ABSTRACT
Objective To investigate the effect of indomethacin on cell cycle and apoptosis of CD34+ cells in blastic phase chronic myeloid leukemia, and explore the role of Wnt/β-catenin signal pathway in the molecular mechanism. Methods CD34+cells were sorted from bone marrow samples of blastic phase chronic myeloid leukemia, chronic phase chronic myeloid leukemia and normal cord blood by immune magnetic bead separation. The purity of CD34+ cells was detected by flow cytometry, and cellular morphology was observed by Wright's staining. The protein expression and location of β-catenin and BCR/ABL in CD34+ cells were analyzed by immunofluorescence assay. The changes of β-catenin protein expression in CD34+ cells treated with indomethacin and imatinib were detected by immunofluorescence, the cell apoptosis and cell cycle were observed by Wright's staining and flow cytometry, the mRNA level of c-myc and cyclin D1 was detected by real-time PCR, and the protein expression of BCR/ABL was detected by flow cytometry and immunofluorescence assay. Results CD34+ cells were successfully acquired with purity over 90%. β-catenin and BCR/ABL highly expressed in those CD34+ cells isolated from blastic phase chronic myeloid leukemia and mainly located in cytoplasm. Indomethacin combined with imatinib significantly suppressed the expression of β-catenin, increased the apoptotic rate and arrested the cell cycle in G0/G1 phase of blastic phase CD34+ cells (P<0.05), decreased the mRNA level of c-myc and cyclin D1 and the expression of BCR/ABL in blastic phase CD34+ cells (P<0.05), but threw no significant effect on normal CD34+ cells. Conclusions Indomethacin may significantly arrest the cell cycle and increase the apoptosis rate of CD34+ cells isolated from blastic phase chronic myeloid leukemia. It enhances the sensitivity of leukemia CD34+ cells to imatinib by inhibiting β-catenin expression, suppressing the transcription of c-myc and cyclin D1 and decreasing the expression of BCR/ABL protein.
ABSTRACT
For present work, 27 clinically diagnosed Chronic Myeloid Leukemia patients were selected, who attended the Out Patient Department of Gujarat Cancer and Research Institute, Ahmedabad. In all these cases relevant history, clinical findings, haematological data and other investigations were noted & bone-marrow samples were obtained for further study, which was done at Genetics Laboratory, B.J. Medical College, Ahmedabad. Samples were cultured, harvested, slides were prepared & photographs were obtained using photomicroscope and Karyotypes were prepared by using conventional cut and paste technique. Cytogenetic evaluation was done to detect the presence of Philadelphia chromosome and/or other chromosomal abnormalities. Out of 27 patients studied, 22 cases were having mild to moderate & remaining 5 cases were having huge splenomegaly. The blood picture showed, 9 were anaemic; 11 having total leukocytic count more than 1 lakh/mm3 ; 8 cases were thrombocytopenic. 25 cases were in chronic and 2 cases were in blastic phase. Cytogenetic evaluation by Karyotypes revealed 13 Ph’ positive cases; 4 Ph’ negative; 3 mosaic & remaining 7 cases came out inconclusive. All relevant parameters including clinical, hematological and cytogenetic were evaluated, analyzed and compared with other similar studies.