The continuing goal of this proposal is to analyze the mechanism by which retinoic acid (RA) inhibits 12-0 tetradecanylphorbol-13- acetate (TRP)-induced ornithine decarboxylase (ODC)-gene transcription. In this study, we plan to test two hypotheses: 1) TPA-induced ODC-gene transcription is mediated through interaction of PKC- medicated phosphorylated proteins (trans-acting factor) with cis-acting elements in 5'-flanking region of ODC gene and RA inhibits TPA-induced ODC-gene transcription by inhibiting the phosphorylation of those proteins; and 2) RA complexed with cellular RA binding protein (CRABP) is transported to specific nuclear receptors and thus acts as a negative regulator of TPA- induced ODC gene transcription. To test the first hypothesis, we plan to: a) Isolate 5'-flanking region of ODC gene and identify by deletion analysis and CAT assay of TPA-responsive enchaner element (cis-acting); b) synthesize TPA- responsive sequences using oligonucleotide synthesizer, and then to test the enhancer ability; c) determine by DNase I footprinting analysis that TPA-modulated trans-acting factors recognize the TPA- responsive enhancer elements; d) isolate TPA-modulated trans-acting factors by specific sequence DNA affinity chromatography; e) determine that TPA-responsive transcription factors (proteins) are substrates for the purified PKC; f) determine whether RA inhibits in vivo the phosphorylation of trans-action factors. To test the second hypothesis, we plan to: a) identify RA responsive sequences in the 5-flanking region of ODC gene; b) determine by DNase I footprinting assay whether there is negative regulatory factor(s) that recognize RA responsive sequences; and, c) synthesize RA-responsive cis-elements and then purify RA responsive proteins(s) by DNA-affinity chromatography. We will analyze that the negative regulatory factor (protein) is a RA nuclear receptor. We have shown that TPA induces the transcription of ODC gene in two in vitro systems (epidermal cells and human bladder carcinoma cell line T24) which will be used for proposed study. This study will provide information about the mechanism by which RA inhibits the induction of ODC, an enzyme associated with tumor promotion, cell proliferation and differentiation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA042585-06
Application #
3184053
Study Section
Metabolic Pathology Study Section (MEP)
Project Start
1986-04-01
Project End
1993-11-30
Budget Start
1991-12-01
Budget End
1993-11-30
Support Year
6
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Jansen, A P; Colburn, N H; Verma, A K (1999) Tumor promoter-induced ornithine decarboxylase gene expression occurs independently of AP-1 activation. Oncogene 18:5806-13
Reddig, P J; Kim, Y J; Verma, A K (1996) Localization of the 12-O-tetradecanoylphorbol-13-acetate response of the human ornithine decarboxylase promoter to the TATA box. Mol Carcinog 17:92-104
Tseng, C P; Verma, A K (1995) Lack of 12-O-tetradecanoylphorbol-13-acetate responsiveness of ornithine decarboxylase introns which have AP-1 consensus sequences. Mol Cell Biochem 146:7-12
Tseng, C P; Kim, Y J; Kumar, R et al. (1994) Involvement of protein kinase C in the transcriptional regulation of 12-O-tetradecanoylphorbol-13-acetate-inducible genes modulated by AP-1 or non-AP-1 transacting factors. Carcinogenesis 15:707-11
Kumar, R; Shoemaker, A R; Verma, A K (1994) Retinoic acid nuclear receptors and tumor promotion: decreased expression of retinoic acid nuclear receptors by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Carcinogenesis 15:701-5
Denning, M F; Verma, A K (1994) The mechanism of the inhibition of squamous differentiation of rat tracheal 2C5 cells by retinoic acid. Carcinogenesis 15:503-7
Kim, Y J; Pan, H; Verma, A K (1994) Non-AP-1 tumor promoter 12-O-tetradecanoylphorbol-13-acetate-responsive sequences in the human ornithine decarboxylase gene. Mol Carcinog 10:169-79
Verma, A K; Shoemaker, A; Simsiman, R et al. (1992) Expression of retinoic acid nuclear receptors and tissue transglutaminase is altered in various tissues of rats fed a vitamin A-deficient diet. J Nutr 122:2144-52
Zachman, R D; Chen, X; Verma, A K et al. (1992) Some effects of vitamin A deficiency on the isolated rat lung alveolar type II cell. Int J Vitam Nutr Res 62:113-20
Denning, M F; Verma, A K (1991) Involvement of retinoic acid nuclear receptors in retinoic acid-induced tissue transglutaminase gene expression in rat tracheal 2C5 cells. Biochem Biophys Res Commun 175:344-50

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