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Biomedical subjects

E Prosperi

Publications and source records attributed to E Prosperi.

At least 19 recordsLinked to original sources
PubMed2003

Anthocyanins protect against DNA damage induced by tert-butyl-hydroperoxide in rat smooth muscle and hepatoma cells.

Anthocyanins are flavonoids present in a variety of pigmented food and, like other flavonoids, seem to play a role in preventing human pathologies related to oxidative stress. In fact, anthocyanins have been shown to exert antiproliferative effects in cell cultures and exhibit antiinflammatory and vasoprotective activities in animal models. Although these biological activities have been related to their antioxidant properties, little is known on the molecular mechanism of action of anthocyanins. The effects of pretreatment with the anthocyanins delphinidin, cyanidin, and their glycoside and rutinoside derivatives against induction of DNA damage induced by tert-butyl-hydroperoxide (TBHP) were evaluated in rat smooth muscle and in rat hepatoma cell lines using alkaline single cell gel electrophoresis (Comet test). In addition, a possible protection exerted by anthocyanins on cell killing, lipid peroxidation, and redox state alterations induced by TBHP was also investigated. It was found that the treatment with TBHP induces the formation of DNA single strand breaks (SSB) and oxidised bases, along with cell killing, lipid peroxidation and redox state alteration. Our data demonstrate that anthocyanins are effective against cytotoxicity, DNA SSB formation and lipid peroxidation induced by TBHP, but they do not have any detectable effect against impairment by TBHP of cellular redox state and on protection against DNA bases oxidation. The presence of a sugar moiety in anthocyanin derivatives reduced this protective effect, mainly in rat hepatoma cells. The different activity of anthocyanins and their derivatives may be explained taking into account a structure/function relationship that could also influence anthocyanin intracellular localisation.

Animals↗
PubMed2001

Early effects of AZT on mitochondrial functions in the absence of mitochondrial DNA depletion in rat myotubes.

Zidovudine (AZT) is a potent inhibitor of human immunodeficiency virus (HIV) replication. In humans, as well as in animal models, long-term treatment with AZT induces a severe myopathy characterised by structural and functional alterations of mitochondria associated with depletion of mitochondrial DNA (mtDNA). In the present work, we compared the effects induced by AZT on mitochondria upon short- or long-term treatments of cultured rat myotubes. Morphological alterations were investigated by electron microscopy, and mtDNA depletion and deletions were analysed by Southern blot. Mitochondrial membrane potential was determined after JC-1 staining by laser-scanning confocal microscopy in whole cells, and by flow cytometry in isolated muscle mitochondria. We found that the early effects of AZT on mitochondrial functions were a marked, yet reversible reduction in mitochondrial membrane potential, in the absence of any effect on mtDNA. The long-term treatment, in addition to mitochondrial membrane potential alterations, induced morphological changes in mitochondria, and a remarkable reduction in the amount of mtDNA, without any significant evidence of mtDNA deletions. In both treatments, a block of the spontaneous contraction of myotubes was observed. To study in more detail the early effects induced by AZT, the ability of the drug to interact with cardiolipin, an important component of internal mitochondrial membrane, was investigated by atomic force microscopy (AFM) in an artificial membrane model system. The results suggest that the primary effects of AZT may be related to a physical interference with the membrane structure leading to a consequent modification of its physical characteristics.

Animals↗
PubMed2001

Specific structural determinants are responsible for the antioxidant activity and the cell cycle effects of resveratrol.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a natural phytoalexin found in grapes and wine, which shows antioxidant and antiproliferative activities. In this study we have investigated whether these properties are dependent on similar or different structural determinants of the molecule. To this purpose, resveratrol derivatives, in which all or each single hydroxylic function were selectively substituted with methyl groups, were synthesized. Analogues with the stilbenic double bond reduced or with the stereoisometry modified were also investigated. The antioxidant activity of these compounds was evaluated by measuring the inhibition of citronellal thermo-oxidation, or the reduction of 2,2-diphenyl-1-picrylhydrazyl radical. In addition, the protection against lipid peroxidation was determined in rat liver microsomes, and in human primary cell cultures. The antiproliferative activity was evaluated by a clonogenic assay, and by analysis of cell cycle progression and DNA synthesis. The results showed that the hydroxyl group in 4' position is not the sole determinant for antioxidant activity. In contrast, the presence of 4'-OH together with stereoisometry in the trans-conformation (4'-hydroxystyryl moiety) was absolutely required for inhibition of cell proliferation. Enzymatic assays in vitro demonstrated that inhibition of DNA synthesis was induced by a direct interaction of resveratrol with DNA polymerases alpha and delta.

Animals↗
PubMed2001

p21(waf1/cip1)-null human fibroblasts are deficient in nucleotide excision repair downstream the recruitment of PCNA to DNA repair sites.

The cyclin-dependent kinase inhibitor p21(waf1/cip1) is known to impair DNA synthesis by binding to PCNA, the co-factor of DNA polymerases delta and epsilon. However, a positive role for p21 in nucleotide excision repair (NER) has been suggested. In this study, the sensitivity to DNA damage and DNA repair efficiency were investigated in p21-null human fibroblasts obtained by targeted homologous recombination. After UV-C irradiation, p21-/- cells showed a threefold reduction in clonogenic survival and an increased susceptibility to apoptosis, as compared with parental p21+/+ cells. Removal of cyclobutane pyrimidine dimers was significantly reduced in p21-/- cells both in the whole genome, and at the level of the rDNA gene cluster, as determined by immunoassay and Southern blot, respectively. After DNA damage, the recruitment of PCNA as detergent-insoluble form associated to DNA repair sites in p21-/- fibroblasts, was comparable to that observed in parental p21+/+ cells. However, PCNA remained associated with DNA for a longer period in p21-/- than in p21+/+ cells. These results suggest that in human cells, p21 is required for NER at a step located downstream the recruitment of PCNA to DNA repair sites.

Apoptosis↗
PubMed2001

Different effects of methotrexate on DNA mismatch repair proficient and deficient cells.

Antifolates exert their antiproliferative activity through the inhibition of dihydrofolate reductase and, as a consequence, of thymidylate synthesis, thereby inducing nucleotide misincorporation and impairment of DNA synthesis. We investigated the processes involved in the repair of antifolate-induced damage and their relationship with cell death. Since misincorporated bases may be removed by DNA mismatch repair (MMR), the study was carried out on the MMR-proficient human cell lines HeLa and HCT116+chr3, and, in parallel, on the MMR-deficient cell lines HeLa cell-clone12, defective in the protein hPMS2, and HCT116, with an inactive hMLH1. After treatment with methotrexate (MTX), we observed that DNA repair synthesis occurs independently of the cellular MMR function. Clear signs of apoptosis such as nuclear shrinkage, chromatin condensation and degradation, DNA laddering, and poly (ADP-ribose) polymerase (PARP) proteolysis, were visible in both MMR(+) and MMR(-) cells. Remarkably, cell viability was lower and the apoptotic process was triggered more efficiently in the MMR-competent cells.

Antimetabolites, Antineoplastic↗
PubMed2001

Etoposide induces the dispersal of DNA ligase I from replication factories.

In eukaryotic cells DNA replication occurs in specific nuclear compartments, called replication factories, that undergo complex rearrangements during S-phase. The molecular mechanisms underlying the dynamics of replication factories are still poorly defined. Here we show that etoposide, an anticancer drug that induces double-strand breaks, triggers the redistribution of DNA ligase I and proliferating cell nuclear antigen from replicative patterns and the ensuing dephosphorylation of DNA ligase I. Moreover, etoposide triggers the formation of RPA foci, distinct from replication factories. The effect of etoposide on DNA ligase I localization is prevented by aphidicolin, an inhibitor of DNA replication, and by staurosporine, a protein kinase inhibitor and checkpoints' abrogator. We suggest that dispersal of DNA ligase I is triggered by an intra-S-phase checkpoint activated when replicative forks meet topoisomerase II-DNA--cleavable complexes. However, etoposide treatment of ataxia telangiectasia cells demonstrated that ataxia-telangiectasia-mutated activity is not required for the disassembly of replication factories and the formation of replication protein A foci.

