Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-05-19
2004-07-27
Helms, Larry R. (Department: 1642)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C536S023100, C536S023500
Reexamination Certificate
active
06767720
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from pending European Patent Application EP 99204512.0 filed on Dec. 23, 1999, the contents of which are herein incorporated by this reference.
TECHNICAL FIELD
The invention relates to the field of drug discovery, diagnosis and treatment of cancer and neurological disorders. More particularly, the invention relates to a new zinc finger protein binding to a member of the a-catenins/vinculin family and discloses that human a-catulin specifically interacts with Rho-GEF Brx/proto-Lbc.
BACKGROUND
Despite extensive knowledge relating to the multitude of cancer forms (varying in appearance from solid tumours and related metastases in distinct parts of the body to leukaemias of blood cells that circulate throughout the body, and varying from being totally benign to being aggressively malignant), effective treatment of cancer remains difficult and, in general, is restricted to three types of treatments: radiation therapy; chemotherapy; and surgical therapy. Possibilities for a more specific therapy, directed against the underlying cause of a specific cancer, or groups of cancers, are currently virtually non-existant. Extensive efforts are currently being directed at providing such specific therapies through drug discovery processes aimed at identifying candidate drugs for treatment of specific cancers, groups of cancer, or neurological disorders.
Development of cancer often starts with changes in a first cell that lead to the unrestricted development and division of that first cell into an ever dividing population of cells. These changes are often an accumulation of mutations or other alterations in key genes that occur chronologically, whereby the mutated cell population looses its original, often specialised character and acquires more and more of a cancerous nature. The normal processes of growth regulation are generally dysfunctional in the altered cells. Transcription of genes that are normally infrequently expressed in non-cancerous cells may no longer be controlled in cancerous cells.
Activation of transcription of genes by transcription factors that would otherwise be dormant in the specific cell type can, for example, lead to the typical unrestricted growth and neoplastic nature of cancer. Examples are mutations in suppressor genes that function normally by generating proteins that suppress transcriptional pathways which are no longer of use in a specialised cell. Mutated suppressor genes no longer help to keep the growth of a cell under control. Drugs directed against or intervening with the specific protein-protein or protein-DNA interactions in transcriptional and/or signalling pathways controlling cell growth, cell differentiation or development can be considered typical candidate drugs for later use in specific therapies for cancer or neurological disorders, especially when the pathways that such drugs target have gone awry, leading to unrestricted growth or aberrant differentiation of cells. In the case of &agr;N-catenin, which is mainly neurally expressed, a broader or additive interpretation in relationship to neural dysfunctions is obvious.
The cadherin superfamily represents several cadherins which function in cell-cell adhesion, morphogenesis and tissue homeostasis (Takeichi, 1991; Kemler, 1992; Suzuki, 1996). The transmembrane glycoprotein E-cadherin is the best-studied prototype of this family and has been identified as a potent suppressor of invasion (Behrens et al., 1989; Frixen et al., 1991; Vleminckx et al., 1991). Recent studies revealed proof for a tumour suppressor role of human E-cadherin, as the encoding gene behaves according to the two-hit model of Knudson (1985) in infiltrative lobular cancers (Berx et al., 1995 and 1996) and diffuse gastric cancers (Becker et al., 1994 and 1996).
Cadherins function as cell-cell adhesion molecules by homophilic interactions with other cadherin molecules, but linkage to the actin cytoskeleton is also essential. The latter is achieved by the catenins (catena means chain) (Ozawa et al., 1990; Cowin, 1994), which comprise the Armadillo proteins (e.g. &bgr;-catenin, plakoglobin and p120
ctn
) and the vinculin-like &agr;-catenins. The Armadillo catenins are proteins, known to be associated with the cytoplasmic domain of cadherins. In turn, the &agr;-catenins link &bgr;-catenin and plakoglobin to the actin cytoskeleton.
These catenins were also found to be associated with APC, a cytoplasmic tumour suppressor gene product (adenomatous polyposis coli) (Peifer, 1993; Su et al., 1993). A typical example of a signal transduction pathway (via &bgr;-catenin) gone wrong and leading to development of cancer, can be found with APC. The APC protein is linked to the microtubular cytoskeleton. Moreover, in the desmosomes, plakoglobin mediates a link between desmosomal cadherins and the cytokeratin cytoskeleton via desmoplakin (Korman et al., 1989; Kowalczyk et al., 1997).
Two subtypes of &agr;-catenin have been identified, &agr;E-catenin (epithelial form) (Nagafuchi et al., 1991; Herrenknecht et al., 1991) and &agr;N-catenin (neural form) (Hirano et al., 1992). Moreover, for both subtypes two isoforms, resulting from alternative splice events, have been identified (Oda et al., 1993; Uchida et al., 1994; Rimm et al., 1994). A tissue specific distribution for either of the subtypes has been reported. The epithelial &agr;E-catenin is expressed in a wide variety of tissues, but only low levels of expression have been observed in the central nervous system (CNS) (Nagafuchi et al., 1991). In contrast, &agr;N-catenin expression is more restricted to particular tissues including the nervous system, in which it is generally expressed (Hirano et al., 1992; Uchida et al., 1994).
A new homologue of the &agr;-catenins was identified and termed &agr;-catulin (Janssens et al., 1999). The &agr;-catulin cDNA has been found to be expressed in most tissues except in neural tissues. The &agr;-catulin protein shows 25% identity with the alpha-catenins, but provides higher sequence conservation in some putative functional domains.
Recently, key regulators of cadherin-mediated adhesiveness were identifed as proteins of the small-GTPase family. This family consists of the subfamilies Ras and Rho (reviewed in Braga et al., 1999). Ras GTPases are involved in growth control and differentiation. Rho GTPases participate in cytoskeletal reorganisation, activation of kinase cascades, induction of gene transcription and DNA synthesis (reviewed by Mackay and Hall, 1998). The Rho family of GTPases consists of Rho, Rac and CDC42 molecules, showing different specific effects (reviewed in Kaibuchi et al., 1999). Rho is involved in formation of stress fibers and focal adhesions, cell morphology and cell aggregation, including cadherin functionality, cell motility, membrane ruffling, smooth muscle contraction, neurite retraction in neuronal cells and cytokinesis. Rac is involved in membrane ruffling, cell motility, actin polymerization and cadherin-mediated adhesion. Cdc42 participates in filopodia formation.
Inside the cell, these GTPases are normally found associated with GDP and therefore in an inactive state. Activation occurs upon binding to GTP, a process that is tightly regulated by activating GEFs (guanine nucleotide exchange factors) and inactivating GAPs (GTPase activating factors). These transitions between GDP-bound and GTP-bound states are important regulatory processes, as for example constitutively active Rho can induce transformation in tissue culture. Moreover, deletions in Rho-GEFs such as Lbc, Vav and Dbl activate small GTPases (reviewed by Cerione and Zheng, 1996).
For the formation of cadherin-dependent cell-cell contacts, activity of endogenous Rho and Rac is required (Braga et al., 1997). Inhibition of Rho or Rac results in removal of cadherins and other molecules involved in cell-cell adhesion (Takaishi et al., 1997). Interestingly, Rac and some Rac-specific regulatory proteins like Tiam-1 and IQGAP are found to be localized to cell-cell contact sites (Habets et al., 1994; Kuroda
Janssens Barbara
van Landschoot Ann
van Roy Frans
Helms Larry R.
TraskBritt
Vlaams Interuniversitair Instituut voor Biotechnologie VZW
Yu Misook
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