Endoplasmic reticulum stress-mediated apoptosis of EBV-transformed B cells by cross-linking of CD70 is dependent upon generation of reactive oxygen species and activation of p38 MAPK and JNK pathway

CD70 is expressed in normal activated immune cells as well as in several types of tumors. It has been established that anti-CD70 mAb induces complement-dependent death of CD70(+) tumor cells, but how anti-CD70 mAb affects the intrinsic signaling is poorly defined. In this report, we show that ligation of CD70 expressed on EBV-transformed B cells using anti-CD70 mAb induced production of reactive oxygen species (ROS) and subsequent apoptosis. We observed an early expression of endoplasmic reticulum (ER) stress response genes that preceded the release of apoptotic molecules from the mitochondria and the cleavage of caspases. CD70-induced apoptosis was inhibited by pretreatment with the ER stress inhibitor salubrinal, ROS quencher N-acetylcysteine, and Ca(2+) chelator BAPTA. We supposed that ROS generation might be the first event of CD70-induced apoptosis because N-acetylcysteine blocked increases of ROS and Ca(2+), but BAPTA did not block ROS generation. We also found that CD70 stimulation activated JNK and p38 MAPK. JNK inhibitor SP600125 and p38 inhibitor SB203580 effectively blocked upregulation of ER stress-related genes and cleavage of caspases. Inhibition of ROS generation completely blocked phosphorylation of JNK and p38 MAPK and induction of ER stress-related genes. Taken together, we concluded that cross-linking of CD70 on EBV-transformed B cells triggered ER stress-mediated apoptosis via ROS generation and JNK and p38 MAPK pathway activation. Our report reveals alternate mechanisms of direct apoptosis through CD70 signaling and provides data supporting CD70 as a viable target for an Ab-based therapy against EBV-related tumors.     

Galectin-9 induces maturation of human monocyte-derived dendritic cells

Maturation of dendritic cells (DCs) is critical for initiation of immune responses and is regulated by various stimulatory signals. We assessed the role of galectin (Gal)-9 in DC maturation. Culture of immature DCs with exogenous Gal-9 markedly increased the surface expression of CD40, CD54, CD80, CD83, CD86, and HLA-DR in a dose-dependent manner, although Gal-9 had no or little effect on differentiation of human monocytes into immature DCs. Gal-9-treated DCs secreted IL-12 but not IL-10, and they elicited the production of Th1 cytokines (IFN-gamma and IL-2) but not that of the Th2 cytokines (IL-4 and IL-5) by allogeneic CD4+ T cells. These effects of Gal-9 on immature DCs were not essentially dependent on its lectin properties, given that they were inhibited only slightly by lactose. We further found that a Gal-9 mutant that lacks beta-galactoside binding activity reproduced the above activities and that an anti-Gal-9 mAb suppressed them. Gal-9 induced phosphorylation of the MAPK p38 and ERK1/2 in DCs, and an inhibitor of p38 signaling, but not inhibitors of signaling by either ERK1/2 or PI3K, blocked Gal-9-induced up-regulation of costimulatory molecule expression and IL-12 production. These findings suggest that Gal-9 plays a role not only in innate immunity but also in acquired immunity by inducing DC maturation and promoting Th1 immune responses.     

Lead and calcium produce rod photoreceptor cell apoptosis by opening the mitochondrial permeability transition pore

