Chen G, Pekary A E, Hershman J M
Endocrinology Research Laboratory, West Los Angeles Veterans Administration Medical Center, California 90073.
Endocrinology. 1992 Aug;131(2):863-70. doi: 10.1210/endo.131.2.1322286.
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.
在研究重组人肿瘤坏死因子-α（TNFα）对FRTL-5大鼠甲状腺细胞系生长和功能的调节作用时，我们注意到随着传代次数增加，几个反应参数发生了显著变化。对于年轻细胞（传代次数小于20），TNFα本身略微增加了[³H]胸腺嘧啶核苷掺入量和DNA含量，对基础¹²⁵I摄取的影响最小。当与促甲状腺激素（TSH）联合使用时，TNFα对TSH刺激的[³H]胸腺嘧啶核苷掺入没有影响，但显著抑制了TSH刺激的¹²⁵I摄取。相比之下，与年轻细胞相比，老化细胞（传代次数大于40）对TNFα产生了高敏感性。TNFα显著刺激[³H]胸腺嘧啶核苷掺入DNA，抑制TSH刺激的每微克DNA的¹²⁵I摄取，但显著降低了总DNA含量和细胞数量。TSH增强了老化细胞中TNFα的作用，导致DNA含量进一步降低。阿非迪霉素是一种与DNA复制相关的DNA聚合酶-α的特异性抑制剂，它显著抑制了年轻和老化细胞中TNFα诱导的[³H]胸腺嘧啶核苷掺入；这表明TNFα对FRTL-5细胞生长的影响与DNA复制有关，而不是与DNA修复有关。FRTL-5细胞的⁵¹Cr释放（一种细胞毒性的测量指标），在老化细胞中，在400 ng/ml TNFα剂量下比基线增加了2倍，而在相同剂量下，年轻细胞中则降至基线的70%。我们还研究了老化细胞（传代次数大于40）中TNFα的蛋白激酶-A（PKA）和蛋白激酶-C（PKC）信号转导机制。TNFα在1 - 40分钟内增加了环磷酸腺苷（cAMP），也增加了相对的PKA和PKC活性。佛波酯肉豆蔻酸乙酸酯（PMA）是PKC的激活剂，增加了[³H]胸腺嘧啶核苷掺入量和DNA含量。PMA不影响TNFα诱导的[³H]胸腺嘧啶核苷掺入增加或其对DNA含量的降低。当用高浓度的PMA（1 μM/24小时）预处理细胞以下调PKC时，TNFα剂量依赖性的[³H]胸腺嘧啶核苷掺入增加和DNA含量降低仅受到轻微抑制，这表明TNFα的主要作用独立于PKC。我们得出结论，FRTL-5细胞对TNFα细胞毒性作用的敏感性随着老化而增加。
