Effects Of β-Catenin On Cd34~+ Cells In Blast Crisis Of CML

Chronic myeloid leukemia is a myeloproliferative disease, which is characterized by BCR/ABL oncogene with a t(9; 22)(q34; q11) chromosomal translocation. The typical BCR/ABL oncoprotein is a strong tyrosine kinase, which plays an important role in occurrence, development and resistance process in CML. Imatinib (Gleevec, IM) is the first-line drug in the treatment of CML, which achieves the purpose of curing diease through the specific inhibition of activity of Bcr-Abl tyrosine kinase. With the clinical applications, more and more patients appear to resistance to IM, especially the presence of leukemia stem cells (LSCs) aggravates the phenomenon of resistance to IM. CML is divided into chronic phase (CP), accelerated phase (AP) and blast crisis (BC) clinically. The basic reason of the transformation from chronic phase to blast phase is the generation and amplification of leukemia stem cells(LSCs).Recent studies have indicated that β-catenin is a key regulatory molecule of Wnt signaling, which can mediate Wnt/(3-catenin signaling pathway to affect the growth, proliferation and differentiation of LSCs, but the mechanisms are not yet elucidated. Because CD34+ cells abound in the bone marrow of blast crisis in CML, and our previous study has confirmed that K562 cells and KU812 cells have a high expression of P-catenin in both protein and mRNA level, so there may be a high expression of β-catenin in CD34+ cells.Indomethacin (IN) is a cyclooxygenase inhibitor, which affects the expression of p-catenin through the internations between PGE2 and Wnt signaling pathway. Therefore, IN was used to inhibit the expression of P-catenin, and combined with IM to achieve the goals of inhibition of proliferation and induction of apoptosis on CD34+cells.In this study, we collected the bone marrow specimens of CML patients and umbilical cord blood, sorted CD34+ cells and detected the expression and distribution of β-catenin and Bcr-Abl. Next, IN was used to inhibit the expression of P-catenin and combined with IM to observe their effects on proliferation, self-renewal, cell cycle and apoptosis, and we preliminary discussed the possible mechanisms.The main methods are as following:1. MACS sorting technology was used to isolate CD34+ cells. The purity was identified by FCM. The morphological difference was observed by Wright’s staining. Flow cytometry, qPCR and immunofluorescence techniques were used to detect the expression and distribution of β-catenin and Bcr-Abl in CD34+ cells.2. CD34+ cells were treated with IN and combination of IM and IN for 48h to inhibit the expression of β-catenin. MTT assay was performed to test the cell growth. Colony-forming assay was used to detect the ability of colony formation. Flow cytometry was utilized to determinate the cell cycle. Wright’s staining and flow cytometry were used to detect the cell apoptosis. qPCR was used to detect the mRNA level of c-myc and cyclinDl.From the above experiments, the results are as following:1. We isolated CD34+ cells from the bone marrow specimens of clinical CML patients and the umbilical cord blood of normal pregnant women with MACS technology successfully. The sorting purity identified by flow cytometry was more than 90%. There was no significant difference among the three types of CD34+ cells by Wright’s staining. The expressions of P-catenin and Bcr-Abl were the highest in the CD34+ cells from CML BP.2. IN could inhibited the expression of β-catenin significantly. MTT test and colony-forming assay showed that IN inhibited cell growth and colony-forming ability. FCM revealed that IN didn’t effect the cell cycle and cell apoptosis, but strengthened the effect of IM on CD34+ cells to make CD34+ cells arrested in G0/G1 phase and cause the cell apoptosis after combined with IM. The mRNA levels of c-myc and cyclinDl were decreased in treated groups.In summary, β-catenin and Bcr-Abl were highly expressed in CD34+ cells from CML BP. After IN inhibited β-catenin and combined with IM the proliferation and self-renewal capacity of CD34+ cells was inhibited, and the apoptosis rised in combination group, whose mechanism might be related to the Wnt/β-catenin signaling pathway.