Calcium overload is suggested to play a fundamental role in the process of rod apoptosis in chemical-induced and inherited retinal degenerations. However, this hypothesis has not been tested directly. We developed an in vitro model utilizing isolated rat retinas to determine the mechanisms underlying Ca(2+)- and/or Pb(2+)-induced retinal degeneration. Confocal microscopy, histological, and biochemical studies established that the elevated [Ca(2+)] and/or [Pb(2+)] were localized to photoreceptors and produced rod-selective apoptosis. Ca(2+) and/or Pb(2+) induced mitochondrial depolarization, swelling, and cytochrome c release. Subsequently caspase-9 and caspase-3 were sequentially activated. Caspase-7 and caspase-8 were not activated. The effects of Ca(2+) and Pb(2+) were additive and blocked completely by the mitochondrial permeability transition pore (PTP) inhibitor cyclosporin A, whereas the calcineurin inhibitor FK506 had no effect. The caspase inhibitors carbobenzoxy-Leu-Glu-His-Asp-CH(2)F and carbobenzoxy-Asp-Glu-Val-Asp-CH(2)F, but not carbobenzoxy-Ile-Glu-Thr-Asp-CH(2)F, differentially blocked post-mitochondrial events. The levels of reduced and oxidized glutathione and pyridine nucleotides in rods were unchanged. Our results demonstrate that rod mitochondria are the target site for Ca(2+) and Pb(2+). Moreover, they suggest that Ca(2+) and Pb(2+) bind to the internal metal (Me(2+)) binding site of the PTP and subsequently open the PTP, which initiates the cytochrome c-caspase cascade of apoptosis in rods.     

Calcium-induced calpain mediates apoptosis via caspase-3 in a mouse photoreceptor cell line

The rd mouse, an accepted animal model for photoreceptor degeneration in retinitis pigmentosa, has a recessive mutation for the gene encoding the beta-subunit of the cGMP phosphodiesterase. This mutation results in high levels of cGMP, which leaves an increased number of the cGMP-gated channels in the open state, thus allowing intracellular calcium (Ca(2+)) to rise to toxic levels, and rapid photoreceptor degeneration follows. To delineate the events in rd photoreceptor degeneration, we demonstrated an increase in calpain and caspase-3 activity, hypothesizing that Ca(2+)-mediated apoptosis in photoreceptors is mediated by calpain, involving mitochondrial depolarization and caspase-3 activation. To examine this hypothesis further, a murine photoreceptor-derived cell line (661W) was treated with the Ca(2+) ionophore A23187, cGMP-gated channel agonist 8-bromo-cGMP, or phosphodiesterase inhibitor isobutylmethylxanthine to mimic the increased Ca(2+) influx seen in the rd photoreceptors. Ca(2+)-induced cell death in 661W cells was found to be mediated by calpain and caspase-3 and could be completely inhibited by the calpain inhibitor SJA6017, implicating both calpain and caspases in the apoptotic process. The apoptotic events correlated in an SJA6017-inhibitable manner with bid cleavage, mitochondrial depolarization, cytochrome c release, and caspase-3 and -9 activation. We concluded that Ca(2+) influx in the rd model of photoreceptor degeneration leads to the activation of the cysteine protease calpain, which executes apoptosis via modulation of caspase-3 activity.     

Mechanism of endothelin-1-induced cytosolic Ca(2+) mobility in cultured H9c2 myocardiac ventricular cells

The effect of endothelin-1 (ET-1) on the intracellular free Ca(2+) ([Ca(2+)](i)) mobility in cultured H9c2 myocardiac ventricular cells was studied after loading with fura-2-AM. In Ca(2+)-containing buffer, ET-1 induced [Ca(2+)](i) rise from 10(-7) to 10(-9) M. ET-1 induced [Ca(2+)](i), which was composed of a first small peak and a secondary persistent plateau. In Ca(2+)-free buffer, pretreatment with 10(-7) M ET-1 inhibited the thapsigargin and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced [Ca(2+)](i) increase. Meanwhile, pretreatment with thapsigargin and CCCP also inhibited ET-1-induced [Ca(2+)](i) rise. In Ca(2+)-containing buffer, the ET(A) receptor antagonist (BQ123) completely abolished the secondary rising peak and plateau. Conversely, the ET(B) receptor antagonist (BQ788) completely inhibited the first small peak and secondary peak plateau. Nifedipine and La(3+) also abolished the 10(-7) M ET-1-induced [Ca(2+)](i) in the first rising peak. The internal Ca(2+) release induced by ET-1 was inhibited by U73122 (phospholipase C inhibitor), propranolol (phospholipase D inhibitor) and aristolochic acid (phospholipase A2 inhibitor). After incubation of 10(-7) M ET-1 in Ca(2+)-free buffer, the addition of 5 mM CaCl(2) increased Ca(2+) influx, implying that release of Ca(2+) from internal stores further induces capacitative Ca(2+) entry. Taken together, these results suggest that both ET(A) and ET(B) receptors are involved in ET-1-induced [Ca(2+)](i) rise in H9c2 myocardiac ventricular cells. Whereas ET(B) receptor seems to mediate the initial Ca(2+) influx via L-type Ca(2+) channel, ET(A) receptor appears to be involved in the subsequent Ca(2+) release from endoplasmic reticulum and mitochondria Ca(2+) stores.     

