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
1999-01-15
2001-09-25
Carlson, Karen Cochrane (Department: 1653)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S068100, C530S350000, C530S395000
Reexamination Certificate
active
06294356
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to two forms of laminin-5. One of these forms promotes the migration of epithelial cells. The other form promotes hemidesmosome assembly. The invention also relates to methods of making and using the two forms of laminin-5 and to products comprising them.
BACKGROUND
Epithelial cells are separated from connective tissue by a basement membrane, which is composed of a variety of extracellular matrix molecules, including proteoglycans, collagen and laminin isoforms. Together these proteins create a framework that is essential for maintaining tissue integrity. However, extracellular matrix proteins play more than just a structural role. They also display a diverse set of biological functions that regulate adhesion, migration, proliferation, differentiation and gene expression of adjacent cells. Roskelly, et al.,
Curr. Op. Cell Biol.
7:736-747 (1995).
Laminin, of which there are at least ten isoforms, is a major component of basement membranes and has been shown to mediate cell-matrix attachment, gene expression, tyrosine phosphorylation of cellular proteins, and branching morphogenesis. See, e.g. Streuli, et al.,
J. Cell Biol.,
129:591-603 (1993); Malinda and Kleinman,
Int. J. Biochem. Cell Biol.
28:957-1959 (1996); Timpl and Brown,
Matrix Biol.
14:275-281 (1994); Tryggvason,
Curr. Op. Cell Biol
5:877-882 (1993); Stahl, et al.,
J. Cell Sci.
110:55-63 (1997). The expression patterns of the laminin isoforms are tissue specific. The laminin-5 isoform (nicein, epiligrin, kalinin) is abundant in transitional epithelium, stratified squamous epithelia, lung mucosa and other epithelial glands. Kallunki, et al.,
J. Cell Biol.
119:679-693 (1992); Jones, et al., unpublished observations. Laminin-5 is a heterotrimer consisting of &agr;3, &bgr;3 and &ggr;2 subunits that associate by means of large &agr;-helical regions to produce a cruciform-shaped molecule. Rousselle, et al.,
J. Cell Biol.
114:567-76 (1991); Baker, et al., J. Cell Sci. 109:2509-2520 (1996).
Laminin-5 is synthesized initially as a 460 kD molecule, which undergoes specific processing to a smaller form after being secreted into the extracellular matrix. Marinkovich, et al.,
J. Biol. Chem.,
267:17900-17906 (1992); Matsui, et al.,
J. Biol. Chem.
270:23496-23503 (1995); Vailly, et al.,
Eur. J. Biochem.
219:209-218 (1994). The size reduction is a result of processing of the &agr;3 and &ggr;2 subunits from 200-190 to 160 kD and from 155 to 105 kD, respectively. Marinkovich, et al.,
J. Biol. Chem.,
267:17900-17906 (1992); Matsui, et al.,
J. Biol. Chem.
270:23496-23503 (1995); Vailly, et al.,
Eur. J. Biochem.
219:209-218 (1994). The proteases involved in these proteolytic events have not been identified.
Laminin-5 has been reported to function in the nucleation of hemidesmosome assembly and as an adhesive factor that retards cell motility. Baker, et al.,
J. Cell Sci.
109:2509-2520 (1996); O'Toole, et al.,
Exp. Cell Res
., in press. In contrast, some authors have provided evidence that laminin-5 enhances cell motility and is expressed at the migrating edges of certain tumor cell populations. Zhang and Kramer,
Exp. Cell Res.
227:309-333 (1996); Pyke, et al.,
Am. J. Pathol.
145:782-791 (1994); Pyke, et al.,
Cancer Res.
55:4132-4139 (1995).
In a number of studies, it has been demonstrated that laminin-5 produced by 804G cells can nucleate the assembly of hemidesmosomes by SCC12, HaCaT and pp126 cells, as well as corneal cells maintained in vitro. Langhofer, et al.,
J. Cell Sci.
105:753-764 (1993); Hormia, et al.,
J. Invest. Dermatol.
105:557-561 (1995); Baker, et al.,
J. Cell Sci.
109:2509-2520 (1996); Baker, et al.,
Exp. Cell Res.
228:262-270 (1996); Tamura, et al.,
J. Periodontal. Res
., in press. Although SCC12, HaCaT and pp126 cells also secrete laminin-5, the laminin-5 that they secrete is incapable of supporting the assembly of hemidesmosomes.