Antineoplastic Agents, Phytogenic↗
PubMed2001

UV-induced DNA incision and proliferating cell nuclear antigen recruitment to repair sites occur independently of p53-replication protein A interaction in p53 wild type and mutant ovarian carcinoma cells.

The tumour suppressor gene TP53 plays an important role in the regulation of DNA repair, and particularly of nucleotide excision repair. The influence of p53 status on the efficiency of the principal steps of this repair pathway was investigated after UV-C irradiation in the human ovarian carcinoma cell line IGROV-1 (expressing wild-type p53) and in the derived clone IGROV-1/Pt1 (with p53 mutations at codons 270 and 282). Clonogenic survival after UV-C irradiation showed that IGROV-1/Pt1 cells were approximately 2-fold more resistant to DNA damage than parental cells. Modulation of p53 protein levels, cell cycle arrest and apoptosis were induced in UV-irradiated IGROV-1 cells, but not in the p53-mutant cell line. Exposure to UV or cisplatin induced down-regulation of p53-replication protein A (RPA) interaction in parental, but not in IGROV-1/Pt1 cells. However, persistent binding of p53 to RPA did not affect the early steps of DNA repair. In fact, both UV-induced DNA incision and the recruitment of proliferating cell nuclear antigen (PCNA) to DNA repair sites occurred to a comparable extent in p53-wild type and -mutant cell lines, although PCNA remained associated with chromatin for a longer period of time in IGROV-1/Pt1 cells. Global genome repair, as detected by immunoblot analysis of cyclobutane pyrimidine dimers, was not significantly different in the two cell lines at 3 h after UV irradiation. In contrast, lesion removal at 24 h was markedly reduced in IGROV-1/Pt1 cells, being approximately 25% of the initial amount of damage, as compared with approximately 50% repair in parental cells. These results indicate that the presence of mutant p53 protein and its persistent interaction with RPA do not affect the early steps of nucleotide excision repair in IGROV-1/Pt1 cells. Thus, repair defects in p53-mutant ovarian carcinoma cells may be attributed to late events, possibly related to a reduced removal/recycling of PCNA at repair sites.

Blotting, Western↗
PubMed2000

c-Myc expression in human anagen hair follicles.

The hair follicle represents a very attractive organ system for studying the precise balance between cell proliferation, growth, differentiation, and death of cells, because it periodically and regularly regenerates, retaining its morphogenetic signals throughout its life. One of the most intriguing oncogenes which is able to induce both cell growth and apoptosis, depending upon the environmental conditions, is c-myc. The aim of the present study was to investigate its presence and localization in human hair follicles by immunohistochemistry and immunofluorescence. Our observations demonstrated the consistent presence of two clusters of c-Myc-expressing cells in anagen follicles, located in two annular regions of the inner root sheath, at the border between cells characterized by putative trichohyalin granules and cells which are keratinized. The lower group belongs to Henle's layer, while the upper group belongs to Huxley's layer. c-Myc oncoprotein seems to favour apoptosis/differentiation and may be a marker for terminal differentiation of trichocytes, at least in the inner root sheath. Our findings agree with the interpretation that the complex morphology of the hair follicle reflects its complex function; the extrusion of a highly organized multicellular structure, the hair shaft, driven by another highly organized multicellular structure, the inner root sheath.

Apoptosis↗
PubMed2000

The antiproliferative effect of beta-carotene requires p21waf1/cip1 in normal human fibroblasts.

