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Biomedical subjects

G Finocchiaro

Publications and source records attributed to G Finocchiaro.

At least 55 records · Page 3Linked to original sources
PubMed1996

Deletion mapping of gliomas suggest the presence of two small regions for candidate tumor-suppressor genes in a 17-cM interval on chromosome 10q.

The loss of genetic material on chromosome 10q is frequent in different tumors and particularly in malignant gliomas. We analyzed 90 of these tumors and found loss of heterozygosity (LOH) in >90% of the informative loci in glioblastoma multiforme (GBM). Initial studies restricted the common LOH region to 10q24-qter. Subsequently, the study of a pediatric GBM suggested D10S221 and D10S209, respectively, as centromeric and telomeric markers of a 4-cM LOH region. It is interesting to note that, in one subset of cells from this tumor, locus D10S209 seems involved in the allelic imbalance of a larger region, with D10S214 as telomeric marker. This 17-cM region contains the D10S587-D10S216 interval of common deletion recently defined on another set of gliomas.

Brain Neoplasms↗
PubMed1996

Partial regression, yet incomplete eradication of mammary tumors in transgenic mice by retrovirally mediated HSVtk transfer 'in vivo'.

Mice transgenic for the activated rat neu oncogene under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) (neu+ mice), develop breast tumors in 100% of cases. We have previously reported that double transgenic mice obtained from crossing neu+ mice with mice transgenic for the herpes simplex virus thymidine kinase (HSVtk) gene can be used as a suitable model to test the 'suicide gene' strategy for mammary tumor gene therapy in vivo. In the present study, we evaluated the efficacy of the HSVtk/ganciclovir (GCV) system in the neu+ mice by inoculating cells producing a retroviral vector bearing the HSVtk gene in the mammary tumors on one side of the animals, and comparing their weight with that of the contralateral tumors, after systemic GCV administration. A statistically significant effect of this therapy was clearly seen (P < 0.001) but complete eradication of the tumors could not be achieved. This was not due to the inefficient delivery of GCV, as no HSVtk expression was detected in the residual tumors, but could be related to the low transduction efficiency (< 10%) and to inability of the 'bystander effect' (probably due to the absence of functional gap-junctions among mammary tumor cells) to kill nontransduced neoplastic cells. These data suggest that results obtained by in vivo models using transplanted tumor cell lines as targets for gene therapy might not be immediately transferable to spontaneously arising tumors in animals or humans.

Adenocarcinoma↗
PubMed1995

Deletion and transfection analysis of the p15/MTS2 gene in malignant gliomas.

We have investigated the status of the MTS2 gene, encoding the cyclin-dependent kinase (CDK) inhibitor p15, in 32 glioblastomas. Semi-quantitative PCR identified 7 tumors in which the amplified material was 18.6% of controls and 7 in which was 48.0%, suggesting the occurrence of homozygous and hemizygous deletions, respectively. Single strand conformation polymorphism analysis identified one polymorphism but no mutations. We also expressed MTS2 and MTS1, encoding the contiguous and highly homologous CDK inhibitor p16, in U-87 human glioblastoma cells. Both genes, either separately or in combination, inhibit significantly the proliferation rate of U-87 cells but such inhibition is progressively lost. As a whole, the data assign a tumor suppressor role to p15 and confirm homozygous deletions as the favorite mechanism for the inactivation of MTS1 and MTS2 in glioblastomas.

Base Sequence↗
PubMed1995

Redefinition of the coding sequence of the MXI1 gene and identification of a polymorphic repeat in the 3' non-coding region that allows the detection of loss of heterozygosity of chromosome 10q25 in glioblastomas.

The MXI1 gene encodes a protein interacting with Max, a regulatory factor of the Myc oncogene, and is located on chromosome 10q25, a region showing frequent loss of heterozygosity in malignant gliomas. We have reassessed the coding sequence of MXI1 and found that, at the 3' end, the open reading frame is 28 codons shorter than previously described. We have also found an AAAAC polymorphic repeat (two alleles, 45% heterozygosity) in the 3' non-coding region of the gene. Six anaplastic astrocytomas and nine glioblastomas, the most malignant form of glioma, were informative for this polymorphism. Loss of heterozygosity was demonstrated in all glioblastomas, but not in the remaining tumors.

