Acrylamide: a probable catalytic topoisomerase II inhibitor

Rheumatoid arthritis-associated HLA-DRB1 genotypes in western Sicily

Metilazione del DNA in artrite reumatoide

Lo stato di metilazione del DNA genomico e del gene PTHrP è stato valutato con tecniche molecolari e citogenetiche in artrite reumatoide (AR), patologia autoimmune caratterizzata anche da alta incidenza di linfomi e da ipercalcemia per overespressione del gene PTHrP. La metilazione del DNA, infatti, ha un ruolo critico nello sviluppo delle malattie neoplastiche; il gene PTHrP avendo tre promotori uno dei quali contiene un’isola CpG è un buon candidato per la deregolazione da alterato pattern di metilazione locale. Le indagini sulla metilazione genomica, condotte su DNA estratto da sangue periferico di pazienti e di donatori e amplificato in reazioni di Methylation-Sensitive Arbitrarily Prim…

Telomerase activity in cells with arsenic-induced genomic instability

It is well known that the occurrence of dicentric chromosomes represent signature of telomere dysfunction and is a clear symptom of genomic instability. V79 Chinese hamster cells, treated with 10μM sodium arsenite for 24h and allowed to grow in drug-free medium (ASO cells), showed genomic instability with aneuploidy and nuclear abnormalities as well as the appearance of dicentric chromosomes since the 90th cell generation. TRAP assay was performed on growing ASO cells and on clones isolated during the course of the expanded growth. As expected, some clones with dicentric chromosomes and severely reduced telomerase activity went to death; surprisingly, other clones also bearing chromosomal e…

Acrilamide: un probabile inibitore della topoisomerasi II

Stato di metilazione del promotore 2 del gene PTHrP in pazienti affetti da mieloma multiplo

Antagonist effects of Acrylamide on clastogenity of VP16

We investigated on the Acrylamide (AA) capability of influencing the clastogenic effects of VP16, the topoisomerase II targeting drug, by performing sequential treatments in V79 Chinese hamster cells. The VP16 cytotoxicity resulted almost completely antagonized by preincubating cells with nontoxic concentrations of AA, as inferred by statistically significant differences versus response with VP16 alone. Moreover, the severe clastogenic effect of VP16, evidenced by the presence of complex structural chromosome aberrations and by high frequencies of micronulei and sister chromatid exchanges, was reduced by AA in a dose-dependent manner. For example, the frequency of micronucleated cells induc…

Genomewide hypomethylation and PTHrP gene hypermethylation as a model for the prediction of cancer risk in rheumatoid arthritis

We have previously shown that PTHrP(38-94)-amide restrains growth and invasion "in vitro", causes striking toxicity and accelerates death of some breast cancer cell lines, the most responsive being MDA-MB231 whose tumorigenesis was also attenuated "in vivo". PTHrP(38-94)-amide contains the domain implicated in the nuclear import of PTHrP. Although the nucleus was identified as a destination for mid-region PTHrP, evidence for direct DNA-binding capability is lacking to date. Here, we examined the localization of PTHrP(38-94)-amide within MDA-MB231 cells and within metaphase spread preparations and characterized its DNA-binding properties, employing a combination of immunocytochemical, cytoge…

Genetic polymorphism of the bitter taste TAS2R38 gene in central Sicily

PTHrP [38-94]-amide is a DNA-binding factor: cytogenetic and molecular evidence and biological effect on normal and neoplastic human breast cells

Persistent genomic instability by arsenic exposure in V79 Chinese hamster cells

Previously, we demonstrated that acute treatment with arsenic leads mammalian cells to exhibit persistent chromosomal instability and DNA hypomethylation, by performing investigations after about 2 months of subculturing. In order to evaluate quantitatively the continuing instability during the expanded growth, we carried out cytogenetic, morphologic and molecular analyses immediately after exposure and every week up to 112 cell generations. Briefly, V79 Chinese hamster cells were treated with 10 µM sodium arsenite (SA) for 24h; at the end of exposure, mitotic rounded-up cells were harvested by shake-off and allowed to grow in drug-free medium. The instability markers, micronucleated and mu…

Biological effects of alpha-pinene in cultured mammalian cells

In this work we report the effects of exposure of mammalian cells to α-pinene, a bicyclic monoterpene founded in essential oils and used in insecticides, solvents, perfumes, etc.. Morphological analysis, performed in V79 cells exposed to increasing doses(25μM up to 50μM) of α-pinene, indicated a increase of dose-related nuclear abnormalities; apoptotic cells were seen at higher doses. Immunofluorescence with anti β- tubulin antibody showed that monoterpene induced genomic instability by interfering with mitotic process; in fact, 50% (vs 19% in the control cells) of irregular mitosis with multipolar or not correctly localized spindles were detected, suggesting that α-pinene affects cell stab…

Arsenite-induced aneuploidy following short and long-term exposure in mammalian cells