In normal human fibroblasts, beta-carotene induces a cell-cycle delay in the G1 phase independent of its provitamin A activity via a mechanism not yet elucidated. In this study we provide biochemical evidence showing that delayed progression through the G1 phase occurs concomitantly with: an increase in both nuclear-bound and total p21waf1/cip1 protein levels; an increase in the amount of p21waf1/cip1 associated with cdk4; the inhibition of cyclin D1-associated cdk4 kinase activity; and a reduction in the levels of hyperphosphorylated forms of retinoblastoma protein, and particularly, in phosphorylated Ser780. The role of p21waf1/cip1 in the antiproliferative effect of the carotenoid was further supported by genetic evidence that neither changes in cell-cycle progression nor in the phosphorylation status of retinoblastoma protein were observed in p21waf1/cip1-deficient human fibroblasts treated with beta-carotene. These results clearly demonstrate that p21waf1/cip1 is involved directly in the molecular pathway by which beta-carotene inhibits cell-cycle progression.

Cell Cycle↗
PubMed1999

Apoptosis-prone phenotype of human colon carcinoma cells with a high level amplification of the c-myc gene.

Although apoptosis can be induced by the enforced expression of exogenously introduced c-myc genes, it is not clear whether overexpression resulting from the amplification of the resident c-myc gene in tumor cells is sufficient to induce apoptosis. We have investigated the relationship between c-myc gene amplification and the propensity of tumor cells to undergo apoptosis, using the SW613-12A1 and SW613-B3 cell lines, which are representatives, respectively, of tumorigenic and non-tumorigenic clones isolated from the SW613-S human colon carcinoma cell line. Tumorigenic clones are characterized by a high level of amplification and expression of the c-myc gene, whereas cells of non-tumorigenic clones have a small number of copies and a lower level of expression of this gene. Analysis of c-myc mRNA level in cells cultured under low serum conditions indicated that the expression of the gene is tightly regulated by serum growth factors in non-tumorigenic B3 cells, whereas it is poorly regulated in tumorigenic 12A1 cells, the level of mRNAs remaining relatively high in serum-starved 12A1 cells. Under these conditions, 12A1 cells showed clear evidence of apoptosis, whereas B3 cells were completely refractory to the induction of apoptosis. Moreover, the study of cell lines derived from non-tumorigenic apoptosis-resistant clones following the introduction by transfection of exogenous c-myc gene copies showed that they have acquired an apoptosisprone phenotype. Altogether, our results strongly suggest that deregulated c-myc expression due to high-level amplification confers an apoptosis-prone phenotype to tumor cells. The possible consequences of these observations for cancer therapy are discussed.

Clone Cells↗
PubMed1999

Differential involvement of DNases in HeLa cell apoptosis induced by etoposide and long term-culture.

We have applied to human HeLa cells two different stimuli of apoptosis: the antitumoral drug etoposide, and a more 'physiological' death condition, obtained by growing cells in the same medium for long time periods, for up to 10 days. Analysis of different parameters demonstrated that in both experimental systems the same apoptotic features are visible. However, the DNA degradation pattern appeared to be different, suggesting the involvement of different DNases. In this view, we have analyzed the activity and expression of Ca2+-Mg2+-dependent and acid DNases. We have observed that DNase I is not modulated during apoptosis. In contrast, the acid L-DNase II (derived from Leukocyte Elastase Inhibitor by post-translational modification), recently identified in our laboratory, is mainly active in the apoptotic pathway induced by long term-culture. Furthermore, we have provided evidence that while caspase 3 is activated by both inducers, caspase 1 is essential only for the etoposide-induced apoptosis.

Antineoplastic Agents, Phytogenic↗
PubMed1998

The cyclin-dependent kinase inhibitors olomoucine and roscovitine arrest human fibroblasts in G1 phase by specific inhibition of CDK2 kinase activity.

