Abstract 2006: Identification and molecular cloning of novel splicing variants of BCL2L12, a new member of the BCL2 family, and their expression analysis in breast, prostate, and colon cancer cells
Authors: Kontos Christos K. , Thomadaki Hellinida , Mavridis Konstantinos , Ardavanis Alexandros , Scorilas Andreas 
Published: August 2012

Abstract
BCL2L12 is a recently discovered member of the apoptosis-related BCL2 family, most members of which are subjected to alternative splicing. Currently, three distinct BCL2L12 variants are known. Alternative splicing may contribute to the etiology of many diseases including cancer, since novel protein isoforms that arise often contain additional functional domains or lack some of the structural motifs of the classical isoform. Hence they acquire new properties or lose some of them, respectively. From a clinical point of view, splicing variants are particularly important in oncology, since they provide selective drug targets or may even serve as a multifactorial marker set for cancer diagnosis and/or prognosis. The aim of the present study was to identify novel spliced variants of the BCL2L12 gene and to study their expression profile in breast, prostate, and colon cancer cell lines. We firstly identified in silico all EST clones displaying homology with the classical BCL2L12 variant. Next, total RNA was extracted from breast, prostate, and colon cancer cells, and first-strand cDNA was synthesized. PCR was carried out using a pair of gene-specific primers for the amplification of the complete coding region plus a small part of the 5α-UTR of the BCL2L12 mRNA transcripts. All PCR products were electrophorized, bands were excised and purified, and PCR amplicons were sequenced directly or after being subcloned. We also performed nested PCR with newly designed variant-specific anti-sense primers, in order to analyze their expression in human cancer cell lines. We identified and cloned ten novel BCL2L12 splicing variants, seven out of which are predicted to encode new protein isoforms, including BH3-only proteins. The remaining three novel splicing variants of BCL2L12 are nonsense-mediated mRNA decay (NMD) candidates. More importantly, notable variations in expression of each BCL2L12 transcript were observed between cancer cell lines, even those originating from the same tissue or organ. For instance, breast cancer BT-20 and BT-474 cell lines present a very similar expression profile of BCL2L12 splicing variants; still, this is very different from the one of the MCF7 cells. Furthermore, great similarities were observed in BCL2L12 expression profiles of colon cancer cells (DLD-1, HT-29 and HCT 116), with the exception of Caco-2 cells, which show a moderate expression of BCL2L12 transcript variant 13. In conclusion, ten novel splicing variants of BCL2L12 were cloned. Subsequent expression analysis in cancer cell lines revealed remarkable differences between their expression profiles.Acknowledgements: This work was supported by a THALIS grant (#224), co-funded by the European Union (European Social Fund) and National Resources (NSRF 2007-2013, Operational Programme “Education and Lifelong Learning”).
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2006. doi:1538-7445.AM2012-2006