KR20060013428A - Inhibition of apoptosis-specific eif-5a( eif-5a1 ) with antisense oligonucleotides and sirans as anti-inflammatory therapeutics - Google Patents

Abstract

The present invention relates to apoptosis specific eucaryotic initiation factor 5A (eIF-5A), referred to as apoptosis-specific eIF-5A or eIF5A1, nucleic acids and polypeptides and methods for inhibiting or suppressing apoptosis in cells using antisense nucleotides or siRNAs to inhibit expression of apoptosis-specific eIF-5A. The invention also relates to suppressing or inhibiting expression of pro-inflammatory cytokines or inhibiting expression of pro-inflammatory cytokines or inhibiting activation of NFkB by inhibiting expression of apoptosis- specific eIF-5A.

Description

Inhibition of apoptosis-specific eIF-5A (＂eIF-5A1) as an anti-inflammatory agent for antisense oligonucleotides and ESAIENA Iii) with antisense oligonucleotides and siRANs as anti-inflammatory therapeutics}

Related Applications

This application is filed on July 23, 2002, filed Jul. 23, 2002, filed July 23, 2001, filed on July 23, 2002, filed on September 7, 2002, filed on September 7, 2002. This is a partial consecutive application of US Patent Application No. 10 / 383,614, filed March 10, 2003, which is part of US Patent Application No. 10 / 277,969, filed Oct. 23, 2002, which is part of US Patent Application No. 10 / 200,148. All are fully incorporated herein. This application also discloses US Provisional Application No. 60 / 476,194, filed June 6, 2003; US Provisional Application No. 60 / 504,731, filed September 22, 2003; US Provisional Application No. 60 / 528,249, filed December 10, 2003; Claims priority of US Provisional Application No. 60 / 557,671, filed March 21, 2004 and US Provisional Application No. 60 / (pending), filed June 2, 2004, all of which are hereby incorporated by reference in their entirety. have.

The present invention relates to apoptosis-specific eukaryotic initiation factor-5A (“eIF-5A”) or deoxyhypusin synthase designated “apoptosis-specific eIF-5A” or eIF-5A1 ”. synthase, DHS) The present invention relates to methods for inhibiting apoptosis-specific eIF-5A and DHS nucleic acids and polypeptides and apoptosis-specific eIF-5A and DHS expression.

Apoptosis is a genetically programmed cellular event characterized by well-defined morphological phenomena such as cell contraction, chromatin condensation, nuclear fragmentation, and membrane blebbing. Kerr et al. (1972) Br. J. Cancer, 26: 239-257; Wyllie et al., (1980) Int. Rev. Cytol., 68: 251-306. It plays an important role in normal tissue development and homeostasis, and defects in the apoptosis program are believed to affect a wide range of human metabolic disorders from neurodegenerative and autoimmune disorders to neoplasms. Thompson (1995) Science, 267: 1456-1462; Mullauer et al. (2001) Mutat. Res, 488: 211-231. Although the morphological properties of apoptotic cells are well characterized, the molecular pathways regulating this process have only begun to be elucidated.

One group of proteins believed to play an important role in apoptosis is the cysteine protease family, caspase, which appears to be required for most pathways of apoptosis. Creagh & Martin (2001) Biochem. Soc. Trans, 29: 696-701; Dales et al. (2001) Leuk. Lymphoma, 41: 247-253. Caspases initiate apoptosis in response to apoptosis stimulation by cleaving a variety of cellular proteins, which cause signs of typical apoptosis, including cell contraction, membrane blebs and DNA mitosis. Chang & Yang (2000) Microbiol. Mol. Biol. Rev., 64: 821-846.

Pro-apoptotic proteins such as Bax or Bak also play an important role in the apoptosis pathway by releasing caspase-activated molecules such as mitochondrial cytochrome c, increasing cell death through apoptosis. Martinou & Green (2001) Nat. Rev. Mol. Cell. Biol., 2: 63-67; Zou et al. (1997) Cell, 90: 405-413. Anti-apoptotic proteins such as Bcl-2 increase cell survival by canceling the activity of pro-apoptotic proteins, Bax and Bak. Tsujimoto (1998) Genes Cells, 3: 697-707; Kroemer (1997) Nature Med., 3: 614-620. The ratio of Bax: Bcl-2 is considered one way in which cell fate is determined; Excess of Bax increases apoptosis and excess of Bcl-2 increases cell survival. Salomons et al. (1997) Int. J. Cancer, 71: 959-965; Wallace-Brodeur & Lowe (1999) Cell Mol. Life Sci., 55: 64-75.

Another protein important in apoptosis is encoded by the tumor suppressor gene p53. The protein is a regulator that regulates cell growth and induces apoptosis in damaged and genetically unstable cells, perhaps through up-regulation of Bax. Bold et al. (1997) Surgical Oncology, 6: 133-142; Ronen et al. (1996); Schuler & Gree (2001) Biochem. Soc. Trans., 29: 684-688; Ryan et al. (2001) Curr. Opin. Cell Biol., 13: 332-337; Zorning et al. (2001) Biochem. Biophys. Acta, 1551, F1-F37.

Alteration of apoptosis pathways is believed to play an important role in many disease processes, including cancer. Wyllie et al. (1980) Int. Rev. Cytol., 68: 251-306; Thompon (1995) Science, 267: 1456-1462; Sen &D'Incalci (1992) FEBS Letters, 307: 122-127; McDonnell et al. (1995) Seminars in Cancer and Biology, 6: 53-60. Investigations of cancer development and progress have typically focused on cell proliferation. However, an important role for apoptosis in oncogenesis has recently become apparent. In fact, much is known about apoptosis because the regulation of apoptosis is constantly changed in some way within tumor cells, using a tumor model. Bold et al. (1997) Surgical Oncology, 6: 133-142.

Cytokines have also been included in the apoptosis pathway. Biological systems require cellular interactions for their regulation and cross-talk between cells generally involves various cytokines. Cytokines are mediators produced in response to a wide variety of stimuli by many different cell types. Cytokines are pleiotropic molecules that can exert many different effects in many different cell types, but are particularly important for the regulation of immune responses and hematopoietic cells and division proliferation and differentiation. The action of cytokines in target cells can advance cell survival, division proliferation, activation, differentiation or apoptosis depending on the specific cytokines, relative concentrations and the presence of other mediators.

The use of anti-cytokines to treat autoimmune disorders (psoriasis, rheumatoid arthritis, Crohn's disease) has gained popularity. Pro-inflammatory cytokines IL-1 and TNF play a large role in the pathology of these chronic diseases and anti-cytokine therapies that reduce the biological action of these two cytokines can provide therapeutic benefits (Dinarello and Abraham). , 2002).

Interleukin 1 (IL-1) is an important cytokine that mediates specific local and systemic inflammatory responses and many disorders, including autoimmune disorders such as vasculitis, osteoporosis, neurodegenerative disorders, diabetes, lupus nephritis and rheumatoid arthritis. In the onset of can cooperate with TNF. The importance of IL-1β in tumor angiogenesis and invasiveness has also been recently demonstrated by resistance of IL-1β knockout mice to metastasis and angiogenesis when infused with melanoma cells (Voronov et al ., 2003).

Interleukin 18 (IL-18) has recently been discovered as one of IL-1 and is linked by structure, receptors and function for IL-1. IL-18 is a major cytokine involved in inflammatory and autoimmune disorders as a result of its ability to induce interferon gamma (IFN-λ), TNF-α and IL-1. Both IL-1β and IL-18 are capable of inducing the production of TNF-α, a cytokine known to contribute to cardiac dysfunction during myocardial ischemia (Maekawa et al ., 2002). Inhibition of IL-18 by neutralization with IL-18 binding protein has been found to reduce ischemia-induced myocardial dysfunction in ischemia / reperfusion models of suprafused human atrial myocardium (Dinarello, 2001). Neutralization of IL-18 using mouse IL-18 binding protein was also possible to reduce IFN-λ, TNF-α and IL-1β transcription levels and to reduce joint damage in collagen-induced arthritis mouse models (Banda et al. , 2003). Reduction of IL-18 production or utility may also be beneficial in controlling metastatic cancer by infusion of IL-18 binding protein in mouse melanoma models that successfully inhibited metastasis (Carrascal et al ., 2003). More important as a pro-inflammatory cytokine, plasma levels of IL-18 were elevated in patients with chronic liver disease and increased levels correlated with disease pain (Ludwiczek et al ., 2002). Similarly, IL-18 and TNF-α were elevated in the serum of diabetic patients with kidney disease. Neuroinflammation with traumatic brain injury was also mediated by inhibition of IL-18 by IL-18 binding proteins with improved neurological recovery in mice with pre-inflammatory cytokines and brain trauma (Yatsiv et al ., 2002).

TNF-α, one of the TNF family of cytokines, is a pro-inflammatory cytokine with pleiotropic effects determined from co-dividing effects in hematopoietic cells, induction of inflammatory responses and induction of apoptosis in many cell types. TNF-α is usually induced by bacterial lipopolysaccharides, parasites, viruses, malignant cells and cytokines and acts to beneficially protect cells from transfection and cancer. However, inadequate induction of TNF-α is a major contributor to disorders resulting from intense and chronic inflammation, such as autoimmune disorders, and may also contribute to cancer, AIDS, heart disease and pneumonia (Aggarwal and Natarajan, 1996; Sharma and Anker, Reconsidered by 2002). The beneficial effects of interfering with TNF-α in human disorders (i.e. inflammatory bowel disease and severe graft-versus-host disease) as well as in experimental animal models of the disease (i.e. pneumonia shock and rheumatoid arthritis) have been described. (Wallach et al ., 1999) Inhibition of TNF-α also suffers from patients suffering from autoimmune disorders such as Crohn's disease (van Deventer, 1999) and rheumatoid arthritis (Richard-Miceli and Dougados, 2001). The ability of TNF-α to enhance the survival and growth of B lymphocytes is also thought to play a role in the development of B-cell chronic lymphocytic leukemia (B-CLL) and within B-CLL. TNF-α levels expressed by T cells are actually correlated with stages of tumor and disease (Bojarska-Junak et al ., 2002) Interleukin-1β (IL-1β) induces TNF-α production It is known to be a cytokine.

Therefore, there is a need for a method of inhibiting or reducing apoptosis as well as reducing levels of cytokines in the body because excessive accumulation of cytokines and TNF-α can lead to deleterious consequences on the body.

Deoxyhyposin synthase (DHS) and hypofucin-containing eukaryotic translation initiation factor-5A (eIF-5A) are known to play an important role in many cellular processes, including cell growth and differentiation. The unique amino acid hypofusin is found in all eukaryotic cells and in some archeobacteria (appears to be associated with eukaryotic cells), but not in eubacteria, and eIF-5A is known only in hypofucin-containing proteins. . Park (1988) J. Biol. Chem., 263: 7447-7449; Schumann & Klink (1989) System. Appl. Microbiol., 11: 103-107; Bartig et al. (1990) System. Appl. Microbiol., 13: 112-116; Gordon et al. (1987a) J. Biol. Chem., 262: 16585-16589. Active eIF-5A is formed in two post-translational steps: The first step is the specific lysine of precursor eIF-5A catalyzed by the deoxyhyfusin synthase of the 4-aminobutyl moiety of spermidine. the formation of deoxyhaifusine residues by migration to the α-amino group; The second step involves the hydroxylation of the present 4-aminobutyl moiety with deoxyhyposin hydroxylase for hypofusin formation.

The amino acid sequence of eIF-5A is well conserved between species and strictly conserved of the amino acid sequence around the hypofusin residue in eIF-5A, indicating that this modification is important for survival. Park et al. (1993) Biofactors 4: 95-104. This assumption is further supported by the observation that inactivation of all isoforms of eIF-5A is observed in yeast data or that the inactivation of the DHS gene, which catalyzes the first step in its activation, blocks cell division. Schnier et al. (1991) Mol. Cell. Biol., 11: 3105-3114; Sasaki et al. (1996) FEBS Lett., 384: 151-154; Park et al. (1998) J. Biol. Chem., 273: 1677-1683. However, depletion of eIF-5A protein in yeast resulted in only a small decrease in total protein synthesis, indicating that eIF-5A is required for translation of specific subsets of mRNA rather than whole protein synthesis. Kang et al. (1993), "Effect of initiation factor eIF-5A depletion on cell proliferation and protein systhesis" in Tuite, M. (ed.), Protein Sythesis and Targeting in Yeast, NATO Series H. Recent discoveries that share highly conserved motifs support the importance of eIF-5A. Xu & Chen (2001) J. Biol. Chem., 276-2555-2561. Moreover, the hypofucin residues of the modified eIF-5A appeared to be essential for sequence-specific binding to RNA, and the binding was not protected from ribonucleases.

Moreover, intracellular depletion of eIF-5A results in significant accumulation of specific mRNAs in the nucleus, indicating that eIF-5A is important for transporting certain kinds of mRNA from the nucleus to the cytoplasm. Liu & Tartakoff (1997) Supplement to Molecular Biology of the Cell, 8: 426a. Abstract No. 2476, 37th American Society for Cell Biology Annual Meeting. Accumulation of eIF-5A in nuclear pore-related intranuclear filaments and their interaction with the general nuclear export receptor further indicates that eIF-5A is a nuclear cytoplasmic transport protein rather than a component of the polysome. Rosorius et al. (1999) J. Cell Science, 112: 2369-2380.

The first cDNA of eIF-5A was Smit-McBrid et al. Was cloned from humans in 1989, and since then the cDNA or gene of eIF-5A has been cloned from various eukaryotic cells, including yeast, rats, chick embryos, alfalfa and tomatoes. Smit-McBrid et al. (1989a) J. Biol. Chem., 264: 1578-1583; Schnier et al. (1991) (yeast); Sano, A. (1995) Imahori, M. et al. (eds), Polyamines, Basic and Clinical Aspects, VNU Science Press, The Netherlands 81-88 (rat); Rinaudo & Park (1992) FASEB J., 6: A453 (chick embryos); Pay et al. (1991) Plant Mol. Biol., 17: 927-929 (alfalfa); Wang et al. (2001) J. Biol. Chem., 276-17541-17549 (tomato).

Expression of eIF-5A mRNA has been investigated in various human tissues and mammalian cell lines. For example, changes in eIF-5A expression were observed in human fibroblasts after serum loss followed by serum addition. Pang & Chen (1994) J. Cell Physiol., 160: 531-538. Aging-related decreases in deoxyhyposin synthase activity and an increase in precursor eIF-5A were observed in aging fibroblasts even though the possibility of reflecting the averaging of differentiation changes of isoforms was not determined. Chen & Chen (1997b) J. Cell Physiol., 170: 248-254.

Studies have shown that eIF-5A targets cells of various proteins such as human immunodeficiency virus type 1 Rev protein and human T cell leukemia virus type 1 Rex protein. Ruhl et al. (1993) J. Cell Biol., 123: 1309-1320; Katahira et al. (1995) J. Virol., 69: 3125-3133. Preliminary studies have targeted RAN by interaction with other RNA-binding proteins such as Rev, indicating that these viral proteins require eIF-5A for viral RNA processing. Liu et al. (1997) Biol. Signals, 6: 166-174.

Thus, although eIF-5A and DHS are known, it remains to be understood how these proteins are involved in the apoptotic pathway as well as cytokine stimulation, which is capable of modulating apoptosis and cytokine expression.

Summary of the Invention

The present invention relates to apoptosis specific eukaryotic initiation factor 5A (eIF-5A), designated "apoptosis specific eIF-5A" or "eIF-5A1". The invention also relates to a method for inhibiting or inhibiting apoptosis in cells using antisense nucleotides or siRNAs to inhibit the expression of apoptosis-specific eIF-5A nucleic acids and polypeptides and apoptosis-specific eIF-5A. . The present invention relates to a method of delivery of siRNA to mammalian lung cells in vivo. The invention also relates to inhibiting or inhibiting the expression of pro-inflammatory cytokines by inhibiting the expression of apoptosis-specific eIF-5A. The invention also relates to inhibiting or inhibiting the expression of p53 by inhibiting the expression of apoptosis specific eIF-5A. The invention also relates to a method of increasing Bcl-2 expression by inhibiting or inhibiting the expression of apoptosis specific eIF-5A1 using antisense nucleotides and siRNAs. The invention also provides a method for inhibiting the production of cytokines, in particular TNF-α in human epithelial cells. In another embodiment of the invention, inhibiting the expression of apoptosis-specific eIF-5A by the use of the desired antisense oligonucleotide in apoptosis-specific eIF-5A is a method of preventing retinal nerve cell death in glaucoma eyes. To provide.

Several isoforms of eukaryotic initiation factor-5A (“eIF-5A”) have been isolated and present in published databanks. These isoforms are known to be functionally redundant. The inventors of the present invention found one isoform that was immediately upregulated prior to induction of apoptosis, which was termed apoptosis-specific eIF-5A or eIF-5A1. It is an object of the present invention to provide apoptosis-specific eIF-5A involved in the activity of eIF-5A as well as DHS.

Apoptosis-specific eIF-5A is likely to be a suitable target for mediation in apoptosis-induced disease states because it appears to act at the level of post-transcriptional regulation of downstream effectors and transcription factors involved in the apoptosis pathway. In particular, apoptosis-specific eIF-5A appears to selectively promote the migration of apoptosis of mRNA encoding downstream effectors and transcription factors from the nucleus to the later translated cytoplasm. The final determination of initiating apoptosis appears to be derived from the complex interaction between internal and external pro-umi anti-apoptotic signals. Lowe & Lin (2000) Carcinogenesis, 21: 485-495. Through this ability to facilitate translation of downstream apoptosis effectors and transcription factors, apoptosis-related eIF-5A appears to overturn the balance between these signals upon apoptosis.

Accordingly, the present invention provides a method of inhibiting or reducing intracellular apoptosis by administering an agent that inhibits or reduces the expression of apoptosis-specific eIF-5A or DHS. By reducing the expression of DHS, less DHS protein is present, making it useful for activating apoptosis-specific eIF-5A. One agent that can inhibit or reduce the expression of apoptosis-specific eIF-5A or freezing of DHS is antisense oligonucleotide. Cellular apoptosis may be delayed or inhibited by reducing the activation of apoptosis-specific eIF-5A or by reducing or inhibiting the expression of apoptosis-specific eIF-5A.

Antisense oligonucleotides have been successfully used to achieve gene-specific inhibition in vivo as well as in vitro. Antisense nucleotides are short, DNA synthesis strands (or DNA analogs) that are antisense (or complementary) to a particular DNA or RNA target. Antisense oligonucleotides have been proposed to prevent the expression of a protein encoded by a DNA or RNA target by binding to the target and stopping expression at the stage of transcription, translation or splicing. To slow down nuclease degradation (Blake et al ., 1985) by using a backbone that is regulated to resist degradation, such as by inversion of phosphodiester bonds in oligonucleotides with phosphorothioate linkages (Blake et al ., 1985). Matzura and Eckstein, 1968) antisense oligonucleotides have been successfully used in cell culture and animal models of disease (Hogrefe, 1999). Other modifications of antisense oligonucleotides to make the oligonucleotides more stable and resistant to degradation are known and understood by those skilled in the art. Antisense oligonucleotides as used herein include oligonucleotides having double or single stranded DNA, double or single stranded RNA, DNA / RNA hybrids, DNA and RNA analogs and base, sugar or backbone modifications. Oligonucleotides are modified by methods known in the art to increase stability and to increase resistance to nuclease degradation. These modifications are known in the art and include, but are not limited to, modifications of oligonucleotide backbones, modifications of sugar moieties, or modifications of bases.

Preferably the antisense oligonucleotides of the invention have a nucleotide sequence that encodes part or all of the coding sequence of an apoptosis-specific eIF-5A polypeptide or a DHS polypeptide. The inventors transfected the viral cell line with antisense nucleotides encoding a portion of the apoptosis-specific eIF-5A polypeptide as described below and measured the number of cells undergoing apoptosis. Cells transfected with antisense oligonucleotides showed a decrease in the number of cells undergoing apoptosis when compared to the same cells not transfected with antisense oligonucleotides. 54-58 show a decrease in the percentage of cells undergoing apoptosis in cells treated with antisense apoptosis-specific eIF-5A oligonucleotides as compared to cells not transfected with antisense apoptosis-specific eIF-5A oligonucleotides. Indicates.

The present invention contemplates the use of many suitable nucleic acid sequences encoding apoptosis-specific eIF-5A polypeptides or DHS polypeptides. For example, the present invention provides the following apoptosis-specific eIF-5A nucleic acid sequences (SEQ ID NOs: 1, 3, 4, 5, 11, 12, 15, 16, 19, 20, and 21) and DHS sequences (SEQ ID NOs: 6, 7) , 8) provides an antisense oligonucleotide. Antisense oligonucleotides of the invention do not require the full length of a given SEQ ID NO. They only need enough length to bind mRNA and inhibit the expression of such mRNA. "Inhibition or reduction of expression" or "inhibition of expression" refers to the absence or detectable reduction in the level of protein and / or mRNA product from a gene of interest, such as apoptosis-specific eIF-5A.

Exemplary antisense oligonucleotides of apoptosis-specific eIF-5A that do not include the entire coding sequence are antisense oligonucleotides of apoptosis-specific eIF-5A having the following SEQ ID NOs: 35, 37 and 39.

Antisense oligonucleotides of apoptosis-specific eIF-5A "include oligonucleotides having substantial sequence identity or substantial homology to apoptosis-specific eIF-5A. Additional antisense of apoptosis-specific eIF-5A of the present invention Oligonucleotides include those having substantial sequence identity (ie 90% homology) to those listed above or those having a sequence that hybridizes to the sequence numbers listed under high stringent conditions. The term “or“ substantial homology ”is used to express that a sequence exhibits substantial structural or functional identity with another sequence. Structural or functional stone differences between sequences with substantial sequence identity or substantial homology are micro margins. It will be (de minimus); that is, they Will not affect the ability of the sequence to function as indicated in the application, the difference may be due to, for example, inherent variations in codon usage between different species, or significant amounts of sequence overlap between two or more different sequences, or Structural differences are considered to be micro margins, even if they have similarities or differ in length or structure, but other sequences have similar physical properties, such as, for example, the ability to hybridize under defined conditions or immunological deadlocks for proteins. Reactivity, similar enzymatic activity, etc. One skilled in the art can readily determine each of these properties by known methods.

Moreover, the two nucleotide sequences are "substantially complementary" when the sequence has at least about 70% or more, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% sequence similarity. . The two amino acid sequences are at least 50%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably 95 between the active or functionally related portions of the polypeptide. It is substantially homologous with more than% similarity.

Sequences are aligned for optimal comparison purposes to determine the percent identity of two sequences (ie, gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous Enemy sequences may be ignored for comparison purposes). In a preferred embodiment at least 30%, 40%, 50%, 60%, 70%, 80% or 90% or more of the reference sequence length is aligned for comparison purposes. Thereafter, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid “identity” refers to an amino acid or nucleic acid “phase) Same sex "). The percent identity between two sequences is a function of the number of identical positions shared by the sequences and takes into account the number of gaps and the length of each gap needed to be introduced for optimal alignment of the two sequences.

Sequence comparison between two sequences and determination of percent identity and similarity can be achieved using a mathematical algorithm (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informations and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).

Nucleic acid and protein sequences of the invention can be further utilized as "question sequences", for example, to perform searches against a sequence database to identify other family members or related sequences. This search is described by Altschul, et al . (1990) J. Mol Biol. It can be executed using the NBLAST and XBLAST programs (version 2.0) of 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program. BLAST protein searches can be performed with the XBLAST program to obtain amino acid sequences homologous to the proteins of the invention. BLASTed BLAST was obtained from Altschul, et al . (1997) Nucleic Acids Res. 25 (17): 3389-3402. When using BLAST and gapped BLAST programs, the default parameters of each program can be used (ie, XBLAST and NBLAST).

The term "apoptosis-specific eIF-5A" includes functional derivatives thereof. The term "functional derivative" of a nucleic acid is used herein to mean homologs or analogs of genes or nucleotide sequences. Functional derivatives retain the function of a given gene, which allows their utility according to the present invention. The "functional derivatives" of the apoptosis-specific eIF-5A polypeptides described herein or the functional derivatives of the antisense oligonucleotides of apoptosis-specific eIF-5A are directed to apoptosis-specific eIF-5A activity or apoptosis-specific eIF-5A. Fragments, variants, analogues or chemical variants of apoptosis-specific eIF-5A that possess immunological cross-reactivity with antibodies specific for. Fragments of apoptosis-specific eIF-5A polypeptides represent any molecular subset.

Functional variants may also include substitutions of similar amino acids that result in no change or insignificant changes in function. Amino acids essential for function can be identified by methods known in the art, such as site-directed mutations or alanine-scanning mutations (Cunningham et al . (1989) Science 244: 1081-1985). The latter method introduces a single alanine mutation at every residue in the molecule. The resulting mutant molecules are tested with assays such as biological activity such as kinase activity or proliferative activity in vitro. Sites important for binding partner / substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al . (1992) J. Mol. Biol. 224: 899-904; de Vos et al . (1992) Science 255: 306-312).

A “variant” is substantially similar to an entire gene or fragment thereof, such as a nucleotide substitution with one or more substituted nucleotides, but retains the ability to encode mRNA transcripts that hybridize to specific genes or hybridize with native DNA. The molecule | numerator is shown. "Homologous" refers to a fragment or variant sequence from another animal genus or species. "Analog" is a non-natural molecule that functions similar or related to the entire molecule, variant or fragment thereof.

Variant peptides include naturally occurring as well as variants prepared by methods well known in the art. Such variants can be readily identified / manufactured using molecular techniques and the sequence information disclosed herein. Such variants can also be readily distinguished from other proteins based on the eIF-5A or DHS protein sequences and / or structural homologs of the invention. The degree of homology / identity present will be based on the evolutionary distance between the ortholog and the amount of difference present in the paralog family, depending on whether the protein is a functional or non-functional variant.

Non-naturally occurring variants of eIF-5A or DHS proteins of the invention can be readily generated using recombinant techniques. Such variants include, but are not limited to deletions, additions, and substitutions in the amino acid sequence of a protein. For example, one kind of substitution is a conserved amino acid substitution. Such substitutions replace a given amino acid of a protein with another amino acid of similar character. In general, conservative substitutions replace the fatty acids Ala, Val, Leu and Ile with each other; Interchange of hydroxyl residues Ser and Thr; Exchange of acidic residues Asp and Glu; Substitution between amide residues Asn and Gln; Exchange of basic residues Lys and Arg; Replacement between the aromatic residues Phe and Tyr. Guidance on which amino acid changes are likely to be phenotypically silent is presented in Bowie et al ., Science 247: 1306-1310 (1990).

The term "hybridization" as used herein is generally used to mean hybridization of nucleic acids under suitable stringency conditions that will be readily apparent to those skilled in the art, depending on the nature of the probe sequence and target sequence. Conditions of hybridization and washing are well known in the art and adjustment of other conditions is readily accomplished depending on the desired stringency by varying incubation time, temperature and / or ionic strength of the solution. Sambrook, J. et al ., Molecular Cloning: A Laboratory Munual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989.

The choice of conditions is defined by the length of the hybridizing sequence, in particular by the length of the probe sequence, the state G-C content of the nucleic acid and the amount of mismatch allowed. Low stringency conditions are desirable when partial hybridization between strands with lower degrees of complementarity is desired. High stringency conditions are preferred when complete or near complete complementarity is desired. For general high stringency conditions the hybridization solution comprises 6 × S.S.C., 0.01 M EDTA, 1 × Denhardt's solution and 0.5% SDS. Hybridization was performed at 68 ° C. for about 3-4 hours for fragments of cloned DNA and for about 12-16 hours for total eukaryotic DNA. At low stringency, the temperature of hybridization is reduced to about 42 ° C. below the melting point (Tm) of the double helix. Tm is known as a function of the G-C content and the length of the double helix as well as the ionic strength of the solution.

The phrase “hybridize to the corresponding portion” of a DNA or RNA molecule as used herein refers to a sufficient sequence that is approximately the same size and sufficient sequence as the molecule to hybridize, i.e., oligonucleotide, polynucleotide, or any nucleotide sequence (in the sense or antisense direction). By similarity it is meant to recognize and hybridize another nucleic acid molecule that exhibits hybridization effects under suitable conditions. For example, a 100 nucleotide length sense molecule will recognize and hybridize nearly 100 nucleotide portions of a nucleotide sequence as long as there is at least 70% sequence similarity between the two sequences. The size of the "corresponding portion" allows for some mismatch in hybridization such that the "corresponding portion" is for example 20-30% larger or smaller than the molecule to hybridize, preferably It is understood that at most 12-15% larger or smaller.

Moreover, functional variants of the polypeptide also include similar amino acid substitutions that result in no or meaningless changes in function. Amino acids essential for function can be identified by methods known in the art, such as site-directed mutations or alanine-scanning mutations (Cunningham et al ., Science 244: 1081-1085 (1989)). The latter method introduces a single alanine mutation at every residue in the molecule. The resulting mutant molecules are tested for biological activity or assays.

The present invention provides other agents that can inhibit or reduce the expression of apoptosis-specific eIF-5A or DHS. One such agent is siRNAs. Small Inhibitory RNAs (siRNAs) represent a viable alternative to antisense oligonucleotides because low concentrations are required to obtain equal or higher expression levels than those obtained with antisense oligonucleotides (Thompson, 2002). Long double helix RNAs have been used to silence the expression of certain genes in various organisms such as plants, nematodes and fruit flies. An enzyme with RNase-III called Dicer processes these long double-stranded RNAs into 21-23 nucleotide low inhibitory RNAs and then integrates into an RNA-induced silencing complex (RISC). Unwinding the siRNA activates RISC and allows single-stranded siRNA to lead the complex to endogenous mRNA by base pairs. Recognition of endogenous mRNA by RISC results in cleavage and as a result is not available for translation. The introduction of long double-stranded RNA into mammalian cells results in a strong antiviral response that can be ignored by the use of siRNAs (Elbashir et al ., 2001). siRNAs have been widely used in cell culture and routinely reduced in specific gene expression of 90% or more.

The use of siRNAs has also gained popularity in inhibiting gene expression in animal models of disease. Recent studies describing siRNAs against luciferases have made it possible to disrupt luciferase expression from plasma co-transfected in a wide variety of organs of pre-birth mice using hydrodynamic infusion release techniques (Lewis et al. , 2002). In the TNF group hydrodynamically injected into the tail vein of mice, siRNA against Fas, a receptor, was much more transfectable than more than 80% of hepatocytes and reduced to 90% Fas expression in the liver until 10 days after the last injection. It was possible (Song et al ., 2003). Fas siRNA was able to protect mice from liver fibrosis and liver necrosis. The progression of pneumonia in mice treated with lethal doses of lipopolysaccharide was inhibited by the use of siRNA directed against TNFα (Sorensen et al ., 2003). SiRNA has the potential to be a very potent drug against the inhibition of expression of specific genes in vivo in terms of cell culture and long lasting effects, the ability to transfect cells in vivo and resistance to degeneration in serum. (Bertrand et al ., 2002).

