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Objective To construct BCR-ABL SH3-T79Y mutant recombinant adenovirus vectors and investigate its effects on apoptosis of K562/G01 cells.Methods SH3-T79Y mutant was amplified by overlapping PCR with pMig210 as template and cloned into recombinant adenovirus vectors .After identifying , packaging and amplifying , the recombinant adenovirus vectors containing SH 3-T79Y mutant was collected .Recombinant adenovirus vectors were transferred into K562/G01 cells.Then transfection efficiency was determinated , changes of cell morphology were observed by Wright 's staining , cell apoptosis was evaluated by flow cytometry , BCR-ABL and CrkL phospho-rylation was detected by Western blot .Results The vectors were successfully constructed .Transfection efficiency was more than 80%after transferring into K562/G01 cells for 72 h;there was obvious apoptosis phenomenon , cell apoptosis significantly increased to 32.46% compared with the control groups ( P<0.05 ) , BCR-ABL and CrkL phosphorylation significantly decreased and so did the expression of BCR-ABL( P<0.05 ) .Conclusions Success-fully constructed the SH 3-T79 Y mutant recombinant adenovirus vectors and it may promote the apoptosis of K 562/G01 cells by inhibiting BCR-ABL and CrkL phosphorylation .
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<p><b>OBJECTIVE</b>To investigate the effect of indomethacin on the proliferation and Wnt/β-catenin pathway in K562 cells.</p><p><b>METHODS</b>The cell growth of K562 cells treated with different concentrations of indomethacin was assessed with MTT assay, and the colony-forming ability of the cells was evaluated by colony-forming assay. The mRNA expressions of BCR/ABL and β-catenin were detected by RT-PCR, and the protein expressions of pBCR/ABL, total BCR/ABL, β-catenin, pGSK-3β and c-myc were analyzed by Western blotting.</p><p><b>RESULTS</b>Indomethacin significantly suppressed the growth and colony-forming ability of K562 cells in a dose-dependent manner. Indomethacin treatment dose-dependently decreased the protein level of pBCR/ABL and total BCR/ABL without affecting bcr-abl mRNA expressions. Compared with the control groups, indomethacin-treated cells showed obviously decreased mRNA and protein expressions of β-catenin and decreased protein expressions of pGSK-3β and c-myc.</p><p><b>CONCLUSION</b>Indomethacin inhibits the proliferation of K562 cells by suppressing the activity of bcr-abl-Wnt/β-catenin pathway.</p>
Subject(s)
Humans , Cell Cycle , Cell Proliferation , Fusion Proteins, bcr-abl , Metabolism , Indomethacin , Pharmacology , K562 Cells , RNA, Messenger , Wnt Signaling Pathway , beta Catenin , MetabolismABSTRACT
Objective To investigate the potential of chronic myeloid leukemia ( CML) cell line KCL22 in indu-cing leukemia in NOD-SCID mice for setting up a basis for constructing a CML mouse transplantation tumor model. Methods 2 ×107 KCL22 cells in logarithmic growth phase were injected via the tail vein into experimental NOD-SCID mice whereas PBS was injected to the mice of control group.General condition of the mice of both groups was observed.Wright staining was used to observe the changes of blood and bone marrow smears.PCR was conducted to detect the transcription level of BCR-ABL, and histology with HE staining was used to evaluate the tumor cell invasion in the liver and spleen. Results Four weeks after the injection of KCL22 cells, the mice in experimental group showed physical signs of decreased reactivity, depression, swollen hindlimb muscles and petechia on the hindlimb femur.Peripheral white blood cells ( WBC) began to increase after 5 weeks, with a significantly increased quantity compared with the control group (P90 days) (P<0.05).Conclusions A NOD-SCID mouse model of CML transplantation tumor is successfully established with leukemia KCL22 cells.
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Objective:To study the influence of protein transduction domain (PTD)-oligomerization domain (OD)-HA fusion proteins on apoptosis of bcr/abl-positive cell lines. Methods:bcr/abl-positive cells were treated with PTD-OD-HA protein. The apoptoses of the cells were detected by flow cytometry (FCM), DNA ladder and transmission electron microscopy (TEM), and the levels of apoptosis-related genes bax and bcl-2 were detected by RT-PCR and Western blotting. Results:FCM examination demonstrated that PTD-OD-HA protein induced the apoptosis of bcr/abl-positive cells; DNA ladder showed that the classic DNA ladders appeared in BaF3-P210 and K562 cells after 48 h treatment with PTD-OD-HA proteins; the apoptoses of BaF3-P210 cells were observed by TEM; the levels of bax in mRNA and protein increased in BaF3-P210 and K562 cells, and bcl-2 decreased. Conclusion:PTD-OD-HA proteins specifically induced the apoptosis of bcr/abl positive cells.