Polymerase chain reaction analysis of parathyroid hormone-related protein gene expression in breast cancer patients and occurrence of bone metastases.

Parathyroid hormone-related protein (PTHrP) is associated with the syndrome of humoral hypercalcemia of malignancy. A high incidence of positive staining for PTHrP is observed in breast cancer and positivity is more frequent in patients who develop bone metastases. We assessed the presence of PTHrP mRNA by using the polymerase chain reaction in 38 normocalcemic breast cancer patients with long-term follow-up (minimum, 5 years) selected for the presence or absence of later bone metastasis development. In all the patients except one, the PTHrP gene was expressed in the breast tumor. The level of amplified PTHrP complementary DNA was inversely related to age (P < 0.02) and positively related to the proportion of invaded nodes (P < 0.02) but was not related to the other usual prognostic factors. The level of PTHrP mRNA was not different between the group of patients without recurrence or metastases (n = 11) and the group of patients who later developed metastases in soft tissues (n = 10). By contrast, patients who subsequently developed bone metastases (n = 17) showed higher PTHrP gene expression than patients in the other two groups (P < 0.001). This study suggests that strong PTHrP gene expression in breast tumors is associated with the onset of bone metastases.

Identification and measurement of calcitonin precursors in serum of patients with malignant diseases.

Previous studies have suggested that molecular species larger than the mature calcitonin (CT) are produced by tumors of different origin. In order to study these species, we developed a monoclonal immunoradiometric assay for calcitonin precursors (CT-pr). This assay was based on both monoclonal antibody KC01 directed to the 1-11 region of katacalcin and monoclonal antibody CT08 directed to the 11-17 portion of CT. The sensitivity of this monoclonal immunoradiometric assay for CT-pr was less than 100 pg/ml. Only one of 131 healthy subjects had CT-pr serum levels greater than 100 pg/ml; this value was therefore selected as the standard serum value in healthy individuals. CT-pr was present in the serum of seven of ten patients with advanced renal failure and in that of 21 of 52 patients (40%) with benign liver disease but was undetectable in sera of patients with other benign diseases. The serum CT-pr level was correlated with that of mature CT in patients with medullary carcinoma of the thyroid. In contrast, the serum CT-pr level was frequently elevated in the absence of a detectable CT level in patients with various malignant tumors and, particularly, in those with either tumors of the neuroendocrine system (60%) or hepatocellular carcinomas (62%). CT-pr was detected in tumor extract from a patient with a hepatocellular carcinoma. Moreover, hybridization experiments with total RNA extracted from this tumor demonstrated the presence of RNAs hybridizing with complementary DNA encoding for common region, calcitonin, and katacalcin sequences. These results show that CT precursors are excreted by numerous cancers and might well be useful biological markers for the follow-up of productive tumors.

c-fos protooncogene is involved in the mitogenic effect of transforming growth factor-beta in osteoblastic cells.

We investigated the contribution of c-fos protooncogene in the mitogenic effect of transforming growth factor-beta (TGF beta) in serum-deprived, confluent rat calvaria osteoblastic cells. The TGF beta-induced growth in these cells was associated with an immediate and transient c-fos mRNA accumulation, similar to the inductive effect of fetal calf serum. To assess the role of c-fos in the response to TGF beta, we used a c-fos antisense (AS) oligonucleotide displaying duplex formation with rat c-fos mRNA. Studies of AS and sense (S) uptake by osteoblastic cells demonstrated that incorporation of labeled oligomers was maximal at 2 h, and the incorporated AS oligonucleotide remained intact for 24 h. Immunofluorescence analysis of c-Fos-labeled cells demonstrated that AS, but not S, oligonucleotide reduced c-Fos protein expression, suggesting specific efficient inhibition of c-fos translation by the AS oligomer. Proliferation assays showed that cell growth induced by fetal calf serum was inhibited by the AS, but not by the S oligonucleotide, in both normal rat osteoblasts and ROS 17/2.8 osteosarcoma cells, demonstrating efficient and specific blockage of cell growth by the AS oligomer. The mitogenic effect of TGF-beta was abolished in cells cultured in the presence of AS, whereas S had no effect, showing that c-fos is required for TGF beta-induced osteoblast cell growth. The results show that the induction of c-fos is implicated in the mitogenic effect of TGF beta in osteoblastic cells and provide a cellular mechanism involved in the response of these cells to TGF beta.

