JP2005192537A - Sphingomyelin (sm) synthase gene and utilization of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an amino acid sequence of an SM synthase and a base sequence encoding the same, and new uses of the same. <P>SOLUTION: This human cDNA (SMS-1) corresponding to the SM synthase activity is isolated by an expression cloning method by using a leukemic cell mutant (WR19L/Fas (-)) of a mouse, lacking the SM synthesis. The WR19L/Fas-SM(-) cells do not express the SM, but the SMS-1 cDNA-expression cells express the SM in a same degree to that of the WR19L/Fas-SM(+) cells, and also since a ceramide and phosphatidylcholine are required for the expression of the SM, it encodes the SM synthase. Also, the SM synthase encoded by the SM-1 cDNA is essential for the cell proliferation. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a sphingomyelin synthase gene and a new use thereof.

  A variety of glycerophospholipids such as diacylglycerol (DAG), phosphatidylinositol, phosphatidic acid are considered bioactive molecules for cell proliferation and survival. Sphingolipids are components of cell membranes and have been known to regulate cell proliferation, cell differentiation, cell death, and the like.

  In recent years, ceramide, a kind of sphingolipid, has come to be considered as a signal mediator of apoptosis, cell differentiation, and secretion. Various stresses such as UV, radiation, heat shock, oxygen deficiency; biological factors such as TNF-α, INF-γ and Fas antibody require ceramide production for apoptosis (Cell Signal 10,685-692 ( 1998)). This suggests that ceramide functions as a signal for inducing apoptosis.

  Many enzymes are involved in the metabolism of ceramide, one of which is sphingomyelin (hereinafter abbreviated as “SM”) synthase, which transfers the choline phosphate part of phosphatidylcholine to ceramide. In addition to producing sphingomyelin, it produces diacylglycerol.

  It has been reported that the activation of SM synthase decreases the level of ceramide, which is regarded as an apoptosis-inducing signal, and subsequently increases the level of diacylglycerol (Biochemistry 21,2753-2759). Diacylglycerol is also an important signal factor for cell growth via protein kinase C (PKC) activation (Science 246,1050) and acts competitively against ceramide-induced apoptosis (Cell Signal 10,685-692).

  Regarding the relationship between ceramide and disease, the amount of ceramide decreased in cells resistant to anticancer drugs, and in leukemia cells collected from anticancer drug resistant leukemia patients, anticancer drug treatment compared to the dedication of anticancer drug sensitive patients. It is known that the increase in ceramide observed is small. This suggests that the mechanism that does not increase the intracellular level of ceramide, an apoptosis-inducing signal, is one of the new drug resistance mechanisms (protein, nucleic acid, enzyme, Vol.47, No.4 ( 2002)).

  Furthermore, sphingomyelin has been reported to inhibit deoxycholic acid-induced apoptosis in a dose-dependent manner, followed by colon epithelial cell hyperproliferation, thus reducing the number of colon crypts (Dig Dis Sci 48,1094-1101 (2003)).

  Thus, it has been clarified that ceramide functions as a signal mediator of apoptosis, and that sphingomyelin inhibits apoptosis. However, since the specific role of sphingomyelin synthase has not been clarified, the usefulness of sphingomyelin synthase has not been determined.

  Here, Non-Patent Document 1 discloses an amino acid sequence of a polypeptide that catalyzes SM synthesis and its reverse reaction in yeast cells, and a base sequence encoding it. In addition, the same literature describes that this polynucleotide is expressed in various human tissues by Northern blotting, and that this polypeptide is localized in the Golgi apparatus of human cells. .

However, Non-Patent Document 1 only describes that the function of this polypeptide catalyzes SM synthesis and its reverse reaction in yeast cells, and the role in actual mammalian cells is clarified. Not.
EMBO J. 2004,1-12 (2004)

  It is a first object of the present invention to provide an amino acid sequence of SM synthase and a base sequence encoding it. Another object of the present invention is to provide a new use of the polypeptide having this amino acid sequence by elucidating the role of cell growth in mammals.

In order to solve the above-mentioned problems, the present inventors have conducted research and obtained the following knowledge.
(1) Human cDNA corresponding to SM synthase activity was isolated by expression cloning using a mouse leukemia cell mutant lacking SM synthesis (WR19L / Fas (-)). This cDNA clone encodes 413 amino acids and was named SMS-1.
(2) WR19L / Fas-SM (-) cells do not express SM, but SMS-1 cDNA expressing cells (WR19L / Fas-SMS-1) are similar to WR19L / Fas-SM (+) cells SM is expressed.
(3) Since SMS-1 expressing cells (WR19L / Fas-SMS-1) require ceramide and phosphatidylcholine for SM expression, it is considered that SMS-1 cDNA encodes SM synthase. It is done.
(4) Although growth of WR19L / Fas-SM (-) cells is suppressed, SMS-1 cDNA expressing cells (WR19L / Fas-SMS-1) are similar to WR19L / Fas-SM (+) cells To proliferate. Therefore, SM synthase encoded by SMS-1 cDNA is essential for cell growth.

