E1A sensitizes cells to tumor necrosis factor alpha by downregulating c-FLIP S

Abstract

Tumor necrosis factor alpha (TNF-alpha) activates both apoptosis and NF-kappaB-dependent survival pathways, the former of which requires inhibition of gene expression to be manifested. c-FLIP is a TNF-alpha-induced gene that inhibits caspase-8 activation during TNF-alpha signaling. Adenovirus infection and E1A expression sensitize cells to TNF-alpha by allowing apoptosis in the absence of inhibitors of gene expression, suggesting that it may be disabling a survival signaling pathway. E1A promoted TNF-alpha-mediated activation of caspase-8, suggesting that sensitivity was occurring at the level of the death-inducing signaling complex. Furthermore, E1A expression downregulated c-FLIP(S) expression and prevented its induction by TNF-alpha. c-FLIP(S) and viral FLIP expression rescued E1A-mediated sensitization to TNF-alpha by restoring the resistance of caspase-8 to activation, thereby preventing cell death. E1A inhibited TNF-alpha-dependent induction of c-FLIP(S) mRNA and stimulated ubiquitination- and proteasome-dependent degradation of c-FLIP(S) protein. Since elevated c-FLIP levels confer resistance to apoptosis and promote tumorigenicity, interference with its induction by NF-kappaB and stimulation of its destruction in the proteasome may provide novel therapeutic approaches for facilitating the elimination of apoptosis-refractory tumor cells.

FIG. 1.

E1A promotes caspase-8 activation by TNF-α, and its function is dependent upon Rb and p300 binding. (A) HeLa cells were mock, Ad5dl309, or Ad5dl337 infected for 24 h and treated with TNF-α for 0, 4, or 12 h. Whole-cell extracts were analyzed by immunoblotting with an anti-caspase-8 antibody. The positions of procaspase-8 and the processed p43 and p24 forms are indicated, (B) HeLa cells infected with adenoviruses containing Rb (928) and/or p300 (RG2) binding point mutations within E1A and treated with TNF-α for 10 h were analyzed by immunoblotting with anti-caspase-8, anti-E1A, and antiactin antibodies. (C) HeLa cells were infected and treated with TNF-α as in the experiments shown in panel B. Viability was assessed by the trypan blue dye exclusion assay.

FIG. 2.

Adenovirus infection prevents c-FLIP_S accumulation during TNF-α signaling. (A) HeLa cells were mock, Ad5dl309, or Ad5dl337 infected for 24 h and treated with TNF-α for 0, 4, or 12 h. Whole-cell extracts were immunoblotted with anti-c-FLIP_L polyclonal (CT) (c-FLIP_L), anti-c-FLIP monoclonal (Dave-2) (c-FLIP_S), anti-IκBα, and antiactin antibodies. The band below IκBα represents a nonspecific cross-reactive band (asterisk). (B) HeLa cells were mock, Ad5dl309, or Ad5dl337 infected for 24 h and treated with TNF/cycloheximide for 0, 4, or 12 h. Whole-cell extracts were immunoblotted with an anti-c-FLIP (Dave-2) monoclonal antibody.

FIG. 3.

v-FLIP and c-FLIP_S rescue TNF-α-mediated E1A-dependent caspase-8 activation and apoptosis. (A) Stable HeLa cell lines expressing either vector control or HA-tagged MC159 were mock, Ad5dl309, or Ad5dl337 infected for 24 h and treated with TNF-α for 10 h. Extracts were analyzed by immunoblotting with anti-caspase-8 and anti-HA antibodies. (B) Viability was assessed by the trypan blue exclusion assay in stable cell lines that were mock or Ad5dl337 infected for 24 h and treated with TNF-α for 10 h. The band above HA-MC159 represents a nonspecific cross-reactive band (asterisk). (C) Vector control- and c-FLIP_S-overexpressing HeLa cells were mock, Ad5dl309, or Ad5dl337 infected for 24 h and treated with TNF-α for 10 h. Extracts were analyzed by immunoblotting with anti-caspase-8 and anti-c-FLIP monoclonal (Dave-2) antibodies. (D) Viability was assessed by the trypan blue exclusion assay in HeLa cells that were mock or Ad5dl337 infected and treated with TNF-α for 10 h.

FIG. 4.

Adenovirus infection prevents NF-κB-mediated induction of c-FLIP and IAP-2 mRNA levels. Real-time RT-PCR was performed on HeLa cells that were mock or Ad5dl309 infected for 24 h and treated with TNF-α for 4 h. (A) The change in mRNA levels of GAPDH, c-FLIP_L, c-FLIP_S, IAP-2, and IκBα was determined as a change in fluorescence relative to that of the untreated mock-infected sample (see Materials and Methods). Fold change is defined as the change in mRNA levels for each sample relative to the untreated mock-infected sample. (B) HeLa cells were mock or 12S E1B⁻ infected for 34 h and treated with TNF-α for 4 h. The change in mRNA levels was assessed for GAPDH, c-FLIP_L, c-FLIP_S, IAP-2, and IκBα as stated above.

FIG. 5.

Adenovirus infection enhances c-FLIP_S degradation through a ubiquitin-dependent proteolysis pathway. (A) HeLa cells were mock or Ad5dl309 infected and treated with epoxomicin and subsequently TNF-α for 10 h. Whole-cell extracts were analyzed by immunoblotting with anti-caspase-8, anti-c-FLIP_L polyclonal (CT) (c-FLIP_L), anti-FLIP monoclonal (Dave-2) (c-FLIP_S), anti-RIP, anti-IAP-1, anti-IAP-2, anti-TRAF2, anti-FADD, and antiactin antibodies. (B) HeLa cells were mock or Ad5dl309 infected and treated with epoxomicin. Whole-cell extracts were analyzed by immunoblotting with anti-FLIP (Dave-2) and antiactin monoclonal antibodies. (C) HeLa cells were mock, Ad5dl309, or Ad5dl337 infected for 24 h and treated with cycloheximide for 0, 4, or 12 h. As a control, mock-infected HeLa cells were treated with TNF/cycloheximide for 0 or 12 h. Whole-cell extracts were immunoblotted with anti-c-FLIP (Dave-2) and antiactin monoclonal antibodies. (D) Whole-cell extracts from mock-, Ad5PAC3-, and 12SE1B⁻-infected HeLa cells were analyzed by immunoblotting with anti-c-FLIP monoclonal (Dave-2) and antiactin antibodies. (E) HeLa cells were transfected with vector control, HA-tagged ubiquitin, c-FLIP_S, or c-FLIP_S and HA-tagged ubiquitin. Transfectants were mock or Ad5dl309 infected for 16 h and treated with epoxomicin for 8 h as indicated. Whole-cell extracts were prepared and immunoblotted with an anti-c-FLIP monoclonal (NF6) antibody. High-molecular-mass forms of c-FLIP_S that correspond to ubiquitinated products are indicated.

FIG. 6.

Epoxomicin blocks caspase-8 processing and TNF/cycloheximide-mediated apoptosis by preventing c-FLIP_S degradation. (A) HeLa cells were incubated with epoxomicin for 12 h and then treated with TNF/cycloheximide or cycloheximide alone for 10 h. Viability was assessed by trypan blue exclusion. (B) Whole-cell extracts were prepared from HeLa cells treated as in panel A and analyzed by immunoblotting with anti-caspase-8 and anti-c-FLIP monoclonal (Dave-2) antibodies.