Effects Of The CUE Domain-containing Protein2on Imatinib Resistance In CML Cells

BackgroundChronic myeloid leukemia (CML) is a kind of comman hematologic malignancies, and is characterized by the formation of the Philadelphia (Ph) chromosome and Bcr-Abl fusion gene. The tyrosine kinase inhibitor Imatinib is a specific targeted molecular drug for the treatment of Ph chromosome-positive CML, particularly in chronic phrase. Although it is considered as one of the most effective drugs and the first-line treatment for CML, resistance to Imatinib seems unavoidable and occurs frequently during it clinical application. In addition to the Bcr-Abl duplication or mutation, abnormal expression of some other gene or alternative signaling pathway activation may also contribute to the resistance to Imatinib.The CUE domain-containing protein2(CUEDC2) is a newly reported protein, and its functions are largely undefined. There is a CUE domain in the protein structure of CUEDC2, which is a moderately conserved ubiquitin-binding domain of～40amino acids found in many eukaryotic proteins. Present researches indicate that CUEDC2is involved in adjusting cell cycle, NF-ΚB signaling pathway and some other events. It is highly expressed in various somatic tumors including breast, ovrarian and kidney tumors, and could reduce the responsiveness of breast cancer cells to endocrine therapies. However, the expression of CUEDC2in CML cells has not been explored yet. NF-ΚB has already been demonstrated to be constitutively active in CML and found to contribute to drug resistance. Whether CUEDC2is involved in the tumorigenesis of CML and drug resistance, the correlation of CUEDC2expression level and NF-ΚB activity in CML cells still need to be comfirmed.ObjectiveThe present study is aimed to detect the expression level of CUEDC2in CML bone marrow samples, normal bone marrow samples and leukemia cell lines and then to explore the correlation between CUEDC2and Imatinib resistance in CML.Methods1. Bone marrow (BM) samples were obtained from58patients with diagnosed CML in our hospital from August1,2011to August1,2012. Bone marrow mononuclear cells were prepared by Ficoll-Hypaque density gradient centrifugation. The expression level of CUEDC2in primary bone marrow cells was assayed by Real time quantitative PCR.2. The expression of CUEDC2in CML cell lines K562(Bcr-Abl positive cell line) and Imatinib-resistant K562cells K562/G01was detected by Real time quantitative PCR.3. shRNA expressing plasmids specifically targeting CUEDC2(termed as CUEDC2-shRNAl and2) were constructed using pGPU6/GFP/Neo vector. An unrelated shRNA sequence with no homology to any human genes was used as a negative control (shNC). These plasmids were then transfected into K562cells and stable clones were selected by G418.4. CUEDC2-pIRES2plasmid was constructed according to molecular cloning, and then stably transfected into K562/G01cells, which made K562/G01cells overexpress CUEDC2.5. MTT assay was used to detected cell viability. Hoechst33258staining and Annexin-V-APC/PI Apoptosis Analysis used to dectect apoptosis induced by Imatinib in CML cells. Western blot was used to detect CUEDC2, NF-ΚB signaling pathway relative proteins and cleaved caspase-3protein expression. Nuclear localization of NF-ΚB p65protein was determined by immunofluorescence assay.Results1. The mean expression level of CUEDC2in CML patients was significantly higher than that in normal control; and the expression level of CUEDC2was significantly correlated with CML clinical stages (P<0.05).2. The expression level of CUEDC2in CML cell lines K562was significantly higher than that in normal control (P<0.05); and the expression of CUEDC2in K562was significantly higher than that in K562/G01cells (P<0.05).3. Our results showed that CUEDC2knockdown in K562cells reduced the sensitivity to Imatinib; CUEDC2overexpression in K562/G01cells increased the sensitivity to Imatinib.4. The results showed that the NF-ΚB signaling pathway activity was upregulated in K562/G01cells compared to sensitive K562cells. CUEDC2knockdown in K562cells increased NF-ΚB signaling pathway activity; NF-ΚB inhibitor Bayl1-7082could block NF-ΚB activity in CUEDC2knockdown K562cells and sensitized these cells to Imatinib. Overexpression of CUEDC2in K562/G01cells decreased NF-ΚB signaling pathway activity.ConclusionsCUEDC2was highly expressed in primary CML bone marrow cells and CML cell line K562and was involved in the Imatinib resistance in CML cells. High expression of CUEDC2conferred K562the sensitivity to Imatinib probably partialy through downregulating the NF-ΚB signaling pathway activity. Decreased expression of CUEDC2contributed to Imatinib resistance in K562/G01cells.