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- Mutat Res 663():iii (2009)
- p53 mutations as fingerprints for aristolochic acid – an environmental carcinogen in endemic (Balkan) nephropathy
- Mutat Res 663():1-6 (2009)
The activation of protooncogenes and inactivation of tumor suppressor genes are considered to be the main molecular events in the multistep process of carcinogenesis. Mutations of the TP53 tumor suppressor gene have been found in nearly all tumor types and are estimated to contribute to more than 50% of all cancers. Most mutations lead to the synthesis of highly stable, inactive proteins that accumulate in the nucleus of cancer cells. Among the 393 codons of the human p53 gene, 222 are targets of 698 different types of mutations. Alterations of codons 175, 248, 273 and 282 correspond to 19% of all mutations and are considered general hot spot mutations. Dietary exposure to aristolochic acid (AA), an established nephrotoxin and human carcinogen found in all Aristolochia species was shown to be the causative agent of aristolochic acid nephropathy (previously called Chinese herbs nephropathy). This syndrome is characterized by proximal tubular damage, renal interstitial f! ibrosis, slow progression to the end stage renal disease and a high prevalence of upper urinary tract urothelial carcinoma (otherwise a highly unusual location). AA preferentially binds to purines in DNA and is associated with a high frequency of A → T transversions in the p53 gene. Rats treated with AA develop A:T → T:A mutations in codon 61. The pathological and clinical features of endemic (Balkan) nephropathy closely resemble those associated with aristolochic acid nephropathy except for the slower progression to end stage renal disease and longer cumulative period before the appearance of urothelial cancer. Recently, we reported the presence of AA-DNA adducts in renal cortex and A → T p53 mutations in tumor tissue of patients from Croatia and Bosnia with endemic nephropathy. These data support the hypothesis that dietary exposure to AA is a major risk factor for endemic (Balkan) nephropathy.
- Site-specific analysis of UV-induced cyclobutane pyrimidine dimers in nucleotide excision repair-proficient and -deficient hamster cells: Lack of correlation with mutational spectra
- Mutat Res 663():7-14 (2009)
Irradiation of cells with UVC light induces two types of mutagenic DNA photoproducts, i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP). To investigate the relationship between the frequency of UV-induced photolesions at specific sites and their ability to induce mutations, we quantified CPD formation at the nucleotide level along exons 3 and 8 of the hprt gene using ligation-mediated PCR, and determined the mutational spectrum of 132 UV-induced hprt mutants in the AA8 hamster cell line and of 165 mutants in its nucleotide excision repair-defective derivative UV5. In AA8 cells, transversions predominated with a strong strand bias towards thymine-containing photolesions in the non-transcribed strand. As hamster AA8 cells are proficient in global genome repair of 6-4PP but selectively repair CPD from the transcribed strand of active genes, most mutations probably resulted from erroneous bypass of CPD in the non-transcribed str! and. However, the relative incidence of CPD and the positions where mutations most frequently arose do not correlate. In fact some major damage sites hardly gave rise to the formation of mutations. In the repair-defective UV5 cells, mutations were almost exclusively C > T transitions caused by photoproducts at PyC sites in the transcribed strand. Even though CPD were formed at high frequencies at some TT sites in UV5, these photoproducts did not contribute to mutation induction at all. We conclude that, even in the absence of repair, large variations in the level of induction of CPD at different sites throughout the two exons do not correspond to frequencies of mutation induction.
