Humoral hypercalcaemia of "benignancy".
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Adolescent↗
Biomedical subjects
D W Burton
Publications and source records attributed to D W Burton.
Expression of parathyroid hormone-related peptide and its receptor messenger ribonucleic acid in human amnion and chorion-decidua: implications for secretion and function.
OBJECTIVE: Our purpose was to define the location and packaging of parathyroid hormone-related peptide in amnion-chorion and the potential target tissues for its action in fetal membranes. STUDY DESIGN: We studied fetal membranes by use of light microscopic immunocytochemistry with three monoclonal antibodies against distinct regions of the parathyroid hormone-related peptide molecule. For electron microscopy immunogold analysis with a monoclonal antibody specific to the 109-141 fragment was used to observe parathyroid hormone-related peptide intracellularly in amnion membrane and in the chorion layers. Multiplex reverse transcriptase-polymerase chain reaction with Southern blotting was used to identify parathyroid hormone/parathyroid hormone-related peptide receptor and control messenger ribonucleic acids in amnion and chorion-decidua. RESULTS: All monoclonal antibodies revealed immunoreactive parathyroid hormone-related peptide in the amniotic epithelial cells and in some fibroblast-like cells embedded in the extracellular matrix of the amnion. Parathyroid hormone-related peptide was also found in the chorion in fibroblast and trophoblast layers and in decidua. Ultrastructurally immunogold particles were evenly distributed throughout the amniotic epithelial cells and were present in apical microvilli and near the basal membranes. Electron microscopy studies of the chorion cytotrophoblast also showed freely dispersed immunogold particles of parathyroid hormone-related peptide with no packaging in secretory granules. Low to undetectable levels of parathyroid hormone/parathyroid hormone-related peptide receptor messenger ribonucleic acid were found in amnion tissue, whereas abundant receptor messenger ribonucleic acid was found in chorion-decidua. CONCLUSIONS: These results suggest the presence of a parathyroid hormone-related peptide paracrine system within the human fetal membranes.
Amnion↗
Identification and characterization of a neuroendocrine-specific 5'-regulatory region of the human chromogranin A gene.
Chromogranin A (CgA) expression is specific to cells of endocrine and neuroendocrine (NE) tissues. Our transfection studies with CgA have identified two DNA regions 5' of the transcription start site that regulate CgA gene transcription: a distal regulatory region (DRR) located between -726 and -455, and a proximal regulatory region (PRR) between -60 and -26. In studies of the DRR using four human NE and six human non-NE cell lines, we demonstrated enhanced transcription of DRR-containing CgA-GH plasmids by the NE cells as a group compared to the non-NE cells. DNase I footprinting identified a protected area in the DRR from -570 to -555 base pairs (bp) composed of the sequence TAATGATGACTAAACA. Centered in this sequence is the simian virus 40 version of the activator protein-1-binding site, TGACTAA. Electrophoretic mobility shift assays (EMSAs) with an oligonucleotide containing the 27 bp of the DRR between -576 and -550, which we refer to as the distal regulatory element (DRE), produced a specific complex with the NE BEN and non-NE COS-1 cell nuclear extracts. The addition of c-Jun and c-Fos antibodies produced strong supershifts of the complex generated by COS-1 extract, but very weak supershifts of the complex formed by BEN extract. These EMSA studies suggest that NE cells such as BEN contain unique nuclear factors distinguishable from activator protein-1 that interact with the DRE. The enhancer effect of the 271-bp DRR could be replaced by the 27-bp DRE in both CgA and calcitonin promoter constructs in BEN cells. Replacement of the DRR with the DRE resulted in a further increase in expression from these plasmids, suggesting the presence of suppressor sequences in the DRR. In transfection studies of the PRR, deletion of its cAMP response element (CRE) dramatically lowered transcription. In addition to demonstrating that its CRE can bind CRE-binding protein, EMSAs with the PRR demonstrated that an intervening sequence between the CRE and the TATA box formed a complex with BEN cell nuclear extract. Our studies demonstrate that both the PRR and DRR are important for high level transcription of the CgA gene in NE cells. The presence of both distal and proximal 5'-regulatory regions in the human CgA gene indicates a complex mechanism of transcriptional regulation. Although the PRR is important for the formation of a functional transcription complex at the TATA region, the DRR is important for the enhancement of CgA gene expression in NE cells.
Base Sequence↗
Parathyroid hormone-related protein expression in human gastric adenocarcinomas not associated with hypercalcemia.
