An investigation of the mechanism of cisplatinum-induced apoptosis in SH-SY5Y neuroblastoma cells
Neuroblastomas are common paediatric tumours derived from the sympathoadrenal lineage. Neuroblastoma cells may arise from neuroblasts, which failed to differentiate or which were not eliminated by apoptosis at an appropriate stage of development. The aim of this thesis was to identify the signalling pathway by which cis-diamminedichloroplatinum (II) (CDDP) a chemotherapeutic agent, triggers caspase activation and apoptosis in the SH-SY5Y neuroblastoma cell line. An understanding of this may prove to be useful for developing better therapeutic agents for treating neuroblastoma and for understanding mechanisms of drug resistance. CDDP was found to induce apoptosis in SH-SY5Y cells via the mitochondrial death pathway, with cytochrome c release and activation of caspases-9 and -3. CDDP, a DNA damaging agent, activates p53 in SH-SY5Y cells and p53 is known to induce apoptosis via the mitochondrial pathway. Bcl-2 family members play a central role in the regulation of the mitochondrial death pathway and may have pro- or antiapoptotic activity. The pattern of expression of members of the Bcl-2 family following CDDP treatment was investigated to determine their regulatory role. PUMA (p53 upregulated modulator of apoptosis), a proapoptotic BH3-only protein and a direct transcriptional target of p53, was found to be upregulated at both the mRNA and protein levels during CDDP-induced apoptosis of SH-SY5Y cells. PUMA has three transcripts that encode PUMA- a, P and 5. PUMA-a and PUMA-(3 are proapoptotic and contain the BH3 domain. PUMA-a was identified as the transcript that increased during CDDP treatment in SH-SY5Y cells. Overexpression of PUMA-a in SH-SY5Y cells was sufficient to induce apoptosis. To identify other genes regulated by CDDP, we performed oligonucleotide microarray analysis using Affymetrix human genome-U133A microarrays and RNA extracted from SH-SY5Y cells treated with DMSO, transplatinum (an isomer of CDDP which does not induce cell death) and CDDP. The results provide a detailed picture of the changes in gene expression that occur following CDDP treatment.