[go: up one dir, main page]

WO2006069000A2 - Klf2 utilise comme mediateur de l'activite des statines - Google Patents

Klf2 utilise comme mediateur de l'activite des statines Download PDF

Info

Publication number
WO2006069000A2
WO2006069000A2 PCT/US2005/045946 US2005045946W WO2006069000A2 WO 2006069000 A2 WO2006069000 A2 WO 2006069000A2 US 2005045946 W US2005045946 W US 2005045946W WO 2006069000 A2 WO2006069000 A2 WO 2006069000A2
Authority
WO
WIPO (PCT)
Prior art keywords
klf2
cells
activity
statin
expression
Prior art date
Application number
PCT/US2005/045946
Other languages
English (en)
Other versions
WO2006069000A3 (fr
Inventor
Mukesh K. Jain
Sucharita Senbanerjee
Pallab Banerjee
Zhiyong Lin
Gillermo Garcia-Cardena
Michael A. Gimbrone Jr.
Original Assignee
The Brigham And Women's Hospital, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Brigham And Women's Hospital, Inc. filed Critical The Brigham And Women's Hospital, Inc.
Publication of WO2006069000A2 publication Critical patent/WO2006069000A2/fr
Publication of WO2006069000A3 publication Critical patent/WO2006069000A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5064—Endothelial cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention is based upon the finding that statins cause their biological effects by increasing the activity of the KLF2 transcription factor.
  • assays of KLF2 can be used to determine if a compound has statin-like activity or antagonizes statin activity.
  • the invention also includes methods for identifying or creating endothelial cells with reduced KLF2 activity. These methods will be of interest to scientists studying disease processes affected by statins, and to clinicians interested in identifying individuals that are likely to be less responsive to statins and that may have a predisposition toward thrombus formation.
  • One way of reducing this predisposition is to increase KLF2 activity by transforming the cells with vectors for expressing EXF2.
  • Increasing KLF2 activity in endothelial cells may also be used as a means of inhibiting angiogenesis.
  • agents that increase KLF2 activity have potential value in the treatment of solid tumors and will be of value to scientists studying tumor growth.
  • statins 3-hydroxy-3- methylglutaryl coenzyme A inhibitors
  • statins increase the accumulation of factors such as endothelial nitric oxide synthase (eNOS) and thrombomodulin (Laufs, et al, Circulation 27:1129-35 (1998); Laufs, et al, J. Biol. Chem. 273:24266-71 (1998); Masamura, et al, Arterioscler. Thromb. Vase. Biol. 23:512-7 (2003)).
  • thrombomodulin is a key endothelial cell surface factor that increases the rate of thrombin-catalyzed protein C activation (Esmon, et al, Proc. Natl. Acad. ScL USA 78:2249-52 (1981)).
  • TM impacts not only coagulation but also imparts anti-inflammatory, anti- thrombotic, and anti-adhesive properties to the endothelium (Esmon, et al, J. Endotoxin Res. 9:192-8 (2003); Esmon, J. Thromb. Haemost. 1: 1343-8 (2003)).
  • statins differentially regulate gene expression remains incompletely understood.
  • small GTP binding proteins ⁇ e.g. Rho
  • certain kinases e.g. Akt
  • statins diminish the activity/function of NF- KB and AP-I - two key pathways regulating the induction of many pro-inflammatory genes (Dichtl, et al, Arterioscler. Thromb. Vase. Biol. 25:58-63 (2003)).
  • statins diminish the activity/function of NF- KB and AP-I - two key pathways regulating the induction of many pro-inflammatory genes (Dichtl, et al, Arterioscler. Thromb. Vase. Biol. 25:58-63 (2003)).
  • statins diminish the activity/function of NF- KB and AP-I - two key pathways regulating the induction of many pro-inflammatory genes (Dichtl, et al, Arterioscler. Thromb. Vase. Biol. 25:58-63 (2003)).
  • statins diminish the activity/function of NF- KB and AP-I - two key pathways regulating the induction of many pro-inflammatory genes (Dich
  • KLF2 Kruppel-like family of transcription factors
  • the invention also encompasses methods of identifying endothelial cells with low KLF2 activity or for making dysfunctional endothelial cells by engineering them in a manner that decreases KLF2 activity. Cells of this type will also be of interest in identifying new potential therapeutic agents and may be used by scientists examining factors that predispose cells to disease or that might make them resistance to certain therapies. Finally, the invention encompasses vectors for expressing KLF2 that may be used to transfect cells and generate a statin-like effect. Thus, cells transformed with the vectors should be, inter alia, more resistant to thrombosis.
  • the invention is directed to an assay for determining if a test compound has either statin-like or statin-antagonizing activity.
