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WO1997004761A1 - Procede et compositions pour traiter des proliferations anormales de cellules - Google Patents

Procede et compositions pour traiter des proliferations anormales de cellules Download PDF

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WO1997004761A1
WO1997004761A1 PCT/US1996/012381 US9612381W WO9704761A1 WO 1997004761 A1 WO1997004761 A1 WO 1997004761A1 US 9612381 W US9612381 W US 9612381W WO 9704761 A1 WO9704761 A1 WO 9704761A1
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cells
protein kinase
cell
activated
ras
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PCT/US1996/012381
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WO1997004761A9 (fr
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Douglas V. Faller
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Trustees Of Boston University
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Publication of WO1997004761A9 publication Critical patent/WO1997004761A9/fr

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    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • 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
    • A61K31/12—Ketones
    • 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
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • 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
    • A61K31/13—Amines
    • A61K31/131—Amines acyclic
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/351—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom not condensed with another ring
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin

Definitions

  • the invention relates to the treatment of proliferative disorders by the administration of agents that induce apoptosis in cells expressing activated oncogenes.
  • the invention also relates to these agents, to methods of screening for new agents effective against specific disorders and to methods for staging neoplastic disorders.
  • Neoplastic cells isolated from animals are frequently clonal in origin. Many of these cells have descended from a single ancestral cell, the founder cell, which lost the ability to regulate its own proliferation. Proliferation in defiance of genetic restraints leads to invasion and colonization of territories reserved for non-neoplastic or otherwise normal cells.
  • Neoplasms which proliferate out of control and grow as single masses are benign. Benign tumors often can be successfully treated by surgical removal of the tumor mass. Neoplastic cells that invade other tissue are malignant. Such cells enter the bloodstream or lymphatic systems and form metastases which are particularly difficult to treat. In fact, many tumors which initially appear benign may already have formed as yet undetectable metastases. Invasion of proximal or distal tissues is characteristic of malignancies, however, uncontrolled proliferation is common to all neoplasms and other proliferative disorders.
  • Normal cells are genetically programmed to self destruct through apoptosis (E. White, Genes & Development 7:2277-84, 1993). Uncontrolled proliferation typically indicates that genetic controls over the cell cycle have been reprogrammed (G.T. Williams et al., Cell 74:777-79, 1993). For example, uncontrolled proliferation can be induced in vitro by the over expression, mutation or activation of a single gene. Genes which have such pleiotropic effects are referred to as dominant oncogenes and, in fact, many spontaneous tumors isolated from humans have been found to contain active dominant oncogenes. Nucleic acid analysis of many of these tumor cells has shown that the most common of the activated oncogenes is ras.
  • Ras was first discovered as the oncogene of a rat sarcoma virus from which its name was derived, ras is also the most highly conserved of all known oncogenes, but is actually only one member of a family of r ⁇ s-related oncogenes. This family includes the functional ras genes, Ha-ras, Ki-r ⁇ s and N-r ⁇ s, and two pseudo ras genes, which are located on human chromosome 6 and the human X chromosome.
  • Ras genes are functionally conserved across phylogenetic lines in addition to being conserved at the protein sequence level.
  • the ras gene or a ras gene homolog has been found in nearly every eukaryotic organism studied including human, mouse, rat, chicken, fruit-fly, mollusk, slime mold, gold fish and yeast.
  • Kas protein has been found in all tissue and cell lineages as well including in the developing embryo.
  • ras activity is involved with the process of growth regulation (S.A. Moodie, Trends. Genet. 10:44-48, 1994). Over expression or mutation of certain dominant oncogenes such as ras causes expressing cells to divide uncontrollably (M.S. Marshall, Trends. Biochem. Sci. 18:250-54, 1993). In vitro studies have shown that ras expression is elevated 5 to 7 fold during the S-phase in tissue culture cells (H.R.L. Bourne et al., Nature 348:678-79, 1990). However, unlike some oncogenes, ras expression does not always correlate with cell division.
  • non-dividing tissue such as brain
  • Mutant ras genes are able to transform mammalian cells in culture. Microinjection of the oncogenic form of the human Ha-ras protein resulted in the rapid proliferation of formerly quiescent cells (J.R. Feramisco et al., Cell 38:109-17, 1984). Further, mammalian ras can support the growth of mutant yeast cells. Studies have also shown that ras proto-oncogene function is required for serum stimulated cell growth of NIH-3T3 cells (L.S. Mulcahy et al., Nature 313:241-43, 1985). Activated ras has also been found in tumors induced by chemical and physical agents.
  • ras activity has been found to be essential for T cell antigen- receptor signal transduction (C.D. Baldari et al., J. Biol. Chem. 268:2693-98, 1993). Ras protein has also been implicated in mitogenic signaling pathways of Swiss mice 3T3 fibroblasts (R.B.S. Roden et al. , Cell Growth Differ.
  • p21ray activity is thought necessary for mediating cell cycle progression, including G 0 to G,, and G, or G 2 to M transitions and ras belongs to a family of G proteins important for the intracellular transmission of cell surface receptor signals (P.H. Howe et al., J. Biol. Chem. 268:21448-52, 1993). High levels of activated p2lras in the absence of cooperating oncogene expression, however, can result in G 2 cell cycle arrest (P. Hirakawa et al. , Proc. Natl. Acad. Sci. USA 85:1519-23, 1988).
  • the proto-oncogene bcl-2 product can protect cells from apoptosis induced by certain biological or chemical reagents (M.I. Walton et al., Cancer Res. 53:1853-61, 1993).
  • Bcl-2 functions in an anti-oxidant pathway to prevent apoptosis (D.M. Hockenbery et al., Cell 75:241-51, 1993).
  • Bcl-2 also blocks chemotherapy-induced apoptosis in human leukemia cell line (T. Miyashita et al., Blood 81:151-57, 1993).
  • Bcl-2 protein has been shown to associate with a human Ras-related protein, R-ras or p23 (M.J. Fernandez-Sarabia et al., Nature 366:274-75, 1993).
