1,25-dihydroxyvitamin D3 receptors in human leukocytes.

A 1,25-dihydroxyvitamin D3 receptor macromolecule was detected in peripheral mononuclear leukocytes from normal humans. This macromolecule was found to be present in monocytes but absent from normal resting peripheral B and T lymphocytes. However, it was present in established lines of malignant B, T, and non-B, non-T human lymphocytes, as well as in T and B lymphocytes obtained from normal humans and activated in vitro.

Parathyroid hormone production in vitro by human parathyroid cells transformed by Simian virus 40.

Cells froma human parathyroid adenoma were infected with simian virus 40 and maintained through 13 subcultures in monolayer tissue culture. For more than 9 months, these "transformed" cells contimed to produce parathyroid hormone which was identified by radioimmunoassay and density-gradient ultra centrifugation.

Chromogranin A: its role in endocrine function and as an endocrine and neuroendocrine tumor marker.

CgA is a 49 kilodalton protein that is present in the secretory granules of most endocrine and many neuroendocrine cells. Detection of CgA in cells by immunocytochemistry and measurement of CgA in serum by immunoassay can serve as tissue and serum markers for CgA-producing tumors. CgA is of diagnostic value in classical endocrine tumors, in hormone-negative tumors, and in endocrine tumors in which other diagnostic procedures have their limitations. Although the biological function of CgA is yet unknown, it may serve as a precursor molecule, like POMC, for a family of biologically active peptides. CgA is an important new tool for the endocrinologist in the diagnosis and management of patients with endocrine and neuroendocrine tumors.

Secretion of parathyroid hormone in patients with medullary thyroid carcinoma.

The secretion of parathyroid hormone (PTH) and calcitonin (CT) was studied in 30 patients with medullary thyroid carcinoma. Most patients with elevated levels of CT were normocalcemic and also had normal basal levels of PTH. Five of six patients with associated hyperparathyroidism were hypercalcemic and had elevated basal PTH levels. Hormone secretion was also studied during infusions with standard and low doses of calcium. PTH unexpectedly increased during 12 of 18 calcium infusions. Such a paradoxical increase in PTH was seen in those patients with the greatest increase in CT and the least increase in calcium during the calcium infusion. Accordingly, increases in PTH concentration during the calcium infusions could be correlated directly with increases in CT and correlated inversely with increases in calcium. These observations suggest that, in some patients with medullary thyroid carcinoma, a further increase in the abnormally elevated CT levels may stimulate PTH secretion. Therefore, at least in acute studies, there may be a functional, as well as a genetic, relationship between the secretion of these two hormones in patients with this thyroid tumor.

The regulation of calcitonin in normal human plasma as assessed by immunoprecipitation and immunoextraction.

We have studied the secretion of calcitonin in normal adults with a new procedure of increased sensitivity for the measurement of the hormone in peripheral plasma. In this method, endogenous calcitonin is immunoprecipitated with specific antibodies from a 10-ml plasma sample. The calcitonin is then dissociated from the antibodies and extracted into alcohol. The alcohol is evaporated, and the calcitonin is recovered in assay diluent for subsequent radioimmunoassay. The procedure produces a fivefold increase in immunoassay sensitivity and eliminates plasma proteins which can produce spurious immunoassay effects. This procedure was used to measure calcitonin in normal adults. The mean (+/- SE) plasma calcitonin in 43 subjects was 10 (+/- 2) pg/ml. A+ the end of a 3-h calcium infusion (12 mg/kg), mean plasma calcitonin in 10 subjects had risen to 114 (+/- 21) pg/ml. In 11 subjects, a 10-min infusion of 150 mg of calcium caused calcitonin to rise to a mean concentration of 28 (+/- 7) pg/ml at 20 min. EDTA infusion (50 mg/kg 2 h) caused a slight decrease in plasma calcitonin. These results are consistent with our previous reports of the low concentrations of calcitonin in adult plasma. Our data may underestimate calcitonin levels since not all of the heterogenous species of hormone may be extracted by this method. In any case, this procedure has allowed us to determine that the low concentrations of plasma calcitonin in normal adults are responsive to perturbations of calcium homeostasis. The immunoextraction method may be applicable to other assays in which it is necessary to increase sensitivity or define specificity.

