Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-12-23
2001-09-25
Huff, Sheela (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023500, C530S350000
Reexamination Certificate
active
06294657
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of inducible gene expression. More specifically, the invention relates to a retinoid-inducible polynucleotide and assays that detect expression of this polynucleotide.
BACKGROUND OF THE INVENTION
Retinoids, which are the compounds comprising vitamin A and its derivatives, play important roles in a variety of biological phenomena. More particularly, retinoids are important for vision, hematopoiesis, bone development and pattern formation during embryogenesis. Retinoids also exhibit antiproliferative activities in certain biological contexts.
Retinoids also have been used extensively as pharmaceutical agents for treating various malignant and non-malignant skin diseases. Malignant skin diseases therapeutically responsive to retinoids include squamous cell carcinoma, actinic keratoses, basal cell carcinoma and Kaposi's sarcoma. Additionally, retinoids are potentially useful as pharmacological agents for the treatment of various epithelial cancers (Peck and DiGiovanna, “Synthetic Retinoids in Dermatology”) in
The Retinoids,
2nd ed., pp 631-658 (1994); Boehm et al.,
Exp. Opin. Invest. Drugs
4:593 (1995); Nagpal and Chandraratna,
Curr. Pharm. Design
2:295 (1996)). Examples of non-malignant skin diseases therapeutically responsive to retinoids include psoriasis and acne. In spite of the demonstrated utility of this class of pharmacological agents, the molecular basis of retinoid action in skin and various cancers is poorly understood.
Two families of nuclear receptors, called the retinoic acid (RA) receptors (RAR-&agr;, -&bgr; and -&ggr;) and the retinoid X receptors (RXR-&agr;, -&bgr; and -&ggr;), mediate pharmacological and physiological retinoid signalling (Chambon,
Sem. in Cell Biol.
5:115 (1994); Mangelsdorf et al., “The Retinoid Receptors” in
The Retinoids,
2nd ed., pp 319-349 (1994); Boehm et al.,
Exp. Opin. Invest. Drugs
4:593 (1995); Nagpal and Chandraratna,
Curr. Pharm. Design
2:295 (1996)). RARs and RXRs, which belong to the superfamily of steroid/thyroid/vitamin D
3
nuclear receptors, readily heterodimerize in vitro (for references see, Nagpal and Chandraratna,
Curr. Pharm. Design
2:295 (1996)) and function as heterodimers in vivo (Nagpal et al.,
EMBO J
12:2349 (1993)). These receptors are ligand-dependent transcription factors which activate the expression of retinoid responsive genes by cooperative action of their activation functions. These activation functions are called AF-1, a ligand-independent activation function, and AF-2, a ligand-dependent activation function (Nagpal et al.,
EMBO J.
12:2349 (1993)).
The two families of retinoid receptors differ from each other with respect to the ligands that bind and activate the receptors. All-trans-RA (RA) binds and activates the RAR family of receptors. A different ligand, 9-cis-RA (9C-RA), binds and activates both the RARs and members of the retinoid X receptor (RXR) family. The retinoid called AGN 190168 (Tazarotene/ethyl 6-[2-(4,4) dimethyl-thiochroman-6-yl] ethynyl-nicotinate) is one example of an RAR-&bgr;/&ggr; selective synthetic retinoid having therapeutic utility. More specifically, this synthetic retinoid can be administered topically to dramatically improve the symptoms associated with psoriasis.
Only a small number of retinoid-inducible gene products have been identified to date. Of these, the gene product encoding a cellular retinoic acid binding protein, called CRABP II, is the only marker known to be induced in vivo by RA in non-diseased skin (Elder et al.,
J Invest. Dermatol.
100:356 (1993)). Interestingly, CRABP II expression was down-regulated by RA in submerged keratinocyte cultures (Elder and Cromie,
J. Toxicol.—Cut. & Ocular Toxicol.
12:173 (1993)) and was overexpressed in cells of tissues that exhibited a psoriatic phenotype (Didierjean et al.,
Biochem. Biophys. Res. Comm.