Base Sequence↗
PubMed1995

The "bystander effect": association of U-87 cell death with ganciclovir-mediated apoptosis of nearby cells and lack of effect in athymic mice.

Cells expressing the herpes simplex-thymidine kinase (HS-TK) gene as a consequence of retroviral transduction, as well as TK-negative (TK-) bystander cells, can be killed by treatment with ganciclovir (GCV). In vitro, this "bystander effect," has been attributed to metabolic cooperation through gap junctions or to the uptake of apoptotic vesicles. We show that GCV treatment kills TK-negative U-87 glioma cells cocultured with cells that express TK (TK+) but that have lost the capacity for releasing retroviral particles. A photometric enzyme immunoassay identifies histone-associated DNA fragments, typical of apoptosis, in the cytosol of GCV-treated TK+ cells, and apoptotic features are also demonstrated by ultrastructural studies. Northern blot analysis and the reverse transcription polymerase chain reaction (PCR) show that connexin 43, a major constituent of gap junctions, is expressed in TK+ and U-87 cells. The size of U-87 tumors in nude mice subsequently injected with TK+ cells and GCV is not significantly different than in untreated animals; whereas, after injecting 1:1 mixtures of U-87 and TK+ cells, GCV treatment only causes a temporary regression of tumor growth. On the contrary, when the injected mixtures contain PA317.STK.SBA (a retroviral producer cell line that can transduce efficiently the HS-TK gene) and U-87 cells, tumors are destroyed effectively by GCV treatment. Thus, an experimental setting in which U-87 gliomas are matched with cells that are able to express, but not to transduce, the HS-TK gene indicates that the bystander effect kills U-87 cells in vitro by mechanisms associated with apoptotic death. In vivo, this effect is not sufficient to restrain the tumor growth taking place in immunodeficient animals.

Animals↗
PubMed1995

Carnitine palmitoyltransferase II deficiency: structure of the gene and characterization of two novel disease-causing mutations.

Carnitine palmitoyltransferase (CPT) II deficiency is the most common inherited disorder of lipid metabolism affecting skeletal muscle. To facilitate the identification of disease-causing mutations in the CPT II gene (CPT1), we have established the genomic organization of this gene. CPT1 spans approximately 20 kb of 1p32 and is composed of five exons ranging from 81 to 1305 bp. The sequences of the exon--intron boundaries were determined for each exon and conformed to the consensus splice junction sequences. The 5' and 3' untranslated regions in exon 1 and 5, respectively, were also determined, including the polyadenylation signal and the polyadenylation site. The mature transcript is predicted to be 3090 nt in length. CPT1 exons from CPT II-deficient patients were amplified and directly sequenced. Two novel disease-causing mutations were identified and characterized. The first mutation was a C-665-to-A transversion in exon 1 resulting in a proline-to-histidine substitution at residue 50 of the protein (P50H). This amino acid substitution occurs within a leucine-proline motif that is highly conserved in acyltransferases from different species. The mutation was detected in both alleles of patient 05SB of Italian ancestry, and in one allele of patients 11EG, 38PG, and 26FD of Italian, Dutch, and French ancestry, respectively. The second mutation was a rare G-2173-to-A transition in exon 5 causing an aspartic-acid-to-asparagine substitution at amino acid 553 (D553N) and the generation of a new MseI site. The mutation was detected only in one allele of patient 15MB, of Italian ancestry, who was also heterozygous for the common S113L substitution. Transfection experiments in COS cells demonstrated that both mutations drastically depressed the catalytic activity of CPT II. Biochemical characterization of P50H mutant CPT II in cultured cells from patient 05SB showed that the mutation does not affect substrate binding sites. Finally, immunoblot analysis demonstrated that both mutations were associated with markedly reduced steady-state level of the protein, thus indicating decreased stability of the mutant CPT II.

Adult↗
PubMed1994

Expression and characterization of human and chimeric human-Paracoccus denitrificans electron transfer flavoproteins.

