Search PubMed⌕ Search

Biomedical subjects

M A Pierotti

Publications and source records attributed to M A Pierotti.

At least 109 records · Page 6Linked to original sources
PubMed1997

Chromosome 1 rearrangements involving the genes TPR and NTRK1 produce structurally different thyroid-specific TRK oncogenes.

The NTRK1 gene in the q arm of chromosome 1 encodes one of the receptors for the nerve growth factor and is frequently activated as an oncogene in papillary thyroid carcinomas. The activation is due to chromosomal rearrangements juxtaposing the NTRK1 tyrosine kinase domain to 5'-end sequences from different genes. The thyroid TRK oncogenes are activated by recombination with at least three different genes: the gene coding for tropomyosin and TPR, both on chromosome 1,and TFG on chromosome 3. In a previous study, we showed that two tumors carrying the TPR/NTRK1 rearrangement contained structurally different oncogenes named TRK-T1 and TRK-T2. In this paper, we report (1) the cDNA structure of TRK-T2, (2) evidence that TRK-T2 is generated by different rearrangements in two thyroid tumors, and (3) a detailed analysis of the three different TPR/NTRK1 rearrangements. With molecular studies based on Southern blot hybridization, cloning, and sequencing, we show that all the rearrangements are nearly balanced, involving deletion, insertion, or duplication of only few nucleotides. In one case, an additional rearrangement involving sequences derived from chromosome 17 was detected.

Base Sequence↗
PubMed1997

Mean age of tumor onset in hereditary nonpolyposis colorectal cancer (HNPCC) families correlates with the presence of mutations in DNA mismatch repair genes.

Fourteen Italian families affected with hereditary nonpolyposis colorectal cancer (HNPCC) were screened for germline mutations at three DNA mismatch repair (MMR) genes, MSH2, MLHI, and GTBP, by using a combination of different methods that included an in vitro synthesized protein assay, single-strand conformation polymorphism analysis, and direct sequencing. DNA alterations were observed in six instances, including a single base deletion in MSH2 exon 14, an A-to-G transition in the splice donor site of MLHI exon 6, and two missense mutations in MLHI exons 5 and 9. A previously reported common mutation affecting the splice donor site of MSH2 exon 5 was identified in two families. No mutations were detected in the GTBP gene. In total, eight of 16 Italian HNPCC families (50%), including two previously reported kindreds, were found to carry a mutation in MMR genes. We compared the mean age of colorectal cancer onset in the index cases (three patients for each family) between the two groups of kindreds, those with identified mutation vs. those without, and found that the first had a significantly lower value (43.0 vs. 53.7 years, P = 0.014). This finding suggests that HNPCC families with a more advanced age of tumor onset are less likely to be associated with known MMR genes.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Molecular genetics of breast cancer.

In the last two decades, molecular studies have enlightened the complexity of the genetic alterations that occur in breast cancer cells. To date, more than 40 different genes or loci have been found to be altered in breast carcinomas. Although some of these genes, as for example ERBB2, appear to be mutated in a high proportion of cases, their mechanism of action and their role in the different stages of cancer development are still poorly understood. More recently, two major determinants of the inherited predisposition to breast cancer, BRCA1 and BRCA2, have been isolated. As a consequence, it is now possible to screen families with a positive history of breast carcinomas for the identification of mutations carriers, in order to address these individuals into adequate programs of cancer surveillance and prevention.

Breast Neoplasms↗
PubMed1996

Genomic organization of the human NTRK1 gene.

