Endogenous CRX expression and IRBP promoter activity in retinoblastoma cells
Purpose: 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...
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| Veröffentlicht in: | Brain research 2001-10, Vol.916 (1), p.136-142 |
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| creator | Boatright, Jeffrey H Borst, Diane E Stodulkova, Eva Nickerson, John M |
| description | Purpose: 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. |
| doi_str_mv | 10.1016/S0006-8993(01)02884-0 |
| format | Article |
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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.</description><identifier>ISSN: 0006-8993</identifier><identifier>EISSN: 1872-6240</identifier><identifier>DOI: 10.1016/S0006-8993(01)02884-0</identifier><identifier>PMID: 11597600</identifier><identifier>CODEN: BRREAP</identifier><language>eng</language><publisher>London: Elsevier B.V</publisher><subject>Antisene ; Base Sequence - genetics ; Biological and medical sciences ; CRX ; CRX protein ; Dose-Response Relationship, Drug ; Eye and associated structures. Visual pathways and centers. Vision ; Eye Proteins ; Fundamental and applied biological sciences. Psychology ; Gene Expression Regulation - drug effects ; Gene Expression Regulation - physiology ; Genes, Reporter - drug effects ; Genes, Reporter - physiology ; Genetic Vectors - physiology ; Glyceraldehyde-3-Phosphate Dehydrogenases - drug effects ; Glyceraldehyde-3-Phosphate Dehydrogenases - genetics ; Homeodomain Proteins - drug effects ; Homeodomain Proteins - genetics ; Humans ; interphotoreceptor retinoid-binding protein ; IRBP ; Molecular Sequence Data ; Nerve Tissue Proteins - drug effects ; Nerve Tissue Proteins - genetics ; Oligonucleotides, Antisense - pharmacology ; Otx Transcription Factors ; Promoter Regions, Genetic - drug effects ; Promoter Regions, Genetic - physiology ; Quantitative PCR ; Retina - drug effects ; Retina - metabolism ; Retinoblastoma ; retinoblastoma cells ; Retinol-Binding Proteins - drug effects ; Retinol-Binding Proteins - genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Ribonuclease H - drug effects ; Ribonuclease H - genetics ; RNA, Messenger - drug effects ; RNA, Messenger - metabolism ; Trans-Activators - drug effects ; Trans-Activators - genetics ; Transcription, Genetic - drug effects ; Transcription, Genetic - genetics ; Tumor Cells, Cultured - drug effects ; Tumor Cells, Cultured - metabolism ; Vertebrates: nervous system and sense organs</subject><ispartof>Brain research, 2001-10, Vol.916 (1), p.136-142</ispartof><rights>2001 Elsevier Science B.V.</rights><rights>2002 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c422t-a98fb33c91ca1bbd85389b1527eb64534da3628bdd908959296cfe9a0b8fb9193</citedby><cites>FETCH-LOGICAL-c422t-a98fb33c91ca1bbd85389b1527eb64534da3628bdd908959296cfe9a0b8fb9193</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0006899301028840$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3536,27903,27904,65309</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=14106779$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/11597600$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Boatright, Jeffrey H</creatorcontrib><creatorcontrib>Borst, Diane E</creatorcontrib><creatorcontrib>Stodulkova, Eva</creatorcontrib><creatorcontrib>Nickerson, John M</creatorcontrib><title>Endogenous CRX expression and IRBP promoter activity in retinoblastoma cells</title><title>Brain research</title><addtitle>Brain Res</addtitle><description>Purpose: 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.