PKB/Akt Activation and Cell Survival with HIV-1 Tat

描述(由申请人提供)： 据估计，全世界有800万人同时患有卡波西氏肉瘤和人类免疫缺陷病毒1型(HIV-1)，对于Kaposi肉瘤(KS)的侵袭性增长没有令人信服的解释。人类疱疹病毒8型(HHV-8)是KS发展所必需的，但除非存在第二个因素，如艾滋病毒-1，否则不足以导致KS。HIV-1转录反式激活因子(TAT)蛋白被认为与KS有关，因为它刺激KS细胞和内皮细胞(ECs)在培养和TAT转基因小鼠中的侵袭和增殖特性。在初步研究中，我们发现TAT激活磷脂酰肌醇3‘-激酶(PI3K)和Akt，后者是肿瘤发生和细胞存活的关键调节因子。我们发现固定化的TAT和生理水平的TAT促进了KS SLK细胞的存活。目前尚不清楚TAT是如何促进细胞存活的，也不知道这种作用在多大程度上促进了KS的生长。我们假设HIV-1Tat激活Akt并促进细胞存活。我们对TAT和我们独有的Faf转基因小鼠品系的初步研究将使我们能够评估TAT在PI3K-Akt激活和细胞存活中的作用。 具体目的1.确定TAT在体外激活Akt的程度，并确定TAT促进细胞存活的信号通路。我们将对HHV-8感染的真皮微血管内皮细胞(HHV8-OMVE)中与TAT相关的Akt磷酸化和Akt kirtase活性进行表征。我们将使用多肽、截短的TAT突变体和抗体来定位TAT中的活性部分。我们将用药物(LY294002和Perifosine)和PI3K-AM显性-阴性突变体来靶向PI3K-Akt途径，以确定该途径在TAT相关细胞生存中的作用。 具体目的2.通过分析裸鼠和TAT转基因小鼠体内表达Tat的细胞，确定Tat在体内激活Akt和促进肿瘤生长的程度。不同大小的TAT将被表达并定位在对Akt磷酸化至关重要的TAT结构域上。我们还将测试设计的Pisk-Akt抑制剂(LY294002和Perifosine)和候选抗癌药物在阻断这一途径和逆转肿瘤生长方面的作用。 这些研究的成功完成将有助于确定TAT的一种新特性，该特性可以刺激在KS和HHV8-DMVE细胞中发挥主要作用的生存机制。这项研究计划将确定旨在阻断这一途径的药物的潜力，该途径与KS有关，可能还与其他与HIV-1感染有关的癌症有关。

DESCRIPTION (provided by applicant): No convincing explanation exists for the aggressive growth of Kaposi's sarcoma (KS) in the estimated 8 million people worldwide afflicted with both this disease and human immunodeficiency virus type 1 (HIV-1). Human herpesvirus 8 (HHV-8) is required for the development of KS but is insufficient to cause it unless a second factor, such as HIV-1, is present. The HIV-1 transactivator of transcription (Tat) protein is implicated in KS because it stimulates the invasive and proliferative properties of KS cells and endothelial cells (ECs) in cultures and in tat transgenic mice. In preliminary studies, we found that Tat activates phosphatidylinositol 3'-kinase (PI3K) and Akt, the latter is a key regulator in tumorigenesis and cell survival. We found that immobilized Tat and physiologic levels of Tat in solution promoted the survival of KS SLK cells. It is not known how Tat promotes cell survival or to what extent this effect promotes growth of KS. We hypothesize that HIV-1 Tat activates Akt and promotes cell survival. Our preliminary studies with Tat and our exclusive faf transgenic mice lines will enable us to evaluate Tat's role in PI3K-Akt activation and cell survival. Specific Aim 1. Determine to what extent Tat activates Akt In vitro, and identify the signaling pathway Tat uses to promote cell survival. We will characterize Tat-related Akt phosphorylation and Akt kirtase activity in HHV-8 infected dermal microvasculature endothelial (HHV8-OMVE) cells. We will map the active moiety in Tat by using peptides, truncated Tat mutants, and antibodies. We will target the PI3K-Akt pathway with drugs (LY294002 and Perifosine) and PI3K-AM dominant-negative mutants to determine the role of this pathway in Tat-related cell survival. Specific Aim 2. Determine to what extent Tat activates Akt In vivo and promotes tumor growth by analyzing Tatexpressing cells In nude mice and tat transgenic mice. Tat of different sizes will be expressed and mapped on Tat domains critical to Akt phosphorylation. We will also test designed PISK-Akt inhibitors (LY294002 and Perifosine) and candidate anticancer agents for their role in blocking this pathway and reversing tumor growth. Successful completion of these studies will help identify a novel property of Tat that stimulates a survival mechanism anticipated to play a major role in KS and HHV8-DMVE cells. This research plan will establish the potential of drugs designed to interrupt this pathway implicated in KS and perhaps in other cancers associated with HIV-1 infection.