Myc Potentiates Apoptosis by Stimulating Bax Activity at the Mitochondria

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.     

Apoptosis Triggered by Myc-Induced Suppression of Bcl-XL or Bcl-2 Is Bypassed during Lymphomagenesis

Enforced Bcl-2 expression inhibits Myc-induced apoptosis and cooperates with Myc in transformation. Here we report that the synergy between Bcl-2 and Myc in transforming hematopoietic cells in fact reflects a Myc-induced pathway that selectively suppresses the expression of the Bcl-X(L) or Bcl-2 antiapoptotic protein. Myc activation suppresses Bcl-X(L) RNA and protein levels in cultures of primary myeloid and lymphoid progenitors, and Bcl-X(L) and Bcl-2 expression is inhibited by Myc in precancerous B cells from Emu-myc transgenic mice. The suppression of bcl-X RNA levels by Myc requires de novo protein synthesis, indicating that repression is indirect. Importantly, the suppression of Bcl-2 or Bcl-X(L) by Myc is corrupted during Myc-induced tumorigenesis, as Bcl-2 and/or Bcl-X(L) levels are markedly elevated in over one-half of all lymphomas arising in Emicro-myc transgenic mice. Bcl-2 and/or Bcl-X(L) overexpression did not correlate with loss of ARF or p53 function in tumor cells, indicating that these two apoptotic pathways are inactivated independently. Therefore, the suppression of Bcl-X(L) or Bcl-2 expression represents a physiological Myc-induced apoptotic pathway that is frequently bypassed during lymphomagenesis.     

Fas-independent apoptosis in T-cell tumours induced by the CD2-myc transgene

Depending on the cellular context, the Myc oncoprotein is capable of promoting cell proliferation or death by apoptosis. These observations suggest that apoptosis in response to deregulated gene expression may represent a natural brake to tumour development. The pathways by which Myc induces apoptosis are as yet poorly characterised although recent observations on rat fibroblasts over-expressing Myc have demonstrated a requirement for the Fas pathway. To investigate the role of Fas in Myc-induced lymphomagenesis we backcrossed CD2-myc mice onto an lpr background. Rates of tumour development and phenotypic properties, including levels of apoptosis were indistinguishable from CD2-myc controls. Further, tumour cell lines derived from mice expressing a regulatable form of Myc showed inducible apoptosis at similar rates regardless of their lpr genotype. These results show that activation of c-myc and loss of Fas do not collaborate in T lymphoma development and that Myc-induced apoptosis in T-cells occurs by Fas-independent pathways.     

Neurokinin A induces c-fos, c-jun, and c-myc expression in L6 rat myoblasts

Neurokinin A (NKA), a neuropeptide belonging to the tachykinin family, induced c-fos proto-oncogene mRNA expression in serum-deprived L6J1 rat skeletal myoblasts in vitro. The marked increase reached maximal levels after 15 to 30 min. In contrast to this, c-jun and c-myc proto-oncogene expression were only slightly induced, with peak levels after 30 min. NKA did not stimulate DNA synthesis or cell proliferation in serum-deprived L6J1 myoblasts. We demonstrate a relationship between NKA treatment and induction of c-fos, c-jun and c-myc mRNA expression in serum-deprived L6J1 rat myoblasts. The results on DNA synthesis and cell proliferation indicate that the induced proto-oncogene expression alone is not enough to induce a cellular response to NKA. Possible mechanisms of action are discussed.     

Activated Ha-ras induces apoptosis by association with phosphorylated Bcl-2 in a mitogen-activated protein kinase-independent manner.

