Biomedical subjects

M A Pierotti

Publications and source records attributed to M A Pierotti.

At least 91 records · Page 5Linked to original sources

PubMed1997

Absence of Fhit protein in primary lung tumors and cell lines with FHIT gene abnormalities.

Genomic alterations and abnormal expression of the FHIT gene at 3p14.2 have been observed in cell lines and primary tumors of the lung. To correlate FHIT locus DNA and RNA lesions with effects on Fhit protein expression, we have analyzed 11 lung cancer cell lines, 15 small cell lung carcinomas, and 38 pairs of non-small cell primary tumors and bronchial mucosa specimens by molecular genetic and immunocytochemical methods. Using specific antibodies against the Fhit protein, we observed concordance between RNA abnormalities and lack of Fhit protein expression in lung tumors and cell lines. In addition, absence of Fhit protein in some precancerous dysplastic lesions suggested that FHIT inactivation may occur at an early phase of lung carcinogenesis.

Acid Anhydride Hydrolases↗

PubMed1997

The multiple endocrine neoplasia type 2B point mutation switches the specificity of the Ret tyrosine kinase towards cellular substrates that are susceptible to interact with Crk and Nck.

The RET proto-oncogene encodes a Tyrosine Kinase Receptor (RTK) which plays an important function in the proliferation and/or differentiation of neuroectodermic cells. Germline mutation of a methionine to a threonine within the RET TK domain predisposes to the Multiple Endocrine Neoplasia type 2B (MEN 2B). It has been demonstrated that, unlike c-Ret, the MEN 2B mutated Ret displays constitutive TK activity, tyrosine autophosphorylation and transforms fibroblasts. However, this oncoprotein is more than a fully activated wild-type (WT) Ret TK since it also displays modified substrate specificity. Change in substrate specificity leads to the tyrosine autophosphorylation of MEN 2B Ret on new sites as well as the phosphorylation of several novel downstream targets. But, none of these substrates have been identified and the ability of MEN 2B Ret phosphoprotein to interact with Src Homology 2 (SH2) domain containing molecules has been poorly investigated. In this report, using a constitutively activated Ret TK form, Ret-ptc 2, we demonstrate that the MEN 2B as the activated WT Ret TK binds to several SH2 signalling proteins such as Shc, Grb-2, Phospholipase Cgamma, Crk and Nck. However, in contrast to the activated WT form, expression of the MEN 2B mutated Ret-ptc 2 results in the tyrosine phosphorylation of a panel of proteins which interestingly interact with Crk and Nck. We identified Paxillin, a cytoskeletal protein as one of the Crk associated proteins that is dramatically phosphorylated in MEN 2B but not in WT Ret expressing cells. These data suggest that MEN 2B mutated Ret triggers distinct signalling pathways that might be related to its transforming power.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Genetic mapping of lung cancer modifier loci specifically affecting tumor initiation and progression.

Mouse inbred strains with inherited predisposition and resistance to lung cancer provide a tool for the dissection of the complex genetics of this disease. In the present report, we have crossed the BALB/c with the SWR/J strain and performed whole-genome scanning for loci affecting lung tumor development in their F2 progeny. Both parental strains carry the pulmonary adenoma susceptibility 1 (Pas1) locus, a major locus affecting predisposition to lung cancer in mice. On distal chromosome 18 and on centromere of chromosome 6, we have mapped two pulmonary adenoma resistance loci (Par2 and Par4, respectively), which reduce lung tumor multiplicity strongly, up to 15-fold. Par2 and Par4, however, do not affect lung tumor size, which is instead controlled by an additional locus that we have mapped on the central region of chromosome 4. We designated this locus as "pulmonary adenoma progression 1" (Papg1), because it specifically modifies lung tumor size but not multiplicity. The present results, therefore, provide evidence for the existence of cancer modifier loci acting on specific stages of lung tumorigenesis.

Adenoma↗

PubMed1997

Chromosomal localizations and molecular analysis of TDG gene-related sequences.

