| Summary: | 碩士 === 國立臺灣大學 === 毒理學研究所 === 106 === Acute myeloid leukemia (AML) is an aggressive type of white blood cell cancer with most patients being beyond 60 years of age. These elderly patients are often present with various comorbidities that make them unsuitable for harsh conventional chemotherapy or curative stem cell transplantation. Decitabine (DAC), a DNA demethylating agent, was found to confer some efficacy to this group of patients in clinical trials. The low-dose epigenetic regimen is generally well-tolerated with mild adverse effects. Previously, our study had shown that DAC at clinically-relevant low doses induces ‘memory-like’ antitumor response in AML cell lines and primary human AML cells. These effects were accompanied by sustained decreases in genome-wide promoter DNA methylation and re-expression of genes in many core signaling pathways. Moreover, we found that DAC was able to alter genome-wide chromatin structure and histone modifications in addition to DNA demethylation. Nevertheless, it remains unclear whether DAC-induced chromatin changes are the results of direct regulatory effects of DAC on chromatin modifiers or indirect responses to global gene expression changes. Thus, the aim of this study is to characterize the dynamic changes of chromatin modifiers in leukemia cells following low-dose DAC treatment using stable isotope labeling with amino acid in cell culture (SILAC)-based quantitative proteomics.
Firstly, Kasumi-1 AML cell line was treated with DAC at a clinically-relevant dose for 3 days (Day 4) followed by a DAC-free growth for 4 days (Day 8), allowing the drug to fully exert its epigenetic effects. Next, whole cell lysate and nuclear proteins were extracted on Day 4 and Day 8, then analyzed with proteomics method. With the whole cell lysate, we identified major biological processes and cell compartment annotation mediated by DAC, such as apoptosis, lysosome, cytoskeleton, proteolysis, etc, which was in consistent with our previous transcriptomic data. Subsequently, we focused on nuclear proteomics since most of the known chromatin modifiers are localized in the nucleus. To gain insight into the dynamic proteomic changes over a time course following DAC treatment, we employed MCODE-protein network clustering bioinformatics analysis. The result showed that on Day 4 following DAC-treatment, nuclear-isolated proteins showed enrichment of pathways related to chromatin modification, chromatin organization and nucleosome assembly. On day 8, the modulated networks shifted to additional pathways such as response to cytokine, type I interferon signaling, oxidation-reduction metabolic process, mitotic cell cycle, transcription elongation, etc. The result of nuclear-isolated proteins also revealed better mass spectrometry identification and resolution on epigenetic-related proteins. Interestingly, in epigenetic proteins, we observed the downregulation of histone acetyl-transferases and deacetylases. On the other hand, more than 30 chromatin remodeling proteins were also differentially expressed, indicating that major changes happened at the chromatin level post-DAC treatment. Furthermore, one of the chromatin modifiers, lymphoid-specific helicase (HELLS) was upregulated on Day 4 and downregulated on Day 8 following DAC treatment which occurred inversely to DNMT1 levels. The differential expression of HELLS was also validated by immunoblot analysis. We will further investigate the role of HELLS on the anti-tumor effect of DAC-treatment in AML cell line. Hence, our result shed some light on the previously unclear modulation of cellular and epigenetic pathways by DAC in a human AML cell line over the time course of DAC treatment.
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