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Deregulation of MYC expression occurs in many haematological malignancies. Previous studies modelling MYC-induced lymphomagenesis in the mouse used transgenic vectors that directed MYC overexpression in a lineage-specific manner. Here, we describe a transgenic mouse strain in which constitutive MYC expression is driven broadly in haemopoiesis by a vector containing regulatory elements of the Vav gene. Healthy young VavP-MYC17 mice had multiple haemopoietic abnormalities, most notably increased size and numbers of B-lymphoid cells, monocytes and megakaryocytes. The mice rapidly developed tumours and, surprisingly, these were exclusively T-cell lymphomas, mostly of mature CD4(+) CD8(-) T cells, a tumour type that is seldom seen in mouse models. To examine tumour development in the absence of the susceptible T cells, we bred VavP-MYC17 mice lacking the Rag1 recombinase. They survived longer and succumbed to tumours of several different haemopoietic cell types: pre-T cells, pro-B cells, macrophages and unusual progenitor cells. Thus, although T-lineage cells have the shortest latent period to transformation, the VavP-MYC17 transgene drives malignant transformation of multiple cell types and VavP-MYC17 mice provide a new model for tumours of multiple haemopoietic lineages.

Gain-of-function mutations in the gene encoding the receptor tyrosine kinase RET have been identified as the aetiological factor for multiple endocrine neoplasia type 2A (MEN2A). MEN2A is a dominantly-inherited cancer predisposition syndrome characterized by medullary thyroid carcinoma, a tumour of the calcitonin-producing thyroid C-cells. There are three isoforms of RET: RET9, RET43 and RET51, and although in vitro evidence suggests they vary in cellular transformation activities, little is known about their function in tumorigenesis in vivo. To address this, we used RET51 cDNA to construct mice in which the most frequent MEN2A mutation, Cys-634-Arg, was expressed under the control of the human calcitonin promoter (CT-2A mice). These mice developed C-cell tumours resembling human MTC and follicular tumours resembling human papillary thyroid carcinoma (PTC) depending on the founder line examined. One founder line developed compound MTC/PTC at low frequency (8%) and pancreatic cystadenocarcinoma. CT-2A mice also displayed a developmental defect in thyroid follicular structure, in which much of the thyroid was occupied by large irregular cystic follicles thought to be derived from the ultimobranchial body, a developmental precursor of the thyroid gland. The CT-2A mice will provide a suitable model to further study the effects of the MEN 2A RET mutation in vivo.

Medullary thyroid carcinoma (MTC) is a neuroendocrine tumor of the calcitonin secreting thyroid C-cells. Somatic and germline mutations in the RET proto-oncogene are associated with sporadic and inherited cases of MTC, respectively. The human MTC cell line, TT, can be differentiated by activated raf-1. This differentiation is characterized, in part, by down-regulation of the RET proto-oncogene. We now show that raf-1 induction is followed by activation of the downstream kinases MEK1/2 and ERK1/2 and that differentiation is dependent on activation of MEK1/2. The concurrent down-regulation of RET appears to involve altered nuclear compartmentalization and transport of RET mRNA. Although RET is down-regulated during raf-1 mediated differentiation, overexpression of activated RET alleles which resist down-regulation does not alter the raf-1 mediated differentiation response. These data suggest that RET down-regulation is associated with, but not required, for raf-1 mediated MTC cell differentiation and that the raf-1 signal transduction pathway plays a dominant role in promoting MTC cell differentiation.

Germline mutations of RET gene, encoding a receptor tyrosine kinase, have been associated with the MEN2A and MEN2B inherited cancer syndromes. In MEN2A mutations affecting cysteine residues in the extracellular domain of the receptor cause constitutive activation of the tyrosine kinase by the formation of disulfide-bonded homodimers. In MEN2B a single mutation in the tyrosine kinase domain (Met918Thr) has been identified. This mutation does not lead to dimer formation, but has been shown (both biologically and biochemically) to cause ligand-independent activation of the Ret protein, but to a lesser extent than MEN2A mutations. Intramolecular activation by cis-autophosphorylation of RetMEN2B monomers has been proposed as a model for activation, although alternative mechanisms can be envisaged. Here we show that the activity of RetMEN2B can be increased by stable dimerization of the receptor. Dimerization was achieved experimentally by constructing a double mutant receptor with a MEN2A mutation (Cys634Arg) in addition to the MEN2B mutation, and by chronic exposure of RetMEN2B-expressing cells to the Ret ligand GDNF. In both cases full activation of RetMEN2B, measured by 'in vitro' transfection assays and biochemical parameters, was seen. These results indicate that the MEN2B phenotype could be influenced by the tissue distribution or concentration of Ret ligand(s).