  • Channel-linked receptors are neurotransmitter-gated ion channels that open and close briefly in response to neurotransmitter binding and thereby transiently alter the electrical excitability of the cell.
  • Catalytic receptors are mainly tyrosine-specific protein kinases that directly phosphorylate target cell proteins on tyrosine residues.
  • G-protein-linked receptors individually activate or inactivate plasma-membrane-bound enzymes or ion channels through GTP binding proteins, G proteins.
  • G protein- linked receptors activates or inactivates adenylate cyclase while others activate a phosphoinositide specific phospholipase C which hydrolizes phosphatidylinositol biophosphate (PIP 2 ) to generate inositol triphosphate and diacylglycerol.
  • Diacylglycerol activates protein kinase C which in turn phosphorylates various cell proteins. Protein kinase C is one of several serine- threonine kinases implicated in cell proliferation and differentiation.
  • protein kinase C When activated by diacylglycerol, protein kinase C transfers the terminal phosphate group from adenosine triphosphate to specific serine or threonine residues on target proteins. In many cells, protein kinase C is thought to phosphorylate and activate the plasma membrane sodium-hydrogen ion exchanger which controls intracellular pH. The highest concentration of protein kinase C is found in the brain, where protein kinase C phosphorylates ion channels in nerve cells and alters their excitability threshold. In some cells, activation of protein kinase C increases the transcription of specific genes. The promoter of at least some of these genes contain a common transcription enhancer sequence, TGANTCA, that is recognized by the gene regulatory protein, jun, whose activity is stimulated by protein kinase C activity.
  • TGANTCA common transcription enhancer sequence
  • the present invention overcomes the problems and disadvantages associated with current strategies and designs and provides new methods and compositions for the treatment and prevention of neoplastic diseases and other disorders of cell proliferation.
  • compositions comprising agents that induce apoptosis of proliferating cells by reducing the activity of a cellular protein kinase.
  • Effective agents include calphostin C, chelerythrine chloride, H7, hexdecyl-methylglycerol, hexadecyl- acetylglycerol, hypericin, K252, RO318220, phloretin, sphingosine, bryostatin and staurosporin.
  • Aberrantly proliferating cells which may be effectively treated include, for example, malignant human neoplastic cells or virally infected cells.
  • Compositions may also contain an agent that regulates intracellular calcium concentrations.
  • Another embodiment of the invention is directed to pharmaceutical compositions comprising agents that regulate intracellular calcium concentrations.
  • Effective pharmaceuticals include agents such as calcium ionophores. Calcium ionophores increase intracellular calcium concentrations by increasing calcium ion transport across cell membranes and, in proliferating cells, induce apoptosis. Effective calcium ionophores include thapsigargin, ionomycin and A23187.
  • Another embodiment of the invention is directed to methods for inducing apoptosis of proliferating cells. Agents that regulate protein kinase activity are administered to aberrantly proliferating cells that express an activated oncogene. Activated oncogenes may be endogenous or introduced by transfection with oncogenic sequences. Suppression of intracellular protein kinase activity induces cells to undergo apoptosis.
  • Another embodiment of the invention is directed to methods for inducing apoptosis of proliferating cells.
  • Agents that regulate intracellular calcium concentrations are administered to cells to increase intracellular calcium concentration. Increased intracellular calcium concentration induces cells expressing activated oncogenes to undergo apoptosis.
  • Another embodiment of the invention is directed to methods for treating disorders comprising the step of administering a therapeutically effective amount of a pharmaceutical agent that induces apoptosis of aberrantly proliferating cells.
  • Disorders which can be treated include neoplasms and infections wherein the aberrant cells express activated oncogenes.
  • a therapeutically effective amount of the agent is that amount which reduces intracellular protein kinase activity or increases calcium concentrations sufficiently to induce apoptosis.
  • Figure 1 Relative cell survival based upon DNA fragmentation of untransformed, activated ras transformed, bcl-2 transformed, and activated ras and bcl-2 transformed Jurkat cell lines.
  • Figure 2 Relative cell survival based upon DNA fragmentation of untransformed, activated ras transformed, bcl-2 transformed, and activated ras and bcl-2 transformed Jurkat cell lines after 24 hours of treatment with PMA.
  • Figure 3 Percent of DNA fragmentation of untransformed, activated ras transformed, bcl-2 transformed, and activated ras and bcl-2 transformed Jurkat cell lines at 48 hours after 24 hours of treatment with PMA.
  • Figure 4 Flow cytometric DNA profiles of untransformed, activated ras transformed, bcl-2 transformed, and activated ras and bcl-2 transformed Jurkat cell lines with and without 24 hours of PKC treatment.
  • Figure 5 Metabolic labeling and co-immunoprecipitation of p2lras and p26bcl-2.
  • Figure 6 Immunoprecipitation and detection of p21r ⁇ s and p26bcl-2 protein using monoclonal antibodies.
  • FIG. 7 RNA blot analysis of ras RNA levels of specific subclones of activate ras transformed Jurkat cells (PHI) following down regulation of PKC.
  • Figure 8 DNA fragmentation analysis of specific subclones of activate ras transformed Jurkat cells (PHI) following down regulation of
  • FIG. 12 DNA fragmentation analysis of Jurkat cell and activate ras transformed Jurkat cells (PHI) in calcium ionophore with or without the addition of calcium chelator.
  • Figure 13 DNA fragmentation of activated ras transformed Jurkat cells (PHI) during serum deprivation.
  • the present invention is directed to agents, compositions and methods for the treatment of cell proliferative disorders and also to methods for typing and for screening for additional agents which may be useful for the treatment of such disorders.
