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Biomedical subjects

F Costanzo

Publications and source records attributed to F Costanzo.

At least 19 recordsLinked to original sources
PubMed2003

BRCA1 expression modulates chemosensitivity of BRCA1-defective HCC1937 human breast cancer cells.

Germline mutations of the tumour suppressor gene BRCA1 are involved in the predisposition and development of breast cancer and account for 20-45% of all hereditary cases. There is an increasing evidence that these tumours are characterised by a specific phenotype and pattern of gene expression. We have hypothesised that differences in chemosensitivity might parallel molecular heterogeneity of hereditary and sporadic breast tumours. To this end, we have investigated the chemosensitivity of the BRCA1-defective HCC1937 breast cancer cell line, and the BRCA1-competent MCF-7 (hormone-sensitive) and MDA-MB231 (hormone-insensitive) breast cancer cell lines using the MTT assay. The 50% inhibitory concentration (IC(50)) for the individual compounds were derived by interpolate plot analysis of the logarithmic scalar concentration curve after a 48 h exposure. HCC1937 cells were significantly (P<0.005) more sensitive to cisplatin (CDDP) (IC(50) : 30-40 microM) compared with MCF-7 (IC(50) : 60-70 microM) and MDA-MB231 (IC(50) : 90-100 microM) cells. On the other hand, BRCA1-defective breast cancer cells were significantly less sensitive to doxorubicin (Dox) (IC(50) : 45-50 microM) compared with MCF-7 (IC(50) : 1-5 microM) and MDA-MB231 (IC(50) : 5-10 microM) (P<0.02), as well as to paclitaxel (Tax) (IC(50) : >2 microM for HCC1937, 0.1-0.2 microM for MCF-7 and 0.01-0.02 microM for MDA-MB231) (P<0.001). Full-length BRCA1 cDNA transfection of BRCA1-defective HCC1937 cells led to the reconstituted expression of BRCA1 protein in HCC1937/(WT)BRCA1-derived cell clone, but did not reduce tumour cell growth in soft agar. BRCA1 reconstitution reverted the hypersensitivity to CDDP (P<0.02), and restored the sensitivity to Dox (P<0.05) and Tax (P<0.001), compared with parental HCC1937 cells. Taken together, our findings suggest a specific chemosensitivity profile of BRCA1-defective cells in vitro, which is dependent on BRCA1 protein expression, and suggest prospective preclinical and clinical investigation for the development of tailored therapeutical approaches in this setting.

Antineoplastic Agents↗
PubMed2002

Transcription factor NF-Y regulates differentiation of CaCo-2 cells.

The CaCo-2 cell line is used to study the molecular mechanisms underlying differentiation of intestinal epithelial cells. These cells undergo a gradual differentiation process that is growth-related and depends on cellular density. CaCo-2 cells acquire a morphological polarity and express such markers of mature enterocytes as sucrase-isomaltase, apolipoproteins, alkaline phosphatase, and H-ferritin. Because the NF-Y transcription factor is required for H-ferritin gene expression, we investigated whether it is involved in the expression of the other CaCo-2 differentiation markers. We observed that subunit NF-YA increases during CaCo-2 differentiation and that the constitutive expression of NF-YA, obtained in stably transfected CaCo-2 cells, results in the expression of differentiation markers. In fact, sucrase-isomaltase, apolipoprotein A1, and H-ferritin were constitutively expressed in NF-YA-transfected cells and their levels did not increase during prolonged culture, while these markers were not expressed in mock-transfected CaCo-2 cells or transfected with an inactive NF-YA expression vector until the onset of differentiation.

Apolipoprotein A-I↗
PubMed2001

Transcriptional regulation of the mismatch repair gene hMLH1.

We have characterized the promoter region of the human gene coding for the MLH1 mismatch repair protein. The total transcriptional activity of the hMLH1 promoter is driven by two positive cis-elements included between nucleotides -300 and -220. The upstream element is a canonical CCAAT box, and it is recognized by the heterotrimeric transcription factor NF-Y. On the other hand, the downstream element is recognized by a nuclear factor of about 120 kDa. Variations in hMLH1 intracellular levels may influence the surveillance of the genome integrity. The identification of the two elements may shad some light on the regulation of the transcriptional regulation of hMLH1 expression.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Evidence of a founder mutation of BRCA1 in a highly homogeneous population from southern Italy with breast/ovarian cancer.

