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.     

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.     

CD72 stimulation modulates anti-IgM induced apoptotic signaling through the pathway of NF-kappaB, c-Myc and p27(Kip1)

Engagement of mIgM induces G1 arrest and apoptosis in immature B cells. The biochemical mechanism(s) regulating the cell death process are poorly understood. Cross-linking of CD72 (a B cell co-receptor) with anti-CD72 antibody was shown to protect B cells from apoptosis. We investigated the molecular mechanism involved in apoptosis preventing signaling mediated by CD72 ligation using a derivative (WEHIdelta) of the WEHI231 cell line which is representative of immature B cells. Apoptotic WEHIdelta cells following cross-linking of mIgM demonstrate a dramatic loss of c-Myc protein after transient up-regulation. In contrast, pre-ligation of CD72 was able to sustain c-Myc expression after transient up-regulation. Cross-linking of mIgM of WEHIdelta cells causes accumulation of the Cdk inhibitor, p27(Kip1). CD72 pre-ligation was shown to inhibit the accumulation of p27(Kip1) protein. Moreover, NF-kappaB activity was not suppressed in WEHIdelta cells after mIgM cross-linking when the cells were pre-treated with anti-CD72 antibody. These results strongly suggest that the apoptosis preventing signal evoked by CD72 ligation is delivered through the pathway of NF-kappaB, c-Myc, p27(Kip1) and cyclin.     

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.     

Rapid loss of intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene c-Myc

Inhibition of the mutationally activated Wnt cascade in colorectal cancer cell lines induces a rapid G1 arrest and subsequent differentiation. This arrest can be overcome by maintaining expression of a single Tcf4 target gene, the proto-oncogene c-Myc. Since colorectal cancer cells share many molecular characteristics with proliferative crypt progenitors, we have assessed the physiological role of c-Myc in adult crypts by conditional gene deletion. c-Myc-deficient crypts are lost within weeks and replaced by c-Myc-proficient crypts through a fission process of crypts that have escaped gene deletion. Although c-Myc(-/-) crypt cells remain in the cell cycle, they are on average much smaller than wild-type cells, cycle slower, and divide at a smaller cell size. c-Myc appears essential for crypt progenitor cells to provide the necessary biosynthetic capacity to successfully progress through the cell cycle.     

c-Myc Blazing a Trail of Death: Coupling of the Mitochondrial and Death Receptor Apoptosis Pathways by c-Myc

TRAIL ligand induces selectively apoptosis in tumor cells by binding to two death receptors (DR4 and DR5) and holds promise as a potential therapeutic agent against cancer. While it has been known for long time that TRAIL receptors are commonly expressed in wide variety of normal tissues, it is not well understood why TRAIL kills tumor cells but leaves normal cells unharmed. The prototypic oncogene c-Myc promotes the cell cycle and simultaneously primes activation of the Bcl-2 family controlled mitochondria apoptosis pathway. A striking reflection of the c-Myc-dependent apoptotic sensitization is the dramatic c-Myc-induced vulnerability of cells to TRAIL and other death receptor ligands. Here we summarize the recent findings regarding the death mechanisms of TRAIL/TRAIL receptor system and the connection of c-Myc to the mitochondrial apoptosis pathway, focusing on our work that couples c-Myc via Bak to the TRAIL death receptor pathway. Finally, we present a mitochondria-priming model to explain how c-Myc-Bak interaction amplifies the TRAIL-induced caspase 8-Bid pathway to induce fullblown apoptosis. We discuss the implications of these findings for understanding the selective cytotoxicity of TRAIL and for the therapeutic exploitation of the death receptor pathway.     

Glutathione influences c-Myc-induced apoptosis in M14 human melanoma cells

The objective of this article is to dissect the mechanisms by which the down-regulation of c-Myc induces programmed cell death in melanoma cells. In stable and doxycycline-inducible M14 melanoma cells, down-regulation of c-Myc induced apoptosis subsequent to a decrease in the intracellular reduced glutathione content and a concomitant accumulation of its oxidized form. This redox alteration was associated with a decrease of the enzyme activities of gamma-glutamyl-cysteine synthetase and NADPH-dependent GSSG reductase, as well as a consequent glutathione release in the extracellular medium. Cytochrome c was released into the cytosol at very early stages of apoptosis induction, long before detectable production of reactive oxygen species and activation of caspase-9 and -3. Macroarray analysis revealed that down-regulation of c-Myc produced striking changes in gene expression in the section related to metabolism, where the expression of gamma-glutamyl-cysteine synthetase and GSSG reductase was found to be significantly reduced. The addition of N-acetyl-l-cysteine or glutathione ethyl ester inhibited the apoptotic process, thus confirming the key role of glutathione in programmed cell death induced by c-Myc.     

