Published online Jun 15, 2002. doi: 10.3748/wjg.v8.i3.469
Revised: February 13, 2002
Accepted: March 5, 2002
Published online: June 15, 2002
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, p53, and p21ras 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, 3H-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 × 10-9 mol·L-1 and 9.9 × 10-8 mol·L-1 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 (20 mg·L-1) could upregulate the expression of N-ras oncogenes and p53 and p21ras in Bel 7402 cells. In the later case, the alteration were 81.1% (12 h) and 97.3% (12 h) 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.