Serum deprivation of Ha-ras-transformed brown adipocyte cell line resulted in a dramatic apoptotic cell death, as detected either by DNA laddering or by an increase in the percentage of hypodiploid cells or by nuclei condensation and fragmentation, as compared with immortalized cell line or primary fetal brown adipocytes. Moreover, transient transfection of immortalized brown adipocytes with a constitutively active ras gene (Ha-raslys12) mimics the high rate of apoptosis detected in the transformed cell line. On the other hand, transient transfection of the dominant-negative construct of raf-1 rescued serum-deprived Ha-ras-transformed brown adipocytes from apoptosis, decreasing the percentage of hypodiploid cells, the external display of phosphatidylserine, and the DNA laddering. However, inhibition of mitogen-activated protein kinase with PD098059 did not preclude apoptosis and in fact increased the rate of apoptosis observed in serum-deprived Ha-ras-transformed cells, indicating that the Ras/Raf-1 pathway induced apoptosis throughout a mitogen-activated protein kinase kinase 1 (MEK-1)-independent pathway. Furthermore, apoptosis in Ha-ras-transformed brown adipocytes is concurrent with an up-regulation in the expression of the pro-apoptotic protein Bcl-xS, the expression of the anti-apoptotic protein Bcl-2 being down-regulated. Finally, an association of Ras and Raf with phosphorylated Bcl-2 protein was demonstrated in immunoprecipitates from apoptotic cells. Thus, we propose a mechanism of apoptosis in Ha-ras-transformed adipocytes under serum deprivation involving Raf-1 association with phosphorylated Bcl-2, down-regulation of Bcl-2 expression, and up-regulation of Bcl-xS expression.     

Activation of cyclin-dependent kinases by Myc mediates induction of cyclin A, but not apoptosis.

The activation of conditional alleles of Myc induces both cell proliferation and apoptosis in serum-deprived RAT1 fibroblasts. Entry into S phase and apoptosis are both preceded by increased levels of cyclin E- and cyclin D1-dependent kinase activities. To assess which, if any, cellular responses to Myc depend on active cyclin-dependent kinases (cdks), we have microinjected expression plasmids encoding the cdk inhibitors p16, p21 or p27, and have used a specific inhibitor of cdk2, roscovitine. Expression of cyclin A, which starts late in G1 phase, served as a marker for cell cycle progression. Our data show that active G1 cyclin/cdk complexes are both necessary and sufficient for induction of cyclin A by Myc. In contrast, neither microinjection of cdk inhibitors nor chemical inhibition of cdk2 affected the ability of Myc to induce apoptosis in serum-starved cells. Further, in isoleucine-deprived cells, Myc induces apoptosis without altering cdk activity. We conclude that Myc acts upstream of cdks in stimulating cell proliferation and also that activation of cdks and induction of apoptosis are largely independent events that occur in response to induction of Myc.     

Direct induction of cyclin D2 by Myc contributes to cell cycle progression and sequestration of p27.

Ectopic expression of Myc induces Cdk2 kinase activity in quiescent cells and antagonizes association of p27(kip1) with Cdk2. The target gene(s) by which Myc mediates this effect is largely unknown. We now show that p27 is rapidly and transiently sequestered by cyclin D2-Cdk4 complexes upon activation of Myc and that cyclin D2 is a direct target gene of Myc. The cyclin D2 promoter is repressed by Mad-Max complexes and de-repressed by Myc via a single highly conserved E-box element. Addition of trichostatin A to quiescent cells mimics activation of Myc and induces cyclin D2 expression, suggesting that cyclin D2 is repressed in a histone deacetylase-dependent manner in quiescent cells. Inhibition of cyclin D2 function in established cell lines, either by ectopic expression of p16 or by antibody injection, inhibits Myc-dependent dissociation of p27 from Cdk2 and Myc-induced cell cycle entry. Primary mouse fibroblasts that are cyclin D2-deficient undergo accelerated senescence in culture and are not immortalized by Myc; induction of apoptosis by Myc is unimpaired in such cells. Our data identify a downstream effector pathway that links Myc directly to cell cycle progression.     

