Wild-type, mitochondrial and ER-restricted Bcl-2 inhibit DNA damage-induced apoptosis but do not affect death receptor-induced apoptosis.

The proto-oncogene Bcl-2 is expressed in membranes of mitochondria and endoplasmic reticulum and mediates resistance against a broad range of apoptotic stimuli. Although several mechanisms of Bcl-2 action have been proposed, its role in different cellular organelles remains elusive. Here, we analyzed the function of Bcl-2 targeted specifically to certain subcellular compartments in Jurkat cells. Bcl-2 expression was restricted to the outer mitochondrial membrane by replacing its membrane anchor with the mitochondrial insertion sequence of ActA (Bcl-2/MT) or the ER-specific sequence of cytochrome b5 (Bcl-2/ER). Additionally, cells expressing wild-type Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/DeltaTM) were employed. Apoptosis induced by ionizing radiation or by the death receptors for CD95L or TRAIL was analyzed by determination of the mitochondrial membrane potential (DeltaPsi(m)) and activation of different caspases. Bcl-2/WT and Bcl-2/MT strongly inhibited radiation-induced apoptosis and caspase activation, whereas Bcl-2/DeltaTM had completely lost its anti-apoptotic effect. Interestingly, Bcl-2/ER conferred protection against radiation-induced mitochondrial damage and apoptosis similarly to Bcl-2/MT. The finding that ER-targeted Bcl-2 interfered with mitochondrial DeltaPsi(m) breakdown and caspase-9 activation indicates the presence of a crosstalk between both organelles in radiation-induced apoptosis. By contrast, Bcl-2 in either subcellular position did not influence CD95- or TRAIL-mediated apoptosis.

A src-like kinase activates outwardly rectifying chloride channels in CFTR-defective lymphocytes.

Defective activation of chloride channels is a hallmark of cystic fibrosis (CF). Recently we have described activation of a volume-sensitive, outwardly rectifying chloride conductance (I(OR)) through the src-like tyrosine kinase p56(lck). Here we show that p56(lck) activates I(OR) independently of CFTR. In lymphocytes from healthy donors, chloride channels could be opened by either intracellular cAMP, p56(lck) or osmotic swelling. In CF lymphocytes, p56(lck) and cell swelling but not cAMP could activate chloride channels. Regulation of I(OR) by p56(lck) thus represents an alternative pathway of stimulating membrane chloride conductance that is left intact in cystic fibrosis.

Radiation sensitivity and apoptosis in human lymphoma cells.

PURPOSE: The impact ofapoptosis on radiation-induced eradication of clonogenic tumour cells is uncertain. The aim was to analyse the relationship of different functional stages during the apoptotic process to cell death and clonogenic cell eradication. MATERIALS AND METHODS: Apoptosis in Jurkat T-cells was studied by morphology, light scatter and caspase activation. Mitochondrial integrity was determined by the mitochondrial membrane potential (delta(phi)m). Cell death was quantified using propidium iodide exclusion. Clonogenic cell death was determined using a dilution survival assay. The influence of Bcl-2 was tested using a Bcl-2 transfected Jurkat clone. RESULTS: Irradiation induced profound apoptosis within 48 h associated with caspase activation and breakdown of delta(phi)m. Inhibition of caspases abrogated the apoptotic morphology with no influence on breakdown of delta(phi)m and survival. Over-expression of Bcl-2 abrogated all hallmarks of apoptosis; delayed cell death, however, had no influence on clonogenic survival after irradiation. CONCLUSION: Based on Bcl-2 as a positional marker, radiation-induced apoptosis can be divided into two stages: the initiation/decision phase, characterized by a breakdown of the mitochondrial membrane potential, and the execution phase, characterized by caspase activation. The execution phase had no influence on survival, whereas the initiation/decision phase controls immediate survival. However, abrogation of both phases did not influence radiation sensitivity.

Tyrosine kinases open lymphocyte chloride channels.

