EP1368376A2 - Pro-apoptotische proteine und dafür kodierende dns-moleküle - Google Patents

Pro-apoptotische proteine und dafür kodierende dns-moleküle

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Publication number
EP1368376A2
EP1368376A2 EP02719729A EP02719729A EP1368376A2 EP 1368376 A2 EP1368376 A2 EP 1368376A2 EP 02719729 A EP02719729 A EP 02719729A EP 02719729 A EP02719729 A EP 02719729A EP 1368376 A2 EP1368376 A2 EP 1368376A2
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European Patent Office
Prior art keywords
polynucleotide
seq
cells
compound
set forth
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English (en)
French (fr)
Inventor
Hartmut Beug
Johannes Hofmann
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Boehringer Ingelheim International GmbH
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Boehringer Ingelheim International GmbH
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Priority to EP02719729A priority Critical patent/EP1368376A2/de
Publication of EP1368376A2 publication Critical patent/EP1368376A2/de
Withdrawn legal-status Critical Current

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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

  • the present invention relates to the therapy and diagnosis of disorders associated with a deregulation of molecular pathways involved in apoptosis.
  • the survival of individual cells in multicellular organisms depends on extracellular signals that are provided by growth factors, cytokines, hormones and extracellular matrix. Furthermore, lineage specificity of survival factors and matrix components can serve to prevent cells from accumulating in ectopic positions (reviewed in Raff, 1996) . Withdrawal of growth factors or removal from matrix contact triggers programmed cell death. Aberrant resistance to apoptosis participates in the development of neoplasia, whereas excessive cell death through apoptosis or necrosis contributes to acute organ failure as well as to chronic diseases.
  • pro-apoptotic and anti- apoptotic regulators include Bcl-2 family members (Chao and Korsmeyer, 1998), caspase family members (Nicholson and Thornberry, 1997, Raff, 1998) and their adaptors like Apaf-1 and Fadd.
  • Caspases are synthesized as dormant proenzymes that, upon proteolytic activation involving interaction with their specific adaptors, acquire the ability to cleave key intracellular substrates. This results in the morphological and biochemical changes associated with apoptosis. Recently it was found that in apoptosis triggered by many stimuli, mitochondria play a pivotal role in coordinating caspase activation through the release of proapoptotic activities (e.g.: cytochrome c, AIF,
  • Diablo/Smac Diablo/Smac . Release of proapoptotic activities from the mitochondria is controlled by proteins of the Bcl-2 family: those that prevent cell death (Bcl-2 and BC1-X ) inhibit release, whereas those that promote death (Bax and Bak) induce this release (reviewed in Desagher and Martinou, 2000) .
  • polysome-bound and polysome free mRNA fractions of FDCP-1 cells were prepared.
  • the polysome-bound fractions were then subjected to suppression subtractive hybridization, SSH, in order to isolate differentially expressed genes.
  • pl2 a novel proapoptotic inducer
  • pl2 protein is indeed differentially expressed in a cytokine-dependent manner.
  • IL-3 removal induces increased translation of the pl2 mRNA.
  • the data obtained in the experiments of the present invention further show that pl2, which is localized in the cytosol in the presence of cytokine, translocates into the mitochondrial fraction in the absence of cytokine and binds to a central component (VDAC) of conductance channels in the outer mitochondrial membrane.
  • VDAC central component
  • the present invention relates to the murine pl2 polypeptide with the amino acid sequence as set forth in SEQ ID NO: 2 or to a polypeptide encoded by a polynucleotide which hybridises under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:l.
  • the present invention relates to an isolated DNA molecule comprising a polynucleotide with the nucleotide sequence as set forth in SEQ ID NO: 1
  • the present invention relates to the human pl2 polypeptide with the amino acid sequence as set forth in SEQ ID NO: or to a polypeptide encoded by a polynucleotide which hybridises under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO: 3.
  • the present invention relates to an isolated DNA molecule comprising a polynucleotide with the nucleotide sequence as set forth in SEQ ID NO: 3 encoding human pl2 polypeptide or an isolated DNA molecule comprising a polynucleotide which hybridises under stringent conditions to a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO: 3.
  • IX SSC 150 mM NaCl, 15mM trisodium citrate
  • 50 mM sodium phosphate pH 7.6
  • 5x Denhardt's solution 10% dextran sulfate
  • 20 ⁇ g/ml denatured, sheared salmon sperm DNA followed by washing the filters in O.lx SSC at about 65°C, or equivalent conditions.
  • the human homologue of murine pl2 is cloned according to standard procedures, e.g. by RT PCR amplification from human RNA extracted from certain tissues or cells (e.g. myeloid cells), using standard PCR protocols. Amplified products are then cloned into suitable vectors for propagation in bacteria, and the DNA sequence of individual amplification products determined and used to identify human pl2 homologues according to the criteria defined above.
  • pl2 refers to both the murine and the human pl2.
  • the present invention relates to isolated pl2 nucleic acid molecules in the form of RNA, such as mRNA.
  • the pl2 DNA molecules include cDNA and genomic DNA obtained by cloning or produced synthetically.
  • the DNA may be double-stranded or single-stranded.
