THE NIP3 FAMILY OF PROTEINS

Abstract: The present invention provides an isolated and purified Nip, Nix and Nox proteins. There is also provided a method for inducing apoptosis in cells by transfecting a cell with a Nip, Nix and Nox proteins. Additionally, there is provided a method for inducing apoptosis in cells by creating antibodies which recognize a specific Nip, Nix and Nox proteins in a muscle or skin of a patient. A method for inducing apoptosis in cells by creating a transgene overexpressing the gene for the Nip, Nix and Nox proteins is also provided. A transgenic animal and progeny thereof is provided having an expression factor consisting essentially of a Nip, Nix and Nox proteins selected from the group consisting essentially of Seq. ID No:1 through 6.

Description: THE NIP3 FAMILY OF PROTEINS BACKGROUND OF THE INVENTION 1. TECHNOLOGICAL FIELD OF THE INVENTION The present invention is in the field of gene therapy to induce program cell death in tumorogenic cells. 2. DESCRIPTION OF RELATED ART Cell death is an important aspect during the embryonic or post-natal development of major organ systems. Apoptosis, or programmed cell demise, also plays a critical role in maintaining homeostasis in many adult tissues. Within vertebrates, bcl-2 is the best understood gene in a cell death pathway and functions as a cell death repressor. Apoptosis is a term used to refer to the process (es) of programmed cell death and has been described in several cell types (Waring et al . (1991) ed. Res. Rev. 11: 219; Williams G.T. (1991) Cell 65: 1097; Williams G.T. (1992) Trends Cell Biol. 2: 263; Yonisch-Rouach et al . (1991) Nature 352: 345) . Apoptosis is likely involved in controlling the amount of and distribution of certain differentiated cell types, such as lymphocytes and other cells of the hematopoietic lineage. The mechanism (s) by which apoptosis is produced in cells is incompletely understood, as are the regulatory pathways by which the induction of apoptosis occurs. Apoptosis was first described as a morphologic pattern of cell death characterized by cell shrinkage, membrane blebbing and chromatin condensation culminating in cell fragmentation (Kerr et al . , 1992). One hallmark pattern early in the process of cell death is internucleosomal DNA cleavage (Wyllie, 1980) . The death-sparing effects of interrupting RNA and protein synthesis and the stereotyped patterns of cell death during development were consistent with a cell autonomous genetic program for cell death (Wyllie et al . (1980) Int. Rev. Cytol . 68: 251; Sulston, J. and Horvitz, H. (1977) Develop. Biol. 56: 110; Abrams et al. (1993) Development 117: 29). The isolation of mutants defective for development cell death in the nematode Caenorhabditis elegans supported this view (Ellis, H. and Horvitz, H. (1986) Cell 44: 817; Hengartner et al . (1992) Nature 365: 494) . The consistency of the morphologic and biochemical patterns defined as apoptosis within different cell types and species, during normal development and as a response to external stimuli are consistent with a common cause of cellular mortality. This thesis is supported by the concept of an endogenous program responsible for cell death and the presence of gene products which are positive and negative regulators of apoptosis. The best studied negative regulator of apoptosis is the bcl-2 proto-oncogene product . It provides the strongest evidence for a shared mammalian pathway of death by its ability to block a wide variety of cell death models. This pattern of morphologic cell death is characterized by a dramatic plasma membrane blebbing, cell volume contraction, nuclear pyknosis, and internucleosomal DNA degradation following the activation of an endonuclease. Over expression of mitochondrial bcl-2 appears to function as an antidote to this process and has the unique function of blocking programmed cell death independent of promoting proliferation. The maintenance of homeostasis in normal tissue, in many respects, reflects a simple balanced equation of input (cellular proliferation and renewal) versus output (cell death) . This is most easily envisioned for encapsulated organs, such as the prostate, but is also true of the recirculating hematopoietic lineages. The maintenance of remarkably invariant cell numbers reflects tightly regulated death pathways as well as controlled proliferation. See for example S.J. Korsmeyer "bcl-2 Initiates a New Category of Oncogenes : Regulators of Cell Death", Blood Vol. 80 No. 4 pp. 879-886, Aug. 15, 1992. Programmed cell death represents a cell autonomous suicide pathway that helps restrict cell numbers. The well-defined loss of specific cells is crucial during embryonic development as part of organogenesis . In the mature tissues, genetically programmed demise regulates the volume of cells. A morphologically distinct and temporally regulated cell death entitled apoptosis has been identified by Wyllie A. H. : "Apoptosis: Cell death in tissue regulation". J. Pathol 153: 313, 1987. Cells dying by apoptosis display marked plasma membrane blebbing, volume contraction, nuclear condensation, and the activation of an endonuclease that cleaves DNA into nucleosomal length fragments. The genetic regulation of cell death is thought to be a central mechanism of cellular homeostasis and development (White, 1996; Chinnaiyan and Dixit, 1996; Jacobson et al . , 1997; Nagata, S. 1997). The Bcl-2 family of genes (White, 1996; Oltvai, et al . , 1994), which are related to ced-9 of C. elegans (Hengartner, et al . , 1994), were originally identified as repressors of cell death. It is known that both pro- and anti-apoptotic Bcl-2 homologs exist, however their exact biochemical function has not been determined. Recent studies suggest that Ced-9 and Bcl-2/Bcl-X L may physically interact with proteins that are required for the execution of apoptosis, Ced-3 and Ced-4 (Chinnaiyan, et al., 1997; Wu, et al . , 1997; Spector, et al . , 1997), however these proteins have not been isolated and purified. Ced-3 is a protease which in mammals is represented by a large family of cysteine proteases which cleave after aspartic acid, now called caspases (Nagata, 1997; Martin, et al . , 1995). In mammalian cells overexpression of -bcl-2 prevents the processing and activation of caspase-3 (CPP32) (Chinnaiyan, Orth, O'Rourke, Duan, Poirier, and Dixit, 1996; Armstrong, et al . , 1996). Bcl-2 family members bear C-terminal transmembrane domains that allow their association with the outer mitochondrial membrane (Krajewski, et al . , 1993) and this mitochondrial localization is important for the suppressive function of Bcl-2 (Tanaka, et al . , 1993; Zhu, et al . , 1996) . There is growing evidence that mitochondrial function is disturbed early in the apoptotic response and may be important in mediating apoptosis (Zamzami, et al . , 1996; Marchetti, et al . , 1996; Liu, et al . , 1996). This is often seen as the loss of mitochondrial membrane potential (Zamzami, et al . , 1996; Marchetti, et al . , 1996) and the release of cytochrome c (Liu, et al . , 1996), and cytochrome c has been implicated in the activation of caspase (Liu, et al., 1996; Yang, et al,. 1997; Kluck, et al . , 1997). Bcl-2 can suppress the release of cytochrome c from mitochondria and prevent caspase activation (Yang, et al . , 1997; Kluck, et al. , 1997) . Additionally, the protein encoded by the bcl-2 proto- oncogene has been reported to be capable of inhibiting apoptosis in many hematopoietic cell systems. The proto- oncogene bcl-2 was isolated and characterized as a result of its frequent translocation adjacent to the immunoglobulin heavy chain enhancer in the t(14;18) chromosome translocation present in more than 80% of human follicular lymphomas (Chen-Levy et al . (1989) Mol. Cell. Biol. 9: 701; Clearly et al . (1986) Cell 47: 19). These neoplasias are characterized by an accumulation of mature resting B cells presumed to result from a block of apoptosis which would normally cause turnover of these cells. Transgenic mice expressing bcl-2 under the control of the Eμ enhancer similarly develop follicular lymphomas which have a high incidence of developing into malignant lymphomas (Hockenbery et al . (1990) Nature 348: 334; McDonnell T.J. and Korsmeyer S.J. (1991) Nature 349: 254; Strasser et al . (1991) Cell 67: 889). The capacity of bcl-2 to enhance cell survival is related to its ability to inhibit apoptosis initiated by several factors, such as cytokine deprivation, radiation exposure, glucocorticoid treatment, and administration of anti-CD-3 antibody (Nunez et al . (1990) op.cit; Hockenbery et al. (1990) op.cit; Vaux et al . (1988) op.cit; Alnemri et al. (1992) Cancer Res. 52: 491; Sentman et al . (1991) Cell 67: 879; Strasser et al . (1991) op.cit). Upregulation of bcl-2 expression also inhibits apoptosis of EBV-infected B- cell lines (Henderson et al . (1991) Cell 65: 1107). The expression of bcl-2 has also been shown to block apoptosis resulting from expression of the positive cell growth regulatory proto-oncogene, c-myc, in the absence of serum or growth factors (Wagner et al . (1993) Mol. Cell. Biol. 13: 2432). However, the precise mechanism(s) by which bcl- 2 is able to inhibit apoptosis is not yet fully defined. The bcl-2 proto-oncogene is rather unique among cellular genes in its ability to block apoptotic deaths in multiple contexts (Korsmeyer, S. (1992) Blood 80: 879). Overexpression of bcl-2 in transgenic models leads to accumulation of cells due to evasion of normal cell death mechanisms (McDonnell et al . (1989) Cell 57: 79). Induction of apoptosis by diverse stimuli, such as radiation, hyperthermia, growth factor withdrawal, glucocorticoids and multiple classes of chemotherapeutic agents is inhibited by bcl-2 in vitro models (Vaux et al . (1988) Nature 335: 440; Tsujimoto, Y. (1989) Oncogene 4: 1331; Nunez et al . (1990) J. Immunol. 144: 3602; Hockenbery et al. (1990) Nature 348: 334; Sentman et al . (1991) Cell 67: 879; Walton et al . (1993) Cancer Res. 53: 1853; Miyashita, T. and Reed, J. (1993) Blood 81: 151). These effects are proportional to the level of bcl-2 expression. Additionally, the endogenous pattern of bcl-2 expression is highly suggestive of a role in the regulation of cell survival in vivo (Hockenbery et al . (1991) Proc. Natl. Acad. Sci. USA 88: 6961; LeBrun et al - (1993) Am. J. Pathol. 