CCDC6 represses CREB1 activity by recruiting histone deacetylase 1 and protein phosphatase 1.

RET/papillary thyroid carcinoma 1 (PTC1) oncogene is frequently activated in human PTCs. It is characterized by the fusion of the intracellular kinase-encoding domain of RET to the first 101 amino acids of CCDC6. The aim of our work is to characterize the function of the CCDC6 protein to better understand the function of its truncation, that results in the loss of the expression of one allele, in the process of thyroid carcinogenesis. Here, we report that CCDC6 interacts with CREB1 and represses its transcriptional activity by recruiting histone deacetylase 1 and protein phosphatase 1 proteins at the CRE site of the CREB1 target genes. Finally, we show an increased CREB1 phosphorylation and activity in PTCs carrying the RET/PTC1 oncogene. Consistently, an increased expression of two known CREB1 target genes, AREG and cyclin A, was observed in this subgroup of thyroid papillary carcinomas. Therefore, the repression of CREB1 activity by CCDC6 has a critical function in the development of human thyroid papillary carcinomas carrying RET/PTC1 activation.

OPN/CD44v6 overexpression in laryngeal dysplasia and correlation with clinical outcome.

Laryngeal dysplasia is a common clinical concern. Despite major advancements, a significant number of patients with this condition progress to invasive squamous cell carcinoma. Osteopontin (OPN) is a secreted glycoprotein, whose expression is markedly elevated in several types of cancers. We explored OPN as a candidate biomarker for laryngeal dysplasia. To this aim, we examined OPN expression in 82 cases of dysplasia and in hyperplastic and normal tissue samples. OPN expression was elevated in all severe dysplasia samples, but not hyperplastic samples, with respect to matched normal mucosa. OPN expression levels correlated positively with degree of dysplasia (P=0.0094) and negatively with disease-free survival (P<0.0001). OPN expression was paralleled by cell surface reactivity for CD44v6, an OPN functional receptor. CD44v6 expression correlated negatively with disease-free survival, as well (P=0.0007). Taken as a whole, our finding identify OPN and CD44v6 as predictive markers of recurrence or aggressiveness in laryngeal intraepithelial neoplasia, and overall, point out an important signalling complex in the evolution of laryngeal dysplasia.

Involvement of H4(D10S170) protein in ATM-dependent response to DNA damage.

H4(D10S170) gene has been identified upon its frequent rearrangement with RET in papillary thyroid tumours (RET/PTC1). The kinase ataxia telangectasia mutated (ATM) phosphorylates a limited number of downstream protein targets in response to DNA damage. We investigated the potential role of H4(D10S170) in DNA damage signaling pathways. We found that in cells treated with etoposide or ionizing radiation (IR), H4(D10S170) underwent ATM-mediated phosphorylation at Thr 434, stabilizing nuclear H4. In ataxia telangectasia cells (A-T), endogenous H4(D10S170) was localized to cytoplasm and was excluded from the nucleus. Moreover, H4(D10S170) was not phosphorylated in ATM-deficient lymphoblasts after ionizing irradiation. Inhibition of ATM kinase interfered with H4(D10S170) apoptotic activity, and expression of H4 with threonine 434 mutated in Alanine, H4(T434A), protected the cells from genotoxic stress-induced apoptosis. Most importantly, after exposure to IR we found that silencing of H4(D10S170) in mammalian cells increased cell survival, as shown by clonogenic assay, allows for DNA synthesis as evaluated by bromodeoxyuridine incorporation and permits cells to progress into mitosis as demonstrated by phosphorylation on Histone H3. Our results suggest that H4(D10S170) is involved in cellular response to DNA damage ATM-mediated, and that the impairment of H4(D10S170) gene function might have a role in thyroid carcinogenesis.

Analysis of cadherin/catenin complexes in transformed thyroid epithelial cells: modulation by beta 1 integrin subunit.

