Analyses of the induction of gene expressions by sonication and their signal transductions

研究表明，来自人前列腺癌的DU145细胞中血红素加氧酶1（HO1）基因表达的诱导依赖于超声的强度和持续时间，此外，线粒体膜电位的降低和细胞内超氧化物浓度的增加也与此相关。在超声前或超声后立即加入可进入细胞的自由基清除剂，可抑制HO1的表达，提示细胞中次生产生的活性氧参与了诱导。同时，在相对于HO1基因mRNA起始位点10 ~ 5.8 kb、5.8 ~ 4.5 kb和4.5 ~ 0 kb的3个亚克隆DNA片段中，发现10 ~ 5.8 kb和4.5 ~ 0 kb片段对超声有应答。这两个片段都含有多个可能参与氧化应激的核苷酸序列基序——应激反应元件（Stress Response Element, StRE）。当突变或缺失应用于基序时，响应超声的启动子活性受到显著影响。因此，我们认为线粒体产生的细胞内氧化应激促进了与StRE相关的转录因子Nrf2的激活，从而诱导了HO1基因的表达。我们克隆了含有环氧化酶-2、flt-1、bmp-7和c-fos基因启动子区域的DNA片段，这些基因与荧光素酶基因相关，参与成骨，构建了基因盒。将基因盒导入人成骨细胞，用1 MHz超声在0.1 ~ 0.5 W/cm^2的强度下，占空比为5 ~ 10%，超声5 ~ 30 min。孵育6 ~ 48小时后，获得启动子的激活。然而，任何诱导荧光素酶活性的方法都被证实，包括环氧化酶-2启动子，该启动子先前在类似条件下超声后显示出诱导作用。

Induction of the heme oxygenase 1(HO1) gene expression of DU145 cells derived from human prostate cancer was shown to be dependent upon intensity and duration of sonication applied, in addition, associated were decrease in mitochondria membrane potential and increase in intracellular superoxide concentration. When free radical scavengers that could enter a cell were added before or immediately after sonication, HO1 expression was suppressed, suggesting that reactive oxygen species secondarily generated in the cell was involved in the induction. At the same time, out of 3 subcloned DNA fragments of 10 to 5.8 kb,5.8 to 4.5 kb and 4.5 to 0 kb relative to the mRNA start site of the HO1 gene, the 10 to 5.8 kb fragment and the 4.5 to 0 kb fragment were found responsive to sonication. The either fragment contains multiple numbers of a nucleotide sequence motif, Stress Response Element (StRE), that may be involved in oxidative stress. When mutations or deletions were applied to the motifs, the promoter activity responsive to sonication was significantly affected. Thus, it was suggested that activation of Nrf2,a transcription factor associating with StRE, was facilitated by the intracellular oxidative stress generated from mitochondria, resulting in induction of HO1 gene expression.We cloned DNA fragments containing promoter regions of cycloxigenase-2,flt-1,bmp-7 and c-fos genes that are involved in osteogenesis and linked to the luciferase gene, constructing gene cassettes. The gene cassettes were introduced into human osteoblast cells and sonicated with 1 MHz ultrasound at intensities of 0.1 to 0.5 W/cm^2, duty cycles of 5 to 10%, for 5 to 30 min. After incubation for 6 to 48 hrs, activations of the promoters were accessed. However, any induction of luciferase activity was confirmed including the cycloxigenase-2 promoter that previously showed induction after sonication in a similar condition.