Osmotic swelling of lymphocytes opens outwardly rectifying Cl(-) channels (ORCC) through the src-like kinase p56(lck). The central role of this tyrosine protein kinase has been shown by genetic and pharmacologic manipulation of the enzyme. Furthermore, p56(lck) activates ORCC independently of cell volume increase. ORCC in lymphocytes and epithelial cells from cystic fibrosis (CF) patients are resistant to activation by cAMP. However, osmotic swelling as well as intracellular purified p56(lck) can activate ORCC in CF lymphocytes. In non-CF lymphocytes ORCC is opened by either, intracellular cAMP, p56(lck) or by osmotic swelling. Osmotic activation of ORCC can be blocked by the tyrosine kinase inhibitor lavendustin in both cell types. Regulation of ORCC by p56(lck) thus represents an alternative pathway of stimulating membrane chloride conductance that is left functional in cystic fibrosis. In addition to osmoregulation these mechanisms could play a major role when cells actively change their volume, i.e. during proliferation and apoptosis. Activation of the tyrosine kinase p56(lck) is an important regulatory step for opening of chloride channels in lymphocytes.

Cell volume in the regulation of cell proliferation and apoptotic cell death.

Cell proliferation must - at some time point - lead to increase of cell volume and one of the hallmarks of apoptosis is cell shrinkage. At constant extracellular osmolarity those alterations of cell volume must reflect respective changes of cellular osmolarity which are hardly possible without the participation of cell volume regulatory mechanisms. Indeed, as shown for ras oncogene expressing 3T3 fibroblasts, cell proliferation is paralleled by activation of Na(+)/H(+) exchange and Na(+),K(+),2Cl(-) cotransport, the major transport systems accomplishing regulatory cell volume increase. Conversely, as evident from CD95-induced apoptotic cell death, apoptosis is paralleled by inhibition of Na(+)/H(+) exchanger and by activation of Cl(-) channels and release of the organic osmolyte taurine, major components of regulatory cell volume decrease. However, ras oncogene activation leads to activation and CD95 receptor triggering to inhibition of K(+) channels. The effects counteract the respective cell volume changes. Presumably, they serve to regulate cell membrane potential, which is decisive for Ca(++) entry through I(CRAC) and the generation of cytosolic Ca(++) oscillations in proliferating cells. As a matter of fact I(CRAC) is activated in ras oncogene expressing cells and inhibited in CD95-triggered cells. Activation of K(+) channels and Na(+)/H(+) exchanger as well as Ca(++) oscillations have been observed in a wide variety of cells upon exposure to diverse mitogenic factors. Conversely, diverse apoptotic factors have been shown to activate Cl(-) channels and organic osmolyte release. Inhibition of K(+) channels is apparently, however, not a constant phenomenon paralleling apoptosis which in some cells may even require the operation of K(+) channels. Moreover, cell proliferation may at some point require activation of Cl(-) channels. In any case, the alterations of cell volume are obviously important for the outcome, as cell shrinkage impedes cell proliferation and apoptosis can be elicited by increase of extracellular osmolarity. At this stage little is known about the interplay of cell volume regulatory mechanisms and the cellular machinery leading to mitosis or death of the cell. Thus, considerable further experimental effort is required in this exciting area of cell physiology.

Inhibition of Jurkat-T-lymphocyte Na+/H+-exchanger by CD95(Fas/Apo-1)-receptor stimulation.

Mitogenic factors are known to stimulate the Na+/H+-exchanger (NHE), leading to cytosolic alkalinization and/or cell swelling. Conversely, a hallmark of apoptosis is cell shrinkage and CD95-induced apoptosis has been reported to be paralleled by cytosolic acidification. To assess whether the CD95-receptor regulates NHE activity in Jurkat T-lymphocytes, we performed conventional BCECF fluorescence measurements and SNARF flow cytometric analysis (FACS). The recoveries from acidifications following application of butyrate or a NH3 pulse were both abolished by a specific NHE-inhibitor, HOE694, indicating that they fully depend on NHE activity. Thus they were taken as a measure of NHE activity. CD95-receptor stimulation caused a cytosolic acidification and blunted the recovery from acidification following application of butyrate or a NH3 pulse. Moreover, the NHE-dependent alkalinization following osmotic cell shrinkage was almost abolished by CD95-receptor stimulation. As apparent from the effect of osmotic cell shrinkage, inhibition of the NHE by CD95-receptor stimulation was absent in Lck56-deficient J-CaM1.6 cells and restored by retransfection of J-CaM1.6-cells with Lck56. CD95-receptor stimulation led within 4 h to a decrease of cellular ATP which could contribute to NHE inhibition. Treatment of Jurkat cells with the NHE inhibitor HOE694 accelerated CD95-induced DNA fragmentation. In conclusion, CD95-receptor stimulation inhibits NHE activity through a mechanism that depends directly or indirectly on the activation of the Src-like kinase Lck56. This effect contributes to CD95-induced cytosolic acidification, DNA fragmentation and cell shrinkage.