The inventors of the present invention transfected cells with apoptosis-specific eIF-5A siRNAs and studied their effect on the expression of apoptosis-specific eIF-5A. FIG. 64 shows cells transfected with apoptosis-specific eIF-5A siRNA less produced than apoptosis-specific eIF-5A protein. 65-67 show apoptosis-specific eIF-5A with a low percentage of cells experiencing apoptosis after exposure to campotesine and TNF-α as compared to cells not transfected with apoptosis-specific eIF-5A siRNAs. Cells transfected with siRNAs are shown. Thus, one embodiment of the present invention provides for inhibiting the expression of apoptosis-specific eIF-5A in a cell by transfecting the cell with a vector comprising siRNA of apoptosis-specific eIF-5A.

Preferred apoptosis-specific siIFs of eIF-5A include those having SEQ ID NOs: 30, 31, 32 and 33. Additional siRNAs include those having substantial sequence identity (ie 90% homology) to those listed or those that hybridize to the listed SEQ ID NO under high stringent conditions. By substantial sequence identity and homology is meant above for the antisense oligonucleotides of the present invention. The term “siRNA of apoptosis-specific eIF-5A” includes functional variants or derivatives as described above for the antisense oligonucleotides of the invention.

Delivery of siRNAs and expression constructs / vectors including siRNAs are known to those skilled in the art. US applications 2004/106567 and 2004/0086884, which are incorporated by reference, describe many expression consensus as well as delivery mechanisms including viral vectors, nonviral vectors, liposome delivery vehicles, plasmid injection systems, artificial viral envelopes, and polylysine conjugates. Provide a truck / vector.

One skilled in the art understands regulatory sequences useful for expression constructs / vectors with antisense oligonucleotides or siRNAs. For example, regulatory sequences are constitutive promoters, inducible promoters, tissue-specific promoters or combinations thereof.

Many important human diseases are due to the non-ideal regulation of apoptosis. This non-ideality can result in either a pathological increase in cell number (eg cancer) or loss of damage to the cell (eg degenerative disease). By way of non-limiting example, the methods and compositions of the present invention can be used to prevent or treat the following apoptosis-related diseases and disorders: neurological / nerve degenerative disorders (eg, Alzheimer's, Parkinson's, Huntington's, muscular dystrophy) Lateral sclerosis (Lou Gehrig's disease), autoimmune disorders (e.g., rheumatoid arthritis, systemic lupus erythematosus (SLE), Duchenne Muscular Dystrophy (DMD), motor neuron disorder, fever, Cardiac fever, chronic heart failure, seizures, infantile spinal muscular atrophy, heart failure, kidney failure, atopic dermatitis, sepsis and pulmonary shock, AIDS, hepatitis, glaucoma, diabetes (type 1 and type 2), asthma, retinal pigmentosa, Osteoporosis, xenograft rejection and burns.

One such disease caused by non-ideality in the regulation of apoptosis is glaucoma. Apoptosis is a fatal factor in blinding patients with glaucoma. Glaucoma is one of the eye conditions that results from damage to the optic nerve resulting in progressive blindness. Apoptosis has been shown to be a direct cause of this optic nerve injury.

Early work in the field of glaucoma research indicated that elevated IOPs interfere with axon migration at the stage of filamentous plate (pore glucosa) that is accompanied by the death of retinal ganglion cells. Quigley and Anderson (1976) Invest . Ophthalmol . Vis . Sci ., 15 , 606-16; Minckler, Bunt and Klock (1978) Invest . Ophthalmol . Vis . Sci ., 17 , 33-50; Anderson and Hendrickson (1974) Invest . Ophthalmol . Vis . Sci ., 13 , 771-83; Quigley et al ., (1980) Invest . Ophthalmol . Vis . Sci ., 19 , 505-17 ;. Animal models of glaucoma and post-human tissue studies suggest that the death of retinal ganglion cells in glaucoma is caused by apoptosis. Garcia-Valenzuela et. al ., (1995) Exp. Eye Res ., 61 , 33-44; Quigley et al ., (1995) Invest . Ophthalmol . Vis . Sci ., 36 , 774-786; Monard (1998) In: Haefliger IO, Flammer J (eds) Nitric Oxide and Endothelin in the Pathogenesis of Glaucoma , New York, NY, Lippincott-Raven, 213-220. Disruption of axon migration as a result of increased IOP may contribute to retinal ganglion cell death due to loss of trophic factors. Quigley (1995) Aust NZJ Ophthalmol , 23 (2) , 85-91. Optic nerve papillary astrocytes in glaucoma eyes have also been found to produce increased levels of some neurotoxic substances. For example, increased production of tumor necrosis factor-α (TNF-α) (Yan et al ., (2000) Arch. Ophthalmol ., 118 , 666-673) and nitric oxide synthesis (Neufeld et al ., (1997) Arch. Ophthalmol ., 115 , 497-503), an enzyme that produces nitric oxide was found in the optic nerve papilla of glaucoma eyes. Moreover, increased expression of nitric oxide synthesis (iNOS) and inducible forms of TNF-α by activated retinal glial cells has been observed in rat models of genetic retinal disease. Cotinet et al ., (1997) Glia , 20 , 59-69; de Kozak et al ., (1997) Ocul. Immunol. Inflamm. , 5 , 85-94; Goureau et al ., (1999) J. Neurochem , 72 , 2506-2515. In glaucoma optic nerve papilla, excess nitric oxide has been linked to the degeneration of exons in retinal glial cells. Arthur and Neufeld, (1999) Surv Ophthalmol , 43 (Suppl 1), S129-S135. Finally, increased production of TNF-α by retinal glial cells in response to premature ischemia or elevated hydrodynamic pressure has been shown to induce apoptosis in symbiotic retinal glial cells. Tezel and Wax, (2000) J. Neurosci,. 20 (23) , 8693-8700.

Antisense oligonucleotides have been used successfully in animal models of eye disease. Caspase 2 expression was increased during ischemia in a model of transient ocular retinal ischemia. It was primarily increased in the inner nuclear and ganglion cell layers of the retina. Caspase expression using antisense nucleotides, as determined by retinal translocation, leads to significant histophysiological and functional improvements. Singh et al ., (2001) J. Neurochem., 77 (2) , 446-75. Another study of the cross section of the optic nerve described retinal ganglion cells that upregulate the pro-apoptotic protein box (Bax) and undergo apoptosis. Repeated infusion of box antisense oligonucleotides into the retina above the rat's temple inhibited the local expression of the box and increased the number of surviving retinal ganglion cells involving optic nerve transactions. Isenmann et al ., (1999) Cell Death Differ ., 6 (7) , 673-82.

Delivery of antisense oligonucleotides to retinal ganglion cells has been improved by carefully protecting the oligonucleotides in liposomes. Oligonucleotides were then coated with the envelope of Hemagglutinating Virus of Japan (HVJ) inactivated by fusion (HVJ liposomes). Intravitreal injection into mice of FITC-labeled antisense oligonucleotides resulted in high fluorescence lasting 3 days within 44% of cells in the ganglion layer, while the fluorescence of the FITC-labeled antisense oligonucleotides disappeared after 1 day Care was made in HVJ liposomes producing. Hangai et al ., (1998) Arch Ophthalmol , 116 (7) , 976.

Methods of interfering or regulating apoptosis of the present invention are not limited to the following, but are direct in controlling apoptosis in cells of the eye such as astrocytes, retinal ganglia, retinal glial cells and filamentous plates. The death of retinal ganglion cells in glaucoma is caused by apoptosis. Thus, there is provided a novel treatment for the prevention of vision loss caused by glaucoma by providing a method of inhibiting apoptosis in retinal ganglion cells or by protecting retinal ganglion cells from degeneration by apoptosis.

The present invention provides a method for preventing retinal ganglion cell death in glaucoma eyes by inhibiting the expression of apoptosis-specific eIF-5A1. Inhibiting the expression of apoptosis-specific eIF-5A1 reduces apoptosis. Apoptosis-specific eIF-5A1 is clearly a strong gene that regulates all apoptic processes. Thus regulating apoptosis in the optic nerve papilla indicates that interfering with the expression of apoptosis-specific eIF-5A1 provides a treatment for glaucoma.

Inhibition of expression of apoptosis-specific eIF-5A is not limited to the following: antisense oligos targeted to human apoptosis-specific eIF5A on cells of the eye, such as filamentous, astrocytic, retinal ganglion or retinal glial cells By administering nucleotides. Antisense oligonucleotides have a nucleotide sequence that encodes in at least one portion of an apoptosis-specific eIF-5A polypeptide as defined above. Antisense oligonucleotides targeted to human apoptosis-specific eIF-5A have a nucleotide sequence that encodes in at least one portion of a human apoptosis-specific eIF-5A polypeptide. Preferably the antisense oligonucleotide consists of SEQ ID NO: 26 or 27 or oligonucleotide bound to a sequence complementary to SEQ ID NOs: 26 and 27 under stringent conditions.

Another embodiment of the present invention provides a method of inhibiting expression of apoptosis-specific eIF-5A1 in filamentous cells, astrocytes, retinal ganglion cells or retinal glial cells. Antisense oligonucleotides were administered to filamentous cells, astrocytic cells, retinal ganglion cells, or retinal glial cells, such as, but not limited to the following: targets 26 and 27 targeted for human apoptosis-specific eIF-5A1. The cell may be human.

In addition to having a role in apoptosis, eIF-5A also plays a role in the immune response. The present invention found that apoptosis-specific eIF-5A levels correlate with elevated levels of two cytokines (interleukin 1-beta "IL-1β" and interleukin 18 "IL-18) in ischemic heart tissue. Thus, it has also been demonstrated that apoptosis-specific eIF-5A is involved in cell death as is present in ischemic heart tissue, and such apoptosis-specific eIF-5A / interleukin correlation is also present in non-ischemic heart tissue. 50a-f and 51. Apoptosis-specific eIF-5A, proliferative eIF-5A ("eIF-5A2"-others known as isoforms), IL-1β and IL-18 using PCR assay Levels were measured and compared in various ischemic heart tissues (from coronary bypass surgery and valve (mitral and atrial valve) replacement patients).

The correlation of apoptosis-specific eIF-5A to this protein interleukin further suggests that the inflammation and apoptosis pathways in ischemia may be regulated via regulating levels of apoptosis-specific eIF-5A. Evidence that apoptosis-specific eIF-5A is involved in the immune response was implied by the fact that human peripheral blood mononuclear cells (PBMCs) usually express very low levels of eIF-5A. However, it is very effectively increased in stimuli with T-lymphocyte-specific stimulus expression of apoptosis-specific eIF-5A (Bevec et al ., 1994). This suggests a role for apoptosis-specific eIF-5A in T-cell proliferation and / or activity. It is also possible that apoptosis-specific eIF-5A may be required as a nucleocytoplasmic shuttle for cytokine mRNAs because it is possible for activated T cells to produce various cytokines. The authors of the above referenced document show that as eIF-5A, increased levels of eIF-5A in PBMCs of HIV-1 patients contributing to effective HIV replication in these cells become cellular binding factors for HIV Rev protein and are required for HIV replication. Was found (Ruhl et al., 1993).

Recently, eIF-5A expression has been found to be increased during dendritic cell maturation. Dendrites are antigen-presenting cells that sensitize secondary and injured T cells to induce T cell-mediated immunogens (Steinman, 1991). Immature dendritic cells lack the ability to stimulate T cells and require adequate stimulation (ie, inflammatory cytokines and / or microbial production) to mature into cells capable of activating T cells. Inhibitors of deoxyhyposin synthase, an enzyme necessary to activate eIF-5A, have been shown to inhibit T lymphocyte activation by dendritic cells by preventing CD83 surface expression (Kruse et al. , 2000). EIF-5A thus promotes dendritic cell ripening by acting as a nucleocytoplasmic shuttle for CD83 mRNA.

In these studies involving the role of eIF-5A in the immune system (Bevec et al ., 1994; Kruse et al ., 2000), the researchers did not enumerate or confirm the isoforms of eIF-5A they were testing. There was no reason for it. Summarizing the above, humans have two isoforms of apoptosis-specific eIF-5A ("eIF5A1") and proliferative eIF-5A ("eIF-5A2") all encoded above in separate staining. Known. Prior to the inventors' findings, it has been believed that all of these isoforms are functionally redundant. The oligonucleotides described by Bevec et al ., Which were used to detect eIF-5A mRNA in stimulated PBMCs, are 100% homologous to human apoptosis-specific eIF-5A1 and cloned by proliferation eIF-5A by study. Has Similarly, the primers described by Kruse et al ., Which were used to detect eIF-5A by reverse transcription polymerization chain reaction during dendritic cell maturation, have 100% homology to human apoptosis-specific eIF-5A. .

The present invention relates to regulating the expression of apoptosis-specific eIF-5A for regulating dendritic cell maturation and PBMC activity, which in turn may regulate T cell-mediated immunity. We studied the role of apoptosis-specific eIF-5A in the differentiation of monocytes into adherent macrophages using a U-937 cell line known to express eIF-5A mRNA (Bevec et al ., 1994). . U-937 is a human mononuclear cell line growing in suspension and will stick and differentiate into macrophages upon stimulation of PMA. When PMA is removed by exchanging the medium, the cells become immobilized and are then able to produce cytokines (Barrios-Rodiles et al ., J. Immunol. 163: 963-969 (1999)). Macrophages produce both TNF-α and IL-β in response to lipopolysaccharide (LPS), a factor based on the outer membrane of many bacteria known to cause a general inflammatory response (Barrios-Rodiles et al . , 1999). See FIG. 78 showing a chart of hepatocellular differentiation of cytokines and resulting products. U-937 cells also produce IL-6 and IL-10 with LPS-stimulation (Izeboud et al ., J. Receptor & Signal Transduction Research, 19 (1-4): 191-202 (1999)).

U-937 cells were used to show that apoptosis-specific eIF-5A is upregulated during monocyte differentiation and TNF-α secretion. See FIG. 77. Accordingly, one aspect of the present invention is to provide a method for inhibiting or delaying the maturation of macrophages to inhibit or reduce the production of cytokines. Such methods include providing a medicament that makes it possible to reduce the expression of either DHS or apoptosis-specific eIF-5A. By reducing or eliminating the expression of DHS, apoptosis-specific eIF-5A activity will be reduced or eliminated. It was believed that this result is essential because apoptosis-specific eIF-5A is upregulated during monocyte differentiation and TNF-α secretion. Thus monocyte differentiation and TNF-α secretion may be reduced or eliminated by reducing or eliminating the activity of apoptosis-specific eIF-5A or by directly reducing or eliminating apoptosis-specific eIF-5A expression. Can be. Any agent capable of reducing the expression of DHS or apoptosis-specific eIF-5A can be used or included, but is not limited to antisense oligonucleotides or siRNAs as described herein.

We use human cell lines known to produce cytokines in response to certain stimuli to act as nuclear cytoplasmic shuttles for cytokine mRNAs in vivo to promote human apoptosis-specific translation of cytokines. We have studied the ability of the enemy eIF-5A. Some recent studies have found that human liver cell lines can respond to cytokine stimulation by inducing the production of other cytokines. HepG2 is a well-characterized human sensitizing cancer cell line known to be cytokine sensitive. In response to IL-1β, HepG2 cells rapidly produce TNF-α mRNA and protein in a dose dependent manner (Frede et al ., 1996; Rowell et al ., 1997: Wordemann et al ., 1998). Therefore HepG2 cells were used as a model system to study the regulation of TNF-α production. We have demonstrated inhibition of human apoptosis-specific eIF-5A expression in HepG2 cells that allow cells to produce less TNF-α after transfection with antisense nucleotides directed towards apoptosis factor 5A.

Thus, one embodiment of the present invention provides a method of reducing the level of cytokines in a cell. This method involves administering a medicament to a cell capable of reducing the expression of apoptosis factor 5A1. Reducing the expression of apoptosis factor 5A1 reduces the expression of cytokines and thus reduces the reduced amount of cytokines produced by the cells. Cytokines are preferably pro-inflammatory cytokines and include, but are not limited to, IL-1, IL-18, IL-6 and TNF-α.

Antisense oligonucleotides are discussed as above. An antisense nucleotide of an exemplary human apoptosis-specific eIF-5A is selected from the group consisting of SEQ ID NOs: 35, 37, and 39 or under high stringent conditions for sequences selected from the group consisting of SEQ ID NOs: 35, 37, and 39 It is an antisense nucleotide that takes up.

The medicament may also be composed of siRNAs having sequences complementary to apoptosis-specific eIF-5A. Preferably the siRNA has a sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32 and 33 or hybridized under high stringent conditions to a sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32 and 33 siRNA. 65-67 show a low percentage of cells undergoing apoptosis after exposure of cells transfected with apoptosis-specific eIF-5A siRNAs to camphortesin and TNF-α. The reagents may also include antisense DHS nucleotides.

The invention also relates to a method of reducing the expression of p53. The method includes administering an agent capable of reducing the expression of apoptosis-specific eIF-5A, such as antisense nucleotides or siRNAs as described above. Reducing the expression of apoptosis-specific eIF-5A reduces the expression of p53 as shown in FIG. 52 and Example 10.

The invention also relates to a method of increasing the expression of Bcl-2. This method involves administering an agent capable of reducing the expression of human apoptosis-specific eIF-5A. Preferred medicaments include the antisense oligonucleotides and siRNAs described above. Reduction of the expression of apoptosis-specific eIF-5A increases the expression of Bcl-2 as shown in FIG. 63 and Example 13. FIG. 63 shows that cells transfected with apoptosis-specific eIF-5A siRNA produce less apoptosis-specific eIF-5A protein and further shows that more Bcl-2 protein is produced. The decrease in apoptosis-specific eIF-5A expression is correlated with the decrease in Bcl-2 expression.

The invention also provides a method of reducing the level of TNF-α in a patient in need thereof comprising administering to the patient any one of the antisense nucleotides or siRNAs described above. Cells transfected with the antisense apoptosis-specific eIF-5A oligonucleotides of the present invention as described in FIG. 69 and Example 14 after TNF-α following induction into IL-1 than cells not transfected with antisense oligonucleotides. Produced less.

The present invention is also directed to increased IL-1, TNF-α, IL-6 and IL-8 levels comprising administering to a mammal in said pathological condition an agent that reduces the expression of apoptosis-specific eIF-5A. Provided are methods for treating a characterized pathological condition.

Known pathological conditions characterized by a decrease in IL-1, TNF-α or IL-6 levels include arthritis-rheumatic and osteoarthritis, asthma, allergies, arteritis, Crohn's disease, inflammatory bowel disease (ibd), ulcerative colitis , Coronary heart disease, cystic fibrosis, diabetes mellitus, lupus, multiple sclerosis, Graves' disease, periodontitis, glaucoma and macular degeneration, ocular surface diseases including keratoconus, tracheal ischemia-heart, kidney, reperfusion injury, sepsis, multiple Myelopathy, rejection of organ transplantation, psoriasis and eczema, including but not limited to. Thus reducing the expression of antisense oligonucleotides, apoptosis-specific eIF-5A with siRNA and the methods of the present invention alleviate these pathological conditions.

The present invention also provides a method for delivering siRNA to mammalian lung cells in vivo. SiRNA directed against apoptosis-specific eIF-5A was administered intranasally (mixed with water). Lipopolysaccharide (LPS) was administered intranasally nasal cavity 24 hours after administration of siRNA to apoptosis-specific eIF-5A. After 24 hours, the right lung was removed and myeloid cell type peroxidase was measured. Mouse apoptosis-specific eIF-5A siRNA inhibited myeloid cell type peroxidase by about 90% compared to control siRNA. There were 5 mice in each group in this study. The results of the present study indicate that siRNA can be successfully delivered in vivo to lung tissue of mammals, and siRNA directed against apoptosis-specific eIF-5A inhibits the expression of apoptosis-specific eIF-5A to cause myeloid cell types. Induces inhibition of peroxidase production. Myeloid cell type peroxidase (“MPO”) is a ribosomal enzyme found in neutrophils. Myeloid cell type peroxidase is an enzyme that utilizes hydrogen peroxidase to convert chlorine into hypochlorous acid. Hypochlorite reacts with and destroys bacteria. In addition, myeloid cell type peroxidase is produced when an artery is inflamed. It is therefore clear that myeloid cell type peroxidase is associated with neutrophils and inflammatory responses. We down-regulate apoptosis-specific eIF-5A with siRNA, resulting in a marked reduction in myeloid cell type peroxidase in lung tissue after exposure to LPS (which typically produces an inflammatory response including production of myeloid cell type peroxidase). Showed. Thus, we have shown that the use of siRNA for apoptosis-specific eIF-5A can reduce or inhibit the amount of myeloid cell type peroxidase in lung tissue, thus reducing or inhibiting the inflammatory response.

LPS is a large molecule cell surface antigen of bacteria that initiates a network of inflammatory responses when applied in vivo. LPS delivery into the nasal cavity causes a decrease in the number of neutrophils in the lung. One of the early events is the activation of mononuclear phagocytes through receptor-mediated processes, which lead to the release of many cytokines, including TNF-α. Eventually increased adhesion of neutrophils to endothelial cells induced by TNF-α leads to massive infiltration into the vP space.

Thus, we have shown not only the correlation between apoptosis-specific eIF-5A and the immune response, but also that siRNA against apoptosis-specific eIF-5A inhibits the production of myeloid cell type peroxidase (ie inflammation). Part of the reaction). We have also shown that it is possible to deliver siRNA into lung tissue by simple intranasal delivery in vivo. siRNA was mixed only with water. This provides a major breakthrough and discovery when one skilled in the art would like to devise an acceptable method of delivery of siRNAs.

The ability to reduce inflammation is a direct importance of many diseases. MPO levels are important predictors of cytokine-induced inflammation caused by heart attack and autoimmune disorders. This ability to reduce or inhibit the inflammatory response is useful for treating inflammatory related disorders such as autoimmune disorders. Moreover, the ability to lower MPO can be a means of protecting patients from ischemic events and heart attacks.

In another experiment mice were similarly treated with siRNAs directed against apoptosis-specific eIF-5A. Lipopolysaccharide (LPS) was administered to mice to induce inflammatory and immune system responses. Under control conditions LPS kills thymic cells, which are important immune system precursor cells produced within the thymus to defend against infection. However, siRNA directed against apoptosis-specific eIF-5A allowed about 90% survival of thymic cells in the presence of LPS. Because thymic cells are precursors of T cells, their destruction causes the body's innate immunity to be unable to produce T cells and thus cannot protect against bacterial infections. Thus the use of siRNA for apoptosis-specific eIF-5A can be used to reduce inflammation without disrupting the body's innate immune defense system (as shown by the low levels of MPO in the first example).

One embodiment of the present invention relates to reducing NFk beta ("NFkB") levels by inhibiting apoptosis-specific eIF-5A with siRNAs targeted to apoptosis-specific eIF-5A. NFk beta is a major cell-signaling molecule for inflammation-its activation leads to expression of COX-2 leading to tissue inflammation. The expression of the COX-2-encoding gene, which is believed to be important for the mass production of prostaglandin at the inflammation site, is transcriptionally regulated by NFkB. NFkB is present in the cytoplasm of cells and binds to inhibitors thereof. Damage and inflammatory stimuli release NFkB from the inhibitor. NFkB migrates into the cell nucleus and activates genes important for COX-2 expression. Thus, salt levels can be reduced by reducing the level of NFk beta.

In one experiment human epithelial cells (HT-29 cells) were treated with siRNAs targeted to apoptosis-specific eIF-5A. Inflammation was then induced by NFkB by addition of TNF or interferon gamma and LPS for 1 hour. The results of this experiment indicate that inhibition of apoptosis-specific eIF-5A expression with siRNA provides a decrease in NFkB levels activated by gamma interferon and LPS. See FIG. 74.

One embodiment of the present invention provides a method for inhibiting expression of endogenous apoptosis-specific eIF-5A in a cell. Expression inhibition is preferably performed by the use of antisense polynucleotides or siRNAs of the apoptosis-specific eIF-5A of the invention described above. Expression of endogenous apoptosis-specific eIF-5A occurs resulting in various effects on cells. For example, reduced expression of various proteins, factors, receptors, cytokines is p53; Pro-inflammatory cytokines (see FIGS. 112, 113, 114); Myeloid cell type peroxidase; Active NFk beta; TLR4 (see FIG. 109); TNFR-1 (see FIG. 111) and iNOS. Reduction of endogenous apoptosis-specific eIF-5A expression increases intracellular Bcl-2 expression levels.

It is understood that the antisense nucleic acid siRNA of the invention for use in animals for prophylactic or therapeutic purposes will be administered in the form of a composition additionally comprising a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more water, saline, phosphate buffered saline, dextrin, glycerol, ethanol as well as combinations thereof. Pharmaceutically acceptable carriers may further consist of a minimum amount of auxiliary material, such as a humectant or emulsifier, preservative or buffer, which increases the shelf life or effectiveness of the binding protein. As known in the art, injection compositions may be formulated to provide fast, sustained or delayed release of the active ingredient after administration into a mammal.

The composition of the present invention may be in various forms. These include, for example, solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions, dispersions or suspensions, liposomes, suppositories, injection and infusion solutions. Preferred forms depend on the intended mode of administration and therapeutic application.

Such compositions can be prepared in a manner well known in the pharmaceutical art. The active ingredient in preparing the composition will generally be enclosed in a carrier which can be mixed with the carrier or diluted by the carrier or can be, for example, in the form of a capsule, bag, paper or other container. When the carrier acts as a diluent it can be a solid, semi-solid or liquid substance which acts as a medium for excipients, additives or active ingredients. The composition thus comprises tablets, rhombic tablets, bags, cachets, elixirs, suspensions, aerosols (solid or liquid media), for example ointments, soft and hard capsules containing up to 10% by weight of active compound, It may be in the form of suppositories, injection solutions, suspensions, sterile packaged powders and topical patches.

In general, what is described in the present invention will be more readily understood through the examples provided by the following illustrative methods. The examples are described to understand the present invention but are not intended to limit the scope thereof. The examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinary skill in the art and are described in many publications. Details of conventional methods as used in the construction of vectors and plasmids, introduction of plasmids into host cells and expression and measurement of genes and gene products are described in Sambrook, J. et al ., (1989) Molecular Cloning: A Laboratory Available in many publications, including Manual, 2nd ed., And Cold Spring Harbor Laboratory Press. All references mentioned herein are included.

1 shows the nucleotide sequence (SEQ ID NO: 11) and the deduced amino acid sequence (SEQ ID NO: 12) at the 3 'end of rat apoptosis-specific eIF-5A.

2 shows the nucleotide sequence (SEQ ID NO: 15) and the deduced amino acid sequence (SEQ ID NO: 16) at the 5 'end of rat apoptosis-specific eIF-5A.

3 shows the nucleotide sequence of rat corpus apoptosis-specific eIF-5A full length cDNA (SEQ ID NO: 1). The amino acid sequence is shown as SEQ ID NO: 2.

4 shows the nucleotide sequence (SEQ ID NO: 6) and the deduced amino acid sequence (SEQ ID NO: 7) at the 3 'end of rat apoptosis-specific DHS cDNA.

5 is an alignment of the full-length nucleotide sequence (SEQ ID NO: 20) of the rat corpus luteum apoptosis-specific eIF-5A cDNA with the nucleotide sequence of human eIF-5A (SEQ ID NO: 3) (accession BC000751 or NM_001970, sequence) Number: 3).

FIG. 6 is an alignment of the full-length nucleotide sequence of rat luteal apoptosis-specific eIF-5A cDNA (SEQ ID NO: 20) and the nucleotide sequence of human eIF-5A (SEQ ID NO: 4) (Accession NM_020390, SEQ ID NO: 4).

FIG. 7 is an alignment of the full-length nucleotide sequence of rat luteal apoptosis-specific eIF-5A cDNA (SEQ ID NO: 20) with the nucleotide sequence of mouse eIF-5A (accession BC003889). Mouse nucleotide sequence (accession BC003889) is SEQ ID NO: 5.

FIG. 8 is an alignment of the inferred full-length amino acid sequence of rat luteal apoptosis-specific eIF-5A (SEQ ID NO: 2) and the inferred amino acid sequence of human eIF-5A (SEQ ID NO: 21) (accession BC000751 or NM_001970).

FIG. 9 is an alignment of the inferred full-length amino acid sequence of rat luteal apoptosis-specific eIF-5A (SEQ ID NO: 2) and the inferred amino acid sequence of human eIF-5A (SEQ ID NO: 22) (Accession NM_020390) .

FIG. 10 is an alignment of the inferred full-length amino acid sequence of rat luteal apoptosis-specific eIF-5A (SEQ ID NO: 2) and the inferred amino acid sequence of mouse eIF-5A (SEQ ID NO: 23) (accession BC003889) .

11 is an alignment of the partial-length nucleotide sequence of rat luteal apoptosis-specific DHS cDNA (residues 1-453 of SEQ ID NO: 6) with the nucleotide sequence of human DHS (SEQ ID NO: 8) (accession BC000333, SEQ ID NO: : 8).

12 is a restriction map of rat corpus luteum apoptosis-specific eIF-5A cDNA.

13 is a restriction map of some-length rat corpus luteum apoptosis-specific DHS cDNA.

FIG. 14 is a Northern blot (top) and ethidium bromide stained gel (bottom) of total RNA probed with the ³² P-dCTP-labeled 3′-end of rat corpus apoptosis-specific eIF-5A cDNA.

FIG. 15 is a Northern blot (top) and ethidium bromide stained gel (bottom) of total RNA probed at the ³² P-dCTP-labeled 3′-end of rat corpus apoptosis-specific DHS cDNA.

FIG. 16 shows the DNA laddering experiments investigated after the degree of apoptosis in the superovulated rat corpus luteum was injected with PGF-2α.

FIG. 17 is an agarose gel of genomic DNA isolated from apoptotic rat corpus luteum showing DNA laddering after rat treatment with PGF F-2α.

FIG. 18 shows DNA laddering experiments investigated in rats treated with spermidine before the degree of apoptosis in the dispersed cells of the hyperovulated rat corpus luteum was exposed to prostagladin F-2α (PGF-2α).

19 shows DNA laddering experiments in which the degree of apoptosis in over-ovulated rat corpus luteum was examined in rats treated with spermidine and / or PGF-2α.

