http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. 2013-06-20T10:58:02Z http://hdl.handle.net/2067/1707 Title: Enhancement of genetic instability in human B cells by Epstein-Barr virus latent infection Authors: Giselico, Luigi; Carloni, Manuela; Palitti, Fabrizio; Mosesso, Pasquale; Alfonsi, Alberto Maria; Gualandi, Giampiero Abstract: The level of genetic instability, as assessed by micronucleus (MN) formation, was higher in Epstein-Barr virus (EBV)-converted B-cell lines with one copy of the EBV genome integrated in each cell than in the parental, EBV-negative, B lymphoma cells. MN induced by EBV latency, as analysed by in situ hybridization, contained mainly centromeric regions, indicating that the presence of EBV affects the segregation of entire chromosomes. The instability was inhibited by treatment with antioxidants. Flow cytometric analysis indicated that there was a higher basal level of peroxides in EBV(+) cells. Direct oxidative stress caused by hydrogen peroxide (which is known to be both apoptogenic and mutagenic) enhanced the number of MN only in an EBV-converted clone. These cells were also resistant to apoptosis, as expected, suggesting that in the parental EBV cells apoptosis may efficiently eliminate cells with genetic damage. These results show for the first time a direct involvement of EBV in the induction of genetic instability, suggesting that it could contribute to tumour progression Description: L'articolo è disponibile sul sito dell'editore: http://mutage.oxfordjournals.org/ 2000-12-31T23:00:00Z http://hdl.handle.net/2067/1674 Title: Effect of storage conditions of blood on radiation-induced chromosomal aberrations and apoptosis in human lymphocytes Authors: Belloni, Paola; Pepe, Gaetano; Palitti, Fabrizio Abstract: To evaluate the effect of storage conditions of blood on the direct relationship between radiation-induced chromosome aberrations and apoptosis in human peripheral blood lymphocytes,whole bloodwas irradi- atedwith 3Gy X-rays. Directly after irradiation, a sample of bloodwas analyzed for chromosome damage and proliferation index, after phytohaemagglutinin stimulation and incubation at 37 ◦ C for 56 h. Blood samples were stored for 48 h at 4 and 20 ◦ C with or without phytohaemagglutinin and analyzed for cell viability and apoptosis at 0, 24 and 48 h storage time. After 48 h of storage, unstimulated cultures were stimulated to proliferate. These samples and cultures stimulated immediately before storage were incubated at 37 ◦ C for 56 h and analyzed for chromosome damage and proliferation index. Metaphases were examined for the presence of dicentrics, excess acentrics, and rings. Storage at 20 ◦ Cwithout phyto- haemagglutinin for 48 h increases significantly the yield of apoptosis and decreases significantly the yield of dicentrics. During 48 h of storage time the presence of phytohaemagglutinin and the temperature of 4 ◦ C protected the irradiated lymphocytes from apoptosis allowing accurate estimation of the real yield of radiation-induced chromosome damage. Therefore these blood-storage conditions enable analysis in metaphase and may offer some advantages for biodosimetry of absorbed radiation dose. Description: L'articolo è disponibile sul sito dell'editore: http://www.sciencedirect.com 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1684 Title: Asynchronously Replicating Eu/Heterochromatic Regions Shape Chromosome Damage. Authors: Di Tommaso, Maria Valeria; Martínez-López, Wilner; Palitti, Fabrizio Abstract: In order to shed more light on the influence of DNA replication on the formation and distribution of chromosome aberrations, breakpoints (BP) produced by UV-C and AluI were assigned either to the early replicating short euchromatic arm (Xpe) or to the late replicating long heterochromatic arm (Xqh) of the Chinese hamster (CHO9) X chromosome. Early (ES) or late (LS) S-phase cells were assessed by pulse incorporating the base analogue 5-bromo-2′-deoxyuridine (BrdU) immediately after UV-C irradiation (30 J/m2) or AluI (20 U) poration followed by BrdU immunodetection with FITC-tagged antibodies in metaphase spreads. Short (30 s) UV-C exposures (1 J/m2/s) induced BP preferentially in Xqh in LS cells and a random distribution of BP along Xpe and Xqh in ES cells. However, BP induced by long (5 min) UV-C exposures (0.1 J/m2/s) clustered according to arm replication time (Xpe during ES and Xqh along LS). Giemsa-stained metaphases showed elevated frequencies of UV-C induced chromatid-type aberrations and gaps, especially in cells exposed to longer UV-C irradiation. BP induced by AluI clustered in Xpe in ES but distributed randomly during LS. In contrast to UV-C, AluI did not produce an increase in the yield of gaps, neither in ES nor in LS cells. Putative mechanisms underlying the observed distributions of chromosome damage in replicating CHO9 cells exposed to UV-C and AluI are discussed. Description: L'articolo è disponibile sul sito dell'editore: http://www.karger.com. 