Differential expression of bcl-2 homologs in human CD34(+) hematopoietic progenitor cells induced to differentiate into erythroid or granulocytic cells

The Bcl-2 family of proteins has been shown to play a central role in the regulation of apoptosis. We have examined the expression of several Bcl-2 homologs upon stimulation of CD34(+) human hematopoietic progenitor cells. CD34(+) cells were induced to differentiate into predominantly erythroid cells in the presence of erythropoietin (Epo) and stem cell factor (SCF), while the addition of G-CSF and SCF led to differentiation predominantly into granulocytic cells, as demonstrated by immunophenotyping and morphological examination of cultured cells. In Epo- and SCF-stimulated cells, we found a marked increase in the level of Bcl-x(L) protein expression and downregulation of Bax expression, apparent from day 4 and more pronounced on days 8 and 21. In contrast, Bcl-x(L) protein expression was downregulated in G-CSF- and SCF-stimulated cells compared with cells cultured in medium alone, whereas there was no sign of change in the level of Bax. Mcl-1 expression showed a biphasic expression pattern in both early erythropoiesis and early granulopoiesis, but with an inverse regulation. Thus, Mcl-1 levels initially decreased in granulocytic progenitor cells and increased in erythroid progenitor cells. Finally, Bcl-2 expression was significantly downregulated in both Epo and SCF and G-CSF- and SCF-stimulated cells. The role of the distinct upregulation of Bcl-x(L) in early erythroid differentiation was further examined by use of specific ribozymes against Bcl-x(L). Addition of Bcl-x(L) ribozymes promoted a clear increase in cell death of Epo- and SCF-stimulated cells, while erythroid differentiation was not affected. In conclusion, we found a distinct regulation of several Bcl-2 family members in CD34(+) cells dependent on the cytokine stimulation given. The use of Bcl-x(L)-specific ribozymes suggested that Bcl-x(L) is important for survival but not for differentiation of erythroid progenitor cells.     

Bcl-x(L) inhibits Bax-induced alterations in mitochondrial respiration and calcium release

Apoptosis is a natural cell elimination process involved in a number of physiological and pathological events. This process can be regulated by members of the Bcl-2 family. Bax, a pro-apoptotic member of this family, accelerates cell death, while the pro-survival member, Bcl-x(L), can antagonize the pro-apoptotic function of Bax to promote cell survival. In the present study, we have evaluated the effect of Bcl-x(L) on Bax-induced alterations in mitochondrial respiration and calcium release. We found that in primary cultured astrocytes, recombinant Bcl-x(L) is able to antagonize Bax-induced decrease in mitochondrial respiration and increase in mitochondrial calcium release. In addition, we found that Bcl-x(L) can lower the calcium store in the endoplasmic reticulum, thus limiting potential calcium flux induced by apoptosis. This regulation of calcium flux by Bcl-x(L) may represent an important mechanism by which this protein promotes cell survival.     

N-terminally cleaved Bcl-xL mediates ischemia-induced neuronal death

Transient global ischemia in rats induces delayed death of hippocampal CA1 neurons. Early events include caspase activation, cleavage of anti-death Bcl-2 family proteins and large mitochondrial channel activity. However, whether these events have a causal role in ischemia-induced neuronal death is unclear. We found that the Bcl-2 and Bcl-x(L) inhibitor ABT-737, which enhances death of tumor cells, protected rats against neuronal death in a clinically relevant model of brain ischemia. Bcl-x(L) is prominently expressed in adult neurons and can be cleaved by caspases to generate a pro-death fragment, ΔN-Bcl-x(L). We found that ABT-737 administered before or after ischemia inhibited ΔN-Bcl-x(L)-induced mitochondrial channel activity and neuronal death. To establish a causal role for ΔN-Bcl-x(L), we generated knock-in mice expressing a caspase-resistant form of Bcl-x(L). The knock-in mice exhibited markedly reduced mitochondrial channel activity and reduced vulnerability to ischemia-induced neuronal death. These findings suggest that truncated Bcl-x(L) could be a potentially important therapeutic target in ischemic brain injury.     