FIG. 20 is a Southern blot of rat genomic DNA probed with ³² P-dCTP-labeled some-length rat corpus luteum apoptosis-specific eIF-5A cDNA.

Figure 21 shows pHM6, mammalian epitope tag expression vector (Roche Molecular Biochemicals).

22 shows Northern blots of total RNA isolated from COS-7 cells following apoptosis induction by withdrawal of serum probed into ³² P-dCTP-labeled 3′-untranslated regions of rat corpus luteum apoptosis-specific DHS cDNA. Top) and ethidium bromide stained gel (bottom).

23 is a flow chart showing the procedure of instantaneous transfection of COS-7 cells.

24 is a western blot of transient expression of foreign proteins in COS-7 cells after transfection with pHM6.

Figure 25 shows increased apoptosis reflected by increased caspase activity when COS-7 cells were transiently transfected with pHM6 containing full-length rat apoptosis-specific eIF-5A in the sense direction.

FIG. 26 shows increased apoptosis reflected by increased DNA mitosis when COS-7 cells were transiently transfected with pHM6 containing full-length rat apoptosis-specific eIF-5A in the sense direction.

Figure 27 shows apoptosis detection reflected by increased nuclear mitosis when COS-7 cells were transiently transfected with pHM6 containing full-length rat apoptosis-specific eIF-5A in the sense direction.

FIG. 28 shows increased apoptosis reflected by increased nuclear mitosis when COS-7 cells were transiently transfected with pHM6 containing full-length rat apoptosis-specific eIF-5A in the sense direction.

29 shows apoptosis detection reflected by phosphatidylserine exposure when COS-7 cells were transiently transfected with pHM6 containing full-length rat apoptosis-specific eIF-5A in the sense direction.

FIG. 30 shows increased apoptosis reflected by increased phosphatidylserine exposure when COS-7 cells were transiently transfected with pHM6 containing full-length rat apoptosis-specific eIF-5A in the sense direction.

FIG. 31 shows increased apoptosis reflected by increased nuclear mitosis when COS-7 cells were transiently transfected with pHM6 containing full-length rat apoptosis-specific eIF-5A in the sense direction.

Figure 32 shows increased apoptosis when COS-7 cells were transiently transfected with pHM6 containing full-length rat apoptosis-specific eIF-5A in the sense direction.

33 shows down-regulation of Bcl-2 when COS-7 cells were transiently transfected with pHM6 containing full-length rat apoptosis-specific eIF-5A in the sense direction. Top picture is Coomassie-blue-stained protein blot; The picture below shows the corresponding Western blot.

FIG. 34 shows Coomassie-blue-stained protein blots of COS-7 transiently transfected with pHM6 containing full-length rat apoptosis-specific eIF-5A in the antisense direction using Bcl-2 as a probe and Corresponding Western blot.

FIG. 35 shows Coomassie-blue-stained protein blot of COS-7 transiently transfected with pHM6 containing full-length rat apoptosis-specific eIF-5A in the sense direction using c-Myc as a probe and Corresponding Western blot.

36 shows Coomassie-blue-stained protein blots of COS-7 transiently transfected with pHM6 containing full-length rat apoptosis-specific eIF-5A in the sense direction when p53 is used as a probe and corresponding Western blot.

FIG. 37 shows Coomassie-blue-stained protein blots and corresponding Western blots of pHM6-full-length rat apoptosis-specific eIF-5A expression in COS-7 cells using anti- [HA] -peroxidase probes. Coomassie-blue-stained protein blot and corresponding western blot of pHM6-full-length rat apoptosis-specific eIF-5A expression in COS-7 cells when a p53 probe is used.

Figure 38 is an alignment of human eIF5A2 (SEQ ID NO: 24) with human eIF5A2 (SEQ ID NO: 22) (GenBank Accession No. XM_113401) isolated from RKO cells. Consensus sequence is shown by SEQ ID NO: 28.

39 is a graph showing the percentage of apoptosis that occurred in RKO and RKO-E6 cells after transient transfection. RKO and RKO-E6 cells were transiently transfected with pHM6-LacZ or pHM6-eIF5A1. RKO cells treated with actinomycin D and transfected with pHM6-eIF5A1 showed a 240% increase in apoptosis compared to cells not treated with actinomycin D and transfected with pHM6-LacZ. RKO-E6 cells treated with actinomycin D and transfected with pHM6-eIF5A1 showed a 105% increase in apoptosis compared to cells transfected with pHM6-LacZ without treatment with actinomycin D.

40 is a graph showing the percentage of apoptosis occurring in RKO cells after transient transfection. RKO and RKO-E6 cells were transiently transfected with pHM6-LacZ, pHM6-eIF5A1, pHM6-eIF5A2 or pHM6-resected eIF5A1. Cells transfected with pHM6-eIF5A1 showed a 25% increase in apoptosis compared to control cells transfected with pHM6-LacZ. This increase was not evident in cells transfected with pHM6-eIF5A2 or pHM6-resected eIF5A1.

41 is a graph showing the percentage of apoptosis that occurred in RKO cells after instantaneous transfection. RKO cells were not transfected or transiently transfected with pHM6-LacZ or pHM6-eIF5A1. 60% of cells transfected with pHM6-eIF5A1 were apoptized after correction for transfection efficiency.

42 provides the results of flow cytometry analysis of RKO cell apoptosis after transient transfection. RKO cells were either transfected or transiently transfected with pHM6-LacZ, pHM6-eIF5A1, pHM6-eIF5A2, or pHM6-resected eIF5A1. The table shows the percentage of cells undergoing apoptosis calculated based on the area under the maximum point of each gate. 80% of cells transfected with pHM6-eIF5A1 exhibited apoptosis after correction of untransfected background background apoptosis and transfection efficiency. Cells transfected with pHM6-LacZ, pHM6-eIF5A2 or pHM6-resected eIF5A1 showed only background levels of apoptosis.

FIG. 43 provides western blots of proteins extracted from RKO cells treated with 0.25 μg / ml of actinomycin D for 0, 3, 37, 24 and 48 hours. Top panel shows Western blot using anti-p53 as primary antibody. Middle panel shows Western blot using anti-eIF5A1 as primary antibody. The lower panel shows the membrane used for anti-eIF5A1 blot stained with Coomassie blue after chemiluminescent detection showing the same loading. Both p53 and eIF5A1 are upregulated by actinomycin D treatment.

FIG. 44 is a bar graph showing that both apoptosis-specific eIF-5A (eIF5A) and proliferative eIF-5A (eIF5b) are expressed in cardiac tissue. Cardiac tissue was taken from patients who received coronary artery bypass graft ("CABG"). Gene expression levels of apoptosis-specific eIF-5A (light gray bars) were compared to proliferative eIF-5A (dark gray bars). X-axis is the number of patient identifiers. Y-axis is ng of pg / 18s (picogram of messenger RNA in nanogram of ribosomal RNA 18S).

45 is a bar graph showing that both apoptosis-specific eIF-5A and proliferative eIF-5A are expressed in cardiac tissue. Heart tissue was taken from a patient who underwent valve replacement. Gene expression levels of apoptosis-specific eIF-5A (light gray bars) were compared to proliferative eIF-5A (dark gray bars). X-axis is the number of patient identifiers. Y-axis is ng of pg / 18s (picogram of messenger RNA in nanogram of ribosomal RNA 18S).

FIG. 46 is a bar graph showing gene expression levels measured by real-time PCR of apoptosis-specific eIF-5A (eIf5a) versus proliferative eIF-5A (eIF5b) in pre-ischemic and post-ischemic heart tissue. . Y-axis is ng of pg / 18s (picogram of messenger RNA in nanogram of ribosomal RNA 18S).

47 schematically shows experiments performed on heart tissue. Cardiac tissue was exposed to normal oxygen levels and expression levels of apoptosis-specific eIF-5A and proliferative eIF-5A were measured. Thereafter, the amount of oxygen delivered to the heart tissue was lowered, thus inducing hypoxia and ischemia, which eventually led to heart attack in the heart tissue. Expression levels of apoptosis-specific eIF-5A and proliferative eIF-5A were measured and compared to the expression levels of cardiac tissue prior to injury by ischemia.

48 shows EKGs of cardiac tissue before and after ischemia is induced.

FIG. 49 shows a lap bench set up with the experiment shown in FIG. 47.

50A-F show patient data where the levels of apoptosis-specific eIF-5A correlate with the levels of IL-1β and IL18. 50A is a chart of data obtained from coronary artery bypass graft (CABG) patients. 50B is a chart of data obtained from prosthetic valve replacement patients. 50C is a graph depicting the correlation of apoptosis-specific eIF-5A with IL-18 in CABG patients. 50D is a graph depicting the correlation of proliferative eIF-5A with IL-18 in CABG patients. 50E is a graph depicting the correlation of apoptosis-specific eIF-5A to IL-18 in prosthetic replacement patients. FIG. 50F is a graph depicting the interrelationship of proliferative eIF-5A against IL-18 in stenosis patients.

FIG. 51 is a data chart of a patient by data of the patient used in FIGS. 50A-F.

FIG. 52 shows the levels of proteins produced by RKO cells after treatment with antisense oligos 1, 2 and 3 (for apoptosis-specific eIF-5A). RKO cells are less produced in apoptosis-specific eIF-5A as well as in p53 after transfection with antisense apoptosis-specific eIF-5A oligonucleotides.

Figure 53 shows uptake of antisense oligonucleotides labeled with fluorescence.

54-58 reduce the percentage of cells undergoing apoptosis in cells treated with antisense apoptosis-specific eIF-5A oligonucleotides as compared to cells not transfected with antisense apoptosis-specific eIF-5A oligonucleotides. Indicates.

FIG. 59 shows treatment of filamentous plate cells with TNF-α and / or campotesin that resulted in an increase in the number of cells undergoing apoptosis.

60 and 61 compare the percentage of cells undergoing apoptosis in cells treated with antisense apoptosis-specific eIF-5A oligonucleotide as compared to cells not transfected with antisense apoptosis-specific eIF-5A oligonucleotide. Indicates.

FIG. 62 shows filamentous cells uptaking labeled siRNA in the presence or absence of serum.

FIG. 63 shows cells transfected with apoptosis-specific eIF-5A siRNA with less apoptosis-specific eIF-5A protein produced and more Bcl-2 protein. Reduction of apoptosis-specific eIF-5A expression is correlated with an increase in BCL-2 expression.

64 shows cells transfected with apoptosis-specific eIF-5A siRAN with less apoptosis factor 5a protein produced.

65-67 show that cells transfected with apoptosis-specific eIF-5A siRNA have a low percentage of cells that undergo apoptosis after exposure to campotesin and TNF-α.

FIG. 68 is a photograph of Hoescht-stained filamentous cell line # 506 transfected with siRNA and treated with campotesine and TNF-α from the experiments described in FIGS. 67 and Example 13. FIG. Apoptotic cells are seen as brighter stained cells. Apoptotic cells have a smaller nucleus due to chromatin condensation and are smaller and irregular in shape.

FIG. 69 shows IL-1 revealing HepG2 cells transfected with apoptosis-specific eIF-5A cells that secrete less TNF-α than non-transfected cells.

FIG. 70 shows the sequence of human apoptosis-specific eIF-5A (SEQ ID NO: 29) and the sequence of siRNAs of the present invention (SEQ ID NOs: 30, 31, 32, 33 and 34).

FIG. 71 shows the sequence of human apoptosis-specific eIF-5A (SEQ ID NO: 29) and the sequence of 3 antisense oligonucleotides of the present invention (SEQ ID NOs: 35, 37 and 39, respectively, in the order shown).

FIG. 72 shows the binding positions of three antisense oligonucleotides (SEQ ID NOs: 25-27, respectively, in the order shown) for human apoptosis-specific eIF-5A1. The full-length nucleotide sequence is SEQ ID NO: 19.

73A and 73B show the nucleotide alignment (SEQ ID NOs: 41 and 42, respectively, in the order shown) and amino acid alignments (SEQ ID NOs: 43 and 22, respectively) of human apoptosis-specific eIF-5A for human proliferating eIF-5A As shown).

FIG. 74A shows a plot of Western blot that inhibited the production of TNF-α in transfected HT-29 cells when siRNAs against apoptosis-specific eIF-5A were not inhibited. 74B shows the result of ELISA.

75 shows the result of ELISA. TNF-α production was reduced in cells treated with siRNAs for apoptosis-specific eIF-5A compared to control cells.

76 shows the time progression of the U-937 differentiation experiment. See Example 16.

FIG. 77 shows the results of Western blot showing up-regulated apoptosis-specific eIF-5A during monocyte differentiation and subsequent TNF-α secretion.

78 shows the use of siRNAs for apoptosis-specific eIF-5A to inhibit hepatocyte differentiation and cytokine production.

FIG. 79 is a bar graph showing IL-8 produced in response to interferon as well as TNF-α. This graph shows that siRNA against apoptosis-specific eIF-5A interferes with all IL-8 produced in response to interferon as well as a significant amount of eIL-8 produced as a result of the combined treatment of interferon and TNF.

FIG. 81 shows Western blot of HT-29 cells treated with IFN gamma for 8 and 24 hours. This blot shows up-regulation (four times in eight hours) of apoptosis-specific eIF-5A in HT-29 cells in response to interferon gamma.

82 shows filamentous cells by immunofluorescence. Filamentous cells (# 506) isolated from the optic nerve papilla of an 83 year old female were shown by immunofluorescence. The primary antibody is a) actin; (b) fibronectin; c) laminin; d) GFAP. All pictures were enlarged 400 times.

FIG. 83 shows apoptosis of filamentous cell line # 506 in response to treatment with campotesin and TNF-α. The filamentous cell line # 506 cells were seeded with 40,000 cells per well on 8-well culture slides. After 3 days, the joined LC cells were treated with either 10 ng / ml TFN-α, 50 μM campotesine or 10 ng / ml TNF-α plus 50 μM campotesine. In solution control for campotesin, an equal volume of DMSO was added to untreated control cells. Cells were stained for 48 hours with Hoescht 33258 and observed by fluorescence microscopy using a UV filter. Brightly stained cells of the condensed or fragmented nuclei were counted as apoptosis.

FIG. 84 shows expression of apoptosis-specific eIF-5A during treatment with either campotethecin or a mixture of TNF-α and campotesin. Filamentous plate # 506 cells were seeded with 40,000 cells per well in 24-well plates. After 3 days LC cells were treated with either 50 μM campotesine or a combination of 10 ng / ml TFN-α and 50 μM campotesine and protein lysate was harvested after 1, 4, 8, 24 hours. As a solution control, equal volume of DMSO was added to the control cells and cell lysate was harvested after 1 and 24 hours. 5 μg of protein from each sample was isolated by SDS-PAGE and transferred to PVDF membrane and western blotted with anti-eIF-5A antibody. Bound antibodies were detected by chemiluminescence and exposed to X-ray film. The membrane was then peeled off and re-blotted with anti-β-actin with internal loading control.

FIG. 85 shows expression of apoptosis-specific eIF-5A in filamentous cell lines # 506 and # 517 with transfection with siRNAs. The filamentous cell lines # 506 and # 517 cells were seeded at 10,000 cells per well on a 24-well plate. After 3 days LC cells were transfected with either GAPDH siRNA, apoptosis-specific eIF-5A siRNAs # 1-4 (SEQ ID NO: 30-33) or control siRNA # 5 (SEQ ID NO: 34). After 3 days of transfection, protein acetate was harvested and 5 μg of protein from each sample was isolated by SDS-PAGE and transferred to PVDF membrane and western blotted with anti-eIF-5A antibody. Bound antibodies were detected by chemiluminescence and exposed to X-ray film. The membrane was then peeled off and re-blotted with anti-β-actin with internal loading control.

86 shows apoptosis of filamentous cell line # 506 cells transfected with apoptosis-specific eIF-5A siRNA and treated with TNF-α and campotesin. Filamentous cell line # 506 cells were seeded at 7,500 cells per well on 8-well culture slides. After 3 days LC cells were transfected with either GAPCH siRNA, apoptosis-specific eIF-5A siRNAs # 1-4 (SEQ ID NO: 30-33) or control siRNA # 5 (SEQ ID NO: 34). After 72 hours of transfection, the transfected cells were treated with the combination of 10 ng / ml TFN-α and 50 μM campotesin. After 24 hours the cells were stained with Hoescht 33258 and observed by fluorescence microscopy using a UV filter. Brightly stained cells of the condensed or fragmented nuclei were counted as apoptosis. This graph shows the mean of n = 4 independent experiments. This figure shows that cells treated with siRNA of apoptosis-specific eIF-5A show a smaller percentage of apoptosis when treated with campotesine and TNF as compared to cells not treated with siRNA of apoptosis-specific eIF-5A. Indicates.

FIG. 87 shows apoptosis of filamentous cell line # 517 cells transfected with apoptosis-specific eIF-5A siRNA # 1 and treated with TNF-α and campotesin. Filamentous cell line # 517 cells were seeded at 7,500 cells per well on an 8-well culture slide. After 3 days LC cells were transfected with either apoptosis-specific eIF-5A siRNA # 1 or control siRNA # 5. After 72 hours of transfection, the transfected cells were treated with the combination of 10 ng / ml TFN-α and 50 μM campotesin. After 24 hours the cells were stained with Hoescht 33258 and observed by fluorescence microscopy using a UV filter. Brightly stained cells of the condensed or fragmented nuclei were counted as apoptosis. The results of two independent experiments are shown here. This indicates that cells treated with siRNA have a low percentage of apoptosis.

FIG. 88 shows TUNEL-labeling of filamentous cell line # 506 cells transfected with apoptosis-specific eIF-5A siRNA # 1 and treated with TNF-α and campotesine. Filamentous cell line # 506 cells were seeded at 7,500 cells per well on 8-well culture slides. After 3 days LC cells were transfected with either apoptosis-specific eIF-5A siRNA # 1 (SEQ ID NO: 30) or control siRNA # 5 (SEQ ID NO: 34). After 72 hours of transfection, the transfected cells were treated with the combination of 10 ng / ml TFN-α and 50 μM campotesin. After 24 hours the cells were stained with Hoescht 33258 and DNA mitosis was assessed in situ using a terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin Nick End Labeling (TUNEL) method. Part A represents the same slide observed through the UV filter to visualize Hoescht-stained nuclei. The results represent two independent experiments. All pictures were enlarged 400 times.

89 shows the design of siRNAs for apoptosis-specific eIF5-A1. siRNAs have SEQ ID NOs: 45, 48, 51, 54 and 56. The full-length nucleotide sequence is shown in SEQ ID NO: 29.

90 shows experimental results showing that siRNAs directed against apoptosis-specific eIF-5A provided a decrease in NKkB activation in the presence of interferon gamma and LPS.

91 shows the time course of PBMC experiments (Example 18).

92 shows western blot of cell lysates from PBMCs collected from two donors over time. PBMCs are stimulated with LPS after treatment with PMA and have increased apoptosis-specific eIF-5A expression.

93 shows that LPS-stimulated PBMCs after treatment with PMA have increased apoptosis-specific eIF-5A expression, consistent with increased TNF production.

94 demonstrates that PBMCs respond to LPS without PMA differentiation.

95 shows that PBMCs transfected with apoptosis-specific eIF-5A siRNA demonstrate inhibition of expression of apoptosis-specific eIF-5A.

FIG. 96 shows that PBMCs transfected with apoptosis-specific eIF-5A siRNA and stimulated with LPS produce less TNF than PBMCs not transfected with apoptosis-specific eIF-5A.

97 shows Western blot of cell lysates from HT-29 cells treated with or without gamma interferon.

FIG. 98 shows Western blot of cell lysates from HT-29 cells transfected with control siRNA or apoptosis-specific eIF-5A siRNA.

FIG. 99 shows that HT-29 cells transfected with apoptosis-specific siRNA have reduced levels of TNF production.

FIG. 100 shows that HT-29 cells transfected with apoptosis-specific eIF-5A siRNA show a decrease in apoptosis compared to control cells. Both control and siRNA-transfected cells were primed with interferon gamma and treated with TNF-α.

FIG. 101 shows that HT-29 cells transfected with apoptosis-specific eIF-5A siRNA express less TLR4 protein than control cells.

FIG. 102 shows that HT-29 cells transfected with apoptosis-specific eIF-5A siRNA express less TNFR1 protein than control cells.

FIG. 103 shows that HT-29 cells transfected with apoptosis-specific eIF-5A siRNA express less iNOS protein than control cells.

FIG. 104 shows that HT-29 cells transfected with apoptosis-specific eIF-5A siRNA express less TLR4 mRNA than control cells.

105 shows the time course of the U937 processing.

106 shows apoptosis-specific eIF-5A is upregulated by PMA in U937 cells.

107 shows that apoptosis-specific eIF-5A is upregulated by LPS in U937 cells.

108 shows that apoptosis-specific eIF-5A protein is reduced after several hours of siRNA treatment.

109 shows that siRNA mediated down-regulation of apoptosis-specific eIF-5A is consistent with a decrease in TLR4.

110 shows that siRNA mediated down-regulation of apoptosis-specific eIF-5A is consistent with the less glycosylated form of interferon gamma receptor in U937 cells.

111 shows that siRNA mediated down-regulation of apoptosis-specific eIF-5A is consistent with a decrease in TNFR1 in U937 cells.

FIG. 112 shows that siRNA mediated down-regulation of apoptosis-specific eIF-5A is consistent with a decrease in LPS-induced TNF-α production in U937 cells.

FIG. 113 shows that siRNA mediated down-regulation of apoptosis-specific eIF-5A is consistent with a decrease in LPS-induced IL-1β production in U937 cells.

FIG. 114 shows that siRNA mediated down-regulation of apoptosis-specific eIF-5A is consistent with a decrease in LPS-induced IL-8 production in U937 cells.

115 shows IL-6 production is independent of siRNA mediated down-regulation of apoptosis-specific eIF-5A in U937 cells.

(Example 1)

Visualization of Apoptosis in Rat Corpus luteum by DNA Ladder

The degree of apoptosis was measured by DNA laddering. Genomic DNA was isolated from dispersed luteal cells and excised luteal tissue using the QIAamp DNA Blood Kit (Qiagen) according to the manufacturer's instructions. The corpus luteum tissue was excised 1 and 24 hours after induction treatment of apoptosis, before induction of apoptosis by treatment with PGF-2α. The isolated DNA was incubated for 30 minutes at room temperature with 500 ng of DNA with 0.2 μCi [α- ³² P] dCTP, 1 mM Tris, 0.5 mM EDTA, 3 units of Klenow enzyme and 0.2 pM of dATP, dGTP and dTTP, respectively. End-labeled. Unintegrated nucleotides were removed by passing the sample through a 1 ml Sepadex G-50 column according to Sambrook et al . The samples were then resolved by Tris-acetate-EDTA (1.8%) gel electrophoresis. The gel was dried under vacuum at room temperature for 30 minutes and exposed to x-ray film at -80 ° C for 24 hours.

In one embodiment the degree of apoptosis in the ovulated rat corpus luteum was investigated 0, 1 or 24 hours after injection with PGF-2α. Ovaries were removed without PGF-2α injection in the 0 hour control group. Laddering of low molecular weight DNA fragments reflecting nuclease activity related to apoptosis is not apparent in resected control corpus luteal tissue prior to treatment with PGF-2α but can be identified 1 hour after apoptosis induction and the apoptosis induction shown in FIG. 16. It turns out after 24 hours. The upper panel in this figure is a radiograph of Northern blots that were propagated into the ³² P-dCTP-labeled 3′-untranslated regions of rat corpus apoptosis-specific DHS cDNA. Sub-panel is ethidium bromide stained gel of total RNA. Each lane contains 10 μg of RNA. The data indicate that there is downregulation of apoptosis-specific eIF-5A transcript after serum withdrawal.

In another embodiment the corresponding control animals were treated with saline instead of PGF-2α. After 15 minutes of treatment with saline or PGF-2α, the corpus luteum was removed from the animals. Genomic DNA was isolated from the corpus luteum 3 and 6 hours after removal of tissue from the animal. DNA laddering and increased terminal labeling of genomic DNA are evident after 6 hours of tissue removal from PGF-2α-treated animals but not after 3 hours of tissue removal. See FIG. 17. DNA laddering reflecting apoptosis is also pronounced when the corpus luteum is treated 15 minutes after PGF-2α and is maintained for 6 hours under in vitro conditions in EBSS (Gibco). Nuclease activity associated with apoptosis is also evident from the broader terminal label of genomic DNA.

In another embodiment hyperovulation was induced by subcutaneous injection with 500 μg PGF-2α. Control rats were treated with an equal amount of saline. After 15-30 minutes the ovary was removed and digested with collagenase. Scattered cells from rats treated with PGF-2α were treated for 1 hour in 10 mM glutamine + 10 mM spermidine, 5 hours more (lane 2) or 10 mM glutamine + 10 mM spurt in 10 mM glutamine without spermidine. Incubate for 1 hour in midine and 5 hours (lane 3) in 10 mM glutamine + 1 mM spermidine. Control cells from rats treated with saline were dispersed with collagenase and incubated 1 and 5 hours further with glutamine alone (lane 1). 500 ng of DNA from each sample was labeled with [α- ³² P] -dCTP using Klenow enzyme, separated on 1.8% agarose gel and exposed to film for 24 hours. The results are shown in FIG.

In another embodiment the overovulated rats were injected subcutaneously at 1 mg spermidine / 100 g body weight and 3 equal doses of 0.333 mg / 100 g body weight 24, 12 and 2 hours prior to subcutaneous injection with 500 μg of PGF-2α. Was delivered. Control rats were separated into 3 sets: no injection; Three injections of spermidine without PGF-2α; And three injections of the same volume of saline before PGF-2α treatment. Ovaries were removed from rats after 1 hour 35 minutes or 3 hours 45 minutes of prostaglandin treatment and used for DNA isolation. 500 ng of DNA from each sample was labeled with [α- ³² P] -dCTP using Klenow enzyme, separated on 1.8% agarose gel and exposed to film for 24 hours: Lane 1, no injection (animal) Was sacrificed at the same time as lanes 3-5); Lane 2, three injections of spermidine (animals sacrificed at the same time as lanes 3-5); Lane 3, PGF-2α injection after 3 saline injections (animals sacrificed 1 hour 35 minutes after PGF-2α treatment); Lane 4, PGF-2α injection after 3 injections of spermidine (animals sacrificed 1 hour 35 minutes after PGF-2α treatment); Lane 5, PGF-2α injection after 3 injections of spermidine (animals sacrificed 1 hour 35 minutes after PGF-2α treatment); Lane 6, PGF-2α injection after 3 injections of spermidine (animals sacrificed 3 hours 45 minutes after PGF-2α treatment); Lane 7, PGF-2α injection after 3 injections of spermidine (animals sacrificed 3 hours 45 minutes after PGF-2α treatment). The results are shown in FIG.

RNA isolation

Total RNA was isolated from luteal tissue from rats at various times after PGF-2α induction of apoptosis. Briefly, the tissue (5 g) was ground in liquid nitrogen. The ground powder was mixed with 30 ml guanidinium buffer (4 M guanidinium isothiocyanate, 2.5 mM NaOAc pH 8.5, 0.8% β-mercaptoethanol). The mixture was filtered through four layers of Miracloth and centrifuged at 10,000 g at 4 ° C. for 30 minutes. The supernatant was centrifuged at a cesium chloride density gradient of 11,200 g for 20 hours. The pelleted RNA was washed with 75% ethanol and resuspended in 600 ml of DEPC-treated water and RNA precipitated with 1.5 ml of 95% ethanol and 60 ml of 3M NaOAc at -70 ° C.

Genomic DNA Isolation and Ladder

Genomic DNA was isolated from the extracted corpus luteum tissue or dispersed corpus luteum tissue using the QIAamp DNA Blood Kit (Qiagen) according to the manufacturer's instructions. DNA was terminated by incubating 500 ng of DNA for 30 minutes at room temperature with 0.2 μCi [α- ³² P] dCTP, 1 mM Tris, 0.5 mM EDTA, 3 units of Klenow enzyme and 0.2 pM of dATP, dGTP and dTTP, respectively. -Covered. Unintegrated nucleotides were removed by passing the sample through a 1 ml Sepadex G-50 column according to Maniatis et al . The samples were then resolved by Tris-acetate-EDTA (2%) gel electrophoresis. The gel was dried under vacuum at room temperature for 30 minutes and exposed to x-ray film at -80 ° C for 24 hours.

Plasmid DNA Isolation, DNA Sequencing

The alkaline lysis method described in Sambrook et al ., Hom. Was used to isolate plasmid DNA. Full-length positive cDNA clones were sequenced using the didioxy sequencing method. Sanger et al ., Proc. Natl. Acad. Sci. USA, 74: 5463-5467. Open reading frames were performed using BLAST search (GenBank, Bethesad, MD) and sequence alignment was achieved using the BCM Search Launcher: Multiple Sequence Alignment Pattern-Induced Multiple Alignment Method (F. Corpet, Nuc. Acids Res. , 16: 10881-10890, (1987)). Sequences and sequence alignments are shown in Figures 5-11.

Northern blot hybridization of rat corpus luteum RNA

At various stages of apoptosis, 20 mg of total RNA isolated from rat corpus luteum was separated on 1% denatured formaldehyde agarose gel and immobilized on a nylon membrane. Full-length rat apoptosis-specific eIF-5A cDNA (SEQ ID NO: 1) labeled with ³² P-dCTP using a random primer kit (Boehringer) was used to probe membrane 7 × 10 ⁷ . Alternatively, full-length rat apoptosis-specific DHS cDNA (SEQ ID NO: 6) labeled with ³² P-dCTP using a random primer kit (Boehringer) was used to probe the membrane (7 × 10 ⁷ cpm). The membrane was washed once with 1 × SSc, 0.1% SDS at room temperature and three times with 0.2 × SSC, 0.1% SDS at 65 ° C. Membrane was dried and exposed to x-ray overnight at -70 ° C.

As shown, both apoptosis-specific eIF-5A and DHS were upregulated in corpus luteum tissue. Expression of apoptosis-specific eIF-5A is significantly increased after induction of apoptosis by treatment with PGF-2α-low at 0 hours but substantially increased within 1 hour of treatment, even more increased within 8 hours of treatment, treatment 24 Slightly increased further in time (FIG. 14). The expression of DHS is low at 0 hours, substantially increased within 1 hour of treatment, even more increased within 8 hours of treatment and slightly higher within 24 hours of treatment (FIG. 15).