Caspase-independent phosphatidylserine exposure during apoptosis of primary T lymphocytes

Exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane is a key feature of apoptosis. As the signals underlying these phenomena are unknown, it is generally assumed that PS exposure is a consequence of caspase activation, another hallmark of apoptosis. In this study we investigated the role of caspases in PS externalization during apoptosis of activated PBL triggered by drugs (etoposide, staurosporine), CD95 engagement, or IL-2 withdrawal. Anti-CD95 mAb induces a rapid activation of caspases, followed by PS exposure and mitochondrial transmembrane potential (DeltaPsim) disruption. In contrast, etoposide (ETO), staurosporine (STS), or IL-2 withdrawal triggers concomitant caspase activation, PS exposure, and DeltaPsim disruption. Such kinetics suggest that PS exposure could be independent of caspase activation. As expected, in activated PBL treated by anti-CD95 mAb, the pan-caspase inhibitor Cbz-Val-Ala-Asp(OMe)-fluoromethylketone and the caspase-8 inhibitor Cbz-Leu-Glu-Thr-Asp(OMe)-fluoromethylketone, but not the caspase-9 inhibitor Cbz-Leu-Glu-His-Asp(OMe)-fluoromethylketone, inhibit PS externalization and DeltaPsim disruption. Surprisingly, during apoptosis induced by ETO, STS, or IL-2 withdrawal, none of those caspase inhibitors prevents PS externalization or DeltaPsim disruption, whereas they all inhibit DNA fragmentation as well as the morphological features of nuclear apoptosis. In Jurkat and H9 T cell lines, as opposed to activated PBL, PS exposure is inhibited by Cbz-Val-Ala-Asp(OMe)-fluoromethylketone during apoptosis induced by CD95 engagement, ETO, or STS. Thus, caspase-independent PS exposure occurs in primary T cells during apoptosis induced by stimuli that do not trigger death receptors.     

hsBAFF regulates proliferation and response in cultured CD4(+) T lymphocytes by upregulation of intracellular free Ca(2+) homeostasis

B cell activating factor belonging to the TNF family (BAFF, also called BLyS, TALL-1, THANK, or zTNF4) is an important survival factor for B cells, and is able to regulate T-cell activation. Recently, we have demonstrated that treatment of mice with human soluble BAFF (hsBAFF) causes a significant increase of percentages of splenic CD4(+) T lymphocytes dose-dependently, but the CD8(+) T lymphocyte percentages maintained unchanged. Here, we show that hsBAFF significantly enhanced CD4(+) T lymphocyte response of cultured mouse splenic cells, and hsBAFF induced the proliferation and IL-2/IFN-γ secretion of purified CD4(+) T lymphocytes suboptimally stimulated through anti-CD3. Of importance, we observed that IL-2 or IFN-γ cytokine has additive effect on the proliferation and activity of hsBAFF-stimulated CD4(+) T lymphocytes. Using Flow cytometry with fluorescent probe, Fluo-3/AM, we found that hsBAFF elicited [Ca(2+)](i) elevation contributing to CD4(+) T cell proliferation. This is evidenced by our finding that pretreatment with BAPTA/AM, an intracellular Ca(2+) chelator, significantly attenuated the proliferation of hsBAFF-stimulated CD4(+) T lymphocytes. Subsequently, we revealed that hsBAFF-stimulated CD4(+) T cell proliferation was markedly suppressed after pretreatment with EGTA, an extracellular Ca(2+) chelator, or with 2-APB, an inhibitor of Ca(2+) influx through CRAC channels, respectively, suggesting that extracellular Ca(2+) influx due to hsBAFF is closely associated with [Ca(2+)](i) elevation contributing to CD4(+) T cell proliferation. In addition, we noticed that hsBAFF-treated cells conferred partial resistance to decrease of cellular viability induced by thapsigargin (Tg), an endoplasmic reticulum (ER) Ca(2+)-ATPase inhibitor. Taken together, our data indicate that hsBAFF may promote CD4(+) T cell proliferation and response by upregulation of [Ca(2+)](i) homeostasis.     