The NTRK1 gene encodes one of the receptors for the Nerve Growth Factors and it is located at 1q21-22. Rearrangements of NTRK1 are frequently detected in human papillary thyroid carcinoma and lead to the formation of chimeric oncogenes, similarly to what observed for the other neurotrophin receptor RET. In addition, the two receptor genes are target of point mutations associated with different human diseases. RET is affected by germ line and somatic mutations in MEN2A, MEN2B tumor syndromes and in the abnormal developmental Hirschsprung disease, whereas mutations of NTRK1 have been reported very recently in patients with congenital insensitivity to pain with anidrosis (CIPA). With the aim to provide a tool for searching mutations along the whole NTRK1 gene, we have determined its genomic organization. Our results demonstrated that NTRK1 is contained within 25 Kb of DNA and is organized in 17 exons, one of which is alternatively spliced. The sequence of the 5' flanking region indicates a high content in C/G, the absence of TATA box, the presence of several putative binding sites for Sp1, AP1, AP2, AP3, ATF and GCF transcription factors.

Alternative Splicing↗
PubMed1996

Mapping of a putative tumor suppressor locus to proximal 7p in Wilms tumors.

Different findings suggest that alterations of chromosome 7 genes play a role in the development of Wilms tumors. To define the positions of these genes, we have accomplished a combined cytogenetic and molecular study on 11 sporadic Wilms tumors. In one case, where both chromosomes 7 were rearranged, the karyotypic picture was consistent with the presence of a tumor suppressor gene at 7p15. To test this hypothesis, a loss of heterozygosity analysis was performed using microsatellite markers. This revealed a common region of allele losses mapped to the proximal short arm of chromosome 7 and defined the position of the gene(s) involved in Wilms tumors within an interval of approximately 25 cM.

Chromosome Mapping↗
PubMed1996

Screening for mutations in exon 11 of the BRCA1 gene in 70 Italian breast and ovarian cancer patients by protein truncation test.

The most common mutations in the familial breast and ovarian cancer susceptibility gene BRCA1 are frameshift and nonsense mutations, which lead to the synthesis of truncated proteins. On this ground, we have analysed BRCA1 exon 11, which includes about 61% of coding region, in germline DNA from 70 Italian breast and/or ovarian cancer patients, using the protein truncation test (PTT). BRCA1 mutations were identified in nine of 29 (approximately 31%) patients with a family history of cancer and in three of 41 (approximately 7%) women with early-onset breast carcinomas, and were subsequently characterized by sequence analysis. In addition, BRCA1 mutations were also detected in six affected relatives of two positive index cases. The observed frequencies of mutations were not significantly different from those expected on the basis of the phenotypic characteristics of patients and their families, indicating that PTT is a rapid and sensitive method that can be used for a first BRCA1 mutational screening. The histological findings in BRCA1 mutated cases showed that eight of nine (approximately 89%) breast carcinomas were of grade III and nine of 9 (100%) ovarian carcinomas were of the endometrioid type (eight of grade III and one of grade II). This suggests that specific histological characteristics may represent additional criteria for selection of cases eligible to BRCA1 mutational analysis.

Adult↗
PubMed1996

4-Hydroxyphenyl retinamide is a highly selective activator of retinoid receptors.

Retinoids have shown promise as anti-cancer and cancer preventative agents. All-trans-N-(4-hydroxyphenyl)retinamide (4HPR) belongs to a new group of retinoids that not only inhibit the proliferation of cancer cells but also can induce apoptosis in certain cancer cells. Because of its increased efficacy against cancer cells and its low toxicity it has been entered into a number of clinical trials. However, its mechanism of action is not known, and it had been assumed that it is not a true retinoid. Here we analyze its ability to function as an activator of nuclear retinoid receptors (RARs and RXRs). We observe that, in transactivation assays, 4HPR is a potent transactivator with RARgamma and a moderate activator with RARbeta but is not an activator with RARalpha and RXRalpha. Furthermore, RARgamma-selective transactivation by 4HPR is enhanced on some response elements and reduced on others when compared to natural retinoids. In contrast to transactivation, 4HPR in transrepression assays functions mostly with RARalpha, RARbeta, and RXRalpha. Optimal receptor activation is seen at 4HPR concentrations at which it is a potent growth inhibitor and inducer of apoptosis. We conclude that 4HPR is a highly selective activator of retinoid receptors. We propose that this selective activation of the nuclear receptors is likely to be the basis for its specific biological activities and its favorable pharmaceutical properties.