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1692 Title: Influence of DMSO on Carbon K ultrasoft X-rays induced chromosome aberrations in V79 Chinese hamster cells Authors: Natarajan, Adayapalam T.; Palitti, Fabrizio; Hill, Mark A.; Stevens, David L.; Ahnström, Gunnar Abstract: Ultrasoft X-rays have been shown to be very efficient in inducing chromosomal aberrations in mammalian cells. The present study was aimed to evaluate the modifying effects of DMSO (a potent scavenger of free radicals) on the frequencies of chromosome aberrations induced by soft X-rays. Confluent held G1 Chinese hamster cells (V79) were irradiated with Carbon K ultrasoft X-rays in the presence and absence of 1 M DMSO and frequencies of chromosome aberrations in the first division cells were determined. DMSO reduced the frequencies of exchange types of aberrations (dicentrics and centric rings) by a factor of 2.1–3.5. The results indicate that free radicals induced by ultrasoft X-rays contribute to a great extent to the induction of chromosome aberrations. The possible implications of these results in interpreting the mechanisms involved in the high efficiency of ultrasoft X-rays in the induction of chromosome aberrations are discussed. Description: L'aricolo è disponibile sul sito dell'editore: http://www.sciencedirect.com 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1699 Title: Distribution of UVC-induced chromosome aberrations along the X chromosome of TCR deficient and proficient Chinese hamster cell lines. Authors: Martínez-López, Wilner; Marotta, Edvige; Di Tommaso, Maria Valeria; Méndez-Acuña, Leticia; Palitti, Fabrizio Abstract: Cells with a transcription coupled repair (TCR) deficiency are characterized by a higher sensitivity to UVC irradiation and by an increase in apoptosis and chromosomal aberration frequencies. It has been claimed that the higher frequency of chromosomal aberrations results from the transcription blockage caused by UVC-lesions located in the transcribed strands of the genome. The distribution of chromosome breakpoints in euchromatic and heterochromatic regions of the X chromosome from TCR deficient and proficient Chinese hamster cell lines was studied. Most UVC-induced breakpoints occurred in euchromatic regions of the X chromosome in both cell lines. No increase of UVC-induced breakpoints in the euchromatic region of the UV61 X chromosome was observed, indicating that TCR failure alone cannot be responsible for the increased frequency of chromosomal aberrations. Differential chromatin remodeling in the TCR defective cell line is proposed as a possible mechanism involved in the distribution of UVC-induced breakpoints along the Chinese hamster X chromosome. A similar explanation for the increase of UVC-induced chromosomal aberrations in TCR defective cells is given Description: L'articolo è disponibile sul sito dell'editore: http://www.sciencedirect.com 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1702 Title: Histone post-translational modifications in DNA damage response Authors: Méndez-Acuña, Leticia; Di Tommaso, Maria Valeria; Palitti, Fabrizio; Martínez-López, Wilner Abstract: The fact that eukaryotic DNA is packed into chromatin constitutes a physical barrier to enzymes and regulatory factors to reach the DNA molecule for replication, transcription, recombination and repair. Although most studies in this field have concentrated on how chromatin regulates transcription, there is a recent emphasis on studying the role of chromatin in the response to DNA damage. Two main chromatin-remodeling mechanisms have been identified, namely, ATP-dependent chromatin-remodeling complexes and histone post-translational modifications (PTMs). PTMs constitute reversible covalent modifications in aminoacidic residues, such as serine and threonine phosphorylation, lysine acetylation, lysine and arginine methylation and lysine ubiquitylation, among others. Moreover, nucleosome composition can be modified by the incorporation of histone variants, which are assembled into nucleosomes independently of DNA replication. The phosphorylation of the histone variant H2AX (gammaH2AX) is one of the best examples of histone PTMs in response to DNA damage induction, but many others have recently been revealed. In this review, we focus on and summarize the best-known histone PTMs observed in excision repair (base excision and nucleotide excision) and double-strand break (non-homologous end-joining and