Basic study for. Cancer Gene Therapy by Antitumor Effects Using Fas-Fas Ligand-Mediated Apoptosis

在免疫特权部位，FasL（配体）组成性表达并导致浸润的淋巴样细胞迅速凋亡。FasL可以保护移植物组织免受免疫介导的损伤。然而，我们和其他研究人员报道，当基因操纵表达FasL时，各种类型的肿瘤在诱导具有广泛中性粒细胞浸润的明显炎症后被排斥。因此，我们研究了使用fasl转染的肿瘤细胞治疗癌症。FasL cdna转染的4种小鼠肿瘤细胞（神经母细胞瘤neuro2a、肺癌3LL、肝癌MH134、纤维肉瘤MethA）注射小鼠后产生排斥反应，并诱导较强的抗肿瘤免疫。因此，FasL的抗肿瘤活性不依赖于肿瘤类型。为了研究表达FasL的肿瘤如何诱导中性粒细胞迁移和消除致瘤性，我们研究了G2 （MHl34+FasL）细胞注射到+/+、lpr^<cg>/lpr^<cg>(lpr^<cg>)和gld/gld Ipr/lpr （gld/lpr）小鼠体内的行为。在lpr^<cg>和gld/lpr小鼠中，细胞周围有大量中性粒细胞浸润后，2个以上的细胞被根除，而在lpr^<cg>和gld/lpr小鼠中则没有这种浸润形成肿瘤。这些结果表明，表达fasl的肿瘤中中性粒细胞的凋亡依赖于Fas，并可能引发中性粒细胞的广泛浸润，导致剧烈炎症，最终导致+小鼠肿瘤细胞的根除。将G2细胞与MH134细胞（Fas/ fasl阴性肿瘤模型）或F6b细胞（MH134+Fas）混合注射小鼠体内，观察其抗肿瘤作用。肿瘤细胞的混合物在其周围广泛浸润中性粒细胞后被根除。G2细胞对F6b细胞的抗肿瘤作用明显强于对亲本细胞（MH134）的抗肿瘤作用。这些结果表明，fasl表达的肿瘤对未转染的肿瘤细胞具有抗肿瘤活性。这似乎对临床应用是有用的，因为并不是所有的肿瘤细胞都能转染转染的cDNA。另一方面，抑制治疗中出现的耐药细胞对肿瘤基因治疗的扩大具有重要意义。为了解决这一问题，我们通过抗fas单抗检测了F6b细胞和抗fas单抗F6b细胞的抗肿瘤作用。在抗Fas单抗的攻击下，通过调节Fas cDNA，使Fas表达减少或耗尽，使耐药细胞存活。综上所述，我们的研究结果表明，将FasL转染到肿瘤细胞中是一种诱导强抗肿瘤作用的好方法，可能对临床应用有很大的帮助。少

In the immunologically privileged sites, FasL (ligand) is constitutively expressed and causes prompt apoptosis of infiltrating lymphoid cells. FasL can protect the grafted tissue from immune-mediated damage. However, we and other researchers reported that various types of tumors, when genetically manipulated to express FasL, were rejected after inducing marked inflammation with extensive neutrophil infiltrate. Thus, we investigated cancer therapy using FasL-transfected tumor cells.Four kinds of FasL cDNA-transfected murine tumor cells (neuroblastoma Neuro-2a, lung carcinoma 3LL, hepatoma MH134, fibrosarcoma MethA) were rejected when injected into mice, and induced strong antitumor immunity. Thus, the antitumor activity of FasL did not depend on tumor type. To address how FasL-expressing tumors induce neutrophil emigration and abrogate tumorigenicity, we investigated the behavior of G2 (MHl34+FasL) cells injected into +/+, lpr^<cg>/lpr^<cg>(lpr^<cg> and gld/gld Ipr/lpr (gld/lpr) mice. G … More 2 cells were eradicated after extensive infiltration of neutrophils around them in +/+ mice but formed tumors without such infiltration in lpr^<cg> and gld/lpr mice. These results indicate that apoptosis of neutrophils with FasL-expressing tumors depends on Fas and may trigger the extensive infiltration of neutrophils resulting in violent inflammation and ultimately in eradication of tumor cells in + mice. The antitumor effect was examined by mixing G2 cells with MH134 cells (model of Fas/FasL-negative tumor) or F6b (MH134+Fas) and injecting into mice. The mixture of tumor cells was eradicated after extensive infiltration of neutrophils around them. The antitumor effect of G2 cells against F6b cells was extremely stronger than that against parent cells (MH134). These results suggested that FasL-expressing tumor exerted antitumor activity against un-transfected tumor cells by-stander effects. This seems to be useful for the clinical application, because all tumor cells could not be always transfected with transfected cDNA. On the other hand, the suppression of resistant cells which appear in treatment is important for augmentation in tumor-gene therapy. To solve this problem, we examined the antitumor effect of F6b cells and anti-Fas Mab-resisant F6b cells by anti-Fas Mab. The resistant cells could survive by modulating Fas cDNA which resulted in the decrease or the depletion of Fas expression for the attack by the anti-Fas Mab.Collectively, our results indicate that transfection of FasL into tumor cells is a good method for the induction of strong antitumor effects and might be much useful for clinical application. Less