142: 743). The bcl-2 protein seems likely to function as an antagonist of a central mechanism operative in cell death. Additionally, it would be beneficial to identify genes which could be generally utilized to induce programmed cell death. While identifying the bcl-2 cell death pathway is significant, a way of regulating the bcl-2 pathway has not been discovered. The ability to down-regulate the effect of bcl-2 would be advantageous in cancer therapy, in controlling hyperplasias such as benign prostatic hypertrophy (BPH) and eliminating self reactive clones in autoimmunity by favoring death effector molecules. Up- regulating the effect of bcl-2 and favoring death repressor molecules would be beneficial in the treatment and diagnosis of immunodeficiency diseases, including AIDs, and in neurodegenerative and ischemic cell death. Many pathological conditions result, at least in part, from aberrant control of cell proliferation, differentiation, and/or apoptosis. For example, neoplasia is characterized by a clonally derived cell population which has a diminished capacity for responding to normal cell proliferation control signals. Oncogenic transformation of cells leads to a number of changes in cellular metabolism, physiology, and morphology. One characteristic alteration of oncogenically transformed cells is a loss of responsiveness to constraints on cell proliferation and differentiation normally imposed by the appropriate expression of cell growth regulator genes. Neurodegenerative diseases (e.g., amyotrophic lateral sclerosis) and HIV-1 pathogenesis have been associated with free radial formation and toxicity, and apoptotic events may be involved in their disease etiologies (Meyaard et al . (1992) Science 257: 217). The presence of bcl-2 in a cell also renders the cell highly resistant to various chemical and physical agents. In particular, bcl-2 renders cancer cells more resistant to chemotherapeutic agents. Recently, various cellular and viral gene products have been identified that share a similar structure or activity with bcl-2. Despite the similarity of bcl-2 to these bcl-2-related proteins, however, no structural feature of the bcl-2 protein has been identified that lends a clue to its mechanism of regulating apoptosis. The precise molecular pathways and secondary changes leading to malignant transformation for many cell types are not clear. However, the characteristic translocation of the apoptosis-associated bcl-2 gene to the immunoglobulin heavy chain locus t(14;18) in more than 80 percent of human follicular B cell lymphomas and 20 percent of diffuse lymphomas and the neoplastic follicular lymphoproliferation present in transgenic mice expressing high levels of bcl-2 indicates that the bcl-2 gene likely is causally involved in neoplastic diseases and other pathological conditions resulting from abnormal cell proliferation, differentiation, and/or apoptosis. Thus, it would be desirable to identify agents which can modify the activity (ies) of the bcl-2 protein so as to modulate cell proliferation, differentiation, and/or apoptosis for therapeutic or prophylactic benefit. Further, such agents can serve as commercial research reagents for control of cell proliferation, differentiation, and/or apoptosis in experimental applications, and/or for controlled proliferation and differentiation of predetermined hematopoietic stem cell or neuronal cell populations in vitro, in ex vivo therapy, or in vivo . It would therefore be useful to develop a treatment of cancers which is based on susceptibility of tumor cell lines to the induction of programmed cell death caused by transfection of the Nip, Nix and Nox proteins. SUMMARY OF THE INVENTION According to the present invention, there is provided an isolated and purified Nip, Nix and Nox proteins. There is also provided a method for inducing apoptosis in cells by transfecting a cell with a Nip, Nix and Nox proteins. Additionally, there is provided a method for inducing apoptosis in cells by creating antibodies which recognize a specific Nip, Nix and Nox proteins in a muscle or skin of a patient. A method for inducing apoptosis in cells by creating transgenes overexpressing the genes for the Nip, Nix and Nox proteins is also provided. A transgenic animal and progeny thereof is provided having an expression factor consisting essentially of a Nip, Nix and Nox proteins selected from the group consisting essentially of Seq. ID No : 1 through 6. DESCRIPTION OF THE DRAWINGS Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein: Figure IA depicts the Nip3 mRNA of 1535 bp with the open reading frame boxed with the predicted protein of 194 amino acids, the putative transmembrane domain between amino acids 164 and 184 of Nip3 protein is shaded, also a mutant lacking the transmembrane sequence was constructed by truncation at amino acid 163 and is called Nip3 163 ; Figure 2A shows an in vi tro Nip3 is expressed as a homodimer, after in vi tro transcription and translation of Nip3 the 35S-labelled protein was separated on SDS-PAGE (lanes 1 and 2) under reducing (R, lane 1) and nonreducing (NR, lane 2) conditions, Nip3 runs as two bands at 60 and 30 kD; Nip3 163 is a truncated mutant in which the terminal 31 amino acids from 164 to 194 containing the putative transmembrane domain have been removed (lanes 3 and 4) and the truncated Nip3163 is expressed as a major band at 29 kD and a minor band at 28 kD under both reducing (lane 3) and nonreducing (lane 4) conditions; Figure 2B shows a comparative peptide mapping of in vi tro translated 35S-labeled 60-kD Nip3 and 28-kD Nip3163 protein; After in vi tro translation, Nip3 and Nip3163 were immunoprecipitated, the 60- and 28-kD bands recovered after SDS-PAGE then trypsin digested, the resulting peptides were separated on the same plate by electrophoresis at pH 1.9 (horizontal dimension with anode to the left) , followed by ascending chromatography, the positions of the origin of Nip3 and Nip3163 are marked by arrows, three [35S] methionine-labeled peptides predicted from the trypsin digest are circled and labeled A, B, and C (Nip3) and A9 , B9, and C9 (Nip3163 ); Peptide A and A9 , amino acids 1-45; B and B9, amino acids 140-146; C and C9, 147-152; All three peptides are represented in both Nip3 and Nip3163, minor spots are similar in the two proteins and likely are partially digested fragments; Figure 2C shows Nip3 homodimerization as identified by the yeast two-hybrid system; (left) Plasmids encoding full- length Nip3 and the COOH-terminal truncated mutant Nip3163 fused to the GAL4 DNA-binding domain were cotransformed with plasmids encoding Nip3 , Nip3163 or empty vector sequences fused to the GAL4 transcriptional activation domain; Protein-protein interactions were determined by growth of yeast in the absence of leucine, tryptophan and histidine; (Right) Growth in the absence of leucine and tryptophan is shown as a control, the results are representative of 3 independent experiments; Figure 3 shows the subcellular localization of Nip3 and Nip3163; Rat-1 cells were transfected with HA-Nip3 and stained with anti-HA antibody using FITC (A) and the mitochondrial protein marker anti-HSP60 antibody using Cy3 ; (B) the stained images were combined to compare the staining pattern of both proteins; (C) and their coincidence is indicated by the conversion of green FITC and red Cy3 stain to yellow throughout the cell cytoplasm, HA-Nip3163 was expressed in Rat-1 cells then stained with anti-HA antibody; (D) or anti-HSP60 antibody; (E) and shown as a combined image using antibodies; (F) as described above; Figure 4A shows that overexpression of Nip3 but not Nip3163 induces apoptosis; Rat-1 or MCF-7 (left) cells were transfected with T7-tagged Nip3 then at different times cells harvested and stained with anti-T7 antibody and FITC anti-mouse IgG antibody to identify cells expressing Nip3 , the frequency of apoptotic cells was quantitated by Hoechst dye staining; Rat-1 cells (right) were then transfected and apoptotic cells expressing (filled circles) and not expressing (hollow circles) Nip3 were quantitated. Rat-1 cells expressing (filled triangles) or not expressing (hollow triangles) Nip3163 were analyzed in the same manner, at least 200 cells were counted in each sample; All assays were repeated three to seven times with identical results; Figure 4B shows a Western blot of Rat-1 cells transfected with T7-Nip3 (left) or T7-Nip3163 (right) , cells were harvested at the times indicated and Western blots developed with anti-T7 antibody; Figure 5. Nip3 sensitizes Rat-1 cells to drug-induced apoptosis; Rat-1 cells were transfected withT7-tagged Nip3 then 12 h later treated with increasing amounts of etoposide, camptothecin or granzyme B and perforin, cells expressing Nip3 were identified by anti-T7 antibody and the nucleus stained with Hoechst dye, apoptotic cells expressing Nip3 (Nip3 1) or cells not expressing Nip3 (Nip3 2) were enumerated; Figure 6 shows that Nip3 overcomes Bcl-2 suppression of apoptosis; (A) (Left) Rat-1 and Rat-l/Bcl-2 cells were transfected with T7-Nip3 and at in-creasing time the cells were harvested and Nip3 -expressing apoptotic cells quantitated as described in Figure 4; (Right) The Rat-1 and Rat-l/Bcl-2 cells were treated with granzyme B as shown at a constant concentration of perforin (125 ng/ml) for 3 h and apoptotic cells quantitated by Hoechst dye as described previously; (B) After transfection of Rat-1 and Rat-l/Bcl-2 cells with T7-Nip3, lysates were harvested at increasing time intervals then Western blotted with anti-T7 antibody. Molecular mass markers are shown on the left; (C) Western blot of Bcl-2 (arrow) in Rat-1 (lane 1) and Rat-l/Bcl-2 (lane 2) cell lines, blots were developed with rabbit anti- human Bcl-2 (PharMingen) , relative molecular mass (kD) is shown on the left; Figure 7A shows the alignment of predicted murine and human Nip3 and Nix proteins; regions of identity with hNix are shaded, Human Nix and murine Nix are 97% identical, hNip3 and mNip3 are 56 and 53% identical to hNix; The predicted transmembrane domains of aligned hNix and mNix from amino acids 196 to 216 are underlined; Figure 7B shows a Northern blot analysis of murine and human and Nix; Human erythroleukemia cells (HEL) (lane 2) and mouse kidney tissue (lane 4) reveal a major 4.5 kb and minor 1.4 kb transcript (arrows); Corresponding ethidium bromide stained total RNA (lanes 1,3) are shown adjacent to the respective Northern blot; Figure 8A depicts an in vi tro transcription and translation of Nix and Nip3 ; 35 S-labelled mNix (lanes 1,2), hNix (lanes 3,4), and mNip3 (lanes 5,6) were electrophoresed on Tris- Tricine SDS PAGE under reducing (R) (Lanes 2,4,6) and non reducing (NR) (lanes 1,3,5) conditions as described herein; Figure 8B shows the mNix expression in vivo; Rat-1 cells were transfected with C-terminally T7-tagged mNix (left) then lysates electrophoresed in Tris-Tricine buffer (see Methods) and Western blotted with anti-T7 antibody; Rat-1 cells treated with the proteasome inhibitor lactacystin were transfected with mNix-T7 in parallel (right) ; Figure 9(A,B) depicts subcellular localization of Nix and the mitochondrial matrix protein HSP60; MCF-7 cells transiently transfected with mNix-T7 were stained with mouse monoclonal anti-T7 antibody and FITC-labeled rabbit anti-mouse IgG (left) and rabbit anti-HSP60 antibody followed by Cy3-conjugated donkey anti-rabbit IgG antibody (right) 12 hours later; (C,D)MCF-7 cells transfected with the Nix ΔTM mutant were stained with anti-T7 antibody (left) or Hoechst dye (right); (E,F) Nix expressing cells undergoing apoptosis 30 hours post transfection, mNix transfected cells stained with anti-T7 antibody (left) and Hoechst dye (right) , note the condensed chromatin in the nucleus in the mNix expressing cells (arrows) ; Figure 10 shows the time course of induction of apoptosis following mNix-T7 (A) or hNip3 (B) transient transfection of MCF-7 (l,D) and Rat-1 (^,0) cells, each tumor cell was transfected in three independent experiments and apoptotic cells quantitated at the times indicated by counting the percentage of apoptotic nuclear morphology by Hoechst staining of Nix or Nip3 expressing cells; Cells transfected with mNix or hNip3 (■, • ) were identified by immunofluorescent staining of anti-T7 antibody, Apoptosis of non-transfected cells ( ,O) was also enumerated; (C) Induction of apoptosis by mNix (•) and mNix ΔTM (O) in Rat-1 cells following transient transfection as described above; (D) Apoptosis of hNip3 (•) and hNip3 ΔTM (O) in Rat-1 cells. The data from three independent experiments were expressed as the mean ± SE for each time point; Figure 11 shows the effect of Bcl-2 or Bcl-X L overexpression of mNix; (A) Time of course of Nix-induced apoptosis in Bcl-2 expressing Rat-1 cells, Rat-1 cells (•) or Rat-1 cells overexpressing Bcl-2 (O) were transfected with mNix and apoptotic cells counted as above; (B) . Transfection of mNix into two cell lines TX5 and TX22 overexpressing Bcl-X L compared to the parental 10T1/2 cell line TC, at different times following transfection, Nix expressing cells (TC-Nix, TX22-Nix, TX5-Nix) undergoing apoptosis were quantitated as described above, cells not expressing Nix (TC-Con, TX22- Con, TX5-Con) were also counted for apoptotic morphology; (C) Apoptosis of the TC, TX5 and TX22 cells lines following staurosporine treatment at the doses indicated for 5 hours, experiments were repeated three times and the mean and SE are shown; (D) Western blot of Bcl-X L (arrow) in cell lines TC, TX5 and TX22, Relative molecular weights (kDa) are shown on the left; and Figures 12 A and B show (A) a comparison of the Nip3 and Nix proteins; (B) a comparison of the Nip3 and Nox proteins . DETAILED DESCRIPTION OF THE INVENTION Generally, the present invention provides a method of producing purified Nip, Nix and Nox proteins, Seq. ID No : 1 through 6, and functional analogs thereof. Also, provided is a method of inducing apoptosis in cells by transfecting a cell with a Nip, Nix and Nox proteins. A detailed description of the isolation protocols and its properties are set forth in the Experimental Section. By functional analogs, it is meant that an analog will generally be at least 70% homologous over any portion that is functionally relevant. In more preferred embodiments the homology will be at least 80% and can approach 95% homology to the Nip, Nix and Nox proteins. The amino acid sequence of an analog may differ from that of the Nip, Nix and Nox proteins when at least one residue is deleted, inserted or substituted. Differences in glycosylation can provide analogs. The molecular weight of the Nip, Nix and Nox proteins can vary between the analog and the present invention due to carbohydrate differences. Standard molecular biology techniques known in the art and not specifically described are generally followed as in Sambrook et al . , Molecular Cloning: A Labora tory Manual , Cold Springs Harbor Laboratory, New York (1989, 1992) , and in Ausubel et al . , Current Protocols in Mol ecular Biology, John Wiley and Sons, Baltimore, Maryland (1989) . Additionally, standard methods in immunology known in the art and not specifically described are generally followed as in Stites et al . (eds), Basic and Clinical Immunology (8 th Edi tion) , Appleton S- Lange, Norwalk, CT (1994) and Mishell and Shiigi (eds) , Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York (1980) . By transfection, it is meant infection by viral nucleic acid. Such methods can be found generally described in Sambrook et al . , Molecular Cloning: A Laboratory Manual , Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al . , Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Chang et al . , Soma tic Gene Therapy, CRC Press, Ann Arbor, MI (1995), Vega et al . , Gene Targeting, CRC Press, Ann Arbor, MI (1995), Vectors : A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988) and Gilboa et al (1986) . Cloning techniques are provided by the present invention. In general, recombinant Nip, Nix and Nox proteins are superior to the native lysosomal enzymes for these applications. Immunoassays are also provided by the present invention. In general, ELISAs are the preferred immunoassays employed to assess a specimen. Both polyclonal and moloclonal antibodies can be used in the assays. The specific assay to be used can be determined by one skilled in the art. Antibody production is provided by the present invention. Antibodies may be prepared against the immunogen, or any portion thereof, for example a synthetic peptide based on the sequence. As stated above, antibodies are used in assays and are therefore used in determining if the appropriate enzyme has been isolated. Antibodies can also be used for removing enzymes from red cell suspensions after enzymatic conversion. Immunogens can be used to produce antibodies by standard antibody production technology well known to those skilled in the art as described generally in Harlow and Lane, Antibodies : A Labora tory Manual , Cold Springs Harbor Laboratory, Cold Spring Harbor, NY, 1988 and Borrebaeck, Antibody Engineering - A Practical Guide, W.H. Freeman and Co., 1992. Antibody fragments may also be prepared from the antibodies and include Fab, F(ab') 2 , and Fv by methods known to those skilled in the art. Nip3 (Nineteen kDa interacting £rotein-3) is an ElB 19K and Bcl-2 binding protein of unknown function. The adenovirus ElB 19K protein is functionally similar to Bcl-2 as a survival factor (White, 1996) . A two hybrid screen of proteins that interact with ElB 19K in the yeast Saccharomyces cerevisiae identified several unique cDNAs named Nipl, Nip2 and Nip3 (Boyd, et al . , 1994). All three proteins interact with discrete conserved domains of ElB 19K protein and Bcl-2 that are involved in suppression of cell death, although a function was not identified. Nip3 is detected as both a 60 kDa and 30 kDa protein in vivo and in vi tro and exhibits strong homologous interaction in a yeast two-hybrid system indicating that it can homodimerize . Nip3 is expressed in mitochondria and a mutant (Nip3 163 ) lacking the putative transmembrane domain and C-terminus does not dimerize or localize to mitochondria. Transient transfection of epitope-tagged Nip3 in Rat-1 fibroblasts and MCF-7 breast carcinoma induces apoptosis within 12 hours while cells transfected with the Nip3 163 mutant have a normal phenotype, suggesting that mitochondrial localization is necessary for induction of cell death. Nip3 overexpression increases the sensitivity to apoptosis induced by granzyme B and topoisomerase I and II inhibitors. Following transfection, both Nip3 and Nip3 163 protein levels decrease steadily over 48 hours indicating that the protein is rapidly degraded and this occurs in the absence of cell death. Bcl-2 overexpression initially delays the onset of apoptosis induced by Nip3 but the resistance is completely overcome in longer periods of incubation. Nip3 protein levels are much higher and persist longer in Bcl-2 expressing cells. Nip3 is an apoptosis-inducing dimeric mitochondrial protein that can overcome Bcl-2 suppression. Nix is a homolog of the ElB 19K/Bcl-2 binding and pro- apoptotic protein Nip3 and is isolated using the same method described for the Nip3 protein. Human and murine Nix have a 56% and 53% amino acid identity to human and murine Nip3 , respectively. Nix encodes a 23.8 kD protein but it is expressed as a 48 kD protein suggesting that it homodimerizes similar to Nip3. The carboxy terminus of Nix, including a transmembrane domain, is highly homologous to Nip3 but it bears a longer and distinct asparagine/proline rich N-terminus. Nix colocalizes with the mitochondrial matrix protein HSP60 and removal of the extreme carboxy terminus including the putative transmembrane domain (TM) results in general cytoplasmic and nuclear expression. Comparisons of the Nip3 , Nix, and Nox proteins are shown in Figures 12 A and B. Further, when transiently expressed, Nix rapidly activates apoptosis and can overcome the apoptosis suppressors Bcl-2 and Bcl-X L although very high levels of Bcl-X L expression will inhibit apoptosis. Removal of the Nix TM domain (ΔTM) had no effect on its ability to induce apoptosis unlike Nip3 ΔTM, which is inactive. Following transfection, Nix protein undergoes progressive proteolysis to a 11 kD C-terminal fragment, which is blocked by the proteasome inhibitor lactacystin. There are a number of therapies which may be accomplished using the Nip, Nix and Nox proteins. For example, the proteins can deliver an apoptotic signal to cancer cells or other cells that one wishes targeted for elimination. It can be delivered ex vivo (bone marrow), by direct intratumoral delivery or via a targeting vector in vivo. Delivery by viral vector or possibly raw DNA, or by small peptides (or molecular derivatives) based on the protein structure can also be done. The anti-apoptotic action of the Nip3 mutant (s) can be used for tissue (eg. bone marrow) protection during chemotherapy or immunotherapy . Ischemic diseases (eg. myocardial infraction, stroke) have an important apoptotic component that may be blocked by the Nip3 mutant . It can be used in organ or tissue transplantations where implanted tissues can be first treated with the gene to prevent subsequent immune attack. Nip3 was originally described by its ability to physically interact with a protein from adenovirus called ElB 19K, which is an apoptosis inhibitor similar to Bcl-2. Also, Nip3 is induced in cells as a consequence of virus infection and this is the mechanism by which some viruses kill cells. Accordingly, the inhibitory protein can also have a potential in control of virally induced cell death. Another use for the Nip3 family genes is to enhance adenovirus based cell death mechanisms which can be used as anti-tumor therapy. There are two mouse monoclonal and one rabbit polyclonal antibody to Nip3. The antibodies recognize a gene product (protein) in muscle and skin of mice and humans . Nip3 is expressed in skin and muscle. The Nix and Nox EST sequences were identified in fetal cDNA libraries and the protein can also be important for fetal development . Transgenics overexpressing the genes are made according to techniques generally known in the art, as well as making mice deficient in the gene expression by homologous recombination (knockout) or by dominant negative transgenes. These mice are models of disease and/or of developmental abnormalities. These transgenics are constructed