Effects of prostaglandins on human hematopoietic osteoclast precursors.

The effect of prostaglandin E2 (PGE2) on osteoclast (OC) differentiation is unclear, either stimulator or inhibitor, depending on the in vitro system used. This probably reflects indirect mechanisms through intermediate cells. We have investigated the direct effect of PGE2 on human OC differentiation from cord blood monocytes (CBMs) in the absence of stromal cells. Macrophages and multinucleated cells (MNCs) resembling OCs form in cultures of CBMs stimulated by 1,25-dihydroxyvitamin D3. In the present study, CBMs were cultured for 3 weeks, as previously described, in the presence or absence of PGE2. The number of MNCs was significantly reduced in the presence of PGE2 as was the proliferation of cultured CBMs, assessed on day 7. Immunohistochemistry was performed to evaluate macrophage markers (CD11b and CD14) and OC marker (beta3-chain). PGE2 significantly increased the numbers of CD11b-positive and CD14-positive cells, whereas the number of beta3-chain-positive cells was significantly decreased. beta3-Chain, c-fos, and human calcitonin receptor (h-CTR) messenger RNA (mRNA) expressions were evaluated by reverse transcription-PCR with RNA extracted from cultured CBMs. In the presence of PGE2, expression of beta3-chain and c-fos mRNA was reduced from the first week of culture. h-CTR mRNA expression was also reduced, and only the h-CTR1 isoform was detected in the presence of PGE2. In addition, when PGE2 was added only during the last week of culture, when no CBM proliferation occurred, the number of CD11b- and beta3-positive cells was unchanged compared to that in the control culture, as were the proportion of MNCs, the fusion index, and the expression of c-fos mRNA. In conclusion, our results suggest that PGE2 has an inhibitory effect on human OC differentiation from CBMs, possibly by reducing precursor proliferation in these cultures. We also hypothesize that PGE2 may reduce OC differentiation by increasing the proportion of precursor cells that differentiate into macrophages. In addition, this may be the result of inhibition of the c-fos expression in CBMs.

Human umbilical cord blood monocytes express calcitonin receptors in culture in the presence of 1,25 dihydroxyvitamin D.

Calcitonin (CT) is a potent inhibitor of bone resorption and there are abundant CT receptors on mature osteoclasts. The relationship between osteoclast precursors and monocyte lineage is far from clear. We recently showed that human cord monocytes in culture, by contrast to adult monocytes, develop some features of osteoclast precursors. We therefore assessed the presence of CT receptors on monocytes. We could not demonstrate any CT receptors on adult monocytes. By contrast, we observed in cultured cord monocytes increased cAMP production in presence of CT. This cAMP response was observed after a 2-week culture and only in the presence of 10(-9) M 1,25-dihydroxyvitamin D. After a 3-week culture, CT 10(-9) to 10(-6) M increased cAMP production dose dependently from 10(-9) M; however the curve was shallower than the one observed in a control CT receptor positive tumoral cell line, MCF7. 125I sCT bound specifically to cord monocytes cultured during 3 weeks; apparent dissociation constant (Kd) was 3.3 +/- 2.2 10(-10) M and average receptor number was 5.1 +/- 0.4 10(4)/cell. On autoradiography all the cells, whether mono or multinucleated, were labeled with 125sCT. One or 2-h exposure to salmon CT did not induce cell contraction. In conclusion, CT receptors can be induced on newborn cord monocytes in the presence of 1,25-dihydroxyvitamin D. This observation shows that osteoclasts and fetal monocytes share a common membrane determinant.

Production of salmon calcitonin I in Oncorhynchus gorbuscha by alternative polyadenylation of two RNA species.

