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WO2001010905A1 - Compositions medicinales destinees a inhiber la proliferation anormale des cellules vasculaires des muscles lisses - Google Patents

Compositions medicinales destinees a inhiber la proliferation anormale des cellules vasculaires des muscles lisses Download PDF

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Publication number
WO2001010905A1
WO2001010905A1 PCT/JP2000/005256 JP0005256W WO0110905A1 WO 2001010905 A1 WO2001010905 A1 WO 2001010905A1 JP 0005256 W JP0005256 W JP 0005256W WO 0110905 A1 WO0110905 A1 WO 0110905A1
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WIPO (PCT)
Prior art keywords
lkb1
smooth muscle
muscle cells
vascular smooth
compound
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PCT/JP2000/005256
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English (en)
Japanese (ja)
Inventor
Jun-Ichi Nezu
Asuka Ose
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Chugai Pharmaceutical Co Ltd
Chugai Research Institute for Molecular Medicine Inc
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Chugai Pharmaceutical Co Ltd
Chugai Research Institute for Molecular Medicine Inc
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Priority to AU63192/00A priority Critical patent/AU6319200A/en
Publication of WO2001010905A1 publication Critical patent/WO2001010905A1/fr
Anticipated expiration legal-status Critical
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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
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to a molecule for suppressing abnormal growth of vascular smooth muscle cells and screening thereof, and a diagnosis of a disease caused by abnormal growth of vascular smooth muscle cells.
  • MIM 175200 PJS
  • PJS Peutz-Jeghers Syndrome
  • PJS is inherited by an autosomal dominant inheritance pattern, the cause gene can be mapped to the chromosome 19 ⁇ 133 region by linkage analysis in a PJS patient's pedigree by the 1997 He recitation inki et al. (He Bandai ink i A et al, Nat Genet 1997 Jan; 15 (l): 87-90).
  • missense mutation was a mutation that lost kinase activity (Mehenni H et al, Am J Hu Genet 1998 Dec 63 (6): 1641-50).
  • the present invention is based on the finding that LKB1 protein is highly expressed and functions in vascular smooth muscle cells, and its purpose is to promote the expression and function of LKB1 protein in vascular smooth muscle cells. To suppress the abnormal growth is there. More specifically, the present invention provides use of an LKB1 protein, an LKB1 gene, and a compound that increases the activity and abundance of the protein for suppressing abnormal proliferation of vascular smooth muscle cells, and a pharmaceutical composition containing these molecules. Offer things. Furthermore, the present invention provides a method for screening for a compound that increases the activity or abundance of LKB1 protein.
  • the present inventors analyzed the tissue distribution of LKB1 gene expression in order to analyze the role of the LKB1 gene in normal cells.
  • expression of mouse LKB1 mRNA in various organs was detected by Northern blotting.
  • expression of the LKB1 gene was observed in all analyzed organs. Therefore, next, the localization of the detailed expression of LKB1 protein in various organs was analyzed by immunohistochemical staining.
  • the present inventors have found that LKB1 protein is strongly expressed not only in smooth muscle cells constituting the mucosal muscle plate, but also particularly in vascular smooth muscle cells. This result was further confirmed by in situ hybridization.
  • the LKB1 gene is a causative gene of PJS, and it is well known that in PJS, polyps are caused by abnormal proliferation of smooth muscle constituting the mucosa of the gastrointestinal tract (Tomlinson IP et al. , J Med Genet. 1997 Dec; 34 (12): 1007-1 Hemminki A. Cell Mol Life Sci. 1999 May; 55 (5): 735-50). Pathologically, the presence of a core of smooth muscle cells in gastrointestinal polyps has become an important diagnostic criterion for PJS.
  • LKB1 protein is strongly expressed in vascular smooth muscle cells
  • LKB1 protein only functions in smooth muscle cells constituting gastrointestinal mucosal muscularis, as shown in the findings of conventional PJS patients. Rather, it suggests that vascular smooth muscle cells also play an important role in regulating proliferation.
  • LKB1 protein in human lesions.
  • the results showed that LKB1 expression was reduced in human coronary atherosclerotic lesions. This fact further supports the importance of LKB 1 in controlling vascular smooth muscle cell proliferation.
