Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2

The oncogene bcl-2 encodes a 26-kD protein localized to intracellular membranes, including the ER, mitochondria, and perinuclear membrane, but its mechanism of action is unknown. We have been investigating the hypothesis that Bcl-2 regulates the movement of calcium ions (Ca2+) through the ER membrane. Earlier findings in this laboratory indicated that Bcl-2 reduces Ca2+ efflux from the ER lumen in WEHI7.2 lymphoma cells treated with the Ca2+-ATPase inhibitor thapsigargin (TG) but does not prevent capacitative entry of extracellular calcium. In this report, we show that sustained elevation of cytosolic Ca2+ due to capacitative entry is not required for induction of apoptosis by TG, suggesting that ER calcium pool depletion may trigger apoptosis. Bcl-2 overexpression maintains Ca2+ uptake in the ER of TG-treated cells and prevents a TG-imposed delay in intralumenal processing of the endogenous glycoprotein cathepsin D. Also, Bcl-2 overexpression preserves the ER Ca2+ pool in untreated cells when extracellular Ca2+ is low. However, low extracellular Ca2+ reduces the antiapoptotic action of Bcl-2, suggesting that cytosolic Ca2+ elevation due to capacitative entry may be required for optimal ER pool filling and apoptosis inhibition by Bcl-2. In summary, the findings suggest that Bcl-2 maintains Ca2+ homeostasis within the ER, thereby inhibiting apoptosis induction by TG.     

Unmethylated CpG-containing oligodeoxynucleotides inhibit apoptosis in WEHI 231 B lymphocytes induced by several agents: evidence for blockade of apoptosis at a distal signalling step

Certain oligodeoxynucleotides (ODN) containing cytosine followed by guanosine (CpG) protect B cells from apoptosis, and induce B-cell proliferation and cytokine production. We investigated the effect of phosphorothioate CpG-containing ODNs (5'-ATAATCGACGTTCAAGCAAG-3' or 5'-TCCATGACGTTCCTGACGTT-3') and control ODNs (which did not contain CpG) on apoptosis and cell growth in WEHI 231 murine B lymphoma cells. Anti-surface (alpha-s)IgM antibody induces 40-60% DNA degradation and growth arrest of WEHI 231 cells in 24 h. Both of these effects were substantially reversed by 30 ng/ml CpG-ODN added up to 8 hr after alpha-sIgM. Control ODNs not containing the CpG motif were without effect. We explored various hypotheses to account for these effects. The phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate, inhibits apoptosis induced by alpha-sIgM, but the anti-apoptotic effect of CpG-ODN was not affected by inhibitors of protein kinase C, indicating that CpG-ODN does not act via protein kinase C. CpG-ODN inhibited apoptosis and growth arrest induced by C2- and C8-ceramide, sphingomyelinase and an intracellular Ca2+ pump inhibitor thapsigargin, indicating that inhibition is not mediated via suppression of the ceramide cycle or suppression of Ca2+ mobilization. CpG-ODN partially inhibited apoptosis induced by okadaic acid, a protein phosphatase inhibitor, and by menadione, a free radical generator. CpG-ODN also inhibited apoptosis and growth arrest induced by ultraviolet-irradiation, glucocorticoid, vinca alkaloids, and doxorubicin. CpG-ODN significantly protected cells from DNA fragmentation induced by alpha-sIgM in the presence of cycloheximide, but cycloheximide itself induces apoptosis which was unaffected by CpG-ODN. These results suggest that CpG-ODNs powerfully modulate the process by which immune cells are committed to death or proliferation by a mechanism acting on distal cell signalling events. CpG-ODNs may be able to decrease immunosuppression in patients undergoing cancer chemotherapy.     

