Renata Cozzi

Resveratrol induces DNA double-strand breaks through human topoisomerase II interaction

Resveratrol, a stilbene found in grapes and wine, is one of the most interesting natural compound due to its role exerted in cancer prevention and therapy. In particular, resveratrol is able to delay cell cycle progression and to induce apoptotic death in several cell lines. Here we report that resveratrol treatment of human glioblastoma cells induces a delay in cell cycle progression during S phase associated with an increase in histone H2AX phosphorylation. Furthermore, with an in vitro assay of topoisomerase IIalpha catalytic activity we show that resveratrol is able to inhibit the ability of recombinant human TOPO IIalpha to decatenate kDNA, so that it could be considered a TOPO II poison.

Oxidative stress induces persistent telomeric DNA damage responsible for nuclear morphology change in Mammalian cells.

One main function of telomeres is to maintain chromosome and genome stability. The rate of telomere shortening can be accelerated significantly by chemical and physical environmental agents. Reactive oxygen species are a source of oxidative stress and can produce modified bases (mainly 8-oxoG) and single strand breaks anywhere in the genome. The high incidence of guanine residues in telomeric DNA sequences makes the telomere a preferred target for oxidative damage. Our aim in this work is to evaluate whether chromosome instability induced by oxidative stress is related specifically to telomeric damage. We treated human primary fibroblasts (MRC-5) in vitro with hydrogen peroxide (100 and 200 µM) for 1 hr and collected data at several time points. To evaluate the persistence of oxidative stress-induced DNA damage up to 24 hrs after treatment, we analysed telomeric and genomic oxidative damage by qPCR and a modified comet assay, respectively. The results demonstrate that the genomic damage is completely repaired, while the telomeric oxidative damage persists. The analysis of telomere length reveals a significant telomere shortening 48 hrs after treatment, leading us to hypothesise that residual telomere damage could be responsible for the telomere shortening observed. Considering the influence of telomere length modulation on genomic stability, we quantified abnormal nuclear morphologies (Nucleoplasmic Bridges, Nuclear Buds and Micronuclei) and observed an increase of chromosome instability in the same time frame as telomere shortening. At subsequent times (72 and 96 hrs), we observed a restoration of telomere length and a reduction of chromosome instability, leaving us to conjecture a correlation between telomere shortening/dysfunction and chromosome instability. We can conclude that oxidative base damage leads to abnormal nuclear morphologies and that telomere dysfunction is an important contributor to this effect.

DNA repair capacity and acute radiotherapy adverse effects in Italian breast cancer patients.

Therapeutic exposure to ionising radiation can induce normal tissue side effects which consistently differ among individuals suggesting a possible genetic control. One approach to elucidate the underlying mechanisms is to analyse the relation between genetic traits, biomarkers of in vitro DNA damage and side toxicity in vivo. 43 breast cancer (BC) patients receiving radiotherapy after a breast-conserving surgery were recruited together with 34 age- and sex-matched healthy controls. Adverse tissue reactions were recorded as indicators of radiotherapy susceptibility. All blood samples from both patients (35) and controls (34) were irradiated in vitro and DNA primary damage and repair kinetic were measured through Comet assay. All study subjects were genotyped for XRCC1, OGG1 and XRCC3 gene polymorphisms. In our small groups we found a positive association between XRCC1 variant allele (399Gln) and the occurrence of breast cancer [p=0.01; OR=2.41, 95%CI (1.24-4.66)]. BC patients showed a higher degree of basal (p<0.001) and X-ray induced DNA damage (p<0.01) when compared to healthy controls. A reduced repair ability was found in BC patients showing high degrees of tissue side effects as classified by Radiation Morbidity Scoring Scheme. BC patients showed an impairment of their DNA repair capacity associated with the development of radiation sensitivity but not with polymorphisms in any of the considered genes.