The involvement of caspases in the CD95(Fas/Apo-1)- but not swelling-induced cellular taurine release from Jurkat T-lymphocytes.

Following a delay of 45 min, stimulation of the CD95 (Fas/Apo-1)-receptor in Jurkat T-lymphocytes leads to the release of the osmolyte taurine, an event coinciding with apoptotic cell shrinkage. The present study has been performed to elucidate the cellular mechanisms involved in CD95-induced taurine release as compared to swelling-induced taurine release, and to explore whether taurine modifies apoptotic DNA fragmentation and cell shrinkage. Taurine release stimulated by osmotic cell swelling is insensitive to the tyrosine kinase inhibitor herbimycin A and the caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) but is blunted in the absence of extracellular Ca2+. Conversely, the Ca2+ ionophore ionomycin stimulates taurine release. However, the taurine release following CD95 stimulation is not paralleled by an increase of cytosolic Ca2+ and not inhibited by complexation of extracellular Ca2+. None of herbimycin A, the phosphatase inhibitor vanadate, spingomyelinase or Lck56 deficiency prevent CD95-induced taurine release. In contrast, the caspase inhibitor zVAD, but not the caspase inhibitor Ac-Tyr-Val-Ala-Asp-chloromethylketone (YVAD), almost abolishes CD95-induced taurine release. Both caspase inhibitors blunt CD95-induced cell shrinkage and DNA fragmentation, zVAD being more effective than YVAD. Preloading of the cells with 40 mM taurine but not with 40 mM mannitol significantly inhibits CD95-induced DNA fragmentation (by 28%) and apoptotic cell shrinkage (by 25%). In conclusion, CD95-receptor triggering leads to caspase-dependent stimulation of cellular taurine release, which facilitates, but is not sufficient for, the triggering of apoptotic DNA fragmentation and cell shrinkage.

Differential role of caspase-8 and BID activation during radiation- and CD95-induced apoptosis.

Activation of the CD95 death receptor as well as ionizing radiation induces apoptotic cell death in human lymphoma cells. The activation of caspases is a hallmark of apoptosis induction irrespective of the apoptotic trigger. In contrast to death receptor signaling, the exact mechanisms of radiation-induced caspase activation are not well understood. We provide evidence that both, radiation and CD95 stimulation, induce the rapid activation of caspase-8 and BID followed by apoptosis in Jurkat T-cells. To analyse the relative position of caspase-8 within the apoptotic cascade we studied caspase activation and apoptosis in Jurkat cells overexpressing Bcl-2 or Bcl-xL. Caspase-8 activation, pro-apoptotic BID cleavage and apoptosis in response to radiation were abrogated in these cells, while the responses to CD95 stimulation were only partially attenuated by overexpression of Bcl-2 family members. In parallel, the breakdown of the mitochondrial transmembrane potential (DeltaPsim) in response to radiation was inhibited by overexpression of Bcl-2/Bcl-xL Jurkat cells genetically deficient for caspase-8 were found to be completely resistant towards CD95. However, radiation-induced apoptotic responses in caspase-8-negative cells displayed only a modest reduction. We conclude that ionizing radiation activates caspase-8 and BID downstream of mitochondrial damage suggesting that, in contrast to CD95, both events function as executioners rather than initiators of the apoptotic process.

Stimulation of CD95 (Fas) blocks T lymphocyte calcium channels through sphingomyelinase and sphingolipids.

Calcium influx through store-operated calcium release-activated calcium channels (CRAC) is required for T cell activation, cytokine synthesis, and proliferation. The CD95 (Apo-1/Fas) receptor plays a role in self-tolerance and tumor immune escape, and it mediates apoptosis in activated T cells. In this paper we show that CD95-stimulation blocks CRAC and Ca(2+) influx in lymphocytes through the activation of acidic sphingomyelinase (ASM) and ceramide release. The block of Ca(2+) entry is lacking in CD95-defective lpr lymphocytes as well as in ASM-defective cells and can be restored by retransfection of ASM. C2 ceramide, C6 ceramide, and sphingosine block CRAC reversibly, whereas the inactive dihydroceramide has no effect. CD95-stimulation or the addition of ceramide prevents store-operated Ca(2+) influx, activation of the transcriptional regulator NFAT, and IL-2 synthesis. The block of CRAC by sphingomyelinase metabolites adds a function to the repertoire of the CD95 receptor inhibiting T cell activation signals.

