Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-05-14
2001-12-04
Clark, Deborah J. R. (Department: 1633)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100
Reexamination Certificate
active
06326484
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of regulation of transcription and more specifically to genes encoding polypeptide regulators of fibroblast growth factor-2 (FGF-2) expression.
BACKGROUND OF THE INVENTION
Many transcription factors are sequence-specific DNA-binding proteins. In general, transcription factors contain two functional domains, one for DNA-binding and one for transcriptional activation or suppression. These functions often reside within structural domains that retain their function when removed form their natural context. The DNA-binding domains of transcription factors falls into several structural families based on their primary amino acid sequence. Examples of families include the helix-tuml-helix, zinc finger, leucine zipper, and helix-loop helix proteins.
Helix-turn-helix proteins have a protein helix which lies across the major grove of the DNA helix and makes contact with exposed base pairs. These proteins contain a second helix that lies across the first and contacts other proteins in the transcription apparatus. Zinc finger proteins have a repeated motif of cysteine and histidine that are thought to fold up into a three-dimensional structure coordinated by a zinc ion. Examples of zinc finger proteins are Sp1, and steroid receptor proteins. Leucine zipper proteins have repeats of four of five leucine residues precisely seven amino acids apart which provide hydrophobic faces though which the proteins can form dimers. Adjacent to the leucine zipper is a domain enriched for positively charged amino acids, arginine and lysine. This DNA-binding domain is amino terminal to the leucine zipper. Examples of leucine zippers are c-fos and c-jun. Helix-loop-helix proteins are similar to the leucine zipper family as they bind DNA as homo- or heterodimers. Helix-loop-helix proteins also have a positively charged domain that recognizes the DNA. Examples of helix-loop-helix proteins are Myol) and c-myc. These categories of transcription factors are not absolute; there are many transcription factors that do not fall into any of these categories.
The regions of transcription factors required for regulating transcription once the DNA-binding domain brings the factor in close contact with the DNA are called “activation domains” or “suppression domains.” These domains are less well characterized than the DNA-binding domains. In some proteins, the activation domain has a net negative charge. However, many potent transcription factors lack acidic regions; the biochemical characteristics of these proteins is not yet understood. At present, most theories of how proteins interact to regulate transcription are highly speculative (for review of transcription factors see: DNA: A Short Course, second edition, J. D. Watson et al. (eds), W. H. Freeman and Co., N.Y., 1992, pp. 161-168).
Fibroblast growth factors are a family of related proteins, most of which initiate fibroblast proliferation. The FGF family includes nine members (FGF1-9), all of which are characterized by an internal 120 amino acid sequence that allows for growth factor binding to cell surface receptors (e.g., Coulier, F., et al., 1994
, Prog. Growith acclor Res
. 5:1). Considerable species crossreactivity has been reported for FGF 1-7 and 9 (e.g., Mathieu, M., et al., 1995
, Ann. Rev. Biochen
. 58:575). FGF-2 (or basic FGF) induces proliferation of fibroblasts, endotlhelial cells, condrocytes, smooth muscle, and melanocytes. It has also been found to induce adipocyte differentiation, stimulate astrocyte migration, and prolong neuron survival (Burgess, W. H., and Maciag, T., 1989
, Ann. Rev. Biochem
. 58:575). It has been proposed that FGF-2 plays a role in angiogenesis (reviewed in: Slavin,
J Cell Biol. Int
. 19:431-444), wound healing, tissue repair, embryonic development, differentiation, neuronal function and neuronal degeneration. Additionally FGF-2 may participate in the production of a variety of pathological conditions resulting from excessive cell proliferation and excessive angiogenesis. For example, FGF-2 is associated with pituitary lactotropli tumorigenesis (Schwcppe, R. E., et al., 1997
, J. Biol. Chem
. 272:30852-9), melanoma (Halaban, R., 1996
, Semin. Oncol
. 23: 673-81)), and astrocytoma (Morrison, R. S., et al., 1994
, J Neuroncol
. 18:207-16).
Wound healing is a complex and protracted process of tissue repair and remodeling involving many different cell types that require a finely tuned control of various biochemical reaction cascades to balance degradative and regenerative processes. Among other things the process comprises the migration of different cell types into the wound region, growth stimulation of epithelial cells and fibroblasts, formation of new blood vessels, and the generation of a new extracellular matrix. At all phases correct functioning critically depends on the biological activities of various cytokines, including chemokines, FGF, FGF-1, FGF-2, IGF, PDGF, and TGF. Animal experiments and clinical experience have demonstrated that the topical administration of various cytokines, including FGF-2, FGF, KGF, PDGF, TGF-&bgr;, either alone or in combination, considerably accelerates wound healing.
SUMMARY OF THE INVENTION
The present invention is based on the discovery of genes that regulate expression of FGF-2. These genes are termed “regulators of FGF-2 transcription (RFT1)”.
In a first embodiment, the invention provides a substantially pure regulator of fibroblast growth factor-2 transcription (RFT)-A polypeptide. RFT-A is a negative regulator of the transcription of FGF-2. Substantially pure variants of RFT-A polypeptide, termed RFT-A′ and RFT-B, are also provided. RFT-A′ is a splice variant of RFT-A, having a deletion of a lysine at position 407 from the amino-terminus. RFT-A′ is a positive regulator of FGF-2 transcription. RFT-B is a splice variant of RFT-A, missing 56 amino acids which are deleted form positions 351-406 of REFT-A. RFT-B is also a positive regulator of the transcription of FGF-2. Also included in the invention are isolated polynucleotides encoding RFT-A, RFT-A′ and RFT-B polypeptide. In another aspect, antibodies that bind to RFT-A, RFT-A′, or RFT-B polypeptides are disclosed.
In another embodiment, the invention provides a method for diagnosis or prognosis of a subject having or at risk of having a disorder associated with FGF-2. The method includes contacting a target cellular component containing FGF-2 in a specimen from the subject with an agent which detects RFT-A (SEQ ID NO:2) or at least one variant thereof; determining the level of RFT-A or at least one variant thereof and comparing the level to a normal specimen, thereby providing a diagnosis or prognosis of the subject. The target cellular component can be nucleic acid or protein, and the agent can be a nucleic acid probe or an antibody, respectively, for example.
In yet another embodiment, the invention provides a method of treating a cell proliferative disorder associated with FGF-2. The method includes administering to a subject with the disorder, a therapeutically effective amount of an agent which modulates FGF-2 expression. Such agents include a polynucleotidc encoding RFT-A polypeptide.
The invention also includes a diagnostic kit useful for the detection of a target cellular component indicative of a disorder associated with FGF-2. The kit includes a carrier means containing one or more containers comprising a first container containing a probe for detection of RFT-A nucleic acid or a probe for detection of RFT-A polypeptide.
In another embodiment, the invention provides a method for identifying a compound that affects RFT-A polypeptide (SEQ ID NO:2) or a variant thereof. The method includes incubating components comprising the compound, RFT-A polypeptide or a variant thereof, and a nucleic acid sequence comprising a 5′-GCCGAAC-3′ motif operably linked to a reporter gene, under conditions sufficient to allow the components to interact; and determining the effect of the compound on expression of the reporter gene.
In anothe
Gage Fred H.
Ueba Tetsuya
Clark Deborah J. R.
Knobbe Martens Olson & Bear LLP
The Salk Institute for Biological Studies
Wilson Michael C.
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