Activation of the CMV-IE Promoter by Hyperthermia In Vitro and In Vivo: Biphasic Heat Induction of Cytosine Deaminase Suicide Gene Expression
Activation of the CMV-IE Promoter by Hyperthermia In Vitro and In Vivo: Biphasic Heat Induction of Cytosine Deaminase Suicide Gene Expression
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DOI：
10.1007/s12033-010-9292-3
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发表时间：
2010-10-01
影响因子：
2.6
通讯作者：
Walther, Wolfgang
中科院分区：
文献类型：
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作者：
Kobelt, Dennis;Aumann, Jutta;Walther, Wolfgang
巨细胞病毒即刻早期(CMV-IE)启动子作为一种强启动子被广泛应用。虽然CMV-IE启动子没有热反应序列，但我们确定了它的热诱导性。我们分析了CMV-IE启动子的体内外热反应和热诱导的可能机制。我们用转基因的SW480人结肠癌细胞(SW480/CMVCD)表达了CMV-IE启动子驱动的细菌胞嘧啶脱氨酶(CD)基因。这些细胞在42A℃下加热，SW480/CMVCD细胞也用于体内研究，其中荷瘤动物接受41.5A℃的高温处理。作为对照，使用SW480(SW480/HSPCD)细胞，其中CD表达由HSP70启动子驱动。在体外，我们观察到加热后SW480/CMVCD细胞中CMV-IE驱动的CD表达的双相，高达25倍的热诱导。在体内，我们发现在SW480/CMVCD荷瘤动物中，高温后CD的表达诱导了2.5倍。对CMV-IE启动子序列的分析揭示了几个转录因子结合位点，它们介导了应激反应。YB-1和C/EBP-β可能介导了CMV-IE启动子的热反应。这些数据指出了使用CMV-IE启动子作为控制系统的热诱导基因治疗研究的局限性。此外，CMV-IE启动子本身可以很好地用于构建热诱导载体。
The cytomegalovirus-immediate early (CMV-IE) promoter is widely used as a strong and constitutively active promoter. Although the CMV-IE promoter does not harbor heat-responsive sequences, we determined its heat inducibility. We analyzed in vitro and in vivo heat responsiveness and possible mechanisms of heat induction of the CMV-IE promoter. We used transfected SW480 human colon carcinoma cells (SW480/CMVCD), expressing CMV-IE promoter-driven bacterial cytosine deaminase (CD) gene. These cells were heated at 42A degrees C. The SW480/CMVCD cells were also used for in vivo studies, in which tumor-bearing animals were treated with hyperthermia at 41.5A degrees C. As controls, SW480 (SW480/HSPCD) cells were used, in which CD expression is driven by the HSP70-promoter. In vitro, we observed a biphasic, up to 25-fold heat induction of CMV-IE-driven CD expression after hyperthermia in SW480/CMVCD cells. In vivo, we found a 2.5-fold induction of CD expression after hyperthermia in SW480/CMVCD tumor-bearing animals. The analysis of the CMV-IE promoter sequence revealed several transcription factor-binding sites, which mediate stress responsiveness. YB-1 and C/EBP-beta might mediate heat responsiveness of the CMV-IE promoter. These data point to limitations in heat-induction gene therapy studies, in which the CMV-IE promoter is used as control system. In addition, the CMV-IE promoter itself could well be used for construction of heat-inducible vectors.