  • statin-like indicates that a compound has the same anti-inflammatory and anti-thrombotic effects on endothelial cells as the statins and this should be reflected in increased KLF2 induced production of eNOS and thrombomodulin.
  • statin-antagonizing indicates that a compound inhibits KLF2 activity and should therefore reduce the effectiveness of statins. Cells with reduced KLF2 activity will require a higher dosage of statin to achieve the same therapeutic effect, or treatment involving the use of other agents that increase KLF2 activity.
  • the assays described above are performed by incubating test cells expressing EXF2 with the test compound and then determining the amount of KLF2 activity in the cells. Any method for assessing activity can be used, including determining the amount of KLF2 mRNA present, or by determining the amount of KLF2 protein present, e.g., using an immunoassay.
  • the results obtained from the test cells are compared with the results from control cells that are incubated in the absence of the test compound. Methods for selecting appropriate control cells are well known in the art.
  • test compound has statin-line activity, hi contrast, if it is found that the test cells have less KLF2 activity than the control cells, it may be concluded that the test compound has statin-antagonizing activity. It has been found that statins increase KLF2 activity by acting at the KLF2 promoter to 'increase transcription. Thus, assays which measure the rate of transcription from this promoter provide another way of determining whether a test compound has statin-like or statin-antagonizing activity.
  • One preferred method for carrying out such assays involves first creating an expression vector in which the KLF2 promoter is operably linked to a marker gene.
  • Appropriate marker genes are well known in the art and typically are selected for ease of detection and quantitation. Host cells are then transfected with the expression vector and incubated in the presence of the test compound. Expression of the marker gene is measured and the results are compared with those of control cells incubated in the absence of the test compound. An increase in marker gene expression is indicative of a test compound that acts in a statin-like manner, and a decrease in marker gene expression is indicative of a statin-antagonizing compound.
  • the invention also includes methods for creating dysfunctional endothelial cells by engineering them to reduce KLF2 activity. This can be done, for example, by using homologous recombination to disrupt the KLF2 promoter or the KLF2 structural sequence. The most preferred method however is to transfect the endothelial cells with a small interfering RNA (SiRNA) that decreases KLF2 activity.
  • SiRNA small interfering RNA
  • the invention is directed to a method for identifying statin- antagonizing endothelial cells characterized by an abnormally low level of KLF2 activity. These cells should be less responsive to treatment using statins and patients having cells of this type may therefore require higher doses of statins or, alternatively, treatment that includes the use of other inducers of KLF2.
  • the assay method involves comparing the KLF2 activity in selected endothelial cells with the amount of activity in a control population of cells.
  • the control population may be endothelial cells derived from individuals known to respond to statins in the normal way or they may simply be cells derived from the general population. Any method for determining KLF2 activity is compatible with the assay method, including assays for determining either mRNA levels or protein levels.
  • the invention also includes methods for increasing statin-like activity in cells by increasing KLF2 activity. This can be accomplished by transforming cells, especially endothelial cells, with an expression vector in which there is a promoter operably linked to a sequence coding for KLF2. By increasing KLF2 expression, the endothelial cell proliferation necessary for angiogenesis is inhibited. Any condition that can benefit by preventing the growth of new blood vessels can be treated using these methods, but it is expected that they will be of particular benefit in the treatment of solid tumors.
  • the inhibition of endothelial cell proliferation by KLF2 appears to be attributable to amino acids 118-150 of KLF2 as shown in SEQ ID NO:1 and separately as SEQ ID NO:5.
  • the invention includes peptides having this sequence, as well as substantially pure polynucleotides that contain a sequence encoding the peptides, and which can be used in their recombinant production.
  • the invention encompasses vectors containing these polynucleotides, preferably operably linked to a promoter, and host cells transformed with the vectors.