  • LKB1 protein and proliferation of vascular smooth muscle cells From the relationship between LKB1 protein and proliferation of vascular smooth muscle cells, it is thought that targeting LKB1 protein enables efficient screening of compounds that control abnormal proliferation of vascular smooth muscle cells. Furthermore, the LKB1 protein and LKB1 gene, and the compounds obtained by such screening, are expected to be applied to the prevention and treatment of various diseases caused by abnormal proliferation of vascular smooth muscle cells. . Further, by detecting the expression of the LKB1 gene, it is possible to diagnose a disease caused by abnormal proliferation of vascular smooth muscle cells.
  • the present invention is based on the above findings, and more specifically,
  • LKB1 protein which is used to suppress abnormal growth of vascular smooth muscle cells
  • a pharmaceutical composition for suppressing abnormal proliferation of vascular smooth muscle cells comprising as an active ingredient the protein according to claim 1, the DNA according to claim 2, or the compound according to claim 3, (5) A method for screening a compound having an activity of suppressing abnormal growth of vascular smooth muscle cells,
  • a method for diagnosing a disease caused by abnormal proliferation of vascular smooth muscle cells (a) preparing a cell sample from a patient with the disease;
  • the present invention provides an LKB1 protein used for suppressing abnormal proliferation of vascular smooth muscle cells and a DNA encoding the protein.
  • the term “used to suppress abnormal proliferation of vascular smooth muscle cells” in the present invention includes the use as a reagent for suppressing abnormal proliferation of vascular smooth muscle cells and the suppression of abnormal proliferation of vascular smooth muscle cells in patients. For use as medicines.
  • the LKB1 protein used for suppressing abnormal proliferation of vascular smooth muscle cells may be a natural protein or a recombinant protein utilizing a genetic recombination technique.
  • These proteins can be prepared by the method described in the gazette (W099 / 28459). That is, the natural protein is subjected to affinity chromatography using an antibody against a partial peptide of the LKB1 protein described in Example 7 of the publication, whereby the LKB1 protein expression is high, for example, testis, It can be isolated and prepared from fetal liver or cultured cells such as K562 cells.
  • the recombinant protein is obtained, for example, by culturing a cell transformed with DNA encoding LKB1 protein (SEQ ID NO: 5 of the publication or mouse LKB1 cDNA of the present application (SEQ ID NO: 1)), and It can be prepared by recovering the expressed protein from the culture supernatant.
  • Examples of cells used for production of recombinant proteins include mammalian cells such as COS cells, CH0 cells, NIH3T3 cells, insect cells such as Sf9 cells, yeast cells, and E. coli.
  • Vectors for expressing recombinant proteins in cells are It varies depending on the cell, but examples of mammalian cell vectors include pcDNA3 (Invitrogen) and pEF-BOS (Nucleic Acids.
  • BAC-to-BAC baculovirus expression system GIBC 0 BRL
  • Pichia Expression Kit Invitrogen
  • pGEX-5X-1 as a vector for E. coli.
  • the vector can be introduced into a host cell by, for example, the calcium phosphate method, the DEAE dextran method, the method using Cationic Liposomal D0TAP (Beilinger Mannheim) or SuperFect (Quiagen), the electroporation method, or the calcium chloride method.
  • the resulting purification of the recombinant protein from the transformant can be performed using literature r
  • the DNA encoding the LKB1 protein used for suppressing abnormal proliferation of vascular smooth muscle cells may be cDNA or genomic DNA.
  • the sequences of LKB1 cDNA and LKB1 genomic DNA are disclosed in the gazette (W099 / 28459) (SEQ ID NO: 5 for LKB1 cDNA, SEQ ID NOs: 1 to 4 for LKB1 genomic DNA).
  • LKB1 cDNA and LKB1 genomic DNA are synthesized by synthesizing oligonucleotides (usually 15 to 50 bases) based on the above-disclosed sequence information, and using this as a primer to convert human fetal liver cDNA into a type II polymerase chain. The reaction can be amplified and prepared.
  • LKB1 cDNA and LKB1 genomic DNA can be prepared.
  • Hybridization can be performed under the conditions described in Example 2.
  • a cDNA library a human fetal liver-derived cDNA library can be used.
  • a commercially available cDNA library and genomic library (CL0NTECH) can also be used.
