Expression of DNA polymerase alpha 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 alpha (pol alpha), 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 alpha 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 alpha-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 alpha-linked fluorescence was observed. On the other hand, the pol alpha-positive population in the saturated culture was ca. 70%, with weaker pol alpha-linked fluorescence. Thus, the population of pol alpha-positive cells and the amount of pol alpha 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 alpha 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 alpha and phenotype-specific proteins.

DNA polymerase α

fluorescein isothiocyanate

phycoerythrin

flow cytometry

paraformaldehyde

periodate‐lysine‐paraformaldehyde

10 mmol/liter sodium phosphate buffer (pH 7.4) containing 0.15 mol/liter NaCl