The beta-thymosins comprise a family of structurally related, highly conserved acidic polypeptides, originally isolated from calf thymus. Recently, we have demonstrated the overexpression of thymosin beta-10 (TB10) in rat thyroid transformed cell lines and in human thyroid carcinoma tissues and cell lines. To verify whether TB10 overexpression is a general event in the process of carcinogenesis, we have analyzed TB10 mRNA levels in human colon carcinomas, germ cell tumors of different histological types, breast carcinomas, ovarian carcinomas, uterine carcinomas, colon and esophageal carcinoma cell lines. Overexpression of the TB10 gene was detected in all of the neoplastic tissues and cell lines compared to the respective normal tissues. Moreover, the mouse model of skin carcinogenesis induced by the combined action of chemical carcinogens and phorbol esters was used to identify the stage of TB10 gene induction. The expression was almost undetectable in normal keratinocytes, its induction occurred even at the papilloma stage, however a further increased expression was observed in the carcinoma derived cell lines. Finally, immunohistochemical analysis of some breast, colon and ovary carcinoma samples by using specific anti-TB10 antibodies revealed the presence of the TB10 protein in all of the neoplastic tissues, but not in the respective normal tissues. Therefore the TB10 detection may be considered a potential tool for the diagnosis of several human neoplasias.

We used subtractive library screening to identify the changes that occur in gene expression during thyroid cell neoplastic transformation. Complementary DNA from normal thyroid cells (HTC 2) was subtracted from a complementary DNA library constructed from a human thyroid papillary carcinoma cell line. The library was screened for genes upregulated in human thyroid papillary carcinoma cell line cells, and several cDNA clones were isolated. One of these clones has a sirtuin core and high homology with the human silent information regulator protein family. This clone, designated 'SIR-T8', was overexpressed in human thyroid carcinoma cell lines and tissues, but not in adenomas. The human SIR-T8 protein has a molecular weight of 39 kDa and is primarily located in the cytoplasm under the nuclear membrane. The SIR-T8 gene is located on chromosome 17q25-1.

Germ-line missense mutations of the receptor-like tyrosine kinase ret are the causative genetic event of the multiple endocrine neoplasia (MEN) type 2A and type 2B syndromes and of the familial medullary thyroid carcinoma. We have used the rat pheochromocytoma cell line, PC12, as a model system to investigate the mechanism or mechanisms by which expression of activated ret alleles contributes to the neoplastic phenotype in neuroendocrine cells. Here we show that stable expression of ret mutants (MEN2A and MEN2B alleles) in PC12 cells causes a dramatic conversion from a round to a flat morphology, accompanied by the induction of genes belonging to the early as well as the delayed response to nerve growth factor. However, in the transfected PC12 cells, the continuous expression of neuronal specific genes is not associated with the suppression of cell proliferation. Furthermore, expression of ret mutants renders PC12 cells unresponsive to nerve growth factor-induced inhibition of proliferation. These results suggest that induction of an aberrant pattern of differentiation, accompanied by unresponsiveness to growthinhibitory physiological signals, may be part of the mechanism of action of activated ret alleles in the pathogenesis of neuroendocrine tumors associated with MEN2 syndromes.Multiple endocrine neoplasia (MEN) types 2A and 2B are hereditary neoplastic syndromes characterized by the presence of medullary thyroid carcinomas and pheochromocytomas. MEN2B is also associated with skeletal abnormalities, ganglioneuromas of the intestinal tract, and mucosal neuromas.Familial medullary thyroid carcinoma is a related cancer disorder characterized by medullary thyroid carcinoma in the absence of pheochromocytoma (1, 2). Mutations in one of five cysteine residues in the extracellular domain of the ret gene, which encodes a ligand-orphan receptor-like tyrosine kinase (3), are the genetic cause of familial medullary thyroid carcinoma and MEN2A syndromes (4, 5). A single point mutation within the ret catalytic domain, which results in a Thr for Met substitution at codon 918, is responsible for the MEN2B syndrome (6, 7). These mutations convert ret into a dominant transforming gene (retMEN2A and retMEN2B alleles) by causing constitutive activation of the intrinsic tyrosine kinase activity of its product (8, 9). However, retMEN2A and retMEN2B differ in their mechanism of activation. In the case of retMEN2A, activation likely results from constitutive receptor dimerization, whereas retMEN2B proteins do not dimerize, but display altered substrate specificity (9, 10).The biological activity of retMEN2 alleles has so far been studied in NIH 3T3 cells, a system prone to "one-hit" transformation. The molecular mechanisms by which ret alleles contribute to the development of neuroendocrine cancer syndromes remain to be elucidated. In MEN2 syndromes, the first step toward neoplastic progression is a generalized hyperplasia of the entire population of thyroid C-cells and of the adrenal medulla chromaffin cells (11). Hyperpla...

The synthesis and cytotoxic evaluation of 3-(alkyl)(alkyl-substituted)spiro[(dihydroimidazo-2,4-dione)-5,3'-(2',3'-dihydrothieno[2,3-b]naphtho-4',9'-dione)]derivatives are described. Evaluation of these analogues against the MCF-7 human breast carcinoma and SW 620 human colon carcinoma cell lines uncovered for most of the compounds a cytotoxic potency comparable to or greater than that of doxorubicin. Compound 15 exhibited remarkable cytotoxic activity against several other human solid tumor cell lines. Interestingly, only a partial cross-resistance to compound 15 in selected tumor cell sublines known to be resistant to doxorubicin (MCF-7/Dx and A2780/Dx) was observed, whereas a total absence of cross-resistance in a tumor cell subline selected for resistance to cisplatin was found (A2780/DDP).