Several genes have been involved in the pathogenesis of hereditary breast/ovarian cancer (BOC), but mutations in the BRCA1 gene are by far the most recurrent. In this study, we report the identification of a founder mutation in a geographically and historically homogeneous population from Calabria, a south Italian region. A screening performed on 24 patients from unrelated families highlighted the high prevalence of a 5083del19 alteration in the BRCA1 gene, which accounts for 33% of the overall gene mutations. The same mutation was also detected in 4 patients, all of Calabrian origin, referred to us by research centres from the north of Italy. Allelotype analysis, performed on probands and unaffected family members revealed the presence a common allele, therefore suggesting a founder effect due to a common ancestor. Our findings underscore the importance of ethnic background homogeneity in patients' selection and highlight the usefulness of founder mutations as a potential tool for optimisation of preclinical diagnosis in gene carriers and therapeutic approaches in affected individuals.

Adult↗
PubMed2000

Diagnosis controversies for adrenal ganglioneuroma.

The authors report a case of adrenal ganglioneuroma which was incidentally diagnosed performing preoperative examination for a sigmoid carcinoma. The authors took this finding as a starting point to underline the rarity of this condition and its chance discovery, this being due to the rare presence of signs and symptoms and its frequent association with other synchronous neoplasms; all this makes differential diagnosis rather difficult.

Adrenal Gland Neoplasms↗
PubMed1999

The B subunit of the CAAT-binding factor NFY binds the central segment of the Co-activator p300.

We report that the heterotrimeric transcription factor NFY or "CAAT-binding factor" binds the -60 region of the human H ferritin promoter, the B site. DNA binding analysis with specific antibodies demonstrates that NFY/B/C subunits tightly bind this site and that NFY/C subunit is masked in vivo by binding with other protein(s). NFY binds the co-activator p300. Specifically, the NFY/B subunit interacts with the central segment of p300 in vivo and in vitro. cAMP substantially increases the formation of the NFY.p300 complex. Taken together these data provide a general model of cAMP induction of non-CRE-containing promoters and suggest that the NFY-B.p300 complex is located at the 5' end of the promoter and the NFY-B.C. TFIIB on the 3' end toward the transcription start site.

Binding Sites↗
PubMed1998

P/CAF/p300 complex binds the promoter for the heavy subunit of ferritin and contributes to its tissue-specific expression.

We analysed the role of the nuclear protein P/CAF in regulating the transcription of the gene for human heavy (H) ferritin in given cell types. P/CAF is a histone acetylase, recruited to specific promoters via interaction with the co-activator molecule p300/CREB-binding protein (CBP). Histone acetylation promoted by P/CAF destabilizes the nucleosome structure, thus contributing to activation of transcription. The transcription of the H ferritin gene is regulated by the transcription factor B-box-binding factor (Bbf), which bridges RNA polymerase II via p300/CBP. Northern blot analyses of RNA species from various human tissues and cell lines demonstrate that the H ferritin gene is expressed at high levels in cells containing high levels of the P/CAF transcript. Moreover, transient overexpression of P/CAF in cells constitutively expressing low levels of this protein activates transcription driven by the region of the H promoter interacting with Bbf. The involvement of p300/CBP in the possible P/CAF-mediated regulation of H promoter was also explored by evaluating the phenomenon in the presence of the oncoprotein E1A. The results of these experiments demonstrate that P/CAF activates the H promoter also in the presence of limited amounts of p300/CBP. We argue that P/CAF is a component of the basal transcription apparatus of the H ferritin gene and that the relative amounts of the P/CAF protein in different cell types could account for the cell-specific control of the H ferritin gene transcription.

Acetyltransferases↗
PubMed1998

Hereditary nonpolyposis coloretal cancer: identification of novel germline mutations in two kindreds not fulfulling the Amsterdam criteria. Mutations in brief no. 203. Online.

Hereditary nonpolyposis colon cancer results from heritable defects in the MLH1, MSH2, PMS1 and PMS2 genes, which encode proteins involved in the mismatch repair process. In this work we report the identification of two novel germline mutations in the MLH1 gene from two unrelated HNPCC families. The two affected families do not fulfill the Amsterdam criteria. In family 1 we found a missense S93G mutation, which lies in a MLH1 domain critical for its MMR functions. In family 2 we found a two nucleotide insertion (AG) in position 523 from the AUG which determines an early stop codon at position 606 (codon 203). In both families the mutant alleles cosegregate with the cancer phenotype.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Growth, spread, and extracellular localization of herpes simplex virus 1 in Vero cells in the presence of an anti-gD plaque inhibiting monoclonal antibody.

