Biomedical subjects

F Palitti

Publications and source records attributed to F Palitti.

At least 37 records · Page 2Linked to original sources

PubMed2000

Werner's syndrome cell lines are hypersensitive to camptothecin-induced chromosomal damage.

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.

Camptothecin↗

PubMed2000

Evidence that camptothecin-induced aberrations in the G(2) phase of cell cycle of Chinese hamster ovary (CHO) cell lines is associated with transcription.

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.

Amanitins↗

PubMed2000

Werner's syndrome lymphoblastoid cells are hypersensitive to topoisomerase II inhibitors in the G2 phase of the cell cycle.

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.

Amsacrine↗

PubMed2000

Catalytic inhibition of topoisomerase II in Werner's syndrome cell lines enhances chromosomal damage induced by X-rays in the G2 phase of the cell cycle.

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.

Catalysis↗

PubMed2000

Clastogenic effects of two tyrosine kinase inhibitors, tyrphostin 23 and tyrphostin 46, on a transformed (CHO-K1) and on a primary embryonic chinese hamster cell line (CHE).

Protein tyrosine kinases (PTKs) play fundamental roles in signal transduction pathways. Many proliferative diseases are characterized by deregulation of PTK activity, therefore PTKs appear as promising targets in the design of anticancer drugs. Tyrphostins are a family of synthetic compounds which efficiently target specific PTKs without competing for ATP and thus are much less cytotoxic with respect to conventional therapeutic agents. We tested two tyrphostin derivatives, Tyrphostin 23 and Tyrphostin 46, on a transformed (CHO-K1) and on a primary embryonic Chinese hamster cell line (CHE) to determine whether these compounds had a genotoxic effect. We found that the tyrphostins increased sister chromatid exchange frequency in both cell lines, but induced chromosomal aberrations only in the transformed CHO-K1 cell line when treatment was in the S phase of the cell cycle, and not in primary CHE cells. Such a result could have important therapeutic implications: it could mean that deregulation of signal transduction pathways in cells which already have a deficit in cell cycle control could cause chromosomal aberrations.

Animals↗

PubMed1999

Cytosine methylation transforms an E2F site in the retinoblastoma gene promoter into a binding site for the general repressor methylcytosine-binding protein 2 (MeCP2).

The CpG-rich promoter of the retinoblastoma tumor suppressor gene (Rb-1) is normally unmethylated. However, aberrant methylation of CpG dinucleotides within the Rb-1 promoter has been depicted in certain tumors, which determines transcriptional inactivity of the gene and absence of the pRb retinoblastoma protein. Here we have concentrated on an E2F-binding site in the Rb-1 promoter. We show that the E2F site is required for cell-cycle regulated Rb-1 transcription in non-transformed cells. The function of the E2F site is associated with its ability to interact with several activating factors of the E2F family. In contrast, in vitro methylation of two tandemly arranged CpGs in the E2F recognition site prevents binding by E2F factors, and determines instead the recruitment of the general repressor methylcytosine-binding protein 2 (MeCP2). These results suggest that the interaction of MeCP2 with the methylated version of the E2F site may represent a step towards Rb-1 promoter inactivity in tumor cells.

3T3 Cells↗

PubMed1999

Lack of effect of caffeine post-treatment on X-ray-induced chromosomal aberrations in Werner's syndrome lymphoblastoid cell lines: a preliminary report.

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.

Caffeine↗

PubMed1999

Chromosome radiosensitivity in human G2 lymphocytes and cell-cycle progression.

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.

Adult↗

PubMed1999

The involvement of chromatin condensation in camptothecin-induced chromosome breaks in G0 human lymphocytes.

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.

Animals↗

PubMed1998

Mechanisms of the origin of chromosomal aberrations.

