甲胎蛋白对人肝癌Bel7402细胞系生长的促进分子机制
The promoting molecular mechanism of alpha-fetoprotein on the growth of human hepatoma Bel7402 cell line.
作者信息
Li Meng-Sen, Li Ping-Feng, He Shi-Peng, Du Guo-Guang, Li Gang
机构信息
Department of Biochemistry, Hainan Medical College, Hainan, China.
出版信息
World J Gastroenterol. 2002 Jun;8(3):469-75. doi: 10.3748/wjg.v8.i3.469.
AIM
The goal of this study was to characterize the AFP receptor, its possible signal transduction pathway and its proliferative functions in human hepatoma cell line Bel 7402.
METHODS
Cell proliferation enhanced by AFP was detected by MTT assay, 3H-thymidine incorporation and S-stage percentage of cell cycle analysis. With radioactive labeled 125I-AFP for receptor binding assay; cAMP accumulation, protein kinase A activity were detected by radioactive immunosorbent assay and the change of intracellular free calcium (Ca2+i) was monitored by scanning fluorescence intensity under TCS-NT confocal microscope. The expression of oncogenes N- ras, p 53, and p21( ras ) in the cultured cells in vitro were detected by Northern blotting and Western blotting respectively.
RESULTS
It was demonstrated that AFP enhanced the proliferation of human hepatoma Bel 7402 cell in a dose dependent fashion as shown in MTT assay, (3)H-thymidine incorporation and S-phase percentage up to 2-fold. Two subtypes of AFP receptors were identified in the cells with Kds of 1.3 x 10(-9)mol.L(-1) and 9.9 x10(-8)mol. (-1)L respectively. Pretreatment of cells with AFP resulted in a significant increase (625%) in cAMP accumulation. The activity of protein kinase A activity were increased up to 37.5, 122.6, 73.7 and 61.2% at treatment time point 2, 6, 12 and 24 hours. The level of intracellular calcium were elevated after the treatment of alpha-fetoprotein and achieved to 204% at 4 min. The results also showed that AFP(20mg.L(-1)) could upregulate the expression of N- ras oncogenes and p 53 and p21( ras ) in Bel 7402 cells. In the later case,the alteration were 81.1%(12h) and 97.3%(12h) respectively compared with control.
CONCLUSION
These results demonstrate that AFP is a potential growth factor to promote the proliferation of human hepatoma Bel 7402 cells. Its growth-regulatory effects are mediated by its specific plasma membrane receptors coupled with its transmembrane signaling transduction through the pathway of cAMP-PKA and intracellular calcium to regulate the expression of oncogenes.
目的
本研究旨在对人肝癌细胞系Bel 7402中的甲胎蛋白（AFP）受体、其可能的信号转导途径及其增殖功能进行特性分析。
方法
采用MTT法、³H-胸腺嘧啶核苷掺入法及细胞周期分析中的S期百分比检测AFP促进的细胞增殖。用放射性标记的¹²⁵I-AFP进行受体结合试验；用放射性免疫吸附试验检测环磷酸腺苷（cAMP）积累及蛋白激酶A活性，在TCS-NT共聚焦显微镜下通过扫描荧光强度监测细胞内游离钙（Ca²⁺i）的变化。分别用Northern印迹法和Western印迹法检测体外培养细胞中癌基因N-ras、p53和p21（ras）的表达。
结果
如MTT法、³H-胸腺嘧啶核苷掺入法及S期百分比所示，AFP以剂量依赖性方式增强人肝癌Bel 7402细胞的增殖，增殖倍数可达2倍。在细胞中鉴定出两种AFP受体亚型，解离常数（Kds）分别为1.3×10⁻⁹mol·L⁻¹和9.9×10⁻⁸mol·L⁻¹。用AFP预处理细胞导致cAMP积累显著增加（625%）。在处理时间点2、6、12和24小时，蛋白激酶A活性分别增加至37.5%、122.6%、73.7%和61.2%。用甲胎蛋白处理后细胞内钙水平升高，4分钟时达到204%。结果还表明，AFP（20mg·L⁻¹）可上调Bel 7402细胞中N-ras癌基因以及p53和p21（ras）的表达。在后一种情况下，与对照相比，变化分别为81.1%（12小时）和97.3%（12小时）。
结论
这些结果表明，AFP是促进人肝癌Bel 7402细胞增殖的潜在生长因子。其生长调节作用由其特异性质膜受体介导，通过cAMP-蛋白激酶A途径和细胞内钙进行跨膜信号转导，以调节癌基因的表达。
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