c-Myc-induced apoptosis in fibroblasts is inhibited by specific cytokines.

We have investigated the mechanism by which deregulated expression of c-Myc induces death by apoptosis in serum-deprived fibroblasts. We demonstrate that Myc-induced apoptosis in low serum is inhibited by a restricted group of cytokines, principally the insulin-like growth factors and PDGF. Cytokine-mediated protection from apoptosis is not linked to the cytokines' abilities to promote growth. Protection from apoptosis is evident in the post-commitment (mitogen-independent) S/G2/M phases of the cell cycle and also in cells that are profoundly blocked in cell cycle progression by drugs. Moreover, IGF-I inhibition of apoptosis occurs in the absence of protein synthesis, and so does not require immediate early gene expression. We conclude that c-Myc-induced apoptosis does not result from a conflict of growth signals but appears to be a normal physiological aspect of c-Myc function whose execution is regulated by the availability of survival factors. We discuss the possible implications of these findings for models of mammalian cell growth in vivo.     

Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression

To explore the role of c-Myc in carcinogenesis, we have developed a reversible transgenic model of pancreatic beta cell oncogenesis using a switchable form of the c-Myc protein. Activation of c-Myc in adult, mature beta cells induces uniform beta cell proliferation but is accompanied by overwhelming apoptosis that rapidly erodes beta cell mass. Thus, the oncogenic potential of c-Myc in beta cells is masked by apoptosis. Upon suppression of c-Myc-induced beta cell apoptosis by coexpression of Bcl-x(L), c-Myc triggers rapid and uniform progression into angiogenic, invasive tumors. Subsequent c-Myc deactivation induces rapid regression associated with vascular degeneration and beta cell apoptosis. Our data indicate that highly complex neoplastic lesions can be both induced and maintained in vivo by a simple combination of two interlocking molecular lesions.     

Specific requirement for Bax, not Bak, in Myc-induced apoptosis and tumor suppression in vivo

Bax and Bak comprise the mitochondrial gateway for apoptosis induced by diverse stimuli. Loss of both bax and bak is necessary to block cell death induced by such stimuli, indicating a great degree of functional overlap between Bax and Bak. Apoptosis is the major intrinsic pathway that limits the oncogenic potential of Myc. Using a switchable mouse model of Myc-induced apoptosis in pancreatic beta cells, we have shown that Myc induces apoptosis in vivo exclusively through Bax but not Bak. Furthermore, blockade of Myc-induced apoptosis by the inactivation of Bax, but not Bak, eliminates all restraints to the oncogenic potential of Myc, allowing the rapid and synchronous progression of invasive, angiogenic tumors.     

Loss of MYC confers resistance to doxorubicin-induced apoptosis by preventing the activation of multiple serine protease- and caspase-mediated pathways

c-Myc plays an essential role in proliferation, differentiation, and apoptosis. Because of its relevance to cancer, most studies have focused on the cellular consequences of c-Myc overexpression. Here, we address the role of physiological levels of c-Myc in drug-induced apoptosis. By using c-MYC null cells we confirm and extend recent reports showing a c-Myc requirement for the induction of apoptosis by a number of anticancer agents. In particular, we show that c-Myc is required for the induction of apoptosis by doxorubicin and etoposide, whereas it is not required for camptothecin-induced cell death. We have investigated the molecular mechanisms involved in executing doxorubicin-induced apoptosis and show caspase-3 activation by both mitochondria-dependent and -independent pathways. Moreover, serine proteases participate in doxorubicin-induced apoptosis partly by contributing to caspase-3 activation. Finally, a complete rescue from doxorubicin-induced apoptosis is obtained only when serine proteases, caspase-3, and mitochondrial activation are inhibited simultaneously. Interestingly, doxorubicin requires c-Myc for the activation of all of these pathways. Our findings therefore support a model in which doxorubicin simultaneously triggers multiple c-Myc-dependent apoptosis pathways.     

The viral nucleocapsid protein of transmissible gastroenteritis coronavirus (TGEV) is cleaved by caspase-6 and -7 during TGEV-induced apoptosis

The transmissible gastroenteritis coronavirus (TGEV), like many other viruses, exerts much of its cytopathic effect through the induction of apoptosis of its host cell. Apoptosis is coordinated by a family of cysteine proteases, called caspases, that are activated during apoptosis and participate in dismantling the cell by cleaving key structural and regulatory proteins. We have explored the caspase activation events that are initiated upon infection of the human rectal tumor cell line HRT18 with TGEV. We show that TGEV infection results in the activation of caspase-3, -6, -7, -8, and -9 and cleavage of the caspase substrates eIF4GI, gelsolin, and alpha-fodrin. Surprisingly, the TGEV nucleoprotein (N) underwent proteolysis in parallel with the activation of caspases within the host cell. Cleavage of the N protein was inhibited by cell-permeative caspase inhibitors, suggesting that this viral structural protein is a target for host cell caspases. We show that the TGEV nucleoprotein is a substrate for both caspase-6 and -7, and using site-directed mutagenesis, we have mapped the cleavage site to VVPD(359) downward arrow. These data demonstrate that viral proteins can be targeted for destruction by the host cell death machinery.     

