Repair-deficient Human Cells Research Articles

Abstract 2075: Systemic small molecule TREX1 inhibitors to selectively activate STING in the TME of metastatic disease

Abstract Genetic evidence from human disease and mouse genetic knock-out studies identify the Stimulator of Interferon Genes (STING) pathway as a critical innate immune sensor for the development of immunity. Tumor cells can evolve to avoid immune recognition through inactivating the STING pathway by diverse mechanisms, indicating that it is important to generating tumor-specific immunity. However, the clinical activity of STING agonists given by intratumoral (IT) administration has not compared well to preclinical studies. The scientific hypothesis supporting these first clinical studies is that localized CD8+ T cell priming would have activity against distal non-injected tumors, but findings that tumors in advanced malignancies have unique antigenic repertoires suggests that this approach may have limited activity against distal tumors. Global innate activation in metastases may therefore be necessary to prime a broadly active CD8+ T cell population targeting diverse antigens, in addition having the benefit of reversing the immune suppressive tumor microenvironment (TME). However, ubiquitous expression of STING presents a significant challenge to achieving a therapeutic index with systemic delivery of direct STING agonists. Selective activation of the STING pathway may be achieved through targeted inhibition of TREX1, a cytosolic DNA exonuclease that modulates cGAS/STING signaling. Expression of TREX1, in contrast to STING, is increased in tumor cells due to elevated levels of cytosolic DNA resulting from genetic instability, DNA repair mutation, inflammatory mediators or DNA-modifying anti-cancer therapies. These observations provide the principal scientific rationale to selectively activate the STING pathway in metastatic disease through targeted inhibition of TREX1. Utilizing published TREX1 X-ray crystal structures to guide medicinal chemistry, we discovered small molecule inhibitors of TREX1 and transformed these molecules from >100 µM leads into a series with drug-like physicochemical properties and picomolar potency against both human and mouse TREX1. We evaluated the activity of lead molecules in cell-based assays, in which TREX1 inhibition enhanced cGAS/STING signaling, and advanced molecules with desired pharmacokinetic profiles to mouse tumor studies. We observed significant anti-tumor activity in mice with CT26 tumors given a combined therapy of low dose doxorubicin to induce dsDNA breaks and increase TME TREX1 expression along with lead series TREX1 molecule inhibitors. Recognizing that TREX1 is a DNA repair enzyme, we also showed that TREX1 inhibitors were cytotoxic in DNA repair deficient human tumor cell lines, informing advancement of this new class of STING therapeutics as a clinical approach to both activate the cGAS/STING pathway to initiate immune recognition, as well as to inhibit DNA repair orthogonal to existing tumor-driver DNA repair mutations. Citation Format: Brian Francica, Dara Burdette, Ryan Clark, Jamie Cope, David Freund, Anja Holtz, Peppi Prasit, Chan Whiting, Thomas W. Dubensky. Systemic small molecule TREX1 inhibitors to selectively activate STING in the TME of metastatic disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2075.

Repair pathways for radiation DNA damage under normoxic and hypoxic conditions: Assessment with a panel of repair-deficient human TK6 cells.

Various types of DNA lesions are produced when cells are exposed to ionizing radiation (IR). The type and yield of IR-induced DNA damage is influenced by the oxygen concentration. Thus, different DNA repair mechanisms may be involved in the response of normoxic and hypoxic cells to irradiation with IR. However, differences between the repair mechanisms of IR-induced DNA damage under normoxic versus hypoxic conditions have not been clarified. Elucidating the relative contribution of individual repair factors to cell survival would give insight into the repair mechanisms operating in irradiated normoxic and hypoxic cells. In the present study, we used a panel of repair-deficient human TK6 cell lines that covered seven repair pathways. Cells were irradiated with X-rays under normoxic and hypoxic conditions, and the sensitivities of each mutant relative to the wild-type (i.e. relative sensitivity) were determined for normoxic and hypoxic conditions. The sensitivity of cells varied depending on the type of repair defects. However, for each repair mutant, the relative sensitivity under normoxic conditions was comparable to that under hypoxic conditions. This result indicates that the relative contribution of individual repair pathways to cell survival is comparable in normoxic and hypoxic cells, although the spectrum of IR-induced DNA damage in hypoxic cells differs from that of normoxic cells.

