Bam - a novel glucocorticoid-regulated BH3-only transcript

Apoptosis is a morphologically defined form of cell death that plays a major role in cell physiology, pathology and cancer therapy. The Bcl-2 family of pro- and anti-apoptotic molecules is a key regulator of this phenomenon, with the sub-family of BH3-only molecules serving as activators and/or facilitators. Apoptosis induced by glucocorticoids (GC) is a central component in the therapy of acute lymphoblastic leukemia (ALL), and defining its molecular basis and that of GC resistance is crucial for therapeutic improvements. We recently identified a novel transcript from the BCL2L11/Bim locus, termed "Bam", that was induced in 7/14 patients (13 children, 1 adult) with ALL during GC monotherapy. In a GC-sensitive ALL cell line, this transcript was induced by GC in a translation-independent manner, suggesting that direct transcriptional induction of this BH3-only molecule by GC might cause apoptosis in at least some ALL children (and perhaps adults). In addition to its possible function in GC-induced apoptosis, this novel BH3 molecule might participate in other death pathways as well. Herein, we propose to determine (1) whether transgenic Bam, conditionally expressed at levels similar to those obtained after GC induction, induces cell death in leukemic ALL cell lines, ex vivo cells from ALL patients and non-lymphoid cells (2) whether conditional Bam knock-down prevents GC-induced apoptosis in the above lymphoid systems, (3) whether the putative Bam promoter region confers GC responsiveness to reporter constructs, (4) where Bam is located in the cell, and (5) whether it is expressed in mice as well. For the proposed studies, we have developed lentiviral expression systems that allow conditional (i.e., tetracycline-regulated) gene over-expression and gene knock-down by RNA interference (RNAi) with small hairpin RNAs. The promoter studies will exploit standard transient transfection assays with wild-type and mutated Bam promoter constructs, location studies will use live cell microscopy, and RACE technology will facilitate identification of Bam transcripts in mice. In conclusion, the proposed research is aimed to characterize a novel putative BH3-only molecule and its role in GC-induced, and perhaps other forms of, apoptosis.

Glucocorticoid (GC) - induced apoptosis is a central component in the therapy of acute lymphoblastic leukemia (ALL), and defining its molecular basis and that of GC resistance is crucial for therapeutic improvements. GC act via their specific receptor, a ligand activated transcription factor of the family of nuclear receptors. Hence, most of the effects of GC are mediated through changes in gene expression, but which gene regulations are responsible for GC-induced apoptosis in leukemia patients is not understood. In the course of an extended expression profiling analysis of experimental systems (leukemia cell lines and mouse thymocytes) and, more importantly, lymphoblasts from children with ALL during systemic GC mono-therapy, we observed that "Bam" was induced in many children during GC therapy. Bam is a predicted 2 exon gene embedded in the BCL2L11/Bim locus. Bim encodes a powerful killer protein of the BH3-only proteins of the BCL2 family. Since the predicted Bam protein contains the same BH3 killer domain as Bim, we proposed to study this gene/protein in more detail. Our work showed that, although Bam mRNA is generated in lymphoid leukemia cells, it is not readily translated into a protein due to lack of a typical translational initiation sequence (Kozak sequence) and additional out-of-frame start codons in its 5`untranslated region. Hence, it is unlikely that Bam is an important mediator of GC-induced cell death. However, we discovered a novel alternative Bim transcript that combines the Bim 5` region with the Bam 3`region leading to a predicted protein sharing the N-terminal amino acids with Bim (including the BH3 domain) but containing an entirely different C-terminus. We show that this novel transcript (termed Bimbam) exists - like Bim - in 3 splice variants, BimbamEL, L and S (extra long, long and short) and is translated into a protein which has similar killing potency as Bim. Since the 2 proteins have very different C-termini and since the Bim C-terminus is responsible for mitochondrial localization and apoptosis induction, the equal killing potency was surprising. We show that Bimbam localizes both to mitochondria and other cytoplasmic membranes (like the endoplasmatic reticulum) and provide evidence that this localization is mediated through the BH3 only domain that presumably interacts with BCL2 family members that are known to locate to these membranes. Bim, in contrast, locates predominantly to the mitochondrial membrane via its unique C-terminus. These findings raise the possibility that Bim and Bimbam might employ distinct killing mechanisms. Moreover, since currently used antibodies do not discriminate between Bim and Bimbam, several findings ascribed to Bim might in reality be the result of Bimbam. What remains to be determined is to what extent Bimbam contributes to GC-induced and/or perhaps other cell death forms.