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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): 819-830
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.
dc.language.isoen
dc.publisherAmsterdam: Elsevier
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=193199e877eb410bf0ca9f0c128a8297
dc.subjectOncology
dc.subjectA431 cells
dc.subjectCell Culture
dc.subjectEpidermal Growth Factor Receptor (EGFR)
dc.subjectOligodeoxynucleotide
dc.subjectPhosphorothioate
dc.subjectSkin cells
dc.subjectIn vitro cultivation
dc.subjectExpression profiling
dc.subjectPrimary tumour
dc.subject.meshCell Division
dc.subject.meshDown-Regulation
dc.subject.meshGene Expression Profiling
dc.subject.meshGene Expression Regulation, Neoplastic
dc.subject.meshGlioma
dc.subject.meshHumans
dc.subject.meshOligonucleotide Array Sequence Analysis
dc.subject.meshOligonucleotides, Antisense
dc.subject.meshRNA, Messenger
dc.subject.meshReceptor, Epidermal Growth Factor
dc.subject.meshRibonuclease H
dc.subject.meshSignal Transduction
dc.subject.meshTumor Cells, Cultured
dc.subject.meshCell Line, Tumor
dc.subject.meshCell Culture
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.
dc.typeJournal article
dc.identifier.journalBiochemical Pharmacology
html.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.


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