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Biomedical subjects

M Fanciulli

Publications and source records attributed to M Fanciulli.

At least 19 recordsLinked to original sources
PubMed2006

Diffusion reaction of oxygen in HfO2/SiO2/Si stacks.

We study the oxidation mechanism of silicon in the presence of a thin HfO2 layer. We performed a set of annealing in 18O2 atmosphere on HfO2/SiO2/Si stacks observing the 18O distribution in the SiO2 layer with time-of-flight secondary ion mass spectrometry (ToF-SIMS). The 18O distribution in HfO2/SiO2/Si stacks upon 18O2 annealing suggests that what is responsible for SiO2 growth is the molecular O2, whereas no contribution is found of the atomic oxygen to the oxidation. By studying the dependence of the oxidation velocity from oxygen partial pressure and annealing temperature, we demonstrate that the rate-determining step of the oxidation is the oxygen exchange at the HfO2/SiO2 interface. When moisture is chemisorbed in HfO2 films, the oxidation of the underlying silicon substrate becomes extremely fast and its kinetics can be described as a wet silicon oxidation process. The silicon oxidation during O2 annealing of the atomic layer deposited HfO2/Si is fast in its early stage due to chemisorbed moisture and becomes slow after the first 10 s.

Journal Article↗
PubMed2004

The artificial zinc finger protein 'Blues' binds the enhancer of the fibroblast growth factor 4 and represses transcription.

The design of novel genes encoding artificial transcription factors represents a powerful tool in biotechnology and medicine. We have engineered a new zinc finger-based transcription factor, named Blues, able to bind and possibly to modify the expression of fibroblast growth factor 4 (FGF-4, K-fgf), originally identified as an oncogene. Blues encodes a three zinc finger peptide and was constructed to target the 9 bp DNA sequence: 5'-GTT-TGG-ATG-3', internal to the murine FGF-4 enhancer, in proximity of Sox-2 and Oct-3 DNA binding sites. Our final aim is to generate a model system based on artificial zinc finger genes to study the biological role of FGF-4 during development and tumorigenesis.

Amino Acid Sequence↗
PubMed2000

The artificial zinc finger coding gene 'Jazz' binds the utrophin promoter and activates transcription.

Up-regulation of utrophin gene expression is recognized as a plausible therapeutic approach in the treatment of Duchenne muscular dystrophy (DMD). We have designed and engineered new zinc finger-based transcription factors capable of binding and activating transcription from the promoter of the dystrophin-related gene, utrophin. Using the recognition 'code' that proposes specific rules between zinc finger primary structure and potential DNA binding sites, we engineered a new gene named 'Jazz' that encodes for a three-zinc finger peptide. Jazz belongs to the Cys2-His2 zinc finger type and was engineered to target the nine base pair DNA sequence: 5'-GCT-GCT-GCG-3', present in the promoter region of both the human and mouse utrophin gene. The entire zinc finger alpha-helix region, containing the amino acid positions that are crucial for DNA binding, was specifically chosen on the basis of the contacts more frequently represented in the available list of the 'code'. Here we demonstrate that Jazz protein binds specifically to the double-stranded DNA target, with a dissociation constant of about 32 nM. Band shift and super-shift experiments confirmed the high affinity and specificity of Jazz protein for its DNA target. Moreover, we show that chimeric proteins, named Gal4-Jazz and Sp1-Jazz, are able to drive the transcription of a test gene from the human utrophin promoter.