The human mismatch-specific thymine DNA glycosylase gene, TDG, encodes a 60-kDa polypeptide able to correct G/T mispairs arising from the deamination of 5-methylcytosine. We localized by FISH three different TDG-related lambda genomic clones, lambda8, lambda11, and lambda12 on chromosome 12. PCR and sequence analyses revealed that only lambda11, localized at 12q24.1, contained the coding gene. We characterized the intron-exon boundaries of the portion of the gene contained in the lambda clone and identified a CA dinucleotide repeat in one intron. Northern blot analysis showed that TDG is expressed at approximately the same level in all human tissues analyzed. SSCP analysis of 50 tumor and corresponding normal tissue DNAs from lung cancer patients did not reveal the presence of any functional mutation. An abnormal SSCP pattern in one sample proved to be a polymorphism after sequencing and RFLP analysis.

Adult↗

PubMed1997

Comparison of the breakpoint regions of ELE1 and RET genes involved in the generation of RET/PTC3 oncogene in sporadic and in radiation-associated papillary thyroid carcinomas.

The RET/PTC3 oncogene is an activated form of the RET protooncogene, which is frequently rearranged in papillary thyroid carcinoma. RET/PTC3 results from a structural rearrangement between the ELE1 and the RET genes, and it has been observed in both sporadic and radiation-associated post-Chernobyl tumors. To understand the molecular basis that predisposes RET and ELE1 genes to be recurrent targets of "illegitimate" recombination, we examined the genomic regions containing the ELE1/RET breakpoints of six sporadic and three post-Chernobyl tumors in two papillary carcinomas of different origins. Our data indicated, in both genes, a clustering of the breakpoints in regions designated ELE1-bcr (1.8 kb) and RET-bcr (1.9 kb). Notably, in all sporadic tumors and in one post-Chernobyl tumor the ELE1/RET recombination corresponded with short sequences of homology (3-7 nt) between the two rearranging genes. In addition, we observed an interesting distribution of the post-Chernobyl breakpoints in ELE1-bcr located within an Alu element, or in between two close Alu elements, and always in A+T-rich regions.

Base Sequence↗

PubMed1997

Association between cigarette smoking and FHIT gene alterations in lung cancer.

Epidemiologic data have strongly indicated that cigarette smoking is linked to the development of lung cancer. However, little is known of the molecular targets of carcinogens contained in tobacco smoke. To identify genetic lesions characteristic of tobacco damage, we undertook a molecular analysis of microsatellite alterations within the FHIT gene and FRA3B, as well as at an independent locus on chromosome 10, D10S197, in lung tumors from heavy smokers and in tumors from never smokers. Loss of heterozygosity affecting at least one locus of the FHIT gene was observed in 41 of 51 tumors in the smokers group (80%) but in only 9 of 40 tumors in nonsmokers (22%). The comparison between the frequency of losses in FHIT in smokers and nonsmokers was statistically significant (P = 0.0001), whereas no difference in loss of heterozygosity rate was observed at D10S197 locus. These findings suggest that FHIT is a candidate molecular target of carcinogens contained in tobacco smoke.

Acid Anhydride Hydrolases↗

PubMed1997

Somatic in frame deletions not involving juxtamembranous cysteine residues strongly activate the RET proto-oncogene.

Somatic RET mutations have been identified in a variable proportion (about 30-70%) of sporadic Medullary Thyroid Carcinoma (MTC) cases. They are represented by the Met918Thr substitution (exon 16) typical of Multiple Endocrine Neoplasia type 2B (MEN2B) and, to a lesser extent, by nucleotide changes occurring at one of five critical cysteine residues (exons 10 and 11) typical of MEN type 2A (MEN2A). An in vitro transforming activity has already been demonstrated for these mutations. A few different MTC somatic mutations have been reported so far whose biological activity has still to be tested. In this paper we report the identification, in two MTC tumor samples, of two interstitial deletions of 48 bp and 6 bp occurred in exons 10 and 11 respectively. Both were somatic heterozygous in frame mutations, not involving any cysteine residue. Moreover, the expression of a full length RET cDNA carrying one of the two deletions demonstrated a strong transforming capacity in NIH3T3 cells.

3T3 Cells↗

PubMed1997

Identification of a novel human kinesin-related gene (HK2) by the cDNA differential display technique.

