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PURPOSE: To examine levels of oxidative DNA base damage and expression of selected genes and proteins related to DNA damage repair in human limbal epithelium engineered ex vivo. METHODS: Cells were expanded from limbal tissue on cell culture-treated inserts in medium containing fetal bovine serum, recombinant growth factors, hormones and cholera toxin (COM) and in medium with human serum as the single growth-promoting additive (HS). Cells were analysed after two, three and four weeks in culture for DNA strand breaks and oxidized purine bases (Comet assay using the enzyme formamidopyrimidine DNA glycosylase, Fpg) and for expression of DNA repair enzymes APE1, OGG1 and Polß by in situ hybridization (ISH) and by immunohistochemistry (IHC). RESULTS: Levels of strand breaks were substantial while levels of net Fpg-sensitive sites (8-oxoguanine and ring-opened FaPy bases) were relatively low in cells engineered in COM and in HS. Both types of medium were found to support expression of base excision repair (BER) enzymes APE1, OGG1 and Polß at the gene level. At the protein level, expression of APE1 and OGG1 was noticeable in both conditions while expression of Polß was low. CONCLUSION: Our findings indicate low levels of oxidative stress and/or efficient DNA purine base damage repair in human limbal epithelium engineered in a medium with human serum as the single growth-promoting additive as well as in traditional medium with xenobiotics.
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Dano ao DNA , DNA/genética , Epitélio Corneano/metabolismo , Proteínas do Olho/metabolismo , Limbo da Córnea/metabolismo , Estresse Oxidativo/fisiologia , Engenharia Tecidual , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Ensaio Cometa , Epitélio Corneano/citologia , Feminino , Humanos , Imuno-Histoquímica , Limbo da Córnea/citologia , Masculino , Pessoa de Meia-IdadeRESUMO
PURPOSE: Coffee is known to contain phytochemicals with antioxidant potential. The aim of this study was to investigate possible antioxidant effects of coffee in healthy human volunteers. METHODS: A placebo-controlled intervention trial was carried out on 160 healthy human subjects, randomised into three groups, receiving 3 or 5 cups of study coffee or water per day, for 8 weeks. Blood samples were taken before, during, and after the intervention. Serum was used for analysis of blood lipids and standard clinical chemistry analytes. Peripheral blood mononuclear cells were isolated, and DNA damage (strand breaks and oxidised bases) was measured with the comet assay. The lipid oxidation product isoprostane 8-iso-PGF2α was assayed in urine samples by LC-MS/MS. RESULTS: There was no significant effect of coffee consumption on the markers of oxidation of DNA and lipids. Creatinine (in serum) increased by a few per cent in all groups, and the liver enzyme γ-glutamyl transaminase was significantly elevated in serum in the 5 cups/day group. Other clinical markers (including glucose and insulin), cholesterol, triacylglycerides, and inflammatory markers were unchanged. There was no effect of coffee on blood pressure. CONCLUSION: In a carefully controlled clinical trial with healthy subjects, up to 5 cups of coffee per day had no detectable effect, either beneficial or harmful, on human health.
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Antioxidantes/uso terapêutico , Café , Dieta Saudável , Hiperlipidemias/prevenção & controle , Neoplasias/prevenção & controle , Estresse Oxidativo , Cooperação do Paciente , Adulto , Antioxidantes/administração & dosagem , Antioxidantes/efeitos adversos , Biomarcadores/sangue , Café/efeitos adversos , Ensaio Cometa , Creatinina/sangue , Registros de Dieta , Feminino , Humanos , Hiperlipidemias/epidemiologia , Hiperlipidemias/etiologia , Hiperlipidemias/metabolismo , Leucócitos Mononucleares/imunologia , Lipídeos/sangue , Lipídeos/urina , Perda de Seguimento , Masculino , Pessoa de Meia-Idade , Neoplasias/epidemiologia , Neoplasias/etiologia , Neoplasias/metabolismo , Países Baixos/epidemiologia , Pacientes Desistentes do Tratamento , RiscoRESUMO