The present invention has been completed based on the above findings, and provides the following polynucleotides and the like.
Item 1. The following polypeptide (1) or (2).
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
(2) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, added or substituted in SEQ ID NO: 1 and having sphingomyelin synthase activity.
Item 2. A cell growth promoter comprising the polypeptide according to Item 1 as an active ingredient.
Item 3. Treatment or improvement of a disease selected from the group consisting of aplastic anemia, myelodysplastic syndrome, hematopoiesis suppression after bone marrow transplantation, liver cirrhosis, myocardial infarction, cerebral infarction, and wound, comprising the polypeptide of item 1 as an active ingredient Agent.
Item 4. The following polynucleotide (3) or (4):
(3) A polynucleotide comprising the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 or a base sequence complementary thereto.
(4) A polypeptide that hybridizes under stringent conditions with a polynucleotide having the nucleotide sequence of nucleotide numbers 338 to 1576 or a nucleotide sequence complementary thereto in SEQ ID NO: 2 and has sphingomyelin synthase activity. Encoding polynucleotide.
Item 5. Item 5. A cell growth promoter comprising the polynucleotide according to Item 4 as an active ingredient.
Item 6. Treatment or improvement of a disease selected from the group consisting of aplastic anemia, myelodysplastic syndrome, hematopoiesis suppression after bone marrow transplantation, liver cirrhosis, myocardial infarction, cerebral infarction, and wound, comprising the polypeptide of item 4 as an active ingredient Agent.
Item 7. A recombinant vector comprising the polynucleotide according to Item 4.
Item 8. Item 8. A transformant having the recombinant vector according to Item 7.
Item 9. Item 10. A method for producing the polypeptide according to Item 1, comprising the step of culturing the transformant according to Item 8, and the step of recovering the polypeptide according to Item 1 from the culture.
Item 10. The following (5) or (6) antisense polynucleotide.
(5) A polynucleotide comprising a base sequence complementary to at least 18 consecutive bases of the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 or a base sequence complementary thereto.
(6) A polynucleotide that hybridizes under stringent conditions with at least 18 consecutive bases of the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 or a base sequence complementary thereto.
Item 11. Item 11. A cell growth inhibitor comprising the antisense polynucleotide according to Item 10 as an active ingredient.
Item 12. Item 11. A therapeutic or ameliorating agent for a disease selected from the group consisting of leukemia, cancer, and autoimmune disease, comprising the antisense polynucleotide according to Item 10 as an active ingredient.
Item 13. The following (7) or (8) siRNA.
(7) An siRNA comprising a pair of an RNA strand complementary to at least 21 consecutive bases of the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 and an RNA strand complementary thereto.
(8) An siRNA comprising a double-stranded RNA that hybridizes under stringent conditions with at least 21 consecutive base sequences of base numbers 338 to 1576 of SEQ ID NO: 2 or a base sequence complementary thereto.
Item 14. Item 14. A cell growth inhibitor comprising the siRNA according to Item 13 as an active ingredient.
Item 15. Item 14. A therapeutic or ameliorating agent for a disease selected from the group consisting of leukemia, cancer, and autoimmune disease, comprising the siRNA according to Item 13 as an active ingredient.
Item 16. The following (9) or (10) ribozyme.
(9) having two base sequence portions complementary to each of two regions consisting of at least 20 consecutive bases in the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 or a base sequence complementary thereto And a ribozyme having an activity of degrading the polynucleotide of Item 4.
(10) Two of the base sequences of base numbers 338 to 1576 described in SEQ ID NO: 2 or two regions that hybridize under stringent conditions to two regions consisting of at least 20 consecutive bases in the base sequence complementary thereto A ribozyme having a base sequence and having an activity of degrading the polynucleotide of Item 4.
Item 17. Item 17. A cell growth inhibitor comprising the siRNA according to Item 16 as an active ingredient.
Item 18. Item 17. A therapeutic or ameliorating agent for a disease selected from the group consisting of leukemia, cancer and autoimmune disease, comprising the siRNA according to Item 16 as an active ingredient.
Item 19. An antibody that specifically recognizes the polypeptide of Item 1.
Item 20. Item 20. A cell growth inhibitor comprising the antibody according to Item 19 as an active ingredient.
Item 21. Item 20. A therapeutic or ameliorating agent for a disease selected from the group consisting of leukemia, cancer, and autoimmune disease, comprising the antibody according to Item 19 as an active ingredient.
Item 22. (a) contacting the test substance with a test cell carrying the recombinant vector according to Item 7,
(b) comparing the sphingomyelin synthase activity or its index value in the test cell with the sphingomyelin synthase activity or its index value in the test cell not contacted with the test substance;
(c) a method for screening a cell growth inhibitor, comprising a step of selecting a test substance that reduces sphingomyelin synthase activity.
Item 23. (a) contacting the test substance with a test cell carrying the recombinant vector according to Item 7,
(b) comparing the sphingomyelin synthase activity or its index value in the test cell with the sphingomyelin synthase activity or its index value in the test cell not contacted with the test substance;
(c) a method for screening a therapeutic or ameliorating agent for a disease selected from the group consisting of leukemia, cancer, and autoimmune disease, comprising a step of selecting a test substance that reduces sphingomyelin synthase activity.
Item 24. (a) contacting the test substance with a test cell carrying the recombinant vector according to Item 7,
(b) comparing the sphingomyelin synthase activity or its index value in the test cell with the sphingomyelin synthase activity or its index value in the test cell not contacted with the test substance;
(c) selecting a test substance that increases sphingomyelin synthase activity, and a method for screening a cell growth promoter.
Item 25. (a) contacting the test substance with a test cell carrying the recombinant vector according to Item 7,
(b) comparing the sphingomyelin synthase activity or its index value in the test cell with the sphingomyelin synthase activity or its index value in the test cell not contacted with the test substance;
(c) a group consisting of aplastic anemia, myelodysplastic syndrome, suppression of hematopoiesis after bone marrow transplantation, cirrhosis, myocardial infarction, cerebral infarction, and wound, including a step of selecting a test substance that increases sphingomyelin synthase activity A method for screening for a therapeutic or ameliorating agent for a more selected disease.

  According to the present invention, an amino acid sequence of a polypeptide having SM synthesis activity and a base sequence encoding the same are provided, and it was confirmed for the first time that a polynucleotide having this base sequence is essential for cell growth.

  As a result, it became clear that the expression inhibitor of this polynucleotide, the activity inhibitor of this polypeptide, antisense, siRNA, ribozyme, antibody and the like are useful for the treatment of cancers that require cell growth suppression. It was. In addition, the polynucleotide, the polypeptide encoded by the polynucleotide, the expression promoter for the polynucleotide, and the activity promoter for the polypeptide may be used for the myelodysplastic syndrome that requires cell proliferation, suppression of hematopoiesis after bone marrow transplantation, etc. It proved useful for treatment.

Hereinafter, the present invention will be described in detail.
(I) Polypeptide The polypeptide of the present invention is the following polypeptide (1) or (2).
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
(2) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, added or substituted in SEQ ID NO: 1 and having sphingomyelin synthase activity.

  In the present invention, the polypeptide includes salts and derivatives thereof in addition to the polypeptide as long as the biological activity is not impaired. Examples of the derivatives include those that have been glycosylated, amidated, phosphorylated, carboxylated, phosphorylated, formylated, acylated, and the like. As the salt, an acid addition salt is preferable. Examples of the acid addition salt include salts with inorganic acids such as hydrochloric acid, phosphoric acid and sulfuric acid; salts with organic acids such as formic acid, acetic acid, propionic acid and liquor acid.

  Examples of the modified polypeptide of (2) include polypeptides of other species corresponding to the human polypeptide of SEQ ID NO: 1. The corresponding polypeptide of another species can be deductively identified from the corresponding gene of another species selected by, for example, NCBI Blast search described below.

  When the polypeptide of (2) is obtained by amino acid substitution of the polypeptide of (1), from the viewpoint of maintaining the structure of the protein, from the standpoint of polarity, charge, solubility, hydrophilicity / hydrophobicity, polarity, etc. It can be substituted with an amino acid having properties similar to those of For example, glycine, alanine, valine, leucine, isoleucine, proline are classified as nonpolar amino acids; serine, threonine, cysteine, methionine, asparagine, glutamine are classified as polar amino acids; phenylalanine, tyrosine, tryptophan Lysine, arginine and histidine are classified as basic amino acids; aspartic acid and glutamic acid are classified as acidic amino acids. Accordingly, substitution can be made by selecting from the same group of amino acids.

The mutant polypeptide of (2) is not limited as long as it has SM synthase activity, but preferably 3 or less, more preferably 1 amino acid is missing in the amino acid sequence of SEQ ID NO: 1. Anything that has been lost, added or replaced may be used.
(II) Polynucleotide The polynucleotide of the present invention is the following polynucleotide (3) or (4).
(3) A polynucleotide comprising the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 or a base sequence complementary thereto.
(4) A polypeptide that hybridizes under stringent conditions with a polynucleotide having the nucleotide sequence of nucleotide numbers 338 to 1576 or a nucleotide sequence complementary thereto in SEQ ID NO: 2 and has sphingomyelin synthase activity. Encoding polynucleotide.