Electron transfer flavoprotein (ETF) is a heterodimer that contains a single equivalent of FAD and accepts electrons from nine flavoprotein dehydrogenases in the mitochondrial matrix. Human ETF was expressed in Escherichia coli using the expression vector previously employed to express Paracoccus denitrificans ETF (Bedzyk, L. A., Escudero, K. W., Gill, R. E., Griffin, K. J., and Frerman, F. E. (1993) J. Biol. Chem. 268, 20211-20217). cDNAs encoding the beta and alpha subunits of the human protein were inserted into the vector, mimicking the arrangement of the P. denitrificans genes in which coding sequences are joined by overlapping termination and initiation codons. A human ETF containing 30% P. denitrificans sequence at the amino terminus of the beta subunit was also expressed and purified. This chimeric ETF has 64% sequence identity with the human sequence in the substituted region. Kinetic constants of medium chain and short chain acyl-CoA dehydrogenases for the chimeric ETFs were slightly changed from those of human ETF; but, there are marked differences in the kinetic constants of sarcosine dehydrogenase and electron transfer flavoprotein-ubiquinone oxidoreductase with the two ETFs. Absorption spectra of the three redox states of human, chimeric, and P. denitrificans ETF flavins are identical. However, the flavin circular dichroism spectra of the three ETFs are characteristic for each species. The spectrum of the chimeric ETF has both human and P. denitrificans ETF features. The amplitude of the 436 nm band is identical to that of the of the human ETF flavin, but the amplitude of the 375 nm band is identical to that of the P. denitrificans ETF flavin. Thus, flavin in the chimeric ETF appears to be exposed to dipoles in the protein framework provided by human and bacterial sequences. These spectral data indicate that the flavin is located in the vicinity of the amino-terminal region of the beta subunit. The kinetic data suggest that the amino-terminal region of the beta subunit comprises part of the docking site for some primary dehydrogenases and electron transfer flavoprotein-ubiquinone oxidoreductase.

Amino Acid Sequence↗
PubMed1994

Mutation rate of the CDKN2 gene in malignant gliomas.

The CDKN2 gene encodes p16, a protein controlling the cell cycle. CDKN2 is deleted in a relevant number of tumor cell lines, but results of the studies in primary tumors are contradictory. We have investigated by using quantitative polymerase chain reaction and single-strand conformation polymorphism analysis the structure of exon 2 of CDKN2 in 32 malignant gliomas. In 11 tumors the amount of amplified material was 21% of that of controls and in 8 tumors it was 42.3%, suggesting the presence of homozygous and hemizygous deletions of the CDKN2 gene, respectively. However, no abnormality could be detected by single-strand conformation polymorphism analysis. The data confirm in primary gliomas that homozygous deletions are a mechanism of CDKN2 inactivation and suggest that another gene in the vicinity could be targeted by mutations.

Base Sequence↗
PubMed1994

Divergent sequences in the 5' region of cDNA suggest alternative splicing as a mechanism for the generation of carnitine acetyltransferases with different subcellular localizations.

We identified two partially overlapping cDNAs containing divergent 5' sequences of human carnitine acetyltransferase (CAT). cDNA lambda SM-1400 extends the sequence of peroxisomal CAT, whereas cDNA lambda SM-1200 creates a new open reading frame encoding a putative mitochondrial leader peptide. An intron is located where sequences diverge, suggesting that mitochondrial, peroxisomal and possibly endoplasmic reticulum CAT mRNAs derive from alternative splicing of the CAT gene.

Alternative Splicing↗
PubMed1994

Identification of 5' regulatory regions of the human carnitine palmitoyltransferase II gene.

We have identified two partially overlapping genomic clones that contain part of the 5' regulatory region of the human carnitine palmitoyltransferase II gene. The 1.2 kb region upstream the transcription start site, as defined by primer extension experiments, shows promoter activity when inserted upstream of a reporter gene and contains a putative insulin responsive element.

Base Sequence↗
PubMed1994

Molecular cloning of cDNAs encoding human carnitine acetyltransferase and mapping of the corresponding gene to chromosome 9q34.1.

Using a combination of PCR screening of cDNA libraries and reverse transcription PCR, we have cloned three overlapping DNA fragments that encode human carnitine acetyltransferase (CAT), a key enzyme for metabolic pathways involved with the control of the acyl-CoA/CoA ratio in mitochondria, peroxisomes, and endoplasmic reticulum. The resulting cDNA (2436 bp) hybridizes to a mRNA species of approximately 2.9 kb that is particularly abundant in skeletal muscle and encodes a 68-kDa protein containing a peroxisomal targeting signal. The sequence matches those of several tryptic peptides obtained from purified human liver CAT and shows striking similarities with other members of the carnitine/choline acetyltransferase family very distant throughout evolution. CAT cDNA has also been used for fluorescence in situ hybridization on metaphase spreads of human chromosomes, and the corresponding gene, CAT1, has been mapped to chromosome 9q34.1.

