Mechanism of immune escape of human osteosarcoma cells through abnormal expressions of Fas and Fas ligand.

骨肉瘤细胞经常转移到肺部。转移过程由几个不同的步骤组成。其中，逃避免疫监视系统是骨肉瘤细胞获得的基本能力之一。近年来研究发现，某些肿瘤细胞可通过Fas受体功能的丧失而逃避细胞毒T淋巴细胞的攻击，并可能通过Fas配体（Fas L）功能的获得而进行反击。本研究旨在探讨Fas和Fas L在人骨肉瘤中的地位和作用，以阐明骨肉瘤免疫逃逸的分子机制.采用RT-PCR和Western blot方法检测人骨肉瘤细胞系HOS、OST和Saos 2及骨肉瘤组织中Fas和Fas L的表达。直接测序未发现结构异常.骨肉瘤组织中Fas呈膜结合型和可溶性。可溶性Fas是F的主要形式 关于我们 如在已建立的骨肉瘤细胞系中产生的，并且只有OST不仅表达可溶性形式，而且表达膜结合形式，这通过免疫荧光研究证实。免疫组化结果显示Fas在22例骨肉瘤中有17例表达。膜型骨肉瘤11例（65%），胞浆型骨肉瘤6例（35%）。3. Fas配体在成骨肉瘤细胞和组织中的表达均为膜结合型，未检测到可溶性Fas L。免疫组化结果显示Fas L过表达17例（85%）。Fas L的表达与临床分期、化疗敏感性及预后无明显相关性。TUNEL-CD8染色显示肿瘤组织中浸润的细胞毒性T淋巴细胞凋亡数与Fas L表达状态无关.将Fas敏感的Jurkat细胞接种于骨肉瘤组织的冰冻切片或已建立的骨肉瘤细胞系的单层上，观察Fas L在骨肉瘤组织和已建立的骨肉瘤细胞系中表达的功能。抗Fas L中和抗体（NOK1）可明显抑制细胞凋亡，表明细胞凋亡是通过Fas/Fas L途径发生的.阿霉素诱导的心肌病是通过Fas过度表达而引起的凋亡机制，提示Fas和Fas L的表达状态可能反映了骨肉瘤的重要现象，骨肉瘤细胞可通过Fas的丢失和FasL的增加而逃避免疫监视，调控Fas和Fas L的表达可能成为骨肉瘤治疗的新策略之一。少

Osteosarcoma cells frequently metastasize to the lungs. Metastatic process is composed of several different steps. Of them, escape from the immune surveilance system is one of the essential ability for osteosarcoma cells to obtain. Recent studies have reported that some carcinoma cells could escape from cytotoxic T-lymphocytes through loss of function of Fas receptor and might counter-attack them through gain of function of Fas ligand (Fas L). We investigated status and function of Fas and Fas L in human osteosarcomas to clarify the molecular mechanism of immune escape working in human osteosarcoma.1. Expressions of Fas and Fas L were demonstrated in established three human osteosarcoma cell lines (HOS, OST and Saos2) and three osteosarcma tissues both at message ad protein levels using RT-PCR and Western blot. No structural abnormality was detected by direct sequencing.2. Osteosarcoma tissues showed Fas of both membrane-bounded and soluble-form. Soluble Fas was a predominant form of F … More as produced in established osteosarcoma cell lines and only OST expressed not only soluble but membrane-bounded form, that was confirmed by immunofluorescent study. Immunohistochemical studies demonstrated expression of Fas in 17 of 22 osteosarcomas. Eleven osteosarcomas (65%) were membrane-type and 6 (35%) in the cytoplasm.3. Fas ligand expressed in both established osteosarcoma cells and tissues was membrane-bounded and no soluble Fas L was detected at all. Immunohistochemistry disclosed overexpression of Fas L in 17 (85%) of 20 cases. No significant correlation was detected between the expression of Fas L and clinicopatholgical parameters including stage, chemosensitivity and prognosis. Status of Fas L expression was not correlated to number of apoptosis of cytotoxic T-lymphocytes infiltrated in the tumor tissues demonstrated by TUNEL-CD8 staining.4. Function of Fas L expressed in osteosarcoma tissues and established osteosarcoma cell lines was demonstrated by the osbervation of apoptosis-induction in Fas-sensitive Jurkat cells which were incuvated on the frozen sections of osteosarcoma tissues or on monolayer of established osteosarcoma cell lines. The apoptosis was significantly inhibited by the incubation with neutralizing anti-Fas L antibody (NOK1), indicating that the apoptosis occurred via Fas/Fas L passway.5. Adriamycin-induced cardiaomyopathy was apoptotic mechanism via overexpession of Fas on the myocardium.These results showed that status of Fas and Fas L might reflect imprtant phenomena in human osteosarcoma and that osteosarcoma cells could escape from immune surveilance through loss of Fas and gain of FasL.Modulation of the Fas and Fas L expressions may be one of new therapeutic strategies against osteosarcomas. Less