EP1052998A4 - Compositions et methodes de modulation de liberation de cytokine en reponse a des agents genotoxiques - Google Patents

Compositions et methodes de modulation de liberation de cytokine en reponse a des agents genotoxiques

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Publication number
EP1052998A4
EP1052998A4 EP99904568A EP99904568A EP1052998A4 EP 1052998 A4 EP1052998 A4 EP 1052998A4 EP 99904568 A EP99904568 A EP 99904568A EP 99904568 A EP99904568 A EP 99904568A EP 1052998 A4 EP1052998 A4 EP 1052998A4
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Prior art keywords
dna
protein kinase
agent
genotoxic
kinase inhibitor
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German (de)
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EP1052998A1 (fr
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Daniel B Yarosh
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Applied Genetics Inc
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Applied Genetics Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates to cytokines and genotoxic agents. More particularly, the invention relates to compositions and methods which can control, e.g., modulate, the release of cytokines by cells in response to exposure to one or more genotoxic agents.
  • cytokines and genotoxic agents More particularly, the invention relates to compositions and methods which can control, e.g., modulate, the release of cytokines by cells in response to exposure to one or more genotoxic agents.
  • genotoxic agents are those chemicals or treatments, such as heat or radiation, that cause or induce damage to DNA, either directly or indirectly. Such damage can lead to mutations, the stoppage of cell cycling, and/or cell death.
  • the damage may be to the nucleic acid bases or to the sugar-phosphate backbone, or may be single- or double -stranded breaks in the DNA chain.
  • the mutations, when they occur, are heritable changes in the DNA sequence or DNA modification patterns that lead to heritable changes in cell function.
  • Genotoxic agents are found in the environment as natural components, such as ultraviolet or ionizing radiation, or as natural contaminants in food, such as aflotoxin, or as man-made pollution such as benzo[a]pyrenes in cigarette smoke or industrial emissions. Genotoxic agents are also used for pharmaceutical and health-related purposes. For example, many anti-cancer radiotherapies and chemotherapeutics are -2-
  • ultraviolet light the genotoxic agent to which humans and animals are most often exposed, can be used for beneficial purposes such as tanning.
  • light or ionizing radiation can be combined with light-sensitizing drugs for dermatological and anti-cancer treatments and to sterilize blood.
  • One of the biological responses to genotoxic agents is the cessation of cell cycling in order to allow time for DNA repair to be undertaken. Such repair may or may not be successful depending on such factors as the level of DNA repair enzymes within the cell, the extent and type of DNA damage, and the like.
  • the cessation of cell cycling serves the important function of preventing acute damage to the genetic material that would result from cell division without repair. If the damage is irreparable then the cell invokes the apoptosis response, that is, pathways of programmed cell death.
  • This general process of molecular signaling within a cell leading to cessation of cell cycling and/or apoptosis has been recently reviewed by P. Herrlich, C. Blattner, A. Knebel, K. Bender and H.
  • tumor promotion changes to tissue containing mutated cells, called tumor promotion, is facilitated by the release of cytokines induced by genotoxic agents and leads to the appearance of tumors.
  • cytokines for review see T. Luger and T. Schwarz, "Epidermal cell-derived cytokines," in Skin Immune System, ed. J.D. Bos, CRC Press Inc., Boca Raton, Fla., 1990, pp257-291).
  • the present invention is concerned with these cytokine -based responses to genotoxic agents.
  • cytokines are a large and varied family of proteins that are released by one cell to influence the activity of other cells and/or itself. Cytokine levels are often modulated in response to perturbations of cell functioning and serve to mediate the response and homeostasis of tissues, organ systems, and whole organisms following exposure to genotoxic agents. Cytokines are not known to be released individually. Rather, they are released as a group that produces a menu of characteristic responses to a genotoxic agent.
  • sunburn caused by solar UV simultaneously induces: (a) the expression of interleukin-1 (IL-1) that causes fever, (b) interleukin-6 (IL-6) that mobilizes liver function, (c) tumor necrosis factor ⁇ (TNF ⁇ ) that induces inflammation, contributes to local antigen-specific immune suppression and activates latent viruses, (d) interleukin-10 (IL-10) that induces suppresser T-cells, (e) a transient decrease followed by an increase in intercellular adhesion molecule 1 (ICAM-1) that controls infiltration of lymphocytes, and (f) a decline in interferon ⁇ (IFN ⁇ ) that modulates immune response, as well as many other cytokines.
  • IL-1 interleukin-1
  • IL-6 interleukin-6
  • TNF ⁇ tumor necrosis factor ⁇
  • IFN ⁇ interferon ⁇
  • cytokine -based extracellular signaling is one of the primary objects of the present invention.
  • cytokine release and “cytokine production” are intended to include extracellular signaling both by actual release of a cytokine from a cell and by extracellular display of a cytokine at the cell surface.
  • cytokine production are intended to include extracellular signaling both by actual release of a cytokine from a cell and by extracellular display of a cytokine at the cell surface.
  • These terms are also intended to be interpreted broadly to include any cellular mechanism which results in increased extracellular cytokine signaling, including, without limitation, de novo synthesis, precursor processing, intracellular transport, extracellular discharge, and surface display of a cytokine.
  • protein kinases are activated by events at the cell membrane, distant from the cell nucleus, and activate additional cytoplasmic kinases that culminate in the phosphorylation of systems of gene-activating factors such as AP-1 and NFKB. DNA binding sites for these modified or released gene-activating factors are often found in the promoter regions of genes -6-
  • cytokines coding for cytokines.
  • many of these binding sites are found in the promoter sequence of the TNF ⁇ gene, as described by S. Takashiba, L. Shapira, S. Amar, and T. Van Dyke, in "Cloning and characterization of human TNFa promoter region," Gene, volume 131, pages 307-308, 1993. It has thus been presumed that the binding of these membrane-activated factors to their binding sites in the promoter regions of the cytokine genes is the pathway by which changes at the cell membrane result in changes in expression of cytokines.
  • Tyrrell summarized the field in "Activation of NFKB in human skin fibroblasts by the oxidative stress generated by UVA radiation", Photochemistry and Photobiology, volume 62, pages 463- 468, 1995: "However, UVC radiation-dependent activation of NFKB is evident in enucleated cells and UVB radiation-dependent activation was shown to occur in nuclear-free cell extracts. Thus it appears that, at least with these two agents, the nucleus is not involved in the activation pathway.”
  • UV-induced lesions can generate signals to activate a Rad3/ATM-like protein in mammalian cells.
  • the Herrlich and Rahmsdorf laboratory repeated their 1997 view of the state of the art in 1998 in C. Blattner, Klaus Bender, Peter Herrlich and Hans Rahmsdorf, "Photoproducts in transcriptionally active DNA induce signal transduction to the delayed U.V.-responsive genes for collagenase and metallothionein," Oncogene. volume 16, pages 2827-2834, 2832, 1998: "It is -9-
  • DNA damage may be an initiating event for induction of cytokine gene expression has largely been ignored.