  In the present invention, polynucleotides (including oligonucleotides) include both DNA and RNA unless otherwise specified. DNA includes cDNA, genomic DNA, and synthetic DNA unless otherwise specified. Unless otherwise stated, RNA includes total RNA, mRNA, rRNA, tRNA and synthetic RNA.

  The polynucleotide of (3) can be obtained by, for example, a method of screening a human cDNA library by hybridization using an oligonucleotide synthesized based on the nucleotide sequence of nucleotide numbers 338 to 1576 of SEQ ID NO: 2 as a probe. It can also be obtained by performing PCR according to a conventional method using a human cDNA library as a template for PCR, for example, using a primer designed based on the nucleotide sequence of nucleotide numbers 338 to 1576 of SEQ ID NO: 2. . It can also be obtained by chemical synthesis.

  The polynucleotide (4) hybridizes under stringent conditions with the polynucleotide having the nucleotide sequence of nucleotide numbers 338 to 1576 described in SEQ ID NO: 2 or a nucleotide sequence complementary thereto. “Polynucleotides that hybridize to a polynucleotide under stringent conditions” include, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, (1989) and the like.

  In the present invention, “stringent conditions” include, for example, heating at 42 ° C. in a solution of 6 × SSC, 0.5% SDS and 50% formamide, and then in a solution of 0.1 × SSC, 0.5% SDS. Examples include conditions under which a positive hybridization signal is observed when washed at 0 ° C.

  Examples of the polynucleotide (4) include polynucleotides of other species such as mice and rats corresponding to the human polynucleotides of SEQ ID NO: 2 and nucleotide numbers 338 to 1576, and natural or non-natural allelic variants. Etc. are included.

  Polynucleotides from other species can be selected, for example, by NCBI blast search (www.ncbi.nlm.nih.gov/BLAST/). Specifically, for example, a partial base sequence of base numbers 338 to 1576 of SEQ ID NO: 2 is subjected to NCBI blast search to search sequences having higher homology than base sequence databases and EST databases of other species. By selecting a base sequence having a homology of, for example, 90% or more in the region corresponding to the partial sequence selected by the search, a corresponding gene of another corresponding species can be selected.

In substituting the nucleotide of the polynucleotide of (3) to determine the polynucleotide of (4), the amino acid obtained after translation is pre-substitution in terms of polarity, charge, solubility, hydrophilicity / hydrophobicity, polarity, etc. Substitution of bases having properties similar to those of the amino acids can be performed. In addition, when one amino acid has several translation codons, it is of course possible to substitute a base within the translation codon.
(III) Recombinant Vector The recombinant vector of the present invention is a vector containing the polynucleotide of the present invention (here, DNA), and can be obtained by incorporating the polynucleotide of the present invention into an appropriate vector DNA.
Vector DNA may be appropriately selected depending on the type of host and intended use. The vector DNA may be a naturally occurring DNA or may be a DNA in which a portion of the DNA other than the portion necessary for growth is missing from the natural DNA. Examples of the vector DNA include, for example, bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses (eg, baculovirus, papovavirus, SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and And vectors derived from genetic elements of bacteriophage (for example, cosmids and phagemids).
In addition, for the production of the polypeptide of the present invention, an expression vector or a cloning vector may be used.
(IV) Transformant A recombinant vector incorporating the polynucleotide of the present invention is transformed into a known host, for example, a bacterium such as Escherichia coli (for example, K12), a bacterium belonging to the genus Bacillus (for example, MI114), yeast (for example, AH22). ), Insect cells (for example, Sf cells), animal cells (for example, COS-7 cells, Vero cells, CHO cells, etc.) and the like by a known method, the transformant of the present invention can be obtained.
As the polynucleotide introduction method, a known method can be used without limitation. Examples of such known polynucleotide introduction methods include calcium phosphate method, DEAE-dextran method, microinjection method, electroporation method and infection.
(V) Polypeptide Production Method The polypeptide of the present invention can be synthesized by a known peptide synthesis method. Examples of such peptide synthesis methods include the method described in “Peptide Synthesis” (Maruzen; issued in 1975). Alternatively, it can be produced using a known chemical synthesizer such as a peptide synthesizer manufactured by Applied Biosystems.
The polypeptide of the present invention can also be produced by a method comprising the step of culturing the transformant of the present invention described above and the step of recovering the polypeptide of the present invention from this culture.

It is preferred to recover the polypeptide of the present invention from the culture and further purify it. Purification may be performed by a known method combining various chromatographies such as gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography.
In addition, the polypeptide of the present invention in the culture is specifically adsorbed and recovered using a polyclonal antibody or a monoclonal antibody specific to this amino acid sequence produced based on the amino acid sequence of the polypeptide of the present invention. You can also.
(VI) Antisense The antisense polynucleotide of the present invention is the following polynucleotide (5) or (6).
(5) A polynucleotide comprising a base sequence complementary to at least 18 consecutive bases of the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 or a base sequence complementary thereto.
(6) A polynucleotide that hybridizes under stringent conditions with at least 18 consecutive bases of the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 or a base sequence complementary thereto.

  The antisense of the present invention inhibits the expression of this polypeptide by hybridizing with the mRNA encoding the polypeptide of the present invention and inhibiting the translation from mRNA to polypeptide, or by degrading the mRNA. .

  The antisense of (5) preferably includes a base sequence complementary to at least 20 consecutive bases of the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 or a base sequence complementary thereto. The antisense of (6) is also preferably one that hybridizes under stringent conditions with at least 20 bases of the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 or a base sequence complementary thereto. .

  The upper limit of the number of antisense bases is not particularly limited, but usually about 30 bases can sufficiently achieve the object. Whether or not the selected sequence inhibits only the expression of the target mRNA may be confirmed by, for example, a BLAST search.

  The region where the polynucleotides (5) and (6) hybridize in the base sequence of base numbers 338 to 1576 of SEQ ID NO: 2 is preferably closer to the 5 ′ end of base numbers 338 to 1576 of SEQ ID NO: 2.

  The antisense polynucleotide of the present invention may be any of single-stranded DNA, single-stranded RNA, double-stranded DNA, or DNA / RNA hybrid. Double-stranded RNA corresponds to siRNA described later.

  The antisense polynucleotide of the present invention may be a derivative of these nucleotides as long as it inhibits the expression of the polypeptide of the present invention. Examples of the derivatives include phosphorothioate DNA and H-phosphonate DNA.

  Inhibition of the expression of the polypeptide of the present invention by the antisense of the present invention can be confirmed, for example, by the following method. Antisense of about 20 to 50 μg / ml is added to human leukemia-derived cells together with known intracellular introduction reagents such as lipofection reagent, lipofectamine reagent, liposome and the like, if necessary. Subsequently, a cell extract is prepared from the cells by a known method, and the expression level of the polypeptide of the present invention may be measured by a known method such as ELISA or Western blot using the antibody of the present invention described later. This expression level is compared with the case where no antisense was added.