  • cancer One of the most difficult disorders to treat effectively and with consistency is cancer. Although thousands of chemical agents have been tested, few have proven to be specifically effective against neoplasia and there is still no broad-based therapy available. It has been discovered, quite surprisingly, that agents effective at reducing cellular protein kinase activity, and specifically protein kinase C (PKC) activity, selectively induce cells that express activated oncogenes to undergo apoptosis. Induction of apoptosis causes neoplastic cells to differentiate, loose their aggressive nature and die. Cells that express an activated oncogene can be selectively induced to undergo apoptosis upon suppression of cellular protein kinase activity. Normal, non-neoplastic cells and neoplastic cells that do not express activated oncogenes are unaffected by treatment.
  • PLC protein kinase C
  • agents that induce apoptosis of cells transformed by activated ras or other activated oncogenes can be effectively used in a wide variety of proliferative disorders as prophylaxis or therapy.
  • agents which increase intracellular calcium concentrations also induce aberrantly proliferating cells to undergo apoptosis.
  • Calcium regulatory agents may also be involved in the regulation of protein kinase activity as most of these protein kinases are calcium dependant.
  • One embodiment of the invention is directed to pharmaceutical agents that selectively induce apoptosis when administered to aberrantly proliferating cells that express an activated oncogene.
  • these agents represent a broadly applicable new approach to treating a wide variety of mammalian disorders including cancer, infection-induced malignancies, hype ⁇ lasia, hypertrophy and most any undesirable cell proliferation.
  • Agents which regulate protein kinase activity either by increasing or decreasing protein kinase activity include phorbols, acylglycerols and nucleic acids. Phorbol esters, for example, first stimulate and subsequently suppress protein kinase activity.
  • the phorbol may be a phorbol ester such as phorbol myristyl acetate, phorbol butyrate, phorbol dibutyrate, deoxyphorbol phenylacetate, tetradecanoyl phorbol acetate, phorbol diacetate, phorbol didecanoate, alpha-phorbol didecanoate and deoxyphorbol isobutyrate.
  • agents which suppress protein kinase activity, and specifically PKC activity include, for example, staurosporine, sphingosine, hypericin, bryostatin, K252, phloretin, RO318220, H7, calphostin C, cheletytfirine chloride and acylglycerol, and derivatives and functional equivalents of these agents such as monoacylglycerol, diacylglycerol, l-oleoyl-2-acetylglycerol, hexadecyl- methylglycerol and hexadecyl-acetylglycerol, and diaoctanoylglycerol.
  • Agents are preferably safe and effective at useful dosages. Although side effects may occur, safe compositions are those wherein the benefits achieved from their use outweigh disadvantages attributable to adverse side effects. Unwanted side effects include nausea, vomiting, hepatic or renal damage or failure, hypersensitivity, allergic reactions, cardiovascular problems, gastrointestinal disturbances, seizures and other central nervous system difficulties, fever, bleeding or hemorrhaging, serum abnormalities and respiratory difficulties.
  • agents are biologically safe at effective doses whereas others may be chemically modified to remove or to add chemical moieties without hindering efficacy. Most agents are safe at effective doses or could be effectively administered at doses which are well-below toxic or mutagenic levels. Transient administration may also prevent or reduce negative consequences attributed to side effects.
  • Therapeutically effective pharmaceuticals may be created by modifying effective agents so that after introduction into the patient, these agents metabolize into active forms which have the desired effect on the patient. Effective pharmaceuticals may also be created which metabolize in a timed-release fashion allowing for a minimal number of introductions which are efficacious for longer periods of time. Combinations of agents can also produce useful new agents from the interaction of d e combination.
  • compositions can be prepared in solution as a dispersion, mixture, liquid, spray, capsule or as a dry solid such as a powder or pill, as appropriate or desired.
  • Solid forms may be processed into tablets or capsules or mixed or dissolved with a liquid such as water, alcohol, saline or other salt solutions, glycerol, saccharides or polysaccharide, oil or a relatively inert solid or liquid.
  • Liquids administered orally may include flavoring agents such as mint, cherry, guava, citrus, cinnamon, orange, mango, or mixed fruit flavors to increase palatability. Pills, capsules or tablets administered orally may also include flavoring agents.
  • compositions may further comprise agents to increase shelf-life, such as buffers, preservatives, anti-oxidants and other components necessary and suitable for manufacture and distribution of the composition.
  • Compositions further comprise a pharmaceutically acceptable carrier.
  • Carriers are chemical or multi-chemical compounds that do not significantly alter or effect the active ingredients of the compositions. Examples include water, alcohols such as glycerol and polyethylene glycol, glycerin, oils, salts such as sodium, potassium, magnesium and ammonium, fatty acids, saccharides or polysaccharides. Carriers may be single substances or chemical or physical combinations of these substances.
  • compositions may also comprise other agents that regulate intracellular calcium concentrations.
  • Calcium regulators may be, for example, calcium ionophores which increase transport of calcium ions across cell membranes. As pharmaceuticals, these agents alter intracellular calcium levels and induce apoptosis. Calcium ionophores that increase intracellular calcium ion levels include A23187, thapsigargin and ionomycin. These agents are also effective at regulating the activity of protein kinase and specifically protein kinase C. Such agents may be administered before, concomitant with or after the administration of other agents which regulate protein kinase activity.
  • Additional agents which are effective at selectively inducing apoptosis include anti-sense nucleic acids such as DNA, RNA or PNA oligonucleotides comprising a sequence complementary to a translated or transcribed sequence of the protein kinase gene.
  • Anti-sense technology exploits the specificity of hybridization reactions between two complementary nucleic acid chains to prevent the expression of that gene. For example, transcription can be inhibited by introducing a single-stranded nucleic acid, complementary to a messenger RNA of the protein kinase or another gene which positively effects kinase activity or expression.
  • Nucleic acids introduced will hybridize, for example, to protein kinase mRNA in the cytoplasm and thereby inhibit translation and subsequent expression of the protein kinase gene.
  • Another method to inhibit protein kinase expression is to introduce anti-sense nucleic acid to protein kinase mRNA or other exogenous oligonucleotides to neoplastic cells.
  • Anti-sense oligonucleotides may be introduced with viral or other vectors which infect or are transfected into target cells.