Cell cycle-dependent tumor necrosis factor apoptosis

To determine if tumor necrosis factor (TNF)-mediated apoptosis affects cells at defined stages of the cell cycle, WEHI-164/2F (WEHI) cells were synchronized at G0-G1 after 3-day cultures in medium containing RPMI 1640 and 0.5% FCS (RPMI-0.5% FCS). The arrested WEHI cells (60-75% in G0-G1) showed increased sensitivity to TNF killing, measured as 48-h 3-(5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assays, and 15-h apoptosis by propidium iodide staining and flow cytometry analysis. The TNF killing kinetics of G0-G1-arrested cells was similar to controls, and TNF did not accelerate or retard cell cycle progression of the arrested cells after feeding with fresh RPMI-0.5% FCS. However, TNF inhibited WEHI DNA synthesis as early as 1 h after treatment, and inhibition was proportionate to sensitivity to TNF-induced apoptosis. WEHI cells treated with TNF showed a higher percentage of cells in S phase with concomitant decrease in G0-G1 and G2-M. When cultured for 3-18 h in fresh RPMI-0.5% FCS to allow progression of the G0-G1-arrested cells toward the G1-S boundary, WEHI cells became more sensitive to TNF killing, especially at the 3-9 h time points. Moreover, TNF did not degrade [125I]5-iodo-2'-deoxyuridine-labeled WEHI DNA if the labeled cells were precultured for 9 h in fresh RPMI-0.5% FCS to allow them to pass S phase before the addition of TNF. These results show that TNF-induced apoptosis of WEHI cells is connected to cell cycle events; WEHI targets receive the TNF cytotoxic signal mainly at the G1-S boundary and begin to die by apoptosis as they exit from S phase.     

Ubiquitin-proteasome pathway mediates intracellular degradation of apolipoprotein B

Newly synthesized apolipoprotein B (apoB) is degraded by a proteolytic process in the pre-Golgi compartment that can be inhibited by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (ALLN) but not by several other protease inhibitors. We have tested the hypothesis that the ubiquitin-proteasome pathway is involved in the intracellular degradation of apoB in liver cells. We found that inhibitors of proteasomes blocked the degradation of apoB in cultured human hepatoma (HepG2) cells. Protein degradation by proteasomes is ATP-dependent, and ATP depletion by dinitrophenol and 2-deoxyglucose also inhibited apoB degradation in these cells. Furthermore, the intracellular human apoB isolated by immunoprecipitation was shown to react specifically with anti-ubiquitin antibody by immunoblotting. This result was corroborated by sequential immunoprecipitation of [35S]methionine-labeled proteins by anti-human apoB and anti-ubiquitin antisera. In contrast, secreted apoB was not ubiquitinated. The amount of intracellular ubiquitinated apoB was increased by the proteasome inhibitors, ALLN and carbobenzoxyl-leucinyl-leucinyl-norvalinal-H (MG115). Our findings suggest that the ubiquitin-proteasome pathway is one mechanism for the intracellular degradation of apoB.     

Extracellular signal-regulated kinase-2, but not c-Jun NH2-terminal kinase, activation correlates with surface IgM-mediated apoptosis in the WEHI 231 B cell line

Both extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) have been implicated in mediating the signaling events that precede apoptosis. We studied the activation of these kinases during apoptosis of WEHI 231 B cells. Surface IgM ligation induces apoptosis of WEHI 231 cells. This effect is augmented by simultaneous engagement of CD95 and is inhibited by costimulation with either CD40 or IL-4R. We determined that surface IgM ligation activates ERK2 to a much greater level than JNK, and that IgM-mediated ERK2 activation is enhanced by costimulation with anti-CD95. Costimulation with either IL-4 or anti-CD40 interferes with anti-IgM-stimulated ERK2 activation. Transient expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) inhibits both ERK2 activation and cell death following stimulation with anti-IgM and the combination of anti-IgM plus anti-CD95. CD40 engagement alone activates JNK, but IL-4 stimulation does not. N-acetyl-L-cysteine pretreatment, which blocks CD40-mediated JNK activation, does not affect the ability of CD40 to inhibit anti-IgM-mediated ERK2 activation and apoptosis. Together, these data suggest that JNK activation is not required for CD40 inhibition of surface IgM-induced cell death and that ERK2 plays an active role in mediating anti-IgM-induced apoptosis of WEHI 231 B cells.     

