Abstract

This study was carried out to demonstrate the mode of ADR-induced cell death(apoptosis) on the light and electron microscopic features, to measure the apoptotic index dependent on various doses of ADR, to investigate the possible mechanism of apoptosis induced by ADR, and to evaluate ISNT method for the detection of DNA strand break. HeLa cells were treated with various doses of ADR 0.1~100.0 microgram/ml and observed under the light and transmission electron microscopes at 6 hours, 1 day and 3 days after ADR treatment. In addition, DNA strand breaks induced by ADR were detected in HeLa cells using the in situ nick translation(ISNT) method. The results were as follows: 1) The cell viability of HeLa cells decreased and the apoptotic index increased following exposure to ADR in a dose-dependent manner, resulting in about 44% of apoptotic index at 100.0 microgram/ml of ADR treatment. 2) Light microscopically, HeLa cells treated with ADR showed shrinkage or condensation of nucleus and cytoplasm. There were various unclear changes showing irregular, large, delineated masses of condensed chromatin abutting on the nuclear envelopes. Later stage of apoptosis revealed contracted and condensed cytoplasm with irregular cell membrane. Electron microscopically, margination of condensed chromatin, dilatation of endoplasmic reticulum under the plasma membrane, aggregation of cytoplasmic organelles with morphologically intact mitochondria, and irregular cell surface with blebbing were observed. 3) ISNT using biotinylated dUTP exhibited strong positive nuclear staining in HeLa cells treated with ADR. There was a marked response at 10.0~20.0 microgram/ml of ADR treatment. It is concluded from the above results that the death of HeLa cells induced by ADR was apoptotic in type based on light and electron microscopic appearance. The apoptotic index correlated with the increasing dose of ADR. ISNT with biotinylated dUTP led to visible evidence of DNA strand breaks following ADR treatment of HeLa cells. ISNT can be used for detection of DNA degradation, caused by activation of endogenous endonuclease, which is an early and specific characteristic of apoptosis.

• Keywords: Adriamycin;Apoptosis;HeLa cell;In situ nick translation