DNA Lesions In Human Cells Research Articles

Cellular Responses to Platinum-Based Anticancer Drugs and UVC: Role of p53 and Implications for Cancer Therapy

Chemotherapy is intended to induce cancer cell death through apoptosis and other avenues. Unfortunately, as discussed in this article, moderate doses of genotoxic drugs such as cisplatin typical of those achieved in the clinic often invoke a cytostatic/dormancy rather than cytotoxic/apoptosis response in solid tumour-derived cell lines. This is commonly manifested by an extended apoptotic threshold, with extensive apoptosis only being seen after very high/supralethal doses of such agents. The dormancy response can be associated with senescence-like features, polyploidy and/or multinucleation, depending in part on the p53 status of the cells. In most solid tumour-derived cells, dormancy represents a long-term survival mechanism, ultimately contributing to disease recurrence. This review highlights the nonlinearity of key aspects of the molecular and cellular responses to bulky DNA lesions in human cells treated with chemotherapeutic drugs (e.g., cisplatin) or ultraviolet light-C (a widely used tool for unraveling details of the DNA damage-response) as a function of the level of genotoxic stress. Such data highlight the growing realization that targeting dormant cancer cells, which frequently emerge following conventional anticancer treatments, may represent a novel strategy to prevent or, at least, significantly suppress cancer recurrence.

Repair and translesion synthesis of O6-alkylguanine DNA lesions in human cells

O6-alkyl-2'-deoxyguanosine (O6-alkyl-dG) lesions are among the most mutagenic and prevalent alkylated DNA lesions that are associated with cancer initiation and progression. In this study, using a shuttle vector-based strand-specific PCR-competitive replication and adduct bypass assay in conjunction with tandem MS for product identification, we systematically assessed the repair and replicative bypass of a series of O6-alkyl-dG lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, in several human cell lines. We found that the extent of replication-blocking effects of these lesions is influenced by the size of the alkyl groups situated on the O6 position of the guanine base. We also noted involvement of distinct DNA repair pathways and translesion synthesis polymerases (Pols) in ameliorating the replication blockage effects elicited by the straight- and branched-chain O6-alkyl-dG lesions. We observed that O6-methylguanine DNA methyltransferase is effective in removing the smaller alkyl groups from the O6 position of guanine, whereas repair of the branched-chain lesions relied on nucleotide excision repair. Moreover, these lesions were highly mutagenic during cellular replication and exclusively directed G→A mutations; Pol η and Pol ζ participated in error-prone bypass of the straight-chain lesions, whereas Pol κ preferentially incorporated the correct dCMP opposite the branched-chain lesions. Together, these results uncover key cellular proteins involved in repair and translesion synthesis of O6-alkyl-dG lesions and provide a better understanding of the roles of these types of lesions in the etiology of human cancer.

APIM-Mediated REV3L⁻PCNA Interaction Important for Error Free TLS Over UV-Induced DNA Lesions in Human Cells.

Proliferating cell nuclear antigen (PCNA) is essential for the organization of DNA replication and the bypass of DNA lesions via translesion synthesis (TLS). TLS is mediated by specialized DNA polymerases, which all interact, directly or indirectly, with PCNA. How interactions between the TLS polymerases and PCNA affects TLS specificity and/or coordination is not fully understood. Here we show that the catalytic subunit of the essential mammalian TLS polymerase POLζ, REV3L, contains a functional AlkB homolog 2 PCNA interacting motif, APIM. APIM from REV3L fused to YFP, and full-length REV3L-YFP colocalizes with PCNA in replication foci. Colocalization of REV3L-YFP with PCNA is strongly reduced when an APIM-CFP construct is overexpressed. We also found that overexpression of full-length REV3L with mutated APIM leads to significantly altered mutation frequencies and mutation spectra, when compared to overexpression of full-length REV3L wild-type (WT) protein in multiple cell lines. Altogether, these data suggest that APIM is a functional PCNA-interacting motif in REV3L, and that the APIM-mediated PCNA interaction is important for the function and specificity of POLζ in TLS. Finally, a PCNA-targeting cell-penetrating peptide, containing APIM, reduced the mutation frequencies and changed the mutation spectra in several cell lines, suggesting that efficient TLS requires coordination mediated by interactions with PCNA.

Replication studies of carboxymethylated DNA lesions in human cells.

