RESUMO
Old rats of the WAG/Rij strain have a high incidence (50%) of medullary thyroid carcinoma, a calcitonin (CT)-secreting tumor. We have characterized and quantified the topographical distribution of [125I]salmon calcitonin (sCT) binding sites in the kidneys of this strain, as compared to Wistar CF rats (2% incidence of spontaneous medullary thyroid carcinoma). We report here that, up to 15 days of postnatal development, the distribution of CT-binding sites in the kidney of the WAG/Rij strain was quite similar to that found in developing and adult Wistar CF rats. However, from the age of 1 month, sCT-binding sites were dramatically reduced in both the medulla and the inner part of the kidney cortex, though plasma CT levels were not significantly different in both strains. Adult WAG/Rij rats bearing a transplanted tumor for 12 weeks had a high level of plasma calcitonin and exhibited an even greater reduction of both medullary and cortical sCT-binding sites. These results suggest that the modification in the CT-binding sites in WAG/Rij rats is not a consequence of a possible down regulation due to elevated circulating hormonal level but could be inherited and possibly associated with the later development of the tumor in this strain.
Assuntos
Calcitonina/metabolismo , Carcinoma/metabolismo , Rim/metabolismo , Ratos Endogâmicos/fisiologia , Receptores de Superfície Celular/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Envelhecimento , Animais , Autorradiografia , Córtex Renal/metabolismo , Medula Renal/metabolismo , Ratos , Receptores da CalcitoninaRESUMO
C-cell tumors (medullary thyroid carcinoma) occur in humans and several other mammalian species. This tumor develops spontaneously with a high incidence (50%) in old Wag/Rij (Wistar-derived strain) rats. We have recently shown that calcitonin binding sites, which are present in the Wistar rats, are lost from renal medulla of the Wag/Rij rats before they reach the age of 1 month. In the present work, we investigated the distribution of calcitonin binding sites in the kidneys of first and second generation hybrids of Wistar x Wag/Rij rats. The absence of calcitonin binding sites from the renal medullas of 25% of F2 hybrids indicates that the deficiency is inherited in a Mendelian fashion and opens the way to establishing inbred strains lacking renal medullary calcitonin binding sites.
Assuntos
Calcitonina/metabolismo , Receptores de Superfície Celular/genética , Neoplasias da Glândula Tireoide/genética , Envelhecimento , Animais , Autorradiografia , Cálcio/farmacologia , Deleção Cromossômica , Cruzamentos Genéticos , Genes , Radioisótopos do Iodo , Medula Renal/metabolismo , Ratos , Ratos Endogâmicos , Receptores da Calcitonina , Receptores de Superfície Celular/análise , Especificidade da Espécie , Glândula Tireoide/metabolismoRESUMO
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.
Assuntos
Genes fos , Osteoblastos/citologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Sequência de Bases , Northern Blotting , Divisão Celular , Imunofluorescência , Regulação da Expressão Gênica , Substâncias de Crescimento/farmacologia , Dados de Sequência Molecular , Osteoblastos/efeitos dos fármacos , Osteossarcoma , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Antissenso/farmacologia , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais , Células Tumorais CultivadasRESUMO
The chemotactic effect of calcitonin (CT) gene products was tested on F9 teratocarcinoma cells, which are an in vitro model of early embryonic development. CT and CT gene-related peptide (CGRP) induce a significant chemotactic response (chemotactic index, 40-50). The order of potency is: chicken CGRP greater than or equal to salmon CT greater than or equal to human CGRP. Human CT is a less potent chemotactic agent (chemotactic index, 15). Compared to other well known peptides with chemotactic activity, such as platelet-derived growth factor (no activity) and transforming growth factor-beta (chemotactic index, 5), CGRP and CT appear to be very active in attracting F9 cells in the Boyden chamber assay. Interestingly, CT and CGRP exhibit little chemotactic effect toward differentiated teratocarcinoma cells (i.e. retinoic acid-treated F9 cells or parietal endodermal PYS cells). While salmon CT and chicken CGRP activate adenylate cyclase activity in F9 cell membranes by 7- to 8-fold, higher concentrations (greater than 10(-10) M) of these peptides are required to stimulate cAMP formation than are required to mediate the chemotactic effect of these peptides. These data imply the possible involvement of CT gene products in regulating cell migration during early embryonic development.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Calcitonina/farmacologia , Fatores Quimiotáticos/farmacologia , Teratoma/patologia , Adenilil Ciclases/metabolismo , Animais , Movimento Celular , Galinhas , Humanos , Salmão , Teratoma/enzimologia , Células Tumorais CultivadasRESUMO
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.
Assuntos
Processamento Alternativo , Calcitonina/biossíntese , RNA Mensageiro/biossíntese , Salmão/genética , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Calcitonina/genética , Primers do DNA , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Transcrição Gênica , Corpo Ultimobranquial/metabolismoRESUMO
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.
Assuntos
Hidroxiprostaglandina Desidrogenases/genética , RNA Mensageiro/genética , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA Complementar , Humanos , Hidroxiprostaglandina Desidrogenases/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
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.
Assuntos
Hidroxiprostaglandina Desidrogenases/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Células HL-60 , Humanos , Isoenzimas/genética , Dados de Sequência Molecular , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
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.
