1,25-Dihydroxyvitamin D3-induced differentiation in a human promyelocytic leukemia cell line (HL-60): receptor-mediated maturation to macrophage-like cells.

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)2D3-exposed HL-60 cells for phagocytic activity, ability to reduce nitroblue tetrazolium, binding of the chemotaxin N-formyl-methionyl-leucyl-[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(-10) 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)2D3-induced differentiation based upon nitroblue tetrazolium reduction and N-formyl-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)2D3: less than or equal to 10(-10) M had no detectable effect, 10(-9) M stimulated growth, and greater than or equal to 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 approximately 4,000 1,25(OH)2D3 receptor molecules per cell, while the nonresponsive HL-60 Blast is endowed with approximately 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)2D3-induced differentiation of HL-60 cells to monocytes/macrophages is occurring via receptor-mediated events.

Dynamic changes in circulating 1,25-dihydroxyvitamin D during reproduction in rats.

The concentrations of 1,25-dihydroxyvitamin D [1,25-(OH)2D], calcium, and phosphorus were measured in the serum of rats during pregnancy and at various stages of lactation. The concentration of 1,25-(OH)2D hormone increased almost two-fold during pregnancy and the latter part of lactation, but decreased to control levels or very low values immediately after birth and weaning, respectively. Furthermore, the concentration of 1,25-(OH)2D was inversely correlated with the concentration of calcium, suggesting that circulating 1,25-(OH)2D fluctuates in concert with calcium demands during the reproductive cycle. Parathyroidectomy in lactating rats caused a 70 percent inhibition of the normally observed 1,25-(OH)2D increase, indicating that parathyroid hormone, in response to changes in serum calcium, is a primary modulator of 1,25-(OH)2D during lactation.

Calcinogenic factor in Solanum malacoxylon: evidence that it is 1,25-dihydroxyvitamin D3-glycoside.

After glycosidic cleavage of the water-soluble vitamin D-like principle of the calcinogenic plant Solanum malacoxylon, the active lipophilic portion was purified by column chromatography and analyzed by combined gas chromatography and mass spectrometry. It was identified as 1,25-dihydroxyvitamin D3, the active form of vitamin D. Thus this active metabolite of vitamin D exists in the plant world, and its presence probably accounts for pathologic calcification in grazing animals ingesting Solanum malacoxylon.

Radioreceptor assay for 1 alpha,25-dihydroxyvitamin D3.

A competitive protein binding assay with a sensitivity of 80 picograms has been developed for 1alpha,25-dihydroxyvitamin D(3), the hormonal form of vitamin D(3). lalpha,25-Dihydroxyvitamin D(3) displaced tritiated hormone from a cytosol-chromatin receptor preparation isolated from chick small intestine, providing a simple assay for the hormone. The concentration of lalpha, 25-dihydroxyvitamin D(3) in human plasma, as determined by this assay, is approximately 6 nanograms per 100 milliliters; in patients with renal disease the concentration of this kidney-produced hormone is significantly lower.

Experimental diabetes reduces circulating 1,25-dihydroxyvitamin D in the rat.

Duodenal calcium absorption and a vitamin D-dependent duodenal calcium-binding protein are depressed in rats with alloxan- or streptozotocin-induced diabetes. To test for possible abnormal vitamin D metabolism in diabetes we measured serum concentrations of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D in control, streptozotocin diabetic, and insulin-treated diabetic rats. The serum concentration of 1,25-dihydroxyvitamin D was depressed in untreated diabetic rats to one-eighth of the level in controls and was restored to control levels by insulin treatment. The serum concentration of 25-hydroxyvitamin D was the same in all three groups. Hence, effects of diabetes on duodenal calcium transport can be explained by reduced concentrations of 1,25-dihydroxyvitamin D resulting either from failure of renal 1alpha-hydroxylation of 25-hydroxyvitamin D or increased catabolism of 1,25-dihydroxyvitamin D.

Molecular cloning of complementary DNA encoding the avian receptor for vitamin D.

