The presence of human coxsackievirus and adenovirus receptor is associated with efficient adenovirus-mediated transgene expression in human melanoma cell cultures
The presence of human coxsackievirus and adenovirus receptor is associated with efficient adenovirus-mediated transgene expression in human melanoma cell cultures
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DOI：
10.1089/hum.1998.9.16-2363
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发表时间：
1998-11-01
影响因子：
4.2
通讯作者：
Dummer, R
中科院分区：
文献类型：
--
作者：
Hemmi, S;Geertsen, R;Dummer, R
腺病毒（AdV）介导的免疫刺激基因表达代表了人类癌症基因治疗的一种有价值的体内方法。治疗基因的表达水平对于此类治疗的功效至关重要。 AdV 的进入依赖于初级腺病毒受体 CAR 和早期鉴定为表面分子整合素家族成员的次级 AdV 受体。我们分析了来自不同阶段的 14 种不同的人类黑色素瘤细胞培养物以及一种黑色素瘤细胞系的 AdV 介导的转导和表达效率。使用不同浓度的重组病毒在不同启动子的控制下表达B7-1共刺激分子，并通过流式细胞术分析B7-1的表达水平，使用商业ELISA测量AdV介导的IL-12表达，将转基因表达水平与HCAR、α(v)β(3)和α(v)β(5)整联蛋白以及HLA I类的表达水平进行比较。在测试的 14 个细胞培养物中，当使用表达 B7-1 和 IL-12 的病毒以相对较低的感染复数 (MOI) 50 时，初级病毒受体 CAR 的存在与高转导效率表型相关，原始活检冰冻切片上的免疫组织化学产生了 CAR 特异性的强信号。相比之下，表达低水平或不可检测水平的 CAR 的细胞培养物需要高 20 至 40 倍的病毒输入才能显示可比的 B7-1 或 IL-12 表达水平。当使用不同的启动子时，转基因的表达水平几乎没有变化，并且没有观察到与HLA I类分子的存在或不存在或整联蛋白的表达水平相关。
Adenovirus (AdV)-mediated gene expression of immune stimulators represents a valuable in vivo approach for gene therapy of human cancer. The expression level of the therapeutic gene is of crucial importance for the efficacy of this type of treatment. Entry of AdV is dependent on the primary adenovirus receptor CAR and the secondary AdV receptor identified earlier to be a member of the integrin family of surface molecules. We have analyzed 14 different human melanoma cell cultures from different stages together with one melanoma cell line for their AdV-mediated transduction and expression efficiency. Recombinant viruses at various concentrations were used for expression of the B7-1 costimulatory molecule under the control of different promoters and the expression levels of B7-1 were analyzed by flow cytometry, AdV-mediated IL-12 expression was measured using a commercial ELISA, Levels of transgene expression were compared with the expression levels of HCAR, the alpha(v)beta(3) and alpha(v)beta(5) integrins, and HLA class I. In 4 of 14 cell cultures tested, the presence of the primary virus receptor CAR was associated with the high transduction efficiency phenotype when using the B7-1- and IL-12-expressing viruses at a relatively low multiplicity of infection (MOI) of 50, Immunohistochemistry on cryosections from the original biopsies yielded a strong signal specific for CAR. In contrast, cell cultures expressing low or undetectable levels of CAR needed a 20- to 40-fold higher viral input to show comparable expression level of B7-1 or IL-12. Expression levels of the transgenes hardly varied when using different promoters and no association was observed with the presence or absence of HLA class I molecules or with the expression levels of integrins.