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WO1999061581A2 - Clonage, expression et procedes d'utilisation de la sphingosine kinase - Google Patents

Clonage, expression et procedes d'utilisation de la sphingosine kinase Download PDF

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WO1999061581A2
WO1999061581A2 PCT/US1999/011521 US9911521W WO9961581A2 WO 1999061581 A2 WO1999061581 A2 WO 1999061581A2 US 9911521 W US9911521 W US 9911521W WO 9961581 A2 WO9961581 A2 WO 9961581A2
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sphingosine kinase
spp
cell
sphk
sphingosine
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PCT/US1999/011521
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WO1999061581A3 (fr
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Sarah Spiegel
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Office Of The Dean Of Research And Graduate Education
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Priority to EP99924494A priority Critical patent/EP1235913A2/fr
Priority to AU40979/99A priority patent/AU4097999A/en
Priority to CA002329124A priority patent/CA2329124A1/fr
Publication of WO1999061581A2 publication Critical patent/WO1999061581A2/fr
Publication of WO1999061581A3 publication Critical patent/WO1999061581A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to nucleic acid molecules encoding sphingosine kinase including mutants, variants, fragments and derivatives thereof, and to vectors and host cells comprising such nucleic acid molecules; methods of using sphingosine kinase due to its effects on cell motility, cell proliferation and cell death; method for screening for inhibitors of sphingosine kinase; and kits comprising the compositions or polypeptides of the invention.
  • the present invention also relates to methods for making sphingosine-1-phosphate using the compositions of the present invention and kits for use in such methods .
  • Sphingosine-1-phosphate SPP
  • SPP Sphingosine-1-phosphate
  • SPHK sphingosine kinase
  • PDGF platelet-derived growth factor
  • NGF NGF
  • vitamin D3 Kleuser et al . , 1998, Cancer Res . 58, 1817
  • PKC PKC
  • cAMP activators Cuvillier et al . , 1996, Nature, 381, 800; Machwate et al., 1998, Mol . Pharmacology 54 , 70
  • calcium mobilization induced by Fc ⁇ Rl, Fc ⁇ Rl, and muscarinic acetylcholine receptors Choi et al . , 1996, supra; Melendez et al . , 1998, supra; Meyer zu Heringdorf et al . , 1998, supra).
  • microinjected SPP mobilizes calcium from internal sources (Meyer zu Heringdorf et al . , 1998, supra), is mitogenic for Swiss 3T3 fibroblasts (Van Brooklyn et al., 1998, J. Cell Biol . 142, 229), and inhibits apoptosis of mouse oocytes induced by the anti-tumor drug doxorubicin (Perez et al . , 1997, Nature Med. 3, 1228) .
  • SPP acts intracellularly to regulate calcium mobilization, cell growth, and survival.
  • SPP receptors couple to three different Gas and ⁇ dimers to signal through cAMP, Ras , and mitogen-activated protein kinase, Rho, phospholipase C, and several protein tyrosine kinases (Goetzl and An, 1998, FASEB J. 12, 1589; Gonda et al . , 1999, Biochem . J. 337, 67; Lee et al . , 1998, Science 279, 1552; Okamoto et al . , 1998, J. Biol . Chem. 273, 27104; Van Brooklyn et al .
  • SPP is stored in high concentrations in human platelets, from which it is released upon activation by physiological stimuli (Yatomi et al . , 1997a, J “ . Biochem . 86, 193; Yatomi et al . , 1997b, J “ . Biol . Chem . 272, 5291), suggesting that SPP can also be considered as an autocrine factor involved in endothelial injury, inflammation, thrombosis, and angiogenesis .
  • the present invention meets the need described above.
  • SPHK to apparent homogeneity from rat kidney (Olivera et al . , 1998, J. Biol . Chem. 273, 12576-12583) and subsequently cloned and characterized the first mammalian SPHKs (murine SPHKla and SPHKlb) (Kohama et al . , 1998, J. Biol . Chem . 273, 23722-23728) .
  • Sequence analyses identified homologs of SPHK in numerous widely disparate organisms, demonstrating that SPHK is a member of a novel but highly conserved gene family and is distinct from other known lipid kinases .
  • SPHK caused a decrease in the levels of sphingosine and, to a lesser extent, ceramide, with a concomitant marked increase in the mass of endogenous SPP (Kohama et al . , 1998, supra) and can be used to clarify its intracellular functions.
  • transfection of cells with a SPHK expression vector confers serum-independent growth, increases proliferation, and suppresses serum-deprivation or ceramide-induced apoptosis .
  • intracellular formation of SPP inhibits motility and chemotaxis, events which were previously ascribed to binding of SPP to cell surface receptors.
  • the DNA fragment is useful as a diagnostic agent, an agent for preparation of sphingosine-1-phosphate, and a therapeutic agent.
  • It is yet another object of the present invention to provide a method for detecting sphingosine kinase in a sample comprising: (i) contacting a sample with antibodies which recognize sphingosine kinase; and
  • detecting the presence or absence of a complex formed between sphingosine kinase and antibodies specific therefor It is a further object of the present invention to provide a diagnostic kit comprising an antibody against sphingosine kinase and ancillary reagents suitable for use in detecting the presence of sphingosine kinase in cells, tissue or serum from yeast, mammals, animals, birds, fish, and plants. It is yet another object of the present invention to provide a method for the detection of sphingosine kinase from a sample using the polymerase chain reaction.
  • It is a further object of the present invention to provide a diagnostic kit comprising primers or oligonucleotides specific for sphingosine kinase RNA or cDNA suitable for hybridization to sphingosine kinase RNA or cDNA and/or amplification of sphingosine kinase sequences and ancillary reagents suitable for use in detecting sphingosine kinase RNA/cDNA in mammalian tissue.
  • the method comprises separating SPP form other phospholipids, converting isolated SPP to sphingosine to produce converted SPP, rephosporylating coverted SPP using a detectably labeled phosphate, and measuring the detectable label produced.
  • Figure 1 Predicted amino acid sequences of two murine sphingosine kinases (SEQ ID NO:l and SEQ ID NO:2). The numbers on the right refer to the amino acid sequence of SPHKla. Sequences homologous to the 8 tryptic peptides (Table 1) that were obtained from purified rat kidney sphingosine kinase are underlined. Calmodulin binding motifs are indicated by dashes . Potential PKC and cAMP-dependent phosphorylation sites are indicated by (*) and (0), respectively.
  • FIG. 1 Tissue specific expression of sphingosine kinase.
  • a sphingosine kinase probe was hybridized to poly(A) + RNA from the indicated mouse tissues (2 ⁇ g/lane) as described in Experimental
  • Lanes 1, heart; 2, brain; 3, spleen; 4, lung; 5, skeletal muscle; 6, liver; 7, kidney; 8, testis.
  • the numbers underneath indicate relative levels of expression compared to background as quantified with the Molecular Dynamics Phosphorlmager.
  • Figure 4 Activity of sphingosine kinase expressed in H ⁇ K293 cells.
  • HEK293 cells were transiently transfected with the pCMVSPORT2 expression vector alone (open circles) or containing either the SPHKla cDNA (pCMVSPORT2sphkla, filled triangles) or the SPHKlb cDNA
  • FIG. 1 Distribution of sphingosine kinase in transfected HEK293 cells. Sphingosine kinase activity in cytosolic (open bars) and membrane (filled bars) fractions. The data are expressed as percent of the total activity.
  • Sphingosine kinase has marked specificity for D-(+)-e ⁇ f ⁇ Vo-sphingosine.
  • HEK293 cells were transfected with pCMVSP0RT2sphkla (filled bars) or pCMVSP0RT2sphklb (open bars) and sphingosine kinase-dependent phosphorylation of various sphingosine analogs or other lipids (50 ⁇ M) was measured in cell lysates as described under "Experimental Procedures". Data are expressed as percentage of phosphorylation of D-erythro- sphingosine.
  • Sphingosine kinase activity in HEK293 cell lysates after transfection with pCMVSP0RT2sphkla was measured with varying concentrations of D-ery thro-sphingosine in the absence (open circles) , or presence of 20 ⁇ M (filled squares) or 40 ⁇ M (filled triangles) N,N-dimethylsphingosine (B) , or presence of 10 ⁇ M (filled squares) or 20 ⁇ M (filled triangles) D, L- threo-dihydrosphingosine (C) .
  • Km ( ⁇ M) and Vmax (nmol/min/mg) values D-(+)- erythro- sphingosine, 2.15 and 46.7. Ki values are 2.12 for D,L- t reo-dihydrosphingosine and 2.64 for N,N-dimethylsphingosine .
  • FIG. 7 Changes in mass levels of sphingolipid metabolites in NIH 3T3 cells overexpressing sphingosine kinase. Mass levels of SPP (A) , sphingosine (B) , and ceramide (C) in NIH 3T3 cells transfected with empty vector (Control) or with pCMVSP0RT2sphkla were measured after 48 h as described under "Experimental Procedures" .
  • FIG. 9 Cellular localization of c-myc- sphingosine kinase.
  • NIH3T3 fibroblasts transiently or stably expressing SPHK and HEK293 cells stably expressing SPHK were incubated with a monoclonal c- myc antibody (20 ⁇ g/ml) , and stained with anti-mouse Texas Red-conjugated IgG (1:100 dilution) (left panels) or anti-mouse FITC-monoclonal IgG (1:10 dilution) (right panels) .
