CYTOTOXIC MANIFESTATIONS OF THE INTERACTION BETWEEN HYPERTHERMIA AND TNF - DNA FRAGMENTATION

The relationship of DNA fragmentation to the greatly enhanced cytotoxi city seen in vitro against tumour cells when recombinant human tumour necrosis factor-alpha (TNF-alpha) is combined with hyperthermia was in vestigated. The TNF-alpha-sensitive L929 and -resistant EMT6 cells wer e treated with 8.8 and 16 ng of TNF-alpha per ml, respectively, and th en heated at 40.5 degrees C for 24 h (L929) or at 43 degrees C for 1 h (L929) or 1.5 h (EMT-6) beginning 1 h later. For both cell lines at b oth temperatures, the addition of heating to the TNF-alpha treatment s ignificantly decreased viability and increased DNA fragmentation at ea rlier time points than seen with either TNF-alpha or heat alone. DNA f ragmentation was further studied using agarose gel electrophoresis to examine the size distribution of the DNA fragments and the ability of intracellular calcium buffering agents BAPTA and quin-2 to inhibit fra gmentation. At 4.5 h after L929 cells were treated with TNF-alpha at 4 3 degrees C, the size distribution of DNA fragments more closely resem bled the oligonucleosome sized apoptotic DNA fragmentation, as seen in irradiated rat thymocytes, than the spectrum of DNA fragments seen in necrotic fragmentation. However, while BAPTA and quin-2 inhibited the calcium-dependent apoptotic fragmentation seen in thymocytes they did not inhibit the DNA fragmentation in L929 cells. In addition, the los s of membrane integrity in both L929 and EMT-6 cells preceded or appro ximated the appearance of DNA fragmentation, whereas loss of membrane integrity usually follows DNA fragmentation in apoptosis. However, mor phological studies showed that apoptotic bodies were present in L929 c ell cultures treated with TNF-alpha and heat, and were distinguishable from necrosing cells. We conclude that both types of DNA fragmentatio n are operant in some cell lines exhibiting a cytotoxic response to TN F-alpha and heat treatments, and that increased fragmentation reflects the greatly enhanced cytotoxic interactions seen with combination tre atments in those cells.