Medulloblastoma is the most commonly occurring malignant brain tumour of childhood. The RASSF1A tumour suppressor is potentially the most important candidate gene identified in medulloblastoma to date, being epigenetically silenced in over 79% of primary tumours. In other tumour types, RASSF1A has been shown to play key roles in both cell cycle regulation and apoptotic signalling pathways. However, its functional role has not been previously addressed in medulloblastoma. UW228-3 medulloblastoma cells, stably expressing RASSF1A displayed increased sensitivity to an anti-CD95 antibody and to the chemotherapeutic agents, etoposide, cisplatin or vincristine, compared to vector control cells. In RASSF1A-expressing UW228-3 cells, increased cleavage of key caspases and of the DNA repair protein, PARP was observed following treatment with anti-CD95 or etoposide in comparison to vector control cells. In addition, inhibition of caspases in RASSF1A-expressing UW228-3 cells by Z-VAD-FMK, prevented apoptotic DNA fragmentation following death receptor activation (by anti-CD95) or DNA damage (induced by etoposide). Increased phosphorylation of H2AX was also observed in RASSF1A-expressing UW228-3 cells during apoptosis, suggesting a possible novel function for this tumour suppressor in the regUlation of histone modification during apoptotic cell death. Increased activation of the pro-apoptotic BCL-2 family member, BAX was also observed in UW228-3 cells stably expressing RASSF1A following activation of both the extrinsic and intrinsic apoptotic pathways. Further investigation of BAX activation in UW228-3 cells revealed that apoptotic signalling can also be promoted in the absence of RASSF1A using the BH3-only mimetic, ABT-737 (BAD-mimetic) in combination with chemotherapeutic agents (etoposide, cisplatin and vincristine). ABT- 737 increased susceptibility to apoptosis induced by DNA damage regardless of RASSF1A expression status through increased activation of BAX. In conclusion, data from this study shows that RASSF1A has a proapoptotic function in vitro and may potentially modulate therapeutic response via the BCL-2 family (and BAX in particular). This family of proteins therefore represent tractable therapeutic targets in medulloblastoma.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:555734 |
| Date | January 2011 |
| Creators | Levesley, Jane |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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