Follow-up genotoxicity assessment of Ames-positive/equivocal chemicals using the improved thymidine kinase gene mutation assay in DNA repair-deficient human TK6 cells.

Genotoxicity testing plays an important role in the safety assessment of pharmaceuticals, pesticides and chemical substances. Among the guidelines for various genotoxicity tests, the in vitro genotoxicity test battery comprises the bacterial Ames test and mammalian cell assays. Several chemicals exhibit conflicting results for the bacterial Ames test and mammalian cell genotoxicity studies, which may stem from the differences in DNA repair capacity or metabolism, between different cell types or species. For better understanding the mechanistic implications regarding conflict outcomes between different assay systems, it is necessary to develop in vitro genotoxicity testing approaches with higher specificity towards DNA-damaging reagents. We have recently established an improved thymidine kinase (TK) gene mutation assay (TK assay) i.e. deficient in DNA excision repair system using human lymphoblastoid TK6 cells lacking XRCC1 and XPA (XRCC1-/-/XPA-/-), the core factors of base excision repair (BER) and nucleotide excision repair (NER), respectively. This DNA repair-deficient TK6 cell line is expected to specifically evaluate the genotoxic potential of chemical substances based on the DNA damage. We focussed on four reagents, N-(1-naphthyl)ethylenediamine dihydrochloride (NEDA), p-phenylenediamine (PPD), auramine and malachite green (MG) as the Ames test-positive chemicals. In our assay, assessment using XRCC1-/-/XPA-/- cells revealed no statistically significant increase in the mutant frequencies after treatment with NEDA, PPD and MG, suggesting the chemicals to be non-genotoxic in humans. The observations were consistent with that of the follow-up in vivo studies. In contrast, the mutant frequency was markedly increased in XRCC1-/-/XPA-/- cells after treatment with auramine. The results suggest that auramine is the genotoxic reagent that preferentially induces DNA damages resolved by BER and/or NER in mammals. Taken together, BER/NER-deficient cell-based genotoxicity testing will contribute to elucidate the mechanism of genotoxicity and therefore play a pivotal role in the accurate safety assessment of chemical substances.

Enhancing the sensitivity of the thymidine kinase assay by using DNA repair-deficient human TK6 cells.

The OECD guidelines define the bioassays of identifying mutagenic chemicals, including the thymidine kinase (TK) assay, which specifically detects the mutations that inactivate the TK gene in the human TK6 lymphoid line. However, the sensitivity of this assay is limited because it detects mutations occurring only in the TK gene but not any other genes. Moreover, the limited sensitivity of the conventional TK assay is caused by the usage of DNA repair‐proficient wild‐type cells, which are capable of accurately repairing DNA damage induced by chemicals. Mutagenic chemicals produce a variety of DNA lesions, including base lesions, sugar damage, crosslinks, and strand breaks. Base damage causes point mutations and is repaired by the base excision repair (BER) and nucleotide excision repair (NER) pathways. To increase the sensitivity of TK assay, we simultaneously disrupted two genes encoding XRCC1, an important BER factor, and XPA, which is essential for NER, generating XRCC1 −/− /XPA −/− cells from TK6 cells. We measured the mutation frequency induced by four typical mutagenic agents, methyl methane sulfonate (MMS), cis‐diamminedichloro‐platinum(II) (cisplatin, CDDP), mitomycin‐C (MMC), and cyclophosphamide (CP) by the conventional TK assay using wild‐type TK6 cells and also by the TK assay using XRCC1 −/− /XPA −/− cells. The usage of XRCC1 −/− /XPA −/− cells increased the sensitivity of detecting the mutagenicity by 8.6 times for MMC, 8.5 times for CDDP, and 2.6 times for MMS in comparison with the conventional TK assay. In conclusion, the usage of XRCC1 −/− /XPA −/− cells will significantly improve TK assay.

Error-prone replication of a 5-formylcytosine-mediated DNA-peptide cross-link in human cells

DNA-protein cross-links can interfere with chromatin architecture, block DNA replication and transcription, and interfere with DNA repair. Here we synthesized a DNA 23-mer containing a site-specific DNA-peptide cross-link (DpC) by cross-linking an 11-mer peptide to the DNA epigenetic mark 5-formylcytosine in synthetic DNA and used it to generate a DpC-containing plasmid construct. Upon replication of the DpC-containing plasmid in HEK 293T cells, approximately 9% of progeny plasmids contained targeted mutations and 5% semitargeted mutations. Targeted mutations included C→T transitions and C deletions, whereas semitargeted mutations included several base substitutions and deletions near the DpC lesion. To identify DNA polymerases involved in DpC bypass, we comparatively studied translesion synthesis (TLS) efficiency and mutagenesis of the DpC in a series of cell lines with TLS polymerase knockouts or knockdowns. Knockdown of either hPol ι or hPol ζ reduced the mutation frequency by nearly 50%. However, the most significant reduction in mutation frequency (50%-70%) was observed upon simultaneous knockout of hPol η and hPol κ with knockdown of hPol ζ, suggesting that these TLS polymerases play a critical role in error-prone DpC bypass. Because TLS efficiency of the DpC construct was not significantly affected in TLS polymerase-deficient cells, we examined a possible role of replicative DNA polymerases in their bypass and determined that hPol δ and hPol ϵ can accurately bypass the DpC. We conclude that both replicative and TLS polymerases can bypass this DpC lesion in human cells but that mutations are induced mainly by TLS polymerases.

Abstract 3368: Insertion and deletion frameshift rates and mutational spectra of tetranucleotide microsatellites in DNA mismatch repair-deficient human cells

Abstract Background. Oxidative DNA damage and inflammatory cytokines cause loss of function of the MMR protein MSH3. MSH3-MSH2 (MutSβ) function is required for correction of postreplicative polymerase errors in the form of frameshifts at tetranucleotide microsatellite sequences to prevent elevated microsatellite alterations at selected tetranucleotides (EMAST). EMAST is increasingly being used as a biomarker for MSH3 deficiency in subtyping colorectal cancers due to its association with metastasis and poor patient outcome. Here, we sought to understand frameshift mutational behavior of tetranucleotide sequences in the absence of MMR. Methods. We generated EGFP-based mutation reporter model systems containing the D9S242 tetranucleotide microsatellite at its native length of (AAAG)18 and at modified lengths of (AAAG)15 and (AAAG)12 to represent other endogenous microsatellite lengths. Plasmid vectors were constructed for each length that placed EGFP +1 bp or -1 bp out-of-frame for protein translation, enabling us to measure deletion and insertion frameshifts. These constructs were stably-integrated into MMR-deficient HCT116 cells, and monoclonal cell lines containing the constructs were isolated to measure mutation rates and spectra. Deletion and insertion frameshift mutations restored the EGFP reading frame to become detectable by flow cytometry. Genomic DNA from mutated cells were isolated and sequenced for insertion/deletion frameshift mutational biases. Results. Combined insertion/deletion frameshift mutation rates at D9S242 locus ranged from 31.6 X 10-4 to 71.1 X 10-4 mutations/cell/generation and was strongly correlated with longer length of tetranucleotide microsatellites (r2 = 0.986, P = 0.0375). We observed slightly higher deletion mutation rates over insertion rates at longer microsatellites, but equivalent insertion and deletion rates at shorter microsatellites. However, accumulation of more deletion frameshifts over time contributed to a distinct mutational bias for deletions for each length, likely due to continual frameshift mutation at insertions. About 79% of all observed frameshifts (insertion or deletion) were one- repeat (e.g., four nucleotides), 16% two-repeat, and 5% three or more repeat mutations that were consistent with a slipped strand mispairing mutation model. Conclusions. Our results suggest that intact MMR recognizes and repairs shorter length tetranucleotide frameshifts more efficiently than larger ones, and preferentially corrects loops on the template strand (preventing deletions) over loops on the newly-synthesized strand (preventing insertions) in human cells. MMR deficiency thus manifests in human tissue with a deletion mutation bias at tetranucleotide sequences (EMAST). Longer tetranucleotide microsatellites have increased propensities for larger DNA slippage errors. Citation Format: Maide O. Raeker, Jovan Pierre-Charles, John M. Carethers. Insertion and deletion frameshift rates and mutational spectra of tetranucleotide microsatellites in DNA mismatch repair-deficient human cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3368.