The tyrosine kinase lck is required for CD95-independent caspase-8 activation and apoptosis in response to ionizing radiation.

Induction of apoptosis is a hallmark of cytostatic drug and radiation-induced cell death in human lymphocytes and lymphoma cells. However, the mechanisms leading to apoptosis are not well understood. We provide evidence that ionizing radiation induces a rapid activation of caspase-8 (FLICE) followed by apoptosis independently of CD95 ligand/receptor interaction. The radiation induced cleavage pattern of procaspase-8 into mature caspase-8 resembled that following CD95 crosslinking and resulted in cleavage of the proapoptotic substrate BID. Overexpression of dominant-negative caspase-8 interfered with radiation-induced apoptosis. Caspase-8 activation by ionizing radiation was not observed in cells genetically defective for the Src-like tyrosine kinase Lck. Cells lacking Lck also displayed a marked resistance towards apoptosis induction upon ionizing radiation. After retransfection of Lck, caspase-8 activation and the capability to undergo apoptosis in response to ionizing radiation was restored. We conclude that radiation activates caspase-8 via an Lck-controlled pathway independently of CD95 ligand expression. This is a novel signaling event required for radiation induced apoptosis in T lymphoma cells.

Cell volume regulatory mechanisms in apoptotic cell death.

One of the hallmarks of apoptosis is cell shrinkage, which--at constant extracellular osmolarity--requires a decrease of cellular osmolarity. Moreover, apoptosis can be elicited by increase of extracellular osmolarity and the resistance of cells towards apoptosis correlates with their ability to regulate their volume in hypertonic environment. On the other hand, CD95-receptor-mediated apoptosis is blunted at moderate increases of extracellular osmolarity. Given the role of cell volume alterations it is not surprising that apoptosis is paralleled by marked alterations of cell volume regulatory mechanisms. Stimulation of the CD95-receptor, which confers apoptosis to a variety of cells, leads to activation of cell volume regulatory anion channel ORCC. However, activation of ORCC is paralleled by inhibition of cell volume regulatory K+ channel Kv1.3. It is only 40 to 60 minutes after triggering of the CD95-receptor when the cells release the organic osmolyte taurine and shrink.

Impact of localized radiotherapy on blood immune cells counts and function in humans.

Immune cells subsets were prospectively analyzed after localized radiotherapy (LRT). LRT reduced the levels of all lymphocyte subsets, with B-cells and naive T-cells being most sensitive. Lymphocyte function was suppressed, but still within the normal range. Rapid recovery of cytotoxic T-cells/natural killer cells after LRT and the functional suppression within normal levels explains the low incidence of infections after LRT.

Intracellular mechanisms of L-selectin induced capping.

Leucocyte adhesion to endothelial cells is a tightly regulated process involving selectins, integrins and immunoglobulin-like proteins. Cell adhesion and communication are controlled by membrane dynamics like receptor capping. Capping of surface receptors is an ubiquitous mechanism but still not well understood. Employing immunofluorescence techniques, we demonstrate that L-selectin triggering results in receptor capping of the L-selectin molecules in lymphocytes. Using pharmacological inhibitors and genetic deficient cell lines we show that this process involves intracellular signalling molecules. L-Selectin capping seems to be independent on activation of p56lck-kinase, but requires the neutral sphingomyelinase, small G proteins and the cytoskeleton. Therefore, capping of L-selectin upon stimulation might play an important role in the very early phase of lymphocyte trafficking.

A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases.

