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E Prosperi

Publications and source records attributed to E Prosperi.

At least 37 records · Page 2Linked to original sources
PubMed1996

Effect of beta-carotene on cell cycle progression of human fibroblasts.

The uptake of beta-carotene (BC) and its effect on the cell cycle progression of normal human fibroblasts in primary culture were investigated by using two different delivery methods: exposure to BC solubilized in the organic solvent tetrahydrofuran (THF) or to BC incorporated into dipalmitoylphosphatidylcholine (DPPC) liposomes. Cell cycle progression was evaluated by immunofluorescence detection and flow cytometric analysis of the proliferating cell nuclear antigen (PCNA). In contrast to THF, which induced a marked reduction in the number of cells in S phase and in the extent of PCNA immunolabeling, DPPC liposomes proved to be an effective delivery system that does not interfere with cell proliferation. Cellular uptake of 0.23 nmol/10(6) cells was found after 24 h incubation in BC-containing DPPC liposomes. This value increased to 1.2 nmol/10(6) cells after 72 h. After the first day of incubation, the number of cells in S phase was reduced by approximately 50%, with a consequent accumulation of cells in G1 phase. This effect was maintained up to 3 days incubation, with no detectable effects on cell viability. This cell cycle delay was found to be reversible, returning the percentage of cells in S phase to the control value 24 h after removal of BC from the medium. In order to determine whether the activity of BC could be attributed to the molecule itself or to its conversion into retinoids, the production of BC metabolites was assessed. Analysis of cellular levels of retinoids failed to demonstrate the presence of retinal, retinol, retinoic acid or retinyl esters during an incubation period of 6 days. These results suggest that in normal human fibroblasts, BC induces a cell cycle delay in the G1 phase and that this effect is independent of conversion to known retinoids.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1996

Sequence of events leading to apoptosis in long term cultured HeLa cells.

We have maintained HeLa cells in culture in the original medium for increasing times to induce growth arrest. Cell viability was evaluated by trypan blue dye exclusion assay. We observed that when cells are maintained in culture for several days, morphological hallmarks of apoptosis become evident. DNA synthesis rate, followed by (3)H-thymidine incorporation slowed down in long term cultured cells. This evidence was supported by the analysis of cell cycle progression determined by proliferating cell nuclear antigen (PCNA) immunostaining. Apoptotic cells have been characterized with respect to the sequential appearance of high molecular weight and internucleosomal DNA fragmentation. We have provided evidence that in this experimental model the first step in DNA degradation is represented by the formation of high molecular weight fragments, whereas nucleosomal DNA ladder is visible later on. The activation of the enzyme poly(ADP-ribose)polymerase, considered a marker of apoptotic death, has been observed. The results suggest that long term culture conditions activate the apoptotic programme.

Journal Article↗
PubMed1996

Immunocytometric determination of the alpha and beta isoforms of human DNA topoisomerase II: influence of different fixatives.

The immunoreactivity of the alpha and beta isoforms (170 and 180 kDa, respectively) of human DNA topoisomerase II (topo II) toward specific monoclonal antibodies (MoAbs), was investigated in HeLa cells. The extent of antigen preservation achieved with the dehydrating fixatives, ethanol and acetone, was compared with that obtained with the cross-linking agent paraformaldehyde. Binding of each MoAb to the relative isozyme was detected by indirect immunofluorescence labelling and quantified by flow cytometry. The amount of antigen detectable was calculated by normalizing the mean fluorescence intensity of samples incubated with specific MoAbs, to the respective background fluorescence. For both isozymes, acetone provided the highest immunofluorescence/background ratio. A strong reduction in the immunoreactivity of the 180-kDa isoform was observed after ethanol, and to a minor extent after paraformaldehyde fixation, while reactivity of the 170-kDa isoform was not significantly affected. Cell cycle distribution of each protein was assessed by dual-parameter analysis of immunofluorescence vs. DNA content. No evident difference in the cell cycle distribution of each isozyme was found with each fixation protocol. At the morphological level, the pattern of immunofluorescence showed that the localization of each isozyme was very similar for all these fixatives. These results confirm the great sensitivity of the 180-kDa isoform to degradation, and indicate that fixation has to be carefully chosen in order to determine the relative amount of topo II isoforms with immunocytometric techniques.