  • substantially pure refers to polynucleotides (or polypeptides) that have been separated from other accompanying biological components.
  • Substantially purified molecules will typically constitute at least 80% of a sample, with greater percentages being preferred. Many means are available for assessing the purity of a protein or nucleic acid within a sample, including analysis of polyacrylamide gel electrophoresis, chromatography and analytical centrifugation.
  • the peptide described above may be used to inhibit the expression of VEGFR2 in endothelial cells. It may therefore be used for the purpose of inhibiting cell proliferation and, ultimately, angiogenesis.
  • an effective amount of the inhibitory agent in this case a peptide, must be brought in contact with the cells.
  • the term "effective amount” refers to sufficient inhibitory agent to significantly reduce, e.g., by at least 20%, the amount of cell proliferation and new blood vessel formation that would occur in the absence of the agent.
  • amino acid or two can be added or deleted with a high likelihood that activity will be maintained.
  • Figure 1 shows a protocol used to assess the effect of control adenovirus
  • Ad-GFP Ad-GFP
  • Ad-K2 KLF2 adenovirus
  • Figure 2 shows photographs of nude mouse ears before (- VEGF) and after (+VEGF) treatment with VEG-A in the presence (Ad-K2) and absence (Ad-GFP) of adenoviral KLF2. The dark blush after VEGF treatment (upper panels) in the control virus
  • KLF2 inhibits VEG-A-mediated angiogenesis.
  • Vector refers to vehicles that can be used for transferring nucleic acid sequences into a host cell.
  • Vectors are typically plasmids or viruses, such as retroviruses, adenoviruses, etc.
  • Expression vector This refers to a vector which is capable of inducing the expression of DNA that has been cloned into it after transformation into a host cell.
  • the cloned DNA is usually placed under the control of (i.e., operably linked to) a promoter.
  • Promoter sequences may be constitutive, inducible or repressible.
  • Host cell refers to any prokaryotic or preferably eukaryotic cell that is the recipient of a vector.
  • the term encompasses cells that have been engineered to incorporate a gene into their genome, as well as cells that maintain transferred nucleic acid outside of the genome. Cells that can serve as hosts as well as techniques for cellular transformation are well known in the art (see, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2 nd ed., Cold Spring Harbor Press (1989)).
  • promoter is a DNA sequence that initiates the transcription of a gene.
  • the most important promoter has the sequence shown as SEQ ID NO:3.
  • expression refers to the process by which a polypeptide is produced from DNA. The process involves the transcription of a coding sequence into mRNA and the translation of this mRNA into a polypeptide. Depending on the context in which it is used, “expression” may refer to the production of mRNA, protein, or both.
  • operably linked refers to genetic elements that are joined in such a manner that enables them to carry out their normal function.
  • a gene is operably linked to a promoter when its transcription is under the control of the promoter and such transcription produces the protein normally encoded by the gene.
  • Gene refers to a nucleic acid sequence that undergoes transcription as the result of promoter activity.
  • KLF2 refers to the human transcription factor that has been described in the art as having the sequence shown as SEQ ID NO.l (Wani, et al, Genomics (50:78-86 (1999); NCBI accession no. AF134053). It is encoded, inter alia, by the DNA sequence shown as SEQ ID NO:2 .
  • Proteins and genes used in assays may be obtained using techniques well known in the art, or can be synthesized using chemical procedures. Guidance concerning sources for all other factors needed in assays may be found in the Examples section, which also provides specific guidance concerning the way in which assays may be performed.
  • the invention includes recombinant DNA molecules in which there is a promoter, typically a mammalian promoter, operably linked to a sequence coding for the KLF2 protein as shown in SEQ ID NO:1.
  • a promoter typically a mammalian promoter
  • Procedures for obtaining promoters and other DNA sequences are well known in the art and standard techniques in molecular biology can be used for constructing DNA molecules with appropriately arranged elements (see, e.g., Sambrook, et ah, Molecular Cloning: A Laboratory Manual, 2 nd ed., Cold Spring Harbor Press (1989)).
  • any type of promoter active in mammalian cells, and particularly endothelial cells, can be used in the invention, including those that are inducible, repressible or constitutive.
  • the KLF2 promoter will be used (see, e.g., SEQ ID NO:3).