Inhibition of the ERCC1-XPF structure-specific endonuclease to overcome cancer chemoresistance.

ERCC1-XPF is a structure-specific endonuclease that is required for the repair of DNA lesions, generated by the widely used platinum-containing cancer chemotherapeutics such as cisplatin, through the Nucleotide Excision Repair and Interstrand Crosslink Repair pathways. Based on mouse xenograft experiments, where ERCC1-deficient melanomas were cured by cisplatin therapy, we proposed that inhibition of ERCC1-XPF could enhance the effectiveness of platinum-based chemotherapy. Here we report the identification and properties of inhibitors against two key targets on ERCC1-XPF. By targeting the ERCC1-XPF interaction domain we proposed that inhibition would disrupt the ERCC1-XPF heterodimer resulting in destabilisation of both proteins. Using in silico screening, we identified an inhibitor that bound to ERCC1-XPF in a biophysical assay, reduced the level of ERCC1-XPF complexes in ovarian cancer cells, inhibited Nucleotide Excision Repair and sensitised melanoma cells to cisplatin. We also utilised high throughput and in silico screening to identify the first reported inhibitors of the other key target, the XPF endonuclease domain. We demonstrate that two of these compounds display specificity in vitro for ERCC1-XPF over two other endonucleases, bind to ERCC1-XPF, inhibit Nucleotide Excision Repair in two independent assays and specifically sensitise Nucleotide Excision Repair-proficient, but not Nucleotide Excision Repair-deficient human and mouse cells to cisplatin.

Generalized Time-Dependent Model of Radiation-Induced Chromosomal Aberrations in Normal and Repair-Deficient Human Cells

We have developed a model that can simulate the yield of radiation-induced chromosomal aberrations (CAs) and unrejoined chromosome breaks in normal and repair-deficient cells. The model predicts the kinetics of chromosomal aberration formation after exposure in the G₀/G₁ phase of the cell cycle to either low- or high-LET radiation. A previously formulated model based on a stochastic Monte Carlo approach was updated to consider the time dependence of DNA double-strand break (DSB) repair (proper or improper), and different cell types were assigned different kinetics of DSB repair. The distribution of the DSB free ends was derived from a mechanistic model that takes into account the structure of chromatin and DSB clustering from high-LET radiation. The kinetics of chromosomal aberration formation were derived from experimental data on DSB repair kinetics in normal and repair-deficient cell lines. We assessed different types of chromosomal aberrations with the focus on simple and complex exchanges, and predicted the DSB rejoining kinetics and misrepair probabilities for different cell types. The results identify major cell-dependent factors, such as a greater yield of chromosome misrepair in ataxia telangiectasia (AT) cells and slower rejoining in Nijmegen (NBS) cells relative to the wild-type. The model's predictions suggest that two mechanisms could exist for the inefficiency of DSB repair in AT and NBS cells, one that depends on the overall speed of joining (either proper or improper) of DNA broken ends, and another that depends on geometric factors, such as the Euclidian distance between DNA broken ends, which influences the relative frequency of misrepair.

Quantitative analysis of the effects of iododeoxyuridine and ionising radiation treatment on the cell cycle dynamics of DNA mismatch repair deficient human colorectal cancer cells

DNA mismatch repair (MMR) is involved in processing DNA damage following treatment with ionising radiation (IR) and various classes of chemotherapy drugs including iododeoxyuridine (IUdR), a known radiosensitiser. In this study, the authors have developed asynchronous probabilistic cell cycle models to assess the isolated effects of IUdR and IR and the combined effects of IUdR + IR treatments on MMR damage processing. The authors used both synchronous and asynchronous MMR-proficient/MMR-deficient cell populations and followed treated cells for up to two cell cycle times. They have observed and quantified differential cell cycle responses to MMR damage processing following IR and IUdR + IR treatments, principally in the duration of both G1 and G2/M cell cycle phases. The models presented in this work form the foundation for the development of an approach to maximise the therapeutic index for IR and IUdR + IR treatments in MMR-deficient (damage tolerant) cancers.