IL-12 provides proliferation and survival signals to murine CD4+ T cells through phosphatidylinositol 3-kinase/Akt signaling pathway

IL-12 is a pleiotropic cytokine that plays an important role in innate and adaptive immunity. IL-12 induces T cell proliferation and IFN-gamma secretion from activated T cells. It was also reported that IL-12 prevents apoptosis of CD4(+) T cells. However, the signaling mechanism that regulates these IL-12-induced responses is poorly understood yet. In this study, we demonstrated that IL-12 activates phosphatidylinositol 3-kinase (PI3K)/Akt pathway in murine CD4(+) T cells, and that this signaling pathway is required for IL-12-induced T cell proliferation and antiapoptotic function, but not for IFN-gamma induction. Through PI3K/Akt pathway, IL-12 up-regulates the expression of cell cycle-related molecule such as cyclin D3, and antiapoptotic molecules such as Bcl-2 and cellular inhibitors of apoptosis proteins-2, followed by down-regulation of active caspase-3. These results suggest that PI3K/Akt pathway is critical for mediating IL-12-induced CD4(+) T cell responses such as T cell proliferation and survival.     

Simultaneously targeting mitochondria and endoplasmic reticulum by photodynamic therapy induces apoptosis in human lymphoma cells

Photodynamic therapy (PDT) and photodetection with protoporphyrin IX (PpIX) precursors have widely been used in the diseases with abnormally proliferative cells, but the mechanism of the modality is not fully understood yet. In this study 70-95% of apoptotic cells after PDT with PpIX precursor, hexaminolevulinate (HAL) in two human lymphoma cell lines, Namalwa and Bjab, were confirmed by fluorescence microscopy, electron microscopy and flow cytometry. HAL-derived PpIX was mainly distributed in the mitochondria and endoplasmic reticulum (ER), both of which were initial targets after light exposure causing two major pathways simultaneously involved in the apoptotic induction. One was the mitochondrial pathway including the release of cytochrome c, cleavage of caspases-9/-3, poly(ADP-ribose) polymerase and DNA fragmentation factor. The other was the ER stress-mediated pathway triggering a transient increase in the cytosolic Ca(2+) level after photodamage to the ER calcium pump protein SERCA2. The released Ca(2+) further initiated the caspase-8 cleavage. The use of both extracellular Ca(2+) chelator EGTA and intracellular Ca(2+) chelator BAPTA-AM confirmed that such cytosolic Ca(2+) originated from the ER rather than extracellular Ca(2+)-containing medium. About 30% of the apoptosis was blocked with BAPTA-AM alone; while a complete inhibition of such apoptosis was achieved with a combination of the caspase-9 inhibitor Z-LEHD-FMK and caspase-8 inhibitor Z-IETD-FMK, thus quantifying each role of the mitochondrial and ER pathways.     