homologous recombination) repair pathways. In brief, the interplay between chromatin remodelers and DNA repair factors is discussed in relation to DNA damage response mechanisms. Copyright 2010 S. Karger AG, Basel. PMID: 20407219 Description: L'articolo è diponibile sul sito dell'editore: http://www.karger.com 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1695 Title: Relationship between DNA Repair and Formation of Sister Chromatid Exchanges and Chromatid Aberrations under the Influence of Poly(ADP-Ribose) Polymerase Inhibition by 3-Aminobenzamide Authors: Filippi, Silvia; Palitti, Fabrizio; Martínez-López, Wilner; Natarajan, Adayapalam T. Abstract: The mechanisms of formation of sister chromatid exchanges (SCEs) and chromosome aberrations following inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide were studied in Chinese hamster ovary cell lines deficient in different repair pathways. The results confirm earlier findings that (a) the 'spontaneous' SCEs are formed due to the incorporated BrdU in the DNA, (b) 'spontaneous' and induced SCEs originate from different mechanisms, and (c) SCEs and chromatid exchanges are formed by different pathways. Description: L'articolo é disponibile sul sito dell'editore: http://www.karger.com 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1712 Title: Catalytic inhibition of topoisomerase II in Werner’s syndrome cell lines enhances chromosomal damage induced by X-rays in the G2 phase of cell cycle Authors: Franchitto, Annapaola; Pichierri, Pietro; Mosesso, Pasquale; Palitti, Fabrizio Abstract: PURPOSE: To investigate whether catalytic topoisomerase II activity by ICRF187, a compound that interferes with the catalytic cycle of topoisomerase II without causing DNA damage, could result in a modulation of X-ray-induced chromosomal damage in Werner's syndrome (WS) cell lines. MATERIALS AND METHODS: Two WS (KO375, DJG) and one normal lymphoblastoid cell line (SNW646) were exposed to X-rays, post-treated with ICRF187 and harvested after various recovery times. Cell progression to mitosis was monitored by 5-bromo-2'-deoxyuridine (BrdUrd) and fluorescent immmunodetection to analyse chromosomal damage in homogeneous treated cell populations in the G1, S or G2 phase of the cell cycle. RESULTS: In WS cell lines, catalytic inhibition of topoisomerase II activity by ICRF187 resulted in potentiation of X-ray- induced chromosomal damage in the G2 phase of the cell cycle. This potentiation was not observed in the G1 or S phases of the cell cycle, neither in WS nor normal cells. CONCLUSION: These results point out the possibility that Werner's syndrome protein (WRNp) might play a role in a G2 recombinational pathway of double-strand break repair, cooperating with topoisomerase II and thus contributing to maintain genomic integrity. Description: L'articolo è disponibile sul sito dell'editore: http://www.taylorandfrancisgroup.com 1999-12-31T23:00:00Z http://hdl.handle.net/2067/1720 Title: Chromosome radiosensitivity in human G2 lymphocytes and cell cycle progression Authors: Palitti, Fabrizio; Pichierri, Pietro; Franchitto, Annapaola; Proietti De Santis, Luca; Mosesso, Pasquale Abstract: PURPOSE: To investigate the possibility that the differential G2-phase radiosensitivity of human peripheral blood lymphocytes, found in normal individuals using the 'G2-phase chromosome radiosensitivity assay', could be attributed to heterogeneity in cellular progression to mitosis rather than differences in radiosensitivity. MATERIALS AND METHODS: Human peripheral blood lymphocytes, from four different donors, were exposed to 50 cGy X-rays and sampled at different times. The progression of cells into mitosis was monitored by 5-bromo 2'-deoxyuridine (BrdUrd) incorporation. RESULTS: The heterogeneous G2-phase chromosome radiosensitivity among different donors was abolished when homogeneous G2-phase cell populations were scored; they contained similar frequencies of cells in early or late G2-phase. CONCLUSIONS: The heterogeneous G2-phase chromosome radiosensitivity, usually found in different normal donors, is caused by the analysis of different cell populations rather than reflecting intrinsic differences in radiosensitivity Description: L'articolo è disponibile sul sito dell'editore: http://informahealthcare.com 1998-12-31T23:00:00Z http://hdl.handle.net/2067/1343 Title: The novel human gene aprataxin is directly involved in DNA single-strand-break repair. Authors: Mosesso, Pasquale; Piane, Maria; Palitti, Fabrizio; Pepe, Gaetano; Penna, Sabrina; Chessa, Luciana Abstract: The cells of an ataxia-oculomotor apraxia type 1 (AOA1) patient, homozygous for a new aprataxin mutation (T739C), were treated with camptothecin, an inhibitor of DNA