using standard methods known in the art and as set forth in United States Patents 5,487,992, 5,464,764, 5,387,742, 5,360,735, 5,347,075, 5,298,422, 5,288,846, 5,221,778, 5,175,385, 5,175,384,5,175,383, 4,736,866 as well as Burke and Olson (1991) , Capecchi (1989) , Davies et al. (1992), Dickinson et al . (1993), Duff and Lincoln (1995), Huxley et al . (1991), Jakobovits et al . (1993), Lamb et al . (1993), Pearson and Choi (1993), Rothstein (1991), Schedl et al . (1993), Strauss et al . (1993). Further, patent applications WO 94/23049, WO 93/14200, WO 94/06908, WO 94/28123 also provide information. The above discussion provides a factual basis for the use of Nip, Nix and Nox proteins in inducing apoptosis. The methods used with and the utility of the present invention can be shown by the following non-limiting examples and accompanying figures. EXAMPLES GENERAL METHODS: General methods in molecular biology: Standard molecular biology techniques known in the art and not specifically described were generally followed as in Sambrook et al . , Molecular Cloning: A Laboratory Manual , Cold Springs Harbor Laboratory, New York (1989, 1992), and in Ausubel et al . , Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989) . Polymerase chain reaction (PCR) was carried out generally as in PCR Protocols : A Guide To Methods And Applica tions , Academic Press, San Diego, CA (1990) . Reactions and manipulations involving other nucleic acid techniques, unless stated otherwise, were performed as generally described in Sambrook et al . , 1989, Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, and methodology as set forth in United States patents 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057 and incorporated herein by reference . General methods in immunology: Standard methods in immunology known in the art and not specifically described were generally followed as in Stites et al . (eds), Basic and Clinical Immunology (8th Edition) , Appleton & Lange, Norwalk, CT (1994) and Mishell and Shiigi (eds) , Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York (1980) . Immunoassays In general, ELISAs are the preferred immunoassays employed to assess a specimen. ELISA assays are well known to those skilled in the art. Both polyclonal and monoclonal antibodies can be used in the assays. Where appropriate other immunoassays, such as radioimmunoassays (RIA) can be used as are known to those in the art . Available immunoassays are extensively described in the patent and scientific literature. See, for example, United States patents 3,791,932; 3,839,153; 3,850,752; 3,850,578 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219 5,011,771 and 5,281,521 as well as Sambrook et al, Molecular Cloning: A Laboratory Manual , Cold Springs Harbor, New York, 1989 Antibody Production Antibody Production: Antibodies may be either monoclonal, polyclonal or recombinant. Conveniently, the antibodies may be prepared against the immunogen or portion thereof for example a synthetic peptide based on the sequence, or prepared recombinantly by cloning techniques or the natural gene product and/or portions thereof may be isolated and used as the immunogen. Immunogens can be used to produce antibodies by standard antibody production technology well known to those skilled in the art as described generally in Harlow and Lane, Antibodies : A Labora tory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988 and Borrebaeck, Antibody Engineering - A Practical Guide, W.H. Freeman and Co., 1992. Antibody fragments may also be prepared from the antibodies and include Fab, F(ab') 2 , and Fv by methods known to those skilled in the art. For producing polyclonal antibodies a host, such as a rabbit or goat, is immunized with the immunogen or immunogen fragment, generally with an adjuvant and, if necessary, coupled to a carrier; antibodies to the immunogen are collected from the sera. Further, the polyclonal antibody can be absorbed such that it is monospecific . That is, the sera can be absorbed against related immunogens so that no cross-reactive antibodies remain in the sera rendering it monospecific . For producing monoclonal antibodies the technique involves hyperimmunization of an appropriate donor with the immunogen, generally a mouse, and isolation of splenic antibody producing cells. These cells are fused to a cell having immortality, such as a myeloma cell, to provide a fused cell hybrid which has immortality and secretes the required antibody. The cells are then cultured, in bulk, and the monoclonal antibodies harvested from the culture media for use . For producing recombinant antibody (see generally Huston et al, 1991; Johnson and Bird, 1991; Mernaugh and Mernaugh, 1995) , messenger RNAs from antibody producing B- lymphocytes of animals, or hybridoma are reverse- transcribed to obtain complimentary DNAs (CDNAs) . Antibody cDNA, which can be full or partial length, is amplified and cloned into a phage or a plasmid. The cDNA can be a partial length of heavy and light chain cDNA, separated or connected by a linker. The antibody, or antibody fragment, is expressed using a suitable expression system to obtain recombinant antibody. Antibody cDNA can also be obtained by screening pertinent expression libraries. The antibody can be bound to a solid support substrate or conjugated with a detectable moiety or be both bound and conjugated as is well known in the art. (For a general discussion of conjugation of fluorescent or enzymatic moieties see Johnstone & Thorpe, I muno chemistry in Practice, Blackwell Scientific Publications, Oxford, 1982.) The binding of antibodies to a solid support substrate is also well known in the art. (see for a general discussion Harlow & Lane Antibodies : A Labora tory Manual , Cold Spring Harbor Laboratory Publications, New York, 1988 and Borrebaeck, Anti-body Engineering - A Practical Guide, W.H. Freeman and Co., 1992) The detectable moieties contemplated with the present invention can include, but are not limited to, fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, β-galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, 14 C and iodination. Recombinant Protein Purification Marshak et al, "Strategies for Protein Purification and Characterization. A laboratory course manual." CSHL Press, 1996. Transgenic and Knockout Methods The present invention provides for transgenic gene and polymorphic gene animal and cellular (cell lines) models as well as for knockout models. For crene therapy: By gene therapy as used herein refers to the transfer of genetic material (e.g DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition phenotype . The genetic material of interest encodes a product (e.g. a protein, polypeptide, peptide or functional RNA) whose production in vivo is desired. For example, the genetic material of interest can encode a hormone, receptor, enzyme, polypeptide or peptide of therapeutic value. For a review see, in general, the text "Gene Therapy" (Advances in Pharmacology 40, Academic Press, 1997) . Two basic approaches to gene therapy have evolved: (1) ex vivo and (2) in vivo gene therapy. In ex vivo gene therapy cells are removed from a patient, and while being cultured are treated in vi tro . Generally, a functional replacement gene is introduced into the cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the host/patient. These genetically reimplanted cells have been shown to produce the transfected gene product in si tu . In in vivo gene therapy, target cells are not removed from the subject rather the gene to be transferred is introduced into the cells of the recipient organism in si tu, that is within the recipient. Alternatively, if the host gene is defective, the gene is repaired in si tu [Culver, 1998] . These genetically altered cells have been shown to produce the transfected gene product in si tu . The gene expression vehicle is capable of delivery/transfer of heterologous nucleic acid into a host cell. The expression vehicle may include elements to control targeting, expression and transcription of the nucleic acid in a cell selective manner as is known in the art. It should be noted that often the 5'UTR and/or 3'UTR of the gene may be replaced by the 5'UTR and/or 3'UTR of the expression vehicle. Therefore as used herein the expression vehicle may, as needed, not include the 5'UTR and/or 3'UTR shown in SEQ ID No : 1 and only include the specific amino acid coding region. The expression vehicle can include a promotor for controlling transcription of the heterologous material and can be either a constitutive or inducible promotor to allow selective transcription. Enhancers that may be required to obtain necessary transcription levels can optionally be included. Enhancers are generally any non-translated DNA sequence which works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter. The expression vehicle can also include a selection gene as described herein below. Vectors can be introduced into cells or tissues by any one of a variety of known methods within the art . Such methods can be found generally described in Sambrook et al . , Molecular Cloning: A Laboratory Manual , Cold Springs Harbor Laboratory, New York (1989, 1992) , in Ausubel et al . , Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Chang et al . , Soma tic Gene Therapy, CRC Press, Ann Arbor, MI (1995), Vega et al . , Gene Targeting, CRC Press, Ann Arbor, MI (1995) , Vectors : A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988) and Gilboa et al (1986) and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see United States patent 4,866,042 for vectors involving the central nervous system and also United States patents 5,464,764 and 5,487,992 for positive-negative selection methods. Introduction of nucleic acids by infection offers several advantages over the other listed methods. Higher efficiency can be obtained due to their infectious nature. Moreover, viruses are very specialized and typically infect and propagate in specific cell types. Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or mixed culture of cells. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events. A specific example of DNA viral vector for introducing and expressing recombinant sequences is the adenovirus derived vector Adenop53TK. This vector expresses a herpes virus thymidine kinase (TK) gene for either positive or negative selection and an expression cassette for desired recombinant sequences. This vector can be used to infect cells that have an adenovirus receptor which includes most cancers of epithelial origin as well as others. This vector as well as others that exhibit similar desired functions can be used to treat a mixed population of cells and can include, for example, an in vi tro or ex vivo culture of cells, a tissue or a human subject. Additional features can be added to the vector to ensure its safety and/or enhance its therapeutic efficacy. Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus. An example of such a negative selection marker is the TK gene described above that confers sensitivity to the antibiotic gancyclovir. Negative selection is therefore a means by which infection can be controlled because it provides inducible suicide through the addition of antibiotic. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or recombinant sequence, cellular transformation will not occur. Features that limit expression to particular cell types can also be included. Such features include, for example, promoter and regulatory elements that are specific for the desired cell type. In addition, recombinant viral vectors are useful for in vivo expression of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity. Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally. This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells. As described above, viruses are very specialized infectious agents that have evolved, in many cases, to elude host defense mechanisms. Typically, viruses infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes its natural specificity to specifically target predetermined cell types and thereby introduce a recombinant gene into the infected cell. The vector to be used in the methods of the invention will depend on desired cell type to be targeted and will be known to those skilled in the art. For example, if breast cancer is to be treated then a vector specific for such epithelial cells would be used. Likewise, if diseases or pathological conditions of the hematopoietic system are to be treated, then a viral vector that is specific for blood cells and their precursors, preferably for the specific type of hematopoietic cell, would be used. Retroviral vectors can be constructed to function either as infectious particles or to undergo only a single initial round of infection. In the former case, the genome of the virus is modified so that it maintains all the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles which are capable of undergoing further rounds of infection. The vector's genome is also engineered to encode and express the desired recombinant gene. In the case of non-infectious viral vectors, the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles. Without such a signal, any particles that are formed will not contain a genome and therefore cannot proceed through subsequent rounds of infection. The specific type of vector will depend upon the intended application. The actual vectors are also known and readily available within the art or can be constructed by one skilled in the art using well-known methodology. The recomnbinant vector can be administered in several ways. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration can provide a quicker and more effective treatment, administration can also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration, especially in the case of neuro-degenerative diseases. Following injection, the viral vectors will circulate until they recognize host cells with the appropriate target specificity for infection. An alternate mode of administration can be by direct inoculation locally at the site of the disease or pathological condition or by inoculation into the vascular system supplying the site with nutrients or into the spinal fluid. Local administration is advantageous because there is no dilution effect and, therefore, a smaller dose is required to achieve expression in a majority of the targeted cells. Additionally, local inoculation can alleviate the targeting requirement required with other forms of administration since a vector can be used that infects all cells in the inoculated area. If expression is desired in only a specific subset of cells within the inoculated area, then promoter and regulatory elements that are specific for the desired subset can be used to accomplish this goal. Such non-targe ing vectors can be, for example, viral vectors, viral genome, plasmids, phagemids and the like. Transfection vehicles such as liposomes can also be used to introduce the non-viral vectors described above into recipient cells within the inoculated area. Such transfection vehicles are known by one skilled within the art. Delivery of gene products/therapeutics (compound) : The compound of the present invention is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners. The pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including but not limited to improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art. In the method of the present invention, the compound of the present invention can be administered in various ways. It should be noted that it can be administered as the compound or as pharmaceutically acceptable salt and can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, diluents, adjuvants and vehicles. The compounds can be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and infusion techniques. Implants of the compounds are also useful. The patient being treated is a warm-blooded animal and, in particular, mammals including man. The pharmaceutically acceptable carriers, diluents, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the invention. It is noted that humans are treated generally longer than the mice or other experimental animals exemplified herein which treatment has a length proportional to the length of the disease process and drug effectiveness. The doses may be single doses or multiple doses over a period of several days, but single doses are preferred. The doses may be single doses or multiple doses over a period of several days. The treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient species being treated. When administering the compound of the present invention parenterally, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion) . The pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Nonaqueous vehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, may also be used as solvent systems for compound compositions. Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol , phenol, sorbic acid, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the compounds . Sterile injectable solutions can be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired. A pharmacological formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicle, adjuvants, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres . Examples of delivery systems useful in the present invention include: 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many other such implants, delivery systems, and modules are well known to those skilled in the art. A pharmacological formulation of the compound utilized in the present invention can be administered orally to the patient. Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable. Known techniques which deliver it orally or intravenously and retain the biological activity are preferred. In one embodiment, the compound of the present invention can be administered initially by intravenous injection to bring blood levels to a suitable level. The patient ' s levels are then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition and as indicated above, can be used. The quantity to be administered will vary for the patient being treated and will vary from about 100 ng/kg of body weight to 100 mg/kg of body weight per day and preferably will be from 10 μg/kg to 10 mg/kg per day. EXAMPLE 1: Materials and Methods Cell cultures and Reagents. Rat-1, Rat-l/Bcl-2 and MCF-7 cells used in this study were cultured in α-minimal essential medium ( -MEM) (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) . Murine monoclonal anti-HA 12CA5 and anti-T7 antibody were purchased by Boehringer Mannheim (Laval, Quebec) and Novagen (Madison, WI) , respectively. Rabbit anti-HSP60 antibody was donated by Dr. Radhey Gupta (McMaster University, Hamilton, Ont . ) . FITC-conjugated goat anti-rabbit IgG (FITC) (Sigma Chemical Co.) and Cy-3 conjugated goat anti-mouse IgG (BioCan Ltd. , Mississauga) were used for fluorescence studies. Cloning and construction of expression plasmids. Following isolation of a Nip3 cDNA, RACE is used to identify both 3' and 5' ends using a premade human brain marathon-ready cDNA according to the manufacturer's instructions (Clontech, Palo Alto, CA) . The Nip3 3' RACE was done using primer 5' CAGCGTTCCAGCCTCGGTTTCT 3' while the 5' RACE was done using three different primers 5' CAAAGGTGCTGGTGGAGGT 3' , 5' TGTGGTGTCTGCGAGCGAGGT 3' , and S' TCCAGCAGTATTTTTTCCA 3' . The appropriate RACE products were TA cloned (Invitrogen, San Diego, CA) and several of the resulting clones were analysed to obtain the described Nip3 sequence . To construct epitope tagged Nip3 in expression plasmids, Kpnl and EcoRI sites were introduced to the 5' and 3 ' end of the Nip3 cDNA encoding only the open reading frame by PCR amplification and its sequence was confirmed by the T7 sequencing Kit (Pharmacia) , the inserted in frame into the Kpni/BamHI sites of plasmid pKM1310 containing the 5' HA-tag. The recombinant cDNA 5'HA-Nip3 was then isolated by EcoRI digestion and blunt end ligated into the plasmid pKG86 creating the plasmid pKG5HA-Nip3 under the control of SP6 promoter which was used for in vi tro transcription/translation of 5 'HA-tagged Nip3 proteins. The plasmid pKG5HA-Nip3 163 was generated by PCR and constructed as described above. For cloning the Nip3 cDNA into the expression vector pcDNA3 (Invitrogen Corp.), the Hindlll site was introduced at the 5' end of HA-Nip3 cDNA by PCR amplification then cloning into the Hindlll/EcoRI sites. The Hindlll/Kpnl fragment containing the HA-tag was inserted into the Hindlll/Kpnl sites of pcDNA3 vector making the pcD-5HA vector for cloning the 5'HA-tagged Nip3 cDNA and its mutants. For 5 'HA-Nip3 and 5 ' HA-Nip3 163 , the cDNA fragments were isolated by KpnI/EcoRI digestion of pKG5HA-Nip3 and pKG5HA-Nip3 163 respectively, then inserted into KpnI/EcoRI sites of pcD-5HA vector, making pcD5HA-Nip3 and pcD5HA- Nip3 163 . For the 3 ' T7 tagged Nip3 and Nip3 163 , a cDNA fragment encoding the T7 epitope was inserted into the EcoRI/XhoI sites of pcDNA3 plasmid. The 3' T7-tagged Nip3 and Nip3 163 open reading frame were cloned into this vector as described above. These recombinant plasmids were used for in vi tro translation and transient transfection into the mammalian cells. For yeast transfection, plasmids encoding Nip3 and Nip3 163 with suitable restriction sites were generated by PCR then ligated in-frame in the GAL4 binding-domain of pASl or pGBT9 and GAL4 transcriptional activating domain of pACTII. The nucleotide sequence of all constructs was confirmed on an ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) . The pASl or pGBT9 and pACTII constructs were co-transformed into the yeast strain KGY37 and colonies selected on SC medium lacking leucine and tryptophan. Protein-protein interactions were determined by growth on SC medium lacking leucine, tryptophan and histidine in the presence of ImM 3 -amino-1, 2 , 4-triazole (3AT) . The relative β-galactosidase activity was measured using both the filter lift (X-Gal) and ONGP methods according to manufacturers instructions. In Vitro translation. 