  • Fluorescence micrographs (60X) were taken using a Nikon Eclipse TE200 inverted fluorescent microscope. Vector transfectants (c-myc- pcDNA3) did not show any significant fluorescence.
  • FIG. 10 Expression of sphingosine kinase stimulates DNA synthesis.
  • NIH 3T3 fibroblasts were transiently transfected with empty vector (pCMV- SPORT2, filled bars) or pCMV-SPORT2-SPHKla (open bars) together with pCEFL-GFP.
  • Cells were serum-starved for 18 h and incubated in serum-free media without (None) or with PDGF (1 ng/ml), FBS (0.1%), or SPP (10 ⁇ M) . After 16 h, BrdU was added for an additional 3 h.
  • Double immunofluorescence was used to visualize transfected cells and BrdU incorporation, and the proportion of cells incorporating BrdU among total transfected cells (expressing the green fluorescent protein GFP) was determined. Data are means ⁇ SD of duplicate cultures from a representative experiment. At least three different fields were scored with a minimum of 100 cells scored per field. Similar results were obtained in three independent experiments .
  • FIG. 11 Expression of sphingosine kinase stimulates growth of NIH 3T3 fibroblasts.
  • A Cells stably transfected with c- myc-pcDNA3 or with c-myc-SPHK-pcDNA3 were plated at low density, washed after 24 h, and cultured in 0.5% calf serum for the indicated days. Media was replaced every two days and the cells were pulsed 18 h after the last media change with [ 3 H] thymidine for an additional 6 h. Data are means ⁇ SD of three independent determinations and are expressed as fold increase of the value determined after 1 day. Similar results were obtained in three independent experiments .
  • B Representative images of cells stably expressing c-myc-SPHK or transfected with vector after 1 and 6 days in 0.5% calf serum. Micrographs were taken using a phase contrast microscope at 40X magnification.
  • FIG. 12 Expression of sphingosine kinase reduces apoptosis.
  • A NIH 3T3 fibroblasts were transiently transfected with vector (filled squares) or SPHK (open squares) , together with pCEFL- GFP, as described in the legend to Fig. 3, and serum starved for the indicated times .
  • Total GFP- expressing cells and GFP-expressing cells displaying fragmented nuclei indicative of apoptosis were counted (Van Brocklyn et al . , 1999, J. Biol . Chem. 274, 4626-4632) .
  • a minimum of 500 cells in each field were scored. Data are mean + SE of three independent experiments, each one done in duplicate or triplicate.
  • CS+Staur Percentages of apoptotic cells were determined by Hoechst staining and visualization by fluorescence microscopy.
  • C Note the typical condensed fragmented nuclei of apoptotic cells in vector but not in SPHK overexpressing cells after serum deprivation.
  • D Mass pools of HEK293 cells stably expressing c-myc-tagged SPHK were incubated in serum-free medium for 30 h, then treated in the absence (None) or presence of 25 ⁇ M C2-ceramide (C2- Cer) or 100 nM staurosporine (Staur) for 24 h and stained with Hoechst. Data are means ⁇ SE of four independent experiments, each one done in triplicate.
  • FIG. 13 Expression of sphingosine kinase inhibits chemotactic motility of NIH 3T3 fibroblasts.
  • NIH 3T3 fibroblasts transfected with empty vector (filled bars) or pCMV-SP0RT2- SPHKla (open bars) were trypsinized 24 h after transfection, washed with serum free media and chemotaxis towards PDGF (60 ng/ml) or 10% serum (FBS) was measured in a Boyden chamber after 24 h (PDGF) or 6 h (FBS) as described in Materials and Methods .
  • NIH 3T3 cells stably transfected with c-myc-pcDNA3 (filled squares) or c-myc-pcDNA3-SPHK (open squares) were detached from the dishes, washed with serum-free media and attachment of cells to collagen I coated plates was determined at the indicated times.
  • D Attachment of NIH 3T3 cells stably transfected with c-myc-pcDNA3 (filled bars) or c-myc-pcDNA3-SPHK (open bars) to collagen I, fibronectin, polylysine, and Matrigel after 30 min was determined as described in Materials and Methods.
  • FIG. 14 Standard curve for SPP.
  • B Standard curves for analysis of SPP. Radioactive spots corresponding to [ 32 P]SPP on the TLC shown in A were quantified with a phosphoimager .
  • Insert Lack of effects of lipids from different sources on SPP determination.
  • FIG. 1 Levels of SPP in serum.
  • A TLC showing [ 32 P] SPP in horse serum (HS) , fetal bovine serum (FBS) or SPP standard (Std) without or with alkaline phosphatase (50 units) treatment. Arrow indicates the location of SPP visualized with molybdenum blue spray, and 0 indicates origin.
  • B Quantitation of levels of SPP in horse serum (HS) , fetal bovine serum (FBS) , calf serum (CS) , or charcoal stripped calf serum (CCS) were determined as described in Materials and Methods. Data are the mean ⁇ SD of triplicate values. Similar results were obtained in three independent experiments.
  • FIG. 16 Levels of SPP and sphingosine in rat tissues.
  • SPP (A) and sphingosine (B) levels were measured in various rat tissues as described in Materials and Methods and are expressed as pmol/mg wet weight. Results are means ⁇ SD of triplicate determinations. Similar results were obtained in two different experiments.
  • Total cellular phospholipids (nmol/mg wet weight) present in lipid extracts from brain, heart, testes, liver, spleen, kidney and eye were 872 ⁇ 59, 231 ⁇ 13, 268 ⁇ 18, 323 ⁇ 18, 260 ⁇ 16, 455 ⁇ 71 and 19 ⁇ 2, respectively.
  • FIG. 17 Measurement of changes in intracellular levels of SPP after treatment of PC12 cells with exogenous sphingosine (A) or NGF (B) .
  • PC12 cells (5 x 10 6 ) were treated with sphingosine (1 ⁇ M) for the indicated time periods. Lipids were extracted and SPP levels were analyzed as described in Materials and Methods.
  • B PC12 cells (5 x 10 6 ) were stimulated without or with NGF (100 ng/ml) for the indicated times. Lipids were extracted and SPP content was determined.
  • the present invention relates to a DNA or cDNA segment which encodes sphingosine kinase.
  • AA011725) containing sequences homologous to 3 of the 8 peptides (peptides 5 (SEQ ID NO:3), 2 (SEQ ID NO: 4), and 4 (SEQ ID NO: 5)) was retrieved.
  • the nucleotide sequences of mouse ESTs AA000819 and AA592274 were then used to search dbEST to obtain EST AA389187.
  • Clones AA107451 and AA389187 were highly homologous at their 3 ' ends , but were slightly divergent at their 5' ends. Sequencing of the full-length cDNAs revealed complete open reading frames coding for 381 and 388 amino acid peptides distributed throughout the protein, and these are thus designated SPHKla (SEQ ID NO:l) and SPHKlb (SEQ ID NO:2) ( Figure 1). In addition, both contained a portion of peptide 8 (SEQ ID NO: 9) .
  • SPHKla and lb have predicted pis of 6.68 and 6.89 and MWs of 42344 and 43254, respectively, in agreement with the MW of purified rat kidney sphingosine kinase (Olivera, et al., J. Biol . Chem. 273, 12576-12583, 1998). Multiple in-frame stop codons were identified in the 5' untranslated region of SPHKla, as well as a Kozak concensus sequence, suggesting that this cDNA clone encodes full-length sphingosine kinase. Because SPHKlb only differs by a few amino acids at the N terminus, it may arise by alternative splicing.
  • Peptide sequences were determined by Edman sequencing or MALDI mass spectrometry. Cysteine residues were alkylated with isopropylacetamide. In peptide 8, the gaps could not be unambiguously defined, although the ion series defined the indicated molecular weights for gaps . 2 Masses represent the average isotopic mass.
  • a database search identified homologs of sphingosine kinase in numerous widely disparate organisms, demonstrating that sphingosine kinase is a member of a novel but highly conserved gene family.
  • a comparison of sequences from Saccharomyces cerviseiae (Bunting, M. et al . , J. Biol . Chem . 271, 10230-10236, 1996) , from C. elegans and S. pombe, reveals the location of several blocks of highly conserved amino acids, one or more of which might constitute critical portions of catalytic or substrate binding sites. Five regions, in particular C1-C5, are highly conserved in all of these sequences. Cl contains an invariant positively charged motif, GGKGK, which may be part of the ATP binding site.
  • one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from (a) a nucleotide sequence encoding a full length SPHKla polypeptide having the complete amino acid sequence in Figure 1, or the complete amino acid sequence encoded by the cloned DNA in GenBank accession no. AF068748 (SEQ ID NO:10); and (b) a nucleotide sequence encoding a full length SPHKlb nucleotide sequence having the complete amino acid sequence in Figure 1, or the complete amino acid sequence encoded by the cloned DNA in GenBank accession no. Af068749 (SEQ ID NO:ll).
  • isolated nucleic acid molecules of the invention include DNA molecules which comprise a sequence substantilly differenct from those described above but which, due to the degeneracy of the genetic code, still encode sphingosine kinase proteins.
  • the genetic code and species-specific codon preferences are well known in the art.
  • Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
  • the DNA may be double-stranded or single-stranded.
  • Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the antisense strand.
  • isolated nucleic acid molecule is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment .
  • recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
  • Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • the present invention is further directed to nucleic acid molecules encoding portions or fragments of the nucleotide sequences described herein. Fragments include portions of the nucleotide sequences of Figure 1 at least 10 contiguous nucleotides in length selected from any two integers, one of which representing a 5' nucleotide position and a second of which representing a 3' nucleotide position, where the first nucleotide for each nucleotide sequence in Figure 1 is position 1.