  • Single-stranded DNA or RNA may be the coding strand, also known as the sense (or plus) strand, or it may be the non-coding strand, also referred to as the antisense (or minus) strand.
  • isolated nucleic acid molecule is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
  • the present invention also relates to pl2 variants, precursors, fragments or derivatives, which may include one or more polynucleotides having a nucleic acid sequence that is complementary to substantially all or a portion of a nucleic acid molecule having a nucleic acid sequence as shown in SEQ ID NO:l or SEQ ID NO: 3.
  • a portion or "a fragment” of a nucleic acid molecule or a polypeptide means a segment of a polynucleotide comprising at least 15 nucleotides (nt), usually at least 18 nt or 25 nt, and may be at least about 50 nt contiguous nucleotides or amino acids of a reference polynucleotide or polypeptide (for example, the polypeptide and polynucleotide shown in SEQ ID NO: 1 or SEQ ID NO: 3, or SEQ ID NO:2 or SEQ ID NO:4, respectively).
  • Double or single stranded pl2 DNA fragments may be obtained by chemically synthesizing oligonucleotides in accordance with conventional methods, by restriction enzyme digestion, by PCR amplification, external deletions, etc . Such small DNA fragments are useful as primers for PCR, hybridization screening probes, etc. Larger DNA fragments, greater than 100 nt are useful for production of the encoded polypeptide. For use in amplification reactions, such as PCR, a pair of primers will be used. The exact composition of the primer sequences is not critical to the invention, but for most applications the primers will hybridize to the subject sequence under stringent conditions, as known in the art.
  • primers that will generate an amplification product of at least about 50 nt, preferably at least about 100 nt .
  • Algorithms for the selection of primer sequences are generally known, and are available in commercial software packages. Amplification primers hybridize to complementary strands of DNA, and will prime towards each other.
  • the invention relates to pl2 DNA fragments that can be used for producing functionally active pl2 fragments, which are useful in therapy.
  • the following experimental approach may be taken:
  • pl2 cDNA fragments of varying length are obtained by external deletion mutagenesis according to standard protocols, e.g. by commercially available tools (Erase-a-Base, Promega) .
  • the fragments are then, as described below for the pl2 versions with modified proapoptotic activity, cloned into suitable expression vectors, expressed in test cells and assessed for the desired proapoptotic activity, e.g. in the TUNEL assay.
  • pl2 fragments may be designed based on secondary structure predictions.
  • predicted alpha helical domains extend from amino acids (aa) 1-4, aa 8-19, aa 34-44, aa 52-84.
  • the C-terminus from amino acids 85-113 is predicted to contain mainly coiled structure.
  • two primers representing the borders of the domain of interest e.g. one of the ⁇ -helical domains, are used for PCR amplification.
  • the thus obtained fragment is subcloned and further tested for proapoptotic activity as described above.
  • the invention also relates to DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the pl2 mouse or human polypeptides . Since the genetic code is well known in the art, it is routine for one of ordinary skill in the art to produce the degenerate variants described above without undue experimentation.
  • the invention relates to pl2 polypeptides which have deviations from the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, caused by the conservative exchange of amino acids, if they are pl2 derivatives or fragments or peptides with the properties which are desirable for their use in therapy or in screening assays described below.
  • the invention also relates to isolated DNA molecules encoding such derivatives with a polynucleotide sequence varying in their sequence from SEQ ID NO:l or SEQ ID NO:3.
  • isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells, and those DNA molecules purified (partially or substantially) from a solution whether produced by recombinant DNA or synthetic chemistry techniques.
  • Isolated RNA molecules include in vivo or in vi tro RNA transcripts of the DNA molecules of the present invention. However, it is intended that "isolated” as used herein does not include the pl2 cDNA present in a cDNA library or in a preparation of purified or isolated genomic DNA containing the pl2 gene or a portion thereof in admixture with one or more other cDNA molecules or DNA fragments.
  • the present invention relates to isolated genomic pl2 DNA molecules containing, besides the coding sequence, the regulatory elements including the promotor sequence.
  • genomic pl2 DNA can be obtained as follows: genomic clones, e.g. cosmid clones, can be isolated according to standard procedures using the pl2 cDNA, or a fragment thereof, as a hybridization probe. Individual subfragments of the obtained clones are tested in transcriptional reporter assays by using expression constructs containing a reporter gene, e.g. lacZ or luciferase, under the control of a minimal promotor sequence, e.g. from the CMV minimal promotor. The subfragments of interest are inserted 5' of the minimal promotor and their effect on reporter gene expression is tested in hematopoietic cells. In a step by step process, further deletions of the subfragments are used to determine the minimal functional promotor sequence.
  • genomic clones e.g. cosmid clones
  • individual subfragments of the obtained clones are tested in transcriptional reporter assays by using expression constructs containing a reporter gene, e.g. lacZ or luciferase, under the
  • the invention relates pl2 DNA or polypeptide homologues, which have been obtained by mutagenesis .
  • the pl2 DNA including flanking promoter regions and coding regions, may be mutated in various ways known in the art to generate targeted changes in promoter strength, sequence of the encoded protein, etc.