Iron-dependent oxidation, ubiquitination, and degradation of iron regulatory protein 2: implications for degradation of oxidized proteins

The ability of iron to catalyze formation of reactive oxygen species significantly contributes to its toxicity in cells and animals. Iron uptake and distribution is regulated tightly in mammalian cells, in part by iron regulatory protein 2 (IRP2), a protein that is degraded efficiently by the proteasome in iron-replete cells. Here, we demonstrate that IRP2 is oxidized and ubiquitinated in cells before degradation. Moreover, iron-dependent oxidation converts IRP2 into a substrate for ubiquitination in vitro. A regulatory pathway is described in which excess iron is sensed by its ability to catalyze site-specific oxidations in IRP2, oxidized IRP2 is ubiquitinated, and ubiquitinated IRP2 subsequently is degraded by the proteasome. Selective targeting and removal of oxidatively modified proteins may contribute to the turnover of many proteins that are degraded by the proteasome.     

Nucleotidase activities of the 26 S proteasome and its regulatory complex

The 26 S proteasome can be assembled from the multicatalytic protease (or 20 S proteasome) and a large multisubunit regulatory complex in an ATP-dependent reaction. The 26 S proteasome and its regulatory complex were purified from rabbit reticulocytes for characterization of their nucleotidase properties. Both particles hydrolyze ATP, CTP, GTP, and UTP to the corresponding nucleoside diphosphate and inorganic phosphate. The Km values for hydrolysis of specific nucleotides by the 26 S proteasome are 15 microM for ATP and CTP, 50 microM for GTP, and 100 microM for UTP; Km values for nucleotide hydrolysis by the regulatory complex are 2-4-fold higher for each nucleotide. Several ATPase inhibitors (erythro-9-[3-(2-hydroxynonyl)]adenine, oligomycin, ouabain, and thapsigargin) had no effect on ATP hydrolysis by either complex whereas known inhibitors of proteolysis by the 26 S enzyme (hemin, N-ethylmaleimide (NEM), and vanadate) significantly reduced ATP hydrolysis by both particles. Hydrolysis of all nucleotides was inhibited by 5 mM NEM but only GTP and UTP hydrolysis was significantly reduced at 50 microM NEM. The 15 microM Km for ATP hydrolysis by the 26 S proteasome is virtually identical to the observed Km of 12 microM ATP for Ub-conjugate degradation. Although nucleotide hydrolysis is required for protein degradation by the 26 S proteasome, nucleotide hydrolysis and peptide bond cleavage are not strictly coupled. Substrate specificity constants (kcat/Km) are similar for hydrolysis of each nucleotide, yet GTP and UTP barely supported Ub-conjugate degradation. Further evidence that nucleotide hydrolysis is not coupled to peptide bond cleavage was obtained using N-acetyl-leucyl-leucyl-norleucinal (LLnL). This compound inhibited peptide hydrolysis by the multicatalytic protease and Ub-conjugate degradation by the 26 S proteasome, but it had little effect on ATP or UTP hydrolysis by the 26 S enzyme.     

Inhibition by dexamethasone of transforming growth factor beta1-induced apoptosis in rat hepatoma cells: a possible association with Bcl-xL induction

