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 less than 20), TNF alpha by itself slightly increased [3H]thymidine incorporation and DNA content, and had a minimal effect on basal 125I uptake. When combined with TSH, TNF alpha had no influence on TSH-stimulated [3H]thymidine incorporation, but significantly inhibited TSH-stimulated 125I uptake. Compared with young cells, aged cells (passage greater than 40), in contrast, developed a high sensitivity to TNF alpha. TNF alpha markedly stimulated [3H]thymidine incorporation into DNA, inhibited TSH-stimulated 125I uptake per micrograms 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 [3H]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. 51Cr 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 greater than 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 [3H]thymidine incorporation and DNA content. PMA did not affect the TNF alpha-induced increase in [3H]thymidine incorporation or its reduction of DNA content. When the cells were pretreated with a high concentration of PMA (1 microM/24 h) to down-regulate PKC, the TNF alpha dose-dependent increase in [3H]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.
Chen
G
G
Endocrinology Research Laboratory, West Los Angeles Veterans Administration Medical Center, California 90073.
Pekary
A E
AE
Hershman
J M
JM
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology*; D016922:Cellular Senescence / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication / Q000187:drug effects; D006801:Humans; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013961:Thyroid Gland / Q000166:cytology* / Q000187:drug effects; D013972:Thyrotropin / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D014409:Tumor Necrosis Factor-alpha; D016590:Aphidicolin; D013972:Thyrotropin; D004247:DNA; D000242:Cyclic AMP; D011494:Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate