Auer K L, Spector M S, Tombes R M, Seth P, Fisher P B, Gao B, Dent P, Kunos G
Department of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298, USA.
FEBS Lett. 1998 Sep 25;436(1):131-8. doi: 10.1016/s0014-5793(98)01074-6.
Activation of alpha1B adrenergic receptors (alpha(1B)AR) promotes DNA synthesis in primary cultures of hepatocytes, yet expression of alpha(1B)AR in hepatocytes rapidly declines during proliferative events. HepG2 human hepatoma cells, which do not express alpha(1B)AR, were stably transfected with a rat alpha1B(AR) cDNA (TFG2 cells), in order to study the effects of maintained alpha(1B)AR expression on hepatoma cell proliferation. TFG2 cells had a decreased rate of growth compared to mock transfected HepG2 cells as revealed by a decrease in [3H]thymidine incorporation into DNA. Stimulation of alpha(1B)AR with phenylephrine caused a further large reduction in TFG2 cell growth, whereas no effect on growth was observed in mock transfected cells. Reduced cell growth correlated with increased percentages of cells found in G0/G1 and G2/M phases of the cell cycle. In TFG2 cells, phenylephrine increased p42MAPkinase activity by 1.5- to 2.0-fold for up to 24 h and increased expression of the cyclin dependent kinase inhibitor protein p21Cip1/WAF1. Treatment of TFG2 cells with the specific MEKI inhibitor PD98059, or infection with a -/- MEK1 recombinant adenovirus permitted phenylephrine to increase rather than decrease [3H]thymidine incorporation. In addition, inhibition of MAP kinase signaling by PD98059 or MEK1 -/- blunted the ability of phenylephrine to increase p21Cip1/WAF1 expression. In agreement with a role for increased p21Cip1/WAF1 expression in causing growth arrest, infection of TFG2 cells with a recombinant adenovirus to express antisense p21Cip1/WAF1 mRNA blocked the ability of phenylephrine to increase p21Cip1/WAF1 expression and to inhibit DNA synthesis. Antisense p21Cip1/WAF1 permitted phenylephrine to stimulate DNA synthesis in TFG2 cells, and abrogated growth arrest. These results suggest that transformed hepatocytes may turn off the expression of alpha1B(ARs) in order to prevent the activation of a growth inhibitory pathway. Activation of this inhibitory pathway via alpha1B(AR) appears to be p42MAPkinase and p21Cip1/WAF1 dependent.
α1B肾上腺素能受体（α(1B)AR）的激活可促进原代培养肝细胞中的DNA合成，然而在增殖过程中，肝细胞中α(1B)AR的表达会迅速下降。为了研究持续表达α(1B)AR对肝癌细胞增殖的影响，将不表达α(1B)AR的HepG2人肝癌细胞用大鼠α1B(AR) cDNA进行稳定转染（TFG2细胞）。与mock转染的HepG2细胞相比，TFG2细胞的生长速率降低，这通过掺入DNA中的[3H]胸苷减少得以体现。用去氧肾上腺素刺激α(1B)AR导致TFG2细胞生长进一步大幅降低，而在mock转染细胞中未观察到对生长的影响。细胞生长减少与细胞周期G0/G1期和G2/M期细胞百分比增加相关。在TFG2细胞中，去氧肾上腺素使p42MAP激酶活性在长达24小时内增加1.5至2.0倍，并增加细胞周期蛋白依赖性激酶抑制剂蛋白p21Cip1/WAF1的表达。用特异性MEK1抑制剂PD98059处理TFG2细胞，或用-/- MEK1重组腺病毒感染，使得去氧肾上腺素增加而非减少[3H]胸苷掺入。此外，PD98059或MEK1 -/-对MAP激酶信号传导的抑制减弱了去氧肾上腺素增加p21Cip1/WAF1表达的能力。与增加的p21Cip1/WAF1表达在导致生长停滞中的作用一致，用重组腺病毒感染TFG2细胞以表达反义p21Cip1/WAF1 mRNA可阻断去氧肾上腺素增加p21Cip1/WAF1表达和抑制DNA合成的能力。反义p21Cip1/WAF1使去氧肾上腺素能够刺激TFG2细胞中的DNA合成，并消除生长停滞。这些结果表明，转化的肝细胞可能关闭α1B(ARs)的表达，以防止生长抑制途径的激活。通过α1B(AR)激活这种抑制途径似乎依赖于p42MAP激酶和p21Cip1/WAF1。