  • the invention includes polynucleotides comprising fragments specified by size, in nucleotides, rather than by nucleotide positions.
  • the invention includes any fragment size, in contiguous nucleotides, selected from intergers between 1- and the entire length of an entire nucleotide sequence minus 1.
  • Preferred sizes include 20-50 nucleotides, 50-300 nucleotides useful as primers and probes.
  • Regions from which typical sequences may be derived include but are not limited to, for example, regions encoding specific epitopes or domains within said sequence, such as domains C1-C5 shown in Figure 2.
  • the invention provides isolated nucleic acid molecules comprising polynucleotides which hybridize under stringent hybridization conditions to a polynucleotide sequence of the present invention described above, for instance, a nucleic acid sequences shown in Figure 1 or a specified fragment thereof.
  • stringent hybridization conditions is intended overnight incubation at 42°C in a solution comprising: 50% formamide, 5X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured sheared salmon sperm DNA, followed by washing the filters in 0.
  • sequences encoding the polypeptides of the present invention or portions thereof may be fused to other sequences which provide additional functions known in the art such as a marker sequence, or a sequence encoding a peptide which facilitates purification of the fused polypeptide, peptides having antigenic determinants known to provide helper T-cell stimulation, peptides encoding sites for post- tranlational modifications, or amino acid sequences which target the fusion protein to a desired location, e.g. a heterologous leader sequence.
  • the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the sphingosine kinase polypeptides shown in Figure 1.
  • Variant may occur naturally, such as a natural allelic variant.
  • allelic variant is intended one of several alternate forms of a gene occupying a given locus of a chromosome of an organism.
  • Non-naturally occuring variants may be produced by known mutagenesis techniques.
  • Such variants include those produced by nucleotide substitution, deletion, or addition of one or more nucleotides in the coding or noncoding regions or both. Alterations in the coding regions may produce conservative or nonconservative amino acid substitutions, deletions, or additions. Especially preferred among these are silent substitutions, additions, and deletions which do not alter the properties and activities of sphingosine kinase polypeptides disclosed herein or portions thereof. Also preferred in this regard are conservative substitutions .
  • Nucleic acid molecules with at least 90-99% identity to a nucleic acid shown in Figure 1 is another aspect of the present invention. These nucleic acids are included irrespective of whether they encode a polypeptide having sphingosine kinase activity.
  • a polypeptide having sphingosine kinase activity is intended polypeptides exhibiting activity similar, but not identical, to an activity of the sphingosine kinase of the invention, as measured in the assays described below.
  • the biological acitivity or function of the polypeptides of the present invention are expected to be similar or identical to polypeptides from other organisms that share a high degree of structural identity/similarity.
  • the present invention relates to a recombinant DNA molecule that includes a vector and a DNA sequence as described above.
  • the vector can take the form of a plasmid, phage, cosmid, YAC, eukaryotic expression vector such as a DNA vector, Pichia pastoris, or a virus vector such as for example, baculovirus vectors, retroviral vectors or adenoviral vectors, and others known in the art.
  • the cloned gene may optionally be placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences, or sequences which may be inducible and/or cell type-specific.
  • Suitable promoters will be known to a person with ordinary skill in the art.
  • the expression construct will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the vectors preferred for use include pCMV- SP0RT2 (Life Technologies, Inc.), pcDNA3 (Invitrogen) , to name a few.
  • the construct into the host cell can be effected by calcium phosphate transfection, electroporation, infection, and other methods known in the art and described in standard laboratory manuals such as Current Protocols in Molecular Biology, Ausubel, F. M. et al . (Eds), Wiley & Sons, Inc. All documents cited herein supra and infra are hereby incorporated in their entirety by referece thereto.
  • the present invention relates to host cells stably transformed or transfected with the above-described recombinant DNA constructs.
  • the host cell can be prokaryotic (for example, bacterial) , lower eukaryotic (for example, yeast or insect) or higher eukaryotic (for example, all mammals, including but not limited to rat and human) . Both prokaryotic and eukaryotic host cells may be used for expression of desired coding sequences when appropriate control sequences which are compatible with the designated host are used. Among prokaryotic hosts, E. coli is most frequently used. Expression control sequences for prokaryotes include promoters, optionally containing operator portions, and ribosome binding sites.
  • Transfer vectors compatible with prokaryotic hosts are commonly derived from, for example, pBR322, a plasmid containing operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, which also contain sequences conferring antibiotic resistance markers . These markers may be used to obtain successful transformants by selection. Please see e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning, Volumes I and II (D. N. Glover ed. 1985) for general cloning methods.
  • the DNA sequence can be present in the vector operably linked to a sequence encoding an IgG molecule, an adjuvant, a carrier, or an agent for aid in purification of SPHK, such as glutathione S- transferase, or a series of histidine residues also known as a histidine tag.
  • the recombinant molecule can be suitable for transfecting eukaryotic cells, for example, mammalian cells and yeast cells in culture systems. Saccharomyces cerevisiae, Saccharomyces carlsbergensis , and Pichia pastoris are the most commonly used yeast hosts, and are convenient fungal hosts. Control sequences for yeast vectors are known in the art.
  • Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC) , such as HEK293 cells , and NIH 3T3 cells, to name a few. Suitable promoters are also known in the art and include viral promoters such as that from SV40, Rous sarcoma virus (RSV) , adenovirus (ADV) , bovine papilloma virus (BPV) , and cytomegalovirus (CMV) . Mammalian cells may also require terminator sequences and poly A addition sequences; enhancer sequences which increase expression may also be included, and sequences which cause amplification of the gene may also be desirable.
  • ATCC American Type Culture Collection
  • Suitable promoters are also known in the art and include viral promoters such as that from SV40, Rous sarcoma virus (RSV) , adenovirus (ADV) , bovine papilloma
  • the transformed or transfected host cells can be used as a source of DNA sequences described above.
  • the transformed or transfected cells can be used as a source of the protein described below.
  • the present invention relates to a SPHKla protein having an amino acid sequence corresponding to GenBank accession no. AF068748 and encompassing 381 amino acids or any allelic variation thereof and SPHKlb protein having an amino acid sequence corresponding to GenBank accession no. AF068749 and encompassing 388 amino acids.
  • a polypeptide or amino acid sequence derived from the amino acid sequences mentioned above refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 2-5 amino acids, and more preferably at least 8- 10 amino acids, and even more preferably at least 11- 15 amino acids, or which is iiranunologically identifiable with a polypeptide encoded in the sequence .
  • a recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid sequence; it may be generated in any manner, including for example, chemical synthesis, or expression of a recombinant expression system.
  • the polypeptide can be fused to other proteins or polypeptides which increase its antigenicity, such as adjuvants for example.
  • the methods of the present invention are suitable for production of any polypeptide of any length, via insertion of the above- described nucleic acid molecules or vectors into a host cell and expression of the nucleotide sequence encoding the polypeptide of interest by the host cell.
  • Introduction of the nucleic acid molecules or vectors into a host cell to produce a transformed host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid- mediated transfection, electroporation, transduction, infection or other methods .
  • Such methods are described in many standard laboratory manuals, such as Davis et al . , Basic Methods In Molecular Biology (1986) .
  • the cells may be cultivated under any physiologically compatible conditions of pH and temperature, in any suitable nutrient medium containing assimilable sources of carbon, nitrogen and essential minerals that support host cell growth.
  • Recombinant polypeptide-producing cultivation conditions will vary according to the type of vector used to transform the host cells.
  • certain expression vectors comprise regulatory regions which require cell growth at certain temperatures, or addition of certain chemicals or inducing agents to the cell growth medium, to initiate the gene expression resulting in the production of the recombinant polypeptide.
  • the term "recombinant polypeptide-producing conditions," as used herein, is not meant to be limited to any one set of cultivation conditions.
  • the polypeptide of interest may be isolated by several techniques .
  • the cells are lysed or ruptured. This lysis may be accomplished by contacting the cells with a hypotonic solution, by treatment with a cell wall-disrupting enzyme such as lysozyme, by sonication, by treatment with high pressure, or by a combination of the above methods.
  • a cell wall-disrupting enzyme such as lysozyme
  • sonication by treatment with high pressure, or by a combination of the above methods.
  • Other methods of bacterial cell disruption and lysis that are known to one of ordinary skill may also be used.
  • the polypeptide may be separated from the cellular debris by any technique suitable for separation of particles in complex mixtures.
  • the polypeptide may then be purified by well known isolation techniques . Suitable techniques for purification include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, electrophoresis, immunoadsorption, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, liquid chromatography (LC) , high performance LC (HPLC) , fast performance LC (FPLC) , hydroxylapatite chromatography and lectin chromatography .
  • LC liquid chromatography
  • HPLC high performance LC
  • FPLC fast performance LC
  • the recombinant or fusion protein can be used as a diagnostic tool and in a method for producing sphingosine-1-phosphate, detectably labeled and unlabeled, and in a method for measuring levels of SPP in samples as described below.
  • the recombinant protein can be used as a therapeutic agent to reduce cell death and/or increase cell proliferation.
  • the transformed host cells can be used to analyze the effectiveness of drugs and agents which inhibit SPHK function, such as host proteins or chemically derived agents or other proteins which may interact with the cell to down-regulate or alter the expression of SPHK, or its cofactors.