  • control of the mechanism, and thus control of cytokine release was clearly impossible.
  • the present invention provides these missing elements in the art.
  • a particular object of the invention is to provide such methods and compositions where the genotoxic agent is ultraviolet light, the most common genotoxic agent to which humans and animals are exposed, and the control (modulation) involves reducing the release of cytokines in response to the genotoxic agent.
  • genotoxic agents e.g., ultraviolet radiation
  • genotoxic agent is an immunosuppressive agent
  • control involves increasing the release of cytokines in response to the genotoxic agent.
  • kinases modulated to modulate the individual's response (sensitivity) to one or more genotoxic agents.
  • DNA-protein kinases a class of enzymes which recognize changes, e.g., double-stranded breaks, in DNA and phosphorylate other proteins and/or themselves, play a role in the release of cytokines in response to genotoxic agents.
  • one or more DNA-protein kinases are required for transcription and/or translation of cytokine genes after exposure to genotoxic agents.
  • a "DNA-protein kinase” is a member of the family of proteins and protein complexes that respond to changes in DNA structure or conformation by phosphorylating other proteins and/or themselves.
  • the characteristics of this family of enzymes has been reviewed by S. Jin, S. Inoue and D. Weaver in “Functions of the DNA Dependent Protein Kinase," Cancer Surveys, volume 29, pages 221-261, 1997. Heretofore these enzymes have only been implicated in (1) the formation of immunocompetent cells especially those that require gene rearrangements involving double -stranded DNA breaks, (2) repair of double stranded breaks in DNA, and (3) regulation of cell cycling and the apoptosis response following DNA damage. These connections have been reviewed by M. Hoekstra in "Responses to DNA damage and regulation of cell cycle -11-
  • PIK kinase Pl3-kinase-related protein kinase family members properties as sensors for cell cycle regulation.
  • Hosoi et al. in their paper entitled “A phosphotidylinisitol 3-kinase inhibitor wortmannin induces radioresistant DNA synthesis and sensitizes cells to bleomycin and ionizing radiation," International Journal of Cancer, volume 78, pages 642-647, 1998, demonstrated that wortmannin is an inhibitor of DNA-protein kinases but ascribed any effects of wortmannin on cytokines to inhibition of the membrane-bound phosphotidylinisitol 3-kinase (PI-3).
  • PI-3 membrane-bound phosphotidylinisitol 3-kinase
  • Cytokine release was ascribed in the prior art to the pathway of the lower dotted box, where the genotoxic agent affects the cell membrane and/or cell membrane receptors, which activate a lipid and protein kinase cascade, which then leads to cytokine gene expression and extracellular cytokine release.
  • Figure 2 schematically shows the genotoxic response pathways in accordance with the invention.
  • a primary difference between Figure 1 and Figure 2 is that the prior art scheme of Figure 1 did not distinguish between early and late events, i.e., events occurring within 3 hours of exposure to a genotoxic agent (early events) and those occurring 6 hours or more after exposure (late events). This time dimension is an important aspect of the discovery which forms the basis of the present invention.
  • An even more fundamental difference between the pathways of Figure 2 and those of Figure 1 is the fact that Figure 2 includes the production of cytokines as one of the effects arising from damage to DNA by genotoxic agents, such production leading to such biological effects as erythema, antigen-specific immunosuppression, melanogenesis and tanning.
  • This cytokine -production effect of genotoxic agents is shown at the lower right of the right hand box of Figure 2, and as indicated in that box, the effect depends on the action of at least one DNA-protein kinase.
  • DNA-PK and ATM double-stranded breaks activate the DNA-protein kinases known generally as DNA-PK and ATM
  • UV-induced photoproducts in DNA activate the DNA-protein kinase known generally as FRAP.
  • FRAP DNA-protein kinase
  • a DNA-protein kinase is central to the recognition of altered DNA and (2) the phosphorylation of downstream proteins results in cytokine gene expression, i.e., cytokine gene transcription and/or translation.
  • cytokine production in response to genotoxic agents can be controlled by modulating the levels or activity of one or more DNA-protein kinases.
  • Such modulation can be achieved by: (1) inhibiting the activity of the one or more DNA-protein kinases using one or more inhibitors, (2) increasing or decreasing the transcription and/or translation of genes involved in the production of the one or more DNA-protein kinases, and/or (3) enhancing the activity of the one or more DNA-protein kinases by, for example, providing the kinase with enhanced levels of the damaged DNA which it responds to and/or with enhanced levels of the substrate which it phosphorylates.
  • compounds that inhibit DNA-protein kinases are used to block induction of cytokines by genotoxic agents.
  • Other inhibitors of DNA-protein kinases include pyrophosphate, wortmannin, 6-dimethylaminopurine, the pyridone derivative OK-1035, and single-stranded DNA, as described by S.P. Lees- Miller, "The DNA-dependent protein kinase, DNA-PK: 10 years and no ends in sight," Biochemistry and Cell Biology, volume 74, pages 503-512, 1996.
  • the exposure to the genotoxic agent may be unintentional, as in exposure to environmental pollution or exposure to sunlight during day-today activities.
  • the exposure may also be intentional, and the side-effects undesirable, as in the case of intentional sun tanning or cancer radiotherapy or chemotherapy.
  • Induction of cytokines may be unwanted because they are immunosuppressive, inflammatory, activate viruses, cause unwanted pigmentation, keloids, adhesions or scarring, or other primary or side-effects of exposure to genotoxic agents.
  • DNA-protein kinase inhibitors e.g., rapamycin or rapamycin-like compounds
  • rapamycin or rapamycin-like compounds e.g., rapamycin or rapamycin-like compounds
  • DNA-protein kinase inhibitors such as rapamycin or rapamycin-like compounds may also be used in combination with cancer chemotherapy drugs or radiotheraphy procedures to reduce the side-effects associated with such treatments, such as, fever, erythema, nausea, vomiting, headaches, chills and abnormal pigmentation.
  • the DNA-protein kinase inhibitor or inhibitors are preferably administered in close temporal proximity to the exposure to the genotoxic agent, such as sunlight, air pollution, chemotherapy, or ionizing radiation, most preferably 30 minutes to one hour prior to exposure.
  • the invention can also be used to avoid the most deleterious side effects of immunosuppressive therapy in transplantation.
  • Organ transplants have become quite common with the introduction of well- tolerated immunosuppressive compounds such as cyclosporin.
  • immunosuppressive compounds such as cyclosporin.