Atm deficiency affects both apoptosis and proliferation to augment Myc-induced lymphomagenesis

Myc oncoproteins are commonly activated in malignancies and are sufficient to provoke many types of cancer. However, the critical mechanisms by which Myc contributes to malignant transformation are not clear. DNA damage seems to be an important initiating event in tumorigenesis. Here, we show that although Myc does not directly induce double-stranded DNA breaks, it does augment activation of the Atm/p53 DNA damage response pathway, suggesting that Atm may function as a guardian against Myc-induced transformation. Indeed, we show that Atm loss augments Myc-induced lymphomagenesis and impairs Myc-induced apoptosis, which normally harnesses Myc-driven tumorigenesis. Surprisingly, Atm loss also augments the proliferative response induced by Myc, and this augmentation is associated with enhanced suppression of the expression of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, regulation of cell proliferation and p27(Kip1) seems to be a contributing mechanism by which Atm holds tumor formation in check.     

c-Myc overexpression sensitizes Bim-mediated Bax activation for apoptosis induced by histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through regulating Bcl-2/Bcl-xL expression

Overexpression of the oncogene c-Myc sensitizes many apoptotic signals through the activation of mitochondrial apoptosis pathway. However, the underling mechanism has not been clearly defined. Here, we investigated the effect of c-Myc expression on histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)-induced apoptosis in rat fibroblast cells possessing various c-Myc levels. In Rat 1a cells overexpressing c-Myc, SAHA-induced enhanced the cell death response relative to the parental cells; whereas Rat 1a cells lacking c-Myc were refractory to SAHA treatment. We demonstrated that SAHA selectively induced the expression of pro-apoptotic BH3-only protein Bim, leading to Bax activation in c-Myc-expressing cells. Where c-Myc was absent, Bim, despite its induction by SAHA, failed to activate Bax and was unable to induce apoptosis. These results indicate that c-Myc is dispensable for Bim induction by SAHA, but is required for subsequent Bax activation. We further show that the expression levels of anti-apoptotic Bcl-2/Bcl2-xL were much elevated in Myc-null cells compared with the c-Myc-expressing cells; furthermore, depletion of Bcl-2/Bcl-xL in these cells restored the ability of SAHA to induce apoptosis by enhancing Bax activation. These data indicate that SAHA induces apoptosis through Bim-triggered Bax activation and that c-Myc regulates this process by modulating Bcl-2/Bcl-xL. Our results provide novel insight into the mechanism whereby Myc sensitizes the apoptotic signals; furthermore, our data suggest that cancer cells with deregulated Myc might be more sensitive to SAHA treatment.     

Reduction of c-myc expression by an antisense approach under Cre/loxP switching induces apoptosis in human liver cancer cells

c-Myc has been documented to be both a positive and a negative signal for the induction of apoptosis. It is well known that overexpression of the c-myc gene induces apoptosis of normal cells, but the result of a reduction in its expression is not fully understood. We examined whether a reduction in c-myc expression would induce apoptosis in human liver cancer cells. Specifically, antisense and sense oligodeoxynucleotides (oligos) against the human c-myc mRNA were synthesized, mixed with a liposome reagent at various ratios, and were applied to the liver cancer-derived cell lines, HCC-T, HepG2, and PLC/PRF/5. To exclude effects resulting from using oligos, plasmid vectors expressing the full-length c-myc cDNA in both sense and antisense orientations under the control of the Cre/loxP system were generated. Monoclonal cell lines including these plasmid vectors were produced and Cre was supplied by adenovirus infection. Apoptosis was determined morphologically and c-Myc and Bcl-2 expression was examined by Western blotting. The antisense myc significantly inhibited the proliferation of the cells within two days, while neither the liposome reagent alone nor sense myc did so. Most of the cells were rounded up by the antisense-treatment and nuclear fragmentation and DNA ladder formation were detected after two days in antisense c-myc-treated cells. Antisense c-myc largely reduced c-Myc and partially Bcl-2 expression; overexpression of Bcl-2 partially rescued from apoptosis in HCC-T and HepG2 cells. These results suggest that the massive reduction in c-myc mRNA induces apoptosis in liver cancer cell lines and consequent decrease in Bcl-2 enhances the cell death. c-Myc reduction under the Cre/loxP switching system may be a useful tool for the clarification of c-myc-related cellular mechanisms in differentiation and proliferation.     