  • Anti-sense nucleic acid may be complementary to the coding region, the 5 1 - and 3'-non-coding regions, or to splice sites of protein kinase mRNA.
  • the anti-sense nucleic acid may also comprise a transcription template, a double stranded DNA with an RNA transcription promoter linked to an anti-sense gene. After this transcription template enters a cell, it can direct the synthesis of anti-sense RNA.
  • the anti ⁇ sense RNA than binds to and inactivates the mRNA of the particular protein kinase causing the cells to undergo apoptosis.
  • Apoptosis can be described as an intracellular mechanism of programmed cell death. It can be characterized by, for example, chromatin condensation, decreased cell volume, untethering of cell membrane from cytoskeleton (zeiosis), quiescence, decreased rate of mitosis and cleavage of DNA. Although the mechanism appears to be largely intracellular, intercellular signaling may be involved to some degree. In contrast, simple necrosis or tissue trauma typically involves cellular cytoplasmic and plasma membrane damage, osmotic swelling and cell lysis. Although an apoptotic cell may have one or more apoptotic characteristic symptoms before death, absolute determinations can be made with observations in comparison to control cells.
  • Apoptosis can be measured by observing certain of these parameters such as the degree of DNA fragmentation. For example, in apoptotic cells as compared to control samples, nucleic acid fragmentation is generally greater than about two fold, preferably greater than about four fold, more preferably greater than about ten fold, and even more preferably greater than about twenty fold.
  • Other parameters which can be used to measure the degree of apoptosis include the rate of mitosis, the amount of chromatin condensation, alterations in cell size or volume or simply decreased metabolism or metabolic rate as measured by the production of metabolic products such as acids or enzymes.
  • Cells that express an activated oncogene are typically abnormal having irregular margins or membranes, unusual mitotic activity, DNA damage or mutations, increased size or volume, unique gene expression patterns, unusual chromatin structure and sub-cellular alterations such as substantially increased numbers of mitochondria.
  • Such abnormal cells demonstrate uncontrolled proliferation which can cause a variety of benign and malignant neoplasms. While it is normal for different cells to divide at different rates in a body, aberrant uncontrolled proliferation is the ability of a cell to divide significantly more often than other cells of the same linage. In the adult, cells such as nerve cells are mostly incapable of further division once development has ceased. Other cells such as liver, thyroid and other glandular cells, once mature seldom divide, but are capable of rapid division on demand such as, for example, after partial destruction of the liver.
  • neoplastic cells retain many features of the specific cell type from which they are derived, many neoplastic cells develop characteristics commonly associated with neoplasms. These characteristics include invasion of the basal lamina, the establishment of metastases, loss of differentiation characteristics, abnormal variability in size and shape, and hypertrophy and hype ⁇ lasia.
  • neoplastic cells are readily evident in vitro. In vitro characteristics include a lack of contact inhibition, growth in soft agar, mutation or over expression of a dominant oncogene, low or no expression of tumor suppression genes, homozygosity of recessive or mutated oncogenes, unstable karyotypes, aneuploidy, anchorage independent growth, chromosomal translocations, double minute chromosomes and defects in nucleic acid repair, and unusual or abnormal transcription or replication, or combinations of these in vitro or other in vitro traits. Many human neoplasms contain cells which express activated oncogenes.
  • An activated oncogene is a gene with a mutation which cause a cell to become neoplastic.
  • the mutation may be of any form in both the coding and non-coding, regions of the gene. Non coding mutations may occur at the 5 1 - or 3' -ends of the gene. These mutations include point mutations, frame shift mutations, amplifications, translocations, insertions, deletions, promoter mutations, enhancer mutations, translocations and capture by virus.
  • Oncogene activations may include more than one mutation.
  • Oncogenes families which contain dominate oncogene mutations include members of the ras family of genes such as Ha-ray, Ki-r ⁇ and N-ras which encode p21ras proteins and are associated with some of the most common forms of human neoplasms.
  • Other oncogenes include abl, crk, dsi, erb, ets, evi, fun, fes/fps, fgr, fins, fos, gli, int, jun, kit, lck, mas, mis, mil/raf, mos, myb, myc, neu, pirn, rel, ros, seq, sis, ski, spi, src, trk and yes. Mutated forms of p53 and related proteins are also considered herein to be expressed from activated oncogenes.
  • Another embodiment of the invention is directed to a method for inducing a population of aberrantly proliferating cells to undergo apoptosis by treatment with agents that regulate protein kinase activity.
  • Cell populations which are treatable can be identified by the expression of activated oncogenes such as, for example, members of the ras, myc, fos, jun, mos and src family of genes. Expression can be from inherent sequences of the cells or from transfected oncogene sequences. Activated oncogenes can be detected by genetic analysis of the cells. Cell samples can be obtained using a wide variety of invasive or non ⁇ invasive techniques.
  • surgical biopsy can be used to remove samples of tissue to make observations, to purify nucleic acid, if necessary, for RFLP (restriction-fragment length polymo ⁇ hism), PCR (polymerase chain reaction) or direct DNA or RNA sequencing, or to culture.
  • RFLP restriction-fragment length polymo ⁇ hism
  • PCR polymerase chain reaction
  • DNA or RNA sequencing or to culture.
  • Confined areas which may contain such cells can be sampled by, for example, needle aspiration. Samples of tissue or fluid can be obtained from various areas of the body and directly analyzed for genetic mutations such as evidence of activated forms of ras.
  • pancreatic fluid will identify treatable forms of pancreatic cancers and stool samples will identify treatable bowel cancers.
  • Other cancers which can be as easily identified include prostate, vaginal, brain, lung, bone marrow and tumors of various organs.
  • mutations including activating mutations may be inherent to the cell, many activating mutations, and consequently neoplasia, may be caused by exposure to chemical mutagens such as cigarette smoke and asbestos, by exposure to electromagnetic radiation such as sunlight and atomic radiation and by exposure to pathogens.
