Res Proj 3: Imaging HTLV-1 Tax Induced Lymphomas

目前的提议建立在前一个项目期启动的研究基础上，这些研究利用了生物柱-- 气味成像(BLI)评估HTLV-1税务淋巴瘤的生物学和治疗。肿瘤移植 建立肿瘤模型和转基因小鼠模型，评价肿瘤细胞的增殖和生长情况。 传播。通过NFKB激活了解HTLV-1税务淋巴瘤的发病机制 与广泛的造血肿瘤直接相关，这些肿瘤也利用NFKB激活。目标是： 目的1.使用高通量筛选来鉴定对税收介导的淋巴瘤细胞至关重要的激酶 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 人类亲属组，以发现有关HTLV-1利用的转化途径的新信息。 目的2.使用BLI评估在肿瘤移植模型中税收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被表达siRNA的慢病毒感染 税务活动的调解人。要调查的税务活动调解人包括NFKB的组成部分 途径、上游激活剂和下游NFKB靶标。从AIM中鉴定的关键激酶介体 1，也将使用新鲜肿瘤移植的模型进行验证。对细胞系进行高密度筛选 荧光素酶活性水平，并皮下注射Rag1小鼠。每隔2-4周对老鼠进行一次成像 在接受或不接受特定小分子抑制剂治疗的情况下，超过12-16周，肿瘤就会 用免疫组织化学方法分析紫杉醇和荧光素酶以及细胞增殖标志(BUDR) 掺入)、血管生成(CD31表达)和细胞凋亡(TUNEL染色)。 目的3.使用BLI在转基因小鼠模型中评估税务肿瘤发生的关键介质的作用。 这个小鼠模型包括Tax和Ltr-荧光素酶转基因。这些小鼠将与小鼠繁殖 表达肿瘤发生关键介质的基因有条件或无条件纯合子缺失， 包括肿瘤抑制因子Arf，NFKB亚基p50和p52，以及Aim 1中发现的激酶。肿瘤将被 通过生物发光成像、病理学、IHC对TAX和荧光素酶、FAC对FcvRII/11、CD4或 CD8，以及增殖、血管生成和凋亡的标志物。 目的4.使用BLI和CGH来评估遗传不稳定性在税务肿瘤发生中的作用。基因的作用 肿瘤起始、维持或进展的不稳定性将在以下转基因动物中进行评估 同时表达与甲虫红融合的CDC25A蛋白。这些发现将与小鼠相关 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.