Simultaneous up-regulation of viral receptor expression and DNA synthesis is required for increasing efficiency of retroviral hepatic gene transfer.
Simultaneous up-regulation of viral receptor expression and DNA synthesis is required for increasing efficiency of retroviral hepatic gene transfer.
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为了提高逆转录病毒肝基因转移的效率，需要同时上调病毒受体表达和 DNA 合成。
DOI：
10.1074/jbc.273.19.11954
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Gupta,S
中科院分区：
文献类型：
--
作者：
Ott,M;Stockert,RJ;Ma,Q;Gagandeep,S;Gupta,S
为了了解逆转录病毒基因转移中病毒受体表达和细胞增殖的相对贡献，我们建立了人肝细胞衍生的HuH-7.MCAT-1细胞系。这些细胞组成型表达鼠亲嗜性逆转录病毒受体MCAT-1，而形态或增殖状态无变化。MCAT-1受体也是一种阳离子氨基酸转运蛋白，HuH-7.MCAT-1.7细胞对阳离子氨基酸l-精氨酸和l-赖氨酸的摄取和稳态积累的Vmax增加。在HuH-7.MCAT-1细胞中，去甲肾上腺素和地塞米松显著上调L-精氨酸摄取，肝细胞生长因子也沿着细胞DNA合成增加L-精氨酸摄取。在与亲嗜性LacZ逆转录病毒孵育的HuH-7.MCAT-1.7细胞中，基因转移也显著增加，并且这进一步增加了激素和肝细胞生长因子。为了确定病毒受体上调本身是否增加了基因转移，细胞周期被表达Mad转录因子（AdMad）的重组腺病毒抑制，所述Mad转录因子是显性负性c-Myc调节因子。这将细胞限制在G 0/G1期，而不减弱MCAT-1活性，如分别通过流式细胞术和l-精氨酸摄取分析所示。当异步循环的HuH-7.MCAT-1.7细胞首先用AdMad病毒感染，然后暴露于亲嗜性LacZ病毒时，基因转移实际上被消除。这些数据表明，虽然病毒受体的上调可以大大增强逆转录病毒介导的基因转移，DNA合成仍然是用这种方法进行肝基因治疗的绝对要求。
To understand the relative contribution of viral receptor expression and cell proliferation in retroviral gene transfer, we created human hepatocyte-derived HuH-7.MCAT-1 cell lines. These cells constitutively express the murine ecotropic retroviral receptor MCAT-1 without changes in morphology or proliferation states. The MCAT-1 receptor is also a cationic amino acid transporter, and the HuH-7.MCAT-1.7 cells showed increasedVmaxof uptake and steady-state accumulation of the cationic amino acidsl-arginine andl-lysine. In HuH-7.MCAT-1 cells,l-arginine uptake was significantly up-regulated by norepinephrine and dexamethasone, and hepatocyte growth factor also increasedl-arginine uptake along with cellular DNA synthesis. Gene transfer was also markedly increased in HuH-7.MCAT-1.7 cells incubated with an ecotropic LacZ retrovirus, and this further increased with hormones and hepatocyte growth factor. To define whether viral receptor up-regulation by itself increased gene transfer, cell cycling was inhibited by a recombinant adenovirus expressing the Mad transcription factor (AdMad), which is a dominant-negative c-Myc regulator. This restricted cells in G0/G1, without attenuating MCAT-1 activity, as shown by flow cytometry andl-arginine uptake analysis, respectively. When asynchronously cycling HuH-7.MCAT-1.7 cells were first infected with the AdMad virus and then exposed to the ecotropic LacZ virus, gene transfer was virtually abolished. The data indicate that while up-regulation of viral receptors can greatly enhance retrovirally mediated gene transfer, DNA synthesis remains an absolute requirement for hepatic gene therapy with this approach.