siRNA-mediated knockdown of Pdcd4 expression causes upregulation of p21(Waf1/Cip1) expression

Abstract

The transformation suppressor gene, programmed cell death gene 4 (Pdcd4), inhibits tumor-promoter-mediated transformation of mouse keratinocytes and has been implicated as a tumor suppressor gene in the development of human cancer. The Pdcd4 protein interacts with translation initiation factors eIF4A and eIF4G and binds to RNA, suggesting that it might be involved in regulating protein translation or other aspects of RNA metabolism. To study the function of Pdcd4 in more detail, we have downregulated Pdcd4 expression in HeLa cells by stable expression of shRNA. We have found that diminished Pdcd4 expression leads to increased expression of p21(Waf1/Cip1) and several other p53-regulated genes. Reporter gene studies demonstrate that Pdcd4 interferes with the activation of p53-responsive promoters genes by p53. Pdcd4 knockdown cells show decreased apoptosis and increased survival after UV irradiation. Taken together, our observations suggest a model in which low Pdcd4 expression after DNA damage favors the survival of cells, which would be eliminated by apoptosis under normal levels of Pdcd4 expression. Our results provide the first evidence that Pdcd4 is important role in the DNA-damage response and suggest that low levels of Pdcd4 expression observed in certain tumor cells contribute to tumorigenesis by affecting the fate of DNA-damaged cells.

Pdcd4 directly binds the coding region of c-myb mRNA and suppresses its translation

“…We have previously shown that Pdcd4 inhibits the activity of transcription factors c-Jun and p53 by interfering with their ability to cooperate with p300 (Bitomsky et al, 2004(Bitomsky et al, , 2008. While studying the effect of Pdcd4 on c-Myb, another transcription factor recruiting p300, we noted that Pdcd4 strongly decreased the amount of c-Myb generated from a cytomegalovirus (CMV) promoter-based c-Myb expression vector.…”

“…Transient transfections were performed in the QT6 fibroblast line as described (Bitomsky et al, 2008). To monitor and normalize transfection efficiencies, the b-galactosidase expression plasmid CMVb (Invitrogen, Karlsruhe, Germany) was included in each transfection.…”

“…C/EBPb was detected with a rabbit antiserum raised against full-length chicken C/EBPb (Mink et al, 1996). Pdcd4 was detected with a rabbit antiserum raised against the N-terminus of human Pdcd4 (Bitomsky et al, 2008) or with a commercial antibody against the C-terminus of Pdcd4 (Upstate Biomol, Hamburg, Germany). Antibodies against GFP were obtained from Roche Diagnostics (Mannheim, Germany).…”

“…On the protein level, p70(S6K) kinase-mediated phosphorylation of Pdcd4 triggers its ubiquitinylation by the E3 ubiquitin ligase complex SCF(betaTRCP) and its subsequent degradation (Dorello et al, 2006). Recent work has suggested that downregulation of Pdcd4 expression contributes to tumor development by stimulating the mobility and metastatic potential of tumor cells Asangani et al, 2008), and by deregulating the cellular DNA damage response (Bitomsky et al, 2008;Singh et al, 2009). …”

“…Pdcd4 affects transcription of certain genes by modulating the activities of specific transcription factors, such as c-Jun (Yang et al, 2003b;Bitomsky et al, 2004), Sp1 and p53 (Bitomsky et al, 2008). As a translation regulator, Pdcd4 interacts with the eukaryotic translation initiation factor eIF4A, a RNA helicase that catalyzes the unwinding of mRNA secondary structures in 5 0 -untranslated regions (UTRs) (Yang et al, 2003a(Yang et al, , 2004.…”