Animals↗
PubMed1995

MRP gene overexpression in a human doxorubicin-resistant SCLC cell line: alterations in cellular pharmacokinetics and in pattern of cross-resistance.

The development of non-P-glycoprotein-mediated multi-drug resistance is a frequent event among lung-cancer cell lines. In an attempt to understand the underlying mechanisms of this phenotype, we have selected a multi-drug-resistant subline (POGB/DX) in vitro for doxorubicin resistance. The original cell line (POGB) was established in vitro from a non-treated patient with a small-cell lung cancer. POGB/DX cells were cross-resistant to other drugs, associated with MDR phenotype. In contrast, they were not resistant to taxol, camptothecin or melphalan, but were instead hypersensitive to 5-fluorouracil. Although expression of the mdr-1 gene was not detected in POGB/DX cells, cellular pharmacokinetics showed a reduced drug accumulation and altered intracellular localization in the POGB/DX cell line. This defect in drug accumulation was associated with overexpression and amplification of the MRP gene. Interestingly, verapamil, a known modulator of P-glycoprotein function, was able to reverse drug resistance and to increase drug accumulation. In Northern-blot analysis no differences in expression of topoisomerase I and II (alpha and beta), DNA polymerase beta, or HSP70 and HSP60 genes were observed between POGB and POGB/DX. Coupled to lack of changes in expression of known resistance factors, overexpression of MRP and modulation by verapamil strongly support a role for this gene product in the development of drug resistance in this SCLC cell system. This study provides evidence that (a) altered cellular pharmacokinetics is related to MRP expression; (b) MRP-mediated phenotype is characterized by a specific pattern of cross-resistance, which does not involve taxol; and (c) verapamil may be effective in modulating the function of the MRP gene product.

Carcinoma, Small Cell↗
PubMed1995

Nuclear binding of cell cycle-related proteins: cyclin A versus proliferating cell nuclear antigen (PCNA).

We have investigated the cell cycle-dependent nuclear binding of cyclin A and of the proliferating cell nuclear antigen (PCNA) in asynchronously growing human fibroblasts. To this purpose, we have applied flow cytometry immunofluorescence, a powerful technique for elucidating the cell cycle phase during which the nuclear binding occurs. We have observed that, in striking contrast with the distribution of nuclear-bound PCNA which is restricted to S phase, the immunofluorescence signal of the nuclear-bound form of cyclin A is high in the G1 and G2 phases of the cell cycle. These results suggest the involvement of nuclear-bound cyclin A in the G1/S and G2/M phase transitions.

Biomarkers↗
PubMed1995

Activation of poly(ADP-ribose)polymerase in apoptotic human cells.

We have studied the effect of the chemotherapeutic drug VP-16 (etoposide) on the metabolism of HeLa cells by analysing different cellular parameters considered as markers of apoptosis. Typical features such as chromatin condensation and internucleosomal DNA cleavage are visible in HeLa cells exposed to VP-16. We investigated whether the appearance of small-sized DNA fragments could regulate the ADP-ribosylation process. To this purpose, we have analysed, by means of the activity gel technique; the structural and catalytical properties of poly(ADP-ribose)polymerase. In extracts from cells where etoposide-induced DNA fragmentation occurred, we have shown that the label of the autoribosylated form of the enzyme is greatly increased even if the amount of the protein remains constant. This phenomenon is completely abolished in cells preincubated with poly(ADP-ribose)polymerase inhibitor, 3-aminobenzamide. After VP-16 administration, we have observed that the level of NAD is not heavily decreased. It is widely agreed that zinc exerts an inhibitory effect on the endonuclease(s) responsible for the fragmentation of DNA during apoptosis. After incubation of cells with zinc/VP-16 we have found the occurrence of apoptotic parameters even in the absence of internucleosomal DNA cleavage. The inhibition of DNA fragmentation prevents the activation of poly(ADP-ribose)polymerase activity. These results indicate that the activation of the enzyme towards the automodification reaction is strictly dependent on the appearance of DNA internucleosomal fragments and could represent a way to control enzyme activity.

Adenosine Monophosphate↗
PubMed1994

Proliferating cell nuclear antigen bound to DNA synthesis sites: phosphorylation and association with cyclin D1 and cyclin A.