  • the present invention relates to monoclonal or polyclonal antibodies specific for the above-described recombinant proteins (or polypeptides) .
  • an antibody can be raised against a peptide described above, or against a portion thereof of at least 10 amino acids, perferrably, 11-15 amino acids.
  • Persons with ordinary skill in the art using standard methodology can raise monoclonal and polyclonal antibodies to the protein (or polypeptide) of the present invention, or a unique portion thereof. Material and methods for producing antibodies are well known in the art (see for example Goding, in, Monoclonal Antibodies: Principles and Practice, Chapter 4, 1986) .
  • the level of expression of SPHK can be detected at several levels .
  • assays for the detection and quantitation of SPHK RNA can be designed, and include northern hybridization assays, in si tu hybridization assays, and PCR assays, among others. Please see e.g., Maniatis, Fitsch and Sambrook, Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning. Volumes I and II (D. N. Glover ed. 1985), or Current Protocols in Molecular Biology, Ausubel, F. M. et al. (Eds), Wiley & Sons, Inc. for general description of methods for nucleic acid hybridization.
  • Polynucleotide probes for the detection of SPHK RNA can be designed from the sequence available at accession numbers AF068748 and/or AF068749 for the mouse sequence [Olivera, A., et al . J " . Biol . Chem. 273:12576-12583].
  • RNA isolated from samples can be coated onto a surface such as a nitrocellulose membrane and prepared for northern hybridization.
  • the tissue sample can be prepared for hybridization by standard methods known in the art and hybridized with polynucleotide sequences which specifically recognize SPHK RNA.
  • the presence of a hybrid formed between the sample RNA and the polynucleotide can be detected by any method known in the art such as radiochemistry, or immunochemistry, to name a few.
  • Any method known in the art such as radiochemistry, or immunochemistry, to name a few.
  • One of skill in the art may find it desirable to prepare probes that are fairly long and/or encompass regions of the amino acid sequence which would have a high degree of redundancy in the corresponding nucleic acid sequences. In other cases, it may be desirable to use two sets of probes simultaneously, each to a different region of the gene.
  • probe sequences are no greater than 500 nucleotides, even more typically they are no greater than 250 nucleotides; they may be no greater than 100 nucleotides, and also may be no greater than 75 nucleotides in length. Longer probe sequences may be necessary to encompass unique polynucleotide regions with differences sufficient to allow related target sequences to be distinguished. For this reason, probes are preferably from about 10 to about 100 nucleotides in length and more preferably from about 20 to about 50 nucleotides.
  • the DNA sequence of SPHK can be used to design primers for use in the detection of SPHK using the polymerase chain reaction (PCR) or reverse transciption PCR (RT-PCR) .
  • PCR polymerase chain reaction
  • RT-PCR reverse transciption PCR
  • the primers can specifically bind to the SPHK cDNA produced by reverse transcription of SPHK RNA, for the purpose of detecting the presence, absence, or quantifying the amount of SPHK by comparison to a standard.
  • the primers can be any length ranging from 7-40 nucleotides, preferably 10-15 nucleotides, most preferably 18-25 nucleotides homologous or complementary to a region of the SPHK sequence. Reagents and controls necessary for PCR or RT-PCR reactions are well known in the art.
  • the amplified products can then be analyzed for the presence or absence of SPHK sequences, for example by gel fractionation, by radiochemistry, and immunochemical techniques. This method is advantageous since it requires a small number of cells.
  • the present invention relates to a diagnostic kit for the detection of SPHK RNA in cells, said kit comprising a package unit having one or more containers of SPHK oligonudeotide primers for detection of SPHK by PCR or RT-PCR or SPHK polynucleotides for the detection of SPHK RNA. in cells by in situ hybridization or northern analysis, and in some kits including containers of various reagents used for the method desired.
  • Kits may also contain one or more of the following items: polymerization enzymes, buffers, instructions, controls, detection labels.
  • Kits may include containers of reagents mixed together in suitable proportions for performing the methods in accordance with the invention.
  • Reagent containers preferably contain reagents in unit quantities that obviate measuring steps when performing the subject methods.
  • the present invention provides a method for identifying and quantifying the level of SPHK present in a particular biological sample. Any of a variety of methods which are capable of identifying (or quantifying) the level of SPHK in a sample can be used for this purpose .
  • Diagnostic assays to detect SPHK may comprise a biopsy or in situ assay of cells from an organ or tissue sections, as well as an aspirate of cells from a tumour or normal tissue.
  • assays may be conducted upon cellular extracts from organs, tissues, cells, urine, or serum or blood or any other body fluid or extract.
  • the assay will comprise, contacting the sample to be assayed with a SPHK ligand, natural or synthetic, or an antibody, polyclonal or monoclonal, which recognizes SPHK, or antiserum capable of detecting SPHK, and detecting the complex formed between SPHK present in the sample and the SPHK ligand or antibody added.
  • SPHK ligands or substrates include for example, sphingosine, in addition to natural and synthetic classes of ligands and their derivatives which can be derived from natural sources such as animal or plant extracts.
  • Other SPHK ligands include calmodulin.
  • SPHK ligands or anti-SPHK antibodies, or fragments of ligand and antibodies capable of detecting SPHK may be labeled using any of a variety of labels and methods of labeling for use in diagnosis and prognosis of disease associated with increased cell proliferation, such as cancer, or reduced cell death.
  • types of labels which can be used in the present invention include, but are not limited to, enzyme labels, radioisotopic labels, non- radioactive isotopic labels, and chemiluminescent labels .
  • suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, delta-5- steroid isomerase, yeast-alcohol dehydrogenase, alpha- glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase , acetylcholine esterase, etc.
  • radioisotopic labels examples include 3 H, U1 ln, 125 I, 32 P, 35 S, 14 C, 57 To, 58 Co, 59 Fe, 75 Se, 152 Eu, 90 Y, 67 Cu, 21 Ci, 211 At, 212 Pb, 47 Sc, 109 Pd, n C, 19 F, 123 I, etc.
  • non-radioactive isotopic labels examples include 157 Gd, 55 Mn, 162 Dy, 52 Tr, 46 Fe, etc.
  • fluorescent labels examples include a I52 Eu label, a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycodyanin label, an allophycocyanin label, a fluorescamine label, etc.
  • chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, etc.
  • the detection of the antibodies (or fragments of antibodies) of the present invention can be improved through the use of carriers .
  • Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes 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 SPHK.
  • 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.
  • suitable carriers for binding monoclonal antibody or will be able to ascertain the same by use of routine experimentation.
  • the ligands or antibodies, or fragments of antibodies or ligands of SPHK discussed above may be used to quantitatively or qualitatively detect the presence of SPHK. Such detection may be accomplished using any of a variety of immunoassays known to persons of ordinary skill in the art such as radioimmunoassays, immunometic assays, etc. Using standard methodology well known in the art, a diagnostic assay can be constucted by coating on a surface (i.e.
  • a solid support for example, a microtitration plate or a membrane (e.g. nitrocelluolose membrane) , antibodies specific for SPHK or a portion of SPHK, and contacting it with a sample from a person suspected of having a SPHK related disease.
  • the presence of a resulting complex formed between SPHK in the sample and antibodies specific therefor can be detected by any of the known detection methods common in the art such as fluorescent antibody spectroscopy or colorimetry.
  • a good description of a radioimmune assay may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S., et al . North Holland Publishing Company, N.Y. (1978) , incorporated by reference herein.
  • Sandwich assays are described by Wide at pages 199-206 of Radioimmune Assay Method, edited by Kirkham and Hunter, E. & S . Livingstone, Edinburgh, 1970.
  • the diagnostic methods of this invention are predictive of proliferation and metastatic potential in patients suffering from cancers including carcinomas of the lung like small cell carcinoma, large cell carcinoma, squamous carcinoma, and adenocarcinoma, stomach carcinoma, prostatic adenocarcinoma, ovarian carcinoma such as serous cystadenocarcinoma and mucinous cytadenocarcinoma, ovarian germ cell tumors, testicular carcinomas, and germ cell tumors, pancreatic adenocarcinoma, biliary adenocarcinoma, heptacellular carcinoma, renal cell adenocarcinoma, endometrial carcinoma including adenocarcinomas and mixed Mullerian tumors (carcinosarcomas) , carcinomas of the endocervix, ectocervix, and vagina such as adenocarcinoma and squamous carcinoma, basal cell carcinoma, melanoma, and skin appendage tumors, es
  • RNA or SPHK protein have increased proliferation and decreased cell death.
  • the protein can be used to identify inhibitors of SPHK activity. Using an enzyme assay as described below in the Examples, natural and synthetic agents and drugs can be discovered which result in a reduction or elimination of SPHK enzymatic activity. Knowledge of the mechanism of action of the inhibitor is not necessary as long as a decrease in the activity of SPHK is detected.
  • Inhibitors may include agents or drugs which either bind or sequester the enzyme's substrate (s) or cofactor(s), or inhibit the enzyme itself, directly, for example by irreversible binding of the agent or drug to the enzyme, or indirectly, for example by introducing an agent which binds the SPHK substrate. Agents or drugs related to this invention may result in partial or complete inhibition of SPHK activity.
  • Inhibitors of SPHK include DL- reo- Dihydrosphingosine (DHS) and the more recently discovered inhibitor N, N-dimethylsphingosine (DMS) described in Edsall, L. C. et al . , 1998, Biochemistry 37, 12892-12898.