Ornithine decarboxylase is a mediator of c-Myc-induced apoptosis.

c-Myc plays a central role in the regulation of cell cycle progression, differentiation, and apoptosis. However, the proteins which mediate c-Myc function(s) remain to be determined. Enforced c-myc expression rapidly induces apoptosis in interleukin-3 (IL-3)-dependent 32D.3 murine myeloid cells following IL-3 withdrawal, and this is associated with the constitutive, growth factor-independent expression of ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis. Here we have examined the role of ODC in c-Myc-induced apoptosis. Enforced expression of ODC, like c-myc, is sufficient to induce accelerated death following IL-3 withdrawal. ODC induced cell death in a dose-dependent fashion, and alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC enzyme activity, effectively blocked ODC-induced cell death. ODC-induced cell death was due to the induction of apoptosis. We also demonstrate that ODC is a mediator of c-Myc-induced apoptosis. 32D.3-derived c-myc clones have augmented levels of ODC enzyme activity, and their rates of death were also a function of their ODC enzyme levels. Importantly, the rates of death of c-myc clones were inhibited by treatment with DFMO. These findings demonstrate that ODC is an important mediator of c-Myc-induced apoptosis and suggest that ODC mediates other c-Myc functions.     

Apoptosis of insulin-secreting cells induced by endoplasmic reticulum stress is amplified by overexpression of group VIA calcium-independent phospholipase A2 (iPLA2 beta) and suppressed by inhibition of iPLA2 beta

The death of insulin-secreting beta-cells that causes type I diabetes mellitus (DM) occurs in part by apoptosis, and apoptosis also contributes to progressive beta-cell dysfunction in type II DM. Recent reports indicate that ER stress-induced apoptosis contributes to beta-cell loss in diabetes. Agents that deplete ER calcium levels induce beta-cell apoptosis by a process that is independent of increases in [Ca(2+)](i). Here we report that the SERCA inhibitor thapsigargin induces apoptosis in INS-1 insulinoma cells and that this is inhibited by a bromoenol lactone (BEL) inhibitor of group VIA calcium-independent phospholipase A(2) (iPLA(2)beta). Overexpression of iPLA(2)beta amplifies thapsigargin-induced apoptosis of INS-1 cells, and this is also suppressed by BEL. The magnitude of thapsigargin-induced INS-1 cell apoptosis correlates with the level of iPLA(2)beta expression in various cell lines, and apoptosis is associated with stimulation of iPLA(2)beta activity, perinuclear accumulation of iPLA(2)beta protein and activity, and caspase-3-catalyzed cleavage of full-length 84 kDa iPLA(2)beta to a 62 kDa product that associates with nuclei. Thapsigargin also induces ceramide accumulation in INS-1 cells, and this response is amplified in cells that overexpress iPLA(2)beta. These findings indicate that iPLA(2)beta participates in ER stress-induced apoptosis, a pathway that promotes beta-cell death in diabetes.     

E2F1 and c-Myc potentiate apoptosis through inhibition of NF-kappaB activity that facilitates MnSOD-mediated ROS elimination

Overexpression of c-Myc or E2F1 sensitizes host cells to various types of apoptosis. Here, we found that overexpressed c-Myc or E2F1 induces accumulation of reactive oxygen species (ROS) and thereby enhances serum-deprived apoptosis in NIH3T3 and Saos-2. During serum deprivation, MnSOD mRNA was induced by NF-kappaB in mock-transfected NIH3T3, while this induction was inhibited in NIH3T3 overexpressing c-Myc or E2F1. In these clones, E2F1 inhibited NF-kappaB activity by binding to its subunit p65 in competition with a heterodimeric partner p50. In addition to overexpressed E2F1, endogenous E2F1 released from Rb was also found to inhibit NF-kappaB activity in a cell cycle-dependent manner by using E2F1(+/+) and E2F1(-/-) murine embryonic fibroblasts. These results indicate that E2F1 promotes apoptosis by inhibiting NF-kappaB activity.