  • Expression vectors which are designed to increase KLF2 activity in cells may utilize any strong promoter which is active in the cells.
  • the human CMV immediate- early promoter Boshart, et ah, Cell ⁇ i:521-530 (1985)
  • Standard vectors for expressing genes in mammalian cells are well known in the art and may be used in conjunction with the present invention.
  • vectors When carrying out in vitro experiments, vectors may be introduced into cells using procedures such as calcium phosphate precipitation, microinjection, electrophoration, liposomal transfer, etc. When transfers are done to host cells in vivo, preferred methods of transformation are by means of a viral vector, liposomal transfer, or through the use of naked DNA.
  • Cells that have incorporated constructs can be identified by assaying for the expressed product, using hybridization techniques, or by using the polymerase chain reaction (PCR) to amplify specific recombinant sequences.
  • PCR polymerase chain reaction
  • the vectors for expressing KLF2 described above may be used in treating solid tumors and other conditions where blocking the growth of new blood vessels has a beneficial therapeutic effect.
  • SEQ ID NO:1 is responsible for KLF2's inhibitory effect on endothelial cell proliferation.
  • This sequence maybe synthesized as a 33 amino acid peptide using standard chemical techniques and administered to patients to inhibit angiogenesis.
  • the dosage administered to a patient can be determined using methods that are well known in pharmacolgy.
  • the peptide may be administered in any phsiologically acceptable form including as a pharmaceutically acceptable salt. It may be included as part of a pharmaceutical composition and administered by any route that does not result in the destruction of peptide activity.
  • KLF2 The structure of human KLF2 is shown in SEQ ID NO:1.
  • This protein as well as a peptide about 33 amino acids in length derived from it (SEQ ID NO: 5) may be used to inhibit angiogenesis in a patient or in an experimental animal (see Figures 1 and 2).
  • Polynucleotides encoding KLF2 or the anti-angiogenesis peptide can be constructed based upon a knowledge of the genetic code and used either therapeutically or to recombinantly produce polypeptide. Many methods are available for accomplishing this such as those described in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2 nd ed., Cold Spring Harbor Press (1989)). However, it is preferred that both nucleic acids and polypeptides be made using standard methods of chemical synthesis.
  • Therapeutic methods may involve either the administration of an anti-angiogensis polypeptide ⁇ i.e., KLF2 or the peptide of SEQ ID NO:5) or the administration of nucleic acids that encode the polypeptide.
  • oligonucleotides designed for the expression of polypeptide may be administered directly to patients.
  • the in vivo transfection of cells has been known for many years and may be accomplished using viral vectors (see e.g. U.S. 6,020,191); liposomes (see e.g., Nicolau, Meth. Enzymol 149:157- 176 (1987)); DNA complexed to agents that facilitate cellular uptake (see e.g., U.S.
  • polypeptide may be directly administered to a patient.
  • the polypeptide may be injected into or near a tumor.
  • the polypeptide should be administered parentally, with administration by injection being preferred.
  • the dosage administered to a patient will be determined by the attending physician based upon clinical considerations and using methods well known in the art.
  • Anti-angiogenesis polypeptides may be administered in either a single or multiple dosage regimen and may be given either alone or in conjunction with other therapeutic agents.
  • Parenteral compositions may be used for intravenous, intraarterial, intramuscular, intraperitoneal, intracutaneous, or subcutaneous delivery. These preparations may be made using conventional techniques and may include isotonic saline, water, polyglycols, Ringer's solution, etc. Topical compositions may also be useful in treating cancers of the skin. All dosage forms may be prepared using methods that are standard in the art (see e.g., Remington's Pharmaceutical Sciences, 16 edition, A. Oslo editor, Easton, PA (1980)).
  • statins as therapeutic agents is well known in the art of clinical medicine. In addition to their known value in the treatment of cardiovascular conditions and inflammation, it has been suggested that statins may also be useful in blocking angiogenesis to prevent tumor growth, and in treating arthritis, osteoporosis, and stroke. Since the induction of KLF2 is the mechanism by which statins act, other inducing factors should have a similar therapeutic effect. Thus, the assays described herein may be used to identify potential therapeutic agents that act like statins. The assays may be based upon a measurement of KLF2 protein, or niRNA. Alternatively, they may measure the ability of test compounds to induce transcription from the KLF2 promoter, the specific mechanism by which the statins appear to increase KLF2 activity.
