The induction of apoptosis in HeLa cells by the loss of LBP-p40
The induction of apoptosis in HeLa cells by the loss of LBP-p40
复制标题
DOI：
10.1038/sj.cdd.4400315
复制
发表时间：
1998-01-01
影响因子：
12.4
通讯作者：
Tanaka, K
中科院分区：
文献类型：
--
作者：
Kaneda, Y;Kaneda, Y;Tanaka, K
为了分析laminin-binding protein前体p40 （LBP-p40）在高等真核细胞中的功能，在LacSwitch系统的控制下，将表达p40反义或正cDNA的质粒DNA导入HeLa细胞，分离稳定转化子，采用Western和Northern blotting检测p40的表达。在添加10%血清的培养基中，用5 mM IPTG诱导反义(AS)-p40，对HeLa细胞转化体p40的表达水平没有影响。然而，在含有5 mM IPTG的无血清培养基中，内源性p40蛋白和信息的表达水平降低到无5 mM IPTG培养基中的30-10%左右。p40的抑制抑制了菌落的形成，AS-p40克隆在含5 mM IPTG的无血清培养基中培养7天后死亡，而不含5 mM IPTG的AS-p40克隆即使在无血清培养基中也未死亡。此外，sense (S)-p40克隆和对照CAT克隆在含5 mM IPTG的无血清培养基中存活超过2周。DNA片段化实验表明，AS-p40减少引起的细胞死亡是由凋亡引起的。将p40 cDNA表达载体转移到AS-p40克隆中，可以部分恢复细胞生长抑制和凋亡细胞死亡。此外，使用自生病毒脂质体引入LBP-p40合成锤头核酶，即使在10%血清存在的情况下，也能抑制LBP-g40的信息，并诱导细胞凋亡。
To analyze the function of the laminin-binding protein precursor p40 (LBP-p40) in higher eukaryotic cells, plasmid DNA expressing antisense or sense cDNA for p40 under the control of the LacSwitch system was introduced into HeLa cells, Stable transformants were isolated, and the expression of p40 was assayed by Western and Northern blotting. The expression level of p40 was not affected in HeLa cell transformants cultured in 10% serum-supplemented media with the induction of antisense (AS)-p40 with 5 mM IPTG. However, both the protein and message for endogenous p40 in serum-depleted media with 5 mM IPTG were reduced to about 30-10% of the expression level in serum-free media without 5 mM IPTG. Colony formation was inhibited with the suppression of p40, AS-p40 clones died in 7 days when cultured in serum-depleted media with 5 mM IPTG, while clones without 5 mM IPTG AS p40 clones never died, even in serum depleted media. Additionally, sense (S)-p40 clones and control CAT clones survived more than 2 weeks in serum-free media with 5 mM IPTG. DNA fragmentation assay revealed that cell death induced by the reduction of AS-p40 resulted from apoptosis. Both the inhibition of cell growth and apoptotic cell death were partially rescued by the transfer of the p40 cDNA expression vector to AS-p40 clones. Moreover, the introduction of a synthetic hammerhead ribozyme for LBP-p40 using a fusigenic viral liposome suppressed the message for LBP-g40 even in the presence of 10% serum, and it also induced apoptosis.