The authors previously reported that transforming growth factor beta1 (TGF-beta1) induces apoptosis in McA-RH7777 (7777) and McA-RH8994 (8994) rat hepatoma cell lines. Although these cell lines exhibit different responses to glucocorticoid treatment in various cellular functions and gene expression, dexamethasone (DEX) inhibited spontaneous and TGF-beta1-induced apoptosis in both. Analysis of analogous hormones in TGF-beta1-induced apoptosis in 8994 cells suggested the inhibitory effect to be glucocorticoid-specific. By cell-cycle analysis and DNA fragmentation assay using sodium butyrate, a G1-arrest-inducing reagent, regulation of apoptosis by TGF-beta1 and DEX was shown independent of the cell cycle. For elucidation of the mechanisms of anti-apoptotic action of DEX, the effects of various chemical probes on this apoptosis model were examined, and various reagents known to exhibit anti-apoptotic activity in other experimental systems were found to be ineffective. The effect of TGF-beta1 and DEX on cellular amounts of several apoptosis-related proteins, members of the Bcl-2 family, Bcl-2, Bcl-xL, Bcl-xS, Bad, and Bax was also examined. DEX drastically increased Bcl-xL in both cell lines irrespective of the presence of TGF-beta1. Bcl-2 and Bcl-xS proteins were not detected, and Bax and Bad content did not change by treatment with TGF-beta1 or DEX. Progesterone (Prog), a partial antagonist for glucocorticoid receptor, inhibited the effects of DEX on apoptosis and Bcl-xL expression in 8994 cells. Thus, Bcl-xL induction by DEX would appear closely associated with its inhibitory effect on spontaneous and TGF-beta1-induced apoptosis in the hepatoma cell lines.     

Calpain involvement in calphostin C-induced apoptosis

A major problem in assessing the role of calpains in apoptosis induction concerns the fact that calpain inhibitors can also impair the activity of the proteasome, also reported to be involved in apoptosis. Herein we showed that apoptosis induced by calphostin C in U937 human promonocytic leukemia cells was associated, at its onset, with enhanced protein (poly)ubiquitination. This observation prompted us to study whether protein degradation through the ubiquitin/proteasome pathway was involved in apoptosis induction. We found that N-acetyl-Leu-Leu-norleucinal (50 microM), a proteasome as well as a calpain inhibitor, was able to reduce calphostin C-induced apoptosis by approximately 60%, whereas lactacystin (10 microM), a specific proteasome inhibitor, was ineffective. These results suggest that calphostin C-induced apoptosis is partly calpain-mediated, but does not require protein degradation through the ubiquitin/proteasome pathway.     

Novel dipeptidyl proteasome inhibitors overcome Bcl-2 protective function and selectively accumulate the cyclin-dependent kinase inhibitor p27 and induce apoptosis in transformed, but not normal, human fibroblasts

It has been suggested that overexpression of the Bcl-2 oncoprotein in human cancer cells contributes to their resistance to apoptosis induced by chemotherapy. We report here that a novel dipeptidyl proteasome inhibitor, CEP1612, at low concentrations rapidly induces apoptosis in human Jurkat T cells overexpressing Bcl-2 and also in all human prostate, breast, tongue and brain tumor cell lines we have tested to date, without exception. In contrast, etoposide, a standard anticancer drug, fails to kill these cells when employed under the same conditions. The apoptosis-inducing abilities of CEP1612 and its analogous compounds match precisely their order for inhibition of the proteasome chymotrypsin-like activity. CEP1612-induced apoptosis is p53-independent, inhibitable by a tetrapeptide caspase inhibitor, and associated with accumulation of the cyclin-dependent kinase inhibitors p21 and p27. Furthermore, CEP1612 selectively accumulates p27 and induces apoptosis in simian virus 40-transformed, but not the parental normal, human fibroblasts. Proteasome inhibitors such as those investigated herein might therefore have potential use as novel anticancer drugs.     

Regulation and function of Bcl-2 during differentiation-induced cell death in HL-60 promyelocytic cells