topoisomerase I which induces DNA single-strand breaks. DNA damage was evaluated by cytogenetic analysis of chromosomal aberrations. The results obtained showed marked and dose-related increases in induced chromosomal aberrations in the patient and her heterozygous mother compared to the intrafamilial wild-type control. The alkaline comet assay confirmed this pattern. Moreover, the AOA1 cells did not show hypersensitivity to ionizing radiation, i.e. X-rays. These findings clearly indicate the direct involvement of aprataxin in the DNA single-strand-break repair machinery. Description: L'articolo è disponibile sul sito dell'editore: http://www.springerlink.com/ 2004-12-31T23:00:00Z http://hdl.handle.net/2067/1721 Title: Distribution of camptothecin-induced breakpoints in Chinese hamster cells treated in late S and G2 phases of the cell cycle. Authors: Bassi, Loredana; Palitti, Fabrizio; Mosesso, Pasquale; Natarajan, Adayapalam T. Abstract: The distribution of camptothecin (CPT)-induced break points in late S or G2 phase of the cell cycle observed in Chinese hamster chromosomes was analysed in 400 metaphases. Contrary to expectation, they were not localized in the heterochromatic regions, suggesting that these chromatid-type aberrations arise by a mechanism which does not involve collision of the CPT-trapped 'cleavable complex' with the replication fork. Since many break points mapped more frequently to light bands (DAPI negative) than dark bands (DAPI positive) with a frequency of 73 and 15% respectively, it could be argued that the presence of the CPT-trapped 'cleavable complex' probably interferes with chromatin condensation. In fact, the euchromatic regions, which are expected to be more actively condensed in G2 phase, were more involved in chromosomal damage. These results do not completely confirm the idea that some residual DNA synthesis occurring in G2 is responsible for the G2 clastogenic effects of CPT as the heterochromatic regions should, in this case, be more involved. Description: L'articolo è disponibile sul sito dell'editore: http://www.oxfordjournals.org 1997-12-31T23:00:00Z http://hdl.handle.net/2067/1713 Title: Evidence that camptothecin-induced aberrations in the G2 phase of cell cycle of Chinese hamster ovary (CHO) cell lines is associated with transcription Authors: Mosesso, Pasquale; Pichierri, Pietro; Franchitto, Annapaola; Palitti, Fabrizio Abstract: It is widely accepted that camptothecin (CPT) is an S-dependent genotoxin. In this study, we aimed to elucidate the 'puzzling' induction of chromosomal damage by CPT in the G(2) phase of CHO cells, where no DNA synthesis is expected, focusing the attention on the possible role of the ongoing RNA synthesis, supposed to cause the conversion of CPT-single stranded cleavage complexes spaced closely on opposite DNA strands into DNA double strand breaks (DSB's) by the action of traversing RNA polymerase.CHO AA8 and its parental mutant EM9 cell lines were pre-treated with alpha-amanitin, which prevents transcription to pre-m-RNA and challenged cells with CPT for the last hour in culture to evaluate whether G(2)-CPT-induced aberrations would have been reduced or abolished in the absence of RNA synthesis compared with G(2)-CPT treatment alone. The results obtained indicated a marked and significant reduction of aberration yields, to almost the control values (alpha-amanitin alone) when inhibition of RNA synthesis was substantial (3h total alpha-amanitin). Partial inhibition of RNA synthesis (2h total alpha-amanitin) slightly reduced the CPT-induced aberrations yield only at the high dose-level employed of CPT (20mM). This finding strongly supports the hypothesis that CPT-single stranded cleavages complexes spaced closely on opposite DNA strands are converted into DNA double strand breaks by the action of traversing RNA polymerase. Description: L'articolo è disponibile sul sito dell'editore: http://www.sciencedirect.com 1999-12-31T23:00:00Z http://hdl.handle.net/2067/1723 Title: Induction of chromosomal aberrations (unstable and stable) by inhibitors of topoisomerase II, m-AMSA and VP16, using conventional Giemsa staining and chromosome painting techniques. Authors: Mosesso, Pasquale; Darroudi, Firouz; Van Der Berg, Marco A.; Vermeulen, Sandra S.; Palitti, Fabrizio; Natarajan, Adayapalam T. Abstract: Frequencies of symmetrical and asymmetrical exchange aberrations induced by two inhibitors of topoisomerase II, namely, 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA) and etoposide (VP16), were estimated in human peripheral blood lymphocytes. The aberrations were scored using conventional Giemsa staining and fluorescence in situ hybridization (FISH) techniques, using chromosome-specific DNA libraries. Stable