Evidence is presented that association of proliferating cell nuclear antigen (PCNA) with nuclear chromatin in human fibroblasts is related to the phosphorylation status of the protein. Using a hypotonic lysis procedure to extract the soluble form of PCNA, it has been shown that the remaining nuclear-bound form, predominantly in S-phase cells, is highly phosphorylated. Cells in early G1, or in G2 + M phases, contain basal levels of the bound form of the protein that is only weakly phosphorylated. Using fractionated immunoprecipitation techniques, PCNA was found to be associated with cyclin A in both soluble and insoluble fractions. In contrast, association of PCNA with cyclin D1 was found in the soluble fraction, while no detectable levels were present in the insoluble fraction. Immunofluorescence labeling and flow cytometric analysis of the cell cycle distribution of cyclin D1 and cyclin A showed that, like PCNA, maximal levels of both proteins were bound to nuclear structures at the G1/S phase boundary. These results suggest that binding of PCNA to DNA synthesis sites occurs after phosphorylation. Association with cyclin D1 and cyclin A might occur in a macromolecular complex assembled at the G1/S phase boundary to drive activation of DNA replication factors.

Binding Sites↗
PubMed1994

DNA stainability with base-specific fluorochromes: dependence on the DNA topology in situ.

The influence of DNA topology on stainability with the externally binding fluorochromes Hoechst 33258 (HO) and mithramycin (MI) was investigated in HeLa nuclei in comparison with the intercalating dye propidium iodide (PI). Changes in DNA topology were induced with a mild DNAse I treatment. Stainability properties of untreated and nuclease-treated nuclei were compared with those of the supercoiled-circular and the relaxed-linear forms of the plasmid pBR322. DNAse-treated nuclei stained with HO showed a higher fluorescence intensity than control samples, independently of the dye concentration, in contrast with the findings obtained with PI. Similar behaviour was observed with the relaxed-linear form of pBR322, compared with the supercoiled-circular molecule. With MI, the stainability of HeLa nuclei did not depend on the DNA topology, whereas the stainability of the plasmid was similar to that of HO. In order to assess whether this discrepancy depended on differences in the availability of DNAse-sensitive sites to the fluorochromes, fluorescence resonance energy transfer (FRET) studies were performed in nuclei stained with HO+PI, or with HO+MI dye pairs. After DNAse I digestion, the relative FRET efficiency between donor (HO) and acceptor molecules (PI or MI) was reduced significantly only when MI was the acceptor. This result may be due to greater stainability of DNAse-sensitive sites with HO than with MI. These findings indicate that DNA stainability with base-specific fluorochromes may be affected by the topology of chromatin regions.

Binding Sites↗
PubMed1994

Expression of the 170-kDa and 180-kDa isoforms of DNA topoisomerase II in resting and proliferating human lymphocytes.

The expression of the 170-kDa (alpha) and the 180-kDa (beta) isoforms of DNA topoisomerase II (topo II) was investigated with two specific monoclonal antibodies (MoAbs) in human peripheral blood lymphocytes (PBL), before and after phytohaemoagglutinin (PHA) stimulation. Binding of each MoAb was detected by indirect immunofluorescence labelling and quantified with flow cytometry. In resting PBL, the intensity of immunostaining was very low for both isozymes; however, topo IIbeta-associated immunofluorescence was about 2.5 times significantly higher (P<0.001) than that associated with the alpha isoform. Between 48 and 72 h of PHA stimulation, when the highest percentage of cells in S and G2+M phases was found, the levels of topo IIalpha and beta increased up to about 30 and 10 times the value measured in resting PBL, respectively. Thus, the two isoforms reached comparable immunofluorescence values. At longer stimulation periods (96-120 h), topo IIalpha immunofluorescence was not significantly changed, while that relative to topo IIbeta declined to about 50% of the peak value (P<0.02). At this time however, topo IIalpha-associated immunofluorescence was not significantly different from that related to the beta isozyme. These results suggest that in resting PBL topo IIalpha is required at levels lower than topo IIbeta, while in proliferating lymphocytes both isoforms are expressed to significantly higher levels.

Cell Cycle↗
PubMed1994

Correlation between ultrastructural and histochemical parameters in lymphokine-activated killer (LAK) cells reacting with target cells in vitro.