  • the DNA sequence or protein product of such a mutation will usually be substantially similar to the sequences as shown in SEQ ID NO:l or SEQ ID NO: 3, or SEQ ID NO: 2 or SEQ ID NO:4, i.e. it will differ by at least one nucleotide or amino acid, respectively, and may differ by at least two but not more than about ten nucleotides or amino acids.
  • the sequence changes may be substitutions, insertions or deletions. Deletions may further include major changes, such as deletions of a domain or exon.
  • Homologues of the pl2 proteins also include versions of the polypeptide containing single or multiple point mutations and/or insertions and/or deletions which are resistant to or mimic post-translational modification. Such mutations may effect the activity or subcellular localisation of the protein.
  • the mutations may be carried out according to standard protocols, e.g. by means of commercially available kits (e.g. QuickChange, Stratagene) , the obtained mutated pl2 cDNA is then inserted into vectors suitable for transfection of test cells, e.g. hematopoietic or other cells in which wildtype pl2 plays an important physiological role. Examples for suitable vectors are retroviral vectors.
  • the proapoptotic activity is assessed in the test cells by standard viability assays, e.g. the TUNEL assay.
  • the subcellular localization of the mutated pl2 may be assessed by immunofluorescence procedures, i.e. using anti-pl2 antibodies and secondary fluorescence-labeled antibodies according to standard protocols.
  • a mutated protein which has a reduced proapoptotic activity and a subcellular localization different from that of the wildtype pl2 may significantly inhibit the proapoptotic activity of the endogenous pl2 by its sequestration or competition with binding partners.
  • pl2 mutants are useful for therapy of conditions in which increased cell death is involved, e.g. myelodysplastic syndromes.
  • mutants with enhanced proapoptotic activity may be obtained.
  • Such mutants may exert their effect by being constitutively localized to mitochondria and can thus be used for the treatment of conditions in which reduced cell death is involved, e.g. cancerous conditions and leukemias.
  • Nucleic acid molecules of the present invention which encode a pl2 polypeptide or a variant, derivative or fragment thereof further include those encoding the amino acid sequence of the polypeptide by itself, together with additional, non-coding sequences, including for example introns and non-coding 5' and 3' sequences, such as the transcribed, untranslated regions (UTRs) or other 5 1 flanking sequences that may play a role in- transcription (e.g. via providing ribosome- or transcription factor-binding sites), mRNA processing (e.g.
  • the coding sequence for the pl2 polypeptide operably linked to a regulatory DNA sequence, particularly a heterologous regulatory DNA sequence such as a promoter or enhancer; and the coding sequence for the pl2 polypeptide linked to one or more sequences which code for amino acids that provide additional functionalities.
  • the DNA encoding the pl2 polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide; e.g. modifications in the form of epitope tagging.
  • the marker amino acid sequence may be a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available.
  • the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al., 1984.
  • Yet another useful marker peptide for facilitation of purification of pl2 is glutathione S-transferase (GST) encoded by the pGEX fusion vector (see, e.g., Winnacker, 1987).
  • GST glutathione S-transferase
  • Other useful tag system is the FLAG system (Jarvik and Telmer, 1998) . For studies of subcellular localization, fusion proteins with green fluorescent proteins (GFPs) may be used.
  • pl2 polypeptides, variants, derivatives or fragments thereof may be expressed in prokaryotic or eukaryotic expression systems according to well-established methods.
  • the pl2 nucleic acids can also be used for identifying homologous or related genes; in identifying compounds that modulate the expression or function of the pl2 protein; mapping functional regions of the protein; and in studying of apoptosis pathways and associated physiological pathways (as described below) .
  • the pl2 promotor sequence is useful for determining whether, in a patient, reduced or absent pl2 expression correlates with a mutation in the promotor sequence.
  • genomic DNA is isolated from patient material, the pl2 promotor region is amplified by PCR and the sequence compared with the authentic promotor sequence from healthy individuals.
  • the pl2 promotor is also useful in screening assays for identifying drugs that modulate, e.g. upregulate, pl2 expression at a transcriptional level.
  • an transcriptional assay uses hematopoietic cells, which have been transfected with a reporter construct in which the reporter gene is under the control of the pl2 promotor sequence. The cells are incubated, in the absence or presence of a test substance, and the effect of the substance on reporter gene expression is measured.
  • a substance upregulating reporter gene expression is a candidate for upregulating pl2 transcription.
  • Transcriptional assays of this type are well-known in the art, they have been described, inter alia, in WO 92/13063.
  • cell-based screening assays in microtiter format can be used to identify drugs that inhibit interaction of pl2 with critical interaction partners required for apoptosis induction (e.g.: VDAC, see Example 3) .
  • VDAC critical interaction partners required for apoptosis induction
  • Such assays are based the well-established two-hybrid system, in which the transcription of a reporter gene depends on the interaction of two binding partners, one of them being fused to a DNA-binding domain (specific for the reporter gene promotor) and the other one to an activation domain.
  • Fusion proteins of the interaction partners of interest e.g pl2 and VDAC (Blachly-Dyson, E., et al., 1993;
  • Messina, A. et al., 2000; Accession No. NM_003374 are expressed in yeast or mammalian cells; interaction of the binding partners leads to expression of a reporter gene product, e.g. an easily detectable marker (e.g.: GFP) , or a resistance gene product (e.g.: Ura3, neomycinR) that allows cell proliferation under selective conditions, depending on the particular system used. Drugs interfering with the interaction will lead to loss of marker expression or loss of cellular proliferation.