Production of Rat Progesterone RT-PCR Products Apoptotic Using Primers Based on Yeast, Fungal, and Human eIF-5A Sequences

Some-length apoptosis-specific eIF-5A sequence (SEQ ID NO: 11) corresponding to the 3′-end of the gene was apoptosis by RT-PCR using oligonucleotide primer pairs designed from yeast, fungal and human eIF-5A sequences. Was generated from rat corpus RNA template. The upstream primer used to isolate the 3'-terminus of the rat apoptosis-specific eIF-5A gene is 20 nucleotide degenerating primers: S is selected from C and G; R is selected from A and G; H is selected from A, T and C; Y is selected from C and T; N is any nucleic acid 5 'TCSAARACHGGNAAGCAYGG 3' (SEQ ID NO: 9). The downstream primers used to isolate the 3 'end of the rat eIF-5A gene include 42 nucleotides: 5' GCGAAGCTTCCATGGCTCGAGTTTTTTTTTTTTTTTTTTTTT 3 '(SEQ ID NO: 10). Reverse transcriptase polymerase chain reaction (RT-PCR) was performed. Briefly, the first strand of cDNA was synthesized using 5 mg downstream primer. The first strand was then used as template in RT-PCR using both upstream and downstream primers.

Separation of RT-PCR product on agarose gel indicated the presence of 900 bp fragment, which was subcloned and sequenced into pBluescript ^™ (Stratagene Cloning Systems, LaJolla, Calif.) Using blunt terminal ligation (SEQ ID NO: 11 ). The 3 'terminal cDNA sequence is SEQ ID NO: 11 and the 3' terminal amino acid sequence is SEQ ID NO: 12. See FIGS. 1-2.

Some-length apoptosis-specific eIF-5A sequence (SEQ ID NO: 15) corresponding to the 5 'end of the gene and overlapping with the 3' end was generated from a rat corpus luteum RNA template that apoptosis by RT-PCR. The 5 'primer is a 24-mer with the sequence designated as the human eIF-5A sequence, 5' CAGGTCTAGAGTTGGAATCGAAGC 3 '(SEQ ID NO: 13). The 3 'primer is a 30-mer with the sequence, 5' ATATCTCGAGCCTTGATTGC AACAGCTGCC 3 '(SEQ ID NO: 14), designed according to the 3' terminal RT-PCR fragment. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed. Briefly, the first strand of cDNA was synthesized using 5 mg downstream primer. The first strand was then used as template in RT-PCR using both upstream and downstream primers.

Isolation of the RT-PCR product on agarose gels indicated the presence of 500 bp fragments, which were then subdivided into pBluescript ^™ (Stratagene Cloning Systems, LsJolla, CA) using XbaI and XhoI cloning sites present in upstream and downstream primers, respectively. It was cloned and sequenced (SEQ ID NO .: 15). The 5 'terminal cDNA sequence is SEQ ID NO: 15 and the 5' terminal amino acid sequence is SEQ ID NO: 16. See FIG. 2.

The sequences at the 3 'and 5' ends of the rat apoptosis-specific eIF-5A (SEQ ID NO: 11 and SEQ ID NO: 15, respectively) overlap with the full-length cDNA sequence (SEQ ID NO: 1) and the full-length cDNA sequence SEQ ID NO: 1) is provided. This full-length sequence was aligned with the sequences in the GenBank database and compared. See FIGS. 1-3. The cDNA clone encodes a 154 amino acid polypeptide (SEQ ID NO: 2) with a calculated molecular weight of 16.8 KDa. Nucleotide sequence for the full-length cDNA of the rat apoptosis-specific corpus luteum eIF-5A gene obtained by RT-PCR, SEQ ID NO: 1 is shown in FIG. 3 and the corresponding deduced amino acid sequence is SEQ ID NO: 9 . The derived full-length amino acid sequence of eIF-5A was aligned with human and mouse eIF-5A sequences. See FIGS. 7-9.

Generation of Rat Progesterone RT-PCR Products Apoptotic with Primers Based on Human DHS Sequences

Partial-length apoptosis-specific DHS sequences (SEQ ID NO: 6) corresponding to the 3 ′ end of the gene were generated from rat corpus RNA templates that apoptized by RT-PCR using oligonucleotide primer pairs designated as human DHS sequences. The 5 'primer is a 20-mer with the sequence, 5' GTCTGTGTATTATTGGGCCC 3 '(SEQ ID NO: 17); The 3 'primer is a 42-mer with sequence, 5' GCGAAGCTTCCATGGCTCGAGTTTTTTTTTTTTTTTTTTTTT 3 '(SEQ ID NO: 18). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed. Briefly, the first strand of cDNA was synthesized using 5 mg downstream primer. The first strand was then used as template in RT-PCR using both upstream and downstream primers.

Separation of RT-PCR product on agarose gel indicated the presence of 606 bp fragment, which was subcloned and sequenced into pBluescript ^™ (Stratagene Cloning Systems, LaJolla, Calif.) Using blunt terminal ligation (SEQ ID NO: 6 ). The nucleotide sequence (SEQ ID NO: 6) for some length cDNA of the rat apoptosis-specific corpus luteum DHS gene obtained by RT-PCR is shown in Figure 4, and the corresponding deduced amino acid sequence is SEQ ID NO: 7.

Isolation and Southern Analysis of Genomic DNA

Genomic DNA was isolated from excised rat ovaries for Southern blots. About 100 mg of ovarian tissue was separated into small pieces and placed in a 15 ml tube. Tissues were washed twice with 1 ml of PBS by slowly shaking the tissue suspension followed by removal of PBS using a pipette. Tissue was resuspended in 2.06 ml of DNA-buffer (0.2 M Tris-HCl pH 8.0 and 0.1 mM EDTA) and 240 μl of 10% SDS and 100 μl of protease K (Boehringer Manheim; 10 mg / ml) Added. The tissue was placed in shaken water bath at 45 ° C. overnight. The next day 100 μl protease K (10 mg / ml) was added and the tissue suspension was incubated in a water bath at 45 ° C. for an additional 4 hours. After incubation the tissue suspension was extracted once with the same volume of phenol: chloroform: iso-amyl alcohol (25: 24: 1) and once with the same volume of chloroform: iso-amyl alcohol (24: 1). After extraction 1 / 10th volume (pH 5.2) of 3 M sodium acetate and 2 volumes of ethanol were added. A glass pipette sealed and formed into a hook using a Bunsen burner was used to draw the DNA seal from the solution and transfer the DNA into a clean microcentrifuge tube. DNA was washed once with 70% ethanol and air-dried for 10 minutes. DNA pellet was dissolved in 500 μl of 10 mM Tris-HCl, pH 8.0, 10 μl of RNase A (10 mg / ml) was added, and DNA was incubated at 37 ° C. for 1 hour. DNA was extracted once with phenol: chloroform: iso-amyl alcohol (25: 24: 1) and DNA was precipitated by adding 1 / 10th volume of 3M sodium acetate (pH 5.2) and 2 volumes of ethanol. DNA was pelleted by centrifugation at 13,000 × g for 10 minutes at 4 ° C. DNA pellet was washed once with 70% ethanol and dissolved in 200 μl of 10 mM Tris-HCl, pH 8.0 by spinning DNA overnight at 4 ° C.

For Southern blot analysis, genomic DNA isolated from rat ovaries was digested with various restriction enzymes that were not cleaved or cut once in endogenous genes. To accomplish this, 10 μg of genomic DNA, 20 μl of 10 × reaction buffer and 100 U restriction enzyme were reacted for 5-6 hours with a total reaction volume of 200 μl. The digested DNA was loaded on a 0.7% agarose gel and electrophoresed at 40 volts for 6 hours or at 15 volts overnight. After electrophoresis, the gel was clarified for 10 min in 0.2 N HCl, then washed twice in 15 ml of denatured solution (0.5 M NaOH, 1.5 M NaCl) and neutralized buffer (1.5 M NaCl, 0.5 M Tris-HCl pH 7.4). Washed twice for 15 minutes. DNA was transferred to nylon membrane and membrane was hybridization solution (40% formamide, 6 X SSC, 5 X Denhart's solution (1 X Denhart's solution is 0.02% Ficoll, 0.02% PVP and 0.02% BSA), 0.5% SDS And 1.5 mg of denatured salmon serum DNA). The 700 bp PCR fragment of the 3 'UTR of the rat eIF-5A cDNA (650 bp of 3' UTR and 50 bp of coding) was labeled [α- ³² P] -dCTP by random priming and 1 × 10 ⁶ cpm / was added to the membrane in ml.

Similarly, 606 bp PCR fragment (450 bp coding and 156 bp 3 ′ UTR) of rat DHS cDNA was randomly primed with [α- ³² P] -dCTP and added at 1 × 10 ⁶ cpm / ml with a second identical membrane It became. Blots were hybridized overnight at 42 ° C. and then washed twice with 2 × SSC and 0.1% SDS at 42 ° C. and twice with 1 × SSC and 0.1% SDS at 42 ° C. Blots were exposed to film for 3-10 days.

Rat corpus genomic DNA was digested with restriction enzymes as shown in FIG. 20 and probed with ³² P-dCTP-labeled full-length eIF-5A cDNA. Hybridization under high stringency conditions showed hybridization of full-length cDNA probes into some restriction fragments for each restriction enzyme digested DNA sample, indicating the presence of some isoform of eIF-5A. In particular, restriction fragments of apoptosis-specific isoforms of eIF-5A were detectable in Southern blots when rat genomic DNA was digested with EcoRV with apoptosis-specific eIF-5A with restriction sites in the open reading frame. Two fragments are shown by the double arrows in FIG. 20. Restriction fragments corresponding to apoptosis-specific isoforms of eIF-5A are indicated by a single arrow in lanes labeled EcoR1 and BamH1, which are restriction enzymes with no cleavage sites within the open reading frame. These results indicate that apoptosis-specific eIF-5A is a single copy in rats. As shown in Figs. 5 to 13, the eIF-5A gene is highly conserved between species, and it is expected that there is a significant amount of conservation between isoforms within any species.

FIG. 21 shows Southern blot of GAT genomic DNA probed with ³² P-dCTP-labeled some-length rat corpus luteum apoptosis-specific DHS cDNA. Genomic DNA was digested with EcoRV, a restriction enzyme that does not cleave some-length cDNA used as a probe. It is clear that two restriction fragments indicate that there are two copies of the gene or that the gene comprises an intron with an EcoRV site.

(Example 2)

This example illustrates apoptosis regulation with apoptosis-specific eIF-5A and DHS.

Culture of COS-7 Cells and Isolation of RNA

An African green monkey kidney fibroblast-like cell line transformed with a mutation of SV40 encoding the wild type T antigen was used for all transformation-based experiments. COS-7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 0.584 g L-glutamine, 4.5 g glucose, 0.37% sodium bicarbonate per liter. Culture medium was supplemented with 10% fetal bovine serum (FBS) and 100 units of penicillin / streptomycin. Cells were grown in a humid environment at 37 ° C. with 5% CO 2 and 95% air. Cells were subcultured every 3-4 days by detaching adherent cells with a solution of 0.25% trypsin and 1 mM EDTA. Desorbed cells were dispersed at a split ratio of 1:10 in fresh culture dishes in fresh medium.

COS-7 cells used for isolation of RNA were grown in 150-mm tissue culture treated Corn. Cells were cultured by detaching them with a solution of trypsin-EDTA. Desorbed cells were collected in centrifuge tubes and pelleted by centrifugation at 3000 rmp for 5 minutes. The supernatant was removed and the cell pellet was snap-frozen in liquid nitrogen. RNA was isolated from frozen cells using the GenElute Mammalian Total RNA Miniprep Kit (Sigma) according to the manufacturer's instructions.

Drying of Recombinant Plasmids and Transfection of COS-7 Cells

The recombinant plasmid carrying the full-length coding sequence of rat apoptosis-specific eIF-5A in the sense direction and the 3 ′ untranslated region (UTR) of the rat apoptosis-specific eIF-5A in the antisense direction is shown in FIG. 21. It was dried using an epitope tag expression vector, pHM6 (Rowhe Molecular Biochemicals). Vectors include: CMV promoter—human cytomegalovirus mediated early promoter / enhancer; Nonapeptide epitope tags from HA-influenza hemagglutinin; BGH pA-bovine growth hormone polyadenylation signal; f1 ori-f1 origin; SV40 ori-SV40 early promoter and origin; Neomycin-neomycin resistance (G418) gene; SV40 pA-SV40 polyadenylation signal; Co1 E1-Co1E1 origin; Ampicillin-Ampicillin resistance gene. The full-length coding sequence of rat apoptosis-specific eIF-5A and the 3 'UTR of rat apoptosis-specific eIF-5A were determined by PCR from the original rat eIF-5A 5A RT-PCR fragment (SEQ ID NO: 1) in pBluescript. Amplified by Primers used to amplify full-length eIF-5A were as follows: forward 5 'GCC AAGCTT AATGGCAGATGATTT GG 3' (SEQ ID NO: 59) (Hind3) and reverse 5 'CT GAATTC CAGT TATTTTGCCATGG 3' (SEQ ID NO: 60) (EcoRl). Primers used to amplify 3 'UTR rat apoptosis-specific eIF-5A were as follows: Forward 5' AAT GAATTC CGCCATGACAGAGGAGGC 3 '(SEQ ID NO: 61) (EcoRl) and reverse 5' GCG AAGCTT CCATGG CTCGAG TTTTTTTTTTTTTTTTTTTTT 3 '(SEQ ID NO: 62) (Hind3).

The full-length rat apoptosis-specific eIF-5A PRC product isolated after agarose gel electrophoresis was 430 bp long while the 3 'UTR rat apoptosis-specific eIF-5A PCR product was 697 bp long. Both PCR products were subcloned into the Hind3 and EcoR1 sites of pHM6 to generate pHM6-full-length apoptosis-specific eIF-5A and pHM6-antisense 3 'UTR eIF-5A. Full-length rat apoptosis-specific eIF-5A PCR products were subcloned in-frame with nonapeptide epitope tags from influenza hemagglutinin (HA) present upstream of the multiple cloning site to anti- [HA]- Detection of recombinant proteins using peroxidase antibodies was enabled. Expression is initiated by human cytomegalovirus mediated early promoter / enhancer to ensure high levels of expression in mammalian cell lines. The plasmid also contains the neomycin-resistant (G418), which allows the selection of stable transfectants, and the SV40 early promoter, which enables episomal replication in cells expressing the SV40 large T antigen, such as COS-7, and It features an origin.

COS-7 cells used for transfection experiments were cultured in 24-well cell culture plates (Corning) for cells used for protein extraction or in 4-chamber culture slides (Falcon) for cells used for staining. Cells were supplemented with 10% FBS but grown to 50-70% confluency in DMEM medium without penicillin / streptomycin. Transfection medium sufficient for one well or culture slide in a 24-well plate was prepared by diluting 0.32 μg of plasmid DNA in 42.5 μl serum free DMEM and incubating the mixture for 15 minutes at room temperature. 1.6 μl of transfection reagent, LipofectAMINE (Gibco, BRL), was diluted in 42.5 μl of serum free DMEM and incubated at room temperature for 5 minutes. After 5 minutes the LipofectAMINE mixture was added to the DNA mixture and incubated together for 30 to 60 minutes at room temperature. Cells to be transfected were washed once with serum free DMEM before overlapping with the transfection medium and the cells were placed back into the growth chamber for 4 hours.

After incubation 0.17 ml DMED + 20% FBS was added to the cells. The cells were further incubated for 40 hours before being induced to undergo apoptosis before staining or harvested for western blot. Mock transfection was performed with plasmid DNA omitted from the transfection medium as a control.

Protein Extraction and Western Blotting

Cells were washed twice in PBS (8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na ₂ HPO ₄ , and 0.24 g / L KH ₂ PO ₄ ) and 150 μl of hot SDS gel-loading buffer Proteins were isolated for western blotting from transfected tax by adding (50 mM Tris-HCl pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue and 10% glycerol). Cell lysates were collected in a microcentrifuge tube and heated at 95 ° C. for 10 minutes and then centrifuged at 13,000 × g for 10 minutes. Supernatants were transferred to fresh microcentrifuge tubes and stored at -20 ° C until use.

2.5 or 5 μg of total protein was isolated on 12% SDS-polyacrylamide gels for western blotting. The isolated protein was transferred to a polyvinylidene difluoride membrane. The membrane was incubated for 1 hour in blocking solution (5% skim milk powder in PBS, 0.02% sodium azide) and washed three times with PBS-T (PBS + 0.05% Tween-20) for 15 minutes. The membrane was stored overnight at 4 ° C. PBS-T. After warming to room temperature the next day the membrane was blocked for 30 seconds in 1 μg / ml polyvinyl alcohol. The membrane was washed five times with deionized water and then blocked for 30 minutes in a 5% milk solution in PBS. Primary antibodies were preincubated for 30 minutes in 5% milk solution in PBS before incubation with the membrane.

Some primary antibodies were used. Anti- [HA] -peroxidase antibodies (Roche Molecular Biochemicals) were used diluted 1: 5000 to detect expression of recombinant proteins. Since these antibodies were conjugated to peroxidase, no secondary antibody was needed, and the blots were washed and developed by chemiluminescence. Other primary antibodies used are monoclonal antibodies from Oncogene that recognize p53 (Ab-6), Bcl-2 (Ab-1) and c-Myc (Ab-2). Monoclonal antibodies against p53 were used diluted to 0.1 μg / ml and monoclonal antibodies against Bcl-2 and c-Myc were both diluted to 0.83 μg / ml. After 60-90 minutes of incubation with the primary antibody, the membranes were washed three times for 15 minutes in PBS-T. The secondary antibody was then diluted in 1% milk in PBS and incubated with the membrane for 60-90 minutes. When p53 (Ab-6) was used as the primary antibody, the secondary antibody used was goat anti-mouse IgG conjugated to alkaline phosphatase (Rockland) diluted 1: 1000. When Bcl-2 (Ab-1) and c-Myc (Ab-2) were used as primary antibodies, rabbit anti-mouse IgG conjugated to peroxidase (Sigma) was used diluted 1: 5000. After incubation with the secondary antibody, the membrane was washed three times with PBS-T.

Two detection methods were used to develop the blots: the colorimetric method and the chemiluminescent method. The staining method was used only when p53 (Ab-6) was used as primary antibody conjugated with alkaline phosphatase-conjugated secondary antibody. Bound antibody was 0.33 mg / ml nitro blue tetrazolium, 0.165 mg / ml 5-bromo-4-chloro-3-indolyl phosphate, 100 mM NaCl, 5 mM MgCl ₂ and 100 mM Tris-HCl, pH 9.5 Visualized by incubating the blots under dark conditions in a solution of. The color reaction was stopped by incubating the blots in 2 mM EDTA in PBS. Chemiluminescent detection methods were used for all other primary antibodies, including anti- [HA] -peroxidase, Bcl-2 (Ab-1) and c-Myc (Ab-2). ECL Plus Western Blotting Detection Kit (Amersham Pharmacia Biotech) was used to detect peroxidase-conjugated binding antibodies. Briefly, the membrane was lightly blotted dried and incubated for 5 minutes in a 40: 1 mixture of reagent A and reagent B. The membrane was blotted dry and placed between acetate sheets and exposed to x-ray film for various periods from 10 seconds to 10 minutes.

Induction of Apoptosis in COS-7 Cells

Two methods of serum deficiency and treatment with Actinomycin D, Streptomyces sp. (Calbiochem) were used to induce apoptosis in transfected COS-7 cells. For both treatments the medium was removed at 40 hours post-transfection. In serum deficiency experiments medium was replaced with serum-free and antibiotic-free DMEM. Cells grown in antibiotic-free DMEM supplemented with 10% FBS were used as controls. In actinomycin induction of apoptosis, the medium was replaced with an antibiotic-DMEM supplemented with actinomycin D dissolved in 10% FBS and 1 μg / ml methanol. Control cells were grown in Antibiotic-DMEM supplemented with 10% FBS and equal volume of methanol. For both methods, the percentage of apoptotic cells was measured after 48 hours by staining with Hoescht or Annexin V-Cy3. Induction of apoptosis was confirmed by Northern blot analysis as shown in FIG. 22.

Hoesht Dyeing

A nuclear stain, Hoesht, was used to label the nuclei of transfected COS-7 cells to identify apoptotic cells based on morphological features such as nuclear mitosis and condensation. A fixative consisting of a mixture 3: 1 of pure methanol and glacial acetic acid was prepared immediately before use. An equal volume of fixative was added to the medium with COS-7 cells growing on the culture slide and incubated for 2 minutes. The medium / fixant mixture was removed from the cells and discarded and 1 ml of fixative was added to the cells. After 5 minutes the fixative was removed and 1 m fresh fixative was added to the cells and incubated for 5 minutes. The fixative was removed and the cells were air-dried for 4 minutes before adding 1 ml of Hoesht stain (0.5 μg / ml Hoescht 33258 in PBS). After 10 minutes of incubation in dark conditions, the staining solution was removed and the slides were washed three times for 1 minute with deionized water. After washing 1 ml of McIvavaine's buffer (0.021 M citric acid, 0.058 M Na ₂ HPO ₄ .7H ₂ O; pH 5.6) was added to the cells and incubated for 20 minutes in the dark. The buffer was removed and the cells were air-dried for 5 minutes in dark conditions and the chamber separating the wells of the culture slide was removed. Several drops of fluorescent Vectashield mounting medium (Vector Laboratories) were added to the slide and covered with coverslips. Stained cells were observed under fluorescence microscopy using a UV filter. Brightly stained cells or non-divided nuclei were scored as apoptosis.

Annexin V-Cy3 Staining

Annexin V-Cy3 Apoptosis Detection Kit (Sigma) was used to fluorescently label phosphatidylserine externalized on apoptotic cells. The kit was used according to the manufacturer's protocol with the following modifications. Briefly, transfected COS-7 cells growing on four chamber culture slides were washed twice with PBS and three times with 1 × binding buffer. 150 μl of staining solution (1 μg / ml AnnCy3 in 1 × binding buffer) was added and cells were incubated for 10 min in the dark. Staining solution was removed and cells were washed 5 times with 1 × binding buffer. The chamber wall was removed from the culture slide and several drops of 1 × binding buffer were placed on the cells and covered with coverslips. Stained cells were analyzed by fluorescence microscopy using a green filter to visualize red fluorescence of positively stained (apoptotic) cells. The total cell population was measured by counting cell numbers under visible light.

(Example 3)

This example illustrates apoptosis regulation of apoptosis-specific eIF-5A and DHS.

Using the general procedure and method described in the above examples, FIG. 23 shows COS in which cells in serum-free medium were incubated for 4 hours in plasmid DNA in LipofectAMINE, serum was added, and cells were incubated for another 40 hours. -7 is a flow chart showing the procedure of instantaneous transfection of cells. Cells are then incubated in serum-containing normal medium for an additional 48 hours prior to assay (i.e., no further treatment) and serum removed for 48 hours to induce apoptosis before analysis or actinomycin D for 48 hours to induce apoptosis before analysis. Is processed.

FIG. 22 is a Western blot showing transient expression of foreign protein in COS-7 after transfection with pHM6. Proteins may be mock transfected or pHM6-LacZ, pHM6-Antisense 3 'rF5A (pHM6-Antisense 3' UTR rat apoptosis-specific eIF-5A) or pHM6-Sense rF5A (pHM6-full length rat apoptosis-specific eIF- After transfection with 5A) it was isolated from COS-7 cells. 5 μg of protein from each sample was fractionated by SDS-PAGE and transferred to PVDF membrane and western blotted with anti- [HA] -peroxidase. Bound antibodies were detected by chemiluminescence and exposed to x-ray film for 30 seconds. Expression of LacZ (lane 2) and sense rat apoptosis-specific eIF-5A (lane 4) is clearly visible.

COS-7 cells were transfected with mock transfection or pHM6-Sense rF5A (pHM6-full length rat apoptosis-specific eIF-5A) as described above. After 40 hours of transfection, cells were induced to undergo apoptosis by withdrawal of serum for 48 hours. Caspase proteolytic activity in the transfected cell extracts was measured using a fluorescence homology caspase assay kit (Roche Diagnostics). DNA mitosis was also measured using the FragEL DNA Cleavage Apoptosis Detection Kit (Oncogene), which labels the 3'-OH terminus of the DNA fragment with fluorescently-labeled deoxynucleotides.

Additional COS-7 cells were transfected with mock transfection or pHM6-Sense rF5A (pHM6-full length rat apoptosis-specific eIF-5A). After 40 hours of transfection, cells are further grown 48 hours in serum-containing normal medium (no further treatment) and induced to undergo apoptosis by withdrawal of serum for 48 hours or 0.5 μg / ml of actinomycin D for 48 hours. Treatment was induced to undergo apoptosis. Cells were stained with Hoescht 33258, indicating nuclear mitosis with apoptosis, or with Annexin V-Cy3, indicating phosphatitylserine exposure with apoptosis. Stained cells were observed under a fluorescence microscope using a green filter and counted to determine the percentage of cells undergoing apoptosis. Total cell population was counted under visible light.

25 shows increased apoptosis reflecting increased caspase activity when COS-7 cells were transiently transfected with pHM6 including full-length rat apoptosis-specific eIF-5A in the sense direction. Expression of rat apoptosis-specific eIF-5A showed a 60% increased caspase activity.

FIG. 26 shows increased apoptosis reflecting increased DNA mitosis when COS-7 cells were transiently transfected with pHM6 with full-length rat apoptosis-specific eIF-5A in the sense direction. Expression of rat apoptosis-specific eIF-5A showed a 273% increased DNA mitosis. FIG. 27 shows increased apoptosis reflecting increased nuclear mitosis when COS-7 cells were transiently transfected with pHM6 including full-length rat apoptosis-specific eIF-5A in the sense direction. There is a greater incidence of mitotic nuclei in cells expressing rat apoptosis-specific eIF-5A. FIG. 28 shows increased apoptosis reflecting increased nuclear mitosis when COS-7 cells were transiently transfected with pHM6 including full-length rat apoptosis-specific eIF-5A in the sense direction. The expression of rat apoptosis-specific eIF-5A increased nuclear mitosis by 27% and 63% than non-serum deficient and serum deficient samples, respectively.

29 shows apoptosis detection reflecting phosphatidylserine exposure when COS-7 cells were transiently transfected with pHM6 containing full-length rat apoptosis-specific eIF-5A in the sense direction. 30 shows increased apoptosis detection reflecting increased phosphatidylserine exposure when COS-7 cells were transiently transfected with pHM6 with full-length rat apoptosis-specific eIF-5A in the sense direction. Expression of rat apoptosis-specific eIF-5A increased phosphatidylserine exposure to 140% and 198% than non-serum deficient and serum deficient samples, respectively.

FIG. 31 shows increased apoptosis detection reflecting increased nuclear mitosis when COS-7 cells were transiently transfected with pHM6 including full-length rat apoptosis-specific eIF-5A in the sense direction. The expression of rat apoptosis-specific eIF-5A increased nuclear mitosis by 115% and 62% than untreated and treated samples, respectively. FIG. 32 shows the increased apoptosis under conditions in which COS-7 cells transiently transfected with pHM6 with full-length rat apoptosis-specific eIF-5A in the sense direction had no further treatment or treatments that induce apoptosis. The comparison is shown.

(Example 4)

This example illustrates the regulation of apoptosis activity after administration of apoptosis-specific eIF-5A and DHS.

Furthermore, COS-7 cells were mock transfected, transfected with pHM6-LacZ or transfected with pHM6-Sense rF5A (pHM6-full length rat apoptosis-specific eIF-5A) and incubated for 40 hours. 5 μg of protein extract from each sample was fractionated by SDS-PAGE and transferred to PVDF membrane and western blotted with monoclonal antibody that recognizes Bcl-2. Rabbit anti-mouse IgG conjugated to peroxidase was used as secondary antibody and bound antibody was detected by chemiluminescence and exposure to x-ray film. The results are shown in FIG. Less Bcl-2 was detected in cells transfected with pHM6-Sense rF5A than transfected with pHM6-LacZ; Thus Bcl-2 is down-regulated.

Additional COS-7 cells were transfected with mock transfection, pHM6-Antisese 3 ′ rF5A (pHM6-antisense 3 ′ UTR of rat apoptosis-specific eIF-5A) or pHM6-Sense rF5A (pHM6-full length rat apoptosis). -Specific eIF-5A). After 40 hours of transfection, cells were induced to undergo apoptosis by withdrawal of serum for 48 hours. 5 μg of protein extract from each sample was fractionated by SDS-PAGE and transferred to PVDF membrane and western blotted with monoclonal antibody that recognizes Bcl-2. Rabbit anti-mouse IgG conjugated to peroxidase was used as secondary antibody and bound antibody was detected by chemiluminescence and exposure to x-ray film.

Additionally additionally, COS-7 cells were mock transfected, transfected with pHM6-LacZ or transfected with pHM6-Sense rF5A (pHM6-full length rat apoptosis-specific eIF-5A) and incubated for 40 hours. It was bait. 5 μg of protein extract from each sample was fractionated by SDS-PAGE and transferred to PVDF membrane and western blotted with monoclonal antibody recognizing p53. Goat anti-mouse IgG conjugated to alkaline phosphatase was used as secondary antibody and bound antibody was detected by color.

Finally, COS-7 cells were mock transfected, transfected with pHM6-LacZ or transfected with pHM6-Sense rF5A (pHM6-full length rat apoptosis-specific eIF-5A) and incubated for 40 hours. . 5 μg of protein extract from each sample was fractionated by SDS-PAGE and transferred to PVDF membrane and western blotted with monoclonal antibody recognizing p53. Corresponding protein blots were probed with anti- [HA] -peroxidase to measure rat apoptosis-specific eIF-5A expression. Goat anti-mouse IgG conjugated to alkaline phosphatase was used as the secondary antibody and the bound antibody was detected by chemiluminescence.

33 shows downregulation of Bcl-2 when COS-7 cells are transiently transfected with pHM6 with full-length rat apoptosis-specific eIF-5A in the sense direction. Top panel shows Coomassie blue-stained protein blot; The lower panel shows the corresponding western blot. Less Bcl-2 was detected in cells transfected with pHM6-Sense rF5A than transfected with pHM6-LacZ.

Figure 34 shows upregulation of Bcl-2 when COS-7 cells were transiently transfected with pHM6 containing full-length rat apoptosis-specific eIF-5A in the antisense direction. Top panel shows Coomassie blue-stained protein blot; The lower panel shows the corresponding western blot. More Bcl-2 was detected in cells transfected with pHM6-antisense 3 ′ rF5A than either mock transfected or transfected with pHM6-Sense rF5A.

35 shows upregulation of c-Myc when COS-7 cells are transiently transfected with pHM6 with full-length rat apoptosis-specific eIF-5A in the sense direction. Top panel shows Coomassie blue-stained protein blot; The lower panel shows the corresponding western blot. More c-Myc was detected in cells transfected with pHM6-Sense rF5A than with pHM6-LacZ or mock controls.

