Expression of DNA Polymerase α and Leu3a Molecules in Growing and Saturated Cultures of Human Leukemic Cells: Phenotype Analysis of Proliferative Cells by Flow Cytometry

A flow cytometric method to analyze phenotypes of proliferative cells was developed using human leukemic cell line MOLT 4. A nuclear protein, DNA polymerase α (pol α), was selected as a marker for proliferative cells, and Leu3a molecule as a cell-surface antigen phenotype marker of the cells. The procedure involved the simultaneous use of fluorescein-conjugated anti-pol α antibody, developed by us, and commercially available phycoerythrin-conjugated anti-Leu3a antibody. The optimal fixative for both proteins was phosphate-buffered 2% paraformaldehyde. The pol α-positive population in logarythmically growing MOLT 4 cells was estimated, by flow cytometry, to be ca. 95%. A sharp flow cytometry histogram with a strong pol α-linked fluorescence was observed. On the other hand, the pol α-positive population in the saturated culture was ca. 70%, with weaker pol α-linked fluorescence. Thus, the population of pol α-positive cells and the amount of pol α in cells was dependent on the cell density of the culture. In contrast, ca. 90% Leu3a-positive populations with similar flow cytometry histograms were seen in either growing or saturated states, suggesting that expression of Leu3a was independent of cell density. The flow cytometric method using fluorescein isothiocyanate-conjugated anti-pol α antibody is useful for detecting proliferative fractions of free tumor cells, such as leukemic cells. Furthermore, analysis of the phenotype of the proliferative or non-proliferative cells became easier by simultaneous labeling with antibodies against pol α and phenotype-speciflc proteins.