Crawford M J, Krishnamoorthy R R, Rudick V L, Collier R J, Kapin M, Aggarwal B B, Al-Ubaidi M R, Agarwal N
Department of Pathology and Anatomy, University of North Texas Health Science Center, Fort Worth, Texas 76107, USA.
Biochem Biophys Res Commun. 2001 Mar;281(5):1304-12. doi: 10.1006/bbrc.2001.4501.
We recently showed that photooxidative stress on cultured photoreceptor cells results in down-modulation of NF-kappaB activity which then leads to apoptosis of cultured 661W photoreceptor cells. In an effort to further delineate the mechanism of photoreceptor cell death, we sought to determine the effects of Bcl-2 overexpression on cell survivability. Wild-type 661W cells were transfected with the plasmid construct pSFFV-neo-Bcl-2 and several clones were isolated. All clones demonstrated increased Bcl-2 mRNA and protein levels, with the B4 clone exhibiting the greatest enhancement. On exposure to visible light the B4 cells were protected from undergoing apoptosis when compared with the mock transfected cells, as ascertained by TUNEL apoptosis assay and formazan based estimation of cell viability. The Bcl-2 overexpressing cells also maintained a higher Bcl-2/Bax ratio, suggesting that this ratio is important in protection from photooxidative stress. Electrophoretic mobility shift assays for NF-kappaB demonstrated higher activity in both nuclear and cytosolic fractions of the B4 photoreceptors compared with the 661W wild-type cells at all light exposure time points. Furthermore, the findings of the gel shift assays were further supported by immunocytochemistry for NF-kappaB which revealed that protein levels of the RelA subunit of NF-kappaB were protected in the nucleus as well as in the cytoplasm of Bcl-2 overexpressing B4 cells exposed to light compared to the 661W cells. These results suggest that Bcl-2 overexpression protects NF-kappaB protein levels and activity in the nucleus, indicating that preservation of NF-kappaB binding activity in the nucleus may be essential for photoreceptor cells to survive photooxidative damage induced apoptosis.
我们最近发现，培养的光感受器细胞上的光氧化应激会导致NF-κB活性下调，进而导致培养的661W光感受器细胞凋亡。为了进一步阐明光感受器细胞死亡的机制，我们试图确定Bcl-2过表达对细胞生存能力的影响。用质粒构建体pSFFV-neo-Bcl-2转染野生型661W细胞，并分离出几个克隆。所有克隆均显示Bcl-2 mRNA和蛋白水平升高，其中B4克隆的增强最为显著。通过TUNEL凋亡检测和基于甲臜的细胞活力评估确定，与mock转染细胞相比，B4细胞在暴露于可见光时可免受凋亡。Bcl-2过表达的细胞也维持较高的Bcl-2/Bax比率，表明该比率在保护免受光氧化应激方面很重要。NF-κB的电泳迁移率变动分析表明，在所有光照时间点，与661W野生型细胞相比，B4光感受器的核和胞质部分中的活性更高。此外，凝胶迁移分析的结果得到了NF-κB免疫细胞化学的进一步支持，该分析表明，与661W细胞相比，在暴露于光的Bcl-2过表达B4细胞的细胞核和细胞质中，NF-κB的RelA亚基的蛋白水平受到保护。这些结果表明，Bcl-2过表达可保护细胞核中的NF-κB蛋白水平和活性，表明保留细胞核中的NF-κB结合活性可能是光感受器细胞在光氧化损伤诱导的凋亡中存活的关键。
