Michele Grieco

PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas

We recently detected a novel activated oncogene by transfection analysis on NIH 3T3 cells in five out of 20 primary human thyroid papillary carcinomas and in the available lymph node metastases. We designated this transforming gene PTC (for papillary thyroid carcinoma). Here we describe the molecular cloning and sequencing of the gene. The new oncogene resulted from the rearrangement of an unknown amino-terminal sequence to the tyrosine kinase domain of the ret proto-oncogene. This gene rearrangement was detected in all of the transfectants and in all of the original tumor DNAs, but not in normal DNA of the same patients, thus indicating that this genetic lesion occurred in vivo and is specific to somatic tumors. Moreover, the transcript coded for by the fused gene was detected in an additional PTC-positive human papillary carcinoma for which mRNA was available.

Ret oncogene activation in human thyroid neoplasms is restricted to the papillary cancer subtype.

We have recently reported the activation of a new oncogene in human papillary thyroid carcinomas. This oncogene, papillary thyroid carcinoma (PTC), is a novel rearranged version of the ret tyrosine-kinase protooncogene. Thyroid neoplasms include a broad spectrum of malignant tumors, ranging from well-differentiated tumors to undifferentiated anaplastic carcinomas. To determine the frequency of ret oncogene activation, we analyzed 286 cases of human thyroid tumors of diverse histologic types. We found the presence of an activated form of the ret oncogene in 33 (19%) of 177 papillary carcinomas. By contrast, none of the other 109 thyroid tumors, which included 37 follicular, 15 anaplastic, and 18 medullary carcinomas, and 34 benign lesions, showed ret activation.

One- and two-step transformations of rat thyroid epithelial cells by retroviral oncogenes.

A system of epithelial cells is described in which it is possible to study the number and the nature of genes capable of conferring the malignant phenotype. Two fully differentiated, hormone-responsive cell lines from rat thyroid glands are presented which are susceptible to one-step or two-step transformation upon infection with several murine acute retroviruses. After infection, both cell lines became independent from their thyrotropic hormone requirement for growth. However, complete transformation was achieved with one of the cell lines (FRTL-5 Cl 2), whereas the other cell line (PC Cl 3) failed to grow in agar and to give rise to tumors in vivo. The latter cell line was susceptible to complete transformation upon cooperation of the v-ras-Ha and the human c-myc oncogenes.

An epidermal growth factor receptor/ret chimera generates mitogenic and transforming signals: evidence for a ret-specific signaling pathway.

A chimeric expression vector which encoded for a molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) and the intracellular domain of the ret kinase (EGFR/ret chimera) was generated. Upon ectopic expression in mammalian cells, the EGFR/ret chimera was correctly synthesized and transported to the cell surface, where it was shown capable of binding EGF and transducing an EGF-dependent signal intracellularly. Thus, the EGFR/ret chimera allows us to study the biological effects and biochemical activities of the ret kinase under controlled conditions of activation. Comparative analysis of the growth-promoting activity of the EGFR/ret chimera expressed in fibroblastic or hematopoietic cells revealed a biological phenotype clearly distinguishable from that of the EGFR, indicating that the two kinases couple with mitogenic pathways which are different to some extent. Analysis of biochemical pathways implicated in the transduction of mitogenic signals also evidenced significant differences between the ret kinase and other receptor tyrosine kinases. Thus, the sum of our results indicates the existence of a ret-specific pathway of mitogenic signaling.

Involvement of RET oncogene in human tumours: specificity of RET activation to thyroid tumours

Non-thyroid neoplasia were analysed by Southern blot of genomic DNA and DNA prepared by reverse transcription and amplification by polymerase chain reaction (RT/PCR) for the activation of the RET oncogene. It is known that the rearrangement of RET occurs in about 10%-20% of human thyroid papillary carcinomas. None of 528 non-thyroid tumours showed rearrangement of the RET proto-oncogene, whereas three out of 30 thyroid papillary carcinomas were positive for RET activation. Therefore the activation of RET seems to be a somatic cell mutation specific to human thyroid carcinomas.

Beta- and gamma-catenin expression in thyroid carcinomas

Cadherins are calcium‐dependent cell–cell adhesion molecules whose intracellular domain forms a complex with proteins required for their function, called catenins. Down‐regulation of cadherins has frequently been detected in many types of human carcinomas, being associated with tumour progression. The present study investigates the immunohistochemical expression of E‐cadherin and beta‐ and gamma‐catenin in 27 human thyroid carcinomas. E‐cadherin immunoreactivity was found to be decreased at cell–cell contacts in 8/15 (53 per cent) papillary, 5/7 (71 per cent) follicular, and 5/5 (100 per cent) anaplastic carcinomas. Beta‐catenin membrane localization was found to be decreased in 6/15 (40 per cent) papillary, 2/7 (28 per cent) follicular, and 5/5 (100 per cent) anaplastic carcinomas. Gamma‐catenin expression was partially or totally lost in 13/15 (86 per cent) papillary, 6/7 (85 per cent) follicular, and 5/5 (100 per cent) anaplastic carcinomas. A normal pattern of expression for these three molecules was observed in areas of normal tissue in each sample. These data indicate that in addition to E‐cadherin, catenins are also down‐regulated at cell–cell junctions in thyroid tumours and could represent potentially useful differentiation and/or transformation markers. The high frequency of alterations of gamma‐catenin expression found in thyroid carcinomas suggests an important role for this gene product in thyroid carcinogenesis.

