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M A Pierotti

Publications and source records attributed to M A Pierotti.

At least 55 records · Page 3Linked to original sources
PubMed2000

TP53 mutations and mdm2 protein overexpression in cholangiocarcinomas.

Tumor suppressor protein p53 is a positive regulator of MDM2 gene expression and the mdm2 protein can bind to p53, preventing the transactivation of p53 responsive genes, thus mimicking TP53 mutation. The authors looked for alterations that could affect, directly and indirectly, p53 function in 13 patients with extrahepatic cholangiocarcinoma. Molecular analysis by single strand conformation polymorphism and DNA sequencing revealed that TP53 gene mutations occurred in only 2 of 13 cholangiocarcinomas. High levels of mdm2 protein were found, by immunohistochemical staining, in 61% of the cholangiocarcinomas and in almost all specimens (70%) displaying stabilized p53 protein in the absence and in the presence of TP53 mutations. The finding of co-overexpressed mdm2 and p53 proteins in cholangiocarcinomas indicates that they can upregulate the expression of mdm2 protein to a level sufficient for binding and accumulating p53 in a presumably inactive complexed form. The presence of TP53 mutations or upregulation of MDM2 gene expression in 9 of the 13 cholangiocarcinomas strongly supports that the impairment of the p53 pathway is an important and specific step in cholangiocarcinoma pathogenesis. At variance with other authors, no alteration of p16ink4/CDKN2 gene was observed in all 13 cholangiocarcinomas.

Aged↗
PubMed2000

Morphologic-cytogenetic analysis of dedifferentiated liposarcomas with an extensive misleading leiomyosarcomatous component.

This report describes two cases of recurrent retroperitoneal dedifferentiated liposarcoma characterized by an extensive leiomyomatous component that prevented the correct diagnosis before the last recurrence. Strong immunoreactivity with smooth muscle and desmin antibodies and ultrastructural features consistent with leiomyosarcoma were observed in the spindle-cell and/or myxoid-like components in all four recurrences in case 1, and in the spindle-cell component of the primary tumor and the first recurrence in case 2. In case 1, the correct diagnosis was suggested by the cytogenetic evidence of ring markers, a hallmark of well-differentiated/dedifferentiated liposarcoma. In case 2, tumor type was yielded mainly by the morphology of the second recurrence, which consisted entirely of a well-differentiated liposarcoma, a sclerosing inflammatory variant, as confirmed by the karyotype. Reevaluation of the first two surgical specimens of each case revealed small areas consistent with well-differentiated liposarcoma that had been previously overlooked. Despite the smooth-muscle antigen profile, both cases retained an mdm2+/p53+/cdk4+ immunophenotype consistent with the genotype.

Aged↗
PubMed1999

The Glu632-Leu633 deletion in cysteine rich domain of Ret induces constitutive dimerization and alters the processing of the receptor protein.

Mutations of the RET gene, encoding a receptor tyrosine kinase, have been associated with the inherited cancer syndromes MEN 2A and MEN 2B. They have also further been associated with both familial and sporadic medullary thyroid carcinomas. Missense mutations affecting cysteine residues within the extracellular domain of the receptor causes constitutive tyrosine kinase activation through the formation of disulfide-bonded homodimers. We have recently reported that a somatic 6 bp in-frame deletion, originally coding for Glu632-Leu633, potently activates the RET gene. This activation is increased with respect to the frequent MEN 2A-associated missense mutation Cys634Arg. This finding specifically correlated to the clinic behavior of the corresponding tumor, which was characterized by an unusually aggressive progression with both multiple and recurrent metastases. By examining the possibility that this deletion acts in a manner similar to cysteine substitution, we have analysed the molecular mechanism by which this oncogenic activation occurs. Phosphorylated dimers of the deleted Ret receptor were detected in immunoprecipitates separated under non-reducing conditions. Like other Cys point mutations, this 6 bp deletion affecting two amino acid residues between two adjacent Cys, is capable of activating the transforming ability of Ret by promoting receptor dimerization. These results suggest that alteration to cysteine residue position or pairing is capable of inducing ligand independent dimerization. Furthermore, we present data demonstrating that the processing and sorting of the Ret membrane receptor to the cell surface is affected by mutation type.