Animals↗
PubMed2000

Identification of a novel partner of RNA polymerase II subunit 11, Che-1, which interacts with and affects the growth suppression function of Rb.

hRPB11 is a core subunit of RNA polymerase II (pol II) specifically down-regulated on doxorubicin (dox) treatment. Levels of this protein profoundly affect cell differentiation, cell proliferation, and tumorigenicity in vivo. Here we describe Che-1, a novel human protein that interacts with hRPB11. Che-1 possesses a domain of high homology with Escherichia coli RNA polymerase final sigma-factor 70 and SV40 large T antigen. In addition, we report that Che-1 interacts with the retinoblastoma susceptibility gene (Rb) by two distinct domains. Functionally, we demonstrate that Che-1 represses the growth suppression function of Rb, counteracting the inhibitory action of Rb on the trans-activation function of E2F1. These results identify a novel protein that binds Rb and the core of pol II, and suggest that Che-1 may be part of transcription regulatory complex.

Amino Acid Sequence↗
PubMed2000

Energy metabolism of human LoVo colon carcinoma cells: correlation to drug resistance and influence of lonidamine.

The relationship between modification of energy metabolism and extent of drug resistance was investigated in two sublines (LoVoDX and LoVoDX10) from human LoVo colon carcinoma cells that exhibit different degrees of resistance to doxorubicin. Results indicated that the extent of alteration in energy metabolism strictly correlated with degree of resistance. In LoVoDX cells, only 14CO2 production was enhanced, whereas in the more resistant LoVoDX10 cells, both 14CO2 and aerobic lactate production were stimulated. The basal and glucose-supported efflux rate and the amount of drug extruded by LoVoDX10 cells were significantly higher than in the resistant LoVoDX cells. Because the expression of surface P-170 glycoprotein was similar in both cell lines, this phenomenon was attributed to increased efflux pump activity resulting from greater ATP availability. Inhibition of 14CO2 production, aerobic glycolysis, and clonogenic activity by lonidamine (LND) increased with enhancement of the energy metabolism. Moreover, LND, by affecting energy-yielding processes, reduced intracellular ATP content, lowered the energy supply to the ATP-driven efflux pump, and inhibited, almost completely, doxorubicin extrusion by resistant LoVo cells. These findings strongly suggest that LND, currently used in tumor therapy, reduces drug resistance by restoring the capacity to accumulate and retain drug of cells with the MDR phenotype that overexpress P-170.

Adenosine Triphosphate↗
PubMed1999

Interaction between the pRb2/p130 C-terminal domain and the N-terminal portion of cyclin D3.

An association between cyclin D3 and the C-terminal domain of pRb2/p130 was demonstrated using the yeast two-hybrid system. Further analysis restricted the epitope responsible for the binding within the 74 N-terminal amino acids of cyclin D3, independent of the LXCXE amino acid motif present in the D-type cyclin N-terminal region. In a coprecipitation assay in T98G cells, a human glioblastoma cell line, the C-terminal domain of pRb2/p130 was able to interact solely with cyclin D3, while the corresponding portion of pRb interacted with either cyclin D3 or cyclin D1. In T98G cells, endogenous cyclin D3-associated kinase activity showed a clear predisposition to phosphorylate preferentially the C-terminal domain of pRb2/p130, rather than that of pRb. This propensity was also confirmed in LAN-5 human neuroblastoma cells, where phosphorylation of the pRb2/p130 C-terminal domain and expression of cyclin D3 also decreased remarkably in the late neural differentiation stages.

Adenovirus E1A Proteins↗
PubMed1999

bcl-2 inhibits mitochondrial metabolism and lonidamine-induced apoptosis in adriamycin-resistant MCF7 cells.

Lonidamine (LND), a selective inhibitor of the energy metabolism of tumor cells, induces apoptosis, independently of the p53 gene, in the adriamycin(ADR)-resistant MCF7 breast-cancer cell line (MCF7 ADR). On the contrary, LND fails to activate the apoptotic program in the parental MCF7-sensitive cell line (MCF7 WT). The extent of bcl-2 expression might account for the different effect of LND on these cell lines. In fact, the MCF7 ADR line shows a low level of bcl-2 protein, whereas MCF7 WT expresses a high level of bcl-2. We therefore investigated the relationship between the amount of bcl-2 and the ability of LND to induce apoptosis, using 4 clones over-expressing bcl-2. The effect of bcl-2 on the energy metabolism was also evaluated. We demonstrated that over-expression of bcl-2 inhibited LND-induced apoptosis, while reducing 14CO2 production, oxygen uptake and ATP content, whereas aerobic lactate production was essentially unaffected. In addition, LND decreased the oxidative metabolism of the MCF7 ADR cells to a greater extent than it did in the bcl-2 transfectants.