Nanogenotoxicity is a crucial endpoint in safety testing of nanomaterials as it addresses potential mutagenicity, which has implications for risks of both genetic disease and carcinogenesis. Within the NanoTEST project, we investigated the genotoxic potential of well-characterised nanoparticles (NPs): titanium dioxide (TiO2) NPs of nominal size 20 nm, iron oxide (8 nm) both uncoated (U-Fe3O4) and oleic acid coated (OC-Fe3O4), rhodamine-labelled amorphous silica 25 (Fl-25 SiO2) and 50 nm (Fl-50 SiO) and polylactic glycolic acid polyethylene oxide polymeric NPs - as well as Endorem® as a negative control for detection of strand breaks and oxidised DNA lesions with the alkaline comet assay. Using primary cells and cell lines derived from blood (human lymphocytes and lymphoblastoid TK6 cells), vascular/central nervous system (human endothelial human cerebral endothelial cells), liver (rat hepatocytes and Kupffer cells), kidney (monkey Cos-1 and human HEK293 cells), lung (human bronchial 16HBE14o cells) and placenta (human BeWo b30), we were interested in which in vitro cell model is sufficient to detect positive (genotoxic) and negative (non-genotoxic) responses. All in vitro studies were harmonized, i.e. NPs from the same batch, and identical dispersion protocols (for TiO2 NPs, two dispersions were used), exposure time, concentration range, culture conditions and time-courses were used. The results from the statistical evaluation show that OC-Fe3O4 and TiO2 NPs are genotoxic in the experimental conditions used. When all NPs were included in the analysis, no differences were seen among cell lines - demonstrating the usefulness of the assay in all cells to identify genotoxic and non-genotoxic NPs. The TK6 cells, human lymphocytes, BeWo b30 and kidney cells seem to be the most reliable for detecting a dose-response.
Assuntos
Testes de Mutagenicidade/métodos , Mutagênicos/toxicidade , Nanopartículas/química , Nanopartículas/toxicidade , Polímeros/toxicidade , Animais , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Ensaio Cometa , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Mutagênicos/química , Polímeros/química , RatosRESUMO
Surface coatings of nanoparticles (NPs) are known to influence advantageous features of NPs as well as potential toxicity. Iron oxide (Fe3O4) NPs are applied for both medical diagnostics and targeted drug delivery. We investigated the potential cytotoxicity and genotoxicity of uncoated iron oxide (U-Fe3O4) NPs in comparison with oleate-coated iron oxide (OC-Fe3O4) NPs. Testing was performed in vitro in human lymphoblastoid TK6 cells and in primary human blood cells. For cytotoxicity testing, relative growth activity, trypan blue exclusion, (3)H-thymidine incorporation and cytokinesis-block proliferation index were assessed. Genotoxicity was evaluated by the alkaline comet assay for detection of strand breaks and oxidized purines. Particle characterization was performed in the culture medium. Cellular uptake, morphology and pathology were evaluated by electron microscopy. U-Fe3O4 NPs were found not to be cytotoxic (considering interference of NPs with proliferation test) or genotoxic under our experimental conditions. In contrast, OC-Fe3O4 NPs were cytotoxic in a dose-dependent manner, and also induced DNA damage, indicating genotoxic potential. Intrinsic properties of sodium oleate were excluded as a cause of the toxic effect. Electron microscopy data were consistent with the cytotoxicity results. Coating clearly changed the behaviour and cellular uptake of the NPs, inducing pathological morphological changes in the cells.
Assuntos
Citotoxinas/química , Citotoxinas/toxicidade , Compostos Férricos/toxicidade , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/toxicidade , Mutagênicos/química , Mutagênicos/toxicidade , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ensaio Cometa , Dano ao DNA , Compostos Férricos/química , Humanos , Propriedades de SuperfícieRESUMO
The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.
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Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardizing the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature, and voltage gradient) are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e., cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls) or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay), or photosensitiser plus light to oxidize guanine (positive control for Fpg- or OGG1-sensitive sites). Reference standards are especially valuable when performing a series of experiments over a long period-for example, analysing samples of white blood cells from a large human biomonitoring trial-to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation.
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This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were collected in the same way and processed using the same blood isolation procedure. The inter-laboratory variation was the prominent contributor to the overall variation. The inter-laboratory coefficient of variation decreased for both DNA strand breaks (from 68 to 26%) and FPG sensitive sites (from 57 to 12%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference between the levels of FPG sensitive sites in PBMC samples from healthy donors in the different centres (0.41-0.52 lesion/10(6) bp).
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Separação Celular/métodos , Dano ao DNA , Laboratórios , Leucócitos Mononucleares/metabolismo , Adulto , Calibragem , Ensaio Cometa , Quebras de DNA de Cadeia Dupla , DNA-Formamidopirimidina Glicosilase/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Testes de Mutagenicidade , Análise de RegressãoRESUMO
The interplay between dietary habits and individual genetic make-up is assumed to influence risk of cancer, via modulation of DNA integrity. Our aim was to characterize internal and external factors that underlie inter-individual variability in DNA damage and repair and to identify dietary habits beneficial for maintaining DNA integrity. Habitual diet was estimated in 340 healthy individuals using a food frequency questionnaire and biomarkers of antioxidant status were quantified in fasting blood samples. Markers of DNA integrity were represented by DNA strand breaks, oxidized purines, oxidized pyrimidines and a sum of all three as total DNA damage. DNA repair was characterized by genetic variants and functional activities of base and nucleotide excision repair pathways. Sex, fruit-based food consumption and XPG genotype were factors significantly associated with the level of DNA damage. DNA damage was higher in women (p=0.035). Fruit consumption was negatively associated with the number of all measured DNA lesions, and this effect was mediated mostly by ß-cryptoxanthin and ß-tocopherol (p<0.05). XPG 1104His homozygotes appeared more vulnerable to DNA damage accumulation (p=0.001). Sex and individual antioxidants were also associated with DNA repair capacity; both the base and nucleotide excision repairs were lower in women and the latter increased with higher plasma levels of ascorbic acid and α-carotene (p<0.05). We have determined genetic and dietary factors that modulate DNA integrity. We propose that the positive health effect of fruit intake is partially mediated via DNA damage suppression and a simultaneous increase in DNA repair capacity.
Assuntos
Antioxidantes/metabolismo , Dano ao DNA/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Comportamento Alimentar , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Adulto , Idoso , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Feminino , Interação Gene-Ambiente , Marcadores Genéticos , Variação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Noruega , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Fatores Sexuais , Fatores de Transcrição/metabolismo , Adulto JovemRESUMO
DNA damage is commonly measured at the level of individual cells using the so-called comet assay (single-cell gel electrophoresis). As the frequency of DNA breaks increases, so does the fraction of the DNA extending towards the anode, forming the comet tail. Comets with almost all DNA in the tail are often referred to as 'hedgehog' comets and are widely assumed to represent apoptotic cells. We review the literature and present theoretical and empirical arguments against this interpretation. The level of DNA damage in these comets is far less than the massive fragmentation that occurs in apoptosis. 'Hedgehog' comets are formed after moderate exposure of cells to, for example, H2O2, but if the cells are incubated for a short period, 'hedgehogs' are no longer seen. We confirm that this is not because DNA has degraded further and been lost from the gel, but because the DNA is repaired. The comet assay may detect the earliest stages of apoptosis, but as it proceeds, comets disappear in a smear of unattached DNA. It is clear that 'hedgehogs' can correspond to one level on a continuum of genotoxic damage, are not diagnostic of apoptosis and should not be regarded as an indicator of cytotoxicity.
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Ensaio Cometa , Dano ao DNA/efeitos dos fármacos , Reparo do DNA , Mutagênicos/toxicidade , Apoptose/efeitos dos fármacos , Apoptose/genética , Ensaio Cometa/métodos , Células HeLa , Humanos , Peróxido de Hidrogênio/toxicidadeRESUMO
AIMS: Dietary antioxidants, including vitamin C, may be in part responsible for the cancer-preventive effects of fruits and vegetables. Human intervention trials with clinical endpoints have failed to confirm their protective effects, and mechanistic studies have given inconsistent results. Our aim was to investigate antioxidant/ pro-oxidant effects of vitamin C at the cellular level. EXPERIMENTAL APPROACH: We have used the comet assay to investigate effects of vitamin C on DNA damage, antioxidant status, and DNA repair, in HeLa (human tumor) cells, and HPLC to measure uptake of vitamin C into cells. RESULTS: Even at concentrations in the medium as high as 200 µM, vitamin C did not increase the background level of strand breaks or of oxidized purines in nuclear DNA. Vitamin C is taken up by HeLa cells and accumulates to mM levels. Preincubation of cells with vitamin C did not render them resistant to strand breakage induced by H2O2 or to purine oxidation by photosensitizer plus light. Vitamin C had no effect on the rate of repair of strand breaks or oxidized bases by HeLa cells. However, vitamin C at a concentration of less than 1 µM, or extract from cells preincubated for 6 h with vitamin C, was able to induce damage (strand breaks) in lysed, histone-depleted nuclei (nucleoids). CONCLUSION: In these cultured human cells, vitamin C displays neither antioxidant nor pro-oxidant properties; nor does it affect DNA strand break or base excision repair.
