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
1999-12-20
2002-04-30
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S243000, C435S252300, C435S254200, C435S320100, C435S325000, C435S410000, C536S023500
Reexamination Certificate
active
06379925
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
The int-3 gene was originally identified based on its oncogenic effects in the mouse mammary gland. int-3 is a frequent target for insertional activation by MMTV proviral DNA in MMTV-induced mammary gland tumors (Gallahan and Callahan, 1987; Robbins et al., 1992; Sarkar et al., 1994). Tumor specific transcripts derived from the int-3 gene encode a protein homologous to the intracellular part of the Notch family of cell surface receptors. Exogenous expression of the int-3 oncoprotein has been shown to affect the growth and development of mammary epithelial cells. Over expression of the int-3 oncoprotein in a mouse mammary epithelial cells (HC11) promotes anchorage independent growth (Robbins et al., 1992). Expression of int-3 as an MMTV-LTR driven transgene in the mouse mammary gland results in abnormal development of the mammary gland and rapid development of undifferentiated mammary carcinomas (Jhappan et al., 1992). In the normal mouse mammary gland, endogenous int-3 protein has been detected in mammary stroma and epithelium (Smith et al., 1995).
Members of the Notch/lin-12 gene family were first identified in Drosophila and
Caenorhabditis elegans
through genetic analysis of mutations that alter cell fate decisions (for review see Artavanis-Tsakonas et al., 1995; Artavanis-Tsakonas and Simpson, 1991; Greenwald and Rubin, 1992). Drosophila Notch regulates multiple cell fate decisions that involve cell-cell interactions during fly development, for instance, control of cell fate decisions involving neural/epidermal specification in proneural clusters (Artavanis-Tsakonas and Simpson, 1991). The
C. elegans
lin-12 and glp-1 proteins are structurally related to Notch, and are also involved in cell fate specifications during development in the nematode (Greenwald, 1985; Yochem and Greenwald, 1989). Genetic analysis of Notch/lin-12 genes suggest that this family of genes controls binary cell fate decisions and inductive signaling that depend on cell-cell interactions (reviewed in Artavanis-Tsakonas et al., 1995; Greenwald, 1994; Greenwald and Rubin, 1992). Alternatively, Notch/lin-12 genes have been proposed to block cell differentiation, thus maintaining the competence of cells for subsequent cell-fate determination (Coffman et al., 1993; Fortini et al., 1993).
Notch/lin-12 genes encode transmembrane receptor proteins characterized by highly repeated, conserved domains. The amino terminus of Notch proteins encodes the extracellular domain and contains as many as 36 repeats of an EGF-like motif involved in ligand binding (Rebay et al., 1993), and three tandem copies of a Notch/lin-12 sequence motif of unknown function. The intracellular portion of Notch proteins is characterized by six tandem copies of a cdc10/ankyrin motif, thought to be a protein-protein interaction domain (Michaely and Bennett, 1992), and a PEST sequence motif which may represent a protein degradation signal (Rogers et al., 1986). In several systems, truncated forms of Notch/lin-12 proteins that contain an intact intracellular domain without most of the extracellular domain behave as constitutively activated receptors (reviewed in Artavanis-Tsakonas et al., 1995; Greenwald, 1994). The human Notch 1 orthologue, TAN-1, was first identified in independently isolated translocation breakpoints in acute T lymphoblastic leukemia and is predicted to encode a truncated product that has an intact intracellular domain but lacks most of the extracellular domain (Ellisen et al., 1991). Similarly, the int-3 oncoprotein encodes the intracellular domain of a Notch-like protein and thus has been proposed to act as an activated Notch receptor (Robbins et al., 1992).
Based on sequence similarity to Drosophila Notch, additional Notch-related genes have been isolated from mammals; including mouse (Franco Del Amo et al., 1993; Lardelli et al., 1994; Lardelli and Lendahl, 1993; Reaume et al., 1992), rat (Weinmaster et al., 1992; Weinmaster et al., 1991), and human (Ellisen et al., 1991; Stifani et al., 1992; Sugaya et al., 1994). To date, three Notch homologues, Notch1, Notch2, and Notch3, have been identified in the mouse and their embryonic expression patterns display partially overlapping but distinct patterns of expression that are consistent with a potential role in the formation of the mesoderm, somites, and nervous system (Williams et al., 1995). Abundant expression of Notch1, Notch2, and Notch3 is found in proliferating neuroepithelium during central nervous system development. Targeted disruption of the Notch1 gene in mice results in embryonic death during the second half of gestation (Conlon et al., 1995; Swiatek et al., 1994) and homozygous mutant embryos display delayed somitogenesis as well as widespread cell death, preferentially in neuroepithelium and neurogenic neural crest (Conlon et al., 1995; Swiatek et al., 1994).
The gene products of Drosophila Delta (Vassin et al., 1987) and Serrate (Fleming et al., 1990), and
C. elegans
Lag-2 (Henderson et al., 1994; Tax et al., 1994) and Apx-1 (Mello et al., 1994) are thought to act as ligands for Notch proteins. In the mouse, the orthologue of Delta, referred to a Dll1 (Delta-like gene 1), is expressed during embryonic development in the paraxial mesoderm and nervous system in a pattern similar to that of mouse Notch1 (Bettenhausen et al., 1995). A murine Serrate-related gene named Jagged has been identified and is partially co-expressed with murine Notch genes in the developing spinal cord (Lindsell et al., 1995).
The identification and expression analysis of a fourth murine Notch homologue is reported here. The fourth murine Notch homologue has been named Notch4 and the int-3 nomenclature has been reserved for the truncated oncogene. Although the intracellular domain of the int-3 oncoprotein shares homology with the Notch/Lin-12 protein family, a comparison of the full length Notch4 protein to that of the int-3 oncoprotein is now provided. The activated int-3 protein encodes only the transmembrane and intracellular domain of the Notch4 protein. The predicted amino acid sequence of Notch4 contains the conserved features of all Notch proteins, however Notch4 has 7 fewer EGF-like repeats compared to Notch
1
and Notch2 and contains a significantly shorter intracellular domain. Notch4 is expressed primarily in embryonic endothelium and in adult endothelium and male germ cells.
This invention uses an established cell line, Rat Brain Microvessel Endothelial cells (RBE4 cells), to test Notch4 activity and to demonstrate that activated Notch4 and Notch4 ligand stimulate angiogenesis in these cells. RBE4 cells grown on collagen coated plates in the absence of bFGF or with low concentrations of bFGF (1 ng/ml), display a cobblestone morphology. In the presence of high concentration of bFGF (5 ng/ml), RBE4 cells exhibit a spindle shape morphology. When RBE4 cells reach confluency, they growth arrest and cells retain their cobblestone morphology. Post-confluent RBE4 cell cultures grown in the presence of 5 ng/ml bFGF, develop multicellular aggregates from the cobblestone monolayer. These three-dimensional structures are sprouts that extend above the monolayer and some of these sprouts will organize into curvilinear and bifurcating structures that float in the cell culture medium. The RBE4 cell sprouts and three-dimensional structures contain high activity of several enzymatic markers that are specific for differentiated microvessels. Thus, bFGF, a known angiogenic agent, induces angiogenesis of RBE4 cells which upon treatment with high concentrations of bFGF develop structur
Kitajewski Jan
Uyttendaele Hendrik
Cooper & Dunham LLP
McKelvey Terry
The Trustees of Columbia University in the City of New York
White John P.
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