New biochemical marker for bone metabolism. Measurement by radioimmunoassay of bone GLA protein in the plasma of normal subjects and patients with bone disease.

gamma-Carboxyglutamic acid-containing protein of bone (BGP) is an abundant noncollagenous protein of mammalian bone. BGP has a molecular weight of 5,800 and contains three residues of the vitamin K-dependent amino acid, gamma-carboxyglutamic acid. We have applied a radioimmunoassay based on calf BGP for the measurement of the protein in the plasma of 109 normal humans and 112 patients with various bone diseases. BGP in human plasma was demonstrated to be indistinguishable from calf BGP by assay dilution studies and gel permeation chromatography. The mean (+/- SE) concentration of BGP in normal subjects was 6.78 (+/- 0.20) ng/ml, 7.89 (+/- 0.32) for males and 4.85 (+/- 0.35) for females. Plasma BGP was increased in patients with Paget's disease of bone, bone metastases, primary hyperparathyroidism, renal osteodystrophy, and osteopenia. Plasma BGP did correlate with plasma alkaline phosphatase (AP) in some instances, but there were dissociations between the two. It was additionally observed that patients with liver disease had normal plasma BGP despite increased plasma AP, a reflection of the lack of specificity of AP measurements for bone disease. Our studies indicate that the radioimmunoassay of plasma BGP can be a useful and specific procedure for evaluating the patient with bone disease.

Parathyroid hormone-like protein is a secretory product of atrial myocytes.

Parathyroid hormone-like protein (PLP) was originally identified from tumors associated with hypercalcemia. Recently, it has been found to be expressed in a stretch-responsive manner in several types of smooth muscle. We studied adult rat heart muscle for the presence of the PLP. Using immunohistology and the PCR, we demonstrated the presence of PLP and its mRNA in all heart chambers. Immunoelectron microscopy demonstrated PLP in secretory vesicles of atrial mycocytes. Using immunoassay, we demonstrated that atria contained a higher concentration of PLP than ventricles. Furthermore, primary cultures of both chambers released PLP into conditioned medium, with atria secreting more than ventricles. Considered with studies of the role of PLP in other tissues, our observations suggest that the production and secretion of PLP by cardiac myocytes represents a calcium-related regulatory function for this stretch-responsive polypeptide in the cardiovascular system. PLP in the heart may be the calcium counterpart for the atrial natriuretic-sodium regulatory axis of the cardiovascular system.

Calcitonin secretion in congenital nongoitrous cretinism.

Plasma calcitonin (CT) was measured in the basal state and/or during provocative tests of hormone secretion in 11 children with congenital non-goitrous cretinism (CNC), in 1 girl with a lingual thyroid, and in 11 normal children. Basal and stimulated CT concentrations were significantly lower in the patients with CNC than in the normal subjects. Mean basal CT (+/- SE) was 41 +/- 4 pg/ml in the normal children, 24 +/- 3 pg/ml in the children with CNC, and 20 +/- 2 pg/ml in the patient with the lingual thyroid. The mean incremental CT responses to calcium infusion were 7.0 +/- 2 pg/ml in the children with CNC, 6.0 pg/ml in the patient with the lingual thyroid, and 146 +/- 47 pg/ml in the normal children. The children with CNC also demonstrated a significant delay in the return of the total serum calcium to basal level after the calcium infusion. The mean incremental CT response after infusion of pentagastrin was 7.6 +/- 2 pg/ml in the children with CNC, 10.0 pg/ml in the child with the lingual thyroid, and 34.4 +/- 11 pg/ml in the normal children. These data indicate that CT deficiency is present in children with CNC and suggest that the deficiency is a consequence of the defective embryologic development of the thyroid gland.

Secretion of calcitonin in hypocalcemic states in man.

The control of calcitonin secretion in humans has been studied extensively only in patients with medullary thyroid carcinoma since the peripheral concentration of the hormone in normal subjects is too low for accurate measurement by existing assay procedures. However, we have recently found that the concentrations of calcitonin in the peripheral plasma of hypocalcemic subjects during provocative tests of hormone secretion were high enough to be measured by radioimmunoassay. Accordingly, the effect of calcium and pentagastrin infusions on plasma calcitonin was studied in nine patients with pseudohypoparathyroidism, seven patients with idiopathic hypoparathyroidism, and six patients with hypocalcemia not due to parathyroid disease. The infusion of calcium in these hypocalcemic subjects resulted in increases in plasma calcitonin to levels that could be readily detected by our radioimmunoassay. Pentagastrin infusion also caused an increase of plasma calcitonin in some subjects, but calcium was approximately 10 times more effective than gastrin in its stimulatory effect on hormone secretion. These results demonstrate that in humans as well as other mammals the secretion of calcitonin by parafollicular cells that are not involved by medullary thyroid carcinoma is directly related to plasma calcium and that gastrin can also stimulate hormone secretion. The results are consistent with the thesis that the secretion of calcitonin by normal human subjects does occur but at peripheral concentrations of the hormone below the detection limits of most existing immunoassays; hypocalcemia leads to increased stores of hormone that can be related by the appropriate stimuli.

