Neutral red uptake and clonogenic survival assays of the hyperthermic sensitization of tumor cells to tumor necrosis factor

• Published in: Radiation research Vol. 119; no. 2; p. 325
• Main Authors: Tomasovic, S P, Barta, M, Klostergaard, J
• Format: Journal Article
• Language: English
• Published: United States 01.08.1989
• Subjects: Animals; Cell Line; Cell Survival - drug effects; Hot Temperature; Humans; Mice; Neutral Red - pharmacokinetics; Recombinant Proteins; Tumor Cells, Cultured - drug effects; Tumor Cells, Cultured - metabolism; Tumor Necrosis Factor-alpha - pharmacology; Tumor Stem Cell Assay
• ISSN: 0033-7587
• DOI: 10.2307/3577623

Vital dye uptake and postfixation dye assays have recently been used to examine the interaction between short-term (24-48 h) exposures to the monokine, tumor necrosis factor (TNF), and hyperthermic treatments with the finding that synergistic increases in cytotoxicity occurred. However, survival measured by these short-term dye assays is not necessarily closely related to eventual loss of clonogenic capacity. Treatment-induced growth delays, delayed cytotoxic effects, or perturbations of vital dye sequestration mechanisms could result in a different measurement of surviving fraction than given by a clonogenic assay. In this study we directly compared the neutral red vital dye uptake and clonogenic survival assays and confirmed in both assays that TNF-sensitive (L-929) and TNF-resistant (EMT-6) phenotypes show greatly reduced survival when treated with combined recombinant human TNF (1.0-0.0005 micrograms/ml) and hyperthermia (1-2 h at 43 degrees C). Moreover, we confirmed that sensitization of the TNF-resistant EMT-6 cells was largely dependent on monokine treatment before hyperthermia and was reduced by the reverse sequence. The greatest sensitization of TNF-responsive L-929 cells also occurred when TNF treatment preceded heating. These results for clonogenic survival are consistent with the hypothesis that hyperthermia used in combination with TNF in vivo is more cytotoxic than TNF or hyperthermia separately.