Vitamin D3 receptors are intracellular proteins that mediate the nuclear action of the active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Two receptor-specific monoclonal antibodies were used to recover the complementary DNA (cDNA) of this regulatory protein from a chicken intestinal lambda gt11 cDNA expression library. The amino acid sequences that were deduced from this cDNA revealed a highly conserved cysteine-rich region that displayed homology with a domain characteristic of other steroid receptors and with the gag-erbA oncogene product of avian erythroblastosis virus. RNA selected via hybridization with this DNA sequence directed the cell-free synthesis of immunoprecipitable vitamin D3 receptor. Northern blot analysis of polyadenylated RNA with these cDNA probes revealed two vitamin D receptor messenger RNAs (mRNAs) of 2.6 and 3.2 kilobases in receptor-containing chicken tissues and a major cross-hybridizing receptor mRNA species of 4.2 kilobases in mouse 3T6 fibroblasts. The 4.2-kilobase species was substantially increased by prior exposure of 3T6 cells to 1,25(OH)2D3. This cDNA represents perhaps the rarest mRNA cloned to date in eukaryotes, as well as the first receptor sequence described for an authentic vitamin.

Vitamin D3--resistant fibroblasts have immunoassayable 1,25-dihydroxyvitamin D3 receptors.

Cultured fibroblasts obtained from patients with tissue resistance to 1,25-dihydroxyvitamin D3 (vitamin D3--dependent rickets, type II) contain normal, low, or undetectable concentrations of this hormone's receptor protein as measured by a ligand-binding assay. Extracts from these cells were evaluated for receptors by immunoassay with a recently developed monoclonal antibody to the chick receptor. The results show that a protein sedimenting at 3.7S and recognizable by the antibody exists in comparable concentrations in cells from both normal and resistant patients, irrespective of the hormone-binding abnormalities of the cells. This implies that deficiencies in hormone binding associated with inherited tissue resistance to 1,25-dihydroxyvitamin D3 probably arise from structural variations in the receptor molecule and not from defective receptor synthesis.

Estrogen binding, receptor mRNA, and biologic response in osteoblast-like osteosarcoma cells.

High specific activity estradiol labeled with iodine-125 was used to detect approximately 200 saturable, high-affinity (dissociation constant approximately equal to 1.0 nM) nuclear binding sites in rat (ROS 17/2.8) and human (HOS TE85) clonal osteoblast-like osteosarcoma cells. Of the steroids tested, only testosterone exhibited significant cross-reactivity with estrogen binding. RNA blot analysis with a complementary DNA probe to the human estrogen receptor revealed putative receptor transcripts of 6 to 6.2 kilobases in both rat and human osteosarcoma cells. Type I procollagen and transforming growth factor-beta messenger RNA levels were enhanced in cultured human osteoblast-like cells treated with 1 nM estradiol. Thus, estrogen can act directly on osteoblasts by a receptor-mediated mechanism and thereby modulate the extracellular matrix and other proteins involved in the maintenance of skeletal mineralization and remodeling.

Evaluation of a role for 1,25-dihydroxyvitamin D3 in the pathogenesis and treatment of X-linked hypophosphatemic rickets and osteomalacia.

