Using new steroidal side-chain-lengthened 26,27-dialkyl analogues of 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3], we manipulated the synthesis of thromboxane and thromboxane-producing enzymes, cyclo-oxygenase and thromboxane synthase, in human promyelocytic leukemia (HL-60) cells in serum-free culture. The order of potency of the analogues for stimulating thromboxane B2 synthetic activity from arachidonic acid (reflecting combined cyclo-oxygenase activity and thromboxane synthase activity) and from prostaglandin H2 (thromboxane synthase activity only) as well as for cyclo-oxygenase induction was 1α,25-(OH)2D3≧1α,25-(OH)2-26,27-(CH3)2D3>1α,25-(OH)2-26,27-(C2H5)2D3≫1α,25-(OH)2-26,27-(C3H7)2D3. These results suggest that there are functional and structural limits to the chain length of C-26 and C-27 dialkyl groups flanking the C-25-OH group in the 1α,25-(OH)2D3 molecule for expressing thromboxane synthetic activity in HL-60 cells. Removal of the C-1α-OH group from 1α,25-(OH)2D3 led to markedly decreased thromboxane synthetic activity in HL-60 cells. These structure-activity relationships indicate that both the C-25-OH and C-1α-OH groups in the 1α,25-(OH)2D3 molecule are essential for expressing thromboxane synthesis in HL-60 cells. Also, the rank order for stimulating thromboxane synthesis correlated well with the binding affinity of these dialkyl analogues for the 1α,25-(OH)2D3 receptor of HL-60 cells, suggesting a 1α,25-(OH)2D3 receptor-mediated induction mechanism. Thromboxane synthesis capacity, a feature of the phenotype of HL-60 cells differentiated along the monocytic lineage, was used as a differentiation marker for the development of more potent 1α,25-(OH)2D3 compounds as potential differentiation-inducing drugs.
- 1α,25-Dihydroxyvitamin D analogues
- HL-60 cells (human)
- Thromboxane synthesis
- Vitamin D receptors