SUMMARY OF THE INVENTION
The invention is based on the discovery that laminin-5 which promotes hemidesmosome assembly (“hemidesmosome-promoting laminin-5”) contains a smaller &agr;3 subunit (“hemidesmosome-promoting &agr;3 subunit”) than laminin-5 which does not promote hemidesmosome assembly. The hemidesmosome-promoting &agr;3 subunit is produced by proteolytic cleavage of the unprocessed &agr;3 subunit. The phrase “unprocessed &agr;3 subunit” is used herein to refer to the &agr;3 subunit as it is initially produced by cells and, of course, prior to the proteolytic processing which produces the hemidesmosome-promoting &agr;3 subunit. The invention is also based on the further discovery that laminin-5 comprising the unprocessed &agr;3 subunit promotes epithelial cell migration.
In particular, the invention provides methods of generating hemidesmosome-promoting laminin-5 comprising proteolytic cleavage of the unprocessed &agr;3 subunit of laminin-5 by plasmin. In one embodiment, the method comprises culturing epithelial cells that do not produce hemidesmosome-promoting laminin-5 under conditions effective so that they produce extracellular matrix protein comprising laminin-5 containing unprocessed &agr;3 subunits and contacting the laminin-5 with plasmin under conditions effective so that the plasmin cleaves the &agr;3 subunits. In another embodiment, the method comprises producing heterotrimeric laminin-5 containing unprocessed &agr;3 subunits by expressing DNA coding for &agr;3, &ggr;2 and &bgr;3 subunits in host cells transformed with the DNA and contacting the laminin-5 with plasmin under conditions effective so that the plasmin cleaves the &agr;3 subunits. In yet another embodiment, the method comprises contacting a material containing unprocessed &agr;3 subunits of laminin-5, but not &ggr;2 subunits or ⊖3 subunits of laminin-5, with plasmin under conditions effective so that the plasmin cleaves the &agr;3 subunits. The cleaved &agr;3 subunits are combined with &ggr;2 and &bgr;3 subunits so that the three subunits combine to form hemidesmosome-promoting laminin-5.
The invention further provides a method of stimulating hemidesmosome assembly by contacting epithelial cells with hemidesmosome-promoting laminin-5 produced by the methods of the invention. It is desirable to grow epithelial cells for various applications on hemidesmosome-promoting laminin-5 since the organization of epithelial cells grown on hemidesmosome-promoting laminin-5 is significantly more advanced and tissue-like than cells grown on laminin-5 which does not promote hemidesmosome formation.
In addition, the invention provides hemidesmosome-promoting laminin-5 and hemidesmosome-promoting &agr;3 subunits of laminin-5. The invention further provides shaped articles coated with hemidesmosome-promoting laminin-5 and pharmaceutical compositions comprising hemidesmosome-promoting laminin-5.
The invention also provides a method of promoting wound healing. The method comprises contacting the wound with an amount of laminin-5 comprising unprocessed &agr;3 subunits which is effective to promote the migration of epithelial cells into the wound. The method may further comprise contacting the wound with an amount of plasmin effective to convert the laminin-5 into hemidesmosome-promoting laminin-5 so that the epithelial cells in the wound are stimulated to assemble hemidesmosomes.
The invention further provides a method of promoting epithelial cell migration. The method comprises contacting epithelial cells with an amount of laminin-5 comprising unprocessed &agr;3 subunits which is effective to promote the migration of the epithelial cells.
In addition, the invention provides a method of promoting epithelialization of a surface. The method comprises contacting the surface with an amount of laminin-5 comprising unprocessed &agr;3 subunits which is effective to promote the migration of epithelial cells over the surface. The method may further comprise contacting the surface with an amount of plasmin effective to convert the laminin-5 into hemidesmosome-promoting laminin-5 so that the epithelial cells on the surface are stimulated to asse
Goldfinger Lawrence E.
Jones Jonathan C. R.
Stack M. Sharon
Carlson Karen Cochrane
Northwestern University
Sheridan & Ross P.C.
Tu Stephen
LandOfFree
Methods and materials for making and using laminin-5 does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Methods and materials for making and using laminin-5, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods and materials for making and using laminin-5 will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2495458