We have used the cDNA differential display technique to isolate genes regulated by the synthetic retinoid N-(4-hydroxyphenyl)-all-trans-retinamide (HPR), a cancer chemopreventive agent in vivo and a powerful inducer of apoptotic cell death in vitro. Here we report the identification of a novel gene, the expression of which is markedly up-regulated in tumor cells after treatment for 30-60 min with HPR. The full-length cDNA of this gene, determined by screening of a human placenta cDNA, is 3.5 kb long and contains an open reading frame of 2037 nt. The gene is > 90% homologous to the mouse KIF2, a gene belonging to the family of kinesin-related motor proteins, and we therefore named it HK2 (human kinesin 2). A shorter form of the HK2 mRNA (HK2s), containing a 57-nt deletion in the open reading frame, has also been detected. Northern analysis revealed that HK2 is widely expressed among hemopoietic and nonhemopoietic cell lines and tissues. By the use of radiation hybrids, HK2 has been localized to chromosome 5q12-q13. Kinesins constitute a superfamily of motor proteins that use energy liberated from ATP hydrolysis to move cargo along microtubules and are implicated in mechanisms of mitosis or meiosis. The role of HK2 in the growth-inhibitory and apoptotic responses elicited by HPR remains to be established.

Amino Acid Sequence↗

PubMed1997

Dissociation between cell cycle arrest and apoptosis can occur in Li-Fraumeni cells heterozygous for p53 gene mutations.

The radiation response was investigated in two lymphoblastoid cell lines (LBC) derived from families with heterozygous germ-line missense mutations of p53 at codon 282 (LBC282) and 286 (LBC286), and compared to cells with wt/wt p53(LBC-N). By gel retardation assays, we show that p53-containing nuclear extracts from irradiated LBC282 and LBC286 markedly differ in their ability to bind to a p53 DNA consensus sequence, the former generating a shifted band whose intensity is 30-40% that of LBC-N, the latter generating an almost undetectable band. Unlike LBC286, which fail to arrest in G1 after irradiation, LBC282 have an apparently normal G1/S checkpoint, as they arrest in G1, like LBC-N. While in LBC-N, accumulation of p53 and transactivation of p21WAF1 increase rapidly and markedly by 3 h after exposure to gamma-radiation, in LBC286 there is only a modest accumulation of p53 and a significantly delayed and quantitatively reduced transactivation of p21WAF1. Instead, in LBC282 while p53 levels rise little after irradiation, p21WAF1 levels increase rapidly and significantly as in normal LBC. Apoptotic cells present 48 h after irradiation account for 32% in LBC-N, 8-9% in LBC282 and 5-7% in LBC286, while the dose of gamma-radiation required for killing 50% of cells (LD50) is 400 rads, 1190 rads and 3190 rads, respectively, hence indicating that the heterozygous mutations of p53 at codon 282 affects radioresistance and survival, but not the G1/S cell cycle control. In all LBC tested, radiation-induced apoptosis occurs in all phases of the cell cycle and appears not to directly involve changes in the levels of the apoptosis-associated proteins bcl-2, bax and mcl-1. Both basal as well as radiation-induced p53 and p21WAF1 proteins are detected by Western blotting of FACS-purified G1, S and G2/M fractions from the three cell lines. p34CDC2-Tyr15, the inactive form of p34CDC2 kinase phosphorylated on Tyr15, is found in S and G2/M fractions, but not in G1. However, 24 h after irradiation, its levels in these fractions diminish appreciably in LBC-N but not in the radioresistant LBC286 and LBC282. Concomitantly, p34CDC2 histone H1 kinase activity increases in the former, but not in the latter cell lines, hence suggesting a role for this protein in radiation-induced cell death. Altogether, this study shows that, in cells harbouring heterozygous mutations of p53, the G1 checkpoint is not necessarily disrupted, and this may be related to the endogenous p53 heterocomplexes having lost or not the capacity to bind DNA (and therefore transactivate target genes). Radiation-induced cell death is not cell cycle phase specific, does not involve the regulation of bcl-2, bax or mcl-1, but is associated with changes in the phosphorylation state and activation of p34CDC2 kinase.

Apoptosis↗

PubMed1997

Identification of Shc docking site on Ret tyrosine kinase.