Antibiotics, Antineoplastic↗
PubMed1999

The RNA polymerase II core subunit 11 interacts with keratin 19, a component of the intermediate filament proteins.

We have previously cloned the human RNA polymerase II subunit 11, as a doxorubicin sensitive gene product. We suggested multiple tasks for this subunit, including structural and regulatory roles. With the aim to clarify the human RNA polymerase II subunit 11 function, we have identified its interacting protein partners using the yeast two-hybrid system. Here, we show that human RNA polymerase II subunit 11 specifically binds keratin 19, a component of the intermediate filament protein family, which is expressed in a tissue and differentiation-specific manner. In particular, keratin 19 is a part of the nuclear matrix intermediate filaments. We provide evidence that human RNA polymerase II subunit 11 interacts with keratin 19 via its N-terminal alpha motif, the same motif necessary for its interaction with the human RNA polymerase II core subunit 3. We found that keratin 19 contains two putative leucine zipper domains sharing peculiar homology with the alpha motif of human RNA polymerase II subunit 3. Finally, we demonstrate that keratin 19 can compete for binding human RNA polymerase II subunit 11/human RNA polymerase II subunit 3 in vitro, suggesting a possible regulatory role for this molecule in RNA polymerase II assembly/activity.

Amino Acid Sequence↗
PubMed1998

Binding properties of the artificial zinc fingers coding gene Sint1.

On the basis of the recognition "code" that suggests specific rules between zinc finger's primary structure and the finger's potential DNA binding sites, we have constructed a new three-zinc finger coding gene to target the nine base pair DNA sequence: 5'-TGG-ATG-GAC-3'. This artificial gene named "Sint1" belongs to the Cys2-His2 zinc finger type. The amino acid positions, crucial for DNA binding, have been specifically chosen on the basis of the amino acid/base contacts more frequently represented in the available list of the proposed recognition "code". Here we demonstrate that Sint1 protein binds specifically the double strand "code" DNA target, with a dissociation constant (Kd) comparable to the Kd of the well known Zif268 protein. Sint1 "code" deduced and the "experimental" selected DNA binding sites share five nucleotide positions. Interestingly, Sint1 shows both high affinity and specificity toward the single strand "code" DNA binding site, with a Kd comparable to the corresponding double strand DNA target. Moreover, we prove that Sint1 is able to bind RNA similarly to several natural zinc finger proteins.

Amino Acid Sequence↗
PubMed1998

Enhancement of doxorubicin content by the antitumor drug lonidamine in resistant Ehrlich ascites tumor cells through modulation of energy metabolism.

The effect of the antitumor drug lonidamine (LND) on respiration, aerobic glycolysis, adenylate pool, doxorubicin (DOX) uptake, and efflux in DOX-resistant and DOX-sensitive Ehrlich tumor cells was investigated. The results may be summarized as follows: 1) In both types of cells, LND inhibited both respiration and glycolysis in a dose-dependent manner and lowered the ATP concentration. The effect was more marked in cells incubated in glucose-free medium; 2) LND raised, to a remarkable extent, the intracellular content of DOX in resistant and sensitive cells respiring on endogenous substrates because of reduced ATP availability, whereas in glucose-supplemented medium, where both respiration and glycolysis contributed to ATP synthesis, the increase was lower; and 3) when LND was added to DOX-loaded cells, it failed to significantly inhibit DOX efflux because of time-dependent phenomena. These findings indicated that LND, a drug currently employed in tumor therapy, might also be useful in reducing or overcoming multidrug resistance (MDR) of those cells with a reduced ability to accumulate and retain antitumor drugs.