The antisense of the present invention can be produced by a known chemical synthesis method.
(VII) siRNA
The siRNA of the present invention is the following (7) or (8) siRNA.
(7) An siRNA comprising a pair of an RNA strand complementary to at least 21 consecutive bases of the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 and an RNA strand complementary thereto.
(8) An siRNA comprising a double-stranded RNA that hybridizes under stringent conditions with at least 21 consecutive base sequences of base numbers 338 to 1576 of SEQ ID NO: 2 or a base sequence complementary thereto.

  Although the mechanism of action of the siRNA of the present invention is not necessarily clear, by hybridizing with the mRNA encoding the polypeptide of the present invention and inhibiting translation from mRNA to polypeptide, or by degrading the mRNA, It is thought to inhibit the expression of this polypeptide.

  The siRNA of (7) preferably includes a base sequence complementary to at least 23 consecutive bases of the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 or a base sequence complementary thereto. The siRNA of (6) is also preferably one that hybridizes under stringent conditions with at least 23 bases of the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 or a base sequence complementary thereto.

  The upper limit of the number of bases of siRNA is not particularly limited, but usually about 30 bases can sufficiently achieve the object. Whether or not the selected sequence inhibits the expression of only the target mRNA may be confirmed by, for example, a BLAST search.

  In addition, the region where the siRNAs (7) and (8) in the base sequence of base numbers 338 to 1576 of SEQ ID NO: 2 hybridize preferably includes a region of about 50 to 100 bases downstream of the start codon. More preferably, it is included in the region.

  The inhibition of the expression of the polypeptide of the present invention by the siRNA of the present invention may be confirmed in the same manner as in the case of antisense.

The siRNA of the present invention can be produced by a known chemical synthesis method.
(VIII) Ribozyme The ribozyme of the present invention is the following ribozyme (9) or (10).
(9) having two base sequence portions complementary to each of two regions consisting of at least 20 consecutive bases in the base sequence of base numbers 338 to 1576 of SEQ ID NO: 2 or a base sequence complementary thereto, and A ribozyme having an activity of degrading the polynucleotide of the present invention.
(10) There are two base sequences that hybridize under stringent conditions in two regions consisting of at least 20 consecutive base sequences of base numbers 338 to 1576 of SEQ ID NO: 2 or a base sequence complementary thereto. And a ribozyme having an activity of degrading the polynucleotide of the present invention.

  Although the upper limit of the number of bases of the ribozyme of the present invention is not particularly limited, it is usually about 100 bases.

  Two regions in the base sequence of base numbers 338 to 1576 of SEQ ID NO: 2 or a base sequence complementary thereto may be adjacent or separated, but there are about 2 to 6 bases between these regions Preferably a base is present. For example, hammerhead ribozymes include cases where 2 bases are present, and hairpin ribozymes include cases where 6 bases are present.

  A ribozyme is an RNA molecule containing an antisense sequence that recognizes a specific site in RNA, and has RNA cleaving enzyme activity. As a result, the ribozyme identifies the target RNA and specifically cleaves a specific portion of the RNA.

  The ribozyme of the present invention may be either a hammerhead type or a hairpin type. The hammerhead type usually recognizes NUX (N is G, U, C, or A, and X is C, U, or A), and cleaves RNA (usually mRNA) at a position 3 'to X. . The hairpin type usually recognizes NNNG / CN * GUCNNNNNNNN (N is G, U, C or A) and cuts RNA (usually mRNA) between N * G.

  Moreover, it is preferable that the ribozyme of the present invention has a motif that interacts with helicase, thereby improving RNA cleavage activity.

  The ribozyme of the present invention is usually composed of RNA, but derivatives such as those containing deoxyribonucleotides and those modified with phosphothioate which are hardly degraded in vivo are also included in the ribozyme of the present invention.

The ribozyme of the present invention can be produced by a known chemical synthesis method or the like.
(IX) Antibody The antibody of the present invention is an antibody that specifically recognizes the polypeptide of the present invention. The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. The antibody of the present invention also includes an antibody having an antigen binding property to a polypeptide consisting of 20 amino acids, preferably 30 amino acids, in the amino acid sequence constituting the polypeptide of the present invention. Furthermore, derivatives of these antibodies (chimeric antibodies, etc.) and those labeled with an enzyme such as peroxidase are also included in the antibodies of the present invention.
Methods for Producing Antibodies These antibodies can be produced according to well-known production methods (for example, Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Sections 11.12 to 11.13).

Specifically, when the antibody of the present invention is a polyclonal antibody, a non-human animal such as a rabbit can be immunized using the polypeptide of the present invention, and can be obtained from the serum of this immunized animal according to a conventional method. . When the antibody of the present invention is a monoclonal antibody, for example, a non-human animal such as a mouse is immunized with the polypeptide of the present invention or a polypeptide having a partial sequence thereof, and the spleen cells and bone marrow of the immunized animal are immunized. It can be obtained from hybridomas obtained by cell fusion with tumor cells (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4-11.11).
(X) Pharmaceutical
As shown in the Examples below, the polypeptide of the present invention has SM synthase activity and is essential for cell growth, so that a disease requiring cell growth, such as aplastic anemia, It can be suitably used for the treatment or improvement of myelodysplastic syndrome, suppression of hematopoiesis after bone marrow transplantation, cirrhosis, myocardial infarction, cerebral infarction, wound and the like. Among them, it can be suitably used for myelodysplastic syndrome requiring lymphocyte proliferation and hematopoiesis suppression after bone marrow transplantation.

  The polypeptide of the present invention is blended with various pharmaceutically acceptable carriers (including excipients, binders, disintegrants, lubricants, etc.) to form a preparation. The formulation may contain conventional additives.

  The form of the preparation is not particularly limited. For example, tablets, pills, capsules, powders, granules, syrups and other oral administration agents; injections, drops, external preparations, parenteral administration agents such as suppositories, etc. Various preparation forms can be mentioned.

The content of the polypeptide of the present invention in the preparation varies depending on the administration route, patient age, weight, symptoms, etc., and cannot be defined unconditionally, but the daily dose of the polypeptide is usually about 10 to 1000 mg, more preferably 100. The amount may be about 500 mg. When it is administered once a day, it is sufficient that this amount is contained in one preparation, and when it is administered three times a day, it is sufficient that this one-third amount is contained in one preparation.
Polynucleotide Since the polypeptide of the present invention has SM synthase activity and is essential for cell proliferation, the polynucleotide encoding the same is also used in, for example, aplastic anemia, myelodysplastic syndrome, suppression of hematopoiesis after bone marrow transplantation. It can be suitably used for the treatment or improvement of cirrhosis, myocardial infarction, cerebral infarction, wound and the like. Among them, it can be suitably used for myelodysplastic syndrome requiring lymphocyte proliferation and hematopoiesis suppression after bone marrow transplantation.
In treating or ameliorating these diseases, the polynucleotide of the present invention may be carried on a vector and directly introduced into the body, or cells may be collected from a human and introduced into the body and then returned to the body. As the vector, any of those known to be usable for gene therapy such as retrovirus, adenovirus, and vaccinia virus can be used without limitation, but a retrovirus is recommended.
Alternatively, the polynucleotide of the present invention can be introduced into cells using microinjection or the like in which liposomes are encapsulated and delivered.

