Immunological identification of 1,25-dihydroxyvitamin D3 receptors in human promyelocytic leukemic cells (HL-60) during homologous regulation.
Immunological techniques were utilized to detect 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) receptor levels and to characterize physical/chemical changes in receptors in human promyelocytic leukemic cells (HL-60) during continuous exposure to hormone. The monoclonal antibody (IVG8C11) raised against the porcine intestinal 1,25-(OH)2D3 receptor immunoprecipitated quantitatively 1,25-(OH)2D3 receptors in nuclear extracts from HL-60 cells. The highly enriched immunoprecipitated receptors were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes, which were probed with 125I-labeled IVG8C11. The basal receptor from the cells treated with 1,25-(OH)2D3 for 2 h was detected as a single form at 53 kDa. Moreover, receptors were shown to be up-regulated at 12 h and down-regulated at 48 and 72 h in the continuous presence of hormone as evidenced by the ratio of density of the bands, 1.0 (2 h):4.2 (12 h):1.2 (48 h):0.9 (72 h), as measured by laser scanning densitometry. The up- and down-regulated receptors were also detected as single forms and had the same molecular mass as the basal receptor. Therefore, the data presented here strongly support the hypothesis of homologous regulation of 1,25-(OH)2D3 receptors in intact human target cells.
Lee
Y
Y
Division of Pharmacology, School of Pharmacy, University of Wisconsin-Madison.
Inaba
M
M
DeLuca
H F
HF
Mellon
W S
WS
D000911:Antibodies, Monoclonal; D002117:Calcitriol / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D003513:Cycloheximide; D002117:Calcitriol