  • Pathogens suspected of inducing neoplastic transformations directly or indirectly include human immunodeficiency virus (HTV), Epstein-Barr virus and other herpes viruses (HZV, HSV-1, HSV-2), hepatitis virus (types A, B, C, etc.), human T cell leukemia virus (HTLV-I, HTLV-II) and other retroviruses, human papilloma and polyoma viruses and many others.
  • HTV human immunodeficiency virus
  • HZV Epstein-Barr virus and other herpes viruses
  • HSV-1, HSV-2 hepatitis virus
  • HTLV-I human T cell leukemia virus
  • HTLV-III
  • Protein kinases are enzymes which transfers the 2-phosphoryl group of adenosine triphosphate (ATP) to some nucleophilic acceptor protein. While ATP is the general phosphate donor, other purine or pyrimidine triphosphates such as guanosine triphosphate, uridine triphosphate, or cytidine triphosphate may also serve as phosphate donors. Protein kinases that are important cellular regulators of growth generally phosphorylates proteins on serines, threonines and tyrosines.
  • kinases include the serine/threonine- specific protein kinases, the serine-specific protein kinases, the tyrosine-specific protein kinases, the cAMP-dependent kinases (A-kinases), the cGMP-dependent kinases (G-kinases), phosphorylase kinases and protein kinase C.
  • Protein kinase C may have a direct connection with ras proteins as both products participate in the G-protein signal transduction pathway.
  • disorders which can be treated include neoplastic and other disorders which contain cells that expresses activated oncogenes such as, for example, ras or an activated r ⁇ y-like oncoprotein.
  • Neoplasms which express activated ras include malignancies, pre-malignancies or diseases that result in a relatively autonomous growth of cells.
  • Some specific neoplastic disorders which are prophylactically or therapeutically treatable include small cell lung cancers and other lung cancers, rhabdomyosarcomas, choriocarcinomas, glioblastoma multiformas (brain tumors), bowel and gastric carcinomas, leukemias, ovarian cancers, cervical cancers, breast cancer, pancreatic cancer, prostate cancers, osteosarcomas or cancers which have metastasized.
  • Non-Hodgkin's lymphomas including the follicular lymphomas, Burkitt's lymphoma, adult T- cell leukemias and lymphomas, hairy-cell leukemia, acute myelogenous, lymphoblastic or other leukemias, chronic myelogenous leukemia, and myelodysplastic syndromes.
  • Additional neoplastic diseases treatable by the compositions of this invention include virally-induced cancers wherein the viral agent is EBV, HPV, HTLV-1 or HBV.
  • treatable cancers include breast cell carcinomas, melanomas and hematoiogic melanomas, ovarian cancers, pancreatic cancers, liver cancers, stomach cancers, colon cancers, bone cancers, squamous cell carcinomas, neurofibromas, testicular cell carcinomas and adenocarcinomas.
  • the patient to be treated may be any mammal such as a human including adults, children, infants and even a fetus. Treatments may be directly or indirectly administered to the patient. Direct administration may be by, for example, oral, parenteral, sublingual, rectal, pulmonary abso ⁇ tion or topical application.
  • Parenteral administration may be by intravenous injection, subcutaneous injection, intramuscular injection, intra-arterial injection, intrathecal injection, intra peritoneal injection or direct injection or other administration to one or more specific sites. Injectable forms of administration are sometimes preferred for maximal effect in, for example, bone marrow.
  • venous access devices such as medi-ports, in-dwelling catheters, or automatic pumping mechanisms are also preferred wherein direct and immediate access is provided to the arteries in and around the heart and other major organs and organ systems.
  • Indirect administration is performed, for example, by administering the composition to cells ex vivo and subsequently introducing the treated cells to the patient.
  • the cells may be obtained from the patient to be treated or from a genetically related or unrelated patient.
  • Related patients offer some advantage by lowering the immunogenic response to the cells to be introduced. For example, using techniques of antigen matching, immunologically compatible donors can be identified and utilized.
  • compositions may be administered as a bolus injection or spray, or administered sequentially over time (episodically) such as every two, four, six or eight hours, every day or every other day.
  • Compositions may also be administered in a timed-release fashion such as by using slow-release resins and other timed or delayed release materials and devices.
  • Orally active compositions are more preferred as oral administration is usually the safest, most convenient and economical mode of drug delivery.
  • Oral administration is usually disadvantageous because compositions are poorly absorbed through the gastrointestinal lining. Agents which are poorly absorbed tend to be highly polar. Consequently, agents which are effective, as described herein, may be made orally bioavailable by reducing or eliminating their polarity.
  • cells may be isolated from a patient and treated before returning the cell to the same or a different patient.
  • This invention may be used, for example, in a bone marrow transplantation to treat a neoplastic disorder.
  • the bone marrow of a patient is first removed and the cells are treated with the agents of this invention. After the patient is treated for the cell proliferative disorder, the treated cells are reintroduced into the patient.
  • bone marrow cells are removed from a patient before chemotherapy and returned to the patient after chemotherapy. While the bone marrow cells are outside the patient, they are often sorted to remove neoplastic or pre-neoplastic cells.
  • the bone marrow cells may be treated with the compositions of this invention ex-vivo before their return into the body. This treatment of this invention may also be used to treat bone marrow cells when the donor and the recipient are different individuals.
  • Another embodiment of the invention is directed to the administration of protein kinase suppressor agents which, in combination with other chemotherapeutic agents, maximize the effect of the compositions in an additive or synergistic manner.
  • Other antineoplastic agents which can be administered with the agents of the invention include differentiating agents such as the butyrates (e.g.
  • alkylating agents such as alkyl sulfonates, aziridine, ethylenimines and methylmelamines, nitrogen mustards, nitrosoureas, antibiotics, antivirals including ganciclovir, antimetabolites such as folic acid analogs, purine analogs, pyrimidine analogs, hormones such as androgens, antiadrenals, antiandrogens, antiestrogens, estrogens, luteinizing hormone and releasing hormone analogs and progestogen.