Amino Acid Sequence↗
PubMed1994

Increasing complexity of the karyotype in 50 human gliomas. Progressive evolution and de novo occurrence of cytogenetic alterations.

We studied the karyotypes of eight differentiated gliomas, 19 anaplastic gliomas, and 23 glioblastomas (GBM). Normal stemlines were present in 70% of the differentiated and anaplastic gliomas; abnormalities were mostly characterized by loss of sex chromosomes. In GBM, on the contrary, only 13% of the stemlines were normal and three groups, 45,XO, near-diploid, and near tetraploid, could be identified. The most frequent alterations among GBM were: total or partial loss of chromosome 10 in nine cases, structural abnormalities of chromosome 9 in seven cases, and loss of the Y chromosome in stemline clones of seven cases. Less frequent abnormalities included chromosomes 7, 1, 3, and 19. Our data support the cytogenetic model of gliomas as multi-stage tumors. GBM, in particular, can originate from the evolution of astrocytomas but can also develop de novo. In both cases loss of genetic material on chromosome 10 seems to play a crucial role.

Adult↗
PubMed1994

A multiple interval physical map of the pericentromeric region of human chromosome 10.

Five intervals in the pericentromeric region of human chromosome 10 have been defined using a panel of somatic cell hybrids carrying portions of the chromosome. The map positions of twelve markers, consisting of four genes and eight anonymous DNA segments, have been localized by assignment to one of the five intervals. Several other markers could be placed in specific intervals by genetic linkage to assigned loci. When previously published data are incorporated, the summary map of the pericentromeric region encompasses thirty-two loci in bands 10p11.2-q11.2.

Base Sequence↗
PubMed1994

p53 mutations and microsatellite analysis of loss of heterozygosity in malignant gliomas.

We have studied alterations of the p53 gene in 27 patients with malignant gliomas. Loss of heterozygosity (LOH) was investigated by microsatellite analysis in 23 patients (22 informative) and detected in nine. Exons 5 through 9 were amplified by polymerase chain reaction (PCR) in these nine patients: alterations were found in five cases (three missense mutations, one non-sense mutation, and one putative deletion), while in four the DNA sequence was normal. In the four patients where LOH could not be studied, the p53 sequence from tumor DNA was normal. These results indicate that microsatellite analysis is a convenient tool for LOH detection at the p53 locus and that mutations of the p53 gene are present in only part of the patients with LOH, implying the possibility that another tumor suppressor gene is located in the proximity of the p53 locus.

Adult↗
PubMed1994

Mutations and polymorphisms of the gene encoding the beta-subunit of the electron transfer flavoprotein in three patients with glutaric acidemia type II.

Electron transfer flavoprotein (ETF) is a heterodimeric enzyme composed of an alpha-subunit and a beta-subunit and contains a single equivalent of FAD per dimer. ETF deficiency can be demonstrated in individuals affected by a severe metabolic disorder, glutaric acidemia type II (GAII). In this study, we have investigated for the first time the molecular basis of beta-ETF deficiency in three GAII patients: two Japanese brothers, P411 and P412, and a third unrelated patient, P485. Molecular analysis of the beta-ETF gene in P411 and P412 demonstrated that both these patients are compound heterozygotes. One allele is carrying a G to A transition at nucleotide 518, causing a missense mutation at codon 164. This point mutation is maternally derived and is not detected in 42 unrelated controls. The other allele carries a G to C transversion at the first nucleotide of the intron donor site, downstream of an exon that is skipped during the splicing event. The sequence analysis of the beta-ETF coding sequence in P485 showed only a C to T transition at nucleotide 488 that causes a Thr154 to Met substitution and the elimination of a HgaI restriction site. HgaI restriction analysis on 63 unrelated controls' genomic DNA demonstrated that the C488T transition identifies a polymorphic site. Finally, transfection of wild-type beta-ETF cDNA into P411 fibroblasts suggests that wild-type beta-ETF cDNA complements the genetic defect and restores the beta-oxidation flux to normal levels.

Amino Acid Metabolism, Inborn Errors↗