Inhibition of Human Natural Killer Cells by Butyltins

丁基锡对人类自然杀伤细胞的抑制

• 批准号: 7597191
• 负责人: MARGARET M WHALEN
• 金额: $ 16.49万
• 依托单位: TENNESSEE STATE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7597191
• 关键词: Activities of Daily Living; Address; Affect; Affinity; Animals; Binding; Biological Assay; Blood; Blood specimen; Categories; Cell physiology; Cell surface; Cells; Class; DNA Binding; DUSP1 gene; Development; ELK1 gene; EMSA; Electrophoretic Mobility Shift Assay; Elements; Environment; Environmental Pollution; Enzyme-Linked Immunosorbent Assay; Exposure to; Flow Cytometry; Genetic Transcription; Goals; Human; Immune; Immune system; Incidence; Individual; Industrial Product; Intake; Japanese Population; Left; Liver; MAP Kinase Kinase Kinase; MAP2K1 gene; MAPK14 gene; MAPK8 gene; Malignant Neoplasms; Marines; Measurable; Measures; Membrane Proteins; Messenger RNA; Methods; Mitogen-Activated Protein Kinase Inhibitor; Mitogen-Activated Protein Kinases; Molecular; Molecular Target; Monitor; Monomeric GTP-Binding Proteins; Natural Killer Cells; Occupational Exposure; Occupations; Organotin Compounds; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Play; Process; Proteins; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Signal Transduction; Signaling Protein; Techniques; Tissues; Toxic effect; Virus Diseases; Western Blotting; Work; cell mediated lymphocytolysis test; cytotoxic; day; design; di-n-butyltin; human MAP2K1 protein; immune function; inhibitor/antagonist; neoplastic cell; protein expression; tributyltin; tumor

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.

拟议研究的目的是阐明丁基锡（BTs）（广泛分布） 环境污染物）降低人自然杀伤（NK）细胞的功能能力。人类 有大量的BT暴露，它们存在于人体血液和其他组织中。BT减少了 人类自然杀伤（NK）细胞的功能，对癌症的主要免疫防御。存在显著 有丝分裂原活化蛋白激酶（MAPK）的BT诱导活化。MAPKs是破坏肿瘤所必需的 NK细胞的功能。因此，假的BT诱导的MAPKs激活可以离开NK细胞， 对随后的肿瘤细胞无反应。MAPK激活途径的改变是 将解决BT诱导的NK功能丧失的核心问题。以下具体目标旨在 研究这个假设：1。确定BT（三丁基锡和二丁基锡）暴露对关键 上游活化信号蛋白的每一类MAPK（p44/42，p38，和JNK）和生理 MAPK（MAPK磷酸酶）抑制剂。待检测的上游激活信号是单体的 GTP结合蛋白（ras和rac）、MAPK激酶激酶（MAP 3 K）（raf-1和ASK-1）和MAPK激酶 （MAP2K）（MEK 1/2、MKK 3/6、MKK 4和MKK 7）。小G蛋白的激活状态将使用 亲和结合测定，然后进行蛋白质印迹;激活MAP 3 K和MAP 2K的磷酸化将 使用蛋白质印迹和激酶测定来测量。将使用磷酸酶测定来监测 MAPK和MAP 2K磷酸酶（PPA-2/PP 1和MKP-1）的活化状态2.确定是否直接 选择性p44/42和p38/JNK通路激活剂激活MAPK通路能够影响NK细胞， 细胞毒性功能、溶细胞蛋白表达和细胞表面蛋白表达，其方式类似于 在BT暴露中观察到。使用铬释放试验、细胞溶解蛋白测定和细胞毒性试验测量细胞毒性功能。 使用蛋白质印迹法测定表达（使用RT-PCR测量mRNA水平），并且细胞表面 使用流式细胞术测量蛋白质表达。3.确定是否直接抑制MAPK途径， 选择性抑制剂能够阻断BT暴露对MAPKs、NK细胞毒性功能、细胞溶解功能和细胞毒性的影响。 蛋白质表达和细胞表面蛋白质表达，使用蛋白质印迹和目的中描述的方法 2. 4.确定BT暴露对MAPKs调控的转录调节因子的影响。这些 包括ELK-1磷酸化、Jun磷酸化以及Jun和Fos总体水平。磷酸化 使用蛋白质印迹和RT-PCR评估转录调节因子的状态和水平; 将使用ELISA和EMSA评估DNA元素。拟议的研究将阐明 环境污染物，如BTs，可能通过其能力增加癌症的易感性， 以减少NK细胞提供的对抗癌症的关键免疫防御。