3T3 Cells↗
PubMed1999

The tumor-suppressor gene FHIT is involved in the regulation of apoptosis and in cell cycle control.

Alteration of the FHIT (fragile histidine triad) gene occurs as an early and frequent event in lung carcinogenesis. FHIT gene transfer into lung cancer cell line H460 lacking Fhit protein expression resulted in reversion of tumorigenicity. To gain insight into the biological function of FHIT, we compared the H460 cell line with its Fhit transfectants (H460/FHIT). A significant inhibition of cell growth was observed in H460/FHIT cells. The analysis of apoptosis by in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling revealed a high rate of apoptosis-induced DNA strand breaks in stable clones. In situ results were confirmed by FACScan analysis that showed an apoptotic rate of 44-47% compared with a 15% level in the control H460 cells. Analysis of cell cycle-phase distribution indicated a significant G(0)/G(1) arrest and the presence of a sub-G(1) peak in the stable clones. No significant changes in Bcl2, BclX, and Bax protein expression level were observed in the transfected clones as compared with the control H460 cells whereas a 2-fold increase in Bak protein levels was noticed. An increased level of p21(waf) protein paralleled by an up-regulation of p21(waf) transcripts also was found in Fhit-expressing clones compared with the H460 cell line. No differences in p53 levels were observed in the same cells, suggesting a p53-independent effect. These data suggest that the observed growth-inhibitory effect in FHIT-reexpressing cells could be related to apoptosis and cell cycle arrest and link the tumor-suppressor activity of FHIT to its proapoptotic function.

Acid Anhydride Hydrolases↗
PubMed1999

Screening for mutations of the APC gene in 66 Italian familial adenomatous polyposis patients: evidence for phenotypic differences in cases with and without identified mutation.

Germline mutations in the APC gene are responsible for familial adenomatous polyposis (FAP), a dominantly inherited syndrome characterized by the development of hundreds to thousands of polyps in the colon and in the rectum of affected individuals and by variable extracolonic manifestations (gastric and duodenal polyps, osteomas, retinal lesions, and desmoid tumors). Through the combined use of single-strand conformation polymorphism (SSCP) analysis and the protein truncation test (PTT), we have screened 66 Italian FAP patients and found 29 different APC mutations in a total of 34 cases. Of the identified mutations, 15 were nonsense, 12 were 1- to 5-bp deletions or insertions and two were complex rearrangements, all leading to the formation of premature stop codons. Only 10 mutations had been already previously described at the germline level, confirming the high heterogeneity of the APC mutational spectrum. The mean age of diagnosis in mutation positive cases and their affected relatives was significantly lower than in cases without identified mutation (30.6 vs 39.1 years, respectively; p = 0.003). In addition, among patients without a family history of polyposis, all mutation-positive cases displayed at least one of the extracolonic manifestations usually associated with FAP, whereas in one-half of the cases without identified mutation, none of these phenotypes was observed. Although a fraction of apparently mutation-negative cases were likely to be due to limitations of the mutation screening strategy, our results suggest, in agreement with previous reports, that allelic and/or genetic heterogeneity might be responsible for the phenotypic variability observed in FAP patients.

Adenomatous Polyposis Coli↗
PubMed1999

Expression of angiogenesis stimulators and inhibitors in human thyroid tumors and correlation with clinical pathological features.

