Maria Teresa Russo

Accumulation of the Oxidative Base Lesion 8-Hydroxyguanine in DNA of Tumor-Prone Mice Defective in Both the Myh and Ogg1 DNA Glycosylases

The OGG1 and MYH DNA glycosylases prevent the accumulation of DNA 8-hydroxyguanine. In Myh−/− mice, there was no time-dependent accumulation of DNA 8-hydroxyguanine in brain, small intestine, lung, spleen, or kidney. Liver was an exception to this general pattern. Inactivation of both MYH and OGG1 caused an age-associated accumulation of DNA 8-hydroxyguanine in lung and small intestine. The effects of abrogated OGG1 and MYH on hepatic DNA 8-hydroxyguanine levels were additive. Because there is an increased incidence of lung and small intestine cancer in Myh−/−/Ogg1−/− mice, these findings support a causal role for unrepaired oxidized DNA bases in cancer development.

The Oxidized Deoxynucleoside Triphosphate Pool Is a Significant Contributor to Genetic Instability in Mismatch Repair-Deficient Cells

ABSTRACT Oxidation is a common form of DNA damage to which purines are particularly susceptible. We previously reported that oxidized dGTP is potentially an important source of DNA 8-oxodGMP in mammalian cells and that the incorporated lesions are removed by DNA mismatch repair (MMR). MMR deficiency is associated with a mutator phenotype and widespread microsatellite instability (MSI). Here, we identify oxidized deoxynucleoside triphosphates (dNTPs) as an important cofactor in this genetic instability. The high spontaneous hprt mutation rate of MMR-defective msh2−/− mouse embryonic fibroblasts was attenuated by expression of the hMTH1 protein, which degrades oxidized purine dNTPs. A high level of hMTH1 abolished their mutator phenotype and restored the hprt mutation rate to normal. Molecular analysis of hprt mutants showed that the presence of hMTH1 reduced the incidence of mutations in all classes, including frameshifts, and also implicated incorporated 2-oxodAMP in the mutator phenotype. In hMSH6-deficient DLD-1 human colorectal carcinoma cells, overexpression of hMTH1 markedly attenuated the spontaneous mutation rate and reduced MSI. It also reduced the incidence of −G and −A frameshifts in the hMLH1-defective DU145 human prostatic cancer cell line. Our findings indicate that incorporation of oxidized purines from the dNTP pool may contribute significantly to the extreme genetic instability of MMR-defective human tumors.

Loss of the CBX7 Gene Expression Correlates with a Highly Malignant Phenotype in Thyroid Cancer

Using gene expression profiling, we found that the CBX7 gene was drastically down-regulated in six thyroid carcinoma cell lines versus control cells. The aims of this study were to determine whether CBX7 is related to the thyroid cancer phenotype and to try to identify new tools for the diagnosis and prognosis of thyroid cancer. We thus evaluated CBX7 expression in various snap-frozen and paraffin-embedded thyroid carcinoma tissues of different degrees of malignancy by quantitative reverse transcription-PCR and immunohistochemistry, respectively. CBX7 expression progressively decreased with malignancy grade and neoplasia stage. Indeed, it decreased in an increasing percentage of cases going from benign adenomas to papillary (PTC), follicular, and anaplastic (ATC) thyroid carcinomas. This finding coincides with results obtained in rat and mouse models of thyroid carcinogenesis. CBX7 loss of heterozygosity occurred in 36.8% of PTC and in 68.7% of ATC. Restoration of CBX7 expression in thyroid cancer cells reduced growth rate, with a retention in the G(1) phase of the cell cycle, suggesting that CBX7 can contribute to the proliferation of the transformed thyroid cells. In conclusion, loss of CBX7 expression correlates with a highly malignant phenotype in thyroid cancer patients.

Comparative study of ZnO and TiO2 nanoparticles: physicochemical characterisation and toxicological effects on human colon carcinoma cells

Abstract Despite human gastrointestinal exposure to nanoparticles (NPs), data on NPs toxicity in intestinal cells are quite scanty. In this study we evaluated the toxicity induced by zinc oxide (ZnO) and titanium dioxide (TiO2) NPs on Caco-2 cells. Only ZnO NPs produced significant cytotoxicity, evaluated by two different assays. The presence of foetal calf serum in culture medium significantly reduced ZnO NPs toxicity as well as ion leakage and NP-cell interaction. The two NPs increased the intracellular amount of reactive oxygen species (ROS) after 6 h treatment. However, only ZnO NPs increased ROS and induced IL-8 release both after 6 and 24 h. Experimental data indicate a main role of chemical composition and solubility in ZnO NPs toxicity. Moreover our results suggest a key role of oxidative stress in ZnO NPs cytotoxicity induction related both to ion leakage and to cell interaction with NPs in serum-free medium.

