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Changes in Actin and Actin-binding Proteins during the Differentiation of HL-60 Leukemia Cells¹

Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06510

Actin and actin-binding proteins form a peripheral network on the cytosolic side of the plasma membrane. These cytoskeleton proteins are involved in functions that require cellular movement and may also have a role in modulating signal transduction during cellular proliferation and differentiation. To measure changes in F-actin and actin-binding proteins during HL-60 differentiation, cells were induced to mature along the granulocytic pathway by exposure to 1 µM retinoic acid (RA) for 5 days and were analyzed for F-actin and actin-binding proteins by flow cytometry. The amounts of F-actin and spectrin in untreated HL-60 cells and in those undergoing differentiation by treatment with the retinoid did not differ. N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin was used to measure F-actin content and a monoclonal antibody followed by fluorescene isothiocyanate-conjugated goat anti-mouse immunoglobulin antibody was used to measure the content of spectrin; cells were analyzed by flow cytometry. In contrast, cells exposed to RA contained larger amounts of -actinin, vinculin, talin, lipocortin I, and lipocortin II, as determined with their respective antibodies followed by flow cytometric analysis as described above. An RA-supersensitive clone of HL-60, designated HL-60/S4, exhibited lower constitutive levels of -actinin, vinculin, and talin but a higher constitutive level of lipocortin II than parental cells. Treatment of HL-60/S4 with RA led to increases in vinculin, talin, lipocortin I, and lipocortin II. An RA-resistant clone, designated HL-60/R3, constitutively expressed larger amounts of -actinin, vinculin, lipocortin I, and lipocortin II than parental HL-60 cells. Treatment of HL-60/R3 with RA resulted in decreases in the amounts of these actin-binding proteins. Changes in actin-binding proteins that occur during the differentiation of HL-60 cells suggest that these proteins may be of importance to the expression of the mature phenotype.

¹ This research was supported in part by U.S. Public Health Service Grant CA-02817 from the National Cancer Institute.

² To whom requests for reprints should be addressed, at Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510.

Received 12/23/91. Accepted 3/20/92.

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