Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-03-21
2004-06-15
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007200
Reexamination Certificate
active
06750015
ABSTRACT:
FIELD OF THE INVENTION
This invention generally relates to expression profiles of genes that are regulated by progesterone receptors, and particularly by progesterone receptor isoforms PR-A and PR-B, and to the use of such genes in methods for identifying progesterone receptor agonist and antagonist ligands, including progesterone receptor isoform-specific ligands and tissue-specific ligands. This invention also relates to methods for determining the profile of genes regulated by progesterone receptors in a tissue sample. In addition, pluralities of polynucleotides transcribed from genes that are regulated by progesterone receptors are disclosed, as are pluralities of antibodies that selectively bind to proteins encoded by such genes.
BACKGROUND OF THE INVENTION
Progesterone is a natural reproductive hormone that targets the breast, uterus, ovaries, brain, bone, blood vessels, immune system, etc. Progestational agents are widely used for oral contraception, menopausal hormone replacement therapy, and cancer treatments. Antiprogestins, which are synthetic ligands that antagonize the actions of progesterone, are in clinical trials for contraception, for induction of labor, and to treat endometriosis, breast cancers and meningiomas. The actions of progesterone are varied and tissue-specific. Even in the normal breast it can have diverse effects: depending on the physiological state of the woman, progesterone can be proliferative, antiproliferative, or differentiative. Additionally, progesterone promotes the development of breast cancers and accelerates the growth of established breast cancers. For example, when used for hormone replacement therapy at menopause, progestins, which are synthetic progestational agents, increase the risk of breast cancer. Paradoxically, they are protective in the uterus and prevent endometrial cancers.
Progesterone, synthetic progestins, and antiprogestins all initially work through the same molecular pathway. These are low molecular weight, lipid soluble “ligands ”. They enter target cells passively, and pass into the nucleus where they bind to progesterone receptors (PRs). Ligand binding activates the PR proteins, which then dimerize, bind to DNA at the promoters of progesterone target genes, and either up- or down-regulate transcription of these genes.
There are two natural isoforms of PR, the A- and B-receptors, also referred to herein as PR-A and PR-B, respectively. The isoforms are derived from two distinct promoters in the single PR gene and are translated from separate translation initiation start sites. PR-B receptors are 933 amino acids in length, which is 164 amino acids longer at the N-terminus than PR-A, and contain a unique transcriptional activation function, AF-3 (Sartorius et al.,
Mol. Endocrinol.
8, 1347-1360 (1994)). Downstream of the additional 164 amino acids of PR-B, the two PRs have the identical primary amino acid content. However, despite this close amino acid composition, the two receptors have dramatically different abilities to activate transcription of progestin-responsive promoters in experimental model systems (Sartorius et al.,
Mol. Endocrinol
. 8, 1347-1360 (1994); Meyer et al.,
J. Biol. Chem
. 267, 10882-10887 (1992); Vegeto et al.,
Mol. Endocrinol
. 7, 1244-1255 (1993); Tung et al.,
Mol Endocrinol
. 7, 1256-1265 (1993); Sartorius et al.,
J. Biol. Chem
. 268, 9262-9266 (1993)). Progestin agonist-liganded PR-B are stronger transactivators than PR-A, although there are cell-type and promoter-dependent exceptions. The antiprogestin RU486 has mixed agonist/antagonist activity on PR-B but not PR-A. Instead, agonist or antagonist-liganded PR-A can dominantly inhibit PR-B and other members of the steroid receptor family, including estrogen receptors (ERs). Thus, PR-A are more likely to be transcriptional repressors than PR-B. (Hovland et al.,
J Biol Chem
273, 5455-60(1998); Vegeto et al.,
Mol. Endocrinol
. 7, 1244-1255 (1993); McDonnell et al.,
J. Biol. Chem
. 269, 11945-11949 (1994)).
