Clesham G J, Browne H, Efstathiou S, Weissberg P L
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, UK.
Circ Res. 1996 Dec;79(6):1188-95. doi: 10.1161/01.res.79.6.1188.
Recombinant adenoviral vectors are being used increasingly for gene transfer studies in mammalian cells and gene therapy protocols in humans. High adenoviral titers are often required for successful transduction of vascular smooth muscle cells (VSMCs), defined as uptake and detectable expression of the foreign gene, but the relative contributions of efficiency of viral uptake and control of transcription are poorly understood. To explore the extent to which a lack of detectable gene expression may be due to inefficient transcription of a successfully transferred gene, we have used a replication-deficient adenovirus expressing beta-galactosidase (RAd35 beta-Gal), under the control of the human cytomegalovirus major immediate-early promoter (CMV-IEP), which contains cAMP and nuclear factor-kappa B response elements, to investigate constitutive and inducible gene expression after gene transfer into human VSMCs. Histochemical staining with 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal), a quantitative spectrophotometric assay, SDS-PAGE, Western blotting, and Northern analysis were used to evaluate beta-galactosidase expression in infected cells. After infection with RAd35 beta-Gal at 30, 100, and 1000 plaque-forming units per cell (pfu/cell), expression of beta-galactosidase was augmented up to 17-, 19-, and 23-fold, respectively, in human VSMCs treated with forskolin and phorbol ester compared with unstimulated cells. After infection, the proportion of detectably transduced cells was increased by enhancer stimulation from 58% to 100% at 100 pfu/cell and from 9% to 62% at 10 pfu/cell, indicating quiescent viral DNA in unstimulated cells. At high adenoviral titers (1000 pfu/cell), the recombinant gene became the most abundant protein in cell extracts. These findings demonstrate that in human VSMCs, limited constitutive expression from the CMV-IEP, rather than failure of translocation of adenoviral DNA, may be responsible for the apparent failure of transduction at a low multiplicity of infection.
重组腺病毒载体越来越多地用于哺乳动物细胞的基因转移研究和人类的基因治疗方案。成功转导血管平滑肌细胞（VSMC）通常需要高腺病毒滴度，成功转导定义为外源基因的摄取和可检测到的表达，但病毒摄取效率和转录控制的相对贡献尚不清楚。为了探究缺乏可检测到的基因表达在多大程度上可能是由于成功转移的基因转录效率低下，我们使用了一种在人巨细胞病毒主要立即早期启动子（CMV-IEP）控制下表达β-半乳糖苷酶的复制缺陷型腺病毒（RAd35 β-Gal），该启动子含有cAMP和核因子-κB反应元件，以研究基因转移到人类VSMC后组成型和诱导型基因表达。用5-溴-4-氯-3-吲哚基β-D-吡喃半乳糖苷（X-gal）进行组织化学染色、定量分光光度测定、SDS-PAGE、蛋白质印迹和Northern分析，以评估感染细胞中β-半乳糖苷酶的表达。在用福斯可林和佛波酯处理的人类VSMC中，与未刺激的细胞相比，在用每细胞30、100和1000个噬斑形成单位（pfu/细胞）的RAd35 β-Gal感染后，β-半乳糖苷酶的表达分别增加了17倍、19倍和23倍。感染后，增强子刺激使可检测到的转导细胞比例在100 pfu/细胞时从58%增加到100%，在10 pfu/细胞时从9%增加到62%，表明未刺激细胞中病毒DNA处于静止状态。在高腺病毒滴度（1000 pfu/细胞）下，重组基因成为细胞提取物中最丰富的蛋白质。这些发现表明，在人类VSMC中，CMV-IEP的有限组成型表达，而非腺病毒DNA转位失败，可能是低感染复数时明显转导失败的原因。
