To observe the effect of solanine on the membrane potential of mitochondria in HepG(2) cells and [Ca(2+)](i) in the cells, and to uncover the mechanism by which solanine induces apoptosis.

HepG(2) cells were double stained with AO/EB, and morphological changes of the cells were observed using laser confocal scanning microscopy (LCSM). HepG(2) cells were stained with TMRE, and change in the membrane potential of mitochondria in the cells were observed using LCSM. HepG(2) cells were double stained with Fluo-3/AM, and change of [Ca(2+)](i) in the cells were observed using LCSM. HepG(2) cells were double stained with TMRE and Fluo-3/AM, and both the change in membrane potential of mitochondria and that of [Ca(2+)](i) in the cells were observed using LCSM.

Cells in treated groups showed typical signs of apoptosis. Staining with TMRE showed that solanine could lower membrane potential; staining with Fluo-3/AM showed that solanine could increase the concentration of Ca(2+) in tumor cells; and those of double staining with TMRE and Fluo-3/AM showed that solanine could increase the concentration of Ca(2+) in the cells at the same time as it lowered the membrane potential of mitochondria.

Solanine opens up the PT channels in the membrane by lowering the membrane po-tential, leading to Ca(2+) being transported down its concentration gradient, which in turn leads to the rise of the concentration of Ca(2+) in the cell, turning on the mechanism for apoptosis.

Osteoclasts are multinucleated cells responsible for bone resorption and play important roles in normal skeletal development, in the maintenance of its integrity throughout life, and in calcium metabolism. During bone resorption, the cytoskeleton of osteoclasts undergoes extensive reorganization, with polarization and formation of ruffled borders to secrete acid and formation of sealing zone to prevent leakage. The differentiation and function of osteoclasts are in turn regulated by osteoblasts, stromal cells, and bone. They are also subjected to negative feedback regulation by extracellular and intracellular calcium concentrations.

Interleukin-3 (IL-3)-dependent myeloid progenitor cell FDC.P2 is induced to undergo apoptotic cell death upon IL-3 depletion. Extracellular adenosine triphosphate (ATP) was found to prevent apoptosis and maintain cell viability of FDC.P2 cells upon IL-3 withdrawal. The antiapoptotic effect of ATP required extracellular Ca2+. Furthermore, FK506, a specific inhibitor of calcium/calmodulin-dependent protein phosphatase calcineurin, inhibited the antiapoptotic effect of ATP. As one of cytokines whose expression is dependent on the activation of calcineurin, interleukin-4 (IL-4) played a critical role in ATP-mediated cell survival of FDC.P2 cells because neutralizing antibody against IL-4 effectively abrogated the antiapoptotic activity of ATP. Moreover, ATP treatment induced a significant amount of secreted IL-4 that was sufficient to maintain cell viability. Taken together, our present results demonstrate that extracellular ATP triggers autocrine production of IL-4 through calcium-dependent activation of calcineurin and secreted IL-4 substitutes IL-3 in protecting FDC.P2 cells from apoptosis even in the absence of IL-3.

Survival and proliferation of cells of a human myelo-erythroid CD34+ leukemia cell line (TF-1) depend on the presence of granulocyte-macrophage colony-stimulating factor or interleukin-3. Upon hormone withdrawal these cells stop proliferating and undergo apoptotic process. In this report we demonstrate that a controlled increase in [Ca2+]i induces hormone-independent survival and proliferation of TF-1 cells. We found that moderate elevation of [Ca2+]i by the addition of cyclopiasonic-acid protected TF1 cells from apoptosis. Furthermore, a higher, but transient elevation of [Ca2+]i by ionomycin treatment induced cell proliferation. In both cases caspase-3 activity was reduced, and Bcl-2 was up-regulated. Higher elevation of [Ca2+]i by ionomycin induced MEK-dependent biphasic ERK1/2 activation, sufficient to move the cells from G0/G1 to S/M phases. Meanwhile, activation of ERK1/2, phosphorylation of the Elk-1 transcription factor, and, consequently, a substantial elevation of Egr-1 and c-Fos levels and AP-1 DNA binding were observed. Moderate elevation of [Ca2+]i, on the other hand, caused a delayed monophasic activation of ERK1/2 and Elk-1 that was accompanied with only a small increase of Egr-1 and c-Fos levels and AP-1 DNA binding. The specific MEK-1 kinase inhibitor, PD98059, inhibited all the effects of increasing [Ca2+]i, indicating that the MAPK/ERK pathway activation is essential for TF-1 cell survival and proliferation. Based on these results we suggest that the elevation of the [Ca2+]i may influence the cytokine dependence of hemopoietic progenitors and may contribute to pathological hematopoiesis.

Jurkat cells undergo apoptosis in response to anti-Fas antibody through a caspase-dependent death cascade in which calcium signaling has been implicated. We have now evaluated the role of calcium during this death cascade at the single cell level in real time utilizing flow cytometric analysis and confocal microscopy. Fluo-3 and propidium iodide were employed to evaluate calcium fluxes and to discriminate between viable and non-viable cells, respectively. Anti-Fas treatment of Jurkat cells resulted in a sustained increase in intracellular calcium commencing between 1 and 2 h after treatment and persisting until subsequent loss of cell membrane integrity. The significance of this rise in calcium was evaluated by buffering intracellular calcium with BAPTA and/or removing calcium from the extracellular medium and monitoring the effects of these manipulations on calcium signaling and components of the apoptotic process. Complete inhibition of the anti-Fas induced rise in intracellular calcium required both chelation of [Ca(2+)](i) and removal of extracellular calcium. Interestingly, this condition did not abrogate several events in Fas-induced apoptosis including cell shrinkage, mitochondrial depolarization, annexin binding, caspase activation, and nuclear poly(A)DP-ribose polymerase cleavage. Furthermore, calcium-free conditions in the absence of anti-Fas antibody weakly induced these apoptotic components. In marked contrast, calcium depletion did not induce DNA degradation in control cells, and inhibited apoptotic DNA degradation in response to anti-Fas. These data support the concept that the rise in intracellular calcium is not a necessary component for the early signal transduction pathways in anti-Fas-induced apoptosis in Jurkat cells, but rather is necessary for the final degradation of chromatin via nuclease activation.

