Apoptotic and necrotic effects of tumour necrosis factor-alpha potentiated with hyperthermia on L929 and tumour necrosis factor-alpha-resistant L929.

Abstract

PURPOSE: The cytotoxic effect of the combination treatment of TNF-alpha and hyperthermia on L929 and TNF-alpha-resistant L929 (rL929) cells was investigated. MATERIALS AND METHODS: L929 cells were treated with TNF-alpha (5 ng/mL), heating at 43 degrees C or the combination of TNF-alpha and heating. The cells were harvested at different time within the 24-hour period. The viability and the type of cell death of the harvested cells were examined. RESULTS: When L929 cells were treated with a combination of TNF-alpha and heating the cells died quickly and apoptosis increased to an overwhelming extent, especially in the group pre-treated with TNF-alpha for 1 h prior to heating. Although rL929 cells were resistant to TNF-alpha alone, the cells became sensitive to TNF-alpha treatment when combined with heating. Similar to the L929 cell, the cells also died rapidly and exhibited apoptosis to a higher extent. Using an Annexin-V-FITC kit and flow cytometer, we found that both necrosis and apoptosis occurred. Agarose gel electrophoresis of DNA extracted from treated cells showed that the DNA fragments were multiples of approximately 200 bp. Furthermore, by studying the kinetics of cell death and apoptosis, we found that the loss of cell membrane integrity preceded the DNA fragmentation in both L929 and rL929 cells. CONCLUSION: The results suggested that hyperthermia may enhance the necrotic and apoptotic effects of TNF-alpha on some tumour cells and overcome the resistance of some tumour cells to TNF-alpha.