This manuscript gives a selective survey of some aspects of the origin of chromosomal aberrations (CA). The following topics are discussed: heterogeneity regarding the induction of initial DNA lesions and their repair among chromosomes or regions of the same chromosome, ratio between symmetrical and asymmetrical exchanges, influence of DNA repeats, chromatin condensation and cell-cycle checkpoints on the formation of chromosome alterations.

Cell Cycle↗

PubMed1998

Influence of pre-existing methylation on the de novo activity of eukaryotic DNA methyltransferase.

Aberrant de novo methylation of CpG island DNA sequences has been observed in cultured cell lines or upon malignant transformation, but the mechanisms underlying this phenomenon are poorly understood. Using eukaryotic DNA (cytosine-5)-methyltransferase (of both human and murine origin), we have studied the in vitro methylation pattern of three CpG islands. Such sequences are intrinsically poor substrates of the enzyme, yet are efficiently methylated when a small amount of 5-methylcytosine is randomly introduced by the M.SssI prokaryotic DNA (cytosine-5)-methyltransferase prior to in vitro methylation by the eukaryotic enzyme. A stimulation was also found with several other double-stranded DNA substrates, either natural or of synthetic origin, such as poly(dG-dC).poly(dG-dC). An A + T-rich plasmid, pHb beta 1S, showed an initial stimulation, followed by a severe inhibition of the activity of DNA (cytosine-5)-methyltransferase. Methylation of poly(dI-dC).poly(dI-dC) was instead inhibited by pre-existing 5-methylcytosines. The extent of stimulation observed with poly(dG-dC).poly(dG-dC) depends on both the number and the distribution of the 5-methylcytosine residues, which probably must not be too closely spaced for the stimulatory effect to be exerted. The activity of the M.SssI prokaryotic DNA methyltransferase was not stimulated, but was inhibited by pre-methylation on either poly(dG-dC).poly(dG-dC) or poly(dI-dC).poly(dI-dC). The prokaryotic and eukaryotic DNA methyltransferases also differed in sensitivity to poly(dG-m5dC).poly(dG-m5dC), which is highly inhibitory for eukaryotic enzymes and almost ineffective on prokaryotic enzymes.

Animals↗

PubMed1998

Interactions with single-stranded and double-stranded DNA-binding factors and alternative promoter conformation upon transcriptional activation of the Htf9-a/RanBP1 and Htf9-c genes.

The murine Htf9-a/RanBP1 and Htf9-c genes are divergently transcribed from a shared TATA-less promoter. Transcription of both genes is initiated on complementary DNA strands and is controlled by cell cycle-dependent mechanisms. The bidirectional promoter harbors a genomic footprint flanking the major transcription start site of both genes. Transient promoter assays showed that the footprinted element is important for transcription of both genes. Protein-binding experiments and antibody assays indicated that members of the retinoid X receptor family interact with the double-stranded site. In addition, distinct factors interact with single DNA strands of the element. Double-stranded binding factors were highly expressed in liver cells, in which neither gene is transcribed, while single-stranded binding proteins were abundant in cycling cells, in which transcription of both genes is efficient. In vivo S1 analysis of the promoter depicted an S1-sensitive organization in cells in which transcription of both genes is active; S1 sensitivity was not detected in conditions of transcriptional repression. Thus, the same element is a target for either retinoid X receptor factors, or for single-stranded binding proteins, and form distinct complexes in different cellular conditions depending on the DNA conformation in the binding site.

3T3 Cells↗

PubMed1998

Induction of chromosomal aberrations (unstable and stable) by inhibitors of topoisomerase II, m-AMSA and VP16, using conventional Giemsa staining and chromosome painting techniques.

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.

Amsacrine↗

PubMed1998

Distribution of camptothecin-induced break points in Chinese hamster cells treated in late S and G2 phases of the cell cycle.

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.

Animals↗

PubMed1998

Caffeine effect on the mitotic delay induced by G2 treatment with UVC or mitomycin C.

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.