  • the above-mentioned polymerase chain reaction-hybridization method can be performed according to a general method described in an experimental book such as "Rsambrook et al., Molecular Cloning (Cold Spring Harbor Lab.)".
  • the DNA fragment amplified by the polymerase chain reaction or the DNA fragment screened by the hybridization method can be subcloned into an appropriate plasmid DNA or the like and used for expression experiments and the like.
  • a compound that promotes LKB1 protein activity (kinase activity), a compound that suppresses LKB1 protein degradation, and a LKB1 gene expression are promoted.
  • Compounds can also be used.
  • immunohistochemical analysis revealed that LKB1 protein was strongly expressed in vascular smooth muscle cells of various organs. From this ⁇ , it is thought that abnormal growth can be suppressed by increasing the amount and function of proteins in vascular smooth muscle cells.
  • a test compound is brought into contact with LKB1 protein, and a compound that increases the kinase activity may be selected. That is, a compound that promotes the kinase activity of LKB1 protein (a) a step of bringing the test sample into contact with the LKB1 protein; (b) a step of detecting the kinase activity of the LKB1 protein; and (c) a kinase activity of the LKB1 protein as compared to the case where the test sample is not brought into contact. And a step of selecting a compound that increases the amount of the compound.
  • the test sample is not particularly limited, and includes, for example, cell extract, cell culture supernatant, fermented microorganism product, marine organism extract, plant extract, purified or crude protein, peptide, non-peptidic compound, synthetic low Molecular compounds and natural compounds.
  • the protein of the present invention to be brought into contact with the test sample may be, for example, a purified protein or a form bound to a carrier.
  • the test sample may be contacted with cells expressing the LKB1 protein.
  • the kinase activity of the LKB1 protein can be detected, for example, as autophosphorylation (autophosphorylation) activity by the LKB1 protein. It said activity, - can be measured with a liquid scintillation child Yonkaun evening Chief as transition amount to the LKB1 protein 32 p from AT p [ ⁇ ].
  • LKB1 expression plasmid DNA pcDNA3 / LKBlmyc
  • SuperFect Qiagen
  • about 10 fi C0S7 cells are sown in a 10 cm dish, cultured for 1 hour, then a mixture of 10 ⁇ g of plasmid DNA and 60% of SuperFect is added, and cultured for about 3 hours.
  • the culture medium is replaced with a new one, and the cells are further cultured for 2 days, and then the cells are removed with a trypsin-EDTA solution and collected.
  • NP40 kinase lysis buffer (10 mM Tris-HCl pH 7.8, 13 ⁇ 4 NP40, 0.15 M sodium chloride, ImM EDTA, 50 ⁇ sodium fluoride, 5 mM sodium pyrophosphate, 10 g / ml aprotinin, ImM PMSF) And solubilize the protein by mixing at 4 ° C for 30 minutes. Protein A / G plus agarose (Santa Cruz) is added to the cell lysate thus obtained and mixed for 30 minutes to remove proteins that are nonspecifically adsorbed to the beads.
  • Kinase Atsu Si is 50mM Tris-HCl (pH 7.8), lmM DTT, (such as Mn) LOMM divalent Kachio down and 10 ⁇ Ci of - performing in the ⁇ 32 P] reaction of the total amount 50 ⁇ 1 including ATP.
  • the reaction is stopped, and the reaction is stopped by adding SDS-PAGE sample buffer and boiling to perform SDS-PAGE. Fix the gel with a methanol / acetic acid solution, dry it, and analyze the image using a BAS200 image analyzer (Fuji Film).
  • Kinase activity is also detected by the method described in the literature (Am J Hum Genet (1988) Dec; 63 (6): 1641-50, Hum Mol Genet (1999) Jan; 8 (l): 45-51). You can also.
  • the compound that suppresses the decomposition of LKB1 protein comprises: (a) a step of bringing a test sample into contact with LKB1 protein;
  • Screening can be performed by a method comprising: (b) a step of detecting the amount of LKB1 protein; and (c) a step of selecting a compound that increases the amount of LKB1 protein as compared to the case where the test sample is not contacted. It is.
  • the test sample is not particularly limited as in the above screening method.
  • the protein of the present invention to be brought into contact with the test sample may be, for example, a purified protein or a form bound to a carrier.
  • the test sample may be brought into contact with cells expressing the LKB1 protein.
