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Biomedical subjects

M Musso

Publications and source records attributed to M Musso.

At least 55 records · Page 3Linked to original sources
PubMed1997

c-Rel and p65 subunits bind to an upstream NF-kappaB site in human granulocyte macrophage-colony stimulating factor promoter involved in phorbol ester response in 5637 cells.

To further clarify the complex transcriptional regulation of the human GM-CSF gene, which was extensively investigated in activated T cells, we have studied the role of an upstream NF-kappaB like site in the 5637 non-lymphoid cell line, which derives from a bladder carcinoma and constitutively produces GM-CSF. This sequence, named the A element, has an active role on GM-CSF transcription and is responsive to the tumor promoter PMA in transient transfection experiments. We describe here a heterodimeric binding complex of NF-kappaB subunits (c-Rel and p65) which is identical to the one obtained using the HIV-LTR-kappaB site as recognition sequence and different from the one (c-Rel and p50) observed with nuclear extracts from Mo T-lymphoid HTLV-II infected cells.

Cell Nucleus↗
PubMed1997

Effects of chain length modification and bis(ethyl) substitution of spermine analogs on purine-purine-pyrimidine triplex DNA stabilization, aggregation, and conformational transitions.

The natural polyamines--putrescine, spermidine, and spermine--are known to stabilize pyrimidine-purine-pyrimidine and purine-purine-pyrimidine triplex DNA formation. We studied the ability of two tetramine and two pentamine analogs of spermine and their bis(ethyl) derivatives to stabilize triplex DNA formation between 5'-TG3TG4TG4TG3T-3' and its target duplex probe, consisting of the oligonucleotides 5'-TCGAAG3AG4AG4AG3A-3' and 5'-TCGATC3TC4TC4TC3T-3'. We used electrophoretic mobility shift assay (EMSA), melting temperature (Tm) measurements, and circular dichroism (CD) spectroscopy to evaluate the effects of these novel polyamine analogs on triplex DNA stability, dissociation constants, aggregation, and conformation. In general, pentamines were more efficacious than tetramines in stabilizing triplex DNA, although most of the polyamines with pendant free amino groups caused DNA aggregation below 50% conversion to triplex DNA. Ethyl substitution of these pendant amino groups lowered their efficacy approximately 2-fold in stabilizing triplex DNA; however, this effect was more than compensated for by the lack of DNA aggregation in the presence of bis(ethyl)polyamines. A concentration-dependent increase in the Tm of triplex DNA was observed in the presence of polyamines. CD spectral measurements showed distinct differences in the conformation of triplex DNA stabilized in the presence of polyamines compared to the CD spectra of the oligonucleotides alone. Temperature-dependent CD spectra of triplex DNA showed monophasic melting in the absence and presence of polyamines, suggesting duplex/triplex --> single-stranded DNA transition. These results indicate that structural modifications of polyamines is an effective strategy to develop triplex DNA-stabilizing ligands, with potential applications in antigene therapeutics.

Circular Dichroism↗
PubMed1996

In vivo persistence of DNA triple helices containing psoralen-conjugated oligodeoxyribonucleotides.

Triple helices represent an attractive method for modulating specific gene expression. In particular, cross-linking between a triplex-forming oligonucleotide (TFO) and its duplex DNA target, typically through the formation of psoralen photoadducts, allows efficient blocking of elongation by RNA polymerases in vitro. However, in vivo, this approach is limited by DNA repair of the photoadduct. Here we describe the use of an oligodeoxyribonucleotide 19mer psoralen-modified TFO to form covalent linkages between an oligonucleotide and both strands of the targeted duplex DNA, thereby efficiently blocking expression of a luciferase reporter gene. Most importantly, we demonstrate that both the psoralen cross-link and the purine-motif triplex remained intact for at least 72 h post-transfection, indicating that such species can persist for an extended period of time in vivo. These findings support the feasibility of an antigene approach for the therapeutic regulation of specific gene expression.

Base Sequence↗
PubMed1996

An upstream positive regulatory element in human GM-CSF promoter is recognized by NF-kappa B/Rel family members.