Ultrastructural and fluorescence data allowed us to study the most important moments of the interaction between lymphokine-activated killer (LAK) cells against target cells (Chang) in vitro. The LAK cells, maintained at low doses of recombinant interleukin-2, were able to recognize, bind and destroy the tumoral cells. Before the attack, the LAK cells were characterized by a cytoplasm with a high ribosomes content; after the identification and the interaction cell-cell, a degeneration of the tumoral cell was observed. These observations allowed us to suppose that the interaction between the two types of cells may be mediated by a receptoral membrane system without the action of lytic enzymes.

Autolysis↗
PubMed1994

Dose-dependent zinc inhibition of DNA ladder in apoptotic HeLa cells regulates the activity of poly(ADP-ribose) polymerase and does not protect from death induced by VP-16.

Zinc ions exert an inhibitory effect on Ca(2+)Mg(2+)-dependent endonuclease which is supposed to be responsible for the fragmentation of DNA during apoptosis. In the experimental system we used, that is HeLa cells treated with VP-16, the protection from internucleosomal DNA degradation is modulated by Zn concentration and appears to be dependent on the time after treatment. This effect does not prevent cell death or occurrence of apoptotic parameters, suggesting that DNA ladder appearance is not a crucial event in apoptosis. The activation of poly(ADP-ribose)polymerase following the administration of VP-16, is not observed in cells in which DNA fragmentation has been abolished by zinc, supporting the hypothesis that this event is regulated by the appearance of small-sized DNA fragments.

Journal Article↗
PubMed1993

A study of cross-resistance pattern and expression of molecular markers of multidrug resistance in a human small-cell lung-cancer cell line selected with doxorubicin.

A doxorubicin-resistant variant of the human small-cell lung-cancer cell line N592 was selected by in vitro continuous exposure to increasing drug concentrations. The aim of this study was to examine the cross-resistance pattern, cellular pharmacokinetics of doxorubicin and expression of molecular factors of resistance. The sub-line N592/DX exhibited a multidrug-resistance phenotype, which was somewhat atypical, since it included cisplatin. Development of doxorubicin resistance could not be attributed to differential doxorubicin uptake or retention. Verapamil partially reverted doxorubicin resistance without affecting cellular pharmacokinetics. These findings are consistent with undetectable levels of mdr-1-gene expression in these cells. A molecular analysis of other putative mechanisms of multidrug resistance indicated no alterations in GSH levels or GSH-related enzymes, but a marginal reduction of topoisomerase II alpha expression in the resistant sub-line. This reduction, which was associated with an increase in topoisomerase I, does not explain the high degree of resistance. This study supports the view that alternative, unidentified mechanisms, which may be of clinical relevance, must be involved in the development of multidrug resistance of small-cell lung cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Increased sensitivity of damaged DNA to digestion with nuclease S1 as assessed in single cells by flow cytometry.

DNA sensitivity to digestion with nuclease S1 was investigated in cells irradiated with gamma rays, or treated with the antitumor drug adriamycin (Adr). The nuclease-resistant DNA fraction was determined by propidium iodide staining. Treated cells were found to be more sensitive to nuclease digestion than the undamaged controls. Gamma ray-induced strand breaks were detectable at doses up to 10 Gy; an increase in the reaction temperature, from 37 degrees to 63 degrees C, was necessary in order to detect higher levels of damage. Nuclease S1 sensitivity in Adr-treated cells showed a single-peak, concentration-dependent relationship, in agreement with the known self-inhibitory effect exerted by high drug doses. Determination of DNA digestion could be performed in combination with other cellular parameters (e.g., protein content). Detection of drug-resistant cells in a heterogeneous population of small-cell lung carcinoma was achieved on the basis of the different sensitivity of the cells to enzymatic digestion. These results indicate that nuclease S1 may be a useful probe for studying in single cells DNA alterations induced by drugs or radiation.

Cell Separation↗
PubMed1993

Proliferating cell nuclear antigen complex formation induced by ultraviolet irradiation in human quiescent fibroblasts as detected by immunostaining and flow cytometry.

