MAPK AND LYTIC FUNCTION IN HUMAN NK CELLS

细胞毒是自然杀伤细胞和T细胞杀伤肿瘤细胞的关键策略，但其分子基础尚不清楚。信号分子分析表明，丝裂原活化蛋白激酶(MAPK)/ERK2关键控制着NK的直接裂解。肿瘤结扎诱导人NK细胞MAPK酪氨酸磷酸化和酶活性。此外，PD098059或显性阴性ERK2的药物抑制MAPK的失活干扰了51-Cr标记的肿瘤细胞的NK裂解，并中断了穿孔素/颗粒酶B的极化。因此，重要的是确定哪些信号分子控制MAPK的激活，目标是一旦确定了这一途径，就可能找到在癌症患者中增强这一途径的方法。初步研究表明，NK利用不依赖ras的MAPK级联途径来杀伤肿瘤细胞。法尼基转移酶抑制剂FTI-277或显性阴性的N17ras表达对肿瘤细胞的NK裂解、穿孔素/颗粒酶B动员或MAPK激活没有不良影响。相反，肿瘤细胞在NK细胞中激活与ras相关的蛋白Rap1a，使其成为GTP形式。阻断香叶化的GGTI-298有效地抑制了NK细胞的裂解能力和Rap1A的激活。这一建议是为了验证Rap1A是MAPK上游效应因子的假设，目的1是确定Rap1A/B-RAF/MEK/MAPK通路是否关键地控制着裂解过程。或者，其他途径也可以送入MAPK来代表重叠或分离的功能。目的2分析PI3K和Syk70在MAPK激活中的作用，目的3提出抑制性NK受体，如KIR，通过MAPK失活功能的假说。目的4是确定MAPK是否通过调节微管重组来控制穿孔素的运动。该实验计划遵循深入的生化和遗传学方法，使用药理学试剂和突变基因结构，以确定MAPK激活的特定作用。明确一种独特的ras非依赖的NK裂解过程可能为针对ras转化的肿瘤而不是患者的NK功能的治疗提供新的策略。

Cytotoxicity is a key strategy utilized by natural killer (NK) and T cells to kill tumor cells, but its molecular basis is unclear. Analysis of signal molecules indicated that mitogen-activated protein kinases (MAPK)/ERK2 critically controlled direct NK lysis. Tumor ligation in human NK cells induced MAPK tyrosine phosphorylation and enzymatic activity. Moreover, MAPK inactivation by pharmacological inhibition with PD098059 or dominant-negative ERK2 expression interfered with NK lysis of 51-Cr labeled tumor cells, and interrupted perforin/granzyme B polarization. It is thus important to define what signal molecules control MAPK activation, with the goal of possibly finding means to enhance this pathway in cancer patients, once it is identified. Preliminary studies indicated that NK utilized a ras-independent MAPK cascade to lyse tumor cells. Farnesyl transferase inhibitor, FTI-277, or dominant-negative N17ras expression, had no deleterious effect on NK lysis of tumor cells, perforin/granzyme B mobilization, or MAPK activation. Instead, tumor cells activate a ras related protein, rap1A, to its GTP form, in NK cells. GGTI-298, which blocks geranylation, a process used by rap1A for membrane localization, effectively inhibited NK lytic capacity and rap1A activation. This proposal is to test the hypothesis that rap1A is an upstream effector of MAPK, and Aim 1 is to identify if the rap1A/B-raf/MEK/MAPK pathway critically controls the lytic process. Alternatively, other pathways may feed into MAPK to represent overlapping or separate functions. Aim 2 is analyze the roles of PI 3-kinase and Syk70 in MAPK activation and Aim 3 addresses the hypothesis that inhibitory NK receptors, such as KIR, inactivate function via MAPK. Aim 4 is to identify if MAPK control of perforin movement is via regulation of microtubule reorganization. The experimental plan follows an in-depth biochemical and genetic approach, using pharmacological agents and mutant gene constructs, to identify specific plays in MAPK activation. Identification of a distinct ras- independent NK lytic process could provide a new strategy for therapeutics that specifically targets ras-transformed tumors and not NK function in patients.