  • Standard two hybrid interaction systems using yeast or mammalian cells are of particular use (e.g. Fotin-Mleczek et al., 2000); such systems are commercially available, e.g. from Promega, Stratagene, Invitrogen, Clontech.
  • the screening method may be based on a cell-free assay.
  • the principle of such an assay is to inhibit the physical interaction of a protein of interest with its binding partner.
  • the assay may be conducted by incubating the binding partners in solution, one of them being fused to a reporter gene product (e.g. alkaline phosphatase or luciferase, which allows for monitoring the interaction by determining the enzymatic activity) and the other one being immobilized on a suitable carrier, e.g. micro titer plates, with the test substance and by determining the effect of a drug on the interaction of the two proteins by measuring the reporter activity.
  • a reporter gene product e.g. alkaline phosphatase or luciferase, which allows for monitoring the interaction by determining the enzymatic activity
  • a suitable carrier e.g. micro titer plates
  • One binding partner may carry a tag resulting from the antecedent purification step, e.g.
  • a GST, His or Myc tag provided binding of the partners is not impaired by the tag.
  • one partner may be specifically immobilized on the carrier via that tag. If a substance is an inhibitor of the interaction, the reporter signal is decreased or abolished after washing off the carrier.
  • larger proteins containing the binding domains may be used, e.g. the full length proteins, or one of the interaction partners is used as a full-length protein and the other one in the form of the binding domain only.
  • one of the interaction partners may be labeled with any other detectable marker, preferably a marker producing a signal that can be easily measured in a high throughput screen, in which the unlabeled interaction molecule is immobilized, e.g. on a microtiter plate surface or on beads.
  • suitable labels are radioactive labels or commercially available fluorescent markers suitable for labeling proteins or peptides.
  • MTT assays cytokine dependent hematopoietic cells in combination with viability assays (MTT assays, luciferase assay; Promega) that allow high throughput screening may be employed.
  • the therapeutic utility of a compound that mimics the action of pl2 in apoptosis induction, e.g. by binding to VDAC, or that increases pl2 expression, is confirmed by measuring its effect on endogenous pl2 expression by means of Northern Blotting and/or conventional nuclear run-on assays.
  • Modulation of the 'pl2 gene activity in vivo is useful for prophylactic and therapeutic purposes, such as treatment of cancer and other hyperproliferative disorders (like myeloid and erythroid leukemias, where pl2 has been shown to exert a proapoptotic activity) .
  • the murine pl2 mRNA demonstrates a tissue specific expression pattern with highest levels observed in heart, liver, kidney, brain and testes and may play a similar proapoptotic role in these tissues as it plays in the hematopoetic system. Due to the high conservation between the murine and the human pl2 protein, the human protein is expected to have a corresponding function, which may be confirmed as described above.
  • the genomic sequences surrounding the pl2 locus may be used to identify cis acting elements required for transcriptional or translational regulation of pl2 expression, especially in different tissues or stages of development, and to identify cis acting sequences and trans acting factors that regulate or mediate pl2 expression.
  • Such transcription or translational control regions may be operably linked to a pl2 gene in order to promote expression of wild type or altered pl2 or other proteins of interest in cultured cells, or in embryonic, fetal or adult tissues, and for gene therapy.
  • Naturally occurring polymorphisms in the promoter region are useful for determining natural variations in expression, particularly those that may be associated with disease.
  • the pl2 DNA may also be used to identify expression of the gene in a biological specimen.
  • the manner in which cells can be probed for the presence of particular nucleotide sequences, as genomic DNA or RNA, is well established in the literature; briefly, DNA or mRNA is isolated from a cell sample. Next, the mRNA is amplified, e.g. by RT-PCR, using reverse transcriptase to form a complementary DNA strand, followed by polymerase chain reaction amplification using primers specific for the subject DNA sequences. Alternatively, the mRNA sample is separated by gel electrophoresis, transferred to a suitable support, e.g. nitrocellulose, nylon, etc., and then probed with a fragment of the subject DNA as a probe.
  • a suitable support e.g. nitrocellulose, nylon, etc.
  • oligonucleotide ligation assays in si tu hybridizations, and hybridization to DNA probes arrayed on a solid chip may also find use. Detection of mRNA hybridizing to the subject sequence is indicative of pl2 gene expression in the sample.
  • the pl2 DNA molecules are useful in gene therapy to treat disorders associated with a deficit in pro- apoptosis proteins, including different states of tumorgenesis .
  • the pl2 DNA molecules of the present invention may be administered, preferably in recombinant form as plasmids, directly or as part of a recombinant virus.
  • Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences.
  • Transcription cassettes may be prepared comprising a transcription initiation region, the pl2 DNA or fragment thereof, and a transcriptional termination region.
  • the transcription cassettes may be introduced into a variety of vectors, e.g. a plasmid; retrovirus, vaccinia virus, adenovirus, listeria monocytogenes; and the like (a summary was provided by Coulie, 1997).
  • synthetic carriers for nucleic acids such as cationic lipids, microspheres, micropellets or liposomes may be used for in vivo administration of a pl2 nucleic acid.