</description><subject>Antisene</subject><subject>Base Sequence - genetics</subject><subject>Biological and medical sciences</subject><subject>CRX</subject><subject>CRX protein</subject><subject>Dose-Response Relationship, Drug</subject><subject>Eye and associated structures. Visual pathways and centers. Vision</subject><subject>Eye Proteins</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Expression Regulation - drug effects</subject><subject>Gene Expression Regulation - physiology</subject><subject>Genes, Reporter - drug effects</subject><subject>Genes, Reporter - physiology</subject><subject>Genetic Vectors - physiology</subject><subject>Glyceraldehyde-3-Phosphate Dehydrogenases - drug effects</subject><subject>Glyceraldehyde-3-Phosphate Dehydrogenases - genetics</subject><subject>Homeodomain Proteins - drug effects</subject><subject>Homeodomain Proteins - genetics</subject><subject>Humans</subject><subject>interphotoreceptor retinoid-binding protein</subject><subject>IRBP</subject><subject>Molecular Sequence Data</subject><subject>Nerve Tissue Proteins - drug effects</subject><subject>Nerve Tissue Proteins - genetics</subject><subject>Oligonucleotides, Antisense - pharmacology</subject><subject>Otx Transcription Factors</subject><subject>Promoter Regions, Genetic - drug effects</subject><subject>Promoter Regions, Genetic - physiology</subject><subject>Quantitative PCR</subject><subject>Retina - drug effects</subject><subject>Retina - metabolism</subject><subject>Retinoblastoma</subject><subject>retinoblastoma cells</subject><subject>Retinol-Binding Proteins - drug effects</subject><subject>Retinol-Binding Proteins - genetics</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>Ribonuclease H - drug effects</subject><subject>Ribonuclease H - genetics</subject><subject>RNA, Messenger - drug effects</subject><subject>RNA, Messenger - metabolism</subject><subject>Trans-Activators - drug effects</subject><subject>Trans-Activators - genetics</subject><subject>Transcription, Genetic - drug effects</subject><subject>Transcription, Genetic - genetics</subject><subject>Tumor Cells, Cultured - drug effects</subject><subject>Tumor Cells, Cultured - metabolism</subject><subject>Vertebrates: nervous system and sense organs</subject><issn>0006-8993</issn><issn>1872-6240</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2001</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkF1rFDEUhoModq3-BCU3Sr2YepLMZJIr0aXWwkKlKngX8jUSmUm2yWxp_72Z7tJe9ioceN6T9zwIvSVwSoDwTz8BgDdCSnYC5CNQIdoGnqEVET1tOG3hOVo9IEfoVSn_6siYhJfoiJBO9hxghTZn0aW_PqZdweurP9jfbrMvJaSIdXT44urrD7zNaUqzz1jbOdyE-Q6HiLOfQ0xm1GVOk8bWj2N5jV4Meiz-zeE9Rr-_nf1af282l-cX6y-bxraUzo2WYjCMWUmsJsY40TEhDelo7w1vO9Y6zTgVxjkJQnaSSm4HLzWYmpNEsmP0Yb-3Nrve-TKrKZSlgY6-HqJ6SmqypU-CRNRtvFvAbg_anErJflDbHCad7xQBtfhW977VIlMBUfe-FdTcu8MHOzN595g6CK7A-wOgi9XjkHW0oTxyLQHe98tJn_ecr95ugs-q2OCj9S5kb2flUniiyn9OUZwA</recordid><startdate>20011019</startdate><enddate>20011019</enddate><creator>Boatright, Jeffrey H</creator><creator>Borst, Diane E</creator><creator>Stodulkova, Eva</creator><creator>Nickerson, John M</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7TK</scope><scope>7X8</scope></search><sort><creationdate>20011019</creationdate><title>Endogenous CRX expression and IRBP promoter activity in retinoblastoma cells</title><author>Boatright, Jeffrey H ; Borst, Diane E ; Stodulkova, Eva ; Nickerson, John M</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c422t-a98fb33c91ca1bbd85389b1527eb64534da3628bdd908959296cfe9a0b8fb9193</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2001</creationdate><topic>Antisene</topic><topic>Base Sequence - genetics</topic><topic>Biological and medical sciences</topic><topic>CRX</topic><topic>CRX protein</topic><topic>Dose-Response Relationship, Drug</topic><topic>Eye and associated structures. Visual pathways and centers. Vision</topic><topic>Eye Proteins</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Expression Regulation - drug effects</topic><topic>Gene Expression Regulation - physiology</topic><topic>Genes, Reporter - drug effects</topic><topic>Genes, Reporter - physiology</topic><topic>Genetic Vectors - physiology</topic><topic>Glyceraldehyde-3-Phosphate Dehydrogenases - drug effects</topic><topic>Glyceraldehyde-3-Phosphate Dehydrogenases - genetics</topic><topic>Homeodomain Proteins - drug effects</topic><topic>Homeodomain Proteins - genetics</topic><topic>Humans</topic><topic>interphotoreceptor retinoid-binding