Abstract:

Objective

To observe the effects of apoptosis of Tca8113 cells induced by Adramycin on the telomerase activity and expression of telomere repeat binding factors （TRF）and probe the mechanism by which telomerase and TRF proteins function during transmitting apoptotic signals.

Methods

The effective concentration of Adramycin in the following experiments was determined by using methylthiazolyl tetrazolium （MTT） assay of Tca8113 cells. Tca8113 cells cultured in the medium were divided into two experiment groups， namely treated group containing Adramycin and untreated one containing sodium saline. After cultured for 5 days and 7 days，apoptosis of Tca8113 cells was examined by flow cytometer method； apoptotic morphology after Giemsa staining was observed under microscope； a qualitative and quantitative analysis of telomerase activity was performed by TRAP （telomeric repeats amplification protocol）-enzymelinked immunosorbent assay； expression and expression level of TRF proteins from Tca8113 cells were detected with immunohistochemical staining and immunofluorescence label assay respectively.

Results

The effective concentration of Adramycin in the subsequent experiments was determined as 5 μg/ml by MTT assay. After 5 days' and 7 days' treatment with Adramycin at 5 μg/ml， apoptotic rates of the treated groups were 36.42±1.70% and 54.24±2.07%，respectively， which were significantly different from those of the untreated groups （P ＜0.05）；apoptotic cells obviously appeared in the treated groups after Giemsa staining； the immunosorbent assay indicated that telomerase activity from the treated groups dropped obviously， which was statistically significant compared with that from the untreated groups （P ＜0.05）； the activity level decreased with increasing duration of Adramycin at 5 μg/ml （P ＜0.05）； expression of TRF proteins which evenly appeared in the nucleus in blue were found in the cells of the two experiment groups； the expression in the plasma and apoptotic debris of induced apoptotic cells was characteristic of blue speckles distributed diffusely； there were no statistically difference on the expression level between the treated and untreated group （P ＞0.05）.

Conclusions

Inducing Tca8113 cells apoptosis by Adramycin inhibited the telomerase activity， changed the expressive characteristics of TRF， but had no influence on the expression level of TRF proteins.

Key words: Adramycin, Tca8113 cells, Apoptosis, Telomerase, Telomere repeat binding factor