  • the antineoplastic agents of the invention may also be administered in conjunction with cytokines such as tumor necrosis factor (TNF), the interleukins fJL-1, IL-2, IL-3, D -4, IL-5, etc.), the interferons (IFN- ⁇ , - ⁇ , - ⁇ ) and growth factors.
  • TNF tumor necrosis factor
  • IFN- ⁇ , - ⁇ , - ⁇ interferons
  • Therapies using combinations of these agents would be safe and effective against mabgnancies and other forms of cancer.
  • Combinations of therapies may also be effective in inducing regression or elimination of a tumor or some other form of cancer such as compositions of the invention plus radiation therapy, toxin or drug conjugated antibody therapy using monoclonal or polyclonal antibodies directed against the transformed cells, gene therapy or specific anti-sense therapy. Effects may be additive, logarithmic, or synergistic, and methods involving combinations of therapies may be simultaneous protocols, intermittent protocols or protocols which are empirically determined.
  • Another embodiment of the invention is directed to a method for screening for agents for their ability to induce apoptosis. These agents will be useful for the treatment of neoplastic and other cell proliferative disorders.
  • Prospective agents may be tested by administering the agent to a cell line and assaying the intracellular protein kinase activity or calcium concentration of the cell line.
  • the agent may be administered to a cell line comprising, for example, activated ras and the cell line monitored for apoptosis.
  • Agents which initially stimulate, but subsequently suppress protein kinase activity may be useful for treatment.
  • Agents which enhance protein kinase activity may also be used to, for example, temporarily stimulate and subsequently inhibit protein kinase activity.
  • Neoplastic cells may be isolated from a surgically removed a biopsy or other specimen, an established cell line, a virally infected cell line or a neoplasm which may be propagated in an immunocompromised host.
  • immunocompromised hosts include nude mice, human immunodeficiency virus infected humans and animals.
  • Nearly neoplastic cells may be pre-neoplastic cells, hype ⁇ lastic cells or hypertrophic cells.
  • Cells to be assayed are treated with an agent which transiently down regulates protein kinase activity and the degree of apoptosis is monitored. If apoptosis occurs, the cell will be diagnosed as having an activated oncogene such as a ray gene or an activated ras functional homolog. If the cell which shows apoptosis after treatment is not cancerous, there will be strong indication that it may be pre-neoplastic and appropriate treatment may be considered.
  • an agent which transiently down regulates protein kinase activity and the degree of apoptosis is monitored. If apoptosis occurs, the cell will be diagnosed as having an activated oncogene such as a ray gene or an activated ras functional homolog. If the cell which shows apoptosis after treatment is not cancerous, there will be strong indication that it may be pre-neoplastic and appropriate treatment may be considered.
  • compositions to be administered contain a therapeutically effective amount of the effective agent.
  • a therapeutical effective amount is that amount which induces apoptosis or, otherwise, has a beneficial effect to the patient by alleviating one or more symptoms of the disorder or simply reduce mortality.
  • a beneficial effect may be a decrease in pain, a decrease in duration, frequency or intensity of blast crises in a leukemia patient, decreased fatigue or an increased strength.
  • a therapeutic amount is that amount of agent that stimulates or enhances the apoptosis of neoplastic or pre-neoplastic cells.
  • a plurality of PKC inhibitors can be administered to a patient in the course of a treatment.
  • Treatments to the patient may be therapeutic or prophylactic.
  • Therapeutic treatment involves administration of one or more compositions of the invention to a patient.
  • Pharmaceutical compositions may also be administered to patients prophylactically.
  • a diagnosis of a neoplastic disorder is often rather difficult for many types of tissues. Many patients in the early stages of pre-neoplastic or neoplastic disease may have no symptoms. Furthermore, a neoplasia may be too small to be detectable with current technology.
  • Some patients such as former cancer patients and patients with disorders such as Beckwith-Wiedemann syndrome, Aniridia, neurofibromatosis, multiple endocrine neoplasm or human immunodeficiency virus infection show a high probably of future neoplastic disorders. These patient can benefit from regular preventive treatment by the methods of this invention. Administration can begin at birth and continue, if necessary, for life. The administration of protein kinase inhibitors to asymptomatic, but high risk patients may help in the destruction of pre-neoplastic or neoplastic cells. Both prophylactic and therapeutic uses are readily acceptable.
  • Another embodiment of the invention is directed to a method for regulating the expression of a protein kinase gene in a mammalian cell. Briefly, the cell is exposed to an effective amount of a agent of the invention. A normally expressed protein kinase gene of the cell, such as protein kinase C, is suppressed to reduced the expression of its product. An effective amount of the composition is that amount which decreases the extent or magnitude of kinase activity.
  • Another embodiment of the invention is directed to a method for down-regulating the proliferation of cells expressing an activated oncogene such as, for example, activated ras. Such cells would be induced to undergo apoptosis or simply die.
  • an effective amount of a composition of the invention is exposed to cells ex vivo or administered to cells in vivo.
  • These cells can be utilized to treat neoplastic disorders by administration to patients.
  • bone marrow removed from a patient may be treated with the compounds of this invention and returned to a patient.
  • Bone marrow cells can be obtained from volunteers or the patients to be treated.
  • the following experiments are offered to illustrate embodiments of the invention, and should not be viewed as limiting the scope of the invention. Examples Example 1 Establishment nf a R ⁇ s and hc.1-2 Transformed Cell Lin
  • DMEM Dulbecco modified Eagles medium
  • Hazelton Research Products, Inc. Lenexa, KA
  • 2 mM L-glutamine 100 U/ml penicillin and 100 ⁇ g/ml streptomycin.
  • Cells were stably transfected by electroporation with a v-Ha-r ⁇ s expressing vector and a Geneticin marker to establish a ras transformed cell line.
  • Jurkat cell lines transfected with the neomycin resistance gene were used as controls. Positive transformants were selected in the same growth medium plus 0.7 mg Geneticin (GIBCO/BRL; Grand Island, NY) per ml.
