Dora Fabbro

TTF-1 gene expression in human lung tumours

Tissue-specific transcription factors control cell determination and differentiation. TTF-1 is a tissue-specific transcription factor expressed in the thyroid and lung. We investigated the expression of TTF-1 in normal human lung, and in various histopathological types of lung cancers by immunohistochemistry. In normal lung, TTF-1 expression was restricted to bronchial and alveolar epithelial cells. TTF-1 expression was found in 7 of the 29 cases of non-small cell lung carcinomas. In these tumours, the expression of TTF-1 did not correlate with the histological degree of differentiation. Results obtained using RNase protection assay confirmed that TTF-1 was expressed only in a subset of non-small cell carcinomas. TTF-1, as expected, was not expressed in neoplasms having a neuroendocrine cell origin, such as carcinoids. Interestingly, TTF-1 was always expressed in small cell lung carcinomas. These findings indicate that: (i) small cell lung carcinomas could originate from the endothermal cell lineage and (ii) dedifferentiation processes that operate in these neoplasms do not affect molecular mechanisms necessary for TTF-1 gene expression.

Immunocytochemical expression of tissue specific transcription factor-1 in lung carcinoma.

AIMS: To investigate the immunocytochemical expression of the tissue specific transcription factor-1 (TTF-1) on cytological specimens of small cell lung carcinoma (SCLC) and to establish its value in the cytological diagnosis of lung cancer. METHODS: For each case, the diagnosis was made on cytological specimens and confirmed on subsequent bronchial biopsy specimens. TTF-1 was detected immunocytochemically using the avidinbiotin complex technique with a rabbit antiserum. Expression of TTF-1 was evaluated in 41 cases of SCLC and 17 cases of non-small cell carcinoma (NSCC). The latter were subdivided into eight cases of adenocarcinomas and nine cases of squamous cell carcinomas (SCC). RESULTS: Positive nuclear immunoreactivity to TTF-1 was identified in 38 (92.7%) of the 41 cases of SCLC, in five (62.5%) of eight cases of adenocarcinoma, and one (11%) of nine cases of SCC. A significant difference was observed between the two main groups, SCLC and NSCC. A comparison between SCLC and adenocarcinoma and SCC showed that TTF-1 expression was significantly different. TTF-1 immunoreactivity was not detected in the inflammatory cells of the same cases. CONCLUSIONS: TTF-1 is strictly associated with SCLC; it was weakly expressed in the various subtypes of NSCC. Although TTF-1 is not specific for SCLC, it can be used to highlight neoplastic cells to good effect when a large inflammatory component is present, and to differentiate SCLC from lymphoid infiltrates.

A molecular code dictates sequence-specific DNA recognition by homeodomains.

Most homeodomains bind to DNA sequences containing the motif 5′‐TAAT‐3′. The homeodomain of thyroid transcription factor 1 (TTF‐1HD) binds to sequences containing a 5′‐CAAG‐3′ core motif, delineating a new mechanism for differential DNA recognition by homeodomains. We investigated the molecular basis of the DNA binding specificity of TTF‐1HD by both structural and functional approaches. As already suggested by the three‐dimensional structure of TTF‐1HD, the DNA binding specificities of the TTF‐1, Antennapedia and Engrailed homeodomains, either wild‐type or mutants, indicated that the amino acid residue in position 54 is involved in the recognition of the nucleotide at the 3′ end of the core motif 5′‐NAAN‐3′. The nucleotide at the 5′ position of this core sequence is recognized by the amino acids located in position 6, 7 and 8 of the TTF‐1 and Antennapedia homeodomains. These data, together with previous suggestions on the role of amino acids in position 50, indicate that the DNA binding specificity of homeodomains can be determined by a combinatorial molecular code. We also show that some specific combinations of the key amino acid residues involved in DNA recognition do not follow a simple, additive rule.

