The analysis of checkpoint mechanism of cell cycle in liver cancer cells

利用AAV载体的基因打靶技术，我们建立了异源敲除的HepG 2细胞克隆（p53+/-），其中内源性TP 53等位基因的外显子2被破坏。然而，尚未获得TP 53 homo敲除的HepG 2细胞克隆。这些结果表明，p53可能是HepG 2细胞增殖所必需的，但使用siRNA敲低p53的表达并不降低细胞增殖。另一方面，我们发现p53+/-细胞持续表达p53的亚型deltaN 40 p53。由于deltaN 40 p53在肝癌中的功能尚不清楚，我们采用多种方法研究了deltaN 40 p53在HepG 2细胞中的功能。我们的研究表明，deltaN 40 p53增强了p21的表达，并诱导HepG 2细胞G1/S期阻滞增加，即deltaN 40 p53弥补了p53的功能，在p53表达降低的情况下抑制肿瘤细胞增殖并诱导细胞衰老。

Using gene targeting with AAV vectors, we established hetero knock-out HepG2 cells clones (p53+/-) of which Exon2 in endogeneous TP53 alleles were destroyed. However, TP53 homo knock-out HepG2 cells clones have not been obtained. These results derived a hypothesis that p53 might be essential for cell proliferation in HepG2 cells, but knock down of expression of p53 using siRNA did not decrease cell proliferation.On the other hand, we found that p53+/- cells constantly expressed deltaN40p53, an isoform of p53. Since the function of deltaN40p53 has been known little in liver cancer, we investigated the function of deltaN40p53 in HepG2 cells by using several methods. Our studies revealed that deltaN40p53 enhanced the expression of p21 and induced an increase of G1/S arrest, that is, deltaN40p53 made up for the function of p53 and suppressed tumor cell proliferation and induced cellular senescence in HepG2 cells when the expression of p53 was reduced.