Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2001-02-08
2002-09-24
LeGuyader, John L. (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S006120, C435S091100, C435S325000, C435S366000, C536S023100, C536S024310, C536S024330, C536S024500
Reexamination Certificate
active
06455307
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides compositions and methods for modulating the expression of Casein kinase 2-alpha prime. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding Casein kinase 2-alpha prime. Such compounds have been shown to modulate the expression of Casein kinase 2-alpha prime.
BACKGROUND OF THE INVENTION
The process of phosphorylation, defined as the attachment of a phosphate moiety to a biological molecule through the action of enzymes called kinases, represents a major course by which intracellular signals are propagated resulting finally in a cellular response. Within the cell, these enzymes are generally classified into a protein-serine/threonine subfamily or a protein-tyrosine subfamily on the basis of phosphorylation substrate specificity. The extent of protein phosphorylation, in turn, is regulated by the opposing action of phosphatases which remove the phosphate moieties. Because phosphorylation is such a ubiquitous process within cells and because cellular phenotypes are largely influenced by the activity of these pathways, it is currently believed that a number of disease states and/or disorders are a result of either aberrant activation of, or functional mutations in, kinases. Consequently, considerable attention has been devoted to the study of kinases and their role in disease processes.
The protein kinases comprise an exceptionally large family of eukaryotic proteins which mediate the responses of cells to external stimuli and to date, in excess of several hundred unique members of the protein kinase family from a wide variety of eukaryotic organisms have been described and characterized at the amino acid sequence level.
Among the enzymes within the protein-serine/threonine kinase subfamily are two distinct casein kinases which have been designated casein kinase I (CKI) and casein kinase II (CKII or CK2) identified by the order of their elution from DEAE-cellulose. The casein kinases are distinguished from other protein kinases by their ability to phosphorylate serine or threonine residues within acidic recognition sequences such as found in casein and each has been thoroughly characterized regarding their physicochemical properties, recognition sequences, substrate specificity and effects on metabolic regulation. These enzymes have been found throughout the cell and their activities have been purified from or found to be associated with cytoplasmic fractions, membranes, nuclei, mitochondria, and cytoskeleton reviewed in (Tuazon and Traugh,
Adv. Second Messenger Phosphoprotein Res
., 1991, 23, 123-164).
Casein kinase 2 (also known generally as casein kinase as well as CKII, CK2 and CSNK2) is a heterotetrameric holoenzyme composed of two catalytic (alpha and/or alpha prime (alpha′)) subunits (Lozeman et al., Biochemistry, 1990, 29, 8436-8447; Meisner et al.,
Biochemistry
, 1989, 28, 4072-4076) and two regulatory beta subunits (Heller-Harrison et al.,
Biochemistry
, 1989, 28, 9053-9058; Jakobi et al.,
Eur. J. Biochem
., 1989, 183, 227-233). While the alpha subunits contain the enzyme's active site, the noncatalytic beta subunit functions to protect the alpha subunit against denaturing agents or conditions and alters the substrate specificity of the enzyme. The alpha subunit protein has also been shown to activate transcription of the beta subunit gene (Robitzki et al.,
J. Biol. Chem
., 1993, 268, 5694-5702).
Casein kinase 2 is unique in that it recognizes phosphoacceptor sites specified by several acidic determinants, it can use both ATP and GTP as phosphoryl donors, it is insensitive to any known second messenger and displays high basal activity.
Casein kinase 2 is constitutively active, highly pleiotropic and its targeting seems to be modulated through association with a variety of cellular proteins, more than 160 of which are known (Pinna and Meggio,
Prog. Cell Cycle Res
., 1997, 3, 77-97). Its expression is abnormally elevated in proliferating and neoplastic tissues and recent studies suggest that mice overexpressing the alpha subunit of casein kinase 2 develop leukemia. Several compounds have been shown to affect the activity of casein kinase 2 with polycationic species stimulating activity and polyanionic species acting as inhibitors (Pinna,
Biochim. Biophys. Acta
, 1990, 1054, 267-284).