3S S-labeled Nip3 protein and mutants were prepared by in vi tro transcription and translation of cDNA cloned in pcD-3HA vector using TnT coupled Reticulocyte Lysate System (Promega, Madison, WI) according to manufacturer's instructions . Two-dimensional peptide mapping. 35 S-methionine-labeled in vi tro translated full length Nip3 and Nip3 163 proteins were immunoprecipitated using mouse monoclonal anti-Nip3 antibody. The proteins were then recovered from unfixed dried SDS-polyacrylamide gels by homogenizing the gel slices then precipitated with trichloroacetic acid and oxidized with performic acid as described previously (Boyle, et al . , 1991). The oxidized proteins were then resuspended in 25 mis of 50 mM NH 4 HC0 3 and digested overnight at 37°C with 10 mg TPCK-treated trypsin. An additional 5 mg of trypsin was then added, and digestion continued for at least another four hr. Following digestion, samples were repeatedly lyophilized (four times) using a Speedvac concentrator. Tryptic digests of the methionine labelled Nip3 proteins were subjected to electrophoresis using pH 1.9 buffer (15% acetic acid, 5% formic acid) followed by ascending chromatography with Scheidtmann buffer (isobutyric acid :pyridine : acetic acid : butanol : water (65:5:3:2:29) as described previously (Scheidtmann, et al . , 1982) . 35 S- methionine-labelled peptides were visualized by autoradiography. Transient transfection and apoptosis assays Rat-1 cells, Rat-l/Bcl-2 or MCF-7 cells were transfected with Nip3 and mutants in the expression plasmid pcD-3HA using LipofectAMINE Reagent (Gibco, Gaithersburg, MD) . Cells (3-8 x 10 5 ) were plated onto 100 x 20 mm tissue culture dishes (Nunc, Roskilde, Denmark) in 12 ml of complete medium 24 hrs . before transfection. The DNA- LipofectAMINE mixture was allowed to incubate in a tissue culture incubator for three hr for Rat-1 or five hours for MCF-7 cells. Aliquots of 5 x 10 5 cells were western blotted as described previously using mouse monoclonal anti-HA antibody 12CA5 or anti-T7 antibody using an ECL detection kit according to manufacturers instructions (Amersham, Amersham, UK) . For some experiments, cells were treated with granzyme B and perforin as previously described (Shi, et al., 1997), etoposide or camptothecin then stained with anti-T7 antibody with FITC-conjugated rabbit anti-mouse antibody and apoptotic cells were enumerated by altered DNA morphology with Hoechst dye staining, counting between 200 and 300 cells per sample (Shi, et al . , 1997). For subcellular localization, cells were grown on cover slips, transfected with HA-Nip3 then fixed with 4% formaldehyde in PBS. Cell were double stained with mouse monoclonal anti-HA antibody 12CA5 and anti-HSP60 antibody and visualized with FITC-conjugated goat anti-mouse IgG secondary antibody and Cy3-conjugated goat anti-rabbit antibody. Fluorescence was visualized using a Zeiss Axiophot microscope equipped with a cooled CCD camera driven by IPLabs Spectrum H-SU2 (version 3.0, Signal Analytics) and Multiprobe 1.1 E (Signal Analytics) software . RNA Isolation and Northern Hybridization Analysis Total RNA from mice tissues was kindly provided by Dr. Robert Shiu (University of Manitoba) . For Northern blot analysis 30 μg of total RNA was hybridized with nick- translated Nip3 probe (1-5 X 10 8 cpm μg DNA) at 42°C for 16- 20 hrs . Following hybridization and washing blots were exposed to Kodak XAR film at -70°C with an intensify screen. RESULTS Ubiquitous Expression of Nip3 Several Nip3 cDNAs were isolated from a human EBV transformed peripheral B lymphocyte library (Clonetech) by PCR and RACE of both 5' and 3' ends, and identified the longest as a 1535 bp sequence (see Genebank accession number Bankltll2877 AF002697 for complete sequence) . This is similar to the cDNA identified earlier (Boyd, et al, 1994) except in the 3' UTR where there is no evidence of a homologous calbindin region in several independent experiments. The RNA has a long 3' UTR and shorter 5' UTR, and the encoded protein contains 194 amino acids and has a predicted molecular weight of about 21.54 kD with a pi of 6.08 (Expasy Tools). There is a putative transmembrane domain between amino acids 164 to 184 (Figure IA) . Nip3 mRNA is expressed in human breast carcinoma MCF-7 as a major transcript of about 1.7 kb and two minor transcripts of 1.5 and 1.3 kb. The level of Nip3 mRNA in mouse tissues was also examined to determine how widely the gene is expressed. Two transcripts of 2.5 and 1.7 kb were identified with the 1.7 kb transcript found in all tissues examined and the larger transcript only certain tissues such as brain, heart, kidney, liver and submaxillary gland (Figure IB) . The identification of more than one Nip3 transcript in human and murine cells may indicate that the gene is alternately spliced or another closely related gene exists . Nip3 protein is expressed as a di er and monomer 35 S-methionine labelled Nip3 were prepared by in vi tro transcription and translation and examined the protein on SDS-PAGE. Nip3 was seen as a major band at approximately 60 kD and a small band of 30 kD (figure 2A) . Nip3 has a predicted molecular weight of about 21.54 kD, much smaller than the size of the two bands estimated from SDS-PAGE, indicating that the protein runs anomalously. The two bands were seen under both reducing and non-reducing conditions. However, when the C-terminus from amino acid 163 was removed (Nip3 163 ) (Figure IA) , the large molecular weight form was absent and a major band of 28 kD and a minor band of 27 kD were detected. These data suggests that the 30 kD protein is a Nip3 monomer while the 60 kD protein is a dimer that was formed by interaction at the C- terminus of the protein. To examine more directly if NIP3 is a homodimer, tryptic peptide fragments of the 60 kDa Nip3 were compared to the 28 kDa Nip3 163 . Following in vi tro translations, 35 S-methionine-labelled Nip3 and Nip3 163 were purified by immunoprecipitation with mouse monoclonal antibody to Nip3 and SDS PAGE. Tryptic peptides were prepared and fragments separated by 2D electrophoresis and chromatography and detected by radioautography as described in Materials and Methods. Three peptide fragments of Nip3 and Nip3 lδ3 contain methionine, while the C-terminal fragments including the truncated region of Nip3 163 do not. Figure 2B illustrates that the two proteins have three major 35 S-methionine-labelled peptides and their positions are very similar following 2D mapping. No other major peptides are detected in the 60 kDa Nip3 band. Minor proteins are also similar and likely represent partially digested Nip3. The ability of Nip3 to homodimerize was next examined in the yeast two hybrid system by inserting the Nip3 cDNA in either the GAL4 binding and activating domains which were then simultaneously expressed in yeast cells (KGY37) . This was compared to Nip3 interaction with non specific protein controls. A strongly positive interaction was found with Nip3 in both vectors indicating that the protein was capable of binding to itself (Table 1) . In contract, the Nip3 163 was unable to interact with Nip3 or with itself (Figure 2C) indicating that the C-terminus was required for homodimerization. Subcellular localization of Nip3 to mitochondria Boyd et al, 1994 reported that HA-Nip3 localizes in discrete cytoplasmic patches which are typical of a mitochondrial distribution. To confirm the mitochondrial localization of Nip3 and to test the hypothesis that it depends on the putative transmembrane domain, Rat-1 cells were transfected with HA-Nip3 or HA-Nip3 163 then stained with FITC-conjugated anti-HA antibody. Cells were simultaneously treated with anti-HSP60 antibody which primarily stains mitochondria (Zhu, et al . , 1996) and Cy3- conjugated second antibody. Both the green Nip3 and red HSP60 fluorescence appeared in a similar punctate pattern, and when the images were combined, a uniform yellow staining pattern indicated virtually complete coincidence of the two stains (Figure 3 A,B,C). In contrast, HA-Nip3 163 stained diffusely throughout the cytoplasm rather than the punctate distribution of the intact Nip3 , however, a small amount of HA staining appeared to colocalize with HSP60 by double fluorescence (Figure 3 D,E,F) . Overexpression of Nip3 but not Nip3 163 induces apoptosis T7 epitope-tagged Nip3 was transiently expressed in Rat-1 or MCF-7 cells. Cells were stained with both anti-T7 antibody to identify cells expressing Nip3 , and Hoechst dye to quantitate apoptosis. Over time, progressively more Nip3 expressing cells became apoptotic starting around 12 hours and reaching completion by 48 to 60 hours (Figure 4A) . Nip3 protein measured by anti-T7 western blot were highest between 12 and 24 hours following transfection then progressively decreased with time paralleling the induction of apoptosis (Figure 4B) . To determine if the transmembrane domain of Nip3 which is necessary for mitochondrial localization was also necessary to induce apoptosis, the experiment was repeated using the Nip3 163 . The mutant was unable to induce apoptosis (Figure 4A,B) . Of additional interest, Nip3 163 protein levels decreased very rapidly with time despite the inability of the mutant to induce apoptosis, thus indicating that the loss of protein in the cell lysates was not released to the death of cells . Nip3 sensitizes Rat-1 cells to apoptosis To determine whether Nip3 can influence apoptosis induced by other cell death signals, Rat-1 cells were transiently transfected with T7-Nip3 for 12 hours then treated with increasing doses of the cytotoxic lymphocyte protease granzyme B, or the topoisomerase I and II inhibitors etoposide or camptothecin. The 12 hour transfection was chosen because of minimal effects of Nip3 on cell survival at this time. Apoptotic cells were then quantitated by Hoechst dye in cells either expressing or not expressing T7-Nip3 protein within the same samples, using immunofluorescence staining by anti-T7 antibody to detect Nip3. The frequency of apoptotic cells was clearly higher in Nip3 expressing cells (Figure 5) . A five- to ten-fold increase in sensitivity to the drugs and a two to three-fold increase in granzyme B apoptosis was observed in three independent experiments. Nip3 overcomes Bcl-2 suppression of apoptosis Since it had been demonstrated that Bcl-2 binds Nip3 (Boyd, et al . , 1994), it was then determined if Bcl-2 was capable of suppressing Nip3 - induced apoptosis. Rat-1 and Rat-l/Bcl-2 cells (Figure 6C) were transfected with T7-Nip3 and apoptotic cells appearing in both populations was determined over time. Apoptotic Rat-1 cells expressing Nip3 were detected within 12 hours and reached maximum levels by 48 hours, while Nip3 did not induce cell death in Rat-l/Bcl-2 cells until 24 hours after