Mitochondrial targeting of JNK/SAPK in the phorbol ester response of myeloid leukemia cells

Treatment of human U-937 myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with activation of the stress-activated protein kinase (SAPK) and induction of terminal monocytic differentiation. The present studies demonstrate that TPA targets SAPK to mitochondria by a mechanism dependent on activation of protein kinase C (PKC) beta. Translocation of SAPK to mitochondria in response to TPA is associated with release of cytochrome c, caspase-3 activation and induction of apoptosis. The results show that TPA induces the association of SAPK with the mitochondrial anti-apoptotic Bcl-x(L) protein. Overexpression of Bcl-x(L) attenuated the apoptotic response to TPA treatment. Moreover, expression of Bcl-x(L) mutated at sites of SAPK phosphorylation (Thr-47, -115) was more effective than wild-type Bcl-x(L) in abrogating TPA-induced cytochrome c release and apoptosis. By contrast, expression of Bcl-x(L) had little effect on induction of the monocytic phenotype. These findings indicate that myeloid leukemia cells respond to TPA with targeting of SAPK to mitochondria and that this response contributes to terminal differentiation through the release of cytochrome c and induction of apoptosis.     

Terminal Myeloid Differentiation is Uncoupled from Cell Cycle Arrest

It has been assumed that terminal myeloid differentiation and cell cycle arrest are coupled processes, and that prohibiting cell cycle arrest blocks differentiation. Previously we have shown that, using the murine M1 myeloid leukemic cell line, deregulated expression of the proto-oncogene c-myc results in cells that cannot be induced to undergo terminal differentiation and continued to proliferate. It has also been shown that ectopic expression of Egr-1 abrogated the c-Myc block in terminal myeloid differentiation, yet there was no accumulation of cells in the G0/G1 phase of the cell cycle. In this study we conclusively demonstrate that M1Myc/Egr-1 cells terminally differentiate while still actively cycling and synthesizing DNA, concluding that the terminal myeloid differentiation program is uncoupled from growth arrest. How deregulated expression/activation of proto-oncogenes that promote cell cycle progression interferes with differentiation and how differentiation is regulated independently of cell cycle control are discussed, as well as the implications with regard to differentiation therapy.     

c-Myc is a critical target for c/EBPalpha in granulopoiesis

CCAAT/enhancer binding protein alpha (C/EBPalpha) is an integral factor in the granulocytic developmental pathway, as myeloblasts from C/EBPalpha-null mice exhibit an early block in differentiation. Since mice deficient for known C/EBPalpha target genes do not exhibit the same block in granulocyte maturation, we sought to identify additional C/EBPalpha target genes essential for myeloid cell development. To identify such genes, we used both representational difference analysis and oligonucleotide array analysis with RNA derived from a C/EBPalpha-inducible myeloid cell line. From each of these independent screens, we identified c-Myc as a C/EBPalpha negatively regulated gene. We mapped an E2F binding site in the c-Myc promoter as the cis-acting element critical for C/EBPalpha negative regulation. The identification of c-Myc as a C/EBPalpha target gene is intriguing, as it has been previously shown that down-regulation of c-Myc can induce myeloid differentiation. Here we show that stable expression of c-Myc from an exogenous promoter not responsive to C/EBPalpha-mediated down-regulation forces myeloblasts to remain in an undifferentiated state. Therefore, C/EBPalpha negative regulation of c-Myc is critical for allowing early myeloid precursors to enter a differentiation pathway. This is the first report to demonstrate that C/EBPalpha directly affects the level of c-Myc expression and, thus, the decision of myeloid blasts to enter into the granulocytic differentiation pathway.     

Inhibition of N-linked glycosylation induces early apoptosis in human promyelocytic HL-60 cells

Inhibition of protein N-glycosylation by tunicamycin induced morphological changes characteristic of apoptosis in human promyelocytic HL-60 cells. Internu-cleosomal DMA fragmentation could be detected after short-time incubation (between 6 and 9 h) of HL-60 cells with low doses of tunicamycin (0.05 μg/ml). Under these conditions the synthesis of glycoproteins was reduced to 17% of control values, while no significant changes in the rates of total protein synthesis could be observed. Tunicamycin ability to induce DNA fragmentation was in good correlation with its potency as glycosylation inhibitor in several myeloid cell lines. Tunicamycin-induced apoptosis was potentiated by activation of protein kinease C (PKC) by phorbol esters and partially prevented by the PKC inhibitor staurosporine. Inhibitors of RNA and protein synthesis displayed a protective effect. Treatment of HL-60 cells with tunicamycin did not elicit the expression of cell surface differentiation antigens or their ability to generate superoxide anion. In contrast, tunicamycin significantly inhibited these processes during dimethyl sulfoxide (DMSO)-induced myeloid differentiation. These observations indicate that the main effect of tunicamycin in HL-60 cells is the induction of apoptosis. © 1995 Wiley-Liss, Inc.     

