A potent cell death activity associated with transient high level expression of BCL-2.
The BCL-2 proto-oncogene contains unusually long untranslated 5' and 3' sequences. Deletion of the sequences flanking the BCL-2 open reading frame dramatically increases the level of protein expression. Transient high level BCL-2 protein expression mediated by plasmid transfection or by infection with recombinant adenovirus results in potent apoptosis of several cell lines. Detailed mutational (deletion and add-back) analysis reveals that both 5'- and 3'-flanking sequences contribute to the negative modulation of protein expression from the BCL-2 open reading frame. It appears that these sequences exert the negative regulatory effect in an orientation-dependent manner. Analysis of BCL-2 RNA levels indicate that elevated levels of mRNA may be the primary cause of elevated levels of protein expression. Apoptosis induced by adenovirus vectors expressing elevated levels of BCL-2 can be readily inhibited by the caspase inhibitor z-VAD-fmk, suggesting that high levels of BCL-2 expression induce apoptosis via the caspase cascade. Mutational analysis of BCL-2 indicates that its pro-apoptotic activity is separable from its anti-apoptosis activity. Our results raise the possibility that oncogenic conversion of BCL-2 may require somatic mutations in the pro-apoptotic activity, in addition to other activating mutations that result in enhanced expression. Consistent with this hypothesis, a somatic mutation of BCL-2 observed in multiple human tumors results in reduced apoptosis activity.
Uhlmann
E J
EJ
Institute for Molecular Virology, Saint Louis University Health Sciences Center, St. Louis, Missouri 63110, USA.
Subramanian
T
T
Vater
C A
CA
Lutz
R
R
Chinnadurai
G
G
D000256:Adenoviridae / Q000235:genetics; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D001483:Base Sequence; D029742:Caenorhabditis elegans Proteins; D020169:Caspases; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D017931:DNA Primers; D019254:Genes, bcl-2; D005822:Genetic Vectors; D006801:Humans; D016296:Mutagenesis; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics
D000590:Amino Acid Chloromethyl Ketones; D029742:Caenorhabditis elegans Proteins; D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D020169:Caspases; D003546:Cysteine Endopeptidases; C089223:ced-3 protein, C elegans