Desmutagenic activity of natural humic acids: inhibition of mitomycin C and maleic hydrazide mutagenicity

Humic acids are natural components of organic matter widespread in the environment. In spite of the incomplete knowledge about their composition, increasing interest in humic acid activity is justified by their ubiquity. Four different humic acids have been tested in Chinese hamster ovary cells in vitro, both alone and in combination with two well-known mutagens (mitomycin C and maleic hydrazide). Data about sister-chromatid exchanges, mitotic and proliferation indices were collected. Our results, on the whole, indicate: (i) a slight mutagenicity and toxicity of tested humic acids, probably due to chlorination during sample preparation; (ii) a desmutagenic rather than antimutagenic activity of the tested humic acids.

Influence of XRCC1 Genetic Polymorphisms on Ionizing Radiation-Induced DNA Damage and Repair

It is well known that ionizing radiation (IR) can damage DNA through a direct action, producing single- and double-strand breaks on DNA double helix, as well as an indirect effect by generating oxygen reactive species in the cells. Mammals have evolved several and distinct DNA repair pathways in order to maintain genomic stability and avoid tumour cell transformation. This review reports important data showing a huge interindividual variability on sensitivity to IR and in susceptibility to developing cancer; this variability is principally represented by genetic polymorphisms, that is, DNA repair gene polymorphisms. In particular we have focussed on single nucleotide polymorphisms (SNPs) of XRCC1, a gene that encodes for a scaffold protein involved basically in Base Excision Repair (BER). In this paper we have reported and presented recent studies that show an influence of XRCC1 variants on DNA repair capacity and susceptibility to breast cancer.

Resveratrol acts as a topoisomerase II poison in human glioma cells

Recently, we demonstrated that Resveratrol (RSV), a well known natural stilbene, is able to induce a delay in S progression with a concomitant increase in γH2AX expression in U87 glioma cells. Furthermore, we showed that it inhibits the ability of recombinant human topoisomerase IIα to decatenate kDNA in vitro. Because proliferating tumor cells express topoisomerases at high levels and these enzymes are important targets of some of the most successful anticancer drugs, we tested whether RSV is able to poison topoisomerase IIα in glioma cells. Then, we monitored the increase of micronuclei in RSV treated U87 cells as a consequence of the conversion of TOPOII/DNA cleavable complexes to permanent DNA damage. Finally, we assayed the ability of RSV in modulating the expression of target proteins involved in DNA damage signalling, namely ATR, ATM, Chk1, Chk2 and γH2AX. Through a molecular modelling here we show that RSV binds at the TOPOII/DNA interface thus establishing several hydrogen bonds. Moreover, we show that RSV poisons TOPOIIα so inducing DNA damage; ATM, Chk2 and γH2AX are involved in the DNA damage signalling after RSV treatment.

Resveratrol and X rays affect gap junction intercellular communications in human glioblastoma cells

Resveratrol (3,4′,5‐trihydroxystilbene) is a polyphenol synthesized by a wide variety of plant species in response to injury, UV irradiation and fungal attack. Many studies have revealed a variety of resveratrol intracellular targets whose modulation gives rise to overlapping responses leading to growth arrest and death. Many authors have reported different human cancer cell lines, treated with resveratrol at micromolar concentrations, arrested their proliferative cycle in the G1/S boundary or in the S phase and this cell cycle arrest was followed by apoptotic death. Less is known about the ability of resveratrol to modify the effect of radiation exposure in normal and cancer cells. Considering that controlled exposure to ionizing radiation is one of the most used treatments in cancer patients and that these schedules are not always effective in medical practice, as in the case of glioma patients, the testing of combined treatment protocols (resveratrol and ionizing radiation) could be of interest, opening the door to future studies which would examine the pharmacological activity of resveratrol. In this study we have looked into whether resveratrol is able to modulate cell cycle progression in human glioblastoma cells and to regulate GJs expression in cancer cells. With this aim in mind we have performed a cytofluorimetric multiparameter assay to quantify the presence of GJs in U87 glioma cells treated with resveratrol and/or X rays. We report that resveratrol induces a delay in cell cycle progression and both alone and in combination with X rays is able to enhance gap junction Intercellular Communications.

