Early spontaneous deficiency of calcitonin renal binding sites in rats with a high incidence of calcitonin-secreting tumors (WAG/Rij).

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.

Loss of calcitonin receptors: a genetically transmitted defect in rats with high incidence of C-cell tumors.

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.

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.

An efficient method to detect calcitonin mRNA in normal and neoplastic rat C-cells (medullary thyroid carcinoma) by in situ hybridization using a digoxigenin-labeled synthetic oligodeoxyribonucleotide probe.

We report here an efficient and rapid method for the specific detection of calcitonin in tumor C-cells of medullary thyroid carcinoma (MTC). This occasionally aggressive tumor arises from the endocrine thyroid C-cells. Its principal marker is calcitonin, the predominant C-cell secretion, which is detected in patients and in our animal model by radioimmunoassay of the plasma, as well as by immunohistochemistry of thyroid tissues. Although calcitonin is easily detectable in normal C-cells, its content is greatly reduced in tumor cells owing to the disappearance of the secretory granules that store the mature peptide. This finding suggests cell dedifferentiation correlated with an increasing aggressivity of the tumor. We therefore developed a rapid detection of calcitonin mRNA by in situ hybridization on routine paraffin sections, using a synthetic oligodeoxyribonucleotide probe labeled with digoxigenin-dUTP. The reaction was detected with an anti-digoxigenin antibody conjugated with alkaline phosphatase, and the enzyme catalyzed the appearance of a dark blue color. The signal was exclusively restricted to the normal, hyperplastic, and tumor C-cells. It was specific, as increasing concentrations of the unlabeled oligonucleotide led to progressive disappearance of the reaction. Its sensitivity was slightly diminished as compared with corresponding frozen sections, but the intensity of the signal was quite acceptable. High levels of calcitonin mRNA were found in all normal and hyperplastic C-cells. They were increased in most of the tumor MTC cells, which did not correlate with the amount of intracellular peptide stores but explained the abnormally high basal levels of circulating calcitonin of the tumor-bearing rats. ISH is therefore of greater value than ICC for an early anatomopathological detection of this tumor. Our data show that the tumor cells are not "dedifferentiated." They only lack the granular compartment storing the mature peptide before exocytosis, but CT biosynthesis and the rest of the secretory process seem to be complete. Our results suggest that factors expressed in malignant C-cells affect basic cell mechanisms involved in the storage of the mature calcitonin, rather than the expression of the CALC gene.

Influences of fixatives on the immunodetection of calcitonin in mouse "C" cells during pre- and post- natal development.

The presence of calcitonin in fetal and postnatal mouse "C" cell was demonstrated by radioimmunoassays of fetal and adult thyroid extracts and by immunohistochemistry following the use of Bouin's fixative from 18 days postcoitum onwards. However, after fixation with formaldehyde no calcitonin could be detected in fetal or early postnatal samples, although it was still easily detectable in adult tissues treated with this fixative, raising the possibility that there may be age-dependent differences in the chemistry or physiological state of intracellular calcitonin. Experiments conducted with several other fixatives and the results of the radioimmunoassays suggest that the loss of calcitonin reactivity is not due to differences in the anti-genic sites of the molecule at the two stages in development. Neither can the positive detection of fetal material treated with Bouin's fixative be due to any action of this liquid. However, the inhibiting action of the aldehydes could be related to the preservation of the granule membranes in the fetal "C" cells: our ultrastructural studies show that the granules can be morphologically classified in dense-core and homogeneous matrix. Such granule membranes are abundant in the young and adult "C" cell and would be reactive, while the small dense-core granules are prevalent in the fetus and would be unreactive, confirming the hypothesis of a difference of structure of the granule membrane and not of calcitonin between fetal and late postnatal "C" cells.

Influence of laminin substratum on cell proliferation and CALC I gene expression in medullary thyroid carcinoma C cell lines.

Medullary thyroid carcinoma (MTC) originates from C cells, which secrete calcitonin (CT) and CT gene-related peptide (CGRP), the two splice peptide products of the CALC I gene. Normal and hyperplastic C cells are intrafollicular, in contact with the basement membrane (BM) that is maintained around the differentiated tumors. To investigate the relationships between MTC evolution and BM constituents, we examined the modifications induced by laminin-1 and -2 (merosin), two isoforms colocalized in the follicular BM, on three MTC cell lines: murine rMTC 6-23 and CA-77 cells, and human TT cells. Laminin exerted a mitogenic activity on rMTC 6-23 and on TT cells, causing a concurrent decrease in both CT and CGRP mRNA levels and production of the peptides. Conversely, laminin reduced the proliferation rate and enhanced CGRP synthesis and secretion in CA-77 cells. This antiproliferative response, which coincides with an increase in differentiation markers, is comparable to that reported in normal cells and also in the neoplastic Caco-2 cell line. This suggests that laminin could exert opposite effects depending on the stage of tumor evolution.

