Miller S C, Taylor A, Watanabe K, Mok K, Torti F M
Signal Transduction Laboratory, Science and Technology Group, SRI International, Menlo Park, California 94025, USA.
Exp Cell Res. 1997 Jan 10;230(1):9-21. doi: 10.1006/excr.1996.3397.
A mouse model system for studying the effect of ultraviolet (uv) radiation on reporter gene expression directed by the human immunodeficiency virus type 1 long-terminal repeat (HIV-LTR) has been developed to address the signals required for LTR trans-activation in cells with the reporter gene stably integrated into the genome. In a stable mouse L cell clone, L-15, NF-kappaB DNA binding activity induced by uv-C (254 nm) but not by tumor necrosis factor-alpha (TNF-alpha) or 12-O-tetra-decanoylphorbol-13-acetate (TPA) correlated with the stimulation of HIV-LTR-directed chloramphenicol acetyltransferase (CAT) activity; uv-C was more effective than uv-B (312 nm), while uv-A (365 nm) had little effect on CAT activity. Inducers of oxidative stress, such as H2O2 treatment up to 200 microM or ionizing radiation up to 20 Gy, also had little effect on CAT expression. Pyrrolidine dithiocarbamate (PDTC) inhibited NF-kappaB DNA binding and stimulation of CAT activity by uv-C in a dose-dependent manner. Unexpectedly, PDTC induced NF-kappaB DNA binding that was additive with the response with TNF. In an effort to separate uv irradiation and uv-induced DNA damage from transactivation of the HIV-LTR we devised a heterokaryon system. The fusion of uv-irradiated human fibroblasts with a mouse L cell clone containing the HIV-LTR-directed lacZ gene resulted in the activation of lacZ activity that was detected in heterokaryons at the single-cell level. These data suggest that uv-induced DNA damage in the chromosomal DNA containing the reporter gene cannot explain activation of the HIV-LTR. This finding demonstrates LTR trans-activation in a nonirradiated genome.
为了研究紫外线（UV）辐射对由人类免疫缺陷病毒1型长末端重复序列（HIV-LTR）指导的报告基因表达的影响，已开发出一种小鼠模型系统，以解决报告基因稳定整合到基因组中的细胞中LTR反式激活所需的信号。在一个稳定的小鼠L细胞克隆L-15中，由uv-C（254nm）诱导而非由肿瘤坏死因子-α（TNF-α）或12-O-十四烷酰佛波醇-13-乙酸酯（TPA）诱导的NF-κB DNA结合活性与HIV-LTR指导的氯霉素乙酰转移酶（CAT）活性的刺激相关；uv-C比uv-B（312nm）更有效，而uv-A（365nm）对CAT活性影响很小。氧化应激诱导剂，如高达200μM的H2O2处理或高达20Gy的电离辐射，对CAT表达也几乎没有影响。吡咯烷二硫代氨基甲酸盐（PDTC）以剂量依赖性方式抑制uv-C诱导的NF-κB DNA结合和CAT活性刺激。出乎意料的是，PDTC诱导的NF-κB DNA结合与TNF反应相加。为了将紫外线照射和紫外线诱导的DNA损伤与HIV-LTR的反式激活分开，我们设计了一种异核体系统。用含有HIV-LTR指导的lacZ基因的小鼠L细胞克隆与紫外线照射的人成纤维细胞融合，导致在异核体的单细胞水平检测到lacZ活性的激活。这些数据表明，含有报告基因的染色体DNA中紫外线诱导的DNA损伤不能解释HIV-LTR的激活。这一发现证明了在未照射的基因组中的LTR反式激活。