transfection. However, by 48 hours virtually all cells expressing Nip3 were apoptotic despite Bcl-2 overexpression (Figure 6A) . That Rat-1 cells expressing Bcl-2 were highly resistant to granzyme B and perforin (Figure 6A) . The Nip3 protein level was considerably higher in Rat-l/Bcl-2 cells than in Rat-1 cells by 12 hours after transfection and remained higher throughout the time course. However, a significant decrease in Nip3 level was detected by 48 to 60 hours in Rat-l/Bcl-2 cells at the same time that recovery of apoptosis was detected (Figure 6B) . DISCUSSION In the present study the ElB 19kDa/Bcl-2 -binding protein Nip3 was identified as a pro-apoptotic protein that localizes to mitochondria. Nip3 protein is unusual in several respects. It migrates on SDS-PAGE at a molecular weight that is much higher than the 21.54 kD predicted by the primary amino acid sequence and appears in vivo following transfection and in vi tro after transcription and translation as 60 kD and 30 kD proteins. The 60 kD band does not dissociate after reduction or reduction and alkylation, nor after treatment with 6M urea (G. Chen and A.H. Greenberg, unpublished data) . Thus Nip3 appears to interact with a second protein very strongly and the evidence favors homodimerization . Three experiments support this conclusion. Firstly, truncation of the C- terminal 31 amino acids results in the loss of the 60 kD Nip3 band and only a lower molecular weight protein is observed. Secondly, 35 S-methionine-labelled tryptic peptide fragments of the 60 kDa Nip3 were identical to the 28 kDa Nip3 163 , and finally, Nip3 exhibits strong interaction with itself in the yeast two hybrid system. The inability to bind Nip3 163 to interact with Nip3 or itself indicates that it cannot dimerize and is consistent with the requirement of the C-terminal portion of Nip3 from homodimerization . The C-terminal domain of Nip3 not only influences dimer formation but also directs its expression to mitochondria. Nip3 colocalized with HSP60 which is primarily expressed in mitochondria (Soltys, et al . 1996) while the mutant Nip3 163 lacking the transmembrane domain was mostly expressed as a free cytosolic protein, a minor component of the Nip3 163 protein was colocalized with the mitochondrial markers suggesting that it may interact by a mechanism other than membrane insertion, perhaps by binding to other mitochondrial proteins. Nip3 163 does not interact with Nip3 or form a homodimer in vi tro so it likely does not homodimerize with endogenous Nip3 in vivo . Nip3 protein expression progressively decreases over time, which may reflect the increasing death of Nip3 expressing cells. Partial Bcl-2 suppression of Nip-3 induced apoptosis and a significant increase in Nip3 protein levels suggests that at least part of the Nip3 is lost by cell death. However, a progressive decrease in protein levels was also observed with the Nip3 163 mutant which does not induce apoptosis, therefore another mechanism is affecting protein turnover. Nip3 contains PEST sequences suggesting that the protein may be susceptible to rapid degradation by other proteases. PEST sequences commonly contain high local concentrations of amino acids P,E,S,T and D flanked by charged amino acids (Rogers, et al . , 1986) and these are abundantly present in Nip3. Thus the posttranslational control of Nip3 expression through rapid protein degradation may constitute a mechanism for regulating the intercellular levels of a potentially lethal protein. Nip3 was originally identified as an ElB 19K interacting protein, but the structure of Nip3 does not closely resemble other death agonists of the Bcl-2 family. Bcl-2 family agonists such as Bax act by heterodimerizing with anti-apoptotic family members and mutations in BH1 and BH2 domains in Bcl-2 or Bcl-X L result in loss of binding and anti-apoptotic activity (Yin, et al . , 1995; Cheng, et al . , 1996) . However, other studies suggest that pro- and anti- apoptotic Bcl-2 family members can act independently (Cheng, et al . , 1996) or through other family members such as BID (Wang, et al . , 1996) . Nip3 does not contain either a BH1 or BH2 domain, although it may have a BH3 domain in which leucine 110 , aspartic acid 115 and isoleucine 117 are conserved based on the critical amino acids of the BH3 domain of BAK that determines BAK-Bcl-X L heterodimerization (Sattler, et al . , 1997). Other BH3 domain-only Bcl-2 family members that are death agonists have been described such as Bik-Nbk (Boyd, et al . , 1995; Han, et al . , 1996), Bid (Wang, et al . , 1996) and, more recently, Hrk (Inohara, et al . , 1997) . Bik and Hrk have putative transmembrane domains while Bid is a cytoplasmic factor that is hypothesized to be recruited to membrane associated Bcl-2 or Bax. Thus it is possible that Nip3 is a death factor of the Bcl-2 family related to Bik/Nbk, Bid or Hrk. Many Bcl-2 family members are integral membrane proteins that bear carboxy terminal regions that allow localization to the mitochondrial outer membrane, nuclear envelope and endoplasmic reticulum (Zhu, et al . , 1996; Hockenbery, et al . , 1990; Akao, et al . , 1994). Localization of Bcl-2 to mitochondria also appears to be important in its suppression of cell death as mutants lacking the transmembrane domain are ineffective when overexpressed (Tanaka, et al . , 1993; Nguyen, et al . , 1994). Nip3 share the ability to localize to mitochondria and recent work suggests that mitochondria may play an important role in the regulation of apoptosis. They are necessary for the apoptotic activity of cytosolic extracts of Xenopus laevis oocytes (Newmeyer, et al . , 1994), and can initiate nuclear destruction when taken from cells undergoing apoptosis in in vi tro reconstituted cell systems (Zamzami, et al . , 1996). Nip3 localization in mitochondria places it in a position to influence mitochondrial function early in the apoptotic response. Nip3 can overcome Bcl-2 suppression of apoptosis so it might act on Bcl-2 or other anti-apoptotic family members that regulate mitochondrial permeability transition pores (Zamzami, et al . , 1996; Marchetti, et al . , 1996) or cytochrome c release (Liu, et al., 1996; Yang, et al . , 1997; Kluck, et al . , 1997). Nip3 preferentially forms homodimers even under reducing conditions and could provide a stable link between Bcl-2 homo- and/or heterodimers and other proteins perhaps forming larger complexes or altering their interaction with regulators such as BAD (Zha, et al . , 1996). In conclusion, the ElB 19k/Bcl-2 binding protein Nip3 is identified as a homodimer localized to mitochondrial membrane. Nip3 expression in mitochondria induces apoptosis and can overcome Bcl-2 suppression of cell death, indicating that Nip3 is a novel pro-apoptotic protein. EXAMPLE 2 MATERIALS AND METHODS Cloning of murine Nix (mNix) , human Nix (hNix) and murine Nip3 (mNip3) . A TBLASTN search of the GeneBank EST database and search of the TIGR database using the human Nip3 cDNA revealed several human and mouse EST clones with limited similarity. Using primers within these EST sequences, PCR fragments were generated using the proof-reading Pfu DNA polymerase (Stratagene) and Taq DNA polymerase (Gibco-BRL) in 1:1 ratio. These fragments were cloned into pCRII vector (Invitrogen, San Diego, CA) and sequenced on an automated DNA sequencer (ABI 310 PRISM, Perkin-Elmer, Foster, CA) confirming that Nix is a new Nip3 homolog. Using primers designed from the EST sequences and primers to the cDNA library vectors, mouse 17 day embryo matchmaker cDNA library was PCRed and human fetal liver 5 '-stretch plus cDNA library (Clontech, Palo Alto, CA) to generate clones of the entire mNix and hNix respectively. Murine Nip3 cDNA was cloned from a λGTll 3T3 fibroblast cDNA library using human Nip3 as a probe. The clones with the largest inserts were sequenced. Expression constructs. Using the full length mNix clone a Kpnl and EcoRI site was introduced by PCR cloning with the 5' primer: 5'- CGGGTACCATGTCTCACTTAGTCGAG and 3 ' primer : 5' -CGGAATTCGTAGGTGCTGGCAGAAGG respectively as described previously (Chen, et al . , 1997). To generate expression vector Nix-T7, this Kpnl -EcoRI fragment containing the mNix ORF was then subcloned into the Kpnl and EcoRI sites of expression vector pcDΝA3-T7, a modified pcDNA3 vector containing a C-terminal T7-tag (Chen, et al . , 1997). Transmembrane domain deleted expression vector mNix TM was generated by PCR using splice overlap extension (Horton, et al., 1993) with the 5' primer: 5'- CTCTGCAGAGTTCCTAAAAAAACGACTGAGCACACC and 3' primer: 5'- GGTGTGCTCAGTCGTTTTTTTAGGAACTCTGCAGAG . Reagents and Cell Lines. Rat-1, Rat-l/Bcl-2, TC (10T1/2 parental cell line), TX5 and TX22 (Bcl-X L transfected 10T1/2 clones) and MCF-7 cells were culture in -minimal essential medium (α- MEM) (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) . Murine monoclonal anti-T7 antibody was purchased from Novagen (Madison, WI) . Rabbit anti- HSP60 antibody was donated by Dr. Radhey Gupta (McMaster University, Hamilton, Ont . ) . FITC-conjugated goat anti- rabbit IgG (FITC) (Sigma Chemical Co.) and Cy3-conjugated goat anti-mouse IgG (BioCan Ltd. , Mississauga) were used for fluorescence studies . Lactacystin was purchased from Calbiochem-Novabiochem (Cambridge, MA) . Northern Analysis. Total RNA (30 μg) from human erythroleukemia (HEL) cells or from mouse kidney was fractionated on a 1% (w/v) agarose/2.2M formaldehyde denaturing gel, and then transferred onto a nitrocellulose membrane and cross-linked as described (Chen, et al . , 1997). Nick-translated human or mouse Nix probes (1-5 x 10 8 cpm/μg DNA) , corresponding to the coding region, were then hybridized to the respective blots at 42°C for 16 hrs and developed. In vitro transcription. 35 S- labeled Nix proteins were prepared by in vi tro transcription and translation of cDNA cloned in pcDNA3 vector using Tnt coupled Reticulocyte Lysate System (Promega, Madison, WI) according to manufacturer's instructions . Western blotting. Aliquots of 5 x 10 5 cells transfected with Nix-T7 were separated by an SDS PAGE method using Tris-Tricine buffer and PVDF membrane that is suitable for detecting small molecular weight peptides (Schagger, et al, . 