To further extend the previous analysis of cis-acting elements and cognate trans-acting factors that contribute to GM-CSF transcriptional regulations, we have examined a promoter region between -1742 and -2010. DNase I footprinting assays showed four protected sequences named A, B, C and D. DNA transfections in the T-lymphoid Mo cell line, which constitutively expresses GM-CSF, indicated that the A element, located between -2002 and -1984, has a positive role on transcription. Further characterization by electrophoretic mobility shift assays in the presence of different competitor oligonucleotides showed that this element binds a factor of the NF-kappa B/Rel family.

Base Sequence↗
PubMed1996

Torsionally-strained DNA and intermolecular purine-purine-pyrimidine triple-helix formation.

A potentially powerful pharmacological approach to modulating the expression of specific, disease-related genes involves the inhibition of transcription factor binding to promoter or enhancer elements through oligonucleotide-mediated triple-helix formation. In vivo, the typical target for intermolecular triplex formation would most likely be torsionally-strained rather than relaxed duplex DNA. To determine the effects of strained DNA on triplex formation, we investigated the interactions between a G/T-rich oligonucleotide and both supercoiled and relaxed plasmid DNA using a restriction endonuclease protection assay. Both the kinetics of formation and dissociation of purine-motif triplexes were unaffected by the conformational state of the duplex DNA. Similarly, the topological state of the plasmid targets was not affected by triplex formation. Taken together, these observations suggest that stable intermolecular triplexes can form in vivo under conditions of moderate torsional strain.

Base Sequence↗
PubMed1996

Comparative activity of idarubicin and idarubicinol in combination with cyclosporin A in multidrug-resistant leukemia cells.

4-Demethoxydaunorubicin (idarubicin, IDA) is an anthracycline that has shown good cytotoxic activity in vitro against tumor cell lines displaying the multidrug-resistant (MDR) phenotype. IDA is converted in the liver into idarubicinol (2HIDA) and, in this form, seems to exert its antitumoral activity in vivo. Recent studies have shown that 2HIDA has tumoricidal activity similar to that of the parent drug when tested in vitro in sensitive neoplastic cells. In this work we compared in vitro the effects of IDA and 2HIDA used alone and in combination with 2 microM cyclosporin A (CyA) in the MDR leukemic cell lines FLCR and K562R and in their sensitive parent cell lines FLC and K562. IDA and 2HIDA showed the same cytotoxic activity in sensitive cells. After 1 h of exposure of cells to each anthracycline, we observed that the cellular uptake of IDA and 2HIDA was also similar. In resistant cells, 2HIDA was 3-4 times less active than IDA. We observed that the intracellular uptake of 2HIDA was lower than that of IDA, and this may be correlated with a greater ability of P-glycoprotein to expel 2HIDA as opposed to IDA. Indeed, when MDR cells were exposed to IDA and 2HIDA in combination with 2 microM CyA, the cytotoxic effect of these anthracyclines was the same, and it was similar to that observed in sensitive cells. These data confirm the utility of the combination of IDA and an MDR-reversing agent in hematological malignancies displaying the MDR phenotype.

Antibiotics, Antineoplastic↗
PubMed1995

Characterization of a distal 5'-flanking region (-2010/-630) of human GM-CSF.

The 5'-flanking region of the human GM-CSF gene was subcloned from the phagic clone lambda J1-16 to detect cis-elements involved in GM-CSF constitutive expression. We determined and sequenced an uncharacterized promoter region of 1381 bp (-2010 to -630), named pPF2000. Putative binding sites of several transcriptional factors were found. Progressive deletion mutants of the PF2000 were analyzed by measuring the linked CAT activities, in constitutive (5637) and inducible (PEU) GM-CSF-producing cells. A positive distal sequence (268 bp), between -2010 and -1742, responsible for the high constitutive expression of GM-CSF in 5637 carcinoma cell line was found.

Base Sequence↗
PubMed1995

Polyamine effects on purine-purine-pyrimidine triple helix formation by phosphodiester and phosphorothioate oligodeoxyribonucleotides.