  • the abundance of the LKB1 protein can be detected, for example, by the Western blotting method.
  • a test sample is brought into contact with cells expressing the LKB1 protein, Thereafter, the cells are lysed and subjected to SDS-PAGE. After SDS-PAGE, transfer the proteins that have been run on the gel to a nitrocellulose membrane.
  • An antibody against the LKB1 protein is brought into contact with the membrane, and further a labeled secondary antibody is brought into contact with the membrane to detect the amount of the LKB1 protein present on the membrane.
  • the compound that promotes the expression of the LKB1 gene comprises: (a) a step of bringing a test sample into contact with cells expressing the endogenous LKB1 gene; (b) a step of detecting the amount of a transcript of the LKB1 gene; (C) a step of selecting a compound that increases the amount of the transcript of the LKB1 gene as compared to the case where the test sample is not brought into contact with the test sample.
  • the test sample is not particularly limited as in the above-mentioned screening.
  • cells expressing the endogenous LKB1 gene for example, cells such as human THP1, K562, SW480, HEK293, and mouse NIH3T3 can be suitably used.
  • the amount of the transcript of the LKB1 gene can be detected using a method known to those skilled in the art, such as a Northern plotting method or an RT-PCR method.
  • a compound that promotes LKB1 gene expression can be screened by a method using the activation of the LKB1 gene expression control region as an index. For efficient screening, it is possible to use the repo overnight gene.
  • the screening is, (a) downstream of the expression control region of LKB1 gene into a cell containing the vector repo Isseki monogenic is operably linked, contacting a test sample, detecting (b) reporter activity And (c) selecting a compound that increases the repo overnight activity as compared to the case where the test sample is not brought into contact with the test sample.
  • the present inventors have clarified the structure of the 5 'upstream region of the LKB1 gene in SEQ ID NO: 1 in the publication (W099 / 28459).
  • the expression control region of the LKB1 gene is, for example, a repo allele downstream of the 5 'upstream region of the LKB1 gene (the DNA consisting of the nucleotide sequence of SEQ ID NO: 1 or a part thereof).
  • the vector can be identified by constructing a vector to which the vector has been ligated, introducing the vector into an appropriate cell, and detecting the activity of the repo overnight gene.
  • the upstream region of the LKB1 gene is cleaved with various restriction enzymes to obtain a fragment of an appropriate size, and this is used as a fragment of the luciferase gene, secretory alkaline phosphatase gene, or chloramphenic acid gene.
  • Produce an expression vector (PicaGen e TM Vector Wako Pure Chemical Industries, Ltd.) that is integrated upstream of the repo overnight gene such as the 1-acetyltransferase (CAT) gene.
  • CAT 1-acetyltransferase
  • this is introduced into appropriate host cells such as COS cells, HEK293 cells, and CH0 cells, and after culturing for a certain period of time, the amount of each reporter gene product inside and outside the cells is measured, whereby the integrated gene fragment is obtained. Can be measured. If an active gene fragment is identified in this way, the same operation is repeated with a smaller fragment, whereby the active site can be specified in a narrower region. Finally, the activity can be measured after the nucleotide sequence of the specified region is changed by site-directed mutagenesis or the like to confirm that it is the active site.
  • a vector is constructed downstream of the repo allele functionally linked (here, “functionally linked” means that the expression control region is The expression control region and the reporter gene are linked so that the reporter gene linked downstream thereof can be expressed in response to the activation.)
  • C As the reporter gene for example, A luciferase gene, a secretory lipophilic phosphatase gene, a chloramphenico-l-acetyltransferase (CAT) gene and the like can be used. This is then introduced into mammalian cells, the test sample is contacted with the cell line, and the repo overnight activity is detected.
  • the test sample is not particularly limited as in the above screening method.
  • the detection of reporter activity can be performed by a known method depending on the type of the reporter gene.
  • the expression of LKB1 gene is promoted by selecting a compound that increases the repo activity overnight compared to the repo-activity in cells that are not brought into contact with the test sample.
  • Compounds that suppress abnormal cell growth can be screened.
  • This screening method is simpler than screening using direct detection methods such as Northern blotting and RT-PCR analysis because it detects LKB1 gene expression using reporter activity as an indicator. It has the feature of.
