WO2001055410A2 - Ceramidase compositions and methods based thereon - Google Patents
Ceramidase compositions and methods based thereon Download PDFInfo
- Publication number
- WO2001055410A2 WO2001055410A2 PCT/US2001/002866 US0102866W WO0155410A2 WO 2001055410 A2 WO2001055410 A2 WO 2001055410A2 US 0102866 W US0102866 W US 0102866W WO 0155410 A2 WO0155410 A2 WO 0155410A2
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- WO
- WIPO (PCT)
- Prior art keywords
- ceramidase
- nucleic acid
- protein
- nucleotide sequence
- gene
- Prior art date
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- G01N2333/98—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
Definitions
- the present invention relates to ceramidase genes, in particular to human mitochondrial ceramidase genes, and their encoded protein products, as well as derivatives and analogs thereof.
- the invention further relates to compositions and methods of diagnosis and therapy for diseases associated with cell overproliferation and sphingolipid signal transduction.
- the invention relates to the regulation of the level of ceramide by inhibiting ceramidase expression or activity.
- the action of a number of extracellular agents as well as stress stimuli such as l ⁇ ,25-dihydroxyvitamin D 3 , tumor necrosis factor , interleukin- l ⁇ , neurotrophins, the Fas ligand, dexamethasone.
- serum withdrawal, chemotherapeutic agents, and ⁇ . -irradiation can cause an elevation in the endogenous levels of ceramide (Hannun, J. Biol. Chem. 269:3125 (1994), Hannun and Obeid, Trends Biochem. Sci. 20:73 (1995), Ballou et al, J. Biol. Chem. 267:20044 (1992), Quintans et al, Biochem. Biophys. Res. Commun.
- ceramide occupies a central position in sphingolipid metabolism.
- Complex sphingolipids can be derived from ceramide through various enzymatic reactions that add various head groups to the 1-hydroxyl position (Hannun, J. Biol. Chem. 269:3125 (1994), Wiegandt in Glycolipids (Weigandt. ed) pp. 199-259, Elsevier, New York (1985), Merrill, Jr. and Jones, Biochim. Biophys.
- Ceramidases hydro lyze ceramide to form sphingosine, which in turn can serve as a substrate for sphingosine kinase, resulting in the formation of sphingosinel -phosphate.
- sphingolipids Hanun et al, 2000, Trends Cell. Biol. 10: 73-80.
- CDases Ceramidases
- SPH sphingosine
- SPP downstream metabolite sphingosine 1 -phosphate
- CDases are the key enzymes to regulate levels of SPH.
- Two reports implicate an alkaline CDase activity in signal transduction. Using cell homogenate of rat glomerular mesangial cells, Coroneos et al. have shown that an alkaline CDase activity was stimulated by the platelet-derived growth factor and not by the inflammatory cytokines (tumor necrosis factor oc and interleukin-1) or the vasoconstrictor peptide endothelin-1 (Coroneos et al, 1995, J. Biol. Chem. 270: 23305-23309.) In another report, Nikolova-Karakashian et al.
- This enzyme is located in the lysosomes, and it plays a role in the catabolic pathway of ceramide, and the inherited deficiency of this enzyme causes Farber disease (Sugita et al., 1972, Science 178: 1100-1102.)
- Farber disease Sugita et al., 1972, Science 178: 1100-1102.
- a neutral activity has been described in liver plasma membranes and in rat intestinal brush border membranes; little is known about this enzyme (Slife et al, 1989, J. Biol. Chem. 264: 10371-10377; Nilsson et al., 1969, Biochim. Biophys.
- Chem. 272 18718-18724.
- both tumor necrosis factor and nitric oxide donors have been shown to stimulate sphingomyelinases, but only nitric oxide donors inhibited ceramidases and resulted in an increase in ceramide levels and the consequent biological effects (Huwiler et al., 1999, J. Biol. Chem.
- the present invention is based upon the identification by the present inventors of a novel human mitochondrial ceramidase (Genbank accession no. NM019893).
- the human mitochondrial ceramidase hydrolyses ceramide to form sphingosine.
- Inhibition of ceramidase activity by a ceramide inhibitor, Urea-C16-ceramide decrease survival and viability in a breast cancer cell line.
- preliminary data show that there is a higher expression of the human mitochondrial ceramidase gene in lung cancer tissues as compared to normal tissues.
- a decreased level of the ceramidase inhibits cell proliferation, viability and survival.
- the ceramidase gene product is involved in the mechanisms underlying the onset and development of diseases associated with cell overproliferation such as cancer and inflammation.
- the present invention relates to nucleotide sequences of ceramidase genes, including, human ceramidase genes and homologs of other species, and amino acid sequences of their encoded proteins. Nucleic acids hybridizable to or complementary to the foregoing nucleotide sequences are also provided.
- the nucleotide sequences can be genomic DNA, cDNA or RNA. In the case of genomic DNA, the nucleotide sequence does not consist of the nucleotide sequence of Genbank sequence accesion no. AC 012131.
- an isolated nucleic acid molecule comprising: (a) the nucleotide sequence of SEQ ID NO: 1; (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2; (c) a ceramidase gene contained in plasmid Mito- CDase-TOPO/BII as deposited with the ATCC on January 25; or (d) the complement of the nucleotide sequence of (a), (b) or (c).
- the invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence that hybridizes to a nucleic acid probe consisting of: (a) the nucleotide sequence of nucleotide position 1 to 1702 or nucleotide position 2289 to 2583 of SEQ ID NO: 1 ; (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of amino acid position 1 to 554 or amino acid position 750-761 of SEQ ID NO: 2;
- the invention provides an isolated nucleic acid comprising a nucleotide sequence that consists of at least 8 consecutive nucleotides of: (a) the nucleotide sequence of nucleotide position 1 to 1702 or nucleotide position 2289 to 2583 of SEQ ID NO: 1; (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of amino acid position 1 to 554 or amino acid position 750 to 761 of SEQ ID NO: 2; (c) a ceramidase gene contained in plasmid Mito-CDase-TOPO/BII as deposited with the ATCC; or (d) the complement of the nucleotide sequence of (a), (b) or (c).
- the invention also provides a nucleic acid probe consisting of at least 8 nucleotides, wherein the nucleic acid probe is hybridizable to at least a portion of: (a) the nucleotide sequence of nucleotide position 1 to 1702 or nucleotide position 2289 to 2583 of SEQ ID NO: 1 ; (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of amino acid position 1 to 554 or 750 to 761 of SEQ ID NO: 2; (c) a ceramidase gene contained in plasmid Mito-CDase-TOPO/BII as deposited with the ATCC; or (d) the complement of the nucleotide sequence of (a), (b) or (c); (under moderately stringent conditions).
- the invention also provides a nucleic acid comprising a nucleotide sequence encoding a fragment of a ceramidase protein that has an amino acid sequence as set forth in
- the invention also provides a nucleic acid molecule comprising the nucleotide sequence of a deletion mutant of : (a) the nucleotide sequence of nucleotide position 1 to nucleotide position 1702 or nucleotide position 2289 to nucleotide position 2583 of SEQ ID NO: 1; (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of amino acid position 1 to amino acid position 554 or amino acid position 750 to amino acid position 761 of SEQ ID NO: 2; or (c) the complement of the nucleotide sequence of (a), or (b); wherein said fragment displays one or more functional activities of ceramidase protein.
- the invention also provides a nucleic acid molecule comprising the nucleotide sequence of a deletion mutant of : (a) the nucleotide sequence of SEQ ID NO: 1 ;
- nucleotide sequence of (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2; or (c) the complement of the nucleotide sequence of (a), or (b); wherein said nucleotide sequence of said deletion mutant comprises more than 791 nucleotides.
- the ceramidase protein is a human protein.
- the invention also relates to fragments (derivatives and analogs thereof) of ceramidase, which comprise one or more domains of a ceramidase protein.
- fragments (derivatives and analogs thereof) of ceramidase which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a full-length (wild-type) ceramidase protein.
- Such functional activities include but are not limited to ceramidase activity, antigenicity
- the invention also provides an isolated polypeptide, the amino acid sequence of which comprises at least one region selected from the group consisting of residues 1 to
- the invention also provides an isolated polypeptide which is at least 60% identical to the ceramidase polypeptide having the amino acid sequence of SEQ ID NO:2, and displays one or more functional activities of ceramidase protein.
- the invention also provides a chimeric protein comprising a fragment of a ceramidase protein consisting of at least 6 amino acids fused via a covalent bond to an amino acid sequence of a second polypeptide.
- the second polypeptide is a signal peptide which facilitate the secretion of the chimeric protein out of the cell.
- Such chimeric protein is particularly useful in the mass production of the chimeric protein since the protein can be harvested out of the cell, such as in a culture media.
- Antibodies, antisense nucleic acid, and ribozyme to ceramidase, and ceramidase derivatives and analogs are additionally provided. Methods of production of the ceramidase proteins, derivatives and analogs, e.g., by recombinant means, are also provided.
- the present invention also encompasses (a) DNA vectors that contain any of the foregoing ceramidase gene, antisense ceramidase gene, and modified ceramidase gene sequences encoding mutant and fusion ceramidase proteins; (b) DNA expression vectors that contain any of the foregoing ceramidase gene, antisense ceramidase gene, and modified ceramidase gene sequences encoding mutant and fusion ceramidase proteins operatively associated with a regulatory element that directs the transcription and/or expression of the foregoing ceramidase gene, antisense ceramidase gene, and modified ceramidase gene sequences encoding mutant and fusion ceramidase proteins; and (c) genetically engineered host cells that contain any of the foregoing DNA vectors or DNA expression vectors.
- compositions which include but are not limited to ceramidase proteins and analogs and derivatives (including fragments) thereof; antibodies, antisense nucleic acids, and ribozymes thereto; nucleic acids encoding the ceramdiase proteins, analogs, or derivatives.
- compositions of the present invention additionally include cloning vectors, including expression vectors, containing the nucleic acid molecules of the invention, and hosts which contain such nucleic acid molecules.
- the invention also provides methods for the diagnosis of hyperproliferative diseases, cardiovascular diseases and inflammation, using antibodies, antisense nucleic acids, and ribozymes thereto; nucleic acids encoding the ceramdiase proteins, analogs, or derivatives.
- the invention provides methods for the prevention and/or treatment of hyperproliferative diseases, cardiovascular diseases and inflammation, using antibodies, antisense nucleic acids, and ribozymes thereto; nucleic acids encoding the ceramdiase proteins, analogs, or derivatives, to modulate the expression of ceramidase gene and/or the activity of ceramidase gene product.
- the invention provides for treatment of disorders of overproliferation (e.g., cancer and hyperproliferative disorders) by administering compounds that inhibit ceramidase activity (e.g., antibodies, antisense nucleic acids, and ribozymes thereto; nucleic acids encoding the ceramdiase proteins, analogs or derivatives; antagonist of ceramidase, particularly those that are active in decreasing cell survival and viability (e.g., as demonstrated in in vitro assays or in breast cancer cell line assays, or can be identified using in vitro assays, animal models, or cell culture assays).
- compounds that inhibit ceramidase activity e.g., antibodies, antisense nucleic acids, and ribozymes thereto; nucleic acids encoding the ceramdiase proteins, analogs or derivatives; antagonist of ceramidase, particularly those that are active in decreasing cell survival and viability (e.g., as demonstrated in in vitro assays or in breast cancer cell line
- the invention also provides methods of treatment of disorders involving deficient cell proliferation or growth, or in which cell proliferation is otherwise desired (e.g., degenerative disorders, growth deficiencies, lesions, physical trauma) by administering compounds that promote ceramidase function (e.g., ceramidase, activator of ceramidase, nucleic acids that encode ceramidase).
- ceramidase function e.g., ceramidase, activator of ceramidase, nucleic acids that encode ceramidase.
- Activating ceramide function can also be done to grow larger animals and plants, e.g., those used as food or material sources.
- the invention also provides a method of identifying a molecule that specifically binds to a ligand selected from the group consisting of a ceramidase protein, a fragment of a ceramidase protein comprising a domain of the protein, and a nucleic acid encoding the protein or fragment, comprising: (a) contacting said ligand with a plurality of molecules under conditions conducive to binding between said ligand and the molecules; and (b) identifying a molecule within said plurality that specifically binds to said ligand.
- a ligand selected from the group consisting of a ceramidase protein, a fragment of a ceramidase protein comprising a domain of the protein, and a nucleic acid encoding the protein or fragment
- the invention also provides a method for identifying compounds that modulate ceramidase gene expression, comprising: (a) contacting a test compound with a cell or cell lysate containing a reporter gene operatively associated with a ceramidase gene regulatory element; and (b) detecting expression of the reporter gene product.
- the invention also provides a method for identifying compounds that modulate ceramidase gene expression comprising: (a) contacting a test compound with a cell or cell lysate containing an expressible ceramidase gene construct; and (b) detecting the transcription and/or translation of the ceramidase gene.
- the invention also provides a method for identifying compounds that modulate the activity of ceramidase gene product or homolog of ceramidase gene product comprising: (a) contacting a test compound with an organism or a cell containing ceramidase gene product or homolog of ceramdiase; and (b) comparing the phenotype of the organism or cell with the phenotype of organism or cell that did not contact the test compound, wherein a change in phenotype indicates that the test compound is capable of modulating the activity of ceramidase gene product or homolog of ceramdiase gene product.
- Figure 1 Illustration of the pathways in sphingolipid metabolism.
- Figure 2. The role of ceramide in cell-cycle arrest and apoptosis induced by tumor necrosis factor (TNF ).
- Figure 3. cDNA and deduced amino acid sequences of human ceramidase.
- the first and second columns indicate the nucleotide (i.e., SEQ ID NO.T) and the deduced amino acid sequences (i.e., SEQ ID NO:2), respectively. Nucleotide and amino acid positions are shown on the right.
- Position amino acid 1 refers to the first nucleotide and amino acid of the ceramidase-predicted coding region.
- Amino acid sequences determined by Edman sequencing of the purified rat brain enzyme are underlined. The putative signal peptide
- Figure 4 Sequence comparison of human ceramidase to putative ceramidase from .4. thaliana, M. tuberculosis, and D. discoideum. Identical amino acids in all four proteins are shaded. Boxed areas indicate gaps introduced to optimize the alignment. Alignment was performed using the Mac Vector, Multiple sequence Alignment program.
- Figure 5. Northern blot analysis of poly(AV " RNAs from human tissues. The labeled 3' -end of human ceramidase was used to probe a human multiple tissue Northern blot; each lane contained 2 ⁇ g of poly(A) + RNA. Size markers are indicated on the right. The major ceramidase band corresponds to a size of 3.5kb.
- Figure 6A HEK293 and MCF7 cells were transfected with vector alone (pcDNA3.1/HisC) or vector containing ceramidase cDNA (pcDNA3.1/HisC-CDase). 48 hours after transfection, ceramidase activity was measured as described in material and methods. Data are the mean of three experiments.
- Figure 6B Cells transfected with empty vector (pEGFPC3, control) or vector containing ceramidase (pEGFPC3 -CDase, overexpression) were lysed, and ceramidase activity was measured on cell lysates.
- Figure 8A Cells were stained with Mitotracker Red and visualized by fluorescence microscopy.
- Figure 8B Cells were stained with TMRM and visualized by confocal microscopy.
- FIG 9A Effect of Urea-C 16 -Ceramide on ceramidase activity using rat brain purified enzyme.
- Ceramidase was purified from rat brain as described in Material and Methods. The effect of Urea-C 16 -ceramide was tested for inhibition using the purifed enzyme (5-10ng of protein in the assay) and [ 3 H]-C 16 -ceramide as substrate in a mixed micelles system as described in Material and Methods.
- Figure 9B Effect of Urea-C 16 -Ceramide on MCF7 cell viability (as measured by MTT assay).