Experimental evidence has shown, both in vitro and in animal models, that neoplastic growth and subsequent metastasis formation depend on the tumor's ability to induce an angiogenic switch. This requires a change in the balance of angiogenic stimulators and inhibitors. To assess the potential role of angiogenesis factors in human thyroid tumor growth and spread, we analyzed their expression by semiquantitative RT-PCR and immunohistochemistry in normal thyroid tissues, benign lesions, and different thyroid carcinomas. Compared to normal tissues, in thyroid neoplasias we observed a consistent increase in vascular endothelial growth factor (VEGF), VEGF-C, and angiopoietin-2 and in their tyrosine kinase receptors KDR, Flt-4, and Tek. In particular, we report the overexpression of angiopoietin-2 and VEGF in thyroid tumor progression from a prevascular to a vascular phase. In fact, we found a strong association between tumor size and high levels of VEGF and angiopoietin-2. Furthermore, our results show an increased expression of VEGF-C in lymph node invasive thyroid tumors and, on the other hand, a decrease of thrombospondin-1, an angioinhibitory factor, in thyroid malignancies capable of hematic spread. These results suggest that, in human thyroid tumors, angiogenesis factors seem involved in neoplastic growth and aggressiveness. Moreover, our findings are in keeping with a recent hypothesis that in the presence of VEGF, angiopoietin-2 may collaborate at the front of invading vascular sprouts, serving as an initial angiogenic signal that accompanies tumor growth.

Angiogenesis Inducing Agents↗
PubMed1999

c-erbB2/neu gene and chromosome 17 analysis in breast cancer by FISH on archival cytological fine-needle aspirates.

The detection of specific genetic alterations in breast cancer is useful for diagnosing, predicting prognosis and planning preoperative treatment. c-erbB2/neu overexpression is usually detected by immunocytochemistry (ICC), although this technique is neither completely reproducible nor highly reliable, owing to specimen and methodologic variability and antibody sensitivity. Here, we combine two well-established techniques, fine-needle aspiration (FNA) and fluorescence in situ hybridization (FISH), to detect c-erbB2/neu amplification in patients candidate to primary chemotherapy and, in part, previously analysed for c-erbB2/neu overexpression. Sixty smears from FNA were used to simultaneously detect c-erbB2/neu and chromosome 17 centromere. FISH was successful in 58 cases and detected 24 amplified cases, three of which were negative by immunophenotyping, 28 negative cases, with evidence of two normal c-erbB2/neu/signals, two cases with deletion of c-erbB2/neu, and four cases with polysomy, thus providing more reliable and informative results than ICC. This study underlines the advantages offered by the FNA and FISH combination which are two rapid, reliable, simple and informative techniques, to analyse one of the most important genetic markers for predicting prognosis and chemotherapy planning for breast carcinoma in particular in the light of the recently proposed trials of primary chemotherapy.

Adult↗
PubMed1999

Esthesioneuroblastoma is not a member of the primitive peripheral neuroectodermal tumour-Ewing's group.

Esthesioneuroblastoma (ENB) is a rare, site-specific, locally aggressive neuronal malignancy so far thought to belong to primitive peripheral neuroectodermal tumour-Ewing's tumour (pPNETs-ETs). Its anatomical location, in addition to morphologic, immunophenotypic and ultrastructural features, suggests its origin in the neuronal or neuroendocrine cells of the olfactory epithelium. However, the cytogenetic and molecular data currently available appear controversial on the presence of the typical translocation t(11;22)(q24;q12) and of trisomy 8, chromosomal changes that characterize the tumours belonging to the pPNETs-ETs. Herein we have analysed five ENB tumour specimens for trisomy 8 by fluorescence in situ hybridization (FISH), for the presence of EWS gene rearrangements by FISH, reverse transcription polymerase chain reaction and Southern blot analyses, as well as for the expression of the Ewing sarcoma-associated MIC2 antigen by immunohistochemistry. Neither EWS/FLI-I, EWS/ERG and EWS/FEV fusion genes nor MIC2 expression were found in any tumour, whereas trisomy 8 was found in one case only. Moreover, DNA from three cases analysed by Southern blot did not show EWS gene rearrangements. Our results support the evidence that ENB is not a member of the pPNETs-ETs.

Adult↗
PubMed1999

Biochemical uncovering of mdm2/p53 complexes in liposarcomas parallels their immunohistochemical detection.

