Proapoptotic Bcl-2 family members provoke cell death by neutralizing their anti-apoptotic relatives, which in turn maintain cell viability by regulating the activation of the cell death effectors, the caspases. In this study we investigate a potential novel splice variant of human BCL2L11/Bim, termed \#8220;Bimbam\#8221;. First evidence for Bimbam was obtained by Affymetrix-based whole genome comparative expression profiling, where a corresponding probe set was found to be induced in 8 of the 13 ALL children and 1 adult during systemic glucocorticoid (GC) monotherapy (Schmidt et al., Blood 107: 2061, 2006). The postulated Bimbam mRNA consists of the 5\#8217; portion of Bim (up to and including the BH3-containing exon 8) and the 3\#8217; portion of Bam, a cDNA originally discovered in a multiple myeloma cDNA library (Claudio et al, 2002). Thus, it encodes a putative protein consisting of the N-terminal region of Bim (including its BH3 domain) and 40 C-terminal amino acids derived from Bam and not present in any known Bim transcript. RT-PCR revealed that the postulated Bimbam mRNA is indeed present in ALL cell lines. The TBP-normalized expression level of Bimbam is similar to that of Bim in 8 leukemia cell lines as analysed by quanitative RT-PCR. Bimbam and Bim proteins have similar molecular masses and could not be distinguished by conventional Western technology. However, the two isoforms were readily discriminated by 2D gel electrophoresis. The subcellular localization of Bimbam, whose C-terminus is entirely different from that of Bim, is currently being investigated in relation to Bim using YFP-fusion proteins. To investigate its function and potential role in GC-induced apoptosis, recombinant Bimbam was cloned into an HIV-derived lentiviral vector for conditional gene expression and subsequently used to infect human T-ALL cells (CEM-C7H2-2C8) that constitutively express a tetracycline responsive transactivator (rtTA). Stable transfected 2C8 subclones were generated and analyzed by real time RT-PCR, Western blotting and FACS analyses. These analyses revealed that recombinant BimBam appears to be a killer protein with potency similar to that of Bim. Thus this novel splice variant may contribute to the anti-leukemic effects of GCs and perhaps other apoptotic responses.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3264.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO