臺灣博碩士論文加值系統

白血病為常見的孩童及老年人癌症，目前以化療、促分化療法及標靶治療為主要治療方針。90%以上的慢性骨髓性白血病 (chronic myeloid leukemia，CML)病患帶有BCR-ABL致癌基因。BCR-ABL融合蛋白俱持續性酪胺酸激酶的活性，因而導致細胞異常增生、分化或存活。Imatinib (商品名: Glivec；中文名:基利克) 為針對帶有BCR-ABL融合蛋白的CML所採用具特異性的酪胺酸激酶抑制劑，是目前第一線治療藥物並有良好的緩解作用。然而，仍有CML病患面臨Imatinib治療反應不佳、產生抗藥性及副作用等挑戰。因此，尋找新的合併治療標靶以增加Imatinib的成效是目前需要的新治療策略。p38 MAPK活性受上游磷酸化cascade的嚴謹調控，實驗室過去的研究中證實蛋白質精氨酸甲基轉移酶 (PRMT1) 會藉由甲基化作用促進 p38α的活化，進而影響紅血球及巨核球的分化。本研究想進一步探討PRMT1/p38α軸線能否影響Imatinib的作用路徑。本研究中，以基因工程的方式改變具BCR-ABL致癌基因的人類CML細胞株K562中p38α或PRMT1的表現程度，並探討對Imatinib藥效的影響。結果指出，下調p38α或其上游磷酸激酶MKK3的表現量，均能顯著增加細胞對Imatinib誘導的抑制細胞總數及促進紅血球分化。此外，下調p38α的表現量能進一步增加 Imatinib誘導的細胞死亡。這些現象均進一步以西方墨點法或流式細胞分析法驗證。然而下調p38β的表現量則無法明顯影響Imatinib的藥效反應，顯現p38 MAPK的異形體在Imatinib的藥效反應扮演不同的角色。此外，以p38α的小分子抑制劑 (VX702) 併用Imatinib後確實能增加Imatinib對K562細胞株的抑制作用。這也證實p38α有潛力成為CML合併療法上的新標靶。此外，若上調PRMT1的表現量能增加細胞對Imatinib誘導的抑制細胞總數及促進紅血球分化，而其中PRMT1的精胺酸甲基轉移酶活性是必要的。此部份的結果亦為過去文獻未提及過的成效，提供未來白血病治療上併用藥物的新策略。

Chronic myeloid leukemia (CML) is caused mainly, in > 90% of patients, by the formation of BCR-ABL oncogene, which possesses unregulated active tyrosine kinase activity leading to abnormal cell proliferating, differentiation and survival. Imatinib is effective for BCR-ABL positive CML, however, patients (20% to 30%) still face the challenges of non-responsiveness, resistance and drug-related side effects. New targets which provide a combined effect with Imatinib is a promising strategy. p38MAPKs, particularly p38α, play a crucial role in hematopoiesis. Our previous studies have unveiled, as a novel regulatory mechanism, that protein methylation via PRMT1 (protein arginine methyltransferases 1) regulates p38α activity and thus participates in regulation of erythroid and megakaryocyte differentiation. Human K562 leukemia cells carrying a characteristic BCR-ABL fusion oncogene are used in this study to examine whether p38α and/or PRMT1 sensitizes cell responses to Imatinib as a proof of concept for a potential combined therapy for CML. In this study, the expression level of p38α or PRMT1 in K562 cells was elevated or reduced. I found that knockdown of p38α and its upstream kinase MKK3 not only enhanced Imatinib-induced cell death/growth, growth inhibition, but also promoted erythroid differentiation, however, knockdown of p38β did not significantly affect the response to Imatinib. These indicated that p38 MAPK isoforms play unique roles upon Imatinib treatment.VX702 is a selective inhibitor of p38. When combined with VX702, Imatinib suppressed cell growth to a significantly higher extent in K562 cells. Overexpression of PRMT1 significantly enhanced Imatinib-induced growth inhibition and erythroid differentiation and the methyltransferase activity of PRMT1 was required for this effect. This study demonstrates that the PRMT1/p38α axis is a potential target for sensitizing CML cells to Imatinib treatment and provides evidence suggesting a novel strategy of combined therapy for CML treatment.