Evaluation of somatostatin biosynthesis, somatostatin receptors and tumor growth in murine medullary thyroid carcinoma.

Spontaneous medullary thyroid carcinomas (MTCs) of old rat thyroids were analyzed for the expression of somatostatin and somatostatin binding sites in tumoral C cells in relation to the stage of tumor development, the mitotic activity of tumoral tissue and calcitonin biosynthesis as a marker of C cell differentiation. High levels of both immunoreactive somatostatin and its mRNA were detected in a subpopulation of tumoral C cells, gathered in areas suggesting a clonal proliferation and located preferentially at the periphery of the tumor. These cells also displayed high levels of calcitonin and its mRNA. However, many calcitonin immunoreactive cells showed no sign of somatostatin synthesis. The proliferative activity of the somatostatin-containing areas was low and slow compared to the areas lacking somatostatin production. However, it increased during the course of tumor growth. Somatostatin binding sites, measured with in vitro receptor autoradiography using 125I-[Tyr3]-octreotide or 125I-[Leu8, dTrp22, Tyr25]SS-28, were not detected in any of the MTCs tested. In rat MTC cells, somatostatin was associated with differentiation and slow proliferation, two parameters inversely correlated with the progression of malignancy. As expected, owing to the highly regulated secretion of the differentiated endocrine cell type, its presence was correlated with low basal calcitonin levels. However, the absence of somatostatin binding sites on any type of MTC cells does not favor a direct autocrine regulation of this peptide in this murine model of human MTC.

Expression of laminin-2 by normal and neoplastic rat C cells during the development of medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) originates from C cells, which secrete calcitonin (CT), their specific marker. C cells are located in contact with the basement membrane (BM) of the thyroid follicles, which is partly made up of the laminin-2 isoform synthesized by thyrocytes. During oncogenesis, proliferation of the C cells, invading the centre of the follicles, leads to a break in their normal contact with the BM. As specific interactions of cells with BM components, especially laminins, are important for proliferation and differentiation, we investigated the relationships of normal and neoplastic C cells with laminin in the Wag/Rij rat model of human MTC. Immunocytochemical studies showed a progressive loss of the laminin layer underlying the hyperplastic C cell nodules around the large dedifferentiated tumours. The alpha2, beta1 and gamma1 chains of the laminin-2 isoform were synthesized and secreted by rat MTC 6-23 cell cultures and the tumours induced by subcutaneous injection of these cells. In situ hybridization combined with anti-CT immunocytochemistry showed a low expression of alpha2 mRNA on differentiated C cells and thyrocytes, but an overexpression on immunonegative spontaneous MTC and induced intrathyroid tumours. The high level of alpha2 gene expression, together with tumour dedifferentiation, suggests a relationship with malignancy.

Calcitonin secretion, C cell differentiation and proliferation during the spontaneous development of murine medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC), a C cell neoplasm, synthesizes large amounts of calcitonin (CT), its biological marker. However, in some cases with a poor prognosis, MTC is associated with low basal CT levels owing to a decrease in the thyroid CT content. Using a murine model of human MTC, we studied the relationships between CT biosynthesis, C cell proliferation, and the circulating CT level during MTC progression. Cell proliferation was revealed by autoradiography of radioactive thymidine incorporation in dividing nuclei, after CT or CT mRNA detection by immunocytochemistry (ICC) or in situ hybridization (ISH). All rat thyroids showed a severe hyperplasia of C cells containing significant amounts of CT and CT mRNA, and a very low mitotic index. Tumours were found in 68% of the thyroids. In the strongly immunoreactive small nodules (ICC+), many labelled nuclei were observed. Subsequently some nodular cells, still containing detectable CT mRNA (ISH+), were not detected by immunocytochemistry (ICC-) owing to a dramatic decrease in secretory granules. Their mitotic index increased, and a rise of the basal CT plasma level was noted. These ISH+, ICC- tumour MTC cells represent a modified aggressive tumour C cell population exhibiting an increased ability to proliferate and were detected by the rise in the basal circulating CT level.