- Cells were seeded in 100mm dishes. The next day, cells were treated with Urea- C 16 -ceramide at the indicated concentration for 18 hours.
- Urea-C 16 -ceramide was delivered in a solution of ethanol containing 2% dodecane. After 18 hours, cell viability was assayed using the MTT assay.
- the present invention relates to nucleotide sequences of ceramidase genes, and amino acid sequences of their encoded proteins.
- the invention further relates to fragments and other derivatives, and analogs, of ceramidase proteins. Nucleic acids encoding such fragments or derivatives are also within the scope of the invention.
- the invention encompasses genes encoding human, and rat ceramidase and related genes
- the inventors of the present invention have recently purified a rat brain membrane-bound nonlysosomal ceramidase (El Bawab et al. 1999, J. Biol. Chem. 274, 27948-27955). Using peptide sequences obtained from the purified rat brain enzyme, the inventors successfully cloned the human isoform.
- the invention provides the nucleotide sequences of ceramidase gene which comprise the cDNA sequences of SEQ ID NO: l ( Figure 3), or nucleotide sequences encoding a ceramidase protein (e.g., a protein having the amino acid sequence of SEQ ID NO:2) ( Figure 3).
- the human ceramidase protein (or ceramidase gene product) as depicted in Figure 3 comprises 763 amino acids and has a molecular weight of 84 KDa. Northern blot analysis of multiple human tissues showed the presence of a major band corresponding to a size of 3.5 kilobase.
- ceramidase protein is ubiquitously expressed but with higher levels in kidney, skeletal muscle, and heart.
- the deduced amino acid sequence of the protein did not show any similarity with proteins of known function but was homologous to three putative proteins from Arabidospis thaliana, Mycobacterium tuberculosis, and Dictyostelium discoideum.
- the ceramidase genes of Arabidospis thaliana accession no. AAD32770), Mycobacterium tuberculosis (accession No. CAB09388), Dictyostelium discoideum
- the ceramidase genes and proteins are from vertebrates, or more particularly, mammals.
- the ceramidase gene and protein are of human origin.
- the ceramidase gene and protein are of rat origin. Production of the foregoing proteins and derivatives, e.g., by recombinant methods, is provided.
- ceramidase gene refers to (a) a nucleic acid molecule comprising the DNA sequence of SEQ ID No. 1 as shown in Figure 3; (b) any nucleic acid molecule consisting essentially of a DNA sequence that encodes the amino acid sequence of SEQ ID No. 2 as shown in Figure 3, or (c) a nucleic acid molecule that hybridizes to another nucleic acid consisting of the DNA sequences that encode the amino acid sequence of SEQ ID No. 2 as shown in Figure 3 or the complement thereof, under medium stringent conditions and encodes a naturally occurring ceramidase protein.
- the ceramidase gene can be cDNA, genomic DNA, or RNA.
- ceramidase gene also includes naturally occurring variants including allelic variants of ceramidase, and degenerate variants of DNA sequences of (a) through (c) as described above.
- a ceramidase gene sequence preferably exhibits at least about 60-80% overall similarity at the nucleotide level to the nucleic acid sequence of SEQ ID NO:l, more preferably exhibits at least about 85-90% overall similarity to the nucleic acid sequence of SEQ ID NO: 1 and most preferably exhibits at least about 95% overall similarity to the nucleic acid sequence of SEQ ID NO:l.
- Ceramidase genes that are less than 60%) overall similar at the nucleotide level to the nucleic acid sequence of SEQ ID NO: 1 are least preferred.
- the degree of similarity can be determined by analyzing sequence data using a computer algorithm, such as those used by the BLAST computer program.
- the ceramidase gene may be a segment of the cDNA molecule, or a genomic DNA molecule that comprises one or more intervening sequences or introns. as well as regulating regions located beyond the 5' and 3' ends of the coding region or within an intron.
- Human EST sequence accession no. AA913512
- human genomic sequence acces no. 2367392
- Any eukaryotic cell potentially can serve as the nucleic acid source for the molecular cloning of the ceramidase gene.
- the nucleic acid sequences encoding ceramidase can be isolated from vertebrate, mammalian, human, porcine, bovine, feline, avian, equine, canine, as well as additional primate sources, insects, plants, etc.
- the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library”), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell.
- Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will contain only exon sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.
- DNA fragments are generated and cloned to form a genomic library. Since some of the sequences encoding related ceramidases are available and can be purified and labeled, the cloned DNA fragments in the genomic DNA library may be screened by nucleic acid hybridization to the labeled probe (Benton, W. and Davis, R., 1977, Science 196:180; Grunstein, M. And Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology to the probe will hybridize. It is also possible to identify the appropriate fragment by restriction enzyme digestion(s) and comparison of fragment sizes with those expected according to a known restriction map if such is available.
- RNA for cDNA cloning of the ceramidase gene can be isolated from cells which express the ceramidase.
- a cDNA library may be generated by methods known in the art and screened by methods, such as those disclosed for screening a genomic DNA library. If an antibody to the ceramidase is available, the ceramidase may be identified by binding of labeled antibody to the putatively ceramidase synthesizing clones.
- DNA fragments are generated, some of which will encode the desired gene.
- the DNA may be cleaved at specific sites using various restriction enzymes.
- DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication.
- the linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.
- identification of the specific DNA fragment containing the desired gene may be accomplished in a number of ways. For example, if an amount of a portion of a ceramidase (of any species) gene or its specific
- RNA or a fragment thereof (see Section 5.1)
- the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton, W. and Davis, R., 1977, Science 196:180; Grunstein, M. And Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology to the probe will hybridize. It is also possible to identify the appropriate fragment by restriction enzyme digestion(s) and comparison of fragment sizes with those expected according to a known restriction map if such is available. Further selection can be carried out on the basis of the properties of the gene.
- the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product.
- cDNA clones, or DNA clones which hybrid-select the proper mRNAs can be selected which produce a protein that, e.g., has similar or identical electrophoretic migration, isolectric focusing behavior, proteolytic digestion maps, kinase activity, inhibition of cell proliferation activity, substrate binding activity, or antigenic properties as known for ceramidase
- the ceramidase protein may be identified by binding of labeled antibody to the putatively ceramidase synthesizing clones, in an ELISA (enzyme-linked immunosorbent assay)-type procedure
- the ceramidase gene can also be identified by mRNA selection by nucleic acid hybridization followed by in vitro translation In this procedure, fragments are used to isolate complementary mRNAs by hybridization Such DNA fragments may represent available, purified ceramidase
- the one embodiment of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that hyb ⁇ dizes to a nucleic acid probe consisting of (a) the nucleotide sequence of nucleotide position 1 to nucleotide position 1702 or nucleotide position 2289 to nucleotide position 2583 of SEQ ID NO 1 , (b) a nucleotide sequence that encodes a polypeptide having the ammo acid sequence of ammo acid position 1 to 554 or ammo acid position 750 to 761 of SEQ ID NO 2, or (c) the complement of the nucleotide sequence of (a), or (b)
- RNA for cDNA cloning of the ceramidase gene can be isolated from cells which express ceramidase Other methods are possible and within the scope of the invention
- an expression library is constructed by methods known in the art For example, mRNA (e g , human) is isolated, cDNA is made and hgated into an expression vector (e g , a bacte ⁇ ophage de ⁇ vative) such that it is capable of being expressed by the host cell into which it is then introduced Va ⁇ ous screening assays can then be used to select for the expressed ceramidase product.
- an expression vector e g , a bacte ⁇ ophage de ⁇ vative
- Va ⁇ ous screening assays can then be used to select for the expressed ceramidase product.
- anti-ceramidase antibodies can be used for selection.
- PCR polymerase chain reaction
- Oligonucleotide primers representing known ceramidase sequences can be used as primers in PCR.
- the oligonucleotide primers represent at least part of the ceramidase conserved segments of strong homology between ceramidase of different species (e.g., transmembrane domains, putative transmembrane domains, signal peptide and low compositional complexity region.
- the synthetic oligonucleotides may be utilized as primers to amplify by PCR sequences from a source (RNA or DNA), preferably a cDNA library, of potential interest.
- PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp TM ).
- the DNA being amplified can include mRNA or cDNA or genomic DNA from any eukaryotic species.
- ceramidase homolog After successful amplification of a segment of a ceramidase homolog, that segment may be molecularly cloned and sequenced, and utilized as a probe to isolate a complete cDNA or genomic clone. This, in turn, will permit the determination of the gene's complete nucleotide sequence, the analysis of its expression, and the production of its protein product for functional analysis, as described infra. In this fashion, additional genes encoding ceramidase proteins and ceramidase analogs may be identified.
- the identified and isolated gene can then be inserted into an appropriate cloning vector.
- vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene).
- the insertion into a cloning vector can, for example, be accomplished by ligatmg the DNA fragment into a cloning vector which has complementary cohesive termini
- the ends of the DNA molecules may be enzymatically modified
- any site desired may be produced by ligatmg nucleotide sequences (linkers) onto the DNA termini, these hgated linkers may comp ⁇ se specific chemically synthesized oligonucleotides encoding rest ⁇ ction endonuclease recognition sequences
- the cleaved vector and ceramidase gene may be modified by homopolyme ⁇ c tailing Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc , so that many copies of the gene sequence are generated
- the desired gene may be identified and isolated after insertion into a suitable cloning vector m a "shot gun" approach En ⁇ chment for the desired gene, for example, by size fractionization, can be done before insertion into the cloning vector
- transformation of host cells with recombinant DNA molecules that incorporate the isolated ceramidase gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene
- the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, ret ⁇ evmg the inserted gene from the isolated recombinant DNA
- the invention provides nucleic acids consisting of at least 8 nucleotides (i e , a hyb ⁇ dizable portion) of a ceramidase gene sequence, in other embodiments, the nucleic acids consist of at least 25 (consecutive) nucleotides, 50 nucleotides, 100 nucleotides, 150 nucleotides, or 200 nucleotides of a ceramidase gene sequence, or a full-length ceramidase coding sequence In another embodiment, the nucleic acids are smaller than 35, 200 or 500 nucleotides in length Such nucleic acids can be single or double stranded The invention also relates to nucleic acids hyb ⁇ dizable to or complementary to the foregoing nucleotide sequences In specific aspects, nucleic acids are provided which comp ⁇ se a nucleotide sequence complementary to at least 8, 10, 25, 50, 100, or 200 nucleotides or the entire coding region of a ceramidas
- the isolated nucleic acid of the present invention compnses a nucleotide sequence that consists of at least 8 consecutive nucleotides of (a) the nucleotide sequence of nucleotide position 1 to 1702 or nucleotide position 2289 to nucleotide position 2583 of SEQ ID NO: 1 ; (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of amino acid position 1 to amino acid position 554 or amino acid position 750 to amino acid position 761 of SEQ ID NO: 2; or (c) the complement of the nucleotide sequence of (a), or (b).
- the present invention further provides a nucleic acid molecule, such as a probe or an oligonucleotide consisting of at least 8 nucleotides, wherein the nucleic acid molecule is hybridizable to at least a portion of: (a) the nucleotide sequence of nucleotide position 1 to 1702 or nucleotide position 2289 to nucleotide position 2583 of SEQ ID NO: 1 ; (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of amino acid position 1 to amino acid position 554 or amino acid position 750 to amino acid position 761 of SEQ ID NO: 2; or (c) the complement of the nucleotide sequence of (a) or (b).
- a nucleic acid molecule such as a probe or an oligonucleotide consisting of at least 8 nucleotides, wherein the nucleic acid molecule is hybridizable to at least a portion of: (a) the nucleo
- a nucleic acid which is hybridizable to a ceramidase gene nucleic acid, or to a nucleic acid encoding a ceramidase derivative, under conditions of low stringency is provided.
- procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci.
- Filters are incubated in hybridization mixture for 18-20 h at 40 °C, and then washed for 1.5 h at 55 °C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60° C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68 °C and reexposed to film. Other conditions of low stringency which may be used are well known in the art (e.g., as employed for cross-species hybridizations).
- a nucleic acid which is hybridizable to a ceramidase gene under conditions of high stringency is provided.
- procedures using such conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65 °C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02%) BSA, and 500 ⁇ g/ml denatured salmon sperm DNA.
- Filters are hybridized for 48 h at 65 °C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X IO 6 cpm of 2 P-labeled probe. Washing of filters is done at 37 °C for 1 h in a solution containing 2X SSC, 0.01% PVP. 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1X SSC at 50°C for 45 min before autoradiography. Other conditions of high stringency which may be used are well known in the art.
- the filters may be prehybridzed in 5ml of ExpressHyb (commercially available from Clonetech) solution at 68 °C overnight.
- the filters may be hybridized in 5-10 ml of the ExpresHyb solution at 68 °C overnight.
- the filters may be washed at room temperature and then followed by another wash at 65 °C for one hour.
- a nucleic acid which is hybridizable to a ceramidase gene under conditions of moderate stringency.
- procedures using such conditions of medium stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 55 °C, overnight in buffer 6X SSC, 5x Denhart's with 0.5%SDS and 100 ⁇ g/ml salmon sperm DNA. Filters are hybridized for 48 h at 55 °C in prehybridization mixture containing 100 ⁇ g/ml denatured salmon sperm DNA and 5-20 X 10 6 cpm of 32 P-labeled probe.
- the filters may be prehybridzed in 5ml of ExpressHyb (commercially available from Clonetech) solution at 60°C overnight.
- the filters may be hybridized in 5-10 ml of the ExpresHyb solution at 60 °C overnight.
- the filters may be washed at room temperature and then followed by another wash at 55 °C for one hour.
- nucleic acids encoding fragments, derivatives and analogs of ceramidase proteins, and ceramidase antisense nucleic acids are additionally provided.
- a "nucleic acid encoding a fragment or portion of a ceramidase protein” shall be construed as referring to a nucleic acid encoding only the recited fragment or portion of the ceramidase protein and not the other contiguous portions of the ceramidase protein as a continuous sequence.
- ceramidase nucleic acids comprising functional domains, and regions conserved between (with homology to) other ceramidase nucleic acids, of the same or different species.
- the ceramidase genes of Arabidospis thaliana (accession no. AAD32770), Mycobacterium tuberculosis (accession No. CAB09388), Dictyostelium discoideum (accession no. 2367392), and putative slug protein (accession no. 2367392) are not within the scope of the present invention.
- Nucleic acids encoding one or more ceramidase domains such as those described in Section 5.3 and in Table 1 are provided.
- the present invention further provides nucleic acid molecules encoding deletion mutants of the ceramidase gene.
- the invention provides a nucleic acid molecule comprising the nucleotide sequence of a deletion mutant of : (a) the nucleotide sequence of nucleotide position 1 to nucleotide position 1702 or nucleotide position 2289 to nucleotide position 2583 of SEQ ID NO: 1; (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of amino acid position 1 to amino acid position 554 or amino acid position 750 to amino acid position 761 of SEQ ID NO: 2; or (c) the complement of the nucleotide sequence of (a), or (b); wherein said fragment displays one or more functional activities of ceramidase protein.
- the present invention also provides a nucleic acid molecule comprising the nucleotide sequence of a deletion mutant of : (a) the nucleotide sequence of SEQ ID NO: 1; (b) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2; or (c) the complement of the nucleotide sequence of (a), or (b); wherein said nucleotide sequence of said deletion mutant comprises more than 791 nucleotides.
- the cloned DNA or cDNA corresponding to the ceramidase gene can be analyzed by methods including but not limited to Southern hybridization (Southern, E.M., 1975, J. Mol. Biol. 98:503-517), Northern hybridization (see e.g., Freeman et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:4094-4098), restriction endonuclease mapping (Maniatis, T., 1982, Molecular Cloning, A Laboratory, Cold Spring Harbor, New York), and DNA sequence analysis. Polymerase chain reaction (PCR; U.S. Patent Nos.
