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.
OBJECTIVE
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.


METHODS
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.
Mullen
Peter
P
Cancer Research UK, Edinburgh Oncology Unit, Western General Hospital, Edinburgh, United Kingdom.
McPhillips
Fiona
F
MacLeod
Kenneth
K
Monia
Brett
B
Smyth
John F
JF
Langdon
Simon P
SP
D001483:Base Sequence; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000493:pharmacokinetics / Q000494:pharmacology*; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000473:pathology*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D019908:Proto-Oncogene Proteins c-raf