Evidence for a direct cell-to-cell virus transfer could be provided by an agent that inhibits plaque formation without interfering with the processes that determine plaque growth in the exit and reinfection pathway of virus transfer. We studied the process of Vero cell infection by herpes simplex virus type 1 laboratory strain F [HSV-1 (F)] in the presence of monoclonal antibody (mAb) F10, an anti gD mAb that inhibits plaque development but does not neutralize virus infectivity in the absence of complement. In virus growth curves, cell associated virus was inhibited at low (0.01) but not at high (10) MOI when all cells are simultaneously infected, showing that the target of the mAb is the process of progressive cells recruitment and not the rate of virus replication. The mAb slightly inhibited virus exit and delayed virus entry. However these two additional inhibitory activities were not responsible for inhibition of virus spread, at least at early time of infection. In fact inhibition of virus spread, as measured by reduction of infectious centers (IC) from infected monolayers, could be appreciated before the appearance of extracellular virus in control cultures. We obtained electron microscope evidence that, both in the absence and in the presence of mAb, extracellular virus was initially concentrated at the interspaces between adjacent cell membranes, with little or no virus present at free cell surfaces. At more advanced stages of infection, only virus at free cell surfaces was found. The results of the study of virus replication in the presence of the mAb confirmed the hypothesis of the existence of a pathway of virus transfer between adjacent cells independent from extracellular virus. However, no electron microscope evidence for a direct cell-to-cell virus passage or for a modification of virus transfer brought about by the plaque inhibiting mAb was obtained. Interestingly, electron microscope studies suggested a targeting of the virions to different extracellular spaces, intercellular spaces and free cell surfaces, in intact and damaged cells respectively.

Animals↗
PubMed1997

Okadaic acid stimulates H ferritin transcription in HeLa cells by increasing the interaction between the p300 CO-activator molecule and the transcription factor Bbf.

The transcription of the human H ferritin gene is regulated by a transcription factor, called Bbf, which binds an enhancer element located in the -100/+1 region of the H promoter. To evaluate a possible role of Bbf phosphorylation on the promoter efficiency, we exposed HeLa cells to the phosphatase inhibitor okadaic acid (OA). The okadaic acid treatment increased about 4-fold the transcription driven by the -100/+1 region of the H promoter. However, the DNA binding activity of Bbf was not modified by OA, as assessed by EMSA. Immunoprecipitation experiments demonstrated that the OA-treatment stimulates and/or stabilizes the complex between Bbf and the nuclear protein p300, most probably by inducing the phosphorylation state of the complex. Bbf depends on the p300 molecule to trigger RNA polymerase II and thus transcription of the H ferritin gene.

Cyclic AMP Response Element-Binding Protein A↗
PubMed1997

A common mechanism underlying the E1A repression and the cAMP stimulation of the H ferritin transcription.

Transcription of the H ferritin gene in vivo is stimulated by cAMP and repressed by the E1A oncoprotein. We report here the identification of the cis-element in the human promoter responsive to both cAMP- and E1A-mediated signals. This promoter region is included between positions -62 to -45 and binds a approximate 120-kDa transcription factor called Bbf. Bbf forms a complex in vivo with the coactivator molecules p300 and CBP. Recombinant E1A protein reduces the formation of these complexes. In vivo overexpression of p300 in HeLa cells reverses the E1A-mediated inhibition of the ferritin promoter transcription driven by Bbf. These data suggest the existence of a common mechanism for the cAMP activation and the E1A-mediated repression of H ferritin transcription.

Adenovirus E1A Proteins↗
PubMed1995

Transcriptional activation of the H-ferritin gene in differentiated Caco-2 cells parallels a change in the activity of the nuclear factor Bbf.