- In vivo treatment with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces organ-specific alterations in in vitro repair of DNA pyridyloxobutylation
- Mutat Res 663():15-21 (2009)
To investigate the mechanisms responsible for inter-organ differences in susceptibility to 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK)-induced carcinogenesis, the objectives were to compare DNA repair activities of extracts from mouse lung and liver towards NNK-induced pyridyloxobutyl (POB) damage to plasmid DNA, and to determine if and the mechanism by whichin vivo NNK treatment of mice alters DNA repair. Repair activity of POB adducts was three times greater in mouse liver than in mouse lung (P < 0.05). Repair activities of lung extracts from mice 4 or 24 h post-NNK treatment were 30–45% those of control (P < 0.05). Conversely, POB adduct repair was 2–3 times higher in liver extracts from NNK treated mice than in controls (4 h, 24 h, P < 0.05). NNK treatment also decreased incision of POB adducts by 92% (4 h, P < 0.05) in lung and increased incision by 169% (24 h, P < 0.05) in liver. NNK decreased immunoreactive levels of the incision protein RPA in lung (! P < 0.05) 4 h post-treatment but increased immunoreactive lung RPA and XPB after 24 h (P < 0.05). In liver, levels of immunoreactive proteins, XPA, XPB and ERCC1 were increased after NNK treatment (24 h, P < 0.05). Binding of XPA and XPB from liver extracts to POB adducts increased following NNK treatment, while binding of XPA and XPB from lung decreased (4 h, 24 h). These results suggest that lower incision activity of nucleotide excision repair and NNK-mediated alterations in levels and activities of key incision proteins contribute to the relative susceptibility of mouse lung to NNK-induced carcinogenesis.
- Divergence to apoptosis from ROS induced cell cycle arrest: Effect of cadmium
- Mutat Res 663():22-31 (2009)
Recently, the role of cadmium (Cd) in immunosupression has gained importance. Nevertheless, the signaling pathways underlying cadmium-induced immune cell death remains largely unclear. In accordance to our previous in vivo report, and to evaluate the further details of the mechanism, we have investigated the effects of cadmium (CdCl2, H2O) on cell cycle regulation and apoptosis in splenocytes in vitro. Our results have revealed that reactive oxygen species (ROS) and p21 are involved in cell cycle arrest in a p53 independent manner but late hour apoptotic response was accompanied by the p53 up-regulation, loss of mitochondrial transmembrane potential (MTP), down-regulation of Bcl-xl, activation of caspase-3 and release of cytochrome c (Cyt c). However, pifithrin alfa (PFT-α), an inhibitor of p53, fails to rescue the cells from the cadmium-induced cell cycle arrest but prevents Bcl-xl down-regulation and loss of Δψm, which indicates that there is an involvement of p53! in apoptosis. In contrast, treatment with N-acetyl cysteine (NAC) can prevent cell cycle arrest and p21 up-regulation at early hours. Although it is clear that, NAC has no effect on apoptosis, p53 expression and MPT changes at late stage events. Taken together, we have demonstrated that cadmium promotes ROS generation, which potently initiates the cell cycle arrest at early hours and finally induces p53-dependent apoptosis at later part of the event.
- DNA repair in modeled microgravity: Double strand break rejoining activity in human lymphocytes irradiated with γ-rays
- Mutat Res 663():32-39 (2009)
Cell response to ionising radiation depends, besides on genetic and physiological features of the biological systems, on environmental conditions occurring during DNA repair. Many data showed that microgravity, experienced by astronauts during space flights or modeled on Earth, causes apoptosis, cytoskeletal alteration, cell growth inhibition, increased frequency of mutations and chromosome aberrations. In this study, we analysed the progression of the rejoining of double strand breaks (DSBs) in human peripheral blood lymphocytes (PBLs) irradiated with γ-rays and incubated in static condition (1g) or in modeled microgravity (MMG). γ-H2AX foci formation and disappearance, monitored during the repair incubation, showed that the kinetics of DSBs rejoining was different in the two gravity conditions. The fraction of foci-positive cells decreased slower in MMG than in 1g at 6 and 24 h after irradiation (P < 0.01) and the mean number of γ-H2AX foci per nucleus was signifi! cantly higher in MMG than in 1g at the same time-points (P < 0.001). In the same samples we determined apoptotic level and the rate of DSB rejoining during post-irradiation incubation. A significant induction of apoptosis was observed in MMG at 24 h after irradiation (P < 0.001), whereas at shorter times the level of apoptosis was slightly higher in MMG respect to 1g. In accordance with the kinetics of γ-H2AX foci, the slower rejoining of radiation-induced DSBs in MMG was observed by DNA fragmentation analyses during the repair incubation; the data of pulsed-field gel electrophoresis assay showed that the fraction of DNA released in the gel was significantly higher in PBL incubated in MMG after irradiation with respect to cells maintained in 1g. Our results provide evidences that MMG incubation during DNA repair delayed the rate of radiation-induced DSB rejoining, and increased, as a consequence, the genotoxic effects of ionising radiation.