Frequency of Factor V, Prothrombin and Methylenetetrahydrofolate Reductase Gene Variants in Preeclampsia

Background: The association between thrombophilic variants (Leiden mutation of the factor V gene, G20210A mutation of the prothrombin gene and C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene) with preeclampsia was investigated in a north-eastern Italian population. Methods: Fifty-eight preeclamptic (PE) women and 74 normal pregnancies were evaluated. Genotypes were determined by polymerase chain reaction. Results: The frequency of heterozygous carriers of the factor V Leiden was similar between PE women (5.2%) compared to the control subjects (4.1%; p 0.76). Also the frequencies of G20210A and C677T mutations were similar between PE and control subjects. Conclusions: In this population, we found no difference in the prevalence of genetic risk factors for thrombosis in women with preeclampsia compared with control subjects.

Association between plasminogen activator inhibitor 1 gene polymorphisms and preeclampsia.

It is known that the plasminogen activator inhibitor 1 (PAI-1) protein levels are increased in placentas of preeclamptic subjects. Therefore, we assessed whether polymorphisms related to the transcriptional control of the PAI-1 gene (–675 4G/5G and –844G/A) are associated with mild preeclampsia. We compared 52 women with preeclampsia to 80 women with a normal pregnancy. None of the preeclamptic women suffered from the severe form of preeclampsia. DNA was extracted from blood, and –675 4G/5G and –844G/A genotypes of the PAI-1 gene were determined. Since it has been shown that the presence of factor V Leiden, prothrombin G20210A, and MTHFR C677T gene variants may be associated with preeclampsia, their frequency was also evaluated in our study groups. The factor V Leiden, PT G20210A, and MTHFR C677T gene variants were not associated with preeclampsia. In the case of the –675 4G/5G polymorphism, genotypes 4G/4G and 5G/5G were more prevalent in the preeclamptic and in the control group, respectively. In the case of –844 G/A polymorphism, genotypes A/A and G/G were more prevalent in the preeclamptic and in the control group, respectively. By using the χ2 test for trend, differences for both genotypes were significant (p = 0.0141 for the –675 genotypes and p = 0.0492 for the –844 genotypes). The frequency of the 4G and 5G alleles of the –675 gene polymorphism was significantly different between preeclamptic and normal women (p = 0.032). Differently, the allelic frequency of the –844 gene polymorphism did not show significant differences between preeclamptic and normal women (p = 0.083). In conclusion, the hypofibrinolytic genotypes 4G/4G and A/A at positions –675 and –844 of the PAI-1 gene are associated with the occurrence of mild preeclampsia independently of thrombophilic mutations of the factor V, prothrombin, and MTHFR genes.

Nucleophosmin is overexpressed in thyroid tumors

Nucleophosmin (NPM) is a protein that contributes to several cell functions. Depending on the context, it can act as an oncogene or tumor suppressor. No data are available on NPM expression in thyroid cells. In this work, we analyzed both NPM mRNA and protein levels in a series of human thyroid tumor tissues and cell lines. By using immunohistochemistry, NPM overexpression was detected in papillary, follicular, undifferentiated thyroid cancer, and also in follicular benign adenomas, indicating it as an early event during thyroid tumorigenesis. In contrast, various levels of NPM mRNA levels as detected by quantitative RT-PCR were observed in tumor tissues, suggesting a dissociation between protein and transcript expression. The same behavior was observed in the normal thyroid FRTL5 cell lines. In these cells, a positive correlation between NPM protein levels, but not mRNA, and proliferation state was detected. By using thyroid tumor cell lines, we demonstrated that such a post-mRNA regulation may depend on NPM binding to p-Akt, whose levels were found to be increased in the tumor cells, in parallel with reduction of PTEN. In conclusion, our present data demonstrate for the first time that nucleophosmin is overexpressed in thyroid tumors, as an early event of thyroid tumorigenesis. It seems as a result of a dysregulation occurring at protein and not transcriptional level related to an increase of p-Akt levels of transformed thyrocytes.