- Northern hybridization analysis can be used to determine the expression of the ceramidase gene as described in Section 6.1.
- Various organelles, cell types, at various states of development or activity can be tested for ceramidase expression.
- the stringency of the hybridization conditions for both Southern and Northern hybridization can be manipulated to ensure detection of nucleic acids with the desired degree of relatedness to the specific ceramidase probe used. Modifications of these methods and other methods commonly known in the art can be used.
- Restriction endonuclease mapping can be used to roughly determine the genetic structure of the ceramidase gene. Restriction maps derived by restriction endonuclease cleavage can be confirmed by DNA sequence analysis. DNA sequence analysis can be performed by any techniques known in the art, including but not limited to the method of Maxam and Gilbert (1980, Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger, F., et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463), the use of T7 DNA polymerase (Tabor and Richardson, U.S. Patent No. 4,795,699), or use of an automated DNA sequenator (e.g., Applied Biosystems, Foster City, CA).
- ceramidase cDNA of the invention was overexpressed in HEK 293 cells and MCF7 cells. Examples of vectors and host cells that were used in these studies are described in Section 6.
- the nucleotide sequence coding for a ceramidase protein or a functionally active analog or fragment or other derivative thereof can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
- a variety of expression vectors may be used in the present invention which include, but are not limited to, plasmids, cosmids, phage, phagemids, or modified viruses.
- such expression vectors also comprise a functional origin of replication for propagation of the vector in an appropriate host cell, one or more restriction endonuclease sites for insertion of the ceramidase gene sequence, and one or more selection markers.
- the necessary transcriptional and translational signals can also be supplied by the native ceramidase gene and/or its flanking regions.
- host-vector systems including but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA, can be used.
- the expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.
- the human ceramidase gene is expressed, or a sequence encoding a functionally active portion of human ceramidase is expressed.
- a fragment of ceramidase comprising a domain of the ceramidase protein is expressed.
- Any method for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a ceramidase gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of nucleic acid sequence encoding a ceramidase protein or peptide fragment may be regulated by a second nucleic acid sequence so that the ceramidase protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a ceramidase protein may be controlled by any promoter/enhancer element known in the art. Vectors based on E.
- Non-limiting examples of regulatory regions that can be used for expression in E. coli may include but not limited to lac, trp, lpp, phoA, recA, tac, T3, T7 and ⁇ P L (Makrides, 1996, Microbiol Rev, 60:512-538).
- Non-limiting examples of prokaryotic expression vectors may include the ⁇ gt vector series such as ⁇ gtl 1 (Huynh et al., 1984 in "DNA Cloning Techniques", Vol. I: A Practical Approach (D. Glover, ed.), pp.
- a potential drawback of a prokaryotic host-vector system is the inability to perform many of the post-translational processing of mammalian cells.
- an eukaryotic host-vector system is preferred, a mammalian host-vector system is more preferred, and a human host-vector system is the most preferred.
- Promoters which may be used to control ceramidase expression include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus
- promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al, 1984, Cell 38:639-646; Omitz et al, 1986, Cold Spring Harbor Symp.
- mice mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al, 1987, Genes and Devel 1:268-276), alpha- fetoprotein gene control region which is active in liver (Krumlauf et al, 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al, 1987, Science 235:53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al, 1987, Genes and Devel.
- beta-globin gene control region which is active in myeloid cells (Mogram et al, 1985, Nature 315:338-340; Kollias et al, 1986, Cell 46:89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al, 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283- 286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al, 1986, Science 234:1372-1378).
- a vector in a specific embodiment, comprises a promoter operably linked to a ceramidase-encoding nucleic acid, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene).
- Expression vectors containing ceramidase gene inserts can be identified by three general approaches: (a) nucleic acid hybridization, (b) presence or absence of "marker" gene functions, and (c) expression of inserted sequences.
- the presence of a ceramidase gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted ceramidase gene.
- the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of a ceramidase gene in the vector.
- certain "marker" gene functions e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.
- recombinant expression vectors can be identified by assaying the ceramidase product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the ceramidase protein in in vitro assay systems, e.g., enzyme activity, binding with anti-ceramidase antibody.
- expression constructs and vectors are introduced into host cells for the purpose of producing the ceramidase.
- Any cell type that can produce mammalian proteins and is compatible with the expression vector may be used, including those that have been cultured in vitro or genetically engineered.
- Host cells may be obtained from normal or affected subjects, including healthy humans, cancer patients, and patients infected with a virus, private laboratory deposits, public culture collections such as the American Type Culture Collection, or from commercial suppliers.
- expression constructs are introduced into cancer cells for the purpose of gene therapy (see Section 5.8).
- Cells into which a ceramidase gene sequence can be introduced for purposes of production of the ceramidase in vivo may include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
- the lentiviral vector was used for transduction of quiescent, primitive human hematopoietic progenitor cells and may provide therapeutically useful levels of gene transfer into human hematopoietic stem cells (Case et al, 1999, Proc. Natl. Acad. Sci. USA 96: p.2988-2993).
- the choice of cell type depends on the type of tumor being treated or prevented, and can be determined by one of skill in the art.
- an expression construct comprising a ceramidase gene sequence is introduced into a preneoplastic or neoplastic cell. Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins.
- a host cell may be chosen which modifies and processes the expressed gene products in a specific fashion similar to the way the recipient processes its ceramidase.
- the type of host cell used in the present invention has been used for expression of heterologous genes, and is reasonably well characterized and developed for large-scale production processes.
- Prefe ⁇ ed mammalian host cells include but are not limited to those derived from humans, monkeys and rodents, (see, for example, Kriegler M. in “Gene Transfer and Expression: A Laboratory Manual", New York, Freeman & Co. 1990).
- a number of viral-based expression systems may also be utilized with mammalian cells to produce ceramidases.
- Vectors using DNA virus backbones have been derived from simian virus 40 (SV40) (Hamer et al, 1979, Cell 17:725), adenovirus (Van Doren et al, 1984, Mol Cell Biol 4:1653), adeno-associated virus (McLaughlin et al, 1988, J Virol 62:1963), and bovine papillomas virus (Zinn et al, 1982, Proc Natl Acad Sci
- SV40 simian virus 40
- adenovirus Van Doren et al, 1984, Mol Cell Biol 4:1653
- adeno-associated virus McLaughlin et al, 1988, J Virol 62:1963
- bovine papillomas virus Zainn et al, 1982, Proc Natl Acad Sci
- the donor DNA sequence may be ligated to an adenovirus transcription translation control complex, e.g., the late promoter and tripartite leader sequence.
- This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing heterologous products in infected hosts.
- a non-essential region of the viral genome e.g., region El or E3
- Bovine papillomavirus can infect many higher vertebrates, including man, and its DNA replicates as an episome.
- a number of shuttle vectors have been developed for recombinant gene expression which exist as stable, multicopy (20-300 copies/cell) extrachromosomal elements in mammalian cells.
- these vectors typically contain a segment of BPV DNA (the entire genome or a 69% transforming fragment), a promoter with a broad host range, a polyadenylation signal, splice signals, a selectable marker, and "poisonless" plasmid sequences that allow the vector to be propagated in E. coli.
- the expression gene construct are transfected into cultured mammalian cells by, for example, the calcium phosphate coprecipitation technique.
- selection of transformants is achieved by use of a dominant selectable marker, such as histidinol and G418 resistance.
- a ceramidase gene sequence can be inserted into BPV vectors, such as pBCMGSNeo and pBCMGHis (Karasuyama et al, ⁇ ur. J. Immunol.
- the vaccinia 7.5K promoter may be used. (See, e.g., Mackett et al, 1982, Proc. Natl Acad. Sci. (USA) 79:7415-7419; Mackett et al, 1984, J. Virol 49:857-864; Panicali et al, 1982, Proc. Natl. Acad. Sci.
- vectors based on the ⁇ pstein-Barr virus ( ⁇ BV) origin (OriP) and ⁇ BV nuclear antigen 1 ( ⁇ BNA-1; a trans-acting replication factor) can be used.
- ⁇ BV ⁇ pstein-Barr virus
- ⁇ BNA-1 ⁇ BV nuclear antigen 1
- Such vectors can be used with a broad range of human host cells, e.g., EBO-pCD (Spickofsky et al, 1990, DNA Prot Eng Tech 2:14-18); pDR2 and ⁇ DR2 (available from Clontech Laboratories).
- Ceramidase may also be made with a retrovirus-based expression system.
- Retro viruses such as Moloney murine leukemia virus, can be used since most of the viral gene sequence can be removed and replaced with ceramidase gene sequence while the missing viral functions can be supplied in trans.
- retroviruses can efficiently infect and transfer genes to a wide range of cell types including, for example, primary hematopoietic cells.
- the host range for infection by a retroviral vector can be manipulated by the choice of envelope used for vector packaging.
- a retroviral vector can comprise a 5' long terminal repeat
- LTR Long Term Evolution
- 3' LTR a packaging signal
- bacterial origin of replication a bacterial origin of replication
- selectable marker a selectable marker.
- the ceramidase DNA is inserted into a position between the 5' LTR and 3' LTR, such that transcription from the 5' LTR promoter transcribes the cloned DNA.
- the 5' LTR comprises a promoter, including but not limited to an LTR promoter, an R region, a U5 region and a primer binding site, in that order. Nucleotide sequences of these LTR elements are well known in the art.
- a heterologous promoter as well as multiple drug selection markers may also be included in the expression vector to facilitate selection of infected cells.
- yeast a number of vectors containing constitutive or inducible promoters may be used with Saccharomyces cerevisiae (baker's yeast), Schizosaccharomyces pombe (fission yeast), Pichia pastoris, and Hansenula polymorpha (methylotropic yeasts).
- Saccharomyces cerevisiae bakeer's yeast
- Schizosaccharomyces pombe Schizosaccharomyces pombe
- Pichia pastoris Pichia pastoris
- Hansenula polymorpha methylotropic yeasts
- Autographa californica nuclear polyhidrosis virus (AcNPV) a baculovirus
- AcNPV Autographa californica nuclear polyhidrosis virus
- the ceramidase gene sequences may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
- AcNPV promoter for example the polyhedrin promoter
- the efficiency of expression of the ceramidase in a host cell may be enhanced by the inclusion of appropriate transcription enhancer elements in the expression vector, such as those found in SV40 virus, Hepatitis B virus, cytomegalovirus, immunoglobulin genes, metallothionein, ⁇ -actin (see Bittner et al, 1987, Methods in Enzymol. 153:516-544; Gorman, 1990, Cu ⁇ . Op. in Biotechnol. 1 :36-47).
- appropriate transcription enhancer elements such as those found in SV40 virus, Hepatitis B virus, cytomegalovirus, immunoglobulin genes, metallothionein, ⁇ -actin (see Bittner et al, 1987, Methods in Enzymol. 153:516-544; Gorman, 1990, Cu ⁇ . Op. in Biotechnol. 1 :36-47).
- the expression vector may also contain sequences that permit maintenance and replication of the vector in more than one type of host cell, or integration of the vector into the host chromosome.
- sequences may include but are not limited to replication origins, autonomously replicating sequences (ARS), centromere DNA, and telomere DNA. It may also be advantageous to use shuttle vectors which can be replicated and maintained in at least two types of host cells.
- the expression vector may contain selectable or screenable marker genes for initially isolating, identifying or tracking host cells that contain DNA encoding a ceramidase.
- selectable or screenable marker genes for initially isolating, identifying or tracking host cells that contain DNA encoding a ceramidase.
- stable expression in mammalian cells is prefe ⁇ ed.
- a number of selection systems may be used for mammalian cells, including but not limited to the Herpes simplex virus thymidine kinase (Wigler et al, 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalski and Szybalski, 1962, Proc. Natl. Acad. Sci.
- adenine phosphoribosyltransferase (Lowy et al, 1980, Cell 22:817) genes can be employed in tk “ , hgprt “ or aprf cells, respectively.
- antimetabolite resistance can be used as the basis of selection for dihydrofolate reductase (dhfr), which confers resistance to methotrexate (Wigler et al, 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al, 1981, Proc. Natl. Acad. Sci.
- any of the cloning and expression vectors described herein may be synthesized and assembled from known DNA sequences by well known techniques in the art.
- the regulatory regions and enhancer elements can be of a variety of origins, both natural and synthetic.
- Some vectors and host cells may be obtained commercially. Non- limiting examples of useful vectors are described in Appendix 5 of Cu ⁇ ent Protocols in Molecular Biology, 1988, ed. Ausubel et al, Greene Publish. Assoc. & Wiley Interscience, which is incorporated herein by reference; and the catalogs of commercial suppliers such as Clontech Laboratories, Stratagene Inc., and Invitrogen, Inc.
- Endogenous ceramidase gene expression can also be reduced by inactivating or "knocking out” the gene or its promoter using targeted homologous recombination.
- endogenous ceramidase gene expression can also be reduced by inactivating or "knocking out” the gene or its promoter using targeted homologous recombination.
- ceramidase gene or a completely unrelated DNA sequence flanked by DNA homologous to the endogenous ceramidase gene (either the coding regions or regulatory regions of the ceramidase gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express ceramidase gene in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the ceramidase gene.
- ES embryonic stem
- inactive ceramidase gene e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra.
- Such techniques can also be utilized to generate animal models. It should be noted that this approach can be adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors, e.g., herpes virus vectors.
- endogenous ceramidase gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the ceramidase gene (i.e., the ceramidase gene promoter and/or enhancers) to form triple helical structures that prevent transcription of the ceramidase gene in target cells in the body.
- deoxyribonucleotide sequences complementary to the regulatory region of the ceramidase gene i.e., the ceramidase gene promoter and/or enhancers
- transgenic non-human, preferably mammals, that have incorporated and express a functional ceramidase gene may be used as animal models of diseases and disorders involving hyperproliferation. Such animals can be used to screen for or test molecules for the ability to inhibit proliferation and thus treat or prevent such diseases and disorders.
- the invention provides ceramidase proteins (interchangeably refe ⁇ ed to as the ceramidase gene product), preferably human ceramidase protein, fragments, derivatives, and analogs thereof which comprise an antigenic determinant (i.e., can be recognized by an antibody) or which are otherwise functionally active, as well as nucleic acid sequences encoding the foregoing.
- the ceramidase proteins are encoded by the ceramdiase nucleic acids described in Section 5.1 supra.
- the amino acid sequence of a ceramidase protein of the invention consists of SEQ ID NO:2 as shown in Fig. 3.
- the amino acid sequence of the full length ceramidase protein comprises one transmembrane domain between amino acids 505 and 525 ( Figure 3) and three other putative transmembrane domains (amino acids, 176-196, 313-333, 431-451, 543-563).
- the sequence also revealed the presence of a signal peptide (amino acids 1-19), and a region of low compositional complexity (amino acids, 38-66). This region of low complexity showed features of a mitochondrial targeting sequence.
- PKC putative phosphorylation sites
- PKA putative phosphorylation sites
- casein kinase 2 putative N- glycosylation sites. The nucleotide position of these active sites are shown in Table 1.
- the ceramidase cDNA of the invention was overexpressed in HEK 293 and MCF7 cells using the pcDNA3.1/ ⁇ is-ceramidase construct, and ceramidase enzyme activity (at pH 9.5) increased by 50- and 12-fold, respectively.
- ceramidase enzyme activity at pH 9.5
- a green fluorescent protein- ceramidase fusion protein was constructed to investigate the localization of this enzyme. The results showed that the green fluorescent protein-ceramidase fusion protein presented a mitochondrial localization pattern and co-localized with mitochondrial specific probes.
- Fragments, or proteins comprising fragments, lacking some or all of the foregoing regions of a ceramidase protein are also provided.