  • the LKB1 protein, the DNA encoding the protein, and the compound isolated by the above screening are used as reagents for suppressing the abnormal growth of vascular smooth muscle cells, and are also used as diseases for causing abnormal growth of vascular smooth muscle cells.
  • Diseases caused by abnormal proliferation of vascular smooth muscle cells include, for example, atherosclerosis, vascular hypertrophy (reste nosis) caused by vascular transplantation, PTCA (percutaneous transluminal coronary angioplasty) therapy, and the like.
  • LKB1 protein When LKB1 protein is used as a reagent for suppressing abnormal proliferation of vascular smooth muscle cells, LKB1 protein can be introduced into vascular smooth muscle cells by a method such as microinjection.
  • the DNA encoding the LKB1 protein When the DNA encoding the LKB1 protein is used as a reagent for suppressing abnormal proliferation of vascular smooth muscle cells, insert the DNA into a vector that can guarantee the expression of the DNA in vascular smooth muscle cells.
  • a vector can be introduced into vascular smooth muscle cells.
  • the vector can be introduced into vascular smooth muscle cells by a method known to those skilled in the art, for example, a calcium phosphate method, a DEAE dextran method, a cationic ribosome method, an electoral poration method, and a lipofection. It is.
  • a microphone opening method for example, a high molecular compound such as protein
  • gene transfer methods such as calcium phosphate method (when the compound is a gene)
  • addition of vascular smooth muscle cells to a culture medium for example, calcium phosphate method, and addition of vascular smooth muscle cells to a culture medium.
  • LKB1 protein or the compound obtained by the above screening is used as a pharmaceutical composition for suppressing abnormal proliferation of vascular smooth muscle cells, in addition to directly administering these to a patient, known pharmaceutical methods may be used.
  • pharmacologically acceptable carriers or vehicles specifically, sterile water or saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives It is possible to formulate a drug product by combining it with an appropriate agent, binder and the like in a unit dosage form generally required for pharmaceutical practice.
  • the amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
  • Additives that can be incorporated into tablets and capsules include, for example, binders such as gelatin, corn starch, tragacanth gum, acacia, excipients such as crystalline cellulose, corn starch, gelatin, and alginic acid. a bulking agent, a lubricant such as magnesium stearate, a sweetening agent such as sucrose, lactose or saccharin, peppermint, if c unit dosage forms flavoring agent such as Akamono oil or cherry one is used is force capsule
  • the above materials may further contain a liquid carrier such as oil and fat.
  • Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.
  • Aqueous injection solutions include, for example, saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizing agents.
  • saline isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizing agents.
  • alcohols specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM) and HC0-50 may be used in combination.
  • the oily liquid includes sesame oil and soybean oil, and may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizer.
  • a buffer for example, a phosphate buffer, a sodium acetate buffer, a soothing agent, for example, proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant may be blended.
  • the prepared injection solution is usually filled into an appropriate ampoule.
  • Administration to patients can be performed, for example, by intraarterial injection, intravenous injection, subcutaneous injection, etc., or intranasally, transbronchially, intramuscularly, transdermally, or orally by a method known to those skilled in the art. It can do better.
  • the dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose.
  • the dosage of the drug of the present invention varies depending on the condition, in the case of oral administration, in an adult (with a body weight of 60 kg), it is generally about 0.1 to 100 mg, preferably about 0.1 mg per day. It is 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • the single dose varies depending on the subject of administration, symptoms, and administration method.
  • injection usually in adults (with a body weight of 60 kg), About 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per day is administered by intravenous injection.
  • the DNA encoding the LKB1 protein When the DNA encoding the LKB1 protein is used as a drug for suppressing abnormal proliferation of vascular smooth muscle cells in patients (for gene therapy use), the DNA encoding the LKB1 protein must be a suitable vector, for example, an adenovirus vector. Adeno-associated virus vector, retrovirus vector, or plasmid DNA And administer it to the patient.
  • a suitable vector for example, an adenovirus vector.
  • Adeno-associated virus vector, retrovirus vector, or plasmid DNA And administer it to the patient.
  • a preferred method can be appropriately selected from methods known to those skilled in the art, such as oral administration, intravenous administration, topical administration to the affected area, and the like.
  • the administration method may be an in vivo method or an ex vivo method.
  • tissue translocation and tissue absorbability can be enhanced by encapsulating the gene in a liposome prepared by converting phospholipids into micelles.