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Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Antioxidantes/metabolismo , Antioxidantes/toxicidade , Ácido Ascórbico/metabolismo , Ácido Ascórbico/toxicidade , Cromatografia Líquida de Alta Pressão , Ensaio Cometa , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Peróxido de Hidrogênio/toxicidade , Oxidantes/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Fármacos Fotossensibilizantes/toxicidade , Fatores de TempoRESUMO
Experiments were conducted to determine the validity of two common genotoxicity testing procedures, the comet assay and the micronucleus (MN) test, when applied to nanoparticles (NP). The comet assay is used to detect strand breaks (SB) induced in cellular DNA. There is a possibility of obtaining false positive results, if residual NP remain in proximity to the virtually naked DNA that results from lysis of agarose-embedded cells, and react with this DNA in ways that do not occur with chromatin in intact cells. However, data showed that if NP are deliberately present at high concentration with lysed cells, there is no change in SB with a range of NP. Only oleic acid-coated Fe3O4 NP induced damage, as these particles also produced equivalent alterations in whole cells. A modification of the comet assay incorporates digestion of DNA with lesion-specific endonucleases, notably formamidopyrimidine DNA glycosylase (FPG), which detects oxidized purines. Again there is a concern regarding the presence of residual NP with DNA of lysed cells, but this time because of the risk of false negative results if NP interfere with the FPG reaction. However, it was found that incubation of cells with NP before treatment with a known 8-oxoguanine-inducing agent does not lead to any decrease in the yield of FPG-sensitive sites. Chromosomal damage is detected with the MN assay, which depends on the use of cytochalasin B (CB) to prevent cell division and accumulates binucleate cells. It is known that CB also inhibits endocytosis, and thus might prevent NP uptake. Data demonstrated that if NP are added to cells together with CB, fewer MN are induced. It is therefore necessary to treat cells with NP prior to CB in order to avoid interference and possible false negative results.
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Teste de Materiais/métodos , Nanopartículas/toxicidade , Testes de Toxicidade/métodos , Animais , Células COS , Chlorocebus aethiops , Aberrações Cromossômicas/induzido quimicamente , Ensaio Cometa/métodos , Citocalasina B/farmacologia , DNA/metabolismo , Quebras de DNA/efeitos dos fármacos , DNA-Formamidopirimidina Glicosilase/metabolismo , Endocitose/efeitos dos fármacos , Endonucleases/metabolismo , Células HeLa , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Testes para Micronúcleos/métodos , Nanopartículas/química , Proteínas de Neoplasias/metabolismo , Tamanho da PartículaRESUMO
PURPOSE: The mechanisms involved in the appearance of a second neoplasia in patients with Hodgkin's disease (HD) are probably related to the genomic damage induced by the treatments administered and its repair. Here, we searched for some constitutive molecular mechanisms that in a basal manner may influence the behavior of HD patients. EXPERIMENTAL DESIGN: We aimed to evaluate with the Comet Assay whether baseline, induced, and unrepaired DNA damage differ between HD patients who did not develop a second neoplasia (HD-NST), HD patients who developed a second tumor (HD-ST), and healthy individuals; and to identify, through cDNA microarray hybridization, an expression signature of genes that could discriminate between the three groups. RESULTS: Baseline, induced, and unrepaired DNA damage was higher in HD-ST than in HD-NST and higher in the second group than in healthy donors. The genomic approach revealed two sets of genes that discriminated between healthy subjects and patients and between the three sets of individuals. Hsp40, RAD50, TPMT, Rap2a, E2F2, EPHX2, TBX21, and BATF were validated by reverse transcription-PCR. CONCLUSIONS: Functional and genomic techniques revealed that alterations in cell cycle, repair, detoxification, and stress response pathways could be involved in the development of HD and in the occurrence of a primary second neoplasia in these patients. Both approaches may be useful as biological markers in the clinical setting.
