Messenger RNA expression profiling of genes involved in epidermal growth factor receptor signalling in human cancer cells treated with scanning array-designed antisense oligonucleotides.

2.50
Hdl Handle:
http://hdl.handle.net/2436/29816
Title:
Messenger RNA expression profiling of genes involved in epidermal growth factor receptor signalling in human cancer cells treated with scanning array-designed antisense oligonucleotides.
Authors:
Petch, Amelia K.; Sohail, Muhammad; Hughes, Marcus D.; Benter, Ibrahim; Darling, John L.; Southern, Edwin M.; Akhtar, Saghir
Abstract:
Scanning oligodeoxynucleotide (ODN) arrays appear promising in vitro tools for the prediction of effective antisense reagents but their usefulness has not yet been reported in mammalian systems. In this study, we have evaluated the use of scanning ODN arrays to predict efficacious antisense ODNs targeting the human epidermal growth factor receptor (EGFR) mRNA in a human epidermoid cancer cell line and in primary human glioma cells. Hybridisation accessibility profile of the first 120nt in the coding region of the human EGFR mRNA was determined by hybridising a radiolabelled EGFR transcript to a scanning array of 2684 antisense sequences ranging from monomers to 27-mers. Two ODNs, AS1 and AS2, complementary to accessible sequences within the EGFR mRNA, were designed and their ability to hybridise to EGFR mRNA was further confirmed by in vitro RNase H-mediated cleavage assays. Phosphorothioate-modified 21-mer AS1 and AS2 ODNs inhibited the growth of an established human A431 cancer cell line as well as primary glioma cells from human subjects when delivered as cationic lipoplexes. In contrast, scrambled controls and AS3-an antisense ODN complementary to an inaccessible site in EGFR mRNA-were inactive. Western blots showed that AS1 ODN exhibited a dose-dependent inhibition of EGFR protein expression in A431 cells in the nanomolar range. Microarray-based gene expression profiling studies of A431 cells treated with the 21-mer phosphorothioate AS1 ODN demonstrated successful inhibition of downstream signalling molecules further confirming the effective inhibition of EGFR expression in human cancer cells by antisense ODNs designed by scanning ODN array technology.
Citation:
Biochemical Pharmacology, 66(5): 819-830
Publisher:
Amsterdam: Elsevier
Journal:
Biochemical Pharmacology
Issue Date:
2003
URI:
http://hdl.handle.net/2436/29816
DOI:
10.1016/S0006-2952(03)00407-6
PubMed ID:
12948863
Additional Links:
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T4P-49621XH-2&_user=1644469&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000054077&_version=1&_urlVersion=0&_userid=1644469&md5=193199e877eb410bf0ca9f0c128a8297
Type:
Article
Language:
en
ISSN:
0006-2952
Appears in Collections:
Cancer Research Group

Full metadata record

DC FieldValue Language
dc.contributor.authorPetch, Amelia K.-
dc.contributor.authorSohail, Muhammad-
dc.contributor.authorHughes, Marcus D.-
dc.contributor.authorBenter, Ibrahim-
dc.contributor.authorDarling, John L.-
dc.contributor.authorSouthern, Edwin M.-
dc.contributor.authorAkhtar, Saghir-
dc.date.accessioned2008-06-10T16:15:44Z-
dc.date.available2008-06-10T16:15:44Z-
dc.date.issued2003-
dc.identifier.citationBiochemical Pharmacology, 66(5): 819-830en
dc.identifier.issn0006-2952-
dc.identifier.pmid12948863-
dc.identifier.doi10.1016/S0006-2952(03)00407-6-
dc.identifier.urihttp://hdl.handle.net/2436/29816-
dc.description.abstractScanning oligodeoxynucleotide (ODN) arrays appear promising in vitro tools for the prediction of effective antisense reagents but their usefulness has not yet been reported in mammalian systems. In this study, we have evaluated the use of scanning ODN arrays to predict efficacious antisense ODNs targeting the human epidermal growth factor receptor (EGFR) mRNA in a human epidermoid cancer cell line and in primary human glioma cells. Hybridisation accessibility profile of the first 120nt in the coding region of the human EGFR mRNA was determined by hybridising a radiolabelled EGFR transcript to a scanning array of 2684 antisense sequences ranging from monomers to 27-mers. Two ODNs, AS1 and AS2, complementary to accessible sequences within the EGFR mRNA, were designed and their ability to hybridise to EGFR mRNA was further confirmed by in vitro RNase H-mediated cleavage assays. Phosphorothioate-modified 21-mer AS1 and AS2 ODNs inhibited the growth of an established human A431 cancer cell line as well as primary glioma cells from human subjects when delivered as cationic lipoplexes. In contrast, scrambled controls and AS3-an antisense ODN complementary to an inaccessible site in EGFR mRNA-were inactive. Western blots showed that AS1 ODN exhibited a dose-dependent inhibition of EGFR protein expression in A431 cells in the nanomolar range. Microarray-based gene expression profiling studies of A431 cells treated with the 21-mer phosphorothioate AS1 ODN demonstrated successful inhibition of downstream signalling molecules further confirming the effective inhibition of EGFR expression in human cancer cells by antisense ODNs designed by scanning ODN array technology.en
dc.language.isoenen
dc.publisherAmsterdam: Elsevieren
dc.relation.urlhttp://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T4P-49621XH-2&_user=1644469&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000054077&_version=1&_urlVersion=0&_userid=1644469&md5=193199e877eb410bf0ca9f0c128a8297en
dc.subjectOncologyen
dc.subjectA431 cellsen
dc.subjectCell Cultureen
dc.subjectEpidermal Growth Factor Receptor (EGFR)en
dc.subjectOligodeoxynucleotideen
dc.subjectPhosphorothioateen
dc.subjectSkin cellsen
dc.subjectIn vitro cultivationen
dc.subjectExpression profilingen
dc.subjectPrimary tumouren
dc.subject.meshCell Divisionen
dc.subject.meshDown-Regulationen
dc.subject.meshGene Expression Profilingen
dc.subject.meshGene Expression Regulation, Neoplasticen
dc.subject.meshGliomaen
dc.subject.meshHumansen
dc.subject.meshOligonucleotide Array Sequence Analysisen
dc.subject.meshOligonucleotides, Antisenseen
dc.subject.meshRNA, Messengeren
dc.subject.meshReceptor, Epidermal Growth Factoren
dc.subject.meshRibonuclease Hen
dc.subject.meshSignal Transductionen
dc.subject.meshTumor Cells, Cultureden
dc.subject.meshCell Line, Tumoren
dc.subject.meshCell Cultureen
dc.titleMessenger RNA expression profiling of genes involved in epidermal growth factor receptor signalling in human cancer cells treated with scanning array-designed antisense oligonucleotides.en
dc.typeArticleen
dc.identifier.journalBiochemical Pharmacologyen

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