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WO1997008184A1 - Procedes et compositions comprenant des agents alterant l'adn et des inhibiteurs ou des activateurs de la tyrosine kinase - Google Patents

Procedes et compositions comprenant des agents alterant l'adn et des inhibiteurs ou des activateurs de la tyrosine kinase Download PDF

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Publication number
WO1997008184A1
WO1997008184A1 PCT/US1996/013922 US9613922W WO9708184A1 WO 1997008184 A1 WO1997008184 A1 WO 1997008184A1 US 9613922 W US9613922 W US 9613922W WO 9708184 A1 WO9708184 A1 WO 9708184A1
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Prior art keywords
abl
dna
cell
gene
cells
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PCT/US1996/013922
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English (en)
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Ralph R. Weichselbaum
Donald W. Kufe
Surender Kharbanda
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Arch Development Corporation
Dana-Farber Cancer Institute
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Application filed by Arch Development Corporation, Dana-Farber Cancer Institute filed Critical Arch Development Corporation
Priority to AU69063/96A priority Critical patent/AU6906396A/en
Publication of WO1997008184A1 publication Critical patent/WO1997008184A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it

Definitions

  • the present invention relates generally to the field of biochemical pathways. More particularly, it concerns the pathways connecting DNA damage, phosphorylation by tyrosine kinases c-abl gene and gene product.
  • Certain cancer treatment methods involve damaging the DNA of the cancer cell.
  • the cellular response to DNA damage includes activation of DNA repair, cell cycle arrest and lethality (Hall, 1988).
  • the signaling events responsible for the regulation of these events remain unclear.
  • p34 cd 34 kD serine/threonine protein kinase
  • PLC protein kinase C
  • protein tyrosine kinase activities Hallahan et al, 1990; Uckun et al, 1993.
  • specific kinases responsible for these activities and their substrates require further study.
  • Mitomycin C is an antitumor antibiotic isolated from Streptomyces caespitosus that covalently binds to DNA (Tomasz et al, 1988). This agent induces both monofunctional and bifunctional DNA lesions (Carrano et al, 1979). Other studies have demonstrated that MMC stimulates the formation of hydroxyl radicals (Dusre et al, 1989). Although the precise mechanism of action of this agent is unclear, MMC-induced cytotoxicity has been attributed to DNA alkylation and the formation of interstrand cross-links (Carrano et al, 1979; Dusre et al, 1989; Tomasz et al, 1988).
  • MMC myelar cells
  • AP-1 is involved in MMC-induced activation of the collagenase enhancer.
  • Protein tyrosine phosphorylation contributes to the regulation of cell growth and differentiation.
  • Protein tyrosine kinases can be divided into receptor-type and nonreceptor-type (Src-like) kinases (Cantley et al, 1991; Hanks et al, 1988; Bonni et al, 1993; Lamer et al, 1993; Ruff- Jamison et al, 1993).
  • Several protein tyrosine kinases have been purified from the cytosolic fractions of various tissues (Nakamura et al, 1988; Wong & Goldberg, 1984; Zioncheck et al, 1986).
  • the Src-like kinases which can associate with receptors at the plasma membrane, induce rapid tyrosine phosphorylation and/or activation of effectors such as phospholipase C- ⁇ l (PLC ⁇ l) (Carter et al, 1991), PLC ⁇ 2 (Hempel et al, 1992), mitogen-activated protein (MAP) kinase (Casillas et al, 1991), GTPase activating protein (GAP) (Gold et al, 1992a) and phosphatidylinositol 3-kinase (PI3-K) (Gold et al, 1992b).
  • PLC ⁇ l phospholipase C- ⁇ l
  • MAP mitogen-activated protein
  • GAP GTPase activating protein
  • PI3-K phosphatidylinositol 3-kinase
  • the damage is potentially lethal because while under normal circumstances it causes cell death, manipulation of the post-irradiation environment can modify the cell response.
  • PLD is repaired and the fraction of cells surviving a given dose of x-rays is increased if conditions are suboptimal for growth, such that cells do not have to undergo mitosis while their chromosomes are damaged.
  • ionizing radiation is useful as a therapy.
  • Cells that are irradiated or treated with D ⁇ A damaging agents halt in the cell cycle at G 2 , so that an inventory of chromosome damage can be taken and repair initiated and completed before mitosis is initiated. By blocking the stress or survival response in these cells, they undergo mitosis with damaged D ⁇ A, express the mutations, and are at a greater risk of dying.
  • the present invention in a general and overall sense, concerns the signalling pathways that connect DNA damage, such as that induced by ionizing radiation or alkylating agents, phosphorylation by tyrosine kinases and the c-Abl gene and gene product. More particularly, the invention involves the use of antisense molecules to selectively inhibit the expression of the c-Abl gene product following exposure of cells to DNA damaging agents, such as mitomycin C or ionizing radiation.
  • DNA damaging agents such as mitomycin C or ionizing radiation.
  • Such an antisense molecule includes a region that is complementary to and capable of hybridizing with a region of the selected gene.
  • the antisense RNA molecules of the present invention are capable of selectively inhibiting the expression of the c-Abl gene product over that of another member of the non-receptor type of tyrosine kinases.
  • the RNA molecule may also comprise a sequence that is complementary to exon region sequences of the c-Abl gene.
  • the invention contemplates nucleic acid molecules that comprise a coding region that expresses an antisense RNA molecule that selectively inhibiting the gene product of the c-Abl gene.
  • This DNA coding region includes an antisense RNA that is complementary to a region of the c-Abl gene.
  • the entire nucleic acid molecule may be a DNA molecule, and this particular embodiment may encode a RNA molecule having a sequence that is complementary to the c-Abl gene sequence, or a portion thereof.
  • the antisense c-Abl gene be prepared to be complementary to an entire c-Abl gene, it is believed that shorter regions of complementary nucleic acid may be employed, so long as the antisense construct can be shown to inhibit expression of the targeted expression product.
  • the nucleic acid molecule of the present invention may comprise a DNA sequence that encodes a RNA antisense molecule having a sequence that is complementary to at least a 2000 base region of the c-Abl gene.
  • the length of this RNA antisense sequence may be a 1000 base, 500 base, 100 base, or even a 10 base region of the c-Abl gene.
  • the antisense RNA of the present invention may be applied directly to cells, in the form of oiigonucleotides incorporating the antisense c-Abl sequences, or nucleic acid sequences may be introduced into the cell that will encode the c-Abl sequence. It has been shown the antisense nucleotides may successfully traverse cell membranes, and that such methods may be successful when liposomes are used to encapsulate the nucleic acid. Other techniques for direct insertion of the antisense construct into cells includes electroporation or calcium phosphate transfection. With these methods, the cells are generally removed from the host organism, treated with the constructs, and returned to the host.
  • the more preferred approach will involve the preparation of vectors that incorporate nucleic acid sequences that encode the c-Abl sequence. It is contemplated that these vectors may either be transiently integrated into the host cell, or may be stably integrated into the host cell genome.
  • An expression vector may comprise a gene encoding a RNA molecule complementary to the c-Abl gene and positioned under the control of a promoter, the gene positioned to effect transcription of the c-Abl gene in an orientation opposite to that of vector transcription.
  • the encoded antisense c-Abl RNA molecule is capable of selectively inhibiting the expression of the c-Abl gene product. This expression preferably occurs in a mammalian cell, and even more preferably, the mammalian cell is a human cell.
  • suitable vectors for use within the scope of the present invention include, but are not limited to, adenovirus, adeno-associated virus, retrovirus or herpes simplex virus 1.
  • the present invention contemplates the preparation of nucleic acid molecules that comprise a coding region that contains regions complementary to and capable of hybridizing with a c-Abl gene sequence.
  • the preferred nucleic acid molecules will be DNA sequences arranged in a vector, such as a virus or plasmid, and positioned under the control of an appropriate promoter.
  • the antisense RNA molecule may itself be an appropriate nucleic acid, such as retrovirus RNA into which the appropriate coding sequences have been incorporated.
  • the nucleic acids may be introduced into cells by means of liposomes, or the like.
  • the particular promoter that is used within the scope of the present invention to control the expression of the antisense RNA in a vector construct is not believed to be particularly crucial, as long as it is capable of expressing the antisense c-Abl DNA in the targeted cell at a rate greater than that of the gene to be inhibited.
  • a human cell it will be preferred to position the antisense RNA coding ' region adjacent to and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter may be of human or viral origin.
  • the most preferred promoters are those that are capable of being expressed in a wide variety of histologic cell types, and which are capable of continuously expressing the antisense RNA. Representative examples include the RSV, Herpes Simplex Virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV) and various retroviral promoters including LTR elements.
  • the invention concerns methods of selectively inhibiting the expression of a gene product of c-Abl in a cell, which includes preparing an antisense RNA molecule having a region that is complementary to and capable of hybridizing with a distinct c-Abl region, followed by introducing the antisense RNA into the cell in an amount effective to inhibit the expression of the c-Abl gene product.
  • the invention also concerns methods of preparing genetic constructs for the expression of antisense c-Abl DNA, which includes incorporation of genomic DNA fragments, as opposed to cDNA, into appropriate vectors for subsequent intracellular incorporation.
  • Also within the scope of the invention are methods of selectively inhibiting the expression of c-Abl in a cell comprising first preparing an antisense RNA molecule that includes a region that is complimentary to and capable of hybridizing with a region of the c-Abl gene, followed by introducing the antisense RNA molecule into the cell in an amount effective to inhibit the expression of the c-Abl gene.
  • Also contemplated are methods of selectively inhibiting the expression of c- Abl while treating a patient with DNA damaging agents that comprises the steps of preparing an antisense RNA molecule that includes a region that is complimentary and capable of hybridizing with a region of the c-Abl gene and administering to the patient the antisense RNA molecule in an amount effective to inhibit the expression of the c-Abl gene.
  • a dose of a DNA damaging agent which may be ionizing radiation, is administered to the patient in an amount effective to produce an increase in c-Abl production, which increase is abrogated by the presence of the antisense construct.
  • the antisense RNA molecule may be introduced into the cell by introduction of a DNA molecule that encodes and is capable of expressing the antisense RNA molecule.
  • the DNA molecule is introduced into ito the cell by a liposome or a virus, which virus may be a retrovirus, adenovirus, or herpes simplex virus.