Anticarcinogenic Agents↗
PubMed1996

Molecular and biochemical analysis of RET/PTC4, a novel oncogenic rearrangement between RET and ELE1 genes, in a post-Chernobyl papillary thyroid cancer.

A post-Chernobyl papillary thyroid cancer, displaying a novel ELE1/RET oncogenic rearrangement with an anomalous fusion transcript, was molecularly characterized. In spite of the presence of a normal breakpoint in exon 5 of the activating ELE1 gene, the sequence of the rearranged genomic DNA showed a previously unreported intra-exonic breakpoint in the RET protooncogene. As a consequence, a cDNA sequence 93 nucleotides larger than the regular one, and with the exon 5 of ELE1 joined to exon 11 instead of exon 12 of RET, is formed. To characterize the product of this new oncogenic ELE1/RET rearrangement, here designated as RET/PTC4, we performed an immunoprecipitation and Western blot analysis on cell extracts from NIH3T3 transfectants. The results showed the presence of two isoforms of the chimeric protein, displaying a constitutive tyrosine phosphorylation. As expected, the molecular weight of this protein was higher than that of RET/ PTC3 protein (p80 and p85, instead of p76 and p81). Previous reports, from our and other laboratories, showed that post-Chernobyl papillary thyroid carcinomas are characterized by a high frequency (about 60%) of RET oncogenic rearrangements (Fugazzola et al., 1995; Klugbauer et al., 1995; Ito et al., 1994). These events predominantly involve ELE1 activating sequence, thus producing RET/PTC3 oncogene (Fugazzola et al., 1995; Klugbauer et al., 1995). Hence, this elevated frequency of RET rearrangements could increase the probability of selecting unusual events as that here described. Alternatively, targeted radiation effects could be responsible for the atypical RET rearrangement producing RET/PTC4 oncogene.

Base Sequence↗
PubMed1996

Aberrant FHIT transcripts in Merkel cell carcinoma.

Merkel cell carcinoma is a rare neuroendocrine carcinoma of the skin which shares several features with small cell lung carcinoma. In a previous study, we reported a high frequency of abnormalities of the FHIT gene, located at 3p14.2, in small cell lung tumors. To determine the role of the FHIT gene in small cell neuroendocrine malignancies, 14 cases of Merkel cell carcinoma were analyzed by reverse transcription of FHIT mRNA followed by PCR amplification and sequencing of products. Eight of 14 tumors (57%) displayed abnormal FHIT products that lacked three or more exons of the FHIT gene. The pattern of abnormal transcripts was similar to that observed in small cell lung tumors, suggesting that FHIT abnormalities might be a common genetic marker of these two types of neuroendocrine tumors.

Acid Anhydride Hydrolases↗
PubMed1996

Microsatellite alterations in bronchial and sputum specimens of lung cancer patients.

Early diagnosis of lung cancer based on conventional screening procedures has been unable thus far to decrease lung cancer mortality. We explored the possibility of using microsatellite instability in the detection and screening of early phases of lung carcinogenesis. We studied tumor, histopathologically normal bronchial mucosa, and cytological specimens of 51 lung cancer patients for the presence of clonal variations at microsatellite polymorphisms. Microsatellite alterations were found in tumor, normal bronchial mucosa and cytological specimens of 25 of 51 (49%) of the patients. The detection of microsatellite alterations in histopathologically normal bronchial specimens and cytological clinical samples with minimal atypia suggests a possible application of this genetic marker in early diagnosis of precancerous lesions and lung cancer.

Adenocarcinoma↗
PubMed1996

The FHIT gene 3p14.2 is abnormal in lung cancer.