Recent observations indicate the existence of pathogenetically distinct groups of well-differentiated (WD) dedifferentiated (DD) liposarcomas. In the retroperitoneal WD-DD liposarcomas, the predominant phenotype is represented by the aberrant (overexpressed) mdm2+/p53+ wild-type profile. At the nonretroperitoneal site, the WD liposarcomas present a wider association of MDM2/P53 gene expression; i.e., mdm2+/p53+, mdm2+/p53-, mdm2-/p53+ and mdm2-/p53-, and TP53 mutations seem to correlate with the dedifferentiation process. A biochemical study of mdm2-p53 association in 11 tumor samples characterized by the presence of different mdm2 and p53 immunophenotypes was performed. Immunoprecipitation assays using a p53-specific antibody were performed on tumor tissue and surrounding normal tissue; the immunoprecipitated material was then investigated for the presence of p53 (control) and of coimmunoprecipitated mdm2. This biochemical analysis showed that, in mdm2+/p53+/wild-type retroperitoneal liposarcomas, a band corresponded to mdm2 protein in the cellular lysates immunoprecipitated with a p53-directed antibody. In contrast, the mdm2+/p53- liposarcoma did not evidence the presence of mdm2 protein nor was p53 protein available to direct immunoprecipitation, as in the p53 mutant tumor samples with mdm2-/p53+ and mdm2-/p53- phenotypes. From the normal counterpart of retroperitoneal liposarcoma lysates, no p53 protein was immunoprecipitated. The findings in this study agree with the molecular data and they show the physical association of mdm2 and p53 in fresh liposarcoma surgical specimens.

Gene Amplification↗
PubMed1999

Mutation analysis reveals novel sequence variants in NTRK1 in sporadic human medullary thyroid carcinoma.

Tyrosine kinase NTRK1 is expressed in neural and nonneuronal tissues. Like RET, NTRK1 is often activated by rearrangements that involve one of at least five other genes in papillary thyroid carcinoma (PTC). Because of similarities in involvement of the two tyrosine kinases RET (rearranged during transfection) and NTRK1 in the pathogenesis of PTC, the obvious parallels between RET and NTRK1 and between PTC and medullary thyroid carcinoma (MTC), NTRK1 seemed to be an excellent candidate gene to play a role in the genesis of MTC. Single-strand conformational polymorphism analysis of 16 exons of NTRK1, from 31 sporadic MTC, revealed variants in five exons (exons 4 and 14-17). Sequence analysis demonstrated one sequence variant each in exons 4, 14, 16, and 17, and four different variants in exon 15. Differential restriction enzyme digestion specific for each variant confirmed the sequencing results. All variants were also present in the corresponding germline DNA. Interestingly, the sequence variants at codon 604 (c1810C>T) and codon 613 (c1838G>T) ofexon 15 always occurred together and might represent linkage disequilibrium. The frequencies of the sequence variants in germline DNA from patients with sporadic MTC did not differ significantly from those in a race-matched control group. Although we did not find any somatic mutations of NTRK1 in sporadic MTC, the single-strand conformational polymorphism conditions reported here, together with the knowledge of the frequency of various sequence variants, may help in future mutation analyses of DNA from other neural and nonneural tissues.

Carcinoma, Medullary↗
PubMed1999

Emergence of p53 mutant cisplatin-resistant ovarian carcinoma cells following drug exposure: spontaneously mutant selection.

