Modulation of Ceramide Induced Apoptosis in Vivo

描述（由申请人提供）：最近的研究提供证据表明，除了DNA损伤外，电离辐射还会作用于细胞膜，引发某些细胞的凋亡。在上一个资助期内产生的遗传、生化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辐射诱导的细胞凋亡信号传导中起关键作用，如卵母细胞和内皮细胞。此外，asmase介导的细胞凋亡对卵巢、胃肠道、肺和肿瘤模型的组织辐射反应至关重要。当前提案的假设是，我们实验室最近发现的跨膜信号机制，涉及神经酰胺驱动的膜筏重组成大型信号平台，提供了辐射诱导内皮细胞凋亡的机制。此外，拟议的研究将测试该过程的基因上调是否有助于肿瘤对辐射敏感。该提案包含3个具体目标。目的1调查单剂量辐射后上皮和非上皮肿瘤异种移植物体内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，因为我们现在已经在3个不同的背景下培育了asmase基因敲除。此外，将通过将肿瘤移植到缺乏DNA损伤修复相关基因（如p53、ATM、SCID）或凋亡相关基因（如Bax、ASMase、BID）的小鼠体内，研究辐射诱导的内皮细胞凋亡的遗传学。目的2在细胞和亚细胞水平上探讨神经酰胺介导的细胞凋亡的促凋亡机制。使用颗粒微束辐照进行的初始单细胞卵母细胞研究将确定SM通路是独立于DNA损伤还是与DNA损伤一起诱导细胞凋亡。后续研究将探讨神经酰胺驱动的微观膜筏重组成大信号平台是否是辐射诱导内皮细胞死亡的机制。Aim 3使用基因治疗方法，通过生成过表达asmase的逆转录病毒和慢病毒载体，调节asmase介导的内皮细胞平台形成和肿瘤细胞凋亡。特别是，骨髓来源的内皮祖细胞将成为调控肿瘤微血管细胞凋亡和影响肿瘤生长速度和辐射反应的目标。如果结果表明过表达ASMase有效地使肿瘤微血管放射增敏，那么将直接使用人类版本的慢病毒ASMase载体来研究其对人类肿瘤放疗的适用性。

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.