[The RET gene in thyroid pathology]. / Le gène RET en pathologie thyroïdienne.

The RET proto-oncogene encodes a receptor tyrosine kinase which plays a crucial role during the embryonic development of the enteric nervous system and of the kidney. Cytogenetic analyses of papillary thyroid carcinoma (PTC), a neoplasm which originates from thyrocytes, have revealed that somatic rearrangements of the RET gene are involved in the etiology of a significant proportion of this tumour. Medullary thyroid carcinoma (MTC) which arises from neural-crest derived C-cells is the cardinal disease feature of multiple endocrine neoplasia type 2 (MEN 2), a dominantly inherited cancer syndrome. Recent studies have provided evidence that germline mutations of the RET gene are the underlying genetic events responsible for MEN 2. This review focuses on the role of RET mutations in the pathogenesis of PTC and MTC and summarizes our present knowledge on the consequences of these alterations on the RET tyrosine kinase function. We further describe a transgenic mouse model for hereditary MTC. Mice carrying a MEN 2A allele of RET under the control of the CGRP/calcitonin promoter develop bilateral and multifocal MTC, morphologically and biologically similar to human MTC.

Development of medullary thyroid carcinoma in transgenic mice expressing the RET protooncogene altered by a multiple endocrine neoplasia type 2A mutation.

Multiple endocrine neoplasia type 2 (MEN 2) is a dominantly inherited cancer syndrome that comprises three clinical subtypes: MEN type 2A (MEN-2A), MEN type 2B (MEN-2B), and familial medullary thyroid carcinoma (FMTC). Medullary thyroid carcinoma (MTC), a malignant tumor arising from calcitonin-secreting thyroid C cells, is the cardinal disease feature of this syndrome, and mortality in affected MEN-2 patients is mainly caused by this malignancy. Germ-line mutations of the RET protooncogene, which encodes a receptor tyrosine kinase, are responsible for these three neoplastic-prone disorders. MEN2 mutations convert the RET protooncogene in a dominantly acting oncogene as a consequence of the ligand-independent activation of the tyrosine kinase. The majority of MEN2A and FMTC mutations are located in the extracellular domain and cause the replacement of one of five juxtamembrane cysteines by a different amino acid. To examine whether expression of a MEN2A allele of RET results in transformation of C cells, we have used the transgenic approach. Expression of the RET gene altered by a MEN2A mutation was targeted in C cells by placing the transgene under the control of the calcitonin gene-related peptide/calcitonin promoter. Animals of three independent transgenic mouse lines, which expressed the transgene in the thyroid, displayed overt bilateral C cell hyperplasia as early as 3 weeks of age and subsequently developed multifocal and bilateral MTC. Moreover, these tumors were morphologically and biologically similar to human MTC which afflicts MEN2 individuals. These findings provide evidence that the MEN2A mutant form of RET is oncogenic in parafollicular C cells and suggest that these transgenic mice should prove a valuable animal model for hereditary MTC.

[Neural crest and multiple endocrinopathies]. / Crête neurale et polyendocrinopathies.

Multiple endocrine neoplasia type 2 (MEN 2) is a cancer syndrome which comprises three related disorders, MEN type 2A (MEN 2A), type 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC), MEN 2A is characterized by the association of MTC, a tumour arising from thyroid C-cells, pheochromocytoma and parathyroid hyperplasia. In addition to the thyroid cancer, MEN 2B associates pheochromocytoma, mucosal neuromas, ganglioneuromatosis of the digestive tract and skeletal abnormalities. In FMTC, the MTC is the sole clinical manifestation. MEN 2 is a dominantly inherited neural crest disorder caused by germline mutations of the RET proto-oncogene. The RET gene encodes a receptor tyrosine kinase, which displays a cadherin-like domain and a cysteine rich motif in its extracellular part. Missense mutations at one of five cysteines clustered in the extra-cytoplasmic domain of RET have been identified in the majority of the MEN 2A families and in two-thirds of FMTC. A single point mutation leading to the replacement of a methionine by a threonine within the tyrosine kinase domain has been detected in almost all cases of MEN 2B. We have screened 170 french MEN 2 families and a germline mutations in the RET gene have been identified in 92% of cases. Moreover, we confirmed the significant correlation between the nature, the position of the RET mutations and the clinical phenotype. The accurate identification by DNA testing of individual predisposed to MEN 2 suggests new protocols of treatment. Thyroidectomy as early as 6 years of age in individuals with MEN 2 mutations has been recently advocated by clinicians. We further provide evidence that MEN 2A and MEN 2B mutations convert the RET proto-oncogene in a dominantly-acting transforming gene due to the ligand-independent constitutive activation of the tyrosine kinase. Finally, we have constructed transgenic mice carrying the RET gene carrying a MEN 2A mutation fused to the calcitonin gene related peptide/calcitonin promoter. Animals of three independent transgenic lines developed C-cell hyperplasia and subsequently MTC with a complete penetrance. Taken together, these findings indicate that MEN 2A form of RET is oncogenic in thyroid C-cells, and suggest that these transgenic animals should prove a valuable model for hereditary MTC. Future work should yield insights in the signaling pathways subverted by the RET-MEN 2 proteins.

