經低劑量地西他濱治療之急性骨髓性白血病細胞以非放射性同位素標定胺基酸細胞培養進行蛋白質體分析

SILAC-based Proteomics Analysis on AML Cell Line with Transient Low-Dose Decitabine Treatment

胡斯萍(Giovanni Audrey Oswita)

指導教授 ： 蔡幸真

國立臺灣大學/醫學院/毒理學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803391

摘要

急性骨髓性白血病（AML）是一種白血球細胞的侵襲性癌症，好發於六十歲以上的長者 。這些病患們常同時罹患其他疾病，故不適合進行較激烈的化學治療或幹細胞移植手術。地西他濱(decitabine)是一種DNA去甲基化藥物，許多臨床試驗已證實這個藥物對年長之急性骨髓性白血病病患具有療效，且多數病患對於此低劑量之表觀遺傳治療具有良好的耐受性，只有輕微的不良反應。我們之前的研究顯示，血癌細胞株和人體初代白血病細胞對於低劑量地西他濱之抑癌效果具有“記憶”效應，這些作用伴隨著全基因體啟動子DNA甲基化的持續降低與許多核心訊息傳導路徑中基因的重新表達。此外，過去研究亦發現地西他濱除了能把DNA去甲基化，還能夠改變全基因體染色質結構和組蛋白修飾，而此染色質變化是因地西他濱對染色質修飾蛋白的直接調節作用或是對基因表達變化的間接反應目前尚不清楚 。因此，本篇研究目的是使用SILAC（以穩定同位素標記之氨基酸進行細胞培養）定量蛋白體學，研究地西他濱治療白血病細胞後染色質修飾蛋白的動態變化。首先，我們使用低劑量的地西他濱處理Kasumi-1白血病細胞株72小時，然後讓細胞株於無藥物之培養基中生長4天，使藥物充份發揮其表觀遺傳效應。接著，在地西他濱處理後的幾個時間點收取細胞以進行全細胞及核蛋白質體學分析。全細胞蛋白質體學發現地西他濱可影響許多細胞分子功能，如細胞凋亡，溶酶體，細胞骨架，蛋白水解等，這與我們過去研究的轉錄體結果一致。接著，我們進行核蛋白質體的分析，因為大多數已知的染色質修飾蛋白位於細胞核內。
為了更深入了解地西他濱處理後不同時間點之蛋白質體動態變化，本研究使用了數種不同的蛋白質體分析軟體來做進一步的分析，如MCODE-蛋白質網絡群集分析與分量回歸分析。結果顯示，在地西他濱連續處理 72 小時後，核蛋白體中與染色質修飾， 染色質組裝及核小體組裝相關的蛋白有顯著增加的現象，而在地西他濱處理後的第八天 (即地西他濱連續處理 72小時後，移除藥物再培養四天)，有顯著變化之蛋白體明顯擴展至各種不同細胞功能網絡，例如對細胞激素的反應，干擾素訊息傳遞，氧化還原代謝反應，細胞有絲分裂等。核蛋白質體分析亦對用藥前後之表觀遺傳相關蛋白變化提供了較佳之質譜鑑定與分辨率。我們發現，白血病細胞經地西他濱處理後，大多數的組蛋白乙醯轉移酶，去乙醯酶及其輔因子表達降低。此外，本研究還觀察到大約30種染色質重塑蛋白於用藥前後有明顯變化，顯示在地西他濱處理後染色質發生了重大變化。總而言之，我的研究深入探討了地西他濱對急性白血病細胞株的表觀遺傳蛋白及其細胞內網路的時序性調控。

關鍵字

地西他濱 ； 急性骨髓性白血病 ； 蛋白質體學 ； 非放射性同位素標定胺基酸細胞培養 ； 表觀遺傳

並列摘要

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.

並列關鍵字

Decitabine ； Acute Myeloid Leukemia (AML) ； Proteomics ； SILAC ； Epigenetics

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