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)被结构性地表达，并引起浸润性淋巴样细胞的迅速凋亡。FasL对移植组织具有免疫保护作用。然而，我们和其他研究人员报告说，当通过基因操作来表达FasL时，各种类型的肿瘤在诱导明显的炎症和广泛的中性粒细胞渗透后被排斥。因此，我们研究了利用FasL基因转导的肿瘤细胞进行肿瘤治疗的研究。4种FasL基因转导的小鼠肿瘤细胞(神经母细胞瘤Neuro-2a、肺癌3LL、肝癌MH134、纤维肉瘤META)注射到小鼠体内后发生排斥反应，并诱导了较强的抗肿瘤免疫。因此，FasL的抗肿瘤活性不依赖于肿瘤类型。为了阐明表达FasL的肿瘤如何诱导中性粒细胞迁移和消除肿瘤的致瘤性，我们研究了G2(MH134FasL)细胞注射到+/+，lpr^&lt；cg&gt；/lpr^&lt；cg&gt；(LPR^&lt；cg&gt；和GLD/GLD IPR/LPR(GLD/LPR)小鼠体内的行为。G…在+/+小鼠中，中性粒细胞在其周围的广泛渗透后被清除，而在LPR^&lt；CG&Gt；和GLD/LPR小鼠中则形成了无此浸润的肿瘤。这些结果表明，FasL表达肿瘤的中性粒细胞的凋亡依赖于Fas，并可能触发中性粒细胞的广泛渗透，导致猛烈的炎症，最终导致+小鼠肿瘤细胞的根除。通过将G2细胞与MH134细胞(Fas/FasL阴性肿瘤模型)或F6B细胞(MH134+Fas)混合后注射到小鼠体内，检测其抗肿瘤作用。在肿瘤细胞周围广泛的中性粒细胞渗透后，肿瘤细胞的混合物被根除。G2细胞对F6B细胞的抗肿瘤作用极强于亲本细胞(MH134)。这些结果提示，表达FasL的肿瘤细胞对未转染的肿瘤细胞具有旁观者效应。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被转基因的cDNAs。另一方面，对治疗中出现的耐药细胞的抑制对于肿瘤基因治疗的增强是重要的。为了解决这一问题，我们检测了抗Fas单抗对F6B细胞和抗Fas单抗耐药的F6B细胞的抗肿瘤作用。在抗Fas单抗的攻击下，通过调控Fas c DNA使Fas的表达减少或消失，从而使耐药细胞存活。综上所述，我们的研究结果表明，将FasL基因导入肿瘤细胞是一种诱导肿瘤细胞产生强大抗肿瘤作用的良好方法，具有较大的临床应用价值。较少

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