Alesci Salvatore, Perera Shiromi M, Lai Edwin W, Kukura Christina, Abu-Asab Mones, Tsokos Maria, Morris John C, Pacak Karel
Clinical Neuroendocrinology Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892, USA.
Endocrinology. 2007 Aug;148(8):3900-7. doi: 10.1210/en.2007-0521. Epub 2007 May 24.
Recombinant adenoviruses (rAd) have been widely used as gene transfer vectors both in the laboratory and in human clinical trials. In the present study, we investigated the effects of adenoviral-mediated gene transfer in primary bovine adrenal chromaffin cells (BACC) and a murine pheochromocytoma cell line (MPC). Cells were infected with one of three nonreplicating E1/E3-deleted (E1(-)/E3(-)) rAd vectors: Ad.GFP, expressing a green fluorescent protein (GFP); Ad.null, expressing no transgene; or Ad.C2.TK, expressing the herpes simplex virus-1 thymidine kinase gene (TK). Forty-eight hours after exposure to Ad.GFP, the percentage of GFP-expressing BACC ranged from 23.5-97% in a dose-dependent manner and similarly from 1.06-84.4% in the MPC, indicating that adrenomedullary cells are a potentially valuable target for adenoviral-mediated gene transfer. Ultrastructural analysis, however, revealed profound changes in the nucleus and mitochondria of cells infected with rAd. Furthermore, infection of BACC with Ad.null was accompanied by a time- and dose-dependent decrease in cell survival due to the vector alone. Specific whole-cell norepinephrine uptake was also decreased in a time- and dose-dependent fashion in BACC. Infection of MPC cells with the Ad.C2.TK vector sensitized them to the cytotoxic effect of the antiviral drug ganciclovir, in direct proportion to the fraction of cells infected with the virus. We conclude that rAd may alter the structural and functional integrity of adrenomedullary cells, potentially interfering with the normal stress response. At the same time, in light of their ability to effectively deliver and express genes in pheochromocytoma cells, they may be applicable to the gene therapy of adrenomedullary tumors.
重组腺病毒（rAd）已在实验室和人体临床试验中广泛用作基因转移载体。在本研究中，我们研究了腺病毒介导的基因转移对原代牛肾上腺嗜铬细胞（BACC）和小鼠嗜铬细胞瘤细胞系（MPC）的影响。细胞用三种非复制型E1/E3缺失（E1(-)/E3(-)）rAd载体之一进行感染：Ad.GFP，表达绿色荧光蛋白（GFP）；Ad.null，不表达转基因；或Ad.C2.TK，表达单纯疱疹病毒1型胸苷激酶基因（TK）。暴露于Ad.GFP 48小时后，表达GFP的BACC百分比以剂量依赖方式在23.5 - 97%范围内，在MPC中类似地在1.06 - 84.4%范围内，表明肾上腺髓质细胞是腺病毒介导的基因转移的潜在有价值靶点。然而，超微结构分析揭示了感染rAd的细胞的细胞核和线粒体有深刻变化。此外，单独的载体Ad.null感染BACC伴随着细胞存活率的时间和剂量依赖性降低。BACC中特异性全细胞去甲肾上腺素摄取也以时间和剂量依赖方式降低。用Ad.C2.TK载体感染MPC细胞使它们对抗病毒药物更昔洛韦的细胞毒性作用敏感，与感染病毒的细胞比例成正比。我们得出结论，rAd可能改变肾上腺髓质细胞的结构和功能完整性，潜在地干扰正常应激反应。同时，鉴于它们在嗜铬细胞瘤细胞中有效递送和表达基因的能力，它们可能适用于肾上腺髓质肿瘤的基因治疗。
