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，逃离免疫监视系统是骨肉瘤细胞获得的基本能力之一。最近的研究报道，一些癌细胞可以通过Fas受体功能的丧失而逃离细胞毒性t淋巴细胞，并可能通过Fas配体功能的获得（Fas L）来进行反击。我们研究了Fas和Fas L在人骨肉瘤中的地位和功能，以阐明免疫逃逸在人骨肉瘤中的分子机制。利用RT-PCR和Western blot技术，在已建立的3种人骨肉瘤细胞系（HOS、OST和Saos2）和3种骨肉瘤组织中证实了Fas和Fas L在信息和蛋白水平上的表达。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两种形态。可溶性Fas是F的主要形式，在已建立的骨肉瘤细胞系中产生，只有OST不仅表达可溶性Fas，而且表达膜结合形式，免疫荧光研究证实了这一点。免疫组化研究显示22例骨肉瘤中有17例表达Fas。11例（65%）骨肉瘤为膜型，6例（35%）为细胞质型。Fas配体在已建立的骨肉瘤细胞和组织中的表达都是膜结合的，完全没有检测到可溶性Fas L。免疫组化显示Fas L过表达17例（85%）。Fas L的表达与分期、化疗敏感性、预后等临床病理指标无明显相关性。TUNEL-CD8染色结果显示，Fas - L的表达状态与肿瘤组织浸润的细胞毒性t淋巴细胞凋亡数量无关。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是通过观察Fas敏感的Jurkat细胞的凋亡诱导，这些细胞分别培养在骨肉瘤组织的冷冻切片或已建立的骨肉瘤细胞系的单层上。中和性抗Fas L抗体（NOK1）孵育可明显抑制细胞凋亡，表明细胞凋亡是通过Fas/Fas L通路发生的。阿霉素诱导心肌病的凋亡机制是通过心肌细胞Fas的过度表达。这些结果表明，Fas和FasL的状态可能反映了人骨肉瘤的重要现象，骨肉瘤细胞可以通过Fas的缺失和FasL的获得来逃避免疫监视。调节Fas和Fas L的表达可能是治疗骨肉瘤的新策略之一。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