H4(D10S170), a gene frequently rearranged with RET in papillary thyroid carcinomas: functional characterization

Human thyroid papillary carcinomas are characterized by rearrangements of the RET protooncogene with a number of heterologous genes, which generate the RET/papillary thyroid carcinoma (PTC) oncogenes. One of the most frequent variants of these recombination events is the fusion of the intracellular kinase-encoding domain of RET to the first 101 amino acids of a gene named H4(D10S170). We have characterized the H4(D10S170) gene product, showing that it is a ubiquitously expressed 55 KDa nuclear and cytosolic protein that is phosphorylated following serum stimulation. This phosphorylation was found to depend on mitogen-activated protein kinase (MAPK) Erk1/2 activity and to be associated to the relocation of H4(D10S170) from the nucleus to the cytosol. Overexpression of the H4(D10S170) gene was able to induce apoptosis of thyroid follicular epithelial cells; conversely a carboxy-terminal truncated H4(D10S170) mutant H4(1–101), corresponding to the portion included in the RET/PTC1 oncoprotein, behaved as dominant negative on the proapoptotic function and nuclear localization of H4(D10S170). Furthermore, conditional expression of the H4(D10S170)-dominant negative truncated mutant protected cells from stress-induced apoptosis. The substitution of serine 244 with alanine abrogated the apoptotic function of H4(D10S170). These data suggest that loss of the H4(D10S170) gene function might have a role in thyroid carcinogenesis by impairing apoptosis.

Thyrotropin receptor gene expression in oncogene-transfected rat thyroid cells: Correlation between transformation, loss of thyrotropin-dependent growth, and loss of thyrotropin receptor gene expression

Rat FRTL-5 and PC-Cl-3 thyroid cells are continuously cultured, clonal lines which require thyrotropin to grow and function. Both can be efficiently transformed when infected with RNA or DNA viruses carrying oncogenes or when directly transfected with activated oncogenes. Transformation, assayed by the appearance of cell growth in agar and by tumorigenicity in syngeneic rats or nude mice, is associated with the loss of thyrotropin-dependent cell division and thyrotropin-regulated functions such as thyroglobulin synthesis. In 16 clones of FRTL-5 or PC-Cl-3 cells transformed with different oncogenes, we show that loss of thyrotropin-dependent growth and function correlates with the loss of thyrotropin receptor gene expression, measured with a rat thyrotropin receptor cDNA probe.

Cooperation between the polyomavirus middle-T-antigen gene and the human c-myc oncogene in a rat thyroid epithelial differentiated cell line: model of in vitro progression.

Two rat thyroid epithelial differentiated cell lines, PC Cl 3 and PC myc, were infected with the polyoma murine leukemia virus (PyMLV) carrying the Middle-T-antigen gene of polyomavirus. After infection, both cell lines acquired the typical markers of neoplastic transformation; however, the PC myc cells showed a greater malignant phenotype. Furthermore, the thyroid differentiated functions were completely suppressed in PC myc cells transformed by PyMLV, whereas they were, at least partially, retained in PC Cl 3 cells transformed by PyMLV, and in particular, thyroglobulin synthesis and secretion were not affected at all. Since no differences in the expression of the middle-T-antigen gene were observed in the two PyMLV-transformed cell lines, the different properties shown by these two infected cell lines must be ascribed to the expression of the c-myc oncogene.

Zinc finger protein 521 antagonizes early B-cell factor 1 and modulates the B-lymphoid differentiation of primary hematopoietic progenitors

Zinc finger protein 521 (EHZF/ZNF521) is a multi-functional transcription co-factor containing 30 zinc fingers and an amino-terminal motif that binds to the nucleosome remodelling and histone deacetylase (NuRD) complex. ZNF521 is believed to be a relevant player in the regulation of the homeostasis of the hematopoietic stem/progenitor cell compartment, however the underlying molecular mechanisms are still largely unknown. Here, we show that this protein plays an important role in the control of B-cell development by inhibiting the activity of early B-cell factor-1 (EBF1), a master factor in B-lineage specification. In particular, our data demonstrate that: (1) ZNF521 binds to EBF1 via its carboxyl-terminal portion and this interaction is required for EBF1 inhibition; (2) NuRD complex recruitment by ZNF521 is not essential for the inhibition of transactivation of EBF1-dependent promoters; (3) ZNF521 represses EBF1 target genes in a human B-lymphoid molecular context; and (4) RNAi-mediated silencing of ZNF521/Zfp521 in primary human and murine hematopoietic progenitors strongly enhances the generation of B-lymphocytes in vitro. Taken together, our data indicate that ZNF521 can antagonize B-cell development and lend support to the notion that it may contribute to conserve the multipotency of primitive lympho-myeloid progenitors by preventing or delaying their EBF1-driven commitment toward the B-cell lineage.