Calcium signaling in cancer and vitamin D

Calcium signals induced by the Ca(2+) regulatory hormone 1,25(OH)(2)D(3) may determine the fate of the cancer cell. We have shown that, in breast cancer cell lines, 1,25(OH)(2)D(3) induces a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) by depleting the endoplasmic reticulum (ER) Ca(2+) stores via inositol 1,4,5-trisphosphate receptor/Ca(2+) release channel and activating Ca(2+) entry from the extracellular space via voltage-insensitive Ca(2+) channels. In normal cells, 1,25(OH)(2)D(3) triggered a transient Ca(2+) response via activation of voltage-dependent Ca(2+) channels, which were absent in breast cancer cells. The normal cells, but not breast cancer cells, expressed the Ca(2+) binding/buffering protein calbindin-D(28k) and were capable of buffering [Ca(2+)](i) increases induced by a mobilizer of the ER Ca(2+) stores, thapsigargin, or a Ca(2+) ionophore, ionomycin. The 1,25(OH)(2)D(3)-induced sustained increase in [Ca(2+)](i) in breast cancer cells was associated with induction of apoptotic cell death, whereas the transient [Ca(2+)](i) increase in normal cells was not. The forced expression of calbindin-D(28k) in cytosol or increase in the cytosolic Ca(2+) buffering capacity with the cell-permeant Ca(2+) buffer BAPTA prevented induction of apoptosis with 1,25(OH)(2)D(3) in cancer cells. The sustained increase in [Ca(2+)](i) in breast cancer cells was associated with activation of the Ca(2+)-dependent apoptotic proteases, mu-calpain and caspase-12, as evaluated with antibodies to active (cleaved) forms of the enzymes and the fluorogenic peptide substrates. Selective inhibition of the Ca(2+) binding sites of mu-calpain decreased apoptotic indices in the cancer cells treated with 1,25(OH)(2)D(3), thapsigargin, or ionomycin. The mu-calpain activation preceded expression/activation of caspase-12, and calpain was required for activation/cleavage of caspase-12. Certain non-calcemic vitamin D analogs (e.g., EB 1089) triggered a sustained [Ca(2+)](i) increase, activated Ca(2+)-dependent apoptotic proteases, and induced apoptosis in breast cancer cells in a fashion similar to that of 1,25(OH)(2)D(3). The 1,25(OH)(2)D(3)-induced transient Ca(2+) response in normal mammary epithelial cells was not accompanied by activation of mu-calpain and caspase-12. In conclusion, we have identified the novel apoptotic pathway in breast carcinoma cells treated with 1,25(OH)(2)D(3): increase in [Ca(2+)](i)-->mu-calpain activation-->caspase-12 activation-->apoptosis. Our results support the hypothesis that 1,25(OH)(2)D(3) directly activates this apoptotic pathway by inducing a sustained increase in [Ca(2+)](i). Differences of Ca(2+) regulatory mechanisms in cancer versus normal cells seem to allow 1,25(OH)(2)D(3) and vitamin D analogs to induce Ca(2+)-mediated apoptosis selectively in breast cancer cells. Thus, deltanoids may prove to be useful in the treatment of tumors susceptible to induction of Ca(2+)-mediated apoptosis.     

Galectin-1 induced activation of the apoptotic death-receptor pathway in human Jurkat T lymphocytes

Galectin-1 (gal-1), a member of the family of β-galactoside binding proteins, participates in several biological processes such as immunomodulation, cell adhesion, regulation of cell growth and apoptosis. The aim of this study was to investigate whether gal-1 interferes with the Fas (Apo-1/CD95)-associated apoptosis cascade in the T-cell lines Jurkat and MOLT-4. Gal-1 and an Apo-1 monoclonal antibody (mAb) induced DNA-fragmentation in Jurkat T-cells whereas MOLT-4 cells were resistant. Gal-1 stimulated DNA-fragmentation could be efficiently inhibited by caspase-8 inhibitor II (Z-IETD-FMK) and a neutralizing Fas mAb. Fas could be identified as a target for gal-1 recognition as demonstrated by immunofluorescence staining, binding of the receptor glycoprotein to immobilized gal-1 and analyses by immunoblotting as well as by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gal-1 stimulates the activation and proteolytic processing of procaspase-8 and downstream procaspase-3 in Jurkat-T cells. Inhibition of gal-1 induced procaspase-8 activation by a neutralizing Fas mAb strongly suggests that gal-1 recognition of Fas is associated with caspase-8 activation. Our data provide the first experimental evidence for targeting of gal-1 to glycotopes on Fas and the subsequent activation of the apoptotic death-receptor pathway.     

