CD69於CML細胞生長、死亡及分化的角色

Chronic myelogenous leukemia(CML)是造血幹細胞不正常增生的疾病，因chromosome translocation t(9;22)產生Philadelphia(Ph) chromosome；Ph chromosome會產生Bcr-Abl fusion gene，進而製造持續活化的tyrosine kinase，Bcr-Abl蛋白質，是造成CML主要致病因子。 Bcr-Abl活化許多訊息傳遞路徑而促進CML細胞的增生、抑制細胞凋亡及分化。本實驗室先前篩選到CD69 蛋白質表現於未分化的CML細胞株K562，因此欲探討CD69是否為Bcr-Abl的下游蛋白質以及CD69在CML細胞中所扮演的角色及功能。首先，利用大量表現Bcr-Abl的穩定細胞株發現，Bcr-Abl可能會透過MEK pathway及NF-κB pathway而活化CD69 promoter activity，所以CD69為Bcr-Abl的downstream protein。此外，利用transient transfection以及Western blot assay的方式證明Bcr-Abl會透過NF-κB與c-jun (AP-1)活化CD69 promoter activity及CD69蛋白質表現量。為了證實CD69參與Bcr-Abl的訊息傳遞，建立大量表現CD69的穩定細胞株(K562-CD69K#2、#3、#4)後，Western blot結果顯示在大量表現CD69的穩定細胞株中發現有蛋白質tyrosine phosphrelation的訊號增加，接著以CD69 monoclonal antibodies(mAbs) crosslinking活化CD69後，蛋白質tyrosine phosphrylation的訊號明顯高於isotype mAbs (IgG)組別，利用目前治療CML最主要的藥物STI571 (Signal Transduction Inhibitor-571；Bcr-Abl tyrosine kinase的inhibitor) 探討細胞的功能性，STI571會造成CML細胞大量進行apoptosis，且與Activin A同樣會誘導K562的erythrocyte分化。實驗結果顯示，大量表現CD69之穩定細胞株無論在有無CD69 monoclonal antibodies(mAbs) crosslinking下，處理STI571或Activin A後皆明顯增加K562細胞的生長活性及抑制細胞凋亡與分化，由上述得知，CD69在CML細胞中可能扮演著增加細胞生長及抑制細胞凋亡與分化的角色，未來會利用siRNA更進一步去證實CD69對CML細胞的生長、凋亡及分化的影響。

關鍵字

慢性髓性白血病； Bcr-Abl ； CD69

並列摘要

Chronic myeloid leukemia (CML) is a clnal bone marrow stem cell disorder. Majority of CML patients have the Philadelphia (Ph) chromosome, a consequence of translocation between chromosomes 9 and 22. The Ph chromosome generates the Bcr-Abl fusion gene, which encodes the Bcr-Abl protein with a constitutive tyrosine kinase activity. Bcr-Abl activate multiple signalings pathways and is the main determinant of the CML cell malignant phenotype,including increased cell proliferation, inhibited apoptosis and differentiation. We have previously shown that CD69 is expressed in the CML cell line-K562 cells. In this study, we investigate whether CD69 is the downstream target of Bcr-Abl and the CD69 functions in CML cells. We found that Bcr-Abl upregulated CD69 promoter activity through MEK pathway and NF-κB pathway in Bcr-Abl overexpressing cells. Transient transfection assay indicated that Bcr-Abl upregulated CD69 promoter may be through NF-κB (p65) or AP-1 transcription factor “c-Jun.”The expression level of CD69 protein was also increased in p65 or c-Jun overexpressing cells. To investigate CD69 function in CML cells, we used CD69 monoclonal antibody to crosslink CD69 in K562 cells stably overexpressing CD69.Our results indicated that CD69 overexpression up-regulate tyrosine phosphrylated signals in K562. Then, more tyrosine phosphrylated signals were increased after CD69 cross-linking .STI571 is an inhibitor of Abl tyrosine kinase and has been proven to be effective for CML patients. After treatment with STI571, CD69 partially increased K562 cells viability and decreases apoptotic cells with or without CD69 cross-linking. STI571 and Activin A both induced human erythroid differentiation. Here we show that after treatment with STI571 or Activin A, CD69 partially inhibits K562 cell differentiation with or without CD69 cross-linking. In conclusion, CD69 is involved in Bcr-Abl signaling to increase cell proliferation and inhibit cell apoptosis and differentiation in CML cell.

並列關鍵字

CML ； Bcr-Abl ； CD69

參考文獻

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CD69於CML細胞生長、死亡及分化的角色

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