- “Functionally active" ceramidase material refers to that material displaying one or more known functional activities associated with a full-length wild-type ceramidase protein, e.g., hydro lyze ceramide to form sphingosine, binding to a ceramidase substrate, antigenicity (binding to an anti-ceramidase antibody), immunogenicity, etc.
- the derivative or analog is functionally active, i.e., capable of exhibiting one or more functional activities associated with a full-length, wild-type ceramidase protein.
- a ceramidase protein lacking a signal peptide is provided, e.g., such a ceramidase protein has the amino acid sequence of 17 to 761 of SEQ ID NO:2.
- a ceramidase protein lacking the mitochondrial targeting sequence is provided.
- Such a ceramidase protein has the amino acid sequence of residues 1 to 37 and residues 67 to 761 of SEQ ID NO:2.
- fragments, derivatives or analogs which have the desired immunogenicity or antigenicity can be used, for example, in immunoassays, for immunization, for inhibition of ceramidase activity, etc.
- derivatives or analogs which are phosphorylated or dephosphorylated are provided.
- Fragments, derivatives or analogs that retain, or alternatively lack or inhibit, a desired ceramidase property of interest e.g., binding to a ceramidase substrate
- a specific embodiment relates to a ceramidase fragment that can be bound by an anti- ceramidase antibody. Fragments, derivatives or analogs of ceramidase can be tested for the desired activity by procedures known in the art, including but not limited to the assays described in Sections 5.10.
- the invention provides fragments of a ceramidase protein consisting of at least 6 amino acids, 10 amino acids, 50 amino acids, or of at least 75 amino acids. In specific embodiments, such fragments are not larger than 35, 100 or 200 amino acids.
- Fragments, derivatives or analogs of ceramidase include but are not limited to those molecules comprising regions that are substantially homologous to ceramidase or fragments thereof (e.g., in various embodiments, at least 60%) or 70% or 80% or 90% or 95% identity over an amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art) or whose encoding nucleic acid is capable of hybridizing to a coding ceramidase sequence, under stringent, moderately stringent, or low stringency conditions.
- Table 2 shows the alignment of the sequenced rat brain peptides to the human cloned protein.
- the peptide sequences obtained from the purified rat brain enzyme were aligned to the peptide sequences deduced from the cloned human ceramidase. Amino acids in bold show difference in sequence.
- the ceramidase proteins, derivatives, or analogs are of ceramidase proteins of animals, e.g., rat, pig, cow, dog, monkey, human, or of plants.
- the mouse homolog of the ceramidase protein is least prefe ⁇ ed.
- the ceramidase protein may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
- the functional properties may be evaluated using any suitable assays as described in Section 6.1.
- native ceramidase proteins can be purified from natural sources, by methods such as those described in Section 6.1 and in El Bawab et al, 1999, J. Biol. Chem. 274(39): 27948-27955.
- the ceramidase proteins can be purified from mitochondria.
- the amino acid sequence of the ceramidase protein can be derived by deduction from the DNA sequence of a ceramidase gene, or alternatively, by direct sequencing of the protein, e.g., with an automated amino acid sequencer (e.g., see Hunkapiller, M., et al, 1984, Nature 310:105-111).
- the ceramidase protein sequence can be further characterized by a hydrophilicity analysis (Hopp, T. and Woods, K., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824).
- a hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of the ceramidase protein and the co ⁇ esponding regions of the gene sequence which encode such regions.
- ceramidase fragments, derivatives or analogs can be made by altering ceramidase sequences by conservative substitutions, additions or deletions that provide for functionally equivalent molecules.
- nucleotide coding sequences other DNA sequences which encode substantially the same amino acid sequence as a ceramidase gene may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of ceramidase genes which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a functionally silent change.
- the ceramidase derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a ceramidase protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a functionally silent change.
- one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
- Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
- the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
- the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
- the positively charged (basic) amino acids include arginine, lysine and histidine.
- the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Accordingly, the present invention provides an isolated polypeptide, the amino acid sequence of which comprises SEQ ID NO: 2 with at least one conservative amino acid substitution.
- the ceramidase derivatives and analogs of the invention can be produced by various methods known in the art.
- the manipulations which result in their production can occur at the gene or protein level.
- the cloned ceramidase gene sequence can be modified by any of numerous strategies known in the art (Maniatis, T., 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
- the sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
- the ceramidase-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
- Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, C, et al, 1978, J. Biol. Chem 253:6551), use of TAB® linkers (Pharmacia), etc.
- ceramidase sequence may also be made at the protein level. Included within the scope of the invention are ceramidase protein fragments or other derivatives or analogs which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
- a ceramidase protein fragment can be made by sequential removal of amino acid residues from the amino and/or carboxyl termainl of the protein.
- analogs and derivatives of ceramidase can be chemically synthesized.
- a peptide co ⁇ esponding to a portion of a ceramidase protein which comprises the desired domain (see Section 5.6.1), or which mediates the desired activity in vitro can be synthesized by use of a peptide synthesizer.
- nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the ceramidase sequence.
- Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, -amino isobutyric acid, 4- aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline.
- cysteic acid t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, C -methyl amino acids, N -methyl amino acids, and amino acid analogs in general.
- the amino acid can be D (dextrorotary) or L (levorotary).
- the ceramidase derivative is a chimeric, or fusion, protein comprising a ceramidase protein or fragment thereof (preferably consisting of at least a domain or motif of the ceramidase protein, or at least 6, 8, or 10 amino acids of the ceramidase protein) joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein.
- a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising a ceramidase-coding sequence joined in-frame to a coding sequence for a different protein).
- Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art.
- a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.
- Chimeric genes comprising portions of ceramidase fused to any heterologous protein-encoding sequences may be constructed.
- a specific embodiment relates to a chimeric protein comprising a fragment of ceramidase of at least six amino acids.
- the present invention provides a chimeric protein comprising a fragment of a ceramidase protein consisting of at least 6 amino acids fused via a covalent bond to an amino acid sequence of a second polypeptide.
- the invention relates to ceramidase derivatives and analogs that comprise one or more domains of a ceramidase protein, including but not limited to the ceramidase transmembrane domains, signal peptide, a region of low compositional complexity, putative myristoylation site, putative phosphorylation sites (PKC, PKA, casein kinase 2) and putative N-glycosylation, functional fragments of any of the foregoing, or any combination of the foregoing.
- PLC putative myristoylation site
- PKA putative phosphorylation sites
- Such domains in human and rat ceramidase proteins are identified in Figure 3.
- the ceramidase derivative can be a molecule comprising multiple regions of homology with a ceramidase protein.
- a first protein region can be considered "homologous" to a ceramidase domain or to a second protein region when the amino acid sequence of the first region is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% identical, when compared to any sequence in the second region of an equal number of amino acids as the number contained in the first region or when compared to an aligned sequence of the second region that has been aligned by a computer homology program known in the art.
- a molecule can comprise one or more regions homologous to a ceramidase domain or a portion thereof.
- the present invention provides an isolated polypeptide which is at least 60% identical to the ceramidase polypeptide having the amino acid sequence of SEQ ID NO:2, and displays one or more functional activities of ceramidase protein.
- the invention provides various phosphorylated and dephosphorylated forms of the ceramidase protein, derivative, or analog. Both phosphorylation and dephosphorylation of ceramidase can occur at different residues.
- Phosphorylation can be carried out by any methods known in the art, e.g., by use of a kinase.
- Dephosphorylation can be carried out by use of any methods known in the art, e.g., by use of a phosphatase.
- a molecule is provided that comprises one or more domains (or functional portion thereof) of a ceramidase protein but that also lacks one or more domains (or functional portion thereof) of a ceramidase protein.
- ceramidase protein derivatives are provided that lack one or more transmembrane domain.
- such a protein may also lack all or a portion of the region of low compositional complexity or the mitochondrial targeting sequence such that the ability of the ceramidase protein to localize to mitochondrial is reduced or lost.
- a molecule is provided that comprises one or more domains (or functional portion thereof) of a ceramidase protein, and that has one or more mutant (e.g., due to deletion or point mutation(s)) domains of a ceramidase protein (e.g., such that the mutant domain has decreased function).
- the active site may be mutated so as to have reduced, absent, or increased ceramidase activity.
- the present invention provides an isolated polypeptide, the amino acid of which comprises at least one region selected from the group consisting of residues 1-19, 38-66, 176-196, 3131-333, 431-451, 505-525, and 543-563 of SEQ ID NO:2.
- the functional activity of ceramidase proteins, derivatives and analogs can be assayed by various methods including those described in Section 6.1.
- various immunoassays known in the art can be used, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
- antibody binding is detected by detecting a label on the primary antibody.
- the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
- the secondary antibody is labelled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
- the antibodies include but are not limited to polyclonal, monoclonal, Fab fragments, single chain antibodies, chimeric antibodies, humanized antibodies, single chain antidobies, Fab fragments, F(ab') 2 fragments, Fv fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
- ceramidase protein may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen.
- Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
- antibodies to a human ceramidase protein are produced.
- antibodies to a domain (e.g., the kinase domain) of a ceramidase protein are produced.
- fragments of a ceramidase protein identified as hydrophilic are used as immunogens for antibody production.
- polyclonal antibodies to a ceramidase protein or derivative or analog may be obtained.
- rabbit polyclonal antibodies to an epitope of a ceramidase protein encoded by a sequence of SEQ ID NO:2, or an immunogenic subsequence thereof can be obtained.
- various host animals can be immunized by injection with the native ceramidase protein, or a synthetic version, or derivative (e.g., fragment) thereof, including but not limited to rabbits, mice, rats, etc.
- adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
- BCG Bacille Calmette-Guerin
- any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
- the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495- 497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al, 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies Colde et al, 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
- monoclonal antibodies can be produced in germ-free animals utilizing recent technology (PCT/US90/02545). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
- the hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently prefe ⁇ ed method of production.
- human antibodies may be used, and such can be obtained by using human hybridomas (Cote et al, 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al, 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).
- human hybridomas Cote et al, 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030
- EBV virus Cold-d Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96
- techniques developed for the production of "chimeric antibodies” (Morrison et al, 1984, Proc. Natl. Acad. Sci. U.S.A.
- a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
- techniques described for the production of single chain antibodies U.S.
- Patent 4,946,778; Bird, 1988, Science 242:423-426; Huston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al, 1989, Nature 334:544-546) can be adapted to produce single chain antibodies against ceramidase gene products.
- Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Ske ⁇ a et al, 1988, Science 242:1038-1041).
- Antibody fragments which recognize specific epitopes may be generated by known techniques.
- such fragments include but are not limited to: the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
- Fab expression libraries may be constructed (Huse et al, 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. Accordingly, the present invention provides a molecule comprising a fragment of the antibody which binds a ceramidase protein.
- Antibody fragments which contain the idiotype of the molecule can be generated by known techniques.
- fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments.
- screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay).
- ELISA enzyme-linked immunosorbent assay
- an antibody that specifically binds a first ceramidase homolog but which does not specifically bind a different ceramidase homolog one can select on the basis of positive binding to the first ceramidase homolog and a lack of binding to the second ceramidase homolog.
- Antibodies specific to a domain of a ceramidase protein are also provided.
- the foregoing antibodies can be used in methods known in the art relating to the localization and activity of the ceramidase protein sequences of the invention, e.g., for imaging these proteins, measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc.
- the present invention relates to the uses of antibodies or fragments thereof capable of specifically recognizing one or more epitopes of the ceramidase gene products, epitopes of conserved variants of the ceramidase gene products, epitopes of mutant ceramidase gene products, or peptide fragments of the ceramidase gene products.
- Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs).
- Such antibodies may be used, for example, in the detection of a ceramidase gene product in an biological sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients may be tested for abnormal levels of ceramidase gene products, and/or for the presence of abnormal forms of the such gene products.
- Such antibodies may also be included as a reagent in a kit for use in a diagnostic or prognostic technique.
- Such antibodies may also be utilized in conjunction with, for example, compound screening schemes, as described, below, for the evaluation of the effect of test compounds on ceramidase gene product levels and/or activity.
- Antibodies to ceramidase gene product may be used in a method for the inhibition of abnormal ceramidase gene product activity.
- antibodies may, therefore, be utilized as part of the treatment methods of the invention. Additionally, such antibodies can be used in conjunction with the gene therapy techniques described, below, to, for example, evaluate the normal and/or engineered ceramidase-expressing cells prior to their introduction into the patient.
- ceramidase is an intracellular protein, it is prefe ⁇ ed that internalizing antibodies be used.
- lipofectin or liposomes can be used to deliver the antibody or a fragment of the Fab region which binds to the ceramidase gene product epitope into cells.
- fragments of the antibody are used, the smallest inhibitory fragment which binds to the ceramidase activation domain(s) is prefe ⁇ ed.
- peptides having an amino acid sequence co ⁇ esponding to the domain of the variable region of the antibody that binds to the ceramidase activation domain(s) can be used.
- Such peptides can be synthesized chemically or produced via recombinant DNA technology using methods well known in the art (e.g., see Creighton, 1983, supra; and Sambrook et al, 1989, above).
- single chain antibodies such as neutralizing antibodies, which bind to intracellular epitopes can also be administered.
- Such single chain antibodies can be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco et al. (1993, Proc. Natl. Acad. Sci. USA 90:7889-7893).
- the use of antisense molecules as inhibitors of gene expression is a specific, genetically based therapeutic approach (for a review, see Stein, in Ch. 69, Section 5 "Cancer: Principle and Practice of Oncology", 4th ed., ed. by DeVita et al, J.B. Lippincott, Philadelphia 1993).
- the present invention provides the therapeutic or prophylactic use of single-stranded nucleic acids of at least six nucleotides that are antisense to a gene or cDNA encoding ceramidase or a portion thereof.
- the invention further provides pharmaceutical compositions comprising an effective amount of the ceramidase antisense nucleic acids of the invention in a pharmaceutically acceptable carrier, as described infra.
- the invention is directed to methods for inhibiting the expression of a ceramidase nucleic acid sequence in a mammalian cell in vitro or in vivo comprising providing the cell with an effective amount of a composition comprising an ceramidase antisense nucleic acid of the invention.
- the antisense nucleic acid of the invention may be complementary to a coding and/or noncoding region of a ceramidase mRNA.
- the antisense molecules will bind to the complementary ceramidase gene mRNA transcripts and reduce or prevent translation. Absolute complementarity, although prefe ⁇ ed, is not required.
- a sequence "complementary" to a portion of an RNA, as refe ⁇ ed to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
- the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid.
- the human ceramidase promoter contains two CT repeats that represent potential triple helix regions (Mavrothalassitis et al, 1990, Oncogene 5:1337-1342). Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be).
- One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
- Nucleic acid molecules that are complementary to the 5' end of the message should work most efficiently at inhibiting translation.
- sequences complementary to the 3' untranslated sequences of mRNAs have recently shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., 1994, Nature 372:333-335.
- Nucleic acid molecules complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon.
- Antisense nucleic acid molecules complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5'-, 3'- or coding region of target or pathway gene mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length.
- the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides, at least 50 nucleotides, or at least 200 nucleotides.
- nucleic acid molecules complementary to either the 5'- or 3'- non-translated, non-coding regions of the ceramidase gene could be used in an antisense approach to inhibit translation of endogenous ceramidase gene mRNA.
- the invention provides antisense ceramidase nucleic acid molecules, preferably RNA molecules, that are essentially single stranded nucleic acid molecules, and comprises a nucleotide sequence complementary to (a) the nucleotide sequence of the sense strand of the polynucleotide (i.e., SEQ ID NO:l) depicted in Figure 3, SEQ ID NO: 1 ; or (b) a nucleotide sequence that encodes the amino acid sequence shown in Figure 3 (i.e., SEQ ID NO:2).
- the antisense ceramidase nucleic acid molecule of the invention is capable of hybridizing in vivo and in vitro to a portion of an ceramidase messenger RNA (mRNA) by virtue of some sequence complementarity.