  • a major side effect of this immunosuppressive therapy has been a rise in skin cancers on sun exposed skin of these patients. See M. Glover, C. Proby and I. Leigh, "Skin cancer in renal transplant patients," Cancer Bulletin, volume 45, pages 220-224, 1993.
  • cyclosporin does not block the release of cytokines following UV-B exposure. See A. Marionnet, Y. Chardonnet, J. Viac and D. Schmitt, "Differences in responses of interleukin-1 and tumor necrosis factor and secretion to cyclosporin-A and ultraviolet B-irradiation by normal and transformed keratinocyte cultures," Experimental Dermatology, volume 6, pages 22-28, 1997. As such, cyclosporin does not have the beneficial effects of a DNA-protein kinase inhibitor, such as rapamycin, in blocking induction of UV-inducible cytokines in sun-exposed skin.
  • a DNA-protein kinase inhibitor such as rapamycin
  • the current invention teaches that genotoxic-exposed organs in general, and sun-exposed skin in particular, should be treated with a DNA- protein kinase inhibitor, such as rapamycin, at a time just prior to or at or following the time of genotoxic exposure, in order to prevent the induction -15-
  • the DNA-protein kinase inhibitor is itself an immunosuppressive agent capable of suppressing rejection of a transplant (such as rapamycin)
  • the inhibitor may be used with or without other immunosuppressive agents and/or other types of drugs or treatments.
  • the regimen of administration and dosage levels of the immunosuppressive agent are selected to take into account both its ability to suppress transplant rejection and its ability to suppress the production of cytokines in response to genotoxic exposure.
  • DNA- protein kinase enhancers compounds that augment the activity of DNA-protein kinases in order to enhance the induction of cytokines following genotoxic treatment.
  • DNA- protein kinase enhancers compounds that augment the activity of DNA-protein kinases in order to enhance the induction of cytokines following genotoxic treatment.
  • some genotoxic treatments are used to induce immunosuppressive responses.
  • psoralen-plus-light is used in skin grafts and psoriasis to induce immunosuppressive cytokines and suppress antigen-specific autoimmune responses.
  • compounds that enhance the activity of DNA-protein kinases can make these immune suppressing therapies more brisk, stronger, and/or more uniform, thus increasing the efficacy of the therapy.
  • compounds that increase DNA-protein kinase activity are used in conjunction with, or in place of, genotoxic agents to enhance the desired immunosuppressive response.
  • one or more compounds that act like UV-irradiated DNA or short pieces of duplex DNA and thus can stimulate DNA-protein kinase activity are applied at the time of the genotoxic treatment, or in place of the genotoxic treatment, to stimulate the release of immunosuppressive cytokines and provide relief from diseases related to autoimmune responses.
  • examples of such compounds include lipid or liposome bound duplex DNA and/or its congeners.
  • Another application of the invention is to identify individuals who need to have the level and/or activity of one or more of their DNA-protein kinases modulated to modulate the individual's response (sensitivity) to one or more genotoxic agents.
  • the DNA-protein kinase activities and/or levels of a patient suffering from a disease in which a genotoxic agent produces an insufficient or excessive cytokine expression are screened to identify the specific DNA-protein kinases responsible for the patient's symptoms.
  • some forms of dermatitis such as atopic dermatitis, lupus erythematosus and porphyria, are caused by immune system over-reaction to environmental UV light or pollution.
  • cell extracts are prepared from tissue samples, and a DNA-protein kinase assay is performed.
  • the assay can, for example, be of the type described below in Example 4, in which antibodies are used to immunoprecipitate the DNA- protein kinase, and then the precipitated DNA-protein kinase is exposed to varying types of DNA damage together with its substrate, such as p53 protein. By measuring the degree of p53 phosphorylation one determines the DNA-protein kinase activity. When compared to normal controls, these studies determine if there is more DNA-protein kinase expressed in the diseased tissue or if the DNA-protein kinase activity is greater in such tissue. This information can then be used in diagnosing disease and selecting therapeutic treatment.
  • assays for DNA-protein kinase levels/activities are required to determine, for example, if a drug is being administered at a level sufficient to inhibit or induce cytokine release.
  • the invention provides effective assays for this purpose in which: (1) a sample of cells is obtained from the subject, (2) a preparation containing DNA-protein -17-
  • kinase(s) is obtained from the sample, (3) the preparation is exposed to DNA damage of the type(s) the kinase (s) is (are) sensitive to together with the appropriate substrate(s) for the kinase(s), and (4) the level of substrate(s) phosphorylation is used as a measure of the level/activity of the kinase (s).
  • Figure 1 is flow diagram showing the understanding in the prior art of the pathways involved in the response of cells to genotoxic agents. As shown therein, DNA damaged by genotoxic agents was thought to activate DNA-protein kinases that then produced phosphorylated substrates, which then triggered intracellular events of cessation of cell cycling and apoptosis.
  • Figure 2 is a flow diagram showing the pathways involved in the response of cells to genotoxic agents in accordance with the present invention.
  • FIG. 3 is a family tree of DNA-protein kinases. The relationships of the known lipid and DNA-protein kinases are shown based on amino acid sequence homologies. The proteins are divided into those with lipid kinase activity and those with protein kinase activity. For each protein the name and the organization of the protein is shown. The thin solid bars indicate regions of non-homology, while the thick solid bars represent regions where small subunits, such as the Ku or FKBP proteins, bind.
  • the open thick bars represent the region of the kinase active site, and the vertically striped region is the carboxy terminus that shows homology among the DNA- protein kinases.
  • Figure 4 is a Western blot of TNF ⁇ protein expression in human keratinocytes.
  • Cells from the human line HaCat were irradiated with 200 J/m 2 UV-B or treated with 1 ⁇ g/ml LPS and incubated for 24 hours at 37°C.
  • Extracts were prepared, electrophoresed in a 15% poly aery lamide gel, transferred to nitrocellulose, and blotted with antibodies against human TNF ⁇ followed by secondary antibodies linked to horseradish peroxidase and exposed using the ECL chemiluminescence system.
  • the lanes are: (1) irradiated; (2) irradiated and treated with 2 ng/ml rapamycin beginning 30 minutes prior to irradiation; (3) treated with LPS; (4) treated with LPS and rapamycin; (5) authentic TNF ⁇ standard.
  • Figure 5 shows the induction of chloramphenicol ace tyltransfe rase
  • CAT DNA-protein kinase inhibitors from the TNF ⁇ promoter following UV exposure in the presence and absence of various DNA-protein kinase inhibitors.
  • XP12BE cells deficient in nucleotide excision repair, were transfected with the TNFcat transgene to form the XPTNF2 cell line that expresses CAT from the TNF ⁇ promoter.
  • the cells were treated with DNA-protein kinase inhibitors beginning 30 minutes prior to irradiation and then exposed to 100 J/m 2 UV-B. After 18 hours, extracts were prepared and assayed for CAT activity using fluorescent chloramphenicol substrate. The reaction products were separated by thin layer chromatography and the fluorescence visualized by UV-A light.