[Uptake and storage of indolamines in the ultimobranchial body of the fetal murine thyroid. 1-Autoradiographic histological study]. / Captage et stockage d'indolamines dans le corps ultimobranchial et la thyroïde de foetus de souris. I. Etude histologique autoradiographique

Incorporation of 3H-5 HTP is studied in ultimobranchial cells and thyroid cells of the mouse foetus from the 13th day to the 18th day of gestation. The APUD characteristics of these cells are first observed on the 14th day when the U.B. bodies are included in the thyroid anlage. The silver granules are then localized on the parafollicular cell cords from which C cells of the adult thyroid arize.

Granular and extra-granular forms of immunoreactive calcitonin in normal rat "C" cells.

The intracellular localization of the intermediate calcitonin-related peptides produced during calcitonin maturation (CT, 3.4 Kd) from the polyprotein precursor (15 Kd) has been studied by means of subcellular fractionation of normal adult rat thyroid using a continuous hypertonic Percoll gradient. A fraction B (Bottom), containing the secretory granules, was separated from a fraction T (Top) constituted of rough and smooth membranes devoid of granules. Immunoreactive calcitonin extracted from both fractions was heterogeneous when analysed by gel electrophoresis followed by radioimmunoassay. In fraction T, the immunoreactive molecules assayed were associated with the membranes. Their apparent molecular weights (lower than 18 Kd) were compatible with the intermediates predicted by the aminoacid sequence of the precursor. However the maturation was not complete, as indicated by the absence of the monomer. The smallest molecular weight detected (4-4.5 Kd) was compatible with a calcitonin-katacalcin uncleaved molecule. These data strongly suggest the occurrence of a cleavage separating the N-terminal part of the molecule during the pre-granular step of calcitonin secretory pathway. In the granular fraction, the monomer constituted the granular matrix, representing 65% of the total immunoreactivity. The remainder was associated with the granules membranes, consisting of the 4-4.5 Kd precursor and some 43 Kd material which has already been demonstrated by others to be covalent polymers of calcitonin [17]. It is thus concluded that a second cleavage could take place in the granules where the major immunoreactive form is the processed calcitonin, either as free monomer or as covalent polymers associated with the membrane of the granules.

Immune response against medullary thyroid carcinoma (MTC) induced by parental and/or interleukin-2-secreting MTC cells in a rat model of human familial medullary thyroid carcinoma.

The existence of inherited aggressive forms of medullary thyroid carcinoma (MTC), and their resistance to all classical therapies, make it a prime candidate for adoptive immunotherapy. As a prelude to a vaccine for the protection of family members at risk of developing the disease, we investigated the immunological antitumour response provoked by the 6/23 rMTC cell line, compared to that of the same cell engineered to secrete interleukin-2 (rMTC-IL2), in an animal model of familial human MTC, the inbred strain of Wag/Rij rats. The rMTC cells developed a tumour that invaded the whole neck 15 days after orthotopic injection (into the thyroid), while the rMTC-IL2 cells were progressively rejected. Co-injection of rMTC-IL2 with the parental cells induced the rejection of the rMTC transplants. When injected, both tumoral cell types showed a similar positive immunoreaction with anti-MHC class I (major histocompatibility complex class I) antibodies. They both recruited natural killer cells and eosinophils at the site of injection. In addition, CD8+ T lymphocytes infiltrated the rMTC-IL2 cells, and eosinophil recruitment was amplified. Neutrophils, macrophages and CD4+ T lymphocytes were scarce. Our results suggest that the CD8+ T lymphocytes are implicated in the anti-tumour reaction elicited by the IL-2-transfected cells. As these effectors are known to induce a specific immunological response, including memory, such a protocol should be tested as a vaccine on the young population genetically at risk of developing a MTC.

Ultrastructural localization of immunoreactive calcitonin in the two cell types of the ultimobranchial gland of the common toad (Bufo bufo L.).