Utilization of oligodeoxyribonucleotides to inhibit specific gene transcription in vivo (antigene strategy) requires the efficient formation of triple helices under physiological conditions. However, pyrimidine-motif triplexes are not favored at physiological pH, and physiological concentrations of potassium cations hamper purine-motif triplex formation. Here we investigated the effects of polyamines on promoting triplex formation by G/T-rich oligodeoxyribonucleotides containing either phosphodiester or a diastereomeric mixture of phosphorothioate linkages. Compared with Mg2+, equimolar concentrations of polyamines greatly facilitated purine-motif triplex formation with the following order of effectiveness: spermine > spermidine > putrescine. At low polyamine concentrations, phosphorothioate oligonucleotides were better at triplex formation than the corresponding phosphodiester oligonucleotides. Kinetic studies indicated that polyamines facilitated triplex formation by increasing the rate of oligonucleotide-duplex DNA association. However, triplex accumulation with either oligonucleotide was still low under physiological conditions (140 mM K+, 10 mM Mg2+, 1 mM spermine). The inhibitory effects of K+ could be partially overcome with high concentrations of Mg2+ or spermine, with phosphodiester oligonucleotides being better able to form triplexes than phosphorothioates under these conditions.

Base Sequence↗
PubMed1995

Phase III comparative trial using CHOP vs CIOP in the treatment of advanced intermediate-grade non-Hodgkin's lymphoma.

Until now, literature data support the fact that the CHOP regimen represents the standard first line treatment for patients with advanced intermediate-grade non-Hodgkin's lymphoma. Recently, idarubicin has been introduced in clinical trials because of its favourable preclinical profile: it is more active than daunorubicin and doxorubicin against a number of experimental tumour systems and is significantly less cardiotoxic in animal models. From March 1991 to June 1993, 115 previously untreated patients with stage II to IV intermediate-grade non-Hodgkin's lymphoma, according to the Kiel classification, were enrolled in a phase III comparative trial. The objectives of the study were to compare the efficacy and safety of using idarubicin instead of doxorubicin in the polychemotherapeutic regimen CHOP (cyclophosphamide, doxorubicin, vincristine, and dexamethasone). Of the 115 patients registered for the trial, 103 were evaluable: 52 received CH (doxorubicin)OP and 51 received CI(Idarubicin)OP. Known prognostic factors were equally distributed among the two groups. There were no significant differences between the 2 groups in the rates of partial and complete response. The overall response rate was 87%, with complete response in 62%: 63% in the CHOP group, and 59% in the CIOP group. At 30 months (median 20 months), 86% of all CR patients were alive without disease in the CHOP group and 85% in the CIOP group. Patients treated with CHOP experienced severe alopecia more frequently (P = .004). Only three patients in the CIOP group showed cardiac adverse events (1 moderate and 2 mild), while in the CHOP group 4 mild, 2 moderate and 1 severe were recorded. LVEF monitoring was carried out in 31 patients of the CHOP group and in 27 of the CIOP group. A median drop of 8.3% of the LVEF was observed in patients treated with CHOP regimen as compared to 4.8% in patients with CIOP regimen (P = .0001). In this trial, the "idarubicin arm" (CIOP regimen) was found to have an equivalent therapeutic efficacy and, slightly, reduced clinical toxicity in comparison to the standard doxorubicin-containing CHOP regimen in patients with intermediate-grade non-Hodgkin's lymphoma.

Adult↗
PubMed1995

[Perforating verruciform collagenoma, an exogenous inclusion skin disease? Apropos of a case induced by calcium chloride].

INTRODUCTION: Perforating verruciform collagenoma, first described in 1963, designates papular and keratotic post-trauma lesions which show an aspect of epidermal perforation microscopically with large fragments of collagen issuing through fistulous chimneys, more or less pycnotic polynuclears and squamating or necrotic epidermal cells. Three publications have presented this diagnosis. CASE REPORT: We observed a patient with multiple lesions with macroscopic and microscopic presentations suggestive of perforating verruciform collagenoma. The lesions appeared after scratches and inoculation with calcium chloride. DISCUSSION: This case is similar to those observed after intradermal inoculation of calcium salts. In the 4 previous cases, perforating verruciform collagenoma was associated with trauma allowing the intradermal penetration of a particular foreign material (glass wool, vegetable debris, drugs or intravenous injections, wound caused by a metallic garbage bin). The exceptional nature of perforating verruciform collagenoma in highly frequent skin wounds would suggest that a very particular post-trauma process is occurring caused by the introduction of an exogenous substance within the derma: calcium chloride in our case, a non-specific material in the four previous cases.

