Identification and Characterization of Nuclear Retionic Acid-Binding Activity in Human Myeloblastic Leukemia HL-60 Cells

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 86; no. 15; pp. 5854 - 5858
• Main Authors: Nervi, Clara, Grippo, Joseph F., Sherman, Michael I., George, Margaret D., Jetten, Anton M.
• Format: Journal Article
• Language: English
• Published: National Academy of Sciences of the United States of America 01-08-1989
• Subjects: Cellular differentiation; COS cells; DNA probes; Genes; Hematopoietic stem cells; Leukemia; Nuclear receptors; Receptors; Retinoids; RNA
• ISSN: 0027-8424; 1091-6490

Specific [3H]retinoic acid (RA)-binding sites in nuclear and cytosolic extracts prepared from human myeloblastic leukemia HL-60 cells have been detected by sucrose density gradient sedimentation and size-exclusion high-performance liquid chromatography (HPLC) analyses. This RA-binding activity migrated as a single peak with an apparent molecular weight of 50,000 and >95% of the total binding activity was associated with the nuclear extract. Nuclear extracts prepared from COS-1 cells transfected with an expression vector for the nuclear RA receptors RARα or RARβ were enriched (20- to 100-fold) with a RA-binding activity that coeluted by size-exclusion HPLC with the putative RAR from HL-60 cells. The HL-60 nuclear receptor exhibited high-affinity binding of RA and its benzoic acid analogs Ch55, Ch30, Ro 13-7410, and SRI 6409-40 and low-affinity binding of retinol, Ro 8-8717, and SRI 5442-60, correlating well with the biological activity of these compounds in HL-60 cells. Saturation binding and Scatchard plot analyses of the binding of RA to the nuclear HL-60 receptor yielded an apparent dissociation constant of ≈ 0.46 nM and 1400 ± 100 receptor sites per cell. Northern blot analyses of poly(A)+ RNA with cDNA probes specific for RARα and RARβ indicated that HL-60 cells contain predominantly transcripts encoded by the RARα gene. Our results suggest that the observed nuclear RA-binding activity in HL-60 cells might mediate the action of RA in these cells.