1987) . Western blots were probed with mouse monoclonal anti-T7 antibody using an ECL detection kit according to manufacturers instructions (Amersham, Amersham, UK) . Transient transfection and apoptosis assays. Cells were transfected with Nix using LipofectAMINE Reagent (Gibco, Gaithersburg, MD) . Aliquots of 5 x 10 5 cells were Western blotted as described above. For apoptosis experiments, Nix expressing cells were detected with mouse anti-T7 antibody and FITC-conjugated rabbit anti-mouse IgG antibody. Apoptotic cells, exhibiting altered DNA morphology following Hoechst dye staining, were enumerated manually counting at least 200 cells per sample in five or more fields as described previously (Chen, et al . , 1997). Independent experiments were then combined and expressed as the mean and SEM. Fluorescence was visualized using a Zeiss Axiophot microscope equipped with a cooled CCD camera (Chen, et al . , 1997). RESULTS AND DISCUSSION Cloning of hNix, mNix, and mNip3- Using the human Nip3 cDNA as a probe, a λGTll mouse fibroblast cDNA library was screened for clones related to Nip3. The clone containing the longest insert of 1311 bp was sequenced and found to contain the entire mouse Nip3 coding region of 187 amino acids (Genbank Accession # AF041054) . The cDNA had overall identity with human Nip3 of 90.2% (Figure IA) . Human and murine ESTs related to but different from Nip3 were identified by searching the Genebank and TIGR databases. Using PCR cloning, as described in Methods, a human 1337 bp and murine 1351 bp cDNA was obtained containing a large ORF of 219 and 218 amino acids, respectively. The protein was named Nix. The predicted human Nix (hNix) (Genebank accession # AF067396) and mouse Nix (mNix) (Genebank accession #AF067395) proteins share 56% and 53% identity with Nip3 of the same species, and are 97% identical to each other (Figure IA) . The highest regions of homology between the four proteins resides in the C- terminus. Nix proteins bear a single hydrophobic alpha helical sequences at the extreme carboxy terminus corresponding to the location of the TM domain of Nip3 (2) (Figure 7A) . Nix has a significantly longer N-terminus that contains an unusual five asparagines in sequence and is proline rich. N-terminal amino acids 1-59 of hNix and 1-32 of hNip3 are the most divergent. Another region from amino acids 97-120 of hNix is also distinct from hNip3 , while the remainder is over 85% homologous with large blocks of identical sequences in both species. Similar to Nip3 (Chen, et al . , 1997; Boyd, et al . , 1994), Nix contains PEST sequences comprised of high frequency stretches of Pro, Glu, Ser, Thr and Asp, flanked by charged amino acids His, Arg or Lys. PEST sequences are associated with proteins having high turnover rates rates (Rogers, et al . , 1986) whose degradation is controlled by the proteasome . hNix and mNix are expressed as 4.5 and 1.4 kb mRNA transcripts, respectively (Figure 7B) . The smaller transcript corresponds to the size of the cDNAs reported above and are likely the complete sequence. The larger transcript has not been identified, however, on sequencing other mNix cDNAs recovered from PCR products, some clones were identified that bear a distinct 5' UTR suggesting alternate splicing of the Nix mRNA. These may represent a larger transcript with an extended 5' UTR. Following in vi tro transcription and translation and Western blotting (Figure 8A) , mNix and hNix run as proteins of 48 kD relative molecular weight under reducing and non reducing conditions using the Tris-Tricine SDS PAGE method (Schagger, et al . , 1987). hNip3 has a 40 kDa relative molecular weight in Tris-Tricine buffered SDS PAGE (Figure 8A) . In contrast, using the Laemmli method, Nix runs as a 70 kDa protein and Nip3 at 60 kDa (Chen, et al . , 1997). The reason for the difference in mobility in the two buffers is not clear, however Nip3 , which is a homodimer that does not dissociate under reducing conditions (Chen, et al . , 1997), runs at its predicted molecular weight of 40 kDa in the Tris-Tricine SDS PAGE. Nix is also unaffected by reduction and is about double its 23.8 kDa predicted mass (Figure 8A) . This suggest that Nix may also be a homodimer that resists reduction. Western blotting of lysates from Rat-1 cells transiently transfected with expression vector Nix-T7 reveals a 48 kDa protein that is progressively degraded over 42 hours (Figure 8B) . Both N- and C-terminal T7- tagged Nix were examined and found that a residual 11 kDa fragment was detected only with the C-terminal tag (Figure 8B) . Nix with an N-terminal tag showed a similar degradation pattern of the 48 kDa band but no smaller fragments were detected. Intermediate fragments were also visible on some transfections at earlier time points or after prolonged development of the blots but only with the C-terminal tag. A similar pattern of degradation was found following transfection with Nip3 in which an 11 kDa fragment was detected using a C- but not an N-terminal tag. The 11 kDa residual Nip3 fragment was only detectable using the Tris Tricine Buffered SDS PAGE, which is optimized for the detection of small peptides. The proteolytic degradation is dependent on a active proteasome. Nix remains as a 48 kD protein when cells were first treated with lactacystin, the proteasome threonine protease inhibitor (Figure 8B) . Nip3 is also degraded rapidly following transfection (Chen, et al . , 1997) and as with Nix, Nip3 degradation is also inhibited in lactacystin treated cells. To determine if Nix is localized to mitochondria, the primary site of Nip3 expression (Chen, et al . , 1997; Yasuda, et al . , 1998), mNix-T7 was transiently transfected into Rat-1 cells and stained with both monoclonal anti-T7 and rabbit anti-HSP60 antibodies. Secondary antibodies FITC-labelled goat anti-mouse IgG and Cy3-labelled goat anti-rabbit IgG were then used to visualize the localization of Nix and HSP60 respectively within the cells after immuno-fluorescence and confocal microscopy. HSP60 is primarily located in mitochondria (Soltys, et al . , 1996) and our results showed that Nix colocalized with HSP60 (Figure 9A) . Since Nip3 requires the TM domain to localize to mitochondria, a mutant Nix lacking the C-terminus was prepared including the putative TM domain (Nix ΔTM) . Nix ΔTM is expressed evenly throughout the cytoplasm and is not localized to any organelle-like structure (Figure 9B) . Unlike Nip3 ΔTM (Chen, et al . , 1997), the Nix mutant was also detected in the nucleus. Because Nip3 induces apoptosis when transiently transfected, the effects of Nix expression in Rat-1 and MCF-7 cells were examined over time. Nix expressing cells undergo apoptosis (Figure 9C) . This was quantitated over a 48 hour period and a progressive increase in the percent of cells that were apoptotic was observed to progressively increase (Figure 10A) and at about the same rate as Nip3 (Figure 10B) . The ΔTM mutant of Nix was examined and found that despite its inability to localize to mitochondria, it was as efficient as intact Nix at inducing apoptosis in Rat-1 cells (Figure IOC) . A similar killing pattern of Nix ΔTM was observed in MCF-7 cells. This killing by Nix ΔTM was unexpected as the apoptotic function of Nip3 is totally ablated by removing its TM domain (Figure 10D) . Thus, Nix has a different mechanism of activating apoptosis than Nip3 and does not require mitochondrial localization to function. Nip3- induced apoptosis is only partially suppressed in Bcl-2 expressing cell lines (Chen, et al . , 1997; Yasuda, et al . , 1998) . Inhibition is detected early in the time course but is overcome at later time points (Chen, et al . , 1997) . Examining the apoptosis rate over a 48 hour period following mNix transfection, there was no significant difference between between Rat-1 and Rat-l/Bcl-2 cell lines (Figure 11A) , that latter being highly resistant to other apoptotic signals such as granzyme B (Chen, et al . , 1997) . At decreasing doses of Nix plasmid from 1.0 to 0.1 μg the transfection efficiency decreased progressively but the apoptosis rate in transfected cells remained constant between the two cells lines. Examining Nix apoptosis in Bcl-X L expressing cell lines TX22 and TX5 , only the higher Bcl-X L expressing cell line TX5 was effective at blocking Nix. In conclusion, Nix, a homolog of the Nip3 protein, shares the ability to induce apoptosis, localized to mitochondria and undergo proteasome-dependent degradation when overexpressed. Unlike Nip3 however, Nix can induce apoptosis as a free cytoplasmic protein when lacking a transmembrane domain. Nip3 and Nix form a new family of mitochondrial cell death proteins. Throughout this application, various publications, including United States patents, are referenced by citation or number. Full citations for the publications are listed below. The disclosures of these publications and patents in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation. Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described. Table 1 : Interaction of Nip3 in Yeast Two Hybrid System Relative level of lacZ expression . 2 Protein X-Gal ONPG pAS1 W 0.7 Nip3 W 1.0 Nip3:Nip3 B 103.3 MK:Nip3 W 0.3 PTP2:Nip3 W 0.5 1. Relative level of interaction is based on β-galactosidase activity. W, white; B, blue 2. Binding domain:Activating domain; pAS1 , empty vector; MK, Myotonin Kinase; PTP2, protein tyrosine phosphatase 2 REFERENCES 1. Akao, Y., Y.S. Otsuki, S. Kataoka, Y. Ito, and Y. Tsujimoto. 1994. 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Claims: CLAIMS What is claimed is : I. Isolated and purified Nip, Nix and Nox proteins. 2. The proteins according to Claim 1, wherein said proteins induce apoptosis in cells. 3. The proteins according to Claim 1, wherein said proteins induce apoptosis in cancer cells. 4. The proteins according to Claim 1, wherein said proteins having a sequence selected from the group consisting essentially of Seq ID No. 1 through 6. 5. A method of inducing apoptosis in cells by transfecting a cell with a Nip, Nix and Nox proteins. 6. The method according to Claim 5, wherein said transfecting step is further defined as transfecting into a cancer cell. 7. The method according to Claim 5, wherein said transfecting step further includes transfecting using a vector. 8. The method according to Claim 4, wherein said transfecting step further includes transfecting using raw DNA. 9. A method of inducing apoptosis in cells by creating antibodies which recognize a specified Nip, Nix and Nox proteins in the muscle or skin of a patient. 10. The method according to Claim 9, wherein said creating step being for creating antibodies for inducing apoptosis in cancer cells. II. A method of inducing apoptosis in cells by creating transgenes overexpressing the genes for the Nip, Nix and Nox proteins. 12. The method according to Claim 11, wherein said creating step further creates transgenes for inducing apoptosis in cancer cells. 13. A transgenic animal and progeny thereof having an expression vector consisting essentially of a Nip, Nix and Nox proteins selected from the group consisting essentially of Seq. ID No : 1 through 6. 14. Isolated and purified antibodies which recognize specified Nip, Nix and Nox proteins in the muscle or skin of a patient . 15. The antibodies according to Claim 14, wherein said antibodies induce apoptosis in cancer cells.

Inventors: GREENBERG ARNOLD H, GREENBERG, ARNOLD, H.
Applicants: GREENBERG ARNOLD H, MANITOBA CANCER TREATMENT & RE, UNIV MANITOBA, GREENBERG, ARNOLD, H., MANITOBA CANCER TREATMENT & RESEARCH FOUNDATION, UNIVERSITY OF MANITOBA
Application year: 1998
Main classification: C12N15
IPC codes: A61K48, C07K14, C12N15