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It is not understood why healthy tissues can exhibit varying levels of sensitivity to the same toxic stimuli. Using BH3 profiling, we find that mitochondria of many adult somatic tissues, including brain, heart, and kidneys, are profoundly refractory to pro-apoptotic signaling, leading to cellular resistance to cytotoxic chemotherapies and ionizing radiation. In contrast, mitochondria from these tissues in young mice and humans are primed for apoptosis, predisposing them to undergo cell death in response to genotoxic damage. While expression of the apoptotic protein machinery is nearly absent by adulthood, in young tissues its expression is driven by c-Myc, linking developmental growth to cell death. These differences may explain why pediatric cancer patients have a higher risk of developing treatment-associated toxicities., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The notion that there might be a cellular basis for aging stems from research that began several decades ago and was proposed to explain the loss of proliferative homeostasis, which is a hallmark of complex animals. Recent years have seen growing support for the idea that two cell fates-apoptosis and cellular senescence, both now well-established tumor suppressor mechanisms-may be important drivers of aging phenotypes and age-related disease. However, there remain many unanswered questions, some quite basic, about how these processes change with age and how they might contribute to aging. It is now clear that failures in apoptosis or senescence can result in hyperproliferative diseases such as cancer. Less is known about whether and how increased apoptosis or senescence can cause tissue degeneration and aging. In addition, there is now a growing recognition that cellular senescence can have cell-nonautonomous effects within tissues. New molecular tools and model organisms, some already on the horizon, will need to be developed to better understand the roles of apoptosis and cellular senescence in age-associated changes in proliferative homeostasis.

Mutations in mitochondrial DNA (mtDNA) accumulate in tissues of mammalian species and have been hypothesized to contribute to aging. We show that mice expressing a proofreading-deficient version of the mitochondrial DNA polymerase g (POLG) accumulate mtDNA mutations and display features of accelerated aging. Accumulation of mtDNA mutations was not associated with increased markers of oxidative stress or a defect in cellular proliferation, but was correlated with the induction of apoptotic markers, particularly in tissues characterized by rapid cellular turnover. The levels of apoptotic markers were also found to increase during aging in normal mice. Thus, accumulation of mtDNA mutations that promote apoptosis may be a central mechanism driving mammalian aging.

Nuclear factor kappaB (NF-kappaB) plays a key role in suppression of tumor necrosis factor (TNF)-mediated apoptosis by inducing a variety of anti-apoptotic genes. Expression of c-Myc has been shown to sensitize cells to TNF-mediated apoptosis by inhibiting NF-kappaB activation. However, the precise step in the NF-kappaB signaling pathway and apoptosis modified by c-Myc has not been identified. Using the inducible c-MycER system and c-Myc null fibroblasts, we found that expression of c-Myc inhibited NF-kappaB activation by interfering with RelA/p65 transactivation but not nuclear translocation of NF-kappaB. Activation of c-Myc promoted TNF-induced release of cytochrome c from mitochondria to the cytosol because of the inhibition of NF-kappaB. Furthermore, we found that NF-kappaB-inducible gene A1 was attenuated by expression of c-Myc and that the restoration of A1 expression suppressed c-Myc-induced TNF sensitization. Our results elucidate the molecular mechanisms by which c-Myc increases cell susceptibility to TNF-mediated apoptosis, indicating that c-Myc may exhibit its pro-apoptotic activities by repression of cell survival genes.

