Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-04-24
2002-10-29
Spector, Lorraine (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024100, C435S069100, C435S183000, C435S194000, C435S015000, C435S021000, C530S350000
Reexamination Certificate
active
06472516
ABSTRACT:
This invention relates to a novel progestin-regulated gene. The protein or polypeptide encoded by this gene appears to have a novel enzymatic activity that may be useful as a readily detectable marker for progestin-responsiveness.
The sex steroid hormone progesterone has two major roles in mammalian physiology. First, progesterone is involved in preparing the uterus for implantation of the fertilized ovum. Second, progestins have proliferative and differentiating effects on mammary epithelium (1, 2). Progesterone is essential for lobuloalveolar development and preparation for lactation: when ovulation is established progesterone, produced by the corpus luteum, stimulates growth of the lobuloalveolar structures and during pregnancy promotes branching of the ductal system and differentiation of alveolar cells into secretory cells ready for milk production. The importance of progestin in these processes is clearly illustrated in progesterone receptor (PR) knockout mice, which fail to develop lobuloalveolar structures (3). Progestins may also have a role in regulating cell proliferation in the human breast. Mitotic activity in breast epithelium varies in a cyclic manner through the menstrual cycle and a role for progesterone in this process is suggested by observations that levels of this hormone and epithelial cell proliferation are both maximal during the late secretory phase (4). Some breast tumours retain progesterone responsiveness and the use of high doses of synthetic progestins are recognised endocrine therapies for PR-positive breast cancers, since in this scenario progestins have an antiproliferative effect (5). Progestins also have predominantly growth inhibitory effects on human breast cancer cell lines in vitro, although under certain conditions they may stimulate growth (2, 6 and references therein). Mechanistic studies have clearly defined both a stimulatory and inhibitory effect of progestins on breast cancer cell cycle progression (6) but the functional consequences of these effects in vivo remain to be defined. This is of considerable importance given the wide spread pharmacological usage of progestins in oral contraceptives and in hormone replacement therapy.
The mechanisms underlying the biological effects of progestins in the normal breast and in breast cancer are only partially understood. Progestin action is mediated primarily via the PR, which upon activation by ligand binding interacts with gene promoter sequences containing progesterone responsive elements (PREs) to regulate gene transcription. Very few mammalian genes have been described that are directly regulated by progestins in this manner: examples include c-jun (7), cfos (8), fatty acid synthetase (FAS) (9), PR (10, 11), and uteroglobin (12, 13). While progestin action ultimately involves changes in the levels of large numbers of mRNAs and proteins, many of these require intermediary de novo protein synthesis. Furthermore, specific genes that mediate the proliferative effects of progestins are likewise poorly defined. Thus much remains to be learned about genes induced as an acute response to progestin treatment and their role in mediating progestin effects on cell proliferation and differentiation.
Several known progestin-regulated genes can be classed as those whose functions are important in differentiation effects mediated by progestin. Examples include FAS (9), alkaline phosphatase (14) and lactate dehydrogenase (15). While these are probably not involved in the proliferative effects of progestin a number of progestin-related genes related to steroid and growth factor action might contribute to these effects at least indirectly. Examples include estrogen receptor (16), PR (11), retinoic acid receptors (17), epidermal growth factor receptor (6, 18), prolactin receptor (19), insulin-like growth factors &agr; and &bgr;1 (6, 8, 23, 24), 17&bgr;-hydroxysteroid dehydrogenase (25) and insulin-like growth factor binding proteins 4 and 5 (26). However, evidence that these gene products are direct mediators of the stimulatory and inhibitory effects of progestins remains to be determined. Potentially of more interest are progestin-regulated genes with known roles in cell cycle control i.e. c-myc, c-fos (6, 8), c-jun (7) and cyclin D1 (27). Progestin induction of c-myc and cyclin D1 is closely related to changes in cell cycle progression (6, 27). While the timing of the induction of c-myc mnRNA, evident after 30 min. of progestin treatment, suggests a potential direct effect of progestins, the slower induction of cyclin D1 MRNA which is maximal at 6 hours could result from the prior induction of other genes that are the primary and specific targets of progestins. Given the established central role for cyclin D1 in steroid and growth factor regulation of breast cancer cell cycle progression (27-30) identification of progestin regulated genes which control cyclin D1 gene expression might link progestin action to the cell cycle.
Using serum-free conditions, studies in this laboratory have shown that T-47D human breast cancer cells which were stimulated to grow with insulin, undergo a transient increase in cell cycle progression in response to progestins. with an increased rate of progression through G1 and a transient increase in the S-phase population. These cells complete a round of replication and thereafter become growth arrested early in G1 phase.
The present invention arose out of a study using this model system to identify novel progestin regulated genes involved in early cell cycle stimulatory responses to progestin or other aspects of progestin action in human breast cancer cells. RNA extracted from T-47D cells grown under serum-free conditions and treated with the synthetic progestin ORG 2058 for 3 hours was used as the template for cDNA synthesis and analysis by the differential display technique (mRNA fingerprinting) (31). Several candidate PCR fragments were identified by this method and characterisation by sequence and Northern analysis of some of these led to the identification and characterisation of a clone. PRG1, which appears to represent a novel progestin-regulated gene.
Thus, in a first aspect, the present invention provides an isolated DNA molecule comprising a nucleotide sequence substantially corresponding or, at least, >80% (more preferably, >90%) homologous to any one of the nucleotide sequences shown at:
(i)
FIG. 2B
from nucleotide 1 to 3018;
(ii)
FIG. 2B
from nucleotide 1 to 470;
(iii)
FIG. 2B
from nucleotide 141 to 3018: and
(iv)
FIG. 2B
from nucleotide 470 to 2103.
Preferably. the isolated DNA molecule is of human origin. More preferably, the isolated DNA molecule is of human kidney cell or breast cancer cell origin, and/or encodes a protein normally expressed in human kidney cells, breast tissue or tumour cells.
The isolated DNA molecule may be incorporated into plasmids or expression vectors. which may then be introduced into suitable bacterial, yeast and mammalian host cells. Such host cells may be used to express the polypeptide encoded by the isolated DNA molecule.
The predicted amino acid sequence of the polypeptide encoded by PRG1 shows substantial homology (~70%) with human liver 6-phosphofructo-2-kinase/fructose 2.6 bisphosphatase (PFK-2/FBPase-2) and it is postulated that the protein encoded by PRG1 may have activities similar to this bifunctional enzyme.
Thus, in a second aspect, the present invention provides a polypeptide in a substantially pure form, said polypeptide comprising an amino acid sequence substantially corresponding to that shown at
FIG. 2B
or an enzymatic portion thereof.
Preferably, the polypeptide of the second aspect is full length.
The polypeptide of the second aspect may be used to raise monoclonal or polyclonal antibodies which may be used, for example, in affinity purification processes or in various ELISA type assays.
Thus, in a third aspect, the present invention provides an antibody specific to the polypeptide of the second aspect.
As will be seen hereinafter, PRG1 appears to be directly regulated by progestin. PRG1 may, ther
Hamilton Jenny Ann
Watts Colin Kenneth William
Lazar-Wesley Eliane
Morrison & Foerster / LLP
Spector Lorraine
The Garvan Institute of Medical Research
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