  • the application may optionally be combined with physical methods, e.g. electroporation.
  • pl2 antisense molecules can be used to down-regulate expression of pl2 in cells or tissues with excessive cell death (as is observed in e.g.: myelodysplastic syndrome) .
  • the antisense reagent may be antisense oligonucleotides (ODN) , particularly synthetic ODN having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA.
  • ODN antisense oligonucleotides
  • the antisense sequence is complementary to the mRNA of the targeted gene, and inhibits expression of the targeted gene products.
  • Antisense molecules inhibit gene expression through various mechanisms, e.g. by reducing the amount of mRNA available for translation, through activation of RNAse H, or steric hindrance.
  • One or a combination of antisense molecules may be administered, where a combination may comprise multiple different sequences.
  • pl2 antisense oligonucleotides may be chemically synthesized by methods known in the art (see Wagner et al., 1996).
  • Preferred oligonucleotides are chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases .
  • Achiral phosphate derivatives include 3 ' -0 ' -5 ' -S-phosphorothioate, 3 ' -S-5 ' -0- phosphorothioate, 3 ' -GH2-5 ' -O-phosphonate and 3 ' -NH-5 ' -O-phosphoroamidate.
  • Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage.
  • Sugar modifications are also used to enhance stability and affinity.
  • the -anomer of deoxyribose may be used, where the base is inverted with respect to the natural 3-anomer.
  • the 2 ' -OH of the ribose sugar may be altered to form 2 ' -O-methyl or 2'-0-allyl sugars, which provides resistance to degradation without comprising affinity. Modification of the heterocyclic bases must maintain proper base pairing.
  • Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl-2 ' - deoxycytidine and 5-bromo-2 ' -deoxycytidine for deoxycytidine.
  • 5-propynyl-2 ' -deoxyuridine and 5-propynyl-2 ' -deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and de
  • the nucleic acids of the invention can also be used to generate transgenic animals or site specific gene modifications in animals or cell lines derived from the transgenic animals.
  • Transgenic animals which are useful for in vivo studies of pl2 derivatives or fragments, may be obtained through homologous recombination, where the normal pl2 locus is altered. Alternatively, a nucleic acid construct is randomly integrated into the genome. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like.
  • Transgenic animals are also useful for verifying that a sequence identified as a minimal promotor sequence also functions in vivo and confers a similar or identical expression pattern as the genomic pl2 locus.
  • the modified cells or animals are useful in the study of pro-apoptotic gene function and regulation.
  • a series of small deletions and/or substitutions may be made in the pl2 gene to determine the role of different epitopes in oncogenesis, signal transduction, etc.
  • pl2 is also useful to construct transgenic animal models for proliferative disorders, e.g. leukemia, where expression of pl2 is specifically reduced or absent.
  • Specific constructs of interest include anti-sense pl2, which will block pl2 expression, and expression of dominant negative pl2 mutations.
  • a detectable marker, such as lac Z may be introduced into the pl2 locus, where upregulation of pl2 expression will' result in an easily detected change in phenotype.
  • pl2 protein or variants thereof By providing expression of pl2 protein or variants thereof in cells in which it is not normally produced, one can induce changes in cell behavior, e.g. increased or decreased cell death.
  • DNA constructs for homologous recombination will comprise at least a portion of the pl2 gene with the desired genetic modification, and will include regions of homology to the target locus. Conveniently, markers for positive and negative selection are included. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For various techniques for transfecting mammalian cells, see Keown et al., 1990.
  • an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder layer or grown in the presence of leukemia inhibiting factor (LIF) .
  • LIF leukemia inhibiting factor
  • ES or embryonic cells may be used to produce transgenic animals. After transformation, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct.
  • Blastocysts are obtained from 4 to 6 week old superovulated females.
  • the ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting offspring screened for the construct.
  • chimeric progeny can be readily detected.
  • the chimeric animals are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. Homozygous null mutants or other mutants may be crossed to mice carrying deletions or mutations in other apoptotic regulators to study genetic interactions. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogeneic or congenic grafts or transplants, or in in vi tro culture.
  • the genetically modified animals may be any non-human mammal, such as laboratory animals, domestic animals, etc. The genetically modified animals may be used in functional studies, drug screening, phenotypic analyses, etc.
  • the availability of a number of proteins that are players in the apoptotic apparatus allows in vi tro reconstruction of the apoptosis pathway.
  • Two or more of the components, including pl2 may be combined in vi tro, and the behavior assessed in terms of activation of cellular organelles that play a central role in apoptosis (like mitochondria) ; modification of protein components, e.g. proteolytic processing, phosphorylation, methylation, etc.; ability of different protein components to bind to each other, etc.
  • the components may be modified by sequence deletion, substitution, etc. to determine the functional role of specific domains.
  • the pl2 nucleic acid sequences may be used to analyze a patient sample for the presence of polymorphisms associated with a disease state or genetic predisposition to a disease state. Biochemical studies may be performed to determine whether a sequence polymorphism in a pl2 coding region, e.g. an SNP (Single Nucleotide Polymorphism) or control regions is associated with the disease.
  • Disease-associated polymorphisms may include deletions, insertions or truncation in the gene, mutations that alter expression level, etc.