Characterization of galectin-9-induced death of Jurkat T cells

Galectin-9, a mammalian lectin with affinity for beta-galactosides, is known as an apoptosis inducer of activated T lymphocytes. In the present study, we examined the properties of galectin-9-mediated cell death of Jurkat T cells. Galectin-9NC (wild-type), consisting of two CRDs (N-terminal and C-terminal carbohydrate recognition domains), and derivatives of it, galectins-9-NN and -9-CC, induced Jurkat T-cell apoptosis. However, a single CRD (galectin-9NT or -CT) had no effect, suggesting the stable dimeric structure of two CRDs is required for the activity. The apoptosis was inhibited by pretreatment with an N-glycan synthesis inhibitor, indicating that the expression of N-glycans in the cells is essential for galectin-9-induced apoptosis. We previously showed that the apoptosis of MOLT-4 cell is mediated by galectin-9 via a Ca(2+)-calpain-caspase-1-dependent pathway. In Jurkat cells, the cell death by galectin-9, was insufficiently suppressed by caspase inhibitors, Ca(2+)-chelator or calpain inhibitor. Furthermore, we observed the loss of mitochondrial membrane potential and significant AIF release in galectin-9-treated cells. These findings suggest that caspase-dependent and-independent death pathways exist in Jurkat cells, and the main pathway might vary with the T-cell type.     

Ce(4+) induced down-regulation of ERK-like MAPK and activation of nucleases during the apoptosis of cultured Taxus cuspidata cells

Ce(4+) (Ce(NH(4))(2)(NO(3))(6)) at 1mM induces apoptosis of suspension cultures of Taxus cuspidata cells; however, the underlying signal mechanisms are unknown. We show here that a 46-kDa ERK (extracellular signal-regulated kinase)-like MAPK appears to be down-regulated at 4h, and remains at low levels for up to 48 h. An inhibitor of superoxide anions (O(2)(-)) generation, diphenyl iodonium (DPI) successfully blocks down-regulation of ERK-like MAPK and degradation of DNA. Moreover, a 41-kDa p38-like MAPK activity remains unchanged from 0.5 to 48 h. The p38 inhibitor SB202190 effectively inhibits p38-like MAPK activity, however, SB202190 fails to modify the apoptotic rate at concentrations up to 100 microM. Three nuclease (34-kDa, 22-kDa and 20-kDa) activities are profoundly enhanced in Ce(4+)-induced T. cuspidata cells. They have an optimum pH at 6.8, and are stimulated by Ca(2+)/Mg(2+). Caspase-3 inhibitor, Ac-DEVD-CHO, does not attenuate the 34-kDa nuclease activity, but inhibits the 22-kDa and the 20-kDa nuclease activities. In addition, inhibition of O(2)(-) generation by DPI significantly reduces the three nuclease activities. In conclusion, the present study suggests that down-regulation of ERK-like MAPK, burst of O(2)(-), activation of caspase-3-like and induction of three nucleases as the key signaling events mediating apoptosis in Ce(4+)-induced cultured T. cuspidata cells.     

p38 MAPK and MSK1 mediate caspase-8 activation in manganese-induced mitochondria-dependent cell death