We have analysed the expression of cadherin/catenin complex molecules in PC C13 rat thyroid cells transformed in vitro with different oncogenes. No significant downregulation of either E-cadherin, alpha-, beta- and gamma-catenin was detected following the introduction of activated forms of myc, adenovirus E1A, ras, raf, myc + ras, E1A + raf. However, ras- and raf-transformed PC C13 cells showed altered adherens junctions. An altered distribution of cadherin/catenin complexes characterized by radially oriented membrane spikes perpendicular to cell edges was the most prominent feature evidenced by immunofluorescence. No beta1 integrin localization was observed in areas where this altered pattern of E-cadherin expression was detected. However, beta1 integrin subunit expression was detected at areas of cell-cell contact where E-cadherin showed a normal pattern of expression. Furthermore, ras- and raf-transformed PC C13 cells showed the ability to migrate in collagen gels, in contrast to their normal untransformed counterpart. Overexpression of beta1 integrin was found to restore normal E-cadherin localization at cell-cell contacts and to partially inhibit the ability to migrate in collagen gels. Finally, two cell lines obtained by ras transformation in vivo, and derived from a rat primary thyroid carcinoma (TK6) and its lung metastasis (MPTK6), were found to have lost gamma-catenin expression. TK6 lost also E-cadherin expression and membrane localization of alpha-catenin. These results suggest that: i) in vitro thyroid cell transformation is associated to a change in cadherin/catenin complexes distribution rather than to a decrease in expression; ii) in vivo transformation is associated to the loss of expression of some of these molecules likely due to tumor progression; iii) alterations in beta1 integrin subunit expression can result in changes in cadherin/catenin function thus implying that an integrin-cadherin synergy may exist in thyroid cells.

Expression of the RET receptor tyrosine kinase and GDNFR-alpha in normal and leukemic human hematopoietic cells and stromal cells of the bone marrow microenvironment.

The RET proto-oncogene product is a receptor tyrosine kinase representing the signal-transducing molecule of a multisubunit surface receptor complex for the glial cell line-derived neurotrophic factor (GDNF), in which a novel glycosyl-phosphatidylinositol (PI)-linked protein (termed GDNFR-alpha) acts as the ligand-binding component. We have analyzed expression of RET and GDNFR-alpha in purified normal hematolymphopoietic cells, leukemia/lymphoma cell lines, and 154 primary samples from patients with hematopoietic malignancies encompassing different lineages and differentiation stages. Relatively low amounts of RET mRNA were found in early CD34+ hematopoietic progenitors, but RET transcripts appeared to increase after myelomonocytic maturation. No expression of RET was found in peripheral blood and tissue B and T lymphocytes. Analysis of human myelomonocytic cell lines was overall consistent with results obtained on purified normal cells. Accordingly, RET expression was mainly confined to acute myeloid leukemias (AMLs) displaying either monocytic (French-American-British M4 and M5) or intermediate-mature myeloid (M2 and M3) phenotypes, being less frequently detected in early myeloid (M0 and M1) AMLs. In contrast, RET mRNA was sporadically detected in B-cell tumors, whereas, among T-cell malignancies, RET transcripts were mainly detected in cells of postthymic and mature T-cell phenotype. RET broad detection in primary tumors was not paralleled by the mutual expression of GDNFR-alpha, which was detected only in 2 isolated primary samples and in 3 leukemia/lymphoma cell lines. However, GDNFR-alpha transcripts, in the absence of RET mRNA, were found in normal bone marrow stromal cells (BMSC), in BM fibroblasts, and in two osteoblast cell lines previously described to support normal hematopoiesis. In the presence of GDNF-receptors derived from BMSC by PI-specific phospholipase C cleavage, GDNF efficiently bound RET-expressing AML blasts and was functionally active by reducing their clonogenic growth and triggering the monocytic maturation of leukemic cells.

Low frequency of p53 mutations in human thyroid tumours; p53 and Ras mutation in two out of fifty-six thyroid tumours.

