kd (see Figure la) were detected in immune complex generated with either anti-T-Ag (PAb419, lane 3) or anti-p53 (PAb421 and PAb246, lanes 2 and 6, respectively) monoclonal antibodies.
  • pi 93 was homologous to a previously identified open reading frame of unknown function isolated from a human immature myeloid cell line (Nomura, N procedura Nagase, T., Miyajima, N., Sazuka, T., Tanaka, A., Sato, S., Seki, N., Kawarabayasi, Y., Ishikawa, K., and Tabata, S. (1994) DNA Res. 1, 251-262). Reverse transcriptase-polymerase chain reaction was used to generate a short cDNA clone spanning the region homologous to the largest pi 93 peptide. This clone was then used to screen an adult mouse heart cDNA library.
  • pl93myc was detected in anti-T-Ag immune complex, and T-Ag was detected in anti-myc immune complex. Neither protein was present in immune complex generated with an AgG subtype-matched nonspecific control antibody. Immune Precipitation analyses of mixtures of in vitro translated pi 93 and recombinant T-Ag were also performed ( Figure 3b). Radiolabeled pi 93 was present in immune complex generated with anti-T-Ag antibody, but not in immune complex generated with an IgG subtype-matched nonspecific control antibody, confirming that the 193 kd T- Ag binding protein was successfully cloned.
  • Northern blots revealed a somewhat restricted pattern of pi 93 expression in adult mouse tissues (Figure 3c). Relatively high levels of pi 93 mRNA were detected in the heart, as might be anticipated given that the protein was originally identified in cell lines derived from cardiac tumors.
  • pi 93 binds to the N-terminus of T-Ag
  • in vitro translation products from a series of T-Ag deletion constructs were mixed with full length in vitro translated pl93, and immune complexes generated with anti-T-Ag antibody were resolved on polyacrylamide gels and visualized by autoradiography (Figure 4).
  • pi 93 was present in immune complex generated with T-Ag mutants with deletions encompassing as much as amino acid residues 147 through 708, indicating that the pi 93 binding site resides within T-Ag amino acid residues 1 through 147.
  • pl93 was not present in immune complex generated with a T-Ag mutant in which amino acid residues 92 through 708 where deleted, indicating that the C-terminal boundary of the binding site lies within T-Ag amino acid residues 107 and 108 which disrupt binding of RB family members did not effect pl93 binding ( Figure 4, construct 1-147 ARB).
  • pl93 binds to the N- terminal region of T-Ag distinct from the RB family member binding site.
  • NIH-3T3 cells were transfected with either CMV- ⁇ GALmyc (an expression construct encoding ⁇ - galactosidase with a myc -epitope tag) or CMV-pl93myc. At 48 hrs.
  • Bax a well characterized pro-apoptosis protein, undergoes a similar cytoplasmic to perinuclear redistribution during apoptosis (Hsu, Y.T., Wolter, K.G., and Youle, R.J. (1997) Proc. Natl. Acad. Sci. USA 94, 3668-3672).
  • pl93myc expressing cells are viable if cell cycle progression is blocked; >90% of pi 93 expressing cells were viable at 40 hrs. post transfection if maintained under low serum conditions. This suggests that some degree of cell cycle progression is needed to actuate apoptosis.
  • NIH-3T3 cells were transfected with CMV-pl93myc alone or co- transfected with CMV-pl93myc and CMV-B1C-X L (a construct encoding human Bcl-XJ.
  • the preponderance of cells transfected with pi 93 alone were dead at 68 hrs. post transfection, whereas co-transfection with BC1-X L markedly antagonized pl93-induced apoptosis (Figure 5f).
  • the CMV-p 193myc expression construct was modified such that amino acid residues 1563 through 1576 were deleted (VRILKAHGDEGLHV). This modification resulted in the deletion of the BH3 motif (amino acid residues 1566- 1572,), and the resulting construct was designated CMV-p 193 [delta symbol]BH.
  • NIH-3T3 cells transfected with CMV-p 193 [delta symbol]BH were viable ( Figure 5f). Indeed, the survival was similar to that obtained for cells co-transfected with CMV-p 193 plus CMV-Bcl-XL.
  • the BH3 domain is required for pi 93- mediated apoptosis.
  • T-Ag is transiently localized in the cytoplasm during M and Gj_
  • pi 93 a T-Ag binding protein present in the AT-2 cardiomyocyte tumor cell line
  • pl93-induced apoptosis can be antagonized by co-expression of pro-survival Cl-2 family members (in our case, Bcl-X
  • pl93 differs markedly in size as compared to other BH3 only family members; the next largest family member, BID, is only 21.95 kd (Wang, K., Yin, X.M., Chao, D.T., Milliman, CL, and Korsmeyer, S.J. (1996) Genes Dev.
  • T-Ag Co-expression of T-Ag antagonizes pl93-induced apoptosis in transiently transfected cells, and results in the cytoplasmic sequestration of both proteins. Moreover, T-Ag is localized in the cytoplasm of AT-2 cardiomyocytes during GI, the same point of the cell cycle where pi 93 induces apoptosis.
  • DnaJ binds to members of the 70 kd heat shock protein family, and this complex facilitates correct protein folding, formation of multi-protein complexes, and protein transport across intracellular membranes (Gething, M.J. and Sambrook, J. (1992) Nature 355, 33-45).
  • BH3 only proteins Bik and BNIP-3 (as well as Bax and Bak, pro-apoptosis proteins containing BH1, BH2 and BH3 domains) are able to bind to adenoviral E1B 19K protein (Farrow, S.N., White, J.H., Martinou, I., Raven, T., Pun, K.T., Grinham, C.J., Martinou, J.C, and Brown, R.
  • pi 93 is a pro-apoptotic T-Ag binding protein, and that T- Ag expression does not elicit an apoptotic response in cardiomyocytes, it will be of interest to determine if abrogation of pi 93 activity can antagonize El A and/or E2F-1 induced cardiomyocyte apoptosis. Abrogation of pi 93 activity may also have a cardioprotective effect under pathophysiological conditions which promote cardiomyocyte apoptosis.
  • apoptotic cardiomyocytes in a variety of cardiovascular diseases including dilated cardiomyopathy, ischemic cardiomyopafhy, arrhythmogenic right ventricular dysplasia, acute myocardial infarction, myocarditis, allograft rejection, and preexcitation syndromes (reviewed in Haunstetter, A. and Izumo, S. (1998) Circulation Research 82, 11 11-1129).
  • apoptosis and resulting cardiac remodeling may contribute to the onset of dilated cardiomyopathy and heart failure (reviewed in Anversa, P., Leri, A., Beltrami, C.A., Guerra, S., and Kajstura, J. (1998) Lab.
  • pi 93 is a new member of the BH3 only pro-apoptosis gene family, pi 93 promotes cell death during Gi, prior to the onset of DNA synthesis.