  • the DNA damaging agents within the scope of the present invention include ionizing radiation and chemical agents, such as alkylating agents.
  • the invention also contemplates methods selectively inhibiting the expression of c-Abl in a cell, including preparing an antisense RNA molecule that comprises a sequence that is complementary to a region of the c-Abl gene and is capable of hybridizing to such a region, preparing a recombinant vector that comprises a nucleic acid sequence capable of expression the antisense RNA in the cell, and introducing the vector into the cell in a manner that allows expression of the encoded antisense RNA at a level sufficient to inhibit gene expression.
  • a target cell with at least one DNA damaging agent and a c-Abl antisense molecule in a combined amount effective to kill the cell.
  • This process may involve contacting the cells with the DNA damaging agent(s) or factor(s) and the antisense c-Abl RNA at the same time.
  • This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the DNA damaging agent and the other composition includes the c-Abl antisense molecule.
  • the target cell may be first exposed to the DNA damaging agent(s) and then contacted with a c-Abl antisense RNA molecule, or vice versa.
  • the DNA damaging agent(s) may be first exposed to the DNA damaging agent(s) and then contacted with a c-Abl antisense RNA molecule, or vice versa.
  • one would contact the cell with both agents within about 12 hours of each other, and more preferably within about 6 hours of each other, with a delay time of only about 4 hours being most preferred. These times are readily ascertained by the skilled artisan.
  • contacted and “exposed”, when applied to a cell are used herein to describe the process by which a DNA damaging agent or antisense RNA molecule are delivered to a target cell or are placed in direct juxtaposition with the target cell.
  • both agents are delivered to a cell in a combined amount effective to kill the cell, i.e., to induce programmed cell death or apoptosis.
  • killing means programmed cell death or apoptosis.
  • apoptosis are used interchangeably in the present text to describe a series of intracellular events that lead to target cell death.
  • kits for use in killing cells such as malignant cells
  • the kits of the invention will generally comprise, in suitable container means, a pharmaceutical formulation of a DNA damaging agent and a pharmaceutical formulation of a c-Abl antisense RNA molecule.
  • These agents may be present within a single container, or these components may be provided in distinct or separate container means.
  • the components of the kit are preferably provided as a liquid solution, or as a dried powder. When the components are provided in a liquid solution, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • kits have been described as part of this invention, it should be noted that the use of ionizing radiation to create DNA damage is an important aspect of the invention not specifically provided in kit form.
  • FIG. IA, FIG. IB, FIG. IC and FIG. ID Activation of Src-like tyrosine kinases by mitomycin C (MMC).
  • MMC mitomycin C
  • HL-60 cells were exposed to 10 M MMC and harvested at 1 h.
  • Cell lysates were subjected to immunoprecipitation with pre ⁇ immune rabbit serum (PIRS) (FIG. IA); anti-Fyn antibodies (FIG. IB); anti-Lyn antibodies (FIG. IC); and anti-Src antibodies (FIG. ID).
  • Phosphorylation reactions were performed in the presence of [ ⁇ P]ATP for 10 min at 30°C.
  • Phosphorylated protein was analyzed by 10% SDS-PAGE and autoradiography.
  • FIG. 2A, FIG. 2B and FIG. 2C Activation of ⁇ 6l ⁇ n kinase by MMC.
  • FIG. 2A HL-60 cells were exposed to the indicated concentrations of MMC for 1 h.
  • FIG. 2B cells were exposed to 10 M MMC for the indicated times. ⁇ s
  • Anti-Lyn immunoprecipitates were incubated with [ ⁇ - P]ATP and enolase. Phosphorylated protein was analyzed by SDS-PAGE and autoradiography. In FIG. 2C, anti-Lyn immunoprecipitates were analyzed by immunoblotting with anti-Lyn.
  • FIG. 3A and FIG. 3B Tyrosine phosphorylation of p56/p53 lyn in MMC-treated cells.
  • HL-60 cells were treated with MMC for 1 h.
  • Cell lysates were immunoprecipitated with anti-Lyn and the immunoprecipitates were subjected to immunoblotting with anti-P-Tyr (FIG. 3A) or anti-Lyn (FIG. 3B).
  • FIG. 4A, FIG. 4B and FIG. 4C MMC-induced p56/p53 lyn activation is sensitive to tyrosine kinase inhibitors and is not a direct effect.
  • FIG. 4A cells were treated with 10 M herbimycin A (H) or genistein (G) for 1 h and then MMC for an additional 1 h.
  • FIG. 4B cells were treated with 5 x 10 "5 M H7 for 1 h and then MMC for 1 h.
  • Anti-Lyn immunoprecipitates were analyzed for phosphorylation of p56/p53 yn and enolase.
  • FIG. 4C cells were treated with MMC for 1 h.
  • Anti-Lyn immunoprecipitates were analyzed for phosphorylation of p56/p53 yn and enolase. Lysates from untreated HL-60 cells were immunoprecipitated with anti-Lyn. MMC (10 M) was added to the kinase reaction and incubated for 15 min. The reaction was analyzed for phosphorylation of p56/p53 yn and enolase.
  • FIG. 5A, FIG. 5B and FIG. 5C Other alkylating agents active p56/p53 ,yn .
  • HL-60 cells were treated with 2 x 10 M adozelesin (FIG. 5 A), 10 M nitrogen mustard (FIG. 5B) and 10 "5 M cis-platinum (FIG. 5C) for 1 h.
  • Anti-Lyn immunoprecipitates were analyzed for phosphorylation of p56/p53 yn and enolase.
  • FIG. 6A and FIG. 6B Association of p56/p53 lyn and p34 cdc2 .
  • HL-60 cells were treated with 10 M MMC for 1 h.
  • FIG. 6A cell lysates were incubated with GST or GST-Lyn proteins immobilized on beads. The resulting complexes were separated by SDS-PAGE and analyzed by immunoblotting with anti-cdc2 antibody.
  • FIG. 6B lysates from control (labeled HL-60) and MMC-treated cells were subjected to immunoprecipitation with anti-cdc2.
  • the immune complexes were assayed for in vitro kinase activity by incubation with One aliquot of the kinase reaction was analyzed by SDS-PAGE and autoradiography. The other aliquot was washed to remove free ATP and boiled in SDS buffer to disrupt complexes. A secondary immunoprecipitation was then performed with anti-Lyn. The anti-Lyn immunoprecipitates were separated by SDS-PAGE and analyzed by autoradiography.
  • FIG. 7 A and FIG. 7B Effects of MMC treatment on tyrosine phosphorylation of p34 cdc .
  • HL-60 cells were exposed to MMC for 1 h.
  • FIG. 7A cell lysates were subjected to immunoprecipitation with anti-cdc2.
  • the immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with anti-P-Tyr.
  • FIG. 7B cell lysates were subjected to immunoprecipitation with anti-cdc2 and immunoblot analysis with anti-cdc2.
  • FIG. 8 Phosphorylation of cdc2 peptides by p56/p53 yn .
  • HL-60 cells were treated with MMC for 1 h.
  • Cell lysates were subjected to immunoprecipitation with anti-Lyn.
  • the immunoprecipitates were assayed for phosphorylation of either a cdc2 (IEKIGEGTYGWYK; SEQ ID NO:3) or mutated cdc2 (mcdc2; Y-15 to F-15) peptide.
  • the results represent the mean ⁇ S.D. of two independent studies each performed in duplicate and are normalized to control phosphorylation of the cdc2 peptide. Control cells (cross hatch); MMC-treated cells (stripes).
  • FIG. 9A, FIG. 9B and FIG. 9C Activation of Src-like protein tyrosine kinases by ionizing radiation.
  • HL-60 cells were exposed to 200 cGy ionizing radiation and harvested at 15 min or 2 hours.
  • FIG. 9A Cell lysates were subjected to immunoprecipitation with anti-Fyn antibodies
  • FIG. 9B cell lysates were subjected to immunoprecipitation with anti-Lyn antibodies
  • FIG. 9C cell lysates were subjected to immunoprecipitation with anti-Lck antibodies.
  • Autophosphorylation reactions were performed by adding [ ⁇ - PJATP for 10 min at 30°C.
  • FIG. 10A and FIG. 10B Activation of p53/56 lyn kinase by ionizing radiation.
  • HL-60 cells were exposed to 200 cGy ionizing radiation for 5 min, 15 min, 30 min, 6 hours, 12 hours, or 24 hours.
  • Cell lysates were subjected to immunoprecipitation with anti-Lyn.
  • FIG. 10 A the immunoprecipitates were analyzed in autophosphorylation reactions.
  • FIG. 10B enolase phosphorylation assays are shown. Samples were separated in 10% SDS-PAGE gels and analyzed by autoradiography. The fold increase of Enolase phosphorylation, increased as measured by scintillation counting of the excised bands, is indicated at the bottom.
  • FIG. 11 Different doses of ionizing radiation induce activation of p53/p56 yn .
  • HL-60 cells were exposed to the indicated doses of ionizing radiation and then harvested at 12 h.
  • Soluble proteins were subjected to immunoprecipitation with anti-Lyn.
  • the immunoprecipitates were analyzed for phosphorylation of p56/p53 yn and enolase. The fold increase in enolase phosphorylation is indicated at the bottom.
  • FIG. 12A and FIG. 12B Effects of H 2 O 2 , NAC and protein tyrosine kinase inhibitors on activation of p56/p53 yn .
  • HL-60 cells were either treated with H 2 O 2 for the indicated times or pretreated with 30 mM NAC for 1 h, irradiated (200 cGy) and harvested at 12 h.
  • FIG. 12B HL-60 cells were treated with 10 ⁇ M herbimycin (H) or 10 ⁇ M genistein (G) for 1 h, irradiated (200 cGy) and then harvested at 12 h.
  • Cell lysates were immunoprecipitated with anti- Lyn and the immunoprecipitates were analyzed for phosphorylation of p56/p53 yn and enolase.
  • FIG. 13A and FIG. 13B Ionizing radiation exposure induces tyrosine phosphorylation of a 34 kD substrate.
  • HL-60 cells were exposed to 200 cGy ionizing radiation and harvested at the indicated times.
  • soluble proteins were subjected to immunoblot (IB) analysis with anti-P-Tyr; and in FIG. 13B soluble proteins were subjected to immunoblot (IB) analysis with anti- p34 cdc2 antibodies.
