APOPTOTIC DEATH IN T LYMPHOCYTES

本项目旨在评估胱天蛋白酶在T细胞死亡中的作用 一般来说。为了更好地研究其功能作用，我们 开发了一种称为PhiPhiLux的新型荧光半胱天冬酶底物， 检测完整凋亡细胞中的半胱天冬酶活化。该试剂是 来自聚ADP-核糖的半胱天冬酶-3切割位点的肽 聚合酶，含有两个在末端被取代的罗丹明染料， 肽，使得它们形成自猝灭的环状化合物， 被蛋白水解裂解破坏。凋亡细胞提取物， 正常胸腺细胞在DEVD后切割该底物， 增加被胱天蛋白酶-3抑制剂选择性地阻断。PhiPhiLux可以 在体外被加载到完整的胸腺细胞中，并且响应于凋亡， 刺激物胸腺细胞中罗丹明浓度随时间增加约10倍 通过流式细胞术测定荧光。这种染色的增加是特别的 通过与半胱天冬酶抑制剂ZVAD-FMK预孵育阻断。双色 用FITC-膜联蛋白-V染色显示PhiPhiLux+细胞是凋亡的 如它们暴露的磷脂酰丝氨酸所示。共聚焦显微镜显示 PhiPhiLux染色是细胞质的，并且在用 星形孢菌素在线粒体PSI丧失后动力学地出现。 在评估胱天蛋白酶在T细胞死亡中的普遍重要性时，我们 发现几乎所有的T细胞死亡都是由传统的凋亡引起的， 标准可以被肽-FMK半胱天冬酶特异性抑制 抑制剂的这包括各种化疗药物的作用。 药物对T细胞肿瘤的作用然而，由于肿瘤细胞死亡的评估， 体外可能与体内肿瘤根除无关，我们已经测试了 半胱天冬酶抑制剂阻断各种药剂的能力 以减少软琼脂中菌落的形成。同时使用 化疗药物和抗Fas，我们一直无法观察到一个 肽-FMK或杆状病毒p35在该测定中的阻断作用 半胱天冬酶抑制剂，即使他们有效地阻止传统的细胞， 在同一个实验中的死亡读数。我们目前正试图 确定这些差异是否是由于不同的动力学 或者是否抑制菌落形成 通过caspase非依赖性途径，即使当触发Fas- 交联

This project aims to assess the role of caspases in T cell death generally. In order to better study their functional role, we have developed a new fluorogenic caspase substrate termed PhiPhiLux which can detect caspase activation in intact apoptotic cells. This reagent is a peptide derived from the caspase-3 cleavage site of poly-ADP- ribose polymerase containing two rhodamine dyes substituted at the ends of the peptide so that they form a self-quenched cyclic compound which is disrupted by proteolytic cleavage. Extracts from apoptotic but not normal thymocytes cleave this substrate after DEVD, and the fluorescence increase is selectively blocked by caspase- 3 inhibitors. PhiPhiLux can be loaded into intact thymocytes in vitro, and in response to apoptotic stimuli thymocytes undergo a time-dependent about 10x increase in rhodamine fluorescence by flow cytometry. This staining increase is specifically blocked by preincubation with the caspase inhibitor ZVAD-FMK. Two-color staining with FITC- annexin-V shows that PhiPhiLux+ cells are apoptotic as seen by their exposed phosphatidyl serine. Confocal microscopy shows that the PhiPhiLux stain is cytoplasmic and in T hybridomas treated with staurosporine appears kinetically after the loss of mitochondrial psi. In assessing the general importance of caspases in T cell death, we have found that virtually all T cell death which is apoptotic by traditional criteria can be specifically inhibited by peptide-FMK caspase inhibitors. This includes the effect of a variety of chemotherapeutic agents on T cell tumors. However, since tumor cell death assessed in vitro may not be relevant to in vivo tumor eradication, we have tested the ability of caspase inhibitors to block the ability of various agents to decrease the formation of colonies in soft agar. Using both chemotherapeutic agents and anti-Fas, we have been unable to observe a blocking effect in this assay by either peptide-FMK or baculovirus p35 caspase inhibitors, even though they efficiently block traditional cell death readouts in the same experiment. We are currently attempting to ascertain whether these differences are due to the different kinetics of the two types of assay, or whether inhibiting colony formation occurs by a caspase-independent pathway even when triggered by Fas- crosslinking.