Phosphorothioate antisense oligonucleotides against basic fibroblast growth factor inhibit anchorage-dependent and anchorage-independent growth of a malignant glioblastoma cell line.
Basic fibroblast growth factor (bFGF) is a broad spectrum mitogen for many cells of neuroectodermal origin, including glial cells. The human malignant glioblastoma cell line U87-MG expresses high steady state levels of the bFGF mRNA and contains abundant stores of biologically active bFGF protein. In the present study we have examined the contribution of endogenous bFGF to the autocrine growth of these cells. Using reverse transcription-polymerase chain reaction, U87-MG cells were shown to express the mRNAs for both bFGF and the bFGF receptor, confirming the existence of the basic requirements for an autocrine loop. Addition of 5 microM bFGF-specific antisense oligonucleotide to U87-MG cultures significantly inhibited the growth rate of these cells within 48 h and blocked proliferation beyond 2 days. The corresponding bFGF-specific sense oligonucleotide did not significantly inhibit cell proliferation over the course of these experiments. Similarly, antisense oligonucleotides significantly inhibited colony formation in soft agar, while the sense sequence was without effect. Western blotting with antihuman bFGF revealed that U87-MG cells synthesize three isoforms of bFGF, approximately 18, 23, and 25 kilodaltons (kDa) in size. The 23- and 25-kDa isoforms together comprise approximately 80% of the total cellular stores of bFGF. Antisense treatment for 4 days reduced the abundance of the 23- and 25-kDa isoforms by 64-74%, but had little effect on the 18-kDa isoform. The inhibitory effect of the antisense oligonucleotides on anchorage-dependent proliferation was reversed by the addition of recombinant 18-kDa human bFGF.(ABSTRACT TRUNCATED AT 250 WORDS)
Murphy
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Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada.
Sato
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Knee
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D001483:Base Sequence; D001932:Brain Neoplasms / Q000378:metabolism / Q000473:pathology*; D002448:Cell Adhesion / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D003470:Culture Media; D005246:Feedback; D016222:Fibroblast Growth Factor 2 / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000494:pharmacology / Q000502:physiology*; D005909:Glioblastoma / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology*; D011519:Proto-Oncogenes; D016372:RNA, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000502:physiology*; D017468:Receptors, Fibroblast Growth Factor; D013873:Thionucleotides
D003470:Culture Media; D009363:Neoplasm Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D017468:Receptors, Fibroblast Growth Factor; D013873:Thionucleotides; D016222:Fibroblast Growth Factor 2