Spontaneous apoptosis in primary cultures of human and rat hepatocytes: molecular mechanisms and regulation by dexamethasone

To elucidate the biochemical pathways leading to spontaneous apoptosis in primary cultures of human and rat hepatocytes, we examined the activation of the caspase cascade, the expression of Bcl-2-related-proteins and heat shock proteins. Comparisons were made before and after dexamethasone (DEX) treatment. We show that DEX inhibited spontaneous apoptosis in a dose-dependent manner. DEX increases the expression of anti-apoptotic Bcl-2 and Bcl-x(L) proteins, decreases the expression of pro-apoptotic Bax and inhibits Bad translocation thereby preventing the release of cytochrome c, the activation of caspases, and cell death. Although, the expression of Hsp27 and Hsp70 proteins remained unchanged, the oncogenic protein c-Myc is upregulated upon DEX-treatment. These results indicate that DEX mediates its survival effect against spontaneous apoptosis by acting upstream of the mitochondrial changes. Thus, the mitochondrial apoptotic pathway plays a major role in regulating spontaneous apoptosis in these cells. Blocking this pathway therefore may assist with organ preservation for transplant, drug screening, and other purposes.     

Bcl-x(S) induces an NGF-inhibitable cytochrome c release

Bcl-x(S), a pro-apoptotic member of the Bcl-2 protein family, is localized in the mitochondrial outer membrane and induces caspase-dependent and nerve growth factor (NGF)-inhibitable apoptosis in PC12 cells. The mechanism of action of Bcl-x(S) and how NGF inhibits this death are not fully understood. It is still unknown whether Bcl-x(S) induces mitochondrial cytochrome c release, and which apoptotic step NGF inhibits. We show that Bcl-x(S) induces cytochrome c release and caspase-3 activation in several cell types, and that in PC12 cells, these events are inhibited by NGF treatment. The survival effect of NGF was inhibited by inhibitors of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI 3-kinase), and the mitogen-activated protein kinase kinase (MEK) inhibitors GF109203X, LY294002, and U0126. These findings show that cytochrome c release and caspase-3 activation participate in Bcl-x(S)-induced apoptosis, and that NGF inhibits Bcl-x(S)-induced apoptosis at the mitochondrial level via the PKC, PI 3-kinase, and MEK signaling pathways.     

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.     

Caspase-mediated Bak activation and cytochrome c release during intrinsic apoptotic cell death in Jurkat cells

Mitochondrial outer membrane permeabilization and the release of intermembrane space proteins, such as cytochrome c, are early events during intrinsic (mitochondria-mediated) apoptotic signaling. Although this process is generally accepted to require the activation of Bak or Bax, the underlying mechanism responsible for their activation during true intrinsic apoptosis is not well understood. In the current study, we investigated the molecular requirements necessary for Bak activation using distinct clones of Bax-deficient Jurkat T-lymphocytes in which the intrinsic pathway had been inhibited. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were equally resistant to apoptosis induced by the DNA-damaging anticancer drug etoposide as determined by phosphatidylserine externalization and caspase activation. Strikingly, characterization of mitochondrial apoptotic events in all three drug-resistant cell lines revealed that, without exception, resistance to apoptosis was associated with an absence of Bak activation, cytochrome c release, and mitochondrial membrane depolarization. Furthermore, we found that etoposide-induced apoptosis and mitochondrial events were inhibited in cells stably overexpressing either full-length X-linked inhibitor of apoptosis protein (XIAP) or the BIR1/BIR2 domains of XIAP. Combined, our findings suggest that caspase-mediated positive amplification of initial mitochondrial changes can determine the threshold for irreversible activation of the intrinsic apoptotic pathway.     

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.