The dose of the polynucleotide of the present invention varies depending on the administration method, patient age, body weight, symptoms, etc., and cannot be defined unconditionally, but the polynucleotide concentration is about 10-100 ng / ml in blood, preferably 20-50. What is necessary is just to administer so that it may be about ng / ml.
Antisense siRNA
Since the antisense and siRNA of the present invention inhibit the expression of the SM synthase gene, it can be suitably used for the treatment or improvement of diseases that require cell growth suppression, such as leukemia, various cancers, and autoimmune diseases. Among these, it can be suitably used for the treatment or improvement of leukemia or cancer.
In treating or ameliorating these diseases, the polynucleotide of the present invention may be introduced into cells using microinjection in which liposomes are encapsulated and delivered, or directly injected or intravenously administered to cells.

The dosage of antisense and siRNA varies depending on the administration method, patient age, weight, symptoms, etc., and cannot be specified unconditionally, but as the amount of antisense and siRNA, the blood concentration is about 10 to 100 ng / ml, preferably 20 to What is necessary is just to administer so that it may be about 50 ng / ml.
Antibody Since the antibody of the present invention inhibits its activity by binding to SM synthase, it is suitably used for the treatment or improvement of diseases requiring suppression of cell proliferation, such as leukemia, various cancers, and autoimmune diseases. it can.

  The antibody of the present invention may be usually an injection such as intravenous injection, intramuscular injection, or subcutaneous injection. The injection may contain conventional additives such as salt, wetting agent and emulsifier, if necessary.

The content of the antibody in the preparation varies depending on the administration route, patient age, weight, symptoms, etc., and cannot be generally specified, but the daily dose of the antibody is usually about 10 to 1000 mg, more preferably about 100 to 500 mg. It can be an amount.
(XI) Screening method for drug The cell growth inhibitor of the present invention, or a screening method for a therapeutic or ameliorating agent for a disease selected from the group consisting of leukemia, cancer and autoimmune disease (first method),
(a) contacting the test substance with a test cell carrying the recombinant vector of the present invention;
(b) comparing the SM synthase activity or its index value in the test cell with the SM synthase activity or its index value in the test cell not contacted with the test substance;
(c) selecting a test substance that reduces SM synthase activity.

The cell growth promoter of the present invention or a therapeutic or ameliorating agent for a disease selected from the group consisting of aplastic anemia, myelodysplastic syndrome, hematopoiesis suppression after bone marrow transplantation, cirrhosis, myocardial infarction, cerebral infarction, and wound The screening method (second method) is:
(a) contacting the test substance with a test cell carrying the recombinant vector of the present invention;
(b) comparing the SM synthase activity or its index value in the test cell with the SM synthase activity or its index value in the test cell not contacted with the test substance;
(c) selecting a test substance that increases SM synthase activity.

  According to the screening method of the present invention, a substance that affects the expression of the polynucleotide of the present invention or the activity of the polypeptide of the present invention indirectly via a cell membrane receptor, etc., is directly taken into the cell and directly A substance that affects its expression by binding to a polynucleotide, or a substance that affects its activity by directly binding to a polypeptide of the present invention, by being taken into cells and acting on other intracellular proteins A substance or the like that indirectly affects the expression of the polynucleotide of the present invention or the activity of the polypeptide of the present invention is selected.

A substance that inhibits the expression of the polynucleotide of the present invention or reduces the activity of the polypeptide of the present invention is useful as a cell growth inhibitor. A substance that promotes the expression of the polynucleotide of the present invention or increases the activity of the polypeptide of the present invention is useful as a cell growth promoter.
Test Cell The test cell for retaining the recombinant vector of the present invention is not limited thereto, but a mammalian cell or an insect cell can be used.

  Examples of mammalian cells that can be used include known cells such as Vero cells, Hela cells, CV1 cells, Namalva cells, COS1 cells, and CHO cells. In addition, human osteosarcoma cell line Saos-2 (ATCC, HTB85), colon cancer cell line DLD-1, human breast cancer cell line MCF-7 (National Food and Drug Institute Cell Bank, JCRB0134), human renal epithelial cell line Cancer cells such as 293 (ATCC, CRL1573) and red leukocyte disease cell line K562 (ATCC, CCL243) can also be used. Since subculture is easy and has many properties of normal cells, cancer cells may be used in the primary screening stage.

As insect cells, known cells such as Sf cells, MG1 cells, and High Five ^™ cells can be used.
Evaluation of SM synthesis activity
The method for confirming the decrease or increase in SM synthase activity is not particularly limited. For example, as shown in Example 6, after incubating cells with radiolabeled amino acids, the extracted lipid is subjected to TLC and corresponds to SM. How to compare the radioactivity of bands, fluorescent substrate NBD? Ceramide NBD? Change to SM BAS on TLC? A method of measuring with a 2000 image analyzer or a known fluorescence measuring device can be used.

Examples of the SM synthase activity index value include lysenin binding ability. When the non-toxic mutant lysenin is used, as shown in Example 2, lysenin binding ability may be measured using an antibody against MBP as lysenin fused to maltose binding protein (MBP). When natural lysenin is used, lysenin binding ability may be evaluated by measuring cell death by lysenin as shown in Example 2.
Test substance The type of test substance is not particularly limited. Examples include proteins, peptides, non-peptidic compounds (nucleotides, amines, carbohydrates, lipids, etc.), organic low molecular compounds, inorganic low molecular compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. It is done.
Contact between the contact / judgment test cell and the test substance can be performed, for example, as follows. That is, the cells carrying the vector are subcultured using a medium suitable for the cells and cultured so as to be in a logarithmic growth phase. When contacting the test substance, the cells are seeded to a cell density of about 5 × 10 ⁵ / ml and cultured for about 24 hours. Next, the test substance is added to the cell culture so as to have a concentration of about 10 to 1000 nM, for example, depending on the type. Next, it is incubated at about 37 ° C. for about 12 to 48 hours. In order to prevent apoptosis from being induced by the test substance, it is preferable to add the apoptosis inhibitor DEVD-CHO at a concentration of about 200 mM.

  Thereafter, the SM synthase activity in the test cell contacted with the test substance is compared with the SM synthase activity in the test cell not contacted with the test substance. The comparison can be made after measuring the SM synthase activity of both cells, or can be confirmed qualitatively by directly comparing the SM synthase activity visually or the like.

If the SM synthase activity is, for example, 75% or less, preferably 50% or less of the control in which the test substance is not contacted by bringing the test substance into contact, it may be determined that the SM synthase activity has decreased. Further, when the SM synthase activity is, for example, 125% or more, preferably 150% or more of the control, it may be determined that the SM synthase activity has increased.
(XII) Examples Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to these examples.
Cell culture In each Example, cells obtained and maintained as follows were used.

  WR19L / Fas cells were obtained from Dr. Yonehara of Kyoto University Virus Research Institute. WR19L / Fas-SM (−) cells and WR19L / Fas-SM (+) cells were isolated from native WR19L / Fas cells by limiting dilution.

Cells were routinely treated at 37 ° C. under 5% CO ₂ and 100% humidity in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 50 μM 2-mercaptoethanol and 75 μg / ml kanamycin. Maintained.

