Identification of a candidate alternative promoter region of human BCL2L11 (BIM) - Characterization of pancreatic cancer stem cells

Abstract I part “Identification of a candidate alternative promoter region of Human BCL2L11 (BIM)”. Background: Despite the importance of the BCL2L11 (BIM) protein in various apoptotic processes in development and disease, little is known of the promoter structure of the human BCL2L11 locus and of the cis-acting elements regulating expression of the human gene. Results: In the search for novel promoter sequences in the human BCL2L11 locus, we have identified previously unrecognized genomic sequences displaying promoter activity and E2F responsiveness, and driving the expression of BCL2L11 coding transcripts. In man, transcripts originating from this novel putative promoter contribute significantly to total BCL2L11 mRNA expression in testis, heart and liver. In HEK293 cells, this novel candidate promoter originates BCL2L11 transcripts whose expression can be modulated by a known modulator of BCL2L11 expression (Trichostatin A) and by E2F, a characterized transcriptional regulator of BCL2L11 expression. Conclusion: The identification of a novel putative human BCL2L11 promoter provides new insights into the structure and regulation of the BCL2L11 locus. Abstract II part “Characterization of pancreatic cancer stem cells” Background: Pancreatic cancer remains a highly aggressive and not curable cancer in spite of the ample research in the last decades. Recent studies have suggested the existence of neoplastic stem cells that sustain the growth of cancer. The aim of the present thesis was to characterize the expression profiles and potential stem-like behavior of Panc-1 cells grown as 3- dimensional spheres. The pancreatic spheres were generated by the addition of well-defined growth factors. The aim of the current study was to link aggressiveness to stemness in sphere growing pancreatic tumour cells. Results: To test the hypothesis that cancer cells growing as spheres demonstrate stem-like properties whereas the cancer cells that grows adhering to the substrate do not, the expression of the reported pancreatic stem cell markers (CD24, CD44 and ESA) has been assessed in PANC1 spheres and adherent cells at the gene and protein level using qRT-PCR and FACS analysis. The higher expression of the metastasis and invasion markers CXCR4, Osteopontin (up to 300 fold), and Sonic Hedgehog pathway elements in the spheres supports the concept that cancer cells able to grow in spheres allegedly possess an increased aggressivity. The different behaviour of spheres vs. adherent cells has been confirmed using a panel of in vitro phenotypic assays as well as in a xenograft mouse model. Conclusion: We have identified and validated a novel research tool which mimics better the real clinical situation for both in vitro as well as for in vivo further study of potential drug candidates. PDF