Association of RET codon 691 polymorphism in radiation-induced human thyroid tumours with C-cell hyperplasia in peritumoural tissue.

The RET proto-oncogene encodes a protein structurally related to transmembrane receptors with an intracellular tyrosine kinase domain. In human thyroid gland, the RET proto-oncogene is normally expressed in parafollicular C-cells. Thyroid C-cell hyperplasia is associated with inherited medullary thyroid carcinomas and is considered as a pre-neoplastic stage of C-cells disease. It has also been observed in thyroid tissues adjacent to follicular and papillary carcinomas. In order to study the relationship between a misfunctioning of the RET proto-oncogene and the presence of C-cell hyperplasia, we compared a series of thyroid glands presenting sporadic or radiation-associated tumours, as well as samples of unrelated normal thyroid tissues, for alteration in exons 10 and 11 of the gene and for the presence or absence of C-cell hyperplasia. Here we report a significantly higher frequency of C-cell hyperplasia present in peritumoural thyroid tissues of radiation-induced epithelial thyroid tumours, than in peritumoural of sporadic thyroid tumours or in control normal thyroid tissues (P=0.001). A G691S RET polymorphism was present with a higher frequency in radiation-induced epithelial thyroid tumours (55%) than in sporadic tumours (20%) and in control normal thyroid tissues (15%). Interestingly, this polymorphism was associated in the majority (88%) of radiation-induced tumours with a C-cell hyperplasia in the peritumoural tissues. Several explanations for this association are discussed.

Role of the cAMP and MAPK pathways in the transformation of mouse 3T3 fibroblasts by a TSHR gene constitutively activated by point mutation.

Constitutive activating mutations of the TSHR gene, have been detected in about 30 per cent of hyperfunctioning human thyroid adenomas and in a minority of differentiated thyroid carcinomas. The mutations activating the TSHR gene(s) in the thyroid carcinomas, were located at the codon 623 changing an Ala to a Ser (GCC-->TCC) or in codon 632 changing a Thr to Ala or Ile (ACC-->GCC or ACC-->ATC). In order to study if the constitutively activated TSHR gene(s) has played a role in the determination of the malignant phenotype presented by these tumors, we investigated: (1) the transforming capacity after transfection of mouse 3T3 cells, of a TSHR cDNA activated by an Ala-->Ser mutation in codon 623 or an Thr-->Ile mutation in codon 632 and (2) the pathway(s) eventually responsible(s) for the malignant phenotype of the cells transformed by these constitutively activated TSHR cDNAs. Our results show that (1) the TSHR(M623) or (M632) cDNAs give rise to 3T3 clones presenting a fully neoplastic phenotype (growth in agar and nude mouse tumorigenesis); this phenotype was weaker in the cells transformed by the 632 cDNA; (2) suggest that the fully transformed phenotype of our 3T3 cells, may be the consequence of the additive effect of the activation of at least two different pathways: the cAMP pathway through G(alpha)s and the Ras dependent MAPK pathway through G(beta)gamma and PI3K and (3) show that the PI3K isoform playing a key role as an effector in the MAPK pathway activation in our 3T3-transformed cells is PI3Kgamma. Signaling from PI3Kgamma to MAPK appears to require in our murine cellular system a tyrosine kinase (still not characterized), Shc, Grb2, Sos, Ras and Raf. It is proposed that the constitutively activated TSHR genes detected in the thyroid carcinomas, may have played an oncogenic role, participating in their development through these two pathways.

Search for NTRK1 proto-oncogene rearrangements in human thyroid tumours originated after therapeutic radiation.