For culture of serum free medium, cells are washed, replated at 1 × 10 ⁵ cells / ml, and 5% CO ₂ in RPMI-1640 medium containing 5 μg / ml human insine and bovine holo type transferrin. The cells were cultured at 37 ° C. After culturing for 48 hours, the number of cells was measured by the dye exclusion method using 0.25% trypan blue.

Confirmation of the presence or absence of SM production in each cell (1) WR19L / Fas-SM (-) cells and WR19L / Fas-SM (+) cells were respectively treated with 2% FBS and L- [ ¹⁴ C] serine (specific activity; 155 mCi / mmol) in RPMI-1640 medium so as to have a density of 5 × 10 ⁵ cells / ml. Cells were labeled by culturing for 2 hours in 5% CO ₂ at 37 ° C.

  WR19L / Fas cells are lymphocyte cells overexpressing Fas antigen, WR19L / Fas-SM (+) cells are natural WR19L / Fas cells expressing SM, WR19L / Fas- SM (-) cells are the SM-deficient mutant.

  Cell lipids of both cells were extracted by the method of Bligh & Dyer (J. Biol. Chem. 264, 19076-19080 (1989)), applied to silica gel thin layer chromatography (TLC) plates (Whatman Maidstone, UK), and methyl acetate. /Propanol/chloroform/methanol/0.25% KCl (25: 25: 25: 10: 9). Since spots having radioactivity were visualized, the radioactivity was quantified using a BAS2000 image analyzer (Fuji Photo Film).

  The result of TLC is shown in FIG. 1A. In FIG. 1A, Cer represents ceramide, GC represents glycosylceramide, PE represents phosphatidylethanolamine, and PS represents phosphatidylserine.

In WR19L / Fas-SM (-) cells, [ ^14C ] serine-labeled SM detected in WR19L / Fas-SM (+) cells was not detected, and [ ^14C ] serine-labeled SM was not synthesized. I understand that.
(2) 50 μg lysate of each of the above cells in a 20 mM Tris / Hcl (Ph.7.5) reaction solution in the presence of 50 μg C6-NBD-ceramide and 500 μg phosphatidylcholine to give a final volume of 100 μl, 37 Incubation was performed at 0 ° C. for 30 minutes, and TLC was performed as described above.

  Fluorescent lipids were visualized with a FluorImager SI system (Molecular Dynamics, Sunnyvale, U.S.A.) at an excitation wavelength of 475 nm and an emission wavelength of 525 nm.

  As shown in FIG.1B, in WR19L / Fas-SM (-) cells, C6-NBD-SM fluorescently labeled with C6-NBD detected in WR19L / Fas-SM (+) cells as a substrate was not detected, It can be seen that SM is not synthesized and there is no SM synthase activity.

Confirmation of the presence of SM on the cell surface side of plasma membrane by FACS analysis (1) Lylyin is an SM-specific cytolytic antibody isolated from earthworms. It is known to make pores and kill cells (J. Biol. Chem. 273, 33787-33794 (1998)).

  In addition, CHO cells expressing SM outside the plasma membrane are sensitive to lysenin-induced cell death, and CHO cell mutants LY-A and LY-B with reduced cell surface SM content. Has been reported to be resistant to lysenin (J. Biol. Chem. 273, 33787-33794 (1998)).

  In this Example (1), a mutant (non-toxic) lysenin (J. Biol. Chem 278, 22762-22770 (2003)) that specifically binds to SM without inducing cell death was used. Thus, the presence of SM localized in the monolayer on the cell surface side of the plasma membrane was confirmed.

  Both cells were incubated for 30 minutes on ice with non-toxic lysenin (MBP-lysenin; provided by Mr. Kobayashi of RIKEN) fused to 100 ng / ml maltose binding protein.

The cells were then washed with ice-cold phosphate buffer saline (PBS) supplemented with 1% FCS and 0.1% NaN ₃ and incubated with rabbit anti-MBP antiserum (New England BioLabs, Berly, UK) on ice for 30 minutes.

  After washing the cells again, the cells were incubated with phycoerythrin (PE) -conjugated anti-rabbit IgG (Sigma-Aldrich) for 30 minutes and using a fluorescence-activating cell-sorter (FACS) calibur (BD Bioscience, San Jose, USA) Were subjected to FACS analysis. Data analysis was performed with Cell Quest software (Becton Dickinson).

The results are shown in FIG. 1C. In WR19L / Fas-SM (−) cells, there was no difference in fluorescence intensity between the case where lysenin treatment was not performed and the case where lysenin treatment was not performed. On the other hand, in WR19L / Fas-SM (+) cells, the fluorescence intensity was increased by lysenin treatment. This shows that the amount of SM on the cell surface side of the plasma membrane is remarkably reduced in WR19L / Fas-SM (−) cells.
(2) Cells are incubated with 500 ng / ml lysenin for 15 minutes at room temperature in the presence of 20 μg / ml propidium iodide (PI; Molecular Probes, Eugene, USA) and FACScalibur (BD Bioscience, San Jose, USA) It was used for the FACS analysis used.

  In addition, propidium iodide is a reagent for staining by being taken up by dead cells having pores in the plasma membrane.

  The results are shown in FIG. 1D. In WR19L / Fas-SM (+) cells, PI uptake increased by lysenin treatment compared to control. On the other hand, in the WR19L / Fas-SM (−) cells, the PI uptake amount was almost the same as that of the control even after lysenin treatment. Thus, since R19L / Fas-SM (−) cells are resistant to lysenin, it can be seen that the amount of SM on the cell surface side of the plasma membrane is significantly reduced.

Confirmation of the presence of SM on the cell surface side of the plasma membrane by confocal microscope observation To visualize SM localized in the monolayer on the cell surface side of the plasma membrane, both cells were coated with poly-L-lysine It was mounted on a slide glass, fixed with 4% formamide, treated with lysenine-MBP at 4 ° C. for 45 minutes, and then treated with anti-MBP antibody. After staining with PE-conjugated anti-rabbit IgG monoclonal antibody, cells were observed using a Zeiss LSM-5 Pascal laser scan confocal microscope (Carl Zeiss, Germany).

  A photomicrograph is shown in FIG. 1C. In WR19L / Fas-SM (+) cells, fluorescence was observed by lysenin treatment. This shows that SM is present on the cell surface side of the plasma membrane in WR19L / Fas-SM (+) cells. In contrast, in WR19L / Fas-SM (−) cells, no fluorescence was observed regardless of the presence or absence of lysenin treatment, indicating that the amount of SM on the cell surface was significantly reduced.

Expression cloning of SMS-1 cDNA (1) SM interacts strongly with cholesterol in biological membranes and model membranes, and SM is a membrane microdomain related to cell functions such as cell death and cell proliferation ( It has been reported that it is required for the formation of lipid rafts). It has also been reported that LY-A cells with reduced SM levels are susceptible to methyl-β-cyclodextrin (MβCD) -induced cell death (J. Biol. Chem 275,34028-34034 (2000). etc).

  Thus, since SM is strongly bound to plasma membrane cholesterol, it is considered that SM is resistant to MβCD-induced cell death.

  In this example, using the pantropic retrovirus transfection system, WR19L / SM (-) cells were transfected with a cDNA library of human Hela cells, and MβCD resistant mutants were selected.