In this paper, we examine the mechanisms that regulate the expression of the heavy (H) ferritin subunit in the colon carcinoma Caco-2 cell line allowed to differentiate spontaneously in vitro. The differentiation process of these cells in continuous culture is accompanied by an accumulation of the mRNA coding for the apoferritin H chain. The analysis of Caco-2 subclones stably transfected with an H-chain promoter-chloramphenicol acetyltransferase (CAT) construct revealed that the mRNA increase is paralleled by an enhanced transcription of the H gene, driven by the -100 to +4 region of the H promoter. The H gene transcriptional activation seems to be a specific feature of differentiated Caco-2 cells, since the activity of other promoters did not change upon differentiation. The -100 to +4 region of the H promoter binds a transcription factor called Bbf (B-box binding factor); electrophoretic-mobility-shift-assay analyses showed that the retarded complex due to Bbf-H promoter interaction is significantly increased in the differentiated cells. We propose that the activation of H-ferritin gene expression may be associated with the establishment of a differentiated phenotype in Caco-2 cells, and that the H-ferritin gene transcriptional up-regulation is accompanied by a modification in the activity of the transcription factor Bbf.

Base Sequence↗
PubMed1995

Negative and positive elements in the promoter region of the human apoferritin L gene.

We have characterized the promoter of the human gene coding for the apoferritin L subunit. Transient transfections of 5' and 3' deletion mutants indicate that the efficiency of the L promoter depends on both negative and positive cis-elements, located upstream and downstream of the transcription start point. DNaseI footprinting analysis of this DNA region revealed the presence of five protected segments. The most upstream one (element 1) corresponds to the negative cis-element and is recognized by factor(s) sharing a GC-sequence specificity. Three positive elements are in the region upstream of the start of transcription; a fifth positive cis-element (element 5) is localized in the first exon of the L gene.

Apoferritins↗
PubMed1995

Cell-type dependent sensitivity of herpes simplex virus 1 mutants to plaque development inhibition by an anti-gD monoclonal antibody.

Herpes simplex virus 1 (HSV-1) mutants selected in Vero cells either for resistance to plaque development inhibition (PDI) (P1, P2 and P3) or for resistance to neutralization (N1, N2, and N3) against an anti-glycoprotein D (gD) monoclonal antibody (mAb) were characterized both in Vero and BHK cells. In Vero cells P mutants were completely resistant to PDI, while N mutants showed from moderate to good resistance. In BHK cells P mutants lost their resistance to PDI, while N mutants became fully resistant. Cell type influenced the plaque size of the mutants as well. In Vero cells P mutant plaques were larger, and N mutant plaques smaller than wild type virus plaques. In BHK cells all plaques were comparable. With one exception (N2 in BHK) resistance to neutralization could be clearly appreciated at high but not at low mAb:virus particles ratio. For most of the mutants the neutralization values remained approximately the same in Vero and BHK cells. P2 and N2 mutants were more resistant to neutralization in BHK than in Vero cells. However, only for N2 mutant did the change in neutralization resistance go in the same direction as the change in PDI resistance. The results show that it is possible to dissociate the neutralizing and the PDI activities of a mAb and that the sensitivity of a virus to plaque inhibition by an anti-gD mAb is cell-type dependent.

Animals↗
PubMed1994

The DNA sequence encompassing the transcription start site of a TATA-less promoter contains enough information to drive neuron-specific transcription.

The FE65 gene encodes a nuclear protein of unknown function that is expressed in several areas of the rat nervous system during development and in the adult animal, particularly in somatic and visceral ganglia. FE65 mRNA is abundant in neuronal cell lines, whereas it is barely detectable in non-neuronal cells. We identified the two transcription start sites of the FE65 gene and we isolated the rat genomic fragment containing one of these two transcriptional start sites. We demonstrate that this fragment contains a promoter able to direct an efficient transcription of a reporter gene in PC12 cells and in NTERA2 cells upon their differentiation with retinoic acid, whereas it functions poorly in non-neuronal cells, such as Rat2 fibroblasts and BRL hepatocytes. This promoter is composed of two regions. The first includes a cis-element whose removal greatly decreases the transcriptional efficiency in all cells examined and which forms similar complexes with proteins from PC12 and Rat2 cells. This cis-element binds Sp1 or another GC-binding factor. The second cis-element encompasses the transcription start site and is still able to direct transcription only in neuronal cells. The DNA-protein complexes formed by this cis-element in neuronal cells differ from those formed in non-neuronal cells. The analysis of point mutations in this region indicates that the proteins that bind to this cis-element interact with both overlapping and distinct nucleotide sequences.

Amino Acid Sequence↗