The effect of hexamethylene‐bis‐acetamide on multiparameter analysis of friend leukemia cells

Abstract

Friend leukemia cells were induced to differentiate by hexamethylene-bis-acetamide (HMBA). The relationship between cell growth cell volume distribution, fluorescence intensity and fluorescence anisotropy of the membrane probe 1,6 diphenyl 1,3,5 hexatriene (DPH) was analyzed in differentiated and undifferentiated cells with the aid of the fluorescence activated cell sorter (FACS II). The induction of a differentiated state was associated with a decrease in cell volume and an increase of fluorescence anFriend leukemia cells (FLC) have been used as a model for the study of erythroid differentiation (7). They can be induced to differentiate and synthesize hemoglobin in vitro when grown in the presence of a variety of structurally unrelated agents (2, 3, 11,14,18,19,22). Among the agents particularly active, hexamethylene-bis-acetamide (HMBA) is one of the most potent inducers (18). The expression of globin genes during cell differentiation can be suppressed by other unrelated agents (16,20, 23, 29). The mechanism whereby these inducers and inhibitors operate is still not known. Studies on the events which occur in induced FLC have shown various changes in membrane properties (4,5,9,12,15,21, 28), synthesis of erythrocyte membrane antigens and restriction of certain other proteins (7,17), whereas, very little work was done on the effect of the inhibitors. In the present work, the effects of an inducer (HMBA) and an inhibitor (dexamethazone) were analyzed. Changes of cell volume and fluorescence Key terms: Cell sorter, hexamethylene-bisacetamide induced Friend leukemia cells, dexamethazoneanisotropy of the membrane probe 1,6 diphenyl 1,3,5 hexatriene (DPH) were observed and their relation to the differentiated state of the cell was analyzed. Materials and MethodsCell culture and benzidine staining: FLC which were derived from a clone of Friend virus transformed cells 745 A were grown as previously described (5). Stimulation of globin synthesis was obtained by growing cells for 4 days in medium supplemerted with 5 mM HMBA kindly provided by Dr. Y. Gazitt.Benzidine staining: Benzidine dihydrochloride (Sigma Chem. Co., St. Louis, MO) 0.1 g was dissolved in 50 ml dilute acetic acid (1.5 ml glacial acetic acid diluted to 50 ml with water) and stored at 4°C in the dark. Just before use, 1 ml of benzidine solution was dispensed into a test tube at 4'C and 20 p1 of 30% H202 were added followed by immediate mixing. To this mixture, 1 ml of a culture containing approximately 3 X lo6 cells/ml was added and swirled rapidly during the addition. After 2 min incubation at 4"C, cells were centrifuged, washed and resuspended in phosphate buffer saline (PBS). The percentage of benzidine reactive cells was dekrmined in a Thoma hemocytometer (Precis, Paris, France) with the aid of the FACS by determining the fluorescence intensity after an excitation with a light source a t 488 nm. As a control the percentage of B+ cells was determined from noninduced cultures.Fluorescence labeling and measurements: The fluorescent hydrocarbon DPH was ...

Comparative uptake of adriamycin and daunorubicin in sensitive and resistant friend leukemia cells measured by flow cytometry

“…It has t o be noted that changes which were OF ADRIAMYCIN AND DAUNORUBICIN 301 cells were rendered resistant to different inducers (12,17,18,25,30 …”