To determine the role of the FHIT gene, which encompasses the fragile site at 3p14.2, we analyzed 59 tumors of the small cell and non-small cell type by reverse transcription of FHIT mRNA, followed by PCR amplification and sequencing of products. Allelic losses affecting the gene were evaluated by microsatellite polymorphism analysis and genomic alterations by hybridization using cDNA and genomic probes. Small cell lung tumors (80%) and non-small cell lung cancers (40%) showed abnormalities in RNA transcripts of FHIT, and 76% of the tumors exhibited loss of FHIT alleles. Abnormal lung tumor transcripts lack two or more exons of the FHIT gene. Small cell lung cancer tumors and cell lines were analyzed by Southern blotting and showed rearranged BamHI fragments. These data suggest a critical role of the FHIT gene in lung carcinogenesis.

Amino Acid Sequence↗
PubMed1996

A comparative study of p53 gene mutations, protein accumulation, and response to cisplatin-based chemotherapy in advanced ovarian carcinoma.

The p53 protein is a multifunctional transcriptional regulator involved in cellular response to DNA damage and has been implicated as a putative determinant of sensitivity of tumor cells to cytotoxic agents. Since the p53 gene becomes inactivated in over one-half of advanced ovarian carcinoma, in this study we have examined the relationships between p53 gene alterations, p53 immunoreactivity, and response to cisplatin-based chemotherapy in ovarian cancer patients. All patients had advanced (FIGO stage III or IV) ovarian carcinoma and, with one exception, were untreated at the time of collection of tumor specimens. After initial debulking surgery, patients received high-dose cisplatin therapy. Tumor samples were analyzed for p53 gene mutations and for p53 protein accumulation, and the findings were correlated with tumor responsiveness. Of the 33 tumors examined, p53 gene mutations were found in 20 cases, including 15 missense mutations, 2 deletions, 2 nonsense mutations, and a base substitution at splice site. Twenty tumors showed positive immunostaining for p53. Only missense mutations were associated with positive immunostaining. In addition, p53 overexpression was detected in five tumors in the absence of mutations. Most (12 of 14) of the missense mutations associated with p53 protein stabilization were found refractory to therapy, as well as tumors overexpressing wild-type p53 (4 of 5). A significant correlation has been found between p53 accumulation, type of mutation (i.e., missense mutations), and pathological response to cisplatin-based therapy. In conclusion, the present results are consistent with a role of p53 as a determinant of chemosensitivity of ovarian carcinoma.

Alleles↗
PubMed1996

Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems.

P53 status may be a determinant of chemosensitivity of tumor cells; however, its involvement in cellular resistance to cisplatin remains uncertain. To investigate the relationships between p53 and the development of resistance to cisplatin, the p53 gene status was studied in ovarian carcinoma cell systems which included two cisplatin-resistant variants (IGROV-1/Pt 0.5 and IGROV-1/Pt 1) selected in vitro after prolonged drug exposure of the cisplatin-sensitive parental IGROV-1 cell line. IGROV-1/Pt 0.5 and IGROV-1/Pt 1 cell lines exhibited a degree of resistance of approximately 6 and 14, respectively, following 96-h exposure to the drug and were cross-resistant to other DNA-damaging agents (ionizing radiation and melphalan). Resistance to cisplatin paralleled a reduced cell susceptibility to cisplatin-induced apoptosis. DNA single-strand conformation polymorphism analysis of exons 5-9 demonstrated the presence of two mutants alleles at exon 8 in the two resistant cell lines, in contrast to the parental IGROV-1 cell line which exhibited the wild-type p53 gene. Direct DNA sequencing revealed that the mutations consist of two nucleotide changes in the DNA-binding domain at codons 270 (T/A) and 282 (C/T). The consecutive levels of p53 protein were lower in IGROV-1 than in IGROV-1/Pt cells. Following exposure to ionizing radiation or cisplatin, accumulation of the p53 protein was markedly enhanced only in the sensitive cells. Concomitantly, the expression of WAF-1 protein was strongly induced in the parental IGROV-1 cells, whereas WAF-1 protein remained undetectable in the IGROV-1/Pt 1 subline after DNA-damaging treatment. Consistent with this finding is the observation that ionizing radiation caused a different pattern of cell cycle perturbation in sensitive and resistant cells. Northern blot analysis demonstrated a marked reduction in bax mRNA levels in IGROV-1/Pt 1 cisplatin-resistant cells. Cotransfection assays with wild-type or mutant p53 expression plasmids and a reporter gene plasmid that utilized the bax gene promoter to drive transcription of chloramphenicol acetyltransferase were consistent with the role of p53 in regulation of bax expression in these cells. Taken together, these observations support a role for mutations of the p53 gene in the development of cisplatin resistance in ovarian cancer as a consequence of loss of the ability of p53 to transactivate bax, an apoptosis-inducing gene.