36 shows upregulation of p53 when COS-7 cells are transiently transfected with pHM6 including full-length rat apoptosis-specific eIF-5A in the sense direction. Top panel shows Coomassie blue-stained protein blot; The lower panel shows the corresponding western blot. More p53 was detected in cells transfected with pHM6-Sense rF5A than those transfected with pHM6-LacZ or mock controls.

37 shows the dependence of p53 upregulation on expression of pHM6-full length rat apoptosis-specific eIF-5A in COS-7 cells. In western blots probed with anti- [HA] -peroxidase, the top panel shows Coomassie blue-stained protein blot and the bottom panel shows the corresponding western blot. More rat apoptosis-specific eIF-5A can be detected in the first transfection than the second transfection. The upper panel of A in western blot probed with anti-p53 shows the corresponding Coomassie blue-stained protein blot and the lower panel shows western blot to p53. For the first transfection more p53 can be detected in cells transfected with pHM6-LacZ or transfected with pHM6-Sense rF5A than the mock control. In the second transfection with little expression of rat apoptosis-specific eIF-5A, there was no detectable differential level of p53 between cells transfected with pHM6-Sense fF5A, pHM6-LacZ or mock controls.

(Example 5)

FIG. 47 shows experiments performed on heart tissue that mimic the beating of a human heart followed by induction of heart failure. 49 shows a set up laboratory bench. Slices of human heart tissue removed during the valve replacement procedure were hooked into the electrode. A small weight was attached to the heart tissue to facilitate the measurement of the strength of the heart beat. The electrode provided the electrical stimulation that caused the tissue to start beating. Gene expression levels of both apoptosis-specific eIF-5A and proliferative eIF-5A were measured in cardiac tissue before ischemia was induced. See FIG. 46. Low levels of apoptosis-specific eIF-5A and proliferative eIF-5A were produced in pre-ischemic heart tissue, and their levels were relatively balanced. Oxygen and carbon dioxide were then delivered to the heart in buffer at 92.5% and 7.5%, respectively. Thereafter, the oxygen level was reduced and the nitrogen level was increased to induce ischemia and eventually to "heart attack". Heart tissue stopped beating. Oxygen levels then returned to normal and heart tissue pulsed again with electrical stimulation to resume heartbeats. After "heart failure" the expression levels of apoptosis-specific eIF-5A and proliferative eIF-5A were measured again. There was a significant increase in the expression level of apoptosis-specific eIF-5A, while the increase in the expression level of proliferative eIF-5A was markedly small. See FIG. 46.

The heart after "heart attack" did not beat strongly as indicated by less compression / migration of the attached weight, thus indicating that cardiac tissue cells are dying rapidly due to the presence of apoptosis-specific eIF-5A. .

EKG is shown in FIG. 48. Normal heartbeat is shown on the left side of the panel (pre-ischemic heart tissue). After re-initiation of "heart attack" (straight) and heart rate, EKG shows reduced activity due to muscle cell death. EKG represents the relative loss of heart rate intensity.

(Example 6)

The following examples provide cell culture conditions.

Human filamentous and astrocyte cell culture

A pair of human eyes were obtained within 48 hours of death at the Eye Bank in Ontario, Canada. Optic nerve papilla (with attached electrode) was removed and placed in Dulbecco's Modified Eagle's medium (DMEM) containing antibiotics / antifungal, glutamine and 10% FBS. The optic nerve papilla (ONH) buttons were recovered from each tissue sample and chopped into four small pieces with precise dissection scissors. Explants were cultured in 12.5 cm 2 plastic culture flasks in DMEM medium. Growth was observed within a month in viable explants. Once the cells reach 90% confluence, the cells trypsinize and require specific secondary cultures to generate filamentous platelets (LC) and astrocytic individuals. Specifically, LC cells were secondary cultured in 25 cm 2 in DMEM containing gentamicin, glutamine and 10% FBS, while astrocytes were developed in 25 cm 2 flasks containing EBM complete media without FBS. FBS was added to astrocytic medium 10 days after the second culture. Cells were supported and secondary cultured by this procedure.

Cell populations obtained by specific secondary cultures were characterized for identity and individual purity using specific fluorescent antibodies stained on 8 well culture slides. Cells were fixed in 10% formalin solution and washed three times with Dulbecco's Phosphate Buffered Saline (DPBS). After blocking with 2% nonfat milk in DPBS the antibody was diluted in 1% BSA in DPBS and applied to the cells at 6 will. The remaining two wells were treated with only 1% bovine serum albumin (BSA) solution and no primary antibody as a control. Cells were incubated with primary antibody for 1 hour at room temperature and then washed three times with DPBS. Appropriate secondary antibodies were diluted in 1% BSA in DPBS and added to each well and incubated for 1 hour. After washing with DPBS the chamber separating the wells of the culture slide was removed from the slide and the slide was immersed in double distilled distilled water and then dried with air. Fluoromount (Vector Laboratories) was applied to each slide and was thinly covered by a 22 x 60 mm coverglass slip.

Immunofluorescence staining was observed by fluorescence microscopy with appropriate filters and compared to control wells not treated with primary antibody. All primary antibodies were obtained from Sigma unless otherwise noted. All secondary antibodies were obtained from Molecular Probes. Primary antibodies used to identify LC cells were as follows: anti-collagen I, anti-collagen IV, anti-laminin, anti-cell fibronectin. Primary antibodies used to identify astrocytes are: anti-galactocerebroside (Chemicon International), anti-A2B5 (Chemicon International), anti-NCAM, anti-human von Willibrand factor (Von Willebrand Factor). Additional antibodies used against both cell populations included anti-glial fibrillary (GFAP) and anti-alpha-strimus muscle actin. If the cell population was stained positive for collagen I, collagen IV, laminin, cellular fibronectin, alpha striated muscle actin and negative for glia fibroblast (GFAP), the cell population was determined to consist of LC cells. If the cell population stained positive for NCAM, glia fibrotic (GFAP) and negative for galactoserevroside, A2B5, human von Willibrand factor, and alpha striated muscle actin, the cell population was determined to be astrocytic It became.

In this preliminary study three sets of human eyes were used to initiate culture. LC cell lines # 506, # 517 and # 524 were obtained from the optic nerve papilla of 83 year old male, 17 year old male and 26 year old female, respectively. All LC cell lines were well characterized and found to contain more than 90% LC cells.

RKO Cell Culture

American Type Culture Collection CRL-2577 (RKO), a human colon cancer cell line expressing wild type p53, was used to test antisense oligonucleotides for their ability to inhibit apoptosis-specific eIF-5A protein expression. RKO was cultured in Minimum Essential Medium Eagle (MEM) containing non-essential amino acids, Earle's salt and L-glutamine. Culture medium was filled with 10% fetal bovine serum (FBS) and 100 units of penicillin / streptomycin. Cells were grown at 37 ° C. in a humid environment of 5% CO ₂ and 95% air. Cells were subcultured on days 3 and 4 by separating adherent cells with 0.25% trypsin and 1 mM EDTA. The isolated cells were distributed into new incubators with split ratios of 1:10 to 1:12 with fresh medium.

HepG2 cell culture

HepG2, a human hepatocellular carcinoma cell line, was used to test the ability of antisense oligos to affect human apoptosis-specific eIF-5A that interferes with the production of TNF-α in response to treatment with IL-1β. HepG2 cells were cultured in DMEM filled with gentamicin, glutamine and 10% FBS and grown at 37 ° C. in a humid environment of 5% CO ₂ and 95% air.

(Example 7)

Induction of apoptosis

Apoptosis was induced into RKO and filamentous cells using actinomycin D, RNA polymerization inhibitors, and camptothecins, topoisomerase inhibitors, respectively. Actinomycin D was used at 0.25 μg / ml concentration and camptothecin was used at 20, 40 or 50 μM concentration. Apoptosis was also induced into filamentous cells using a combination of camptothecin (50 μΜ) and TNF-α (10 ng / ml). It has been found that the combination of camptothecin and TNF-α is more effective in inducing apoptosis than using either camptothecin or TNF-α.

Antisense oligonucleotides

Three antisense oligonucleotide sets targeted for human apoptosis-specific eIF-5A were designed and obtained by Molecula Research Labs. The sequence of the first antisense oligonucleotide targeted to human apoptosis-specific eIF-5A (# 1) was 5'CCT GTC TCG AAG TCC AAG TC 3 '(SEQ ID NO: 63). The sequence of the second antisense oligonucleotide targeted to human apoptosis-specific eIF-5A (# 2) was 5 'GGA CCT TGG CGT GGC CGT GC 3' (SEQ ID NO: 64). The sequence of the third antisense oligonucleotide targeted to human apoptosis-specific eIF-5A (# 3) was 5'CTC GTA CCT CCC CGC TCT CC 3 '(SEQ ID NO: 65). The control oligonucleotide was SEQ ID NO: 5'CGT ACC GGT ACG GTT CCA GG 3 '(SEQ ID NO: 66). Fluescein isothiocyanate (FITC) -labeled antisense oligonucleotides (Molecula Research Labs) were used to monitor transfection efficiency and the sequence was 5 'GGA CCT TGG CGT GGC CGT GCX 3' (SEQ ID NO: 67). Where X represents the FITC label. All antisense oligonucleotides were fully phosphorothiate.

Transfection of Antisense Nucleotides

The ability of apoptosis-specific eIF-5A antisense oligonucleotides to interfere with apoptosis-specific eIF-5A protein expression was tested in RKO cells. RKO cells were transfected with antisense oligonucleotides using Oligofectamin (Invitrogen), a transfection reagent. Twenty four hours prior to transfection, cells were filled up with 10% FBS but penicillin / streptomycin was isolated from 157,000 wells into 24 well plates in MEM medium lacking. After 24 hours the cells generally reached about 50% confluence. RKO cells were either transfected fictitiously or transfected with 100 nM or 200 nM antisense oligonucleotides. Sufficient amount of transfection medium for one well of a 24-well plate may be obtained by diluting 0 μl, 0.125 μl or 2.5 μl of 20 μM stock antisense oligonucleotide with serum-free MEM to a final volume of 42.5 μl. Was prepared and the mixture was incubated at room temperature for 15 minutes. 1.5 μl Oligofectamine was diluted in 6 μl serum-free MEM and incubated for 7.5 minutes at room temperature. After 5 minutes the diluted oligofectamine mixture was added to the DNA mixture and incubated together for 20 minutes at room temperature. The cells were washed once with serum-free MEM before adding 200 μl of MEM to the cells and thinly covering 50 μl of transfection medium. The cells were placed in dark conditions in the growth chamber for 4 hours. After incubation 125 μL of MEM + 30% FBS was added to the cells. Cells were treated with 0.25 μg / ml actiniocin D for 24 hours and then further incubated for 48 hours before cell extracts were harvested by Western blot analysis.

Transfection of filamentous cells was also tested using 100 nM and 200 nM antisense oligonucleotides and oligofectamines in the same order described for RKO cells. However, effective transfection of filamentous cells can be achieved by simply adding antisense oligonucleotides diluted from 1 μM to 10 μM in serum-free medium to cells for 24 hours and then serum-containing every 24 hours for a total of 2-5 days. This was done by replacing the medium with fresh antisense oligonucleotides diluted in the medium.

The efficiency of antisense oligonucleotide transfection was performed by transfection with FITC-labeled antisense oligonucleotides, although not conjugated to FITC at the 3 'end with the same sequence as apoptosis-specific eIF-5A antisense oligonucleotide # 2. To be utilized or monitored. RKO and filamentous cells were transfected with FITC-labeled antisense oligonucleotides on 8-well culture slides. After 48 hours the cells were washed with PBS and fixed for 10 minutes in 3.7% formaldehyde in PBS. The wells were removed and mounting medium (Vectashield) was added and covered with coverslips. The cells were then visualized under UV light over fluorescence microscopy using a fluorescein filter (Green H546, filter set 48915) and it was determined that cells that emit bright green fluorescence take oligonucleotides.

Detection of apoptosis

Apoptosis in cells treated with either control antisense oligonucleotides or antisense oligonucleotide apoptosis-specific eIF-5A SEQ ID NO: 26 following transfection of filament plates with antisense oligonucleotides and introduction of apoptosis with campotesin The percentage of cells undergoing was determined. Two methods were used to detect apoptotic filamentous cells—Hoescht staining and DeadEnd ^™ Fluorometric TUNEL. Nuclear staining was used for Hoescht staining to label the nuclei of filamentous cells to identify apoptotic cells based on morphological features such as nuclear mitosis and condensation. Dye fixatives consisting of pure methanol and crystalline acetic acid in a ratio of 3: 1 were prepared immediately before use. An equal volume of dye fixative was added to the medium of growing cells on the culture slide and incubated for 2 minutes. The medium / dye fixative mixture was removed from the cells and discarded and 1 ml of the dye fixative was added to the cells. After 5 minutes the dye fixer was discarded and 1 ml of fresh dye fixer was added to the cells and incubated for 5 minutes. The dye fixative was discarded and the cells were air-dried for 4 minutes before 1 ml of Hoescht staining (0.5 μg / ml Hoescht 33258 in PBS) was added. After 10 minutes of incubation in dark conditions, the stained solution was discarded, the chamber separating the wells of the culture slide was removed and the slide was washed three times for 1 minute with deionized water. After washing, McIlvaine buffer (0.021 M citric acid, 0.058 M Na ₂ HPO ₄ .7H ₂ O; pH 5.6) was added to the cells and covered with coverslips. Stained cells were observed under a fluorescence microscope using a UV filter. Cells with brightly stained or destroyed nuclei were recorded apoptotic. At least 200 cells were counted per well.

DeadEnd ^™ Fluorometric TUNEL (Promega) was used to detect DNA breakdown, a characteristic feature of apoptotic cells. After Hoescht staining, the culture slides were simply washed with distilled water and further washed by dipping the slides twice for 5 minutes in PBS (137 mM NaCl, 2.68 mM KCl, 1.47 mM KH ₂ PO ₄ , 8.1 mM Na ₂ HPO ₄ ). . The slides were blotted onto a paper towel between two washes. Cells were permeabilize by soaking in 0.2% Triton X-100 in PBS for 5 minutes. The cells were then washed again by soaking the slides twice in PBS for 5 minutes and blotted onto the paper towel between two washes. Equilibration buffer [200 mM potassium cacodylate (pH 6.6), 25 mM Tris-HCl (pH 6.6), 0.2 mM dithithreitol, 0.25 mg / ml bovine serum albumin and 2.5 mM cobalt chloride 25 [mu] l was added per well and incubated for 5-10 minutes. Equilibration buffer, nucleotide mix [50 μΜ Fluorescein-12-dUTP, 100 μΜ dATP, 10 mM Tris-HCl, pH 7.6 and 1 mM EDTA] and terminal deoxynucleotidyl transferase enzyme, 30 μl of reaction mixture with Tdt, 25 U / μl), respectively, in a ratio of 45: 5: 1, was prepared for each well during equilibrium. After incubation in equilibration buffer 30 μl of reaction mixture was added per well and covered with coverslip. The reaction was continued under dark conditions at 37 ° C. for 1 hour. The reaction was terminated by dipping the slides in 2 × SSC [0.3 M NaCl and 30 mM sodium citrate, pH 7.0) and incubated for 15 minutes. Slides were then washed by soaking in PBS three times for 5 minutes. PBS was removed by sucking around the wells with Kim wipe and a drop of mounting medium (Oncogene research project, JA1750-4ML) was added to each well and the slides covered with coverslips. Cells were observed under a fluorescence microscope using a UV filter (UVG 365, filter set 487902) to count the number of Hoescht-stained nuclei. Any cell with a brightly stained or destroyed nucleus was counted apoptotic. At the same time the cells were then observed using a fluorescein filter and any nuclei that fluoresced in bright green were counted as apoptotic. The percentage of apoptotic cells in the field of view was calculated by dividing the number of bright green nuclei calculated using the fluorescein filter by the total number of nuclei calculated under the UV filter. At least 200 cells were counted per well.

54-57 show the results of this study. The percentage of apoptotic cells in the samples transfected with apoptosis-specific eIF-5A was clearly much less than that seen in cells transfected with control oligonucleotides.

Protein Extraction and Western Blot

Proteins from transfected RKO cells were washed by washing the cells with PBS and adding hot cytolysis buffer [0.5% SDS, 1 mM dithiothreitol, 50 mM Tris-HCl (pH 8.0)] per well. Harvested for blot analysis. The cells were discarded and the extraction result was transferred to a microfuse tube, boiled for 5 minutes and stored at 20 ° C. Protein was measured using a Bio-Rad Protein Assay (Bio-Rad) according to the manufacturer's instructions.

For Western blot 5 μg total protein was isolated on 12% SDS-polyacrylamide gel. The separated protein was transferred to a polyvinylidene difluoride membrane. The membrane was incubated for 1 hour in blocking solution (5% skim milk powder in PBS) and washed three times for 15 minutes with 0.05% Tween-20 / PBS. The membrane was stored in 4 ° C. PBS-T overnight. After warming to room temperature the next day the membrane was blocked for 30 seconds in 1 μg / ml polyvinyl alcohol. The membrane was washed five times with deionized water and then blocked for 30 minutes in a 5% milk solution in 0.025% Tween-20 / PBS. Primary antibodies were preincubated for 30 minutes in a 5% milk solution in 0.025% Tween-20 / PBS before incubation with the membrane.

Some primary antibodies were used. Monoclonal antibodies in Oncogene that recognize p53 (Ab-6) and polyclonal antibodies are synthetic peptides (amino-CRLPEGDLGKEIEQKYD-carboxy) homologous to the c-terminus of human apoptosis-specific eIF-5A induced in chickens (SEQ ID NO: 68). (Gallus Immunotech). Anti-β-actin antibody (Oncogene) was also used to account for the same loading of proteins. Monoclonal antibodies against p53 were used diluted at 0.05 μg / ml, antibodies against apoptosis-specific eIF-5A were diluted 1: 1000 and antibodies against actin were diluted 1: 20,000. After incubation with primary antibody for 60-90 minutes, the membrane was washed three times for 15 minutes in Tween-20 / PBS. The secondary antibody was then diluted with 1% milk in 0.025% Tween-20 / PBS and incubated with the membrane for 60-90 minutes. When p53 (Ab-6) was used as the primary antibody, the secondary antibody used was rabbit anti-mouse IgG conjugated to peroxidase (Sigma) diluted 1: 5000. When anti-apoptotic-specific eIF-5A was used as the primary antibody, rabbit-anti-chicken IgY conjugated to peroxidase (Gallus Immunotech) was used diluted 1: 5000. The secondary antibody used with actin was goat anti-mouse IgM conjugated to peroxidase (Calbiochem) used diluted 1: 5000. After incubation with the secondary antibody, the membrane was washed three times in PBS-.

ECL Plus Western Blotting Detection Kit (Amersham Pharmacia Biotech) was used to detect peroxidase-conjugates bound to antibodies. In brief, the membrane was lightly blotted dried and incubated for 5 minutes under a 40: 1 mixture of reagent A and reagent B. The membrane was blotted dry and placed between acetate sheets and exposed to X-ray film for various periods from 10 seconds to 30 minutes. The membrane was removed by immersing the membrane in stripping buffer [100 mM 2-mercaptoethanol, 2% SDS and 62.5 mM Tris-HCl, pH 6.7] and incubated at 50 ° C. for 30 minutes. It became. The membrane was then rinsed with deionized water and washed twice in a large volume of 0.05% Tween-20 / PBS for 10 minutes. The membrane was removed and reblotted three times.

(Example 8)

construction of siRNA

Small inhibitory RNAs (siRNAs) that affect human apoptosis-specific eIF-5A have been used to specifically inhibit the expression of apoptosis-specific eIF-5A in RKO and filamentous cells. Six siRNAs were generated by in vivo transcription using Silencer ^™ siRNA construction kit (Ambion Inc.). Four siRNAs were generated for human apoptosis-specific eIF-5A (siRNAs # 1 to # 4) (SEQ ID NOs: 30 to 33). See FIG. 70. Two siRNAs were used as controls; SiRNA directed against GAPDH supplied into the kit, itself does not target apoptosis-specific eIF-5A but has an inverse sequence of apoptosis-specific eIF-5A siRNA # 1 (SEQ ID NO: 30) (siRNA # 5) (SEQ ID NO: 34). These siRNAs were generated according to the manufacturer's protocol. In short, DNA oligonucleotides encoding the required siRNA strands were used as templates for T7 RNA polymerase after annealing of T7 promoter primers and fulfilling reaction with Klenow fragments to generate individual strands of siRNA. After transcriptional reactions to both the sense strand and the antisense strand, the reaction was bound and the two siRNA strands were annealed and treated with DNase and RNase and the column was clarified. The sequence of the DNA oligonucleotide (underlined T7 primer annealing portion) used to generate siRNAs was as follows: siRNA # 1 antisense 5 'AAAGGAATGACTTCCAGCTGA CCTGTCT 3' (SEQ ID NO: 69) and siRNA # 1 sense 5 'AATCAGCTGGAAGTCATTCCT CCTGTCTC 3 '(SEQ ID NO: 70); siRNA # 2 antisense 5 'AAGATCGTCGAGATGTCTACT CCTGTCTC 3' (SEQ ID NO: 71) and siRNA # 2 sense 5 'AAAGTAGACATCTCGACGATC CCTGTCTC 3' (SEQ ID NO: 72); siRNA # 3 antisense 5 'AAGGTCCATCTGGTTGGTATT CCTGTCTC 3' (SEQ ID NO: 73) and siRNA # 3 sense 5 'AAAATACCAACCAGATGGACC CCTGTCTC 3' (SEQ ID NO: 74); siRNA # 4 antisense 5 'AAGCTGGACTCCTCCTACACA CCTGTCTC 3' (SEQ ID NO: 75) and siRNA # 4 sense 5 'AATGTGTAGGAGGAGTCCAGC CCTGTCTC 3' (SEQ ID NO: 76); siRNA # 5 antisense 5 'AAAGTCGACCTTCAGTAAGGA CCTGTCTC 3' (SEQ ID NO: 77) and siRNA # 5 sense 5 'AATCCTTACTGAAGGTCGACT CCTGTCTC 3' (SEQ ID NO: 78).

Silencer ^™ siRNA Label Kit-FAM (Ambion) was used to label GAPDH siRNA with FAM to monitor the uptake of siRNA into RKO and filamentous cells. After transfection on 8-well culture slides, cells were washed with PBS and fixed in 3.7% formaldehyde in PBS for 10 minutes. Wells were removed and mounting medium (Vectashiled) was added and covered with coverslips. Uptake of FAM-labeled siRNA was observed under fluorescence microscopy under UV light using a fluorescein filter. GAPDH siRNA was labeled according to the manufacturer's protocol.

siRNA transfection

RKO cells and filamentous cells were transfected with siRNA using the same transfection protocol. RKO cells were sown the day before transfection in 8-well media slides or 24-well plates at a density of 46,000 cells and 105,800 cells per well, respectively. The filamentous cells were transfected at 40-70% cell confluence and sprinkled with generally 7500-10,000 cells per well on 8-well culture slides for 3 days prior to transfection. Sufficient transfection medium for one well of an 8-well culture slide was prepared by diluting siRNA 25.5 pmole in Poti-Mem (Sigma) to a final volume of 21.2 μl. 0.425 μl Lipofectamine 2000 was diluted in Opti-Mem to a final volume of 21.2 μl and incubated for 7-10 minutes at room temperature. The diluted Lipofectamine 2000 mixture was then added to the diluted siRNA mixture and incubated together at room temperature for 20-30 minutes. Cells were washed once with serum-free medium before 135 ml of serum-free medium was added and 42.4 ml of transfection medium was covered. The cells were placed back into the growth chamber for 4 hours. After incubation, 65 ml of serum-free medium + 30% FBS was added to the cells. Transfection of siRNA into cells used for Western blot analysis was performed in 24-well plates under the same conditions as transfection in 8-well slides except that the volume increased 2.3-fold.

After transfection, RKO and filamentous cells were incubated for 72 hours before collecting the cell extract for Western blot analysis. To determine the effectiveness of siRNAs affecting apoptosis-specific eIF-5A that interferes with apoptosis, filamentous cells were treated with 50 μM of Campotethecin (Sigma) and 48 hours or 72 hours after transfection to induce apoptosis. TNF-α was treated with 10 ng / ml. Cells were stained with Hoescht after 24 or 48 hours to determine the percentage of cells undergoing apoptosis.

(Example 9)

(Example 9)

Quantification of HepG2 TNF-α Production

HepG2 cells were plated at 20,000 cells per well on 48-well plates. After 72 hours the medium was removed and cells were added with fresh medium containing either 2.5 μM of control antisense oligonucleotide or antisense oligonucleotide apoptosis-specific eIF-5A # 2. Fresh medium containing antisense oligonucleotides was added after 24 hours. Incubation with Oligonucleotides After a total of 48 hours, the medium was replaced with medium containing Interleukin 1β (IK-1β, 1000 pg / ml; Leinco Technologies) and incubated for 6 hours. Media was collected and frozen for TNF-α quantification. Additional comparable incubation of untreated cells with cells treated only with IL-1β was used for control. All treatments were performed twice. TNF-α released into the medium was measured by an ELISA assay (Assay Designs Inc.) according to the manufacturer's protocol.

(Example 10)

The following experiment shows that antisense apoptosis-specific eIF-5A nucleotides are capable of inhibiting the expression of apoptosis-specific eIF-5A as well as p53.

RKO cells were either untransfected, mock transfected or transfected with 200 nM of antisense oligonucleotide apoptosis-specific eIF-5A # 1, # 2 or # 3 (SEQ ID NOs 25, 26 and 27). RKO cells were also transfected with 100 nM of antisense oligonucleotide apoptosis-specific eIF-5A # 2 (Sur. 26). After 24 hours of transfection, cell extracts were harvested and 5 μg of protein from each sample was separated on SDS-PAGE gels, transferred to PVDF membrane and western blotted with antibodies against apoptosis-specific eIF-5A. After chemiluminescence detection, the membrane was removed and probed again with an antibody against p53. After chemiluminescence detection, the membrane was removed again and probed again with antibodies to actin. See FIG. 52 showing levels of protein produced by RKO cells after treatment with antisense oligos 1, 2 and 3 (for apoptosis-specific eIF-5A) (SEQ ID NOs 25, 26 and 27, respectively). RKO cells not only produced less p53 after being transfected with antisense apoptosis-specific eIF-5A nucleotides but also produced less apoptosis-specific eIF-5A.

(Example 11)

The following experiment shows that antisense apoptosis-specific eIF-5A nucleotides are capable of reducing apoptosis.

In one experiment, filamentous cell line # 506 was transfected with FITC-labeled antisense oligonucleotide 100 nM using (A) oligofectamine transfection reagent or (B) FITC as diluted directly in serum-free medium. -Transfected with labeled antisense oligonucleotide. After 24 hours, medium containing 10% FBS and fresh antisense oligonucleotides diluted to 10 μM were added to the cells. The cells of (A) and (B) were fixed after 48 hours in total and observed under fluorescence microscopy under UV light using a fluorescein filter. Figure 53 shows uptake of fluorescently labeled antisense oligonucleotides.

In another experiment, filamentous cell line # 506 was transfected with 10 μM of control antisense oligonucleotide or antisense oligonucleotide apoptosis-specific eIF-5A # 2 (SEQ ID NO: 26) for a total of 4 days. 48 hours after starting antisense oligonucleotide treatment, cells were treated with 20 μM or 40 μM campotesine for 48 hours. Antisense oligonucleotides and campotesin-containing medium were replaced daily. The percentage of apoptotic cells was determined to label the cells with Hoescht and TUNEL. See FIG. 54.

In another experiment, filamentous cell line # 506 was transfected with 10 μM of control antisense oligonucleotide or antisense oligonucleotide apoptosis-specific eIF-5A # 2 (SEQ ID NO: 26). After 24 hours the medium was replaced and fresh antisense oligonucleotide was added. 48 hours after initiation of antisense oligonucleotide treatment, the antisense oligonucleotide was removed and cells were treated with 20 μM of campotesin for 3 days. Campotesine-containing medium was changed daily. The percentage of apoptotic cells was determined by labeling cells with Hoescht and TUNEL. See FIG. 55.

In another experiment, filamentous cell line # 517 was transfected with 1 μM of control antisense oligonucleotide or antisense oligonucleotide apoptosis-specific eIF-5A # 2 (SEQ ID NO: 26) for a total of 5 days. 48 hours after the start of antisense oligonucleotide treatment, cells were treated with 20 μM of campotesin for 3 or 4 days. Antisense oligonucleotides and campotesin-containing medium were replaced daily. The percentage of apoptotic cells was determined by labeling cells with Hoescht and TUNEL. See FIG. 56.

In another experiment, filamentous cell line # 517 was transfected with 2.5 μM of control antisense oligonucleotide or antisense oligonucleotide apoptosis-specific eIF-5A # 2 (SEQ ID NO: 26) for a total of 5 days. 48 hours after the start of antisense oligonucleotide treatment, cells were treated with 40 μM campotesine for 3 days. Antisense oligonucleotides and campotesin-containing medium were replaced daily. The percentage of apoptotic cells was determined by labeling cells with Hoescht and TUNEL. See FIG. 57.

In another experiment, filamentous cell line # 517 was transfected with 1 μM or 2.5 μM of control antisense oligonucleotide or antisense oligonucleotide apoptosis-specific eIF-5A # 2 (SEQ ID NO: 26) for a total of 5 days. 48 hours after initiation of antisense oligonucleotide treatment, cells were treated with 40 μM campotesine for 3 days. Antisense oligonucleotides and campotesin-containing medium were replaced daily. The percentage of apoptotic cells was determined by labeling cells with Hoescht and TUNEL. See FIG. 58.

In another experiment, filamentous cell line # 517 was either untreated or treated with TNF-α 10 ng / ml, campotesin 50 μM or TNF-α 10 ng / ml, campotesine 50 μM. The percentage of apoptotic cells was determined to label the cells with Hoescht. See FIG. 59.

In another experiment, filamentous cell lines # 506 and # 517 were transfected with 2.5 μM or 5 μM of control antisense oligonucleotide or antisense oligonucleotide apoptosis-specific eIF-5A # 2 (SEQ ID NO: 26) for a total of 2 days. . Fresh medium containing antisense oligonucleotides was added after 24 hours. 48 hours after initiation of antisense oligonucleotides cells were treated with 50 μM of campotesine and 10 ng / ml TNF-α for 2 days. The percentage of apoptotic cells was determined to label the cells with Hoescht. See FIG. 60.