- mRNA messenger RNA
- Such hybridization conditions may be highly stringent as exemplified above, or moderately stringent, e.g., washing in 0.2xSSC/0.1% SDS at 42°C (Ausubel F.M. et al, eds., 1989, Cu ⁇ ent Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York, at page 2.10.3).
- nucleic acid molecules are deoxyoligonucleotides ("oligos")
- highly stringent conditions may refer, e.g., to washing in 6xSSC/0.05%> sodium pyrophosphate at 37°C (for 14-base oligos), 48°C (for 17-base oligos), 55°C (for 20-base oligos), and 60°C (for 23 -base oligos).
- Antisense nucleic acid molecules may be synthesized chemically or enzymatically, and delivered to cells that requires treatment by injection.
- antisense ceramidase RNA molecules can be synthesized in a cell by inserting the ceramidase gene or a fragment thereof in a manner such that the antisense RNA molecules are made, preferably in a controllable fashion.
- double-stranded RNA which has been shown to effectively block gene expression can also be used. Genetic interference by double-stranded RNA (RNA interference or RNA-i) has been successfully used to determine both the role of a specific gene and cells that express the specific gene (Misquitta and Paterson, 1999, Proc. Natl. Acad. Sc , 96: 1451-1456; Fire et al, 1998, Nature, 391 :
- nucleic acid molecules may be used to interfere with ceramidase gene regulation, so as to modulate, for example, the phenotype and ceramide level of cells. Further, such sequences may be used as part of ribozyme and/or triple helix sequences, also useful for ceramidase gene regulation.
- control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.
- the antisense molecule can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
- the antisense molecule can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
- the antisense molecule may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al, 1987, Proc. Natl. Acad. Sci.
- the antisense molecule may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
- the antisense molecule may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine,
- the antisense molecule may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
- the antisense molecule comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
- the antisense molecule is an ⁇ -anomeric oligonucleotide.
- oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al, 1987, Nucl. Acids Res. 15:6625-6641).
- the oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al, 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al, 1987, FEBS Lett. 215:327-330).
- antisense molecules of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
- an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
- phosphorothioate oligonucleotides may be synthesized by the method of Stein et al (1988, Nucl. Acids Res. 16:3209)
- methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al, 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
- antisense nucleotides complementary to the ceramidase coding region such as the ones described in Section 6.1, could be used, those complementary to the transcribed untranslated region are also prefe ⁇ ed.
- the present invention provides a nucleic acid molecule comprising a nucleotide sequence that hybridizes to the complement of the nucleic acid sequence of SEQ ID NO:l and encodes a polypeptide with one or more activities of a ceramidase protein, linked uninterrupted by stop codons to a coding sequence that encodes a heterologous protein or peptide.
- the invention further provides a nucleic acid molecule that is single- stranded, and that hybridizes under highly stringent conditions to a nucleic acid probe having the nucleotide sequence of SEQ ID NO:l .
- the ceramidase antisense nucleic acids can be used to treat or prevent a disease or condition involving a cell type that expresses, or preferably overexpresses, ceramidase. Cell types which express or overexpress ceramidase RNA can be identified by various methods known in the art.
- Such methods include but are not limited to hybridization with a ceramidase-specific nucleic acid (e.g., by Northern hybridization, dot blot hybridization, in situ hybridization), detection of ceramidase gene product by immunoassays, etc.
- a ceramidase-specific nucleic acid e.g., by Northern hybridization, dot blot hybridization, in situ hybridization
- detection of ceramidase gene product by immunoassays etc.
- primary tissue from a patient can be assayed for ceramidase expression prior to treatment, e.g., by immunocytochemistry or in situ hybridization.
- Pharmaceutical compositions of the invention comprising an effective amount of a ceramidase antisense nucleic acid in a pharmaceutically acceptable carrier, can be administered to a patient having a disease or disorder which is of a type that expresses or overexpresses ceramidase RNA or protein.
- the amount of ceramidase antisense nucleic acid which will be effective in the treatment of a particular disorder or condition will depend on the nature of the cancer or condition, and can be determined by standard clinical techniques. Where possible, it is desirable to determine the antisense cytotoxicity of the tumor type to be treated in vitro, and then in useful animal model systems prior to testing and use in humans.
- the antisense molecules should be delivered to cells which express the ceramidase gene in vivo.
- a number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense molecule linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.
- Antisense molecules can be delivered to the desired cell population via a delivery complex.
- compositions comprising ceramidase antisense nucleic acids are administered via biopolymers (e.g., poly- ⁇ -l->4-N-acetylglucosamine polysaccharide), liposomes, microparticles, or microcapsules.
- biopolymers e.g., poly- ⁇ -l->4-N-acetylglucosamine polysaccharide
- liposomes e.g., poly- ⁇ -l->4-N-acetylglucosamine polysaccharide
- it may be desirable to utilize liposomes targeted via antibodies to specific identifiable tumor antigens Leonetti et al, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2448-2451; Renneisen et al. 1990, J. Biol. Chem. 265:16337-16342).
- Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA (For a review see, for example Rossi, J., 1994, Cu ⁇ ent Biology 4:469- 471).
- the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage.
- the composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see U.S. Pat. No. 5,093,246, which is incorporated by reference herein in its entirety.
- engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences encoding target gene proteins.
- Ribozyme molecules designed to auto-catalytically cleave ceramidase gene mRNA transcripts can also be used to prevent translation of ceramidase gene mRNA and expression of ceramidase target genes. (See, e.g., PCT International Publication
- the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the ceramidase gene mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non- functional mRNA transcripts.
- the ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes”) such as the one which occurs naturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al, 1984, Science, 224:574-578;
- Cech-type ribozymes such as the one which occurs naturally in Tetrahymena Thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al, 1984, Science, 224:574-578;
- the Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.
- the invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in an ceramidase gene.
- the ribozymes can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express the ceramidase gene in vivo.
- a prefe ⁇ ed method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous ceramidase gene messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
- Ribozyme, and triple helix molecules of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides or by in vitro and in vivo transcription of DNA sequences encoding the ribozyme or triple helix molecule.
- the present invention provides a nucleic acid comprising a nucleotide sequence encoding a ceramidase-specific ribozyme, which comprises an autocatalytic cleaving ribozyme, and a region that hybridizes under highly stringent conditions to a nucleic acid having the nucleotide sequence of SEQ ID NO: 1.
- the present invention provides a variety of methods for the diagnostic and prognostic evaluation of cell hyperproliferation, vascular diseases, and inflammation and diseases associated with sphingolipid signal transduction.
- Such methods may, for example, utilize reagents such as the ceramidase nucleotide sequences, fusion protein (GFP-fusion protein), and antibodies directed against ceramidase gene products, including peptide fragments thereof, as described, above.
- reagents such as the ceramidase nucleotide sequences, fusion protein (GFP-fusion protein), and antibodies directed against ceramidase gene products, including peptide fragments thereof, as described, above.
- such reagents may be used, for example, for: (1) the detection of the presence of ceramidase gene mutations, or the detection of either over- or under- expression of ceramidase gene mRNA in diseased cells relative to normal cells, or the qualitative or quantitative detection of other alleic forms of ceramidase transcripts which may co ⁇ elate with the phenotypes of various diseases, and (2) the detection of an over- abundance of ceramidase gene product relative to the non-disease state or the presence of a modified (e.g., less than full length) ceramidase gene product which co ⁇ elates with a diseased phenotype.
- a modified (e.g., less than full length) ceramidase gene product which co ⁇ elates with a diseased phenotype.
- the present invention provides a method of diagnosing a disease or disorder characterized by an abe ⁇ ant level of ceramidase RNA or protein in a subject, comprising measuring the level of ceramidase RNA or protein in a sample derived from the subject, in which an increase or decrease in the level of ceramidase RNA or protein, relative to the level of ceramidase RNA or protein found in an analogous sample not having the disease or disorder indicates the presence of the disease or disorder in the subject.
- One embodiment of the present invention is directed to a method of diagnosing or screening for the presence of or a predisposition for developing a disease or disorder involving cell ove ⁇ roliferation or dysfunctional sphingolipid signal transduction in a subject comprising measuring the level of ceramidase protein, ceramidase RNA or ceramidase functional activity in a sample derived from the subject, in which a decrease in the level of ceramidase protein, ceramidase RNA, or ceramidase functional activity in the sample, relative to the level of ceramidase protein, ceramidase RNA, or ceramidase functional activity found in an analogous sample not having the disease or disorder or a predisposition for developing the disease or disorder, indicates the presence of the disease or disorder or a predisposition for developing the disease or disorder.
- Another embodiment of the present invention is directed to a method of diagnosing or screening for the presence of or a predisposition for developing a disease or disorder involving cell ove ⁇ roliferation or dysfunctional sphingolipid signal transduction in a subject comprising detecting one or more mutations in ceramidase DNA, RNA or protein derived from the subject in which the presence of said one or more mutations indicates the presence of the disease or disorder or a predisposition for developing the disease or disorder.
- the methods described herein may be applied to samples of cells or cellular materials taken directly from a patient. Any method known in the art for collection or isolation of the desired cells or materials can be used.
- the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic test kits comprising at least one specific ceramidase nucleic acid or anti-ceramidase gene antibody reagent described herein, which may be conveniently used, e.g., in clinical settings or in home settings, to diagnose patients exhibiting preneoplastic or neoplastic abnormalities, and to screen and identify those individuals exhibiting a predisposition to such neoplastic changes.
- the present invention is useful for the diagnosis and prognosis of malignant diseases in which the ceramidase gene or gene product is implicated or is suspected to be implicated.
- malignancies include but are not limited to cancer of the liver, ovary, breast, lung, bladder, kidney, colon, rectum, prostate gland and cervix.
- the invention relates to a method for detecting in a sample the presence of a ceramidase nucleic acid, said method comprising: (a) contacting the sample with a nucleic acid probe capable of hybridizing to at least a portion of the nucleic acid molecule of claim 1 under hybridizing conditions; and (b) measuring the hybridization of the probe to the nucleic acids of the sample, thereby detecting the presence of the ceramidase nucleic acid.
- the invention in another embodiment, relates to a method for detecting in a sample the presence of the ceramidase nucleic acid, said method comprising: (a) contacting the sample with two diffferent nucleic acid primers capable of hybridizing to at least a portion of the nucleic acid molecule of claim 1 under hybridizing conditions; (b) selectively amplifying the portion of the nucleic acid molecule of claim 1 flanked by the two nucleic acid primers; and (c) detecting the amplified nucleic acid, thereby detecting the presence of the ceramidase nucleic acid.
- ceramidase gene expression can also be assayed.
- Nucleic acid from any nucleated cell can be used as the starting point for such assay techniques, and may be isolated according to standard nucleic acid preparation procedures which are well known to those of skill in the art.
- any nucleated cell can be used as a starting source for genomic nucleic acid.
- any cell type or tissue in which the ceramidase gene is expressed such as, for example, breast cancer cells or cells from inflamed tissues, may be utilized.
- Diagnostic methods for the detection of ceramidase gene specific nucleic acid molecules, in patient samples or other appropriate cell sources may involve the amplification of specific gene sequences, e.g., by the polymerase chain reaction (PCR; see Mullis, K.B., 1987, U.S. Patent No. 4,683,202), followed by the analysis of the amplified molecules using techniques well known to those of skill in the art.
- PCR polymerase chain reaction
- the isolated cells can be derived from cell culture or from a patient.
- the analysis of cells taken from culture may be a necessary step in the assessment of cells to be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the ceramidase gene.
- Such analyses may reveal both quantitative and qualitative aspects of the expression pattern of the ceramidase gene, including activation or inactivation of ceramidase gene expression and presence of mutations.
- a cDNA molecule is synthesized from an RNA molecule of interest by reverse transcription. All or part of the resulting cDNA is then used as the template for a nucleic acid amplification reaction, such as a PCR or the like.
- the nucleic acid reagents used as synthesis initiation reagents (e.g., primers) in the reverse transcription and nucleic acid amplification steps of this method are chosen from among the ceramidase gene nucleic acid reagents described in Section 5.1.
- the prefe ⁇ ed lengths of such nucleic acid reagents are at least 9-30 nucleotides.
- the nucleic acid amplification may be performed using radioactively or non-radioactively labeled nucleotides. In some cases, enough amplified product may be made such that the product may be visualized by standard ethidium bromide staining or by utilizing any other suitable nucleic acid staining method.
- RT-PCR techniques can be utilized to detect differences in ceramidase transcript size which may be due to normal or abnormal alternative splicing. Additionally, such techniques can be performed using standard techniques to detect quantitative differences between levels of full length and/or alternatively spliced ceramidase transcripts detected in normal individuals relative to those individuals having cancer or exhibiting a predisposition toward neoplastic changes.
- primers and/or hybridization probes can be used, such that, in the absence of such sequence, no amplification would occur.
- primer pairs may be chosen utilizing the sequence data depicted in Figure 3 to choose primers which will yield fragments of differing size depending on whether a particular exon is present or absent from the ceramidase transcript, or the choice of poly A signal being utilized.
- ceramidase gene expression assays "in situ", 1 e , directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid pu ⁇ fication is necessary
- Nucleic acid reagents such as those desc ⁇ bed m Section 5 1 may be used as probes and/or p ⁇ mers for such m situ procedures (see, for example, Nuovo, G J , 1992, "PCR In Situ Hyb ⁇ dization Protocols And Applications", Raven Press, NY)
- results obtained by the methods desc ⁇ bed herein may be combined with diagnostic test results based on other genes that are also implicated in the pathology of the cancer For example, K-tas and p53 mutations are often observed in patients
- Antibodies directed against wild type or mutant ceramidase gene products or conserved va ⁇ ants or peptide fragments thereof, which are discussed above, may also be used as diagnostics and prognostics, as desc ⁇ bed herein
- GFP- ceramdiase fusion protein may be used as diagnostics and prognostics
- Such diagnostic methods may be used to detect abnormalities m the level of ceramidase gene expression, or abnormalities in the structure and/or temporal, tissue, cellular, or subcellular location of ceramidase gene product
- the tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the ceramidase gene, such as, for example, breast cancer cells or mflammed cells
- the protein isolation methods employed herein may, for example, be such as those desc ⁇ bed in Harlow and Lane (Harlow, E and Lane, D , 1988, "Antibodies A Laboratory Manual", Cold Sp ⁇ ng Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, New York), which is mco ⁇ orated herein by reference in its entirety
- the isolated cells can be de ⁇ ved from cell culture or from a patient
- the analysis of cell taken from culture may be a necessary step to test the effect of compounds on the expression of the ceramidase gene
- Prefe ⁇ ed diagnostic methods for the detection of ceramidase gene products or conserved va ⁇ ants or peptide fragments thereof may involve, for example, immunoassays wherein the ceramidase gene products or conserved variants, including gene products which are the result of alternatively spliced transcripts, or peptide fragments are detected by their interaction with an anti-ceramidase gene product-specific antibody.
- antibodies, or fragments of antibodies, such as those described, above, in Section 5.4, useful in the present invention may be used to quantitatively or qualitatively detect the presence of ceramidase gene products or conserved variants or peptide fragments thereof in a sample, inside a cell or even inside an organelle, such as the mitochondria.
- the antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of ceramidase gene products or conserved variants or peptide fragments thereof. In situ detection may be accomplished by removing a histological specimen from a patient, such as paraffin embedded sections of breast tissues and applying thereto a labeled antibody of the present invention.
- the antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. It may also be desirable to introduce the antibody inside the cell, for example, by making the cell membrane permeable. Through the use of such a procedure, it is possible to determine not only the presence of the ceramidase gene product, or conserved variants or peptide fragments, but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection. Similarly, GFP-ceramidase fusion protein may also be used for the detection of ceramidase gene product in tissues.