We have previously shown that p53 mutations are associated with cisplatin resistance in ovarian carcinoma IGROV-1/Pt 1 cells. The relationship between p53 status and the development of resistance has not been completely elucidated; in particular, the biological mechanisms behind the acquired drug-resistant p53-mutant phenotype were not clearly explained. Thus, in this study, we investigated whether the p53 mutations found in IGROV-1/Pt 1 cells (270 and 282 codons) resulted from selection, under the selective pressure of the cytotoxic treatment, of a spontaneously mutant cell population preexistent in the cisplatin-sensitive parental cell line (IGROV-1) or were induced by drug (genotoxic) treatment. For this purpose, an allele-specific PCR approach was used. Primers carrying the desired mutations (T-->A codon 270, C-->T codon 282) in the 3' terminus, and the corresponding wild-type primers were used to amplify genomic DNA from the original IGROV-1 cell line used to select the mutant IGROV-1/Pt 1. To increase sensitivity, we hybridized blots of the PCRs with the radiolabeled PCR fragment from IGROV-1/Pt 1. Amplification was obtained for IGROV-1 DNA with the mutated allele-specific primers, indicating the preexistence of a mutated population in the IGROV-1 cell line. Titration experiments suggested that the frequency of the mutated alleles was <0.1%. Single-strand conformation polymorphism and allele-specific PCR analysis of the IGROV-1/Pt 0.1 cells, which are less resistant to cisplatin than IGROV-1/Pt 1 cells and which carry both mutant and wild-type p53 alleles with a wild-type predominance, suggested a progressive selection of the mutant population by cisplatin treatment. This is the first observation that indicates that a subpopulation of p53 mutant cells can occasionally be selected by cisplatin treatment. Thus, considering the susceptibility to spontaneous mutations of the p53 gene in advanced ovarian carcinoma, the selection process resulting in emergence of p53 mutant tumors is a possible origin of resistance of ovarian carcinoma to DNA-damaging agents. The survival advantage of p53 mutant cells in the presence of genotoxic agents could be related to a loss of susceptibility to p53-dependent apoptosis and to defects in checkpoints pathways, resulting in genomic instability.

Alleles↗
PubMed1999

Detection of microsatellite alterations in plasma DNA of non-small cell lung cancer patients: a prospect for early diagnosis.

A major problem in lung cancer is the lack of clinically useful tests for early diagnosis and screening of an asymptomatic population by non-invasive diagnostic procedures. Recent studies have demonstrated the possibility to detect genetic alterations in plasma or serum DNA from patients with various cancers. However, these data rely on small series of aggressive tumors with advanced-stage disease. To determine whether genetic changes in plasma are also detectable in patients with limited disease and thereby potentially useful for early detection, we looked for microsatellite instability (allele shift) and loss of heterozygosity in plasma DNA of 87 stage I-III non-small cell lung cancers and 14 controls. Combining two markers with a high rate of instability (D21S1245) and loss of heterozygosity (FHIT locus), a microsatellite alteration was observed in 49 of 87 (56%) non-small cell lung cancer tumors and in 35 of 87 (40%) plasma samples. Thirty of 49 (61%) of the cases showing tumor alterations also displayed a change in plasma DNA; in addition, 5 patients displayed alterations in plasma samples only. None of the control individuals had genetic changes in plasma. No association was found between the frequency of microsatellite alterations in plasma and tumor stage or histology. Of interest, plasma DNA abnormalities were detectable in 43% of pathological stage I cases and in 45% of tumors up to 2 cm in maximum diameter. These findings highlight new prospects for early tumor detection by noninvasive screening procedures based on the analysis of genetic changes in plasma.

Adult↗
PubMed1998

Loss of FHIT function in lung cancer and preinvasive bronchial lesions.

We previously cloned and characterized the tumor suppressor gene FHIT (fragile histidine triad) at chromosome 3p14.2 and found that this gene is altered by deletions in human tumors, including lung cancer. To assess the frequency and specificity of inactivation and its relevance in a clinical setting, we have produced antibodies against the Fhit protein and studied its expression in a series of non-small cell lung cancers and normal bronchial mucosa and a spectrum of preinvasive lesions by immunohistochemistry. The data indicate that the loss of Fhit protein is the most frequent alteration in non-small cell lung cancer (73%) and precancerous lesions (93%), is significantly higher in the tumors of smokers (75%) than in those of nonsmokers (39%; P < 0.0005), and is an independent and more frequent event than p53 overexpression in tumors and precancerous lesions (73 versus 46%). The percentage of cases lacking Fhit expression was higher in the squamous type compared to adenocarcinoma (87 versus 57%; P < 0.00001), whereas other histotypes (large cell, mucoepidermal) showed an intermediate value (69%). Loss of Fhit expression in a very high percentage of primary lung carcinomas and precancerous lesions supports the notion that FHIT alterations play an important role in the growth control of bronchial cells. FHIT inactivation is particularly important in squamous cell carcinomas that are often associated with precursor dysplastic lesions. The overall high frequency and precocity of Fhit loss in lung carcinogenesis and the development of the presently described immunohistochemical approach suggest a potential use of this gene in the early detection of lung cancer and in chemopreventive studies as an intermediate biomarker.