RNAs of ultimobranchial bodies (U.B.) from the pink salmon, Oncorhynchus gorbuscha, were studied using the polymerase chain reaction (PCR) with specific oligodeoxyribonucleotides (oligos) of the salmon calcitonin (sCT) mRNA selected in exon 2 or 3 and a poly(T) oligo. We observed two amplified DNA fragments, differing by 200 bp which hybridized with a specific exon 4 probe. Sequence analysis indicated that they both encoded exon 4, but differed in the length of their 3' non-coding regions by use of a putative polyadenylation signal situated 200 bp upstream from the established polyadenylation site. These two polyadenylation signals very likely were regulated differently, as the larger expressed transcript was predominant. To date, such use of an alternative polyadenylation signal in a CT mRNA has not been described in other vertebrates, and only the chicken CT mRNA possesses a second classical polyadenylation signal which is not known to be used. This characteristic of sCT biosynthesis appears to be typical in lower vertebrates and is of phylogenic interest. Moreover, it engenders a hypothesis of a relationship between the high concentration of the peptide observed in females of this species and their capacity to produce sCT by different biosynthetic pathways.

Molecular characterization of two novel isoforms of the human calcitonin receptor.

Calcitonin inhibits bone resorption by acting on osteoclasts via a specific receptor. The calcitonin receptor (CTR) is also found in many other normal and malignant tissues and cell lines. It has been cloned and sequenced in several species including humans. It belongs to a subclass of seven-transmembrane G protein-coupled receptors. Four human CTR (H-CTR) isoforms generated by alternatively spliced mRNA have previously been described. Two H-CTR encoding DNAs containing an unidentified 50-bp insert are now reported from T47D cells. The 50-bp insert corresponds to a DNA region located between exon 9 and exon 10, and appears to originate from an alternative splicing process. The two H-CTR cDNAs encode 274 and 290 aa long isoforms. Both are deleted from the putative fourth transmembrane domain to C-tail. They differ by the presence (H-CTR5) or absence (H-CTR6) of a previously known 16-aa insert in the putative first intracellular loop. Cell- and tissue-distribution analysis using RT-PCR demonstrates that the shorter one, HCTR6, is more prevalent. The mRNA of both isoforms was detected in giant cell tumor, whereas only H-CTR6 mRNA was detected in TT cells and kidney tissue. Neither H-CTR5 nor H-CTR6 could be detected in peripheral blood mononuclear cells cultured in the presence of RANKL, in MCF7 cells, and in cortical brain and ovarian tissues. When H-CTR6 was transiently expressed in HEK293 cells, CT failed to induce production of cAMP or to bind to the receptor. These suggest either an intrinsic loss of ligand binding function, or an altered intracellular trafficking. Our findings therefore indicate the existence of two novel splice variants of the H-CTR and confirm that multiple splicing patterns could be involved in the post-transcriptional regulation of the gene.

Cloning and sequencing of a new 15-hydroxyprostaglandin dehydrogenase related mRNA.

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH) inactivates prostaglandins. We recently reported an mRNA sequence coding for a predicted isomer (PGDH(rI)) of this enzyme. The TT cell line, derived from medullary thyroid carcinoma (MTC), expresses mRNAs for both isomers. We report here the expression by TT cells and MTC of a third 15-PGDH related mRNA (PGDH(rII)), 241 nt shorter than type-I 15-PGDH. RNase protection assays confirmed that TT cells expressed this mRNA (PGDH(rII)). Thus different splicing patterns could be involved in the post-transcriptional regulation of type-I 15-PGDH gene in MTC.

Sequence of a novel mRNA coding for a C-terminal-truncated form of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

We amplified, using the polymerase chain reaction (PCR) and NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (type-I 15-PGDH)-specific primers, RNA extracted from the HL-60 cell line. Two bands, differing in size by approx. 160 bp, were detected with ethidium bromide staining after electrophoresis of amplification products and hybridization with a 15-PGDH-specific probe. Sequencing these DNA bands revealed that the largest corresponded to the 15-PGDH cloned from human placenta [Ensor et al., J. Biol. Chem. 265 (1990) 14888-14891]. The smaller sequence coded for a predicted C-terminal-truncated form of 15-PGDH. This subtype of the type-I 15-PGDH mRNA was also found using RT-PCR in human liver, placenta and a cell line derived from a human medullary thyroid carcinoma (TT cells). Hybridization studies using specific probes indicated that this new mRNA form probably corresponded to the 3.4-kb mRNA, one of the two 15-PGDH mRNAs previously detected in Northern blot analysis.

