Quantitative Determination of Bcl-2 Expression in AML Cell Lines and in Normal and Leukemic Progenitor Cell Compartments by Laser Scanning Cytometry: Comparison with Flow Cytometry and Western Blot

Abstract

Laser Scanning Cytometry (LSC) is a newly developed microscope-based and computerized system that directly analyzes up to 3- color fluorescence simultaneously from single cells attached to slides. It permits correlations of quantitative cytometric data with morphology and with cytogenetic abnormalities determined by FISH. The data generated by LSC is equivalent to those obtained by flow cytometry in principle. In this study, Bcl-2 expression was measured in five leukemia cell lines by flow cytometry (FCM), LSC, and western blot. The order of Bcl-2 expression in cell lines from highest to lowest is HL60- DOX, OCI/AML3, HL60, MO7E, and TF-1. There was an excellent correlation of results of LSC vs. western blot, R2=0.97; FCM vs. western blot, R2=0.85; FCM vs. LSC, R2=0.83 (all p values <0.05). We then studied Bcl-2 expression in normal and AML progenitor cell populations. Bcl-2 expression was measured by LSC and western blot for CD34+/CD38+ and CD34+/CD38- cells after FACS-sorting. In normal bone marrow, Bcl-2 was significantly higher in CD34+/CD38+ than in CD34+/CD38- cells (p=0.009). Significant differences in Bcl-2 expression levels were noted in CD34+/CD38- cells between AML and normal bone marrow (NBM) populations (p=0.02), and AML-PB and NBM (p=0.005). All sorted populations contained 25% to 76.5% leukemia cells, based on FISH analysis. We conclude that LSC is a powerful technique for the analysis of gene expression in small numbers of FACS-sorted progenitor cells. It also allows correlations with morphology and cytogenetic abnormalities. The differential overexpression of Bcl-2 protein in CD34+/CD38- AML vs. normal bone marrow cells supports the concept of Bcl-2 as a novel target in the treatment of AML.