Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-07-07
2001-03-27
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S069100, C536S023500, C530S399000
Reexamination Certificate
active
06207375
ABSTRACT:
BACKGROUND OF THE INVENTION
Breast and endometrial carcinomas remain major oncological problems. Both types of cancers are considered to be responsive to endocrine therapy. This is particularly true in the case of breast cancer, whether one considers ablative or additive therapy. However, tumor responses to steroid therapy vary significantly among these tumors. In general, for breast carcinoma, only half of the tumors of estrogen receptor (ER)-positive breast cancer patients, assessed by the dextran-coated charcoal assay (DCC assay) (see U.S. Pat. No. 5,030,417; Thibodeau et al.,
Clin. Chem.,
27, 687 (1981)), respond to estrogen (E) therapy. Moreover, 80% of all breast cancer metastases to bone are ER positive. Although the ER and PR (progesterone receptor; Thibodeau et al.,
Clin. Chem.,
27, 687 (1981)) assays currently in use clinically have improved a physician's ability to predict a response to hormonal therapy, the DCC assay has a relatively high false-positive rate (Ingle,
Cancer,
53, 766 (1984)). Furthermore, the lack of response to estrogen therapy by many ER-positive patients indicates the need for an assay with estrogen-related markers that have a substantially higher predictive index than the DCC assay.
The breast cancer genes BRCA-1 and BRCA-2 appear to be involved in the inherited susceptibility of breast cancer. Both BRCA-1 and BRCA-2 genes encode polypeptides having homology domains and properties of a granin family of proteins (Schuard et al.,
Endocrine Reviews,
14, 659 (1993); Steeg,
Nat. Gene.,
12, 223 (1996); Jensen et al.,
Nat. Genet.,
12, 303 (1996); Holt et al.,
Nat. Genet.,
12, 298 (1996); Wooster et al.,
Science,
265, 2088 (1995)). Overexpresssion of the BRCA genes inhibits, and underexpression encourages, tumor growth. The p53 gene, a tumor suppressor gene, and the BRCA-1 gene, which exhibits properties of a tumor suppressor gene, are regulated by estrogen in breast cancer cells.
Transforming growth factor-beta (TGB-&bgr;) is produced by breast cancer cells and can inhibit breast cancer cell growth (Valverius et al.,
Cancer Res.,
49, 6269 (1989); Knabbe et al.,
Cell,
48, 417 (1987)). The production and activation of TGF-&bgr; is regulated by estrogen, parathyroid hormone, glucocorticoids, and other bone regulatory agents including TGF-&bgr; itself (Oursler et al.,
Endocrinology,
129, 3313 (1991); Oursler et al.,
Endocrinology,
133, 2187 (1993); Subramaniam et al.,
J. Cell. Biochem.,
57, 52 (1995)). More recently, TGF-&bgr; has been implicated in the antiestrogen-induced apoptosis of breast cancer cells (Chen et al.,
J. Cell, Biochem.,
61, 9 (1996)).
Thus, a continuing and urgent need exists for an accurate determinant marker effective to ascertain the most efficacious therapy of breast cancers. In particular, there is a need to identify and isolate estrogen and TGF-&bgr; regulated genes, the expression of which are predictive of a breast or endometrial cancer patient's response to steroid therapy or the metastatic potential of these cancers. Moreover, there is also a need to identify and isolate genes that may be tumor suppressor genes for both breast and endometrial carcinomas.
SUMMARY OF THE INVENTION
The present invention provides an isolated and purified DNA molecule comprising a DNA sequence encoding TGF-&bgr; inducible early factor-1 (TIEF-1), or a biologically active subunit or variant thereof. A preferred embodiment of the invention is a DNA sequence, eg., SEQ ID NO:1, that encodes a polypeptide having an amino acid sequence comprising SEQ ID NO:2. Also provided is an isolated and purified DNA molecule which is complementary to the DNA molecules described hereinabove.