protein</topic><topic>IRBP</topic><topic>Molecular Sequence Data</topic><topic>Nerve Tissue Proteins - drug effects</topic><topic>Nerve Tissue Proteins - genetics</topic><topic>Oligonucleotides, Antisense - pharmacology</topic><topic>Otx Transcription Factors</topic><topic>Promoter Regions, Genetic - drug effects</topic><topic>Promoter Regions, Genetic - physiology</topic><topic>Quantitative PCR</topic><topic>Retina - drug effects</topic><topic>Retina - metabolism</topic><topic>Retinoblastoma</topic><topic>retinoblastoma cells</topic><topic>Retinol-Binding Proteins - drug effects</topic><topic>Retinol-Binding Proteins - genetics</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>Ribonuclease H - drug effects</topic><topic>Ribonuclease H - genetics</topic><topic>RNA, Messenger - drug effects</topic><topic>RNA, Messenger - metabolism</topic><topic>Trans-Activators - drug effects</topic><topic>Trans-Activators - genetics</topic><topic>Transcription, Genetic - drug effects</topic><topic>Transcription, Genetic - genetics</topic><topic>Tumor Cells, Cultured - drug effects</topic><topic>Tumor Cells, Cultured - metabolism</topic><topic>Vertebrates: nervous system and sense organs</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Boatright, Jeffrey H</creatorcontrib><creatorcontrib>Borst, Diane E</creatorcontrib><creatorcontrib>Stodulkova, Eva</creatorcontrib><creatorcontrib>Nickerson, John M</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Neurosciences Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Brain research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Boatright, Jeffrey H</au><au>Borst, Diane E</au><au>Stodulkova, Eva</au><au>Nickerson, John M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Endogenous CRX expression and IRBP promoter activity in retinoblastoma cells</atitle><jtitle>Brain research</jtitle><addtitle>Brain Res</addtitle><date>2001-10-19</date><risdate>2001</risdate><volume>916</volume><issue>1</issue><spage>136</spage><epage>142</epage><pages>136-142</pages><issn>0006-8993</issn><eissn>1872-6240</eissn><coden>BRREAP</coden><abstract>Purpose: 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.</abstract><cop>London</cop><cop>Amsterdam</cop><cop>New York, NY</cop><pub>Elsevier B.V</pub><pmid>11597600</pmid><doi>10.1016/S0006-8993(01)02884-0</doi><tpages>7</tpages></addata></record> |
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| subjects | Antisene Base Sequence - genetics Biological and medical sciences CRX CRX protein Dose-Response Relationship, Drug Eye and associated structures. Visual pathways and centers. Vision Eye Proteins Fundamental and applied biological sciences. Psychology Gene Expression Regulation - drug effects Gene Expression Regulation - physiology Genes, Reporter - drug effects Genes, Reporter - physiology Genetic Vectors - physiology Glyceraldehyde-3-Phosphate Dehydrogenases - drug effects Glyceraldehyde-3-Phosphate Dehydrogenases - genetics Homeodomain Proteins - drug effects Homeodomain Proteins - genetics Humans interphotoreceptor retinoid-binding protein IRBP Molecular Sequence Data Nerve Tissue Proteins - drug effects Nerve Tissue Proteins - genetics Oligonucleotides, Antisense - pharmacology Otx Transcription Factors Promoter Regions, Genetic - drug effects Promoter Regions, Genetic - physiology Quantitative PCR Retina - drug effects Retina - metabolism Retinoblastoma retinoblastoma cells Retinol-Binding Proteins - drug effects Retinol-Binding Proteins - genetics Reverse Transcriptase Polymerase Chain Reaction Ribonuclease H - drug effects Ribonuclease H - genetics RNA, Messenger - drug effects RNA, Messenger - metabolism Trans-Activators - drug effects Trans-Activators - genetics Transcription, Genetic - drug effects Transcription, Genetic - genetics Tumor Cells, Cultured - drug effects Tumor Cells, Cultured - metabolism Vertebrates: nervous system and sense organs |
| title | Endogenous CRX expression and IRBP promoter activity in retinoblastoma cells |
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