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

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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.