Adenine Nucleotides↗
PubMed1998

The interacting RNA polymerase II subunits, hRPB11 and hRPB3, are coordinately expressed in adult human tissues and down-regulated by doxorubicin.

We previously isolated the human RPB11 cDNA, encoding the 13.3 kDa subunit of RNA polymerase II, and demonstrated that expression of this subunit is modulated by doxorubicin. Using hRPB11 as bait in a yeast two-hybrid system, two cDNA variants encoding a second RNA polymerase II subunit, hRPB3, have now been isolated and characterized. These two hRPB3 mRNA species differed in 3' UTR region length, the longer transcript containing the AU-rich sequence motif that mediates mRNA degradation. Both hRPB11 and hRPB3 transcripts share a similar pattern of distribution in human adult tissues, with particularly high levels in both heart and skeletal muscle, and the expression of both is down-regulated by doxorubicin as found previously for the hRPB11 subunit. Taken together, these findings suggest that the interaction between hRPB3 and hRPB11 is fundamental for their function and that this heterodimer is involved in doxorubicin toxicity.

Adult↗
PubMed1998

Levels of expression of hRPB11, a core subassembly subunit of human RNA polymerase II, affect doxorubicin sensitivity and cellular differentiation.

We have previously shown that the human RNA polymerase II subunit 11 (hRPB11) is among the proteins specifically downregulated upon Doxorubicin (Dox) treatment of human cancer cell lines, and that Dox resistant clones derived upon drug selection express about 20% of the protein present in the original parental cell line. Given the prominent role that this subunit appears to have in eukaryotic cells, and the fact that its deletion causes lethality in yeast, we wanted to test the effect of the reintroduction of parental cell line levels of this subunit in Dox resistant colon cancer cells (LoVoDX). Stable transfectants of LoVoDX expressing parental (LoVoH) levels of hRPB11 showed a reduced sensitivity to the drug without changing the response of these cells to other chemotherapeutic agents, confirming a specific inverse correlation between cellular Dox sensitivity anti-hRPB11 levels of expression. In addition we show here that the levels of expression of this same RNA polymerase II subunit directly affect cellular differentiation, reducing the rate of cell proliferation, clonogenicity and increasing the expression of E-cadherin, a marker of epithelial cell differentiation. As expected from cells with these characteristics, upon in vivo administration of these clones in nude mice, we detected a significant reduction in the size and time of appearance of the primary tumors and overall metastatic capability. Finally, the role played by hRPB11 in regulating the transcription of specific genes is underlined by transient transfection experiments that show transactivation of the E-cadherin promoter by this protein.

Animals↗
PubMed1998

Identification of novel mRNA transcripts of the nm23-M1 gene that are modulated during mouse embryo development and are differently expressed in adult murine tissues.

The nm23-M1, a putative metastasis-suppressor gene, and its homologs are involved in development and differentiation. We have shown previously that in vitro neuronal cell proliferation and differentiation can be modulated by nm23-M1 expression levels. In the present study, by the yeast two-hybrid system, we have shown that, at the onset of mouse tissue differentiation, the Nm23-M1 protein forms either homodimers, or heterodimers with Nm23-M2. Furthermore, we have isolated two cDNA variants of the nm23-M1 gene in the 3'-untranslated region (UTR). The two variants related to novel mRNA transcripts that are modulated in mouse embryo and are differently expressed in adult murine tissues.

Animals↗
PubMed1998

Effect of local anesthetic ropivacaine on the energy metabolism of Ehrlich ascites tumor cells.