The association of the proliferating cell nuclear antigen (PCNA) to DNA synthesis sites was investigated in human quiescent fibroblasts after UV irradiation. Associated PCNA was detected with the monoclonal antibody (MoAb) PC10 and by immunofluorescence assessment with flow cytometry, after a hypotonic lysis step in order to release unbound molecules. Immunofluorescence levels, relatively low in untreated control cells, increased by about threefold after uv irradiation. The time course of PCNA complex formation showed a maximum after about 30 min from irradiation and was found to be dose-dependent up to about 10 J/m2, after that it reached a plateau. Formation of the PCNA-chromatin complex was neither significantly affected by the topoisomerase II inhibitor VP-16, nor by the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide. In contrast, a significant reduction was obtained either after ATP depletion or after incubation with the protein-kinase inhibitor staurosporine. Immunoprecipitation studies on nuclear extracts from 32P-labeled cells showed that PCNA bound to DNA synthesis sites was phosphorylated. The results indicate that PCNA associated to DNA repair synthesis sites may be detected with PC10 MoAb after a hypotonic lysis step, and provide evidence that the transition from soluble to chromatin-associated form of the protein is dependent on a phosphorylation mechanism.

Alkaloids↗
PubMed1993

Flow cytometric analysis of DNA sensitivity to nuclease S1 in UVC-irradiated human fibroblasts.

The sensitivity of human fibroblast DNA towards the activity of nuclease S1 was investigated in situ after irradiation of the cells with UVC light. DNA digestion was assessed using flow cytometry by staining the nuclease-resistant DNA fraction with the specific fluorochrome propidium iodide (PI). The results showed a non-linear dependence of DNA digestion on the UVC irradiation dose between 0 and 20 J m-2. About 70% of the UVC-induced DNA sensitivity to nuclease S1 was lost after 30 min of repair. These results suggest that flow cytometry may be useful for assessing the heterogeneity of cell response to UV damage.

Cells, Cultured↗
PubMed1993

Involvement of proliferating cell nuclear antigen in DNA repair after damage induced by genotoxic agents in human fibroblasts.

The association of the proliferating cell nuclear antigen (PCNA) to DNA synthesis sites during the process of DNA repair, was investigated in human diploid fibroblasts after treatment with different genotoxic agents. For this purpose, confluent cultures were treated with agents that form primarily DNA adducts, such as u.v.-C, 8-methoxypsoralen (8-MOP) and 4,4',6-trimethylangelicin (TMA), or with agents that induce strand breaks, such as X-rays or bleomycin (BLM). Chromatin-associated PCNA was detected with a monoclonal antibody and indirect immunofluorescence labelling. Quantitative analysis was performed by flow cytometry. Not all of the tested agents were able to activate the association of PCNA with chromatin. X-rays, u.v.-C and BLM induced a significant increase in PCNA complex as compared to the control samples. In contrast, 8-MOP and TMA did not show any detectable effect on the levels of associated PCNA, even at post-incubation repair times as long as 10 h. However, these drugs damaged DNA, as shown by the formation of micronucleated cells 48 h after treatment. The lack of PCNA activation was not due to an inhibition of the repair mechanism, since in TMA-treated fibroblasts, subsequent irradiation with u.v.-C induced an increase in PCNA levels comparable to that found in cells treated with u.v.-C alone. These results indicate that PCNA is involved in DNA excision repair of genotoxic agents, but suggest that similar types of lesions may be repaired with alternative pathways not requiring PCNA.

Bleomycin↗
PubMed1992

Comparative studies on the effects of doxorubicin and differentiation inducing agents on B16 melanoma cells.

The differentiation-inducing activity of doxorubicin on B16 melanoma cells grown in vitro was compared with that of other known differentiation inducers, such as theophylline, retinoic acid, and melanocyte-stimulating hormone (MSH). At drug concentrations resulting in cytostatic effects, doxorubicin and theophylline induced morphological changes (dendritic-like structures with a terminal melanin granule) with an enhancement of total melanin content and tyrosinase activity. Retinoic acid did not alter melanin content and cell morphology, although it affected cell growth. MSH enhanced total melanin content and tyrosinase activity, with no significant morphological changes. Flow cytometric analysis showed that MSH led to an accumulation of cells in G1 phase whereas doxorubicin induced an accumulation of cells in G2 + M. Studies on DNA content in doxorubicin-treated cells, selected on the basis of a morphologically differentiated pattern, showed a clustering of these cells in G2 + M, probably due to a cytokinesis block. Thus doxorubicin can induce cell differentiation comparable with other differentiation inducers.

Animals↗