Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1999-04-06
2000-03-07
Johnson, Nancy A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
435 71, 435 72, 435 721, 435 723, 435 78, 435 41, 435 691, 435 694, 435 697, 435 701, 435 703, C12N 1509, C12N 1516, C12Q 100, C12Q 102
Patent
active
060338431
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to methods for preventing the growth of tumours, and for assays for compounds useful in the prevention of tumours.
BACKGROUND OF THE INVENTION
The cyclins are a class of polypeptides which are involved in the control of the cell cycle. Three closely related human D-type cyclins have been identified, all of which interact with and activate cyclin dependent kinases (CDK) 4 and 6, although they have specialized function in distinct cell types. The cyclin D1 gene has been found to be overexpressed and/or deregulated by clonal chromosome rearrangements or by amplification in B cell lymphoma, in parathyroid adenoma, and in breast and squamous cell cancer. It has also recently been shown that cyclin D1 deficient mice have a defect in estrogen-mediated proliferation of breast epithelium during pregnancy (Sicinski et al, 1995, Cell 82; 621-630; Fantl et al, 1995, Genes & Development 9; 2364-2372).
DISCLOSURE OF THE INVENTION
We have investigated the mechanisms of cyclin D1 regulation of cell growth and found that this protein interacts directly with the estrogen receptor (ER) and potentiates the transcription of estrogen receptor-regulated genes. Transcription is increased by formation of cyclin D1-ER complex which binds to the estrogen response element (ERE) found upstream of estrogen responsive genes. This finding provides a target for the control of cell proliferation, particularly in those cells which grow in response to stimulation by estrogen, e.g. breast tumour cells. Thus the present invention is useful for assaying for potential inhibitors of the growth of estrogen responsive tumour cells, particularly those in which cyclin D1 is found at elevated levels. Elevated levels of cyclin D1 may occur for a variety of reasons, e.g. over expression of a single cyclin D1 gene or by gene amplification. Thus in a first aspect the present invention provides an assay for inhibitors of estrogen responsive tumour cells which comprises: inhibitor compound under conditions where the cyclin D1 and the estrogen receptor, in the absence of inhibitor, are capable of forming a complex which is capable of binding to an estrogen response element; of binding and transcriptionally activating; and activation caused by said inhibitor compound.
The present invention further provides an assay for an inhibitor of estrogen responsive tumour cells which comprises: inhibitor compound under conditions where the cyclin and the estrogen receptor, in the absence of inhibitor, are capable of forming a complex; and inhibitor compound.
In a further aspect, the invention provides compounds obtainable by such an assay, for example peptide compounds based on the portions of cyclin D1 or the estrogen receptor which interact with each other.
The assay of the invention is optionally performed in the presence of an estrogen which is capable of binding to the estrogen receptor.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1E shows that cyclin D1 potentiates ERE responsive gene transcription in the presence of 17.beta.-estradiol.
FIGS. 2A-2C show cyclin D1 enhances ERE responsive gene transcription independently of ligand activation of ER.
FIGS. 3A-3C show the potentiation of gene activation by cyclin D1 is ER-mediated.
FIGS. 4A-4C show cyclin D1 interacts with (un)liganded ER.
FIG. 5 shows that activation of ER by cyclin D1 does not require sequence specific DNA binding and cyclin D1 specifically activates the EF region of ER.
DETAILED DESCRIPTION OF THE INVENTION
The cyclin D1 may be any suitable mammalian cyclin D1, particularly human cyclin D1. Human D1 cyclin has been cloned and sources of the gene can be readily identified by those of skill in the art. See for example, Xiong et al, 1991, Cell 65; 691-699 and Xiong et al, 1992, Genomics 13; 575-84. Murine D1 cyclin has also been cloned. Other mammalian cyclins can be obtained using routine cloning methods analogous to those described in the aforementioned references.
Although wild-type cyclin D1 is preferred mutants of D1 which still retain the ability to intera
REFERENCES:
Zwijsen et al., Cell, 88(3):405-415 (1997).
Van Diest et al., Am. J. Pathol., 150(2):705-711 (1997).
Wilcken et al., Clin. Cancer Res., 3(6):849-854 (1997).
International Search Report.
Bernards Rene
Michalides Rob J. A. M.
Zwijsen Renate M. L.
Harris Alana M.
Johnson Nancy A.
Prolifix Limited
Williams Kathleen M.
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