  • the assays are also useful in identifying agents that reduce KLF2 activity. Agents of this type should typically be avoided by patients undergoing treatment with statins. In addition, such compounds may be of interest to researchers studying thrombosis and attempting to identify the exact biological effects of anti-thrombotic drugs. In this regard, the ability to create endothelial cells with reduced KLF2 activity will also be useful.
  • assays of endothelial cells to determine whether they have normal levels of KLF2 activity will be useful for the reasons alluded to above. Specifically, patients having cells with reduced activity will typically require higher doses of statins to achieve a desired therapeutic effect. People with abnormally low levels of KLF2 may also be predisposed to development of conditions that respond to statin therapy, particularly cardiovascular diseases. Thus, assays of KLF2 activity in endothelial cells may provide a way of identifying individuals in need of preventive therapy, i.e., treatment with an agent that raises KLF2 activity.
  • any agent known in the art that increases KLF2 activity in endothelial cells should be of potential clinical value as a therapeutic agent.
  • One suitable agent is an expression vector for KLF2.
  • these expression vectors will provide researchers with a means for engineering cells in vitro that can be used to help to better define the biological effects of statins and endothelial cell pathologies.
  • Example 1 KLF2 as a Mediator of Statin Activity A. Summary
  • statin induced expression of eNOS and TM is KLF2 dependent.
  • KLF2 mRNA was induced by treatment with multiple statins in a concentration-dependent manner. This effect was inhibited by both mevalonate and geranylgeranylpyrophosphate but not by farnesylpyrophosphate.
  • Mevastatin induces KLF2 promoter activity through a single MEF binding site and mutation of this site abrogates the inductive effect.
  • siRNA mediated knockdown of KLF2 strongly attenuates the ability of mevastatin to increase eNOS and TM accumulation in endothelial cells.
  • HAVECs Human umbilical vein endothelial cells
  • Cambrex Bioscience Walkersville, MD
  • All statins were purchased from Calbiochem and prepared according to manufacturer's recommendations.
  • the thrombomodulin and MEF2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); the eNOS antibody was from BD Biosciences (Palo Alto, CA); the ⁇ - tubulin antibody was from Sigma (St. Louis, MO).
  • the adenoviral constructs were generated by the Harvard Gene Therapy Initiative (Boston, MA).
  • the MEF2A and MEF2C plasmids and the -1.7kB-KLF2-Luc promoter were gifts from colleagues. All deletion constructs of the KLF2 promoter were generated by PCR and cloned into the PGL2 basic vector. Mutation of the MEF site was accomplished by using the QuikChange mutagenesis kit following the manufacture's instruction (Stratagene).
  • MEF2A and MEF2C protein were generated by in vitro transcription and translation
  • siRNA mediated knockdown studies Small interfering RNA oligonucleotides were purchased from Dharmacon (Lafayette, CO) and knockdown performed as previously described with minor modifications (Gonzalez, et al., J Biol Chem. 279:40659- 69 (2004)). HUVECs were plated one day before transfection in antibiotic-free EBM-2 medium. On the day of transfection, 10OnM of specific siRNA targeting human KLF2 or non-specific siRNA was incubated with Lipofectamine 2000 (Invitrogen) at room temperature for thirty minutes before adding to the HUVECs in OPTI-MEM (Invitrogen). Three hours later the medium was replaced by EBM-2 and cultured for an additional 48 hours. Cells were treated with or without mevastatin for 24 hours and harvested for protein.
  • statins can induce factors such as eNOS and TM
  • Adenoviral over expression of KLF2 in HUVECs alters the expression of endothelial products such as eNOS (SenBanerjee, et al., J. Exp. Med. 199:1305-1315 (2004)) and TM (unpublished observation).
  • eNOS endothelial products
  • Ad- GFP control infected cells
  • Ad-GFP-K2 adenoviral overexpression of KLF2
  • statins cause cells to be depleted in mevalonate.
  • statins cause cells to be depleted in mevalonate.
  • HUVECs were incubated with mevastatin in the presence or absence of mevalonate. It was found that supplementation with mevalonate completely blocked statin-dependent induction of KLF2.
  • Mevalonate is a precursor for cholesterol as well as isoprenoid intermediates such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGP).
