Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-06-21
2002-12-10
Nguyen, Dave T. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C536S024100, C435S320100
Reexamination Certificate
active
06492344
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of cancer biology and therapy. Specifically, the invention is directed to methods for altering the differentiated state of a cell by altering syndecan-1 expression. The method allows for the normalization of the growth rate and differentiation state of malignant cells, and is based on the stimulation of syndecan-1 expression in the malignant cells. Re-expression of syndecan-1 in such malignant cells promotes their normal differentiated phenotype and prevents their formation into tumors. This method may also be applied to normal cells to maintain their expression of genes characteristic of the differentiated state, e.g., the method may be used to prevent baldness by maintaining keratin production.
In addition, the invention is directed to transcription regulatory elements associated with the syndecan-1 gene, such as the FGF-inducible Response Element (FiRE) and to the use of these transcription regulatory elements for controlling gene expression. The FiRE can also be used to target expression of a reporter gene to wound sites in vivo.
BACKGROUND OF THE INVENTION
Cell surface proteoglycans play an important role in the regulation of cell behavior (Ruoslahti et al.,
Cell
64:867-869 (1991)). Through their covalently bound glycosaminoglycan side chains, such proteoglycans can bind various extracellular effector molecules (Jalkanen, et al., in
Receptors for Extracellular Matrix,
J. MacDonald & R. Mecham, eds., Academic Press, San Diego, pp. 1-37 (1991)). One central challenge in proteoglycan biology is to understand the biological consequences which result from the binding of different effector molecules to cell surface proteoglycans. It is important to determine the intracellular responses triggered by effector binding and how these responses lead to altered cellular behavior. One way to investigate these matters is to create biological models which are dependent on the expression of specific proteoglycans.
Syndecan-1 is the best characterized cell surface proteoglycan (Saunders et al.,
J. Cell Biol.
108:1547-1556(1989); Mali et al.,
J. Biol. Chem.
265:6884-6889 (1990)). It was originally isolated from mouse mammary epithelial (NMuMG) cells as a hybrid proteoglycan containing both heparan sulfate and chondroitin sulfate glycosaminoglycan side chains (Rapraeger et al.,
J. Biol. Chem.
260:11046-11052 (1985)). Recent studies have revealed its expression, not only on epithelial cells but also on differentiating fibroblasts of developing tooth (Thesleff et al.,
Dev. Biol.
129:565-572 (1988); Vainio et al.,
J. Cell Biol.
108:1945-1964 (1989)), on endothelial cells of sprouting capillaries (Elenius et al.,
J. Cell Biol.
114:585-596 (1991)) and on the surface of lymphocyte subpopulations (Sanderson et al.,
Cell Regul.
1:27-35 (1989)). This suggests that syndecan-1 function can vary from one cell type to another. Syndecan belongs to a family of proteoglycans with conserved plasma membrane and cytoplasmic domains but with dissimilar ectodomains (Mali et al.,
J. Biol. Chem.
265:6884-6889 (1990)). The conserved structure of syndecans suggests that it could participate in signal transduction through the plasma membrane.
Syndecan-1 binds several extracellular effector molecules but does so in a selective manner. For example, syndecan binds interstitial collagens and fibronectin but does not bind vitronectin or laminin (Koda et al.,
J. Biol. Chem.
260:8156-8162 (1985)); Saunders et al.,
J. Cell Biol.
106:423-430 (1988); Elenius et al.,
J. Biol. Chem.
265:17837-17843 (1990)). Moreover, syndecan-1 isolated from tooth mesenchyme has revealed selective binding to tenascin not observed for syndecan from NMuMG cells (Salmivirta et al.,
J. Biol. Chem.
266:7733-7739 (1991)). This suggests that variations in syndecan glycosylation alters the binding properties of syndecan. Polymorphism of syndecan-1 glycosylation has also been observed in simple and stratified epithelia (Sanderson et al.,
Proc. Natl. Acad Sci. USA
85:9562-9566 (1988)); but whether these changes also reflect altered ligand recognition by syndecan remains unknown. Syndecan-1 also binds growth factors, such as basic fibroblast growth factor (Kiefer et al.,
Proc. Natl. Acad. Sci. USA
87:6985-6989 (1990); Elenius et al.,
J. Biol. Chem.
267:6435-6441 (1992)).