Rearrangements of NTRK1 proto-oncogene were detected in 'spontaneous' papillary thyroid carcinomas with a frequency varying from 5 to 25% in different studies. These rearrangements result in the formation of chimaeric genes composed of the tyrosine kinase domain of NTRK1 fused to 5' sequences of different genes. To investigate if the NTRK1 gene plays a role in radiation-induced thyroid carcinogenesis, we looked for the presence of NTRK1-activating rearrangements in 32 human thyroid tumours (16 follicular adenomas, 14 papillary carcinomas and two lymph-node metastases of papillary thyroid carcinomas) from patients who had received external radiation, using the reverse transcription polymerase chain reaction, Southern blot and direct sequencing techniques. These data were compared with those obtained in a series of 28 'spontaneous' benign and malignant thyroid tumours, collected from patients without a history of radiation exposure and four in vitro culture cell lines derived from 'spontaneous' thyroid cancers. Our results concerning the radiation-associated tumours showed that only rearrangements between NTRK1 and TPM3 genes (TRK oncogene) were detected in 2/14 papillary carcinomas and in one lymph-node metastasis of one of these papillary thyroid carcinomas. All the radiation-associated adenomas were negative. In the 'spontaneous' tumours, only one of the 14 papillary carcinomas and one of the four in vitro culture cell lines, derived from a papillary carcinoma, presented a NTRK1 rearrangement also with the TPM3 gene. Twenty-five of this series of radiation-associated tumours were previously studied for the ras and RET/PTC oncogenes. In conclusion, our data: (a) show that the overall frequency of NTRK1 rearrangements is similar between radiation-associated (2/31: 6%) and 'spontaneous' epithelial thyroid tumours (2/32: 6%). The frequency, if we consider exclusively the papillary carcinomas, is in both cases 12%; (b) show that the TRK oncogene plays a role in the development of a minority of radiation-associated papillary thyroid carcinomas but not in adenomas; and (c) confirm that RET/PTC rearrangements are the major genetic alteration associated with ionizing radiation-induced thyroid tumorigenesis.

Iodide symporter gene expression in human thyroid tumors.

Expression of the Na+/I- symporter (NIS) gene was investigated by RT-PCR in a selected series of 26 primary thyroid carcinomas (19 papillary, 5 follicular, and 2 anaplastic). Fifteen follicular adenomas (11 "cold" and 4 "hot" adenomas) were also studied. Five of 19 papillary thyroid cancer did not express NIS messenger ribonucleic acid (mRNA). In all but 1 follicular cancer, NIS transcript was fully detected. In anaplastic tissue, NIS mRNA was only barely detected in 1 case. All of the follicular thyroid adenomas except 1 expressed the NIS gene. In contrast, all tumors studied excluding the anaplastic histotype fully expressed thyroglobulin and thyroid peroxidase mRNA transcripts. In 2 patients, a lower expression (3- to 5-fold) of NIS mRNA was found in metastasis by dot blot analysis compared with those in both normal and primary neoplastic thyroid tissue. Four of 8 differentiated thyroid cancer patients selected for the presence of metastases with negative posttherapy 131I total body scan showed the lack of NIS gene expression in their primary cancer. This defect, at least in these cases, is a somatic and intrinsic lesion of the primary cancer cells and is not due to a dedifferentiation process in the metastatic tissue. The early detection of the loss of NIS gene expression in the primary cancer, therefore, may provide useful information for the management of differentiated thyroid cancer patients.

Thyrotropin receptor gene alterations in thyroid hyperfunctioning adenomas.

Forty-four thyroid autonomously hyperfunctioning adenomas were analyzed to assess the frequency of mutations occurring in the TSH receptor (TSHR). PCR-amplified fragments encompassing the entire exon 10 of the TSHR gene were obtained from the genomic DNA extracted from the tumors and their adjacent normal tissues and were examined by direct nucleotide sequencing. Point mutations were found in 9 of the 44 adenomas examined (20%). One mutation occurred in codon 619 (Asp to Gly), four in codon 623 (three were Ala to Ser, one Ala to Val substitution), two in codon 632 (both Thr to Ile), and two in codon 633 (Asp to Tyr or His). All the alterations were located in a part of the gene coding for an area including the third intracellular loop and the sixth transmembrane domain of the TSH receptor. All mutations were somatic and heterozygotic, and none was simultaneous with alterations of ras or gsp oncogenes. Thus, our data show that in our series of 44 hyperfunctioning thyroid adenomas, a somatic mutation of the TSHR, responsible for the constitutive activation of the cAMP pathway, occurs in 20% of the tumors.

Pattern of ras and gsp oncogene mutations in radiation-associated human thyroid tumors.

The preferential activation of the Ki-ras oncogene in follicular radiation-associated human thyroid carcinomas, has been suggested by Wright et al. (1991). However, only 12 thyroid tumors were analysed in this study. In order to confirm if radiation favours, in human thyroid tumorigenesis, the appearance of a particular molecular lesion, we studied 33 benign and malignant human radiation-associated thyroid tumors. We used polymerase chain reaction (PCR) amplification and allele-specific hybridization with mutant-specific probes for the three ras genes and the gsp oncogene. Compared to 85 'spontaneous' human thyroid tumors, the radiation-associated cases: (1) show a similar overall frequency of ras and gsp mutations (about 30% and 6% respectively); (2) present a similar frequency of mutation of the three ras genes without any predominance in adenomas and papillary carcinomas and (3) all Ki-ras mutations were found in papillary carcinomas (4/15). ras and gsp genes were never found mutated simultaneously, suggesting an alternative role for both oncogenes in the thyroid tumorigenic radiation-associated process.

gsp mutations in human thyroid tumours.