OBJECTIVE: Our evaluation of a patient with a parathyroid hormone-related protein (PTHrP)-producing gastric adenocarcinoma and hypercalcemia prompted us to study the expression of PTHrP in 13 additional patients with gastric cancer and in 10 control cases. Our objective was to investigate by immunohistology the expression of PTHrP in gastric cancer. METHODS: Immunohistology studies were conducted with two murine monoclonal antibodies to synthetic peptides of human PTHrP, 9H7, and 8B12. The 9H7 antibody was raised to the carboxy-terminal amino acid fragment (109-141) of PTHrP, and the 8B12 antibody was raised to the amino-terminal amino acid fragment (1-34) of PTHrP. RESULTS: Paraffin-embedded tumor specimens from 13 of 14 cases of gastric adenocarcinoma stained positively with the antibody to the carboxy terminus of the PTHrP peptide, but none stained positively with the antibody directed against the amino terminus. None of 10 control cases stained positively with either antibody. The staining was predominately evident in the cytoplasm of the tumor cells. Except for the sentinel case, none of the other patients with gastric adenocarcinoma had hypercalcemia. Thus, gastric adenocarcinoma seems to rarely result in systemic hypercalcemia. CONCLUSIONS: Our studies demonstrated that abnormal PTHrP production can occur in malignant cells without producing hypercalcemia. PTHrP may play a role in the pathogenesis of gastric adenocarcinoma that is independent of its hypercalcemic effects. PTHrP measurements may be clinically valuable in patients with cancer who are not hypercalcemic.
Adenocarcinoma↗
Alveolar epithelial cells express and secrete parathyroid hormone-related protein.
Parathyroid hormone-related protein (PTHrP) was discovered as a hypercalcemia-inducing product of malignant cells and has since been demonstrated to be a product of many tissues. Although it is robustly expressed in fetal lung, PTHrP expression has not been assigned to alveolar epithelial cells in adult lung. We have shown that PTHrP is expressed in the adult rat lung and by cultured rat alveolar type II epithelial cells with sensitive and specific immunoassays and immunohistochemical techniques. Immunoassay of cell extracts demonstrated that freshly isolated type II cells contained PTHrP (136 pg/10(7) cells), whereas freshly isolated alveolar macrophages and cultured macrophages did not express PTHrP. Cultured type II cells secreted PTHrP into medium, 202 +/- 11 fg PTHrP/micrograms cell protein in 24 h. Basal secretion remained stable up to 7 days in culture. Treatment with phorbol myristate acetate or 1-oleoyl-2-acetyl-sn-glycerol produced a dose-related, 2- to 4-fold increase in PTHrP secretion. However, forskolin, ionomycin, ATP, phenylephrine, capsaicin, and bradykinin had no effect. Thus, PTHrP secretion appeared to be regulated by a protein kinase C-dependent pathway. PTHrP could also be demonstrated in pulmonary lavage fluid. Although the function of PTHrP in the adult lung is unknown, it could involve control of cell growth and differentiation or control of surfactant lipid secretion. Further studies are necessary to elucidate the function of PTHrP in the lung.
Animals↗
Parathyroid hormone-related protein in the cardiovascular system.
PTH-related protein (PTHrP) is expressed in a stretch-responsive manner in several types of smooth muscle. We previously demonstrated the production of PTHrP in adult rat heart muscle. In this study, we demonstrate the production of PTHrP in the cardiovascular systems of several mammalian species, including human. We demonstrate PTHrP by immunohistology, using a panel of murine monoclonal antibodies to PTHrP epitopes that span the entire length of the human PTHrP amino acid sequence, quantitate the concentration of PTHrP in the rat cardiovascular system by region-specific RIAs, and measure the relative levels of PTHrP messenger RNA (mRNA) by competitive polymerase chain reaction. Immunohistology studies demonstrated the presence of PTHrP in the cardiovascular systems of humans, rats, pigs, and rabbits. The most robust expression was found in atria, followed by the large vessels, then ventricles. No difference was seen between the left and right sides of the heart. Double staining procedures revealed that PTHrP and atrial naturietic peptide were coexpressed in some cells. Using RIAs and polymerase chain reaction, we demonstrated that atria contained a higher concentration of PTHrP than ventricles and that the relative PTHrP concentrations correlated to its mRNA concentrations in these two tissues. The concentrations of PTHrP in the smooth muscle surrounding the aorta and vena cava were comparable to those in atria. However, in these large vessels, the higher PTHrP levels did not correspond to its mRNA levels. Whereas the immunoreactive concentrations of PTHrP were similar in the atria, aorta, and vena cava, the mRNA levels in the aorta and vena cava were 3-fold lower than those in the atria. Certain PTHrP epitopes appeared to be differentially expressed in specific cardiovascular tissues. A comparison of region-specific assays showed that immunoreactivity measured by immunoassays to PTHrP-(38-64) and PTHrP-(109-141) were 3- to 5-fold greater than that determined by an immunoassay to PTHrP-(1-34). Our observations demonstrate that the atria, aorta, and vena cava contain the greatest amounts of PTHrP in the cardiovascular system. The discrepancy between the concentrations of PTHrP and its mRNA present in the aorta and vena cava suggest that the two may be regulated differently in these tissues. The widespread distribution of PTHrP suggests an important function for the protein in the cardiovascular system, possibly functioning as the calcium counterpart for the atrial natriuretic-sodium regulatory axis.