OBJECTIVE: p53 is a well-known nuclear phosphoprotein encoded by a suppressor gene know to be mutated in various kinds of human tumours. A relationship between p53 gene mutation and tumour progression seems to be a common feature of several neoplasias. DESIGN: In order to investigate the role of p53 mutations in human thyroid tumours, DNA samples derived from fifty-six neoplastic tissues, ranging from benign adenomas to undifferentiated carcinomas, were examined for the presence of p53 gene mutations. METHODS: The analysis has been conducted using polymerase chain reaction (PCR) amplification of the exons 5-9 of the p53 gene followed by single strand conformation polymorphism (SSCP) and sequence analyses. RESULTS: One anaplastic carcinoma and one papillary carcinoma showed p53 gene mutations in exons 5 and 8, respectively. A cell line established from the papillary carcinoma showed the same mutation present in the original tumour. Both p53 mutations were heterozygous. The p53 positive samples were analysed for other genetic alterations frequently detected in human thyroid carcinomas (mutations of the RET, TRK, and ras oncogenes): both p53-mutated samples proved to be mutated at level of codon 13 of the c-Ki-ras gene. CONCLUSIONS: Our data confirm that p53 gene alterations are rare in well-differentiated thyroid tumours, that they are an important requirement for the establishment in culture of human thyroid carcinoma cell lines, and that they can be associated with other genetic alterations, namely ras mutations, in the malignant progression of thyroid tumours.

Selection of activating mutations of c-fms in FDC-P1 cells.

FDC-P1 haemopoietic cells were used to select mutations of c-fms that constitutively activate the receptor for macrophage-colony stimulating factor (M-CSF or CSF-1). One mutation changed Ser 929 to Gly within a Ser/Gly rich region of the C-terminal tail and a second changed a nearby, highly conserved Leu 926 for Pro. A third mutation (D802V) changed Asp 802 to Val within the alpha L12/beta 9 region of the tyrosine kinase domain, so supporting the crystallographic evidence that this region triggers kinase activation. A c-kit mutation exactly equivalent to D802V was previously identified in a leukamic cell line and was demonstrated here to be transforming. Surprisingly, although D802V potently transformed FDC-P1 cells, it could not induce Rat-2 fibroblast foci, even in the presence of M-CSF. It is suggested that the accelerated receptor degradation induced by D802V may account for its cell specific effect.

Characteristic patterns of HOX gene expression in different types of human leukemia.

Homeobox-containing genes are a network of genes encoding DNA-binding proteins highly conserved throughout evolution. They are involved in the control of normal development as well as in the regulation of gene expression in adult differentiating systems, including hematopoiesis. Aberrant expression of homeobox-containing genes has recently been related to leukemic phenotype. Human homeobox-containing genes of the HOX family are organized into 4 large clusters. We have analyzed the expression of HOX genes in different types of human leukemia to investigate whether the physical organization of HOX loci reflects a regulatory hierarchy involved in the differentiation of hematopoietic cells or whether HOX gene expression might contribute to the leukemic phenotype. Our results show that HOX genes are coordinately regulated in blocks in myeloid cells whereas they appear to function as isolated genes in lymphoid cells. Six contiguous genes of the HOX2 locus, highly expressed in acute non-lymphocytic leukemia, are switched off in chronic myelogenous leukemia, suggesting that down-regulation of HOX2 genes might be required for cell maturation of the myeloid lineages. In contrast, a few scattered genes are active in lymphoid populations. These observations suggest that hematopoietic cells express a repertoire of HOX genes characteristic of a particular cell lineage at a specific stage of differentiation. The characteristic patterns of HOX gene expression may reflect the potentially important role that these genes play in cell lineage determination during both normal and leukemic hematopoiesis.

Coordinate regulation of HOX genes in human hematopoietic cells.

Hematopoiesis is a continuous process in which precursor cells proliferate and differentiate throughout life. However, the molecular mechanisms that govern this process are not clearly defined. Homeobox-containing genes, encoding DNA-binding homeodomains, are a network of genes highly conserved throughout evolution. They are organized in clusters expressed in the developing embryo with a positional hierarchy. We have analyzed expression of the four human HOX loci in erythroleukemic, promyelocytic, and monocytic cell lines to investigate whether the physical organization of human HOX genes reflects a regulatory hierarchy involved in the differentiation process of hematopoietic cells. Our results demonstrate that cells representing various stages of hematopoietic differentiation display differential patterns of HOX gene expression and that HOX genes are coordinately switched on or off in blocks that may include entire loci. The entire HOX4 locus is silent in all lines analyzed and almost all the HOX2 genes are active in erythroleukemic cells and turned off in myeloid-restricted cells. Our observations provide information about the regulation of HOX genes and suggest that the coordinate regulation of these genes may play an important role in lineage determination during early steps of hematopoiesis.