  • tissue transportability and tissue absorbability can be enhanced by adding a cationic lipid to form a complex with gene DNA. This makes it possible to replace the mutated LKB1 gene in the patient with a normal gene, or to administer the normal gene additionally to the patient, thereby causing abnormalities in the patient's vascular smooth muscle cells. It is thought that proliferation can be suppressed.
  • the present inventors have also found a relationship between the expression of the LKB1 gene and abnormal proliferation of vascular smooth muscle cells. Accordingly, the present invention provides a method for diagnosing a disease caused by abnormal proliferation of vascular smooth muscle cells using the expression of the LKB1 gene as an index.
  • the diagnostic method of the present invention can be carried out by preparing a cell sample from a patient (including a person who has not yet developed the disease) of the disease and detecting the expression of the LKB1 gene in the cell.
  • the disease to be diagnosed according to the present invention is not particularly limited as long as it is a disease caused by abnormal proliferation of vascular smooth muscle cells, and examples include coronary atherosclerosis.
  • the cell sample prepared from the patient is usually a cell of a patient's lesion or a cell that may become a lesion, including vascular smooth muscle cells.
  • LKB1 gene in cells can be detected by methods known to those skilled in the art. That is, it can be detected at the transcription level, for example, by the Northern blotting method RT-PCR method, and at the protein level, for example, by the Western blotting method.
  • the patient is determined to be suffering from or predisposed to the disease.
  • the present invention makes the above diagnosis of a disease caused by abnormal proliferation of vascular smooth muscle cells.
  • a diagnostic agent is a diagnostic agent that hybridizes to LKBlcDNA and contains DNA having a chain length of at least 15 bases.
  • Such MAs include probe DNA and primer DNA for measuring the amount of transcript of LKB1 gene. Those skilled in the art can design and synthesize these DNAs based on the base sequence of the LKB1 gene.
  • the primer DNA is usually 15 to 100 bases, preferably 17 to 30 bases.
  • an example of a method for detecting the expression of the LKB1 gene using the primer DNA is an RT-PCR method.
  • the probe DNA usually has a chain length of at least 15 bases or more.
  • the probe DNA is appropriately labeled and used.
  • the labeling method include a method of labeling by phosphorylating the 5 'end of the oligonucleotide with 32 P using T4 polynucleotide kinase, and a random hexamer-oligonucleotide using a DNA polymerase such as Klenow enzyme.
  • a method of incorporating a substrate base labeled with an isotope such as 32 P, a fluorescent dye, or biotin may be mentioned.
  • Northern blotting can be exemplified as a method for detecting the expression of the LKB1 gene using the probe DNA.
  • Another embodiment of the diagnostic agent of the present invention is a diagnostic agent comprising an antibody that binds to LKB1 protein.
  • Antibodies that bind to LKB1 protein can be prepared by methods known to those skilled in the art.
  • a Western blotting method can be exemplified. Simple explanation of figure
  • FIG. 1 is a diagram showing a multiple alignment of amino acid sequences of human (Human), mouse (Mouse), and African Xenopus (Xenopus) LKB1. Identical amino acids are shaded. Putative ATP binding sites are indicated by *, and putative serine / threonine kinase active catalytic sites are indicated by +. In addition, two protein kinase A phosphorylation sites conserved in all species are indicated by #.
  • FIG. 2 is a photograph showing the results of Northern analysis of mouse LKB1 expression.
  • FIG. 3 is a photograph showing the results of Western analysis.
  • Mouse LKB1 expressed in C0S7 cells was stained with anti-LKB1 P6 antibody (LKB1) and anti-c-myc antibody (c-myc), respectively. It can be seen that mouse LKB1 protein was stained with high specificity by the anti-LKB1 P6 antibody.
  • LLB1 P6 antibody anti-LKB1 P6 antibody
  • c-myc anti-c-myc antibody
  • FIG. 4 is a photograph showing the results of immunohistological staining. Each mouse section was stained with an anti-LKB1 P6 antibody (a to: f, except for c). c is a negative control using normal IgG instead of anti-LKB1 P6 antibody.
  • a kidney, b, c: heart, d: rectum, e: gland stomach, f: bone marrow. It can be seen that the expression is strongly localized in smooth muscle cells such as vascular smooth muscle cells and mucosal muscle plates.