  • Changes in the promoter or enhancer sequence that may affect expression levels of pl2 can be compared to expression levels of the normal allele by various methods known in the art.
  • Methods for determining promoter or enhancer strength include quantitation of the expressed natural protein; insertion of the variant control element into a vector with a reporter gene such as ⁇ -galactosidase, luciferase chloramphenicol acetyltransferase, etc . that provides for convenient quantitation; and the like.
  • a number of methods are available for analyzing nucleic acids for the presence of a specific sequence, e.g. a disease associated polymorphism. Where large amounts of DNA are available, genomic DNA is used directly.
  • the region of interest is cloned into a suitable vector and grown in sufficient quantity for analysis.
  • Cells that express pl2 may be used as a source of mRNA, which may be assayed directly or reverse transcribed into cDNA for analysis.
  • the nucleic acid may be amplified by conventional techniques, such as the polymerase chain reaction (PCR) , to provide sufficient amounts for analysis.
  • PCR polymerase chain reaction
  • the use of the polymerase chain reaction is described in Saiki, et al., 1985, and a review of techniques may be found in Sambrook, et al . , 1989.
  • various methods are known in the art that utilize oligonucleotide ligation as a means of detecting polymorphisms, for examples see Riley et al., 1990, and Delahunty et al., 1996.
  • the pl2 protein is also useful as an immunogen for producing specific antibodies.
  • a still further aspect of the present invention relates to antibodies and antibody preparations specifically reactive with an epitope of the pl2 polypeptide .
  • Polyclonal anti-pl2 antibodies are conventionally obtained by immunising animals, particularly rabbits and goats, by injecting the pl2 antigen or fragments thereof and subsequently purifying the immunoglobulin .
  • Monoclonal anti-pl2 antibodies may be obtained by standard procedures following the principle described by K ⁇ hler and Milstein, 1975, by immunising animals, particularly mice, then immortalising antibody- producing cells from the immunised animals, e.g. by fusion with myeloma cells, and screening the supernatant of the hybridomas obtained by immunological standard assays for monoclonal anti-pl2 antibodies.
  • these animal antibodies may optionally be chimerised in the conventional way (Neuberger et al., 1984; Boulianne et al., 1984) or humanised (Riechmann et al., 1988; Graziano et al., 1995).
  • Antibodies specific for a pl2 may be used in staining or in immunoassays of samples from an individual to be analysed for pl2 and for screening human conditions for P12 mediated pathologies.
  • Samples used in these assays include biological fluids such as semen, blood, cerebrospinal fluid, tears, saliva, lymph, dialysis fluid and the like; organ or tissue culture derived fluids; and fluids extracted from physiological tissues. Also included are derivatives and fractions of such fluids.
  • the cells may be dissociated, in the case of solid tissues, or tissue sections may be analyzed. Alternatively a lysate of the cells may be prepared.
  • a screening procedure was developed to identify potential proapoptotic activities in myeloid IL-3 dependent progenitors.
  • the screen is based on previous data suggesting that novel protein synthesis is required for proper induction of apoptosis in this cell system (Williams et al., 1990).
  • Goal of the screen is to identify mRNAs that are translated preferentially in cytokine depleted cells as compared to cells grown in the presence of cytokine. It is pointed out that under these experimental conditions the bulk of cellular transcription and translation drops.
  • Identification of the genes of the present invention was done by using polysome-bound mRNA preparations for differential screening, allowing to simultaneously assess differential expression of genes at multiple, important levels, i.e. transcriptional control, mRNA processing, nuclear export, mRNA stability and translational control.
  • This method for identifying and isolating genes differentially expressed with respect to protein levels produced comprises the following steps:
  • cytosol e.g. on sucrose gradients
  • fractions containing either the ribosome-free mRNA compartment or mRNA bound to polyribosomes of increasing size e.g. on sucrose gradients
  • c-DNAs subjecting the c-DNAs to the different procedures available for differential screening, i.e. by PCR-based subtractive hybridization methods such as suppression subtration hybridization (SSH) or hybridizing the cDNAs with arrays or chips of multiple known or unknown c-DNAs
  • SSH suppression subtration hybridization
  • c-DNAs differentially expressed genes
  • step f selectively amplifying and cloning the cDNAs identified in step f ) .
  • IL-3 dependent murine myeloid progenitor FDCP-1 cells were used in this screen.
  • Cells were grown in RPMI medium (Beug et al., 1995) or serum-free StemPro34TM (Life Technologies) according to the instructions of the supplier.
  • IL-3 was added as recombinant protein (murine recombinant IL-3, Pepro Tech, 1 ng/ml) .
  • Cytokine depletion was done as follows: after 200x10 ⁇ cells had been obtained, one aliquot of 100x10 ⁇ cells was washed twice with PBS and reseeded in medium without IL-3 for 8 hours. The other aliquot was maintained in IL-3 for the same time.
  • Cells were lysed at 4°C in extraction buffer containing 10 mM Tris-HCl (pH 8.0), 140 mM NaCl, 1.5 mM MgCl2, 0.5% Nonidet-P-40, 500 U/ml RNasin, 20 mM dithiothreitol, 150 ⁇ g/ml cycloheximide and 1 mM phenylmethylsulfonyl fluoride. Nuclei were removed by centrifugation at 3000 g for 2 min at 4°C.
