Background: To distinguish between subgroups of patients with acute leukemia, the rate of spontaneous (culture-induced) apoptosis of leukemic cells was evaluated using five methods.Methods: Leukemic cells (cells) from the bone marrow of children with acute lymphoblastic leukemia (ALL, n ؍ 112) and acute myeloid leukemia (AML, n ؍ 30) were cultured for 20 h in vitro. The level of apoptosis was detected by fluorescent microscopy after staining with acridine orange (AO) or by flow cytometry after staining using PI, JC-1, the APO-BRDU kit, or the AnnexinV-FITC kit.Results: ALL cells were significantly more sensitive to spontaneous apoptosis versus AML cells, as was detected by all methods. The least sensitive technique was apoptosis detection by sub-G1-peak/PI-staining.
No difference in the rate of apoptosis in cells was determined between T-and
Resistance to chemotherapy is an obstacle to the successful treatment of oncohematological malignances. Failure of therapeutic treatment may be due to the development of multidrug resistance (MDR), the mechanisms of which include upregulation of membrane-resident transporters that efflux chemotherapeutic drugs from tumor cells. Deregulation may occur at different levels: gene or protein expression or function depletion. Childhood acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) cells and chronic lymphocytic leukemia (CLL) cells of adults were studied. ABCB1 (P-gp) and ABCG2 (BCRP) expression were determined by flow cytometry, rhodamine 123 (Rho123) and mitoxantrone were used for functional activity study of MDR proteins, sensitization of leukemic cells to drugs was quantified by methyl thiazolyl tetrazolium (MTT) assays. Appropriate gene expression was determined using semi-quantitative RT-PCR. No differences between expression of P-gp and BCRP and genes in primary and relapsed acute leukemia (AL) cells as well as in de novo and treated CLL samples were established. Higher expression of P-gp and BCRP proteins was detected in CLL lymphocytes compared to blast cells. Increased P-gp protein expression and function was detected in cells of CLL patients who had more aggressive therapy regimen. Doxorubicine, rubomycinum and L-asparaginase resistance correlates with P-gp overexpression and increased function in pediatric AL whereas vincristine resistance might be associated with P-gp protein expression in AL samples and impared P-gp function in CLL lymphocytes only. A tendency for the decreased doxorubicin cytotoxic activity was shown in BCRP-overexpressing cells both in children and adults leukemia. Multifactorial ANOVA showed that P-gp/MDR1 and BCRP as well as their function could not be used as unconditional and universal predictors of leukemia cell drug resistance in vitro. These results suggest that studied MDR transporter-proteins have a limited role per se in vitro and admittedly in vivo drug resistance estimated in leukemia patients or it is not yet fully understood unless would not be studied in aggregate. In any event, the expression and function studies of the proteins under investigation when singularly considered do not have a crucial significance for impact on drug resistance evaluation in all leukemia patients.
In view of obscure clinical and biological significance of leukemic cells heterogeneity, we studied the efficacy of apoptosis, proliferation, and expression levels of the Bcl-2, MDR1, LRP, and BCRP genes in sorted CD34+ and CD34- subpopulations of childhood AML leukemic samples. In five out of nine cases, CD34+ cells were less sensitive to spontaneous apoptosis and had from 1.2- to 5.0-fold higher expression levels of Bcl-2 (eight of ten) and from 1.5- to 28.7-fold higher expression levels of MDR1 (eight of ten). The expression levels of the LRP gene were from 1.1- to 1.8-fold higher in CD34+ subpopulations (five of ten cases), and the expression levels of the BCRP gene were from 1.1- to 22.4-fold higher in CD34+ leukemic cells (six of ten). In all M4 cases, the expression levels of LRP were higher in the CD34- subpopulation. Significant differences in the patterns of genes expression between patients do not allow us to conclude that the CD34+ fractions have more resistant phenotype than the CD34- subpopulations. Nevertheless, distinctions between CD34+ and CD34- cells may lead to different chemosensitivities between leukemic subpopulations in vivo and may determine the alteration of the leukemic immunophenotype during treatment and in relapse.
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