Apoptosis induced by hyperthermia in Dunn osteosarcoma cell line in vitro

The effect of hyperthermia at 43.5 degrees C for 1 h on Dunn osteosarcoma cells was studied. With sham-heated cells (37 degrees C, 1 h) as the control, the hyperthermia treated cells were divided into five groups. Time 0 group was the cells that were harvested immediately after heated at 43.5 degrees C for 1 h. Whereas time 3, 6, 12, and 24 h groups were the cells that were collected respectively after reincubation at 37 degrees C for the above different time periods. The appearance of hyperthermia-induced apoptosis of Dunn osteosarcoma cells was demonstrated to be time dependent. With the confocal microscopic study and TUNEL staining, the morphological characteristics of apoptosis, condensed nuclei and fragmented nuclei were obvious when reincubated at 37 degrees C for 6 h after hyperthermic treatment. This hyperthermia-induced apoptosis was further confirmed by flow cytometric analysis on DNA contents. The sub-G1 region that was proposed as a marker of apoptotic cells was most significantly elevated at 6 h after hyperthermic treatment and, thereafter, decreased to the levels of control values by 24 h, as the apoptotic cells underwent secondary necrosis and degraded to debris. The DNA strand breaks, considered as the key biochemical event of apoptosis, were detected by the TUNEL assay. This study indicated that hyperthermia (43.5 degrees C for 1 h) can induce apoptotic changes on osteosarcoma cells in vitro very rapidly (within 6 h after treatment), and its occurrence might not be detected if the samples are not taken at several early time points after hyperthermia.