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

Growth in vitro and ultrastructure of cells from a medullary carcinoma of the human thyroid gland: transformation by simian virus 40 and evidenc of thyrocalcitonin and prostaglandins.

PIP: Continuous single layer tissue cultures were established with explants from a metastatic nodule of medullary carcinoma primary in the thyroid gland. Some cultures were infected with simian virus 40 and the cells acquired morphologic and growth characteristics associated with viral transformation. Thyrocalcitonin activity was detected by radioimmunoassay of culture fluids as late as 7 months after initiation of the single layers. Substances with the chemical and biological properties of (PG) prostaglandins E2 and F2alpha were extracted from culture fluids after 4 months. The original carcinoma cells contained abundant cyctoplasmic secretory granules, which were demonstrated by electron microscopy. These granules disappeared during subculture of the monolayers, and electron-dense secretory substance accumulated within dilated cisternae of the ribosome-bound endoplasmic reticulum. Ultrastructural differences in surface organization of the virus transformed and uninfected culture cells were noted. Amyloid was present in the original tumor stroma and tissue culture explants, but could not be identified in those serial subcultures derived from the monolayer outgrowths.^ieng

Parathyroid hormone-like protein: alternative messenger RNA splicing pathways in human cancer cell lines.

Parathyroid hormone-like protein (PLP) is expressed in a wide variety of cancers and exerts diverse biological effects in addition to hypercalcemia. We studied the expression of the gene for PLP in cancer cell lines derived from different tissues that produce PLP. We used the polymerase chain reaction to evaluate the PLP mRNA species produced in these various cell lines by differential transcription initiation and alternative splicing pathways. A series of exon-specific oligonucleotide primers that hybridize to DNA sequences adjacent to exon-intron splice junctions throughout the PLP gene were designed. These primers were used to evaluate steady state levels of PLP mRNA species. Our analysis with promoter-specific primers demonstrated expression from all three putative transcription start sites of PLP, designated P1, P2, and P3. P2-initiated transcripts were present in all of the cell lines, whereas the presence of P1- and P3-initiated messages were cell line specific. Our analysis with carboxy-terminal coding-specific primers demonstrated the utilization of the alternative splicing pathways that produce all three mature PLP polypeptides, PLP-139, -1-173, and -1-141. The 1-139 mRNA species was found in all of the cell lines, whereas the 1-141 and 1-173 mRNA species were cell line specific. These studies demonstrate the prevalence of the P2-initiated mRNA and the PLP1-139 alternative splicing pathway in the tumor cell lines studied and suggest that other pathways of PLP gene expression may be regulated in a cell line-dependent manner. The particular form of PLP expressed by a given cell can influence its biological effects.

Parathyroid hormone-related protein induces interleukin 8 production by prostate cancer cells via a novel intracrine mechanism not mediated by its classical nuclear localization sequence.

PTHrP (parathyroid hormone-related protein) overexpression by prostate carcinoma cells has been implicated in tumor progression. Although the biological effects of PTHrP can be mediated by the G-protein-coupled PTH/PTHrP receptor, PTHrP also has intracrine actions mediated by a nuclear localization sequence at residues 87-107. We investigated the effect of PTHrP transfection and treatment on production by prostate carcinoma cells of IL (interleukin)-8, which can regulate prostate cancer growth by angiogenic activity and growth-promoting effects. Six prostate cancer cell lines exhibited constitutive expression of PTHrP and IL-8 that were significantly correlated (r = 0.93; P < 0.01). We transfected wild-type and mutant PTHrP into these cells. Wild-type PTHrP1-173 and PTHrP33-173 lacking the PTH/PTHrP receptor-binding domain induced a 3-fold stimulation of IL-8 production but not production of another angiogenic factor, vascular endothelial growth factor. Transfection of the COOH-terminal truncation mutant PTHrP1-87 induced a 5-fold simulation of IL-8 and a 3-fold increase in IL-8 mRNA. Cells transfected with PTHrP1-87 and 1-173 also showed increased cell proliferation. In contrast, exogenous PTHrP1-34 and 1-86 peptides did not significantly affect IL-8 production; moreover, PTHrP-neutralizing antibodies did not inhibit the production of IL-8 by transfected PTHrP. Additional transfection studies with progressively COOH-terminally truncated PTHrP1-87 defined a 23-amino acid sequence, PTHrP65-87, required for PTHrP1-87 to robustly stimulate IL-8 in prostate cancer cells. Confocal microscopy and immunoassay demonstrated PTHrP1-87 nuclear localization. Our results demonstrate that PTHrP acts to induce IL-8 production in prostate cancer cells via an intracrine pathway independent of its classical nuclear localization sequence. This novel pathway could mediate the effects of PTHrP on the progression of prostate cancer.