  • the arrow indicates the position of 34 kD signals.
  • FIG. 14A and FIG. 14B Different doses of ionizing radiation induce tyrosine phosphorylation of the 34 kD protein.
  • HL-60 cells were exposed to the indicated doses of ionizing radiation and then harvested at 5 min.
  • soluble proteins were subjected to immunoblot (IB) analysis with anti-P-Tyr; and in FIG. 14B, soluble proteins were subjected to immunoblot (IB) analysis with anti- p34 cd antibodies.
  • the arrows indicate the position of the 34 kD signals.
  • FIG. 15A and FIG. 15B Ionizing radiation induces tyrosine phosphorylation of p34 cdc .
  • HL-60 cells were exposed to 50 cGy ionizing radiation and harvested at 5 min.
  • Cell lysates from control and irradiated cells were subjected to immunoprecipitation (IP) with p34 antiserum and protein A- Sepharose.
  • IP immunoprecipitation
  • FIG. 15 A the immunoprecipitates were subjected to immunoblot (IB) analysis with anti-P-Tyr antibodies; and in FIG. 15B, the immunoprecipitates were subjected to immunoblot (IB) analysis with anti-p34 c antibodies.
  • FIG. 16A, FIG. 16B, FIG. 16C, FIG. 16D, FIG 16E, and FIG 16F Activation of c-Abl by diverse DNA damaging agents.
  • FIG. 16A, FIG. 16B, and FIG. 16C U-937 or NIH3T3 cells were treated with 2 Gy ionizing radiation (IR) and harvested at 1 h. Nuclei were isolated and the nuclear proteins subjected to immunoprecipitation with anti- Abl (K-12, Santa Cruz Biotechnology, San Diego, CA). In vitro immune complex kinase assays were performed using a GST-Crk( 120-225) fusion protein as substrate (U-937, FIG. 16A, lanes 1 and 2; NIH3T3, FIG 16B). GST-Crk( 120-212) fusion protein (which lacks the critical Y221) was used as a negative control (lane 3). The anti- Abl immunoprecipitates were also analyzed by immunoblotting with anti-Abl (FIG. 16C).
  • FIG. 16D U-937 cells were treated with 2 Gy IR and harvested at the indicated times. Nuclear proteins were then subjected to immunoprecipitation with anti-Abl antibody. Immunoprecipitations were also performed with preimmune rabbit serum (PIRS) from cells exposed to 2 Gy IR and harvested at 1 h. In vitro immune complex kinase assays were performed using the c-Abl substrate EAIYAAPFAKKK (SEQ ID NO:5). The data (percent control phosphorylation) represent the mean + S.E of three separate studies.
  • PIRS preimmune rabbit serum
  • FIG. 16E and FIG. 16F NIH3T3 cells were treated with 10 ⁇ M CDDP for .30 min, 10 ⁇ M MMC for 1 h or 2 Gy IR (harvested at 1 h). Nuclear proteins were subjected to immunoprecipitation with anti-Abl. Kinase assays were performed using GST-Crk( 120-225) fusion protein (FIG. 16E) or EAIYAAPFAKKK (SEQ ID NO:5) peptide (FIG. 16F) as substrates.
  • FIG. 17A and FIG. 17B Activation of SAP kinase activity by DNA damaging agents.
  • FIG. 17A NIH3T3 cells were treated with 20 Gy IR (harvested at 1 h), 10 ⁇ M CDDP for 2 h or 10 ⁇ M MMC for 2 h. Total lysates were immunoprecipitated with anti-SAP kinase antibody and in vitro immune complex kinase reactions containing GST-Jun(2-100) fusion protein were analyzed by 10% SDS-PAGE and autoradiography.
  • FIG. 17B Abl-/- cells were treated with 20 Gy IR (harvested at 1 h), 10 ⁇ M CDDP for 2 h or 10 ⁇ M MMC for 2 h.
  • NIH3T3 cells were also treated with 10 ⁇ M MMC for 2 h as a positive control.
  • Total cell lysates were immunoprecipitated with anti- SAP kinase and in vitro immune complex kinase assays were performed using GST-Jun(2-100) as substrate.
  • FIG. 18 A, FIG. 18B and FIG. 18C Activation of c-Abl and SAP kinase by DNA-damaging agents in c-Abl reconstituted Abl-/- (Abl+) cells.
  • FIG. 18 A Nuclear proteins isolated from NIH3T3, Abl-/- abd ABL+ cells were subjected to immunoprecipitation with anti-Abl. The immunoprecipitates were analyzed by immunoblotting with anti-Abl.
  • FIG. 18B NIH3T3, Abl-/- and Abl+ cells were treated with 2 Gy IR and harvested at 1 h. Nuclei were isolated and the nuclear proteins subjected to immunoprecipitation with anti-Abl.
  • FIG. 18C Abl+ cells were treated with either IR (20 Gy and harvested at 1 h) or MMC (10 ⁇ M for 2 h). Total cell lysates were immunoprecipitated with anti-SAP kinase antibody and in vitro immune complex kinase assays containing GST-Jun (2-100) fusion protein were analyzed by 10% SDS-PAGE and autoradiography.
  • FIG. 19A and FIG. 19B Activation of SAP kinase by TNF is independent of c-Abl.
  • FIG. 19A NIH3T3 cells were treated with 2 Gy IR (harvested at 1 h) or 10 ng/ml TNF for 15 min. Nuclear lysates were immunoprecipitated with anti-Abl antibody and in vitro immune complex kinase assays were performed using peptide as substrate.
  • FIG. 19B NIH3T3 or Abl-/- cells were treated with 10 ng/ml TNF for 30 min or 20 Gy IR. Total cell lysates were immunoprecipitated with anti-SAP kinase and immune complex kinase assays were performed using GST-Jun (2-100) as substrate.
  • the product of the c-Abl gene is a non-receptor tyrosine kinase that is localized to the nucleus and cytoplasm.
  • the present invention demonstrates that ionizing radiation (IR) activates c-Abl. Similar results were obtained with the alkylating agents cisplatinum and mitomycin C.
  • the inventors also demonstrate that cells deficient in c-Abl fail to activate Jun kinase (JNK/SAP kinase) following IR or alkylating agent exposure and that reconstitution of c-Abl in these cells restores that response. In contrast, the stress response to tumor necrosis factor is stimulated by a c-Abl-independent mechanism.
  • c-Abl contains actin binding and DNA binding domains.
  • Rb retinoblastoma
  • c-Abl activation could play a role in regulating these responses to genotoxic stress.
  • overexpression of c-Abl arrests cells in G- phase
  • activation of c-Abl by DNA damage may regulate distinct stress pathways that include SAP kinase.
  • Antisense constructs are oligo- or polynucleotides comprising complementary nucleotides to the control regions or coding segments of a DNA molecule, such as a gene or cDNA. Such constructs may include antisense versions of both the promoter and other control regions, exons, introns and exonrintron boundaries of a gene. Antisense molecules are designed to inhibit the transcription, translation or both, of a given gene or construct, such that the levels of the resultant protein product are reduced or diminished.
  • Nucleic acid sequences which comprise "complementary nucleotides” are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, that the larger purines will base pair with the smaller pyrimidines to form only combinations of Guanine paired with Cytosine (G:C) and Adenine paired with either Thymine (A:T), in the case of DNA, or Adenine paired with Uracil (A:U) in the case of RNA.
  • complementary and/or antisense sequences mean nucleic acid sequences that are substantially complementary over their entire length and have very few base mismatches. For example, nucleic acid sequences of fifteen bases in length may be termed complementary when they have a complementary nucleotide at thirteen or fourteen positions with only a single mismatch. Nucleic acid sequences which are "completely complementary” will be nucleic acid sequences which are entirely complementary throughout their entire length and have no base mismatches. In general, the longer the sequence, the larger the number of mis-matches that are tolerated.
  • Antisense RNA constructs may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
  • the antisense constructs have evident utility in gene inhibition embodiments.
  • U.S. Patent 4,740,463, incorporated herein by reference describes in general methods for antagonizing the effects of an oncogene using oppositely transcribed oncogene DNA segments.
  • the methodology generally disclosed in U.S. Patent 4,740,463 may be used in connection with the DNA damaging and c-abl inhibition methods and compositions of the present invention.
  • PCT Patent Application WO 95/10265 also describes methods useful for the delivery of antisense oligos, which methods utilize a surface active non-ionic copolymer (a block copolymer). Such delivery methods may also be used in the context of the present invention.
  • the anti- ⁇ / constructs may be linked to a cell-specific binding agent for enhanced delivery, as described in PCT Patent Application WO 94/23050.
  • Oiigonucleotides which contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression (Wagner et al, 1993).
  • Oligos modified according to EP 431,523 are also contemplated for use.
  • Adenoviruses have been widely studied and well-characterized as a model system for eukaryotic gene expression. Adenoviruses are easy to grow and manipulate, and they exhibit broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10 -10 plaque-forming unit (PFU)/ml, and they are highly infective. The life cycle of Adenoviruses does not require integration into the host cell genome. The foreign genes delivered by Adenovirus vectors are expressed episomally, and therefore, have low genotoxicity to host cells. Adenoviruses appear to be linked only to relatively mild diseases, since there is no known association of human malignancies with Adenovirus infection. Moreover, no side effects have been reported in studies of vaccination with wild-type Adenovirus (Couch et al, 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
  • PFU plaque-forming unit
  • Adenovirus vectors have been successfully used in eukaryotic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies demonstrated that recombinant Adenoviruses could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al, 1990; Rich et al, 1993).
  • Adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury, et al, 1987), providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the El and E3 regions, the maximum capacity of the current
  • Adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the Adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity.
  • the term "recombinant" cell is intended to refer to a cell into which a recombinant gene, such as a gene from the adenoviral genome has been introduced. Therefore, recombinant cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced gene. Recombinant cells are thus cells having a gene or genes introduced through the hand of man.
  • the recombinantly introduced genes encode radiation sensitizing or radiation protecting factors and are inserted in the El or E3 region of the adenovirus genome. It is recognized that the present invention also encompasses genes that are inserted into other regions of the adenovirus genome, for example the E2 region.
  • the adenovirus vector construct may therefore, comprise at least 10 kb or at least 20 kb or even about 30 kb of heterologous DNA and still replicate in a helper cell.