Serum Deprivation and Protein Synthesis Inhibition Induce Two Different Apoptotic Processes in N18 Neuroblastoma Cells

N18 are murine neuroblastoma cells that underwent cell death upon serum deprivation or inhibition of protein synthesis by means of cycloheximide (CHX). In both cases, an ultrastructural morphology and an internucleosomal pattern of DNA fragmentation typical of apoptosis were found. However, electron microscopy revealed abundant lipid vesicles in the cytoplasm of CHX-treated cells that were not found in their serum-deprived counterparts. In addition, when both types of apoptotic cells were compared by means of flow cytometry and chromatin staining with propidium iodide, the former showed consistently less fluorescence than the latter. Therefore, in N18 cells, both apoptotic processes seemed to differ at a structural level. At a functional level, we found that apoptosis was blocked by the protease inhibitor TLCK in CHX-treated but not in serum-deprived cells. On the other hand, we generated N18 clones that overexpressed Bcl-2 protein. After a period of 48 h we found that identical levels of Bcl-2 protein were able to block apoptosis in serum-deprived but not in CHX-treated cells. In conclusion, two different biochemical pathways leading to apoptosis seem to coexist in N18 neuroblastoma cells.     

c-myc induces autophagy in rat 3Y1 fibroblast cells

The proto-oncogene c-myc is a multifunctional gene that regulates cell division, cell growth, and apoptosis. Here we report a new function of c-myc: induction of autophagy. Autophagy is a bulk degradation system for intracellular proteins. Autophagy proceeds with characteristic morphologies, which begins with the formation of a double-membrane structure called the autophagosome surrounding a portion of the cytoplasm, after which its outer membrane then fuses with the lysosomal membrane to become an autolysosome. Autophagosomes and autolysosomes are generally called autophagic vacuoles. When c-Myc protein was overexpressed in rat 3Y1 fibroblasts or when the chimeric protein c-MycER was activated by estrogen, the number of autophagic vacuoles in cells increased significantly. The formation of autophagic vacuoles induced by c-Myc was completely blocked by a specific inhibitor of autophagosome formation, 3-methyladenine. A c-Myc mutant lacking Myc Box II induced neither apoptosis nor oncogenic transformation, but still stimulated autophagy. An inhibitor of caspases suppressed apoptosis but not autophagy. These results suggest that the autophagy caused by c-myc is not due to the apoptosis or tumorigenesis induced by c-myc. Taken together, our results suggest that the induction of autophagy is a novel function of c-myc.     

Myc,Cdk2 and cellular senescence: Old players,new game

The aberrant activation of oncogenic pathways promotes tumor progression, but concomitantly elicits compensatory tumor-suppressive responses, such as apoptosis or senescence. For example, Ras induces senescence, while Myc generally triggers apoptosis. Myc is in fact viewed as an anti-senescence oncogene, as it is a potent inducer of cell proliferation and immortalization, bypasses growth-inhibitory signals, and cooperates with Ras in cellular transformation. Recent reports prompt re-evaluation of Myc-induced senescence, and of its role in tumor progression and therapy. We have shown that the cyclin-dependent kinase Cdk2, although redundant for cell cycle progression, has a unique role in suppressing a Myc-induced senescence program: Myc activation elicited expression of p16^INK4a and p21^Cip1, and caused senescence in cell lacking Cdk2, but not in Cdk2-proficient cells. Additional cellular activities have been identified that suppress Myc-induced senescence, including the Wrn helicase, Telomerase and Miz1. These senescence-suppressing activities were critical for tumor progression, as deficiency in Cdk2, telomerase or Miz1 reduced the onset of Myc-induced lymphoma in transgenic mice. Other gene products like p53, SUV39H1 or TGFß promoted senescence, which together with apoptosis contributed to tumor suppression. Paradoxically, Myc directly counteracted the very same senescence program that it potentially elicits, since it positively regulated Wrn, Telomerase and Cdk2 activity, and Cdk2 inhibition re-activated the latent senescence program in Myc expressing cells. Hence, while these molecules are instrumental to the oncogenic action of Myc, they may simultaneously constitute its Achille's heel for therapeutic development.