  Specifically, pantropic retrovirus particles containing the vesicular stomatitis virus (VSV-G) G glycoprotein were prepared using a human Hela cDNA retrovirus expression library kit and GP2-293 packaging cells (BDBiosciences Clontech).

  24 hours after infection, WR19L / Fas-SM (−) cells were cultured overnight in RPMI-1640 medium containing 2% FBS. After washing the cells with serum-free RPMI-1640 medium, the cells are incubated in RPMI-1640 medium containing 1.5 mM MβCD for 5 minutes at 37 ° C. and supplemented with normal medium so that the final FBS concentration is 5%. And then cultured at 37 ° C. for 60 hours.

  Cells were replated in RPMI-1640 medium containing 2% FBS, cultured overnight, and treated again with an appropriate concentration of MβCD. By performing 1.5 mM MβCD treatment twice, 3 mM MβCD treatment twice, and 5 mM MβCD treatment twice, WR19L / SM (−) cell MβCD resistant mutants were isolated by limiting dilution.

  PCR using pLIB expression vector specific primers (5 'and 3' pLIB primers, BD Biosciences Clontech) was used to amplify a 2.0 kb cDNA inserted into the genome of MβCD resistant cells and pGEM-T easy vector ( Promega, Madison, WI, USA).

  Using a DNA sequencer, determine the base sequence of the region considered to encode SM synthase in the above 2.0 kb cDNA of MβCD resistant cells (WR19L / Fas-SMS-1 cells) The amino acid sequence of the peptide was deduced.

  The base sequence and amino acid sequence are shown in FIG. 2A. MβCD resistant cells incorporated a 1967 bp human cDNA encoding 413 amino acids into the genome. The strile alpha motif (SAM) region sequence and the transmembrane (TM) region sequence shown in FIG. 2A are predicted using the BLAST algorithm and the SOSUI program, respectively.

Further, FIG. 2B shows a Hydropathy plot of the amino acid sequence of SMS-1 analyzed by the method of Kyte & Doolittle (J. Mol. Biol. 157, 105-132 (1982)). The SMS-1 peptide has an estimated molecular weight of 48.6 kDa. From the BLAST algorithm and the SOSUI program, it can be seen that SMS-1 has one strile alpha motif (SAM) region sequence in the N-terminal region and four transmembrane (TM) region sequences.
(2) DNA was extracted from WR19L / Fas-SM (-) cells and WR19L / Fas-SMS-1 cells according to conventional methods, and subjected to agarose gel electrophoresis. A gel photograph is shown in FIG. 2C. In WR19L / Fas-SM (-) cells, a 2 kb band not detected in WR19L / Fas-SMS-1 cells was detected, and SMS-1 cDNA was introduced into WR19L / Fas-SM (-) cells. It was confirmed.

Measurement of viability of cells exposed to MβCD After sequencing and computer analysis, the cDNA was subcloned into a pLIB expression vector and transfected into WR19L / Fas-SA (-) cells using VSV-G retrovirus particles . The resulting cells were treated with 5 mM MβCD as follows to enrich for MβCD resistant cells (WR19L / Fas-SMS-1 cells).

That is, 1 × 10 ⁶ WR19L / Fas-SM (+) cells, WR19L / Fas-SM (−) cells and WR19L / Fas-SMS-1 cells were washed, and 1 ml of serum-free RPMI-1640 medium was used. The suspension was treated with MβCD at an appropriate concentration and incubated at 37 ° C. for 5 minutes in 5% CO ₂ . After adding 1 ml of normal culture solution, the cells were further incubated for 12 hours. The cell viability was measured using a cell viability kit (Nacalai Tesque) using WST-8.

  The results are shown in FIG. 2D. In WR19L / Fas-SMS-1 cells in which SMS-1 cDNA is introduced into WR19L / Fas-SM (−) cells, the resistance to MβCD-induced cell death is restored. From this, it can be seen that SM was introduced by the introduction of the cDNA.

Measurement of SM synthase activity in WR19L / Fas-SMS-1 cells (1) In the same manner as in (1) of Example 1, WR19L / Fas-SM (-) cells and WR19L / Fas-SMS-1 cells were -After incubation with [ ¹⁴ C] serine, lipids were extracted and subjected to TLC. The results are shown in FIG. 3A. In WR19L / Fas-SMS-1 cells, SM labeled with [ ¹⁴ C] serine was detected, indicating that SM synthesis was restored.
(2) lysate of WR19L / Fas-SM (-) cells and WR19L / Fas-SMS-1 cells in the presence of C6-NBD-ceramide and phosphatidylcholine in the same manner as (2) of Example 1 After incubation, TLC was performed. The results are shown in FIG. 3B. C6-NBD-SM is detected in WR19L / Fas-SMS-1 cells, indicating that SM synthesis is restored.

From the above results, it can be seen that SMS-1 cDNA is essential for SM synthesis in mammalian cells.
(3) WR19L / Fas-SMS-1 cells were homogenized in an ice-cold buffer containing 20 mM Tris HCl (pH 7.4), 2 mM EDTA, 10 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 2.5 μg / ml leupeputin. Lysate containing 500 μg of cellular protein was added to 10 mM Tris HCl (pH 7.5), 1 mM EDTA, 20 μM C6-NBD-ceramide, 120 μM [N-methyl- ¹⁴ C] PC (specific activity; 57 μCi / mmol) or [methyl − ¹⁴ C] CDP-choline (specific activity; 54 μCi / mmol) was added to the reaction solution and incubated at 37 ° C. for 30 minutes.

Extract lipids by Bligh &Dyer's method (J.Biol.Chem 264,19076-19080 (1989)), apply to TLC plate, and develop with solution containing chloroform / methanol / 12mM MgCl ₂ aqueous solution (65: 25: 4) did. Radioactivity was visualized using the BAS2000 system.

  The results are shown in FIG. 3C. In WR19L / Fas-SMS-1 cells, SM is synthesized when incubated in the presence of ceramide and PC, indicating that PC is used as a donor for phosphatidylcholine.

  Biochemistry 21,2753-2759 (1982) describes that SM synthase uses phosphatidylcholine as a phosphocholine donor, and the above results indicate that SMS-1 cDNA encodes SM synthase. It confirms that it is something to do.

Effect of lysenin on survival of WR19L / Fas-SMS-1 cells 7 × 10 ⁵ WR19L / Fas-SMS-1 cells were washed, resuspended in prewarmed PBS and treated with 0,100,500 and 1000 ng / ml lysenin And incubated for 1 hour at 37 ° C. in 5% CO ₂ . After addition of FBS, the number of viable cells was counted by dye exclusion method using 0.25% trypan blue.

  The results are shown in FIG. 4A. The number of surviving WR19L / Fas-SMS-1 cells decreases in a lysenin concentration-dependent manner. This indicates that the amount of SM in the plasma membrane increases due to overexpression of SMS-1 cDNA.

Effect of SMS-1 cDNA on cell survival
After culturing WR19L / Fas-SM (+) cells, WR19L / Fas-SM (-) cells and WR19L / Fas-SMS-1 cells in serum-free medium for 48 hours, the number of viable cells is 0.25%. Counted by try exclusion method using trypan blue.