  • DHS DL- reo- Dihydrosphingosine
  • DMS N-dimethylsphingosine
  • Inhibitors of SPHK may be used in the treatment or amelioration of diseases such as cancer, artherosclerosis , neurodegenerative disorders, i.e. stroke, Alzheimer's.
  • Agents which decrease the level of SPHK (i.e. in a human or an animal) or reduce or inhibit SPHK activity may be used in the therapy of any disease associated with the elevated levels of SPHK or diseases associated with increased cell proliferation such as cancer.
  • An increase in the level of SPHK is determined when the level of SPHK in a tumor cell is about 2-3 times the level of SPHK in the normal cell, up to about 10-100 times the amount of SPHK in a normal cell.
  • Agents which decrease SPHK R ⁇ A include, but are not limited to, one or more ribozymes capable of digesting SPHK R ⁇ A, or antisense oligonucleotides capable of hybridizing to SPHK R ⁇ A such that the translation of SPHK is inhibited or reduced resulting in a decrease in the level of SPHK.
  • antisense oligonucleotides can be administered as DNA, as DNA entrapped in proteoliposomes containing viral envelope receptor proteins (Kanoda, Y. et al . , 1989, Science 243, 375) or as part of a vector which can be expressed in the target cell such that the antisence DNA or RNA is made.
  • Vectors which are expressed in particular cell types are known in the art, for example, for the mammary gland, please see Furth, (1997) (J " . Mammary Gland Biol . Neopl . 2, 373) for examples of conditional control of gene expression in the mammary gland.
  • the DNA can be injected along with a carrier.
  • a carrier can be a protein such as a cytokine, for example interleukin 2, or polylysine-glycoprotein carrier.
  • cytokine for example interleukin 2, or polylysine-glycoprotein carrier.
  • carrier proteins and vectors and methods of using same are known in the art.
  • the DNA could be coated onto tiny gold beads and said beads introduced into the skin with, for example, a gene gun (Ulmer, J. B. et al . , 1993, Science 259, 1745).
  • antibodies, or compounds capable of reducing or inhibiting SPHK, that is reducing or inhibiting either the expression, production or activity of SPHK, such as antagonists can be provided as an isolated and substantially purified protein, or as part of an expression vector capable of being expressed in the target cell such that the SPHK- reducing or inhibiting agent is produced.
  • co-factors such as various ions, i.e. Ca2 + or factors which affect the stability of the enzyme can be administered to modulate the expression and function of SPHK.
  • These formulations can be administered by standard routes. In general, the combinations may be administered by the topical, transdermal, intraperitoneal, oral, rectal, or parenteral (e.g. intravenous, subcutaneous, or intramuscular) route.
  • SPHK-inhibiting compounds may be incorporated into biodegradable polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor or implanted so that the SPHK-inhibiting compound is slowly released systemically.
  • biodegradable polymers and their use are described, for example, in detail in Brem et al . (1991) J “ . Neurosurg. 74, 441-446. These compounds are intended to be provided to recipient subjects in an amount sufficient to effect the inhibition of SPHK.
  • agents which are capable of negatively affecting the expression, production, stability or function of SPHK are intended to be provided to recipient subjects in an amount sufficient to effect the inhibition of SPHK.
  • agents which stimulate the level of SPHK such as agonists of SPHK, may be used in the therapy of any disease associated with a decrease of SPHK, or a decrease in cell proliferation, wherein
  • SPHK is capable of increasing such proliferation, e.g. developmental retardation.
  • the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of agent which is in the range of from about 1 pg/kg to 10 mg/kg (body weight of patient) , although a lower or higher dosage may be administered.
  • a composition is said to be "pharmacologically acceptable” if its administration can be tolerated by a recipient patient.
  • Such an agent is said to be administered in a "therapeutically effective amount” if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.
  • the compounds of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle.
  • a pharmaceutically acceptable carrier vehicle e.g., water, alcohol, and water.
  • suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin are described, for example, in Remington's Pharmaceutical Sciences [16th ed. , Osol, A. ed., Mack Easton PA. (1980)].
  • a pharmaceutically acceptable composition suitable for effective administration such compositions will contain an effective amount of the above-described compounds together with a suitable amount of carrier vehicle.
  • Control release preparations may be achieved through the use of polymers to complex or absorb the compounds .
  • the controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the method of incorporation in order to control release.
  • Another possible method to control the duration of action by controlled release preparations is to incorporate the compounds of the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers .
  • microcapsules prepared, for example, interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacrylate) microcapsules , respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions .
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions .
  • kits for use in the diagnostic or therapeutic methods described above.
  • Kits according to this aspect of the invention may comprise one or more containers, such as vials, tubes, ampules, bottles and the like, which may comprise one or more of the compositions of the invention.
  • kits of the invention may comprise one or more of the following components, one or more compounds or compositions of the invention, and one or more excipient, diluent, or adjuvant.
  • SPP has previously been quantified by mass spectrometry after phenylisothiocyanate derivatization (Van Veldhoven, P. P. et al., 1994, FEBS Lett . 350, 91-95) and by a liquid chromatography/ion spray ionization tandem mass spectrometric method (Mano, N. et al . , 1997, Anal. Biochem. 244, 291-300), methods which provide sensitive analyses in the fmol range and also allow quantitation of multiple sphingolipid metabolites simultaneously.
  • the sphingosine kinase described above is highly specific for the naturally occuring D- (+) - ery thro- trans-isomer of sphingosine (Kohama, T. et al . , 1998, J. Bio . Chem. 273, 23722-23728).
  • D- (+) - ery thro- dihydrosphingosine is also phosphorylated, ableit to a lesser extent, whereas other related sphingolipids are not substrates (Kohama, et al . , 1998, supra).
  • Use of the recombinant SPHK described above provides an enzymatic method which is more specific than other procedures (Van Veldhoven et al . , 1994, supra; Yatomi, et al., 1995, supra) which non-selectively modify the amino group of this sphingoid base.
  • a method for the measurement of SPP in a sample comprising (a) partitioning the sphingosine from the sphingosine kinase in the sample. This can be achieved by extracting lipids from said sample such that SPP is in the aqueous phase, or any method of separation including HPLC, column chromatography and others known to people in the art .
  • the method can be applied to any sample, tissue, serum or cells.
  • tissue it is preferable if the tissue is homogenized prior to lipid extraction.
  • the solution should be adjusted to a workable volume by addition of a salt, such as 1 M NaCl . such that the organic and inorganic phases are separated.
  • the lipids can be extracted by any method as long as the SPP is in a separate phase from sphingosine.
  • a solution of chloroform/methanol/3N NaOH at a 1:1:0.1, v/v has been used in the examples below.
  • Other methods and solutions will be known to people in the art and can be used to separate SPP from sphingosine.
  • the phase containing the SPP is retained, and incubated under conditions which allow the removal of the phosphate group to produce a dephosphorylated SPP.
  • a phosphatase can be used to remove the phosphate group, for example, alkaline phosphatase. Other methods from removing the phosphate group can be applied, such as chemical hydrolysis, use of a nonspecific phosphatase, and other methods known to people in the art .
  • the dephosphorylated SPP is subjected to rephosphorylation in the presence of recombinat SPHK of the present invention and a detectably labeled phosphate source such that the phosphate added to the SPP is detectable.
  • [ ⁇ - 32 P]ATP, or [ ⁇ - 33 P]ATP for example, [ ⁇ - 32 P]ATP, or [ ⁇ - 33 P]ATP, to name a few.
  • Any recombinant SPHK can be used, SPHKla, SPHKlb, or any portion or variant of these proteins retaining kinase function, as well as any sphingosine kinase from other sources, mammalian, eukaryotic, or prokaryotic, which is capable of selectively adding a phosphate group to sphingosine.
  • the level of SPP is determined by measuring the detectable label using methods known in the art depending on which label is utilized. Methods for measurment of label' can be by a phosphorimaging, scintillation counting, colorimetric measurements, chemiluminescence, and immunostaining, to name a few. A concentration range from 25.0 fmol to 250 umol of SPP can be detected using the method of the present invention.
  • Serum, medium, and G418 were obtained from Biofluids, Inc. (Rockville, MD) . Restriction enzymes were from New England Biolabs (Beverly, MA) . Poly (A) + RNA blots of multiple mouse adult tissues were purchased from Clontech (Palo Alto, CA) . Lipofectamine PLUSTM and Lipofectamine were from Life Technologies, Inc. (Gaithersburg, MD) . Monoclonal antibodies against c-myc were from Zymed (San Francisco, CA) , and anti-mouse Texas Red dye- conjugated goat antibody was from Jackson Immunoresearch (West Grove, PA) . The Anti-Fade kit was from Molecular Probes (Eugene, OR) .
  • the bromodeoxyuridine incorporation detection kit and anti-mouse FITC-conjugated IgG were obtained from Boehringer Mannheim (Indianapolis, IN).
  • Bisbenzimide hydrochloride (Hoechst #33258) was from Calbiochem
  • Silica Gel 60 G plates were from EM Sciences (Cherry Hill, NJ) .
  • Mouse 2.5S nerve growth factor was obtained from Upstate Biotechnology Inc . (Lake Placid, NY) .
  • Sprague Dawley rat tissues were from Pelfreeze (Rogers, AR) .
