Abstract
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.
Highlights
It is well established that the BCL-2 proto-oncogene suppresses apoptosis induced by a multitude of stimuli in a variety of cell types [1, 2]
We show that deletion of the 5Јand 3Ј-flanking sequences results in a large increase in BCL-2 protein expression, which results in cell death via the CED-3/ ICE family caspase cascade
We have identified a surprising cell death activity of BCL-2 manifested during high levels of transient protein expression
Summary
It is well established that the BCL-2 proto-oncogene suppresses apoptosis induced by a multitude of stimuli in a variety of cell types [1, 2]. Transfection of the pcDNA3 vector or the vector expressing the parental cDNA (pcDNA3-Bcl2) did not induce significant cell death (Fig. 1B). The observed cell death activity of BCL-2 was confirmed with two different independently derived clones that lack the flanking sequences (not shown).
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