aberrations (translocations) were detected using two cocktails of DNA libraries specific for three chromosomes, namely 1, 3 and X and 2, 4 and 8, representing approximately 40% of the whole human genome. The frequencies of dicentrics and translocations increased in a dose-dependent manner, however, m-AMSA was found to be a more potent inducer of chromosomal aberrations in comparison with VP16 (at concentrations at which comparable frequencies of aberrations were induced) by 20- to 30-fold. When corrected for DNA content of chromosomes in each cocktail, a higher frequency of translocations with the cocktail consisting of chromosomes 2, 4 and 8 in comparison with 1, 3 and X was evident. The genomic translocation frequency calculated from chromosome painting analysis for m-AMSA exceeded that estimated for dicentrics by approximately 2-fold. However, for VP16 almost equal frequencies of both types of chromosome exchange were found Description: L'articolo è disponibile sul sito dell'editore: http://www.oxfordjournals.org 1997-12-31T23:00:00Z http://hdl.handle.net/2067/1349 Title: Potassium Bromate but not X-ray cause unexpectedly elevated levels of DNA breakage similar to those induced by ultraviolet light in Cockayne syndrome (CS-B) fibroblast Authors: Mosesso, Pasquale; Penna, Sabrina; Pepe, Gaetano; Lorenti Garcia, Claudia; Palitti, Fabrizio Abstract: It has been previously reported that the elevated accumulation of repair incision intermediates in cells from patients with combined characteristics of xeroderma pigmentosum complementation group D (XP-D) and Cockayne syndrome (CS) XP-D/CS fibroblasts following UV irradiation is caused by an "uncontrolled" incision of undamaged genomic DNA induced by UV-DNA-lesions which apparently are not removed. This could be an explanation for the extreme sensitivity of these cells to UV light. In the present study, we confirm the immediate DNA breakage following UV irradiation also for CS group B (CS-B) fibroblasts by DNA migration in the "comet assay" and extend these findings to other lesions such as 8-oxodeoxyguanosine (8-oxodG), selectively induced by KBrO3 treatment. In contrast, X-ray exposure does not induce differential DNA breakage. This indicates that additional lesions other than the UV-induced photoproducts (cyclobutane pyrimidine dimers, CPD, and 6-pyrimidine-4-pyrimidone products, 6-4 PP), such as 8-oxodG, specifically induced by KBrO3, are likely to trigger "uncontrolled" DNA breakage in the undamaged genomic DNA in the CS-B fibroblasts, thus accounting for some of the clinical features of these patients Description: L'articolo é disponibile sul sito dell'editore: http://www.karger.com 2003-12-31T23:00:00Z http://hdl.handle.net/2067/1348 Title: Relationship between chromatin structure, DNA damage and repair following X-irradiation of human lymphocytes Authors: Mosesso, Pasquale; Palitti, Fabrizio; Pepe, Gaetano; Piñero, Joaquin; Bellacima, Raffaela; Ahnström, Gunnar; Natarajan, Adayapalam T. Abstract: Earlier studies using the technique of premature chromosome condensation (PCC) have shown that in human lymphocytes, exchange type of aberrations are formed immediately following low doses (<2 Gy) of X-rays, whereas at higher doses these aberrations increase with the duration of recovery. This reflects the relative roles of slow and fast repair in the formation of exchange aberrations. The underlying basis for slow and fast repairing components of the DNA repair may be related to differential localization of the initial damage in the genome, i.e., between relaxed and condensed chromatin. We have tried to gain some insight into this problem by (a) X-irradiating lymphocytes in the presence of dimethyl sulfoxide (DMSO) a potent scavenger of radiation-induced .OH radicals followed by PCC and (b) probing the damage and repair in two specific chromosomes, 18 and 19, which are relatively poor and rich in transcribing genes by COMET-FISH, a combination of Comet assay and fluorescence in situ hybridization (FISH) techniques. Results obtained show (a) that both fast appearing and slowly formed exchange aberrations seem to take place in relaxed chromatin, since they are affected to a similar extent by DMSO, (b) significant differential DNA breakage of chromosome 18 compared to chromosome 19 in both G0 and G1 phases of the cell cycle as detected by Comet assay, indicating that relaxed chromatin containing high densities of transcriptionally active genes shows less fragmentation due to fast repair (chromosome 19) compared to chromosome 18, and (c) that relaxed chromatin is repaired or mis-repaired faster than more compact chromatin Description: L'articolo é disponibile sul sito dell'editore: http://www.sciencedirect.com 