- Suppression of topoisomerase IIα expression and function in human cells decreases chromosomal radiosensitivity
- Mutat Res 663():40-45 (2009)
The mechanism behind chromatid break formation is as yet unclear, although it is known that DNA double-strand breaks (DSBs) are the initiating lesions. Chromatid breaks formed in cells in the G2-phase of the cell-cycle disappear ('rejoin') as a function of time between radiation exposure and cell fixation. However, the kinetics of disappearance of chromatid breaks does not correspond to those of DSB rejoining, leading us to seek alternative models. We have proposed that chromatid breaks could be formed indirectly from DSB and that the mechanism involves topoisomerase IIα. In support of this hypothesis we have recently shown that frequencies of radiation-induced chromatid breaks are lower in two variant human promyelocytic leukaemic cell lines with reduced topoisomerase IIα expression. Here we report that suppression of topoisomerase IIα in human hTERT-RPE1 cells, either by its abrogation using specific siRNA or by inhibition of its catalytic activity with the in! hibitor ICRF-193, causes a reduction in frequency of chromatid breaks in radiation-exposed cells. The findings support our hypothesis for the involvement of topoisomerase IIα in the formation of radiation-induced chromatid breaks, and could help explain inter-individual variation in human chromosomal radiosensitivity; elevation of which has been linked with cancer susceptibility.
- Mitochondrial mutant cells are hypersensitive to ionizing radiation, phleomycin and mitomycin C
- Mutat Res 663():46-51 (2009)
Mitochondrial DNA (mtDNA) is an important contributor to the ATP-generating oxidative phosphorylation complex. Single nucleotide mutations in mitochondrial genes involved in ATP synthesis result in a broad range of diseases. Leber optic atrophy and Leigh's syndrome are two such diseases arising from point mutations in the mitochondrial genome. Here, ionizing radiation, phleomycin and mitomycin C (MMC) were used to induce structural chromosomal aberrations in Leber's and Leigh's cells to investigate how these mitochondrial mutations affect the cell's DNA repair processes. Because of the energy deprivation that results from mitochondrial mutations, we hypothesized that these mutant cells would demonstrate hypersensitivity when exposed to oxidative and genotoxic stress and we also expected that these cells would not be able to repair nuclear DNA damage as efficiently as normal cells. As a consequence, these mutant cells are expected to show increased levels of DNA damage,! longer cell cycle delays and increased levels of cell death. Following acute radiation exposure these mutant cells showed an increase in the number of chromosomal aberrations and decreased mitotic indices when compared with normal human lymphoblastoid cells with wild-type mtDNA. When exposed to phleomycin or MMC, the mitochondrial mutant cells again showed hypersensitivity and decreased mitotic indices compared to normal cells. These results suggest that Leber's and Leigh's cells have an impaired ability to cope with oxidative and genotoxic stress. These observations may help explain the role of ATP generation in understanding the enhanced sensitivity of mitochondrial mutant cells to cancer therapeutic agents and to adverse environmental exposure, suggesting that individuals with mtDNA mutations may be at a greater risk for cancer and other diseases that result from an accumulation of nuclear DNA damage.