The specificity and the temporal location of cell cycle arrest induced by the cyclin-dependent kinase (CDK) inhibitors olomoucine and roscovitine were investigated in normal human fibroblasts. Effects on the cell cycle were compared with those induced by the kinase inhibitor staurosporine, which arrests normal cells in early G1 phase by acting upstream of CDK2. Consistent with their in vitro activity, olomoucine and roscovitine, but not the related compound iso-olomoucine, induced a dose-dependent arrest in G1 phase. Following removal of CDK inhibitors, cells resumed cycle progression entering S phase with a kinetics faster than staurosporine-treated samples. Cellular levels of PCNA, cyclin D1, and cyclin E were not affected by the CDK inhibitors. In contrast, staurosporine significantly reduced the levels of these proteins, as determined by immunocytometry and Western blot analysis. Cyclin A was detectable only in some cells remaining in the G2 + M compartment of samples treated with CDK inhibitors, but not in samples treated with staurosporine. Significant reduction in the hyperphosphorylated forms of retinoblastoma protein was found in samples treated with CDK inhibitors, while only hypophosphorylated forms were observed in staurosporine-treated samples. Concomitantly, CDK2, but not CDK4, activity immunoprecipitated from cells treated with olomoucine or roscovitine was markedly inhibited. These results suggest that in normal cells, CDK2 kinase activity is the specific target of olomoucine and roscovitine.

CDC2-CDC28 Kinases↗
PubMed1998

Involvement of the proliferating cell nuclear antigen (PCNA) in DNA repair induced by alkylating agents and oxidative damage in human fibroblasts.

The involvement of the proliferating cell nuclear antigen (PCNA) in the process of DNA repair induced by alkylating agents or by oxidative damage was investigated in human quiescent fibroblasts by immunofluorescence and flow cytometry. Transition from soluble to the DNA-bound form of PCNA, was taken as the parameter to determine its involvement in repair DNA synthesis. Treatment with the alkylating agents methylmethane sulfonate and N-methyl-N'-nitro-N-nitrosoguanidine resulted in the rapid and dose-dependent increase in the nuclear binding of PCNA. Similar results were obtained with compounds such as hydrogen peroxide or tert-butyl hydroperoxide, which are known to induce oxidative DNA damage. Tert-butyl hydroperoxide may also generate malondialdehyde through a reaction of lipid peroxidation. This mutagenic and carcinogenic product has been previously shown to form adducts with DNA. Therefore, the possibility that tert-butyl hydroperoxide could induce DNA damage through this pathway was investigated by incubating cells directly in the presence of malondialdehyde. Such treatment resulted in an increase in immunofluorescence associated with nuclear-bound PCNA. The ability of oxidative and alkylating agents to induce the nuclear binding of PCNA was also assessed in proliferating cells. In these conditions, treatment with hydrogen peroxide or methylmethane sulfonate, resulted in an increase in nuclear-bound PCNA in the G1 and in the G2 + M compartments, but not in S phase. At longer times after treatment, PCNA immunostaining was reduced to basal levels, while an increase in nuclear binding of p21(waf1/cip1) protein was found in concomitance with cell-cycle arrest. These results indicate that agents inducing DNA base alterations in vivo, promote the nuclear binding of PCNA. These lines of evidence support the role of a PCNA-dependent reaction in the base excision repair system.

Alkylating Agents↗
PubMed1997

Nuclear association of cyclin D1 in human fibroblasts: tight binding to nuclear structures and modulation by protein kinase inhibitors.

The association of cyclin D1 with nuclear structures was investigated in normal human fibroblasts by using hypotonic detergent extraction procedures, immunofluorescence quantitation with flow cytometry, and Western blot analysis. About 20% of the total cellular levels of cyclin D1 was found to be tightly bound to nuclear structures, being the complex formation resistant to DNase I treatment and to high salt extraction. Maximal levels of the insoluble form of the protein were found in the middle to late G1 phase of the cell cycle. Cell fractionation and immunoprecipitation techniques after in vivo 32P-labeling showed that both soluble and nuclear-bound forms of cyclin D1 were phosphorylated. Both fractions were reactive to an anti-phosphotyrosine antibody, while only the latter was detectable with an anti-phosphoserine antibody. Treatment with the protein kinase inhibitor staurosporine, which induces a cell cycle arrest in early G1 phase, strongly reduced cyclin D1 phosphorylation. Concomitantly, the ratio of nuclear-bound/total cyclin D1 levels was reduced by about 60%, compared with the control value. The protein kinase A specific inhibitor isoquinoline-sulfonamide (H-89) induced a similar reduction in the ratio, with no significant modification in the total amount of protein. In contrast, both calphostin C and bisindolylmaleimide, specific inhibitors of protein kinase C, consistently increased by 30-50% the ratio of nuclear-bound/total amount of the cyclin protein. These results suggest that, during the G1 phase, formation of an insoluble complex of cyclin D1 occurs at nuclear matrix structures and that this association is mediated by a protein kinase A-dependent pathway.

