Regulation of interphotoreceptor retinoid-binding protein (IRBP) gene expression by cAMP in differentiated retinoblastoma cells

Azza E.B. El-Remessy, Ahmad M. Rabie, Mamdouh M. El-Shishtawy, Laila A. Eissa, Gregory I. Liou

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Purpose: To determine the mechanism of cyclic AMP (cAMP) regulation of the interphotoreceptor retinoid-binding protein (IRBP) gene in retinoblastoma cells. Methods: WERI-Rb1 cells pretreated with laminin or grown on poly-D-lysine-coated substratum for three days were treated with forskolin/3-isobutyl-1-methylxanthin (IBMX) or with dimethyl sulfoxide (DMSO). During a time course of 96 h, cell morphologies were determined by light microscopy, cellular cAMP levels measured by radioimmunoassay, and IRBP and β-actin gene expression determined by Northern blot and RNase protection analyses. IRBP expression and β-actin gene expression in these cells were also determined in the presence or absence of actinomycin D or cycloheximide. Results: After laminin treatment for 3 days, 27-34% of WERI-Rb1 cells differentiated into spindle shapes. Further forskolin treatment in the presence of IBMX for 5 days resulted in many cells exhibiting the formation of long, ramifying, neuritelike processes that were abolished by an inhibitor of protein kinase A. Cells grown on poly-D-lysine-coated substratum treated with forskolin remained undifferentiated. Treatment of laminin-pretreated cells with forskolin/IBMX, but not with DMSO/IBMX, raised the cAMP level 15-fold within the first hour of treatment. Northern blot analysis of these cells showed a rapid increase of IRBP mRNA, but not β-actin mRNA, reaching a maximum of about 3-fold at 6-8 h. A similar increase of IRBP mRNA was observed using RNase protection analysis except that the maximum was observed at 2-4 h. Actinomycin D blocked this IRBP mRNA induction. Cycloheximide had no effect in this induction. Conclusions: These results demonstrate that laminin induces WERI-Rb1 cell differentiation and cAMP provokes the formation of long ramifying neurite-like processes. Forskolin selectively induces IRBP gene expression in the laminin-treated cells through a cAMP-mediated pathway without de novo protein synthesis.

Original languageEnglish (US)
Pages (from-to)243-251
Number of pages9
JournalMolecular Vision
Volume6
Issue number33
StatePublished - Dec 1 2000

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Retinoblastoma
Cyclic AMP
Gene Expression
Laminin
Colforsin
Actins
Messenger RNA
Dactinomycin
Cycloheximide
Ribonucleases
Dimethyl Sulfoxide
Northern Blotting
Lysine
interstitial retinol-binding protein
Retinoblastoma Genes
Neurites
Cyclic AMP-Dependent Protein Kinases
Radioimmunoassay
Cell Differentiation
Microscopy

ASJC Scopus subject areas

  • Ophthalmology

Cite this

El-Remessy, A. E. B., Rabie, A. M., El-Shishtawy, M. M., Eissa, L. A., & Liou, G. I. (2000). Regulation of interphotoreceptor retinoid-binding protein (IRBP) gene expression by cAMP in differentiated retinoblastoma cells. Molecular Vision, 6(33), 243-251.

Regulation of interphotoreceptor retinoid-binding protein (IRBP) gene expression by cAMP in differentiated retinoblastoma cells. / El-Remessy, Azza E.B.; Rabie, Ahmad M.; El-Shishtawy, Mamdouh M.; Eissa, Laila A.; Liou, Gregory I.

In: Molecular Vision, Vol. 6, No. 33, 01.12.2000, p. 243-251.

Research output: Contribution to journalArticle

El-Remessy, AEB, Rabie, AM, El-Shishtawy, MM, Eissa, LA & Liou, GI 2000, 'Regulation of interphotoreceptor retinoid-binding protein (IRBP) gene expression by cAMP in differentiated retinoblastoma cells', Molecular Vision, vol. 6, no. 33, pp. 243-251.
El-Remessy, Azza E.B. ; Rabie, Ahmad M. ; El-Shishtawy, Mamdouh M. ; Eissa, Laila A. ; Liou, Gregory I. / Regulation of interphotoreceptor retinoid-binding protein (IRBP) gene expression by cAMP in differentiated retinoblastoma cells. In: Molecular Vision. 2000 ; Vol. 6, No. 33. pp. 243-251.
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abstract = "Purpose: To determine the mechanism of cyclic AMP (cAMP) regulation of the interphotoreceptor retinoid-binding protein (IRBP) gene in retinoblastoma cells. Methods: WERI-Rb1 cells pretreated with laminin or grown on poly-D-lysine-coated substratum for three days were treated with forskolin/3-isobutyl-1-methylxanthin (IBMX) or with dimethyl sulfoxide (DMSO). During a time course of 96 h, cell morphologies were determined by light microscopy, cellular cAMP levels measured by radioimmunoassay, and IRBP and β-actin gene expression determined by Northern blot and RNase protection analyses. IRBP expression and β-actin gene expression in these cells were also determined in the presence or absence of actinomycin D or cycloheximide. Results: After laminin treatment for 3 days, 27-34{\%} of WERI-Rb1 cells differentiated into spindle shapes. Further forskolin treatment in the presence of IBMX for 5 days resulted in many cells exhibiting the formation of long, ramifying, neuritelike processes that were abolished by an inhibitor of protein kinase A. Cells grown on poly-D-lysine-coated substratum treated with forskolin remained undifferentiated. Treatment of laminin-pretreated cells with forskolin/IBMX, but not with DMSO/IBMX, raised the cAMP level 15-fold within the first hour of treatment. Northern blot analysis of these cells showed a rapid increase of IRBP mRNA, but not β-actin mRNA, reaching a maximum of about 3-fold at 6-8 h. A similar increase of IRBP mRNA was observed using RNase protection analysis except that the maximum was observed at 2-4 h. Actinomycin D blocked this IRBP mRNA induction. Cycloheximide had no effect in this induction. Conclusions: These results demonstrate that laminin induces WERI-Rb1 cell differentiation and cAMP provokes the formation of long ramifying neurite-like processes. Forskolin selectively induces IRBP gene expression in the laminin-treated cells through a cAMP-mediated pathway without de novo protein synthesis.",
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AU - El-Remessy, Azza E.B.

