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. Transie nt high level BCL-2 protein expression mediated by plasmid transfectio n or by infection with recombinant adenovirus results in potent apopto sis of several cell lines. Detailed mutational (deletion and add-back) analysis reveals that both 5'- and 3'-flanking sequences con tribute to the negative modulation of protein expression from the BCL-2 open r eading frame. It appears that these sequences exert the negative regul atory 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 adenov irus vectors expressing elevated levels of BCL-2 can be readily inhibi ted 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 separa ble from its antiapoptosis activity. Our results raise the possibility that oncogenic conversion of BCL-2 may require somatic mutations in t he pro-apoptotic activity, in addition to other activating mutations t hat result in enhanced expression. Consistent with this hypothesis, a somatic mutation of BCL-2 observed in multiple human tumors results in reduced apoptosis activity.