Increased p27Kip1 cyclin-dependent kinase inhibitor gene expression following anti-IgM treatment promotes apoptosis of WEHI 231 B cells

Engagement of the B cell receptor of WEHI 231 immature B cells leads sequentially to a drop in c-Myc, to induction of the cyclin-dependent kinase inhibitor p27Kip1, and finally to apoptosis. Recently we demonstrated that the drop in c-Myc expression promotes cell death, whereas the induction of p27 has been shown to lead to growth arrest. In this paper, we demonstrate that increased p27 expression also promotes apoptosis of WEHI 231 B cells. The rescue of WEHI 231 cells by CD40 ligand engagement of its receptor prevented the increase in p27 induction. Inhibition of p27-ablated apoptosis induced upon expression of antisense c-myc RNA. Furthermore, specific induction of p27 gene expression resulted in apoptosis of WEHI 231 cells. Lastly, inhibition of expression of c-Myc, upon induction of an antisense c-myc RNA vector, was sufficient to induce increased p27 levels and apoptosis. Thus, these findings define a signaling pathway during B cell receptor engagement in which the drop in c-Myc levels leads to an increase in p27 levels that promotes apoptosis.     

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.     

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.     

TNFalpha-mediated cell death is independent of cdc25A

Tumor necrosis factor (TNFs) have been shown to be synthesized by ovarian carcinomas, and may therefore affect tumor cells in an autocrine manner. Therefore, we investigated the effects of recombinant TNFs on ovarian carcinoma cells N.1 and examined expression of the proto-oncogenes c-myc and cdc25A which are known to play a prominent role in apoptosis. TNFalpha elicited apoptosis in N.1 cells within 72 h which was shown by typical morphological changes, DNA fragmentation and signature type cleavage of poly(ADP-ribose) polymerase into a 89 kDa proteolytic peptide. TNFalpha-induced apoptosis was accompanied by constitutive c-Myc expression, although the mRNA level of phosphatase cdc25A was suppressed within 24 h of TNFalpha treatment and the protein level decreased after 48 h. Cdc25A tyrosine phosphatase is an activator of the cdk2-cyclin E complex which allows for cell cycle progression. As expected, we found TNFalpha-mediated Cdc25A down-regulation to inhibit Cdk2 activity. Cdc25A suppression was related to TNFalpha-induced apoptosis but not to a TNFalpha-induced G0 arrest because cyclin D1 expression was unaffected and the gene gas6 (growth arrest specific 6) was not induced. Arresting cells by treatment with genistein prevented TNFalpha-triggered apoptosis and inhibited c-myc expression. TNFalpha-induced apoptosis is not accompanied by cell cycle arrest which may be due to constitutive c-Myc expression, although Cdc25A and Cdk2 activity is also down-regulated. High c-Myc and low Cdc25A activity might present conflicting signals to the cell cycle machinery which are incompatible with cell survival.     

c-Myc protein synthesis is initiated from the internal ribosome entry segment during apoptosis

Recent studies have shown that during apoptosis protein synthesis is inhibited and that this is in part due to the proteolytic cleavage of eukaryotic initiation factor 4G (eIF4G). Initiation of translation can occur either by a cap-dependent mechanism or by internal ribosome entry. The latter mechanism is dependent on a complex structural element located in the 5' untranslated region of the mRNA which is termed an internal ribosome entry segment (IRES). In general, IRES-mediated translation does not require eIF4E or full-length eIF4G. In order to investigate whether cap-dependent and cap-independent translation are reduced during apoptosis, we examined the expression of c-Myc during this process, since we have shown previously that the 5' untranslated region of the c-myc proto-oncogene contains an IRES. c-Myc expression was determined in HeLa cells during apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand. We have demonstrated that the c-Myc protein is still expressed when more than 90% of the cells are apoptotic. The presence of the protein in apoptotic cells does not result from either an increase in protein stability or an increase in expression of c-myc mRNA. Furthermore, we show that during apoptosis initiation of c-myc translation occurs by internal ribosome entry. We have investigated the signaling pathways that are involved in this response, and cotransfection with plasmids which harbor either wild-type or constitutively active MKK6, a specific immediate upstream activator of p38 mitogen-activated protein kinase (MAPK), increases IRES-mediated translation. In addition, the c-myc IRES is inhibited by SB203580, a specific inhibitor of p38 MAPK. Our data, therefore, strongly suggest that the initiation of translation via the c-myc IRES during apoptosis is mediated by the p38 MAPK pathway.     

Regulation of c-Myc through phosphorylation at Ser-62 and Ser-71 by c-Jun N-terminal kinase.

