Warmann Steven W, Armeanu Sorin, Frank Heike, Buck Heike, Graepler Florian, Lemken Marie-Luise, Heitmann Heike, Seitz Guido, Lauer Ulrich M, Bitzer Michael, Fuchs Jörg
Department of Pediatric Surgery, University of Tübingen, Hoppe-Seyler-Street 3, 72076 Tübingen, Germany.
Pediatr Surg Int. 2006 Jan;22(1):16-23. doi: 10.1007/s00383-005-1573-8.
Poor treatment results in advanced hepatoblastoma (HB) made alternative treatment approaches desirable. Gene-directed tumor therapy is increasingly investigated in different malignancies. The aim of this study was to analyze possible alternatives of gene transfer into HB cells and to study therapeutic applications based on different strategies. Liposomal transfection of HB cells was assessed using liver-specific promoters, and adenovirus and Sendai virus transductions were performed in vitro. Transfer efficiencies were measured via flow cytometry determining expression of vector-encoded marker gene green fluorescent protein. Gene silencing of the anti-apoptotic bcl-2 gene in HUH6 cells was performed using lipofection of small interfering RNA (siRNA). Additionally, suicide gene therapy was carried out through a yeast-derived cytosine deaminase (YCD)-combined yeast uracil phosphoribosyltransferase (YUPRT)-based adenovirus-mediated gene transfer, leading to a potent intracellular prodrug transformation of 5-fluorocytosine into 5-fluorouracil. Treatment efficiencies were monitored via MTT viability assay. Highest gene transfer rates (86%) were observed using adenovirus transduction. We furthermore observed a significant therapeutic effect of adenovirus-mediated YCD::YUPRT suicide gene transfer. Liposomal-mediated anti-bcl-2 siRNA transfer led to a significant improvement of cisplatin treatment in HUH6 cells. Liver-specific promoters were found to be strongly active in HUH6 cells (mixed HB-derived), but less active in HepT1 cells (embryonal HB-derived). Liposomal transfection and viral transduction are effective approaches to genetically manipulate HB cells in vitro. For the first time, we demonstrate a positive effect of siRNA gene silencing in this malignancy. Additionally, we successfully investigated a model of adenovirus-based suicide gene therapy in HB cell cultures. Our data strongly encourage further studies assessing these alternative treatment approaches.
晚期肝母细胞瘤（HB）的治疗效果不佳，使得人们期望采用替代治疗方法。基因导向肿瘤治疗在不同恶性肿瘤中的研究日益增多。本研究的目的是分析将基因导入HB细胞的可能替代方法，并基于不同策略研究其治疗应用。使用肝脏特异性启动子评估HB细胞的脂质体转染，并在体外进行腺病毒和仙台病毒转导。通过流式细胞术测量转染效率，以确定载体编码的标记基因绿色荧光蛋白的表达。使用小干扰RNA（siRNA）脂质体转染在HUH6细胞中进行抗凋亡bcl-2基因的基因沉默。此外，通过基于酵母衍生的胞嘧啶脱氨酶（YCD）联合酵母尿嘧啶磷酸核糖转移酶（YUPRT）的腺病毒介导的基因转移进行自杀基因治疗，导致5-氟胞嘧啶有效转化为5-氟尿嘧啶的细胞内前药转化。通过MTT活力测定监测治疗效率。使用腺病毒转导观察到最高的基因转移率（86%）。我们还观察到腺病毒介导的YCD::YUPRT自杀基因转移具有显著的治疗效果。脂质体介导的抗bcl-2 siRNA转移导致HUH6细胞中顺铂治疗效果显著改善。发现肝脏特异性启动子在HUH6细胞（混合HB来源）中具有强活性，但在HepT1细胞（胚胎HB来源）中活性较低。脂质体转染和病毒转导是在体外对HB细胞进行基因操作的有效方法。我们首次证明了siRNA基因沉默在这种恶性肿瘤中的积极作用。此外，我们成功地在HB细胞培养物中研究了基于腺病毒的自杀基因治疗模型。我们的数据强烈鼓励进一步研究评估这些替代治疗方法。
