Antisense oligonucleotide targeting of Raf-1: importance of raf-1 mRNA expression levels and raf-1-dependent signaling in determining growth response in ovarian cancer

Peter Mullen, Fiona McPhillips, Kenneth MacLeod, Brett Monia, John F Smyth, Simon P Langdon

Research output: Contribution to journalArticlepeer-review

27 Citations (Scopus)

Abstract

PURPOSE: We sought to identify determinants of growth response to the Raf-1-targeted antisense oligonucleotide (ASO; ISIS 5132) using a large panel of ovarian cancer cell lines.

EXPERIMENTAL DESIGN: First-(ISIS 5132) and second-generation (ISIS 13650) anti-Raf 1 ASOs were compared with control oligonucleotides. Growth was assessed by cell counts; apoptosis was assessed by poly(ADP-ribose) polymerase cleavage; and cell cycle analysis was assessed by flow cytometry. Protein expression was detected by Western blot analysis, and mRNA expression was detected by quantitative reverse transcription-PCR. Raf-1 kinase activity was detected by anti-Raf-1 immunoprecipitation, followed by myelin basic protein phosphorylation.

RESULTS: A panel of 15 ovarian cancer cell lines was used to model a range of growth responses to ASOs targeting Raf-1 mRNA. Growth inhibition varied from 10% to >90% inhibition. Growth inhibition was associated with increased apoptosis and accumulation of cells in the G(2)-M and S phases of the cell cycle. Growth response was not related to level of Raf-1 protein expression, Raf-1 kinase activity, intracellular ASO uptake, or degree of Raf-1 protein inhibition. However, ASO growth response was associated with a high proportion of Raf-1 mRNA [relative to total (i.e., Raf-1 + A-Raf + B-Raf) Raf mRNA] and significantly higher Raf-1 kinase activity induction following growth factor (transforming growth factor alpha) stimulation in the cell lines consistent with dependency of these cell lines on Raf-1.

CONCLUSIONS: These data indicate that ovarian cancers demonstrate differential sensitivity to ASOs targeted against Raf-1, and target expression levels and degree of utilization of Raf-1 signaling are implicated. Clinically sensitive tumors could feasibly be identified.

Original languageEnglish
Pages (from-to)2100-8
Number of pages9
JournalClinical Cancer Research
Volume10
Issue number6
DOIs
Publication statusPublished - 15 Mar 2004

Keywords

  • Base Sequence
  • Cell Cycle/drug effects
  • Cell Division/drug effects
  • Cell Line, Tumor
  • Female
  • Gene Expression Regulation, Enzymologic/drug effects
  • Gene Expression Regulation, Neoplastic/drug effects
  • Humans
  • Oligonucleotides, Antisense/pharmacokinetics
  • Ovarian Neoplasms/genetics
  • Proto-Oncogene Proteins c-raf/genetics
  • RNA, Messenger/genetics
  • Transcription, Genetic/genetics

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