- Immunoassays for ceramidase gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of identifying ceramidase gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
- the biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
- a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
- the support may then be washed with suitable buffers followed by treatment with the detectably labeled ceramidase gene specific antibody or a GFP-ceramidase fusion protein.
- the solid phase support may then be washed with the buffer a second time to remove unbound antibody or fusion protein.
- the amount of bound label on solid support may then be detected by conventional means.
- solid phase support or carrier any support capable of binding an antigen or an antibody.
- supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
- the nature of the carrier can be either soluble to some extent or insoluble for the pu ⁇ oses of the present invention.
- the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
- the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
- the surface may be flat such as a sheet, test strip, etc.
- Prefe ⁇ ed supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation. The binding activity of a given lot of anti-ceramidase gene product antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
- the present invention provides the measurement of ceramidase gene products, and the uses of such measurements in clinical applications.
- the measurement of ceramidase gene product of the invention can be valuable in detecting hype ⁇ roliferative disease, cardiovascular disease or inflammation in a subject, in screening of hype ⁇ roliferative disease, cardiovascular disease or inflammation in a population, in differential diagnosis of the physiological condition of a subject, and in monitoring the effect of a therapeutic treatment on a subject.
- the present invention also provides for the detecting, or diagnosing of cancer, or the monitoring of treatment of cancer by measuring in addition to ceramidase gene product at least one other marker, such as receptors or differentiation antigens.
- at least one other marker such as receptors or differentiation antigens.
- serum markers selected from, for example but not limited to, carcinoembryonic antigen (CEA), and prostate specific antigen (PSA) can be measured in combination with ceramidase gene product to detect, diagnose, stage, or monitor treatment of prostate cancer.
- the prognostic indicator is the observed change in different marker levels relative to one another, rather than the absolute levels of the markers present at any one time. These measurements can also aid in predicting therapeutic outcome and in evaluating and monitoring the overall disease status of a subject.
- the invention provides for treatment or prevention of various diseases and disorders by administration of a therapeutic compound (termed herein "Therapeutic”).
- a therapeutic compound include but are not limited to: ceramidase proteins and analogs and derivatives (including fragments) thereof; antibodies thereto; nucleic acids encoding the ceramidase proteins, analogs, or derivatives; ceramidase antisense nucleic acids, ribozyme, triplex DNA, and ceramidase agonists and antagonists.
- Ceramide modulates a number of biochemical and cellular responses to stress, including apoptosis, cell-cycle a ⁇ est and cell senescence.
- Several extracellular agents and stress stimuli such as tumor necrosis factor ⁇ , chemotherapeutic agents and heat are known to cause ceramide accumulation.
- One way of accumulating ceramide is accomplished by regulating enzymes such as ceramidase in its metabolism. Also, a large number of agonists and stress signals increase the level of ceramide.
- ceramide concentration is sufficient to reproduce many of the biological effects of cytokines and stress inducers that are coupled to ceramide accumulation.
- the accumulation of ceramides also reproduce most of the features of cell senescence.
- ceramides cause cell differentiation, both mo ⁇ hologically and through the activation of biochemical programmes of cell differentiation. Ceramide also causes apoptosis in most cancer cells which can be accompanied by cell-cycle a ⁇ est. Furthermore, there is evidence which suggests that ceramide is closely associated with
- TNF ⁇ -induced apoptosis TNF ⁇ -induced apoptosis.
- modulation of the levels of ceramide or sphingosine through the methods of the present invention can bring about treatment and prevention of diseases that are related to stress response and apoptosis.
- diseases and disorders are disclosed below which may be treated or prevented by the methods of the present invention.
- Disorders involving cell ove ⁇ roliferation or dysfunctional sphingolipid signal transduction are treated or prevented by administration of a Therapeutic that inhibits ceramidase function.
- Disorders in which cell proliferation is deficient or is desired can be treated or prevented by administration of a Therapeutic that promotes ceramidase function.
- a human ceramidase protein, derivative, or analog, or nucleic acid, or an antibody to a human ceramidase protein is therapeutically or prophylactically administered to a human patient. All such methods involve modulating ceramidase gene activity and/or expression which in turn modulate the level of ceramide, for example, in a cell or an organelle. The above is described in detail in the subsections below. Descriptions and sources of Therapeutics that can be used according to the invention are found in Sections 5.1 through 5.7 herein.
- the present invention provides a method of increasing the level of ceramide in a cell comprising contacting the cell with a compound that inhibits the ceramidase activity.
- the invention relates to a method of inhibiting the formation of sphingosine in a cell comprising contacting the cell with a compound that inhibits the ceramidase activity such that the amount of sphingosine formed as a result of conversion from ceramide is reduced.
- the invention relates to a method of increasing the intracellular levels of ceramide in an animal comprising administering to the animal an effective amount of a compound that inhibits the ceramidase activity of the ceramidase protein in the animal's cells.
- the invention in another embodiment, relates to a method of inhibiting the intracellular formation of sphingosine in an animal comprising administering to said animal an effective amount of compound that inhibits the ceramidase activity of the ceramidase protein in the animal's cells.
- Diseases and disorders involving cell ove ⁇ roliferation that can be treated or prevented include but are not limited to malignancies, premalignant conditions (e.g., hype ⁇ lasia, metaplasia, dysplasia), benign tumors, hype ⁇ roliferative disorders, benign dysproliferative disorders, etc.
- Cancer is characterized primarily by an increase in the number of abnormal cells derived from a given normal tissue, invasion of adjacent tissues by these abnormal cells, and lymphatic or blood-borne.
- Diseases and disorders involving cell ove ⁇ roliferation such as cancer are treated or prevented administration of a Therapeutic that inhibits (i e , decreases) ceramidase function
- a Therapeutic that inhibits (i e , decreases) ceramidase function
- Examples of such a Therapeutic include but are not limited to ceramidase proteins, de ⁇ atives, or fragments that are functionally inactive
- Other Therapeutics that can be used, e g , ceramidase antagonists can be identified using in vitro assays or animal models
- Therapeutics that inhibits ceramidase function are administered therapeutically (including prophylactically) (1) in diseases or disorders involving an increased (relat ⁇ e to normal or desired) level of ceramidase protein or function, for example, in patients where ceramidase protein is biologically overactive or overexpressed, or (2) in diseases or disorders wherein in vitro (or in vivo) assays (see infra) indicate the utility of ceramidase antagonist administration
- the increased level in ceramidase protein or function can be readily detected, e g , by obtaining a patient tissue sample (e g , from biopsy tissue) and assaying it in vitro for RNA or protein levels, structure and/or activity of the expressed ceramidase RNA or protein
- Many methods standard in the art can be thus employed, including but not limited to ceramidase enzyme assays, immunoassays to detect and/or visualize ceramidase protein (e g , Western blot, lmmunoprecipit
- malignancy or dysproliferative changes are treated or prevented in the bladder, breast, colon, lung, melanoma, pancreas, prostate or uterus.
- sarcoma, or leukemia is treated or prevented.
- the Therapeutics of the invention that inhibits ceramidase activity can also be administered to treat premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders listed in Table 3.
- Such prophylactic or therapeutic use is indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hype ⁇ lasia, metaplasia, or most particularly, dysplasia has occu ⁇ ed (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79.)
- the presence of one or more characteristics of a transformed phenotype, or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample from a patient can indicate the desirability of prophylactic/therapeutic administration of a Therapeutic that inhibits ceramidase function.
- Such characteristics of a transformed phenotype include mo ⁇ hology changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, etc. (see also id., at pp. 84-90 for characteristics associated with a transformed or malignant phenotype).
- Other disorders of proliferation that may benefit from inhibition of ceramidase include: psoriasis, autoimmune disorders such as lupus nephritis, glomerular diseases (with mesangial cell proliferation).
- vascular interventions including angioplasty, stenting, atherectomy and grafting for the treatment of cardiovascular diseases are often complicated by undesirable effects.
- One of the adverse reactions to vascular intervention include endothelial and smooth muscle cell proliferation which can lead to hype ⁇ lasia, or more specifically, restenosis which is the re-clogging of the artery, occlusion of blood vessels, reperfusion injury, platelet aggregation, and calcification.
- an injurious stimulus induces expression of growth-stimulatory cytokines such as interleukin 1 and tumor necrosis factor.
- ceramide inhibit the growth of endothelia and smooth muscle cells of the coronary artery.
- the outcome of a treatment is to at least produce in a treated subject a healthful benefit, which in the case of cardiovascular diseases, includes but is not limited to a reduced risk of re-clogging of arteries after a vascular intervention procedure, and improved circulation.
- Interleukin-1 is a major inducer of inflammation and TNF is an important regulator of the reaction.
- Both cytokines can activate ceramidase, and thus inhibiting the activity of ceramidase can result in an anti-inflammatory effect. This may involve the prevention of the formation of sphingosine and sphingosine phosphate which have pro- inflammatory effects. Also, inhibition of ceramidase may prevent the hype ⁇ roliferation of immune cells that are important for inflammation. There is evidence which suggests that an increase in ceramide and a decrease in sphingosine leads to a decrease in sphingosine phosphate.
- ceramide level can be increased by administering ceramidase antagonists such as a compound that inhibits ceramidase activity, ceramidase-antibodies, antisense and ribozyme molecules, and triple helix-forming molecules.
- ceramidase antagonists such as a compound that inhibits ceramidase activity, ceramidase-antibodies, antisense and ribozyme molecules, and triple helix-forming molecules.
- the present invention also relates to the treatment of disorders involving deficient cell proliferation (growth) or in which cell proliferation is otherwise desired (e.g., degenerative disorders, growth deficiencies, lesions, physical trauma) by administering compounds that agonize, (promote) ceramidase function (e.g., ceramidase, agonist of ceramidase, nucleic acids that encode ceramidase).
- Other disorders that may benefit from activation of cermidase are neurodegenerative disorders (e.g., Alzheimer's disease), and disorders of
- the gene of the human ceramidase of the invention is localized on chromosome 10 (lOql l)(i.e., LOC6392). Base on this location, ceramidase may be involved in diseases associated with this region, in addition to the disease and disorder discussed above, which include adenocarcinoma (thyroid), acute myeloid leukemia, and squamous cell cancer, especially that which is associated with the Nasopharynx region.
- diseases associated with this region in addition to the disease and disorder discussed above, which include adenocarcinoma (thyroid), acute myeloid leukemia, and squamous cell cancer, especially that which is associated with the Nasopharynx region.
- ceramidase activity can be decreased by, for example, directly decreasing ceramidase gene product activity and/or by decreasing the level of ceramidase gene expression.
- compounds such as those identified through assays described in Section 5.10 which decrease ceramidase activity can be used in accordance with the invention to treat cardiovascular diseases such as restenosis.
- such molecules can include, but are not limited to peptides, including soluble peptides, and small organic or inorganic molecules, and can be refe ⁇ ed to as ceramidase antagonists.
- antisense and ribozyme molecules which inhibit ceramidase gene expression can also be used in accordance with the invention to reduce the level of ceramidase gene expression, thus effectively reducing the level of ceramidase gene product present, thereby decreasing the level of ceramidase activity.
- triple helix molecules can be utilized in reducing the level of ceramidase gene activity.
- Such molecules can be designed to reduce or inhibit either wild type, or if appropriate, mutant ceramidase activity. Techniques for the production and use of such molecules are well known to those of skill in the art.
- the present invention relates to a method of treating a disease or disorder associated with cell ove ⁇ roliferation or sphingolipid signal transduction in an animal comprising administering to said animal a compound that inhibits the ceramidase activity in an amount sufficient to effect said inhibition.
- the present invention relates to a method of treating a disease or disorder associated with cell ove ⁇ roliferation or sphingolipid signal transduction in an animal comprising administering to the animal an effective amount of the nucleic acid molecule of claim 13 that targets ceramidase transcripts, and interferes with translation of ceramidase transcripts.
- the present invention relates to a method of treating a disease or disorder associated with cell ove ⁇ roliferation or sphingolipid signal transduction in an animal comprising administering to the animal an effective amount of the nucleic acid molecule of claim 14 that targets ceramidase transcripts, and interferes with translation of ceramidase transcripts.
- a delivery complex can comprise an appropriate nucleic acid molecule and a targeting means.
- targeting means can comprise, for example, sterols, lipids, viruses or target cell specific binding agents.
- Viral vectors that can be used with recombiant viruses include, but are not limited to adenovirus, adeno-associated virus, he ⁇ es simplex virus, vaccinia virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes.
- Gene therapy refers to treatment or prevention performed by the administration of a nucleic acid to a subject.
- the therapeutic nucleic acid produces intracellularly an antisense nucleic acid molecules that mediates a therapeutic effect by inhibiting ceramidase expression.
- the therapeutic nucleic acid comprises an antisense ceramidase nucleic acid that is part of an expression vector that produces the antisense molecule in a suitable host.
- a nucleic acid has a promoter operably linked to the antisense ceramidase sequence, said promoter being inducible or constitutive, and, optionally, tissue-specific.
- a nucleic acid molecule is used in which the antisense ceramidase sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antisense ceramidase nucleic acid (Koller and Smithies,
- nucleic acid into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector or a delivery complex, or indirect, in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient.
- direct in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector or a delivery complex
- indirect in which case, cells are first transformed with the nucleic acid in vitro, then transplanted into the patient.
- the present invention provides a delivery complex comprising an expression construct comprising the nucleic acid sequence of ceramidase gene, antisense, ribozyme, variant, or analog, wherein the nucleotide sequence is operatively associated with a regulatory nucleotide sequence containing transcriptional and/or translational regulatory signals that controls expression of the nucleotide sequence in a host cell, and a targeting means.
- the nucleic acid is directly administered in vivo, where it is expressed to produce the antisense nucleic acid molecule or encoded nonfunctional ceramidase gene product.
- a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
- the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated April 16, 1992 (Wu et al); WO 92/22635 dated December 23, 1992 (Wilson et al); WO92/20316 dated November 26, 1992 (Findeis et al); WO93/14188 dated July 22, 1993 (Clarke et al), WO 93/20221 dated October 14, 1993 (Young)).
- a specific receptor see, e.g., PCT Publications WO 92/06180 dated April 16, 1992 (Wu et al); WO 92/22635 dated December 23, 1992 (Wilson et al); WO92/20316 dated November 26, 1992 (Findeis et al); WO93/14188 dated July 22, 1993 (Clarke et al), WO 93/20221 dated October 14, 1993 (Young)
- the nucleic acid can be introduced intracellularly and inco ⁇ orated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al, 1989, Nature 342:435-438). It is observed that it can be difficult to achieve intracellular concentrations of the antisense sufficient to suppress translation of endogenous mRNAs. Therefore a prefe ⁇ ed approach utilizes a recombinant DNA construct in which the antisense oligonucleotide or polynucleotide is placed under the control of a strong promoter, some of which are described supra.
- Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
- Such vectors can be constructed by recombinant DNA technology methods standard in the art.
- Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells.
- a viral vector that contains the antisense ceramidase nucleic acid is used.
- a retroviral vector can be used (see Miller et al, 1993, Meth. Enzymol. 217:581-599). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA.
- the antisense ceramidase nucleic acid to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene into a patient.
- retroviral vectors More detail about retroviral vectors can be found in Boesen et al, 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
- Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al, 1994, J. Clin. Invest. 93:644-651; Kiem et al, 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Cu ⁇ . Opin. in Genetics and Devel 3:110-114.
- Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993, Cu ⁇ ent Opinion in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy.
- Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al, 1993, Proc. Soc. Exp. Biol. Med. 204:289-300.
- the form and amount of therapeutic nucleic acid envisioned for use depends on the cancer, desired effect, patient state, etc., and can be determined by one skilled in the art.
- the compounds and nucleic acid sequences described herein can be administered to a patient at therapeutically effective doses to treat or prevent diseases and disorder discussed above.
