C-Myc Augments the Apoptotic Activity of Cytosolic Death Receptor Signaling Proteins by Engaging the Mitochondrial Apoptotic Pathway

Juha Klefstrom,Emmy W Verschuren,Gerard Evan

doi:10.1074/jbc.m206967200

Juha Klefstrom, Emmy W Verschuren + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m206967200

Copy DOI

Abstract

Activation of c-Myc sensitizes cells to apoptosis induction by ligand-activated death receptors. Such sensitization to death receptors by oncogenes may well be the mechanism underlying tumor cell sensitivity to tumor necrosis factor (TNF) or TNF-related apoptosis-inducing ligand (TRAIL). The mechanism by which this c-Myc-induced sensitization occurs is unclear but could involve modulation of expression of death receptors or their ligands or potentiation of the sensitivity of mitochondria to release pro-apoptotic effectors such as holocytochrome c. Here, we show that ectopic expression of the death receptor signaling protein RIP (receptor-interactive protein) triggers apoptosis via a FAS-associated death domain protein (FADD) and caspase 8-dependent pathway. Induction of apoptosis by this intracellular activation of the death receptor signaling pathway is significantly augmented by c-Myc expression. Moreover, c-Myc expression strongly promotes the potential of RIP to induce cytochrome c release from mitochondria. This implicates the mitochondrial apoptotic pathway in this synergy, a notion confirmed by the inability of c-Myc to sensitize to RIP killing in cells lacking the obligate mitochondrial apoptotic effectors Bax and Bak. We conclude that the lethality of the RIP-activated cytosolic caspase 8 pathway is augmented by c-Myc priming mitochondria to release cytochrome c. This places the intersection of apoptotic synergy between c-Myc and death receptor signaling downstream of the death receptors.

Highlights

Aspect of c-Myc over its proliferative functions offers the potential for selective ablation of tumor cells
We show that the apoptotic activity of RIP, which is strictly dependent on FASassociated death domain protein (FADD) and caspase 8, is contingent on c-Myc priming of mitochondrial BH3 proteins to release cyt c
Our results demonstrate that the mitochondrial effects of c-Myc, which promote cyt c release from mitochondria to the cytosol, amplify the apoptotic activity of a FADD and caspase 8-dependent death receptor signaling pathway

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Primary Cell Cultures—Rat-1-MycERTAM and Rat-1Myc⌬ERTAM cells have been previously published [21]. Wild type RIP and RIP mutants were subsequently cloned into pcDNA 3.1ϩ (Invitrogen) and retroviral pBMN-ires-GFP expression vectors Protein expression from these constructs was verified from total lysates of transfected or transduced cells by Western blot analysis using specific antibodies. The mean green fluorescence values (arbitrary units) obtained for pBMN-ires-GFP-transfected cells were 41, 59, 73, 85, 96, and 142 when 0.0, 0.05, 0.1, 0.2, 0.4, and 0.8 ␮g, respectively, of pBMN-ires-GFP was included in the transfection mixture (in each case the total plasmid concentration was balanced to 1 ␮g with pcDNA3.1ϩ DNA). Samples were incubated at 37 °C and read at 405 nm with an automated plate-reading spectrophotometer (Wallac) in 1-h intervals

RESULTS

DISCUSSION