A role for caspase-8 and c-FLIP L in proliferation and cell-cycle progression of primary hepatocytes

Abstract

Growth factors are known to favor both proliferation and survival of hepatocytes. In the present study, we investigated if c-FLIP(L) (cellular FLICE-inhibitory protein, long isoform) could be involved in epidermal growth factor (EGF)-stimulated proliferation of rat hepatocytes since c-FLIP(L) regulates both cell proliferation and procaspase-8 maturation. Treatment with MEK inhibitors prevented induction of c-FLIP(L) by EGF along with total inhibition of DNA replication. However, EGF failed to inhibit processing of procaspase-8 in the presence of EGF suggesting that c-FLIP(L) does not play its canonical anti-apoptotic role in this model. Downregulation of c-FLIP expression using siRNA oligonucleotides strongly reduced DNA replication but did not result in enhanced apoptosis. Moreover, intermediate cleavage products of c-FLIP(L) and caspase-8 were found in EGF-treated hepatocytes in the absence of caspase-3 maturation and cell death. To determine whether the Fas/FADD/caspase-8/c-FLIP(L) complex was required for this activity, Fas, procaspase-8 and Fas-associated death domain protein (FADD) expression or function was inhibited using siRNA or constructs encoding dominant negative mutant proteins. Inhibition of any of these components of the Fas/FADD/caspase-8 pathway decreased DNA replication suggesting a function of these proteins in cell-cycle arrest. Similar results were obtained when the IETD-like caspase activity detectable in EGF-treated hepatocytes was inhibited by the pan-caspase inhibitor, z-ASP. Finally, we demonstrated co-immunoprecipitation between EGFR and Fas within 15 min following EGF stimulation. In conclusion, our results indicate that the Fas/FADD/c-FLIP(L)/caspase-8 pathway positively controls the G(1)/S transition in EGF-stimulated hepatocytes. Our data provide new insights into the mechanisms by which apoptotic proteins participate to mitogenic signals during the G(1) phase.

Old, new and emerging functions of caspases

“…However, the DISC complex comprising Fas/ FADD/c-FLIP L /caspase-8 also controls cell-cycle progression of hepatocytes following EGF stimulation. 122 Like caspase-8, caspase-1 and -6 have also been shown to regulate B-cell proliferation and maintain lymphocyte homeostasis. 123,124 In a contrasting role, caspase-3-deficient B cells show hyperproliferation that is dependent on p21, a cyclindependent kinase inhibitor.…”

Pharmacological inhibition or small interfering RNA targeting acid ceramidase sensitizes hepatoma cells to chemotherapy and reduces tumor growth in vivo

“…Lipofectamine 2000 was used for siRNA transfection of primary hepatocytes as previously described (Gilot et al, 2005). Cells were assayed usually 36-72 h after siRNAs duplexes transfection.…”

A Polymorphic −844T/C in FasL Promoter Predicts Survival and Relapse in Non–Small Cell Lung Cancer

“…This observation seems to be explained by the suppression of T-cell immunity via increased apoptosis of tumor-infiltrating lymphocytes (2-5), which consequently contributes to tumor progression and lymph node metastasis (29,40,41). Interestingly, tumor-derived FasL has shown a tumor growth-promoting effect operating via the Fas receptor (8)(9)(10)(11)(12)(13)(14)(15)(16). A recent animal model study further indicated that human FasL, but not murine FasL neutralized by antibody, resulted in significantly reduced tumor load and colony formation efficacy (9).…”

“…However, many cancer cells resist Fas-mediated apoptosis via overexpression of apoptosis-signaling inhibitors, such as c-FLIP L , which is highly competitive and homologous to procaspases-8 and -10 but does not induce further apoptotic signaling by procaspases (6)(7)(8). Both in vitro and in vivo studies have provided overwhelming evidence of activation of tumor progression by tumor-derived FasL via the AKT, NF-kB, and c-jun signaling pathway (8)(9)(10)(11)(12)(13)(14)(15)(16). Tumor size and the number of tumor nodules have also been significantly reduced in mice injected with FasL antibody (9).…”