- a therapeutically effective dose refers to that amount of a compound sufficient to result in a healthful benefit in the treated subject.
- Formulations and methods of administration that can be employed when the therapeutic composition comprises a nucleic acid are described supra.
- Toxicity and therapeutic efficacy of compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50%> of the population).
- the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
- Compounds which exhibit large therapeutic indices are prefe ⁇ ed. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
- the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
- the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
- the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
- the therapeutically effective dose can be estimated initially from cell culture assays.
- a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
- IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
- levels in plasma can be measured, for example, by high performance liquid chromatography.
- compositions for use in accordance with the present invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
- the compounds and their physiologically acceptable salts and solvents can be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
- the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpy ⁇ olidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
- binding agents e.g., pregelatinised maize starch, polyvinylpy ⁇ olidone or hydroxypropyl methylcellulose
- fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
- lubricants e.g., magnesium stearate, talc or silica
- disintegrants e.g., potato
- Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
- Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
- the preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
- Preparations for oral administration can be suitably formulated to give controlled release of the active compound.
- compositions can take the form of tablets or lozenges formulated in conventional manner.
- the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluo
- the compounds can be formulated for parenteral administration (i.e., intravenous or intramuscular) by injection, via, for example, bolus injection or continuous infusion.
- parenteral administration i.e., intravenous or intramuscular
- Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
- the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
- the compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
- the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
- the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. 5.9.3 MONITORING THE EFFECT
- the present invention provides a method for monitoring the effect of a therapeutic treatment on a subject who has undergone the therapeutic treatment.
- Ceramidase gene product can be identified and detected in patients with hype ⁇ roliferative disease, cardiovascular disease or inflammation, different manifestations of disease, providing a sensitive assay to monitor therapy.
- the therapeutic treatments which may be evaluated according to the present invention include but are not limited to radiotherapy, surgery, chemotherapy, vaccine administration, endocrine therapy, immunotherapy, and gene therapy, etc.
- the chemotherapeutic regimens include, but are not limited to administration of drugs such as, for example, fluorouracil and taxol
- the method of the invention comprises measuring at suitable time intervals before, during, or after therapy, the amount of a ceramidase gene product. Any change or absence of change in the amount of the ceramidase gene product can be identified and co ⁇ elated with the effect of the treatment on the subject, such as, for example, a reduction of the disease phenotype of the patient.
- the approach that can be taken is to determine the levels of ceramidase gene product levels at different time points and to compare these values with a baseline level
- the baseline level can be either the level of the marker present in normal, disease free individuals; and/or the levels present prior to treatment, or during remission of disease, or during periods of stability. These levels can then be co ⁇ elated with the disease course or treatment outcome. Elevated levels of ceramidase gene product relative to the baseline level indicate a poor response to treatment.
- the present invention further provides methods for the identification of compounds that may, through its interaction with the ceramidase gene or ceramidase gene product, provide a therapeutic benefit to the recipient, especially one suffers from cancer, cardiovascular disease, or inflammatory conditions. It is known that ceramidase. the substrate; and sphingosine, the product, bind the enzyme ceramidase. In addition, some phosphatidic acid and cardiolipin can modulate the activity of ceramidase. Hence, these compounds may bind to ceramidase.
- the following assays are designed to identify: (i) compounds that inhibit ceramidase activity; (ii) compounds that bind to ceramidase gene products, including mammalian and non-mammalian homologs of ceramidase; (iii) compounds that bind to other intracellular proteins and/or segments of nucleic acid that interact with a ceramidase gene product, including mammalian and non-mammalian homologs of ceramidase; (iv) compounds that interfere with the interaction of the ceramidase gene product, including mammalian and non-mammalian homologs of ceramidase, with other intracellular proteins and/or segments of nucleic acid; and (iv) compounds that modulate the activity of ceramidase gene (i.e., modulate the level of ceramidase gene expression and/or modulate the level of ceramidase activity).
- Assays may additionally be utilized which identify compounds which bind to ceramidase gene regulatory sequences (e.g., promoter sequences). See e.g., Platt, 1994, J. Biol. Chem. 269:28558-28562, which is inco ⁇ orated herein by reference in its entirety. Also provided is a method for identifying compounds that modulate ceramidase gene expression, comprising: (a) contacting a test compound with a cell or cell lysate containing a reporter gene operatively associated with a ceramidase gene regulatory element; and (b) detecting expression of the reporter gene product.
- ceramidase gene regulatory sequences e.g., promoter sequences
- Also provided is another method for identifying compounds that modulate ceramidase gene expression comprising: (a) contacting a test compound with a cell or cell lysate containing ceramidase transcripts; and (b) detecting the translation of the ceramidase transcript.
- Any reporter gene known in the art can be used, such as but limited to, green fluorescent protein, ⁇ -galactosidase, alkaline phosphatase, chloramphenicol acetyltransferase, etc.
- an embodiment of the present invention is related to a method of identifying a compound that binds to a ligand selected from the group consisting of a ceramidase protein, a fragment of a ceramidase protein comprising a domain of the protein, and a nucleic acid encoding the protein or fragment, comprising: (a) contacting said ligand with a plurality of molecules under conditions conducive to binding between said ligand and the molecules; and (b) identifying a molecule within said plurality that binds to said ligand.
- One embodiment of the present invention is related to a method for identifying compounds that modulate ceramidase gene expression comprising: (a) contacting a test compound with a cell or cell lysate comprising an expression construct comprising a ceramidase gene; and (b) detecting the transcription or translation of the nucleotide sequence of ceradmidase.
- Another embodiment of the present invention is related to a method for identifying compounds that modulate ceramidase gene expression, comprising: (a) contacting a test compound with a cell or cell lysate containing a reporter gene operatively associated with the regulatory element of a ceradmidase gene; and (b) detecting expression of the reporter gene product.
- An embodiment of the present invention relates to a method for identifying compounds that modulate the activity of ceramidase gene product or homolog of ceramidase gene product comprising: (a) contacting a test compound with an organism or a cell containing ceramidase gene product or homolog of ceramdiase; and (b) comparing the phenotype of the organism or cell with the phenotype of organism or cell that did not contact the test compound, wherein a change in phenotype indicates that the test compound is capable of modulating the activity of ceramidase gene product or homolog of ceramdiase gene product.
- Enzyme assays may be used to detect or measure the ceramidase activity of a test substance, see for example, Section 6.1.
- the test substance may be a patient sample, cell lysate. a purified preparation of the enzyme, a mutant, a variant, or an analog of ceramidase. This is useful in evaluating whether a given substance has ceramidase activity. For example, this assay may be used to detect whether a given mutant, variant, analog of ceramidase has the ability to hydro lyse ceramide into sphingosine.
- the principle of the assays involves preparing a reaction mixture of the test substance and ceramide under conditions and for a time sufficient to allow the substance to convert the ceramide into sphingosine, if the substance has any ceramidase activity.
- the level of ceramide or sphingosine may be detected in the reaction mixture to determine the amount of ceramidase activity present in the test substance.
- the assays may be used in screening for compounds that inhibit the enzyme activity.
- the enzyme assay can be carried out using purified enzyme or a preparation containing ceramidase activity in screening.
- the test compound can be added directly to the assay reaction, or can be pre-incubated with the ceramidase enzyme. If the ceramidase activity deceases in the presence of a test compound, the test compound has an inhibitory effect on the ceramidase enzyme. If the ceramidase activity increases in the presence of a test compound, the test compound has an agonistic effect on the ceramidase enzyme. Any method, technique or assay format, including high throughput methods known in the art may be used together with this assay for drug screening. It is contemplated that the ceramidase enzyme assays of the invention are used to identify specific inhibitors of the ceramidase protein. Non-specific inhibitors, such as dithiothreitol, are less prefe ⁇ ed.
- In vitro systems may be designed to identify compounds capable of interacting with, e.g., binding to, the ceramidase gene products of the invention and homologs of ceramidase.
- Compounds identified may be useful, for example, in modulating the activity of wild type and/or mutant ceramidase gene products, may be useful in elaborating the biological function of the ceramidase gene product, may be utilized in screens for identifying compounds that disrupt normal ceramidase gene product interactions, or may in themselves disrupt such interactions.
- the principle of the assays used to identify compounds that interact with the ceramidase gene product involves preparing a reaction mixture of the ceramidase gene product, or fragments thereof and the test compound under conditions and for a time sufficient to allow the two components to interact with, e.g., bind to, thus forming a complex, which can represent a transient complex, which can be removed and/or detected in the reaction mixture.
- These assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring ceramidase gene product or the test substance onto a solid phase and detecting ceramidase gene product/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the ceramidase gene product or fragment thereof may be anchored onto a solid surface, and the test compound, which is not anchored, may be labeled, either directly or indirectly.
- microtitre plates may conveniently be utilized as the solid phase.
- the anchored component may be immobilized by non-covalent or covalent attachments.
- Non-covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying.
- an immobilized antibody preferably a monoclonal antibody, specific for the protein to be immobilized may be used to anchor the protein to the solid surface.
- the surfaces may be prepared in advance and stored.
- the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface.
- the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
- an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously nonimmobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).
- a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for ceramidase gene product or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes.
- Any method suitable for detecting protein-protein interactions may be employed for identifying ceramidase protein-intracellular protein interactions, especially interactions mediated by the various domain of the ceramidase protein.
- Another method that detects protein interactions in vivo is the two-hybrid system, which is described here for illustration only and not by way of limitation.
- This approach has been described (Chi en, et al., 1991, Proc. Natl Acad. Sci. USA, 88:9578-9582) and a kit is commercially available from Clontech (Palo Alto, CA).
- the two-hybrid system or related methodologies may be used to screen activation domain libraries for proteins that interact with a "bait" gene product.
- ceramidase gene products may be used as the bait gene product.
- Total genomic or cDNA sequences are fused to the DNA encoding an activation domain.
- This library and a plasmid encoding a hybrid of a bait ceramidase gene product fused to the DNA-binding domain are co-transformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene.
- a bait ceramidase gene sequence such as the open reading frame of the ceramidase gene, can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing is then used to identify the proteins encoded by the library plasmids.
- a cDNA library of the cell line from which proteins that interact with bait ceramidase gene product are to be detected can be made using methods routinely practiced in the art.
- the cDNA fragments can be inserted into a vector such that they are translationally fused to the transcriptional activation domain of GAL4.
- Such a library can be co-transformed along with the bait ceramidase gene-GAL4 fusion plasmid into a yeast strain that contains a lacZ gene driven by a promoter that contains GAL4 activation sequence.
- a cDNA encoded protein, fused to a GAL4 transcriptional activation domain that interacts with bait ceramidase gene product will reconstitute an active GAL4 protein and thereby drive expression of the HIS3 gene.
- Colonies that express HIS3 can be detected by their growth on petri dishes containing semi-solid agar based media lacking histidine. The cDNA can then be purified from these strains, and used to produce and isolate the bait ceramidase gene product-interacting protein using techniques routinely practiced in the art.
- the ceramidase gene products of the invention, fragments thereof, and homologs of ceramidase may, in vivo, interact with one or more intracellular macromolecules, such as proteins and nucleic acid molecules.
- intracellular macromolecules may include, but are not limited to DNA, RNA (including polyadenylated (poly(A)) RNA and RNA with the 5' cap structure) and those proteins identified via methods such as those described, above, in Section 5.6.2.
- intracellular macromolecules are refe ⁇ ed to herein as "interacting partners”.
- Compounds that disrupt ceramidase interactions in this way may be useful in regulating the activity of the ceramidase gene product, including mutant ceramidase gene products.
- Such compounds may include, but are not limited to molecules such as peptides, and the like, which would be capable of gaining access to the intracellular ceramidase gene product.
- the basic principle of the assay systems used to identify compounds that interfere with the interaction between the ceramidase gene product and its intracellular interacting partner or partners involves preparing a reaction mixture containing the ceramidase gene product, or fragments thereof, and the interacting partner under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex.
- the reaction mixture is prepared in the presence and absence of the test compound.
- the test compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of ceramidase gene product and its intracellular interacting partner. Control reaction mixtures are incubated without the test compound or with a vehicle or carrier.
- any complexes between the ceramidase gene product or fragments thereof and the intracellular interacting partner is then detected.
- the formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the ceramidase gene protein and the interacting partner.
- complex formation within reaction mixtures containing the test compound and normal ceramidase gene protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant ceramidase gene protein. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal ceramidase gene proteins.
- Cell-based methods are presented herein which identify compounds capable of treating cancer cardiovasular diseases. Inflammation, or hypoproliferative disorders by modulating ceramidase activity. Specifically, such assays identify compounds which affect ceramidase-dependent processes, such as but not limited to changes in cell mo ⁇ hology, cell division, differentiation, adhesion, motility, or tumorigenicity.
- the cell-based assays are based on expression of the ceramidase gene product in a mammalian cell and measuring the ceramidase-dependent process.
- Any mammalian cells that can express the ceramidase gene and allow the functioning of the ceramidase gene product can be used, in particular, cancer cells derived from the prostate gland.
- Other cancer cell lines such as those derived from prostate, liver, ovary, breast, lung, rectum, kidney and non-erythroid hemopoietic cells, may also be used provided that a detectable ceramidase gene product is produced. Recombinant expression of the ceramidase gene in these cells or other normal cells can be achieved by methods described above.
- cells producing functional ceramidase gene products are exposed to a test compound for an interval sufficient for the compound to modulate the activity of the ceramidase gene product.
- the activity of ceramidase gene product can be measured directly or indirectly through the detection or measurement of ceramidase- dependent cellular processes such as, for example, the manifestation of a transformed phenotype.
- a cell not producing the ceramidase gene product may be used for comparisons.
- any techniques known in the art may be applied to detect or measure it.
- ceramidase Using peptide sequences obtained from a purified rat brain ceramidase, a human isoform of ceramidase is identified.
- This example demonstrates the cloning and expression of human ceramidase gene; and nucleic acid and amino acid sequence analysis of the human ceramidase gene.
- the human ceramidase gene product was found to be localized in the mitochondria. The experiment also showed that a ceramidase inhibitor inhibited ceramidase activity and reduced the viability of human breast cancer cells.
- MATERIALS AND METHODS Frozen rat brains were purchased from Pel-Freez Biologicals (Rogers, AK).
- Hitrap Q-Sepharose high performance Hitrap blue Sepharose high performance.
- MonoS (H/R 5/5), MonoP (HR 5/5), and Superose 12 (HR 10/30) columns and phenyl-Sepharose high performance, polybuffer 96, and polybuffer 74 media were purchased from Amersham Pharmacia Biotech. Centriprep and Centricon sample concentrators were from Amicon, Inc. (Beverly, MA). Pro-blue staining and silver staining kits were from Owl Separation Systems (Portsmouth, NH). Triton X-100 was from Sigma.
- Frozen rat brains (53-58g) were thawed in 150 ml of 20mM cold phosphate buffer (pH7.4) containing 0.25M sucrose, ImM EDTA, and 0.2mM phenylmethylsulfonyl fluoride (homogenization buffer). Brains were then homogenized using a Dounce homogenizer. The homogenate was centrifuged at 1 ,000 X g for 10 mins, and the pellet of this centrifugation was homogenized again using 80 ml of homogenization buffer. After centrifugation at 1,000 X g for 10 mins, the pellet was washed twice with 50 ml of homogenization buffer.
- the triton X-100 solubilized fraction was applied to a Q-Sepharose column equilibrated with buffer A. After washing the column, CDase activity was eluted with a linear gradient from 0 to 0.3M NaCl in buffer A. Fractions of 5ml were collected.