  • the isoprenoids are important post-translational lipid modifications of a variety of proteins such as Ras and Rho. Ras proteins are predominantly farnesylated whereas the Rho proteins are mainly geranylgeranylated.
  • Ras or Rho have a role in the statin-dependent induction of KLF2
  • HUVECs were incubated with mevastatin in the presence of the isoprenoid intermediates FPP and GGPP.
  • GGPP was found to partially reverse the mevastatin-mediated induction of KLF2 mRNA. In contrast, no effect was seen with FPP.
  • Statins induce the KLF2 promoter via a MEF binding site — To gain greater insight regarding the molecular basis for statin-mediated induction of KLF2, we assessed the effect of these agents on KLF2 promoter activity. It was found that mevastatin treatment induced the -1.7kB-Luc KLF2 promoter activity about threefold. Using a series of deletion mutants, we found that the majority of the inductive effect was maintained using the -221bp-Luc promoter fragment but lost with further deletion to the -114bp-Luc construct. Thus, treatment with statins induces the KLF2 promoter through a critical 107 bp region.
  • MEF2A and MEF2C have also been implicated as regulators of endothelial cell biology (Wang, et ah, Science 302:1578- 81 (2003); Lin, et ah, Development i25:4565-74(1998); Hayashi, et ah, J. Clin. Invest. 113:1138-48 (2004.
  • statins induce KLF2 expression and that a reduction in KLF2 expression attenuates statin-mediated accumulation of eNOS and TM levels.
  • Our data also implicate another family of transcription factors - the MEFs - as being involved in the induction of KLF2.
  • statins one of the most important effects of statins is to increase eNOS and TM levels in endothelial cells. This is thought to occur through both transcriptional and post- transcriptional means. Studies from our laboratory indicate that KLF2 can induce eNOS and TM mRNA and promoter activity suggesting that transcription events underlie, at least in part, the ability of this factor to induce these targets. However, this does not exclude the possibility that the KLF2 induction of eNOS and TM accumulation in endothelial cells may also be, in part, through post-transcriptional effects as well as effects on protein synthesis and stability. Indeed, the requirement for KLF2 in the ability of mevastatin to induce eNOS and TM strongly suggest that additional mechanisms are likely involved.
  • Example 2 KLF2 in Angiogenesis The present Example describes evidence indicating that the induction of KLF2 leads to an inhibition of VEGF-mediated angiogenesis.
  • a primary strategy in cancer treatment has been to block the development of the blood vessels which tumors need to sustain their growth.
  • Angiogenesis requires the proliferation of endothelial cells, and this proliferation is induced by vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • the main receptor responsible for mediating the effect of VEGF in promoting endothelial cell proliferation is VEGFR2.
  • VEGF is also a pro-inflammatory agent and inflammation is considered a key feature in pathologic angiogenesis.
  • VEGF treatment of FIUVECs is known to induce key adhesion molecules (VCAM-I), coagulant proteins (tissue factor), and other factors (e.g., COX-2). These factors have been implicated in a number of malignancies such as lung cancer and breast cancer.
  • VCAM-I key adhesion molecules
  • tissue factor tissue factor
  • COX-2 coagulant proteins
  • VEGF-A Animals were injected with control (Ad-GFP) or KLF2 adenovirus (Ad-KLF2) and then exposed to VEGF-A (see Figure 1 for protocol). It was found that adenoviral overexpression of KLF2 prevented the VEGF-mediated induction of angiogenesis (see ⁇ Figure 2). This was verified by staining for blood vessels.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pathology (AREA)
  • Organic Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Wood Science & Technology (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des méthodes d'identification de composés imitant ou inhibant l'effet thérapeutique des statines. Les composés de l'invention sont identifiés par des dosages du facteur de transcription KLF2. L'invention concerne également des méthodes de création ou d'identification de cellules endothéliales présentant une faible activité de KLF2, lesquelles cellules sont résistantes au traitement par les statines. Elle concerne des méthodes permettant d'augmenter l'activité de KLF2 par transfection de cellules avec des vecteurs d'expression.
PCT/US2005/045946 2004-12-21 2005-12-20 Klf2 utilise comme mediateur de l'activite des statines WO2006069000A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US63766404P 2004-12-21 2004-12-21
US60/637,664 2004-12-21
US65738205P 2005-03-02 2005-03-02
US60/657,382 2005-03-02

