Modulation of Ceramide Induced Apoptosis in Vivo

描述（由申请人提供）：最近的研究提供了证据，除了DNA损伤外，电离辐射作用于细胞膜，引发某些细胞的凋亡性死亡。在上一个资助期间产生的遗传、生物化学和细胞生物学数据确定了酸性鞘磷脂酶（ASMase）介导的神经酰胺生成在特定细胞（如卵母细胞和内皮细胞）中信号辐射诱导的细胞凋亡中的关键作用。 此外，ASMase介导的细胞凋亡对于卵巢、胃肠道、肺和肿瘤模型中的组织辐射反应至关重要。 目前的建议的假设是，跨膜信号传导机制，最近发现的我们的实验室，涉及神经酰胺驱动重组膜筏到大的信号平台，提供了一种机制，辐射诱导内皮细胞凋亡。 此外，拟议的研究将测试这一过程的遗传上调是否有助于肿瘤对辐射敏感。 该提案包含三个具体目标。 目的1：研究单次剂量辐射后上皮和非上皮肿瘤异种移植物在体内发生内皮细胞凋亡的程度。 微血管功能障碍在胃肠道和肿瘤反应中的作用将在多个小鼠品系中确定，因为我们现在已经将asmase敲除培育到3个不同的背景中。 此外，将通过将肿瘤移植到缺乏参与DNA损伤修复（即p53、ATM、SCID）或凋亡（即Bax、ASMase、BID）的基因的小鼠中来研究辐射诱导的内皮细胞凋亡的遗传学。 目的2从细胞和亚细胞水平探讨神经酰胺介导的细胞凋亡的促凋亡机制。 最初的单细胞卵母细胞研究，使用粒子微束照射将确定是否SM途径诱导细胞凋亡独立或与DNA损伤一致。 后续研究将探讨神经酰胺驱动的微观膜筏重组为大信号平台是否是辐射诱导的内皮细胞死亡的机制。 目的3使用基因治疗方法来调节ASMase介导的内皮细胞平台形成和肿瘤内的凋亡，使用逆转录病毒和慢病毒载体产生过表达asmase。 特别地，骨髓来源的内皮祖细胞将被靶向以操纵发育中的肿瘤微血管系统内的细胞凋亡并影响肿瘤生长速率和辐射反应。 如果结果表明过表达ASMase有效地使肿瘤微血管系统放射增敏，则将使用慢病毒ASMase载体的人版本直接检查对人肿瘤放射疗法的适用性。

DESCRIPTION (provided by applicant): Recent studies provided evidence that, in addition to DNA damage, ionizing radiation acts upon cellular membranes to initiate apoptotic death in some cells. Genetic, biochemical and cell biologic data generated during the last funding period established a critical role for acid sphingomyelinase (ASMase)-mediated ceramide generation in signaling radiation-induced apoptosis in select cells such as oocytes and endothelium. Further, ASMase-mediated apoptosis was crucial for tissue radiation responses in the ovary, GI tract, lung and tumor models. The hypothesis of the current proposal is that a transmembrane signaling mechanism, recently discovered by our laboratory, involving ceramidedriven re-organization of membrane rafts into large signaling platforms, affords a mechanism by which radiation induces endothelial apoptosis. Furthermore, the proposed research will test whether genetic up-regulation of this process serves to sensitize tumors to radiation. The proposal contains 3 specific aims. Aim 1 surveys the extent to which epithelial and non-epithelial tumor xenografts undergo endothelial cell apoptosis in vivo after single dose radiation. The role of microvascular dysfunction in GI and tumor responses will be determined in multiple mouse strains, as we have now bred the asmase knockout onto 3 separate backgrounds. Further, the genetics of radiationinduced endothelial apoptosis will be investigated by transplanting tumors into mice lacking genes involved in DNA damage repair (i.e. p53, ATM, SCID) or apoptosis (i.e. Bax, ASMase, BID). Aim 2 addresses the pro-apoptotic mechanism of ceramide-mediated apoptosis at the cellular and sub-cellular level. Initial single cell oocyte studies using particle microbeam irradiation will establish whether the SM Pathway induces apoptosis independent of or in concert with DNA damage. Subsequent studies will address whether ceramide-driven re-organization of microscopic membrane rafts into large signaling platforms is the mechanism of radiation-induced endothelial cell death. Aim 3 uses a gene therapy approach to modulate ASMase-mediated endothelial cell platform formation and apoptosis within tumors using retroviral and lentiviral vectors generated to overexpress asmase. In particular, bone-marrow-derived endothelial progenitors will be targeted to manipulate apoptosis within developing tumor microvasculature and influence tumor growth rate and radiation response. If results indicate that overexpressing ASMase effectively radiosensitizes tumor microvasculature, applicability to human tumor radiotherapy will be examined directly using human versions of lentiviral asmase vectors.