Growth inhibition and antioxidative status induced by selenium-enriched broccoli extract and selenocompounds in DNA mismatch repair-deficient human colon cancer cells

The effects of enzymatic-digested Se-enriched broccoli extracts (SeB) and selenocompounds on growth and antioxidative status in human colon cancer cells was investigated in this study. HCT116 and HCT116+Chr.3 cells were treated with selenocompounds (sodium selenite, sodium selenate, Se-Met, MeSeCys) or SeB [high-Se (H-SeB) or low-Se (L-SeB)]. The cytotoxicity induced by selenocompounds in HCT116 cells was not associated with cellular H2O2 level, while the differential cytotoxicity observed by sodium selenite between HCT116 and HCT116+Chr.3 cell lines was related to cellular H2O2 production with the change in antioxidative enzyme activity, and the restoration of chromosome 3. H-SeB was found to reduce the cellular H2O2 content in HCT116+Chr.3 cells. The results in this study indicate that regardless of Se content, the cytotoxicity in HCT116 cells of both SeB forms appeared to be H2O2-independent, whereas the cytotoxicity in HCT116+Chr.3 of either SeB form appeared to be H2O2-dependent with an increase in antioxidative ability for H-SeB.

The induction and repair of inter-strand DNA cross-links (ICL) with sulphur mustard (SM) in normal and DNA repair deficient human cells

The induction and repair of inter-strand DNA cross-links (ICL) with sulphur mustard (SM) in normal and DNA repair deficient human cells

Excision Repair Cross-Complementing-1 for Small Cell Lung Cancer

We read the article by Rossi et al.1Rossi G Nannini N Tiseo M ERCC1 and small cell lung cancer.J Thorac Oncol. 2010; 5: 1876-1877Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar in the November issue of the Journal of Thoracic Oncology with great interest. In the article, the authors summarized six studies, including our previous study,2Kim YH Ishii G Goto K et al.Expression of breast cancer resistance protein is associated with a poor clinical outcome in patients with small-cell lung cancer.Lung Cancer. 2009; 65: 105-111Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar regarding the expression of excision repair cross-complementing-1 (ERCC1) in small cell lung cancer. Overall, the expression of ERCC1 was not correlated with the response to platinum-based chemotherapy; however, low expression of ERCC1 was significantly associated with good prognosis in patients with limited disease (LD) in three studies. Based on these data, they speculated that ERCC1 expression is a prognostic rather than a predictive marker of response to platinum-based chemotherapy only in patients with LD; however, they did not take account of the influence of radiotherapy. The proportion of patients who received thoracic radiotherapy was reported in two of the three studies, excluding the study by Rossi et al.,1Rossi G Nannini N Tiseo M ERCC1 and small cell lung cancer.J Thorac Oncol. 2010; 5: 1876-1877Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar with 35 of 40 (87%) and 38 of 45 (84%) patients with LD receiving thoracic radiotherapy in the study by Lee et al.3Lee HW Han JH Kim JH et al.Expression of excision repair cross-complementation group 1 protein predicts poor outcome in patients with small cell lung cancer.Lung Cancer. 2008; 59: 95-104Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar and Ceppi et al.,4Ceppi P Longo M Volante M et al.Excision repair cross complementing-1 and topoisomerase IIalpha gene expression in small-cell lung cancer patients treated with platinum and etoposide: a retrospective study.J Thorac Oncol. 2008; 3: 583-589Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar respectively. Although the precise mechanism remains to be elucidated, previous in vitro studies have shown that low ERCC1 expression is associated with a high sensitivity to radiation5Murray D Vallee-Lucic L Rosenberg E et al.Sensitivity of nucleotide excision repair-deficient human cells to ionizing radiation and cyclophosphamide.Anticancer Res. 2002; 22: 21-26PubMed Google Scholar; therefore, patients who received thoracic radiotherapy were excluded from analysis in our study.2Kim YH Ishii G Goto K et al.Expression of breast cancer resistance protein is associated with a poor clinical outcome in patients with small-cell lung cancer.Lung Cancer. 2009; 65: 105-111Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar To date, many studies have been reported concerning the expression of ERCC1 also in non-small cell lung cancer; however, the results are diverse, there is no consensus, and it has not been accepted for clinical use. The role of ERCC1 is complex, and further studies are necessary for better understanding of the function of ERCC1 in both chemoresistance and radioresistance.