Publications (2)

Publication Number Publication Date
WO2006069000A2 true WO2006069000A2 (fr) 2006-06-29
WO2006069000A3 WO2006069000A3 (fr) 2007-08-09

Family

ID=36602241

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2005/045946 WO2006069000A2 (fr) 2004-12-21 2005-12-20 Klf2 utilise comme mediateur de l'activite des statines

Country Status (1)

Country Link
WO (1) WO2006069000A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1614429A1 (fr) * 2003-04-17 2006-01-11 Kowa Co. Ltd. Promoteur de l'expression du gene lklf/klf2
WO2008132458A1 (fr) * 2007-04-30 2008-11-06 Inion Limited Compositions utiles dans la modulation de réponses immunitaires et le traitement ou la prévention de réponses inflammatoires et procédés apparentés

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004039956A2 (fr) * 2002-10-29 2004-05-13 Genentech, Inc. Compositions et methodes de traitement de maladies liees au systeme immunitaire

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004039956A2 (fr) * 2002-10-29 2004-05-13 Genentech, Inc. Compositions et methodes de traitement de maladies liees au systeme immunitaire

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DEKKER ET AL.: 'Prolonged fluid shear stress induces a distinct set of endothelial cell genes most specifically lung Kruppel-factor (KLF2)' BLOOD vol. 100, no. 5, September 2002, pages 1689 - 1698 *
WANI ET AL.: 'cDNA isolation, genomic structure, regulation, and chromosomal localization of human lung Kruppel-like factor' GENOMICS vol. 60, 1999, pages 78 - 86 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1614429A1 (fr) * 2003-04-17 2006-01-11 Kowa Co. Ltd. Promoteur de l'expression du gene lklf/klf2
EP1614429A4 (fr) * 2003-04-17 2007-05-02 Kowa Co Promoteur de l'expression du gene lklf/klf2
WO2008132458A1 (fr) * 2007-04-30 2008-11-06 Inion Limited Compositions utiles dans la modulation de réponses immunitaires et le traitement ou la prévention de réponses inflammatoires et procédés apparentés

Also Published As

Publication number Publication date
WO2006069000A3 (fr) 2007-08-09

Similar Documents

Publication Publication Date Title
JP4351896B2 (ja) ｐ３８／ＪＴＶ−１を有効成分とする癌治療用薬学的組成物及び癌治療用薬学的組成物のスクリーニング方法
Hino et al. Caveolin-1 as tumor suppressor gene in breast cancer
Wang et al. Microrna-26b attenuates monocrotaline-induced pulmonary vascular remodeling via targeting connective tissue growth factor (CTGF) and cyclin D1 (CCND1)
CN109022461B (zh) 一种PTEN基因的上游开放阅读框45aa-uORF核苷酸序列及其编码的多肽的应用
WO2007138334A2 (fr) Méthodes de traitement
WO2003057204A2 (fr) Prevention ou attenuation de l'auto-immunite
Zeng et al. Inhibition of EphA2 protects against atherosclerosis by synergizing with statins to mitigate macrophage inflammation
WO2006069000A2 (fr) Klf2 utilise comme mediateur de l'activite des statines
EP2243828A1 (fr) Utilisation de polypeptides "mixed lineage kinase like" (polypeptides MLKL) pour le traitement contre le cancer
CN101309928A (zh) E2epf泛素载体蛋白与von hippel-lindau的互作及其应用
WO2002016658A1 (fr) Procedes et compositions modulant l'expression de la transcriptase inverse de la telomerase (tert)
US20070122401A1 (en) Methods and compositions for modulating telomerase reverse transcriptase (tert) expression
JP2004535775A5 (fr)
EP2493492A1 (fr) Twist1 phosphorylé et cancer
US20230375528A1 (en) Screening method for the identification of novel therapeutic compounds
EP1263951A2 (fr) Proteine transactivatrice transcriptionnelle
CN114585742B (zh) 治疗
CN110819718B (zh) miR-154-5p在前列腺癌骨转移的新应用
WO2010018764A1 (fr) Agent anti-angiogénique, procédé de criblage et kit de criblage associés
WO2006122090A2 (fr) Dosages de criblage bases sur les cellules, destines aux composes qui regulent l'expression d'un marqueur de tumeur dj-1
CN110156877B (zh) 抑制肿瘤的多肽类似物UBE2Z1-330aa及其编码核酸、引物对、表达载体和应用
JP7244913B2 (ja) Ｅｇｆｒ遺伝子変異陽性非小細胞肺癌の検出方法
CN116144776B (zh) Cdc25a作为srsf10靶向剪切位点在制备肝癌治疗药物中的应用
EP1151094A1 (fr) Traitement du cancer du col de l'uterus
WO2003038441A2 (fr) Procedes de criblage a base de proteines de la famille des cited

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05854623

Country of ref document: EP

Kind code of ref document: A2