AGING OF FRTL-5 RAT-THYROID CELLS CAUSES SENSITIVITY TO CYTOTOXICITY INDUCED BY TUMOR-NECROSIS-FACTOR-ALPHA

While investigating the modulation of the growth and function of the FRTL-5 rat thyroid cell line by recombinant human tumor necrosis factor-alpha (TNF-alpha), we noticed that pronounced changes in several response parameters occurred with increasing passage number. For young cells (passage <20), TNF-alpha by itself slightly increased [H-3]thymidine incorporation and DNA content, and had a minimal effect on basal I-125 uptake. When combined with TSH, TNF-alpha had no influence on TSH-stimulated [H-3]thymidine incorporation, but significantly inhibited TSH-stimulated I-125 uptake. Compared with young cells, aged cells (passage >40), in contrast, developed a high sensitivity to TNF-alpha. TNF-alpha markedly stimulated [H-3]thymidine incorporation into DNA, inhibited TSH-stimulated I-125 uptake per mu-g DNA, but dramatically decreased the total DNA content and cell number. TSH augmented the TNF-alpha effect in aged cells, resulting in a further reduction of DNA content. Aphidicolin, a specific inhibitor of DNA polymerase-alpha which is associated with DNA replication, dramatically inhibited TNF-alpha-induced [H-3]thymidine incorporation in both young and aged cells; this suggested that the effect of TNF-alpha on FRTL-5 cell growth is related to DNA replication, rather than DNA repair. Cr-51 release from FRTL-5 cells, a measure of cytotoxicity, increased 2-fold over baseline in aged cells at a dose of 400 ng/ml TNF-alpha and decreased to 70% of baseline in young cells at this same dose. The protein kinase-A (PKA) and protein kinase-C (PKC) signal transduction mechanisms of TNF-alpha in aged cells (passage >40) were also studied. TNF-alpha increased cAMP and also increased relative PKA and PKC activity in 1-40 min. Phorbol myristate acetate (PMA), an activator of PKC, increased [H-3]thymidine incorporation and DNA content. PMA did not affect the TNF-alpha-induced increase in [H-3]thymidine incorporation or its reduction of DNA content. When the cells were pretreated with a high concentration of PMA (1-mu-M/24 h) to down-regulate PKC, the TNF-alpha dose-dependent increase in [H-3]thymidine incorporation and decrease in DNA content were only slightly inhibited, suggesting that the main effects of TNF-alpha are independent of PKC. We conclude that the sensitivity of FRTL-5 cells to the cytotoxic effect of TNF-alpha increases with aging.