Although a defect in renal transport of phosphate seems well established as the primary abnormality underlying the pathogenesis of X-linked hypophosphatemic rickets and osteomalacia, several observations indicate that renal phosphate wasting and hypophosphatemia cannot solely account for the spectrum of abnormalities characteristic of this disease. Thus, in the present study, we investigated the potential role of abnormal vitamin D metabolism in the pathogenesis of this disorder and the effect of 1,25-dihydroxyvitamin D(3) therapy on both the biochemical abnormalities characteristic of this disease and the osteomalacia. Four untreated patients, ages 14-30 yr, had normocalcemia (9.22+/-0.06 mg/dl); hypophosphatemia (2.25+/-0.11 mg/dl); a decreased renal tubular maximum for the reabsorption of phosphate per liter of glomerular filtrate (2.12+/-0.09 mg/dl); normal serum immunoreactive parathyroid hormone concentration; negative phosphate balance; and bone biopsy evidence of osteomalacia. The serum 25-hydroxyvitamin D(3) concentration was 33.9+/-7.2 ng/ml and, despite hypophosphatemia, the serum level of 1,25-dihydroxyvitamin D(3) was not increased, but was normal at 30.3+/-2.8 pg/ml. These data suggested that abnormal homeostasis of vitamin D metabolism might be a second defect central to the phenotypic expression of X-linked hypophosphatemic rickets/osteomalacia. This hypothesis was supported by evaluation of the long-term response to pharmacological amounts of 1,25-dihydroxyvitamin D(3) therapy in three subjects. The treatment regimen resulted in elevation of the serum 1,25-dihydroxyvitamin D levels to values in the supraphysiological range. Moreover, the serum phosphate and renal tubular maximum for the reabsorption of phosphate per liter of glomerular filtrate increased towards normal whereas the phosphate balance became markedly positive. Most importantly, however, repeat bone biopsies revealed that therapy had positively affected the osteomalacic component of the disease, resulting in normalization of the mineralization front activity. Indeed, a central role for 1,25-dihydroxyvitamin D(3) in the mineralization of the osteomalacic bone is suggested by the linear relationship between the serum level of this active vitamin D metabolite and the mineralization front activity. We, therefore, suggest that a relative deficiency of 1,25-dihydroxyvitamin D(3) is a factor in the pathogenesis of X-linked hypophosphatemic rickets and osteomalacia and may modulate the phenotypic expression of this disease.

Radioligand receptor assay for 25-hydroxyvitamin D2/D3 and 1 alpha, 25-dihydroxyvitamin D2/D3.

A competitive protein binding assay for measurement of the plasma concentration of 1 alpha, 25-dihydroxyvitamin D3 [1alpha, 25-(OH)2D3] has been extended to include the immediate precursor of this hormone, 25-hydroxyvitamin D3 (25-OHD3). In addition, the assay system is capable of measuring the two metabolic products of ergocalciferol, namely. 25-hydroxyvitamin D2 (25-OHD2) and 1alpha, 25-dihydroxyvitamin D2 [1alpha, 25-(OH)2D2]. The target tissue assay system consists of a high affinity cytosol receptor protein that binds the vitamin D metabolites and a limited number of acceptor sites on the nuclear chromatin. By utilizing a series of chromatographic purification steps, a single plasma sample can be assayed for any of the four vitamin D metabolites either individually or combined. Therefore, the assay procedure allows for both the quantitative and qualitative assessment of the total active vitamin D level in a given plasma sample. To show that the binding assay was capable of measuring 1alpha, 25-(OH)2D2 as well as 1alpha, 25 (OH)2D3, two groups of rats were raised. One group, supplemented with vitamin D3, produced assayable material that represented 1alpha, 25-(OH)2D3. The other group, fed only vitamin D2 in the diet, yielded plasma containing only 1alpha, 25-(OH)2D2 as the hormonal form of the vitamin. The circulating concentrations of the two active sterols were nearly identical (15 ng/100 ml) in both groups, indicating that the competitive binding assay can be used to measure both hormonal forms in plasma. In a separate experiment, 1alpha, 25-(OH)2D2 was generated in an in vitro kidney homogenate system using 25-OHD2 as substrate. Comparison of this sterol with 1alpha, 25-(OH)2D3 in the assay system showed very similar binding curves; the D2 form was slightly less efficient (77%). Comparison of the respective 25-hydroxy forms (25-OHD2 vs. 25-OHD3) at concentrations 500-fold that of 1alpha, 25-(OH)2D3, again suggested that the binding of the D2 metabolite was slightly less efficient (71%). Finally, the assay was employed to measure the total active vitamin D metabolite pools in the plasma of normal subjects and patients with varying degrees of hypervitaminosis D. The normal plasma levels of 25-OHD and 1alpha, 25-(OH)2D measured in Tucson adults were 25-40 ng/ml and 2.1-4.5 ng/100 ml, respectively. Both sterols were predominately (greater than 90%) in the form of vitamin D3 metabolites in this environment. Typical cases of hypervitaminosis D exhibited approximately a 15-fold increase in the plasma 25-OHD concentration, and a dramatic changeover to virtually all metabolites existing in the form of D2 vitamins. In contrast, the circulating concentration of 1alpha, 25-(OH)2D was not substantially enhanced in vitamin D-intoxicated patients. We therefore conclude that hypervitaminosis D is not a result of abnormal plasma levels of 1alpha, 25-(OH)2D but may be cuased by an excessive circulating concentration of 25-OHD.