Germline mutations in the RET proto-oncogene are seen in the majority of patients with the dominantly inherited cancer syndromes multiple endocrine neoplasia type 2 (MEN 2). The clinical subtypes of MEN 2 (MEN 2A, MEN 2B and familial MTC) all have medullary thyroid carcinoma, but vary in the involvement of pheochromocytoma, parathyroid adenoma/hyperplasia and developmental abnormalities. A single RET mutation, resulting in the substitution M918T, has been identified in 94% of cases of MEN 2B (which consists of MTC, pheochromocytoma and developmental abnormalities). Here we report the identification of a new germline RET mutation (A883F) in two de novo cases of MEN 2B. Identification of this new mutation will contribute to understanding the molecular basis of MEN 2B, and will assist in the clinical management of families harbouring this mutation.

Activating germline mutations of the RET receptor tyrosine kinase are found in the majority of cases of inherited cancer syndrome MEN 2, and inactivating mutations in some cases of dominantly inherited Hirschsprung disease. Using RET activated by a MEN 2 mutation, we show that both the SH2 and PTB domains of the adaptor protein Shc interact with RET, and we identify the PTB domain interaction site. Interaction with both the SH2 and PTB domains of Shc contributes to the transcriptional activation of a serum response element. RET alternate splicing affects the strength of interaction with both the Shc SH2 and PTB domains. In addition, a splice isoform-specific HSCR missense mutation, which does not inactivate the RET kinase activity, decreases the strength of the PTB domain interaction and the level of RET-dependent Shc phosphorylation.

The RET proto-oncogene encodes a receptor tyrosine kinase (TK). It has been shown that distinct germline mutations in the RET proto-oncogene are associated with the dominantly inherited cancer syndromes multiple endocrine neoplasia type 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma (FMTC) as well as Hirschsprung disease (HSCR), a congenital disorder characterised by absent enteric innervation. In this study, we have transfected NIH3T3 and PC12 phaeochromocytoma cells with MEN2A (Cys634-> Arg) and MEN2B (Met918-> Thr) RET constructs. Both caused transformation of the NIH3T3 cells and differentiation of PC12 cells. The Ret (MEN2A) and Ret (MEN2B) proteins were constitutively phosphorylated on tyrosine, and their in vitro kinase activity was significantly higher than that of the wild type protein. The MTC cell line TT carries a CYs634-> Trp MEN2A mutation, and we have shown by immunoelectronmicroscopy that Ret is clustered on the cell surface in a manner reminiscent of ligand-induced aggregation of cell surface receptors. RET is activated, as RET/PTC oncogene, by somatic rearrangements which link the TK domain to a constitutive dimerization interface in papillary thyroid carcinomas. We have compared the biological and biochemical activity of the TK domains of the wild type and MEN 2B Ret in the context of the RET/PTC. The results show that the MEN 2B mutation significantly increases the TK domain enzymatic activity suggesting that dimerization may be still necessary for MEN 2B Ret to express its full activity.

The RET proto-oncogene encodes a receptor tyrosine kinase. We and others have recently shown that distinct germline mutations of the RET proto-oncogene account for the majority of cases of the dominantly inherited multiple endocrine neoplasia (MEN) type 2 syndromes, and can cause a dominantly inherited form of Hirschsprung disease, a disorder of development of the autonomic innervation of the gut. RET is also oncogenically activated in some sporadic thyroid and adrenal tumours. Here we report the characterisation of multiple mRNA isoforms of RET generated by alternate splicing. Two isoforms are predicted to encode membrane-spanning receptors with a truncated extracellular ligand-binding domain. A third isoform is predicted to encode a soluble, secreted form of the receptor. These mRNA isoforms are expressed in both normal and tumour tissues.

Germline mutations within one of six codons of the RET proto-oncogene account for the majority of cases of multiple endocrine neoplasia (MEN) type 2A and type 2B and familial medullary thyroid carcinoma (FMTC). MEN 2A and FMTC mutations characterised thus far occur exclusively in the cysteine-rich domain of the extracellular region of RET. We now report a missense mutation in the intracellular tyrosine kinase domain of RET in the germline of a family with FMTC that does not have a cysteine codon mutation. In this family, the mutation, which alters GAG (Glu) to GAC (Asp) at codon 768, segregates with the FMTC phenotype. The same mutation was also detected in sporadic MTC but not in corresponding constitutional DNA, confirming that it is likely to be of pathological significance rather than a rare polymorphism.