The ultimobranchial (UB) glands of the common toad Bufo bufo consist of several cellular masses containing two quite different cell types which line a central lumen filled with amorphous material. The morphologically defined Type I cell is akin to a typical calcitonin secretory cell as observed for all vertebrates, with small dense-core secretory granules. On the contrary the Type II cell displays large apical dense bodies which may be related to the secretion and/or absorption of the amorphous material. Cells morphologically related to Type II cells have been described in the UB glands of Sauropsidea and in the UB follicles of mammalian thyroid gland. An immunocytochemical stain using an antiserum raised against synthetic salmon calcitonin demonstrated the specific localization of an immunoreactive product in both Type I and Type II cell granules, suggesting that both cell types could be involved in calcitonin metabolism. Moreover, the presence of immunoreactive calcitonin in the Type II cells of the Bufo UB gland raises the question of the function of the morphologically equivalent cells of other species.

[Immunohistochemical demonstration of calcitonin cells in the fetal thyroid of mice]. / Démonstration immunohistochimique des cellules à calcitonine de la thyroïde foetale de souris.

Calcitonin was revealed by means of an indirect immunohistochemical method using a serum against synthetic human calcitonin. After the use of the Bouin-Hollande fixative liquid, the hormone was detected in cells located in the central part of the thyroïdian wings. They are few at the 18th day of fetal life, their number increases regularly until reaching ten times the initial number at birth. The immunological reaction is negative if only aldehyde fixatives are used.

Treatment of medullary thyroid carcinoma by combined expression of suicide and interleukin-2 genes.

Inherited medullary thyroid carcinomas (MTC) are aggressive and resistant to conventional chemo- and radiotherapies. We evaluated a novel strategy for treatment of MTC, combining "suicide" and interleukin-2 (IL-2) gene therapies. Tumors were produced in Wag/Rij rats by orthotopic injection of the rMTC 6-23 cell line, and/or derivatives expressing the herpes simplex virus 1 thymidine kinase (HSV1-TK) gene (rMTC-TK). Ganciclovir, a nucleoside analog selectively transformed to a toxic metabolite by HSV1-TK, totally eradicated rMTC-TK tumors in 60% of the animals. 1:1 rMTC and rMTC-TK mixed tumors were also strongly inhibited by ganciclovir (P < 0.05), indicating the occurrence of an efficient "bystander" effect in vivo. Double labelling of rMTC cell membranes and apoptotic nuclei revealed that, as with the TK+ cells, some TK- cells died by apoptosis. A 1:1 mixture of rMTC and rMTC-TK cells was administered to produce established tumors and then rMTC cells, transfected to express the IL-2 gene (rMTC-IL2), were inoculated. Combined ganciclovir and IL-2 treatment improved the inhibition of tumor growth compared to that following ganciclovir alone (86% compared to 54%, P < 0.05). This treatment also significantly enhanced macrophage activation and tumor infiltration by CD8+ and CD4+ T lymphocytes. These results open an avenue for combining suicide and immunoregulatory gene therapies for MTC management in man.

Influence of calcium and vitamin D deficient diet on calcitonin gene expression in the ultimobranchial cells of the developing chicken.

Recently hatched chickens were fed a vitamin D and calcium deficient diet for 4 weeks. Calcitonin (CT) biosynthesis in the ultimobranchial glands (UBG) was studied during the treatment by means of in situ hybridization of specific CT mRNAs and immunocytochemical detection of the CT intracellular stores. Circulating CT levels were measured by radioimmunoassay. Within 1 week of the start of treatment, the deficient animals had significantly lowered plasma calcium concentrations and a dramatic fall of plasma CT levels, but the UBGs were not much affected. From week 2 to week 4, the UBG underwent a gradual atrophy. The tissue became lacunar due to the presence of an abnormally developed cystic component. Although calcemia returned to normal at week 4, the cellular endocrine cords were dramatically reduced, corresponding to the undetectable circulating CT levels. However, the UB glands always contained persistent CT-secreting cells, mainly at the periphery of the tissue or in contact with enlarged parathyroid tissue inclusions. These endocrine UB cells contained large amounts of hybridizable CT mRNA and immunodetectable stores of the mature hormone, and their ultrastructural features were quite unaffected compared to normal ones. Thus, we conclude that, in the chicken, severe calcium malnutrition led to a striking reduction of CT biosynthesis in the UB glands by decreasing the number of secretory cells and not by triggering modifications of the biosynthetic activity of the UB endocrine cells.

Localization of a salmon calcitonin-like molecule in one type of ultimobranchial cells in the freshwater turtle Pseudemys scripta.

