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 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-p40 even in the presence of 10% serum, and it also induced apoptosis.
Kaneda
Y
Y
Institute for Molecular and Cellular Biology, Osaka University, Suita, Osaka 565, Japan. kaneday@imcb.osaka-u.ac.jp
Kinoshita
K
K
Sato
M
M
Saeki
Y
Y
Yamada
R
R
Wataya-Kaneda
M
M
Tanaka
K
K
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015152:Blotting, Northern; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002467:Cell Nucleus / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D002795:Choline O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D010957:Plasmids; D011498:Protein Precursors / Q000032:analysis / Q000235:genetics*; D016372:RNA, Antisense; D016337:RNA, Catalytic / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D017474:Receptors, Laminin / Q000032:analysis / Q000235:genetics*; D014170:Transformation, Genetic
D011498:Protein Precursors; D016372:RNA, Antisense; D016337:RNA, Catalytic; D012333:RNA, Messenger; D017474:Receptors, Laminin; D007544:Isopropyl Thiogalactoside; D002795:Choline O-Acetyltransferase