  • FIG. 5 is a photograph showing the results of in situ hybridization using normal mouse liver sections.
  • FIG. 6 is a photograph showing the results of staining of human coronary atherosclerotic lesions with an anti-LKB1 P6 antibody.
  • the site consisting of normal smooth muscle cells and the site consisting of proliferative smooth muscle cells are shown.
  • mice By searching the database using the sequence of the human LKB1 cDNA, we found several ESTs (Expressed Sequence Tags) derived from mice that showed very high homology to this. From these sequences, an MPJ 85 primer (5 ′ GATGAATTCCGAAGGACAGA GGACAAAGAGTGG 3, / SEQ ID NO: 3) and a LK E2 primer (5, GATGAATTCTTAGAG GTCTTCTTCTGAGATGAGCTTCTGCTCCTGCTGCTTGCAGGCCGA 3, / SEQ ID NO: 4) were prepared, and a mouse embryo on day 17 of Mar.
  • an MPJ 85 primer (5 ′ GATGAATTCCGAAGGACAGA GGACAAAGAGTGG 3, / SEQ ID NO: 3
  • a LK E2 primer 5, GATGAATTCTTAGAG GTCTTCTTCTGAGATGAGCTTCTGCTCCTGCTGCTTGCAGGCCGA 3, / SEQ ID NO: 4
  • TM cDNA (CL0NTECH) was subjected to type I PCR to amplify cDNA containing the entire open reading frame (0RF) of mouse LKB1. This was cut with EcoRI added to both ends of the primer, and subcloned into pcDNA3 vector (Invitrogen). The sequence of the gene was determined by sequencing a plurality of clones. The 5 'side of mouse LKB1 cDNA was cloned by 5' RACE (Rapid Amplification of cDNA Ends method).
  • Marathon Ready TM cDNA (CL0NTECH) derived from mouse day 17 embryo was type III, MPJ15 primer (5 'TGCGCAGCTTTTTCTTCTTGAGGA 3, / SEQ ID NO: 5) and adapter-primer attached to the kit.
  • PCR using (API primer) was performed, and the amplified fragment of about 400 bp was subcloned into pT7Blue_T vector (Novagen) by TA cloning method.
  • the sequence on the 5 'side of mouse LKB1 cDNA was determined by sequencing a plurality of the obtained subclones.
  • the sequence of mouse LKB1 cDNA thus obtained is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 1. No .: shown in 2.
  • Figure 1 shows the multiple alignment of the LKB1 amino acid sequences of Human, Mouse, and Xenopus LKB1.
  • mouse LKB1 cDNA (corresponding to base numbers 21 to 309 of GenBank accession number AB015801) was amplified by PCR using the following primers.
  • MP J15 primer (5, TGCGCAGCTTTTTCTTCTTGAGGA 3, / SEQ ID NO: 5)
  • Example 4 Western analysis Amplification of a mouse LKB1 cDNA fragment having a C-myc epitope sequence (EQKLISEEDL / SEQ ID NO: 8) at the C-terminus was amplified by PCR using the MPJ85 primer and LK E2 primer as described above, and expressed for mammalian cells.
  • the vector, pcDNA3 vector (In vitrogen) was incorporated into the EcoRI site.
  • a clone (pcDNA3 / MLKmyc) having no PCR error was selected by sequencing the obtained subclones and used for expression experiments.
  • mice C57 lack / 6J, female, 10 weeks old, Claire were flushed with periodate-lysine-paraformaldehyde (PLP) containing 4% paraformaldehyde; The tissue was removed and fixed in a PLP solution at 4 ° C for 16 hours.
  • the femur was similarly fixed in a PLP solution at 4 ° C for 16 hours, and then decalcified by immersion in an EDTA-glycerol solution at 4 ° C for 2 weeks. Next, it was dehydrated by immersing it in acetone for 4 hours at 4 ° C to remove acetone, and then embedded in paraffin (AMeX method) (Sa to Y et al, Am J Pathol.
  • PLP periodate-lysine-paraformaldehyde
  • the clone (pcDNA3 / MLK) in which the cDNA containing all 0RF of mouse LKB1 prepared in Example 1 was integrated into a pcDNA3 vector was used as a type III for preparing an RNA probe.
