Potent vitamin D3 analogs: their abilities to enhance transactivation and to bind to the vitamin D3 response element

1,25 dihydroxyvitamin D3 [1,25(OH)2D3] mediates its biological activities through specific binding to the vitamin D3 receptor (VDR) and subsequent association with vitamin D3 responsive elements (VDRE) in genes modulated by 1,25(OH)2D3. Several novel vitamin D3 compounds (Cmpds) have recently been identified which have 5- to 1000-fold greater abilities to induce differentiation and to inhibit proliferation of HL-60 leukemic blast cells as compared to the parental 1,25(OH)2D3 (code name, Cmpd C). To clarify the mechanism by which five of these vitamin D3 analogs [1,25(OH)2-16ene-D3, (Cmpd HM); 1,25(OH)2-16ene-23yne-D3, (Cmpd V); 1,25(OH)2-16ene-23yne-26,27 F6-D3; 22-Oxa-1,25(OH)2D3; 1,25(OH)2-23yne-D3] mediate their remarkably potent biological activities, we have investigated their abilities in HL-60 cells to transactivate a chloramphenicol acetyl transferase (CAT) reporter gene containing a VDRE from the human osteocalcin gene attached to a thymidine kinase minimal promoter. Also, the abilities of the analogs to enhance the binding of the human recombinant VDR/retinoic X receptor alpha (RXR alpha) heterodimer to the VDRE were examined in gel mobility shift assays. In serumless cultures, a series of potent vitamin D3 analogs had comparable abilities to transactivate the reporter gene as did the biologically less potent 1,25(OH)2D3 (approximately 15-20-fold stimulation in cultures containing 2 x 10(-8)M of vitamin D3 cmpds). Biologically very weak inducers of differentiation of HL-60 [24R,25(OH)2D3; 25(OH)-16ene-23yne-D3] had markedly diminished abilities to induce transactivation. Dose-response studies of Cmpds C, V, HM (10(-7)-10(-11)M) showed that in serumless culture conditions, transactivation of the VDRE-CAT was similar; however, in the presence of serum, Cmpd C at 10(-9)M had 20-fold less activity than analogs V and HM. These results may reflect increased binding of Cmpd C to the D binding protein (DBP) in serum as compared to the lower binding affinities for DBP by Cmpds HM and V. Affinities of the biologically potent analogs for VDR did not parallel their abilities either to transactivate VDRE-CAT or to mediate a biological affect on HL-60 cells. In further studies, gel mobility shift assays showed that VDR alone did not have detectable binding to VDRE; likewise, VDR plus RXR had little binding to VDRE in the absence of ligand. In contrast, biologically active vitamin D3 compounds (Cmpds HM, C, V) in a dose-dependent fashion enhanced the VDR/RXR (retinoid X receptor)-VDRE retarded band.(ABSTRACT TRUNCATED AT 400 WORDS)