  • the samples are: (1) substrate alone; (2) untreated cells; (3) UV irradiated cells; (4) cells UV irradiated and treated with rapamycin; (5) cells treated with rapamycin alone; (6) untreated cells; (7) UV irradiated cells; (8) cells UV irradiated and treated with wortmannin; (9) untreated cells; (10) UV irradiated cells; (11) cells UV irradiated and treated with staurosporine; (12) LPS treated cells; and (13) cells treated with LPS and rapamycin. -19-
  • FIG. 6 shows induction of chloramphenicol acetyltransferase (CAT) from the TNF ⁇ promoter following LPS treatment.
  • XPTNF2 cells were treated as described in Figure 5, except for exposure to 1 ⁇ g/ml LPS instead of UV.
  • CAT activity was calculated from the amount of protein extract and the amount of product formed in 30 minutes, quantified by computerized image analysis of the fluorescent thin layer chromatography plate.
  • Figure 7 is a Western blot of p70 S6K phosphorylation in human keratinocytes.
  • Cells from the human line HaCat were irradiated with 200 J/m 2 UV-B or treated with 1 ⁇ g/ml LPS and incubated for 24 hours at 37°C. Extracts were prepared, electrophoresed in a 10% polyacrylamide gel, transferred to nitrocellulose and blotted with antibodies against the serine and threonine phosphorylated form of human p70 S6K followed by secondary antibodies linked to horseradish peroxidase and exposed using the ECL chemiluminescence system.
  • the lanes are: (1) unirradiated; (2) irradiated; (3) irradiated and treated with 2 ng/ml rapamycin beginning 30 minutes prior to irradiation; (4) treated with LPS; and (5) treated with LPS and rapamycin.
  • Figure 8 shows FRAP and ATM kinase activity on p53 peptide. Extracts were prepared from the human keratinocyte line HaCat. The extracts were incubated with antibody against FRAP (black bars) or ATM (gray bars) and the bound antibody-kinase product was precipitated with Protein G agarose beads by centrifugation. The bead-bound FRAP kinase was mixed with the FKBP protein and both FRAP and ATM were incubated with a peptide portion of the p53 protein. To this mixture were added various DNAs and inhibitors, as shown.
  • reaction products were diluted eight-fold, added to ELISA plates and developed with antibodies against phosphoserine-modified protein and phosphothreonine- modified protein, and alkaline phosphatase secondary antibodies with nitrophenyl phosphate substrate.
  • Controls included phosphoserine- and phosphothreonine -bovine serum albumin.
  • Phosphorylated proteins were measured by optical density at 405 nm. -20-
  • Figure 9 is a dose response curve showing the level of inhibition of TNFcat expression for different concentrations of rapamycin.
  • the CAT activity was calculated from the fraction of acetylated chloramphenicol as described in Figure 5 and compared to the activity in the absence of rapamycin.
  • DNA-protein kinases play a central role in the release of cytokines by cells in response to genotoxic agents
  • DNA-Protein Kinases DNA-protein kinases were originally recognized by animal and human mutants that lacked activity in one member of this family of enzymes.
  • mice with SCID failed to generate a complete immune system due to failure of immunoglobulin gene rearrangements.
  • SCID severe combined immunodeficiency disease
  • mice were found to have a genetic mutation that inactivated a DNA protein kinase activity essential to a process of immunoglobulin gene rearrangement involving double stranded DNA breaks. In this way, an intermediate in development of immune cells, double-stranded breaks, resembles DNA damage. See the review by S. Jackson, "DNA-dependent protein kinase," in the International Journal of Biochemistry and Cell Biology, volume 29, pages 935-938, 1997. -21-
  • AT ataxia telangiectasia
  • ATM ATM
  • DNA-protein kinases are found in all eucaryotic organisms from yeast to humans. Each cell has more than one type of DNA-protein kinase, drawn from this large family of similar enzymes.
  • the family of amino acid sequence related DNA-protein kinases includes, among others, the original DNA-PKcs and Ku subunits related to the SCID mutation, ATM, ATR, TEL1, MEC1, MEI41, FRAP, TORI, TOR2, and RAD3, as described in Jin, Inoue and Weaver, 1997, supra. This family of DNA-protein kinases and its sequence homology are shown in Figure 3. Research is on-going to identify additional members of the family.
  • DNA-protein kinases are in general comprised of two subunits, one much larger than the other. The smaller subunit is not shown in Figure 3. Both the SCID and AT diseases result from mutations in the gene coding for the smaller subunit. These genetic mutants, as well as inhibitors that block DNA-protein kinase activity, have been used to analyze the functions which DNA-protein kinases perform.
  • the present invention is concerned with compounds that interfere with the activity of one or more DNA-protein kinases, whether by directly -22-
  • DNA-protein kinase inactivating the active site or sites of the DNA-protein kinase, by directly interfering with binding of the DNA-protein kinase to DNA and/or the substrate or substrates which the DNA-protein kinase phosphorylates, by competing with the DNA-protein kinase for binding to DNA and/or the substrate(s), or by interfering with assembly of the subunits of the DNA- protein kinase.
  • DNA-protein kinase inhibitors which can be used in the practice of the invention include rapamycin, pyrophosphate, wortmannin, 6-dimethylaminopurine, OK-1035, staurosporine, and single-stranded DNA. These inhibitors, when combined with suitable vehicles known in the art, can be administered to a subject in various forms, including orally, by injection, and topically. The level of administration will depend on the specific subject, inhibitor, and genotoxic agent, and can be determined in accordance with standard medical practices for therapeutic treatments.
  • a level of inhibitor administration is selected which can be tolerated by the patient and which achieves a reduction in DNA-protein kinase activity sufficient to reduce the expression of cytokines in response to one or more genotoxic agents.
  • Cytokine expression levels can be measured using standard techniques known in the art. Suitable inhibitor dose levels and administration regimens can thus be selected by monitoring cytokine expression in response to genotoxic agents as the dose/regimen is varied. For example, levels of TNF- ⁇ can be monitored after UV exposure for subjects receiving one or more DNA-protein kinase inhibitors.
  • secondary cytokines can have more complex kinetics, e.g., levels of interferon- ⁇ can fall and levels of ICAM-1 can fall and then rise in response to a genotoxic agent, such as UV. If such a secondary cytokine is used to determine doses/regimens for a DNA-protein kinase inhibitor, these more complex kinetics need to be taken into account, e.g., a level of inhibitor may be selected which maintains the level of interferon- ⁇ activity at a predetermined value after genotoxic exposure. -23-
  • the level/regimen of inhibitor administration is selected by assaying for DNA-protein kinase activity, e.g., by assaying for DNA-protein kinase activity using the assays discussed herein.
