Interaction between signaling molecules and HSP90 on apoptosis pathway in HSG cells

近年来，研究发现许多信号分子都受HSP家族蛋白的调控，这些蛋白具有分子伴侣的功能。本研究旨在探讨信号分子与热休克蛋白90（HSP 90）在人涎腺腺癌细胞（HSG）凋亡途径中的相互作用。另一方面，单独的激动性Fas抗体在30%的细胞中诱导凋亡，TNF-α处理后，激动性Fas抗体在60%的细胞中诱导凋亡。RT-PCR分析TNF-α处理后细胞凋亡信号分子Fas的高表达。提示TNF-α诱导的Fas表达增强了Fas抗体介导的细胞凋亡。caspase-8和caspase-3均参与Fas抗体诱导的HSG细胞凋亡。虽然激动性Fas抗体显示30%的细胞凋亡，但格尔德霉素和激动性Fas抗体的组合增加到90%的细胞。提示HSP 90参与了细胞凋亡信号分子的调控。Western blotting分析显示，Fas抗体刺激后，HSP 90 β蛋白表达增加，而HSP 90 α蛋白表达无明显变化。此外，对HSG细胞进行蛋白质组分析。双向凝胶电泳上观察到的HSP 90蛋白斑点在激动性Fas抗体刺激后向更酸性的方向移动，表明HSP 90的磷酸化。

Recently, it has been reported that a number of signaling molecules are regulated by the HSP family proteins which function as molecular chaperones. The present study focused on the interaction between signaling molecules and HSP90 on apoptosis pathway in the human salivary gland adenocarcinoma cell line (HSG).In HSG cells, TNF-α alone did not induce apoptosis. On the other hand, agonistic Fas antibody alone induced apoptosis in 30 % of the cells and TNF-α treatment followed by agonistic Fas antibody induced apoptosis in 60 % of the cells. RT-PCR analysis of apoptotic signaling molecules after TNF-α treatment revealed the highly expression of Fas. Thus, it was suggested that Fas increased by TNF-α enhanced the agonistic Fas antibody-mediated apoptosis. From the results of the experiment with inhibitors for caspases which play important roles for apoptotic pathway, both caspase-8 and caspase-3 are involved in the agonistic Fas antibody-induced apoptosis.HSG cells were treated with geldanamycin which is a specific inhibitor for HSP90. While agonistic Fas antibody showed apoptosis in 30 % of the cells, the combination of geldanamycin and agonistic Fas antibody increased to 90 % of the cells. This result suggested that HSP90 participates in the regulation of apoptotic signaling molecules. Western blotting analysis after the agonistic Fas antibody stimulation showed that the protein level of HSP90β isoform increased while that of HSP90α did not changed. Furthermore, proteome analysis of HSG cells was performed. Protein spots of HSP90 observed on the two-dimensional gel electrophoresis were shifted to the more acidic after the agonistic Fas antibody stimulation, suggesting the phosphorylation of HSP90.