  The results are shown in FIG. 4B. The growth of WR19L / Fas-SM (-) cells is suppressed, but WR19L / Fas-SMS-1 cells are proliferating to the same extent as WR19L / Fas-SM (+) cells. This shows that SM synthase is essential for cell growth.

A and B are diagrams showing TLC patterns of lipids of WR19 / Fas-SM (−) cells and WR19 / Fas-SM (+) cells. C and D are diagrams showing the presence or absence of SM on the surface of WR19 / Fas-SM (−) cells and WR19 / Fas-SM (+) cells. A is a figure which shows the base sequence of SMS-1 cDNA, and the amino acid sequence which it codes. B is a diagram showing the SMS-1 peptide hydropathy. FIG. C shows the presence or absence of SMS-1 cDNA in the genomes of WR19 / Fas-SM (−) cells and WR19 / Fas-SMS-1 cells. D shows that WR19 / Fas-SMS-1 cells have acquired resistance to MβCD. A and B are diagrams showing that WR19 / Fas-SMS-1 cells have SM synthesis activity. C shows that SMS-1 cDNA uses phosphatidylcholine as a phosphocholine donor. A is a figure which shows that WR19 / Fas-SMS-1 cell overexpresses SM. B shows that WR19 / Fas-SMS-1 cells grow to the same extent as WR19 / Fas-(+) cells.

Claims (25)

The following polypeptide (1) or (2).
(1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1.
(2) A polypeptide having an amino acid sequence in which one or more amino acids are deleted, added or substituted in SEQ ID NO: 1 and having sphingomyelin synthase activity. A cell growth promoter comprising the polypeptide according to claim 1 as an active ingredient. Treatment of a disease selected from the group consisting of aplastic anemia, myelodysplastic syndrome, hematopoiesis suppression after bone marrow transplantation, cirrhosis, myocardial infarction, cerebral infarction, wound, comprising the polypeptide of claim 1 as an active ingredient Improver. The following polynucleotide (3) or (4):
(3) A polynucleotide comprising the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 or a base sequence complementary thereto.
(4) A polypeptide that hybridizes under stringent conditions with a polynucleotide having the nucleotide sequence of nucleotide numbers 338 to 1576 or a nucleotide sequence complementary thereto in SEQ ID NO: 2 and has sphingomyelin synthase activity. Encoding polynucleotide. A cell growth promoter comprising the polynucleotide according to claim 4 as an active ingredient. Treatment of a disease selected from the group consisting of aplastic anemia, myelodysplastic syndrome, hematopoietic suppression after bone marrow transplantation, cirrhosis, myocardial infarction, cerebral infarction, wound, comprising the polypeptide according to claim 4 as an active ingredient Improver. A recombinant vector comprising the polynucleotide according to claim 4. A transformant carrying the recombinant vector according to claim 7. A method for producing the polypeptide according to claim 1, comprising a step of culturing the transformant according to claim 8, and a step of recovering the polypeptide according to claim 1 from the culture. The following (5) or (6) antisense polynucleotide.
(5) A polynucleotide comprising a base sequence complementary to at least 18 consecutive bases of the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 or a base sequence complementary thereto.
(6) A polynucleotide that hybridizes under stringent conditions with at least 18 consecutive bases of the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 or a base sequence complementary thereto. A cell growth inhibitor comprising the antisense polynucleotide according to claim 10 as an active ingredient. A therapeutic or ameliorating agent for a disease selected from the group consisting of leukemia, cancer and autoimmune disease, comprising the antisense polynucleotide according to claim 10 as an active ingredient. The following (7) or (8) siRNA.
(7) An siRNA comprising a pair of an RNA strand complementary to at least 21 consecutive bases of the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 and an RNA strand complementary thereto.
(8) An siRNA comprising a double-stranded RNA that hybridizes under stringent conditions with at least 21 consecutive base sequences of base numbers 338 to 1576 of SEQ ID NO: 2 or a base sequence complementary thereto. A cell growth inhibitor comprising the siRNA according to claim 13 as an active ingredient. A therapeutic or ameliorating agent for a disease selected from the group consisting of leukemia, cancer and autoimmune disease, comprising the siRNA according to claim 13 as an active ingredient. The following (9) or (10) ribozyme.
(9) having two base sequence portions complementary to each of two regions consisting of at least 20 consecutive bases in the base sequence of base numbers 338 to 1576 described in SEQ ID NO: 2 or a base sequence complementary thereto And the ribozyme which has the activity which decomposes | disassembles the polynucleotide of Claim 4.
(10) Two of the base sequences of base numbers 338 to 1576 described in SEQ ID NO: 2 or two regions that hybridize under stringent conditions to two regions consisting of at least 20 consecutive bases in the base sequence complementary thereto A ribozyme having a base sequence and having an activity of degrading the polynucleotide of claim 4. A cell growth inhibitor comprising the siRNA according to claim 16 as an active ingredient. A therapeutic or ameliorating agent for a disease selected from the group consisting of leukemia, cancer and autoimmune disease, comprising the siRNA according to claim 16 as an active ingredient. An antibody that specifically recognizes the polypeptide according to claim 1. A cell growth inhibitor comprising the antibody according to claim 19 as an active ingredient. A therapeutic or ameliorating agent for a disease selected from the group consisting of leukemia, cancer and autoimmune disease, comprising the antibody according to claim 19 as an active ingredient. (a) contacting the test substance with a test cell carrying the recombinant vector according to claim 7;
(b) comparing the sphingomyelin synthase activity or its index value in the test cell with the sphingomyelin synthase activity or its index value in the test cell not contacted with the test substance;
(c) a method for screening a cell growth inhibitor, comprising a step of selecting a test substance that reduces sphingomyelin synthase activity. (a) contacting the test substance with a test cell carrying the recombinant vector according to claim 7;
(b) comparing the sphingomyelin synthase activity or its index value in the test cell with the sphingomyelin synthase activity or its index value in the test cell not contacted with the test substance;
(c) a method for screening a therapeutic or ameliorating agent for a disease selected from the group consisting of leukemia, cancer, and autoimmune disease, comprising a step of selecting a test substance that reduces sphingomyelin synthase activity. (a) contacting the test substance with a test cell carrying the recombinant vector according to claim 7;
(b) comparing the sphingomyelin synthase activity or its index value in the test cell with the sphingomyelin synthase activity or its index value in the test cell not contacted with the test substance;
(c) selecting a test substance that increases sphingomyelin synthase activity, and a method for screening a cell growth promoter. (a) contacting the test substance with a test cell carrying the recombinant vector according to claim 7;
(b) comparing the sphingomyelin synthase activity or its index value in the test cell with the sphingomyelin synthase activity or its index value in the test cell not contacted with the test substance;
(c) a group consisting of aplastic anemia, myelodysplastic syndrome, suppression of hematopoiesis after bone marrow transplantation, cirrhosis, myocardial infarction, cerebral infarction, and wound, including a step of selecting a test substance that increases sphingomyelin synthase activity A method for screening for a therapeutic or ameliorating agent for a more selected disease.

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