Metabolic activation of some N-nitroso compounds (NOCs), an important class of DNA damaging agents, can induce the carboxymethylation of nucleobases in DNA. Very little was previously known about how the carboxymethylated DNA lesions perturb DNA replication in human cells. Here, we investigated the effects of five carboxymethylated DNA lesions, i.e. O6-CMdG, N6-CMdA, N4-CMdC, N3-CMdT and O4-CMdT on the efficiency and fidelity of DNA replication in HEK293T human embryonic kidney cells. We found that, while neither N6-CMdA nor N4-CMdC blocked DNA replication or induced mutations, N3-CMdT, O4-CMdT and O6-CMdG moderately blocked DNA replication and induced substantial frequencies of T→A (81%), T→C (68%) and G→A (6.4%) mutations, respectively. In addition, our results revealed that CRISPR-Cas9-mediated depletion of Pol η resulted in significant drops in bypass efficiencies of N4-CMdC and N3-CMdT. Diminution in bypass efficiencies was also observed for N6-CMdA and O6-CMdG upon depletion of Pol κ, and for O6-CMdG upon removal of Pol ζ. Together, our study provided molecular-level insights into the impacts of the carboxymethylated DNA lesions on DNA replication in human cells, revealed the roles of individual translesion synthesis DNA polymerases in bypassing these lesions, and suggested the contributions of O6-CMdG, N3-CMdT and O4-CMdT to the mutations found in p53 gene of human gastrointestinal cancers.

Role of base excision repair genes and proteins in gamma-irradiated resting human peripheral blood mononuclear cells.

Ionising radiation induces several isolated and clustered DNA lesions in human cells. Depending on the type of lesions, DNA repair pathways get activated to maintain the integrity of the genome. Base excision repair (BER) pathway is known to repair single-strand breaks and base damages through short- and long-patch genes and proteins. In the present study, attempt has been made to study the role of BER genes and proteins in resting human peripheral blood mononuclear cells (PBMCs) exposed to gamma radiation. Venous blood samples were collected from 20 random and healthy individuals with written informed consent. Dose-response and time-dependent changes at the level of DNA damage, transcription and protein expression were studied in PBMC. Dose-response studies were done in PBMC exposed between 0.1 and 2.0 Gy, whereas time-dependent changes in post-irradiated PBMC were studied up to 240 min. Our results have shown a significant (P ≤ 0.05) dose-dependent increase in the percentage of DNA in tail (%T) among the individuals studied. At transcriptional level, LIGASE3, MBD4 and LIGASE1 showed significant up-regulation (P ≤ 0.05) at 4h compared to 0h. Short-patch BER proteins such as OGG1 and LIGASE3 showed significant increase (P ≤ 0.05) in expression at lower doses (<0.6 Gy), whereas long-patch BER proteins such as MBD4, FEN1 and LIGASE1 showed an increase in expression at higher doses (1.0 and 2.0 Gy), suggesting dose-dependent and pathway-specific role of BER proteins in human PBMCs at G0/G1. In conclusion, BER genes and proteins play an active role in repairing radiation-induced DNA damage in resting PBMC, which has important biological significance in terms of DNA repair process in humans.

UKEMS/Dutch EMS-sponsored workshop on biomarkers of exposure and oxidative DNA damage &amp; 7th GUM-32P-postlabelling workshop, University of Munster, Munster, Germany, 28-29 March 2011

Environmental exposures are a major concern for human cancer. However, the precise contribution of specific risk factors and their interactions, both with each other and with genotype, continue to be difficult to elucidate. The exposome is the comprehensive characterisation of an individual's lifetime exposure history (Wild, C. P. (2009) Environmental exposure measurement in cancer epidemiology. Mutagenesis, 24, 117-125). Unravelling complex environmental and genetic aetiologies in order to plan effective public health interventions demands that both environmental exposures and genetic variations are reliably measured. The development, validation and application of biomarkers of exposure are manifestly critical to the future of cancer epidemiology. The aim of this workshop at the University of Münster was to discuss the current status of exposure biomarkers in cancer molecular epidemiology as well as new findings achieved by applying the methods to studies of mechanisms of human cancer. Day 1 focused on biomarkers of exposure (i.e. carcinogen DNA adducts), effect and susceptibility to gain greater understanding of environmental cancer risks and their modulation. Day 2 focused on the role of oxidative stress and DNA damage in human carcinogenesis including methodologies used for the measurement of oxidatively induced DNA lesions in human cells or tissues and the possible use of these lesions as cancer biomarkers.

Fibroblast senescence

Fibroblast senescence

Expression levels of the DNA repair enzyme HAP1 do not correlate with the radiosensitivities of human or HAP1-transfected rat cell lines.