Assuntos
Calcitonina/análise , Calcitonina/genética , Precursores de Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Centrifugação com Gradiente de Concentração , Clonagem Molecular , DNA/análise , Plasmídeos , RNA Mensageiro/análiseRESUMO
Renal binding sites for labelled salmon calcitonin (sCT) were studied using cryostat sections and autoradiography. Increasing concentrations of unlabelled sCT inhibited 125I-sCT binding. 125I-sCT bound to a single site with a Kd of 2 nM and a number of sites of 220 fmol/mg protein. Mammalian calcitonins had low affinities and peptides unrelated to CT were devoid of any significant affinity for 125I-sCT receptors. Autoradiograms disclosed a high concentration of 125I-sCT receptors mainly located in the outer medulla and heterogeneously in the renal cortex. The distribution of specific binding sites is in agreement with the current concepts of renal action of calcitonin.
Assuntos
Rim/análise , Receptores de Superfície Celular/análise , Animais , Ligação Competitiva , Humanos , Masculino , Ratos , Ratos Endogâmicos , Receptores da Calcitonina , Salmão , Especificidade da Espécie , SuínosRESUMO
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.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Fígado/metabolismo , Processamento Alternativo , Sequência de Bases , Células Cultivadas , Primers do DNA , Humanos , Fígado/citologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RadioimunoensaioRESUMO
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.
Assuntos
Calcitonina/genética , Galinhas/genética , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Peptídeo Relacionado com Gene de Calcitonina , GenesRESUMO
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.
Assuntos
Calcitonina/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Peptídeos/farmacologia , Receptores de Glucagon/biossíntese , Animais , Sequência de Bases , Peptídeo Relacionado com Gene de Calcitonina/biossíntese , Linhagem Celular , Primers do DNA , Glucagon/farmacologia , Peptídeo 1 Semelhante ao Glucagon , Receptor do Peptídeo Semelhante ao Glucagon 1 , Peptídeos Semelhantes ao Glucagon/farmacologia , Cinética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Fragmentos de Peptídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Ratos , Neoplasias da Glândula Tireoide , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
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.
Assuntos
Calcitonina/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Animais , Calcitonina/genética , Sistema Livre de Células , Galinhas , Eletroforese em Gel de Poliacrilamida , Imunoquímica , Poli A/isolamento & purificaçãoRESUMO
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.
Assuntos
Calcitonina/genética , Fígado/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , DNA Complementar , Expressão Gênica , Humanos , Fígado/citologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RadioimunoensaioRESUMO
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.
Assuntos
Calcitonina/genética , Galinhas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/fisiologia , Regulação da Expressão Gênica , Genes , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
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.
Assuntos
Calcitonina/genética , Calcitriol/farmacologia , Animais , Cálcio/sangue , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos , Glândula Tireoide/fisiologia , Fatores de TempoRESUMO
Two genes code for calcitonin gene-related peptides (CGRPs). One expresses by tissue-specific alternate splicing calcitonin and CGRP I mRNAs, the other CGRP II mRNA. Calcitonin is the marker of sporadic or hereditary human medullary thyroid carcinoma (MTC). CGRP II expression is not well established in normal or tumoral thyroid. After amplification by polymerase chain reaction, CGRP I and II mRNAs were detected in six cases of MTC associated with other endocrine neoplasia (MEN IIa) and in two cases of isolated MTC. CGRP I was detected in all non-C cell tumoral thyroids (6 samples), CGRP II was barely detectable in three out of six cases. CGRP II could be a specific tumoral marker of MTC.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/genética , Calcitonina/genética , Carcinoma/genética , Neoplasias da Glândula Tireoide/genética , Sequência de Bases , Southern Blotting , Expressão Gênica , Humanos , Dados de Sequência Molecular , Oligonucleotídeos/química , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Neoplásico/genética , Glândula Tireoide/fisiologiaRESUMO
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.
Assuntos
Carcinoma Medular/metabolismo , Receptores da Calcitonina/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Carcinoma Medular/genética , Primers do DNA/genética , Expressão Gênica , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos , Receptores da Calcitonina/classificação , Receptores da Calcitonina/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Suínos , Neoplasias da Glândula Tireoide/genética , Células Tumorais Cultivadas/metabolismoRESUMO
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.
Assuntos
Calcitonina/análise , Precursores de Proteínas/análise , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/análise , Humanos , Plasmídeos , RNA Mensageiro/análise , Glândula Tireoide/análise , Neoplasias da Glândula Tireoide/análiseRESUMO
We studied the hormonal secretion of a human mixed follicular and medullary carcinoma. Thyroglobulin (Tg) secretion, especially by large cells and sometimes by small ones, was visualized with immunoenzymatic staining. Calcitonin (CT) was produced by small spindle-shaped cells. Moreover, immunofluorescence double staining performed on the resected thyroid tissue showed the secretion of both Tg and CT in a small number of cells. The cells lost their hormonal secretion after 2 months of culture. Hormonal secretion was modulated by different additives in the medium. Tg secretion was induced when TSH was added to the culture medium; the maximal effect was produced with the addition of 1 mU TSH/ml and 1 microM cortisol, which potentiated the effect of TSH on Tg production. A durable Tg secretion was obtained by embedding the cells in Engelbretch-Hohn-Swarn (EHS) tumour matrix. The CT production was reinduced by the addition of 4 mM Ca2+, 1 microM glucagon and 1 microM cortisol to the culture medium. These findings show that different cells are found in a mixed follicular and medullary carcinoma, some of which can secrete both CT and Tg. They can remain differentiated for a long period after being embedded in EHS tumour matrix with Ca2+ and hormonal components.