The different members of the vascular endothelial growth factor (VEGF) family act as key regulators of endothelial cell function controlling vasculogenesis, angiogenesis, vascular permeability and endothelial cell survival. In this study, we have functionally characterized a novel member of the VEGF family, designated VEGF-E. VEGF-E sequences are encoded by the parapoxvirus Orf virus (OV). They carry the characteristic cysteine knot motif present in all mammalian VEGFs, while forming a microheterogenic group distinct from previously described members of this family. VEGF-E was expressed as the native protein in mammalian cells or as a recombinant protein in Escherichia coli and was shown to act as a heat-stable, secreted dimer. VEGF-E and VEGF-A were found to possess similar bioactivities, i.e. both factors stimulate the release of tissue factor (TF), the proliferation, chemotaxis and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo. Like VEGF-A, VEGF-E was found to bind with high affinity to VEGF receptor-2 (KDR) resulting in receptor autophosphorylation and a biphasic rise in free intracellular Ca2+ concentration, whilst in contrast to VEGF-A, VEGF-E did not bind to VEGF receptor-1 (Flt-1). VEGF-E is thus a potent angiogenic factor selectively binding to VEGF receptor-2. These data strongly indicate that activation of VEGF receptor-2 alone can efficiently stimulate angiogenesis.

Physiology of Receptor-Mediated Lymphocyte Apoptosis.

Apoptosis (programmed cell death), a physiological mechanism eliminating abundant and potentially harmful cells, is triggered by a variety of stimuli including activation of distinct receptors. The machinery mediating CD95 receptor-induced apoptosis includes caspases, ceramide, kinases, Ras and Rac, formation of O(2)(-), mitochondrial proteins, inhibition of K(+) channels, activation of Cl(-) channels, and osmolyte release.

Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis in T lymphocytes.

CD95/Fas/APO-1 mediated apoptosis is an important mechanism in the regulation of the immune response. Here, we show that CD95 receptor triggering activates an outwardly rectifying chloride channel (ORCC) in Jurkat T lymphocytes. Ceramide, a lipid metabolite synthesized upon CD95 receptor triggering, also induces activation of ORCC in cell-attached patch clamp experiments. Activation is mediated by Src-like tyrosine kinases, because it is abolished by the tyrosine kinase inhibitor herbimycin A or by genetic deficiency of p56lck. In vitro incubation of excised patches with purified p56lck results in activation of ORCC, which is partially reversed upon addition of anti-phosphotyrosine antibody. Inhibition of ORCC by four different drugs correlates with a 30-65% inhibition of apoptosis. Intracellular acidification observed upon CD95 triggering is abolished by inhibition of either ORCC or p56lck. The results suggest that tyrosine kinase-mediated activation of ORCC may play a role in CD95-induced cell death in T lymphocytes.

The tyrosine kinase p56lck mediates activation of swelling-induced chloride channels in lymphocytes.

Osmotic cell swelling activates Cl- channels to achieve anion efflux. In this study, we find that both the tyrosine kinase inhibitor herbimycin A and genetic knockout of p56lck, a src-like tyrosine kinase, block regulatory volume decrease (RVD) in a human T cell line. Activation of a swelling-activated chloride current (ICl-swell) by osmotic swelling in whole-cell patch-clamp experiments is blocked by herbimycin A and lavendustin. Osmotic activation of ICl-swell is defective in p56lck-deficient cells. Retransfection of p56lck restores osmotic current activation. Furthermore, tyrosine kinase activity is sufficient for activation of ICl-swell. Addition of purified p56lck to excised patches activates an outwardly rectifying chloride channel with 31 pS unitary conductance. Purified p56lck washed into the cytoplasm activates ICl-swell in native and p56lck-deficient cells even when hypotonic intracellular solutions lead to cell shrinkage. When whole-cell currents are activated either by swelling or by p56lck, slow single-channel gating events can be observed revealing a unitary conductance of 25-28 pS. In accordance with our patch-clamp data, osmotic swelling increases activity of immunoprecipitated p56lck. We conclude that osmotic swelling activates ICl-swell in lymphocytes via the tyrosine kinase p56lck.

The CD40 ligand directly activates T-lymphocytes via tyrosine phosphorylation dependent PKC activation.

The activation of B-lymphocytes depends critically on the interaction of the CD40 receptor with its ligand. Here, we provide evidence that the CD40 ligand (CD40L) also functions as a direct stimulatory molecule for T-lymphocytes. Activation of T-lymphocytes via CD40L induces tyrosine phosphorylation of cellular proteins including PLC gamma. Tyrosine phosphorylation of PLC gamma correlates with an IP3- and Ca(2+)-release and an activation of PKC. Inhibition of src-like tyrosine kinases by Herbimycin A prevents these activation events suggesting a crucial role of tyrosine phosphorylation in T-lymphocyte activation via CD40L.