Indirect data suggest that the two PR isoforms have physiologically different functions. They are unequally expressed in different tissues and physiological states. For instance, increasing ratios of PR-A to PR-B in the chick oviduct in late winter, or in aged, nonlaying hens, resulted in measurable decreases in PR functional activity (Boyd-Leinen et al.,
Endocrinology
111,30-36 (1982); Spelsberg et al.,
Endocrinology
107, 1234-44 (1980)). There are stage-specific and region-specific variations in the PR-A:PR-B ratio in the developing rat brain (Kato et al.,
J Steroid Biochem Mol Biol
47, 173-82 (1993)) and studies in primates show that PR-B predominates in the estrogen treated hypothalamus, while expression of the PR-A isoform predominates in the pituitary (Baez et al.,
J Biol Chem
262, 6582-8 (1987); Bethea et al.,
Endocrinology
139, 677-87 (1998)). In the human endometrium, absolute levels and the ratio of PR-A to PR-B vary extensively during the menstrual cycle (Mote et al.,
Hum Reprod
15 Suppl 3, 48-56 (2000); Mote et al.,
J Clin Endocrinol Metab
84, 2963-71 (1999); Mangal et al.,
J Steroid Biochem Mol Biol
63, 195-202 (1997); Feil et al.,
Endocrinology
123, 2506-2513 (1988)). In addition, uncontrolled, or over-expressed PR-B levels are associated with a highly malignant phenotype in endometrial, cervical and ovarian cancers (Farr et al.,
Mamm. Genome
4, 577-584 (1993); Fujimoto et al.,
J Steroid Biochem Mol Biol
62, 449-54 (1997)).
In the normal breast, progesterone is both proliferative and differentiative [reviewed in\(Clarke et al.,
Endocr. Rev.
11, 266-301 (1990))]. Breast epithelium mitoses increase during the menstrual cycle and peak in the late luteal phase, coincident with high circulating levels of progesterone. Progesterone induces lobular-alveolar outgrowth during each menstrual cycle and during pregnancy induces further lobular-alveolar development in preparation for the terminal differentiative event of lactation. PR null mice exhibit incomplete mammary gland ductal branching and failure of lobulo-alveolar development, as well as failure to ovulate and to exhibit sexual behavior (Lydon et al.,
Genes Develop.
9, 2266-2278 (1995)).
Little is known about cyclic changes in PR-A and PR-B in the normal human breast. However, in the mouse mammary gland, evidence supports a critical and unique role for each of the two PR isoforms. It has been reported that a 3:1 overexpression of PR-A over PR-B results in extensive mammary gland epithelial cell hyperplasia, excessive ductal branching, and a disorganized basement membrane; all features associated with neoplasia (Shyamala et al.,
Proc Natl Acad Sci USA
95, 696-701 (1998)). In contrast, when PR-B is overexpressed, ductal growth prematurely arrests and inappropriate lobulo-alveolar formation is observed (Shyamala et al.,
Proc Natl Acad Sci USA
97, 3044-9 (2000)). However, when the PR-A isoform was selectively knocked out, leaving only PR-B, the mammary gland appeared to develop normally in response to estradiol and progesterone. In contrast, decidualization of the endometrium and the normal antiproliferative effect of progesterone in the uterus were absent (Mulac-Jericevic et al.,
Science
289, 1751-4 (2000)). Such data indicate that PR-A and PR-B have different tissue-specific effects.
In human breast cancers the presence of PR in estrogen receptor (ER) positive tumors indicates that responsiveness to endocrine therapies is likely, while absence of PR is associated with hormone resistance. thus, PR are routinely measured in breast cancers as a guide to treatment (Horwitz et al.,
Recent Prog. Horm. Res
. 41, 249-316 (1985); Horwitz et al.,
J Biol Chem
253, 8185-91 (1978); McGuire,
Semin. Oncol
. 5, 428-433 (1978)). PR are also direct targets of second-line progestin therapies in patients whose tumors have developed antiestrogen resistance (Kimmick et al.,
Cancer Treat Res
94, 231-54 (1998); Howell et al.,
Recent Results Cancer Res
152, 227-44 (1998)). Nothing is known, however, about the role of PR-A vs. PR-B in breast ca
Horwitz Kathryn B.
Richer Jennifer
Kemmerer Elizabeth
Li Ruixiang
Sheridan & Ross P.C.
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