The RET proto-oncogene encodes two isoforms of a receptor type tyrosine kinase which plays a role in neural crest and kidney development. Distinct germ-line mutations of RET have been associated with the inherited cancer syndromes MEN2A, MEN2B and FMTC as well as with the congenital disorder Hirschsprung disease (HSCR), whereas somatic rearrangements (RET/PTCs) have been frequently detected in the papillary thyroid carcinoma. Despite these findings, suggesting a relevant role for RET product in development and neoplastic processes, little is known about the signalling triggered by this receptor. In this study, we have demonstrated that the transducing adaptor molecule Shc is recruited and activated by both Ret isoforms and by the rearranged cytoplasmatic Ret/ptc2 oncoproteins as well as by the membrane bound receptor activated by MEN2A or by MEN2B associated mutations. Moreover, our analysis has identified the Ret tyrosine residue and the Shc domains involved in the interaction. In fact, here we show that both the phosphotyrosine binding domains of Shc, PTB and SH2, interact with Ret/ptc2 in vitro. However, PTB domain binds 20 folds higher amount of Ret/ptc2 than SH2. The putative binding site for either SH2 and PTB domains has been identified as Tyr586 of Ret/ptc2 (Tyr1062 on proto-Ret). In keeping with this finding, by using RET/PTC2-Y586F mutant, we have demonstrated that this tyrosine residue, the last amino acid but one before the divergence of the two Ret isoforms, is the docking site for Shc.

3T3 Cells↗

PubMed1997

Distinct mdm2/p53 expression patterns in liposarcoma subgroups: implications for different pathogenetic mechanisms.

Recent findings have indicated that TP53 inactivation in sarcomas may result from mutation and/or deletion of the TP53 gene or, alternatively, from binding to the MDM2 gene products. To investigate further a possible role of the two genes in sarcomas, 24 large and deep-seated lipomas and 74 liposarcomas of various subtypes were analysed for mdm2 and p53 overexpression by immunocytochemistry. Nineteen cases of the same series were also molecularly analysed for both MDM2 gene amplification and TP53 mutations, and a further ten cases for non-random chromosomal abnormalities. In the retroperitoneal well-differentiated-dedifferentiated (WD-DD) group, 15/16 WD and 8/8 DD liposarcomas displayed the mdm2+/p53+ phenotype, consistent with MDM2 gene amplification in the absence of TP53 mutations. In the non-retroperitoneal WD-DD group, 5/11 WD liposarcomas also retained the mdm2+/p53+ phenotype whereas all DD liposarcomas showed an immunophenotype and, when assessed, a genotype consistent with mutant TP53. Null mdm2 immunophenotype, coupled with evidence of a specific chromosome translocation t(12;16), was constantly observed in both the usual and the cellular subtypes of myxoid liposarcoma, three cases of which also showed TP53 alterations at the genetic or protein level. Neither mdm2 nor p53 overexpression was observed in the lipomas. The results show the existence of three main pathogenetically distinct groups of liposarcoma. The first retroperitoneal WD-DD group, which represents a novel class of tumours within a single histological category of sarcoma, where MDM2-mediated inactivation of p53 could be related to the pathogenetic mechanism. The second is the non-retroperitoneal WD-DD group, where the TP53 mutations appear to correlate with the dedifferentiation process. The third is the myxoid group, which is characterized by its own unique cytogenetic profile and never shows any involvement of TP53 or MDM2 genes. As for diagnostic significance, the absence of mdm2 and p53 reactivity in lipomas seems to represent a useful marker for differential diagnosis from lipoma-like WD liposarcomas.

Adolescent↗

PubMed1997

Detection of p53 point mutations by double-gradient, denaturing gradient gel electrophoresis.

Genetic instability is a typical feature of tumor cells. This evidence has stimulated the development of rapid methods for detection of gene mutations. A new, improved protocol for denaturing gradient gel electrophoresis (DGGE), to screen for point mutations in genomic DNA, is reported: double gradient (DG) DGGE. In this technique, to the primary, denaturing gradient (typically 30-80% or 40-80% urea/formamide) a secondary gradient, colinear with the first, is superimposed: a porosity gradient (typically 6.5-12% polyacrylamide). The secondary gradient acts by recompacting smeared and diffuse bands of heteroduplexes, which are often indistinguishable from background fluorescence, and by augmenting the resolution between closely spaced homoduplex zones. This allows proper densitometric quantitation of the ratio of the two homoduplex bands. The reliability of this technique has been documented by detection of a number of mutations in exons 6 and 8 of the p53 gene which had escaped revelation by single-strand conformational polymorphism (SSCP) analysis. Additionally, the precise assessment of ratio of the doublet of homoduplex bands has allowed quantitation of the extent of p53 mutation in a mixed cell population extracted from a tumor specimen.