Treatment of cells with the anti-cancer drug camptothecin (CPT) induces topoisomerase I (Top1)-mediated DNA damage, which in turn affects cell proliferation and survival. In this report, we demonstrate that treatment of the wild-type HCT116 (wt HCT116) human colon cancer cell line and the isogenic p53(-/-) HCT116 and p21(-/-) HCT116 cell lines with a high concentration (250 nm) of CPT resulted in apoptosis, indicating that apoptosis occurred by a p53- and p21-independent mechanism. In contrast, treatment with a low concentration (20 nm) of CPT induced cell cycle arrest and senescence of the wt HCT116 cells, but apoptosis of the p53(-/-) HCT116 and p21(-/-) HCT116 cells. Further investigations indicated that p53-dependent expression of p21 blocked apoptosis of wt HCT116 cells treated with 20 nm, but not 250 nm CPT. Interestingly, blocking of the apoptotic pathway, by Z-VAD-FMK, in p21(-/-) HCT116 cells following treatment with 20 nm CPT did not permit the cells to develop properties of senescence. These observations demonstrated that p21 was required for senescence development of HCT116 cells following treatment with low concentrations of CPT.

The c-myc proto-oncogene encodes a transcription factor that participates in the regulation of cellular proliferation, differentiation, and apoptosis. Ectopic overexpression of c-Myc has been shown to sensitize cells to apoptosis. We report here that cells lacking c-Myc activity due to disruption of the c-myc gene by targeted homologous recombination are defective in DNA damage-initiated apoptosis in the G(2) phase of the cell cycle. The downstream effector of c-Myc is cyclin A, whose ectopic expression in c-myc(-/-) cells rescues the apoptosis defect. The kinetics of the G(2) response indicate that the induction of cyclin A and the concomitant activation of Cdk2 represent an early step during commitment to apoptosis. In contrast, expression of cyclins E and D1 does not rescue the apoptosis defect, and apoptotic processes in G(1) phase are not affected in c-myc(-/-) cells. These observations link DNA damage-induced apoptosis with cell cycle progression and implicate c-Myc in the functioning of a subset of these pathways.

The ability of the c-Myc oncoprotein to potentiate apoptosis has been well documented; however, the mechanism of action remains ill defined. We have previously identified spatially distinct apoptotic pathways within the same cell that are differentially inhibited by Bcl-2 targeted to either the mitochondria (Bcl-acta) or the endoplasmic reticulum (Bcl-cb5). We show here that in Rat1 cells expressing an exogenous c-myc allele, distinct apoptotic pathways can be inhibited by Bcl-2 or Bcl-acta yet be distinguished by their sensitivity to Bcl-cb5 as either susceptible (serum withdrawal, taxol, and ceramide) or refractory (etoposide and doxorubicin). Myc expression and apoptosis were universally associated with Bcl-acta and not Bcl-cb5, suggesting that Myc acts downstream at a point common to these distinct apoptotic signaling cascades. Analysis of Rat1 c-myc null cells shows these same death stimuli induce apoptosis with characteristic features of nuclear condensation, membrane blebbing, poly (ADP-ribose) polymerase cleavage, and DNA fragmentation; however, this Myc-independent apoptosis is not inhibited by Bcl-2. During apoptosis, Bax translocation to the mitochondria occurs in the presence or absence of Myc expression. Moreover, Bax mRNA and protein expression remain unchanged in the presence or absence of Myc. However, in the absence of Myc, Bax is not activated and cytochrome c is not released into the cytoplasm. Reintroduction of Myc into the c-myc null cells restores Bax activation, cytochrome c release, and inhibition of apoptosis by Bcl-2. These results demonstrate a role for Myc in the regulation of Bax activation during apoptosis. Moreover, apoptosis that can be triggered in the absence of Myc provides evidence that signaling pathways exist which circumvent Bax activation and cytochrome c release to trigger caspase activation. Thus, Myc increases the cellular competence to die by enhancing disparate apoptotic signals at a common mitochondrial amplification step involving Bax activation and cytochrome c release.

c-myc nullizygous fibroblasts (KO cells) were used to compare the abilities of c-myc, N-myc and L-myc oncoproteins to accelerate growth, promote apoptosis, revert morphology, and regulate the expression of previously described c-myc target genes. All three myc oncoproteins were expressed following retroviral transduction of KO cells. The proteins all enhanced the growth rate of KO cells and significantly shortened the cell cycle transition time. They also accelerated apoptosis following serum deprivation, reverted the abnormal KO cell morphology, and modulated the expression of previously described c-myc target genes. In most cases, L-myc was equivalent to c-myc and N-myc in restoring all of the c-myc-dependent activities. These findings contrast with the previously reported weak transforming and transactivating properties of L-myc. Myc oncoproteins may thus impart both highly similar as well as dissimilar signals to the cells in which they are expressed.

