Induction of monocytic differentiation by calcitriol (1,25-dihydroxyvitamin D3) in the human promyelocytic leukemic cell line (HL-60) in serum-free medium.
The effect of calcitriol on the induction of differentiation in human promyelocytic leukemic cell line (HL-60) cultured in serum-free chemically defined medium (SFM) was investigated. The utilization of SFM containing RPMI-1640 basal medium supplemented with insulin (5 micrograms/ml), transferrin (5 micrograms/ml), sodium selenite (5 ng/ml), and bovine serum albumin (0.5 micrograms/ml), transferrin examination of the cellular/molecular mechanism of calcitriol's action in HL-60 cell differentiation without interference of components present in serum. HL-60 cells grown in SFM were induced to differentiate into monocytes/macrophages by calcitriol as indicated by induction of differentiation-associated biological and biochemical parameters: chemiluminescent (CL) responsiveness, lysozyme activity, nonspecific esterase, expression of cell surface antigens, and reduced proliferation. The exposure of HL-60 cells in SFM to calcitriol (from 10(-10) to 10(-8)M) resulted in dose-dependent induction of these parameters, which was similar to those obtained with cells grown in 10% fetal calf serum containing medium (10% SCM). However, calcitriol was 5-fold more potent for HL-60 cells cultured in SFM than those cultured in 10% SCM as indicated by shifts in dose-response curves for induction of CL responsiveness and lysozyme activity. The effect of calcitriol on the proliferation and acquisition of several monocyte-associated cell surface antigens was also more sensitive for HL-60 cells cultured in SFM than for cells grown in 10% SCM. We characterized and quantitated calcitriol receptors in HL-60 cells cultured in SFM in comparison to those in 10% SCM after exposing intact cells to radiolabeled calcitriol. Cells cultured in either SFM or 10% SCM exhibited calcitriol receptors that migrated at 3.4S as a single peak on sucrose gradients and elicited inherent DNA binding ability. There was essentially no difference in the apparent dissociation constants (Kd) nor in the number of calcitriol binding sites per HL-60 cell, that is approximately 6.0 X 10(-11) M and approximately 3000 binding sites/cell respectively. It is concluded that culturing HL-60 cells in SFM results in full expression of calcitriol-induced phenotypic changes excluding the possibility that such changes result from the indirect effect of calcitriol mediated by identified and/or unidentified components present in serum.
Lee
Y
Y
School of Pharmacy, University of Wisconsin, Madison 53706.
Dunlap
B E
BE
Mellon
W S
WS
D002117:Calcitriol / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003470:Culture Media; D004950:Esterases / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000473:pathology*; D008163:Luminescent Measurements; D009000:Monocytes / Q000187:drug effects* / Q000473:pathology; D009113:Muramidase / Q000378:metabolism; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000378:metabolism
D003470:Culture Media; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D004950:Esterases; D009113:Muramidase; D002117:Calcitriol