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Biomarcadores Tumorais/metabolismo , Doença de Hodgkin/metabolismo , Segunda Neoplasia Primária/metabolismo , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/genética , Criança , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Feminino , Doença de Hodgkin/genética , Humanos , Masculino , Pessoa de Meia-Idade , Segunda Neoplasia Primária/genética , Análise de Sequência com Séries de Oligonucleotídeos , Adulto JovemRESUMO
The comet assay (single-cell gel electrophoresis) is a sensitive and simple method for measuring DNA damage. An early modification of the assay involved the application of specific repair endonucleases to convert lesions to breaks; thus, for example, endonuclease III was used to measure oxidized pyrimidines. This concept has now been extended to produce an in vitro assay for DNA repair activity in a cell-free extract, for example from lymphocytes. The extract is incubated with substrate DNA containing specific base damage, and repair incision is detected as breaks in this DNA. We have recently been studying effects of phytochemicals in cultured cells, whether as antioxidants or as potential modulators of DNA repair. We realized that there is a need to check that observed effects that appear as an enhancement of repair (i.e. increased breaks in substrate DNA) are not simply due to a direct damaging effect of the phytochemical or to induction of non-specific nucleases. Here, we describe a rigorous approach to testing for this possibility, which we recommend to anyone carrying out similar experiments.
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Ensaio Cometa/instrumentação , Ensaio Cometa/métodos , Dano ao DNA , Antioxidantes/toxicidade , Ácido Ascórbico/metabolismo , Células Cultivadas , DNA/efeitos dos fármacos , Células HeLa , Humanos , Testes de Mutagenicidade/instrumentação , Testes de Mutagenicidade/métodos , Mutagênicos/análise , Reprodutibilidade dos TestesRESUMO
The role of dietary antioxidants in human health remains controversial. Fruits and vegetables in the diet are associated with lower rates of chronic disease, and this is often attributed to their content of antioxidants, and a resulting protection against oxidative stress. However, large-scale human trials with antioxidant supplements have shown, if anything, an increase in mortality. We have investigated the biological properties of beta-cryptoxanthin, a common carotenoid, in cell culture model systems, using the comet assay to measure DNA damage. At low concentrations, close to those found in plasma, beta-cryptoxanthin does not itself cause damage, but protects transformed human cells (HeLa and Caco-2) from damage induced by H(2)O(2) or by visible light in the presence of a photosensitizer. In addition, it has a striking effect on DNA repair, measured in different ways. Incubation of H(2)O(2)-treated cells with beta-cryptoxanthin led to a doubling of the rate of rejoining of strand breaks and had a similar effect on the rate of removal of oxidized purines by base excision repair. The latter effect was confirmed with an in vitro assay: cells were incubated with or without beta-cryptoxanthin before preparing an extract, which was then incubated with substrate DNA containing 8-oxo-7,8-dihydroguanine; incision was more rapid with the extract prepared from carotenoid-preincubated cells. No significant increases were seen in protein content of human 8-oxoguanine DNA glycosylase 1 or apurinic endonuclease 1. The apparent cancer-preventive effects of dietary carotenoids may depend on the enhancement of DNA repair as well as antioxidant protection against damage.
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Antioxidantes/farmacologia , Carotenoides/farmacologia , Dano ao DNA , Reparo do DNA , Xantofilas/farmacologia , Células CACO-2 , Ensaio Cometa , Criptoxantinas , DNA Glicosilases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Guanina/análogos & derivados , Guanina/biossíntese , Células HeLa , Humanos , OxirreduçãoRESUMO
The secreted mitogen vascular endothelial growth factor, VEGF, plays a pivotal role in angiogenesis. Hypoxia, inactivation of p53 and oncogenic K-Ras induce VEGF expression. Other factors such as p73 may also affect VEGF levels. Curiously, p73 may also regulate angiogenesis by affecting the expression of the pigment epithelium-derived factor, PEDF. Additionally, VEGF might harbor additional functions through the activation of E2F transcription factors. Recently, a new VEGF variant formed by alternative splicing, VEGF(165)b, has been described as exerting anti-angiogenic activity. We study here whether p73 isoforms levels -TAp73 and DeltaTAp73- and p53 and K-Ras status affect the expression of the above-mentioned angiogenesis-related genes (through the correlation between their expressions), the prognostic value of VEGF(165)b and PEDF and the correlation between VEGF and E2F-1 levels. Tumor and normal tissue of 112 colorectal cancer patients was analyzed to evaluate: (i) levels of TAp73, DeltaTAp73, VEGF, VEGF(165)b, PEDF and E2F-1 by quantitative real-time RT-PCR, (ii) p53 status by immunohistochemistry and (iii) mutations in the first exon of K-Ras by PCR-SSCP. Tumor characteristics were examined in each patient. Associations were observed between: (i) specific p73 isoforms and VEGF and VEGF(165)b expression; (ii) DeltaEx2p73 variant and downregulation of PEDF; (iii) VEGF and PEDF expression; (iv) inactive p53 and VEGF(165)b levels; (v) oncogenic K-Ras and PEDF downregulation; (vi) E2F-1 and VEGF expressions; (vii) VEGF(165)b downregulation and poor prognosis parameters of tumors. We conclude that the levels of p73 isoforms could affect the expression of VEGF, VEGF(165)b and PEDF. This scenario becomes complicated because a feedback between VEGF and PEDF may exist. VEGF may activate the E2F-1 factor. Mutations in K-Ras could negatively regulate PEDF expression. p53 inactivation might result in compensatory mechanisms such as over-expression of VEGF(165)b. Our data support the role of VEGF(165)b as a tumor suppressor factor in colorectal carcinogenesis and its possible prognosis value.