  • Protein sequencing of sphingosine kinase was electrophoresed on SDS-PAGE and the Coomassie-stained 49 kd band excised. After S-carboxyamidomethylation, this band was subjected to in-gel tryptic digestion as described (Rani, C.S. et al . , 1997, J. Biol . Chem. 272, 10777- 10783) . The resulting peptide mixture was separated by microbore high performance liquid chromatography on a Zorbax C18 1.0 mm by 150-mm reverse-phase column in a Hewlett-Packard 1090 HPLC with al040 diode array detector.
  • MCF-7 human breast cancer cells were cultured as recommended by the American Type Culture Collection
  • ATCC Human monoblastic leukemia U937, Jurkat, and HL60 cells were obtained from ATCC (Rockville, MD) and grown in RPMI 1640 containing 10% fetal bovine serum (FBS) at 37 °C in a humidified 5% C0 2 atmosphere and routinely subcultured every 2-3 days.
  • PC12 cells were a generous gift from Dr. Gordon Guroff (NICHD, NIH, Bethesda, MD) and were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum and 5% FBS.
  • Human embryonic kidney cells (HEK293, ATCC CRL-1573) were cultured in DMEM supplemented with 2 mM glutamine containing 10% FBS, 100 units/ml penicillin and 100 ⁇ g/ml streptomycin.
  • NIH 3T3 fibroblasts (ATCC CRL-1658) and Swiss 3T3 cells (ATCC CCL-92) were grown in high glucose Dulbecco ' s modified Eagle's medium (DMEM) containing 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 2 mM L-glutamine supplemented with 10% calf serum (Kohama et al . , 1998, supra).
  • DMEM Dulbecco ' s modified Eagle's medium
  • Transient expression of sphingosine kinase Transfections of various cell types were carried out using Lipofectamine PLUS reagent, essentially as described by the manufacturer.
  • sphingosine kinase Transfections of various cell types were carried out using Lipofectamine PLUS reagent, essentially as described by the manufacturer.
  • pCMV- SPORT2 Vectors containing sphingosine kinas
  • cells were seeded at 3 x 10 5 per well in collagen-coated 6 well clusters. After 24 h, cells were transfected with a mixture of Lipofectamine PLUS (8 ⁇ l) and lipofectamine (8 ⁇ l) and vector alone (2 ⁇ g) or sphingosine kinase constructs (2 ⁇ g) . In some cases, as a measure of transfection efficiency, cells were co-transfected with 0.5 ⁇ g of pCEFLGFP (a kind gift of Dr. Silvio Gutkind) , which encodes the green fluorescent protein, and were visualized with a fluorescence microscope .
  • pCEFLGFP a kind gift of Dr. Silvio Gutkind
  • cell lysates were fractionated into cytosol and membrane fractions by centrifugation at 100,000 x g for 60 min (4"C) .
  • Sphingosine kinase activity was determined in the presence of 50 ⁇ M sphingosine, 0.25% Triton X-100, and [ 32 P]ATP (10 ⁇ Ci, 1 mM) containing MgCl 2 (10 mM) in buffer A as previously described (Meye zu Heringdorf, D. et al., 1998, EMBO J. 17, 2830-2837).
  • the labeled SPP was separated by TLC on silica gel G60 with 1- butanol/ethanol/acetic acid/water (80:20:10:20, v/v) and visualized by autoradiography .
  • the radioactive spots corresponding to authentic SPP were identified as described (Yamamura, S. et al . , 1997, Biochemistry 36, 10751-10759) and quantified with a Molecular Dynamics Storm Phosphoimager (Sunnyvale, CA) .
  • Sphingosine kinase specific activity was expressed as pmol of SPP formed per min per mg of protein.
  • Lipid extractions-Cells were washed with PBS and scraped in 1 ml of methanol containing 2.5 ⁇ l cone . HC1. Lipids were extracted by adding 2 ml chloroform/1M NaCl (1:1) and 100 ⁇ l 3N NaOH or lipids were extracted with 5 ml of chloroform/methanol/1 M NaCl (2:1:2, v/v) and 40 ⁇ l cone. NH 4 0H and phases separated. Ceramide, sphingosine and phospholipid levels were measured in the organic layer. The basic aqueous phase containing SPP, devoid of the majority of phospholipids and sphingosine, was transferred to a siliconized glass tube.
  • the organic phases were re- extracted with 1 ml methanol/lM NaCl (1:1) plus 50 ⁇ l 3N NaOH, and the aqueous fractions combined. Extractions of lipids from various serum samples were performed in a similar fashion. To 0.5 ml of serum, 0.5 ml of 1M NaCl was added and lipids extracted as described above. Tissue samples were homogenized in 25 mM HCl/1 M NaCl, and aliquots were taken for protein analysis and SPP extraction.
  • sphingosine- Sphingosine was measured by the sphingosine kinase method as previously described (van Koppen, C. J. et al . , 1996, J. Biol . Chem. 271, 2082-2087). Briefly, aliquots of the organic phase containing 5-10 nmol of total phospholipids were dried under nitrogen and then resuspended in 0.25% Triton X-100. Reactions were started by addition of of (10 ⁇ Ci, 10 mM) containing 10 mM MgCl 2 , 30,000 U of partially purified rat kidney sphingosine kinase (Meyer zu Heringdorf et al . , 1998, supra), and then incubated for 30 min at 37°C. Labeled SPP was quantified as described above. For each experiment, known amounts of sphingosine were used to generate a standard curve .
  • coli diacylglycerol kinase (0.88 U/ml) , 20 ⁇ l [ ⁇ - 32 P]ATP (10 ⁇ Ci, 10 mM) and 100 ⁇ l reaction buffer (100 mM imidazole (pH 6.6), 100 mM NaCl, 25 mM MgCl 2 , and 2 mM EGTA) . After incubation for 1 h at room temperature, lipids were extracted with 0.5 ml chloroform/methanol/conc. HCl (100:100:1, v/v) and 85 ⁇ l of 1 M KCl.
  • RNA blots containing 2 ⁇ g of poly (A) + RNA per lane from multiple mouse adult tissues were purchased from Clontech. Blots were hybridized with the 0.8 kb Sail fragment of pCMV-Sport2-mSPHKl, which was gel-purified and labeled with [ 32 P]dCTP by random priming. Hybridization in ExpressHybTM buffer (Clontech) at 65°C overnight was carried out according to the manufacturer's protocol. Bands were quantified using the Molecular Dynamics Phosphorlmager.
  • sphingosine kinase -HEK293 cells were plated on collagen coated 100 mm dishes and transfected with plasmid vectors containing sphingosine kinase (pCMV-SP0RT2sphkla) as previously described (Kohama et al . , 1998, supra).
  • Buffer A 200 mM Tris (pH 7.4) containing 20% glycerol, 1 mM mercaptoethanol, 1 mM EDTA, plus phosphatase inhibitors (1 mM sodium orthovanadate and 15 mM NaF) and protease inhibitors (10 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin, and 1 mM PMSF) and the pyridoxal phosphate analog 4-deoxypyridoxine (0.5 mM) to inhibit pyridoxal-dependent SPP lyase] .
  • Buffer A 200 mM Tris (pH 7.4) containing 20% glycerol, 1 mM mercaptoethanol, 1 mM EDTA, plus phosphatase inhibitors (1 mM sodium orthovanadate and 15 mM NaF) and protease inhibitors (10 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin, and 1
  • sphingosine (5 ⁇ M) was added to the media 10 min prior to the termination of the 24 h incubation period. The media was then collected and the cells scraped from the dishes.
  • Cellular and secreted [ 32 P]SPP were extracted in alkaline conditions as described above, followed by acidic extraction with chloroform/methanol/conc. HCl (100:100:1, v/v), to partition [ 32 P]SPP into the organic phase.
  • [ 32 P]SPP was resolved on TLC with 1- butanol/ethanol/acetic acid/water (80:20:10:20, v/v), visualized and quantified as described (Olivera et al., 1998, J. Biol . Chem. 273, 12576-12583).
  • NIH 3T3 cells were serum-starved in DMEM supplemented with 2 ⁇ g/ml insulin, 2 ⁇ g/ml transferrin, and 20 ⁇ g/ml BSA, and then stimulated with various agents. After 16 h, cells were incubated for 3 h with bromodeoxyuridine (BrdU) (10 ⁇ M) , and then fixed in 4% paraformaldehyde containing 5% sucrose (pH 7.0) for 20 min at room temperature.
  • bromodeoxyuridine BrdU
  • cultures were pulsed with 1 ⁇ Ci of [ 3 H] thymidine for 6 h and radioactivity incorporated into trichloroacetic acid-insoluble material measured as previously described (Olivera and Spiegel, 1993, Nature 365, 557-560) . Values are the means of triplicate determinations and standard deviations were routinely less than 10% of the mean.
  • Staining of apoptotic nuclei - Transiently transfected NIH 3T3 cells were cultured in serum-free medium for 1 or 2 days. Cells expressing GFP were examined with an inverted fluorescent microscope . Apoptotic cells were distinguished by condensed, fragmented nuclear regions.
  • the percentage of intact and apoptotic nuclei in cells expressing GFP fluorescence were determined. A minimum of 500 cells were scored. Stably transfected NIH 3T3 fibroblasts were washed and incubated in serum free media containing the indicated agents for 24 h, and stably transfected HEK293 cells were serum-starved for 30 h, washed and incubated in serum free media containing the indicated agents . Cells were then fixed with 3.7% formalin, washed with PBS and treated with bisbenzimide trihydrochloride (24 ⁇ g/ml; Hoechst #33258) for 10 minutes. Stained apoptotic nuclei were scored.