Gliotoxin, a potential etiologic agent which is synthesized by Aspergillus fumigatus and other pathogenic fungi, exhibits a variety of immunosuppressive activities. We have found that gliotoxin markedly inhibits both perforin-dependent and Fas ligand-dependent cytotoxic T-lymphocyte (CTL)-mediated cytotoxicity. Gliotoxin blocked granule exocytosis and the production of inositol phosphates in response to anti-CD3 stimulation. Apparently, activation signals were not efficiently received by the gliotoxin-treated CTL clone, perhaps because gliotoxin profoundly disturbed CTL cell attachment to immobilized anti-CD3. Although the expression of surface molecules of the CTL clone such as CD3 was unaffected by gliotoxin, the effector/target conjugate formation was inhibited dose-dependently by gliotoxin treatment of the effector CTL clone. These results suggest that gliotoxin prevents CTL from interacting with target cells.

Topoisomerase I inhibitors are promising new chemotherapeutic agents for the treatment of certain malignancies. The present study investigated the impact of the topoisomerase I inhibitor camptothecin on cell death in cardiomyocytes and sought to determine whether the sesquiterpene gamma-lactone--thapsigargin, that alter sarcoplasmic reticulum calcium flux, modulates the effect of camptothecin on the cardiomyocyte. Camptothecin-induced cell death was demonstrated in cardiomyocytes maintained in culture, from 7 day old embryonic chick hearts, by the trypan blue and the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, two independent indicators of the loss of cell viability. The type of cell death was attributed to apoptosis based on cell structure, DNA fragmentation and flow cytometry studies. Camptothecin-treated cardiomyocytes were shrunken with membrane blebs and nuclear fragmentation. Camptothecin produced a dose-dependent increase in DNA fragments of 180 base pairs, or multiples thereof, which are characteristic of apoptosis. A two-fold increase in this type of DNA fragmentation was produced by camptothecin (10 microM) compared to control (diluent-treated) cells. Flow cytometry analysis of populations of 10,000 cardiomyocytes stained with propidium iodide demonstrated a significant increase in the proportion of the population with alterations of DNA content consistent with apoptosis. Pretreatment of cells with thapsigargin, which selectively inhibits sarcoplasmic reticulum and endoplasmic reticulum Ca+2-dependent ATPase, significantly augmented camptothecin-induced apoptosis. Exploring further the role of calcium in camptothecin-induced cell death, we found that the Ca+2 chelator EGTA decreased camptothecin-induced DNA fragmentation. These data indicate the potential for cardiotoxicity from camptothecin through the process of apoptosis and suggest that agents which affect cellular calcium regulation enhance camptothecin-induced apoptosis.

Overexpression of wild-type p53 in M1 myeloid leukemia cells induces apoptotic cell death that was suppressed by the calcium ionophore A23187 and the calcium ATPase inhibitor thapsigargin (TG). This suppression of apoptosis by A23187 or TG was associated with suppression of caspase activation but not with suppression of wild-type-p53-induced expression of WAF-1, mdm-2, or FAS. In contrast to suppression of apoptosis by the cytokines interleukin 6 (IL-6) and interferon gamma, a protease inhibitor, or an antioxidant, suppression of apoptosis by A23187 or TG required extracellular Ca2+ and was specifically abolished by the calcineurin inhibitor cyclosporin A. IL-6 induced immediate early activation of junB and zif/268 (Egr-1) but A23187 and TG did not. A23187 and TG also suppressed induction of apoptosis by doxorubicin or vincristine in M1 cells that did not express p53 by a cyclosporin A-sensitive mechanism. Suppression of apoptosis by A23187 or TG was not associated with autocrine production of IL-6. Apoptosis induced in IL-6-primed M1 cells after IL-6 withdrawal was not suppressed by A23187 or TG but was suppressed by the cytokines IL-6, IL-3, or interferon gamma. The results indicate that these Ca2+-mobilizing compounds can suppress some pathways of apoptosis suppressed by cytokines but do so by a different mechanism.

Osteoclasts which derive from hemopoietic cells are multinucleated cells responsible for bone resorption. We found that cyclopiazonic acid (CPA), thapsigargin (TG), and 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) induced osteoclast-like cell (OCL) formation in cocultures of mouse calvaria-derived stromal cells and hemopoietic cells such as bone marrow cells and spleen cells. OCLs induced by these compounds showed typical characteristics of osteoclasts such as tartrate-resistant acid phosphatase activity and pit forming activity. These compounds are known as endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitors that increase intracellular Ca2+ levels by inhibiting Ca2+-ATPase activity located in the membrane of ER/SR. Ca2+-ionophores such as ionomycin which increase intracellular Ca2+ levels also stimulated OCL formation in the cocultures. Differentiation of hemopoietic cells into OCLs induced by these compounds required the presence of calvarial cells. These results indicate that an increase of intracellular Ca2+ levels may be a part of signaling pathways to induce osteoclast differentiation in the presence of calvarial cells.