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Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/metabolismo , Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas Nucleares/metabolismo , Serpinas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Idoso , Idoso de 80 Anos ou mais , Regulação para Baixo , Fator de Transcrição E2F1/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Genes ras , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mutação , Neovascularização Patológica , Valor Preditivo dos Testes , Prognóstico , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
MicroRNAs (miRNAs) are noncoding RNAs that regulate expression of target mRNAs and are controlled by tumor suppressors and oncogenes. Altered expression of specific miRNAs in several tumor types and its association with poor prognosis parameters have been reported. Fewer data are available on its impact on patients' survival. We studied the impact of the expression of miR-17-5p, miR-106a, and miR-126 on survival and its correlation with the levels of their target mRNAs and host gene and TP53 alterations. We assessed in 110 colon cancer patients the levels of miR-17-5p, miR-106a, miR-126, E2F1, and EGFL7 by quantitative real-time RT-PCR and loss of heterozygosity (LOH) in the TP53 region. Tumor characteristics, disease-free survival (DFS), and overall survival (OS) were examined in each patient. Altered expression of miR-17-5p, miR-106a, and EGFL7 was associated with pathological tumor features of poor prognosis. Downregulation of miR-106a predicted shortened DFS (P = 0.03) and OS (P = 0.04). miR-17-5p correlated with DFS only at early stages (P = 0.07). Inverse correlations were found between miR-17-5p and miR-106a levels and their target expression, E2F1 (P = 0.04 and P = 0.03, respectively). No correlation was found between miR-126 expression and its host gene levels, EGFL7. miR-106a deregulation was revealed as a marker of DFS and OS independent of tumor stage. The lack of association between expression of miR-126 and its host gene EGFL7 suggests their regulation by independent stimuli. Inverse correlation between miR-17-5p and miR-106a and E2F1 levels supports E2F1 as a target mRNA for the two miRNAs.
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Neoplasias do Colo/mortalidade , MicroRNAs/metabolismo , Proteínas de Ligação ao Cálcio , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Família de Proteínas EGF , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Perda de Heterozigosidade , MicroRNAs/genética , RNA Neoplásico/metabolismo , Análise de SobrevidaRESUMO
PURPOSE: The regulator of angiogenesis most extensively studied is VEGF. VEGF mRNA in plasma from patients with colorectal cancer was analyzed as a possible surrogate marker of tumor angiogenesis. METHODS: VEGF mRNA was measured by quantitative PCR in plasma, tumors and circulating tumor cells from colorectal cancer patients. Circulating VEGF protein was analyzed by ELISA. Microvessel density was determined. RESULTS: Levels of VEGF mRNA and protein in plasma were higher in patients than in controls. VEGF mRNA was overexpressed in tumors with respect to normal tissues. Levels of VEGF protein were associated with VEGF mRNA in plasma, but no associations with tumor samples were found. A trend to statistical significance was shown between high VEGF mRNA and vascular invasion. MVD was not related to VEGF mRNA in plasma. CONCLUSIONS: Thus, VEGF mRNA could be a marker similar to VEGF protein in plasma.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neovascularização Patológica/genética , RNA Mensageiro/genética , Fator A de Crescimento do Endotélio Vascular/genética , Neoplasias Colorretais/cirurgia , Primers do DNA , Marcadores Genéticos , Humanos , Neovascularização Patológica/patologia , Reação em Cadeia da Polimerase , RNA Neoplásico/sangue , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificaçãoRESUMO
We studied by real-time PCR cyclin D1 and thymidylate synthase (TS) mRNA in plasma as possible markers of clinical outcome in breast cancer. We observed poor outcome in patients with presence of cyclin D1 mRNA in good-prognosis groups, such as negative vascular invasion. Presence of both markers was associated with non-response to treatment after relapse. In patients treated with tamoxifen, a trend to significant relation between poor outcome and cyclin D1 mRNA was found. Cyclin D1 mRNA in plasma could identify patients with poor overall survival in good-prognosis groups and patients non-responsive to tamoxifen.