Transcriptional activation by c-Myc through specific E box elements is thought to be essential for its biological role. However, c-MycS is unable to activate transcription through these elements and yet retains the ability to stimulate proliferation, induce anchorage-independent growth, and induce apoptosis. In addition, c-MycS retains the ability to repress transcription of several specific promoters. Furthermore, c-MycS can rescue the c-myc null phenotype in fibroblasts with homozygous deletion of c-myc. Taken together, our data argue against the paradigm that all of the biological functions of c-Myc are mediated by transcriptional activation of specific target genes through E box elements.

Many vertebrate cells are resistant to apoptotic stimuli, whose variety and the mechanisms involved are not fully understood. Endothelin-1 is an endothelium-derived vasoactive peptide that mediates many physiological functions, such as vasoconstriction and cell proliferation. Deregulated expression of c-Myc induces apoptosis in serum-deprived fibroblasts. Using a panel of isogenic fibroblast cell lines with differential c-myc expression levels, we demonstrate that low doses of endothelin-1 protect fibroblasts against serum deprivation-induced apoptosis, which occurs through a c-Myc-dependent process. The endothelin-1-induced cell survival was mediated by the ET(A) receptor and was not linked to the ability of endothelin-1 to induce cell proliferation. The survival function of endothelin-1 was abrogated by inhibiting the mitogen-activated protein kinase pathway. These results demonstrate a hitherto unappreciated role of endothelin-1 as a potent survival factor for c-Myc-dependent apoptosis, a process mediated by the ET(A) receptor and the mitogen-activated protein kinase pathway.

Here we investigate the role of the Raf-1 kinase in transformation by the v-abl oncogene. Raf-1 can activate a transforming signalling cascade comprising the consecutive activation of Mek and extracellular-signal-regulated kinases (Erks). In v-abl-transformed cells the endogenous Raf-1 protein was phosphorylated on tyrosine and displayed high constitutive kinase activity. The activities of the Erks were constitutively elevated in both v-raf- and v-abl-transformed cells. In both cell types the activities of Raf-1 and v-raf were almost completely suppressed after activation of the cyclic AMP-dependent kinase (protein kinase A [PKA]), whereas the v-abl kinase was not affected. Raf inhibition substantially diminished the activities of Erks in v-raf-transformed cells but not in v-abl-transformed cells, indicating that v-abl can activate Erks by a Raf-1-independent pathway. PKA activation induced apoptosis in v-abl-transformed cells while reverting v-raf transformation without severe cytopathic effects. Overexpression of Raf-1 in v-abl-transformed cells partially protected the cells from apoptosis induced by PKA activation. In contrast to PKA activators, a Mek inhibitor did not induce apoptosis. The diverse biological responses correlated with the status of c-myc gene expression. v-abl-transformed cells featured high constitutive levels of expression of c-myc, which were not reduced following PKA activation. Myc activation has been previously shown to be essential for transformation by oncogenic Abl proteins. Using estrogen-regulated c-myc and temperature-sensitive Raf-1 mutants, we found that Raf-1 activation could protect cells from c-myc-induced apoptosis. In conclusion, these results suggest (i) that Raf-1 participates in v-abl transformation via an Erk-independent pathway by providing a survival signal which complements c-myc in transformation, and (ii) that cAMP agonists might become useful for the treatment of malignancies where abl oncogenes are involved, such as chronic myeloid leukemias.