Endogenous CRX expression and IRBP promoter activity in retinoblastoma cells.
OBJECTIVE
To determine whether antisense oligonucleotides (AODNs) targeted against CRX, a photoreceptor-specific trans-acting factor, suppress CRX expression and interphotoreceptor retinoid binding protein (IRBP) promoter activity.


METHODS
Cultures of human retinoblastoma cells were transfected with chloramphenicol acetyltransferase (CAT) reporter plasmids containing a mouse IRBP promoter and AODNs directed against CRX. RT-PCR using primers specific to CRX, OTX2, GAPDH, or RNase H was conducted on total RNA isolated from retinoblastoma cells at various times following transfection with AODNs.


RESULTS
Transfection of retinoblastoma cells with IRBP promoter CAT constructs alone produced high activity. Co-transfection with AODNs suppressed IRBP promoter activity in a concentration-dependent manner, with half-maximal effect produced at about 2 nM AODN concentration. Transfection with CAT constructs containing an SV40 promoter produced high activity that was unaffected by co-transfection with AODNs. RT-PCR products were obtained for all target sequences. CRX RT-PCR product from cells transfected with AODNs was greatly diminished following transfection with an AODN whereas control transcripts, including that of OTX2, were relatively unaffected.


CONCLUSIONS
The CRX-specific AODNs specifically and potently suppressed CRX expression and IRBP promoter activity, as measured by RT-PCR and transient transfection assays, respectively. Little or no effect was seen on controls. These data suggest that endogenous CRX is required for IRBP promoter activity in retinoblastoma cells.
Boatright
J H
JH
Emory Eye Center, Atlanta, GA, USA. jboatri@emory.edu
Borst
D E
DE
Stodulkova
E
E
Nickerson
J M
JM
D001483:Base Sequence / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005136:Eye Proteins; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D017930:Genes, Reporter / Q000187:drug effects / Q000502:physiology; D005822:Genetic Vectors / Q000502:physiology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000187:drug effects / Q000235:genetics; D018398:Homeodomain Proteins / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D051857:Otx Transcription Factors; D011401:Promoter Regions, Genetic / Q000187:drug effects* / Q000502:physiology; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D012160:Retina / Q000187:drug effects* / Q000378:metabolism; D012175:Retinoblastoma; D012177:Retinol-Binding Proteins / Q000187:drug effects* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016914:Ribonuclease H / Q000187:drug effects / Q000235:genetics; D015534:Trans-Activators / Q000187:drug effects* / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects* / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D000099214:Retinol-Binding Proteins, Interstitial
D005136:Eye Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D016376:Oligonucleotides, Antisense; D051857:Otx Transcription Factors; D012333:RNA, Messenger; D012177:Retinol-Binding Proteins; D016914:Ribonuclease H; D015534:Trans-Activators; D000099214:Retinol-Binding Proteins, Interstitial; C497659:OTX2 protein, human; C109288:cone rod homeobox protein