Polymorphonuclear leukocytes are generated by differentiation of early myeloid precursors. Once fully differentiated, blood neutrophils are programmed to die rapidly and are removed by tissue macrophages. In normal myeloid cells, the death mechanism seems to be coupled to the differentiation pathway and is accomplished by a process termed apoptosis. In the present study, we have examined the role of Bcl-2 in the differentiation pathways of the promyelocytic cell line HL-60. Treatment of HL-60 with retinoic acid or phorbol ester, which induced neutrophil or macrophage-like cell differentiation, respectively, resulted in progressive loss of cellular viability and internucleosomal DNA degradation. In HL-60, differentiation and apoptosis were coupled to down-regulation of the Bcl-2 protein. Overexpression of Bcl-2 by gene transfer inhibited apoptosis triggered by terminal differentiation of HL-60. Yet, Bcl-2 did not alter the expression of surface markers or other phenotypic changes that are induced upon myeloid differentiation. In contrast to HL-60, another immature myeloid cell line, K562, did not produce Bcl-2 but expressed a related protein, Bcl-xL, that functions as a repressor of apoptotic cell death. K562 has been shown to be relatively resistant to a variety of apoptotic stimuli. Incubation of HL-60 and K562 with inhibitors of macromolecular synthesis induced apoptosis, which appeared earlier in HL-60 than in K562. Interestingly, Bcl-2 overexpression protected K562 cells from apoptosis induced by inhibitor of macromolecular synthesis but it had little or no effect on HL-60 cells. We conclude that although differentiation and apoptosis proceed simultaneously, they can be uncoupled by expression of Bcl-2. Down-regulation of Bcl-2 appears to be part of the differentiation pathway and may serve to facilitate the apoptotic response.     

Involvement of valosin-containing protein, an ATPase Co-purified with IkappaBalpha and 26 S proteasome, in ubiquitin-proteasome-mediated degradation of IkappaBalpha

The inactivation of the prototype NF-kappaB inhibitor, IkappaBalpha, occurs through a series of ordered processes including phosphorylation, ubiquitin conjugation, and proteasome-mediated degradation. We identify valosin-containing protein (VCP), an AAA (ATPases associated with a variety of cellular activities) family member, that co-precipitates with IkappaBalpha immune complexes. The ubiquitinated IkappaBalpha conjugates readily associate with VCP both in vivo and in vitro, and this complex appears dissociated from NF-kappaB. In ultracentrifugation analysis, physically associated VCP and ubiquitinated IkappaBalpha complexes sediment in the 19 S fractions, while the unmodified IkappaBalpha sediments in the 4.5 S fractions deficient in VCP. Phosphorylation and ubiquitination of IkappaBalpha are critical for VCP binding, which in turn is necessary but not sufficient for IkappaBalpha degradation; while the N-terminal domain of IkappaBalpha is required in all three reactions, both N- and C-terminal domains are required in degradation. Further, VCP co-purifies with the 26 S proteasome on two-dimensional gels and co-immunoprecipitates with subunits of the 26 S proteasome. Our results suggest that VCP may provide a physical and functional link between IkappaBalpha and the 26 S proteasome and play an important role in the proteasome-mediated degradation of IkappaBalpha.     

Mtd, a novel Bcl-2 family member activates apoptosis in the absence of heterodimerization with Bcl-2 and Bcl-XL

We have identified and characterized Mtd, a novel regulator of apoptosis. Sequence analysis revealed that Mtd is a member of the Bcl-2 family of proteins containing conserved BH1, BH2, BH3, and BH4 regions and a carboxyl-terminal hydrophobic domain. In adult tissues, Mtd mRNA was predominantly detected in the brain, liver, and lymphoid tissues, while in the embryo Mtd mRNA was detected in the liver, thymus, lung, and intestinal epithelium. Expression of Mtd promoted the death of primary sensory neurons, 293T cells and HeLa cells, indicating that Mtd is a proapoptotic protein. Unlike all other known death agonists of the Bcl-2 family, Mtd did not bind significantly to the survival-promoting proteins Bcl-2 or Bcl-XL. Furthermore, apoptosis induced by Mtd was not inhibited by Bcl-2 or Bcl-XL. A Mtd mutant with glutamine substitutions of highly conserved amino acids in the BH3 domain retained its ability to promote apoptosis, further indicating that Mtd does not promote apoptosis by heterodimerizing with Bcl-2 or Bcl-XL. Mtd-induced apoptosis was not blocked by broad range synthetic caspase inhibitors z-VAD-fmk or a viral protein CrmA. Mtd is the first example of a naturally occurring Bcl-2 family member that can activate apoptosis independently of heterodimerization with survival-promoting Bcl-2 and Bcl-XL.