Galloway D C, Blake D G, Shepherd A G, McLellan L I
Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Scotland, U.K.
Biochem J. 1997 Nov 15;328 ( Pt 1)(Pt 1):99-104. doi: 10.1042/bj3280099.
We have shown that in HepG2 cells treatment with 75 microM t-butylhydroquinone (tBHQ) results in a 2.5-fold increase in glutathione concentration, as part of an adaptive response to chemical stress. In these cells the elevation in intracellular glutathione level was found to be accompanied by an increase of between 2-fold and 3-fold in the level of the 73 kDa catalytic subunit of gamma-glutamylcysteine synthetase (heavy subunit, GCSh) and the 31 kDa regulatory subunit (light subunit, GCSl). Levels of GCSh and GCSl mRNA were increased by up to 5-fold in HepG2 cells in response to tBHQ. To study the transcriptional regulation of GCSl, we subcloned 6.7 kb of the upstream region of the human GCSl gene (GLCLR) from a genomic clone isolated from a P1 lymphoblastoid cell line genomic library. HepG2 cells were transfected with GLCLR promoter reporter constructs and treated with tBHQ. This resulted in an induction of between 1.5-fold and 3.5-fold in reporter activity, indicating that transcriptional regulation of GLCLR is likely to contribute to the induction of GCSl by tBHQ in HepG2 cells. Sequence analysis of the promoter region demonstrated the presence of putative enhancer elements including AP-1 sites and an antioxidant-responsive element, which might be involved in the observed induction of the GLCLR promoter.
我们已经表明，在HepG2细胞中用75微摩尔叔丁基对苯二酚（tBHQ）处理会导致谷胱甘肽浓度增加2.5倍，这是对化学应激的适应性反应的一部分。在这些细胞中，发现细胞内谷胱甘肽水平的升高伴随着γ-谷氨酰半胱氨酸合成酶73 kDa催化亚基（重亚基，GCSh）和31 kDa调节亚基（轻亚基，GCSl）水平增加2至3倍。响应tBHQ，HepG2细胞中GCSh和GCSl mRNA水平增加高达5倍。为了研究GCSl的转录调控，我们从从P1淋巴母细胞系基因组文库中分离的基因组克隆中克隆了人GCSl基因（GLCLR）上游区域的6.7 kb片段。用GLCLR启动子报告构建体转染HepG2细胞并用tBHQ处理。这导致报告活性诱导1.5至3.5倍，表明GLCLR的转录调控可能有助于tBHQ在HepG2细胞中诱导GCSl。启动子区域的序列分析表明存在推定的增强子元件，包括AP-1位点和抗氧化反应元件，这可能参与了观察到的GLCLR启动子的诱导。