Evidence for in vivo upregulation of the intestinal vitamin D receptor during dietary calcium restriction in the rat.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] increases intestinal calcium absorption through events that include binding of 1,25(OH)2D3 to the intracellular vitamin D receptor. In vitro studies using mammalian cell cultures reveal an increase in vitamin D receptor content after exposure to 1,25(OH)2D3. To test the hypothesis that 1,25(OH)2D3 increases enterocyte vitamin D receptor content in vivo, male rats were fed either a normal calcium diet (NCD, 1.2% Ca) or low calcium diet (LCD, 0.002% Ca). After 21 d LCD increased serum 1,25(OH)2D3 levels (27 +/- 3 vs. 181 +/- 17 pg/ml, P less than 0.001) and increased transepithelial mucosal to serosal calcium fluxes (Jms) across duodenum (65 +/- 21 vs. 204 +/- 47 nmol/cm2.h, NCD vs. LCD, P less than 0.01) and jejunum (23 +/- 3 vs. 46 +/- 4, P less than 0.007). No change in serosal to mucosal calcium fluxes (Jsm) were observed. LCD increased 1,25(OH)2D3 receptor number threefold in duodenum (32.9 +/- 6.7 vs. 98.7 +/- 13.7 fmol 1,25(OH)2D3/mg protein) and jejunum (34.1 +/- 9.5 vs. 84.9 +/- 7.7) without a change in the receptor affinity for 1,25(OH)2D3 (Kd is 0.17 +/- 0.06 vs. 0.21 +/- 0.02 nM for NCD and LCD duodenum, respectively). Duodenal polyadenylated vitamin D receptor mRNA determined by Northern blot analysis did not increase appreciably during LCD, suggesting that upregulation in vivo may not be due primarily to increased receptor synthesis. The results of this study indicate that under physiologic conditions as during chronic dietary calcium restriction, increased intestinal vitamin D receptor content accompanies increased calcium active transport. Upregulation of the vitamin D receptor by 1,25(OH)2D3 may result primarily from posttranslational processes that decrease degradation of the receptor with increased receptor synthesis responsible for a negligible portion of the accumulation.

The role of 1 alpha, 25-dihydroxyvitamin D in the mediation of intestinal hyperabsorption of calcium in primary hyperparathyroidism and absorptive hypercalciuria.