The presence of gsp mutations at codons 201 and 227 in the gene coding for the alpha subunit of the GTP-binding Gs protein which stimulates adenylyl cyclase (AC) has been investigated in 31 samples of differentiated thyroid tumours, which had been previously characterized with respect to their adenylyl cyclase activity (ACA) before and after stimulation by thyroid-stimulating hormone (TSH). Polymerase chain reaction (PCR) amplification of DNA extracted from these tumours, followed by high stringency oligonucleotide probing, enabled the detection of mutations in three samples originating from tumours with high constitutive ACA, which was not significantly further stimulated by TSH. Two mutations were at codon 227 and replaced Gln227 by His or Lys, and one was at codon 201, with the substitution of Arg201 by Ser. Because thyrocytes belong to the subset of differentiated cells which are programmed to proliferate in response to elevated cAMP levels, the gsp mutations observed in some differentiated thyroid carcinomas probably contributed to their tumorigenic phenotype.

Presence of mutations in all three ras genes in human thyroid tumors.

Polymerase chain reaction (PCR) amplification followed by oligonucleotide probing was used to investigate the presence of ras genes mutations in human thyroid adenomas and carcinomas. The results confirm the frequent occurrence of mutations in all three ras genes in both adenomas and carcinomas, in agreement with the hypothesis that the ras mutations may constitute early steps in thyroid tumorigenesis. No evident correlation between the frequency of ras mutations, the identity of the mutated ras gene, the position affected in the ras gene or the type of mutation and the pathological features is apparent. However, definitive conclusion on this point is precluded because of the small number of tumors examined at the present time.

Detection of activated ras oncogenes in human thyroid carcinomas.

Focus formation following DNA transfection of mouse 3T3-Vill cells was used to search for the presence of activated oncogenes in human thyroid tumors. Oncogenes belonging to the ras family were detected in four out of six thyroid carcinomas (Ki-ras in one anaplastic tumor and one follicular moderately differentiated tumor and Ha-ras and N-ras in two papillary tumors). Normal thyroid tissue samples obtained from two patients, one with an anaplastic tumor and one with a benign adenoma, and samples from 4 benign adenomas and from one toxic goiter of a patient with Graves' disease gave negative results. In one case, restriction enzyme analysis demonstrated the presence of a mutation in codon 12 of the activated Ha-ras oncogene. Our data show that all three ras proto-oncogenes can become activated in malignant thyroid tumors.

Differential adhesiveness of rhabdomyosarcoma-derived cloned metastatic cell lines to vascular endothelial monolayers.

A series of 11 cloned cell lines derived from a primary, nickel-induced rat rhabdomyosarcoma was evaluated for their metastatic capacity (number of lung colonies following i.v. injection) and attachment kinetics to confluent pig endothelial cell monolayers grown in vitro. The morphology of the adhering cells was also studied by optical and scanning electron microscopy. Cells from all lines tested began to attach to the endothelial monolayers within 15 min of incubation at 37 degrees C, with 64% to 93% of the cells adhering after 2 h. Attachment rates at 30 min ranged from 29 to 48% for four lines classed as "weakly adhesive" (attachment, less than 50%) and from 53 to 78% for seven lines classed as "highly adhesive" (attachment, greater than 50%). Four clones of five displaying low lung-colonizing capacities also showed low attachment rates to endothelial monolayers in vitro. All of six highly colonizing lines studied had high attachment rates. A degree of positive correlation was observed between the amount of cell surface fibronectin as evaluated by immunofluorescence and the early phase attachment rates (and lung-colonizing capabilities) of the different cloned cell lines. Early (15 min) attachment of tumor cells to isolated extracellular matrix preparations proceeded at higher rates than to endothelial monolayers, and previously detected differences between high- and low-colonizing clones were less evident with these matrix substrates. Our results suggest possible interrelationships between specific cell adhesion properties and the metastatic potential of blood-borne tumor cells.

[In vivo regulation by calcium of thyroid metabolism of iodine in rats]. / Régulation, in vivo, par le calcium, du métabolisme thyroïdien de l'iode, chez le rat.

In vivo experiments using a calcium deficient or a normal diet, proved that calcium enhance iodine entry and concentration into the thyroid gland of the rat and the intra-thyroidal T4 pool, T4 level in serum was also increased. Peripheral metabolism of T4, serum TSH levels and pituitary TSH contents were unaffected. Calcium has a local regulatory function on the thyroid gland.