The human-derived promyelocytic leukemia cell line, HL-60, is known to differentiate into mature myeloid cells in the presence of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) . We investigated differentiation by monitoring 1,25(OH)2 D3-exposed HL-60 cells for phagocytic activity, ability to reduce nitroblue tetrazolium, binding of the chemotaxin N-formyl-methionylleucyl-[ 3H]phenylalanine, development of nonspecific acid esterase activity, and morphological maturation of Wright-Giemsa-stained cells. 1,25(OH)2D3 concentrations as low as 10-'0 M caused significant development of phagocytosis, nitroblue tetrazolium reduction, and the emergence of differentiated myeloid cells that had morphological characteristics of both metamyelocytes and monocytes . These cells were conclusively identified as monocytes/ macrophages based upon their adherence to the plastic flasks and their content of the macrophage-characteristic nonspecific acid esterase enzyme . The estimated ED50 for 1,25(OH)2 D3-induced differentiation based upon nitroblue tetrazolium reduction and Nformyl-methionyl-leucyl-[ 3H]phenylalanine binding was 5.7 x 10-9 M. HL-60 cells exhibited a complex growth response with various levels of 1,25(OH)2 D3: :510-1' M had no detectable effect, 10-9 M stimulated growth, and 2:10-8 M sharply inhibited proliferation . We also detected and quantitated the specific receptor for 1,25(OH)2D3 in HL-60 and HL-60 Blast, a sub-clone resistant to the growth and differentiation effects of 1,25(OH)2D3. The receptor in both lines was characterized as a DNA-binding protein that migrated at 3.3S on high-salt sucrose gradients . Unequivocal identification was provided by selective dissociation of the 1,25(OH) 2D3-receptor complex with the mercurial reagent, p-chloromercuribenzenesulfonic acid, and by a shift in its sedimentation position upon complexing with anti-receptor monoclonal antibody. On the basis of labeling of whole cells with 1,25(OH)2 [3H]D3 in culture, we found that HL-60 contains 4,000 1,25(OH)2D3 receptor molecules per cell, while the nonresponsive HL-60 Blast is endowed with -8% of that number. The concentration of 1,25(OH) 2D3 (5 x 10-9 M) in complete culture medium, which facilitates the saturation of receptors in HL-60 cells, is virtually identical to the ED50 for the sterol's induction of differentiation . This correspondence, plus the resistance of the relatively receptor-poor HL-60 Blast, indicates that 1,25(OH)2 D3-induced differentiation of HL-60 cells to monocytes/ macrophages is occurring via receptor-mediated events .The induced differentiation of the human promyelocytic leutamin D3 (1,25(OH]2D3), is of great current interest (1-4). kemia, HL-60, by a variety of compounds such as phorbolThe most potent inducer of differentiation in HL-60 cells is esters, DMSO, retinoic acid (RA),' and la,25-dihydroxyvivarious concentrations of KCI; NBT, nitroblue tetrazolium; PBS, 'Abbreviations used in this paper: FMLP, N-formyl-methionyl-leuphosphate-buffered saline ; PCMBS, p-chloromercuribenzene sulfonic cyl-phenylala...

Using both clonal Chinese hamster ovary (CHO) cells in culture and the hen ovary, we have searched for the presence of specific 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors. Receptor analyses were carried out on high salt cytosols of CHO cells and high salt extracts of hen ovarian nuclei that were originally isolated in low salt buffer. Both CHO and hen ovary contain a specific high affinity 1,25-(OH)2D3 receptor (Kd = 10(-10) - 10(-11) M), which sediments (3.3S) in high salt sucrose gradients identically to the chick intestinal receptor for 1,25-(OH)2D3. This 3.3S macromolecule from both sources absorbed to DNA-cellulose at 0.1 M KCl and eluted during a linear salt gradient at 0.2-0.22 M KCl, a property characteristic of the 1,25-(OH)2D3 receptor. Saturation analysis indicated that there are approximately 2000 copies of the receptor molecule per CHO cell. We also investigated the effect of 1,25-(OH)2D3 on ovarian cell growth in monolayer culture of CHO cells. Significant inhibition of CHO cell growth (up to 60%) was observed in the presence of physiological (100 pM) levels of 1,25-(OH)2D3 in the culture medium. This inhibition of growth was dose dependent and was accompanied by a parallel decrease in total cell protein. Concentrations of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 as high as 1 nM did not affect CHO cell growth, indicating that, like receptor binding, cell proliferation is selectively influenced by 1,25-(OH)2D3 over other vitamin D metabolites. These data demonstrate that ovarian cells in mammals and birds possess the 1,25-(OH)2D3 receptor which may play a role in the effect of 1,25-(OH)2D3 on the growth of these cells in culture.