  • the supernatant was further supplemented with 665 ⁇ g/ml heparin, centrifuged at 12000 g for 10 min at 4°C to eliminate mitochondria and layered onto a 10 ml linear sucrose gradient (15-40% sucrose [w/v] supplemented with 10 mM dithiothreitol, 100 ⁇ g/ml cycloheximide and 0.5 mg/ml heparin). Centrifugation was performed in a SW41Ti rotor at 38000 rpm for 120 min at 4°C.
  • RNA was analyzed by electrophoresis on denaturing 1% formaldehyde agarose gels and subsequent Northern blotting. After UV-crosslinking of RNA to the nylone membranes (GeneScreen, DuPont) , the distribution of 18S and 28S rRNA was visualized by staining of filters with methylene blue.
  • SDS sodiumdodecylsulfate
  • 10 mM EDTA 10 mM EDTA
  • 200 ⁇ g/ml proteinase K for 30 min at 37°C.
  • the RNA was analyzed by electrophoresis on denaturing 1% formaldehyde agarose gels and subsequent Northern blotting. After UV-crosslinking of RNA to the nylone membranes (GeneScreen, DuPont) , the distribution of 18S and 28S rRNA was visualized by staining of filters with methylene blue.
  • RNA fractions 1-8 or 1-9 (depending on the cell type, see examples) of each sucrose gradient, representing the ribosome-free RNA population and RNA fractions 11-20 or 12-20, corresponding to the ribosome-bound RNA compartment, were pooled and subjected to poly (A) mRNA selection. Only polysome- bound mRNA fractions were used for further steps of the differential screening. Extracts from cells maintained in IL-3 or withdrawn from IL-3 for 4 and 8 hours were also fractionated on sucrose gradients, for these, however, mRNA was separately isolated from each fraction to verify translational control of candidate genes isolated in the SSH screen.
  • Poly (A) mRNA of unbound and polysome-associated RNA species was selected using the Oligotex-dT purification system (Quiagen), however, poly (A) mRNA selection can be performed with various poly (A) mRNA isolation procedures within kits provided by different manufacturers.
  • Poly (A) mRNA of highest quality must be sufficiently enriched in terms of decreasing the portion of ribosomal RNA in the poly (A) mRNA preparation to ⁇ 20% when compared to the portion of ribosomal RNA present in the cytoplasmic RNA preparation, poly (A) mRNA is quantified by standard OD260 _rea ding . Limitations: at least 0.3-0.4 ⁇ g of poly (A) mRNA is required for cDNA synthesis described in this protocol.
  • BLAST searches were performed on the following databases: Genebank non-redundant cds, EST databases, Drosophila genome database, C.elegans genome database.
  • two known genes and one EST showed clear differential expression after rescreening by Northern blots.
  • One of these genes was previously known to be involved in apoptosis ("requiem", Gabig et al., 1994) therefore it was not further characterized.
  • the second gene, cyclophilin is a potential member of the permeability transition complex of mitochondria, its role in apoptosis in hematopoietic cells however is not yet clear.
  • the third differentially regulated sequence herein designated pl2 had no homology to any known gene or described gene functions.
  • a full length cDNA of pl2 was produced by 5' RACE using purified mRNA from
  • FDCP-1 cells and the SMART RACE cDNA Amplification kit Cloning and sequencing of this gene revealed an open reading frame of 12 kD. A number of identical sequences were found in the mouse EST databases. Significant homology of the mouse pl2 gene was found to ESTs from human (e.g.: AAF28946.1) and rat (e.g.: AA892572), representing approximately 60% sequence identity at the protein level. Similar sequences were also present in Drosophila (AE003682) and C. elegans (P34341) genome databases, suggesting the existence of homologs with similar functions in invertebrates.
  • human e.g.: AAF28946.1
  • rat e.g.: AA892572
  • Probes for further northern blot analysis were prepared from gel purified cDNA fragments using Gibco/Life Technologies Random Primers Labeling System according to instructions of the manufacturer.
  • the candidate genes isolated from the SSH screen were first tested by Northern blot, using combined polysome-bound fractions of mRNA from FDCP-1 cells maintained in IL-3 or withdrawn from IL-3 for 8 hours. Out of the 4 mRNAs that were differentially expressed, the pl2 mRNA appeared to be significantly regulated at the translational level since it was significantly enriched in the polysome bound fraction of IL-3 deprived cells (Fig. 1A) .
  • the cDNA hybridized equally well to total mRNA from FDCP-1 cells maintained in IL-3 or withdrawn from IL-3 for 8 hours. Only 16 hours after IL-3 withdrawal, where most cells had already entered apoptosis, synthesis of the total cellular mRNA was downregulated (Fig. IB) .
  • the labelled cDNA was hybridized to a Northern blot containing the mRNAs from individual fractions of polysome gradients from FDCP-1 cells withdrawn from
  • IL-3 for 4 and 8 hours as well as those maintained in IL-3 (- IL-3, Oh, see Fig. 1C) .
  • IL-3 IL-3 for 4 and 8 hours as well as those maintained in IL-3 (- IL-3, Oh, see Fig. 1C) .