Prostate cancer progression, metastasis, and gene expression in transgenic mice.

We previously reported that a transgenic mouse line containing the fetal globin promoter linked to the SV40 T antigen (T Ag) viral oncogene (Ggamma/T-15) resulted in prostate tumors. In this study, we further explored tumor origin, frequency, invasiveness, androgen sensitivity, and gene expression pattern. T Ag was detected in adult but not fetal and neonatal prostates, suggesting a role for androgens in tumor progression. However, castration shortly after prostate morphogenesis did not prevent tumor development, suggesting an androgen-independent phenotype. Tumors originated within ventral or dorsal prostate lobes and involved intraepithelial neoplasia, rapid growth in the pelvic region, and metastasis to lymph nodes and distant sites. In addition, the primary cancers could be propagated in nude mice or nontransgenic mice. Seventy-five percent of hemizygous and 100% of homozygous transgenic males developed prostate tumors, suggesting a T Ag dosage effect. Biochemical characterization of advanced tumors revealed markers of both neuroendocrine and epithelial phenotypes; markers of terminal differentiation are lost early in tumorigenesis. Tumor suppressor genes (p53 and Rb), normally bound to T Ag, were up-regulated; bcl-2 proto-oncogene, which prevents apoptosis, was slightly up-regulated. Myc, a stimulus to cell cycle progression, was unchanged. We propose the Ggamma/T-15 transgenic line as a model of highly aggressive androgen-independent metastatic prostate carcinoma with features similar to end-stage prostate cancer in humans.

Immunohistochemical localization of parathyroid hormone-related protein in human prostate cancer.

Parathyroid hormone-related protein (PTHrP) is produced by a variety of malignant tumors and has been implicated as a major cause of humoral hypercalcemia of malignancy. Expression of PTHrP in prostate cancer tissue was studied immunohistochemically using 33 radical prostatectomy specimens from patients with clinically localized carcinoma of the prostate. None of these patients demonstrated hypercalcemia prior to the surgery. Acetone-methyl benzoate-xylene-processed, paraffin-embedded tissues were stained with a validated mouse monoclonal antibody to an amino acid fragment, PTHrP(109-141), using the streptavidin-peroxidase enzyme conjugate method. All cases (33 of 33; 100%) studied demonstrated some degree of immunoreactivity throughout the cytoplasm of the tumor cells, but immunostaining was absent from inflammatory and stromal cells. The intensity of the staining appeared to directly correlate with increasing tumor grade. The widespread immunohistochemical localization of PTHrP in carcinoma of the prostate suggests that PTHrP may play some local role in the growth of transformed cells in the prostate. Furthermore, overexpression of PTHrP may be a possible marker to evaluate the malignant potential of carcinoma of the prostate.

Parathyroid hormone-related protein in patients with primary breast cancer and eucalcemia.

Parathyroid hormone-related protein (PTHrP) is a causative factor of humoral hypercalcemia in breast cancer and other malignancies. We studied circulating PTHrP levels with three different immunoassays directed against different parts of the PTHrP molecule in 48 patients with breast cancer and eucalcemia. The methods used were: (a) a RIA with antibodies directed toward the midregion (63-78); (b) an immunofluorometric assay with two antibodies against 1-34 and 38-67; and (c) an immunoradiometric assay with antibodies against 1-40 and 1-72. Although most patients had PTHrP levels indistinguishable from normal when measured by all three methods, four patients had increased serum levels in the IFMA. PTHrP was detected by immunohistochemistry in tumors from nearly all patients. One patient with elevated PTHrP in plasma measured by IFMA showed intense staining of tumor by immunohistochemistry; the tumor was histologically graded as III (severe) and was the largest of all tumors in this patient group. The IFMA can identify increased serum PTHrP in some patients with breast cancer who are not hypercalcemic. This assay may be especially useful in screening patients for this tumor during a relative early phase of the disease.