Cell Division↗
PubMed1997

Multiple roles of the proliferating cell nuclear antigen: DNA replication, repair and cell cycle control.

The proliferating cell nuclear antigen (PCNA), the auxiliary protein of DNA polymerase delta and epsilon, is involved in DNA replication and repair. This protein forms a homotrimeric structure which, encircling DNA, loads the polymerase on the DNA template. A role for PCNA in the cell cycle control is recognised on the basis of the interaction with cyclins, cyclin-dependent kinases (cdks) and the cdk-inhibitor p21 waf1/cip1/sdi1 protein. Association with the growth-arrest and DNA-damage inducible proteins gadd45 and MyD118, further demonstrates the role of PCNA as a component of the cell cycle control apparatus.

Animals↗
PubMed1997

A variant of the Nijmegen breakage syndrome with unusual cytogenetic features and intermediate cellular radiosensitivity.

We report the first Italian case of Nijmegen breakage syndrome (NBS). The proband is an immunodeficient, microcephalic, 11 year old boy with a "bird-like" face. He developed a T cell rich B cell lymphoma. Spontaneous chromosomal instability was detected in T and B lymphocytes and fibroblasts; chromosomes 7 and 14 were only sporadically involved in the rearrangements and no clonal abnormality was present. The patient appeared to be sensitive both to ionising radiation and to bleomycin, although his sensitivity did not reach the level of AT reference cells. After bleomycin treatment, inhibition of DNA synthesis was low when compared with normal cells, but higher than observed in an AT reference strain. Moreover, cell cycle analysis, after drug exposure, showed a progressive reduction in the percentage of S phase cells, but the G1 arrest, found in normal cells, was not observed. On clinical evaluation our patient shares features with NBS subjects, but cytogenetic and cell biological data do not completely overlap with those reported in Nijmegen breakage syndrome. The ethnic origin of our patient might account for these differences, as expression of different allelic forms at the NBS locus.

Antigens, Surface↗
PubMed1996

p21waf1/cip1 protein associates with the detergent-insoluble form of PCNA concomitantly with disassembly of PCNA at nucleotide excision repair sites.

Evidence is presented that after exposure of normal human fibroblasts to UV-C light, nuclear binding of the proliferating cell nuclear antigen (PCNA) required for nucleotide excision repair, was rapidly triggered in the G1 and G2 phases of the cell cycle. Association to repair sites of the detergent-insoluble form of PCNA reached a peak 15-30 min after irradiation, and then decreased to basal levels within 24-48 h. In contrast, the nuclear association of p21 protein showed a slower kinetics, reaching maximal levels between 24 and 48 h but, similarly to PCNA, occurring only in G1 and G2 phases. Although the two proteins are known to be associated as detergent-soluble proteins, it is unknown whether they associate also in the detergent-insoluble form. To address this question, the chromatin-bound form of PCNA was released by using DNAse I. DNA digestion resulted in the almost complete release of PCNA from its binding sites, while only about 60% of nuclear-bound p21 could be solubilized. Immunoprecipitation of PCNA and p21 released by enzymatic digestion showed that p21 was associated with PCNA bound to late DNA repair sites. These results indicate that during nucleotide excision repair, nuclear binding of PCNA precedes that of p21 protein, and suggest that temporal association of p21 with the detergent-insoluble fraction is coincident with the disassembly of PCNA from DNA repair sites.

Cell Cycle↗