Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-03-30
2001-05-15
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C530S387900, C536S023500, C536S024310
Reexamination Certificate
active
06232064
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to the field of growth factors and specifically to Connective Tissue Growth Factor (CTGF), a polynucleotide encoding this factor and methods of use for CTGF.
2. Related Art
Growth factors are a class of secreted polypeptides that stimulate target cells to proliferate, differentiate and organize in developing tissues. The action of growth factors is dependent on their binding to specific receptors which stimulates a signalling event within the cell. Examples of some well-studied growth factors include platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor beta (TGF-&bgr;), transforming growth factor alpha (TGF-&agr;), epidermal growth factor (EGF), and fibroblast growth factor (FGF).
PDGF is a cationic, heat-stable protein found in the alpha-granules of circulating platelets and is known to be a mitogen and a chemotactic agent for connective tissue cells such as fibroblasts and smooth muscle cells. Because of the activities of this molecule, PDGF is believed to be a major factor involved in the normal healing of wounds and pathologically contributing to such diseases as atherosclerosis and fibrotic diseases. PDGF is a dimeric molecule consisting of an A chain and a B chain. The chains form heterodimers or homodimers and all combinations isolated to date are biologically active.
Studies on the role of various growth factors in tissue regeneration and repair have led to the discovery of PDGF-like proteins. These proteins share both immunological and biological activities with PDGF and can be blocked with antibodies specific to PDGF.
These new growth factors may play a significant role in the normal development, growth, and repair of human tissue. Therapeutic agents derived from these molecules may be useful in augmenting normal or impaired growth processes involving connective tissues in certain clinical states, e.g., wound healing. When these growth factors are involved pathologically in diseases, therapeutic developments from these proteins may be used to control or ameliorate uncontrolled tissue growth.
The formation of new and regenerating tissue requires the coordinate regulation of various genes that produce both regulatory and structural molecules which participate in the process of cell growth and tissue organization. Transforming growth factor beta (TGF-&bgr;) appears to be a central regulatory component of this process. TGF-&bgr; is released by platelets, macrophages and neutrophils which are present in the initial phases of the repair process. TGF-&bgr; can act as a growth stimulatory factor for mesenchymal cells and as a growth inhibitory factor for endothelial and epithelial cells. The growth stimulatory action of TGF-&bgr; appears to be mediated via an indirect mechanism involving autocrine growth factors such as PDGF BB or PDGF AA or connective tissue growth factor (CTGF).
Several members of the TGF-&bgr; superfamily possess activities suggesting possible applications for the treatment of cell proliferative disorders, such as cancer. In particular, TGF-&bgr; has been shown to be potent growth inhibitor for a variety of cell types (Massague,
Cell
49:437, 1987), MIS has been shown to inhibit the growth of human endometrial carcinoma tumors in nude mice (Donahoe, et al.,
Ann. Surg.
194:472, 1981), and inhibin &agr; has been shown to suppress the development of tumors both in the ovary and in the testis (Matzuk, et al.,
Nature,
360:313, 1992).
Many of the members of the TGF-&bgr; family are also important mediators of tissue repair. TGF-&bgr; has been shown to have marked effects on the formation of collagen and causes of striking angiogenic response in the newborn mouse (Roberts, el al.,
Proc. Natl. Acad. Sci., USA
83:4167,1986). The bone morphogenic proteins (BMPs) can induce new bone growth and are effective for the treatment of fractures and other skeletal defects (Glowacki, el al.,
Lancet,
1:959, 1981; Ferguson, et al.,
Clin. Orthoped. Relat. Res.,
227:265, 1988; Johnson, et al.,
Clin Orthoped. Relat. Res.,
230:257, 1988).
The isolation of growth factors and the genes encoding them is important in the development of diagnostics and therapeutics for various connective tissue-related disorders. The present invention provides such an invention.
SUMMARY OF THE INVENTION
Various cell types produce and secrete PDGF and PDGF-related molecules. In an attempt to identify the type of PDGF dimers present in the growth media of cultured endothelial cells, a new growth factor was discovered. This previously unknown factor, termed Connective Tissue Growth Factor (CTGF), is related immunologically and biologically to PDGF, however it is the product of a distinct gene.
In a first aspect, the present invention provides a polypeptide growth factor for connective tissue cells. The polypeptide is a mitogenic agent and a chemotactic agent for cells.
In a second aspect, the present invention provides a polynucleotide encoding a connective tissue growth factor characterized as encoding a protein (1) existing as a monomer of approximately 36-38 kD molecular weight, and (2) capable of binding to a PDGF receptor.
In a further aspect, the invention provides a method for accelerating wound healing in a subject by applying to the wound an effective amount of a composition which contains CTGF.
In yet another aspect, the invention provides a method of diagnosing pathological states in a subject suspected of having pathology characterized by a cell proliferative disorder which comprises, (1) obtaining a sample suspected of containing CTGF from the subject, (2) determining the level of CTGF in the sample, and (3) comparing the level of CTGF in the sample to the level of CTGF in normal tissues.
A method of ameliorating diseases characterized by a cell proliferative disorder, by treating the site of the disease with an effective amount of a CTGF reactive agent is also provided.
The present invention identifies a TGF-&bgr; responsive or regulatory element in the 5′ untranslated nucleotides of the CTGF gene (or −157 to −145). Based on the identification of this element, the invention now provides a method for identifying a composition which affects CTGF expression comprising incubating components comprising the composition and TGF-&bgr; regulatory element (T&bgr;RE), in the presence of a TGF-&bgr; factor which regulates T&bgr;RE, and measuring the effect of the composition on CTGF expression. Thus, the invention provides a means for drug discovery for treatment of fibrotic diseases.
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Bradham, Jr. Douglass M.
Grotendorst Gary R.
Gray Cary Ware & Freidenrich LLP
Haile Lisa A.
Spector Lorraine
University of South Florida
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