  • This clone was cleaved with HindII and antisense RNA was synthesized using SP6 RNA polymerase.
  • the clone was digested with NotI, and sense RNA was synthesized using T7 polymerase. By adding bases labeled with digoxigenin (DIG) during RNA synthesis, each RNA was labeled with DIG and used as a probe.
  • DIG digoxigenin
  • paraffin sections of mouse liver were deparaffinized with EtOH, the sections were treated with 0.2N HCl at room temperature for 20 minutes, and further treated with 5 ⁇ g / ml proteinase K at 37 ° C. for 15 minutes.
  • fixation was performed by treating with 4% paraformaldehyde / PBS for 5 minutes at room temperature, and blocking was performed twice by treating with 2 mg / ml glycine for 15 minutes at room temperature.
  • RNaseA treatment treatment was performed at 37 ° for 60 minutes with 100 ⁇ g / ml RNaseA.
  • Hybridization was performed in 50% formaldehyde, lOmM Tris'HCl (pH 8.0), 0.2 ⁇ g // l yeast tRNA, lx Denhardt's solution, 10% dextran sulfate, 600mM NaC lmM EDTA, 0.253 ⁇ 4 SDS, Performed overnight at 45 ° C. After washing, the hybridized signal was detected by DIG antibody labeled with alkaline phosphatase.
  • the LKB1 gene was strongly expressed in vascular smooth muscle cells. This confirmed that the LKB1 gene was strongly expressed in vascular smooth muscle cells not only by immunohistochemical staining (Example 5) but also by in situ hybridization.
  • Example 7 Immunohistochemical staining of human tissue Formalin-fixed, valaffin-embedded sections of coronary atherosclerotic lesions removed from a 70-year-old man were used for staining. Staining was performed using an anti-LKB1 P6 antibody at a concentration of l ⁇ g / ml to 2.5 ⁇ g / ml. Detection was performed using the ABC-AP kit from Vector and a Red substrate kit. The results showed that staining of human atherosclerotic lesions reduced LKB1 expression in actual human lesions. This is a finding that further supports the importance of LKB1 in controlling the growth of vascular smooth muscle cells. Industrial applicability
  • an LKB1 protein for suppressing abnormal growth of vascular smooth muscle cells a DNA encoding the protein, and a compound that increases the function or abundance of the LKB1 protein are provided.
  • These molecules can be used as reagents for suppressing abnormal proliferation of vascular smooth muscle cells, and pharmaceutical compositions for preventing and treating diseases associated with abnormal proliferation of vascular smooth muscle cells such as arteriosclerosis. Is expected to be applied as

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Abstract

Il a été mis en évidence que la protéine LKB1 est fortement exprimée, notamment dans les cellules vasculaires des muscles lisses, outre le fait que ces cellules constituent des myotomes. L'invention propose que, dans les cas de prolifération anormale des cellules vasculaires des muscles lisses, cette prolifération soit inhibée par induction de l'expression de la protéine LKB1 ou de la fonction de celle-ci.
PCT/JP2000/005256 1999-08-06 2000-08-04 Compositions medicinales destinees a inhiber la proliferation anormale des cellules vasculaires des muscles lisses Ceased WO2001010905A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002006520A1 (fr) * 2000-07-19 2002-01-24 Chugai Seiyaku Kabushiki Kaisha Methode de criblage de compose regulant la transduction de signaux mek/erk et utilisation medicale dudit compose

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DIETER E. JENNE ET AL.: "Peutz-Jeghers syndrome is caused by mutations in a novel serine threonine kinase", NAT. GENET., vol. 18, no. 1, 1998, pages 38 - 43, XP002934256 *
I.P.M. TOMLINSON ET AL.: "Peutz-Jeghers syndrome", J. MED. GENET., vol. 34, no. 12, 1997, pages 1007 - 1011, XP002934258 *
SEUNG MYUNG DONG ET AL.: "Frequent somatic mutations in serine/threonine kinase 11/Peutz-Jeghers syndrome gene in left-sided codon cancer", CANCER RES., vol. 58, no. 17, 1998, pages 3787 - 3790, XP002934257 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002006520A1 (fr) * 2000-07-19 2002-01-24 Chugai Seiyaku Kabushiki Kaisha Methode de criblage de compose regulant la transduction de signaux mek/erk et utilisation medicale dudit compose

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