  • a level/regimen of inhibitor administration is selected which produces a substantial decrease (e.g., a decrease of 10%, preferably 50%) in the activity of one or more DNA- protein kinases.
  • levels of FRAP activity can be monitored and selected so as to reduce the release of cytokines upon exposure to one or more genotoxic agents.
  • UV exposure can be the genotoxic agent, with the level of FRAP being selected to minimize TNF- ⁇ release.
  • the DNA-protein kinase inhibitor can be administered either continuously or, preferably, in connection with the exposure of the patient to the genotoxic agent(s). Most preferably, the DNA-protein kinase inhibitor is administered in advance of exposure to the genotoxic agent(s), e.g., 30 minutes before exposure, with the administration being continued through exposure and for a period thereafter, e.g., 24 hours after exposure. Administration at less than before, during, and after exposure can also be used in the practice of the invention, but is less preferred.
  • Rapamycin A particularly important inhibitor of DNA-protein kinase activity is rapamycin. This inhibitor specifically inhibits the assembly of the large and small subunits of FRAP, as described by E. Brown, P. Beal, C.
  • wortmannin another important inhibitor of DNA-protein kinase activity, blocks FRAP by altering the key amino acids involved in phosphorylation, as described by M. Wymann, G. Bulgarelli-Leva, M. Zvelebil, L. Pirola, B. Vanhaesebroeck, M. Waterfield, and G. Panayotou, "Wortmannin inactivates phosphoinisitide 3-kinase by -24-
  • Rapamycin is used clinically today to suppress the immune system during grafting and transplantation because it blocks the proliferation of traditional immune cells, e.g. T cells, as described in M. Suthanthiran and T Strom, "Immunoregulatory drugs: mechanistic basis for use in organ transplantation", Pediatric Nephrology, volume 11, pages 651-657, 1997. It has not been used in conjunction with short term exposure to genotoxic agents, but rather has been used for extended periods in order to retain transplants. Significantly, patients receiving immunosuppressive therapy, including patients receiving rapamycin, are explicitly directed to avoid genotoxic exposure such as sunlight during the entire extended periods of immunosuppressive therapy which can and usually does continue for years. See M. Glover, C. Proby and I. Leigh, "Skin cancer in renal transplant patients", Cancer Bulletin, volume 45, pages 220-224, 1993.
  • Rapamune® sirolimus/rapamycin
  • rapamycin When delivered by the oral route, typical dose ranges for rapamycin are 2 to 5 mg per day. When delivered by the topical route, typical concentrations are in the range of 0.2% w/v. When delivered by the intravenous route, maximally tolerated doses are in the range of 25 mg per kg of body weight. Clinically, doses for immunosuppressive purposes are in the range of 0.5 to 25 mg per square meter of skin surface per day.
  • a loading dose of rapamycin of approximating 21 to 24 mg per m 2 body surface area is initially delivered intravenously, as described by C.G. Groth, C. Brattstrom, and L. Backman, "New trails in transplantation: how to exploit the potential of sirolimus in clinical -25-
  • tissue levels are insufficient to modulate DNA- protein kinase activities after genotoxic exposure and thus modulate cytokine release in response to such exposure.
  • concentrations higher than 2 ⁇ g/ml (or about 2 nM) are necessary in order to achieve substantial inactivation of DNA-protein kinases, specifically, FRAP kinase.
  • UV induction of TNF ⁇ is insensitive to rapamycin at doses less than 2 ⁇ g/ml.
  • the current practice of administering rapamycin to achieve whole blood levels of 10-30 ⁇ g/ml do not achieve rapamycin levels in the plasma or tissues that are sufficient to inactivate FRAP according to the invention.
  • rapamycin has been determined solely by the ability to prevent rejection of transplanted organs, and not on the doses required to inhibit FRAP activity in genotoxically-exposed non- immune cells.
  • the doses are prescribed for extended, long-term use and are not modulated for exposure to genotoxic agents.
  • rapamycin is used to reduce the release of immunosuppressive cytokines, such as, IL-1, IL-6, IL-10, ICAM-1 and -26-
  • rapamycin is used at dosage levels high enough to achieve such reduction in the release of cytokines, i.e., at levels higher than those used to prevent transplant rejection.
  • rapamycin is used to preserve, instead of suppress, the immune system, specifically, to preserve the immune system after genotoxic exposure.
  • Enhancers of DNA-protein kinase activity include (1) short segments (e.g., segments having a length of less than about 25 thousand base pairs) of double-stranded DNA, i.e., segments of DNA which have ends to which the DNA-protein kinase can bind, (2) damaged double -stranded DNA which can be short or long strands, (3) DNA-protein kinase subunits in situations where such subunits have been depleted, (4) the substrate or substrates which the DNA-protein kinase phosphorylates, and (5) ATP.
  • short segments e.g., segments having a length of less than about 25 thousand base pairs
  • double-stranded DNA i.e., segments of DNA which have ends to which the DNA-protein kinase can bind
  • damaged double -stranded DNA which can be short or long strands
  • DNA-protein kinase subunits in situations where such subunits have been depleted (4) the substrate or substrates which the DNA
  • the DNA-protein kinase enhancers can be administered in the same manner as discussed above in connection with DNA-protein kinase inhibitors except that, since the enhancers are biologicals, vehicles/carriers which preserve the enhancer's biological activity and promote its delivery to target tissue should be used.
  • vehicles/carriers which preserve the enhancer's biological activity and promote its delivery to target tissue should be used.
  • cationic lipids can be used to deliver the DNA into cells
  • topical administration can be performed using a liposome delivery system. See Yarosh, U.S. Patent No. 5,190,762. -27-
  • Doses/regimens for enhancers can similarly be determined using the procedures discussed above with regard to inhibitors.
  • the enhancer will normally be administered prior to the exposure to the genotoxic agent, with the administration being continued during the exposure and for a period of time thereafter.
  • monitoring of cytokine and/or DNA- protein kinase levels can be used to determine appropriate doses and regimens for particular subjects, enhancers, and genotoxic agents.
  • E. Modulation of Cytokine Release As discussed above, in accordance with the invention, cytokine release following genotoxic exposure can be either decreased or increased by inhibiting or enhancing DNA-protein kinase activity. In most cases, a decrease in cytokine release will be desired, the most common example being reducing cytokine release as a result of UV exposure.
  • DNA-protein kinase inhibitors are administered to a patient undergoing such therapy to reduce the side-effects of the therapy.
  • the one or more inhibitors are preferably administered in advance of a therapy session, with the administration being continue for a period after the session, e.g., for a day or so.
  • the DNA-protein kinase inhibitor or inhibitors are delivered in such a manner that they reach the tissues that have suffered the undesired genotoxic damage from the therapy.
