Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1996-07-26
2001-07-03
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007400, C435S007900, C435S019000, C436S503000
Reexamination Certificate
active
06255057
ABSTRACT:
FIELD OF THE INVENTION
The present invention is in the field of biological assays for the detection of ethanol exposure in mammals and for the evaluation of drugs that modify the cellular effects of ethanol consumption.
BACKGROUND OF THE INVENTION
The abuse of ethanol remains a major public health problem in the U.S. and throughout the world. It is of interest to provide methods for the detection of ethanol exposure in individuals so as to monitor compliance with abuse treatment regimens. It is also of interest to provide assays useful for the evaluation of drugs that may be used to treat one or more of the adverse effects of chronic ethanol over-consumption. In order to provide such methods and assays, it is necessary to gain detailed understanding of the biochemical effects of ethanol exposure at a cellular level. Recent evidence suggests that ethanol modifies the function of certain proteins and signal transduction pathways, thereby producing changes in second messenger concentrations, protein kinases, and gene expression. This observation does not provide a specific test which enables the effects of ethanol to be readily determined. Recently, it has been shown that the specificity of protein kinases appears to correlate with their localization within the cell. Mochly-Rosen, 1995
, Science
268:247.
Chronic alcoholism causes functional and pathologic changes in many organs, particularly the brain, but the molecular mechanisms which account for these effects are not well understood. It would be desirable to provide methods for monitoring the effect of chronic ethanol exposure on mammalian cells, and further desirable to provide methods for determining if an individual has been actively consuming ethanol for extended periods of time.
The present invention relates to an assay for detecting the effects of ethanol, particularly the chronic exposure of ethanol, on animal cells, particularly human or other mammalian cells. This assay can be used both in a diagnostic test to determine ethanol consumption in an individual, or in screening for drugs or treatments which moderate, inhibit, reverse or enhance the effects of ethanol consumption.
SUMMARY OF THE INVENTION
The present invention relates, in part, to the discovery that exposure to ethanol alters dramatically the subcellular localization of the catalytic C&agr; subunit of the cAMP dependent protein kinase (PKA) and the &dgr;- and &egr;-subunits of protein kinase C (PKC). For example, the catalytic C&agr; subunit of PKA, which is normally localized to the Golgi apparatus area, appears to translocate to the nucleus upon exposure of a cell to ethanol. Ethanol also has been shown to cause translocation of PKC activity from cytosolic to membrane fraction in astroglial cells and human lymphocytes and epidermal keratinocytes. The present invention further relates to the discovery that the detectable amount of the regulatory subunit RI of PKA decreases, and the amounts of the &agr;-, &dgr;- and &egr;-subunits of PKC increase in certain cell types, including but not limited to, NG108-15 cells (&agr;-, &dgr;-, and &egr;-subunit) or PC12 cells (&dgr;- and &egr;-subunit), upon the exposure to ethanol. These discoveries provide the basis for assays that may be used to detect the exposure of cells to ethanol and further for assays that may be used for the screening of drugs or treatment to modulate the effects of ethanol consumption.
One aspect of the invention is to provide assays that provide an indication of the exposure of a cell or an individual to ethanol by identifying at least one cell component, e.g., a protein, that has a cellular localization (distribution) that varies in correlation with the exposure of the cell to ethanol, and determining the distribution of that cell component within a cell of a sample to be tested. In one preferred embodiment, the cell component comprises a subunit of the cAMP dependent protein kinase, PKA, the C&agr; subunit being particularly preferred. In another preferred embodiment, the cellular component comprises an isozyme protein kinase C, PKC, wherein the &dgr; or the &egr; isozyme of protein kinase C is particularly preferred.
Another aspect of the invention is to provide assays that provide an indication of exposure of a cell or an individual to ethanol by measuring the amount of protein that varies in amount in a relationship with the exposure of the cell to ethanol. In one preferred embodiment, the decrease of the detectable amount of the regulatory subunit RI of PKA in response to ethanol exposure is determined. In another preferred embodiment, the increase of the detectable amounts of &agr;PKC, &dgr;PKC, or &egr;PKC in response to ethanol exposure is measured.
