Quantitative assessment of BAX transcript and flow cytometric expression in acute myeloid leukemia: a prospective study.

OBJECTIVE: Quantitative assessment of BAX transcripts and protein in acute myeloid leukemia (AML). METHODS: We quantitatively evaluated BAX gene transcripts by real-time polymerase chain reaction (TaqMan probe chemistry) and protein expression by flow cytometry. RESULTS: Consecutive 112 AML patients with a median age of 16 (1-59) years were recruited in the study. By flow cytometry, the percentage expression was in linear correlation with relative median fluorescent intensity (RMFI; R = 0.4425; P < 0.001). However, there was no linear relationship between the transcript copies of the BAX with its RMFI (R = -0.0559; P = 0.586). The expression of the BAX at both protein and transcript level was significantly higher in AML patients as compared with normal control. RMFI of the BAX were higher in the cohort with lower white blood cell count (P = 0.029). None of the other baseline characteristics correlated with either the BAX transcript or the RMFI. BAX expression did not correlate with complete remission rate, event free, disease free, and overall survival. CONCLUSION: BAX gene expression in AML was evaluated first time with two different methods but did not correlate with the survival outcome.

Assessment of the normal or leukemic nature of CD34+ cells in acute myeloid leukemia with low percentages of CD34 cells.

BACKGROUND AND OBJECTIVES: The percentages of CD34+ cells in the bone marrow of patients with acute myeloid leukemia (AML) vary widely. Especially in the low range (<5% CD34+ cells), the nature (normal or malignant) of the CD34+ cells is uncertain. Since only in a minority of cases are molecular techniques applicable, in this study we explored a multiparameter approach using phenotypic and functional characteristics to discriminate normal CD34+ cells from malignant ones. DESIGN AND METHODS: CD34+ cells from 24 AML patients with <5% CD34+ cells and from 3 patients with >50% CD34+ cells were studied immunophenotypically for aberrant phenotypes, CD133 and CD90 expression and for P-glycoprotein activity. RESULTS: In the low (0.02-0.7%) CD34+ range, our approach offered strong evidence for a normal origin of the CD34+ cells in 18/19 cases, which was confirmed by interphase fluorescent in situ hybridization on sorted CD34+ cells in 3 cases, which had concomitant presence of cytogenetic abnormalities in the CD34- blasts. In contrast, in the intermediate (1.6-3.5%) CD34+ range, the CD34+ cells appeared as normal in only 1/5 cases. In the high (51-67%) CD34+ range, as expected the majority of CD34+ cells were malignant, although in 2/3 cases a small subpopulation (i.e. 0.15% and 0.20%) of CD34+ cells were of normal origin. INTERPRETATION AND CONCLUSIONS: Our multiparameter approach enabled us to define the nature of CD34+ cells in AML. This has implications for studies dealing with the characterization of primitive malignant cells. Moreover, it enabled identification of truly CD34 negative AML, which would be eligible for CD34-based immunological purging of autologous stem cell transplants.

Leukotriene D4-induced homologous desensitization in basal and differentiated U-937 cells: characterization with the partial agonist leukotriene E4 and assessment of receptor reserve.

U-937 cells express receptors for leukotriene D4, and LTD4 stimulates receptor-mediated Ca++ mobilization with an EC50 of 5 nM. In these cells, LTE4 produces Ca++ mobilization with an EC50 of 500 nM and a maximum response that is 15 to 20% of the LTD4 response. LTE4 was shown to be a partial agonist, with a Kp of 400 nM and a linear relationship between effect and receptor occupancy, and was used as a tool to study LTD4-induced desensitization. Pretreatment of U-937 cells with LTD4 resulted in a concentration-dependent shift to the right and a decrease in the maximum of LTE4 concentration-response curves, indicating that desensitization had occurred. LTD4-induced desensitization was homologous for LTE4 and LTD4; Ca++ mobilization produced by LTB4, N-formyl-L-methionyl-L-leucyl-L-Phe and platelet-activating factor was not affected. LTD4-pretreatment produced a concentration-related desensitization of LTE4-induced Ca++ mobilization in both basal and dimethyl sulfoxide-differentiated U-937 cells. However, the maximal desensitization was greater in basal cells (66% +/- 4%) than in differentiated cells (33% +/- 7%). LTD4 pretreatment resulted in a greater percentage of reduction of Ca++ mobilization produced by LTE4 than that produced by LTD4, suggesting that a receptor reserve exists for LTD4. The extent of receptor reserve was assessed in basal and differentiated U-937 cells by comparing LTD4 concentration-response curves from control and desensitized cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of the sensitivity of leukaemic cells to cytotoxic drugs by bioluminescence measurement of ATP in cultured cells.

A short-term test in vitro is described, which can be used to detect resistance to cytostatic agents in leukaemic cells. Leukaemic cell suspensions were incubated with cytostatic agents and the resulting intracellular ATP concentrations were measured by a bioluminescence ATP assay. There was a clear dose-effect relationship in acute leukaemia and chronic lymphocytic leukaemia cells for drugs used in the treatment of leukaemias. A good correlation was found between the ATP content of leukaemic cells and cell viability as determined by the trypan blue dye exclusion test. Preliminary individual clinical correlations suggest a correlation between the chemosensitivity in vitro and the response patterns in vivo in leukaemia patients. This simple, fast and sensitive method may have application for determination of drug-induced cytotoxicity in leukaemic cells in vitro.