Animals↗
Chromogranin A peptide-specific antisera and high-performance size exclusion chromatography demonstrate amino-terminal and carboxy-terminal fragments of the native molecule in human cell lines.
We have studied the pattern of amino-terminal and carboxy-terminal protein processing of chromogranin A (CgA) in five human cell lines, four derived from lung cancers: NCI-H478, NCI-H1011, NCI-H727, and BEN; and one derived from human medullary thyroid carcinoma: TT. This was accomplished by fractionation of cell extracts by high-performance size exclusion chromatography (HPSEC) and measurement of CgA in the fractions by radioimmunoassay (RIA). Three RIA's were used, one specific for the amino-terminus of CgA, one specific for the carboxy-terminal region, and a monoclonal antibody-based assay that recognizes only the native CgA molecule. We demonstrated the presence of different amino- and carboxy-terminal immunoreactive species of CgA in the different cell lines. The amino-terminal assay demonstrated distinct low-molecular-size species in the NCI-H478 and NCI-H1011 cell lines, and a similar peak in the TT cells. The amino-terminal assay did not recognize any distinct species in BEN and NCI-H727 cell lines. The carboxy-regional assay demonstrated distinct low-molecular-size species in the NCI-H478 and NCI-H101 cell lines and high-molecular-size species in the NCI-H727 and BEN cells. Our studies demonstrate with region-specific RIA's the presence of both amino- and carboxy- forms of CgA in human cells that secrete this protein. These results provide direct evidence that CgA-producing cells produce, probably through endoproteolytic processing of the native molecule, amino- and carboxy-terminal forms of the protein.
Amino Acid Sequence↗
Neoplastic hormone-producing cells of the placenta produce and secrete parathyroid hormone-related protein. Studies by immunohistology, immunoassay, and polymerase chain reaction.
BACKGROUND: Parathyroid hormone-related protein (PTHrP) was originally discovered as a product of tumors associated with hypercalcemia. Subsequent studies have demonstrated the widespread production of PTHrP in both fetal and adult tissues, among them the placenta, where it has been identified in amniotic epithelial cells and trophoblasts. However, studies of PTHrP production by the neoplastic placenta have not been reported. EXPERIMENTAL DESIGN: We have studied placental cells and tissues for PTHrP by immunohistology, immunoassay, and the polymerase chain reaction. We studied normal placenta, placenta neoplasia, and placental cell lines grown in culture. RESULTS: Our studies in abnormal placenta demonstrate that PTHrP is produced by chorioangioma, choriocarcinomas, and hydatidiform moles, indicating that PTHrP may be a marker for neoplasia of the placenta. Our studies in normal placenta demonstrate that PTHrP is produced by several placental cell types, including, syncytiotrophoblast, intermediate trophoblasts, cytotrophoblasts, and amniotic epithelium, suggesting an important role for this protein in several aspects of placental function. Cell lines derived from trophoblasts and amniotic epithelium also produced PTHrP and secreted it into culture medium. Polymerase chain reaction with PTHrP coding sequence primers demonstrated the appropriate polymerase chain reaction product in these placental cells. CONCLUSIONS: The production of PTHrP by several normal and abnormal placental cell types is evidence for an important function for PTHrP in normal and neoplastic placenta.
Cell Line↗
Associations and dissociations between serum bone Gla protein and alkaline phosphatase in skeletal metabolism.
Serum bone Gla protein (BGP) and alkaline phosphatase (AP) activity were compared for the assessment of skeletal status in 11 normal pregnant women, 12 normal women on days 3 and 13 of the menstrual cycle, five postmenopausal women before and after 1 month of treatment with ethinyl estradiol (20 micrograms/day), five patients with cancer and hypercalcemia during treatment with calcitonin, and one patient with Paget disease during treatment with Plicamycin. BGP and AP correlated with each other only in the pregnant women. In all other circumstances, there was no correlation between these two serum osteoblast products. Furthermore, there were conditions in which the two measurements became discordant. These studies demonstrate that BGP and AP commonly are dissociated when used as measurements of skeletal status. Although both are osteoblast products, BGP and AP probably reflect different aspects of osteoblast differentiation and function.
