Identification and characterization of nuclear retinoic acid-binding activity in human myeloblastic leukemia HL-60 cells.
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 greater than 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 RAR alpha or RAR beta 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 approximately 0.46 nM and 1400 +/- 100 receptor sites per cell. Northern blot analyses of poly(A)+ RNA with cDNA probes specific for RAR alpha and RAR beta indicated that HL-60 cells contain predominantly transcripts encoded by the RAR alpha gene. Our results suggest that the observed nuclear RA-binding activity in HL-60 cells might mediate the action of RA in these cells.
Nervi
C
C
Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
Grippo
J F
JF
Sherman
M I
MI
George
M D
MD
Jetten
A M
AM
D000818:Animals; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008970:Molecular Weight; D010957:Plasmids; D018168:Receptors, Retinoic Acid; D014162:Transfection; D014212:Tretinoin / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology
D002352:Carrier Proteins; D018168:Receptors, Retinoic Acid; D014212:Tretinoin