  • a retroviral expression vector containing bcl-2 and hygromycin resistance gene was transfected into the amphotropic murine retroviral vector packaging line DAMP to establish a bcl-2 cell line.
  • Introduction of the bcl-2 gene into Jurkat cells was performed by infecting Jurkat cells with the packaged retrovirus in the presence of polybrene at 40 ⁇ g/ml.
  • Jurkat cells with stable integration of bcl-2 was selected with growth medium containing 200 ⁇ g/ml hygromycin.
  • the division times of different cell lines were measured in growth media with and without phorbol esters.
  • Bcl-2 protein In vivo phosphorylation of Bcl-2 protein: Cells (2 x IO 7 ) were prepared for phosphorylation by growth in phosphate free medium for 6 hours containing 2.5 mCi of [ 32 P]PO 4 . Both normal cells and cell grown under 500 nM of PMA were phosphorylated. To isolate phosphorylated Bcl-2 protein, cell lysates were normalized for protein and incubated with an anti-human Bcl-2 antibody (Pharmingen; San Diego, CA).
  • Immunocomplexes containing Bcl-2 were coupled to protein A and washed 5 times with 1 ml of lysis buffer (1 % Triton X-100, 50 mM HEPES, pH 7.4, 100 mM NaCl 2 , 1 mg/ml BSA, 10 mM benzamindine, 10 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin and 10 ⁇ g/ml soybean trypsin inhibitor). Immunoprecipitates were analyzed by SDS-PAGE. Bcl-2 was detected after electrophoresis by autoradiography of the dried gel.
  • Immunoprecipitation and westem blot Cells (2 x IO 7 ) were washed twice in PBS in preparation for immunoprecipitation. Soluble protein were extracted by treating the cells with 1 % Triton X-100, 50 mM HEPES, pH 7.4, 100 mM NaCl 2 , 1 mg/ml BSA, 10 mM benzamindine, 10 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin and 10 ⁇ g/ml soybean trypsin inhibitor.
  • the supematant fraction was adjusted to 0.5 M NaCl, 0.5% deoxycholate and 0.05 % sodium dodecyl sulphate.
  • a duplicate cell lysate preparation was made for immunoprecipitation and Westem Blot experiments. 100 ⁇ g of cell lysate were used for each immunoprecipitation with either pan-r ⁇ s antibody (Oncogene Science), or anti-human Bcl-2 antibody (DAKO; Malvern, PA). Lysates were co-incubated with antibodies for two hours, coupled to protein A and washed five times with 1 ml of lysis buffer. Immunoprecipitates were analyzed by 12.5% SDS-PAGE. After electrophoresis, proteins were detected by immunoblotting with specific antibodies and developed with an anti- mouse immunoglobulin alkaline phosphatase agent (Oncogene Science).
  • Immunoblot and kinase assay Cells (2 x IO 7 ), under normal condition or after down-regulation of PKC activity, were permeabilized with 1 ml of kinase buffer containing 5 ⁇ Ci of [ 32 P]ATP for 10 minutes at 37°C. For inhibition experiments, 0.1 ⁇ M of staurosporin or 200 ⁇ M genistein was added to the kinase buffer. Proteins were extracted from lysed cells and analyzed by SDS-PAGE. After electrophoresis, gels either were subjected to autoradiography directly or to westem blot analysis and subsequently to autoradiography .
  • [ 35 S]-metabolic labeling of cellular proteins Cells (2 x IO 7 ) were labeled by growth in 50 ⁇ Ci of [ 35 S]-Methionine and 50 ⁇ Ci of [ 35 S]-Cysteine for 3 hours. Labeled proteins were immunoprecipitated with specific antibodies and separated by 12.5% SDS-PAGE. Gels were fixed with 10% glacial acetic acid and 30% methanol for 30 minutes, treated with an enhancing solution and subjected to autoradiography.
  • RNA blot analysis of total cellular RNA Total cellular RNA was isolated by guanidine thiocyanate phenol RNA extraction, quantified, separated by electrophoresis on formaldehyde agarose gels and transferred to nitrocellulose. Hybridizations were performed at 68 °C with 6xSSC, 2x
  • [ 32 P]-DNA probes [ 32 P]-labeled DNA probes were made by the random oligonucleotide primer method.
  • the actin probe consisted of a 0.7 kb Pst I fragment encoding murine ⁇ -actin.
  • the ras probe consisted of a 700-kb
  • Example 4 Transfected Constructs have nn Effect nn Cell Survival.
  • Jurkat cells were transformed with the constructs of Example 1 and their growth monitored to determine the effects of the various constructs on survival. Untransformed, activated ras transformed (pHl), bcl-2 transformed and activated ras and bcl-2 transformed cells were monitored and their growth plotted ( Figure 1). All four cell types show similar division times and survival rates. The transformation of the construct and the expression of the transgene appear to have no effect on survival and growth of cells under routine in vitro culture.
  • Example 5 Cell Lines Expressing ras are Induced intn Apnptnsis hy Phnrhnl
  • Example 6 Decreased Survival nf Cells Transferred with Activated rnx.
  • Staurosporin an inhibitor of PKC, staurosporin. Staurosporin treatment activated apoptosis in activated ras containing cells with faster kinetics than did chronic exposure to high-dose PMA, as might be expected from the more rapid action of staurosporin on inhibition of PKC. Twelve hours after exposure to staurosporin, over 26% of activated ras containing cells demonstrated significant nuclear DNA fragmentation, whereas only 8% of the parental Jurkat cells exhibited this indicator of programmed cell death.
  • a human bcl-2 gene was introduced into Jurkat cells (Jurkat/ c/- 2) and double transfected (activated ras/bcl-2) cells made to investigate the effect of bcl-2 over expression on the cell death induced by activated p2 ⁇ ras after inhibition of PKC activity.