Electrophoresis, Polyacrylamide Gel↗

PubMed1997

Pas1 is a common lung cancer susceptibility locus in three mouse strains.

Inherited predisposition to lung cancer is a phenotypic trait shared by different mouse inbred strains that show either a high or an intermediate predisposition. Other strains are instead genetically resistant. The Pas1 locus is the major determinant of lung cancer predisposition in the A/J strain (Gariboldi et al. 1993). To define the determinants of susceptibility to lung tumorigenesis in the highly susceptible SWR/J and in the intermediately susceptible BALB/c mice, we analyzed (BALB/c x SWR/J)F2 and (BALB/c x C3H/He)F2 crosses by genetic linkage experiments. The present results provide unequivocal evidence that the same Pas1/+ allele that leads to lung cancer predisposition is shared by A/J, SWR/J, and BALB/c strains. The intermediate susceptibility of the BALB/c strain would result by interaction of Pas1 locus with lung cancer resistance loci.

Animals↗

PubMed1997

Relevance of cytogenetic and fluorescent in situ hybridization analyses in the clinical assessment of soft tissue sarcoma.

Soft tissue sarcomas are a heterogeneous group of malignant tumors displaying a wide range of clinical presentations, morphological features, and biological behaviors. These characteristics and the recent development of differentiated treatment regimens for the different types of soft tissue sarcomas call for refined histological classification using additional ancillary approaches such as cytogenetic and molecular genetic analyses. We coupled classical cytogenetics and fluorescent in situ hybridization (FISH) on both metaphases and interphase nuclei to show the feasibility of this approach to characterize tumor type-specific chromosome rearrangements in soft tissue sarcomas of different histotype. In 35 cases analyzed, we detected the presence of specific chromosome rearrangements such as t(X;18) in synovial sarcoma (SS), t(12;16) in myxoid liposarcoma (MLS), t(11;22) in peripheral primitive neuroectodermal tumors (pPNET), t(2;13) in alveolar rhabdomyosarcoma (ARMS) and ring chromosomes in dermatofibrosarcoma protuberans (DFSP). In several cases, the presence of these cytogenetic rearrangements was of help for a differential diagnosis. The FISH analysis using painting probes not only confirmed the cytogenetic results but also allowed the identification of tumor-specific chromosome changes in those cases presenting low mitotic index or with poor quality chromosomes. Moreover, in the absence of analysable metaphases, FISH was successfully performed on interphase nuclei. Taken together, these results indicate both the diagnostic and clinical relevance of a molecular cytogenetic analysis in the study of soft tissue sarcomas.

Biomarkers, Tumor↗

PubMed1997

Association of chromosome 12p genetic polymorphisms with lung adenocarcinoma risk and prognosis.

The mapping near Kras2 of pulmonary adenoma susceptibility 1 (Pas1), a major locus affecting inherited predisposition to lung cancer in mice prompted us to test the homologous human region (12p12) for association with lung adenocarcinoma, by a population-based study. We genotyped 213 lung adenocarcinoma patients and 219 healthy blood donor subjects for five polymorphic markers mapping in the putative region of interest. Three marker polymorphisms, located in a region spanning approximately 700 kb, were significantly associated with lung adenocarcinoma risk. Furthermore, polymorphisms in KRAS2 and PTHLH loci were also associated with tumor prognosis. These results suggest the existence of a human Pas1 homologous locus on chromosome 12p12.

Adenocarcinoma↗

PubMed1997

Role of antioxidants and intracellular free radicals in retinamide-induced cell death.