  • Transfected NIH 3T3 cells were harvested by trypsinization, washed twice with DMEM containing 0.1% BSA, and added to the upper chamber at 5xl0 5 cells per well.
  • the chambers were incubated in a humidified incubator at 37 °C in 5% C ⁇ 2/95% air for 6 or 24 h, as indicated.
  • the cells which traversed the collagen and spread on the lower surface of the filter were fixed in methanol for 8 min and stained with crystal violet .
  • Nonmigratory cells on the upper membrane surface were removed with a cotton swab.
  • the number of migratory cells per membrane was enumerated using light microscopy at 40X magnification (Wang et al . , 1999, supra).
  • Adhesion assay - Collagen I (0.1 mg/ml), fibronectin (0.5 mg/ml), polylysine (0.1 mg/ml), or Matrigel (1:10 dilution) were added into wells of a 6- well culture plate and incubated for 45-60 min at room temperature. Plates were then incubated with 3% BSA in PBS for 30 min to block non-specific binding sites followed by three washes with PBS. NIH 3T3 fibroblasts were harvested by scraping in PBS/10 mM EDTA, washed and resuspended in DMEM/BSA at 10 5 cells/ml, and 2 ml suspensions were then added to each well and incubated at 37 °C for the indicated time.
  • Non-adherent cells were removed and attached cells fixed with 70% ethanol for 20 min and stained with crystal violet. Wells were gently rinsed three times with water and allowed to dry. Incorporated dye was dissolved in 100 ⁇ l/well of 0.1M sodium citrate in 50% ethanol (pH 4.2) and the absorbance measured at 540 nm (Wang et al . , 1999, supra).
  • Example 1
  • Clones AA107451 and AA389187 were highly homologous at their 3' ends, but were slightly divergent at their 5 'ends. Sequencing of the full-length cDNAs revealed apparent open reading frames coding for 381 and 388 amino acid polypeptides containing sequences highly homologous to seven isolated peptides distributed throughout the protein, and these are thus designated SPHKla and SPHKlb (Fig. 1) . In addition, both contained a portion of peptide 8. SPHKla and lb have predicted pis of 6.68 and 6.89 and MWs of 42344 and 43254, respectively, in agreement with the MW of purified rat kidney sphingosine kinase (Olivera et al., 1998, supra).
  • SPHKlb only differs by a few amino acids at the N terminus, it may arise by alternative splicing. However, both sequences lacked Kozak concensus sequences, suggesting that these cDNAs may not include the actual initiation sequences.
  • SPHKla has 2 overlapping calcium/calmodulin binding concensus sequences of the 1-8-14 Type B motif ( (FILVW)xxxxxx(FAILVW)xxxxx(FILVW) , containing net positive charges of 2-4) (Rhoads, A. R. , and Friedberg, F., 1997, FASEB J. 11, 331-340).
  • SPHKla contains 2 overlapping calcium/calmodulin binding consensus sequences, one of Type B and one of Type A ( (FIL )xxx(FAILVW)xx(FAILVW)xxxxx(FILVW) ) containing net positive charges of 3 - 6) (Rhoads and Friedberg,
  • SPHKlb contains all the above calcium/calmodulin binding consensus sequences as well as an additional Type B motif at the N-terminus.
  • the existence of multiple calcium/calmodulin binding motifs supports our previous observations that purified rat kidney sphingosine kinase binds tightly to calmodulin- sepharose in the presence of calcium (Olivera et al . ,
  • Sphingosine kinase is thought to be mainly a cytosolic enzyme (Olivera et al . , 1998, supra). Consistent with this , a hydropathy plot indicates that SPHKla does not contain signal peptide or hydrophobic transmembrane sequences (data not shown) .
  • Sphingosine kinase appears to be remarkably devoid of recognizable regulatory domains, including SH2 , SH3 , or PH domains .
  • a domain reminiscent of a proline-rich SH3 binding site (Ren, R. et al., 1993, Science 259, 1157-1161) is present at the N-terminus.
  • sphingosine kinase and phosphatidylinositol-3 kinase or phosphatidylinositol- 4 kinase.
  • sphingosine kinase is activated by acidic phospholipids including phosphatidic acid, phosphatidyl inositol, phosphatidyl inositol 4, 5-bisphosphate, and particularly phosphatidyl serine, yet bears no similarity to proteins known to bind these phospholipids, such as phosphatidic acid phosphohydrolase, phospholipase C, or protein kinase C.
  • a data base search identified homologs of sphingosine kinase in numerous widely disparate organisms, demonstrating that sphingosine kinase is a member of a novel but highly conserved gene family (Fig.
  • LCB4 and LCB5 Two of these genes, named LCB4 and LCB5 , were recently shown to code for sphingosine kinases from Saccharomyces cerevisiae (Nafiec, M. M. et al . , 1998, J. Biol. Chem. 273, 19437-19442). Most of the other cDNA sequences were from ESTs or putative open reading frames identified from C. elegans and S. pombe genomic sequences . A comparison of these sequences reveals the location of several blocks of highly conserved amino acids, one or more of which might constitute critical portions of catalytic or substrate binding sites. Five regions, in particular (Cl to C5) , are highly conserved in all of these sequences.
  • Cl contains an invariant positively charged motif, GGKGK, which may be part of the ATP binding site. Moreover, the Cl and C3 subdomains of sphingosine kinase show high amino acid similarity to residues 296-315 and 378-389 of human diacylglycerol kinase ⁇ with 35% and 58% identity, respectively (Bunting, M. et al., 1996, J “ . Biol . Chem. 271, 10230-10236; Ding, L. et al., 1997, Proc . Natl . Acad . Sci . U. S. A . 94, 5519-5524) .
  • subdomain 1 C4-a
  • diacylglycerol kinase family members Klauck, T. M. et al., 1996, J. Biol . Chem . 271, 19781-19788; Tang, W. et al., 1996, J. Biol . Chem. 271, 10237-10241.
  • subdomain 1 in diacylglycerol kinases contains a GXGXXG box which is similar to the GXGXXGX 12 _ 14 K motif, known to participate in ATP binding to protein kinases, the downstream K is missing.
  • the GXGXXG box Sakane, F. et al .
  • Example 2 Tissue distribution of sphingosine kinase The tissue distribution of sphingosine kinase mRNA expression in adult mouse tissues was analyzed by Northern blotting (Fig. 3). In most tissues, including adult brain, heart, spleen, lung, kidney, and testis, a predominant 2.4 kb mRNA species was detected, indicating ubiquitous expression of sphingosine kinase. However, the level of expression was markedly variable among the different tissues.
  • mRNA levels were highest in adult lung and spleen and there were barely detectable levels in skeletal muscle and liver.
  • the mSPHKl mRNA detected by Northern blotting was slightly larger than the size of the cDNA (1.9 kb) .
  • expression of mSPHKla mRNAs in various mouse tissues did not closely correlate with the relative sphingosine kinase activities in rat tissues, as we previously found that spleen and kidney have higher specific activities than liver, which has about twice the activity of brain (Olivera et al . , 1998, supra).
  • Example 3 Recombinant sphingosine kinase activity
  • HEK293 cells were transiently transfected with pCMVSP0RT2 expression vectors containing either SPHKla or SPHKlb cDNAs and sphingosine kinase activity was measured. Modest levels of endogenous sphingosine kinase activity were present in control cells (either untransfected or transfected with an empty vector) (Fig. 4) .
  • the substrate specificity of sphingosine kinase activity from cells transfected with either SPHKla or SPHKlb was the same as found previously for purified rat kidney sphingosine kinase (Olivera et al . , 1998, supra).
  • the naturally occurring D- (+) -erytro-trans- isomer was the best substrate.
  • D, L-eryfchro- dihydrosphingosine was phosphorylated to a similar extent, whereas, phyto-sphingosine, D,L- fchreo- dihydrosphingosine, ceramide, diacylglycerol, and phosphatidylinositol were not phosphorylated (Fig.
  • SPHKla is active in intact cells, and overexpression alters the balance of sphingolipid metabolites within cells .
  • PDGF stimulated cytosolic c-myc-SPHK activity in transfected NIH 3T3 fibroblasts to a similar extent as its effect on endogenous SPHK (Table 3), indicating that c-myc-SPHK activity is regulated by the signaling pathways triggered by growth factors in the same manner as the native enzyme.
  • NIH 3T3 cells were transfected with c-myc-pcDNA3-SPHKla or c- myc-pcDNA3 , incubated in serum-free media containing fatty acid free BSA ( 20 ⁇ g/ml ) , transferrin ( 2 ⁇ g/ml ) and insulin ( 2 ⁇ g/ml ) for 18 h, and then stimulated with the indicated concentrations of PDGF for 10 min . Cytosolic SPHK activity was measured as described in Materials and Methods and standard deviations were ⁇ 10% .
  • Example 7 Effect of sphingosine kinase overexpression on programmed cell death - SPP has been shown to suppress apoptosis induced by cytokines, such as TNF and Fas ligand (Cuvillier et al., 1996, supra), serum deprivation (Edsall et al . ,
  • chemotaxis measurements were routinely performed over a 24 h period with a potent chemoattractant such as PDGF, the inhibitory effect of SPP was discernible as soon as 6 h (Fig. 13A) .
  • expression of SPHK also inhibited chemokinesis, or random cell motility, albeit to a lesser extent (Fig. 13B) .