- the active fraction obtained from Q-sepharose NaCl peak
- the active fractions obtained from blue Sepharose were adjusted to 0.6M NaCl and applied to a phenyl-Sepharose column equilibrated with buffer C. After washing the column with buffer C and eluting more proteins by decreasing the NaCl concentration, CDase activity was eluted with a Triton X-100 gradient (0-0.5%) in lOmM Tris buffer, pH 7.5. Fractions of 1 ml were collected. CDase activity and proteins were measured as described.
- Rat brain enzyme was purified as described above and also in El Bawab et al, 1999, J. biol Chem. 274: 27948-27955. Three preparations of 100-120 rat brains each were used.
- the purified protein from the last column was subjected to SDS-polyacrylamide gel electrophoresis, the gel was stained directly with Coomassie Blue or transfe ⁇ ed to polyvinylidene difluoride membrane using CAPS buffer, pH 11 , as transfer buffer, and the membrane was then stained.
- the ceramidase (CDase) band was excised from the gel or from the membrane and subjected to digestion using AspN.
- the digest mixture was separated by microcapillary reversed phase HPLC, and selected peptides were submitted to Edman degradation and sequencing.
- ceramidase Cloning of ceramidase The sequences of the obtained peptides were used to search the Genbank data base. The peptides identified a putative slug protein (accession no. 2367392) and two human sequences (accession no. AA913512 and AC012131). The following primers were synthesized: forward primer based on the AC012131, CTGAGTGGCACTCACACTCATTCAGGT; and the reverse primer based on the EST AA913512, GGCTTCAGAATGTCCTGCTTCCGA. PCR amplification was performed using the human kidney RACE library as a template. A 1.8-kb fragment was obtained.
- New primers were then designated on the 5'- (reverse, ACCTGAATGAGTGTGAGTGCCACTCAG) and 3'- (forward, TTCGGGGATGTCCTGCAGCCAGCAAAACCTGAATACAG) ends of the 1.8-kb fragment to perform touch down PCR.
- a 5 '-end fragment of 0.7kb and a 3'-end fragment of 0.6 kb were obtained.
- Assembling the 1.8-kb fragment and the 5'- and 3 '-ends fragments resulted in a fragment of around 2.5 kb, with a putative open reading frame of 2289 base pairs.
- the full-length CDase fragment was generated by PCR using the forward primer ATGAGTGCCATCACAGTGGCCCTTCTC starting at the longest start codon and the reverse primer ACTAAATAGTTACAACTTCAAAAGCCGGG.
- the forward primer also contained the Kpnl site sequence, and the reverse primer contained the Apal site sequence.
- PCR amplification was performed at a denaturing temperature of 94 °C for 1 min followed by annealing at 65 °C for 2.5 min and extension at 72 °C for a total of 35 cycles.
- the amplified fragment (2289 base pairs) was separated by electrophoresis on 1.5% agarose gel. After purification, the full-length cDNA was subcloned into TOPO blunt end cloning vector. Sequencing, using T7 and Ml 3 reverse primers of the TOPO inserts, revealed multiple full-length clones in the sense and in the antisense direction.
- the sense fragments were named TOPO-CDase and were used to construct the pcDNA3.1/ ⁇ isC-CDase vector.
- TOPO-CDase vector was digested with Kpnl and Apal overnight. The resulting fragment was gel-isolated and subcloned into the same sites in pcDNA3.1/HisC vector, the His-tag being at the N terminus of ceramidase.
- pEGFPC3-CDase vector To construct pEGFPC3-CDase vector, the open reading frame of CDase cDNA was first amplified as described above. The amplified product was then digested by restriction enzymes Kpnl and Apal and cloned into Kpnl and Apal sites of the vector pEGFPC3, thus generating a GFP tag at the N terminus of ceramidase protein. The sequence and orientation of the fragments were then confirmed by sequencing.
- the radioactive probe was then denaturated by boiling for 2 mm and added to the blot in ExpressHyb solution. Hybridization was carried out overnight at 65 °C. After washing, the blot was exposed to x-ray film for 5 days at - 80°C.
- HEK 293 cells, human embryonic kidney cells, and MCF-7 cells were seeded at 10 5 cells/dish.
- Transfection with vector alone (pcDNA3.1/ ⁇ isC) or vector containing full-length CDase (pcDNA3.1/HisC-CDase) was performed using Superfect and 3 ⁇ g of each plasmid/dish. After 3-4 h of incubation with the mixture, the cells were washed with phosphate-buffered saline and fresh medium was added. After 48 h, CDase activity was measured.
- lysis buffer 50 mM Tris, pH 7.4, 5 mM EDTA, 1% Triton X-100. 300mM NaCI, 1 mM phenylmethylsulfonyl fluoride, and 2 ⁇ g/ml of leupeptin and aprotinin
- lysates were centrifuged at 12,000 x g for 15 min. Samples (10 ⁇ g of lysates) were then boiled for 5 min, loaded onto a 7.5% SDS-polyacrylamide gel, electrophoresed, and transfe ⁇ ed to a nitrocellulose membrane.
- the GFP-CDase fusion protein was detected by using anti-GFP affinity purified antibody at a dilution of 1 : 1000 and a anti-rabbit secondary antibody at a dilution of 1 :3000.
- cell lysates were first precleared by incubating with 30 ⁇ l of a mixture of protein A/protein G agarose beads for 30 min followed by centrifugation at 12,000 x g for 1 min. The cleared lysates were then rocked in the presence of 5 ⁇ g of anti-GFP antibody or 5 ⁇ g of control IgG complexed to a mixture of protein
- CDase activity was measured as described in El Bawab et al, 1999, J. Biol. Chem. 274(39): 27948-27955; and Yavin et al, 1969, Biochem. 8: 1692-1698, using [ 3 H]C 16 -ceramide as substrate in a mixed micelle assay system. Briefly, lOnmol of [ 3 H]C 16 - ceramide were mixed with 1 OO ⁇ l of Triton X- 100 (0.2%) and 1 OO ⁇ l of sodium cholate
- Cells were plated on 35-mm diameter glass coverslips. They were transfected with 1 ⁇ g of empty vector or vector-containing ceramidase as described above. After 48 h, the cells were loaded with 25 nM Mitotracker Red for 20 min and then washed with phosphate-buffered saline and fixed. For confocal microscopy, images were collected by Zeiss 410 LSCM system equipped with krypton Argon laser and a 60 X oil merge lens (N.A 1.4). After 48 h of transfection, cells plated on glass coverslips were mounted on a microscopy stage and maintained in phosphate-buffered saline buffer.
- GFP images were collected by excitation at 488 nm and emission at 516-560 nm. To label mitochondria, cells were subsequently co-loaded with 50 nM TMRM. The TMRM images were then taken by excitation at 568 nm and emission at 590 nm long-path emission filter. To avoid fluorescent cross-talking, green GFP and red TMRM fluorescence were taken sequentially.
- Results are expressed as % of control value, control being the activity in the absence of the Urea-C 16-ceramide.
- MTT 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl tetrazolium bromide
- Day 1 50 ul cells (-5,000 cell) were plated into each well of 96 well- plate; day 2: 50 ul of 2x concentration of Urea-C 16-Ceramide were added into each well; day 3: 25 ul of MTT from a stock of 5 mg/ml in PBS were added to each well and incubated for 5 hours; 100 ul of lysing buffer which contains: 20% SDS (w/v), 50% N,N Dimethyl formamide (v/v), 1% acetic acid (v/v), and 50 % water (v/v) were added to each well and let stand overnight; day 4: absorbance was read using a spectrophotometer at 595 nm.
- a rat brain CDase was purified to homogeneity with a pH optimum in the neutral to alkaline range. El Bawab et al, 1999, J. Biol. Chem. 274:27948-27955. The scale up of the purification protocol was optimized to obtain high amounts of the protein. In each preparation (100-120 rat brains), 1-10 ⁇ g of CDase protein were obtained (visible by Coomassie Blue). After digestion and HPLC separation of the AspN digest, three peptide sequences of 14-17 amino acids (Table 2) were obtained. The data base of the GenBankTM was searched using peptides 1 and 2, and the same human EST sequence (accession no.
- AA913512 was identified by both peptides. Using the human EST, all three peptides were searched and identified a putative slug protein (accession no. 2367392). The human EST aligned at the C terminus of the slug protein. Next, the slug protein was used to search the GenBankTM Data base, and this yielded a human genomic sequence of 15,960 kb (accession no. AC012131), which aligned with a region close to the N terminus of the slug protein.
- New primers of both ends of the 1.8-kb human fragment were synthesized, and RACE-PCR was performed using a human kidney RACE library as template. After two rounds of PCR, a 5'-end fragment of 0.7 kb and a 3'-end fragment of 0.6 kb were obtained. These fragments were then gel-isolated, subcloned into TOPO vector, and sequenced. The fragments contained the primer sequences, part of the 1.8 kb-fragment, and did not identify any EST in the GenBankTM of known function, indicating that these fragments most probably co ⁇ espond to the extension of the 5'-end and the 3'-end of the 1.8-kb human fragment. The 5 '-end fragment contained multiple start ATG codons in frame, and the
- 3 '-end contained two stop codons next to each other. Taking the first ATG (longest) as start codon and the double stop codon at the 3 '-end as stop codon, an open reading frame of 2289 base pairs encoding a protein of 84 kDa was predicted.
- the CDase amino acid sequence showed no similarity to any known mammalian protein.
- the protein was homologous to three putative proteins from Arabidospis thaliana (accession no. AAD32770), Mycobacterium tuberculosis (accession no. CAB09388), and Dictyostelium discoideum (accession no. 2367392) (Fig. 4), indicating that these proteins may be ceramidases in those organisms. There were several blocks highly conserved in all of these proteins, and the overall homology between the human and those proteins ranged between 30 and 50%.
- ceramidase To determine tissue distribution of this ceramidase, we performed Northern blot analysis using the 3 '-end of CDase cDNA (0.67 kb) as a probe and a human premade multitissue Northern blot, Fig. 5 shows the presence of a minor high size band at around 7 kb, a major band of 3.5 kb, and two other minor bands of 3.1 and 2.4 kb. The presence of multiple bands could be the result of alternative splicing. The major 3.5-kb ceramidase band was ubiquitously expressed in all tissue represented on the blot, with the highest expression in kidney, skeletal muscle, and heart.
- a GFP- tagged ceramidase was constructed, in which the GFP was at the N terminus of ceramidase protein.
- 293 cells were transfected with this construct and performed Western blot and immunoprecipitation experiments using GFP antibody.
- Fig. 6B cells overexpressing the fusion protein contain a GFP -positive band at around 123 kDa, this band being absent in control cells transfected with the pEGFPC3 empty vector. Based on GFP molecular mass (27 kDa), CDase molecular mass was deduced to be around 96 kDa.
- Fig. 4C shows the pH profile of the human CDase.
- the enzyme catalyzed the hydrolysis of ceramide in a relatively broad range with a pH optimum between pH 7.5 and 9.5.
- the effect of EDTA, MgCl 2 , and CaCl 2 (all at 10 mM) were tested and found that they did not affect significantly ceramidase activity.
- Dithiothreitol at 20 mM was found to inhibit the activity by 75% (Fig. 6D).
- the predicted isoelectric point value was 6.69. All these properties are in close agreement with the purified rat brain enzyme.
- FIG. 9A is a graph expressed as ceramidase activity measured in % of control ceramidase activity verses concentration of Urea-C 16 -ceramide.
- the effect of Urea-C 16 -ceramide was tested for inhibition using the purifed enzyme (5-10ng of protein in the assay) and [ 3 H]-C 16 -ceramide as substrate in a mixed micelles system as described above. The result shows that Urea-C, 6 - ceramide is an inhibitor of rat brain ceramidase. Increase in concentration of inhibitor decreases the ceramidase activity.
- the first mammalian mitochondrial ceramidase was cloned and characterized as described above.
- the enzyme has characteristics similar to the rat brain purified enzyme in its estimated molecular mass, isoelectric point, optimum pH, and dependence on cations (El Bawab et al, 1999, J. Biol. Chem. 274: 27948-27955).
- ceramidase enzymes in bacteria, plant, and mammals have been reported.
- Okino et al. published the cloning of an alkaline ceramidase from Pseudomonas aeruginosa (accession no. 6594292).
- Tani et al. published the purification of a ceramidase protein from mouse liver (Tani et al, 2000, J. Biol. Chem. 275: 3462-3468). The sequence of this protein was also homologous to the M. tuberculosis GenBankTM putative protein.
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WO2002051439A2 (en) * | 2000-12-22 | 2002-07-04 | Medlyte, Inc. | Compositions and methods for the treatment and prevention of cardiovascular diseases and disorders, and for identifying agents therapeutic therefor |
US7829674B2 (en) | 2006-10-27 | 2010-11-09 | Lpath, Inc. | Compositions and methods for binding sphingosine-1-phosphate |
US8871202B2 (en) | 2008-10-24 | 2014-10-28 | Lpath, Inc. | Prevention and treatment of pain using antibodies to sphingosine-1-phosphate |
WO2015177329A1 (en) * | 2014-05-23 | 2015-11-26 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for determining whether a patient will achieve a response after radiation therapy |
US9217749B2 (en) | 2006-05-31 | 2015-12-22 | Lpath, Inc. | Immune-derived moieties reactive against lysophosphatidic acid |
US9274130B2 (en) | 2006-05-31 | 2016-03-01 | Lpath, Inc. | Prevention and treatment of pain using antibodies to lysophosphatidic acid |
US9274129B2 (en) | 2006-05-31 | 2016-03-01 | Lpath, Inc. | Methods and reagents for detecting bioactive lipids |
CN111285930A (en) * | 2020-03-11 | 2020-06-16 | 北京诺思兰德生物技术股份有限公司 | Nucleic acid molecule for coding parathyroid hormone protein or fragment thereof |
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US9217749B2 (en) | 2006-05-31 | 2015-12-22 | Lpath, Inc. | Immune-derived moieties reactive against lysophosphatidic acid |
US9274129B2 (en) | 2006-05-31 | 2016-03-01 | Lpath, Inc. | Methods and reagents for detecting bioactive lipids |
US9274130B2 (en) | 2006-05-31 | 2016-03-01 | Lpath, Inc. | Prevention and treatment of pain using antibodies to lysophosphatidic acid |
US8025877B2 (en) | 2006-10-27 | 2011-09-27 | Lpath, Inc. | Methods of using humanized antibodies and compositions for binding sphingosine-1-phosphate |
US8067549B2 (en) | 2006-10-27 | 2011-11-29 | Lpath, Inc. | Humanized antibodies and compositions for binding sphingosine-1-phosphate |
US8026342B2 (en) | 2006-10-27 | 2011-09-27 | Lpath, Inc. | Compositions and methods for binding sphingosine-1-phosphate |
US7956173B2 (en) | 2006-10-27 | 2011-06-07 | Lpath, Inc. | Nucleic acids coding for humanized antibodies for binding sphingosine-1-phosphate |
US7829674B2 (en) | 2006-10-27 | 2010-11-09 | Lpath, Inc. | Compositions and methods for binding sphingosine-1-phosphate |
US8871202B2 (en) | 2008-10-24 | 2014-10-28 | Lpath, Inc. | Prevention and treatment of pain using antibodies to sphingosine-1-phosphate |
WO2015177329A1 (en) * | 2014-05-23 | 2015-11-26 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for determining whether a patient will achieve a response after radiation therapy |
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CN111285930A (en) * | 2020-03-11 | 2020-06-16 | 北京诺思兰德生物技术股份有限公司 | Nucleic acid molecule for coding parathyroid hormone protein or fragment thereof |
CN111285930B (en) * | 2020-03-11 | 2022-02-18 | 北京诺思兰德生物技术股份有限公司 | Nucleic acid molecule for coding parathyroid hormone protein or fragment thereof |
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WO2001055410A9 (en) | 2002-10-17 |
WO2001055410A3 (en) | 2002-03-14 |
AU2001233086A1 (en) | 2001-08-07 |
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