- Functional polymorphisms of cyclooxygenase-2 (COX-2) gene and risk for esophageal squmaous cell carcinoma
- Mutat Res 663():52-59 (2009)
Cyclooxygenase-2 (COX-2) influences carcinogenesis through regulation of angiogenesis, apoptosis and cytokine expression. We aimed to evaluate association of COX-2 polymorphisms with predisposition to esophageal squamous cell carcinoma (ESCC), its phenotype variability and modulation of environmental risk in northern Indian population. We genotyped 174 patients with ESCC and 216 controls for COX-2 gene polymorphisms (−765G>C; −1195G>A; −1290A>G; 3′UTR 8473T>C) using PCR-RFLP. Data were statistically analyzed using chi-square test and logistic regression model. COX-2 −765C allele carriers were at increased risk for ESCC (OR = 1.66; 95% CI = 1.08–2.54; P = 0.004). However, −1195G>A; −1290A>G; 3′UTR 8473T>C polymorphisms of COX-2 gene were not significantly associated with ESCC. We observed significantly enhanced risk for ESCC due to interaction between COX-2 −1195GA × −765GC + CC genotypes (OR = 4.60; 95% CI = 1.63–13.01; P = 0.004). High risk to! ESCC was also observed with respect to COX-2 haplotypes, A−1290G−1195C−765T8473 and A−1290A−1195C−765T8473 [OR = 3.35; 95% CI = 0.83–13.44; P = 0.089; OR = 4.28; 95% CI = 0.43–42.40; P = 0.246] however, it was not statistically significant. Stratification of subjects based on gender showed that females were at higher risk for ESCC due to COX-2 −765C carrier genotypes (OR = 2.97; 95% CI = 1.23–7.18; P = 0.016). In association of genotypes with clinical characteristics, −765C carrier genotype conferred risk of ESCC in middle third of esophagus (OR = 1.78; 95% CI = 1.08–2.93; P = 0.023). In case-only analysis, interaction of environmental risk factors and COX-2 genotypes did not further modulate the risk for ESCC. In summary, COX-2 −765G>C polymorphism confers ESCC susceptibility particularly in females and patients with middle third anatomical location of the tumor. Interaction of COX-2 −1195GA and −765C carrier genotypes also modulates ESCC ris! k.
- The RAD9-dependent gene trans-activation is required for excision repair of active genes but not for repair of non-transcribed DNA
- Mutat Res 663():60-68 (2009)
The Saccharomyces cerevisiae RAD9 and RAD24 are two cell cycle checkpoint genes required for UV-dependent up-regulation of a battery of genes involved in different metabolic pathways. RAD9 is also implicated in nucleotide excision repair (NER); however, its precise role is still unclear. For the present study, we made use of the high-resolution primer extension technique to show that the RAD9-deleted cells are deficient in the repair of both strands of the URA3 gene. Interestingly, this defect was suppressed by over-expressing the RAD24 gene, suggesting that the role of RAD9 in NER is indirect probably through the UV-dependent trans-activation of some NER factors. Accordingly, we present evidence that the inhibition of UV-related de novo protein synthesis by cycloheximide has no effect on the rad9Δ mutant while it suppresses the correcting effect of RAD24 over-expression. Importantly, we have also shown that RAD9 has no role in repair of transcriptionally inactive DNA! sequences (URA3 promoter and transcriptionally silent GAL10 gene). Furthermore, de novo protein synthesis was not required for NER in the absence of transcription-coupled NER. This implies that RAD9-dependent gene up-regulation is required for NER only when this process is coupled to transcription.
- The anti-apoptotic role for p53 following exposure to ultraviolet light does not involve DDB2
- Mutat Res 663():69-76 (2009)
The p53 tumour suppressor is a transcription factor that can either activate or repress the expression of specific genes in response to cellular stresses such as exposure to ultraviolet light. The p53 protein can exert both pro- and anti-apoptotic effects depending on cellular context. In primary human fibroblasts, p53 protects cells from UV-induced apoptosis at moderate doses but this is greatly affected by the nucleotide excision repair (NER) capacity of the cells. The damage-specific DNA binding protein 2 (DDB2) is involved in NER and is associated with xeroderma pigmentosum subgroup E (XP-E). Importantly, DDB2 is also positively regulated by the p53 protein. To study the potential interplay between DDB2 and p53 in determining the apoptotic response of primary fibroblasts exposed to UV light, the expression of these proteins was manipulated in primary normal and XP-E fibroblast strains using human papillomavirus E6 protein (HPV-E6), RNA interference and recombinant ! adenoviruses expressing either p53 or DDB2. Normal and XP-E fibroblast strains were equally sensitive to UV-induced apoptosis over a broad range of doses and disruption of p53 in these strains using HPV-E6 or RNA interference led to a similar increase in apoptosis following exposure to UV light. In contrast, forced expression of p53 or DDB2 did not affect UV-induced apoptosis greatly in these normal or XP-E fibroblast strains. Collectively, these results indicate that p53 is primarily protective against UV-induced apoptosis in primary human fibroblasts and this activity of p53 does not require DDB2.
