2185-Pos Board B155 Extracellular ATP Mediates FasL-induced Necrosis of Lymphoid Cells Via P2X7 Activation Mauricio Henriquez, Andrew Quest. FONDAP Center for Molecular Studies of the Cell (CEMC), Institute of Biomedical Sciences (ICBM), Faculty de Medicine, University of Chile, Santiago, Chile. We demonstrated previously that FasL triggers both necrotic and apoptotic death in lymphoid cells. The execution of apoptosis was linked to activation of caspase-8 and caspase-3, while necrotic cell death required only activation of caspase-8 associated with delayed increases in ceramides. However, the pre- cise signaling mechanisms involved in deciding between FasL-induced apopto- tic or necrotic cell death in lymphoid cells remained unknown. On the other hand, many studies have implicated extracellular ATP as a mediator of cell death by necrosis, as well as apoptosis. In particular, ATP-dependent activation of purinergic P2X7 receptors has been suggested to trigger both forms of cell death. Hence, the objective here was to evaluate whether ATP/P2X7 partici- pated in cell death induced by FasL in lymphoid cells. Flow cytometric analysis following staining with propidium iodide, in human Jurkat cells demonstrated that oxidized ATP, a specific P2X7 antagonist, selectively inhibited necrotic, but not apoptotic cell death induced by Fas-L. ATP, the physiological P2X7 ligand, was released from Jurkat cells following incubation with FasL. Further- more, FasL-stimulated intracellular calcium-transients in Jurkat cells (mea- sured using the Fluo3-AM probe) were blocked either when extracellular cal- cium was chelated or when cells were preincubated with oxidized ATP. The presence of P2X7 receptors in Jurkat cells was corroborated by Western blot- ting. This study represents the first demonstration of cross-talk between the two cell death receptors Fas and P2X7 in lymphoid cells. 2186-Pos Board B156 T Cell Receptor Regulation Of Fas-mediated Apoptotic Calcium Release Askar M. Akimzhanov, Xinmin Wang, Darren Boehning. University of Texas Medical Branch, Galveston, TX, USA. Although alterations in calcium levels are known to play an important role in a variety of physiological processes, only in past few years has the role of cal- cium signaling in regulation of apoptosis been well recognized. It has been shown that apoptotic stimuli provoke rapid elevation of cytosolic calcium con- centration, which in turn orchestrates the release and activation of multiple pro-apoptotic factors. Our previous results in T cells demonstrated that Fas- mediated apoptosis requires calcium release, which was dependent upon phospholipase C-g1 (PLC-g1) activation and calcium release from inositol 1,4,5-trisphosphate receptor (IP 3 R) channels. Here, we show that PLC-g1 acti- vation after Fas receptor ligation requires canonical components of the T cell receptor complex. Specifically, the active form of the Src family tyrosine ki- nase Lck and PLC-g1 become associated with the death-inducing signaling complex (DISC) in a stimulus-dependent manner. We found that Lck activates PLC-g1 indirectly via Zap70 tyrosine kinase and other members of the T cell receptor signaling pathway. Moreover, in absence of a functional T cell recep- tor complex, Fas stimulation failed to induce calcium release. This led to sig- nificantly inhibited effector caspase activation and delayed cell death. These findings strongly suggest a vital interplay between Fas and the T cell receptor complex, which has significant implications for T cell regulation. 2187-Pos Board B157 Novel K þ Channel Blocker Induces Apoptosis Via Ca 2þ Release From ER Stores Elena Zaks-Makhina 1 , Chandra Vignere 1 , Vicenta Salvador-Recatala 2 , Edwin S. Levitan 1 . 1 University of Pittsburgh, Pittsburgh, PA, USA, 2 University of Pennsylvania, Philadelphia, PA, USA. In search of novel K þ channel modulators we have undertaken HTS of chem- ical libraries. The primary screen using yeast whose growth in low [K þ ] me- dium depends on expression of the inwardly rectifying K þ channel Kir2.1 was followed by K þ current measurements in mammalian cells. One of the identified compounds, 22G ({1-[3-(4-chloro-phenyl)-adamantan-1-yl]-ethyl}- (1-ethyl-piperidin-4-yl)-amine), inhibits Kir2.1-dependent yeast growth, de- creases 86 Rb þ flux via Kir2.1 and acutely blocks the whole-cell Kir2.1 current (EC 50 ¼25 mM) in transfected HEK293 cells. As a part of specificity character- ization of 22G, we examined its effect on 86 Rb þ efflux via recombinant and native Ca 2þ -activated K þ channel (IKCa) in HEK293 cells and IEC-6 entero- cytes, respectively. Surprisingly, 22G induced rapid K þ efflux via IKCa. Because previously we have established that IKCa-dependent K þ efflux is es- sential for apoptosis, we tested whether 22G is capable of inducing apoptosis. When applied at 25 mM for 10 min, 22G caused activation of caspases in 90% of IEC-6 cells, as determined by staining with the fluorescent pan-caspase in- hibitor FAM-VAD-FMK. Blockade of 22G-induced K þ current by the