http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. Wed, 19 Jun 2013 06:09:55 GMT 2013-06-19T06:09:55Z 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 Fri, 31 Dec 1999 23:00:00 GMT http://hdl.handle.net/2067/1716 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 Wed, 31 Dec 1997 23:00:00 GMT http://hdl.handle.net/2067/1722 1997-12-31T23:00:00Z 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 Thu, 31 Dec 1998 23:00:00 GMT http://hdl.handle.net/2067/1720 1998-12-31T23:00:00Z 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 Fri, 31 Dec 1999 23:00:00 GMT http://hdl.handle.net/2067/1712 1999-12-31T23:00:00Z 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 Fri, 31 Dec 1999 23:00:00 GMT http://hdl.handle.net/2067/1713 1999-12-31T23:00:00Z http://hdl.handle.net/2067/1717 Title: Lack of effect of caffeine post-treatment on X-rays chromosomal aberrations in Werner's syndrome lymphoblastoid cell lines: A Preliminary Report Authors: Franchitto, Annapaola; Proietti De Santis, Luca; Pichierri, Pietro Abstract: PURPOSE: To investigate whether in Werner's syndrome cells the G2 phase of the cell cycle has some abnormal response to post-treatment with agents such as caffeine and hydroxyurea known to interfere with cellular response to DNA damage. MATERIALS AND METHODS: Two Werner's syndrome lymphoblastoid cell lines (KO375 and DJG) and the normal cell line SNW646 were exposed to 50 cGy of X-rays or mitomycin-C and posttreated with caffeine or hydroxyurea in the G2 phase of the cell cycle. RESULTS: Hydroxyurea post-treatment potentiated the X-ray-induced aberration levels both in the normal and Werner's syndrome (KO375 and DJG) cell lines; in contrast caffeine was only effective in the normal cell line. Similar results were observed when Werner's syndrome cells were treated in the G1 phase with the S-dependent agent mitomycin-C and post-treated with caffeine in G2, extending the observation that Werner's syndrome cells are unaffected by caffeine G2 post-treatment. CONCLUSIONS: These results show a lack of caffeine effect in Werner's syndrome cells, suggesting an involvement of the Werner's syndrome protein in the signal transduction pathway by which caffeine could override the DNA damage induced G2 checkpoint Description: L'articolo è disponibile sul sito dell'editore: http://informahealthcare.com Thu, 31 Dec 1998 23:00:00 GMT http://hdl.handle.net/2067/1717 1998-12-31T23:00:00Z 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 Fri, 31 Dec 1999 23:00:00 GMT http://hdl.handle.net/2067/1715 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 Sun, 31 Dec 2000 23:00:00 GMT http://hdl.handle.net/2067/1710 2000-12-31T23:00:00Z