Heavy metals are important regulators of cell apoptosis. Manganese (Mn(2+)) is a potent inducer of apoptosis in different cell types, but the precise mechanisms that mediate such effects are not well defined. We previously reported that Mn(2+) was a potent apoptotic agent in human B cells, including lymphoma B cell lines. We show here that Mn(2+)-induced cell death in human B cells is associated with caspase-8-dependent mitochondrial activation leading to caspase-3 activity and apoptosis. We used specific caspase-8 interfering shRNAs to reduce caspase-8 expression, and this also reduced Mn(2+)-induced caspase-3 activation and apoptosis. Mn(2+)-triggered caspase-8 activation is associated with a specific pathway, which is independent of Fas-associated death domain protein, and dependent on the sequential activation of p38-mitogen-activated protein kinase (p38 MAPK) and mitogen- and stress-response kinase 1 (MSK1). Inhibition of p38 activity using either pharmacological inhibitors or dominant-negative mutant forms of p38 blocked Mn(2+)-mediated phosphorylation of MSK1 and blocked subsequent caspase-8 activation. However, specific inhibitors and the expression of a dominant-interfering mutant of MSK1 only inhibited caspase-8 activation, but not p38 activity. These findings suggest a novel model for the regulation of caspase-8 during Mn(2+)-induced apoptosis based on the sequential activation of p38 MAPK, MSK1, caspase-8 and mitochondria, respectively.     

Galectin-9 Enhances Cytokine Secretion,but Suppresses Survival and Degranulation,in Human Mast Cell Line

Galectin-9 (Gal-9), a lectin having a β-galactoside-binding domain, can induce apoptosis of Th1 cells by binding to TIM-3. In addition, Gal-9 inhibits IgE/Ag-mediated degranulation of mast cell/basophilic cell lines by binding to IgE, thus blocking IgE/Ag complex formation. However, the role of Gal-9 in mast cell function in the absence of IgE is not fully understood. Here, we found that recombinant Gal-9 directly induced phosphorylation of Erk1/2 but not p38 MAPK in a human mast cell line, HMC-1, which does not express FcεRI. Gal-9 induced apoptosis and inhibited PMA/ionomycin-mediated degranulation of HMC-1 cells. On the other hand, Gal-9 induced cytokine and/or chemokine production by HMC-1 cells, dependent on activation of ERK1/2 but not p38 MAPK. In addition, the lectin activity of Gal-9 was required for Gal-9-mediated cytokine secretion by HMC-1 cells. These observations suggest that Gal-9 has dual properties as both a regulator and an activator of mast cells.     

Both calcium and ROS as common signals mediate Na(2)SeO(3)-induced apoptosis in SW480 human colonic carcinoma cells

Recent studies have shown that reactive oxygen species (ROS) play a crucial role in Se-induced cell apoptosis. A number of studies have demonstrated that perturbed cellular calcium homeostasis has been implicated in apoptosis. The main objective of this study was to evaluate the role of Ca(2+) in Na(2)SeO(3)-induced apoptosis and the relationship between Ca(2+) and ROS in human colonic carcinoma cells SW480. When SW480 cells were exposed to 25-100 microM Na(2)SeO(3), both cell apoptosis and growth inhibition were observed by flow cytometric analysis and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Na(2)SeO(3) was able to induce increase of [Ca(2+)](i) and ROS production and disrupt mitochondrial membrane potential (Delta Psi m) in SW480 cells monitored by using a confocal laser scanning microscope. Ca(2+) channel inhibitor CoCl(2) and an intracellular Ca(2+) chelator o-phtalaldehyde, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA) completely inhibited [Ca(2+)](i) increase, but catalase had no effect on Na(2)SeO(3)-induced increase of [Ca(2+)](i). BAPTA-AM, CoCl(2), and mitochondrial Ca(2+) uptake inhibitor ruthenium red blocked Delta Psi m dissipation. The increase of ROS was also suppressed by CoCl(2), BAPTA, ruthenium red, N-acetylcysteine and catalase, respectively. The mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) completely inhibited Na(2)SeO(3)-induced ROS increase. This showed that ROS increase is due to mitochondrial Ca(2+) overload. The Na(2)SeO(3)-induced apoptosis of SW480 cells was also inhibited by CoCl(2), BAPTA, ruthenium red, N-acetylcysteine, and catalase, respectively. The results mentioned above imply that both calcium and Ca(2+)-dependent ROS as a signal molecule mediate apoptosis induced by Na(2)SeO(3) in SW480 cells.     