  • replicate in a helper cell it is meant that the vector encodes all the necessary cis elements for replication of the vector DNA, expression of the viral coat structural proteins, packaging of the replicated DNA into the viral capsid and cell lysis, and further that the trans elements are provided by the helper cell DNA.
  • Replication is determined by contacting a layer of uninfected cells with virus particles and incubating said cells. The formation of viral plaques, or cell free areas in the cell layers is indicative of viral replication.
  • the adenoviral DNA that stably resides in the helper cell may comprise a viral vector such as an Herpes Simplex virus vector, or it may comprise a plasmid or any other form of episomal DNA that is stable, non-cytotoxic and replicates in the helper cell.
  • heterologous DNA DNA derived from a source other than the adenovirus genome which provides the backbone for the vector.
  • This heterologous DNA may be derived from a prokaryotic or a eukaryotic source such as a bacterium, a virus, a yeast, a plant or animal.
  • the heterologous DNA may also be derived from more than one source.
  • a promoter may be derived from a virus and may control the expression of a structural gene from a different source such as a mammal.
  • Preferred promoters include viral promoters such as the SV40 late promoter from simian virus 40, the Baculovirus polyhedron enhancer/promoter element, RSV, Herpes Simplex Virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV) and various retroviral promoters including LTR elements.
  • viral promoters such as the SV40 late promoter from simian virus 40, the Baculovirus polyhedron enhancer/promoter element, RSV, Herpes Simplex Virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV) and various retroviral promoters including LTR elements.
  • the promoters and enhancers that comprise the heterologous DNA will be those that control the transcription of protein encoding genes in mammalian cells may be composed of multiple genetic elements.
  • the term promoter refers to a group of transeriptional control modules that are clustered around the initiation site for RNA polymerase II. Promoters are believed to be composed of discrete functional modules, each comprising approximately 7-20 bp of DNA, and containing one or more recognition sites for transeriptional activator proteins. At least one module in each promoter functions to position the start site for RNA synthesis.
  • TATA box In some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV 40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transeriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between elements is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.
  • the heterologous DNA of the present invention may also comprise an enhancer.
  • enhancers and promoters are operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Aside from this operational distinction, enhancers and promoters are very similar entities. They have the same general function of activating transcription in the cell. They are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • enhancers and promoters are homologous entities and that the transeriptional activator proteins bound to these sequences may interact with the cellular transeriptional machinery in fundamentally the same way. It is understood that any such promoter or promoter/enhancer combination may be included in the heterologous DNA of the adenoviral vector to control expression of the heterologous gene regions.
  • the heterologous DNA may include more than one structural gene under the control of the same or different promoters.
  • the heterologous DNA may also include ribosome binding sites and polyadenylation sites or any necessary elements for the expression of the DNA in a eukaryotic or a mammalian cell. These vector constructs are created by methods well known and routinely practiced in the art such as restriction enzyme digestion followed by DNA ligase directed splicing of the various genetic elements.
  • the heterologous DNA may further comprise a constitutive promoter.
  • a constitutive promoter is a promoter that exhibits a basal level of activity that is not under environmental control.
  • constitutive promoters include, but are not limited to, intermediate-early CMV enhancer/promoter, RSV enhancer-promoter, SV40 early and SV-40 late enhancer/promoter, MMSV LTR, SFFV enhancer/promoter, EBV origin of replication, or the Egr enhancer/promoter.
  • intermediate-early CMV enhancer/promoter RSV enhancer-promoter
  • SV40 early and SV-40 late enhancer/promoter SV LTR
  • SFFV enhancer/promoter EBV origin of replication
  • Egr enhancer/promoter Egr enhancer/promoter
  • tissue specific promoter is a promoter that is active preferentially in a cell of a particular tissue type, such as in the liver, the muscle, endothelia and the like.
  • tissue specific promoters include the RSV promoter to be expressed in the liver or the surfactin promoter to be expressed in the lung, with the muscle creatine kinase enhancer combined with the human cytomegalovirus immediate early promoter being the most preferred for expression in muscle tissue, for example.
  • the cellular exposure to ionizing radiation is associated with transeriptional activation of certain immediate early genes that encode transcription factors (Weichselbaum et al, 1991). These genes include members of the jun-fos, NF-kB and early growth response (EGR-1) gene families (Hallahan et al, 1992; Datta et al, 1992; Brach et al, 1991). The induction of these genes following x-irradiation may represent cellular responses to oxidative stress (Datta et al, 1992; Brach et al, 1991; Datta et al, 1993).
  • EGR-1 early growth response
  • the present invention is a method of expressing an antisense RNA molecule in a mammalian cell. This method would comprise the steps of obtaining an adenoviral vector construct comprising more than 7.5 kb of heterologous DNA, replicating the adenoviral vector construct in a helper cell, obtaining virion particles produced by the helper cells and infecting mammalian cells with the virion particles.
  • the c-Abl antisense RNA molecule to be expressed as described in the preceding paragraph may of any origin, for example, an animal or a human gene.
  • the adenovirus vector construct contains a deletion in the El or E3 region of the genome and the foreign gene is inserted in its place.
  • the virion plaques that would be produced by the replicating viral vector and would thus lyse the host cell can be obtained by any acceptable means. Such means would include filtration, centrifugation or preferably physical touching of viral plaques. All such methods of obtaining virion particles and infecting mammalian cells with the particles are well known to those of skill in the art.
  • adenovirus type 5 virus was selected because a great deal of biochemical and genetic information about the virus is known, and it has historically been used for most constructions employing adenovirus as a vector. It is understood, however, the adenovirus may be of any of the 42 different known serotypes of subgroups A-F.
  • Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the method of the present invention.
  • the level and pattern of expression of c-Abl antisense RNA following infection can be optimized. For example, selection of a promoter which is active specifically in certain cell types will permit tissue-specific expression of the antisense molecule.
  • the invention relates to a method for increasing c-Abl antisense RNA levels in a subject comprising administering to the subject an effective amount of a pharmaceutical composition which includes the adenovirus vector/c-Abl antisense RNA construct.
  • the inventors propose that an effective o amount of the vector construct will involve the administration of from about 10 to 10 virus particles, which may be given either as a single bolus injection directly into the tumor or as a systemic intravenous infusion over several hours.
  • the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
  • Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication- defective adenovirus vector for use in the method of the present invention. This is because Adenovirus type 5 is a human adenovirus of which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
  • the invention relates to pharmaceutical compositions wherein the adenovirus vector/c-Abl antisense RNA gene construct is dispersed in a pharmacologically acceptable solution or buffer.
  • Preferred solutions include neutral saline solutions buffered with phosphate, lactate, or Tris, containing sucrose or glycerol, and the like.
  • a preferred means of purifying the vector involves the use of buoyant density gradients, such as cesium chloride gradient centrifugation.
  • adenovirus is a virus that infects humans
  • an immunological reaction is believed to be a possibility
  • Such a test could be performed in a variety of accepted manners, for example, through a simple skin test or through a test of the circulating blood levels of adenovirus-neutralizing antibodies.
  • the particular cell line used to propagate the recombinant adenoviruses of the present invention is not critical to the present invention.
  • the recombinant adenovirus vectors can be propagated on, e.g., human 293 cells, or in other cell lines that are permissive for conditional replication-defective adenovirus infection, e.g., those which express adenovirus EIA gene products "in trans" so as to complement the defect in a conditional replication-defective vector.
  • the cells can be propagated either on plastic dishes or in suspension culture, in order to obtain virus stocks thereof.
  • Liposomes have been used for more than a decade to introduce exogenous DNA into cells (Mukherjee et al, 1978; Nicolau et al, 1983).
  • the term liposome is used to describe different forms of surfactant vesicles consisting of one or more concentric lipid bilayer spheroids surrounding an aqueous space.
  • Classical liposomes consist of fatty acid esters and fat-alcohol ethers of glycerol phosphatides. Their net charge is negative under physiological pH conditions due to phosphate groups.
  • liposomes bearing a positive charge derived from quaternary ammonium groups such as N-(l-(2,3-dioleoyloxy)propyl)-N,NN- r trimethylammonium chloride (DOTMA) (Feigner et al, 1987) have been introduced. These cationic liposomes interact strongly with cellular membrane which are themselves negatively charged. In contrast to classic liposomes they do not encapsulate or entrap " DNA but bind it at their surface. Another group of liposomes consists of nonionic surfactant vesicles. While classical phospholipid- based liposomes are of low toxicity, the toxicity and antigenicity of the partially synthetic cationic and nonionic liposomes have not been rigorously evaluated.
  • DOTMA N-(l-(2,3-dioleoyloxy)propyl)-N,NN- r trimethylammonium chloride
  • exemplary liposome preparations include, but are not limited to DOTMA, l,2-dioleyloxypropyl-3 -trimethyl ammonium bromide; DOPE, dioleoylphosphatidylethanolamine; POPE, palmitoyloleoylphosphatidylethanolamine; DMPE, dimyristoylphosphatidylethanolamine; DPPE, dipalmitoylphosphatidylethanolamine;
  • DOPE dioleoylphosphatidylethanolamine
  • POPE palmitoyloleoylphosphatidylethanolamine
  • DMPE dimyristoylphosphatidylethanolamine
  • DPPE dipalmitoylphosphatidylethanolamine
  • DSPE distearoylphosphatidylethanolamine
  • PMME dioleoylphosphatidylmonomethylethanolamine
  • PDME dioleoylphosphatidyldimethylethanolamine
  • DOPC dioleoylphosphatidylcholine
  • CPE dioleoylphosphatidylcaprylamine
  • DPE dioleoylphosphaqtidyldodecylamine
  • DORI l,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide
  • the present invention also embodies a method using HSV-1 for delivering genes for gene therapy.
  • the method involves combining the gene used for gene therapy with the HSV-1 virus rendered non- pathogenic.
  • the gene and the virus are then combined with a pharmacologically acceptable carrier in order to form a pharmaceutical composition.
  • This pharmaceutical composition is then administered in such a way that the mutated virus containing the gene for therapy, or the HSV-1 wild type virus containing the gene, can be incorporated into cells at an appropriate area.
  • the use of the HSV-1 virus with a specific mutation in the ⁇ *34.5 gene provides a method of therapeutic treatment of tumorigenic diseases both in the CNS and in all other parts of the body (Chou 1992).