Selectively targeting Mcl-1 for the treatment of acute myelogenous leukemia and solid tumors

Bcl-2, Bcl-x(L), Mcl-1, and A1 are the predominant anti-apoptotic members of the Bcl-2 family in somatic cells. Malignant B lymphocytes are critically dependent on Bcl-2 or Bcl-x(L) for survival. In contrast, a new study by Glaser and colleagues in the January 15, 2012, issue of Genes & Development (pp. 120-125) demonstrates that Mcl-1 is essential for development and survival of acute myelogenous leukemia cells. These results provide new impetus for the generation of selective Mcl-1 inhibitors.     

Anti-apoptotic Mcl-1 is essential for the development and sustained growth of acute myeloid leukemia

Acute myeloid leukemia (AML) frequently relapses after initial treatment. Drug resistance in AML has been attributed to high levels of the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1. Here we report that removal of Mcl-1, but not loss or pharmacological blockade of Bcl-x(L), Bcl-2, or Bcl-w, caused the death of transformed AML and could cure disease in AML-afflicted mice. Enforced expression of selective inhibitors of prosurvival Bcl-2 family members revealed that Mcl-1 is critical for survival of human AML cells. Thus, targeting of Mcl-1 or regulators of its expression may be a useful strategy for the treatment of AML.     

IL-6 up-regulates mcl-1 in human myeloma cells through JAK / STAT rather than ras / MAP kinase pathway

Mcl-1 is an anti-apoptotic member of the Bcl-2 family which is tightly regulated during myeloid and B cell differentiation. We have recently reported that Mcl-1 is expressed in human myeloma cells and that Mcl-1 and Bcl-x(L) expression are correlated. In the current study, we demonstrate that IL-6, a survival factor for the human myeloma cell line MDN, rapidly up-regulates Mcl-1 whereas it has no effect on Bcl-2 protein level. In MDN cells, IL-6 induces both extracellular signal-regulated protein kinase (ERK)1,2 and STAT3 activation whereas STAT1 and STAT5 activation remains undetectable. Furthermore, while investigating the IL-6 signaling pathway leading to Mcl-1 up-regulation, we show that a janus kinase (JAK)-2 inhibitor is able to inhibit both STAT3 activation and Mcl-1 up-regulation whereas an MAP/ERK kinase (MEK) inhibitor has no effect. In conclusion, our data suggest the involvement of the JAK / STAT pathway but not of the Ras / mitogen-activated protein (MAP) kinase pathway in IL-6-induced Mcl-1 up-regulation.     

Lipopolysaccharide induces CXCL2/macrophage inflammatory protein-2 gene expression in enterocytes via NF-kappaB activation: independence from endogenous TNF-alpha and platelet-activating factor

CXCL2 (macrophage inflammatory protein-2 (MIP-2)), a critical chemokine for neutrophils, has been shown to be produced in the rat intestine in response to platelet-activating factor (PAF) and to mediate intestinal inflammation and injury. The intestinal epithelium, constantly exposed to bacterial products, is the first line of defence against micro-organisms. It has been reported that enterocytes produce proinflammatory mediators, including tumour necrosis factor (TNF) and PAF, and we showed that lipopolysaccharide (LPS) and TNF activate nuclear factor (NF)-kappaB in enterocytes. However, it remains elusive whether enterocytes release CXCL2 in response to LPS and TNF via a NF-kappaB-dependent pathway and whether this involves the endogenous production of TNF and PAF. In this study, we found that TNF and LPS markedly induced CXCL2 gene expression in IEC-6 cells, TNF within 30 min, peaking at 45 min, while LPS more slowly, peaking after 2 hr. TNF- and LPS- induced CXCL2 gene expression and protein release were completely blocked by pyrrolidine dithiocarbamate (PDTC) and helenalin, two potent NF-kappaB inhibitors. NEMO-binding domain peptide, a specific inhibitor of inhibitor protein kappaB kinase (IKK) activation, a major upstream kinase mediating NF-kappaB activation, significantly blocked CXCL2 gene expression and protein release induced by LPS. WEB2170 (PAF antagonist) and anti-TNF antibodies had no effect on LPS-induced CXCL2 expression. In conclusion, CXCL2 gene is expressed in enterocytes in response to both TNF and LPS. LPS-induced CXCL2 expression is dependent on NF-kappaB activation via the IKK pathway. The effect of LPS is independent of endogenous TNF and PAF.     