In another experiment, filamentous cell lines # 506, # 517 and # 524 were transfected with 2.5 μM of control antisense oligonucleotide or antisense oligonucleotide apoptosis-specific eIF-5A # 2 (SEQ ID NO: 26) for a total of 2 days. . Fresh medium containing antisense oligonucleotides was added after 24 hours. 48 hours after initiation of antisense oligonucleotides cells were treated with 50 μM of campotesine and 10 ng / ml TNF-α for 2 days. The percentage of apoptotic cells was determined to label the cells with Hoescht. See FIG. 61.

(Example 12)

The following experiments show cells transfected with siRNAs targeted against apoptosis-specific eIF-5A with less apoptosis-specific eIF-5A. This experiment also shows that siRNAs targeted for apoptosis-specific eIF-5A are capable of reducing apoptosis.

In one experiment, filamentous cell line # 517 was transfected with FAM-labeled siRNA 100 nM using (A) serum or (B) lipofectamine 2000 transfection reagent without serum during transfection. . The cells of (A) and (B) were fixed after a total of 24 hours and observed under a fluorescence microscope under UV light using a fluorescein filter. See FIG. 62.

In another experiment, RKO cells were transfected with siRNA 100nM with or without serum during transfection. Two control siRNAs (siRNA # 5 (SEQ ID NO: 34) and four targeted for GAPDH) and four siRNAs (siRNA # 1- # 4) (SEQ ID NOs: 30-33) targeted for apoptosis-specific eIF-5A 6 siRNAs were transfected. 72 hours after transfection, cell extracts were harvested and 5 μg of protein from each sample was separated on an SDS-PAGE gel and transferred to the PVDF membrane and western blotted with antibodies against apoptosis-specific eIF-5A. After chemiluminescence detection, the membrane was removed and probed again with an antibody against bcl-2. After chemiluminescence detection, the membrane was removed again and probed again with antibodies to actin. See FIG. 63.

In another experiment, filamentous cell lines # 506 and # 517 were transfected with siRNA 100nM. Two control siRNAs (siRNA # 5 (SEQ ID NO: 34) and four targeted for GAPDH) and four siRNAs (siRNA # 1- # 4) (SEQ ID NOs: 30-33) targeted for apoptosis-specific eIF-5A 6 siRNAs were transfected. 72 hours after transfection, cell extracts were harvested and 5 μg of protein from each sample was separated on an SDS-PAGE gel and transferred to the PVDF membrane and Western blotted with antibodies against apoptosis-specific eIF-5A. After chemiluminescence detection, the membrane was removed and probed again with antibodies to actin. See FIG. 64.

In another experiment filamentous cell line # 506 was transfected with siRNA 100 nM. Two control siRNAs (siRNA # 5 (SEQ ID NO: 34) and four targeted for GAPDH) and four siRNAs (siRNA # 1-# 4) targeted to apoptosis-specific eIF-5A (SEQ ID NO: 30 A total of six siRNAs were transfected with ˜33). 48 hours after transfection, the medium was replaced with medium containing 50 μM of campotesin and 10 ng / ml of TNF-α. After 24 hours, the percentage of apoptotic cells was determined to label the cells with Hoescht. See FIG. 65.

In another experiment filamentous cell line # 506 was transfected with siRNA 100 nM. Two control siRNAs (siRNA # 5 (SEQ ID NO: 34) and four targeted for GAPDH) and four siRNAs (siRNA # 1- # 4) (SEQ ID NOs: 30-33) targeted for apoptosis-specific eIF-5A 6 siRNAs were transfected. After 72 hours of transfection, the medium was replaced with a medium containing 50 μM of campotesin and 10 ng / ml of TNF-α. After 24 hours, the percentage of apoptotic cells was determined to label the cells with Hoescht. See FIG. 66.

In another experiment filamentous cell line # 506 was either untransfected or transfected with siRNA 100 nM. Two control siRNAs (siRNA # 5 (SEQ ID NO: 34) and four targeted for GAPDH) and four siRNAs (siRNA # 1- # 4) (SEQ ID NOs: 30-33) targeted for apoptosis-specific eIF-5A 6 siRNAs were transfected. After 72 hours of transfection, the medium was replaced with a medium containing 50 μM of campotesin and 10 ng / ml of TNF-α. Fresh medium was also added to the untransfected untreated control cells. After 48 hours, the percentage of apoptotic cells was determined to label the cells with Hoescht. See FIG. 67.

Photographs of Hoescht-stained filamentous cell line # 506 transfected with siRNA from the experiment and treated with campotesin and TNF-α are described in FIG. 67 and Example 13. See FIG. 68.

(Example 13)

This experiment shows that treating human cell lines with antisense oligonucleotides that affect apoptosis-specific eIF-5A results in cells producing less TNF-α.

HepG2 cells were treated with 2.5 μM of control antisense oligonucleotides or antisense oligonucleotide apoptosis-specific eIF-5A # 2 for a total of 2 days. Fresh medium containing antisense oligonucleotides was added after 24 hours. The added cells remained untreated for 2 days. 24 hours after the start of treatment, cells were treated with IL-1β (1000 pg / ml) in fresh medium for 6 hours. At the end of the experiment medium was collected for TNF-α quantification and frozen (-20 ° C.). TNF-α released into the medium was measured by an ELISA assay (Assay Designs Inc.). See FIG. 69. Cells transfected with antisense oligonucleotides of apoptosis-specific eIF-5A produced less TNF-α.

(Example 14)

HT-29 cells (human colon adenocarcinoma) were transfected with siRNA or a reverse siRNA for apoptosis-specific eIF-5A. The siRNA used was as follows:

Position 690 (3'UTR)% G / C = 48

5 'AAGCUGGACUCCUCCUACAC 3' (SEQ ID NO: 79)

The control sequences used were as follows:

% G / C = 39

5 'AAACACAUCCUCCUCAGGUCG 3' (SEQ ID NO: 80)

After 48 hours the cells were treated with interferon-gamma (IFN-γ) for 16 hours. After 16 hours the cells were washed with fresh medium and treated with lipopolysaccharide (LPS) for 8 or 24 hours. At each time point (8 hours or 24 hours) the cell culture medium was removed from the cells and frozen and TNF-α present in the medium was quantified by ELISA. Cell lysates were also harvested and quantified for the protein and used to regulate TNF-α values for pg / mg protein (to control for cell number differences in other wells). The results of western blot and ELISA are shown in Figures 74A and B. 75 shows the results of the same experiment excluding cells at high density.

(Example 15)

Tissue Culture Conditions of U-937 Cell Line

U-937 is a human mononuclear cell line that grows suspended and attaches or differentiates into macrophages by stimulation of PMA (ATCC No. CRL-1593.2) (not cells obtained directly from TACC). Cells with 2 mM L-glutamine, 1.5 g / L sodium bicarbonate, 4.5 g / L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, and 10% FBS (fetal bovine serum) in CO ₂ (5%) at 37 ° C. It was maintained in RPMI 1640 medium. Cells were distributed into fresh medium twice weekly (distribution ratio of 1: 4 or 1: 5) and cell density was always maintained between 10 ⁵ cells / ml and 2 × 10 ⁶ cells / ml. Cells were suspended and cultured in tissue culture-treated plastic T-25 flasks and experiments were performed in 24-well plates.

Time lapse experiment

Two days before the start of the experiment, the cell density was adjusted with 3 × 10 ⁵ cells / ml medium. On the day of the experiment, cells were harvested in the log phase. Cell suspension was transferred to a 15 ml tube and centrifuged at 400 × g for 10 minutes at room temperature. Supernatants were aspirated and cell pellets were washed / resuspended with fresh medium. Cells were again centrifuged at 400 × g for 10 minutes, supernatant was aspirated and cell pellets finally resuspended in fresh medium. The cell suspension and trypan blue solution (0.4% trypan blue dye in PBS) were mixed in equal volume and live cells were calculated using a hemocytometer and microscope. Cells were diluted to 4 × 10 ⁵ cells / ml.

24-well plates were prepared by adding PMA or DMSO (solution control) to each well. 1 ml of cell suspension was added to each well so that each well contained 400,000 cells, 0.1% DMSO +/− 162 nM PMA. Cells were maintained in 37 ° C. CO ₂ (5%) incubator. Each well of cells was harvested at 0, 24, 48, 72, 96, 99 and 102 hours. See FIG. 76 for an experiment time point and a further overview.

Medium was replaced at 72 hours. Because some cells were attached and others were suspended, care was needed to prevent detachment of the attached cells. The medium of each well was carefully transferred into the corresponding microcentrifuge tube and the tube centrifuged at 14,000 × g for 3 minutes. The tube was aspirated and the cell pellet was resuspended in fresh medium (1 ml, (-) DMSO, (-) PMA) and returned to the original well. The cells were immobilized in fresh medium without PMA. At 96 hours, LPS (100 ng / ml) was added and cells were harvested after 3 hours (99 hours) and after 6 hours (102 hours).

At this point the suspended cells and medium were transferred from each well to a microcentrifuge tube. Cells were pelleted at 14,000 × g for 3 minutes. Medium (supernatant) was transferred to a clean tube and stored for ELISA / cytokine analysis (-20 ° C.). The cells remaining in the wells were washed with PBS (1 ml, 37 ° C.) and this PBS was also used to wash the cell pellet in the corresponding microcentrifuge tubes. The cells were pelleted again at 14,000 × g for 3 minutes. Cells were lysed in boiling cytolysis buffer (50 mM Tris pH 7.4 and 2% SDS). Cells and suspended cells attached to each well were pooled. Samples were boiled and stored at -20 ° C.

Western blotting

Protein concentration in each cell sample was determined by bicinchoninic acid (BCA) method using BSA (bobbin serum albumin) as reference protein. Protein samples (5 μg total protein) were separated by 12% SDS-PAGE electrophoresis and transferred to PVDF membrane. The membrane was blocked with polyvinyl alcohol (1 μg / ml, 30 seconds) and 5% skim milk in PBS-t (1 hour). The membrane was probed with mouse monoclonal antibodies raised against human eIF-5A (BD Biosciences cat # 611976l; 1: 20,000 in 5% Scheme Milk, 1 hour). The membrane was washed with PBS-t for 3 x 10 minutes. The secondary antibody was horseradish peroxidase-conjugated anti mouse antibody (Sigma, 1: 5000 in 1% Scheme Milk, 1 hour). The membrane was washed with PBS-T for 3 x 10 minutes. Protein bands were visualized by chemiluminescence (ECL detection system, Amersham Pharmacia Biotech).

Membranes were removed and probed again for actin to account for similar amounts of protein loaded into each gel lane. The membrane was removed (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-Hcl pH 6.7; 30 min at 50 ° C.) and washed as above. The membrane was probed with actin primary antibody (actin monoclonal antibody prepared in mice; Oncogene, Ab-1; 1: 20,000 in 5% steam milk). Secondary antibodies were washed and detected in the same manner as above.

FIG. 77 shows that eIF-5A is upregulated during monocyte (U-397) differentiation followed by TNF-α secretion.

(Example 16) Inhibition of Il-8 production in response to interferon gamma by apoptosis-specific eIF-5A siRNA

HT-29 (human colon adenocarcinoma) cells were transfected with siRNAs that affect apoptosis-specific eIF-5A. After about 48 hours of transfection, the medium was replaced so that a portion of the test sample had a medium with interferon gamma and a portion of the sample had a medium without interferon gamma. Sixteen hours after interferon gamma addition, cells were washed and media with or without TFN-α was placed over the cells. Media (used for ELISA detection of IL-8) and cell lysates were harvested after 8 and 24 hours.

79 and 80 show IL-8 produced in response not only to interferon but also to TNF-α. Priming the cells with interferon gamma prior to TNF treatment causes the cells to produce more IL-8 than either treatment. This may be due to known upregulation of TNF receptor 1 in response to interferon. Thus, 'priming' a cell with interferon allows the cell to respond better to TNF because the cell has more receptors. SiRNA against apoptosis-specific eIF-5A has no effect on IL-8 production, which responds only to TNF (previous experiments), but siRNA produces significant amounts of IL-8 produced as a result of combining interferon and TNF. In addition, it blocks most of the IL-8 produced in response to interferon. These results show that we blocked the interferon signaling pathway leading to IL-8 by using siRNAs that did not block the TNF pathway but affected apoptosis-specific eIF-5A. FIG. 81 shows Western blot showing upregulation (4 fold in 8 hours) of apoptosis-specific eIF-5A in response to interferon gamma in HT-29 cells.

(Example 17)

Human filament culture

A pair of human eyes were prepared within 48 hours post mortem from the Eye Bank of Ontario, Canada. Optic nerve papilla (with attached electrode) was removed and placed in Dulbecco's Modified Eagle's medium (DMEM) containing antibiotics / antifungal, glutamine and 10% FBS. The optic nerve papilla (ONH) buttons were recovered from each tissue sample and chopped into four small pieces with precise dissection scissors. Explants were cultured in 12.5 cm 2 plastic culture flasks in DMEM medium. Growth was observed within a month in viable explants. Once the cells reach 90% confluence, the cells trypsinize and require specific secondary cultures to generate filamentous platelets (LC) and astrocytic individuals. Specifically, LC cells were subcultured in 25 cm 2 in DMEM containing gentamycin, glutamine and 10% FBS, while astrocytes were developed in 25 cm 2 flasks containing EBM Clonetics without FBS. Cells were supported and secondary cultured by this procedure.

Identification and individual purity of the cell individuals obtained by specific secondary cultures were characterized using specific fluorescent antibody staining in 8-well culture slads. Cells were fixed in 10% formalin solution and washed three times with Dulbecco's Phosphate Buffered Saline (DPBS). After blocking with 2% nonfat milk in DPBS the antibody was diluted in 1% BSA in DPBS and applied to cells in 6 wells. The remaining two wells were treated with only 1% bovine albumin (BSA) solution and there was no primary antibody as a control. Cells were incubated with primary antibody for 1 hour at room temperature and then washed three times with DPBS. Appropriate secondary antibodies were diluted in 1% BSA in DPBS and added to each well and incubated for 1 hour. After washing with DPBS the slides were washed in water, air dried and covered with Vector Laboratories. Immunofluorescence staining was observed by fluorescence microscopy with appropriate filters and compared to control wells not treated with primary antibody. All primary antibodies were obtained from Sigma unless otherwise noted. All secondary antibodies were obtained from Molecular Probes. Primary antibodies used to identify LC cells were: anti-collagen I, anti-collagen IV, anti-laminin, anti-cellular fibronectin, anti-glial fibrillary acidic protein ) And anti-alpha-smooth muscle actin. If the cell population was stained positive for collagen I, collagen IV, laminin, cellular fibronectin, alpha smooth muscle actin and negative for glia fibroblast (GFAP), it was determined that the cell population consisted of LC cells. In this study, two sets of human eyes were used to initiate culture. LC cell lines # 506 and # 517 were obtained from optic nerve papillas of 83 and 17 year old men, respectively. All LC cell lines were well characterized and found to contain more than 90% LC cells.

Treatment of LC Cells

Apoptosis was induced into filamentous cells using a compound of 50 μM campotesine (Sigma) and 10 ng / ml TNF-α (Leinco Technologies). It has been found that compounds of campotesine and TNF-α are much more effective in inducing apoptosis than when either capotesin or TNF-α are used alone.

Construction and Transfection of siRNAs

Small inhibitory RNAs (siRNAs) that affect human apoptosis-specific eIF-5A have been used to specifically inhibit the expression of eIF-5A in filamentous cells. Six siRNAs were generated by in vivo transcription using Silencer ^™ siRNA construction kit (Ambion Inc.). Four siRNAs were generated for human apoptosis-specific eIF-5A (siRNAs # 1 to # 4). Two siRNAs were used as controls; SiRNAs affecting GAPDH supplied into the kit, siRNAs themselves that do not target eIF-5A but have a reverse sequence of apoptosis-specific eIF-5A siRNA # 1 (siRNA # 5). These siRNAs were generated according to the manufacturer's protocol. The eIF-5A and control siRNA targets were sequenced as follows: siRNA # 1 5 'AAAGGAATGACTTCCAGCTGA 3' (SEQ ID NO: 81); siRNA # 2 5 'AAGATCGTCGAGATGTCTACT 3' (SEQ ID NO: 82); siRNA # 3 5 'AAGGTCCATCTGGTTGGTATT 3' (SEQ ID NO: 83); siRNA # 4 5 'AAGCTGGACTCCTCCTACACA 3' (SEQ ID NO: 84); siRNA # 5 5 'AAAGTCGACCTTCAGTAAGGA 3' (SEQ ID NO: 85). The filamentous cells were transfected with siRNA using LipofectAMINE 2000.

The filamentous cells were transfected at 40-70% cell confluence and were generally scattered on 8-well culture slides with 7500 cells per well for 3 days prior to transfection. A sufficient amount of transfection medium for one well of an 8-well culture slide was prepared by diluting siRNA 25.5 pmole in Opti-Mem (Sigma) until the final volume was 21.2 μl. 0.425 μl Lipofectamine 2000 was diluted in Opti-Mem to a final volume of 21.2 μl and incubated for 7-10 minutes at room temperature. The diluted Lipofectamine 2000 mixture was then added to the diluted siRNA mixture and incubated for 20-30 minutes at room temperature. Cells were washed once with serum-free medium before 135 μl of serum-free medium was added to the cells and covered with 42.4 μl of transfection medium. The cells were placed back into the growth chamber for 4 hours. After incubation, 65 μl of serum-free medium + 30% of FBS was added to the cells. Transfection of siRNA into cells for use in Western blot analysis was performed in 24-well plates using the same conditions as transfection in 8-well slides except for a 2.3-fold increase in volume. After transfection, filamentous cells were incubated for 72 hours prior to treatment with 50 μM campotesine (Sigma) and 10 ng / μl TNF-α (Leinco Technologies) to induce apoptosis. Cell lysates were then harvested for western blot or tested for apoptosis.

Detection of Apoptotic Cells

Transfected cells treated with TNF-α and campotesin for 24 hours were stained with Hoeschtt 33258 to determine the percentage of cells undergoing apoptosis. In brief, the cells were fixed with a mixture of pure methanol and crystalline acetic acid in a ratio of 3: 1 and then incubated with Hoescht staining (0.5 μg / ml Hoescht 33258 in PBS). After 10 minutes of incubation in dark conditions, the stained solution was discarded and the chamber separating the wells of the culture slide was removed and the slide washed three times for 1 minute with deionized water. After washing, McIlvaine buffer (0.021 M citric acid, 0.058 M Na ₂ HPO ₄ .7H ₂ O; pH 5.6) was added to the cells and covered with coverslip. Stained cells were observed under a fluorescence microscope using a UV filter. Cells with brightly stained or destroyed nuclei were recorded apoptotic. At least 200 cells were counted per well. DeadEnd ^™ Fluorometric TUNEL (Promega) was used to detect DNA breakdown, a characteristic feature of apoptotic cells. After Hoescht staining, the culture slides were simply washed with distilled water and further washed by dipping the slides twice for 5 minutes in PBS (137 mM NaCl, 2.68 mM KCl, 1.47 mM KH ₂ PO ₄ , 8.1 mM Na ₂ HPO ₄ ). And blot the slides on a paper towel between two washes. Cells were permeabilize by soaking in 0.2% Triton X-100 in PBS for 5 minutes. The cells were then washed again by soaking the slides twice in PBS for 5 minutes and blotted onto the paper towel between two washes. 25 μl of equilibration buffer [200 mM Cargodylate (pH 6.6), 25 mM Tris-HCl (pH 6.6), 0.2 mM dithiothreitol, 0.25 mg / ml bovine serum albumin and 2.5 mM cobalt chloride] was added per well. Incubate for 5-10 minutes. Equilibration buffer, nucleotide mix [50 μΜ Fluorescein-12-dUTP, 100 μΜ dATP, 10 mM Tris-HCl, pH 7.6 and 1 mM EDTA] and terminal deoxynucleotidyl transferase enzyme, Tdt 30 U / μl), respectively, in a ratio of 45: 5: 1, 30 μl of the reaction mixture was prepared for each well during equilibration. After incubation in equilibration buffer 30 μl of reaction mixture was added per well and covered with coverslip. The reaction was continued under dark conditions at 37 ° C. for 1 hour. The reaction was terminated by dipping the slides in 2 × SSC [0.3 M NaCl and 30 mM sodium citrate, pH 7.0) and incubated for 15 minutes. Slides were then washed by soaking in PBS three times for 5 minutes. PBS was removed by sucking around the wells with Kim wipe and a drop of mounting medium (Oncogene research project, JA1750-4ML) was added to each well and the slides covered with coverslips. Cells were observed under fluorescence microscopy using a UV filter (UV-G 365, filter set 487902) to count the number of Hoescht-stained nuclei. Any cell with a brightly stained or destroyed nucleus was counted apoptotic. At the same time the cells were then observed using a fluorescein filter (green H546, filter set 48915) and any nuclei that fluoresced bright green were counted as apoptotic. The percentage of apoptotic cells in the field of view was calculated by dividing the number of bright green nuclei calculated using the fluorescein filter by the total number of nuclei calculated under the UV filter. At least 200 cells were counted per well.

Protein Extraction and Western Blot Analysis

The protein was collected from 2 cells in PBS (8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na ₂ HPO ₄ and 0.24 g / L KH ₂ PO ₄ ) from filamentous cells grown on 24-well plates. After washing several times, it was isolated by Western blot by adding 50 [mu] l of cell lysis buffer [2% SDS, 50 mM Tris-HCl, pH 7.4]. Cell lysates were collected in microcentrifuge tubes, boiled for 5 minutes, and stored at -20 ° C until used. Protein concentration was measured using the Bicinchoninic Acid Kit (BCA; Sigma). For Western blot 5 μg of total protein was separated on a 12% SDS-polyacrylamide gel. The separated protein was transferred to a polyvinylidene difluoride membrane. Membranes were incubated for 1 hour with blocking solution (scheme milk powder in PBS, 0.02% sodium azide) and washed three times for 15 minutes in PBS-T (PBS + 0.05% Tween-20). The membrane was stored overnight in PBS-T at 4 ° C. After warming to room temperature the next day, the membrane was blocked for 30 seconds with 1 μg / ml polyvinyl alcohol. The membrane was rinsed five times in deionized water and then blocked for 30 minutes in a 5% milk solution in PBS. Primary antibodies were preincubated for 30 minutes in 5% milk solution in PBS prior to incubation with the membrane. Primary antibodies used were 1: 20,000 anti-eIF-5A (BD Transduction Laboratories) and anti-β-actin (Oncogene). The membrane was washed three times with PBS-T and incubated for 1 hour with a suitable HRP-conjugate secondary antibody diluted in 1% milk in PBS. Blots were washed and an ECL Plus Western blotting detection kit (Amersharm Pharmacia Biotech) was used to detect peroxidase-conjugated antibodies.

result

Two filamentous (LC) cell lines were established from optic nerve papilla obtained from male donors over the ages of 83 years (# 506) to 17 years (# 517). Cells isolated from human filaments had the same broad and flat shape as the prominent nucleus, which had been studied in other studies (Lambert et al ., 2001). Consistent with the characteristics of the other groups, LC cells exhibited alpha smooth muscle actin as well as many extracellular matrix proteins, including cellular probronectin (FIG. 82 b), laminin (FIG. 82 c), collagen I and collagen IV (data not shown). 82a) (Clark et al ., 1995; Hernandez et al ., 1998; Hernandez and Yang, 2000; Lambert et al ., 2001). Negative immunoreactivity of LC cells with glial fibrin protein (GFAP) was also observed consistent with previous findings (FIG. 82D) (Lambert et al ., 2001). These findings support the identification of isolated cells as LC cells rather than optic nerve papillary stellate cells.

Because TNF-α appears to play an important role during the glaucoma process, the sensitivity of LC cells to the cytotoxic effects of TNF-α was investigated. Confluent LC cells were exposed for 48 hours to the binding of camptothecin and TNF-α or campotesine and TNF-α (FIG. 83). Hoesht staining showed that TNF-α alone was not cytotoxic to LC cells. Treatment with campotesin caused about 30% cell death of LC cells. However, a synergistic increase in apoptosis was observed when LC cells were treated with both campotesine and TNF-α, which resulted in 45% killing of LC cells by 48 hours. These results indicate that LC cells can respond to the cytotoxic effects of TNF-α when primed for apoptosis by campotesin.

EIF-5A is a nuclear cytoplasmic shuttle protein known to be required for cell division and has recently been suggested to be involved in apoptosis. Apoptosis-specific eIF-5A protein expression was examined in LC cells induced to undergo apoptosis by either campotethecin or campotesin plus TNF-α. The expression of apoptosis-specific eIF-5A did not change significantly upon treatment with campotesin except for a slight decrease (FIG. 84A). However, significant upregulation of apoptosis-specific eIF-5A protein was observed after 8 and 24 hours of camptothecin plus TNF-α treatment (FIG. 84B). These results indicate that apoptosis-specific eIF-5A expression is specifically induced by exposure to TNF-α and the expression is correlated to the induction of apoptosis. This indicates the role of apoptosis-specific eIF-5A within the apoptosis pathway downstream of TNF-α receptor binding.

To investigate the importance of apoptosis-specific eIF-5A expression during TNF-α-induced apoptosis in LC cells, a series of four siRNAs (siRNAs # 1 to # 4) targeting eIF-5A were designed and in vitro transcription. Synthesized by To determine the effectiveness of siRNAs in inhibiting apoptosis-specific eIF-5A protein expression, LC cell lines # 506 and # 517 were transfected with respective siRNAs and apoptosis-specific eIF-5A protein expression in cell lysates was 72 It was investigated after time (Figure 85). Also for comparison cells were transfected with siRNA and / or control siRNA (siRNA # 5) for GAPDH that had the same chemical composition as siRNA # 1 but did not recognize apoptosis-specific eIF-5A. All siRNAs directed against apoptosis-specific eIF-5A were able to significantly inhibit apoptosis-specific eIF-5A expression in all LC cell lines (FIG. 85). GAPDH siRNA was used as an additional control because GAPDH siRNA is an active siRNA that can inhibit the expression of its target protein GAPDH, unlike the control siRNA # 5, which simply has the reverse sequence of siRNA # 1 but no cell target. Does not appear). In addition, all four siRNAs for apoptosis-specific eIF-5A were able to protect transfected LC cells (# 506) from apoptosis induced by 24-hour treatment with TNF-α and campotene (FIG. 86). ). Using Hoesht staining to detect cell death, siRNAs (siRNAs # 1 to # 4) were 59% (siRNA # 1), 35% (siRNA # 2), 50% (siRNA # 3) and 69% (siRNA # 4). It has been shown that apoptosis of LC cells can be reduced). Interestingly, siRNA against GAPDH was also able to reduce apoptosis of LC cells by 42% (FIG. 86). GAPDH is known to have cellular functions in addition to its role as glycolysis enzymes, including the proposed function during apoptosis of cerebellar neurons (Ishitani and Chung, 1996; Ishitani et al , 1996a; Ishitani et al ., 1996b). In a similar experiment, siRNA # 1 could reduce apoptosis of LC cell line # 517 by 53% in response to TNF-α and camptothecin, for LC cells in which apoptosis-specific eIF-5A siRNA was isolated from other optic nerve papillas. Proved to be protective (FIG. 87). These results indicate that apoptosis-specific eIF-5A has a function during apoptosis and is an important mediator in the pathway leading to TNF-α-induced apoptosis in LC cells.

To confirm that LC cells exposed to TNF-α and campotesin die by normal apoptosis, DNA fragmentation was performed by terminal deoxynucleotidyl transferase-mediated dUPT-digoxigenin nick end markers (TUNEL). In-situ using the method. LC cells (# 506) were treated with TNF-α and campotesin for 24 hours after 3 days of transfection with apoptosis-specific eIF-5A siRNA (siRNA # 1) or control siRNA (siRNA # 5). The cells were also stained with Hoescht to facilitate visualization of the nucleus. 46% of LC cells transfected with control siRNA were positive for TUNEL staining, while only 8% of LC cells transfected with apoptosis-specific eIF-5A siRNA # 1 were positively labeled, indicating that they were apoptosis-specific eIF-5A. siRNA showed at least 80% protection from apoptosis (FIG. 88). Similar results were obtained with apoptosis-specific eIF-5A siRNA # 4, which provided at least 60% protection from apoptosis against control siRNA (data not shown).

(Example 18)

Blood Collection and Preparation of PBMCs

About 10 ml of blood was collected from each healthy donor. Blood was collected by venipuncture in a vacuum collector containing sodium citrate as an anticoagulant. Samples were processed within 24 hours of collection.

60% SIP (9 part v / v Percoll with 1 part v / v 1.5 M NaCl) was cured on the bottom of 15 ml conical tube. The blood was then superimposed on with a minimal mix of blood and Percoll cushions. Samples were centrifuged for a total of 30 minutes at 1000xg with slow acceleration for the first 5 minutes and slow deceleration for the last 5 minutes. Purified serum at the top of the obtained gradient was removed and a white cushion of PBMCs (1-2 ml) was collected and added dropwise to a tube containing 10 ml warm RPMI + 15 FBS. PBMCs were pelleted and counted.

Stimulation to Induce Cytokine Production in PBMCs Over Time

PBMCs were separated was seed (seed) at 2x10 ⁵ ~5x10 ⁵ cells / well. Cells were treated with phorbol 12-myristate 13-acetate (PMA; 100 ng / well). The medium was replaced at 72 hours and did not contain any stimulating factors. Lipopolysaccharide (LPS; 100 ng / well; from E. coli, serotype 0111) was then added to the wells 96 hours after PMA addition into PBMCs. As outlined in FIG. 91, samples were collected at various times before and after LPS addition (96h). Both adherent cells (easy to monocytes and macrophages) were collected along with floating cells (easy to lymphocytes). To collect samples for cytokine secretion analysis, the media from each well was transferred to a clean microcentrifuge tube and debris was removed by centrifugation at 13000 xg for 3 minutes. The pellet obtained was collected with adherent cells. Medium was stored at -20 ° C with 200-250 μl aliquots before analysis. The cells were washed with 1 ml of 37 ° C. phosphate buffered saline (PBS) and then lysed in boiled cytolysis buffer (50 mM Tris pH 7, 2% SDS per well; 100 μl). Cell lysates were boiled and stored frozen at -20 ° C. Western blot is shown in FIG. 92 and the corresponding ELISA is shown in FIG. 93.

PBMC Stimulation Induces Apoptosis-Specific eIF-5A Expression

PBMCs were seeded at 2x10 ⁵ ~5x10 ⁵ cells / well. To determine which stimulants induce apoptosis-specific eIF-5A, as well as whether they co-operate, phytohemagglutinin (PHA; 100 ng / ml), phorbol 12-mi Stimulated with state 13-acetate (PMA; 100 ng / ml), lipopolysaccharide (LPS; 100 ng / ml) or all three. Samples were collected 12 and 36 hours after stimulation and analyzed for apoptosis-specific eIF-5A expression (FIG. 94).