The cuase for the intestinal hyperabsorptionof calcium (Ca) in various forms of hypercalciurias was explored by a careful measurement of plasma 1 alpha, 25-dihydroxycholecalciferol [1 alpha, 25-(OH)I D] and by an assessment of intestinal Ca absorption and of parathyroid function. In 18 cases of primary hyperparathyroidism (PHPT), the mean plasma concentration of 1 alpha, 25-(OH)2D was significantly increased (4.9 +/- 2.2 SD ng/dl vs. 3.4 +/- 0.9 ng/dl for the control group), and was significantly correlated with fractional Ca absorption (alpha) (r = 0.80, P less than 0.001). Plasma 1 alpha, 25-(OH)2D was also correlated with urinary Ca (P less than 0.05), but not with serum Ca or phosphorus (P), P clearance, urinary cyclic AMP, or serum immunoreactive parathyroid hormone. In 21 cases of absorptive hypercalciuria (AH), plasma 1 alpha, 25-(OH)2D was elevated in one-third of cases, and the mean value of 4.5 +/- 1.1 ng/dl was significantly higher than that of the control group (P less than 0.01). Since relative hypoparathyroidism may be present, the normal absolute value of plasma 1 alpha, 25-(OH)2D, found in two-thirds of cases of AH, may be considered to be inappropriately high. Moreover, in the majority of cases of AH, the data points relating plasma 1 alpha, 25-(OH)2D and alpha fell within 95% confidence limits of values found in non-AH groups (including PHPT). The results suggest that the intestinal hyperabsorption of Ca in PHPT aw AH may be vitamin D dependent. However, the disturbance in vitamin D metabolism may not be the sole cause for the high Ca absorption in AH, since in some patients with AH, the intestinal Ca absorption appears to be inapp

Identification and regulation of 1,25-dihydroxyvitamin D3 receptor activity and biosynthesis of 1,25-dihydroxyvitamin D3. Studies in cultured bovine aortic endothelial cells and human dermal capillaries.

Because 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to play roles in both proliferation and differentiation of novel target cells, the potential expression of 1,25(OH)2D3 receptor (VDR) activity was investigated in cultured bovine aortic endothelial cells (BAEC). Receptor binding assays performed on nuclear extracts of BAEC revealed a single class of specific, high-affinity VDR that displayed a 4.5-fold increase in maximal ligand binding (Nmax) in rapidly proliferating BAEC compared with confluent, density-arrested cells. When confluent BAEC were incubated with activators of protein kinase C (PKC), Nmax increased 2.5-fold within 6-24 h and this upregulation was prevented by sphingosine, an inhibitor of PKC, as well as by actinomycin D or cycloheximide. Immunohistochemical visualization using a specific MAb disclosed nuclear localized VDR in venular and capillary endothelial cells of human skin biopsies, documenting the expression of VDR, in vivo, and validating the BAEC model. Finally, additional experiments indicated that BAEC formed the 1,25(OH)2D3 hormonal metabolite from 25(OH)D3 substrate, in vitro, and growth curves of BAEC maintained in the presence of 10(-8) M 1,25(OH)2D3 showed a 36% decrease in saturation density. These data provide evidence for the presence of a vitamin D microendocrine system in endothelial cells, consisting of the VDR and a 1 alpha-hydroxylase enzyme capable of producing 1,25(OH)2D3. That both components of this system are coordinately regulated, and that BAEC respond to the 1,25(OH)2D3 hormone by modulating growth kinetics, suggests the existence of a vitamin D autocrine loop in endothelium that may play a role in the development and/or functions of this pathophysiologically significant cell population.

A composite element binding the vitamin D receptor, retinoid X receptor alpha, and a member of the CTF/NF-1 family of transcription factors mediates the vitamin D responsiveness of the c-fos promoter.

The hormonal form of vitamin D, 1 alpha,25-dihydroxyvitamin D3 [1,25- (OH)2D3], transiently stimulates the transcription of the c-fos proto-oncogene in osteoblastic cells. We have identified and characterized a vitamin D response element (VDRE) in the promoter of c-fos. The 1,25-(OH)2D3-responsive region was delineated between residues -178 and -144 upstream of the c-fos transcription start site. A mutation that inhibited binding to the sequence concomitantly abolished 1,25-(OH)2D3-induced transcriptional responsiveness; similarly, cloning to the site upstream of a heterologous promoter conferred copy-number-dependent vitamin D responsiveness to a reporter gene, demonstrating that we have identified a functional response element. The structure of the c-fos VDRE was found to be unusual. Mutational analysis revealed that the c-fos VDRE does not conform to the direct repeat configuration in which hexameric core-binding sites are spaced by a few nucleotide residues. In contrast, the entire 36-bp sequence was essential for binding. We identified the vitamin D receptor and the retinoid X receptor alpha as components of the complex that bound the c-fos VDRE. However, our results also show that a putative CCAAT-binding transcription factor/nuclear factor 1 (CTF/NF-1) family member bound the response element in conjunction with the nuclear hormone receptors. The expression of this CTF/NF-1 family member appeared restricted to bone cells. These data hint at new molecular mechanisms of action for vitamin D.