Levodopa therapy reduces DNA damage in peripheral blood cells of patients with Parkinson’s disease

Oxidative stress seems to play a major role in the pathogenesis of neurodegeneration. In Parkinson’s disease (PD) patients, the dopaminergic neurons are subjected to oxidative stress resulting from reduced levels of antioxidant defenses such as glutathione and high amount of intracellular iron. Levodopa (LD) is widely used for the symptomatic treatment of PD, but its role in oxidative damage control is still unclear. The aim of this study was to analyze the presence of DNA damage in peripheral blood lymphocytes (PBL) of PD patients, during a washout and a controlled LD dosage and to evaluate the oxidative damage fluctuation after LD intake. The standard and the Fpg-modified version of Comet assay were applied in analyzing DNA damage in PBL from blood samples of nine PD patients and nine matched controls. Due to the limited number of patients we cannot reach definite conclusions even if our data confirm the accumulation of DNA lesions in PD patients; these lesions decrease after LD intake.

DNA strand methylation and sister chromatid exchanges in mammalian cells in vitro.

Among other targets, DNA demethylating agents are known to affect the sister chromatid exchange (SCE) frequency in mammalian cells in vitro. The SCE increase appears to be maintained for many (10-16) cell cycles after the end of the pulse in a given cell population, unlike SCEs induced by DNA damaging agents. Yet, epigenetic changes (such as demethylation) would not be expected to affect SCE at all. In the present report we challenge the working hypothesis of a relation between SCEs and demethylation by comparing SCE induction during different rounds of replication when the parental strands were normally methylated or demethylated. Azacytidine (AZA), ethionine (ETH), mitomycin-C (MMC), UV-irradiation (UV) and hydrogen peroxide (H(2)O(2)) were tested for SCE induction in a Chinese hamster ovary cell line after a single pulse, one or two cell cycles before fixation. Whereas MMC, UV and H(2)O(2) induce SCE in both protocols, AZA and ETH show an effect on SCEs only if administered two cycles before fixation. Because two cell cycles are needed in order to achieve demethylation of the parental DNA strand, the data reported here support our working hypothesis that demethylation in the parental DNA strand, at the level of the replication fork (i.e., the region where SCEs are formed), is responsible for an increase in mistaken ligations of processed damage, eventually yielding an increase in SCEs.

Evaluation of Levodopa and Carbidopa Antioxidant Activity in Normal Human Lymphocytes In Vitro: Implication for Oxidative Stress in Parkinson’s Disease

The main pathochemical hallmark of Parkinson’s disease (PD) is the loss of dopamine in the striatum of the brain, and the oral administration of levodopa (l-dopa) is a treatment that partially restores the dopaminergic transmission. In vitro assays have demonstrated both toxic and protective effects of l-dopa on dopaminergic cells, while in vivo studies have not provided any convincing data. The peripheral metabolic pathways significantly decrease the amount of l-dopa reaching the brain; therefore, all of the current commercial formulations require an association with an inhibitor of dopa-decarboxylase, such as carbidopa. However, the dosage and the actual effectiveness of carbidopa have not yet been well defined. PD patients exhibit a reduced efficiency of the endogenous antioxidant system, and peripheral blood lymphocytes (PBLs) represent a dopaminergic system for use as a cellular model to study the pharmacological treatments of neurodegenerative disorders in addition to analysing the systemic oxidative stress. According to our previous studies demonstrating a protective effect of both l-dopa and carbidopa on neuroblastoma cells in vitro, we used the PBLs of healthy donors to evaluate the modulation of DNA damage by different concentrations of l-dopa and carbidopa in the presence of oxidative stress that was exogenously induced by H2O2. We utilised a TAS assay to evaluate the in vitro direct scavenging activity of l-dopa and carbidopa and analysed the expression of genes that were involved in cellular oxidative metabolism. Our data demonstrate the antioxidant capacity of l-dopa and carbidopa and their ability to protect DNA against oxidative-induced damage that derives from different mechanisms of action.