Fibroblast growth factors (FGFs) are a family of heparin-binding peptides comprising 9 known members. Basic fibroblast growth factor (FGF-2 or bFGF) is synthesized by, and acts on various cell types and tissues. In vitro, it is a strong mitogen for cells of mesodermal origin, can modulate cell motility and differentiation, is a potent angiogenic factor, and potentiates neovascularization in vivo (Burgess and Magiac,
Ann. Rev. Biochem.
58:575-606 (1989); Mason,
Cell
78:547-552 (1994)). Keratinocyte growth factor (FGF-7 or KGF) is produced solely by cells of mesodermal origin. FGF-7 is proliferative for various epithelial cells (Basilico and Moscatelli,
Adv. Cancer Res.
59:115-65 (1992); Rubin et al.,
Cell Biol. Int.
19:399-411 (1995)), and is also an important mediator of hair follicle growth and differentiation (Danilenko et al.,
Am. J. Pathol.
147:145-54 (1995); Guo et al.,
Genes
&
Develop.
10:165-75 (1996)). Both FGF-2 and FGF-7 are involved in wound healing, where they act as both autocrinic and paracrinic factors. In wounded skin FGF-2 is found in fibroblasts and endothelial cells. This growth factor stimulates proliferation of most cell types involved in wound healing, e.g., keratinocytes, fibroblasts, and vascular and capillary endothelial cells (Bennett et al.,
Am. J. Surg.
165:728-737 (1993)). FGF-7, which is synthesized only by fibroblasts and is induced during wound healing (Wemer et al.,
Science
266:819-22 (1994); Werner et al.,
Proc. Natl. Acad. Sci.
89:6898-6900 (1992), and acts as a paracrinic factor on keratinocytes, inducing their proliferation and migration (Bennett et al., supra). FGF-7 is important for normal wound reepithelialization (Werner et al. (1994), supra). However, recent data with FGF-7 knockout mice indicate that KGF may not be required for normal wound healing (Guo et al.,
Genes
&
Develop.
10:165-175 (1996).
Growth factors are involved in the initiation, control, and termination processes of wound healing in an autocrinic and paracrinic manner. FGF-2 is produced by fibroblasts and is also found in association with extracellular matrix and basement membranes where it can be released by proteolytic activity. FGF-2 enhances the accumulation and proliferation of fibroblasts, keratinocytes, endothelial cells, and macrophages. In animal models it induces neovascularization, cell migration, and granulation tissue formation. It has been shown to accelerate wound healing in several different situations, e.g., incisions, burns, and diabetic wounds (See Bennett and Schulz,
Am. J. Surg.
165: 728-737 (1993)).
Several AP-1 regulated genes are expressed during wound healing. Fos is rapidly activated on the wound healing edge (Martin and Nobes, Mech. Dev. 38: 209-215 (1992)). Jun may also be activated during wounding or wounding induced tumorigenesis (Marshall et al.,
Virology
188(1): 373-379 (1992). Cancerous cells are also known to be able to activate the AP-1 complex, and c-fos is required for malignant tumor progression (Saez et al.,
Cell
82:721-732 (1995)).
Yayon and coworkers (Yayon et al.,
Cell
64:841-848 (1991)) and Rapraeger and coworkers (Rapraeger et al.,
Science
252:1705-1708 (1991)) have shown that heparin-like molecules are required for the binding of FGF-2 to its high affinity receptor, indicating that syndecan-like molecules can also modulate the growth factor response. It has been observed that heparin is required for oligomerization of FGF-1 molecules leading to FGFR dimerization or further oligomerization and further signaling (Spivak-Kroizman et al.,
Cell
79:1015-24 (1994); Ornitz et al.,
Mol. Cell Biol.
12:240-47 (1992)). Several mechanisms, for both negative and positive regulation for FGF action by proteoglycans have been postu
Jaakkola Panu
Jalkanen Markku
Vihinen Tapani
Biotie Therapies Corp.
Nguyen Dave T.
Shukla Ram R.
Sterne Kessler Goldstein & Fox P.L.L.C.
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