| Chronic myeloid leukemia is a clonal hematopoietic neoplasm derivedfrom leukemia stem cell which is characterized by BCR/ABL oncogenewith a t (9;22)(q34; q11) chromosomal translocation. The typicalBCR/ABL oncoprotein is a strong tyrosine kinase, leading to infiniteproliferation, apoptosis inhibition, and disease progression of leukemiccells by activating a number of downstream signal pathways. CML isdivided into chronic phase (CP), accelerated phase (AP) and blast crisis(BC). The blast crisis of CML which is related to high mortality is themajor challenge because the lack of effective therapies. β-Catenin plays animportant role in the progression of CML blasts, whereas the relationshipbetween β-catenin and the key pathways activated by BCR/ABL have notyet been elucidated. In CML blast crisis, c-myc and cyclinD1are thecommon targets in the down-stream of PI3K-AKT and Wnt/β-cateninpathways, which regulate the proliferation and apoptosis of leukemic cells.GSK-3β is a critical factor that connect PI3K-AKT pathway toWnt/β-catenin pathway, indicating that PI3K-AKT and Wnt/β-catenin mayinteract with each other.In this study, we first detected the expression of β-catenin in CML-CP and BC to assure that β-catenin is overexpression in CML-BC. Next,LY294002, a specific inhibitor of PI3K-AKT pathway, was used to assaythe proliferation and cell cycles of K562cells. At last, we discussed thepossible mechanism of LY294002inhibits the proliferation of CML blasticcells. The main methods are as following:1. Western blot and qPCR were used to analyze the expression ofBCR/ABL and β-catenin in CML patients in CP and BC. Moreover, theprotein expression of β-catenin in CML cell lines was also detected.2. LY294002was used on K562cells. Cell growth was detected byMTT assay. Cell-colony ability was determined by colony-forming test.Cell cycles were assessed by flow cytometry.3. K562cells were treated with LY294002for24h, Western blot wasperformed to assay the protein levels of pβ-catenin（S33/S37/T41）, totalβ-catenin, pGSK-3β(S9). qPCR was used to detected the mRNA ofβ-catenin. The proteins in cytoplasm and nucleus were separated from totallysate, nuclear and cytoplasmic sections were analyzed by Western blotwith a β-catenin antibody. Immunofluorescence analysis was performed toconfirm the distribution of β-catenin. Cycloheximide was used to analyzethe stability of β-catenin. Western blot and qPCR were used to detect themRNA and protein of c-myc, cyclinD1.From the above experiments, the results are as following:1. We successfully detected the expression of β-catenin. Compared with the patients in CP, The mRNA and protein levels of BCR/ABL andβ-catenin were higher in patients in BC. Next, we found that the proteinlevel of β-catenin is higher in K562, KU812cells than those in BP210,32DP cells and BCR/ABL-negative cells BaF3and32D.2. MTT test and colony-forming assay showed that LY294002inhibited cell growth and colony-forming ability in a dose-dependentmanner. The outcome of FCM revealed that cells in G1phase rised,whereas cells in S phase and G2phase reduced.3. Western blot revealed that pβ-catenin （S33/S37/T41）, totalβ-catenin, pGSK-3β(S9) were decreased after LY294002treatment. Noevident changes were observed for the mRNA of β-catenin. LY294002induced reductive levels of β-catenin in both nuclear and cytoplasmicsections compared with control groups. β-Catenin degraded in atime-dependent manner and faster in inhibitor pre-treated groups. ThemRNA and protein expression of c-myc and cyclinD1were decreased.In summary, our data indicated that the protein level of BCR/ABL andβ-catenin was significantly increased in BC of CML. The blockade ofPI3K-AKT signaling inhibits the proliferation and cell cycle of CMLblastic cells and the mechanism may be related to down-regulatedexpression of Wnt/β-catenin pathway. |
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