Calcitonin mRNA activity in young obese (fa/fa) Zucker rats.

Alterations in both calcitonin (CT) secretion and plasma calcium were recently described in adult obese Zucker rats. We have investigated the CT biosynthetic activity of thyroid glands in 30-day-old obese Zucker rats (fa/fa), and their controls (Lean). Plasma calcium level was significantly increased (+0.6 mg/dl) in obese animals, but plasma phosphate was unchanged. Plasma CT levels measured by radioimmunoassay (RIA) were significantly decreased in fatty (0.50 +/- 0.03 vs 0.68 +/- 0.03 ng/ml in Leans; P less than 0.001), but thyroidal hormone content was not different between Lean and fatty rats (68.7 +/- 5.1 in Leans vs 60.5 +/- 3.6 ng/gland in fatty rats). mRNA was extracted from 10 thyroids, and translated in a rabbit reticulocyte lysate (NEN) in the presence of [35S]methionine. After polyacrylamide gel electrophoresis, specific immunoprecipitates were autoradiographed and quantified by integration. A 50% decrease in translatable CT mRNA was observed in fatty rats. In basal conditions, the biosynthetic activity of C cells in obese rats correlates with the secretion rate of the hormone in the face of unchanged thyroidal CT contents.

CGRP is expressed in primary cultures of human hepatocytes and in normal liver.

We recently reported that human liver and primary cultures of hepatocytes express calcitonin. We therefore studied the expression of calcitonin gene related peptide (CGRP), the alternative splicing product of the calcitonin gene, in hepatocytes and liver. We used polymerase chain reaction amplification with specific primers to detect the presence of CGRP I and II messengers and a specific radioimmunoassay to measure the peptide. We report here that CGRP is synthesized by primary cultures of hepatocytes and in liver. As liver also possesses specific receptors for CGRP in non-parenchymal cells, a paracrine system could be involved in liver metabolism.

Structure of chicken calcitonin predicted by partial nucleotide sequence of its precursor.

DNA complementary to chicken ultimobranchial gland mRNA was cloned into the Pst I site of plasmid vector pBR322. A plasmid was selected by DNA-mRNA hybridization. We report here the partial nucleotide sequence of chicken calcitonin mRNA and the deduced complete amino acid sequence of chicken calcitonin.

Cell free translation of chicken calcitonin messenger RNA.

The primary step of calcitonin biosynthesis was studied in a normal organ: chicken ultimobranchial gland, a tissue particularly rich in calcitonin secretory cells. Poly(A)-rich RNA was extracted and purified from ultimobranchial organs and translated in a reticulocyte lysate in the presence of labelled methionine. Polyacrylamide gel electrophoresis of specific immunoprecipitates revealed a major band of Mr 14 500 and a band of Mr 13 300. Thus, in chicken the precursor of calcitonin is a Mr 14 500 polypeptide. The minor component of Mr 13 300 could represent limited processing by the reticulocyte lysate.

Calcitonin gene expression in normal human liver.

Immunoreactive calcitonin (CT) is present in liver. This could represent hormone synthesized by liver cells, degraded or bound to specific receptors reported in this organ. We report here that the calcitonin gene is expressed in liver. We proved this by demonstrating, by PCR amplification using specific primers, the presence of calcitonin messenger in human liver and in primary cultures of human hepatocytes and detected by radioimmunoassay CT in hepatic tissues and cells. The synthesis of hormone by liver that also possesses specific receptors for CT favors the presence of an autocrine or paracrine system involving calcitonin in this organ.

Isolation and partial characterization of the calcitonin gene in a lower vertebrate. Predicted structure of avian calcitonin gene-related peptide.

The gene coding for calcitonin was isolated and characterized in a non-mammalian vertebrate, chicken. Sequencing of the 3'-end of this gene revealed after the calcitonin coding exon, an intron followed by an exon coding for a calcitonin gene-related peptide, which displays significant amino acid sequence homology with mammalian CGRPs so far sequenced.

Expression of glucagon-like peptide 1 receptor in a murine C cell line: regulation of calcitonin gene by glucagon-like peptide 1.