  • the " ⁇ - 34.5 minus” virus can induce apoptosis and thereby cause the death of the host cell, but this virus cannot replicate and spread (Chou 1992). Therefore, given the ability to target tumors within the CNS, the ⁇ * 34.5 minus virus has proven a powerful therapeutic agent for hitherto virtually untreatable forms of CNS cancer. Furthermore, use of substances, other than a virus, which inhibit or block expression of genes with anti-apoptotic effects in target tumor cells can also serve as a significant development in tumor therapy and in the treatment of herpes virus infection, as well as treatment of infection by other viruses whose neuro virulence is dependent upon an interference with the host cells' programmed cell death mechanisms. The procedures to generate the above recombinant viruses are those published by Post and Roizman (1981), and U.S. Patent No. 4,769,331, incorporated herein by reference.
  • Retroviruses may also be used to deliver the antisense RNA constructs to the host target tissues.
  • These viruses in which the 3' LTR (linear transfer region) has been inactivated. They are enhancerless 3'LTR's, often referred to as self- inactivating viruses because after productive infection into the host cell, the 3 'LTR is transferred to the 5' end and both viral LTR's are inactive with respect to transeriptional activity.
  • a use of these viruses well known to those skilled in the art is to clone genes for which the regulatory elements of the cloned gene are inserted in the space between the two LTR's.
  • An advantage of a viral infection system is that it allows for a very high level of infection into the appropriate recipient cell.
  • the present invention contemplates a pharmaceutical composition comprising a therapeutically effective amount of at least one genetic construct of the present invention and a physiologically acceptable carrier.
  • a therapeutically effective amount of a genetic construct that is combined with a carrier to produce a single dosage form varies depending upon the host treated and the particular mode of administration.
  • a specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy.
  • a composition of the present invention is typically administered orally or parenterally in dosage unit formulations containing standard, well known nontoxic physiologically acceptable carriers, adjuvants, and vehicles as desired.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intraarterial injection, or infusion techniques.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions are formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • Suitable vehicles and solvents that may be employed are water, Ringer' s solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or di ⁇ glycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • a genetic construct of the present invention can also be complexed with a poly(L-Lysine)(PLL)-protein conjugate such as a transferrin-PLL conjugate or an asialoorosomucoid-PLL conjugate.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, syrups, solutions, suspensions, and elixirs containing inert diluents commonly used in the art, such as water.
  • Such compositions can also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
  • HL-60 cells were grown in RPMI- 1640 medium containing 15% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate and 1 mM non-essential amino acids. Cells were treated with MMC (Sigma
  • Immune complex kinase assays Cells (2-3 x 10 ) were washed twice with ice cold phosphate buffered saline (PBS) and lysed in 2 ml of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT and 10 mg/ml of leupeptin and aprotinin). After incubation on ice for 30 min, insoluble material was removed by centrifugation at 14000 rpm for 10 min at 4°C. Soluble proteins were precleared by incubating with 5 mg/ml rabbit-anti-mouse IgG for 1 h at 4°C and then for an additional 30 min after addition of protein A-sepharose.
  • PBS phosphate buffered saline
  • the supernatant fraction was incubated with pre-immune rabbit serum, anti-Fyn, anti-Lyn, anti-Src (UBI, Lake Placid, NY) or anti-cdc2 (sc-54, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies for 1 h at 4°C followed by 30 min after addition of protein A-sepharose.
  • the immune complexes were washed three times with lysis buffer and once with kinase buffer (20 mM HEPES, pH 7.0, 10 mM MnCl 2 and 10 mM MgCl 2 ) and resuspended in 30 ml of kinase buffer containing 1 mCi/ml [ ⁇ - 32 P]ATP (3000 Ci/mmol; NEN, Boston, MA) with and without 5-8 mg of acid-treated enolase (Sigma). The reaction was incubated for 15 min at 30°C and terminated by the addition of 2x SDS sample buffer. The proteins were separated in 10% SDS-polyacrylamide gels and analyzed by autoradiography. Radioactive bands were excised from certain gels and quantitated by scintillation counting.
  • Immune complexes were also resuspended in 30 ml kinase buffer containing 1 mCi/ml [ ⁇ - 32 P]ATP and either 100 mM cdc2 peptide (amino acids 7 to 20; IEKIGEGTYGVVYK; SEQ ID NO:3) or 100 mM mutated cdc2 peptide with Phe- 15 substituted for Tyr- 15 (IEKIGEGTFGVVYK; SEQ ID NO:4).
  • the reactions were incubated for 15 min at 30°C and terminated by spotting on P81 phosphocellulose discs (GIBCO/BRL). The discs were washed twice with 1% phosphoric acid and twice with water before analysis by liquid scintillation counting.
  • Immunoblot analysis Immune complexes bound to protein A-sepharose were prepared as for the autophosphorylation assays. Proteins were separated in 10% SDS-polyacrylamide gels and transferred to nitrocellulose paper. The residual binding sites were blocked by incubating the filters in 5% dry milk in PBST (PBS/0.05% Tween-20) for 1 h at room temperature. The blots were subsequently incubated with anti-cdc2 or anti-phosphotyrosine (anti-P-Tyr; MAb 4G10, UBI).
  • the filters were incubated for 1 h at room temperature with anti-mouse IgG (whole molecule) peroxidase conjugate (Sigma) in 5% milk/PBST. The filters were then washed and the antigen-antibody complexes visualized by the ECL detection system (Amersham, Arlington Heights, IL).
  • anti-mouse IgG whole molecule peroxidase conjugate
  • Immunoprecipitations were performed with anti-p34 c c2 at 5 mg/ml cell lysate. Immune complexes were collected on protein A-Sepharose beads (Pharmacia), washed three times with lysis buffer and twice with kinase buffer, resuspended in kinase buffer and then incubated for 10 min at 30°C in the presence of 1 mCi ml [ ⁇ - P]ATP. One aliquot of the kinase reaction was subjected to SDS-PAGE and autoradiography.
  • the plasmid encoding a glutathione S-transferase (GST)-Lyn (amino acids 1 to 243) fusion protein was obtained from T. Pawson, Toronto, Canada and transfected into E. coli DH5a (Pleiman et al, 1993).
  • the fusion protein was induced with IPTG, purified by affinity chromatography using glutathione-Sepharose beads (Pharmacia) and equilibrated in lysis buffer.
  • HL-60 cell lysates were incubated with 50 mg immobilized GST or GST-Lyn for 2 h at 4°C.
  • the protein complexes were washed three times with lysis buffer and boiled for 5 min in SDS sample buffer. The complexes were then separated in 10% SDS-PAGE and subjected to silver staining or immunoblot analysis with anti-cdc2.
  • HL-60 cells express the p59 yn , p56/p53 yn and pp60 c"src tyrosine kinases (Barnekow & Gessler, 1986; Gee et al, 1986; Katagiri et al, 1991).
  • the inventors have shown that certain of these tyrosine kinases are activated during treatment of HL-60 cells with MMC.
  • p56/p53 yn Activation of p56/p53 yn was confirmed at different concentrations of MMC and by assaying for phosphorylation of the substrate protein enolase. Increases in p56/p53 yn activity were found at 10 and 10 " M MMC, while more pronounced stimulation of this kinase was apparent at 10 and 10 M (FIG. 2A). The results further demonstrate that p56/p53 yn activity is rapidly induced in MMC-treated cells. Increases in MMC-induced phosphorylation of p56/p53 yn and enolase were first detectable at 30 min (4.2-fold increase for enolase) and persisted through at least 12 h (4.1 -fold for enolase) of drug exposure (FIG. 2B). The induction of p56/p53 yn activity was not related to cell death since viability as determined by trypan blue exclusion was >90% at 12 h of MMC treatment.
  • MMC acts as a monofunctional and bifunctional alkylating agent (Carrano et al, 1979). Consequently, adozelesin, another monofunctional but structurally distinct alkylating agent (Bhuyan et al, 1992; Hurley et al, 1984), was investigated. The results demonstrate that treatment of HL-60 cells with adozelesin is similarly associated with stimulation of p56/p53 yn and enolase phosphorylation (FIG. 5A). Other studies were performed with agents that also induce the formation of DNA cross-links.
  • Nitrogen mustard an agent that forms monoadducts and DNA interstrand cross-links (Ewig & Khon, 1977; Hartley et al, 1992), was effective in inducing p56/p53 lyn activity (FIG. 5B).
  • treatment of cells with cis-platinum an agent that forms intrastrand cross-links (Sherman & Lippard, 1987), was associated with stimulation of the p56/p53 yn kinase (FIG. 5C).
  • the inventors assayed the adsorbates for reactivity with anti-cdc2.
  • the results indicate that p34 associates with the GST-Lyn fusion protein and not the GST control (FIG. 6A).
  • the potential interaction between p56/p53 lyn and p34 cdc2 was further examined in coimmunoprecipitation studies. Lysates of control and MMC-treated cells were subjected to immunoprecipitation with anti-cdc2 and the immunoprecipitates were assayed for autophosphorylation (FIG. 6B). One aliquot of the in vitro kinase reaction was assayed by SDS-PAGE and autoradiography.
  • the lyn gene encodes two forms of the tyrosine kinase, p56 yn and p53 yn , due to alternate mRNA splicing (Yamanashi et al, 1987; Yamanashi et al, 1989).
  • p56/p53 yn is related to pp60 c"src and p59 f n (Cantley et al, 1991).
  • p56/p53 lyn was activated in MMC-treated cells. These kinases are often associated with cell surface receptors at the interface between the cell membrane and cytoplasm.
  • alkylating agents such as MMC are generally attributed to DNA damage, their action may be related to alkylation of RNA or protein.
  • MMC treatment of intact cells is associated with activation of p56/p53 yn raised the possibility that this effect might be due to direct alteration of Lyn protein.
  • p56/p53 yn activity was however decreased in vitro by incubation of anti-Lyn immune complexes with MMC.
  • adozelesin In order to address the possibility that MMC-induced activation of p56/p53 yn is related to formation of DNA lesions, another agent, adozelesin, was used that covalently binds to the N-3 of adenine within the minor groove of DNA (Bhuyan et al, 1992; Hurley et al, 1984). Adozelesin also induces ⁇ 56/p53 lyn activity.
