Part I
BCR-ABL fusion oncogene results fromt(9;22)(q34;q11) translocation of chromosome is the most common genetic abnormality found in chronic myeloid leukemia (CML) cells . The encoded protein of this fusion gene exhibits constitutively active tyrosinekinase activity which is required for the pathogenesis of CML. We addressed how BCR-ABL oncoprotein increased Skp2 expression. Treatment of Imatinib or LY294002 reduced Skp2mRNA in BCR-ABL-positive K562 cells. Knockdown of AKT by small hairpin RNAalso reduced Skp2 expression. We found that BCR-ABL up-regulated Skp2 via Sp1 because (1) the Sp1 site located at the −386/−380 promoter region was important for BCR-ABL-induced Skp2 promoter activity, (2) chromatin immunoprecipitation assay demonstrated that Imatinib inhibited the recruitment of p300 to the Sp1 site of Skp2 promoter and (3) knockdown of Sp1 reduced Skp2 expression in K562 cells. These results suggest that BCR-ABL controls Skp2 gene transcription via the PI3K/AKT/Sp1 pathway. In addition to transcriptional regulation of Skp2, Bcr-Abl also modulates Skp2 protein stability in these cells. Treatment of Bcr-Abl kinase inhibitor imatinib led to G1 growth arrest accompanied with reduced Skp2 expression. Interestingly, reduction of Skp2 protein occurred prior to down-regulation of Skp2 mRNA suggesting a post-translational control. The half-life of Skp2 protein was significantly attenuated in imatinib-treated cells. Knockdown of Bcr-Abl similarly caused Skp2 protein instability. The decrease of Skp2 was induced by increased protein degradation through the ubiquitin/ proteasome pathway. Our results demonstrated that imatinib treatment or Bcr-Abl knockdown reduced Emi1, an endogenous inhibitor of the E3 ligase APC/Cdh1 which mediated the degradation of Skp2 protein. We found that Emi1 stability was regulated by phosphorylation and mutation of tyrosine 142 significantly reduced the stability. Lines of evidence suggested Bcr-Abl-induced Emi1 phosphorylation was mediated by Src kinase. (1) Src inhibitor SU6656 inhibited Emi1 tyrosine phosphorylation in Bcr-Abl-positive K562 cells. (2) Transfection of v-Src rescued the reduction of Emi1 by imatinib. (3) Mutation of tyrosine 142 to phenylalanine (Y142F) abolished the phosphorylation of Emi1 by recombinant Src kinase. In addition, ectopic expression of wild type but not Y142F mutant Emi1 could counteract imatinib-caused G1 growth arrest. Collectively, our results suggest that Bcr-Abl fusion oncogene increases Emi1 phosphorylation and stability to prevent Skp2 protein degradation via APC/Cdh1-induced ubiquitination and to enhance proliferation of CML cells.
Part II
Although imatinib therapy of chronic myelogenous leukemia is effective, the resistance to imatinib challenges the treatment of this disease. Therefore, search of novel drugs to overcome imatinib resistance is a critical issue in clinic. Withaferin A (WA), an extract of Withania somniferia, exhibits anti-cancer activity on a number of solid tumors. In this study, we investigate the effect of WA on imatinib-sensitive and -resistant CML cells. WA at low concentrations induced autophagy in imatinib-sensitive K562 cells. Co-treatment of chloroquine suppressed autophagy and switched WA-treated K562 cells to apoptosis. This data indicated that autophagy protected K562 cells from apoptosis induced by WA. However, we found that WA triggered caspase activation and apoptosis in imatinib-resistant T315I-positive cells and this effect was associated with down-regulation of Akt activity. Treatment of the AKT inhibitor LY294002 also caused apoptosis in imatinib-resistant T315I-positive cells. Ectopic expression of constitutively active Akt reversed WA-induced apoptosis and caspase activation in imatinib-resistant T315I-positive cells. Molecular study demonstrates that WA repressed the Akt signaling pathway by decreasing Akt expression. We found that WA abolished formation of the hsp90/cdc37/Akt complex to cause Akt degradation through the ubiquitin- and proteasome-dependent pathway. More importantly, WA also induced AKT down-regulation and apoptosis in primary CML cells. Taken together, our results suggested that imatinib-resistant T315I-positive cells were more addicted to Akt-dependent survival pathway and were more sensitive to WA. Therefore, WA could be useful for the treatment of imatinib-resistant CML.
Part III
Suberoylanilide hydroxamic acid (SAHA) is undergoing clinical trial for the treatment of various cancers including chronic myeloid leukemia (CML). We study the potential miRNAs which involved in the anti-cancer effect of SAHA. Microarray analysis revealed that the expression of 57 and 63 miRNAs was significantly changed in K562 cells treated with SAHA for 8h and 24h respectively. Five miRNAs(miR-92a, miR-199b-5p, miR-223, miR-627 and miR-675) were highly expressed in K562 cells and continuously repressed by SAHA. miR-92a and miR-223 known to play important roles in normal and hematopoisis were further characterized. Up-regulation of miR-92a was found in K562 cells and in primary CML cells. Inhibition of miR-92a with SAHA led to increase of the tumor suppressor Fbxw7. Conversely, ectopic expression of pri-miR-92a reversed SAHA-induced apoptosis of K562 cells, increase of Fbxw7 3¡¦-UTR reporter activity and up-regulation of Fbxw7. Collecively, miR-92a is up-regulated in CML cells, and SAHA downregulated the expression of miR-92a to result in apoptosis of CML cells.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0802110-181153 |
| Date | 02 August 2010 |
| Creators | Chen, Jing-yi |
| Contributors | Yow-Ling Shiue, Yeou-Lih Huang, Long-sen Chang, Shinne-Ren Lin, Wen-Chun Hung |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0802110-181153 |
| Rights | withheld, Copyright information available at source archive |
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