THE INDUCTION OF APOPTOSIS IN HELA-CELLS BY THE LOSS OF LBP-P40

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 in troduced into HeLa cells, Stable transformants were isolated, and the expression of p40 was assayed by Western and Northern blotting. The ex pression level of p40 was not affected in HeLa cell transformants cult ured in 10% serum-supplemented media with the induction of antisense ( AS)-p40 with 5 mM IPTG. However, both the protein and message for endo genous p40 in serum-depleted media with 5 mM IPTG were reduced to abou t 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 c lones died in 7 days when cultured in serum-depleted media with 5 mM I PTG, while clones without 5 mM IPTG AS p40 clones never died, even in serum depleted media. Additionally, sense (S)-p40 clones and control C AT clones survived more than 2 weeks in serum-free media with 5 mM IPT G. DNA fragmentation assay revealed that cell death induced by the red uction 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 int roduction of a synthetic hammerhead ribozyme for LBP-p40 using a fusig enic viral liposome suppressed the message for LBP-g40 even in the pre sence of 10% serum, and it also induced apoptosis.