  • HL-60 cells also respond similarly to other alkylating agents, such as nitrogen mustard which reacts predominantly with guanines by alkylation of their N-7 positions or forms DNA interstrand cross-links (Ewig & Khon, 1977; Hartley et al, 1992). Moreover, p56/p53 yn activity was stimulated by cis-platinum which induces intrastrand cross-links (Sherman & Lippard, 1987). Thus, structurally distinct agents that damage DNA by diverse mechanisms are capable of inducing p56/p53 yn activity.
  • the p34 cd serine/threonine protein kinase controls entry of cells into mitosis (Nurse, 1990; Pines & Hunter, 1990). This kinase is regulated by networks of kinases and phosphatases that appear to respond to the state of DNA replication. Activation of p34 cdc2 involves association with cyclin B and posttransiational modifications of the p34 /cyclin B complex (Norbury & Nurse, 1992).
  • pl07 weel dual-specificity kinase is responsible for phosphorylation of p34 on Tyr- 15 (Featherstone & Russell,
  • pl07 weel appears to control p34 activity to ensure completion of S-phase, other studies suggest that pl07 wee is not required for the DNA-damage-dependent mitotic checkpoint. In this context, normal mitotic arrest has been observed after irradiation of Schizosaccharomyces pombe cells with a defective or missing weel gene (Barbet & Carr, 1993). Other studies have shown that p34 is phosphorylated on tyrosine in yeast weel minus mutants (Gould et al, 1990). The present results in mammalian cells suggest that regulation of p34 2 following exposure to alkylating agents involves activation of p56/p53 yn .
  • Lyn tyrosine kinase Treatment of human HL-60 myeloid leukemia cells with ionizing radiation is associated with activation of the Lyn tyrosine kinase.
  • the lyn gene encodes two forms of this kinase, p56 yn and p53 yn , as a result of alternate splicing (Yamanashi et al, 1987; 1989). Both p56/p53 lyn , but not certain other Src-related kinases, are activated in irradiated HL-60 cells. Activation of p56/p53 yn represents a signaling pathway distinct from those involved in X-ray-induced early response gene expression.
  • HL-60 myeloid leukemia cells were grown in RPMI- 1640 medium containing 15% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, 2mM L-glutamine, ImM sodium pyruvate and ImM non-essential amino acids. Cells in logarithmic growth phase were suspended in complete RPMI-1640 medium with 0.5% FBS 18 hours prior to irradiation.
  • FBS heat-inactivated fetal bovine serum
  • HL-60 cells were also treated with 50 mM H 2 O 2 (Sigma Chemical Co., St. Louis, MO), 30 mM N-acetyl cysteine (NAC; Sigma), 10 ⁇ M genistein (GIBCO/BRL, Gaithersburg, MD) or 10 ⁇ M herbimycin A (GIBCO/BRL).
  • the supematants were incubated with 2.5 ⁇ l of anti-human Fyn, 2 ⁇ l of anti-human Lyn, 3 ⁇ l of anti-human Lyk (N-terminal) or 3 ⁇ l of anti-Src antibody (UBI, Lake Placid, NY) for 1 hour at 4°C followed by 30 min with protein A- sepharose.
  • the immune complexes were washed three times with lysis buffer, once with kinase buffer (20 mM HEPES, pH 7.0, 10 mM MnCl 2 and 10 mM MgCl 2 )
  • Immune complexes as prepared for autophosphorylation assays were washed three times with lysis buffer and once with kinase buffer. The beads were resuspended in 30 ⁇ l of kinase buffer containing 1 mCi/ml [ ⁇ - pjATP and 3-5 mg of acid treated enolase (Sigma). The reaction was incubated for 10 min at 30°C and terminated by the addition of 2 x SDS sample buffer. The proteins were resolved by 10% SDS-PAGE. Equal loading of the enolase was determined by staining with Coomassie blue. The gels were then destained and analyzed by autoradiography. Radioactive bands were also excised from the gel and quantitated by scintillation counting.
  • HL-60 cells were also irradiated with 200 cGy and immunoprecipitates assayed for both p56/p53 yn autophosphorylation and enolase (a substrate protein) phosphorylation. Irradiation was associated with an increase in p56/p53 yn autophosphorylation at 5 min that persisted through 12 hours (FIG. 10A). However, assays at 24 hours after X-ray treatment revealed declines in p56/p53 yn signals (FIG. 10A).
  • FIG. 10B This increase in activity was rapid and sustained for at least 12 hours (FIG. 10B). Quantitation of P-incorporation into enolase by scintillation counting demonstrated X-ray-induced increases in p56/p53 yn activity of approximately 3-fold at 15 min to 12 hours (FIG. 10B). As observed in autophosphorylation studies, enolase phosphorylation was also decreased at 24 hours (FIG. 10B).
  • FIG. 11 Similar studies were performed at different doses of ionizing radiation (FIG. 11). Treatment with 25 cGy had little if any effect on phosphorylation of p56/p53 yn or enolase. Doses of 50 cGy, however, were associated with increases in p56/p53 yn activity (FIG. 11). Moreover, on the basis of enolase phosphory ⁇ lation there was an apparent dose-dependent stimulation of this kinase (FIG. 11). The cellular effects of ionizing radiation are believed to be related to direct interaction of X-rays with DNA or through the formation of reactive oxygen intermediates (ROIs) which damage DNA and cell membranes (Hall, 1988).
  • ROIs reactive oxygen intermediates
  • HL-60 cells were either treated with H 2 O 2 for the indicated times or pretreated with 30 mM NAC for 1 hour, irradiated (200 cGy) and harvested at 12 hours. Irradiated HL-60 cells treated with H 2 O 2 did not show a detectable increase in phosphorylation of p56/p53 yn or enolase (FIG. 12A). Cells were also treated with the antioxidant NAC (Roederer et al, 1990; Staal et al, 1990), an agent that abrogates oxidative stress by scavenging certain ROIs and increasing intracellular glutathione levels (Aruoma et al, 1989; Burgunder et al, 1989).
  • NAC had little effect on X-ray-induced p56/p53 lyn activity (FIG. 12 A), while this agent completely blocks induction of c-jun and EGR-1 gene expression in irradiated HL- 60 cells (Datta et al, 1992b; 1993).
  • HL-60 cells were treated with 10 ⁇ M herbimycin (H) or 10 ⁇ M genistein (G) for 1 hour, irradiated (200 cGy) and then harvested at 12 hours.
  • Cell lysates were immunoprecipitated with anti-Lyn and the immunoprecipitates were analyzed for phosphorylation of p56/p53 yn and enolase.
  • the tyrosine kinase inhibitors, herbimycin and genistein inhibited X-ray-induced p56/p53 yn activity (FIG. 12B).
  • Src-like proteins may be activated through dephosphorylation by tyrosine phosphatases (Mustalin & Altman, 1990; Cantley et al, 1991; Hartwell & Weinart, 1989) and potentially other mechanisms (Cantley et al, 1991; Hartwell & Weinart, 1989).
  • HL-60 cells were grown in RPMI 1640 medium containing 15% heat-inactivated total bovine serum supplemented with 100 units/ml penicillin, 100 ⁇ g/ml streptomycin and 2mM L-glutamine. Exponentially growing cells were suspended in serum free media 18 h prior to irradiation. Irradiation was performed at room temperature using a Gammacell 1000 (Atomic Energy of Canada, Ottawa) with a 117 Cs source emitting at a fixed dose rate of 13.3 Gy/min as determined by dosimetry.
  • Gammacell 1000 Atomic Energy of Canada, Ottawa
  • Soluble proteins (50 ⁇ g) were separated by electrophoresis in 10% SDS- polyacrylamide gels and then transferred to nitrocellulose paper. The residual binding sites were blocked by incubating the filter in 5% dry milk in PBST (PBS/0.05% Tween 20) for 1 h at room temperature. The filters were then incubated for 1 h with either mouse anti-phosphotyrosine (anti-P-Tyr; 4G10) monoclonal antibody (4G10, UBI, Lake Placid, NY) or a mouse anti-p34 cdc2 monoclonal antibody which is unreactive with other cyclin-dependent kinases (sc- 54; Santa Cruz Biotechnology, Santa Cruz, CA).
  • anti-P-Tyr 4G10 monoclonal antibody
  • sc- 54 mouse anti-p34 cdc2 monoclonal antibody which is unreactive with other cyclin-dependent kinases
  • the blots were incubated with anti-mouse or anti-rabbit IgG peroxidase conjugate (Sigma Chemical Co., St. Louis, Mo).
  • the antigen-antibody complexes were visualized by chemiluminescence (ECL detection system, Amersham, Arlington Heights, IL).
  • Immunoprecipitations were performed with anti-P-Tyr or anti-p34 at 5 ⁇ g/ml cell lysate. Immune complexes were collected with protein A-Sepharose (Pharmacia) and immunoprecipitates were analyzed by 10% SDS-PAGE. After transfer to nitrocellulose and blocking, immunoblot analysis was performed with either anti-p34 or anti-P-Tyr and detected with the appropriate HRP-conjugated second antibody using the ECL system.
  • HL-60 cells were exposed to 200 cGy ionizing radiation and monitored for proteins with increased levels of phosphotyrosine.
  • an anti-P-Tyr antibody in immunoblot analyses, reactivity with a protein of approximately 34 kD was increased at 1 min after ionizing radiation treatment (FIG. 13 A). Similar findings were obtained at 5 and 10 min, while reactivity was decreased at 15 min (FIG. 13 A).
  • the filters were washed and reprobed with an anti-p34 cdc2 antibody.
  • the anti-P-Tyr and anti-p34 signals were superimposable. Moreover, there was little detectable change in p34 cdc2 protein levels following exposure to ionizing radiation (FIG. 13B).
  • Extracts of irradiated cells were subjected to immunoprecipitation with anti- p34 c .
  • the immunoprecipitates were then monitored by immunoblotting with anti-P-Tyr.
  • the signal for p34 cdc2 was increased in irradiated as compared to control cells (FIG. 15A). While this result further supported increased tyrosine phosphorylation of p34 , the filter was washed and reprobed with anti-p34 cdc2 to assay for levels of p34 cdc2 protein.
  • the finding that the anti-p34 cdc2 signals were similar in control and irradiated cells (FIG. 15B) indicated that p34 cdc2 undergoes increased phosphorylation on tyrosine following ionizing radiation exposure.