Animals↗

PubMed1996

AZT-induced hypermethylation of human thymidine kinase gene in the absence of total DNA hypermethylation.

Genome-wide DNA hypermethylation induced by 3'-azido-3'-deoxythymidine (AZT) has been suggested to be involved in the development of AZT resistance. We used a CD4 T-lymphoblastoid CEM line and its AZT-resistant MT500 variant with reduced thymidine kinase activity. Evaluation of total DNA methylation, after AZT treatment, failed to show an increase in the 5-methylcytosine level in both parental and AZT-resistant cells. The effect was instead observed at a more specific gene level, on the three HpaII sites present in exon 1 of the human thymidine kinase gene. These results suggest that AZT treatment can induce site-specific hypermethylation, even in the absence of a more general DNA hypermethylating effect.

5-Methylcytosine↗

PubMed1996

Development and validation of alternative metabolic systems for mutagenicity testing in short-term assays.

We present here the results obtained within the framework of an EU funded project aimed to develop and validate alternative metabolic activating systems to be used in short-term mutagenicity assays, in order to reduce the use of laboratory animals for toxicology testing. The activating systems studied were established cell lines (Hep G2, CHEL), genetically engineered V79 cell lines expressing specific rat cytochromes P450, erythrocyte-derived systems, CYP-mimetic chemical systems and plant homogenates. The metabolically competent cell lines were used as indicator cells for genotoxic effects as well as for the preparation of external activating systems using other indicator cells. The following endpoints were used: micronuclei, chromosomal aberrations and sister chromatid exchanges, mutations at the hprt locus, gene mutations in bacteria (Ames test), unscheduled DNA synthesis and DNA breaks detected in the comet assay. All metabolic systems employed activated some promutagens. With some of them, promutagens belonging to many different classes of chemicals were activated to genotoxicants, including carcinogens negative in liver S9-mediated assays. In other cases, the use of the new activating systems allowed the detection of mutagens at much lower substrate concentrations than in liver S9-mediated assays. Therefore, the alternative metabolizing systems, which do not require the use of laboratory animals, have a substantial potential in in vitro toxicology, in the basic genotoxicity testing as well as in the elucidation of activation mechanisms. However, since the data basis is much smaller for the new systems than for the activating systems produced from subcellular liver preparations, the overlapping use of both systems is recommended for the present and near future. For example, liver S9 preparations may be used with some indicator systems (e.g., bacterial mutagenicity), and metabolically competent mammalian cell lines may be used with other indicator systems (e.g., a cytogenetic endpoint) in a battery of basic tests.

Animals↗

PubMed1996

The diplochromosome of endoreduplicated cells: a new approach to highlight the mechanism of sister chromatid exchange.

Chinese hamster lung embryonic cells (CL1) were treated with colchicine in order to induce endoreduplication and subsequently with mitomycin-C (MMC) to induce exchanges within the diplochromosome. The use of chromosomal differential staining through incorporation of 5-bromodeoxyuridine, resulting in only one stained chromatid, has allowed the analysis of all classes of exchanges among the four chromatids of the diplochromosome. Three classes of exchanges may occur: intradiplochromatid exchanges (ICEs) between the two inner chromatids, cousin chromatid exchanges (CCEs) between one inner and one outer chromatid, and sister chromatid exchanges (SCEs) between the two sister chromatids of the diplochromosome. The results show that MMC treatment, in the last cell cycle of endoreduplication, as expected, significantly increases only the frequency of SCEs, whereas the frequency of ICEs and CCEs remains unchanged. This result supports replication models of formation of SCEs. Furthermore the fact that the number of ICEs does not increase means that the molecular mechanism of somatic crossing over is not related to that of SCE formation, or very rarely. The results also indicate a statistically significant lower induction of SCEs in endoreduplicated metaphases as compared with diploid ones both in control and MMC-treated cells. Such a result may be due to structural restrictions within the diplochromosome.

Animals↗