Adult↗
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.
Animals↗
The parathyroid hormone-related protein (PTHrP) gene preferentially utilizes a GC-rich promoter and the PTHrP 1-139 coding pathway in normal human amnion.
Parathyroid hormone-related protein (PTHrP) is an oncofetal protein that is widely expressed in mammalian tissues. The PTHrP gene is a complex with three transcriptional start-sites, two TATA boxes and a GC-rich region, and three predicted polypeptide products, PTHrP 1-141, PTHrP 1-139, and PTHrP 1-173. The originally discovered form of PTHrP, PTHrP 1-141, and the classical TATA box promoters are generally assumed to be the major pathways of PTHrP gene expression. We have used the polymerase chain reaction (PCR) to study PTHrP gene expression in the human amnion. Our studies demonstrate that the GC-rich promoter is preferentially used and that PTHrP 1-139 is the major PTHrP mRNA expressed in human amnion. PTHrP 1-139 lacks the carboxy-terminal arginine and histidine residues of PTHrP 1-141; these two basic amino acids could have significant effects on the biological activity of PTHrP. These preferential pathways for PTHrP gene expression are shared by malignant and normal human tissues.
Amnion↗
Flow-cytometric analysis of the effects of triethylenemelamine on somatic and testicular tissues of the rat.
The effects of short-term (24 h) exposure to triethylenemelamine on cellular DNA in five tissues (bone marrow, spleen, kidney, large intestine, and testis) of the rat were studied using flow cytometry. Mean coefficients of variation of the G1 peaks were increased in both the low and high dosage groups relative to controls. Bone marrow exhibited the highest degree of effect, possibly due to the rapid rate of cell division in that tissue, and spleen was next highest. Thus, hematopoietic tissues are highly responsive to short-term, acute exposure to this mutagen. The results of the flow-cytometry assay closely paralleled a simultaneous chromosomal assay conducted on bone marrow of the same rats. These data are interpreted to be consistent with the hypothesis that the observed increase in mean coefficients of variation is due to the clastogenic effects of the mutagen and subsequent unequal distribution of DNA among the daughters of affected cells.
Animals↗
Two-site assays of bone gla protein (osteocalcin) demonstrate immunochemical heterogeneity of the intact molecule.
We developed a panel of monoclonal antibodies to human bone gla protein (BGP; osteocalcin) peptides that span the linear sequence of the molecule, specifically BGP 1-12 (N-terminal), BGP 15-30 (midregion), and BGP 38-49 (C-terminal). These antibodies were evaluated in various combinations of two-site formats in studies of serum BGP concentrations. For clinical studies, we selected from a panel of antibodies the two most sensitive antibody pairs for the intact molecule (N-C); we also used a polyclonal RIA based on BGP-C. For the two-site format, we used two N-terminal antibodies, 029 and 052, adsorbed to polystyrene beads, and radioiodinated a C-terminal antibody, 663. The standard for each of the assays was purified human BGP. The following BGP serum concentrations (microgram/L, mean +/- SE) were measured with the various assays: by the 029-663 assay, results for normal subjects were 7 +/- 3, for patients with renal failure 25 +/- 8, and for patients with Paget disease 12 +/-4; by the 052-663 assay, the respective results were 22 +/- 4, 44 +/- 12, and 31 +/- 7; by the polyclonal assay, the results were 3 +/- 0.2, 13 +/- 2, and 5 +/- 1. The two intact (N-C) assays were significantly (P < 0.01) correlated (r = 0.94), but their serum values differed by more than twofold in terms of the same BGP standard. The polyclonal assay significantly correlated with each of the intact assays (r = 0.83, 0.77), but it, too, gave different serum values for BGP. These studies demonstrate the immunochemical heterogeneity of circulating BGP, heterogeneity that is manifest even in immunoassays specific for the same region of the molecule.
Antibodies, Monoclonal↗
All major lung cancer cell types produce parathyroid hormone-like protein: heterogeneity assessed by high performance liquid chromatography.