  • p34 cdc Activation of p34 cdc requires association with cyclin B (Pines & Hunter, 1989; Russel & Nurse, 1987) and certain posttransiational modifications.
  • cyclin B Pines & Hunter, 1989; Russel & Nurse, 1987
  • the p34 cdc2 /cyclin B complex is inactivated by phosphorylation of p34 c2 on tyrosine 15 by Weel (Featherstone & Russell, 1991; Parker et al, 1991; 1992; Gould & Nurse, 1989).
  • Dephosphorylation of p34 cdc2 on Tyr- 15 by the cdc25 gene product is necessary for activation of p34 cdc2 and entry into mitosis (Gould et al, 1989; Enoch & Nurse, 1990).
  • the weel and cdc25 gene products thus determine the timing of entry into mitosis by a series of phosphorylations and dephosphorylations of p34 .
  • Other work in S. pombe has demonstrated that mitotic checkpoints monitor DNA synthesis and the presence of DNA damage (Al-Khodairy & Carr, 1992; Rowley et al, 1992; Lock & Ross, 1990).
  • the DNA damage checkpoint evidently regulates p34 by mechanisms distinct from those induced by the replication checkpoint (Rowley et al, 1992; Lock & Ross, 1990).
  • Other studies have demonstrated that p34 c kinase activity is decreased when CHO cells are exposed to 8 Gy ionizing radiation (Uckun et al, 1992b).
  • the present invention discloses activation of Src-like tyrosine kinases and phosphorylation of tyrosine kinase substrates, such as p34 , as a rapid response to ionizing radiation. Inhibition of the radiation-induced activation of those tyrosine kinases prevents or inhibits substrate phosphorylation.
  • the present invention contemplates a process to alter the response of cell to radiation, the process comprising inhibiting tyrosine kinase activity.
  • the tyrosine kinase is a Src-like tyrosine kinase of the lyn family.
  • the cells were swelled in 2 ml of ice cold hypotonic lysis buffer [1 mM EGTA, 1 mM EDTA, 10 mM ⁇ -glycerophosphate, 2 mM MgCl 2 , 10 mM KCI, 1 mM sodium orthovanadatam, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 10 ⁇ g/ml each of pepstatin, leupeptin and aprotinin] for 30 min and then subjected to Dounce homogenization (15-25 strokes, tight pestle A).
  • ice cold hypotonic lysis buffer [1 mM EGTA, 1 mM EDTA, 10 mM ⁇ -glycerophosphate, 2 mM MgCl 2 , 10 mM KCI, 1 mM sodium orthovanadatam, 1 mM phenylmethylsulfonyl fluoride, 1 mM D
  • the resulting lysate was loaded onto 1.5 ml of buffer A [1 M sucrose in hypotonic lysis buffer containing the protease and phosphatase inhibitors] and centrifuged at 1600g for 15 min to pellet nuclei. The pellet was washed and solubilized in buffer A containing 1% NP-40.
  • Anti-c-Abl immunoprecipitations were performed by adding the K12 anti-Abl antibody and Protein A-Sepharose for 2h at 4°C.
  • Immune complex kinase assays were performed by incubating the resulting protein complexes in kinase buffer [50 mM Tris, pH 7.5, 10 mM MnCl 2 , 1 mM DTT], with either 5 ⁇ g GST-Crk( 120-225) or GST-Crk(120- 212), 2-5 ⁇ Ci[y- 32 P]ATP (New England Nuclear, Boston, MA) for 30 min at 28°C and analyzed by 10% SDS-PAGE and autoradiography.
  • kinase buffer 50 mM Tris, pH 7.5, 10 mM MnCl 2 , 1 mM DTT
  • peptide phosphorylation assays In the peptide phosphorylation assays, immune complexes were incubated in kinase buffer with 20 ⁇ M peptide [EAIYAAPFAKKK; SEQ ID NO:5], 10 ⁇ M ATP and 2-5 ⁇ Ci[ ⁇ - 32 P]ATP for 4 min at 25°C. After incubation, 25 ⁇ l was spotted onto phosphocellulose discs, followed by washing with 1% phosphoric acid and then distilled water. The incorporated [ 19 P] phosphate was determined by scintillation counting.
  • the c-Crk protein contains an N-terminal SH2 domain followed by two SH3 domains.
  • c-Abl binds to the N-terminal SH3 domain of Crk and phosphorylates Tyr221 (Feller et al, 1994; Ren et al, 1994).
  • NIH3T3 fibroblasts were used in similar studies to determine whether activation of c-Abl is detectable in different cell types. The results demonstrate that IR treatment is also associated with stimulation of c-Abl activity in these cells (FIG. 16B).
  • the inventors used a peptide (EAIYAAPFAKKK; SEQ ID NO:5) recently identified as a specific substrate for c-Abl activity (Songyang et al, 1995).
  • Anti-Abl immunoprecipitates from irradiated U-937 cells exhibited maximal (nearly 5-fold) phosphorylation of the peptide substrate at 1 h (FIG. 16D). In contrast, immunoprecipitates prepared with preimmune rabbit serum failed to exhibit IR-induced phosphorylation of the peptide (FIG. 16D).
  • IR IR induces single and double DNA strand breaks
  • the inventors asked whether treatment with other agents that damage DNA is also associated with c-Abl activation.
  • Cisplatinum (CDDP) forms DNA intrastrand crosslinks (Sherman and Lippard, 1987), while mitomycin C (MMC) forms monofunctional and bifunctional DNA lesions (Tomasz et al, 1988).
  • MMC mitomycin C
  • Treatment of NIH3T3 cells with these alkylating agents was associated with an increase in c-Abl activity which was similar to that obtained following IR exposure (FIG. 16E, FIG. 16F).
  • SAP stress-activated protein
  • the inventors stably expressed c-Abl in the Abl-/- cells (designated Abl+).
  • Abl-/- cells were reconstituted with the c-Abl gene by retroviral transduction.
  • the c-Abl (murine type IV) gene was subcloned into the pBaBe-puro retroviral expression vector (Morgenstern and Land, 1990).
  • Helper-free retrovirus was generated and used to infect Abl-/- cells as described (Pear et al, 1993). Puromycin selected cells were used in experiments following removal from drug for 18 h.
  • the level of c-Abl expression in the Abl+ cells was readily detectable, but somewhat lower than that in NIH3T3 cells (FIG. 18 A).
  • IR treatment of the Abl+ cells was associated with stimulation of c-Abl activity (FIG. 18B). More importantly, exposure of the Abl+ cells to IR was associated with increases in SAP kinase activity (FIG. 18C).
  • MMC treatment of the Abl+ cells also resulted in activation of both c-Abl and SAP kinase activities (FIG. 18C). Taken together, these results provide definitive evidence that c-Abl mediates signals in the SAP kinase stress response pathway.
  • DNA damaging agents may be used with the tyrosine kinase inhibitors, as provided by this invention. This includes agents that directly crosslink DNA, agents that intercalate into DNA, and agents that lead to chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
  • Mitomycin C is an extremely toxic antitumor antibiotic that is cell cycle phase-
  • Cisplatin which has also been widely used to treat cancer, with efficacious doses used in clinical applications of 20 mg/m for 5 days every three weeks for a total of three courses.
  • Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.
  • Agents that damage DNA also include compounds that interfere with DNA replication, mitosis, and chromosomal segregation.
  • these compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the " like.
  • adriamycin also known as doxorubicin
  • etoposide verapamil
  • podophyllotoxin and the " like.
  • these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m2 at 21 day intervals for adriamycin, to 35-50 mg/m2 for etoposide, intravenously or double the intravenous dose orally.
  • nucleic acid precursors that disrupt the synthesis and fidelity of nucleic acid precursors, and subunits also lead to DNA damage.
  • nucleic acid precursors have been developed.
  • agents that have undergone extensive testing and are readily available are particularly useful.
  • agents such as 5-fluorouracil (5-FU) are preferentially used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic cells.
  • 5-FU is applicable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg/kg/day being commonly used.
  • the DNA damaging agents or factors defined herein include any chemical compound or treatment method that induces DNA damage when applied to a cell.
  • agents and factors include ionizing radiation and waves that induce DNA damage, such as, ⁇ -irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like.
  • chemotherapeutic agents function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein.
  • Chemotherapeutic agents contemplated to be of use include, e.g., alkylating agents such as mitomycin C, adozelesin, cis-platinum, and nitrogen mustard.
  • the invention also encompasses the use of a combination of one or more DNA damaging agents, whether ionizing radiation-based or actual compounds, with one or more tyrosine kinase inhibitors.
  • Tyrosine protein kinase activities are known to be associated with oncogene products of the retroviral src gene family, and also with several cellular growth factor receptors such as that for epidermal growth factor (EGF).
  • EGF epidermal growth factor
  • Activation of protein tyrosine phosphorylation by p56/p53 yn in the present studies demonstrates that the lyn protein is associated with the cell cycle regulatory protein p34 , contributing to mitotic arrest. If this association is blocked, such as by use of protein tyrosine kinase inhibitors such as genistein or herbimycin A, the cells are unable to arrest in the G 2 phase, forcing cell cycle traverse and expression of potentially lethal damage.
  • DNA damaging agents such as ionizing radiation or alkylating agents with tyrosine kinase inhibitors is a novel approach to enhancing cell killing.
  • Genistein a natural isoflavonoid phytoestrogen, has been reported to exhibit specific inhibitory activity against tyrosine kinases of EGF receptor, pp60 v"src and ppl lO gag" . It has been generally shown to block a number of EGF dependent phenomena, including both receptor autophosphorylation and histone phosphorylation.
  • Herbimycin A has also been shown to inhibit the autophosphorylation of EGF-stimulated receptors in intact cells in a time and dose dependent manner. Herbimycin A both decreases the receptor quantity and the EGF-stimulated receptor kinase activity.
  • tyrosine kinase inhibitors may also be used, for example, those isolated from natural sources.
  • One such compound is erbstatin (Umezawa and Imoto M, 1991; Sugata et al., 1993) and its analogues, e.g., RG 14921 (Hsu et al, 1992).
  • Lavendustin A from Streptomyces griseolavendus (Onoda et al, 1989), which is about 50 times more inhibitory than erbstatin, and analogues thereof, are also contemplated for use as protein-tyrosine kinase inhibitors (Smyth et al. , 1993b).
