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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 G2-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 α) 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.

c-Raf is an essential component of the extracellular related kinase (ERK) signal transduction pathway. Immunohistochemical staining indicated that c-Raf was present in 49/53 ovarian adenocarcinomas investigated and high c-Raf expression correlated significantly with poor survival (P = 0.002). c-Raf protein was detected in 15 ovarian cancer cell lines. Antisense oligodeoxynucleotides (ODNs) (ISIS 5132 and ISIS 13650) reduced c-Raf protein levels and inhibited cell proliferation in vitro. Selectivity was demonstrated by the lack of effect of ISIS 5132 on A-Raf or ERK, while a random ODN produced only minor effects on growth and did not influence c-Raf expression. ISIS 5132 produced enhanced apoptosis and cells accumulated in S and G 2/M phases of the cell cycle. In vivo, ISIS 5132 inhibited growth of the s.c. SKOV-3 xenograft while a mismatch ODN had no effect. These data indicate that high levels of c-Raf expression may be important in ovarian cancer and use of antisense ODNs targeted to c-Raf could provide a strategy for the treatment of this disease. © 2001 Cancer Research Campaign http://www.bjcancer.com

The lipoxygenase-derived eicosanoids leukotrienes and lipoxins are well defined regulators of hemeodynamics and leukocyte recruitment in inflammatory conditions. Here, we describe a novel bioaction of lipoxin A4(LXA4), namely inhibition of leukotriene D4 (LTD4)-induced human renal mesangial cell proliferation, and investigate the signal transduction mechanisms involved. LXA4 blocked LTD4-stimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity in parallel to inhibition of LTD4-induced mesangial cell proliferation. Screening of a human mesangial cell cDNA library revealed expression of the recently described cys-leukotriene1/LTD4 receptor. LTD4-induced mesangial cell proliferation required both extracellular-related signal regulated kinase (erk) and PI 3-kinase activation and may involve platelet-derived growth factor receptor transactivation. LTD4-stimulated the MAP kinases erk and p38 via a pertussis toxin (PTX)-sensitive pathway dependent on PI 3-kinase and protein kinase C activation. On screening a cDNA library, mesangial cells were found to express the previously described LXA4 receptor. In contrast to LTD4, LXA4 showed differential activation of erk and p38. LXA4 activation of erk was insensitive to PTX and PI 3-kinase inhibition, whereas LXA4 activation of p38 was sensitive to PTX and could be blocked by the LTD4 receptor antagonist SKF 104353. These data suggest that LXA4stimulation of the MAP kinase superfamily involves two distinct receptors: one shared with LTD4 and coupled to a PTX-sensitive G protein (Gi) and a second coupled via an alternative G protein, such as Gq or G12, to erk activation. These data expand on the spectrum of LXA4bioactions within an inflammatory milieu.

Human skin is exposed daily to solar ultraviolet (UV) radiation. UV induces the matrix metalloproteinases collagenase, 92-kD gelatinase, and stromelysin, which degrade skin connective tissue and may contribute to premature skin aging (photoaging). Pretreatment of skin with all-trans retinoic acid (tRA) inhibits UV induction of matrix metalloproteinases. We investigated upstream signal transduction pathways and the mechanism of tRA inhibition of UV induction of matrix metalloproteinases in human skin in vivo. Exposure of human skin in vivo to low doses of UV activated EGF receptors, the GTP-binding regulatory protein p21Ras, and stimulated mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38. Both JNK and p38 phosphorylated, and thereby activated transcription factors c-Jun and activating transcription factor 2 (ATF-2), which bound to the c-Jun promoter and upregulated c-Jun gene expression. Elevated c-Jun, in association with constitutively expressed c-Fos, formed increased levels of transcription factor activator protein (AP) 1, which is required for transcription of matrix metalloproteinases. Pretreatment of human skin with tRA inhibited UV induction of c-Jun protein and, consequently, AP-1. c-Jun protein inhibition occurred via a posttranscriptional mechanism, since tRA did not inhibit UV induction of c-Jun mRNA. These data demonstrate, for the first time, activation of MAP kinase pathways in humans in vivo, and reveal a novel posttranscriptional mechanism by which tRA antagonizes UV activation of AP-1 by inhibiting c-Jun protein induction. Inhibition of c-Jun induction likely contributes to the previously reported prevention by tRA of UV induction of AP-1-regulated matrix-degrading metalloproteinases in human skin.

There is currently much interest in the role of the Raf family in cancer, particularly since mutated B-Raf has been shown to be oncogenic in certain disease types. In this study we have explored the expression, signaling and function of the three known Raf isoforms (Raf-1, A-Raf and B-Raf) in patients with ovarian cancer. While increased expression of Raf-1 was associated with poor survival, increased expression of B-Raf was associated with improved survival. Using a panel of ovarian cancer cell lines, all three isoforms were shown to be involved in growth factor initiated signaling. Antisense inhibition of function in ovarian cancer cell lines indicated that both Raf-1 and A-Raf, but not B-Raf, were linked to cell proliferation. Raf-1 (but not A-Raf or B-Raf) was also associated with reduced apoptosis. While individual Raf reduction by isoform-targeted antisense oligonucleotides (ODNs) produced growth inhibition in some cell lines, similar use of the MEK inhibitor UO126 produced growth inhibition in all cell lines tested. These data suggest that Raf-1 is the predominant Raf isoform responsible for regulating cellular growth in ovarian cancer cells and may be particularly important in high grade serous ovarian cancers.

In this study, we characterize the uptake and specificity of a first-generation Raf-1 antisense oligonucleotide (ASO) (ISIS 5132) and compare it with a second-generation ASO (ISIS 13650) and an RNA interference approach. All three approaches resulted in inhibition of both Raf-1 expression and cellular growth. Specificity of the Raf-1 ASOs was confirmed by comparison with ASOs targeted against another Raf isoform (B-Raf) as well as mismatch sequences. Cellular uptake studies with FAM-labelled ISIS 5132 revealed that whilst the majority of cells treated at a low-intermediate plating density were labelled within 3 hr, cells treated at high density demonstrated neither Raf-1 protein knockout nor significant growth inhibition, following similar treatment. This lack of response at high cell densities was associated with reduced pERK and Raf-1 inhibition. Cell cycle analysis revealed that whilst SKOV-3 cells both accumulated in the S-phase of the cell cycle and showed enhanced annexin V levels, following Raf-1 ASO treatment; these effects were also demonstrated with first-generation but not second-generation mismatch oligonucleotides. Bromodeoxyuridine incorporation analysis suggested that these effects may indeed be partly attributable to sequence nonspecific effects. Finally, the combination of ISIS 5132 with either carboplatin or taxol showed enhanced growth inhibition, supporting the view that such ASOs may have a more effective clinical role when used in combination with cytotoxic agents.

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.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.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% to90% 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.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.