Studies of the downstream effectors of Akt in Multiple Myeloma cell lines

• Main Authors: Wei-Feng Huang, 黃偉峰
• Other Authors: Jung-Hsin Hsu
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/20229131126305567838

碩士 === 國立東華大學 === 生命科學系 === 99 === Multiple myeloma (MM) is a disease caused by abnormal plasma cells. Interleukin-6 (IL-6) is an essential growth factor for MM cells and it can activate PI3K/AKT pathway, JAK/STAT pathway and RAS /MAPK pathway. These pathways affect anti-apoptosis and proliferation in MM cells. AKT plays an important role in the PI3K/AKT pathway. By combining comparative proteomic approaches with MALDI-TOF MS, we have identified many putative downstream effectors of the PI3K/AKT pathway. Several interesting proteins were identified including β-actin, cyclophilin A (CypA), E3 SUMO-protein ligase PIAS-NY protein, heat shock protein 27 (HSP27), promyelocytic leukemia protein (PML) and transforming growth factor β-2. Previously, we have shown that β-actin is the downstream effector of the PI3K/AKT pathway and is regulated by the AKT activity in AF-10 cells. In this study using U266 cells, we proved that β-actin is the substrate of AKT and phosphorylation levels of β-actin are positively correlated with the AKT activity in MM cells. Cell migration ability decrease in the AKT knockdowned AF-10 cells.This result indicates that IL-6 induces cell migration ability of myeloma cells, and phosphorylation of β-actin probably participates in the process. CypA is not only the Akt substrate but also the peptidylprolyl cis-trans isomerase (PPIase A) that catalyzing the cis-trans transformation of the peptidyl-prolyl bond. To characterze the interacting partners of CypA, we pulled down proteins with co-immunoprecipitation and subsequently separated them by 2D-gel electrophoresis followed by MALDI-TOF MS analysis. IFN-α induces apoptosis in MM cells by the PML nuclear bodies (NBs) but not all MM cells are IFN-α sensitive. AF-10 is an IFN-α sensitive cell line and the mRNAs of PML isoforms I, II, VI express without any treatment. After IFN-α treatment for 48h, the mRNAs of PML isoforms I, II, III, VI express in AF-10. The PML protein decreased after AF-10 cells treated briefly with IL-6. This result suggests that PML may be degraded in the cytoplasm.