Assuntos
Neoplasias da Mama/sangue , Ciclina D1/análise , RNA Mensageiro/sangue , Timidilato Sintase/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Ciclina D1/sangue , Intervalo Livre de Doença , Feminino , Humanos , Prognóstico , Estudos Prospectivos , Análise de Sobrevida , Tamoxifeno/uso terapêutico , Timidilato Sintase/sangue , Resultado do TratamentoRESUMO
PURPOSE: LISCH7 is a gene potentially regulated by p53 that is up-regulated in metastasis development. Our hypothesis was that the expression of LISCH7 in primary colorectal tumors determined certain characteristics of the tumors, as well as their behavior, and that its identification in plasma could serve as a prognostic marker. EXPERIMENTAL DESIGN: We tested this hypothesis in a series of 115 tumors and normal tissues and in 83 plasmas from patients with sporadic colorectal carcinomas, as well as in 20 healthy control plasmas in which the expression levels of the gene were measured by real-time PCR. The expression data were contrasted with clinicopathologic variables. RESULTS: Although LISCH7 expression was not detected in any control plasma samples, it was positive in 25 (30.1%) plasmas from patients (P = 0.002). LISCH7 mRNA in plasma was significantly associated with the pathologic stage (P = 0.019), with lymph node metastasis (P = 0.008) and with vascular invasion (P = 0.005). Expression was not detected in any normal tissues but was detected in 80 tumor tissues, with a clear association found with vascular invasion (P = 0.027). Moreover, we show that LISCH7 was specifically expressed by the epithelial tumor cells. The adjusted overall survival study showed independent prognostic values for LISCH7 expression levels in tumor tissues (hazard ratio, 3.45; 95% confidence interval, 1.19-9.98). CONCLUSIONS: Our results suggest that LISCH7 is a good tumor marker whose expression levels could be considered as a poor prognosis factor in human colon cancer. Furthermore, plasma is suggested as a feasible source of nucleic acids for subsequent noninvasive prognostic studies.
Assuntos
Neoplasias do Colo/sangue , Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , RNA Mensageiro/metabolismo , Receptores de LDL/sangue , Receptores de LDL/metabolismo , Receptores de Lipoproteínas/biossíntese , Receptores de Lipoproteínas/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Idoso , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Metástase Linfática , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , Resultado do Tratamento , Proteína Supressora de Tumor p53/metabolismoRESUMO
INTRODUCTION: Deregulation of Polycomb member Bmi-1 is involved in cell proliferation and human oncogenesis. Modulation of Bmi-1 is found in several tumor tissues, including primary breast carcinomas; however, analysis of Bmi-1 in plasma of cancer patients has not been reported. This is the first study that evaluates Bmi-1 in plasma by using a large series of primary breast carcinomas to investigate the presence at diagnosis of detectable Bmi-1 mRNA in plasma and possible correlations between this event and a series of clinical-pathological parameters of the tumors. METHODS: Bmi-1 expression levels were quantified in plasma of 111 breast cancer patients and in 20 healthy controls by real-time quantitative polymerase chain reaction. RESULTS: Cancer patients with the presence of Bmi-1 mRNA in plasma had higher levels of Bmi-1 expression than healthy controls with Bmi-1 mRNA in plasma. The higher expression levels of Bmi-1 correlated with well-established markers of poor clinical outcome in breast cancer such as positive p53 immunostaining and negative progesterone receptors. Moreover, we described for the first time a statistically significant correlation between Bmi-1 expression in plasma of breast cancer patients and disease-free and overall survival in advanced stages. CONCLUSION: Our results suggest that levels of Bmi-1 expression may be a surrogate marker of poor prognosis and may become clinically useful as noninvasive diagnostic markers.