Adult↗
PubMed1995

A new approach in the evaluation of gonadotropins for the diagnosis of polycystic ovary syndrome.

The new immunometric methods using monoclonal antibodies to detect gonadotropins revealed a reduction in the absolute values of these hormones, especially LH, due to a decrease in cross-reaction between gonadotropin subunits. Therefore, reference values of the LH/FSH ratio and their diagnostic significance in the polycystic ovary syndrome (PCOS) need to be defined again. We evaluated the LH/-FSH ratio in basal conditions and after administration of GnRH (100 micrograms as i.v. bolus) in seventy patients with PCOS employing an immunoenzymatic method. In PCOS patients the LH/FSH ratio was greater than 1 in 70% when evaluated on a single sample and in 88% when evaluated on a pooled serum from four samples every 30 minutes; in the control group the ratio was always lower than 1. The LH/-FSH ratio evaluated on the peak values in response to GnRH was greater than 2 in all patients and lower than 2 in the control group. Our results indicate that the LH/FSH ratio is still an important diagnostic tool in PCOS, especially when evaluated on the peak values in response to GnRH, even if its reference values are lower using these new monoclonal methods.

Adolescent↗
PubMed1994

c-Krox, a transcriptional regulator of type I collagen gene expression, is preferentially expressed in skin.

We have cloned a mouse cDNA that is a member of the Krox gene family and encodes a protein we have named c-Krox. The c-Krox protein contains three zinc fingers of the Cys2His2 type. c-Krox binds specifically to a guanine-rich cis-acting element present twice in the promoter element of the mouse alpha 1(I) collagen gene. Study of c-Krox gene expression shows that c-Krox is markedly enriched in skin, one of the two major sites of type I collagen synthesis, but is absent in bone, the other main type I collagen-producing tissue, indicating that type I collagen gene expression is differentially regulated in skin and bone. DNA transfection experiments in mouse NIH 3T3 fibroblasts, cells that express the c-Krox gene, or in Drosophila S2 cells, which do not express c-Krox, reveal that c-Krox can activate transcription of a reporter gene linked to several copies of its binding site in the alpha 1(I) collagen promoter. Thus, c-Krox is only the second member of the Krox family for which a target gene has been identified. The selective spatial pattern of expression of its mRNA and its transcription activation ability suggest that c-Krox may be an important regulator of type I collagen skin specific expression in physiologic conditions and in fibrotic diseases such as scleroderma.

3T3 Cells↗
PubMed1993

H and L ferritin gene expression in U937 cells induced to macrophage differentiation.

Ferritin is an ubiquitous protein that has been shown to regulate cell differentiation in several experimental systems. In this study we have investigated the expression of ferritin genes encoding the heavy (H) and light (L) chains in t'B U937 cell line, induced to differentiate to macrophage-like cells by 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA) or 1-beta-D-arabinofuranosylcytosine (Ara-C). An increase in the level of H ferritin mRNA was detected in U937 cells that had been incubated with Ara-C. Treatment of U937 cells with Actinomycin D suggested that the H ferritin mRNA increase was mediated by post-transcriptional mechanisms. The L ferritin mRNA level increased only following stimulation of U937 cells with RA. Immunophenotypic and cytochemical analyses showed that Ara-C was the strongest inducer of the macrophagic differentiation of U937 cells. These results suggest that the increase of H ferritin mRNA expression may represent a sensitive marker of myeloid cells differentiating along the monocyte-macrophage lineage.

Antigens, CD↗
PubMed1992

Lactoferrin binding sites and nuclear localization in K562(S) cells.

Lactoferrin, a single chain cationic glycoprotein, present in the secondary granules of neutrophils, acts as a negative feedback regulator of myelopoiesis. Specific receptors for lactoferrin were detected on the surface of different hematopoietic cell types. The influence of lactoferrin on cell growth in culture has been reported. Interactions of lactoferrin with DNA were also demonstrated. In the present paper we confirm the presence of lactoferrin specific binding sites on K562 cells and we estimate the number of binding sites and the dissociation constant. By Western blotting analysis performed on K562 lysates we find a band of about 120 kDa responsible for specific binding of lactoferrin. We also show that lactoferrin, after binding at the cell surface, is internalized in a temperature dependent way and is immunologically detectable as a DNA-linked protein in nuclear extracts.

Binding Sites↗