Role of Mismatch Repair and MGMT in Response to Anticancer Therapies

Tumor resistance to cytotoxic chemotherapy drugs and their toxicity to normal cells are major clinical obstacles to anticancer therapy effectiveness. Alterations in various DNA repair pathways play a key role in the development of both mechanisms of drug resistance and toxicity. Since deregulation of the DNA damage response and alterations in DNA repair pathways are relatively common in human cancer, the knowledge of these alterations in cancer cells would be an important predictive factor for the clinical response to chemotherapy and a useful guide in designing an appropriate therapeutic strategy. This review is focused on the mismatch repair (MMR) pathway and the O(6)-methylguanine-DNA-methyltransferase (MGMT) repair protein. In particular, we examine how inactivation of these DNA repair mechanisms might affect the response of tumor cells to chemotherapy, with a special emphasis on agents inducing methylation and oxidative DNA damage and interstrand DNA cross-links (ICLs). In addition, we provide novel experimental evidence indicating that MMR is required for efficient repair of ICLs via stabilization of RAD51 containing repair intermediates. Finally, we discuss possible emerging therapeutical strategies for treating MMR-defective tumors.

A role for oxidized DNA precursors in Huntington's disease-like striatal neurodegeneration.

Several human neurodegenerative disorders are characterized by the accumulation of 8-oxo-7,8-dihydroguanine (8-oxodG) in the DNA of affected neurons. This can occur either through direct oxidation of DNA guanine or via incorporation of the oxidized nucleotide during replication. Hydrolases that degrade oxidized purine nucleoside triphosphates normally minimize this incorporation. hMTH1 is the major human hydrolase. It degrades both 8-oxodGTP and 8-oxoGTP to the corresponding monophosphates. To investigate whether the incorporation of oxidized nucleic acid precursors contributes to neurodegeneration, we constructed a transgenic mouse in which the human hMTH1 8-oxodGTPase is expressed. hMTH1 expression protected embryonic fibroblasts and mouse tissues against the effects of oxidants. Wild-type mice exposed to 3-nitropropionic acid develop neuropathological and behavioural symptoms that resemble those of Huntingtons disease. hMTH1 transgene expression conferred a dramatic protection against these Huntingtons disease–like symptoms, including weight loss, dystonia and gait abnormalities, striatal degeneration, and death. In a complementary approach, an in vitro genetic model for Huntingtons disease was also used. hMTH1 expression protected progenitor striatal cells containing an expanded CAG repeat of the huntingtin gene from toxicity associated with expression of the mutant huntingtin. The findings implicate oxidized nucleic acid precursors in the neuropathological features of Huntingtons disease and identify the utilization of oxidized nucleoside triphosphates by striatal cells as a significant contributor to the pathogenesis of this disorder.

Role of MUTYH and MSH2 in the Control of Oxidative DNA Damage, Genetic Instability, and Tumorigenesis

Mismatch repair is the major pathway controlling genetic stability by removing mispairs caused by faulty replication and/or mismatches containing oxidized bases. Thus, inactivation of the Msh2 mismatch repair gene is associated with a mutator phenotype and increased cancer susceptibility. The base excision repair gene Mutyh is also involved in the maintenance of genomic integrity by repairing premutagenic lesions induced by oxidative DNA damage. Because evidence in bacteria suggested that Msh2 and Mutyh repair factors might have some overlapping functions, we investigated the biological consequences of their single and double inactivation in vitro and in vivo. Msh2(-/-) mouse embryo fibroblasts (MEF) showed a strong mutator phenotype at the hprt gene, whereas Mutyh inactivation was associated with a milder phenotype (2.9 x 10(-6) and 3.3 x 10(-7) mutation/cell/generation, respectively). The value of 2.7 x 10(-6) mutation/cell/generation in Msh2(-/-)Mutyh(-/-) MEFs did not differ significantly from Msh2(-/-) cells. When steady-state levels of DNA 8-oxo-7,8-dihydroguanine (8-oxoG) were measured in MEFs of different genotypes, single gene inactivation resulted in increases similar to those observed in doubly defective cells. In contrast, a synergistic accumulation of 8-oxoG was observed in several organs of Msh2(-/-)Mutyh(-/-) animals, suggesting that in vivo Msh2 and Mutyh provide separate repair functions and contribute independently to the control of oxidative DNA damage. Finally, a strong delay in lymphomagenesis was observed in Msh2(-/-)Mutyh(-/-) when compared with Msh2(-/-) animals. The immunophenotype of these tumors indicate that both genotypes develop B-cell lymphoblastic lymphomas displaying microsatellite instability. This suggests that a large fraction of the cancer-prone phenotype of Msh2(-/-) mice depends on Mutyh activity.