  • Piceatannol (3,4,3',5'-tetrahydroxy-trans-stilbene; Geahlen and McLaughlin, 1989) and polyhydroxylated stilbene analogues thereof (Thakkar et al, 1993) may also be used.
  • emodin 3-methyl-l,6,8-trihydroxyanthraquinone
  • desmal 8-formyl-2,5,7-trihydroxy-6-methylflavanone
  • the chlorosulfolipid, malhamensilpin A isolated from the cultured chrysophyte Poterioochromonas malhamensis (Chen et al., 1994); flavonoids obtained from Koelreuteria henryi (Abou-Shoer et al, 1991); fetuin, a natural tyrosine kinase inhibitor of the insulin receptor (Rauth et al, 1992).
  • tyrphostins Another group of compounds known to be tyrosine kinase inhibitors are the tyrphostins, which are low molecular weight synthetic inhibitors (Gazit et al, 1989).
  • the tyrphostins AG17, AG18, T23 and T47 have been shown to inhibit pancreatic cancer cell growth in vitro (Gillespie et al, 1993).
  • Tyrphostins have also been shown to have antiproliferative effects on human squamous cell carcinoma in vitro and in vivo (Yoneda et al, 1991).
  • RG-13022 and RG-14620 were found to suppress cancer cell proliferation in vitro and tumor growth in nude mice.
  • Another active tyrphostin is AG879 (Ohmichi et al, 1993).
  • BE-23372M (E)-3-(3,4-dihydroxybenzylidene)-5-(3,4-dihydroxyphenyl)- 2(3H)-furanone, is also a tyrosine kinase inhibitor (Tanaka et al, 1994a). This may be synthesized from 3-(3,4-dimethoxybenzoyl)propionic acid and veratraldehyde or 3,4-diacetoxy-benzaldehyde, as described by Tanaka et al.
  • BE-23372M may also be isolated from the culture broth of a Rhizoctonia solani fungus (strain F23372) using acetone and then purified by solvent extraction and column chromatography (Okabe et al, 1994).
  • tyrosine kinase inhibitors that may be used include 4,5-Dianilinophthalimide, which has, alone, been shown to have in vivo antitumor activity (Buchdunger et al, 1994). Hydroxylated 2-(5'-salicyl)naphthalenes form another group of inhibitors that could be used in the present invention, and may be prepared as described by Smyth et al. (1993 a).
  • a DNA damaging agent and a tyrosine kinase inhibitor in a combined amount effective to kill the cell.
  • the term "in a combined amount effective to kill the cell” means that the amount of the DNA damaging agent and inhibitor are sufficient so that, when combined within the cell, cell death is induced.
  • the combined effective amount of the two agents will preferably be an amount that induces more cell death than the use of either element alone, and even one that induces synergistic cell death in comparison to the effects observed using either agent alone.
  • a number of in vitro parameters may be used to determine the effect produced by the compositions and methods of the present invention. These parameters include, for example, the observation of net cell numbers before and after exposure to the compositions described herein.
  • a “therapeutically effective amount” is an amount of a DNA damaging agent and tyrosine kinase inhibitor that, when administered to an animal in combination, is effective to kill cells within the animal. This is particularly evidenced by the killing of cancer cells within an animal or human subject that has a tumor. "Therapeutically effective combinations” are thus generally combined amounts of DNA damaging agents and tyrosine kinase inhibitors that function to kill more cells than either element alone and that reduce the tumor burden.
  • the present invention generally relates to methods of inhibiting or down- regulating the expression of the c-Abl gene through the preparation and use of antisense constructs that are complementary to distinct regions of the c-Abl gene.
  • the nucleotide sequences of the c-Abl gene are set forth in SEQ ID NO: 1 and SEQ ID NO: 2.
  • a preferred method for cloning c-Abl sequences is through the application of PCR-amplified cloning.
  • oligonucleotide primers complementary to c-Abl as may be determined from the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, that allow the specific amplification of the c-Abl gene sequence.
  • Recombinant clones that inco ⁇ orate c-Abl DNA are readily achieved through the PCR amplification of the distinct coding region using primers incorporating the amplified DNA into a recombinant clone, and selecting recombinant clones that have received the c-Abl DNA bearing clones.
  • the c-Abl DNA containing clones are then purified, and preferably, the cloned DNA is sequenced sufficiently to ensure that it contains the desired sequences.
  • c-Abl DNA is then removed from the vector employed for c-Abl DNA cloning, and used in the construction of appropriate antisense vectors. This will entail, of course, placing the c-Abl DNA in an antisense direction behind an appropriate promoter and positioned so as to bring the expression of the antisense c-Abl under control of the promoter.
  • primers for c-Abl amplification When selecting primers for c-Abl amplification, one typically desires to use primers such that at least about 40-50 and preferably about 100-200 nucleotides of the c-Abl gene are amplified and thereby cloned. It is generally believed that the larger the region of c-Abl gene sequence that is cloned, the better the down ⁇ regulation of the targeted gene.
  • vectors may be a retrovirus, adenovirus, or HSV-1.
  • neoplastic disease Patients exhibiting neoplastic disease are treated with a protein kinase inhibitor, for example genistein, at a concentration of between 1 and lOO ⁇ M, or herbimycin A at a concentration of between about 1 and 1 OO ⁇ M, for 6 hours prior to exposure to a DNA damaging agent.
  • a protein kinase inhibitor for example genistein
  • herbimycin A at a concentration of between about 1 and 1 OO ⁇ M
  • Patients are exposed to ionizing radiation (2 gy/day for up to 35 days), or an approximate a total dosage of 700 gy. 3)
  • patients are treated with a single intravenous dose of mitomycin C at a dose of 20 mg/m .
  • mitomycin C treatment in combination with tyrosine protein kinase inhibitors will be effective against cancer of the stomach, pancreas, oral cavity, breast and head/neck.
  • RNA molecule that comprises a sequence that is complementary to a region of the c-Abl gene and hybridizes to such a region.
  • This antisense RNA molecule may be in combination with a recombinant vector that comprises a nucleic acid sequence capable of expressing the antisense RNA in the cell.
  • the vector is introduced into the cell in a manner that allows expression of the encoded antisense RNA at a level sufficient to inhibit gene expression.
  • patients are treated with a single intravenous dose of mitomycin C at a dose of 20 mg/m 2 .
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • CCCCGTTCCC CTTTCCACGT TGCCATCAGC ATCCTCGGCC TTGGCAGGGG ACCAGCCGTC 3360
  • AACCTGACTC CAAAACCCCT CCGGCGGCAG GTCACCGTGG CCCCTGCCTC GGGCCTCCCC 2640
  • CTGCCCTCCC GCACCTTCCT CCTCCCCGCT CCGTCTCTGT CCTCGAATTT TATCTGTGGA 3780

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Abstract

La présente invention concerne des voies de signalisation reliant les lésions de l'ADN, telles que celles induites par rayonnements ionisants ou des agents alkylants, la phosphorylation par la tyrosine kinase, le gène c-Abl et le produit de ce gène. Plus particulièrement, l'invention a pour objet l'utilisation de molécules antisens pour inhiber, de manière sélective, l'expression du produit du gène c-Abl après l'exposition de cellules à des agents altérant l'ADN, tels que la mitomycine C ou les rayonnements ionisants.
PCT/US1996/013922 1995-08-30 1996-08-30 Procedes et compositions comprenant des agents alterant l'adn et des inhibiteurs ou des activateurs de la tyrosine kinase WO1997008184A1 (fr)

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EP1068870A4 (fr) * 1998-04-03 2003-02-12 Ajinomoto Kk Agents antitumoraux
US6992106B2 (en) 1998-04-03 2006-01-31 Ajinomoto Co., Inc. Anti-tumor composition
US7973076B2 (en) 1998-04-03 2011-07-05 Ajinomoto Co., Inc. Anti-tumor composition
US7655696B2 (en) 1998-04-03 2010-02-02 Ajinomoto Co., Inc. Anti-tumor composition
US7592319B2 (en) 1998-06-30 2009-09-22 Sloan-Kettering Institute For Cancer Research Uses of DNA-PK
JP2002519010A (ja) * 1998-06-30 2002-07-02 スローン − ケタリング・インスティテュート・フォー・キャンサー・リサーチ Ｄｎａ−ｐｋの使用法
EP1090146A4 (fr) * 1998-06-30 2002-04-17 Sloan Kettering Inst Cancer Utilisations d'adn-pk
EP1832663A3 (fr) * 1998-06-30 2007-09-19 Sloan-Kettering Institute For Cancer Research Utilisations de ADN-PK
US7700568B2 (en) 1998-06-30 2010-04-20 Sloan-Kettering Institute For Cancer Research Uses of DNA-PK
US7118862B2 (en) 2001-04-18 2006-10-10 Dana-Farber Cancer Institute, Inc. Induction of apoptosis by cellular stress
US7556935B2 (en) 2001-04-18 2009-07-07 Dana-Farber Cancer Institute, Inc. Induction of apoptosis by cellular stress
US8586291B2 (en) 2001-06-14 2013-11-19 The Regents Of The University Of California Mutations in the Bcr-Abl tyrosine kinase associated with resistance to ST1-571
US8697348B2 (en) 2001-06-14 2014-04-15 The Regent Of The University Of California Mutations in the Bcr-Abl tyrosine kinase associated with resistance to STI-571
US9056924B2 (en) 2001-06-14 2015-06-16 The Regents Of The University Of California Mutations in the BCR-ABL tyrosine kinase associated with resistance to STI-571
US9085644B2 (en) 2001-06-14 2015-07-21 The Regents Of The University Of California Mutations in the Bcr-Abl tyrosine kinase associated with resistance to STI-571
US9994910B2 (en) 2001-06-14 2018-06-12 The Regents Of The University Of California Mutations in the Bcr-Abl tyrosine kinase associated with resistance to STI-571
US7592142B2 (en) 2001-10-05 2009-09-22 Oregon Health And Science University Detection of gleevec resistance
US7416873B2 (en) 2001-10-05 2008-08-26 Oregon Health & Science University Detection of gleevec resistant mutations
US7326534B2 (en) * 2001-10-05 2008-02-05 Oregon Health And Science University Detection of gleevec resistant mutations

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