Elucidation of the role of the anti-apoptotic Bcl-2 family A1 in haematopoietic cell development and tumorgenesis by using RNA interference in teh mouse
Zusammenfassung der Projektergebnisse
Apoptosis or programmed cell death is an important process for tissue homeostasis and maintenance. While too much apoptosis can lead to neuronal degeneration for example, too little results in diseases like cancer or autoimmunity. Key players of the apoptotic process are the Bcl-2 proteins, which can be subdivided into the pro-survival and proapoptotic members. Whereas the former are able to induce apoptosis the latter are able to antagonise this by binding and neutralising the pro-apoptotic proteins. Gene-targeting studies in mice have identified the essential roles of most pro-survival Bcl-2 family members in normal physiology and in cancer development or maintenance. However, the function of the pro-survival family member A1 has only been studied by overexpression studies. This is due to the fact that the genetic locus encoding for A1 underwent quadruplication generating 3 functional isoforms (A1a, A1b and A1d) and one pseudogene (A1c). To overcome this problem and increase our knowledge of A1 in vivo, we decided to knock-down all functional A1 isoforms by RNA interference. Since A1a, b and d are more than 96% homologues on DNA level, we identified one shRNA sequence targeting all functional A1 isoforms at once. Following in vitro validation, we incorporated the shRNA into a lentiviral backbone and generated transgenic mice. As it is not known, whether A1 plays a critical survival role for the developing embryo, we decided to use a tetracycline inducible system for A1 specific shRNA expression allowing for spatial and temporal knock-down. Expression studies have shown that A1 is up-regulated during the early stages of T cell development. Consistent with these findings, we observed an impairment of developing thymocytes in the A1 knockdown mice. As previously reported, ectopic expression of A1 can rescue the survival of mature B-lymphocytes lacking the signalling molecule PLC gamma, we observed a lower frequency of those cells in the absence of A1. Furthermore, as predicted from the mice lacking one isoform of A1 (A1a), granulocytes showed increased spontaneous death in culture and failed to accumulate in normal numbers in vivo. This model highlights the critical role of A1 in leukocyte development and homeostasis. Importantly, we will now be able to use these A1 knockdown mice to address a role of endogenous A1 in autoimmunity as well as the development, maintenance and drug sensitivity of lymphomas. This may lead to the development of A1 specific inhibitory compounds for the treatment of patients suffering from lymphoma or autoimmune disease.
Projektbezogene Publikationen (Auswahl)
- Maximal killing of lymphoma cells by DNA damage-inducing therapy requires not only the p53 targets Puma and Noxa, but also Bim. Blood. 2010. 116(24):p. 5256-67
Happo L, Cragg MS, Phipson B, Haga JM, Jansen ES, Herold MJ, Dewson G, Michalak EM, Vandenberg CJ, Smyth GK, Strasser A, Cory S, Scott CL