- DNA polymerase ζ is essential for hexavalent chromium-induced mutagenesis
- Mutat Res 663():77-83 (2009)
Translesion synthesis (TLS) is a unique DNA damage tolerance mechanism involved in the replicative bypass of genetic lesions in favor of uninterrupted DNA replication. TLS is critical for the generation of mutations by many different chemical and physical agents, however, there is no information available regarding the role of TLS in carcinogenic metal-induced mutagenesis. Hexavalent chromium (Cr(VI))-containing compounds are highly complex genotoxins possessing both mutagenic and clastogenic activities. The focus of this work was to determine the impact that TLS has on Cr(VI)-induced mutagenesis in Saccharomyces cerevisiae. Wild-type yeast and strains deficient in TLS polymerases (i.e. Polζ (rev3), Polη (rad30)) were exposed to Cr(VI) and monitored for cell survival and forward mutagenesis at the CAN1 locus. In general, TLS deficiency had little impact on Cr(VI)-induced clonogenic lethality or cell growth. rad30 yeast exhibited higher levels of basal and induced mut! agenesis compared to Wt and rev3 yeast. In contrast, rev3 yeast displayed attenuated Cr(VI)-induced mutagenesis. Moreover, deletion of REV3 in rad30 yeast (rad30 rev3) resulted in a significant decrease in basal and Cr(VI) mutagenesis relative to Wt and rad30 single mutants indicating that mutagenesis primarily depended upon Polζ. Interestingly, rev3 yeast were similar to Wt yeast in susceptibility to Cr(VI)-induced frameshift mutations. Mutational analysis of the CAN1 gene revealed that Cr(VI)-induced base substitution mutations accounted for 83.9% and 100.0% of the total mutations in Wt and rev3 yeast, respectively. Insertions and deletions comprised 16.1% of the total mutations in Cr(VI) treated Wt yeast but were not observed rev3 yeast. This work provides novel information regarding the molecular mechanisms of Cr(VI)-induced mutagenesis and is the first report demonstrating a role for TLS in the fixation of mutations induced by a carcinogenic metal.
- cDNA analysis demonstrates that the BRCA2 intronic variant IVS4-12del5 is a deleterious mutation
- Mutat Res 663():84-89 (2009)
Mutation screening of the breast and ovarian cancer predisposition genes BRCA1 and BRCA2 is becoming an increasingly important part of clinical practice. Classification of rare non-truncating sequence variants in the BRCA1 and BRCA2 genes is problematic because it is not known whether these subtle changes alter function sufficiently to predispose cells to cancer development. Several studies have reported the biochemical analysis of BRCA2 variants, which disrupted the 5′ and 3′splicing consensus elements (the GU-AG rule). However, little has been done to look into the consequences of variants located outside the 5′ and 3′ consensus splice sites. cDNA analysis demonstrates that the BRCA2*IVS4-12del5 splice site variant results in the deletion of exon 5, and the gene putatively produces a truncated BRCA2 protein of 164 amino acids instead of 3418 with the incorporation of 22 out of frame amino acids. The pattern of breast, melanoma, and pancreatic cancers in the p! aternal kindred is consistent with autosomal dominant inheritance of a deleterious BRCA2 mutation. Analysis of a tumor specimen indicates loss of heterozygosity (LOH). Sequence alignment indicates the deleted region is well conserved across different species. These results support the conclusion that BRCA2 IVS4-12del5 is a deleterious mutation. This study will shed light on the reclassification of intronic variants that do not disrupt the 5′ and 3′ splice sites (the GU-AG rule).
- Mutat Res 663():IV (2009)