Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-05-25
2004-07-27
Helms, Larry R. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007800, C435S008000
Reexamination Certificate
active
06767705
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is related to changes in cellular gene expression and compounds that produce changes in cellular gene expression. In particular, the invention is related to the identification of genes the expression of which is modulated by a class of compounds known as retinoids, being chemically related to retinoic acid and Vitamin A, More specifically, the invention provides methods for identifying compounds other than retinoids that modulate expression of these cellular genes. The invention also provides reagents that are recombinant mammalian cells containing recombinant expression constructs that express a reporter gene under the transcriptional control of a promoter for a gene that is regulated by retinoids, and methods for using such cells for identifying compounds other than retinoids that modulate expression of these cellular genes.
2. Summary of the Related Art
Retinoids are naturally-occurring or synthetic derivatives of vitamin A. They comprise a class of clinically important differentiation agents that regulate cell growth and differentiation at the level of transcription, by binding to nuclear receptors that act as ligand-dependent transcription factors. These compounds induce cellular differentiation or terminal proliferation arrest in a number of tumor cell types in vivo and in vitro that express retinoid receptors (Warrell, 1997, in
CANCER. PRINCIPLES
&
PRACTICE OF ONOCOLOGY
(DeVita et al., eds.), pp. 483-490 (Lippincott-Raven, Philadelphia), making them useful for treating some cancers, such as acute promyelocytic leukemia and cancer chemoprevention.
The target of retinoid action is the cell nucleus, where retinoids bind to two types of receptors, termed RARs (retinoic acid receptors) and RXRs (retinoid X receptors) (Mangelsdorf et al., 1994, “The retinoid receptors,” in: The Retinoids biology, chemistry, and medicine, Spain et al., eds., New York: Raven Press, pp. 319-351) Retinoid-bound receptor molecules form homo- (RXR—RXR) and heterodimers (RAR-RXR) that act as transcription factors. These dimers bind to specific cis-regulatory sequences in the promoters of retinoid-responsive target genes, termed RARE (Retinoic Acid Response Elements), regulating their transcription.
A RARE sequence has a minimal half-site consensus sequence that is generally well conserved as AGGTCA or AGTTCA (Mangelsdorf et al., 1994, “The retinoid receptors,” in: The Retinoids: biology, chemistry, and medicine, Sporn et al., eds., New York: Raven Press, pp. 327-331). RAREs are typically configured into one of three structured motifs: direct repeats, palindromes, and complex elements without an obvious consensus structure. The direct repeat requires a lesser amount of receptor to activate a retinoid-inducible gene than the other configurations. In addition, direct repeats separated by 5 nucleotides have demonstrated the strongest responses while moderate responses are typically generated by direct repeats separated by 2 nucleotides. (See Mangelsdorf et al., 1994, “The retinoid receptors,” in: The Retinoids: biology, chemistry, and medicine, Sporn et a!., eds., New York: Raven Press, pp. 327-331.)
The resulting changes in gene expression are caused either directly by retinoid receptor regulation of target gene expression, or indirectly through the action of retinoid-activated signal transduction pathways, for example, pathways activated by the transcription factor AP-l. These gene expression changes are ultimately responsible for the growth-inhibitory effect of retinoids (Warrell, Id.).
Although retinoids have had some clinical success in cancer treatment, their use has been limited by at least two factors: development of resistance (Miller et al., 1998,
Cancer
83:1471-1482) or toxicity. Development of resistance is due in part to alterations of retinoic acid receptors and retinoid receptor-mediated pathways (Miller et al., ibid.). Toxicity is generally attributed to the broad physiologic consequences of retinoids, resulting from pleiotropic changes in gene expression produced by treatment with retinoids.
Several growth-inhibitory genes have been previously found to be inducible by retinoids in epithelial cells. None of these genes, however, was shown to be solely responsible for the growth-inhibitory effect of retinoids.
Adamo et al., 1992,
Endocrinology
131: 1858-1866 disclosed induction of insulin-like growth factor binding protein 3 (IGFBP-3) in breast carcinoma cell lines.
Swisshelm et al., 1995,
Proc. Natl. Acad. Sci. USA
92: 4472-4476 identified another insulin-like growth factor binding protein, IGFBP-7 (also known as insulin-like growth factor binding protein related protein 1, or IGFBP-rP1, and mac25) as a protein induced by treatment of cells with fenretinide (4-hydroxyphenylretinamide, 4-HPR). This protein was also shown to be down-regulated in mammary carcinoma cell lines.
Kato et al., 1996,
Oncogene
12: 1361-1364 showed that introduction of mac25 cDNA into an osteosarcoma cell line inhibited growth.
Gucev et al., 1996,
Cancer Res.
56: 1545-1550 identified IGFBP-3 as a protein induced in breast carcinoma cells both by all-trans retinoic acid (RA) and by transforming growth factor &bgr; (TGF-&bgr;). RA-mediated growth inhibition in these cells was alleviated by introducing an antisense oligonucleotide into the cells that inhibited IGFBP-3 expression, or by introducing exogenous IGFBP-3 into the cells.
DiSepio et al., 1998,
Proc. Natl. Acad. Sci. USA
95: 14811-14815 showed that tazarotene-induced gene 3 (TIG-3, also known as retinoid-inducible gene 1, RIG-1), a putative tumor suppressor gene, is induced in primary human keratinocytes. TIG-3 shows decreased expression in cancer cells, inhibits the growth of cancer cells when expressed, and shares sequence homology with a known tumor suppressor gene, H-rev 107.
Huang et al., 2000,
Molec. Cell. Endocrinol.
159: 15-24 showed that TIG-3 was induced by retinoids in a gastric carcinoma cell line in vitro.
Liu et al., 2000,
J. Cancer Res. Clin. Oncol.
126: 85-90 reported that RA-induced expression of a metastasis suppressor gene, nm23-H1 in a hepatocarcinoma cell line.
The teachings of the prior art suggest that one mechanism of retinoid-mediated growth inhibition is the activation (or re-activation) of tumor suppressor genes and other growth-inhibitory genes that have been repressed or whose expression has been down-regulated in tumor cells. However, the reports in the art fail to indicate the identity or number of growth-inhibitory genes that are activated under the conditions of retinoid-induced growth arrest. Such reports also fail to indicate if retinoid-induced genes are induced by retinoids directly, through RARE sites in their promoters, or indirectly. In the latter case, it should be possible to activate such growth-inhibitory genes even in the cells that are not responsive to retinoids.
There remains a need in this art to identify genes whose expression is modulated by retinoids, and especially growth-inhibitory genes that are induced by retinoids indirectly. There is also a need in this art to develop methods for assessing the effects of compounds on expression of retinoid-modulated cellular genes, particularly growth-inhibitory genes. There is an additional need to develop alternative compounds that mimic the effects of retinoids on cellular gene expression, to which resistance is not so easily developed and that lack the toxicity and other systemic side-effects of retinoids in current clinical use.
SUMMARY OF THE INVENTION
This invention provides genes whose expression is modulated by retinoids and reagents and methods for identifying compounds that mimic the effects of retinoids without producing resistance or toxicity to said compounds.
In a first aspect, the invention provides a recombinant expression construct encoding a reporter gene operably linked to a promoter from a gene the expression of which is induced by a retinoid and which does not contain a RARE site. In preferred embodiments, the reporter gene encodes firefly luciferase, chl
Chang Bey-Dih
Dokmanovic Milos
Roninson Igor B.
Helms Larry R.
McDonnell & Boehnen Hulbert & Berghoff
The Board of Trustees of the University of Illinois
Yu Misook
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