Retinoid X receptors stimulate and 9-cis retinoic acid inhibits 1,25-dihydroxyvitamin D3-activated expression of the rat osteocalcin gene.

The vitamin D receptor (VDR) binds the vitamin D-responsive element (VDRE) as a heterodimer with an unidentified receptor auxiliary factor (RAF) present in mammalian cell nuclear extracts. VDR also interacts with the retinoid X receptors (RXRs), implying that RAF may be related to the RXRs. Here we demonstrate that highly purified HeLa cell RAF contained RXR beta immunoreactivity and that both activities copurified and precisely coeluted in high-resolution hydroxylapatite chromatography. Furthermore, an RXR beta-specific antibody disrupted VDR-RAF-VDRE complexes in mobility shift assays. These data strongly indicate that HeLa RAF is highly related to or is identical to RXR beta. Consequently, the effect of the 9-cis retinoic acid ligand for RXRs was examined in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated gene expression systems. Increasing concentrations of 9-cis retinoic acid (1 nM to 1 microM) markedly reduced 1,25(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells. All-trans retinoic acid also interfered with vitamin D responsiveness, but it was consistently less potent than the 9-cis isomer. Transient transfection studies revealed that attenuation by 9-cis retinoic acid was at the transcriptional level and was mediated through interactions at the osteocalcin VDRE. Furthermore, overexpression of both RXR beta and RXR alpha augmented 1,25(OH)2D3 responsiveness in transient expression studies. Direct analysis of VDRE binding in mobility shift assays demonstrated that heteromeric interactions between VDR and RXR were enhanced by 1,25(OH)2D3 and were not affected appreciably by 9-cis retinoic acid, except that inhibition was observed at high retinoid concentrations. These data suggest a regulatory mechanism for osteocalcin gene expression that involves 1,25(OH)2D3-induced heterodimerization of VDR and unliganded RXR. 9-cis retinoic acid may attenuate 1,25(OH)2D3 responsiveness by diverting RXRs away from VDR-mediated transcription and towards other RXR-dependent transcriptional pathways.

Influence of 1,25-dihydroxyvitamin D3 on cultured osteogenic sarcoma cells: correlation with the 1,25-dihydroxyvitamin D3 receptor.

We have characterized the effects of the steroid hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on a series of rat osteogenic sarcoma cell lines of increasing osteoblastic-like nature (ROS 24/1, ROS 2/3, and ROS 17/2.8). When these cells were grown in monolayer culture in the presence of 10 nM 1,25-(OH)2D3, there was a dramatic and selective inhibition of proliferation in the ROS 17/2.8 line. Similar concentrations of other vitamin D metabolites did not elicit this effect. Furthermore, the aggregated cuboidal ROS 17/2.8 cells showed a marked change after 6 days of treatment with 10 nM 1,25-(OH)2D3 to an apparently less transformed spindle-like morphology. In contrast, ROS 2/3 displayed only a slight morphological alteration, and ROS 24/1 was unchanged by treatment with 1,25-(OH)2D3. Anchorage-independent growth studies performed in soft agar indicated that 1,25-(OH)2D3 inhibited colony formation to the greatest degree in ROS 17/2.8, with a lesser effect in ROS 2/3. Based upon analyses by sucrose gradient centrifugation, DNA cellulose chromatography, and saturation of specific binding, the level of the 1,25-(OH)2D3 receptor was quantitated in these cells. ROS 17/2.8 cells possess 18,000 copies of the receptor per cell, while ROS 2/3 contains only 500 binding sites per cell, and no detectable high-affinity 1,25-(OH)2D3 receptor is found in ROS 24/1. The receptor in ROS cells is indistinguishable from other mammalian 1,25-(OH)2D3 receptors in that it is a DNA-binding protein that sediments on sucrose gradients at 3.3S, and specifically binds the hormone with high affinity (Kd = 2 to 3 X 10(-11) M). Since the biological responses of these three cell lines to 1,25-(OH)2D3 exhibit a strong correlation with the respective number of receptor molecules per cell, we propose that the actions of this hormone are mediated by the specific 1,25-(OH)2D3 receptor.

