Inhibition of Human Natural Killer Cells by Butyltins

拟议研究的目的是阐明丁基锡(BTS)(广泛存在)的机制 环境污染物)会降低人类自然杀伤(NK)细胞的功能。人类 大量接触BTS，在人类血液和其他组织中发现BTS。BTS降低 人类自然杀伤(NK)细胞的功能，这是对抗癌症的主要免疫防御。有重要意义的是 Bt诱导的丝裂原活化蛋白激酶(MAPKs)的激活。MAPK是摧毁肿瘤所必需的 NK细胞的功能。因此，虚假的Bt诱导的MAPKs激活可能会离开NK细胞 对后续的肿瘤细胞无反应。假设MAPK激活通路的改变是 Bt诱导的NK功能丧失的核心将被解决。以下具体目标旨在 研究这一假设：1.确定Bt(三丁基锡和二丁基锡)暴露对Key的影响(S) 每一类MAPK(p44/42、p38和JNK)的上游激活信号蛋白及其生理活性 MAPK的抑制剂(MAPK磷酸酶)。待检测的上游激活信号为单体 GTP结合蛋白(ras和rac)、MAPK激酶(MAP3K)(RAF-1和ASK-1)和MAPK激酶 (MAP2K)(MEK1/2、MKK3/6、MKK4和MKK7)。小G蛋白的激活状态将使用 亲和结合分析和免疫印迹；激活MAP3Ks和MAP2Ks的磷酸化将 用蛋白印迹和蛋白水解酶分析进行检测。将使用磷酸酶检测来监测 MAPK和MAP2K磷酸酶(PPA-2/PP1和MKP-1)的激活状态2.确定是否直接 选择性p44/42和p38/JNK途径激活剂激活MAPK通路对NK细胞的影响 细胞毒功能、细胞溶解蛋白表达和细胞表面蛋白表达类似于 与Bt接触有关。细胞毒性功能的测定使用铬释放试验，细胞溶解蛋白 用免疫印迹法(用RT-PCR法测定mRNA水平)检测表达，细胞表面 用流式细胞术检测蛋白质的表达。3.确定是否通过直接抑制MAPK通路 选择性抑制剂能阻断Bt暴露对MAPKs、NK细胞毒功能、细胞溶解功能的影响 蛋白质表达和细胞表面蛋白表达，使用蛋白质印迹和AIM中描述的方法 2.4.确定Bt暴露对MAPKs调控的转录调控因子的影响。这些 包括ELK-1磷酸化、JUN磷酸化以及JUN和Fos的总体水平。磷酸化 转录调节子的状态和水平将使用蛋白质印迹和RT-PCR进行评估；结合的能力 DNA成分将使用ELISA法和EMSA法进行评估。拟议的研究将阐明这一角色 环境污染物，如BTS，可能通过它们的能力在增加癌症易感性方面发挥作用 以削弱NK细胞提供的对癌症的关键免疫防御。

The objective of the proposed studies is to elucidate the mechanism by which butyltins (BTs) (widespread environmental contaminants) decrease the functional capacity of human natural killer (NK) cells. Humans have significant exposure to BTs, and they are found in human blood and other tissues. BTs reduce the function of human natural killer (NK) cells, a primary immune defense against cancer. There is significant BT-induced activation of mitogen activated protein kinases (MAPKs). MAPKs are required for the tumordestroying function of NK cells. Thus, spurious BT-induced activation of MAPKs could leave the NK cell unresponsive to subsequent tumor cells. The hypothesis that alterations of MAPK activation pathways are central to the BT-induced loss of NK function will be addressed. The following specific aims are designed to investigate this hypothesis: 1. Determine the effect(s) of BT (tributyltin and dibutyltin) exposures on key upstream activating signaling proteins for each class of MAPK (p44/42, p38, and JNK) and the physiological inhibitors of MAPKs (MAPK phosphatases). The upstream activating signals to be examined are monomeric GTP-binding proteins (ras and rac), MAPK kinase kinases (MAP3K) (raf-1 and ASK-1), and MAPK kinases (MAP2K) (MEK1/2, MKK3/6, MKK4, and MKK7). Activation states of small G proteins will be monitored using an affinity binding assay followed by western blot; activating phosphorylations of MAP3Ks and MAP2Ks will be measured using western blot and kinase assays. A phosphatase assay will be used to monitor the activation state of the MAPK and MAP2K phosphatases (PPA-2/PP1 and MKP-1) 2. Determine if direct activation of MAPK pathways by selective p44/42 an p38/JNK pathway activators is able to affect NK cytotoxic function, cytolytic protein expression, and cell surface protein expression in a manner similar to that seen with BT exposures. Cytotoxic function is measured using a chromium release assay, cytolytic protein expression is determined using western blot (mRNA levels are measured using RT-PCR), and cell surface protein expression is measured using flow cytometry. 3. Determine if direct inhibition of MAPK pathways by selective inhibitors is able to block the effects of BT exposures on MAPKs, NK cytotoxic funciton, cytolytic protein expression, and cell surface protein expression, using western blot and the methods described in aim 2. 4. Determine the effects of BT exposures on transcription regulators regulated by MAPKs. These include, ELK-1 phosphorylation, Jun phosphorylation, and overall levels of Jun and Fos. Phosphorylation state and levels of transcription regulators will be assessed using western blot and RT-PCR; ability to bind DNA elements will be assessed using an ELISA and EMSA. The proposed studies will elucidate the role that environmental contaminants, such as BTs, may play in increasing susceptiblity to cancer by their ability to diminish the critical immune defense against cancer that is provided by NK cells.