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

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    Authors
    Petch, Amelia K.
    Sohail, Muhammad
    Hughes, Marcus D.
    Benter, Ibrahim
    Darling, John L.
    Southern, Edwin M.
    Akhtar, Saghir
    Issue Date
    2003
    
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    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
    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
    Journal article
    Language
    en
    ISSN
    0006-2952
    ae974a485f413a2113503eed53cd6c53
    10.1016/S0006-2952(03)00407-6
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