The Na+/H+ exchanger controls deoxycholic acid-induced apoptosis by a H+-activated, Na+-dependent ionic shift in esophageal cells

Apoptosis resistance is a hallmark of cancer cells. Typically, bile acids induce apoptosis. However during gastrointestinal (GI) tumorigenesis the cancer cells develop resistance to bile acid-induced cell death. To understand how bile acids induce apoptosis resistance we first need to identify the molecular pathways that initiate apoptosis in response to bile acid exposure. In this study we examined the mechanism of deoxycholic acid (DCA)-induced apoptosis, specifically the role of Na(+)/H(+) exchanger (NHE) and Na(+) influx in esophageal cells. In vitro studies revealed that the exposure of esophageal cells (JH-EsoAd1, CP-A) to DCA (0.2 mM-0.5 mM) caused lysosomal membrane perturbation and transient cytoplasmic acidification. Fluorescence microscopy in conjunction with atomic absorption spectrophotometry demonstrated that this effect on lysosomes correlated with influx of Na(+), subsequent loss of intracellular K(+), an increase of Ca(2+) and apoptosis. However, ethylisopropyl-amiloride (EIPA), a selective inhibitor of NHE, prevented Na(+), K(+) and Ca(2+) changes and caspase 3/7 activation induced by DCA. Ouabain and amphotericin B, two drugs that increase intracellular Na(+) levels, induced similar changes as DCA (ion imbalance, caspase3/7 activation). On the contrary, DCA-induced cell death was inhibited by medium with low a Na(+) concentrations. In the same experiments, we exposed rat ileum ex-vivo to DCA with or without EIPA. Severe tissue damage and caspase-3 activation was observed after DCA treatment, but EIPA almost fully prevented this response. In summary, NHE-mediated Na(+) influx is a critical step leading to DCA-induced apoptosis. Cells tolerate acidification but evade DCA-induced apoptosis if NHE is inhibited. Our data suggests that suppression of NHE by endogenous or exogenous inhibitors may lead to apoptosis resistance during GI tumorigenesis.     

Expression of the ryanodine receptor isoforms in immune cells.

Ryanodine receptor (RYR) is a Ca(2+) channel that mediates Ca(2+) release from intracellular stores. We have used RT-PCR analysis and examined its expression in primary peripheral mononuclear cells (PBMCs) and in 164 hemopoietic cell lines. In PBMCs, type 1 RYR (RYR1) was expressed in CD19(+) B lymphocytes, but less frequently in CD3(+) T lymphocytes and in CD14(+) monocytes. Type 2 RYR (RYR2) was mainly detected in CD3(+) T cells. Induction of RYR1 and/or RYR2 mRNA was found after treatment with stromal cell-derived factor 1, macrophage-inflammatory protein-1alpha (MIP1alpha) or TGF-beta. Type 3 RYR (RYR3) was not detected in PBMCs. Many hemopoietic cell lines expressed not only RYR1 or RYR2 but also RYR3. The expression of the isoforms was not associated with specific cell lineage. We showed that the RYR-stimulating agent 4-chloro-m-cresol (4CmC) induced Ca(2+) release and thereby confirmed functional expression of the RYR in the cell lines expressing RYR mRNA. Moreover, concordant induction of RYR mRNA with Ca(2+) channel function was found in Jurkat T cells. In untreated Jurkat T cells, 4CmC (>1 mM) had no effect on Ca(2+) release, whereas 4CmC (<400 microM) caused Ca(2+) release after the induction of RYR2 and RYR3 that occurred after treatment with stromal cell-derived factor 1, macrophage-inflammatory protein-1alpha, or TGF-beta. Our results demonstrate expression of all three isoforms of RYR mRNA in hemopoietic cells. Induction of RYRs in response to chemokines and TGF-beta suggests roles in regulating Ca(2+)-mediated cellular responses during the immune response.