  • Fig. 13C The time-course of adhesion to plastic (not shown) or collagen I coated-wells was similar for vector and SPHK-transfeeted cells (Fig. 13C) .
  • Example 9 Isolation, detection and quantification of SPP - Conditions were optimized to obtain the highest recovery of SPP during the extraction and enzymatic reactions. Extraction and dephosphorylation efficiencies were monitored with [ 32 P]SPP or [ 3 H] sphingosine. SPP partitions into the aqueous phase at alkaline pH with high recovery (Van Veldhoven, et al . , 1994, supra; Yatomi, et al . , 1995, supra) with the added advantage of excluding the majority of cellular phospholipids and sphingolipids, including sphingosine, which remain in the organic phase.
  • Alkaline extracted SPP was rapidly and reproducibly converted to sphingosine after digestion with bovine intestinal mucosa alkaline phosphatase.
  • Alkaline phosphatase treatment most effectively cleaved the phosphate group from SPP, which was remarkably resistant to acidic or alkaline hydrolysis. Less than 20% hydrolysis of [ 32 P]SPP occurred even after 4 h at 70 °C with 1 M KOH or 1 M HCl.
  • the substrate selectivity of recombinant sphingosine kinase makes our enzymatic method much more specific than other procedures (Van Veldhoven, et al . , 1994, supra; Yatomi, et al . , 1995, supra) which non-selectively modify the amino group of this sphingoid base.
  • the 32 P-labeled product detected by TLC had the same mobility as standard SPP (Fig. 14A) .
  • the amount of [ 32 P]SPP formed was proportional to the amount of SPP standard added over a wide concentration range, from 250 fmol to 2500 pmol (Fig. 14B and data not shown) .
  • Example 10 SPP in serum - Because previously it has been reported that serum contains high levels of SPP (Yatomi, Y. et al . , 1997, J. Biochem . 121, 969-973), we measured SPP levels in serum from various animals (Fig. 15) . Indeed, SPP was readily detected in serum, the highest levels occurring in horse serum (0.6 ⁇ M) , with lesser amounts in fetal bovine serum and calf serum where the concentration is similar to that previously found in human serum (Yatomi et al . , 1997, supra) . To verify that the presence of sphingosine did not interfere with SPP measurements, SPP in lipid extracts from horse serum and FBS was measured before and after alkaline phosphatase treatment (Fig.
  • Example 11 Mass level determination of SPP and sphingosine in rat tissues -
  • Several methods have previously been used to measure the sphingosine and SPP content of rat tissues (Kobayashi, T. et al . , 1988, Eur. J. Biochem . 172, 747-752; Merrill, A. H. et al., 1988, Anal . Biochem. Ill , 373-381; Yatomi, Y. et al., 1997, FEBS Lett . 404, 173-4).
  • SPP and sphingosine partition into different phases, thus enabling us to concurrently determine the levels of these sphingolipid metabolites. As shown in Fig.
  • rat spleen contains the highest level of sphingosine. Moreover, the sphingosine levels in spleen, kidney and liver were in excellent agreement with previously reported mass levels determined by either HPLC, mass spectrometry, or by our previous enzymatic method (Van Veldhoven and Mannaerts, 1987, supra; Merrill et al . , 1988, supra; Van Veldhoven, P. P. et al . , 1989, Anal. Biochem . 183, 177-189).
  • testes contain only a relatively small amount of SPP, similar to the level in liver and heart (Fig. 16A) . It is interesting to note that in tissues with the highest SPP content, such as brain and eye, the ratio of sphingosine to SPP is approximately 2:1. This contrasts with the other tissues examined in which sphingosine levels are nearly 10-20 fold greater than SPP.
  • Example 12 SPP and sphingosine in cultured cells - While sphingosine levels have been determined in a number of cell types (Olivera et al . , 1994a, supra;, Van Veldhoven et al . , 1989, supra; Wilson, E. et al . , 1988, J. Biol . Chem. 263, 9304-9309; Ohta, H. et al . , 1994, Anal . Biochem. 222, 489-494), there have only been scattered reports of SPP measurements.
  • Example 13 Changes in levels of SPP in PC12 cells after treatment with exogenous sphingosine or
  • a useful enzymatic assay to measure SPP and sphingosine has been developed which utilizes recombinant sphingosine kinase.
  • the SPP assay described here is similar to our previous method (Olivera et al . , 1994a, supra; Olivera and Spiegel, 1998, supra) in which sphingosine kinase in fibroblast extracts was used to determine mass levels of sphingosine.
  • To quantitatively determine cellular levels of SPP using the sphingosine kinase assay it is necessary to first separate SPP from sphingosine and other cellular lipids, which can be effectively done using the alkaline lipid extraction procedure described here.
  • a major advantage of this assay is that due to the high degree of specificity of sphingosine kinase, D- ery thro SPP levels can selectively be measured in lipid extracts . Furthermore, this method is highly sensitive and can be used to measure SPP and sphingosine levels that could only previously be measured using mass spectrometry or HPLC. Since levels of SPP in cultured cells are extremely low (0.1-1 pmol/ 10 6 cells), our method to determine levels of this important sphingolipid second messenger should be very useful.
  • SPHK the key enzyme responsible for the formation of SPP, whether it functions inter or intracellularly.
  • Activated platelets release intracellularly stored SPP, in a PKC-dependent manner (Yatomi et al . , 1997b, J". Biol . Chem. 272, 5291-5297).
  • SPHK overexpressing cell lines including
  • NIH 3T3 fibroblasts and HEK293 cells do not secrete SPP although they have markedly increased intracellular SPP levels, suggesting that only certain cell types are capable of secreting SPP and that these transfected cells are useful tools to study the intracellular roles of SPP.
  • SPHK activity and SPP levels can be regulated by external signals (Spiegel et al . , 1998a, Biochemistry (Mosc) 63, 69-73; Spiegel and Merrill, 1996, FASEB J. 10, 1388-1397).
  • the levels of SPP in cells overexpressing SPHK were maximally increased only 4-8 fold. This level was similar to the level of SPP produced in the same parental cells after stimulation by growth factors, addition of sphingosine, or even SPP itself (Olivera and Spiegel, 1993, supra).
  • SPP lyase and/or SPP phosphatase may also play an important role in determining the steady state levels of SPP.
  • SPP activity and SPP levels Another possible explanation for the lack of correlation of SPHK activity and SPP levels is that the substrate for the overexpressed SPHK, sphingosine, may be located in a different subcellular compartment. Indeed, SPHK is mainly cytosolic, whereas sphingosine is mainly membrane associated. However, availability of sphingosine may not be the only limiting factor for the production of SPP, because addition of exogenous sphingosine increased synthesis of SPP in parental cells and in cells expressing SPHK by the same proportion.
  • SPHK activity has been found in the cytosolic fraction of most cells [Olivera, 1993 #700; Louie, 1976 #2138; Hirschberg, 1970 #2438; Stoffel, 1973 # 2450] and in the membrane fraction of certain tissues and organs (Buehrer and Bell, 1992, supra; Keenan, 1972, Biochim. Biophys . Acta . 270, 383-396; Nagiec et al . , 1998, J. Biol . Chem. 273, 19437- 19442) .
  • SPP has been proposed as an intracellular mediator of cell growth (Spiegel and Merrill, 1996, supra) , and in agreement, microinjection of SPP (Van Brocklyn et al . , 1998, supra) and caged SPP (Qiao et al., 1998, Bioorg. Med. Chem. 8, 711-714) is mitogenic for Swiss 3T3 fibroblasts.
  • SPP has been shown to inhibit cell motility, chemoinvasion, and haptotactic motility (Sadahira et al., 1994, FEBS Lett . 340, 99-103) of human B16 melanoma cells and other cell lines in a low concentration range (10 to 100 nM) (Sadahira et al . , 1992, supra), and these effects seem to be mediated through a cell surface receptor since SPP immobilized on glass beads, which can not traverse the cell membrane, mimicked the effects of SPP (Yamamura et al., 1997, supra). SPP also inhibits chemotactic motility and trans-endothelial migration of human neutrophils (Kawa et al .
  • SPP may act intracellularly, and not by binding to a putative cell surface receptor, to inhibit motility and invasion of MDA-MB-231 cells (Wang et al . , 1998, supra) .
  • the mechanism by which SPP elicits this anti- motogenic effect is not yet known.
  • Intracellular SPP has been shown to induce rapid reorganization of the actin cytoskeleton (Bornfeldt et al . , 1995, supra; Wang et al . , 1997, supra), resulting in stress fiber formation and concomitant assembly of focal adhesions as well as tyrosine phosphorylation of both focal adhesion kinase (FAK) and paxillin in Swiss 3T3 fibroblasts (Wang et al . , 1997, supra). It is still not clear whether SPP acts extracellularly through an Edg family receptor to inhibit motility.
  • SPHK-derived SPP as a second messenger in cell proliferation, survival, and motility.
  • SPHK belongs to a new class of lipid kinases, different in structure and biochemical properties than the PI kinase family, but similar in the broad spectrum of signals and in vital and versatile cell functions that they regulate.

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Abstract

L'invention concerne des acides nucléiques codant la sphingosine kinase (SPHK), ainsi que des procédés d'utilisation de celle-ci dans la production de sphingosine-1-phosphate (SPP), dans le criblage des inhibiteurs de SPHK et en tant qu'agent thérapeutique.
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EP1235913A2 (fr) 2002-09-04
WO1999061581A3 (fr) 2002-07-11
AU4097999A (en) 1999-12-13

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