  • the DNA-protein kinase inhibitor or inhibitors are typically administered in the same manner as the chemotherapy agent, but they may also be specifically directed to, for example, the gastrointestinal track (oral administration), the scalp (topical administration), and/or the site of injection of the chemotherapy agent (topical or subcutaneous administration) where the side-effects of chemotherapy are most common.
  • the gastrointestinal track oral administration
  • the scalp topical administration
  • the site of injection of the chemotherapy agent topical or subcutaneous administration
  • DNA-protein kinase inhibitors specifically, rapamycin
  • rapamycin are used in such transplant rejection therapy, these uses do not achieve protection from genotoxic exposure.
  • the present invention teaches that the DNA-protein kinase inhibitors should be delivered in different forms and in different ways from the forms and ways in which they have been used in the transplant field.
  • these inhibitors should be given immediately before and for a short time after genotoxic exposure.
  • additional localized application of DNA-protein kinase inhibitors should be used to alleviate the effects of genotoxic exposure.
  • one or more inhibitors should be applied topically to those areas of the skin which have been exposed to the sun light.
  • the doses should be adjusted to achieve inhibition of cytokine release as a result of DNA damage. This means that for short periods of time surrounding the genotoxic exposure the dosage level of one or more DNA- protein kinase inhibitors, such as rapamycin, will be higher than at other times during transplant rejection therapy.
  • one or more transplant rejection drugs which are DNA-protein kinase inhibitors, e.g., rapamycin and its analogs (such as SDZ RAD), are used in conjunction with one or more transplant rejection drugs which are not such inhibitors, e.g., cyclosporin A or ascomycin. Since drugs of these two types generally do not have overlapping toxicities, this combination allows for greater flexibility in achieving the goal of immunosuppression, while at the same time, allowing -29-
  • cytokine production for protection of the patient from cytokine production as a result of exposure to one or more genotoxic agents.
  • the transplant rejection drug or drugs which are DNA-protein kinase inhibitors are used at a level which contributes at least some immunosuppression, but more importantly, are used at a level and/or in a manner which inhibits cytokine release in response to genotoxic agents.
  • the relative amounts of the two types of immunosuppressive drugs are determined for each patient based on the patient's sensitivities to the drugs and on the amount of DNA- protein kinase inhibitor(s) required to achieve a desired level of protection from cytokine release.
  • the invention can be used to enhance cytokine production in response to genotoxic agents.
  • the invention can be used in the treatment of a variety of diseases, including autoimmune diseases, which respond to immunosuppressive genotoxic agents.
  • enhanced cytokine production can be used in the treatment of psoriasis, a skin disease characterized by keratinocyte hyperproliferation and T-cell infiltration into the skin.
  • Two common genotoxic agents used to treat this disease are coal tar and psoralen-plus- light.
  • the genotoxic treatment causes cytokines to be released which suppress the T-cell activation and thus alleviate the disease symptoms.
  • DNA-protein kinase enhancers are used to increase the level of cytokine release in response to the genotoxic agent.
  • one or more DNA-protein kinase enhancers are administered to the patient just before or, preferably, at the time of administration of the immunosuppressive genotoxic agent(s).
  • the use of these enhancers can permit reduction in the amount of genotoxic agent which needs to be administered and, in some cases, the enhancer(s) alone or -30-
  • T-cell activation can be suppressed by the appropriate immunosuppressive cytokines, and the DNA-protein kinase enhancers of the invention serve to augment the formation of those immunosuppressive cytokines upon exposure to genotoxic agents.
  • DNA-protein kinases are a central biological link between genotoxic agents and cytokine release allows those kinases to serve as measurement points (biological endpoints) for (1) sensitivity of individuals to genotoxic agents and (2) the effectiveness of modulators of cytokine release.
  • DNA-protein kinase activity by measuring the level of a DNA-protein kinase activity of an individual, one can determine the level of sensitivity of that individual to genotoxic agent(s) which produce the type of DNA damage to which the DNA-protein kinase responds. For example, to measure the sensitivity to UV, one would measure the level of FRAP activity, while to measure the sensitivity to ionizing radiation (x-rays), one would measure the level of ATM activity. A high measured level of DNA-protein kinase activity indicates an individual who will respond to the genotoxic agent(s) with a high level of cytokine release, and a low measured level indicates an individual who will respond to the agent(s) with low levels of cytokine release.
  • an inhibitor or inhibitors for that DNA-protein kinase can be used as described above to modulate the individual's cytokine response to the -31-
  • genotoxic agent(s) associated with that kinase For individuals with low levels of DNA-protein kinase activity, further diagnostic procedures can be undertaken to determine the source of the low level of activity, e.g., a genetic screening can be performed. Such a DNA-protein kinase assay can provide a basis for undertaking a screening which otherwise would not be conducted.
  • assays for DNA-protein kinase activity can be used to obtain a desired level of the immunosuppressive agent.
  • an assay for DNA-protein kinase activity more directly monitors the biological effectiveness of the compound and any of its metabolites.
  • Assays for levels of DNA-protein kinase activity which can be used in the practice of the invention include those which employ a radiolabeled ATP substrate, e.g., 32 P-ATP, a peptide substrate, gel electrophoresis, and an autoradiographic readout. See, for example, D. Price, J. Grove, V. Calvo, J. Avruch and B. Bierer, "Rapamycin-Induced Inhibition of the 70-Kilodalton S6 Protein Kinase," Science, volume 257, pages 973-977, 1992.
  • Example 4 A preferred assay which avoids the use of radiolabeled reactants and which can simultaneously process many more samples than the Price et al. procedure is illustrated in Example 4 below.
  • DNA-protein kinase level of a subject comprises the steps of: (1) -32-
  • a sample of cells is obtained from the subject, (2) a preparation containing DNA-protein kinase(s) is obtained from the sample using anti-DNA-protein kinase antibodies, (3) the preparation is exposed to DNA damage of the type(s) the kinase(s) is (are) sensitive to together with the appropriate substrate(s) for the kinase(s), and (4) the level of phosphorylation of the substrate(s) is used as a measure of the level/activity of the kinase(s). The level of substrate phosphorylation is quantified using standard ELISA methods with antibodies specific to the substrate when phosphorylated by the DNA-protein kinase under assay.
  • the specificity and sensitivity of the assay can be modified by changing or combining the types and or concentrations of the anti-DNA- protein kinase antibodies and/or the substrates used in performing the assay.
  • levels of DNA-protein kinase activity for a plurality of kinases can be determined simultaneously by forming mixtures of anti- DNA-protein kinase antibodies and substrates in steps (2) and (3), respectively.
  • the sensitivity of the assay can be modified by increasing antibody and/or substrate concentrations and/or reaction times.
  • the DNA-protein kinase inhibitors of the invention may be formulated alone with suitable vehicles or they can be combined with each other and/or with other pharmaceutical ingredients, e.g., genoprotective agents which are not DNA-protein kinase inhibitors.
