レーザーサイトメーターを用いたheat shock protein 72の加温および抗癌剤添加による動態の検討

Abstract

Heat shock proteins (HSPs) are regarded as the proteins most related to the development of thermotolerance. Recently, not only their role in thermotolerance, but also their role in resistance to anticancer agents is gathering concern. In this study, the kinetics of hsp 72 in HeLa cells treated with heating and/or anticancer agents were studied. Hsp 72 was immunostained by the indirect fluorescent technique using a monoclonal anti-hsp 72 antibody (Amersham). The staining pattern was observed and analyzed using a laser cytometer, ACAS 570 (Meridian). Hsp 72 was normally found in the cytoplasm at 37°C and moved rapidly into the nucleus with heating at 43°C for 2 hours. It then returned to the cytoplasm 4 to 6 hours after heating. The hsp 72 content reached a peak at 8 hours after heating. Hsp 72 was induced in all cells treated with cisplatin, adriamycin, peplomycin, or etoposide for 48 hours. In the cells treated with both heating at 43°C for 2 hours and these anticancer agents, hsp 72 induction was most suppressed by adriamycin. However, translocation of hsp 72 to the nucleus was specific for heating and was not affected by the anticancer agents. By laser cytometry the intracellular localization of hsp 72 and the changes of its content were simultaneously detected. Moreover, the change pattern of hsp 72 content measured by laser cytometry coincided with that measured by the Western blotting procedure.