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

人前列腺癌DU145细胞的血红素加氧酶1(HO1)基因表达的诱导与辐射强度和时间有关，并与线粒体膜电位降低和细胞内超氧阴离子浓度升高有关。当加入可进入细胞的自由基清除剂时，HO1的表达被抑制，这表明细胞内次级产生的活性氧参与了诱导。同时，在HO1基因的3个亚克隆片段中，分别有10~5.8kb、5.8~4.5kb和4.5~0kb的亚克隆片段，其中10~5.8kb的片段和4.5~0kb的片段响应超声处理。这两个片段都包含多个核苷酸序列基序，即应激反应元件(STRE)，它可能与氧化应激有关。当对基序进行突变或缺失时，启动子对超声波的反应活性会受到显著影响。我们克隆了与成骨相关的环氧合酶-2、Flt-1、BMP-7和c-fos基因启动子区域的DNA片段，构建了基因盒。将基因盒导入人成骨细胞，用频率为1 MHz、功率为0.1~0.5W/cm2、占空比为5~10%的超声波作用5~30min。孵育6-48小时后，获得启动子的激活。然而，任何荧光素酶活性的诱导都得到了证实，包括以前在类似条件下超声波作用后显示的环氧酶-2启动子的诱导。

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.