180:204 (1991)). Those having ordinary skill in the art will appreciate that psoriasis is a hyperproliferative and inflammatory condition of the skin (Krueger and Duvic,
J Invest. Dermatol.
102:14S (1994)) which clinically responds to retinoid treatment (Esgleyes-Ribot et al.,
J Am. Acad. Dermatol.
30:581 (1994); Weinstein,
Brit. J Dermatol.
135(Suppl. 49):32 (1996)).
Two novel genes, called Tazarotene-induced gene 1 (TIG1) and Tazarotene-induced gene 2 (TIG2), were recently identified by virtue of their inducible expression in skin raft cultures treated with Tazarotene (Nagpal et al.,
J Invest. Dermatol.
106:269 (1996); Nagpal et al., submitted (1997)). TIG1 was also shown to be induced by Tazarotene in foreskin keratinocyte and fibroblast cultures. Significantly, both TIG1 and TIG2 were induced in vivo by topical treatment of psoriatic lesions with Tazarotene.
Herein we disclose the discovery and utility of a novel retinoid-induced polynucleotide that is unrelated to either TIG1 or TIG2.
SUMMARY OF THE INVENTION
One aspect of the present invention regards an isolated polynucleotide that encodes a protein having the polypeptide sequence of SEQ ID NO:12. In a preferred embodiment the polynucleotide has the sequence of SEQ ID NO:11.
A second aspect of the invention regards a method of identifying a test compound for treatment of a hyperproliferative disorder of skin. According to the invented method, a negative control sample containing RNA isolated from an untreated control culture of cells derived from skin is first obtained. The cells of this control culture have not been treated with an inducer. Next, a test sample containing RNA isolated from a test culture of said cells derived from skin is obtained. Cells in this test culture will have been treated with the test compound. After the two samples of RNA have been obtained, the amount of Tazarotene Inducible Gene-3 (TIG3) RNA present in each of the samples is quantitated. The TIG3 RNA is an RNA having a polynucleotide sequence corresponding to the sequence of SEQ ID NO:11. Finally, the amount of TIG3 RNA in each of the samples is compared to determine if the amount of TIG3 RNA in the test sample is greater or lesser than the amount of TIG3 RNA in the negative control sample. A compound will be identified as a test compound for the treatment of the hyperproliferative disorder if the amount of TIG3 RNA in the test sample is greater than four-fold more than the amount of TIG3 RNA in the negative control sample. In a preferred embodiment, the step for quantitating TIG3 mRNA will involve hybridizing the negative control sample and the test sample with a labeled probe having a sufficient number of consecutive nucleotides complementary to the sequence of SEQ ID NO:11 to specifically hybridize with TIG3 mRNA under high stringency conditions (0.1×SSPE/1% SDS at 65° C.), and then quantitating the amount of hybridization between the probe and each of the samples. In another preferred embodiment, the negative control sample is derived from keratinocytes or fibroblasts. In yet other preferred embodiments, the TIG3 probe used in the procedures is labeled with a radioactive label and the amount of hybridized probe is quantitated by autoradiography. RNA contained in the negative control sample and RNA contained in the test sample can be immobilized to a solid support prior to the hybridizing step. In a different embodiment of the invented method, the step for quantitating the amount of TIG3 RNA present in negative control and test samples is accomplished by: (i) reverse transcribing mRNA present in each of the samples, wherein the product of reverse transcription has a polynucleotide sequence corresponding to a segment of the sequence given by SEQ ID NO:11, the product being TIG3 cDNA; (ii) amplifying specifically any TIG3 cDNA produced in step (i) by a polymerase chain reaction to result in the production of TIG3 amplification products; and (iii) quantitating the TIG3 amplification products produced when negative control and test samples are separately used as sources of RNA templates for the reverse transcribing step. In the practice of this method, the results of
Chandraratna Roshantha A.
DiSepio Daniel
Nagpal Sunil
Allergan Sales Inc.
Baran Robert J.
Fisher Carlos A.
Huff Sheela
Voet Martin A.
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