IKCa inhibitor clotrimazole prevented the induction of apoptosis. Thus, 22G is a po- tent inducer of IKCa-mediated apoptosis. To test whether 22G activates pro-ap- optotic IKCa current by elevating the cytosolic [Ca 2þ ], we measured Ca 2þ levels in 22G-treated HEK293, CHO and IEC-6 cells loaded with the Ca 2þ in- dicator fluo-4. At 50 mM, 22G induced increase in cytosolic Ca 2þ in Ca 2þ -free solution in all three cell lines. Depletion of ER Ca 2þ stores with thapsigargin and cyclopiazonic acid abolished 22G-induced surge of cytosolic [Ca 2þ ] and IKCa current. Therefore, 22G activates IKCa current via Ca 2þ release from ER stores. We suggest using compound 22G as a reliable apoptogen, a tool for modulation of intracellular [Ca 2þ ] and inhibitor of Kir2.1 current in patch-clamp experiments. 2188-Pos Board B158 The Amino-terminal Peptide Of Bax Perturbs Intracellular Ca 2þ Homeo- stasis To Enhance Apoptosis In Prostate Cancer Cells Na Li, Peihui Lin, Chuanxi Cai, Zui Pan, Noah Weisleder, Jianjie Ma. UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA. Targeting the interconnected cellular pathways controlling apoptosis and regu- lation of Ca 2þ homeostasis are two avenues for treatment of human cancers. During apoptosis, proteolytic cleavage of Bax at the amino-terminus generates a truncated Bax of ~18 kDa (p18Bax) and an amino-terminal peptide of ~3 kDa (p3Bax). Extensive studies have shown that p18Bax behaves like a BH3 protein with enhanced pro-apoptotic function over the full-length Bax (p21Bax), little is known about the function of p3Bax in apoptosis. We have previously shown that Bax and Ca 2þ synergistically amplifying apoptosis signaling (Pan, et al. J Biol Chem 276: 32257, 2001), and that store-operated Ca 2þ entry (SOCE) con- tributes to Bax-mediated apoptosis in prostate cancer cells (Li, et al. J Cell Physiol 216: 172, 2008). Here we test if p3Bax can contribute to regulation of Ca 2þ signaling during apoptosis, through a membrane penetrating peptide (TAT) to facilitate delivery of recombinant p3Bax into NRP-154 cells, a pros- tate epithelial cell line with tumorigenic capacity. We find that TAT-p3Bax fusion peptide can enhance thapsigargin-induced apoptosis in NRP-154 cells, elevate SOCE activity and increase IP 3 sensitive intracellular Ca 2þ stores. Our data indicates that p3Bax can modulate the entry of extracellular Ca 2þ , and thus regulate the amplification of apoptosis in prostate cancer cells. An- other unique observation of this study is that TAT-p3Bax is not toxic to NRP-Bax cells under resting conditions, it only enhances the process of apopto- sis initiated by exposure to TG. This is particularly important when considering the exogenous p3Bax peptide as a therapeutic agent for prostate cancer. In such a case, p3Bax would not produce cytotoxic effects in cells with normal Ca 2þ homeostasis, it could be used in combination with other cytotoxic agents to am- plify apoptosis in targeted cancer cells. 2189-Pos Board B159 The Anti-apoptotic Mitochondrial Membrane Protein Bcl-2; An Achilles Heel Of Cancer Cells? Marcus Wallgren 1 , Marc-Antoine Sani 1 , Henrik Vestin 1 , Erick J. Dufourc 2 , Gerhard Gro¨bner 1 . 1 Department of Chemistry, Umea˚, Sweden, 2 CNRS-Universite´ Bordeaux, Bordeaux, France. Escape from programmed cell death, apoptosis, is one of the main hallmarks of cancer.. The anti-apoptotic protein Bcl-2 belongs to the Bcl-2 protein family, which function as a major gatekeeper in the mitochondrial pathway. Bcl-2 is found to a great extent in many breast cancers and is highly involved in the in- herent resistance to anti-cancer drugs. This protein is mitochondrial membrane- associated and we will use NMR spectroscopy to provide structural information of the membrane-mediated mechanism underlying the action of Bcl-2 as a po- tent inhibitor of cell death. For this purpose we express currently the full length protein and carry out var- ious preliminary studies of the membrane dependent behaviour of synthesized segments of the proteins by a range of biophysical methods ranging from CD (Circular Dichroism), ATR (Attenuated Total Reflection), Calorimetry to solid state NMR spectroscopy.At present, we study the impact of the unique BH4 do- main of the pro-survival Bcl-2 protein on the mitochondrial membranes, since this interaction seems to be essential to block any apoptotic activation with its connected pore-formation and cytochrome c release. Our first results reveal that the BH4 domain requires cardiolipin to be able to convert into an a-helix con- formation. In contact with neutral membranes, the peptide seems to aggregate on the surface. Bcl-2’s counterplayer is Bax protein which upon activation tran- locates to the mitochondrial membrane. In this process the first helix localized at the N-terminus of Bax (Bax-a1) can act as an addressing sequence, which upon activation directs the protein from the cytosol towards the mitochondrial surface. Our biophysical approach provided insight into the molecular 424a Tuesday, March 3, 2009