  • T-Ag is localized in the cytoplasm during the same phase of the cell cycle, and co-expression of T-Ag antagonizes pl93-induced cell death and results in the cytoplasmic localization of both proteins, pi 93 binds to the N-terminus of T-Ag in a region which contributes to transforming activity in some cell types.
  • Colony growth assay in NIH-3T3 cells indicate that decreased pi 93 activity as a consequence of anti-sense expression results in increased rates of cell growth (Figure 7).
  • a priori, expression of dominant negative variants of pl93 should also result in increased rates of cell growth.
  • the cDNAs were subcloned into a CMV expression vector.
  • the structure of the pi 93 variants are depicted in Figure 8A.
  • the expression vectors also carried a neomycin-resistance cassette.
  • NEH-3T3 cells were transfected with the various expression vectors, and the cells were cultured in the presence of G418.
  • FIG. 8B Representative cultures of cells transfected with the various constructs are shown in Figure 8B.
  • Cells transfected with the CMV-null vector represent the negative control (this reflects the rate of growth in the absence of any positive or negative cell cycle regulators, see culture plate A). Consistent with the pro-apoptotic activity of pi 93, no colonies were observed in cultures transfected with full-length pi 93 (amino acid residues 1-1689; culture plate B). A slight enhancement in cell growth was detected in cells transfected with a vector expressing pi 93 amino acid residues 1 through 1342 (culture plate C).
  • NIH-3T3 cells were transfected with a CMV-null expression construct, or a CMV-p 193dn expression construct, and stable cell lines were generated. The cells were then incubated in growth medium supplemented with MMS (0 mM, 0.1 mM, 0.5 mM or 1 mM) for 3 hrs. at 37°C Cells were then harvested and apoptosis was measured by determining the degree of DNA fragmentation (nucleosomal cleavage of DNA is diagnostic for apoptosis).
  • MMS methyl methanesulfonate
  • MHC myosin heavy chain
  • a cDNA encoding pl93dn was inserted downstream of the promoter, followed by the SV40 early region transcription terminator (SV40 nucleotide residues #2586-2452, see Reddy, V. B., B. Thimmappaya, R. Dhar, K. N. Subramanian, B. S. Zain, J. Pan, P. K. Ghosh, M. L. Celma, and S. M. Weissman (1978) The genome of simian virus 40. Science 200:494-502.).
  • the resulting transgene was designated MHC-pl93dn.
  • a schematic diagram of the transgene is presented in Figure 9.
  • transgene DNA was digested with restriction enzymes to separate the MHC-pl93 sequences from the vector, and the insert purified from an agarose gel using Geneclean glass beads (Bio 101, Vista CA). Purified insert DNA was microinjected into inbred C3HeB/FeJ (Jackson Laboratories,Bar Harbor MA) zygotes using standard methodologies [3]. The microinjected embryos were cultured in vitro to the two cell stage, and then implanted into pseudopregnant SW/Taconic (Taconic Farms, Germantown NY) female mice. For all surgeries, mice were anesthetized with 2.5% Avertin (0.015 ml/g bodyweight IP, Fluka Biochemicals, Ronkomkoma NY). All manipulations were performed according to NIH and Institutional Animal Care and Use Guidelines.
  • Myocardial damage in response to chronic isoproterenol infusion was monitored in control and MHC-pl93dn transgenic mice to determine if transgene expression was cardioprotective.
  • Non-transgenic control and MHC-pl93dn transgenic mice were identified and sequestered until they reached 11 weeks of age.
  • Continuous isoproterenol infusion was administered using implanted osmotic mini-pumps (model 2001, Alzet, Palo Alto CA, flow rate of 1 ⁇ l/hr) filled with 0.028 g/ml isoproterenol (dissolved in saline). After seven days of treatment the mice were sacrificed, the hearts harvested, cryoprotected and sectioned using standard histologic techniques (Bullock, G. R.
  • Panels A and B depict sections of a nontransgenic heart after seven days of isoproterenol infusion. Abundant Sirius red staining is apparent throughout the ventricular myocardium (panel A shows the left ventricular myocardium near the apex of the heart, panel B shows the ventricle myocardium near the base of the).
  • Adenoviral El A oncoprotein can reactivate cell cycle ion cardiomyocytes, but this reactivation is immediately followed by apoptotic cardiomyocyte death (Kirshenbaum, L. A. and M. D. Schneider. Adenovirus El A represses cardiac gene transcription and reactivates DNA synthesis in ventricular myocytes, via alternative pocket protein- and p300-binding domains, J. Biol. Chem. (1995) 270: 7791-7794). Moreover, blocking the p53-regulated apoptotic pathway only partially rescues the cardiomyocytes.
  • the experiment utilized a previously described technique to generate enriched cardiomyocyte cultures from differentiating ES cells (U.S. Patent Nos. 5,602,301 and 5,733,727 to Field et al.; and Klug, M. G., M. H. Soonpaa, G. Y. Koh, and L. J. Field (1996) Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts, J. Clin. Invest, 98: 216-224).
  • Undifferentiated ES cells were transfected with an MHC-neor/pGK-hygror transgene alone or in combination with a MHC-E1A, MHC-pl93dn and/or MHC- p53dn transgenes. Transfected undifferentiated ES cells were then selected on the basis of hygromycin resistance.
  • the cultures were induced to differentiate. Once cardiomyocytes were apparent in the culture (as evidenced by the presence of beating cells, which usually occurs at 8 days post-induction), the cultures were subjected to G418 selection. Since the neor cassette is under the regulation of the cardiac MHC promoter, only cardiomyocytes survive this selection procedure. After 60 days of G418 selection, the cultures were fixed and stained with PAS to permit visualization of the cardiomyocytes. Control plates (transfected with the MHC-neor/pGK-hygror transgene alone) gave rise to numerous colonies of beating myocytes (see the control plate, Figure 12).
  • hypertrophic stimuli are in fact mitogenic stimuli, and that in the mature cardiac myocyte the response to such stimuli is to first increase cell size, and then transit Gl/S.
  • Our data clearly indicate that two pro-apoptotic pathways (the p53 and pi 93 pathways) are activated in cardiomyocytes which are experimentally induced to proliferate. The apoptotic response observed during the process of decompensation might result from the initiation of cell cycle activity in the presence of active p53 and pi 93 pathways.