  • suitable vehicles e.g., genoprotective agents which are not DNA-protein kinase inhibitors.
  • genoprotective agents which are not DNA-protein kinase inhibitors.
  • One such example is a combination of rapamycin with one or more sunscreens, e.g., titanium dioxide, and/or one or more DNA repair enzyme(s), e.g., T4 endonuclease V, in topical formulations, to be used prior to, during or after exposure to one or more genotoxic agents, such as solar UV.
  • sunscreens e.g., titanium dioxide
  • DNA repair enzyme(s) e.g., T4 endonuclease V
  • DNA-protein kinase inhibitors in such formulations are selected as described above, e.g., using a DNA-protein kinase activity assay. -33-
  • the levels of the other active ingredients in the formulation will generally correspond to the levels of the ingredient when used by itself.
  • the DNA-protein kinase inhibitors of the invention e.g., wortmannin
  • a genotoxic chemotherapy agent e.g., mitomycin C
  • DNA-protein kinase enhancers of the invention may also be formulated alone with suitable vehicles or they can be combined with each other and or with pharmaceutical agents which are not DNA-protein kinase enhancers, e.g., with genotoxic agents.
  • suitable vehicles e.g., a combination of damaged DNA, psoralen, and a suitable vehicle for application to the skin of a psoriasis patient.
  • the human immortalized HaCat cell line was from Dr. Jonathan Garlick, State University of New York at Stony Brook.
  • the XPTNF2 cell line was prepared by transfection of the XP group A SV-40-transformed fibroblast cell line XP12BE with PCATTNF, as described by J. Kibitel, V. Hejmadi, L. Alas, A. O'Connor, B. Sutherland and D. Yarosh in "UV-DNA Damage in mouse and human cells induces the expression of tumor necrosis factor ⁇ ", Photochemistry and Photobiology, volume 67, pages 541-546, 1998.
  • This cell line expresses the chloramphenicol acetyltransferase gene from the mouse TNF ⁇ promoter.
  • Non-transformed human keratinocytes were purchased from Clonetics Corporation, San -34-
  • Rapamycin, wortmannin, staurosporine and lipopolysaccharide (LPS) were from Sigma Chemical Company.
  • the drugs were prepared at 1, 000-fold concentration and diluted into media just before use. Rapamycin-, wortmannin- or staurosporine- treated cells were pretreated for 30 minutes before UV irradiation, and for 18 hours after irradiation. For LPS treatment, the LPS was added to the cells for one hour at 37°C, and then the cells were refed with fresh media for 18 hours.
  • the UV was delivered from a Westinghouse FS-40 unfiltered sunlamp at 3.2 J/m 2 /sec.
  • the media was removed, and the cells were irradiated and then refed with the same media for 18 hours.
  • Normal human epidermal keratinocytes were grown to 90% confluence in 10-cm plates, and then treated with drugs and genotoxic agents. After incubation, the cells were collected with a standard running buffer containing sodium dodecyl sulfate (SDS), sonicated for 2 seconds with a Heat Systems Ultrasonic Sonicator at 70% power, boiled in water for 5 minutes and stored at -70°C.
  • SDS sodium dodecyl sulfate
  • the monoclonal antibody against human TNF ⁇ was from Boehringer Mannheim Biochemicals. The blots were then washed and incubated with goat anti-mouse IgG linked to biotin and then avidin-horse radish peroxidase, and then developed with the ECL kit as above.
  • the CAT assays were performed as described in Kibitel et al., 1998. Briefly, the XPTNF2 cells were treated with drugs and genotoxic agents and incubated for 18-24 hours. Extracts were prepared by three rounds of freezing and thawing and centrifugation, and 50 ⁇ g of each extract was mixed with 5 nmoles of BODIPY-chlorampenicol (Molecular Probes, Eugene, Oregon) and 0.5mM acetyl-coA (Sigma Chemical Company). After 30 minutes incubation at 37°C, the reaction products were extracted with cold ethyl acetate, and analyzed by thin layer chromatography. The fluorescent substrate and the acetylation products were visualized by UV-A, and the digitized image was analyzed by computerized image analysis to calculate the fraction of acetylated chloramphenicol and thus the CAT activity.
  • BODIPY-chlorampenicol Molecular Probes, Eugene, Oregon
  • acetyl-coA
  • HaCat cells were grown in 10-cm dishes to near confluence. They were then sonicated for 10 sec with the Heat Systems Ultrasonicator microtip at 70% power in TGN Buffer (50 mM Tris, pH 7.5, 50 mM glycerophosphate, -36-
  • the agarose beads were collected by centrifugation, washed with TGN buffer, then high salt buffer (100 mM Tris, pH 7.4, 500 mM LiCl), then PK- buffer (25 mM HEPES-KOH, pH 7.9, 50 mM KC1, 10 mM MgCl 2 , 1 mM DTT, 0.5 ⁇ l l Sigma P8340 protease inhibitor cocktail, 2 nM microclystin LR, 200 ⁇ M ATP), and resuspended in 50 ⁇ l PK- buffer.
  • high salt buffer 100 mM Tris, pH 7.4, 500 mM LiCl
  • PK- buffer 25 mM HEPES-KOH, pH 7.9, 50 mM KC1, 10 mM MgCl 2 , 1 mM DTT, 0.5 ⁇ l l Sigma P8340 protease inhibitor cocktail, 2 nM microclystin LR, 200 ⁇ M ATP
  • Modifiers of the reaction included 0.5 ⁇ g ⁇ -DNA or ⁇ -DNA irradiated with 250 J/m 2 UV-C from a G15T germicidal lamp, and rapamycin at 40 ng/ml.
  • 2.6 ⁇ g FKBP Sigma Chemical Company
  • 50 ⁇ g p53 peptide amino acids 1-393, Santa Cruz Biotechnology
  • 1 nmol ATP were added to all the DNA protein kinase reactions. After 2 hours incubation at 30°C, the reaction products were separated from the agarose beads by centrifugation.
  • reaction products were diluted 8-fold into standard ELISA Coating Buffer (1.59 g/L Na 2 CO 3 , 2.93 g/L NaHCO 3 , 0.1 g/L thimerisol), and 200 ⁇ l were placed in wells of a Immulon 2HB 96-well ELISA plate (Dynex
  • TBS/NonI 25 ml/L 2M Tris pH 8, 30 ml L 5 M NaCl, 0.1% Nonidet P-40
  • the wells were washed and incubated with 100 ⁇ l of a mixture of 10 ⁇ l avadin-alkaline phosphatase (Sigma Chemical Company) in 10 ml TBS/NonI for 60 minutes at room temperature.