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1719 Title: The involvement of chromatin condensation in campthotecin-induced chromosome breaks in G0 human lymphocytes Authors: Mosesso, Pasquale; Fonti, Enrica; Bassi, Loredana; Lorenti Garcia, Claudia; Palitti, Fabrizio Abstract: In the present study we evaluated campthotecin (CPT)-induced chromosomal damage in human lymphocytes in the G0 phase of the cell cycle as revealed by the premature chromosome condensation technique. The results obtained here indicate that CPT was able to induce chromosome fragments in the G0 phase of the cell cycle of human lymphocytes as detected in prematurely condensed chromosomes. This result appears to be rather surprising, since the DNA lesions produced by CPT (e.g. 'protein concealed' DNA single-strand breaks) should not produce any damage in G0. A possible explanation for this result could come from much evidence to suggest that chromatin condensation processes are significantly involved in the conversion of DNA lesions into chromosome breaks in prematurely condensed chromosomes. The unexpected clastogenic behaviour of CPT can be explained taking into account the chromosome condensation induced by mitosis promoting factors when human lymphocytes are fused in G0, thus converting the 'protein concealed' DNA single-strand breaks induced by CPT into chromosome breaks. The same perspective should be taken into consideration for breaks induced by CPT under normal physiological conditions in the G2 phase of the cell cycle. Description: L'articolo é disponibile sul sito dell'editore: http://www.oxfordjournals.org 1998-12-31T23:00:00Z http://hdl.handle.net/2067/1345 Title: The protective effect of L-Carnitine in peripheral blood human lymphocytes exposed to oxidative agents Authors: Lorenti Garcia, Claudia; Filippi, Silvia; Mosesso, Pasquale; Menotti Calvani, R.; Nicolai, Raffaella; Mosconi, Luigi; Palitti, Fabrizio Abstract: Literature data indicate l-carnitine (LC), a trans-mitochondrial carrier of acetyl and long chain groups, as an agent possessing protective effects against oxidative stress in mammalian cells. However, the major factor involved in the protective mechanism is not known. The protection activity exerted by this agent against reactive oxygen species induced by hydrogen peroxide (H2O2) and t-butylhydroperoxide (t-butyl-OOH) treatment in isolated human peripheral blood lymphocytes (PBLs) has been studied. Human lymphocytes cells were isolated and pre-incubated with 5 mM lc before H2O2 (100 µM) and t-butyl-OOH (400 µM) treatment. The protective effect of lc on treated PBLs was measured by single cell gel electrophoresis and the analysis of chromosomal aberrations. Results show that lc treated cells exhibited a significant decrease in the number of oxidative induced single-strand breaks and chromosomal aberrations. Description: L'articolo é disponibile sul sito dell'editore: http://www.oxfordjournals.org 2005-12-31T23:00:00Z http://hdl.handle.net/2067/1715 Title: Werner's syndrome cell lines are hypersensitive to camptothecin-induced chromosomal damage. Authors: Pichierri, Pietro; Franchitto, Annapaola; Mosesso, Pasquale; Palitti, Fabrizio Abstract: Werner's syndrome (WS) is a recessive human genetic disorder associated with an elevated incidence of many types of cancer. The WS gene product, WRNp, belongs to the RecQ family of DNA helicases and is required for the maintenance of genomic stability in human cells. A possible interaction between helicases and topoisomerases that could co-operate in many aspects of DNA metabolism such as progression of the replication forks, recombination and repair has been recently suggested. In addition, sgs1 gene product in yeast, homologous to WS gene, has been shown to physically interact with topoisomerase types I and II. Earlier data from our laboratory suggested that WRN helicase might play a role in a G2 recombinational pathway of double strand breaks (DSBs) repair, co-operating with topoisomerase II. In this work, the effect of the topoisomerase I inhibitor camptothecin in WS cells has been investigated at the chromosomal level. The data from the present work suggest that the inhibition of topoisomerase I activity by camptothecin results in a higher induction of chromosomal damage in WS cell lines in the G2-phase and in the S-phase of the cell cycle compared to normal cells, perhaps associated with the defects in DNA replication synthesis. Description: L'articolo é disponibile sul sito dell'editore: http://www.sciencedirect.com 1999-12-31T23:00:00Z http://hdl.handle.net/2067/1710 Title: Werner‘s syndrome protein is required for correct recovery after replication arrest and DNA damage induced in S-phase of cell cycle