Thyroid pathologies in transgenic mice expressing a human activated Ras gene driven by a thyroglobulin promoter.

Four transgenic mice carrying the human activated c-Ha-Ras gene, the expression of which was driven into the thyroid gland by a bovine thyroglobulin promoter, have been produced. The M1 and M2 mice developed papillary thyroid carcinomas and the M2 mouse also developed a lung carcinoma, however none of them transmitted the transgene. Both the M3 and the M4 mice gave rise to transgenic lines. M3 progeny mice develop a goitre with morphological aspects of hyperplasia as well as a thymus hyperplasia. M4 developed a papillary thyroid carcinoma and a lung carcinoma. Lung tumors but not thyroid tumors were observed in M4 adult transgenic progeny. In this M4 line, thyroid dysgenesis leading to growth retardation and premature death was observed upon serial backcross that enhanced the DBA/2J genetic background. The development of thyroid tumors in M1, M2, M4 transgenic mice demonstrates the oncogenic potential of activated Ras gene in the thyroid gland. The M4 line raises interesting questions relative to the interference between the Ras-mediated signal transduction pathway and thyroid morphogenesis.

Oncogenic potential of guanine nucleotide stimulatory factor alpha subunit in thyroid glands of transgenic mice.

Transgenic mice have been used to address the issue of the oncogenic potential of mutant guanine nucleotide stimulatory factor (Gs) alpha subunit in the thyroid gland. The expression of the mutant Arg-201-->His Gs alpha subunit transgene has been directed to murine thyroid epithelial cells by bovine thyroglobulin promoter. The transgenic animals develop hyperfunctioning thyroid adenomas with increased intracellular cAMP levels and high uptake of [125I]iodine and produced elevated levels of circulating triiodothyronine and thyroxine. These animals demonstrate that the mutant form of Gs alpha subunit carries an oncogenic activity, thus supporting the model that deregulation of cAMP level alters growth control in thyroid epithelium. These animals represent models for humans with autonomously functioning thyroid nodules.

Biological effects of asbestos fibres on rat lung maintained in vitro.

The in vitro systems used to determine whether asbestos acts as an initiator or as a promoter have failed to give definitive answers. We studied the effect of chrysotile and crocidolite in an initiation-promotion model on the Fischer rat embryo lung. Two assay systems were used in succession: organ culture of the lung cultured for 24 days and epithelial cell culture derived from treated or untreated explants cultured for 25 passages. Apart from the control groups, three major groups were analysed: (1) fibres with complete carcinogenic potency: explants and/or cells treated with fibres alone; (2) fibres with initiating potency; short treatment with fibres, followed by treatment with the classical promoter TPA; (3) fibres with promoting potency: short benzo[a]pyrene treatment followed by treatment with the fibres. In organ culture, fibres alone induce only cytotoxic lesions; in the 'fibres with promoting potency' group, precancerous lesions were observed. In epithelial cell culture, several transformation criteria are analysed. Our results with the cell system confirm that fibres act as a promoter, but also as a complete carcinogen. However, for equal doses, crocidolite needs a longer treatment time than chrysotile. These different assays failed to demonstrate any initiating activity of the fibres. The use of organ and cell culture in succession makes it possible to demonstrate the in vitro promoting effect of chrysotile and crocidolite.