Apigenin, a dietary flavonoid, sensitizes human T cells for activation-induced cell death by inhibiting PKB/Akt and NF-kappaB activation pathway

Resistance of T cells to activation-induced cell death (AICD) is associated with autoimmunity and lymphoproliferation. We found that apigenin (4',5,7-trihydroxyflavone), a non-mutagenic dietary flavonoid, augmented both extrinsic and intrinsic pathways of apoptosis in recurrently activated, but not in primarily stimulated, human blood CD4+ T cells. Apigenin potentiated AICD by inhibiting NF-kappaB activation and suppressing NF-kappaB-regulated anti-apoptotic molecules, cFLIP, Bcl-x(L), Mcl-1, XIAP and IAP, but not Bcl-2. Apigenin suppressed NF-kappaB translocation to nucleus and inhibited IkappaBalpha phosphorylation and degradation in response to TCR stimulation in reactivated peripheral blood CD4 T cells, as well as in leukemic Jurkat T cell lines. Among the pathways that lead to NF-kappaB activation upon TCR stimulation, apigenin selectively inhibited PI3K-PKB/Akt, but not PKC-theta activation in the human T cells, and synergized with a PI3K inhibitor to markedly augment AICD. Apigenin also suppressed expression of anti-apoptotic cyclooxygenase 2 (COX-2) protein in activated human T cells, but it did not affect activation of Erk MAPKinase. Thus, in chronically activated human T cells, relatively non-toxic apigenin can suppress anti-apoptotic pathways involving NF-kappaB activation, and especially cFLIP and COX-2 expression that are important for functioning and maintenance of immune cells in inflammation, autoimmunity and lymphoproliferation.     

Inhibition of IKK down-regulates antigen + IgE-induced TNF production by mast cells: a role for the IKK-IkappaB-NF-kappaB pathway in IgE-dependent mast cell activation

Mast cells (MC) are major effector cells for allergic diseases. Cross-linking of immunoglobulin E (IgE) and its high-affinity receptor, FcepsilonRI, by antigen initiates a cascade of signaling events leading to nuclear factor (NF)-kappaB activation and tumor necrosis factor (TNF) production. Here, we demonstrated that inhibition of inhibitor of kappaB (IkappaB) kinase (IKK) by a peptide IKK inhibitor or by four individual chemical IKK inhibitors including 15-deoxy-prostaglandin J(2), BMS-345541, SC-514, or sulindac significantly blocked IgE + trinitrophenyl (TNP)-induced TNF production by mouse bone marrow-derived MC (BMMC). Moreover, IgE + TNP induced a rapid phosphorylation of IKKalpha but not IKKbeta in BMMC. IgE + TNP-induced phosphorylation of IKKalpha was accompanied with phosphorylation and degradation of IkappaBalpha, subsequent NF-kappaB activation, and TNF production. Inhibition of IKK by sulindac decreased IKKalpha phosphorylation, IkappaBalpha phosphorylation and degradation, NF-kappaB activation, and TNF production by BMMC. It is interesting that IgE + TNP stimulation also induced a prominent synthesis of IKKalpha and IkappaBalpha. Inhibition of NF-kappaB activity by pyrrolidine dithiocarbomate (PDTC) blocked IgE + TNP-induced IkappaBalpha synthesis. NF-kappaB activity and TNF production were also inhibited when PDTC was used even after IgE + TNP stimulation, suggesting a potential role for the newly synthesized IkappaBalpha in MC activation. In addition, IgE + TNP-induced IKKalpha and IkappaBalpha phosphorylation was inhibited by a protein kinase C (PKC) inhibitor Ro 31-8220. Taken together, our results support a role for the IKK-IkappaB-NF-kappaB pathway, which likely involves PKC in IgE-dependent TNF production by MC. Thus, IKK may serve as a new target for the regulation of MC function in allergy.     