Physiologically, casein kinase 2 has been shown to play a role in cell cycle progression (Marshak and Russo,
Cell. Mol. Biol. Res
., 1994, 40, 513-517), liver regeneration (Perez et al.,
FEBS Lett
., 1988, 238, 273-276), viral replication (Dupuy et al.,
J. Virol
., 1999, 73, 8384-8392; Lenard,
Pharmacol. Ther
., 1999, 83, 39-48; Schubert et al.,
J. Mol. Biol
., 1994, 236, 16-25; Wadd et al.,
J. Biol. Chem
., 1999, 274, 28991-28998), apoptosis (Watabe et al.,
Cell. Growth Differ
., 1997, 8, 871-879), transduction of growth signals (Tawfic and Ahmed,
J. Biol. Chem
., 1994, 269, 24615-24620), prostate cancer (Yenice et al.,
Prostate
, 1994, 24, 11-16), breast cancer (O'Brien et al.,
Biochem. Biophys. Res. Commun
., 1999, 260, 658-664), insulin signaling (Singh,
Mol. Cell. Biochem
., 1993, 121, 167-174) and Alzheimer's disease (Blanquet,
Neuroscience
, 1998, 86, 739-749).
Inhibitors of the enzyme, as a whole or subunit-specific, may have therapeutic potential and consequently, modulation of casein kinase 2 activity and/or expression is believed to be an appropriate point of therapeutic intervention in pathological conditions.
To date, investigative strategies aimed at modulating casein kinase 2 function have involved the use of antibodies, antisense oligonucleotides, and chemical inhibitors.
Disclosed in U.S. Pat. No. 5,171,217 are methods of delivering inhibitors of protein kinases, including casein kinase 2, to an affected intramural site (March et al., 1992).
Antisense oligonucleotides targeting each of the subunits of casein kinase 2 in various organisms have been reported in the art. Chen et al. designed one antisense oligonucleotide targeting the beta subunit of casein kinase 2 in Xenopus to investigate oocyte maturation (Chen and Cooper,
Proc. Natl. Acad. Sci. U.S.A
., 1997, 94, 9136-9140). This oligonucleotide targeted residues 175-182 and contained phosphodiester links with the three phosphodiester links at the 3′ end replaced by phosphorothioate links.
Shayan et al. report the use of an antisense oligonucleotide targeting the alpha subunit of casein kinase 2 in viral infected bovine lymphoblastoid cells (Shayan and Ahmed,
Parasitol. Res
., 1997, 83, 526-532). In the mouse, antisense oligonucleotides targeting the start codon of each of the three subunits have been designed and used to investigate the process of neuritogenesis (Ulloa et al.,
Embo J
., 1993, 12, 1633-1640; Ulloa et al.,
Cell. Mol. Neurobiol
., 1994, 14, 407-414).
Antisense oligonucleotides, targeting the start codon of each of the subunits of human casein kinase 2, have also been reported (Pepperkok et al.,
Exp. Cell. Res
., 1991, 197, 245-253; Pyerin et al.,
Ann. N.Y. Acad. Sci
., 1992, 660, 295-297). In these studies of human fibroblasts (IMR-90 cells), antisense oligonucleotides were used to investigate the role of casein kinase 2 in early growth stimulation by epidermal growth factor (Pepperkok et al.,
Exp. Cell. Res
., 1991, 197, 245-253; Pyerin et al.,
Ann. N.Y. Acad. Sci
., 1992, 660, 295-297).
In MDA231 breast carcinoma cell lines, an antisense oligonucleotide targeting the start codon of the alpha prime subunit of human casein kinase 2 was used to investigate alternative splicing patterns in the CD44 gene (Formby and Stern,
Mol. Cell. Biochem
., 1998, 187, 23-31). And in human Ca9-22 cells derived from squamous cell carcinomas of the head and neck (SCCHN), transfection with a phosphodiester antisense oligonucleotide targeting the alpha subunit of human casein kinase 2 resulted in growth inhibition of the carcinoma cell line (Faust et al.,
Head Neck
, 2000, 22, 341-346).
Antisense technology is emerging as an effective means
Freier Susan M.
McKay Robert
Wyatt Jacqueline
ISIS Pharmaceuticals Inc.
LeGuyader John L.
Licata & Tyrrell P.C.
Schmidt M
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