DNA polymerase β-dependent long patch base excision repair in living cells

We examined a role for DNA polymerase β (Pol β) in mammalian long patch base excision repair (LP BER). Although a role for Pol β is well known in single-nucleotide BER, information on this enzyme in the context of LP BER has been limited. To examine the question of Pol β involvement in LP BER, we made use of nucleotide excision repair-deficient human XPA cells expressing UVDE (XPA-UVDE), which introduces a nick directly 5′ to the cyclobutane pyrimidine dimer or 6-4 photoproduct, leaving ends with 3′-OH and 5′-phosphorylated UV lesion. We observed recruitment of GFP-fused Pol β to focal sites of nuclear UV irradiation, consistent with a role of Pol β in repair of UV-induced photoproducts adjacent to a strand break. This was the first evidence of Pol β recruitment in LP BER in vivo. In cell extract, a 5′-blocked oligodeoxynucleotide substrate containing a nicked 5′-cyclobutane pyrimidine dimer was repaired by Pol β-dependent LP BER. We also demonstrated Pol β involvement in LP BER by making use of mouse cells that are double null for XPA and Pol β. These results were extended by experiments with oligodeoxynucleotide substrates and purified human Pol β.

Effect of the anti-neoplastic drug doxorubicin on XPD-mutated DNA repair-deficient human cells

Doxorubicin (DOX), a member of the anthracycline group, is a widely used drug in cancer therapy. The mechanisms of DOX action include topoisomerase II-poisoning, free radical release, DNA adducts and interstrand cross-link (ICL) formation. Nucleotide excision repair (NER) is involved in the removal of helix-distorting lesions and chemical adducts, however, little is known about the response of NER-deficient cell lines to anti-tumoral drugs like DOX. Wild type and XPD-mutated cells, harbouring mutations in different regions of this gene and leading to XP-D, XP/CS or TTD diseases, were treated with this drug and analyzed for cell cycle arrest and DNA damage by comet assay. The formation of DSBs was also investigated by determination of γH2AX foci. Our results indicate that all three NER-deficient cell lines tested are more sensitive to DOX treatment, when compared to wild type cells or XP cells complemented by the wild type XPD cDNA, suggesting that NER is involved in the removal of DOX-induced lesions. The cell cycle analysis showed the characteristic G2 arrest in repair-proficient MRC5 cell line after DOX treatment, whereas the repair-deficient cell lines presented significant increase in sub-G1 fraction. The NER-deficient cell lines do not show different patterns of DNA damage formation as assayed by comet assay and phosphorylated H2AX foci formation. Knock-down of topoisomerase IIα with siRNA leads to increased survival in both MRC5 and XP cells, however, XP cell line still remained significantly more sensitive to the treatment by DOX. Our study suggests that the enhanced sensitivity is due to DOX-induced DNA damage that is subject to NER, as we observed decreased unscheduled DNA synthesis in XP-deficient cells upon DOX treatment. Furthermore, the complementation of the XPD-function abolished the observed sensitivity at lower DOX concentrations, suggesting that the XPD helicase activity is involved in the repair of DOX-induced lesions.