Fludarabine-based induction therapy does not overcome the negative effect of ABCG2 (BCRP) over-expression in adult acute myeloid leukemia patients

Over-expression of multidrug resistance (MDR) proteins PGP and BCRP has a negative prognostic impact in acute myeloid leukemia (AML) patients. Inclusion of fludarabine in induction chemotherapy increases remission rate in PGP over-expressing cases. We investigated the role of BCRP in 138 adult AML patients receiving induction therapy with fludarabine. None of the MDR-related proteins influenced complete remission attainment. Conversely, high levels of BCRP significantly affected disease-free survival, as higher relapse rates (48.5% vs 28.5%) and earlier relapse occurred in BCRP+ patients. Also overall survival was affected by BCRP positivity, and survival significantly worsened in case of concomitant PGP and BCRP over-expression.

High periostin expression correlates with aggressiveness in papillary thyroid carcinomas

Periostin is a mesenchyme-specific gene product, which acts as an adhesion molecule during bone formation and supports osteoblastic cell line attachment and spreading. However, periostin expression is activated in a large variety of epithelial human tumors and correlates with their aggressiveness. Knowledge of expression of periostin in thyroid tumors is still scanty. The aim of the present work was to investigate periostin expression in differentiated neoplasms of the thyroid and to correlate it with several clinical and molecular features of these tumors. Periostin expression was evaluated by quantitative PCR and immunohistochemistry in normal thyroid tissues, papillary thyroid carcinomas (PTCs), follicular thyroid carcinomas (FTCs), and follicular adenomas (FAs). Periostin mRNA levels were also evaluated in several thyroid tumor cell lines. PTCs show mean periostin mRNA levels significantly higher than corresponding normal tissues. In five PTCs, periostin mRNA values were at least 30-fold higher than corresponding normal tissues. Conversely, mean periostin mRNA levels of FTCs and FAs were similar to those of normal tissues. Consistent with mRNA studies, periostin was detectable by immunohistochemistry in cancerous epithelial cells only in several cases of PTCs but not in normal tissue, FTCs, and FAs. In PTCs, periostin mRNA levels positively correlate with extrathyroidal invasion, distant metastasis, and higher grade staging. A negative correlation between periostin and expression of some markers of the thyroid-differentiated phenotype (thyroglobulin, thyrotropin receptor) was also present in the PTCs. These results indicate that an increase in periostin gene expression is present in several PTCs, in which it appears as a marker of aggressiveness. Experiments in thyroid tumor cell lines indicate that high levels of periostin mRNA are due, at least in part, to the increase in periostin promoter activity.

A novel de novo germ‐line V292M mutation in the extracellular region of RET in a patient with phaeochromocytoma and medullary thyroid carcinoma: functional characterization

Context  In multiple endocrine neoplasia (MEN), rearranged during transfection (RET), gene testing has been extensively exploited to characterize tumour aggressiveness and optimize the diagnostic and clinical management.

Osteopontin expression correlates with nuclear factor-κB activation and apoptosis downregulation in clear cell renal cell carcinoma.

Osteopontin (OPN) is a phosphoglycoprotein implicated in tumorigenesis and tumor cell metastasis. Apoptosis inhibition is one of the mechanisms that contribute to development and progression of cancer, and might be initiated by OPN interaction with tumor cells. The aim of this study was to analyze the relation between OPN and nuclear factor-kappa B (NF-κB) expression in clear cell renal cell carcinoma (CCRCC), as well as their relation to apoptotic activity of tumor cells. Expression of OPN protein and p65 NF-κB subunit was analyzed immunohistochemically in 87 CCRCC samples, and compared mutually and with apoptotic index. Expression of OPN mRNA was analyzed using quantitative real-time PCR and compared with OPN and NF-κB protein expression in 22 CCRCC samples. Statistical analysis showed an association of p65 NF-κB with OPN mRNA (p=0.015) and protein (p<0.001). Also, we found an inverse relationship of OPN with NF-κB protein expression and apoptotic activity of tumor cells (p=0.006 and p=0.022, respectively). Our results indicate that p65 NF-κB signaling pathway may be involved in OPN-mediated CCRCC progression, partly by protecting tumor cells from apoptosis. Therefore, both molecules can constitute potential targets for therapeutic intervention in CCRCC.