The involvement of the apoptosis-modulating proteins ERK 1/2, Bcl-xL and Bax in the resolution of acute inflammation in vivo

Inflammatory cell recruitment, activation, and apoptosis are highly regulated processes involving several checkpoints controlling the resolution of inflammation. We investigated the role of the mitogen-activated protein kinase (MAPK) signaling pathway (ie, ERK1/2) and apoptosis-regulating Bcl-2 family members (ie, Bcl-x(L) and Bax) in the resolution of a rat carrageenan-induced pleurisy model. The specific ERK1/2 inhibitor PD98059 enhanced the resolution of inflammation, whereas the MEK1/2 inhibitor U0126 had no effect and the flavonoid apigenin, a nonspecific inhibitor of ERK1/2 and COX-2, augmented inflammation. Specifically, PD98059 significantly decreased the total number of macrophages and neutrophils in the pleural cavity, mainly by increasing the rate of neutrophil apoptosis, as measured by Annexin V labeling and morphological analysis. Conversely, a specific inhibitor of proapoptotic Bax (V5) increased inflammation, indicating that by preventing apoptosis in vivo, resolution of inflammation is delayed. This was associated with a decrease in neutrophil apoptosis and an increase in macrophage and neutrophil numbers perpetuating the inflammatory response. In conclusion, this study shows that ERK1/2, Bax, and Bcl-x(L) play important functional roles in the resolution phase of the acute inflammatory response in vivo by influencing apoptosis. Importantly, these data may provide novel therapeutic targets for the treatment of inflammatory diseases.     

Anti-rheumatic compound aurothioglucose inhibits tumor necrosis factor-alpha-induced HIV-1 replication in latently infected OM10.1 and Ach2 cells

NF-kappaB is a potent cellular activator of HIV-1 gene expression. Down-regulation of NF-kappaB activation is known to inhibit HIV replication from the latently infected cells. Gold compounds have been effectively used for many decades in the treatment of rheumatoid arthritis. We previously reported that gold compounds, especially aurothioglucose (AuTG) containing monovalent gold ion, inhibited the DNA-binding of NF-kappaB in vitro. In this report we have examined the efficacy of the gold compound AuTG as an inhibitor of HIV replication in latently infected OM10.1 and Ach2 cells. Tumor necrosis factor (TNF)-alpha-induced HIV-1 replication in OM10.1 or Ach2 cells was significantly inhibited by non-cytotoxic doses of AuTG (>10 microM in OM10.1 cells and >25 F.M in Ach2 cells), while 25 microM of the counter-anion thioglucose (TG) or gold compound containing divalent gold ion, HAuCl3, had no effect. The effect of AuTG on NF-kappaB-dependent gene expression was confirmed by a transient CAT assay. Specific staining as well as electron microscopic examinations revealed the accumulation of metal gold in the cells, supporting our previous hypothesis that gold ions could block NF-kappaB-DNA binding by a redox mechanism. These observations indicate that the monovalent gold compound AuTG is a potentially useful drug for the treatment of patients infected with HIV.     

Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins

Commitment of cells to apoptosis is governed largely by the interaction between members of the Bcl-2 protein family. Its three subfamilies have distinct roles: The BH3-only proteins trigger apoptosis by binding via their BH3 domain to prosurvival relatives, while the proapoptotic Bax and Bak have an essential downstream role involving permeabilization of organellar membranes and induction of caspase activation. We have investigated the regulation of Bak and find that, in healthy cells, Bak associates with Mcl-1 and Bcl-x(L) but surprisingly not Bcl-2, Bcl-w, or A1. These interactions require the Bak BH3 domain, which is also necessary for Bak dimerization and killing activity. When cytotoxic signals activate BH3-only proteins that can engage both Mcl-1 and Bcl-x(L) (such as Noxa plus Bad), Bak is displaced and induces cell death. Accordingly, the BH3-only protein Noxa could bind to Mcl-1, displace Bak, and promote Mcl-1 degradation, but Bak-mediated cell death also required neutralization of Bcl-x(L) by other BH3-only proteins. The results indicate that Bak is held in check solely by Mcl-1 and Bcl-x(L) and induces apoptosis only if freed from both. The finding that different prosurvival proteins have selective roles has notable implications for the design of anti-cancer drugs that target the Bcl-2 family.     

CD30 ligation differentially affects CXCR4-dependent HIV-1 replication and soluble CD30 secretion in non-Hodgkin cell lines and in gamma delta T lymphocytes

We studied whether signaling through CD30, a member of the TNF receptor family, affected acute infection with HIV-1, encompassing its entire replicative cycle. Several non-Hodgkin cell lines, targets of CXCR4-dependent (X4) HIV-1 infection, were positive for CD30 expression. CD30 ligation induced up-regulation of viral replication only in certain CD30+ cell lines. Enhancement of X4 virus replication by CD30 engagement inversely correlated with both CD30 surface density and constitutive NF-kappaB activation. Conversely, expression of CD30, but not of other members of the TNF receptor family, was proportional to constitutive NF-kappaB binding. Concomitantly, secretion of soluble (s) CD30 increased in all cell lines by CD30 ligation. sCD30 release was enhanced by engagement of CD30 alone and, to a greater extent, by co-engagement of CD3 also in primary gamma delta T lymphocytes, along with complementary modulations of their surface CD30 expression. sCD30-containing supernatant specifically inhibited HIV-1 expression induced by CD30 engagement in chronically infected ACH-2 T cells; thus sCD30 may act as a negative feed-back molecule. In conclusion, we have delineated novel features of CD30 biology and underline the peculiar link of CD30 expression to constitutive NF-kappaB activation which is pivotal to both HIV replication and cell survival.     

Bax, Bcl-2, and NF-kappaB expression in sanguinarine induced bimodal cell death

The apoptosis related proteins Bax, Bcl-2, and NF-kappaB were analyzed in sanguinarine induced apoptosis and blister cell death (BCD) of K562 erythroleukemia cells and in sanguinarine treated high Bcl-2 expressing JM1 pre-B lymphoblastic cells, utilizing immunofluorescence-flow cytometry. Sanguinarine induced apoptosis of K562 cells was found to have increased Bax expression and decreased NF-kappaB, whereas BCD showed a decrease in Bax expression and an increase in NF-kappaB. In contrast, high Bcl-2 expressing JM1 cells, when exposed to the same concentrations (and duration) of sanguinarine that induced PCD and BCD in K562 cells, failed to show the respective morphologies while showing a concomitant increase in Bcl-2. Results from studies with K562 cells suggest that Bax is pro-apoptotic and also that NF-kappaB activation may be associated with BCD. Results from studies with JM1 cells suggest that Bcl-2 is anti-apoptotic and anti-BCD. Results from JM1 cells strengthen the assumption in the literature of the central role Bcl-2 plays in chemoresistance by assuming an anti-PCD role. These results also suggest that, in JM1 cells, Bcl-2 may further complicate chemoresistance by being anti-BCD in nature, in addition to its anti-PCD role.