Another aspect of the invention is to provide assays for screening therapeutic compounds that modulate the effects of ethanol on a cell. These screening assays measure the effects of a compound of interest to interfere with one or more of the cellular effects of ethanol described herein, i.e., changes in the localization of C&agr;, &dgr;PKC, or &egr;PKC, decreases in the amount of RI, increases in the amount of &agr;PKC, &dgr;PKC, or &egr;PKC, changes in the set of proteins phosphorylated by or expressed in response to C&agr;, &dgr;PKC and &egr;PKC. For example, C&agr; may induce phosphorylation of CREB and thereby its activation, resulting in the induction of CRE-regulated gene expression. Another aspect of the invention is to provide kits for detecting the exposure of cells to ethanol. Kits of the invention may include labeled antibodies capable of specifically binding to C&agr; or RI of PKA or to the c&agr;-, &dgr;- and &egr;-subunits of PKC, respectively.
REFERENCES:
patent: 5069895 (1991-12-01), Diamond et al.
Gayer, G.G., et al., 1991, “Ethanol Increases Tyrosine Hydroxylase Gene Expression in N1E-115 Neuroblastoma Cells,”J. Biol. Chem.266:22279-22284.
Gerstin, E., et al., 1996, “Protein Kinase C Isozymes Required for Up-regulation of L-type Calcium Channels by Ethanol,”Supp. Alcholism Clinical and Experimental Research20:102A.
Gordon et al., 1986, “Ethanol Regulation of Adenosine Receptor-stimulated cAMP Levels in a Clonal Neural Cell Line: An in vitro Model of Cellular Tolerance to Ethanol,”Proc. Natl. Acad. Sci USA83:2105.
Heberlein, U., et al., 1993, “Star is Required for Neuronal Differentation in the Drosophila Retina and Displays Dosage-Sensitive Interactions with Ras1,”Dev. Biol.160:51-63.
Hundle, B., et al., 1995, “An &egr;-PKC-derived Peptide Fragment Prevents Enhancement of NGF-induced Neurite Outgrowth by Phorbol Esters and Ethanol,”9th International Conference on Second Messengers&Phosproteins151.
Hundle, B., et al., 1995, “Overexpression of &egr;-Protein Kinase C Enhances Nerve Growth Factor-induced Phosphorylation of Mitogen-activated Protein Kinases and Neurite Outgrowth,”J. Biol. Chem.270:30134-30140.
Messing, et al., 1990, “Protein Kinase C Participates in Up-Regulation of Dihydropyridine-Sensitive Calcium Channels by Ethanol,”J. of Neurochem.55:1383-1389.
Messing, et al., 1991, “Chronic Ethanol Exposure Increases Levels of Protein Kinase C &dgr; and &egr; and Protein Kinase C-mediated Phosphorylation in Cultured Neural Cells,”J. Biol. Chem.266:23428-23432.
Miles, M.F., et al., 1991, “Mechanisms of Neuronal Adaptation to Ethanol,”J. Biol. Chem.266:2409-2414.
Miles, M.F., et al., 1993, “Phosducin-like Protein: An Ethanol-responsive Potential Modulator of Guanine Nucleotide-binding Protein Function,”Proc. Natl. Acad. Sci. USA90:10831-10835.
Mochly-Rosen, D., et al., 1988, “Chronic Ethanol Causes Heterologous Desensitization of Receptors by Reducing &agr;sMessenger RNA,”Nature333:848-850.
Mochly-Rosen, 1995, “Localization of Protein Kinases by Anchoring Proteins: A Theme in Signal Transduction,”Science268:247.
Nagy, L.E., et al., 1989, “Adenosine is Required for Ethanol-induced Heterologous Desensitization,”Mol. Pharm.36:744-748.
Nagy, L.E., et al., 1991, “cAMP-dependent Protein Kinase Regulates Inhibition of Adenosine Transport by Ethanol,”Mol. Phar.40:812-817.
Nagy, L.E., et al., 1988, “Cultured Lymphocytes from
Diamond Ivan
Dohrman Doug Paul
Gordon Adrienne Sue
Cooley & Godward LLP
Ernest Gallo Clinic and Research Center
Hayes Robert C.
Kunz Gary L.
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