目錄
目錄 i
縮寫表 iii
中英文對照表 v
圖次目錄 vi
中文摘要 viii
英文摘要 ix
研究目標 x
緒論 1
實驗材料 10
實驗方法 14
結果 19
一、探討K562細胞株中p38α的表現量對Imatinib藥效的影響 19
1. 下調p38α的表現量能增加細胞對Imatinib的藥效反應 19
2.下調p38α的表現量可以增加Imatinib誘導的DNA片斷化 20
3.下調p38α的表現量可以增加Imatinib誘導的Caspase 3活性 21
4.下調p38α的表現量可以增加Imatinib誘導的紅血球分化 21
5.以VX702合併Imatinib能增加藥物對細胞的抑制作用 22
二、探討K562細胞株中MKK3的表現量對Imatinib藥效的影響 22
1.下調MKK3的表現量能增加細胞對Imatinib的藥效反應 22
2.下調MKK3的表現量可以增加Imatinib誘導的紅血球分化 23
三、探討K562細胞株中p38β的表現量對Imatinib藥效的影響 24
1. 下調p38β的表現量僅能些微影響細胞對Imatinib的藥效反應 24
2.下調p38β的表現量些微影響Imatinib誘導的紅血球分化 25
四、K562細胞株中改變PRMT1的表現量對Imatinib藥效的影響 25
1.上調PRMT1的表現量能增加細胞對Imatinib的藥效反應 25
2.上調PRMT1的表現量可以增加Imatinib誘導的紅血球分化 26
3.PRMT1的甲基轉移酶活性在Imatinib誘導的紅血球分化中是必要的 26
4.下調PRMT1的表現量能些微影響細胞對Imatinib的藥效反應 27
5.下調PRMT1的表現量些微影響Imatinib誘導的紅血球分化 28
討論 29
參考文獻 34
圖次 40

圖次目錄
Figure 1. Cellular responses of K562 cells to Imatinib 40
Figure 2. Knockdown of p38α enhanced cellular responses to Imatinib. 41
Figure 3. shRNA control had no effect on cellular responses to Imatinib…………....43
Figure 4. Analysis of cell size and granularity in K562 and P2B6 upon Imatinib……... treatment……………………………………………………………...……….…44
Figure 5. Knockdown of p38α enhanced DNA fragmentation of cells treated with
Imatinib……………………………………………………………..…………... 45
Figure 6. Knockdown of p38α enhanced activation of Caspase 3 in cells treated with Imatinib…………………………………………………………………………. 47
Figure 7. Knockdown of p38α enhanced Imatinib-induced erythroid differentiation..48
Figure 8. Pharmacological inhibition of p38α enhanced cellular responses to Imatinib. 49
Figure 9. Knockdown of MKK3 enhanced cellular responses to Imatinib. 51
Figure 10. Knockdown of MKK3 enhanced Imatinib-induced erythroid differentiation 52
Figure 11. Knockdown of p38β slightly affected cellular responses to Imatinib 53
Figure 12. Knockdown of p38β slightly affected Imatinib-induced erythroid differentiation………………………………….....……………………………...55
Figure 13. Overexpression of PRMT1 enhanced cellular responses to Imatinib 56
Figure 14. Overexpression of PRMT1 enhanced Imatinib-induced erythroid differentiation………………………………………………….……………….. 57
Figure 15. The methyltransferase activity of PRMT1 is required for Imatinib-induced erythroid differentiation…………………………………………..…………….58
Figure 16. Knockdown of PRMT1 slightly affected cellular responses to Imatinib. 59

Figure 17. Knockdown of PRMT1 slightly affected Imatinib-induced erythroid differentiation………………………………………...…………………………60
Figure S1. Knockdown of p38α in K562 cells by shRNA…………………………...61

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