Apoptosis↗
PubMed1996

Variant translocation t(6;10)(p21;q22) in pulmonary chondroid hamartoma.

Banding cytogenetics and fluorescence in situ hybridization analysis of a pulmonary chondroid hamartoma (PHC) showed the presence of a t(6;10)(p21;q22). A cytogenetically identical translocation has previously been found in another case of PHC, suggesting that it could represent a variant form of the standard t(6;14)(p21;q24).

Chromosome Banding↗
PubMed1996

Cytogenetics and molecular genetics of carcinomas arising from thyroid epithelial follicular cells.

Cytogenetic and molecular analyses of thyroid tumors have indicated that these neoplasms represent a good model for analyzing human epithelial cell multistep carcinogenesis. They comprise, in fact, a broad spectrum of lesions with different phenotypes and variable biological and clinical behavior. Molecular analysis has detected specific genetic alterations in the different types of thyroid tumors. In particular, the well-differentiated carcinomas of the papillary type are characterized by activation of the receptor tyrosine kinases (RTKs), RET and NTRK1 proto-oncogenes. Cytogenetic analysis of these tumors has contributed to defining the chromosomal mechanisms leading to RTK oncogenic activation. In the majority of cases, intrachromosomal inversions of chromosome 10 and chromosome 1 led to the formation of RET-derived and NTRK1-derived oncogenes, respectively. Interestingly, molecular analysis of these oncogenes revealed their nature of chimeric fusion proteins all sharing the tyrosine kinase (TK) domains of the respective proto-oncogenes. Moreover, the sequencing of the oncogenic rearrangements led to the identification of a breakpoint cluster region in both RTK proto-oncogenes. Exposure to ionizing radiation is associated with papillary carcinomas and RET activation has been suggested to be related to this event. Conversely, RAS point mutations are frequently observed in tumors with follicular histology and have been associated with metastatic dissemination. Iodide-deficient areas seem to provide a higher frequency of RAS positive follicular carcinomas. Finally, a high prevalence of TPS3 point mutations has been detected only in undifferentiated or anaplastic carcinomas and found to correlate inversely with 8CL2 expression. All of these findings are contributing to the definition of genetic and environmental factors relevant for the pathogenesis of thyroid tumors. Moreover, the characterization of specific genetic lesions could provide significant molecular tools for a better differential diagnosis and for the development of novel therapeutic avenues for thyroid cancer.

Carcinoma, Papillary↗
PubMed1996

Downexpression of aldehyde dehydrogenase 1 in murine lung tumors.

We analyzed the expression of the cytosolic aldehyde dehydrogenase 1 (Aldh1) gene in mouse lung tumors by northern blotting and immunocytochemical analysis. Aldh1 was abundantly expressed in normal lung tissue, with a predominant cellular localization on bronchiolar cells. However, expression of Aldh1 was strongly reduced (more than tenfold) in lung tumors. As aldehyde dehydrogenases metabolize some antitumor alkylating drugs to inactive compounds, the low expression of Aldh1 in lung tumors may account for the drug sensitivity of these tumors to chemotherapeutic agents.

Aldehyde Dehydrogenase↗