The cancer chemopreventive synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) possesses antiproliferative and apoptotic activity at pharmacological doses. In this study we show that addition of antioxidants to HL-60 cells cultured in the presence of 3 microM HPR, markedly suppresses the apoptopic effect of the retinoid and significantly prolongs cell survival (48-96 h). We also show, by the use of the oxidation-sensitive probe 2',7'-dichlorofluorescin diacetate (DCF-DA) and in combination with flow cytometric and spectrofluorimetric analysis, that treatment of cells with 3 microM HPR results in an immediate and sustained production of intracellular free radicals, most likely hydroperoxides. Interestingly, the formation of these HPR-induced free radicals is effectively blocked by the water soluble antioxidants L-ascorbic acid and N-acetyl-L-cysteine. Neither 3-15 microM N-(4-methoxyphenyl) retinamide (MPR), the structurally similar but biologically inert analog of HPR, nor 3 microM doses of the retinoids all-trans retinoic acid, 9-cis-retinoic acid, TTNPB and SR11237 induce intracellular free radicals, thus indicating that the specificity of this phenomenon is restricted to HPR. Altogether, we provide the first direct evidence that HPR stimulates the generation of intracellular free radicals, which appear to have a causative role in the induction of apoptosis in vitro. Our findings raise the possibility that the therapeutic efficacy of HPR may, at least in part, depend on these apoptosis-inducing oxidative phenomena.

Antioxidants↗

PubMed1997

Insular carcinoma: a distinct de novo entity among follicular carcinomas of the thyroid gland.

We reclassified 720 nonmedullary invasive thyroid carcinomas diagnosed and treated between 1975 and 1993. Twenty-seven cases met the criteria of insular carcinoma and 29 cases those of widely invasive follicular carcinoma. Comparison of these histotypes with respect to pathologic stage and overall, relative, and visceral metastasis-free survival showed a significant association between histotype and pT and pN categories. In particular, pT4 (p < 0.001) and pN1 (p < 0.001) categories were more frequent in the insular carcinoma histotype. By contrast, no significant differences in overall, relative, or visceral metastasis-free survival were observed between insular carcinoma and widely invasive follicular carcinoma. Molecular analysis by polymerase chain reaction-single-strand conformation polymorphism demonstrated RAS gene family point mutations in five of eight cases analyzed in each of the two histotypes, with a high proportion of CAA-->AAA transversion at codon 61 of the N-RAS gene in insular carcinoma. These findings suggest that insular carcinoma represents a de novo entity distinct from widely invasive follicular carcinoma, that widely invasive follicular carcinoma has biologic characteristics more consistent with poorly differentiated than well-differentiated carcinomas, and that both insular carcinoma and widely invasive follicular carcinoma share similar molecular alterations.

Abdominal Neoplasms↗

PubMed1997

PCR analysis of IgH and BCL2 gene rearrangement in the diagnosis of follicular lymphoma in lymph node fine-needle aspiration. A critical appraisal.

In order to improve the cytomorphologic diagnosis of malignant lymphoma on lymph node fine-needle aspiration (FNA), and to make a confident discrimination between low-grade follicular non-Hodgkin's lymphoma (NHL) and lymphoid hyperplasia, polymerase chain reaction (PCR) analysis was performed of the Ig CDR3 region and BCL2 breakpoint region in 25 nonselected cases of malignant lymphoma (17 NHL and 8 Hodgkin's disease [HD]) with histologic control, and 22 cases of lymph nodal hyperplasia with histologic and/or clinical control. Among lymphomas, IgH monoclonality was detected in 7 (77%) of 9 NHLs and BCL2 rearrangement in 3 (17.6%) of 17 NHLs, all of which were follicular centroblastic-centrocytic (FCBCC). Three BCL2/JH negative FCBCC cases were monoclonal for CDR3. Neither IgH monoclonality nor BCL2 rearrangement were found in HD. Among cytologically diagnosed lymphoid hyperplasias, one IgH polyclonal case was considered false-negative, being histologically diagnosed as lymphoplasmacytic NHL on the subsequent excisional biopsy. Another 4 cases (2 BCL2 rearranged and 2 monoclonal for IgH) were considered false-positive on the basis of histologic features or clinical control. These data indicate that the combined PCR analysis of IgH and BCL2 rearrangements can confirm a cytologic diagnosis of lymphoma in FNAs while, due to the occurrence of both false-positive and false-negative results, it is of limited value in the distinction between follicular lymphoma and lymphoid hyperplasia without morphologic or clinical support.

Biopsy, Needle↗