Different mechanisms are involved in oxidative DNA damage and genotoxicity induction by ZnO and TiO2 nanoparticles in human colon carcinoma cells.

In this work we investigated the genotoxicity of zinc oxide and titanium dioxide nanoparticles (ZnO NPs; TiO2 NPs) induced by oxidative stress on human colon carcinoma cells (Caco-2 cells). We measured free radical production in acellular conditions by Electron Paramagnetic Resonance technique and genotoxicity by micronucleus and Comet assays. Oxidative DNA damage was assessed by modified Comet assay and by measuring 8-oxodG steady state levels. The repair kinetics of DNA oxidation as well as the expression levels of hOGG1 were also analyzed. Even if both NPs were able to produce ROS in acellular conditions and to increase 8-oxodG levels in Caco-2 cells, only ZnO NPs resulted genotoxic inducing micronuclei and DNA damage. Furthermore, Caco-2 cells exposed to ZnO NPs were not able to repair the oxidative DNA damage that was efficiently repaired after TiO2 NPs treatment, through OGG1 involvement. These results indicate that the high oxidant environment caused by ZnO NPs in our cellular model can induce DNA damage and affect the repair pathways.

MUTYH mutations associated with familial adenomatous polyposis: functional characterization by a mammalian cell-based assay†

MUTYH‐associated polyposis (MAP) is a colorectal cancer syndrome, due to biallelic mutations of MUTYH. This Base Excision Repair gene encodes for a DNA glycosylase that specifically mitigates the high mutagenic potential of the 8‐hydroxyguanine (8‐oxodG) along the DNA. Aim of this study was to characterize the biological effects, in a mammalian cell background, of human MUTYH mutations identified in MAP patients (137insIW [c.411_416dupATGGAT; p.137insIleTrp]; R171W [c.511C>T; p.Arg171Trp]; E466del [c.1395_1397delGGA; p.Glu466del]; Y165C [c.494A>G; p.Tyr165Cys]; and G382D [c.1145G>A; p.Gly382Asp]). We set up a novel assay in which the human proteins were expressed in Mutyh−/− mouse defective cells. Several parameters, including accumulation of 8‐oxodG in the genome and hypersensitivity to oxidative stress, were then used to evaluate the consequences of MUTYH expression. Human proteins were also obtained from Escherichia coli and their glycosylase activity was tested in vitro. The cell‐based analysis demonstrated that all MUTYH variants we investigated were dysfunctional in Base Excision Repair. In vitro data complemented the in vivo observations, with the exception of the G382D mutant, which showed a glycosylase activity very similar to the wild‐type protein. Our cell‐based assay can provide useful information on the significance of MUTYH variants, improving molecular diagnosis and genetic counseling in families with mutations of uncertain pathogenicity. Hum Mutat 30:1–8, 2009.

The Base Excision Repair Pathway Is Required for Efficient Lentivirus Integration

An siRNA screen has identified several proteins throughout the base excision repair (BER) pathway of oxidative DNA damage as important for efficient HIV infection. The proteins identified included early repair factors such as the base damage recognition glycosylases OGG1 and MYH and the late repair factor POLß, implicating the entire BER pathway. Murine cells with deletions of the genes Ogg1, Myh, Neil1 and Polß recapitulate the defect of HIV infection in the absence of BER. Defective infection in the absence of BER proteins was also seen with the lentivirus FIV, but not the gammaretrovirus MMLV. BER proteins do not affect HIV infection through its accessory genes nor the central polypurine tract. HIV reverse transcription and nuclear entry appear unaffected by the absence of BER proteins. However, HIV integration to the host chromosome is reduced in the absence of BER proteins. Pre-integration complexes from BER deficient cell lines show reduced integration activity in vitro. Integration activity is restored by addition of recombinant BER protein POLß. Lentiviral infection and integration efficiency appears to depend on the presence of BER proteins.