Res Proj 3: Imaging HTLV-1 Tax Induced Lymphomas

目前的建议是建立在前一个项目期间开始的研究基础上的，这些研究利用生物发光- 气味成像（BLI），以评估HTLV-1 Tax淋巴瘤的生物学和治疗。肿瘤移植 模型和转基因小鼠模型用于成像以评估肿瘤细胞增殖， 传播。了解HTLV-1 Tax淋巴瘤的发病机制，通过NF κ B活化， 与也利用NF κ B活化的广泛的造血肿瘤直接相关。目标是： 目标1.高通量筛选Tax介导的淋巴瘤细胞关键激酶 增殖和/或存活。我们将使用Tax转基因肿瘤细胞系来筛选siRNA文库， 人类激酶组，以揭示有关HTLV-1利用的转化途径的新信息。 目标二。使用BLI评估肿瘤移植模型中Tax肿瘤发生的关键介质的作用。 表达荧光素酶的肿瘤细胞系将用表达siRNA的慢病毒感染，以产生可能的免疫应答。 税务活动的中介。要调查的税务活动中介包括NFKB的组成部分 途径，上游激活剂和下游NF κ B靶点。从Aim中识别的关键激酶介质 1，也将使用具有新鲜肿瘤移植物的模型进行验证。筛选细胞系的高 水平的荧光素酶活性，并皮下注射到Rag 1小鼠中。每2-4周对小鼠成像 用或不用特异性小分子抑制剂治疗12-16周，然后 通过免疫组织化学分析Tax和荧光素酶，以及细胞增殖标记物（BUDR 掺入）、血管生成（CD 31表达）和细胞凋亡（Tunel染色）。 目标3：使用BLI评估转基因小鼠模型中Tax肿瘤发生的关键介质的作用。 该小鼠模型包括Tax和LTR-荧光素酶转基因。这些老鼠将与 表达肿瘤发生关键介质的基因的条件性或非条件性纯合缺失， 包括肿瘤抑制因子Arf、NF κ B亚基p50和p52以及在Aim 1中鉴定的激酶。肿瘤将是 通过生物发光成像、病理学、Tax和荧光素酶的IHC、FcvRII/III、CD 4或 CD 8和增殖、血管生成和凋亡的标志物。 目标4。使用BLI和CGH评估遗传不稳定性在Tax肿瘤发生中的作用。基因的作用 将在Tax转基因动物中评价肿瘤起始、维持或进展的不稳定性，Tax转基因动物 也表达融合于甲虫红的CDC 25 A蛋白。这些发现将与小鼠 比较基因组杂交（CGH）阵列。

The current proposal builds upon studies initiated in the previous project period that utilized biolumine- scent imaging (BLI) to assess the biology and treatment of HTLV-1 Tax lymphomas. A tumor transplant model and a transgenic mouse model were established for imaging to assess tumor cell proliferation and dissemination. Understanding the pathogenesis of HTLV-1 Tax lymphomas, through NFKB activation, has direct relevance to a wide range of hematopoietic neoplasms that also utilize NFKB activation. The aimsare: Aim 1. Use of high throughput screen to identify kinases that are critical for Tax-mediatedlymphoma cell proliferation and/or survival. We will use a Tax transgenic tumor cell line for to screen the siRNA library to the human kinome to uncover new information about the transformation pathways exploited by HTLV-1. Aim 2. Use of BLI to assess the role of critical mediators of Tax tumorigenesis in a tumor transplant model. Tumor cell lines expressing luciferase will be infected with a lentivirus expressing siRNAto a possible mediator of Tax activity. Mediators of Tax activityto be investigated include components of the NFKB pathway, upstream activators, and downstream NFKB targets. Critical kinase mediators, identified from Aim 1, will also be validated using a model with fresh tumor transplants. The cell lines are screened for high levels of luciferase activity, and injected subcutaneously into Rag1 mice. Mice are imaged every 2-4 wks over 12-16 wks with or without treatment with specific small molecule inhibitors, and tumors are then analyzed by immunohistochemistry for Tax and luciferase, as well as markers of cell proliferation (BUDR incorporation), angiogenesis (CD31 expression), and apoptosis (Tunel stain). Aim 3. Use of BLI to assess the role of critical mediators of Tax tumorigenesis in a transgenic mouse model. This mouse model includes Tax and LTR-luciferase transgenes. These mice will be bred with mice with conditional or non-conditional homozygous deletions of genes expressing critical mediators of tumorigenesis, including tumor suppressor Arf, NFKB subunits p50 and p52, and kinases identified in Aim 1. Tumors will be evaluated by bioluminescent imaging, pathology, IHC for Tax and luciferase, FACSfor FcvRII/lll, CD4, or CD8, and markers of proliferation, angiogenesis, and apoptosis. Aim 4. Use of BLI and CGH to assess the role of genetic instability in Tax tumorigenesis. The role of genetic instability in tumor initiation, maintenance, or progression will be evaluated in Tax transgenic animals that also express the CDC25A protein fused to click beetle red. These findings will be correlated to mouse comparative genomic hybridization (CGH) arrays.