| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1990: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
Thiol protease inhibitors (TPIs) were isolated from culture media and cell extracts of two human lung cancer cell lines and were characterized by biochemical and biological means to understand the relationship between growth and TPI.
1) High molecular weight (HMW-) TPI and low molecular weight (LMW-) TPI were purified from culture media of human lung cancer cell line LK-2 (squamous cell carcinoma) by gel filtration on Sephadex G-100 and ion exchange chromatography on DEAE-Sephacel. Their molecular weight (MW) were estimated to be 90,000 and 10,000 respectively, by gel filtration. LMW-TPI was dominant in TPI activity. LMW-TPI is stable below 60^ﾟC and in a range of pH6-10. LMW-TPI was a specific inhibitor of thiol protease. From LK-2 cell extract, only LMW-TPI (MW10,000) could be detected.
2) None of TPIs, purified from culture media or cells of LK-2, reacted with anti-UTPI rabbit IgG or anti-alpha_2-TPI rabbit IgG by double immunodiffusion method.
3) From a human lung adenocarcinoma cell line, luci-10, buth HMW-TPI and LMW-TPI were purified from culture media but only LMW-TPI from cell extracts.
4) Daily examination of TPI activity in culture media of Luci-10 showed that cancer cells at stationary phase released TPI two to three times more than at logarithmic growth phase.
5) LMW-TPI, purified from culture media of LK-2, suppressed incorporation of ^3H-Thymidine of LK-2 cells, Other TPIs (alpha_2 -TPI in serum, UTPIin urine, a microbial protease inhibitor E64) suppressed it. On the other hand, Serine Protease inhibitors such as UTI in urine and soybean trypsin inhibitor had no effect.
6) Thiol proteases such as papain, ficin, and cathepsin B stimulated incorporation of ^3H-Thymidine of LK-2 cell.
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