Transfection of PBMCs

PBMCs were transfected the day of preparation. Cells were seeded at 2x10 ⁵ ~5x10 ⁵ cells / well (150 ㎕ per well for transfection in the 240-well plate). They were transfected individually in each well (providers 77, 78 and 79; FIGS. 95 and 96) or all at once in conical tubes before seeding (providers 80 and 84; FIG. 96). For each well of transfected cells, 15 pmole of siRNA was diluted in 50 μl Opti-MEM (Sigma). 1 μl Lipofectamine 2000 (Invitrogen) was diluted in 49 μl Opti-MEM (Sigma), incubated for 7-10 minutes, added to the diluted siRNA and incubated for 25 minutes. Transfection medium was applied onto the cells and placed in a 37 ° C. gross chamber for 4 hours. Final transfection medium contained 9% serum. After incubation, 250 μl of serum-free RPMI + 21% FBS was added into the cells to bring the final serum concentration to 15%.

Stimulation to Induce Cytokine Production in Transfection After PBMCs

Lipopolysaccharide (LPS; 100 ng / well; from E. col, serotype 0111) was added to cells in 500 μl of media 72 hours after transfection of PBMCs as described above. Samples were collected 24 hours post stimulation. Both wells treated with LPS and only transfected (ie, non-irritating) were collected. To collect samples for analysis of cytokine secretion, the medium from each well was transferred to a clean microcentrifuge tube and debris was removed by centrifugation at 13000 × g for 3 minutes. The pellet obtained was collected with adherent cells. Medium was stored at -20 ° C with 200-250 μl aliquots before analysis. The cells were washed with 1 ml of 37 ° C. phosphate buffered saline (PBS) and then lysed in boiled cytolysis buffer (50 mM Tris pH 7, 2% SDS per well; 100 μl). Cell lysates were boiled and stored frozen at −20 ° C. for BCA protein quantification.

(Example 19)

Cell culture

HT-29, a human colon adenocarcinoma cell line, was maintained in RPMI with 0% fetal bovine serum (FBS). Tissue lymphoma cell line U937 was grown in suspension in RPMI with 10% FBS. Both cell lines were maintained in a humid environment at 37 ° C. and 5% CO ₂ . For experiments with U937 cells cells were counted and adjusted to 3 × 10 ⁵ cells / ml 2 days before the start of the experiment. On the first day of the experiment, cells were collected by centrifugation at 400 × g for 10 minutes and cell pellets were resuspended in fresh RPMI medium with 10% FBS, centrifugation was repeated and the repellet cells were fresh RPMI medium without FBS. Resuspended within. Cells were counted and adjusted to 2 × 10 ⁶ cells / ml.

siRNA

이었다. 상응하는 이중나선 siRNA 서열은: siRNAs were designed based on human apoptosis-specific eIF-5A sequences and synthesized by Dharmacon RNA Technologies. Apoptosis-specific eIF-5A siRNA (h5A1) target sequences are:

It was. The corresponding double helix siRNA sequence is:

이었다.

It was.

이었다. 상응하는 이중나선 siRNA 서열은:The control siRNA (h control) sequence is

It was. The corresponding double helix siRNA sequence is:

이었다.

It was.

Transfection of HT-29 Cells

HT-29 cells were seeded at 105,000 cells per well on 24-well plates the day before transfection. 25.5 pmole of siRNA was diluted in 50 μl Opti-Mem (Sigma) for each well of transfected cells. 1 μl Lipofectamine 2000 (Invitrogen) was diluted in 49 μl Opti-MEM (Sigma), incubated for 7-10 minutes, added to the diluted siRNA and incubated for 25 minutes. The cells to be transfected were washed once with serum-free RPMI and applied to 100 µl of transfection medium before adding 300 µl of serum-free RPMI. The cells were placed in a 37 ° C. gross chamber for 4 hours. After incubation, 300 μl of serum-free RPMI + 30% FBS was added into the cells.

Electroporation of U937 Cells

Apoptosis-specific eIF-5A and control siRNA were diluted in Opti-Mem medium (Sigma). 400 μl of cells (800,000 cells) and 100 pmole siRNA were mixed in a 0.4 mm electroporation cuvette. Cells were electroporated at 300 V, 10 mSec, 1 pulse with an ECM 830 Electrosquare porator (BTX, San Diego, Calif.). After electroporation the cells were mixed slowly and added into wells containing RPMI and concentrated FBS to a final FBS concentration of 10%.

Treatment of HT-29 Cells

TNF-α production was induced into HT-29 cells according to the method developed by Suzuki et al., 2003. HT-29 cells were primed with 200 unit / ml interferon gamma (Roche Diagnostics) 48 hours after transfection. After 16 hours of interferon gamma (IFNγ) priming the cells were washed with medium and lipopolysaccharide (LPS; 100 ng / ml; from E. coli, serotype 0111; Sigma) was added at 100 μg / ml. After 8 or 24 hours of LPS stimulation, the medium from each well was transferred to a microcentrifuge tube and stored at −20 ° C. until analyzed for TNFα by ELISA. The cells were washed with 1 ml of phosphate buffered saline (PBS) heated to 37 ° C. and then lysed in boiled cytolysis buffer (50 mM Tris pH 7, 2% SDS). Cell lysates were boiled and stored frozen at -20 ° C. Protein concentration in cell lysates was measured by bicinchoninic acid assay (BCA) with bovine serum albumin used as reference.

IL-8 production was induced in HT-29 cells by treatment of IFNγ. HT-29 cells were treated with 200 unit / ml IFNγ 48 hours after transfection. After 24 hours of treatment, the medium from each well was transferred to a microcentrifuge tube and stored at −20 ° C. until analyzed for IL-8 by liquid-phase electrochemilumiescence (ECL). The cells were washed with 1 ml of phosphate buffered saline (PBS) heated to 37 ° C. and then lysed in boiled cytolysis buffer (50 mM Tris pH 7, 2% SDS). Cell lysates were boiled and stored frozen at -20 ° C. Protein concentration in cell lysates was measured by bicinchoninic acid assay (BCA) with bovine serum albumin used as reference.

Induce differentiation in U937 cells

U937 cells were collected and counted 16 hours after electroporation. 200,000 cells in 1 ml of medium were added to each well of a 24-well plate. Macrophage differentiation was stimulated by adding Probol 12-myristate 13-acetate (PMA; 100 ng / ml). After 48 hours into PMA,> 80 monocytes were transformed from cells in suspension (monocytes) to adherent cells (macrophages). At 48 hours medium and non-adherent cells were removed and fresh RPMI medium (1 ml per well) with 10% FBS was added. Cells were placed for 24 hours in fresh medium to stop.

Stimulation to Induce Cytokine Production in U937 Cells

72 hours after PMA addition into U937 cells, lipopolysaccharide (LPS; 100 ng / ml; from E. coli, serotype 0111), interferon γ (IFNγ; 100 units / ml) or binding of LPS and IFNγ to wells Added. Samples were collected at various times before and after stimulus addition (72h) as described in FIG. 108. To collect samples for cytokine secretion analysis, the media from each well was transferred to a clean microcentrifuge tube and debris was removed by centrifugation at 13000 × g for 3 minutes. Medium was stored at -20 ° C with 200-250 μl aliquots before analysis. Cells were washed with 1 ml of 37 ° C. phosphate buffered saline (PBS) and then lysed in boiled cytolysis buffer (50 mM Tris pH 7, 2% SDS per well; 75 μl). Similar wells were pooled. Cell lysates were boiled and stored frozen at -20 ° C.

Cytokine Quantification

All media samples were stored frozen at -20 ° C. TNFα was quantified using an ELISA kit from Assay Design according to the manufacturer's instructions supplied with standards for 0-250 pg TNFα / ml. For U937 experiments, the medium samples for TNFα were diluted 20-fold (0h, 3h LPS) or 80-fold (6h, 24h, 30h LPS) with RPMI + 10% FBS. IL-1β, IL-8 and IL-6 were quantified by liquid-phase electrochemiluminescence (ECL). The medium from the HT-29 experiment was not diluted. All cytokine measurement results were corrected for the amount of total cellular protein per well (mg).

IL-8, IL-1β and IL-6 were analyzed by liquid phase. Briefly, purified monoclonal mouse anti-mouse anti-human IL-8, IL-1β and IL-6 (R & D Systems) were labeled with biotin (Igen, Inc., Gaithersburg, MD). Furthermore, goat anti-human IL-8, IL-1β and IL-6 antibodies (R & D) were labeled with ruthenium (Igen) according to the manufacturer's instructions. Biotinylated antibodies were diluted to a final concentration of 1 mg / ml in pH 7.4 PBS (ECL buffer) containing 0.25% BSA, 0.5% Tween-20 and 0.01% azide. 25 ml of biotinylated antibody per assay tube was pre-incubated with 25 ml of 1 mg / ml streptavidin-coated paramagnetic bead solution (Dynal Corp., Lake Success, NY) for 30 minutes with strong shaking at room temperature. It became. Test specimens (25 ml) and standards diluted in RPMI were added to the tubes and then to 25 ml rutheniumized antibody (final concentration 1 mg / ml, diluted in ECL buffer). The tube was then shaken for an additional 2 hours. The reaction was stopped by the addition of 200 ml / tube of PBS and the amount of chemiluminescence was measured using the Origen Analyzer (Igen).

SDS-PAGE and Western Blotting

Protein concentrations in cell lysates were measured by bicincronic acid analysis (BCA) with bovine serum albumin used as standard. 5 μg of total cellular protein was isolated by 10% or 14% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). 10% gels were used for the 50 kDa (TLR4, IFNγ, TNF-R1, iNOS) protein assays while 14% gels were used for apoptosis-specific eIF-5A (17 kDa). Gels were transferred to polyvinylidene fluoride (PVDF) membrane in transfer buffer (48 mM Tris, 39 mM glycine, 1.3 mM SDS, pH 9.2; 15 V for 18 minutes) using a semi-dry transfer kit (Bio-Rad). lost. The membrane was blocked for 1 hour with 5% scheme milk in PBS-t (PBS with 0.1% Tween 20). Primary antibodies were diluted in blocking solution and all blots were incubated at room temperature with shaking. The primary antibodies used were apoptosis-specific eIF-5A (BD Bioscience; 1: 20,000, 1 hour incubation; recognition of both apoptosis-specific eIF-5A and eIF-5A2), TLR4 (Santa Cruz Biotechnology Inc; TLR4 ( H-80): sc-10741; 1: 1000; 2 hours incubation), IFN-γRα (Santa Cruz Biotechonology Inc; IFN-γRα (C-20): sc-700; 1: 1000; 1 hour incubation) , TNF-R1 (Santa Cruz Biotechonology Inc; TNF-R1 (H-5): sc-8436; 1: 200; 3 hour incubation), iNOS (BD Trandsuction Laboratories: 610431; 1: 10,000; 1 hour incubation) And β-actin (Oncogene; actin (Ab-1); 1: 20,000; 1 hour incubation). After primary antibody incubation the blots were washed three times for 5-10 minutes with PBS-t. Horseradish peroxidase-conjugated (HRP) secondary antibody was diluted with 1% scheme milk and incubated with the membrane for 1 hour. Secondary antibodies used were anti-mouse IgG-HRP (Sigma; 1: 5000; for apoptosis-specific eIF-5A and TNF-R1), anti-rabbit IgG-HRP (Amersham Pharmacia Biotech; 1: 2500; TLR4 and IFNγ-Rα), anti-mouse IgM-HRP (Calbiochem; 1: 5000; for actin). After secondary antibody incubation the blots were washed four times for 5-10 minutes with PBS-t. Blots were developed with increased chemiluminescence detection reagent (ECL; Amersham Pharamcia Biotech) according to the manufacturer's instructions and the bands visualized with X-ray film (Fuji).

RT-PCR

In order to observe changes in TLR4 mRNA in HT-29 transfected in response to IFNγ, RT-PCR was assayed by Medvedev et al. It was carried out according to 2002. Expression of GAPDH was used as a control to indicate that the same amount of cDNA was used between samples. PCR cycle increases (20, 25, 30 and 35) were used to determine the optimal number of cycles resulting from amplified production detectable under unsaturated conditions. PCR products were detected by ethidium bromide-introduction and separated by agarose gel electrophoresis. RT-PCR of total mRNA isolated from siRNA-transfected HT-29 cells treated or untreated for 6 hours with IFNγ was used to detect TLR4 and GAPDH transcripts. HT-29 cells were transfected with siRNA as described above. 48 hours after transfection, cells were treated with 200 unit / ml IFNγ. Control cells not treated with IFNγ had only media exchange. Total mRNA was isolated using GenElute Mammalian RNA miniprep kit (Sigma) according to the manufacturer's protocol for adherent cells. Media was removed and cells washed twice with warm PBS. Lysis buffer was added to the cells, lysate was transferred to a microcentrifuge tube and total RNA was isolated according to the manufacturer's protocol.

Primers for TLR4 (NM_003266) are:

Front

Reverse

Expected fragment size: 674 bp

It was.

Primers for GAPDH (BC023632) are:

Front

Reverse

Expected fragment size: 599 bp

It was.

Total RNA was reverse transcribed using the following conditions.

Single PCR reactions were performed using the following conditions:

2.0 μl 10X Tsg buffer

0.4 μl dNTP (10 mM)

0.4 μl forward primer (25 pmol / μl)

0.4 μl reverse primer (25 pmol / μl)

2.0 μl MgCl ₂ (15 mM)

0.8 μl cDNA

13.88 μl H ₂ O

0.12 μl Tsg polymerase

PCR conditions for TLR4:

95 ℃ 5 minutes heating

20, 25, 30 or 35 cycles: 95 ° C. 1 minute

                            55 ℃ 1 minute

                            72 ℃ 2 minutes

10 minutes extension at 72 ℃

Immersion at 4 ℃

PCR conditions for GAPDH:

95 ℃ 5 minutes heating

20, 25, 30 or 35 cycles: 95 ° C. 1 minute

                            57 ° C 1 minute

                            72 ℃ 2 minutes

10 minutes extension at 72 ℃

Immersion at 4 ℃

<110> SENESCO TECHNOLOGIES, INC. <120> Inhibition of apoptosis-specific eIF-5A ("eIF-5A1") with antisense          oligonucleotides and siRNAs as anti-inflammatory therapeutics <150> US60 / 476,194 <151> 2003-06-06 <150> US60 / 504,731 <151> 2003-09-22 <150> US60 / 528,249 <151> 2003-12-10 <150> US60 / 557,671 <151> 2004-03-31 <150> US60 / 575,814 <151> 2004-06-02 <160> 95 <170> KopatentIn 1.71 <210> 1 <211> 1139 <212> DNA <213> Rattus sp. <220> <221> CDS (222) (33) .. (494) <400> 1 caggtctaga gttggaatcg aagcctctta aa atg gca gat gat ttg 47                                             Met Ala Asp Asp Leu                                               1 5 gac ttc gag aca gga gat gca ggg gcc tca gcc acc ttc cca atg cag 95 Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala Thr Phe Pro Met Gln                  10 15 20 tgc tca gca tta cgt aag aat ggt ttt gtg gtg ctc aag ggc cgg cca 143 Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val Leu Lys Gly Arg Pro              25 30 35 tgt aag atc gtc gag atg tct act tcg aag act ggc aag cat ggc cat 191 Cys Lys Ile Val Glu Met Ser Thr Ser Lys Thr Gly Lys His Gly His          40 45 50 gcc aag gtc cat ctg gtt ggt att gat att ttt act ggg aag aaa tat 239 Ala Lys Val His Leu Val Gly Ile Asp Ile Phe Thr Gly Lys Lys Tyr      55 60 65 gaa gat atc tgc ccg tcg act cat aac atg gat gtc ccc aac atc aaa 287 Glu Asp Ile Cys Pro Ser Thr His Asn Met Asp Val Pro Asn Ile Lys  70 75 80 85 agg aat gat ttc cag ctg att ggc atc cag gat ggg tac cta tcc ctg 335 Arg Asn Asp Phe Gln Leu Ile Gly Ile Gln Asp Gly Tyr Leu Ser Leu                  90 95 100 ctc cag gac agt ggg gag gta cga gag gac ctt cgt ctg cct gag gga 383 Leu Gln Asp Ser Gly Glu Val Arg Glu Asp Leu Arg Leu Pro Glu Gly             105 110 115 gac ctt ggc aag gag att gag cag aag tat gac tgt gga gaa gag atc 431 Asp Leu Gly Lys Glu Ile Glu Gln Lys Tyr Asp Cys Gly Glu Glu Ile         120 125 130 ctg atc aca gtg ctg tcc gcc atg aca gag gag gca gct gtt gca atc 479 Leu Ile Thr Val Leu Ser Ala Met Thr Glu Glu Ala Ala Val Ala Ile     135 140 145 aag gcc atg gca aaa taactg gcttccaggg tggcggtggt ggcagcagtg 530 Lys Ala Met Ala Lys 150 atccatgagc ctacagaggc ccctccccca gctctggctg ggcccttggc tggactccta 590 tccaatttat ttgacgtttt attttggttt tcctcacccc ttcaaactgt cggggagacc 650 ctgcccttca cctagctccc ttggccaggc atgagggagc catggccttg gtgaagctac 710 ctgcctcttc tctcgcagcc ctgatggggg aaagggagtg ggtactgcct gtggtttagg 770 ttcccctctc cctttttctt tttaattcaa tttggaatca gaaagctgtg gattctggca 830 aatggtcttg tgtcctttat cccactcaaa cccatctggt cccctgttct ccatagtcct 890 tcacccccaa gcaccactga cagactgggg accagccccc ttccctgcct gtgtctcttc 950 ccaaacccct ctataggggt gacaagaaga ggaggggggg aggggacacg atccctcctc 1010 aggcatctgg gaaggccttg cccccatggg ctttaccctt tcctgtgggc tttctccctg 1070 acacatttgt taaaaatcaa acctgaataa aactacaagt ttaatatgaa aaaaaaaaaa 1130 aaaaaaaaa 1139 <210> 2 <211> 154 <212> PRT <213> Rattus sp. <400> 2 Met Ala Asp Asp Leu Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala   1 5 10 15 Thr Phe Pro Met Gln Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val              20 25 30 Leu Lys Gly Arg Pro Cys Lys Ile Val Glu Met Ser Thr Ser Lys Thr          35 40 45 Gly Lys His Gly His Ala Lys Val His Leu Val Gly Ile Asp Ile Phe      50 55 60 Thr Gly Lys Lys Tyr Glu Asp Ile Cys Pro Ser Thr His Asn Met Asp  65 70 75 80 Val Pro Asn Ile Lys Arg Asn Asp Phe Gln Leu Ile Gly Ile Gln Asp                  85 90 95 Gly Tyr Leu Ser Leu Leu Gln Asp Ser Gly Glu Val Arg Glu Asp Leu             100 105 110 Arg Leu Pro Glu Gly Asp Leu Gly Lys Glu Ile Glu Gln Lys Tyr Asp         115 120 125 Cys Gly Glu Glu Ile Leu Ile Thr Val Leu Ser Ala Met Thr Glu Glu     130 135 140 Ala Ala Val Ala Ile Lys Ala Met Ala Lys 145 150 <210> 3 <211> 462 <212> DNA <213> Homo sapiens <400> 3 atggcagatg acttggactt cgagacagga gatgcagggg cctcagccac cttcccaatg 60 cagtgctcag cattacgtaa gaatggcttt gtggtgctca aaggccggcc atgtaagatc 120 gtcgagatgt ctacttcgaa gactggcaag cacggccacg ccaaggtcca tctggttggt 180 attgacatct ttactgggaa gaaatatgaa gatatctgcc cgtcaactca taatatggat 240 gtccccaaca tcaaaaggaa tgacttccag ctgattggca tccaggatgg gtacctatca 300 ctgctccagg acagcgggga ggtacgagag gaccttcgtc tccctgaggg agaccttggc 360 aaggagattg agcagaagta cgactgtgga gaagagatcc tgatcacggt gctgtctgcc 420 atgacagagg aggcagctgt tgcaatcaag gccatggcaa aa 462 <210> 4 <211> 460 <212> DNA <213> Homo sapiens <400> 4 atggcagacg aaattgattt cactactgga gatgccgggg cttccagcac ttaccctatg 60 cagtgctcgg ccttgcgcaa aaacggcttc gtggtgctca aaggacgacc atgcaaaata 120 gtggagatgt caacttccaa aactggaaag catggtcatg ccaaggttca ccttgttgga 180 attgatattt tcacgggcaa aaaatatgaa gatatttgtc cttctactca caacatggat 240 gttccaaata ttaagagaaa tgattatcaa ctgatatgca ttcaagatgg ttacctttcc 300 ctgctgacag aaactggtga agttcgtgag gatcttaaac tgccagaagg tgaactaggc 360 aaagaaatag agggaaaata caatgcaggt gaagatgtac aggtgtctgt catgtgtgca 420 atgagtgaag aatatgctgt agccataaaa ccctgcaaat 460 <210> 5 <211> 462 <212> DNA <213> Rattus sp. <400> 5 atggcagatg atttggactt cgagacagga gatgcagggg cctcagccac cttcccaatg 60 cagtgctcag cattacgtaa gaatggtttt gtggtgctca aaggccggcc atgtaagatc 120 gtcgagatgt ctacttcgaa gactggcaag catggccatg ccaaggtcca tctggttggc 180 attgacattt ttactgggaa gaaatatgaa gatatctgcc cgtcgactca taatatggat 240 gtccccaaca tcaaacggaa tgacttccag ctgattggca tccaggatgg gtacctatcc 300 ctgctccagg acagtgggga ggtacgagag gaccttcgtc tgcctgaagg agaccttggc 360 aaggagattg agcagaagta tgactgtgga gaagagatcc tgatcacagt gctgtctgcc 420 atgacagagg aggcagctgt tgcaatcaag gccatggcaa aa 462 <210> 6 <211> 606 <212> DNA <213> Rattus sp. <220> <221> CDS (222) (1) .. (453) <400> 6 gct gtg tat tat tgg gcc cat aag aac cac ata cct gtg ctg agt cct 48 Ala Val Tyr Tyr Trp Ala His Lys Asn His Ile Pro Val Leu Ser Pro   1 5 10 15 gca ctc aca gac ggc tca ctg ggt gac atg atc ttt ttc cat tcc tat 96 Ala Leu Thr Asp Gly Ser Leu Gly Asp Met Ile Phe Phe His Ser Tyr              20 25 30 aaa aac cca ggc ttg gtc ctg gac atc gtt gaa gac ctg cgg ctc atc 144 Lys Asn Pro Gly Leu Val Leu Asp Ile Val Glu Asp Leu Arg Leu Ile          35 40 45 aac atg cag gcc att ttc gcc aag cgc act ggg atg atc atc ctg ggt 192 Asn Met Gln Ala Ile Phe Ala Lys Arg Thr Gly Met Ile Ile Leu Gly      50 55 60 gga ggc gtg gtc aag cac cac atc gcc aat gct aac ctc atg cgg aat 240 Gly Gly Val Val Lys His His Ile Ala Asn Ala Asn Leu Met Arg Asn  65 70 75 80 gga gct gac tac gct gtt tat atc aac aca gcc cag gag ttt gat ggc 288 Gly Ala Asp Tyr Ala Val Tyr Ile Asn Thr Ala Gln Glu Phe Asp Gly                  85 90 95 tca gac tca gga gcc cgg cca gat gag gct gtc tcc tgg ggc aag atc 336 Ser Asp Ser Gly Ala Arg Pro Asp Glu Ala Val Ser Trp Gly Lys Ile             100 105 110 cgg atg gat gca cag cca gta aag gtc tat gct gat gca tct ctg gtt 384 Arg Met Asp Ala Gln Pro Val Lys Val Tyr Ala Asp Ala Ser Leu Val         115 120 125 ttc ccc ttg ctg gtg gct gag aca ttc gcc caa aag gca gat gcc ttc 432 Phe Pro Leu Leu Val Ala Glu Thr Phe Ala Gln Lys Ala Asp Ala Phe     130 135 140 aga gct gag aag aat gag gac tgagcag atgggtaaag acggaggctt 480 Arg Ala Glu Lys Asn Glu Asp 145 150 ctgccacacc tttatttatt atttgcatac caacccctcc tgggccctct ccttggtcag 540 cagcatcttg agaataaatg gcctttttgt tggtttctgt aaaaaaagga ctttaaaaaa 600 aaaaaa 606 <210> 7 <211> 151 <212> PRT <213> Rattus sp. <400> 7 Ala Val Tyr Tyr Trp Ala His Lys Asn His Ile Pro Val Leu Ser Pro   1 5 10 15 Ala Leu Thr Asp Gly Ser Leu Gly Asp Met Ile Phe Phe His Ser Tyr              20 25 30 Lys Asn Pro Gly Leu Val Leu Asp Ile Val Glu Asp Leu Arg Leu Ile          35 40 45 Asn Met Gln Ala Ile Phe Ala Lys Arg Thr Gly Met Ile Ile Leu Gly      50 55 60 Gly Gly Val Val Lys His His Ile Ala Asn Ala Asn Leu Met Arg Asn  65 70 75 80 Gly Ala Asp Tyr Ala Val Tyr Ile Asn Thr Ala Gln Glu Phe Asp Gly                  85 90 95 Ser Asp Ser Gly Ala Arg Pro Asp Glu Ala Val Ser Trp Gly Lys Ile             100 105 110 Arg Met Asp Ala Gln Pro Val Lys Val Tyr Ala Asp Ala Ser Leu Val         115 120 125 Phe Pro Leu Leu Val Ala Glu Thr Phe Ala Gln Lys Ala Asp Ala Phe     130 135 140 Arg Ala Glu Lys Asn Glu Asp 145 150 <210> 8 <211> 453 <212> DNA <213> Homo sapiens <400> 8 tccgtgtatt actgggccca gaagaaccac atccctgtgt ttagtcccgc acttacagac 60 ggctcgctgg gcgacatgat cttcttccat tcctacaaga acccgggcct ggtcctggac 120 atcgttgagg acctgaggct catcaacaca caggccatct ttgccaagtg cactgggatg 180 atcattctgg gcgggggcgt ggtcaagcac cacattgcca atgccaacct catgcggaac 240 ggggccgact acgctgttta catcaacaca gcccaggagt ttgatggctc tgactcaggt 300 gcccgaccag acgaggctgt ctcctggggc aagatccggg tggatgcaca gcccgtcaag 360 gtctatgctg acgcctccct ggtcttcccc ctgcttgtgg ctgaaacctt tgcccagaag 420 atggatgcct tcatgcatga gaagaacgag gac 453 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Primer <220> <221> modified_base <222> (12) <223> a, t, c or g <400> 9 tcsaarachg gnaagcaygg 20 <210> 10 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Primer <400> 10 gcgaagcttc catggctcga gttttttttt tttttttttt tt 42 <210> 11 <211> 972 <212> DNA <213> Rattus sp. <220> <221> CDS (222) (1) .. (327) <400> 11 tcg aag acc ggt aag cac ggc cat gcc aag gtc cat ctg gtt ggt att 48 Ser Lys Thr Gly Lys His Gly His Ala Lys Val His Leu Val Gly Ile   1 5 10 15 gat att ttt act ggg aag aaa tat gaa gat atc tgc ccg tcg act cat 96 Asp Ile Phe Thr Gly Lys Lys Tyr Glu Asp Ile Cys Pro Ser Thr His              20 25 30 aac atg gat gtc ccc aac atc aaa agg aat gat ttc cag ctg att ggc 144 Asn Met Asp Val Pro Asn Ile Lys Arg Asn Asp Phe Gln Leu Ile Gly          35 40 45 atc cag gat ggg tac cta tcc ctg ctc cag gac agt ggg gag gta cga 192 Ile Gln Asp Gly Tyr Leu Ser Leu Leu Gln Asp Ser Gly Glu Val Arg      50 55 60 gag gac ctt cgt ctg cct gag gga gac ctt ggc aag gag att gag cag 240 Glu Asp Leu Arg Leu Pro Glu Gly Asp Leu Gly Lys Glu Ile Glu Gln  65 70 75 80 aag tat gac tgt gga gaa gag atc ctg atc aca gtg ctg tcc gcc atg 288 Lys Tyr Asp Cys Gly Glu Glu Ile Leu Ile Thr Val Leu Ser Ala Met                  85 90 95 aca gag gag gca gct gtt gca atc aag gcc atg gca aaa taa 330 Thr Glu Glu Ala Ala Val Ala Ile Lys Ala Met Ala Lys             100 105 ctggcttcca gggtggcggt ggtggcagca gtgatccatg agcctacaga ggcccctccc 390 ccagctctgg ctgggccctt ggctggactc ctatccaatt tatttgacgt tttattttgg 450 ttttcctcac cccttcaaac tgtcggggag accctgccct tcacctagct cccttggcca 510 ggcatgaggg agccatggcc ttggtgaagc tacctgcctc ttctctcgca gccctgatgg 570 gggaaaggga gtgggtactg cctgtggttt aggttcccct ctcccttttt ctttttaatt 630 caatttggaa tcagaaagct gtggattctg gcaaatggtc ttgtgtcctt tatcccactc 690 aaacccatct ggtcccctgt tctccatagt ccttcacccc caagcaccac tgacagactg 750 gggaccagcc cccttccctg cctgtgtctc ttcccaaacc cctctatagg ggtgacaaga 810 agaggagggg gggaggggac acgatccctc ctcaggcatc tgggaaggcc ttgcccccat 870 gggctttacc ctttcctgtg ggctttctcc ctgacacatt tgttaaaaat caaacctgaa 930 Taaaactaca Agtttaatat Gaaaaaaaaa Aaaaaaaaaaa Aa 972 <210> 12 <211> 109 <212> PRT <213> Rattus sp. <400> 12 Ser Lys Thr Gly Lys His Gly His Ala Lys Val His Leu Val Gly Ile   1 5 10 15 Asp Ile Phe Thr Gly Lys Lys Tyr Glu Asp Ile Cys Pro Ser Thr His              20 25 30 Asn Met Asp Val Pro Asn Ile Lys Arg Asn Asp Phe Gln Leu Ile Gly          35 40 45 Ile Gln Asp Gly Tyr Leu Ser Leu Leu Gln Asp Ser Gly Glu Val Arg      50 55 60 Glu Asp Leu Arg Leu Pro Glu Gly Asp Leu Gly Lys Glu Ile Glu Gln  65 70 75 80 Lys Tyr Asp Cys Gly Glu Glu Ile Leu Ile Thr Val Leu Ser Ala Met                  85 90 95 Thr Glu Glu Ala Ala Val Ala Ile Lys Ala Met Ala Lys             100 105 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Primer <400> 13 caggtctaga gttggaatcg aagc 24 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Primer <400> 14 atatctcgag ccttgattgc aacagctgcc 30 <210> 15 <211> 489 <212> DNA <213> Rattus sp. <220> <221> CDS (222) (33) .. (485) <400> 15 caggtctaga gttggaatcg aagcctctta aa atg gca gat gat ttg 47                                             Met Ala Asp Asp Leu                                               1 5 gac ttc gag aca gga gat gca ggg gcc tca gcc acc ttc cca atg cag 95 Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala Thr Phe Pro Met Gln                  10 15 20 tgc tca gca tta cgt aag aat ggt ttt gtg gtg ctc aag ggc cgg cca 143 Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val Leu Lys Gly Arg Pro              25 30 35 tgt aag atc gtc gag atg tct act tcg aag act ggc aag cat ggc cat 191 Cys Lys Ile Val Glu Met Ser Thr Ser Lys Thr Gly Lys His Gly His          40 45 50 gcc aag gtc cat ctg gtt ggt att gat att ttt act ggg aag aaa tat 239 Ala Lys Val His Leu Val Gly Ile Asp Ile Phe Thr Gly Lys Lys Tyr      55 60 65 gaa gat atc tgc ccg tcg act cat aac atg gat gtc ccc aac atc aaa 287 Glu Asp Ile Cys Pro Ser Thr His Asn Met Asp Val Pro Asn Ile Lys  70 75 80 85 agg aat gat ttc cag ctg att ggc atc cag gat ggg tac cta tcc ctg 335 Arg Asn Asp Phe Gln Leu Ile Gly Ile Gln Asp Gly Tyr Leu Ser Leu                  90 95 100 ctc cag gac agt ggg gag gta cga gag gac ctt cgt ctg cct gag gga 383 Leu Gln Asp Ser Gly Glu Val Arg Glu Asp Leu Arg Leu Pro Glu Gly             105 110 115 gac ctt ggc aag gag att gag cag aag tat gac tgt gga gaa gag atc 431 Asp Leu Gly Lys Glu Ile Glu Gln Lys Tyr Asp Cys Gly Glu Glu Ile         120 125 130 ctg atc aca gtg ctg tcc gcc atg aca gag gag gca gct gtt gca atc 479 Leu Ile Thr Val Leu Ser Ala Met Thr Glu Glu Ala Ala Val Ala Ile     135 140 145 aag gct cgag 489 Lys ala 150 <210> 16 <211> 151 <212> PRT <213> Rattus sp. <400> 16 Met Ala Asp Asp Leu Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala   1 5 10 15 Thr Phe Pro Met Gln Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val              20 25 30 Leu Lys Gly Arg Pro Cys Lys Ile Val Glu Met Ser Thr Ser Lys Thr          35 40 45 Gly Lys His Gly His Ala Lys Val His Leu Val Gly Ile Asp Ile Phe      50 55 60 Thr Gly Lys Lys Tyr Glu Asp Ile Cys Pro Ser Thr His Asn Met Asp  65 70 75 80 Val Pro Asn Ile Lys Arg Asn Asp Phe Gln Leu Ile Gly Ile Gln Asp                  85 90 95 Gly Tyr Leu Ser Leu Leu Gln Asp Ser Gly Glu Val Arg Glu Asp Leu             100 105 110 Arg Leu Pro Glu Gly Asp Leu Gly Lys Glu Ile Glu Gln Lys Tyr Asp         115 120 125 Cys Gly Glu Glu Ile Leu Ile Thr Val Leu Ser Ala Met Thr Glu Glu     130 135 140 Ala Ala Val Ala Ile Lys Ala 145 150 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Primer <400> 17 gtctgtgtat tattgggccc 20 <210> 18 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Primer <400> 18 gcgaagcttc catggctcga gttttttttt tttttttttt tt 42 <210> 19 <211> 1299 <212> DNA <213> Homo sapiens <400> 19 ggcacgaggg cggcggcggc ggtagaggcg gcggcggcgg cggcagcggg ctcggaggca 60 gcggttgggc tcgcggcgag cggacggggt cgagtcagtg cgttcgcgcg agttggaatc 120 gaagcctctt aaaatggcag atgacttgga cttcgagaca ggagatgcag gggcctcagc 180 caccttccca atgcagtgct cagcattacg taagaatggc tttgtggtgc tcaaaggccg 240 gccatgtaag atcgtcgaga tgtctacttc gaagactggc aagcacggcc acgccaaggt 300 ccatctggtt ggtattgaca tctttactgg gaagaaatat gaagatatct gcccgtcaac 360 tcataatatg gatgtcccca acatcaaaag gaatgacttc cagctgattg gcatccagga 420 tgggtaccta tcactgctcc aggacagcgg ggaggtacga gaggaccttc gtctccctga 480 gggagacctt ggcaaggaga ttgagcagaa gtacgactgt ggagaagaga tcctgatcac 540 ggtgctgtct gccatgacag aggaggcagc tgttgcaatc aaggccatgg caaaataact 600 ggctcccagg atggcggtgg tggcagcagt gatcctctga acctgcagag gccccctccc 660 cgagcctggc ctggctctgg cccggtccta agctggactc ctcctacaca atttatttga 720 cgttttattt tggttttccc caccccctca atctgtcggg gagcccctgc ccttcaccta 780 gctcccttgg ccaggagcga gcgaagctgt ggccttggtg aagctgccct cctcttctcc 840 cctcacacta cagccctggt gggggagaag ggggtgggtg ctgcttgtgg tttagtcttt 900 tttttttttt tttttttttt tttaaattca atctggaatc agaaagcggt ggattctggc 960 aaatggtcct tgtgccctcc ccactcatcc ctggtctggt cccctgttgc ccatagccct 1020 ttaccctgag caccacccca acagactggg gaccagcccc ctcgcctgcc tgtgtctctc 1080 cccaaacccc tttagatggg gagggaagag gaggagaggg gaggggacct gccccctcct 1140 caggcatctg ggagggccct gcccccatgg gctttaccct tccctgcggg ctctctcccc 1200 gacacatttg ttaaaatcaa acctgaataa aactacaagt ttaatatgaa aaaaaaaaaa 1260 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1299 <210> 20 <211> 462 <212> DNA <213> Rattus sp. <400> 20 atggcagatg atttggactt cgagacagga gatgcagggg cctcagccac cttcccaatg 60 cagtgctcag cattacgtaa gaatggtttt gtggtgctca agggccggcc atgtaagatc 120 gtcgagatgt ctacttcgaa gactggcaag catggccatg ccaaggtcca tctggttggt 180 attgatattt ttactgggaa gaaatatgaa gatatctgcc cgtcgactca taacatggat 240 gtccccaaca tcaaaaggaa tgatttccag ctgattggca tccaggatgg gtacctatcc 300 ctgctccagg acagtgggga ggtacgagag gaccttcgtc tgcctgaggg agaccttggc 360 aaggagattg agcagaagta tgactgtgga gaagagatcc tgatcacagt gctgtccgcc 420 atgacagagg aggcagctgt tgcaatcaag gccatggcaa aa 462 <210> 21 <211> 154 <212> PRT <213> Homo sapiens <400> 21 Met Ala Asp Asp Leu Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala   1 5 10 15 Thr Phe Pro Met Gln Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val              20 25 30 Leu Lys Gly Arg Pro Cys Lys Ile Val Glu Met Ser Thr Ser Lys Thr          35 40 45 Gly Lys His Gly His Ala Lys Val His Leu Val Gly Ile Asp Ile Phe      50 55 60 Thr Gly Lys Lys Tyr Glu Asp Ile Cys Pro Ser Thr His Asn Met Asp  65 70 75 80 Val Pro Asn Ile Lys Arg Asn Asp Phe Gln Leu Ile Gly Ile Gln Asp                  85 90 95 Gly Tyr Leu Ser Leu Leu Gln Asp Ser Gly Glu Val Arg Glu Asp Leu             100 105 110 Arg Leu Pro Glu Gly Asp Leu Gly Lys Glu Ile Glu Gln Lys Tyr Asp         115 120 125 Cys Gly Glu Glu Ile Leu Ile Thr Val Leu Ser Ala Met Thr Glu Glu     130 135 140 Ala Ala Val Ala Ile Lys Ala Met Ala Lys 145 150 <210> 22 <211> 153 <212> PRT <213> Homo sapiens <400> 22 Met Ala Asp Glu Ile Asp Phe Thr Thr Gly Asp Ala Gly Ala Ser Ser   1 5 10 15 Thr Tyr Pro Met Gln Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val              20 25 30 Leu Lys Gly Arg Pro Cys Lys Ile Val Glu Met Ser Thr Ser Lys Thr          35 40 45 Gly Lys His Gly His Ala Lys Val His Leu Val Gly Ile Asp Ile Phe      50 55 60 Thr Gly Lys Lys Tyr Glu Asp Ile Cys Pro Ser Thr His Asn Met Asp  65 70 75 80 Val Pro Asn Ile Lys Arg Asn Asp Tyr Gln Leu Ile Cys Ile Gln Asp                  85 90 95 Gly Tyr Leu Ser Leu Leu Thr Glu Thr Gly Glu Val Arg Glu Asp Leu             100 105 110 Lys Leu Pro Glu Gly Glu Leu Gly Lys Glu Ile Glu Gly Lys Tyr Asn         115 120 125 Ala Gly Glu Asp Val Gln Val Ser Val Met Cys Ala Met Ser Glu Glu     130 135 140 Tyr Ala Val Ala Ile Lys Pro Cys Lys 145 150 <210> 23 <211> 154 <212> PRT <213> Mus musculus <400> 23 Met Ala Asp Asp Leu Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala   1 5 10 15 Thr Phe Pro Met Gln Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val              20 25 30 Leu Lys Gly Arg Pro Cys Lys Ile Val Glu Met Ser Thr Ser Lys Thr          35 40 45 Gly Lys His Gly His Ala Lys Val His Leu Val Gly Ile Asp Ile Phe      50 55 60 Thr Gly Lys Lys Tyr Glu Asp Ile Cys Pro Ser Thr His Asn Met Asp  65 70 75 80 Val Pro Asn Ile Lys Arg Asn Asp Phe Gln Leu Ile Gly Ile Gln Asp                  85 90 95 Gly Tyr Leu Ser Leu Leu Gln Asp Ser Gly Glu Val Arg Glu Asp Leu             100 105 110 Arg Leu Pro Glu Gly Asp Leu Gly Lys Glu Ile Glu Gln Lys Tyr Asp         115 120 125 Cys Gly Glu Glu Ile Leu Ile Thr Val Leu Ser Ala Met Thr Glu Glu     130 135 140 Ala Ala Val Ala Ile Lys Ala Met Ala Lys 145 150 <210> 24 <211> 153 <212> PRT <213> Homo sapiens <400> 24 Met Ala Asp Glu Ile Asp Phe Thr Thr Gly Asp Ala Gly Ala Ser Ser   1 5 10 15 Thr Tyr Pro Met Gln Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val              20 25 30 Leu Lys Gly Arg Pro Cys Lys Ile Val Glu Met Ser Thr Ser Lys Thr          35 40 45 Gly Lys His Gly His Ala Lys Val His Leu Val Gly Ile Asp Ile Phe      50 55 60 Thr Gly Lys Lys Tyr Glu Asp Ile Cys Pro Ser Thr His Asn Met Asp  65 70 75 80 Val Pro Asn Ile Lys Arg Asn Asp Tyr Gln Leu Ile Cys Ile Gln Asp                  85 90 95 Gly Cys Leu Ser Leu Leu Thr Glu Thr Gly Glu Val Arg Glu Asp Leu             100 105 110 Lys Leu Pro Glu Gly Glu Leu Gly Lys Glu Ile Glu Gly Lys Tyr Asn         115 120 125 Ala Gly Glu Asp Val Gln Val Ser Val Met Cys Ala Met Ser Glu Glu     130 135 140 Tyr Ala Val Ala Ile Lys Pro Cys Lys 145 150 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 25 gacttggact tcgagacagg 20 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 26 gcacggccac gccaaggtc 19 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 27 ggacagcggg gaggtacgag 20 <210> 28 <211> 153 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Illustrative consensus          sequence <400> 28 Met Ala Asp Glu Ile Asp Phe Thr Thr Gly Asp Ala Gly Ala Ser Ser   1 5 10 15 Thr Tyr Pro Met Gln Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val              20 25 30 Leu Lys Gly Arg Pro Cys Lys Ile Val Glu Met Ser Thr Ser Lys Thr          35 40 45 Gly Lys His Gly His Ala Lys Val His Leu Val Gly Ile Asp Ile Phe      50 55 60 Thr Gly Lys Lys Tyr Glu Asp Ile Cys Pro Ser Thr His Asn Met Asp  65 70 75 80 Val Pro Asn Ile Lys Arg Asn Asp Tyr Gln Leu Ile Cys Ile Gln Asp                  85 90 95 Gly Cys Leu Ser Leu Leu Thr Glu Thr Gly Glu Val Arg Glu Asp Leu             100 105 110 Lys Leu Pro Glu Gly Glu Leu Gly Lys Glu Ile Glu Gly Lys Tyr Asn         115 120 125 Ala Gly Glu Asp Val Gln Val Ser Val Met Cys Ala Met Ser Glu Glu     130 135 140 Tyr Ala Val Ala Ile Lys Pro Cys Lys 145 150 <210> 29 <211> 1309 <212> DNA <213> Homo sapiens <400> 29 ggcacgaggg tagaggcggc ggcggcggcg gcagcgggct cggaggcagc ggttgggctc 60 gcggcgagcg gacggggtcg agtcagtgcg ttcgcgcgag ttggaatcga agcctcttaa 120 aatggcagat gacttggact tcgagacagg agatgcaggg gcctcagcca ccttcccaat 180 gcagtgctca gcattacgta agaatggctt tgtggtgctc aaaggccggc catgtaagat 240 cgtcgagatg tctacttcga agactggcaa gcacggccac gccaaggtcc atctggttgg 300 tattgacatc tttactggga agaaatatga agatatctgc ccgtcaactc ataatatgga 360 tgtccccaac atcaaaagga atgacttcca gctgattggc atccaggatg ggtacctatc 420 actgctccag gacagcgggg aggtacgaga ggaccttcgt ctccctgagg gagaccttgg 480 caaggagatt gagcagaagt acgactgtgg agaagagatc ctgatcacgg tgctgtctgc 540 catgacagag gaggcagctg ttgcaatcaa ggccatggca aaataactgg ctcccaggat 600 ggcggtggtg gcagcagtga tcctctgaac ctgcagaggc cccctccccg agcctggcct 660 ggctctggcc cggtcctaag ctggactcct cctacacaat ttatttgacg ttttattttg 720 gttttcccca ccccctcaat ctgtcgggga gcccctgccc ttcacctagc tcccttggcc 780 aggagcgagc gaagctgtgg ccttggtgaa gctgccctcc tcttctcccc tcacactaca 840 gccctggtgg gggagaaggg ggtgggtgct gcttgtggtt tagtcttttt tttttttttt 900 tttttttttt aaattcaatc tggaatcaga aagcggtgga ttctggcaaa tggtccttgt 960 gccctcccca ctcatccctg gtctggtccc ctgttgccca tagcccttta ccctgagcac 1020 caccccaaca gactggggac cagccccctc gcctgcctgt gtctctcccc aaaccccttt 1080 agatggggag ggaagaggag gagaggggag gggacctgcc ccctcctcag gcatctggga 1140 gggccctgcc cccatgggct ttacccttcc ctgcgggctc tctccccgac acatttgtta 1200 aaatcaaacc tgaataaaac tacaagttta atatgaaaaa aaaaaaaaaa aaaaaaaaaa 1260 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1309 <210> 30 <211> 23 <212> DNA <213> Homo sapiens <400> 30 aaaggaatga cttccagctg att 23 <210> 31 <211> 23 <212> DNA <213> Homo sapiens <400> 31 aagatcgtcg agatgtctac ttc 23 <210> 32 <211> 23 <212> DNA <213> Homo sapiens <400> 32 aaggtccatc tggttggtat tga 23 <210> 33 <211> 23 <212> DNA <213> Homo sapiens <400> 33 aagctggact cctcctacac aat 23 <210> 34 <211> 23 <212> DNA <213> Homo sapiens <400> 34 aaagtcgacc ttcagtaagg att 23 <210> 35 <211> 20 <212> DNA <213> Homo sapiens <400> 35 cctgtctcga agtccaagtc 20 <210> 36 <211> 20 <212> DNA <213> Homo sapiens <400> 36 gacttggact tcgagacagg 20 <210> 37 <211> 20 <212> DNA <213> Homo sapiens <400> 37 ggaccttggc gtggccgtgc 20 <210> 38 <211> 20 <212> DNA <213> Homo sapiens <400> 38 gcacggccac gccaaggtcc 20 <210> 39 <211> 20 <212> DNA <213> Homo sapiens <400> 39 ctcgtacctc cccgctctcc 20 <210> 40 <211> 19 <212> DNA <213> Homo sapiens <400> 40 ggacagcggg gaggtacga 19 <210> 41 <211> 465 <212> DNA <213> Homo sapiens <400> 41 atggcagatg acttggactt cgagacagga gatgcagggg cctcagccac cttcccaatg 60 cagtgctcag cattacgtaa gaatggcttt gtggtgctca aaggccggcc atgtaagatc 120 gtcgagatgt ctacttcgaa gactggcaag cacggccacg ccaaggtcca tctggttggt 180 attgacatct ttactgggaa gaaatatgaa gatatctgcc cgtcaactca taatatggat 240 gtccccaaca tcaaaaggaa tgacttccag ctgattggca tccaggatgg gtacctatca 300 ctgctccagg acagcgggga ggtacgagag gaccttcgtc tccctgaggg agaccttggc 360 aaggagattg agcagaagta cgactgtgga gaagagatcc tgatcacggt gctgtctgcc 420 atgacagagg aggcagctgt tgcaatcaag gccatggcaa aataa 465 <210> 42 <211> 462 <212> DNA <213> Homo sapiens <400> 42 atggcagacg aaattgattt cactactgga gatgccgggg cttccagcac ttaccctatg 60 cagtgctcgg ccttgcgcaa aaacggcttc gtggtgctca aaggacgacc atgcaaaata 120 gtggagatgt caacttccaa aactggaaag catggtcatg ccaaggttca ccttgttgga 180 attgatattt tcacgggcaa aaaatatgaa gatatttgtc cttctactca caacatggat 240 gttccaaata ttaagagaaa tgattatcaa ctgatatgca ttcaagatgg ttacctttcc 300 ctgctgacag aaactggtga agttcgtgag gatcttaaac tgccagaagg tgaactaggc 360 aaagaaatag agggaaaata caatgcaggt gaagatgtac aggtgtctgt catgtgtgca 420 atgagtgaag aatatgctgt agccataaaa ccctgcaaat aa 462 <210> 43 <211> 154 <212> PRT <213> Homo sapiens <400> 43 Met Ala Asp Asp Leu Asp Phe Glu Thr Gly Asp Ala Gly Ala Ser Ala   1 5 10 15 Thr Phe Pro Met Gln Cys Ser Ala Leu Arg Lys Asn Gly Phe Val Val              20 25 30 Leu Lys Gly Trp Pro Cys Lys Ile Val Glu Met Ser Ala Ser Lys Thr          35 40 45 Gly Lys His Gly His Ala Lys Val His Leu Val Gly Ile Asp Ile Phe      50 55 60 Thr Gly Lys Lys Tyr Glu Asp Ile Cys Pro Ser Thr His Asn Met Asp  65 70 75 80 Val Pro Asn Ile Lys Arg Asn Asp Phe Gln Leu Ile Gly Ile Gln Asp                  85 90 95 Gly Tyr Leu Ser Leu Leu Gln Asp Ser Gly Glu Val Pro Glu Asp Leu             100 105 110 Arg Leu Pro Glu Gly Asp Leu Gly Lys Glu Ile Glu Gln Lys Tyr Asp         115 120 125 Cys Gly Glu Glu Ile Leu Ile Thr Leu Leu Ser Ala Met Thr Glu Glu     130 135 140 Ala Ala Val Ala Ile Lys Ala Met Ala Lys 145 150 <210> 44 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 44 aaaggaatga cttccagctg a 21 <210> 45 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <400> 45 aaaggaauga cuuccagcug att 23 <210> 46 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <400> 46 ucagcuggaa gucauuccuu utt 23 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 47 aagatcgtcg agatgtctac t 21 <210> 48 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <400> 48 aagaucgucg agaugucuac utt 23 <210> 49 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <400> 49 aguagacauc ucgacgaucu utt 23 <210> 50 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 50 aaggtccatc tggttggtat t 21 <210> 51 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <400> 51 aagguccauc ugguugguau utt 23 <210> 52 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <400> 52 aauaccaacc agauggaccu utt 23 <210> 53 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 53 aagctggact cctcctacac a 21 <210> 54 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <400> 54 aagcuggacu ccuccuacac att 23 <210> 55 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <400> 55 uguguaggag gaguccagcu utt 23 <210> 56 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 56 aaagtcgacc ttcagtaagg a 21 <210> 57 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <400> 57 aaagucgacc uucaguaagg att 23 <210> 58 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <400> 58 uccuuacuga aggucgacuu utt 23 <210> 59 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 59 gccaagctta atggcagatg atttgg 26 <210> 60 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 60 ctgaattcca gttattttgc catgg 25 <210> 61 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 61 aatgaattcc gccatgacag aggaggc 27 <210> 62 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 62 gcgaagcttc catggctcga gttttttttt tttttttttt tt 42 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 63 cctgtctcga agtccaagtc 20 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 64 ggaccttggc gtggccgtgc 20 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 65 ctcgtacctc cccgctctcc 20 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 66 cgtaccggta cggttccagg 20 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 67 ggaccttggc gtggccgtgc 20 <210> 68 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 68 Cys Arg Leu Pro Glu Gly Asp Leu Gly Lys Glu Ile Glu Gln Lys Tyr   1 5 10 15 Asp     <210> 69 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 69 aaaggaatga cttccagctg acctgtctc 29 <210> 70 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 70 aatcagctgg aagtcattcc tcctgtctc 29 <210> 71 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 71 aagatcgtcg agatgtctac tcctgtctc 29 <210> 72 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 72 aaagtagaca tctcgacgat ccctgtctc 29 <210> 73 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 73 aaggtccatc tggttggtat tcctgtctc 29 <210> 74 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 74 aaaataccaa ccagatggac ccctgtctc 29 <210> 75 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 75 aagctggact cctcctacac acctgtctc 29 <210> 76 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 76 aatgtgtagg aggagtccag ccctgtctc 29 <210> 77 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 77 aaagtcgacc ttcagtaagg acctgtctc 29 <210> 78 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 78 aatccttact gaaggtcgac tcctgtctc 29 <210> 79 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 79 aagcuggacu ccuccuacac 20 <210> 80 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 80 aaacacaucc uccucagguc g 21 <210> 81 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 81 aaaggaatga cttccagctg a 21 <210> 82 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 82 aagatcgtcg agatgtctac t 21 <210> 83 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 83 aaggtccatc tggttggtat t 21 <210> 84 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 84 aagctggact cctcctacac a 21 <210> 85 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 85 aaagtcgacc ttcagtaagg a 21 <210> 86 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic siRNA sequence <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <220> <221> misc_feature (222) (1) .. (2) <223> a, t, c, g or u <400> 86 nngcuggacu ccuccuacac a 21 <210> 87 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic siRNA sequence <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <400> 87 gcuggacucc uccuacacat t 21 <210> 88 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic siRNA sequence <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <400> 88 uguguaggag gaguccagct t 21 <210> 89 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic siRNA sequence <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <220> <221> misc_feature (222) (1) .. (2) <223> a, t, c, g or u <400> 89 nnacacaucc uccucagguc g 21 <210> 90 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic siRNA sequence <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <400> 90 acacauccuc cucaggucgt t 21 <210> 91 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic siRNA sequence <220> <223> Description of Combined DNA / RNA Molecule: Synthetic          oligonucleotide <400> 91 cgaccugagg aggaugugut t 21 <210> 92 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic siRNA sequence <400> 92 cggatggcaa catttagaat tagt 24 <210> 93 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 93 tgattgagac tgtaatcaag aacc 24 <210> 94 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 94 ctgatgcccc catgttcgtc at 22 <210> 95 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 95 ccaccaccct gttgctgtag 20

Claims (28)

Antisense oligonucleotides of apoptosis-specific eIF-5A characterized by inhibiting endogenous expression of apoptosis-specific eIF-5A in cells 2. The antisense oligonucleotide of claim 1, wherein said apoptosis-specific eIF-5A is encoded by the nucleotide sequence of SEQ ID NO: 20. 2. The antisense oligonucleotide of claim 1, wherein said apoptosis-specific eIF-5A is encoded by the nucleotide sequence of SEQ ID 41. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide has the sequence shown in SEQ ID NO: 35. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide has the sequence shown in SEQ ID NO: 37. The antisense oligonucleotide of claim 1, wherein the antisense oligonucleotide has the sequence shown in SEQ ID NO: 39. 3. Expression vector for transfection of a mammalian cell comprising the antisense oligonucleotide of claim 1 and a regulatory sequence operably linked to an antisense oligonucleotide to enable transcription of the antisense oligonucleotide in the cell Expression vector for transfection of a mammalian cell comprising the antisense oligonucleotide of claim 4 and a regulatory sequence operably linked to an antisense oligonucleotide to enable transcription of the antisense oligonucleotide in the cell Expression vector for transfection of a mammalian cell comprising the antisense oligonucleotide of claim 5 and a regulatory sequence operably linked to an antisense oligonucleotide to enable transcription of the antisense oligonucleotide in the cell Expression vector for transfection of a mammalian cell comprising the antisense oligonucleotide of claim 6 and a regulatory sequence operably linked to an antisense oligonucleotide to enable transcription of the antisense oligonucleotide in the cell Intracellular, characterized in that the apoptosis-specific eIF-5A antisense oligonucleotide inhibits the expression of endogenous apoptosis-specific eIF-5A in the cell, comprising administering the expression vector of claim 7 into the cell. Methods of Inhibiting Apoptosis-Specific eIF-5A Expression The apoptosis-specific eIF-5A antisense oligonucleotide in said cell comprising the step of administering the expression vector of claim 8, 9 or 10 into said cell results in the expression of endogenous apoptosis-specific eIF-5A. A method of inhibiting apoptosis-specific eIF-5A expression in cells characterized by inhibition The method of claim 11, wherein the inhibition of endogenous apoptosis-specific eIF-5A expression in the cell inhibits the intracellular apoptosis, reduces the expression of p53 in the cell, and produces cytoplasm produced in the cell. Decreases the level of Cain, increases the expression of Bcl-2 in the cell, reduces the level of myeloid cell type peroxidase produced in the cell, decreases the level of active NFk beta in the cell, and Has an effect on cells selected from the group consisting of reducing the level of intracellular TLR4, reducing the level of intracellular TNFR-1, and reducing the level of intracellular iNOS. The method of claim 12, wherein the inhibition of endogenous apoptosis-specific eIF-5A expression in the cell inhibits the intracellular apoptosis, reduces the expression of p53 in the cell, and produces cytoplasm produced in the cell. Decreases the level of Cain, increases the expression of Bcl-2 in the cell, reduces the level of myeloid cell type peroxidase produced in the cell, decreases the level of active NFk beta in the cell, and Has an effect on cells selected from the group consisting of reducing the level of intracellular TLR4, reducing the level of intracellular TNFR-1, and reducing the level of intracellular iNOS. Methods of delivering siRNA into mammalian lung cells in vivo, comprising mixing the siRNA with water and delivering it to the mammalian nasal cavity SiRNA of apoptosis-specific eIF-5A, characterized in that siRNA inhibits endogenous expression of apoptosis-specific eIF-5A in cells The siRNA of claim 16, wherein the apoptosis-specific eIF-5A is encoded by the nucleotide sequence of SEQ ID NO: 20. 18. The siRNA of claim 16, wherein the apoptosis-specific eIF-5A is encoded by the nucleotide sequence of SEQ ID 41. The siRNA of claim 16, wherein the siRNA has the sequence shown in SEQ ID NO: 30. The siRNA of claim 16, wherein the siRNA has the sequence shown in SEQ ID NO: 31. 18. The siRNA of claim 16, wherein the siRNA has the sequence shown in SEQ ID NO: 32. 18. The siRNA of claim 16, wherein the siRNA has the sequence shown in SEQ ID NO: 33. Intracellular apoptosis-specific characterized in that the apoptosis-specific eIF-5A siRNA inhibits the expression of endogenous apoptosis-specific eIF-5A in the cell, comprising administering the siRNA of claim 16 into the cell. Methods of Inhibiting Red EIF-5A Expression The apoptosis-specific eIF-5A siRNA in said cell comprises the step of administering the siRNA of claim 19, 20, 21 or 22 into the cell. Method of inhibiting apoptosis-specific eIF-5A expression in cells characterized by inhibiting expression Intracellular apoptosis-specific characterized in that the apoptosis-specific eIF-5A siRNA inhibits the expression of endogenous apoptosis-specific eIF-5A in the cell, comprising administering the siRNA of claim 16 into the cell. Methods of Inhibiting Red EIF-5A Expression 23. Expression of endogenous apoptosis-specific eIF-5A by said apoptosis-specific eIF-5A siRNA in said cell, comprising administering the siRNA of claim 19, 20, 21 or 22 into the cell. Method of inhibiting apoptosis-specific eIF-5A expression in cells characterized by 26. The cytosol of claim 25, wherein the inhibition of endogenous apoptosis-specific eIF-5A expression in said cell inhibits said intracellular apoptosis, reduces the expression of p53 in said cell, and produces cytoplasm produced in said cell. Decreases the level of Cain, increases the expression of Bcl-2 in the cell, reduces the level of myeloid cell type peroxidase produced in the cell, reduces the level of active NFk beta in the cell, and Has an effect on cells selected from the group consisting of reducing the level of intracellular TLR4, reducing the level of intracellular TNFR-1, and reducing the level of intracellular iNOS. 27. The cytosol of claim 26, wherein the inhibition of endogenous apoptosis-specific eIF-5A expression in the cell inhibits the intracellular apoptosis, reduces the expression of p53 in the cell, and produces cytoplasm produced in the cell. Decreases the level of Cain, increases the expression of Bcl-2 in the cell, reduces the level of myeloid cell type peroxidase produced in the cell, decreases the level of active NFk beta in the cell, and Has an effect on cells selected from the group consisting of reducing the level of intracellular TLR4, reducing the level of intracellular TNFR-1, and reducing the level of intracellular iNOS.

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