Suppression of c-Myc induces apoptosis via an AMPK/mTOR-dependent pathway by 4-<Emphasis Type="Italic">O</Emphasis>-methyl-ascochlorin in leukemia cells

4-O-Methyl-ascochlorin (MAC) is a methylated derivative of the prenyl-phenol antibiotic ascochlorin, which was isolated from an incomplete fungus, Ascochyta viciae. Although the effects of MAC on apoptosis have been reported, the underlying mechanisms remain unknown. Here, we show that MAC promoted apoptotic cell death and downregulated c-Myc expression in K562 human leukemia cells. The effect of MAC on apoptosis was similar to that of 10058-F4 (a c-Myc inhibitor) or c-Myc siRNA, suggesting that the downregulation of c-Myc expression plays a role in the apoptotic effect of MAC. Further investigation showed that MAC downregulated c-Myc by inhibiting protein synthesis. MAC promoted the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and its target proteins, including p70S6 K and 4E-BP-1. Treatment of cells with AICAR (an AMPK activator), rapamycin (an mTOR inhibitor), or mTOR siRNA downregulated c-Myc expression and induced apoptosis to a similar extent to that of MAC. These results suggest that the effect of MAC on apoptosis induction in human leukemia cells is mediated by the suppression of c-Myc protein synthesis via an AMPK/mTOR-dependent mechanism.     

Reversible activation of c-Myc in skin: induction of a complex neoplastic phenotype by a single oncogenic lesion.

The protooncogene c-myc regulates cell growth, differentiation, and apoptosis, and its aberrant expression is frequently observed in human cancer. However, the consequences of activating c-Myc in an adult tissue, in which these cellular processes are part of normal homeostasis, remain unknown. In order to achieve this, we have targeted expression of a switchable form of the c-Myc protein to the skin epidermis, a well characterized homeostatic tissue. We show that activation of c-MycER in adult suprabasal epidermis rapidly triggers proliferation and disrupts differentiation of postmitotic keratinocytes. Sustained activation of c-Myc is sufficient to induce papillomatosis together with angiogenesis--changes that resemble hyperplastic actinic keratosis, a commonly observed human precancerous epithelial lesion. All these premalignant changes spontaneously regress upon deactivation of c-MycER.     

Myc-mediated apoptosis is blocked by ectopic expression of Bcl-2.

The product of the c-myc proto-oncogene is an important positive regulator of cell growth and proliferation. Recently, c-Myc has also been demonstrated to be a potent inducer of apoptosis when expressed in the absence of serum or growth factors. To further examine Myc-induced apoptosis, we coexpressed the proto-oncogene bcl2, which has been shown to block apoptosis in other systems, with c-myc in serum-deprived Rat 1a fibroblasts. Here we report that ectopic expression of bcl2 specifically blocks apoptosis induced by constitutive c-myc expression. Constitutive c-myc expression in serum-deprived Rat 1a cells caused a > 15-fold increase in the number of dead cells, accompanied by DNA fragmentation. However, coexpression of bcl2 with c-myc in these cells led to a 10-fold increase in the number of live cells and a significant decrease in DNA fragmentation. Thus, Bcl-2 effectively inhibits Myc-induced apoptosis in serum-deprived Rat 1a fibroblasts without blocking entry into the cell cycle. These results imply that apoptosis serves as a protective mechanism to prevent tumorigenicity elicited by deregulated Myc expression. This protective mechanism is abrogated, however, by Bcl-2 and therefore may explain the synergism between Myc and Bcl-2 observed in certain tumor cells.     

Akt and c-Myc Differentially Activate Cellular Metabolic Programs and Prime Cells to Bioenergetic Inhibition

The high glucose consumption of tumor cells even in an oxygen-rich environment, referred to as the Warburg effect, has been noted as a nearly universal biochemical characteristic of cancer cells. Targeting the glycolysis pathway has been explored as an anti-cancer therapeutic strategy to eradicate cancer based on this fundamental biochemical property of cancer cells. Oncoproteins such as Akt and c-Myc regulate cell metabolism. Accumulating studies have uncovered various molecular mechanisms by which oncoproteins affect cellular metabolism, raising a concern as to whether targeting glycolysis will be equally effective in treating cancers arising from different oncogenic activities. Here, we established a dual-regulatable FL5.12 pre-B cell line in which myristoylated Akt is expressed under the control of doxycycline, and c-Myc, fused to the hormone-binding domain of the human estrogen receptor, is activated by 4-hydroxytamoxifen. Using this system, we directly compared the effect of these oncoproteins on cell metabolism in an isogenic background. Activation of either Akt or c-Myc leads to the Warburg effect as indicated by increased cellular glucose uptake, glycolysis, and lactate generation. When cells are treated with glycolysis inhibitors, Akt sensitizes cells to apoptosis, whereas c-Myc does not. In contrast, c-Myc but not Akt sensitizes cells to the inhibition of mitochondrial function. This is correlated with enhanced mitochondrial activities in c-Myc cells. Hence, although both Akt and c-Myc promote aerobic glycolysis, they differentially affect mitochondrial functions and render cells susceptible to the perturbation of cellular metabolic programs.