Regulation of interphotoreceptor retinoid-binding protein (IRBP) gene expression by cAMP in differentiated retinoblastoma cells.
OBJECTIVE
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 b-actin gene expression determined by Northern blot and RNase protection analyses. IRBP expression and b-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, neurite-like 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 b-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.
El-Remessy
A E
AE
Department of Ophthalmology, Medical College of Georgia, Augusta, GA, USA.
Rabie
A M
AM
El-Shishtawy
M M
MM
Eissa
L A
LA
Liou
G I
GI
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000199:Actins / Q000235:genetics; D015152:Blotting, Northern; D002454:Cell Differentiation / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D005136:Eye Proteins / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007797:Laminin / Q000494:pharmacology; D023282:Nuclease Protection Assays; D012333:RNA, Messenger / Q000378:metabolism; D011863:Radioimmunoassay; D019572:Retinal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D012175:Retinoblastoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D012177:Retinol-Binding Proteins / Q000235:genetics*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D000099214:Retinol-Binding Proteins, Interstitial
D015056:1-Methyl-3-isobutylxanthine; D000199:Actins; D005576:Colforsin; D000242:Cyclic AMP; D003513:Cycloheximide; D003609:Dactinomycin; D005136:Eye Proteins; D007797:Laminin; D012333:RNA, Messenger; D012177:Retinol-Binding Proteins; D000099214:Retinol-Binding Proteins, Interstitial