We studied the long-term progression of chromosomal instability in V79 cells treated acutely with arsenite (10mM, 24 hr) followed by growth in arsenic-free medium for 120 cell generations. Indirect immunostaining using anti-ß-tubulin antibody showed severe alterations in spindle morphology after only 6 h treatment and cytogenetic investigations carried out at the end of treatment revealed that the percentage of cells with 21 chromosomes (modal number of the cell line) decreased, making way for aneuploid cells. The acquired instability remained and propagated within the cell population. Moreover, we treated V79-derived G12 cells with sub-lethal doses (0.1-1.0 μM) of arsenite for 10 days foll…

POLIMORFISMI DEI GENI CYP2A6 E CYP2E1 IN RELAZIONE A STILI DI VITA IN UNA POPOLAZIONE DELLA SICILIA CENTRO-OCCIDENTALE

Le più recenti stime dell’OMS testimoniano che le patologie correlate all’abitudine al fumo e al consumo di alcool sono tra le principali cause di morte nei soggetti adulti ed è noto che la diversità interindividuale nell’adottare tali stili di vita è attribuibile, oltre che a fattori psico-sociali, anche a polimorfismi dei geni per i citocromi P450. Questo studio mira a verificare se esista una correlazione fra i genotipi CYP2A6 -CYP2E1 e abitudine al fumo e consumo d’alcool nella Sicilia centro-occidentale. I risultati ottenuti dall’analisi della distribuzione di alcuni alleli di entrambi i geni in 65 soggetti (41 donne e 24 uomini) hanno evidenziato che i genotipi CYP2A6*1/*4A e CYP2A6*1…

Early and late effects of arsenic exposure in mammalian cells

Previously we demonstrated that V79 Chinese hamster cells underwent either early genetic instability or apoptosis When exposed to sodium arsenite (SA). Genetic instability was evidenced by aneuploid and morphologically abnormal cells, but not by cells with chromosome aberrations. As dividing cells turned out to be the most sensitive to SA exposure, due to the arsenics direct action on the mitotic spindle assembly, we later ascertained the fate of genetically unstable cells escaping apoptosis, by harvesting mitotic rounded-up cells at the end of a 24 h treatment. The progeny of the exposed Chinese hamster cells showed an increased level of mutations related to genome DNA hypomethylation indu…

Mid-region parathyroid hormone-related protein (PTHrP) binds chromatin of MDA-MB231 breast cancer cells and isolated oligonucleotides “in vitro”

We have previously shown that PTHrP(38-94)-amide restrains growth and invasion "in vitro", causes striking toxicity and accelerates death of some breast cancer cell lines, the most responsive being MDA-MB231 whose tumorigenesis was also attenuated "in vivo". PTHrP(38-94)-amide contains the domain implicated in the nuclear import of PTHrP. Although the nucleus was identified as a destination for mid-region PTHrP, evidence for direct DNA-binding capability is lacking to date. Here, we examined the localization of PTHrP(38-94)-amide within MDA-MB231 cells and within metaphase spread preparations and characterized its DNA-binding properties, employing a combination of immunocytochemical, cytoge…

Telomere dysfunction in cells with arsenic-induced genomic instability

It is well known that the occurrence of dicentric chromosomes represent signature of telomere dysfunction and is a clear symptom of genomic instability. V79 Chinese hamster cells, treated with 10µM sodium arsenite for 24h and allowed to grow in drug-free medium (ASO cells), showed genomic instability with aneuploidy and nuclear abnormalities as well as the appearance of dicentric chromosomes since the 90th cell generation. TRAP assay was performed on growing ASO cells and on clones isolated during the course of the expanded growth. As expected, some clones with dicentric chromosomes and severely reduced telomerase activity went to death; surprisingly, other clones also bearing chromosomal e…

Effect of inorganic arsenic on rat cortical astrocytes in culture

Although inorganic arsenic is a well known poisonous metalloid, the cellular and molecular mechanisms of its action remain elusive. The present study was aimed at analyzing the effects of NaAsO2 on primary cultures of rat astrocytes by determining DNA damage by comet assay, and by evaluating possible changes of the concentration of some conserved heat shock proteins. Cells treated with inorganic arsenic underwent induction of Hsp70, demonstrating a state of stress. Moreover, although micromolar NaAsO2 treatments (60 μM) only reduced cell viability to 60% respect to untreated cells, high DNA damage was already observed after 24h treatment with 10 μM arsenite. Since arsenic is known to be not…

In vivo and in vitro inhibitory effects of acrylamide on DNA topoisomerase II

Acrylamide (AA), a chemical produced in several foodstuffs when cooked at a high temperature, is considered a probable human carcinogen, but the molecular mechanism underlying its genotoxicity has not fully known. Numerous authors have reported the induction by AA of DNA double strand breaks and sister chromatid exchange (SCE); we here confirmed the acrylamide capability of damaging DNA by utilizing Comet assay, which showed a dose-dependent increase of tail lenght, in metabolically non competent V79 Chinese hamster cells. Moreover, we observed that Acrylamide (AA) was able to antagonize in vivo the citotoxicity of well know poison etoposide; this suggested that topoisomerase II activity wa…