Acid Anhydride Hydrolases↗
PubMed1998

Transforming activity of the chimeric sequence formed by the fusion of collagen gene COL1A1 and the platelet derived growth factor b-chain gene in dermatofibrosarcoma protuberans.

As a consequence of a reciprocal translocation t(17;22)(q22;q13) and of supernumerary ring chromosomes derived from the t(17;22), a fusion between the platelet-derived growth factor b-chain (PDGF, c-sis proto-oncogene) and the collagen type 1A1 (COL1A1) genes has been recently described in dermatofibrosarcoma protuberans (DP), an infiltrating skin tumor (Simon et al., 1997). Although PDGFB has been implicated in transforming processes via autocrine and paracrine pathways, by the activation of the cognate receptor, no direct evidence of its involvement in neoplastic transformation of human tumours has been so far provided. In this report, we have tested the DNA from four DPs in the classical DNA transfection assay onto NIH3T3 fibroblast cell line. All the DNAs induced the formation of transformed foci in the transfected cultures whose derived cell lines were shown to contain a fused sequence comprising the human COL1A1 and PDGF genes. The relative breakpoint regions have been sequenced revealing that this gene fusion deleted exon 1 of PDGF and released the growth factor from its normal regulation. All the biochemical and biological assays were consistent with the model of an autocrine mechanism for NIH3T3 transformation by the human rearranged PDGFB gene involving the activation of the endogenous PDGF receptor.

3T3 Cells↗
PubMed1998

Grb2 binding to the different isoforms of Ret tyrosine kinase.

The RET proto-oncogene encodes two isoforms of a receptor tyrosine kinase which plays a role in neural crest and kidney development. Ret ligands have been recently identified as the neuron survival factor GDNF (Glial-Derived Neurotrophic Factor) and Neurturin. Somatic rearrangements of RET, designated RET/PTCs, have been frequently detected in papillary thyroid carcinomas. In addition, distinct germ-line mutations of RET gene have been associated with the inherited cancer syndromes MEN (Multiple Endocrine Neoplasia) 2A, 2B and FMTC (Familial Medullar Thyroid Carcinomas) as well as with the congenital megacolon or Hirschsprung's disease, thus enlightening a significant role of this receptor gene in diverse human pathologic conditions. In this study, by performing classical inhibition experiments using synthetic phosphopeptides and by site-directed mutagenesis of the putative docking site, we have determined that for Grb2 the latter is provided by the tyrosine 620 of Ret/ptc2 long isoform (corresponding to Tyr 1096 on proto-Ret). However, in intact cells, the interaction of Grb2 with the two short and long Ret isoforms expressed separately is of similar strength, thus suggesting that Ret short isoform interaction with Grb2 could be mediated not only by Shc but also by a molecule that binds preferentially to this isoform. This possibility is supported by the evidence that the mutant Ret/ptc2Y620F long isoform displays a weak coimmunoprecipitation with Grb2 and that this mutant, lacking the docking site for Grb2 but owing all the others phosphotyrosines, surprisingly displays a reduced transforming activity compared to that of the two WTs oncogenes. We thus conclude that in intact cells both Ret isoforms bind to Grb2, although with different modalities. In addition, the present results are in agreement with the possibility that different signal transduction pathways are associated with the two isoforms of Ret.

3T3 Cells↗