Apurinic/apyrimidinic (AP) sites in DNA are potentially lethal and mutagenic. They can arise spontaneously or following DNA damage from reactive oxygen species or alkylating agents, and they constitute a significant product of DNA damage following cellular exposure to ionizing radiation. The major AP endonuclease responsible for initiating the repair of these and other DNA lesions in human cells is HAP1, which also possesses a redox function. We have determined the cellular levels of this enzyme in 11 human tumour and fibroblast cell lines in relation to clonogenic survival following ionizing radiation. Cellular HAP1 levels and surviving fraction at 2 Gy (SF2) varied five- and tenfold respectively. However, no correlation was found between these two parameters following exposure to γ-irradiation at low (1.1 cGy per min) or high (108 cGy per min) dose rates. To examine this further, wild-type and mutant versions of HAP1 were overexpressed, using an inducible HAP1 cDNA expression vector system, in the rat C6 glioma cell line which has low endogenous AP endonuclease activity. Induction of wild-type HAP1 expression caused a > fivefold increase in the capacity of cellular extracts to cleave an oligonucleotide substrate containing a single abasic site, but increased expression did not confer increased resistance to γ-irradiation at high- or low-dose rates, or to the methylating agent methyl methanesulphonate (MMS). Expression in C6 cell lines of mutant forms of HAP1 deleted for either the redox activator or DNA repair functions displayed no apparent titrational or dominant negative effects. These studies suggest that the levels of endogenous AP endonuclease activities in the various cell lines examined are not limiting for efficient repair in cells following exposure to ionizing radiation or MMS. This contrasts with the correlation we have found between HAP1 levels and radiosensitivity in cervix carcinomas (Herring et al (1998) Br J Cancer 78: 1128–1133), indicating that HAP1 levels in this case assume a critical survival role and hence that established cell lines might not be a suitable model for such studies. © 1999 Cancer Research Campaign

Effect of arsenic and cadmium on the persistence of mutagen‐induced DNA lesions in human cells

Effect of arsenic and cadmium on the persistence of mutagen‐induced DNA lesions in human cells

Effect of arsenic and cadmium on the persistence of mutagen-induced DNA lesions in human cells

The alkaline single cell gel electrophoresis (SCG test or comet assay) was used to characterize the influence of sodium arsenite (NaAsO2) and cadmium sulphate (CdSO4) on the persistence of mutagen-induced DNA lesions. Human blood and SV4O-transformed fibroblasts (MRC5CV1) were treated for 2 hr with methyl methanesulphonate (MMS) or benzo(a)pyrene (BaP). MMS induced concentration-related DNA damage in white Blood cells (WBC) and fibroblasts in similar concentrations. For the induction of DNA damage in white blood cells (WBC) and fibroblasts in similar concentrations. For the induction of DNA damage by BaP, higher concentrations had to be applied to WBC than to the fibroblast cell line. To study the influence of metal ions on the persistence of DNA lesions, treated cells were further incubated for 2 hr in the absence (postincubation) or presence (posttreatment) of NaAsO2 or CdSO4. After postincubation, MMS and BaP-induced DNA effects were reduced in both cell types, indicating that repair of DNA lesions had taken place. When the cells were posttreated with NaAsO2 or CdSO4, BaP- and MMS-induced DNA lesions persisted in both cell types, indicating an inhibition of DNA repair by these metals. The results suggest a strong interaction of arsenic and cadmium with BaP- and MMS-induced DNA repair processes.

Use of shuttle vectors to study the molecular processing of defined carcinogen-induced DNA damage: mutagenicity of single O4-ethylthymine adducts in HeLa cells

We developed a simian virus 40 based shuttle vector system to study the molecular consequences of distinct carcinogen-induced DNA lesions in human cells. To establish the mutagenicity of O4-ethylthymine adducts, oligonucleotides carrying a single O4-ethylthymine adduct at a unique position were ligated into the vector molecules. Following replication in HeLa cells on average 23% of the progeny molecules carried a mutation in the region of modification. The vast majority of these mutations represented single T----C transitions at the position of the modified base, most probably as a consequence of mispairing of the O4-ethylthymine residues during replication. To a minor extent the O4-ethylthymine adduct may also induce T----A transversions or double point mutations. The in vivo mutation frequency of the adduct was found to be comparable to that of a C-A mismatch at the same position, but was lower than that expected from in vitro experiments with adducted DNA templates and purified DNA polymerases.