We examined the production of parathyroid hormone-like protein (PLP) by all major lung cancer cell types, including epidermoid, small cell, and adenocarcinoma. We demonstrated that cell lines derived from each cell type released PLP into culture medium. To determine the predominant species of PLP produced by the cell lines, guanidine hydrochloride extracts were prepared and analyzed for PLP by high performance size exclusion chromatography under denaturing conditions. Cell lines from each of the major lung tumors produced multiple and different forms of PLP. The estimated molecular sizes of the PLP species ranged from 3.5-11.7 kilodaltons. Our results demonstrate that PLP is produced by all major lung cancer cell types and extend these observations to adenocarcinoma and large cell carcinomas of the lung. Furthermore, we observed PLP heterogeneity among these different cell types, which may represent tissue-specific PLP processing among different human lung tumors.
Chromatography, High Pressure Liquid↗
Distinct patterns of chromogranin A-related species can be demonstrated in endocrine cells.
We have studied the pattern of chromogranin A (CgA)-related species in different human endocrine cells that produce CgA and also express the calcitonin gene. Antibodies against CgA peptides that span its linear sequence were used in Western analysis of cell lines derived from medullary thyroid carcinoma (MTC), small cell lung cancers (SCLC), epidermoid cell lung cancer (ECLC) and a pulmonary carcinoid tumor (CRND). Each of the cell lines demonstrated a distinct pattern of CgA-related species. Gel filtration studies also revealed multiple and different forms of immunoreactive CgA in the cell lines. Although proteolysis may contribute to our results, these observations suggest that native CgA is processed to smaller species in a tissue-specific pattern by different endocrine cells. More conclusive studies, however, are necessary to establish that cell processing leads to the specific CgA moieties that we have observed.
Blotting, Western↗
A high-fat diet increases calcitonin secretion in the rat.
We have studied the effects of diet-induced obesity on thyroidal calcitonin, plasma calcitonin, calcium and phosphorus in rats. Twelve 9-week-old female rats were randomly divided into two groups. One group was fed a low-fat diet while the other was fed a high-fat diet. Both diets had 0.76% Ca, 0.56% P and 2.2 U/g vitamin D; however, the high-fat diet had hydrogenated vegetable oil added at 405 g/kg. All rats were pair-fed and consumed 11 g/day per rat for 27 weeks at which time the rats were fasted overnight and exsanguinated. The rats on the high-fat diet weighted 406 +/- 21 g (mean +/- SEM) versus 292 +/- 13 g for controls and had higher levels of serum calcitonin (104 +/- 12 versus 57 +/- 9 pg/ml). The obese rats also had increased thyroidal calcitonin by radioimmunoassay and increased thyroidal C-cells by immunohistology. The increased calcitonin levels occurred without a concomitant increase in calcium levels. These data indicate that a high-fat diet in rats stimulates C-cell growth and calcitonin secretion.
Animals↗
Demonstration of Chromogranin A in human neuroendocrine cell lines by immunohistology and immunoassay.
We have used immunohistology and radioimmunoassay procedures to study Chromogranin A (CgA) in human neuroendocrine tumor cell lines, especially small cell lung cancers (SCLC). By immunohistology, CgA could be detected in 11 of 18 classical SCLC cell lines, in a medullary thyroid carcinoma (MTC) cell line, and in only one of 13 variant- or non-SCLC cell lines. By radioimmunoassay, CgA could be detected in the cells and culture media of all of the classical SCLC cell lines tested. Many of the classical SCLC cell lines also produced calcitonin (CT). These studies demonstrate that CgA production is a common feature of SCLC cell lines, especially those with neuroendocrine characteristics.
Calcitonin↗
The effects of forskolin and calcium ionophore A23187 on secretion and cytoplasmic RNA levels of Chromogranin-A and calcitonin.
We have studied the regulation of the secretion and cytoplasmic RNA levels of calcitonin (CT) and Chromogranin-A (CgA) to determine if the biosynthesis and secretion of these two substances are controlled in a coordinated fashion. The studies were conducted in two cell lines, a medullary thyroid carcinoma (MTC) cell line and a lung tumor (BEN) cell line. Both cell types secrete CT and CgA. Forskolin treatment resulted in a significant increase in the secretion of CT and CgA in each cell line and in CT-specific cytoplasmic RNA in the MTC cell line. Treatment with calcium ionophore A23187 resulted in significantly increased secretion of both substances in the lung tumor cells but not in the medullary thyroid carcinoma cells. A significant increase in CT-specific or CgA-specific cytoplasmic RNA was not seen in either cell line. We conclude that the secretion of CT and CgA are regulated in a coordinated fashion in these cell lines through processes that are calcium-mediated and processes that involve cyclic AMP-dependent protein kinase A. However, each of these regulatory pathways is not always operative in a given tissue. The coordinate regulation of the secretion of CT and CgA supports the hypothesis that CgA participates in the secretory process of its associated hormones.
Calcimycin↗