The DNA molecules of the invention are double-stranded or single-stranded, preferably, they are cDNA. A preferred embodiment of the invention includes a DNA molecule that has at least about 80%, preferably at least about 90%, and more preferably at least about 95%, identity to the DNA sequence comprising SEQ ID NO:1. Thus, a preferred embodiment of the invention includes a DNA molecule which has at least about 80%, preferably at least about 90%, and more preferably at least about 95%, identity to SEQ ID NO:1.
As described hereinbelow, TIEF-1 expression is rapidly induced by TGF-&bgr;, and estrogen (E), administration. The induction of TIEF-1 expression by estrogen is dependent on the presence of the estrogen receptor (ER) in cells. The TIEF-1 gene encodes three zinc finger motifs, domains of which are known to bind nucleic acid. Moreover, the C-terminal region of TIEF-1 contains a proline-rich region, which may have a transcriptional activating function.
As used herein, “TIEF-1” is preferably a polypeptide comprising SEQ ID NO:2, as well as variants of SEQ ID NO:2 which have at least about 80%, preferably at least about 90%, and more preferably at least about 95%, identity or homology to SEQ ID NO:2, or a biologically active subunit thereof. Biologically active subunits of TIEF-1, e.g., peptides comprising SEQ ID NO:4 or SEQ ID NO:5, variant TIEF-1 polypeptides and biologically active subunits thereof, falling within the scope of the invention have at least about 10%, preferably at least about 50%, and more preferably at least about 90%, the activity of the polypeptide comprising SEQ ID NO:2. The activity of a polypeptide or peptide of the invention can be measured by methods well known to the art including, but not limited to, the ability of the polypeptide or peptide to elicit a sequence-specific immunologic response when the peptide is administered to a mammal, e.g., goat, rabbit, sheep or mice (see Example 3).
An isolated and purified DNA molecule of the invention, such as a probe or a primer, of at least seven nucleotide bases which hybridizes to the DNA molecules, or RNA molecules derived from these DNA molecules, is useful to detect, quantify and amplify complementary DNA stands in eukaryotic tissue samples comprising TIEF-1 sequences or sequences related to TIEF-1. The cDNAs of the present invention are useful for detecting the expression of TIEF-1, for detecting related DNA molecules and for amplifying nucleic acid sequences, wherein said sequences fall within the scope of the present invention.
The invention also provides an expression cassette comprising a preselected DNA sequence encoding TIEF-1, or a biologically active subunit or variant thereof, which is operably linked to a promoter functional in a host cell. Such expression cassettes can be incorporated into expression vectors which can then be employed to transform prokaryotic or eukaryotic host cells, so as to result in expression of TIEF-1 polypeptide. The present vectors can also contain a functional DNA sequence which is a selectable marker gene and/or reporter gene, as described below. Therefore, therapeutic compositions containing TIEF-1 polypeptide, subunits of TIEF-1, or variants thereof, are also within the scope of the invention.
Also provided is an expression cassette comprising: a preselected DNA segment that is complementary to a DNA sequence encoding TIEF-1, e.g., SEQ ID NO:1, which is operably linked to a promoter functional in a host cell. Thus, the present invention provides an expression cassette which expresses an “anti-sense” mRNA transcript of a DNA sequence of the invention. When this transcript is introduced into a host cell, it can alter TIEF-1 expression, as well as cell growth and/or differentiation of the host cell.
Further provided is an isolated and purified TIEF-1 polypeptide, or a biologically active subunit or variant thereof. Also provided are isolated, purified peptides of TIEF-1, or biologically active variants thereof e.g., a peptide comprising SEQ ID NO:4 or SEQ ID NO:5. A preferred TIEF-1 polypeptide comprises an amino acid sequence corresponding to SEQ ID NO:2. TIEF-1 polypeptide or a TIEF-1 peptide can be employed to prepare antibodies specific for TIEF-1.
Thus, the invention also provides a purified antibody, or a preparation of polyclonal or monoclonal antibodies, that specifically reacts with TIEF-1 protein or polypeptide. A preferre
Roche Patrick C.
Spelsberg Thomas C.
Subramaniam Malayannan
Jones W. Gary
Mayo Foundation for Medical Educational & Research
Schwegman Lundberg Woessner & Kluth P.A.
Taylor Janell E.
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