The expression of c-myc promotes cell proliferation and also sensitizes cells to various extracellular apoptotic stimuli. However, signal pathways regulating the function of Myc proteins during apoptosis are unknown. c-Jun N-terminal kinase (JNK) is activated by various apoptotic stimuli, but neither the target molecule(s) or the action of JNK has been identified in Myc-mediated apoptosis. Here, we found that JNK selectively interacted with, and phosphorylated, c-Myc at Ser-62 and Ser-71 as confirmed with phospho-c-Myc-specific antibodies. Interestingly, dominant negative mutant JNK(APF) impaired the c-Myc-dependent apoptosis, but not mutated c-Myc (S62A/S71A)-dependent apoptosis triggered by UV irradiation. Furthermore, c-Myc (S62A/S71A)-expressing NIH3T3 cells were not sensitized like wild type c-Myc-expressing NIH3T3 cells to JNK-activating apoptotic stimuli, such as UV and Taxol. These results indicate that the JNK pathway is selectively involved in the c-Myc-mediated apoptosis and that the apoptotic function of c-Myc is directly regulated by JNK pathway through phosphorylation at Ser-62 and Ser-71.     

A role for c-myc in chemically induced renal-cell death.

A variety of genes, including c-myc, are activated by chemical toxicants in vivo and in vitro. Although enforced c-myc expression induces apoptosis after withdrawing survival factors, it is not clear if activation of the endogenous c-myc gene is an apoptotic signal after toxicant exposure. The renal tubular epithelium is a target for many toxicants. c-myc expression is activated by tubular damage. In quiescent LLC-PK1 renal epithelial cells, c-myc but not max or mad mRNA is induced by the nephrotoxicant S-(1,2-dichlorovinyl)-L-cysteine (DCVC). The kinetics of DCVC-induced c-myc expression and apoptosis suggested an association between cell death and prolonged activation of c-myc expression after toxicant exposure. Accordingly, prolonged activation of an estrogen receptor-Myc fusion construct, but not a construct in which a c-Myc transactivation domain had been deleted, was sufficient to induce apoptosis in LLC-PK1 cells. Moreover, under conditions in which necrosis was the predominant cell death pathway caused by DCVC in parental cells, overexpressing c-myc biased the cell death pathway toward apoptosis. DCVC also induced ornithine decarboxylase (odc) mRNA and activated the odc promoter. Activation of the odc promoter by DCVC required consensus c-Myc-Max binding sites in odc intron 1. Inhibiting ODC activity with alpha-difluoromethylornithine delayed DCVC-induced cell death. Therefore, odc is a target gene in the DCVC apoptotic pathway involving c-myc activation and contributes to apoptosis. Finally, a structurally related cytotoxic but nongenotoxic analog of DCVC did not induce c-myc and did not activate the odc promoter or induce apoptosis. The data support the hypothesis that activation of apoptotic cell death in quiescent renal epithelial cells involves induction of c-myc. This is the first study to demonstrate that c-myc induction by a specific nephrotoxicant leads to gene activation and cell death.     

Pathogenesis of Burkitt lymphoma: expression of an activated c-myc oncogene causes the tumorigenic conversion of EBV-infected human B lymphoblasts

To study the pathogenesis of Burkitt lymphoma, we introduced activated c-myc genes into human EBV-infected lymphoblastoid cells derived from in vitro infection of normal cord blood or directly from infected peripheral blood from AIDS patients. In both cell types the constitutive expression of exogenous c-myc caused negative regulation of endogenous c-myc expression, changes in growth properties typical of transformed cells, and acquisition of tumorigenicity in immunodeficient mice. In all myc-transfected populations the degree of malignancy directly correlated with the level of c-myc mRNA. EBV infection and c-myc activation are thus sufficient for the tumorigenic conversion of human B cells in vitro, strongly supporting the hypothesis that these same two pathogenetic steps may be involved in the in vivo development of Burkitt lymphoma.     

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.     

Involvement of NF-kappaB and c-myc signaling pathways in the apoptosis of HL-60 cells induced by alkaloids of Tripterygium hypoglaucum (levl.) Hutch.

Tripterygium hypoglaucum (levl.) Hutch (Celastraceae) (THH) root is a Chinese medicinal herb commonly used for treating autoimmune diseases. In the present study, alkaloids of THH were prepared and their cytotoxicity against the HL-60 cell was investigated. THH-induced apoptosis was observed using flow cytometry, confocal fluorescence microscope, and DNA laddering and caspase assays. The molecular mechanism involved in the induction of HL-60 cell apoptosis by THH alkaloids was examined using cDNA microarrays containing 3000 human genes derived from a leukocyte cDNA library. Sixteen genes were identified to be differentially expressed in HL-60 cells upon THH treatment. Several genes related to the NF-kappaB signaling pathway and cell apoptosis (such as NFKBIB, PRG1 and B2M) were up-regulated. In addition, c-myc binding protein and apoptosis-related cysteine proteases caspase-3 and caspase-8 were also regulated. The changes in c-Myc RNA expression and c-myc protein level were further confirmed by RT-PCR and Western blot analysis. The results demonstrated that THH alkaloids induced apoptosis of HL-60 cells though c-myc and NF-kappaB signaling pathways.