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)
EP00957721A 1999-08-23 2000-08-23 P193 proteine und nuklene säure, und deren anwendung Withdrawn EP1212418A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US15026699P 1999-08-23 1999-08-23
US150266P 1999-08-23
PCT/US2000/023161 WO2001014418A2 (en) 1999-08-23 2000-08-23 p193 PROTEINS AND NUCLEIC ACIDS, AND USES THEREOF

Publications (1)

Publication Number Publication Date
EP1212418A2 true EP1212418A2 (de) 2002-06-12

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP00957721A Withdrawn EP1212418A2 (de) 1999-08-23 2000-08-23 P193 proteine und nuklene säure, und deren anwendung

Country Status (6)

Country Link
EP (1) EP1212418A2 (de)
JP (1) JP2003520025A (de)
AU (1) AU6930100A (de)
CA (1) CA2383371A1 (de)
IL (1) IL148286A0 (de)
WO (1) WO2001014418A2 (de)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0114418A2 *

Also Published As

Publication number Publication date
IL148286A0 (en) 2002-09-12
CA2383371A1 (en) 2001-03-01
WO2001014418A2 (en) 2001-03-01
WO2001014418A3 (en) 2002-02-14
JP2003520025A (ja) 2003-07-02
AU6930100A (en) 2001-03-19

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