| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
We established several FAK cDNA-transfected cells and found that FAK-transfected HL-60 (HL-60/FAK) cells are resistant to apoptosis induced with hydrogen peroxide, etoposide and radiation compared with the parental HL-60 or the vector-transfected (HL-60/Vect) cells. We carried out proteome analysis to study the mechanism of resistance to apoptosis in HL-60/FAK cells. Increased proteins included stress proteins such as hsp90, ribosomal proteins, and antioxidant enzymes such as peroxyredoxin 2. Some of these proteins are assumed to contribute to the antiapoptotic action of FAK.
To identify target genes that are involved in HL-60/FAK cells, we performed cDNA microarray screening using apoptosis-chip. There, we identified the decrease of glutathione peroxidase. This result prompted us to investigate the changes of antioxidant enzymes. Here, we demonstrate that lipid peroxidation was suppressed after treatment with hydrogen peroxide in HL-60/FAK cells but not vector-transfected HL-60 (HL-60/
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Vect) cells. Basal activity and mRNA expression of antioxidant enzymes, particularly of GSH reductase (GRe), phospholpid hydroperoxide glutathione peroxidase (PHGPx) were markedly elevated in HL-60/FAK cells. In contrast, glutathione peroxidase (GPx) and catalase levels were decreased in HL-60/FAK cells.
HL-60/FAK cells are highly resistant to TRAIL-induced apoptosis. TRAIL at 500 ng/ml induced significant DNA fragmentation, activation of caspase-8 and 3, the processing of a proapaptotic BID, and mitochondrial release of cytochrome c in HL-60/Vect cells, whereas no such events were observed in the HL-60/FAK cells. In particular, the expression of procaspase-8 gene and subsequent cleavage of caspase-8 were markedly reduced in HL-60/FAK cells, while expression of TRAIL-receptor 2 and 3, TRADD, and FADD was equivalent in both types of cells. The introduction of FAK siRNA in HL-60/FAK cells sensitized them against TRAIL-induced apoptosis, confirming that overexpressed FAK downregulates procaspase-8 expression, which subsequently inhibits downstream apoptosis pathway in the HL-60/FAK cells. Less
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