  • IL-3 IL-3 for 4 and 8 hours as well as those maintained in IL-3 (- IL-3, Oh, see Fig. 1C) .
  • a significant fraction of the mRNA had shifted from the free mRNA pool or from monosomes to the polysome pool. This effect was even stronger after 8 hours of IL-3 withdrawal.
  • GPDH housekeeping gene
  • Antisera directed against pl2 were produced by injecting rabbits with KLH coupled to a 12 amino acid long carboxy-terminal peptide whose sequence was derived from the predicted ORF of the murine pl2 cDNA. Injection was repeated four times in two weeks intervals .
  • Antisera were first tested on the bacterially produced GST-pl2 fusion protein containing the full length pl2 fused to the C-terminus of GST. In bacterial cell lysates, the antisera specifically recognize the GST-pl2 fusion but not GST alone (Fig. 2A) . In order to verify that expression of the pl2 protein was indeed induced in FDCP-1 cells by IL-3 withdrawal, western blots from cell lysates of FDCP-1 cells maintained in IL-3 or withdrawn from IL-3 for different time intervals were probed with anti-pl2 antisera. Fig. 2B demonstrates that a 12kd protein is weakly expressed in the proliferating cells, but upregulated 8 hours after IL-3 withdrawal.
  • the pl2 gene product was further analyzed at the functional level, to determine if it had a role in apoptosis induced by cytokine withdrawal.
  • the cDNA was introduced into a retroviral vector allowing conditional expression via a tetracyclin/doxycylin inducible Tet-repressor-VP16 fusion protein (Paulus et al., 1996; retro-Tet-off vector, Clontech) .
  • Infected FDCP-1 cell clones were maintained in supersaturating concentrations of IL-3 and in the presence of doxycylin, which represses expression of pl2 from the retroviral construct, in order to protect cells from the potential pro-apoptotic activity of pl2.
  • doxycylin was removed from the medium and viability in the cultures was measured using the TUNEL assay.
  • As a control clones infected with empty retrovirus were used under identical experimental conditions. pl2 expressing clones died after removal of doxycyclin (Fig. 3, black bars).
  • apoptosis induction was dependent on IL-3 concentration (1 ng/ml to 0.1 ng/ml) and almost complete at the lowest concentration tested (0.1 ng/ml) .
  • doxycylin induction of apoptosis was seen at an intermediate level, due to the leakiness of the promoter (Fig. 3, grey bars) .
  • Control clone cultures contained very few apoptotic cells even under low IL-3 concentrations (Fig. 3, white bars) .
  • pl2 induces apoptosis in other cell types.
  • Mouse fibroblasts were transiently transfected using Lipofectamine (Gibco BRL) with an expression construct co-expressing green fluorescent protein (GFP) and the pl2 cDNA. 24 and 48 hours after transfection, the cells were analyzed for GFP expression and apoptosis induction using the TUNEL assay by FACS analysis. No induction of apoptosis was detected in the GFP-expressing cells at either time point (data not shown) . Together with the tissue specific expression of pl2 mRNA (not shown) these results suggest that the pl2 protein induces apoptosis in a cell type specific manner.
  • cell lysates from exponentially growing and cytokine deprived FDCP-1 cells were separated in cytoplasmic, nuclear and mitochondrial fractions by differential centrifugation: cells were lysed by douncing in hopotonic lysis buffer contained 10 mM Hepes pH 7.3, 200 mM mannitol, 70 mM sucrose, 1 mM EGTA, 1 mM NaF, 0.1 mM Na x V04y an d protease inhibitor Complete TM (Roche Molecular Biochemicals) .
  • Nuclear fractions were collected in low speed pellets (750 g) , mitochondrial fractions in high speed pellets (15000 g) and cytoplasmic fractions are represented by the high speed supernatant. These fractions were subsequently subjected to western-blotting using the antiserum directed against the C-terminus of pl2.
  • the bulk of cellular pl2 was found in the cytosol of cells grown in the presence of IL-3. After cytokine removal the total cellular pl2-level increases as expected but now the majority of cellular pl2 was found in the mitochondrial fraction (Fig. 4).
  • the apparent absence of proteolytic processing of pl2 suggests that it is not imported into mitochondria but rather binds to the outer mitochondrial membrane
  • the quality of subcellular fractionations was controlled by the mitochondrial marker porin and the cytoplasmic localized caspase-2 (Fig. 4).
  • cell lysates were prepared from cells grown in the presence or absence of cytokine and pl2 was immmunoprecipitated from the cell lysates (lysis buffer contained 20 mM Hepes pH7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1 % TritonX-100, ImM DTT, 1 mM NaF, 0.1 mM Na x V04 y ) using antisera directed against the pl2 C-terminus and protein A-sepharose beads (Pharmacia).
  • the immunoprecipitates were subjected to western blotting and filters were probed with the following antisera: rabbit anti pl2 antisera as described above, anti VDAC (31 HL, Calbiochem) . Whereas no stable interactions of pl2 with Bcl-2 family members was detectable (not shown) , the outer mitochondrial membrane component VDAC specifically coprecipitated with pl2 from cytokine starved cell-lysates but not from lysates of cells grown in the presence of cytokine (Fig. 5) .
  • VDAC voltage dependent anion channel

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