Immunohistochemical methods using affinity adsorbed antibodies raised against the three families of calcitonins (CT) were applied to ultimobranchial (UB) cells in situ to investigate the nature of the Chelonian calcitonin molecule and its distribution in the ultimobranchial bodies of the freshwater turtle, Pseudemys scripta. In this species, the UB glands were present on both sides and consisted of scattered cell clumps between epithelial vesicular structures. The neighboring parathyroid tissue also contained two components, the majority being composed of similar vesicles, with occasional solid cell cords evenly distributed. Calcitonin immunoreactivity was found in the cell clumps of the UB gland and in the cell cords of the associated parathyroid, but not in the epithelial component lining the vesicles or in the amorphous material which sometimes filled the lumen. Turtle calcitonin was exclusively of the salmon type, as determined by the negative results obtained in situ after the use of antibodies raised against human and porcine molecules. The salmon-like calcitonin content of the ultimobranchial area was estimated as 15.2 ng; however, the molecule was undetectable in the circulation. In this work we localize the quantitate a salmon-like CT molecule in one type of ultimobranchial and parathyroid cell of a reptile for the first time.

Distribution of renal calcitonin binding sites in mammalian and nonmammalian vertebrates.

Calcitonin (CT), the hypocalcemic hypophosphatemic hormone, is present in many vertebrate species. The principal target organs for CT are, in mammals, kidney and bone, and in fish, bone and gill. We have investigated the presence of renal calcitonin binding sites in a fish (Salmo gairdneri), two amphibians (Bufo bufo and Rana esculenta), two reptiles (Pseudemys scripta and Gekko gecko), and two birds (Gallus domesticus and Cothurnix japonica). We compared their distribution to a mammal, the rat (Wistar strain), using quantitative autoradiography methods. Moreover, CT binding sites were also characterized in the chicken kidney by a membrane assay. No renal 125I-sCT binding sites were detected in the fish, the amphibians, and the reptiles studied. In the rat, binding sites were present in the outer medulla and in the cortex. In the chicken and in the quail, scattered binding sites were also observed in the medulla and in the cortex, whose pattern seemed to follow the distribution of the glomeruli and/or the collecting tubules. Chicken kidney membranes were also purified by sucrose gradient centrifugation. Scatchard analysis of the binding data revealed the presence of one type of 125I-sCT binding site with an apparent dissociation constant of 4.3 nM and a number of sites of 73 fmol/mg/protein. The present results suggest that calcitonin renal receptors appeared late in evolution and thus that a regulation of renal function by calcitonin was only effective in birds.

Ultimobranchial gland of the domestic fowl. Two types of secretory cells involved in calcitonin metabolism.

The ultimobranchial gland (UBG) of birds is particularly rich in calcitonin, the hypocalcaemic hypophosphataemic hormone, that is secreted by the C-cells of the mammalian thyroid. The principal cells of the UBG have a striking resemblance with the mammalian C-cells, i.e., they possess small intracytoplasmic dense-core secretory granules, 150-300 nm in diameter. The gland also contains a second, morphologically distinct, endocrine cell type with larger granules, 500-800 nm in diameter. A sensitive immunocytochemical reaction was developed with the use of antibodies against salmon calcitonin. By means of this technique the presence of calcitonin-immunoreactive molecules was demonstrated in both secretory cell types of the UB gland of the chicken. This gland can thus be considered as a homogeneous calcitonin-producing tissue. Whether the secretory products are identical is discussed and differences in the secretory pathways are suggested.

Elevated levels of calcitonin mRNA: a marker for the spontaneous development of medullary thyroid carcinoma in rats.

The aging WAG/Rij rats (a Wistar derived strain) develop spontaneously medullary thyroid carcinoma with a high frequency (50%). We have studied calcitonin biosynthesis in Wistar and WAG/Rij rats strains in order to determine if early changes in this parameter occurred in the WAG/Rij strain. Thyroidal and plasma CT levels were measured in three months old WAG/Rij and Wistar rats before and after acute calcium challenge. Total RNA was extracted from thyroid glands and specific CT messenger RNA levels estimated by dot and Northern blot analysis with a 32P-labeled probe specific for CT mRNA. The capacity of mRNA to direct synthesis of CT precursor was also measured by translation in an in vitro system. Though mean basal circulating CT levels were equivalent in both strains, CT release after calcium stimulation was much increased in the WAG/Rij rat. CT content of the glands and CT mRNA levels were two fold higher in the WAG/Rij strain. Thus, in this strain, CT biosynthesis and secretion were increased long before the development of a C-cell carcinoma.