Immunocytochemical detection of 1,25-dihydroxyvitamin D3 receptor in breast cancer.

We have developed an immunocytochemical technique to visualize the receptor for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in cryostat sections of human breast tumors and normal human breast tissue utilizing a monoclonal antibody (9A7 gamma) to chick intestinal receptor which recognizes mammalian 1,25(OH)2D3 receptor. Specific staining was observed in the nuclei of tumor cells. Previous studies by our group have shown that a high proportion of breast tumors bind radiolabeled 1,25(OH)2D3 and we have confirmed this, demonstrating immunocytochemical 1,25(OH)2D3 receptor in 43 of 55 (78%) of breast carcinomas. No correlation with the presence of immunostainable estrogen receptor was found in these breast cancer specimens. Sections of normal breast showed immunoreactivity in the nuclei of epithelial cells of the lobules and ducts. Our results demonstrate that the receptor for 1,25(OH)2D3 resides predominantly in the nucleus of breast carcinoma cells. The reason for its prominent expression in breast cancers is not yet known but may be related to growth regulation.

Phenotypic and functional analysis of 1,25-dihydroxyvitamin D3 receptor mediated modulation of the human myeloma cell line RPMI 8226.

Several recent studies have demonstrated the presence of specific receptors for the 1,25-dihydroxyvitamin D3 (calcitriol) in activated normal lymphocytes. By DNA cellulose chromatography, we show evidence of such specific receptors in the human myeloma cell line RPMI 8226. Nanomolar concentrations of 1,25-dihydroxyvitamin D3 reduce the proliferation of RPMI 8226 cells significantly and simultaneously induce the appearance of both new properties and phenotype expression, such as butyrate esterase, enhanced expression of CD20 (B1), CD15 (Leu-M1) antigens and lambda chains, and decreased expression of the PC1 antigen using microfluorometric analysis. But such an increased expression of membrane lambda chains was not associated with an enhanced secretion of lambda chains. Furthermore, the bone resorbing activity produced normally by RPMI 8226 cells was reduced significantly after 1,25-dihydroxyvitamin D3 treatment. The possible mechanisms and significance of these new functional and phenotypic properties are discussed with respect to the B-cell lineage.

Immunocytochemical determination of estrogen receptor, progesterone receptor, and 1,25-dihydroxyvitamin D3 receptor in breast cancer and relationship to prognosis.

We have determined the estrogen receptor, progesterone receptor (PR), and 1,25-dihydroxyvitamin D3 receptor content of 136 breast carcinomas by an immunocytochemical method. The presence of the three receptors was not related to clinical features of presentation such as T-stage or to age or menopausal status. However, each of the three receptors has a different relationship to the course of the disease in these patients. The presence of PR was significantly associated with an improved overall survival (chi 2 = 4.61, P = 0.032). Patients whose tumors contained immunocytochemically detectable 1,25-dihydroxyvitamin D3 receptor had a longer disease-free interval than those patients with negative tumors (chi 2 = 4.01, P = 0.045). The presence of estrogen receptor and PR were found to correlate with an increased survival between relapse and death (P = 0.027 and P = 0.09, respectively). The relationships between estrogen receptor and PR and prognosis are more apparent when the degree of cell staining is considered. Combined receptor analysis improves our ability to predict the course of the disease and may therefore facilitate better management of the patients.

Identification and quantitation of intracellular retinol and retinoic acid binding proteins in cultured cells.

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.