AU - Rabie, Ahmad M.

AU - El-Shishtawy, Mamdouh M.

AU - Eissa, Laila A.

AU - Liou, Gregory I.

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N2 - Purpose: To determine the mechanism of cyclic AMP (cAMP) regulation of the interphotoreceptor retinoid-binding protein (IRBP) gene in retinoblastoma cells. Methods: WERI-Rb1 cells pretreated with laminin or grown on poly-D-lysine-coated substratum for three days were treated with forskolin/3-isobutyl-1-methylxanthin (IBMX) or with dimethyl sulfoxide (DMSO). During a time course of 96 h, cell morphologies were determined by light microscopy, cellular cAMP levels measured by radioimmunoassay, and IRBP and β-actin gene expression determined by Northern blot and RNase protection analyses. IRBP expression and β-actin gene expression in these cells were also determined in the presence or absence of actinomycin D or cycloheximide. Results: After laminin treatment for 3 days, 27-34% of WERI-Rb1 cells differentiated into spindle shapes. Further forskolin treatment in the presence of IBMX for 5 days resulted in many cells exhibiting the formation of long, ramifying, neuritelike processes that were abolished by an inhibitor of protein kinase A. Cells grown on poly-D-lysine-coated substratum treated with forskolin remained undifferentiated. Treatment of laminin-pretreated cells with forskolin/IBMX, but not with DMSO/IBMX, raised the cAMP level 15-fold within the first hour of treatment. Northern blot analysis of these cells showed a rapid increase of IRBP mRNA, but not β-actin mRNA, reaching a maximum of about 3-fold at 6-8 h. A similar increase of IRBP mRNA was observed using RNase protection analysis except that the maximum was observed at 2-4 h. Actinomycin D blocked this IRBP mRNA induction. Cycloheximide had no effect in this induction. Conclusions: These results demonstrate that laminin induces WERI-Rb1 cell differentiation and cAMP provokes the formation of long ramifying neurite-like processes. Forskolin selectively induces IRBP gene expression in the laminin-treated cells through a cAMP-mediated pathway without de novo protein synthesis.

AB - Purpose: To determine the mechanism of cyclic AMP (cAMP) regulation of the interphotoreceptor retinoid-binding protein (IRBP) gene in retinoblastoma cells. Methods: WERI-Rb1 cells pretreated with laminin or grown on poly-D-lysine-coated substratum for three days were treated with forskolin/3-isobutyl-1-methylxanthin (IBMX) or with dimethyl sulfoxide (DMSO). During a time course of 96 h, cell morphologies were determined by light microscopy, cellular cAMP levels measured by radioimmunoassay, and IRBP and β-actin gene expression determined by Northern blot and RNase protection analyses. IRBP expression and β-actin gene expression in these cells were also determined in the presence or absence of actinomycin D or cycloheximide. Results: After laminin treatment for 3 days, 27-34% of WERI-Rb1 cells differentiated into spindle shapes. Further forskolin treatment in the presence of IBMX for 5 days resulted in many cells exhibiting the formation of long, ramifying, neuritelike processes that were abolished by an inhibitor of protein kinase A. Cells grown on poly-D-lysine-coated substratum treated with forskolin remained undifferentiated. Treatment of laminin-pretreated cells with forskolin/IBMX, but not with DMSO/IBMX, raised the cAMP level 15-fold within the first hour of treatment. Northern blot analysis of these cells showed a rapid increase of IRBP mRNA, but not β-actin mRNA, reaching a maximum of about 3-fold at 6-8 h. A similar increase of IRBP mRNA was observed using RNase protection analysis except that the maximum was observed at 2-4 h. Actinomycin D blocked this IRBP mRNA induction. Cycloheximide had no effect in this induction. Conclusions: These results demonstrate that laminin induces WERI-Rb1 cell differentiation and cAMP provokes the formation of long ramifying neurite-like processes. Forskolin selectively induces IRBP gene expression in the laminin-treated cells through a cAMP-mediated pathway without de novo protein synthesis.

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