We have characterized, by RT-PCR amplification using specific primers, the presence of glucagon-like peptide-1 (GLP-I) receptor mRNA in CA-77 cells, a C cell line derived from a rat medullary thyroid carcinoma. Down-regulation of the GLP-1 receptor mRNA was observed after exposure of CA-77 C cells with GLP-1 (7-37). Increased secretion of both calcitonin gene-related peptide (CGRP) and calcitonin (CT) occurred after treatment with GLP-1 (7-37) associated with elevated steady-state levels of CGRP and CT mRNA. GLP-1 (7-37) increased cAMP formation in CA-77 cells in a dose-dependent manner; exendin (9-39), a GLP-1 receptor antagonist, inhibited cAMP production. The GLP-1 peptide which is produced by intestinal cells could be involved in the control of CT secretion through an entero-thyroidal axis implying GLP-1 receptor and increased CT gene expression.

The complete sequence of human preprocalcitonin.

DNA complementary to mRNA extracted from the thyroid glands of patients suffering from medullary carcinoma of the thyroid (MCT), a calcitonin-producing tumour, was inserted in the Pst site of pBR 322 by G-C tailing. The recombinant plasmids were used to transform Escherichia coli DP 50. Ampicillin-resistant clones were screened using a 32P-labelled cDNA to mRNA extracted from a case of MCT particularly rich in calcitonin (CT) mRNA. Positive clones were subsequently rescreened using a 32P poly(T) probe. Eighty clones were thus purified, and the inserts obtained by digestion with PstI were subjected to positive hybridization selection with subsequent translation in vitro. An insert stimulating synthesis of the protein and containing restriction sites compatible with the previously published complete sequence of calcitonin mRNA from rat was sequenced. This cDNA insert contained the entire coding region of 426 bp, 70 bp at the 5'-end, and 295 bp upstream from the poly(A) tail. The complete amino acid sequence of human preprocalcitonin could thus be deduced.

An isoform of the human calcitonin receptor is expressed in TT cells and in medullary carcinoma of the thyroid.

We amplified, using the polymerase chain reaction and calcitonin receptor (CTR) specific primers, RNA extracted from medullary thyroid carcinoma (MTC) and the derived TT cell line. Both secrete large amounts of calcitonin. Electrophoresis of amplification products revealed, in both cases, an ethidium bromide-stained band that hybridized to a CTR probe. Sequencing the band amplified from TT cells revealed an open reading frame identical to the sequence of H-CTR but lacking 16 amino acids in the first intracellular loop. This demonstrates the existence of an mRNA coding for a subtype of H-CTR which is expressed in TT cells and MTC.

Increased level of calcitonin mRNA after 1,25-dihydroxyvitamin D3 injection in the rat.

Vitamin D metabolites are able to change plasma calcitonin (CT) levels, but nothing is known about a possible effect at the CT gene level. Here we have investigated the acute effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on the CT biosynthetic activity of thyroid glands from adult rats. Plasma CT levels were significantly increased (X2) 1 and 2 h after 1,25-(OH)2D3 injection in the face of unchanged plasma calcium values. The thyroidal CT content also was unchanged. A 2-fold increase in CT mRNA level measured by dot-blot hybridization occurred 1 and 2 h after 1,25-(OH)2D3 administration. Expression of CT gene products was examined in the rabbit reticulocyte lysate cell-free translation assay. After polyacrylamide gel electrophoresis, specific immunoprecipitates were autoradiographed and quantified by integration. A single precursor of Mr approximately equal to 15 000 could be specifically immunoprecipitated with CT antisera. A 3-4-fold rise in translatable CT mRNA activity was observed 1 and 2 h after 1,25-(OH)2D3 injection. Thus, parallel changes in CT mRNA level, CT mRNA activity and plasma CT levels were observed in adult female rats after administration of 1,25-(OH)2D3. These findings demonstrate for the first time that 1,25-(OH)2D3 enhanced CT gene expression in the face of unchanged plasma calcium levels.

Sequence and expression of the chicken calcitonin gene.

The avian calcitonin gene was isolated and sequenced; two mRNAs are expressed by tissue-specific alternate splicing. The peptides encoded by the mRNAs are the protein precursors of either calcitonin or calcitonin gene-related peptide (CGRP). Calcitonin is expressed predominantly in ultimobranchial bodies and CGRP in brain.