The effect of local anesthetic ropivacaine on the energy metabolism of Ehrlich ascites tumor cells has been investigated. Ropivacaine impaired energy metabolism of Ehrlich ascites tumor cells by affecting primarily mitochondrial metabolism. Even at low concentrations ropivacaine decreased the rate of oxygen uptake, but its effect was remarkably higher on the uncoupled respiration and, in both cases, it was strongly enhanced by hydrophobic anion tetraphenylboron (TPB-). The decrease of oxygen uptake was ascribed to an impairment of electron transport from site 1- and 2-entering substrates to respiratory chain. The inhibition of respiration, coupled to a true uncoupling mechanism by an electrophoretic mechanism, impaired ADP phosphorylation, decreased ATP content, and collapsed mitochondrial membrane potential. Ropivacaine, at all concentrations tested, stimulated aerobic lactate production, and this increase, in addition to the inhibition of respiration, was also due to an activation of mitochondrial ATPase.

Adenosine Triphosphate↗
PubMed1996

Cloning of a novel human RNA polymerase II subunit downregulated by doxorubicin: new potential mechanisms of drug related toxicity.

Using the differential display PCR method, we have isolated an mRNA downregulated in doxorubicin resistant human cell lines. The full length cDNA clone was identified as the human homologue of yeast RPB11 subunit of RNA polymerase II. Northern blot analysis of normal tissues detected a particularly high expression of RPB11 mRNA in heart and skeletal muscle. Reduction of this mRNA expression was observed in all the cell lines tested after drug treatment and was paralleled by a similar decrease of the protein levels. These findings suggest that doxorubicin may exert in vivo specific inhibitory effects on a major component of the transcription machinery.

Amino Acid Sequence↗
PubMed1996

Energy metabolism of adriamycin-sensitive and -resistant Ehrlich ascites tumor cells.

Respiration, glycolysis, utilization of carbon from 14C-labeled glucose and the activities of some regulatory enzymes of the Krebs cycle and glycolysis in adriamycin-sensitive (EH-WT) and -resistant (EH-ADR) Ehrlich ascites tumor cells have been investigated. The following summarizes the results: 1. Compared with wild-type cells, EH-ADR cells exhibited an enhanced rate of oxygen consumption as well as of ATP production (2-fold). 2. When the cells were supplied with glucose as the only added energy source, the aerobic lactate production was 30% higher in EH-ADR cells. However, in spite of the enhanced glycolysis, 50% of total cell ATP was still supplied by oxidative phosphorylation, whereas in EH-WT cells 65% of ATP was derived from glycolysis. 3. The activities of the regulatory enzymes were remarkably more elevated in EH-ADR cells. 4. The amount of glucose carbon atoms metabolized through the Krebs cycle and pentose phosphate pathway in EH-ADR cells was significantly higher than in EH-WT cells. 5. These differences confirmed a modified energy metabolism in resistant cells and reflected metabolic adaptations associated with the development of multidrug resistance.

Animals↗
PubMed1996

Effect of the antitumor drug lonidamine on glucose metabolism of adriamycin-sensitive and -resistant human breast cancer cells.

The effect of lonidamine on glucose metabolism, hexokinase activity and adenylate pool of MCF-7 human breast cancer cells sensitive and resistant to adriamycin has been investigated. The following summarizes the results: 1. In both cell types the greatest part of glucose was metabolized to lactate, whereas only a small proportion of glucose carbon atoms was incorporated into CO2, lipids, nucleic acids, and supporting structures. 2. Glucose utilization, lactate production, and ATP content were higher in resistant cells due to a greater activity of mitochondrial hexokinase. 3. Lonidamine decreased glucose utilization, aerobic glycolysis and ATP content in both cell types and the effect was significantly higher on resistant cells. 4. The extent of inhibition in sensitive and resistant cells overlapped that found for mitochondrially bound hexokinase, thus indicating that the greater sensitivity of resistant cells to lonidamine was due to their higher amount of bound hexokinase. These findings confirmed a modified glucose metabolism in cells with resistant phenotype and suggested that lonidamine might be usefully used to reduce or overcome multidrug resistance of those cells with a reduced ability to accumulate and retain antitumor drugs.

Adenine Nucleotides↗