Philadelphia positive (Ph+) Acute Lymphoblastic Leukaemia (ALL) is characterised by the presence of the BCR-ABL fusion gene, which encodes a protein tyrosine kinase with aberrant activity. Imatinib, a chemical Bcr-Abl inhibitor, is rarely effective in Ph+ ALL patients as a single agent. In this study, insight into molecular and signalling changes occurring in Ph+ ALL during Imatinib therapy were investigated using cDNA microarrays. An optimal microarray assay was established to examine the gene expression changes in leukaemic cells from Ph+ ALL patients treated with Imatinib. Over 500 genes with ≥1.5-fold up- or down-regulation were identified. Based on gene ontology and novelty to Bcr-Abl signalling, six genes were selected and expression changes in five of these genes (PKCε, PINK1, SPRY2, ATF4 and PECAM1) confirmed by real time RT-PCR in Imatinib treated primary Ph+ ALL cells or the SUP-B15 cell line. The functional role of Protein Kinase C epsilon (PKCε) in response to Imatinib was further investigated using the Ph+ lymphoid and myeloid cell lines, SUP-B15 and K562. Detection of Imatinib-induced apoptosis by annexin V and PI staining demonstrated that SUP-B15 cells were less sensitive to Imatinib compared to K562 cells. PKCε mRNA was 50-fold higher in Ph+ ALL cells than Ph+ myeloid cells. In SUP-B15 cells, Imatinib upregulated PKCε mRNA but the protein was reduced by proteolytic cleavage. Inhibition of caspases showed that this cleaved product was not required for Imatinib induced-apoptosis. The treatment of SUP-B15 and primary Ph+ ALL cells with TAT-εV1-2 peptide, a specific inhibitor of PKCε, increased Imatinib-induced apoptosis. While the forced overexpression of PKCε in K562 cells reduced Imatinib-induced apoptosis. This increased expression of PKCε was associated with the increase of survival and anti-apoptotic proteins, Akt and Bcl-2. In summary, Gene expression profiling of Ph+ ALL cells during Imatinib therapy identified PKCε as an Imatinib responsive gene. A novel role of PKCε in Ph+ ALL response to imatinib is proposed. Experimental data presented in this thesis indicate that PKCε mediates pro-survival/anti-apoptosis signals in Ph+ ALL thereby reducing Imatinib-induced death. Thus, targeting PKCε during Imatinib therapy may be beneficial for the future treatment of Ph+ ALL.
| Identifer | oai:union.ndltd.org:ADTP/258343 |
| Date | January 2008 |
| Creators | Loi, To Ha, Clinical School - St Vincent's Hospital, Faculty of Medicine, UNSW |
| Publisher | Publisher:University of New South Wales. Clinical School - St Vincent's Hospital |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://unsworks.unsw.edu.au/copyright, http://unsworks.unsw.edu.au/copyright |
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