Abrogation of c-Raf expression induces apoptosis in tumor cells

Abstract

Signal transduction pathways involving the c-Raf protein kinase are frequently activated in tumor cells. We have addressed the relevance of this activation by a loss-offunction approach. An anti-sense phosphorothioate oligonucleotide (ODN) speci®cally targeted against craf mRNA (Monia et al., 1996a) was used to block cRaf protein expression in four di erent cell lines derived from lung, cervical, prostate and colon carcinomas. Concomitant with the abrogation of c-Raf expression we observed the occurrence of classical apoptotic markers, including chromatin condensation, inter-nucleosomal DNA cleavage, annexin V binding and cleavage of PARP, which was followed by cell death, a ecting most of the cell population. This induction of apoptosis occurred independent of the p53 status of the cell. These ®ndings demonstrate that c-Raf can protect tumor cells from undergoing programmed cell death, and suggest that the interference with c-Raf expression or function by ODNs or speci®c drugs could represent a powerful means for improving the e cacy of anti-cancer therapy.Keywords: apoptosis; programmed cell death; Raf; antisense; phosphorothiate ODN The occurrence of pro-apoptotic lesions, including the deregulation of Myc or the loss of pRB, is a hallmark of oncogenesis (Evan et al., 1992). The onset of programmed cell death (PCD) is prevented, however, by the concomitant activation of anti-apoptotic mechanisms, such as the deregulation of speci®c signal transduction cascades, the loss of p53 (Kinzler and Vogelstein, 1994) or the activation of protective Bcl-2 family members (Korsmeyer, 1995;Reed, 1996; Kroemer, 1997). Any manipulation that would shift this intricate balance between pro-and anti-apoptotic pathways could have profound e ects on the fate of a tumor cell, either on its own or in combination with conventional anti-cancer drugs. Since the Raf-Ras pathway is activated in many human tumors, we have been interested in studying its role in controlling PCD in tumor cells. This interest was fostered by two recent reports (Monia et al., 1996a, b) which showed that an anti-sense phosphorothioate oligonucleotide (ODN) speci®cally targeted against c-raf mRNA (ISIS5132) had profound e ects on the growth of the human lung adenocarcinoma cell line A549 both in culture and on xenografts in nude mice. The underlying mechanisms remained, however, unclear.In a ®rst set of experiments, we investigated whether the anti-sense ODN ISIS5132 (Monia et al., 1996a) (subsequently referred to as AS) would also reduce the expression of c-Raf protein in, and a ect the growth of, other tumor cell lines. As shown by the immunoblot in Figure 1, treatment of the lung tumor line A549, the cervical carcinoma cell line HeLa, the prostate carcinoma cell line LNCaP or the colon tumor cell line SW620 with AS led to a dramatic reduction of cRaf protein to levels below the detection limit of the assay. In contrast, a mismatched version of this ODN (Monia et al., 1996a) (termed MM in the present study) had no detectable e ect relative to untreated cont...

Intracellular production of DNA enzyme by a novel single-stranded DNA expression vector

“…In conclusion, the results demonstrated here as well as our earlier data [10][11][12] suggest that this ssDNA expression technology may offer an important research tool in gene function study and has potential applications in gene target validation and drug development.…”

“…11 Furthermore, suppression of c-raf gene expression induced cell apoptosis as indicated by increasing genomic DNA fragmentation in cells, which agrees with the observation by others using an antisense approach. 12,13 In this study, we report the construction of an improved version of single vector system. We tested this new ssDNA expression vector for generating DNA enzyme molecules that specifically cleaves b-gal mRNA at protein translation starting site (ATG).…”

Activation of Stress-activated Protein Kinase/c-Jun NH2-terminal Kinase and p38 Kinase in Calphostin C-induced Apoptosis Requires Caspase-3-like Proteases but Is Dispensable for Cell Death

“…The phosphorylated Bad is sequestered in the cytosol bound to 14-3-3, and only unphosphorylated Bad heterodimerizes with Bcl-2 or Bcl-x L to promote cell death (47). However, the cross-talk between Bcl-2 and Raf1 may be little developed in calphostin C-induced apoptosis because of the down-regulation of Bcl-2 and the inactivation of Raf1, but abrogation of c-Raf expression is reported to induce apoptosis in tumor cells (48). Direct and selective activation of the ERK pathway may actually suppress the effects of factors that mediate apoptosis (22,23), and Raf1-ERK signaling pathway plays a pivotal role in suppressing apoptotic death probably due to rapid up-regulation of bcl-2 and bcl-x L (27,28), both suggesting that Raf1-ERK signaling is a survival pathway.…”

Cooperation of Ras‐ and c‐Myc–dependent pathways in regulating the growth and invasiveness of synovial fibroblasts in rheumatoid arthritis

“…In this context, Ras-mediated signaling together with c-Raf-1, the primary upstream activator of ERKs, is of particular interest. Depending on the cell cycle and costimulatory events, activation of c-Raf-1 can result in a variety of counteracting cellular responses such as proliferation, growth arrest, induction of apoptosis, or differentiation (11)(12)(13). Of interest in RA, c-Raf-1 has also been associated with the up-regulation of adhesion molecules such as integrins (14).…”