  • the growth curves of the two bcl-2 transfected cell lines were similar to the untransfected parental cell lines under normal growth conditions ( Figure 1). Over expressed bcl-2 dramatically inhibited the apoptosis induced by constitutively-activated p21r ⁇ s after down-regulation of PKC ( Figures 2 and 3).
  • Bcl-2 protein was metabolically-labeled with 32 P-PO 4 under normal conditions or after down-regulation of PKC activity.
  • Bcl-2 protein was immunoprecipitated using an anti-Bcl-2 antibody and resolved on denaturing gels ( Figure 5). Simultaneously, a fraction of the total lysate from each cell type was also resolve by electrophoresis to verify successful and equivalent labeling of proteins in each cell type.
  • Bcl-2 protein (26 kDa) was phosphorylated in activated ras and bcl-2 transfected cells after the suppression of PKC activity.
  • Bcl-2 protein in phosphorylated in response to the change in the intracellular environment engendered by activated p21ras and the suppression of the PKC activity.
  • Example 9 /toy-Dependent In Vitrn Phosphorylation of Bcl-2.
  • Bcl-2 protein in vitro was studied to determine relative kinase activities on Bcl-2 in response to PKC down-regulation in the presence of activated p21ras.
  • the 26 kDa phosphoiylated Bcl-2 protein was detected in immune complexes at increased levels only from permeabilized activated ras and bcl-2 transfected cells and only after down-regulation of PKC activity (Figure 5, lane 5).
  • a 21 kDa newly-kinased protein species was also detected under these conditions, possibly representing the Bcl-2 partner, Bax.
  • this labeled protein in the co-precipitations after in vitro, but not in vivo labeling may reflect differences in the turnover of this labeled species of intrinsic differences in the two experimental techniques. Phosphorylation of these proteins was inhibited by the serine threonine kinase inhibitor, staurosporin, which suggests the involvement of a serine threonine kinase in Bcl- 2 phosphorylation during stimulation of ras-induced apoptosis. In contrast, the tyrosine protein kinase inhibitor genistein had no effect on the phosphorylation of Bcl-2 in this permeabilzed cell system.
  • Clones 6 and 20 which express lower levels of ras mRNA, had lower percentages of v-ray-induced nuclear DNA fragmentation (20-25 %). In contrast, clones 7 and 19 demonstrated greater than 40% DNA fragmentation (Figure 8). These data suggest that the extent of the apoptosis induced by activated p21ray is relative to the level of ras expression, and indicates that apoptosis observed is due to the ras gene product.
  • Example 11 Protein Synthesis Required for Apoptosis Induced hy Activated Completion of pathways leading to nuclear DNA fragmentation and apoptosis are in some cases dependent upon new synthesis of macromolecules.
  • the dependence of the DNA fragmentation observed after down-regulation of protein kinase C in the presence of activated ras genes was determined by exposure of the cells to the protein synthesis inhibitor cycloheximide at a concentration that inhibited the incorporation of [ 35 S]- methionine and [ 35 S]-cysteine into new protein by > 90% .
  • This protein synthesis inhibitor was added to the cell lines after down regulation of PKC, and nuclear DNA fragmentation was analyzed.
  • Apoptotic pathways leading to nuclear DNA fragmentation in lymphocytes can be active by signals which result in calcium mobilization, either from intracellular pools or from extracellular sources. Because an activated ras gene has been reported to alter calcium flux in lymphocytes, the effects of calcium ionophore on the growth and viability of these cell lines were examined. Calcium ionophore (at a final concentration of 2 ⁇ M) was added to Jurkat and activated ras transfected cell cultures. Treated cells were assayed for cell growth kinetics and nuclear DNA fragmentation (Figure 10), Growth curves of both cell lines in the presence of the calcium ionophore were similar. Survival decreased rapidly over the first two days in both cell types.
  • EGTA a chelator of extracellular calcium, prevented the induction of nuclear DNA fragmentation by the ionophore, demonstrating that the action of the ionophore was dependent on extracellular calcium (Figure 12).
  • EGTA was used to determine if the ras-induced apoptosis in activated ras containing cells was dependent on extracellular calcium.
  • EGTA moderately reduced the percentage of DNA fragmentation in activated ras containing cells when added during the last 6 hours of PKC down regulation ( Figure 10), but not to the same extent as it inhibited ionophore-induced nuclear DNA fragmentation ( Figure 12).

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Abstract

L'invention concerne des agents et des compositions pharmaceutiques contenant des agents qui régulent l'activité de la protéine kinase cellulaire. Ces compositions, lorsqu'elles sont administrées à des cellules exprimant le gène ras, induisent une apoptose de ces cellules. Ces compositions et ces agents peuvent être utilisés pour traiter des maladies associées à l'activité de la protéine kinase, en particulier les carcinomes, les sarcomes, les lymphosarcomes, les leucémies et d'autres maladies qui s'accompagnent d'une augmentation de l'activité de la protéine kinase et en particulier de la protéine kinase C. L'invention concerne également des procédés pour déterminer l'efficacité d'agents chimiothérapeutiques et des procédés pour caractériser des tumeurs, par la détermination de la diminution de l'activité protéine kinase.
PCT/US1996/012381 1995-07-28 1996-07-29 Procede et compositions pour traiter des proliferations anormales de cellules WO1997004761A1 (fr)

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WO1999010004A3 (fr) * 1997-08-29 1999-05-27 Ontogeny Inc Regulation de tissus musculaires par des polypeptides 'hedgehog', et formulations et utilisations associees
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US7265153B2 (en) 1998-02-11 2007-09-04 Faller Douglas V Compositions and methods for the treatment of cystic fibrosis
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US7510830B2 (en) * 2000-07-28 2009-03-31 Cancer Research Technology Limited Cancer treatment by combination therapy
US7863322B2 (en) 2001-09-03 2011-01-04 Cancer Research Technology Limited Anti-cancer combinations
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EP2014651A1 (fr) * 2007-07-12 2009-01-14 Exonhit Therapeutics SA Composants et procédés de modulation de Rho GTPases
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