The parathyroid hormone-related protein associated with malignancy is secreted by neuroendocrine tumors.

We have demonstrated the production of the PTH-related protein (PTHrP) associated with hypercalcemia of malignancy by human neuroendocrine cell lines that also produce calcitonin gene products and chromogranin A. PTHrP was demonstrable in the cells by immunocytochemistry and immunoassay and Northern analysis of the cells revealed the presence of multiple mRNAs for PTHrP. The cell lines also secreted PTHrP in a regulated fashion, with the most potent secretagogue being phorbol. Thus, PTHrP is secreted by neuroendocrine cells and it may have neuroectodermal lineage. The coexpression of calcitonin gene products and chromogranin A, also neuroendocrine, with PTHRP may influence its secretion and ultimate biological effects in vivo.

Glucocorticoid regulation of parathyroid hormone-related peptide gene transcription in a human neuroendocrine cell line.

A PTH-related peptide (PTHRP) has been identified and its cDNA cloned from tumors associated with the syndrome of humoral hypercalcemia of malignancy. The PTHRP and PTH genes appear to represent members of a gene family. Whereas the PTH gene is expressed exclusively in the parathyroids, the PTHRP gene appears to be widely expressed, but little is known concerning the regulation of its expression in any site. We studied the regulation of PTHRP gene expression in a human carcinoid cell line (NCI-H727) which has neuroendocrine features and also produces calcitonin, calcitonin gene-related peptide, and chromogranin-A. We found that the synthetic glucocorticoid triamcinolone produced time- and dose-dependent decreases in steady state PTHRP and calcitonin mRNA levels in NCI-H727 cells. This effect was blocked by the competitive glucocorticoid inhibitor RU-486. Messenger RNA stability and transcription run-off experiments revealed that triamcinolone decreased PTHRP and calcitonin expression by repressing the transcription rates of both genes.

Expression of messenger ribonucleic acids encoding a parathyroid hormone-like peptide in normal human and animal tissues with abnormal expression in human parathyroid adenomas.

A novel PTH-like peptide has recently been purified and cloned from human tumors associated with the syndrome of humoral hypercalcemia of malignancy. We surveyed the expression of mRNAs encoding this peptide in normal tissues by Northern analysis. One or more low abundance hybridizing transcripts was identified in poly(A)+ RNA prepared from human keratinocytes, thyroid, bone marrow, and fibroblasts, from bovine hypothalamus, pituitary, parathyroid, adrenal cortex, and adrenal medulla, and from rat brain, stomach mucosa, and fetal but not adult liver. One or more hybridizing transcripts was also identified in poly(A)+ RNA prepared from a number of established lines, including rat pituitary (GH4), rat pheochromocytoma (PC 12), human osteosarcoma (TE-85), and human medullary carcinoma (TT) cells. Northern analysis of mRNAs from abnormal human parathyroid tissue revealed an overexpression of transcripts for the PTH-like peptide which appeared to be specific for adenomatous or autonomous glands. These findings suggest that the PTH-like peptide is expressed in a number of endocrine and nonendocrine tissues, that it is developmentally expressed in at least one tissue (fetal liver), and that the regulation of its expression is abnormal in human parathyroid adenomas.

Vitamin D3 compounds regulate human immunodeficiency virus type 1 replication in U937 monoblastoid cells and in monocyte-derived macrophages.

We studied the effects of vitamin D3 compounds on the replication of human immunodeficiency virus type 1 (HIV-1) in the monoblastoid cell line U937 and in primary monocyte-derived macrophage cultures to understand how modulators of monocyte/macrophage effector function might affect the pathogenesis of HIV-1 infection. U937 cell cultures exposed to 1, alpha 25-dihydroxyvitamin D3 prior to HIV-1 infection showed enhanced virus replication that was apparently due to increased cellular resistance to viral cytopathic effects; a marked inhibition of virus replication was noted in cells exposed to 1 alpha,25-dihydroxyvitamin D3 subsequent to infection. Exposure of blood-derived monocyte/macrophages to vitamin D3 compounds prior to infection also affected virus growth; in most cases, substantial inhibition of HIV-1 replication was noted in vitamin D3-treated macrophage cultures. Our results demonstrate that vitamin D3 compounds with recognized abilities to induce cellular differentiation can modulate HIV-1 infection of human macrophages.