  • the plates were washed, developed with phospho- nitrophenylphosphate in 0.1 M diethanolamine pH 10, and read at 405 n m by a microtiter plate reader.
  • Example 1 This example demonstrates that TNF ⁇ is expressed by normal human keratinocytes after UV exposure and that this expression is inhibited by rapamycin.
  • UV induces TNF ⁇ , as can be seen in lane 1.
  • Rapamycin at 2 ng/ml inhibited the expression of TNF ⁇ , as seen by the great reduction in this band in lane 2.
  • lipopolysaccharide (LPS) induces TNF ⁇ without DNA damage by binding to the cell surface membrane receptor CD 14.
  • LPS lipopolysaccharide
  • Example 2 This example demonstrates that the induction of TNF ⁇ mRNA expression by UV is inhibited by rapamycin.
  • Substrate alone is shown in lane 1 and background levels of acetylation by untreated cell extracts is shown in lanes 2, 6 and 9.
  • CAT expression from the TNF ⁇ promoter is reflected by increasing acetylation of the fluorescent chloramphenicol substrate, resulting in faster migrating species (from bottom to top) in the thin layer chromatography assay.
  • TNF ⁇ is induced by genotoxic treatments, such as UV (lanes 3,7 and 10), and by non-genotoxic treatments, such as treatment with LPS (lane 12). UV induction of TNF ⁇ is inhibited by rapamycin (lane 5), demonstrating that the DNA-protein kinase FRAP is required for transduction of the signal of DNA damage into expression of the cytokine TNF ⁇ gene.
  • TNF ⁇ induction is also inhibited by wortmannin at 500 nM, a dose that inhibits DNA-protein kinases (lane 8), and also by staurosporine at 200 nM (lane 11), a dose that specifically inhibits serine phosphorylation, a characteristic type of phosphorylation by DNA-protein kinases.
  • Example 3 As known in the art, downstream in the pathway following activation of the FRAP kinase is phosphorylation of the p70S6K kinase, which phosphorylates ribosomal proteins and alters translation of gene transcripts.
  • One measure of UV-specific activation of the FRAP kinase is thus phosphorylation of p70S6K. This example uses this measure to demonstrate such activation.
  • Human keratinocytes were pre-treated with 2 ng/ml rapamycin, then treated with UV-irradiation or LPS and extracted as described above (see Example 1 and Materials and Methods).
  • the extracts were electrophoresed in a 10% poly aery lamide gel and then probed with antibodies specific for the serine/threonine phosphorylated forms of p70S6K in a Western blot.
  • the polyacrylamide gel was stained with Coomassie blue to identify total protein loaded in each lane.
  • UV irradiation increased phosphorylation of p70 S6K (compare lanes 1 and 2) and this phosphorylation was blocked by pre-treatment with rapamycin (lane 3).
  • the LPS induced phosphorylation of p70S6K (lane 4) was comparatively insensitive to rapamycin (compare lanes 4 and 5).
  • the loading control bands shown at the bottom of this figure demonstrate the equivalent loading of protein in the gel.
  • Example 4 This example demonstrates the direct activation of DNA-protein kinase activity by damaged or broken DNA. -40-
  • ATM or FRAP in extracts of the human keratinocyte cell line HaCat were immunoprecipitated by incubation with (1) antibodies against either ATM or FRAP and (2) immunoprecipitating anti-antibodies linked to agarose beads.
  • the bound ATM or FRAP was collected by centrifugation, and then mixed with a phosphorylation substrate polypeptide derived from the p53 protein, and in the case of FRAP, with its small subunit protein FKBP.
  • bacteriophage ⁇ DNA, UV-irradiated DNA, or rapamycin were added to the reactions. Adenosine triphosphate was then added and the reactants incubated for 2 hours at 30°C.
  • reaction products were then bound to an ELISA plate and probed with antibodies specific for phosphoserine and phosphothreonine.
  • the binding of these antibodies was detected by (1) secondary antibodies linked to alkaline phosphatase and (2) nitrophenyl phosphate substrate.
  • the resulting yellow color was measured by optical density at 405 nm using a multiwell plate reader.
  • Example 5 This example shows that rapamycin's ability to reduce induction of TNF ⁇ by UV is dose dependent.
  • XPTNF2 cells irradiated with 100 J/m 2 UV-B to induce expression of the CAT gene from the TNF ⁇ promoter were incubated with increasing -41-

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Abstract

L'invention concerne un mécanisme inconnu auparavant de libération de cytokine en réponse à des agents génotoxiques, ainsi que des procédés et techniques thérapeutiques et diagnostiques sur la base de ce mécanisme. Le mécanisme consiste en la reconnaissance d'ADN endommagé par des protéines d'ADN kinases et la phosphorylation ultérieure de substrats entraînant une libération de cytokine. L'invention concerne également des méthodes de modulation de l'activité de protéines d'ADN kinases et donc de la libération de cytokines en réponse à des agents génotoxiques. L'invention concerne enfin des dosages de l'activité de protéines d'ADN kinases, pouvant s'utiliser pour contrôler une telle modulation.
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Title
BASU S ET AL: "The DNA-dependent protein kinase participates in the activation of NF kappa B following DNA damage.", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. UNITED STATES 9 JUN 1998, vol. 247, no. 1, 9 June 1998 (1998-06-09), pages 79 - 83, XP001097650, ISSN: 0006-291X *
HOSOI Y ET AL: "A phosphatidylinositol 3-kinase inhibitor wortmannin induces radioresistant DNA synthesis and sensitizes cells to bleomycin and ionizing radiation.", INTERNATIONAL JOURNAL OF CANCER. JOURNAL INTERNATIONAL DU CANCER. UNITED STATES 23 NOV 1998, vol. 78, no. 5, 23 November 1998 (1998-11-23), pages 642 - 647, XP002919460, ISSN: 0020-7136 *
MOLNAR-KIMBER K L ET AL: "Comparison of effects of sirolimus on cytokine dependent and cytokine independent proliferation.", INFLAMMATION RESEARCH: OFFICIAL JOURNAL OF THE EUROPEAN HISTAMINE RESEARCH SOCIETY... [ET AL.]. SWITZERLAND AUG 1995, vol. 44 Suppl 2, August 1995 (1995-08-01), pages S189 - S190, XP001070575, ISSN: 1023-3830 *
ROSENZWEIG K E ET AL: "Radiosensitization of human tumor cells by the phosphatidylinositol3-kinase inhibitors wortmannin and LY294002 correlates with inhibition of DNA-dependent protein kinase and prolonged G2-M delay.", CLINICAL CANCER RESEARCH: AN OFFICIAL JOURNAL OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH. UNITED STATES JUL 1997, vol. 3, no. 7, July 1997 (1997-07-01), pages 1149 - 1156, XP001069065, ISSN: 1078-0432 *
See also references of WO9939720A1 *

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