Establishment of a Method for Analyzing Translesion DNA Synthesis across a Single Bulky Adduct in Human Cells

Translesion DNA synthesis (TLS) is a tolerance pathway of replication block caused by DNA lesions. To measure the efficiency and fidelity of TLS in human cells, we established a shuttle vector assay by modifying of a bacterial TLS assay system. The assay consists of transfection of DNA repair-deficient human cells with a plasmid possessing a single DNA adduct, and transformation of indicator bacteria with plasmids extracted from the cells. We show that plasmid replication was suppressed to 1/9 by a single aminobiphenyl-dG adduct, and the mutant frequency of TLS-operated plasmids was 0.31, of which the major mutation (78%) was G to T transversion. The results demonstrate that this assay is applicable in practice for investigating TLS in human cells.

Kinetics of H2AX phosphorylation after exposure to cisplatin

Cisplatin is a widely used cancer chemotherapeutic drug that causes DNA crosslinking and stimulates H2AX phosphorylation. Our goal was to assess the potential of gammaH2AX to help predict tumor response to cisplatin treatment. The kinetics of cisplatin-induced DNA interstrand crosslinks was measured using the alkaline comet assay and compared with gammaH2AX formation and clonogenic cell survival in several DNA repair proficient or deficient human and rodent cell lines. The comet assay was effective in ranking cell lines according to their relative sensitivity to cisplatin based on reduced crosslink formation measured 6 h after drug exposure or by the failure of irs3 and UV41 cell lines to subsequently remove crosslinks. In comparison, the initial rate of phosphorylation of H2AX measured over the first 6 h after cisplatin treatment was unrelated to drug sensitivity or crosslinking proficiency. However, for proliferating cell cultures, the fraction of cells that retained gammaH2AX foci 24 h after cisplatin treatment was correlated with the fraction of cells that lost clonogenic potential (slope = 1.1, r(2) = 0.85). H2AX phosphorylation occurs in response to replication fork damage caused by cisplatin induced DNA lesions, probably interstrand crosslinks. Although early kinetics of gammaH2AX formation was uninformative, retention of gammaH2AX foci 24 h after treatment was shown to be a useful indicator of cell response to killing by cisplatin. However, for gammaH2AX to serve as an indicator of cell viability after cisplatin treatment, cells must have the opportunity to transit S phase during the recovery period.

Induction of gamma-H2AX Foci in Lymphocytes, Fibroblasts and Tumor Cells by Single and Pulsed High Dose Rate X-rays

Purpose and background: Variation in radiosensitivity of normal tissues is related to genetic susceptibility (Svensson et al. 2006). Persistence of gamma-H2AX foci after ionizing radiation is higher in DNA-DSB repair deficient cells than in normal cells. We investigated whether differences in number of ?-H2AX foci may be useful as a predictor of radiosensitivity. Materials and methods: Induction of gamma-H2AX foci was measured in various cell types: freshly isolated lymphocytes, fibroblast cells (AT+/+, AT+/-, AT-/-), DNA-DSB repair deficient and normal hamster cells and human tumour cells (lung carcinoma, SW-1573 and prostate cancer cell lines with different TP53 status: LNCaP: TP53 wt, PC3: TP53 null and DU145: TP53 mutant). A radiation dose of 2.4 Gy was given as a single dose or as pulsed doses of 100 mGy in 24 h (PDR: 240 pulses of 100mGy, 1 pulse every 6 min). The number of gamma-H2AX foci was determined 30 min and 24 h after single dose and directly after PDR. Results: Maximal number of γ-H2AX foci occurred after single dose at 30 min after irradiation without significant differences in numbers of foci between different cell lines. The number of gamma-H2AX foci was radiation dose dependent and linearly correlated with clonogenic survival. Both 24 h after a single dose and after PDR more remaining foci are observed in repair deficient cells than in normal cells. In addition the TP53 null cells have more remaining foci than the mutant and wt TP53 cells. Conclusions: The detection gamma-H2AX after in vitro irradiation of lymphocytes and cell lines is simple, reproducible and sensitive. The number of residual gamma-H2AX foci after single dose as well as after PDR enables us to distinguish cells with differences in DNA-DSB repair capacities. However, it remains to be established whether gamma-H2AX assay applied in lymphocytes can be used to predict for late normal tissue toxicity after radiotherapy. Sponsored by the Dutch Cancer Foundation UVA2008-4019

γ-H2AX formation in response to interstrand crosslinks requires XPF in human cells