Specific germline mutations of the receptor tyrosine kinase, Ret, predispose to multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma. The mechanisms by which different Ret isoforms (Ret-2A and Ret-2B) cause distinct neoplastic diseases remain largely unknown. On the other hand, forced expression of these mutated versions of Ret induces the rat pheochromocytoma cell line, PC12, to differentiate. Here we used an inducible vector encoding a dominantnegative Ras (Ras p21 N17 ) to investigate the contributions of the Ras pathway to the phenotype induced in PC12 cells by the expression of either Ret-2A or Ret-2B mutants. We show that the Ret-induced molecular and morphological changes are both mediated by Ras-dependent pathways. However, even though inhibition of Ras activity was sufficient to revert Ret-induced differentiation, the kinetics of morphological reversion of the Ret-2B-was more rapid than the Ret-2A-transfected cells. Further, we show that in Ret-transfected cells the suc1-associated neurotrophic factor-induced tyrosine phosphorylation target, SNT, is chronically phosphorylated in tyrosine residues, and associates with the Sos substrate. These results indicate the activation of the Ras cascade as an essential pathway triggered by the chronic active Ret mutants in PC12 cells. Moreover, our data indicate SNT as a substrate for both Ret mutants, which might mediate the activation of this cascade.

Thyroid papillary carcinomas are characterized by RET/ PTC rearrangements that cause the tyrosine kinase domain of the RET receptor to fuse with N-terminal sequences encoded by heterologous genes. This results in the aberrant expression of a ligand-independent and constitutively active RET kinase. We analysed actin reorganization induced by the RET/PTC1 oncogene in PC Cl 3 rat thyroid epithelial cells. Di erently from oncogenes Src, Ras and Raf, RET/PTC1 caused actin ®laments to form prominent stress ®bers. Moreover, stress ®bers were identi®ed in human thyroid papillary carcinoma cell lines harboring RET/PTC1 rearrangements but not in thyroid carcinoma cells negative for RET/PTC rearrangements. RET/MEN 2A, a constitutively active but unrearranged membrane-bound RET oncoprotein, did not induce stress ®bers in PC Cl 3 cells. Induction of stress ®bers by RET/PTC1 was restricted to thyroid cells; it did not occur in NIH3T3 ®broblasts or MCF7 mammary cells. RET/PTC1-mediated stress ®ber formation depended on Rho but not Rac small GTPase activity. In addition, inhibition of Rho, but not of Rac, caused apoptosis of RET/PTC1-expressing thyroid cells. We conclude that Rho is implicated in the actin reorganization and cell survival mediated by the chimeric RET/PTC1 oncogene in thyroid epithelial cells, both phenotypes being cell type-and oncogene typespeci®c. Oncogene (2001) 20, 6973 ± 6982.

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Oncogenic variants of the receptor tyrosine kinase, Ret, cause formation of tumors of neuroendocrine derivation in the multiple endocrine neoplasia type 2 and, thus, likely interfere with antiproliferative and/or differentiative extracellular signals. Here we took advantage of two rat pheochromocytoma-derived cell lines (PC12/MEN2A and PC12/MEN2B) to investigate whether Ret-induced nerve growth factor (NGF) unresponsiveness might involve impairment of ERK signaling. In fact, these cells, stably transfected with distinct forms of the active ret oncogene, fail to block proliferation, even upon NGF stimulation. In these cells we show the presence of both chronic ERKs activity and high expression levels of MKP-3, an ERK-specific phosphatase. Despite the presence of MKP-3, ERK activity can be further stimulated by NGF, but it fails to translocate into the nucleus and consequently to induce immediate-early gene transcription. Because of the presence of MKP-3, our results suggest the existence of a negative regulatory feedback acting on ERKs as a mechanism responsible for the abrogation of NGF-induced terminal differentiation. Indeed, MKP-3 seems to be implicated in the persistence of ERKs in cell cytoplasm. This interpretation is further supported by the observation that in ret-transfected cells, forced expression of an active form of MEK-1 may overcome this block; it restores transcription from the c-fos promoter, induces translocation of ERKs into the nucleus, and inhibits cell proliferation.Ret is a receptor tyrosine kinase whose expression is restricted to neuronal cells of the central and peripheral nervous system. Recently four ligands have been described for this receptor as follows: glial cell-derived neurotrophic factor, neurturin, artemin, and persephin (1).Germ line mutations of the receptor tyrosine kinase, Ret, are responsible for the multiple endocrine neoplasia (MEN) 1 type 2A and 2B syndrome and for the familial medullary thyroid carcinoma (2-5). MEN-2A and MEN-2B are distinct hereditary neoplastic syndromes both characterized by the presence of medullary thyroid carcinomas and pheochromocytomas (6, 7). Missense mutations at one of five cysteine residues (Cys-609, -611, -618, -620, and -634) clustered in the extra cytoplasmic domain of ret are the most frequent causative genetic events of familial medullary thyroid carcinoma and the MEN-2A syndrome (8). A single point mutation, which results in a Thr for Met substitution at codon 918 within the Ret catalytic domain, is responsible for the MEN-2B syndrome (8). These mutations convert ret into a dominant transforming gene and cause constitutive activation of its intrinsic tyrosine kinase activity, although their mechanism of activation differs (8 -10). In MEN-2 syndromes, the molecular mechanisms by which the mutated Ret contribute to the development of neuroendocrine neoplasms remain largely unknown. Indeed, the inheritance of mutated ret alleles implicates them in the pathogenesis of a generalized hyperplasia of the entire population of thyroid C-cells...