Characterization of novel phorbol ester- and serum-responsive sequences of the rat ornithine decarboxylase gene promoter

P. K. Mar, Addanki P Kumar, D. C. Kang, B. Zhao, L. A. Martinez, R. L. Montgomery, L. Anderson, A. P. Butler

Research output: Contribution to journalArticle

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Abstract

Ornithine decarboxylase (ODC), the key regulatory enzyme in mammalian polyamine biosynthesis, is rapidly induced by mitogens and tumor promoters. We used transient expression assays and DNA-protein binding studies to examine the regulation of ODC promoter activity by phorbol esters and serum growth factors. A fragment of the ODC 5' flanking region (nt -1156 to +13) was sufficient to confer 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive expression to a luciferase reporter gene when transfected into H35 cells. However, induction by TPA was not observed in Rat2 fibroblasts, although refeeding of serum-starved Rat2 cells with fresh serum-containing medium rapidly induced a fivefold to sixfold increase in ODC promoter activity, maximal about 8 h after refeeding. Deletion analysis demonstrated that several sequences contributed to basal ODC promoter activity but that nt -92 to +13 was sufficient for induction by TPA or by serum. This sequence lacked canonical TPA-responsive elements, and an activator protein-1 (AP-1) consensus oligonucleotide failed to compete effectively for proteins binding to this region. Two of four protein complexes observed by gel-shift analysis of nt -92 to +13 were competitively inhibited by wild-type but not mutant oligonucleotides encompassing a variant cyclic AMP-response element (CRE) (ODC nt -50 to -42); however, a consensus CRE did not compete. Mutagenesis of this site demonstrated that it contributes to basal expression of the ODC promoter but not to TPA or serum responsiveness. Thus, we conclude that the proximal ODC promoter (nt -92 to +13) responds to TPA and serum stimulation in a cell-type-specific manner that is not mediated by canonical AP-1 elements.

Original languageEnglish (US)
Pages (from-to)240-250
Number of pages11
JournalMolecular Carcinogenesis
Volume14
Issue number4
DOIs
StatePublished - 1995
Externally publishedYes

Fingerprint

Ornithine Decarboxylase
Phorbol Esters
Tetradecanoylphorbol Acetate
Serum
Genes
Transcription Factor AP-1
Response Elements
Oligonucleotides
Cyclic AMP
5' Flanking Region
DNA-Binding Proteins
Polyamines
Electrophoretic Mobility Shift Assay
Luciferases
Reporter Genes
Mitogens
Protein Binding
Mutagenesis
Carcinogens
Intercellular Signaling Peptides and Proteins

Keywords

  • Ornithine decarboxylase
  • Phorbol ester
  • Transcription

ASJC Scopus subject areas

  • Cancer Research
  • Molecular Biology

Cite this

Characterization of novel phorbol ester- and serum-responsive sequences of the rat ornithine decarboxylase gene promoter. / Mar, P. K.; Kumar, Addanki P; Kang, D. C.; Zhao, B.; Martinez, L. A.; Montgomery, R. L.; Anderson, L.; Butler, A. P.

In: Molecular Carcinogenesis, Vol. 14, No. 4, 1995, p. 240-250.

Research output: Contribution to journalArticle

Mar, P. K. ; Kumar, Addanki P ; Kang, D. C. ; Zhao, B. ; Martinez, L. A. ; Montgomery, R. L. ; Anderson, L. ; Butler, A. P. / Characterization of novel phorbol ester- and serum-responsive sequences of the rat ornithine decarboxylase gene promoter. In: Molecular Carcinogenesis. 1995 ; Vol. 14, No. 4. pp. 240-250.
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AU - Zhao, B.

AU - Martinez, L. A.

AU - Montgomery, R. L.

AU - Anderson, L.

AU - Butler, A. P.

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AB - Ornithine decarboxylase (ODC), the key regulatory enzyme in mammalian polyamine biosynthesis, is rapidly induced by mitogens and tumor promoters. We used transient expression assays and DNA-protein binding studies to examine the regulation of ODC promoter activity by phorbol esters and serum growth factors. A fragment of the ODC 5' flanking region (nt -1156 to +13) was sufficient to confer 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive expression to a luciferase reporter gene when transfected into H35 cells. However, induction by TPA was not observed in Rat2 fibroblasts, although refeeding of serum-starved Rat2 cells with fresh serum-containing medium rapidly induced a fivefold to sixfold increase in ODC promoter activity, maximal about 8 h after refeeding. Deletion analysis demonstrated that several sequences contributed to basal ODC promoter activity but that nt -92 to +13 was sufficient for induction by TPA or by serum. This sequence lacked canonical TPA-responsive elements, and an activator protein-1 (AP-1) consensus oligonucleotide failed to compete effectively for proteins binding to this region. Two of four protein complexes observed by gel-shift analysis of nt -92 to +13 were competitively inhibited by wild-type but not mutant oligonucleotides encompassing a variant cyclic AMP-response element (CRE) (ODC nt -50 to -42); however, a consensus CRE did not compete. Mutagenesis of this site demonstrated that it contributes to basal expression of the ODC promoter but not to TPA or serum responsiveness. Thus, we conclude that the proximal ODC promoter (nt -92 to +13) responds to TPA and serum stimulation in a cell-type-specific manner that is not mediated by canonical AP-1 elements.

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