To further define the molecular mechanisms involved in processing interstrand crosslinks, we monitored the formation of phosphorylated histone H2AX (γ-H2AX), which is generated in chromatin near double strand break sites, following DNA damage in normal and repair-deficient human cells. Following treatment with a psoralen derivative and ultraviolet A radiation doses that produce significant numbers of crosslinks, γ-H2AX levels in nucleotide excision repair-deficient XP-A fibroblasts (XP12RO-SV) increased to levels that were twice those observed in normal control GM637 fibroblasts. A partial XPA revertant cell line (XP129) that is proficient in crosslink removal, exhibited reduced γ-H2AX levels that were intermediate between those of GM637 and XP-A cells. XP-F fibroblasts (XP2YO-SV and XP3YO) that are also repair-deficient exhibited γ-H2AX levels below even control fibroblasts following treatment with psoralen and ultraviolet A radiation. Similarly, another crosslinking agent, mitomycin C, did not induce γ-H2AX in XP-F cells, although it did induce equivalent levels of γ-H2AX in XPA and control GM637 cells. Ectopic expression of XPF in XP-F fibroblasts restored γ-H2AX induction following treatment with crosslinking agents. Angelicin, a furocoumarin which forms only monoadducts and not crosslinks following ultraviolet A radiation, as well as ultraviolet C radiation, resulted only in weak induction of γ-H2AX in all cells, suggesting that the double strand breaks observed with psoralen and ultraviolet A treatment result preferentially following crosslink formation. These results indicate that XPF is required to form γ-H2AX and likely double strand breaks in response to interstrand crosslinks in human cells. Furthermore, XPA may be important to allow psoralen interstrand crosslinks to be processed without forming a double strand break intermediate.

Recruitment of DNA Damage Checkpoint Proteins to Damage in Transcribed and Nontranscribed Sequences

We developed a chromatin immunoprecipitation method for analyzing the binding of repair and checkpoint proteins to DNA base lesions in any region of the human genome. Using this method, we investigated the recruitment of DNA damage checkpoint proteins RPA, Rad9, and ATR to base damage induced by UV and acetoxyacetylaminofluorene in transcribed and nontranscribed regions in wild-type and excision repair-deficient human cells in G1 and S phases of the cell cycle. We find that all 3 damage sensors tested assemble at the site or in the vicinity of damage in the absence of DNA replication or repair and that transcription enhances recruitment of checkpoint proteins to the damage site. Furthermore, we find that UV irradiation of human cells defective in excision repair leads to phosphorylation of Chk1 kinase in both G1 and S phase of the cell cycle, suggesting that primary DNA lesions as well as stalled transcription complexes may act as signals to initiate the DNA damage checkpoint response.

Effects of camptothecin on double-strand break repair by non-homologous end-joining in DNA mismatch repair-deficient human colorectal cancer cell lines

Loss of a functional mismatch repair (MMR) system in colorectal cancer (CRC) cells is associated with microsatellite instability and increased sensitivity to topoisomerase inhibitors. In this study, we have investigated whether a defect in double-strand break (DSB) repair by non-homologous end-joining (NHEJ) could explain why MMR-deficient CRC cells are hypersensitive to camptothecin (CPT), a topoisomerase I inhibitor. To evaluate the efficiency and the fidelity of DSB repair, we have transiently transfected plasmids containing cohesive or non-complementary ends in cells with various MMR defects. We have observed that the repair efficiency of DSB with cohesive and non-complementary ends is comparable in all cell lines. In contrast to the MMR-proficient cell line HT29, the MMR-deficient cell lines were highly accurate in repairing DSB with cohesive ends, but this characteristic could not be directly assigned to the primary MMR deficiency. Furthermore, CPT treatment had no detectable effect on the repair of cohesive ends but significantly decreased the repair efficiency of non-complementary DSB. In conclusion, although our observations show that DSB repair efficiency by NHEJ decreases upon treatment with CPT, which possibly contributes to its cytotoxicity, it is quite unlikely that it accounts for the hypersensitivity of MMR-deficient cells to topoisomerase inhibitors.