Authors: Pichierri, Pietro; Franchitto, Annapaola; Mosesso, Pasquale; Palitti, Fabrizio Abstract: Werner's syndrome (WS) is a rare autosomal recessive disorder that arises as a consequence of mutations in a gene coding for a protein that is a member of RecQ family of DNA helicases, WRN. The cellular function of WRN is still unclear, but on the basis of the cellular phenotypes of WS and of RecQ yeast mutants, its possible role in controlling recombination and/or in maintenance of genomic integrity during S-phase has been envisaged. With the use of two drugs, camptothecin and hydroxyurea, which produce replication-associated DNA damage and/or inhibit replication fork progression, we find that WS cells have a slower rate of repair associated with DNA damage induced in the S-phase and a reduced induction of RAD51 foci. As a consequence, WS cells undergo apoptotic cell death more than normal cells, even if they arrest and resume DNA synthesis at an apparently normal rate. Furthermore, we report that WS cells show a higher background level of DNA strand breaks and an elevated spontaneous induction of RAD51 foci. Our findings support the hypothesis that WRN could be involved in the correct resolution of recombinational intermediates that arise from replication arrest due to either DNA damage or replication fork collapse. Description: L'articolo é disponibile sul sito dell'editore: http://www.molbiolcell.org 2000-12-31T23:00:00Z http://hdl.handle.net/2067/1716 Title: Werner’s sindrome lymphoblastoid cells are hypersensitive to topoisomerase II inhibitors in the G2 phase of the cell cycle Authors: Pichierri, Pietro; Franchitto, Annapaola; Mosesso, Pasquale; Proietti De Santis, Luca; Balajee, Adayabalam S.; Palitti, Fabrizio Abstract: Werner's syndrome (WS) is a rare autosomal recessive human disorder and the patients exhibit many symptoms of accelerated ageing in their early adulthood. The gene (WRN) responsible for WS has been biochemically characterised as a 3'-5' helicase and is homologous to a number of RecQ superfamily of helicases. The yeast SGS1 helicase is considered as a human WRN homologue and SGS1 physically interacts with topoisomerases II and III. In view of this, it has been hypothesised that the WRN gene may also interact with topoisomerases II and III. The purpose of this study is to determine whether the loss of function of WRN protein alters the sensitivity of WS cells to agents that block the action of topoisomerase II. This study deals with the comparison of the chromosomal damage induced by the two anti-topoisomerase II drugs, VP-16 and amsacrine, in both G1 and G2 phases of the cell cycle, in lymphoblastoid cells from WS patients and from a healthy donor. Our results show that the WS cell lines are hypersensitive to chromosome damage induced by VP-16 and amsacrine only in the G2 phase of the cell cycle. No difference either in the yield of the induced aberrations or SCEs was found after treatment of cells at G1 stage. These data might suggest that in WS cells, because of the mutation of the WRN protein, the inhibition of topoisomerase II activity results in a higher rate of misrepair, probably due to some compromised G2 phase processes involving the WRN protein. Description: L'articolo é disponibile sul sito dell'editore: http://www.sciencedirect.com 1999-12-31T23:00:00Z http://hdl.handle.net/2067/1722 Title: Caffeine effect on the mitotic delay induced by G2-treatment with UVC or mitomycin-C. Authors: Franchitto, Annapaola; Pichierri, Pietro; Mosesso, Pasquale; Palitti, Fabrizio Abstract: It is well established that DNA lesions trigger cell cycle check-points causing a mitotic delay that is required for their repair before cells enter the mitotic phase. Caffeine, in some cases, can remove this delay and consequently potentiates the yield of induced chromosome aberrations. The objective of this study was to test the effect of a G2 treatment with S-dependent agents (UV light and mitomycin C) on the cell kinetics of a G2 cell population and evaluate whether post-treatments with caffeine could modulate removal of the expected cell cycle delay. Cell kinetics were monitored by analysing the mitotic index (MI) values in combination with the 5-bromo-2'-deoxyuridine (BrdUrd) labelling technique. Chinese hamster fibroblast cultures (AA8) were treated in G2 phase of the cell cycle with 8 and 15 J/m2 UV light or 0.1 and 0.6 microgram/ml mitomycin C for 1.5 h. Post-treatments with caffeine were performed at dose levels and recovery times where the mitotic indices were substantially reduced. The results obtained showed that both UV light and mitomycin C induced a G2 arrest, as indicated by MI values and the absence of BrdUrd-labelled metaphases. For UV light the