Neuroblastoma cells are a good model for neuronal development because of their ability to extend neurites in response to various stimuli, including retinoic acid. In the present experiments, we have examined five human neuroblastoma cell lines (LA-N-1, IMR-32, LA-N-5, SK-N-MC, and CHP-100) for the presence of cellular retinoic acid binding protein (CRABP), a receptor-like protein implicated in the molecular functioning of vitamin A. CRABP is identified and quantitated by sucrose gradient centrifugation, selective inhibition by the mercurial reagent pchloromercuribenzene sulfonic acid (PCMBS), and saturation analysis. All five lines contain significant levels of cytosolic CRABP (2.5-7.5 pmol/mg of protein), which display typical properties of specific high affinity retinoic acid binding, a sedimentation coefficient of 2 S, and inhibition by PCMBS. Three of the lines (LA-N-1, IMR-32, and LA-N-5) are strongly growth inhibited by 1 ,LM retinoic acid in monolayer culture, whereas two (LA-N-1 and LA-N-5) undergo marked differentiation to a stellate, fusiform morphology with characteristic neurite outgrowths. The SK-N-MC and CHP-100 lines are relatively resistant to the antiproliferative effects of retinoic acid under these conditions. Nevertheless, all five lines are effectively inhibited by retinoic acid in their ability to form anchorage-independent colonies in soft agar. Thus, although CRABP is not necessarily correlated with growth inhibition in monolayer culture, it is associated with retinoic acid's ability to inhibit neuroblastoma colony formation in soft agar. More experiments will be required to determine if this effect on growth in soft agar reflects the putative ability of retinoic acid to convert tumorigenic neuroblastoma cell lines into the normal differentiated phenotype.

Four hybridomas that secrete monoclonal antibodies to the chicken intestinal cytoplasmic la,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor have been obtained. Splenic lymphocytes, derived from two male Lewis rats expressing serum antireceptor activity after repeated immunization with a partially purified preparation of this protein, were fused with two nonsecreting murine myeloma cell lines, SP2/0-Agl4 and P3-X63-Ag8.653. Viable hybrids were screened for anti-chicken intestinal 1,25-(OH)2D3 receptor activity by incubation of hybrid media with receptor-hormone complex; this was followed by immunoprecipitation with rabbit anti-rat IgG. Of 1,724 hybridomas assayed by this technique, 4 were positive (2 derived from each animal) for the secretion of an antireceptor immunoglobulin molecule. After cloning by limiting dilution, the cell lines (designated SP2/0-4A5, P3-8C8, SP2/0-8D3, and SP2/0-9A7) were expanded into suspension culture. Antibody-induced alterations in the sedimentation pattern of the native 1,25-(OH)2D3 receptor, coupled with Ouchterlony double-diffusion techniques, indicate that SP2/0-4A5 secretes an IgG2a, SP2/0-9A7 produces an IgG2b, and P3-8C8 secretes an IgG. In contrast, SP2/0-8D3 was found to synthesize an IgM. The monoclonal antibodies react with both occupied and unoccupied chicken intestinal receptor and nuclear receptors, and they crossreact with 1,25-(OH)2D3 receptors from a wide variety of tissue and cultured cell types, including human 1,25-(OH)2D3 receptors. These immunological reagents should prove valuable in the elucidation of the molecular action of 1,25-(OH)2D3.The mechanism of action of the secosteroid la,25-dihydroxyvitamin D3 [1,2D3] is currently considered to be similar, if not identical, to that of other typical genomic-acting steroid hormones (1, 2). Consistent with this hypothesis is the fact that all known target tissues of vitamin D3, including intestine (3), bone (4), and kidney (5), contain specific, high-affinity cytosolic receptor proteins that interact with chromatin (6) and are thought to bind to DNA (7). This association apparently alters specific transcriptional events in favor of mineral-regulating proteins such as vitamin D3-dependent calcium-binding protein (8).A prerequisite to the elucidation ofvitamin D action is a more complete understanding of the biochemical and physicochemical nature of the 1,25-(OH)2D3 receptor molecule. Because of the lack ofcellular abundance and inherent chemical instability of the receptor, its purification from chicken intestine has proven to be difficult (7, 9). We have described elsewhere (10) the use of a partially purified chicken intestinal receptor preparation, containing a predominant protein of 64,000 daltons, as immunogen in eliciting serum antireceptor response in two different male Lewis rats. We report here the successful polyethylene glycol-mediated fusion of mouse myeloma cells with splenic lymphoid cells from these two rats to obtain stable hybridomas that secrete monoclonal antibodies to the chicken intestinal...

Although the mechanism whereby vitamin A mediates normal cell differentiation and inhibits tumor cell proliferation is unknown, intracellular receptor-like proteins for retinol and retinoic acid have been implicated in the molecular action of vitamin A. We have assayed these two binding proteins, cellular retinol binding protein (protein R) and cellular retinoic acid binding protein (protein RA), in the cytosolic fraction of various normal and tumor cells via sucrose density gradient centrifugation and saturation analysis. Employing charcoal separation of bound and free tritiated retinoid, the saturation analysis yields an approximate Kd for ligand binding and an estimate of the number of protein R and protein RA molecules per cell. Unique protein R and protein RA macromolecules sedimenting at 2 S with Kd values of 7-42 nM are detected in murine cells (1 degree epidermal, 3T6 fibroblasts and melanoma) and human neuroblastoma cells. Concentrations of the intracellular binding proteins range from 55 000 to 3 000 000 copies per cell. When one cell line (C-127 mouse mammary) is transformed by bovine papilloma virus, protein RA levels increase from undetectable to 193 000 copies per cell. Assessment of growth inhibition by 10(-6) M retinol or retinoic acid in the culture medium reveals that there exists a partial, but not absolute, correlation between the presence of protein R or protein RA and the antiproliferative effect of the particular retinoid in the tested cell lines. We conclude that the 2 S intracellular binding proteins for the retinoids are present in most vitamin A responsive cells, but may not be essential for biologic actions of the vitamin such as growth inhibition in monolayer culture.