G2 block was observed at lower and higher dose levels after 1.5 h, while for mitomycin C it was observed only at the higher dose level after 1 h. However, in both cases the block lasted approximately 1 h, after which, even though slowed down, the cell population entered mitosis, as indicated by increased MI values. This block was not removed by caffeine post-treatment. In contrast, caffeine G2 post-treatment was able to remove G2 arrest induced by G1-S treatments. Accordingly, our results suggest that both UV light- and mitomycin C-induced damage must be processed during S phase to allow caffeine to remove induced G2 blocks. Description: L'articolo è disponibile sul sito dell'editore: http://www.oxfordjournals.org 1997-12-31T23:00:00Z http://hdl.handle.net/2067/1704 Title: Relation between DNA repair, apoptosis and chromosomal aberrations in presence of pifithrin-alpha, an inhibitor of p53. Authors: Meschini, Roberta; Berni, Andrea; Ortenzi, Vincenza; Mancinelli, Pierluigi; Palitti, Fabrizio Abstract: The aim of this study was to investigate the impact of inhibition of p53 in X-irradiated human peripheral blood lymphocytes (HPBL) in the G0 phase of the cell cycle in the presence or absence of pifithrin-alpha (PFT-alpha), a specific inhibitor of p53, on repair of DNA damage or induced apoptosis. Lymphocyte (HPBL) cultures were X-irradiated with 3 Gy in the absence or presence of PFT-alpha. In order to distinguish the effects of PFT-alpha either on DNA repair or on apoptosis, PFT-alpha was added to the cultures employing different protocols namely, a) "continuous treatment", where PFT-alpha was added four hours before X-irradiation and left until the end of the experiment, b) "pre-treatment", where PFT-alpha was added four hours before X-irradiation and removed by washing the cells with phosphate buffered saline (PBS) four hours after irradiation and c) "post-treatment", where PFT-alpha was added four hours after irradiation and left in the medium until the harvest. At various times after irradiation of lymphocytes, Single Cell Gel Electrophoresis was performed to detect DNA damage in individual cells. Apoptosis and chromosomal aberrations were quantified at later sampling times following irradiation. The results presented here strengthen the known role of p53 protein in priming apoptotic cell death in HPBL following X-irradiation. Furthermore, our data suggest that the inhibition of p53 by PFT-alpha affects the repair kinetics of X-ray induced DNA lesions leading to mis-repair events and consequently to an enhancement of cytogenetic damage in HPBL. Description: L'articolo é disponibile sul sito dell'editore: http://www.sciencedirect.com 2009-12-31T23:00:00Z http://hdl.handle.net/2067/1694 Title: Relationship between spontaneous or radiation-induced apoptosis and telomere shortening in G(0) human lymphocytes Authors: Belloni, Paola; Latini, Paolo; Palitti, Fabrizio Abstract: To examine the correlation between spontaneous or radiation-induced apoptosis and telomere shortening, G0 human peripheral blood lymphocytes were irradiated with X-rays and analyzed for viability, apoptosis, and telomere length. Part of the lymphocytes was kept under liquid-holding conditions for 48 h, and then loaded onto Ficoll-Paque medium to separate apoptotic (high-density) from normal (normal-density) cells. Then all samples were examined for the same three end-points. To determine whether expression of p53 influences the telomere shortening associated with a spontaneous or radiation-induced apoptotic process, the lymphocytes were also analyzed for expression of p53 at 0 and 48 h recovery times (non-irradiated and irradiated samples) and after 2 weeks in liquid-holding conditions (non-irradiated sample). After 48 h in liquid-holding, the p53-dependent apoptotic lymphocytes in the irradiated cultures presented shortened telomeres. After a 2-week recovery time, non-irradiated cells showed a p53-dependent spontaneous apoptosis, but no telomere shortening. These results demonstrate that radiation-induced apoptosis correlates with shortened telomeres in G0 human lymphocytes. Spontaneous and radiation-induced apoptosis are dependent on expression of p53. In contrast, p53 may not play an effective role in telomere shortening, because spontaneous apoptosis did not correlate with telomere shortening. As most tumours are compromised with respect to p53 function, our findings on the role of p53 in telomere shortening may prove critical for applying therapeutic modalities in the clinic, and may facilitate the design of agents that selectively disrupt telomere integrity in tumour cells. Description: L'articolo é disponibile sul sito dell'editore: http://www.sciencedirect.com 2009-12-31T23:00:00Z