Monitoring Repair of UV-Induced 6-4-Photoproducts with a Purified DDB2 Protein Complex

Because cells are constantly subjected to DNA damaging insults, DNA repair pathways are critical for genome integrity [1]. DNA damage recognition protein complexes (DRCs) recognize DNA damage and initiate DNA repair. The DNA-Damage Binding protein 2 (DDB2) complex is a DRC that initiates nucleotide excision repair (NER) of DNA damage caused by ultraviolet light (UV) [2]-[4]. Using a purified DDB2 DRC, we created a probe ("DDB2 proteo-probe") that hybridizes to nuclei of cells irradiated with UV and not to cells exposed to other genotoxins. The DDB2 proteo-probe recognized UV-irradiated DNA in classical laboratory assays, including cyto- and histo-chemistry, flow cytometry, and slot-blotting. When immobilized, the proteo-probe also bound soluble UV-irradiated DNA in ELISA-like and DNA pull-down assays. In vitro, the DDB2 proteo-probe preferentially bound 6-4-photoproducts [(6-4)PPs] rather than cyclobutane pyrimidine dimers (CPDs). We followed UV-damage repair by cyto-chemistry in cells fixed at different time after UV irradiation, using either the DDB2 proteo-probe or antibodies against CPDs, or (6-4)PPs. The signals obtained with the DDB2 proteo-probe and with the antibody against (6-4)PPs decreased in a nearly identical manner. Since (6-4)PPs are repaired only by nucleotide excision repair (NER), our results strongly suggest the DDB2 proteo-probe hybridizes to DNA containing (6-4)PPs and allows monitoring of their removal during NER. We discuss the general use of purified DRCs as probes, in lieu of antibodies, to recognize and monitor DNA damage and repair

Rapid turnover of DNA methylation in human cells

Recent studies demonstrated that cytosine methylation in the genome can be reversed without DNA replication by enzymatic mechanisms based on base excision-repair pathways. Both enzymatic methylation and demethylation mechanisms are active in the cell nucleus at the same time. One can hypothesize that the actual level of CpG methylation could be the result of a balance between the two antagonistic processes with a rapid turnover. In the present study, we used mass spectrometry to measure the total methyl-cytosine content of the genome in cultured human cells after short incubation with the known methyltransferase inhibitor 5-deoxy-azacytidine. A significant decrease of the DNA methylation was observed. Indeed, the inhibition of the methylation can only result in a rapid reduction of the overall methyl-cytosine level if the process of demethylation is simultaneous. These observations suggest that the enzymatic mechanisms responsible of the opposing reactions of DNA methylation and demethylation act simultaneously and may result in a continuous and rapid turnover of methylated cytosines. This conclusion is supported by the observation that 5-deoxy-azacytidine was incorporated in the genomic DNA of non-dividing cells and could be detected as soon as after two hours of incubation, hence providing a mechanistic explanation to the inhibition of methyltransferases. The observations are compatible with the idea that the enzymatic mechanisms that bring together of the opposing reactions of DNA methylation and demethylation act simultaneously and may result in a continuous and unsuspected rapid turnover of DNA methylation. This conclusion is at odds with the generally accepted view of high stability of cytosine methylation where the role of enzymatic demethylation is considered as limited to some special situations such as transcription. It places DNA methylation in the same category as other epigenetic modifications with covalent modifications dynamically added to and removed from the chromatin with high turnover rate

A robust prognostic gene expression signature for early stage lung adenocarcinoma

Background: Stage I lung adenocarcinoma is usually not treated with adjuvant chemotherapy; however, around half of these patients do not survive 5 years. Therefore, a reliable prognostic biomarker for early stage patients would be critical to identify those most likely to benefit from early additional treatments. Several studies have searched for gene expression prognostic biomarkers for lung adenocarcinoma, but these have not yielded a widely accepted prognosticator. Results: We analyzed gene expression from seven published lung adenocarcinoma cohorts for which we included only stage I and II patients who were not given adjuvant therapy. Seven genes consistently obtained statistical significance in Cox regression for overall survival. The combined signature has a weighted mean hazard ratio of 3.2 in all cohorts and 3.0 (C.I. 1.3–7.4, p < 0.01) in an independent validation cohort and is strongly correlated with previously published signatures of chromosomal instability and cell cycle progression. Conclusions: The new prognostic signature, if validated prospectively, may enable better stratification and treatment of early stage lung cancer patients. Electronic supplementary material The online version of this article (doi:10.1186/s40364-016-0058-3) contains supplementary material, which is available to authorized users

Additional file 1: Figure S1A. of A robust prognostic gene expression signature for early stage lung adenocarcinoma

TCGA LUAD RNAseq − stage I and II ESLA − 7, DFS. Figure S1B: TCGA LUAD RNAseq − stage I and II ESLA − 7, DFS − no treatment. Figure S1C: TCGA LUAD RNAseq − stage I and II CIN25, DFS. Figure S1D: TCGA LUAD RNAseq − stage I and II CIN25, DFS − no treatment. Figure S1E: TCGA LUAD RNAseq − stage I and II CCP, DFS. Figure S1F: TCGA LUAD RNAseq − stage I and II CCP, DFS − no treatment. (PDF 20 kb

Additional file 3: of A robust prognostic gene expression signature for early stage lung adenocarcinoma

TCGA LUSC clinical data. (TXT 124 kb

A purified DDB2 protein complex can be used to detect UV-induced DNA damage.

<p>(<b>A</b>) Experimental strategy to prepare the DDB2 proteo-probe. (<b>B</b>) Signal obtained by hybridization of the DDB2 proteo-probe onto fibroblasts with or without damaging treatments. Hybridized DDB2 proteo-probe is revealed by anti-HA immunofluorescence. Nuclei are visualized by DAPI staining. Nuclei are delineated based on DAPI staining and using CellProfiler <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085896#pone.0085896-Carpenter1" target="_blank">[26]</a>.</p

The DDB2 proteo-probe recognizes 6-4-photoproducts <i>in vitro</i>.

<p>(<b>A</b>) The DDB2 proteo-probe signal increases linearly with fluence (J/m²). Fibroblasts were irradiated with different doses of UV-C. Each point is an average of three replicas. Each replica represents an average of at least 60 cells. Dashed line: linear fit (R² = 0.94). Error bars: s.e.m. (<b>B</b>) The DDB2 proteo-probe signal is DNA-dependent. Fibroblasts were irradiated with UV-C (10 J/m²), and untreated or treated with DNase. Nuclei are visualized by DAPI staining. (<b>C</b>) The DDB2 proteo-probe signal can be competed with UV-treated plasmid DNA. Fibroblasts and plasmid DNA were irradiated with UV-C (10 J/m² and 300 J/m², respectively). The DDB2 proteo-probe was incubated with plasmid DNA prior to hybridization onto irradiated fibroblasts. Dashed line: no plasmid control proteo-probe signal level. Each point is an average of three replicas. Each replica represents an average of at least 400 cells. Error bars: s.e.m. (<b>D</b>) The DDB2 proteo-probe binds preferentially to 6-4-photoproducts [(6-4)PP] over cyclobutane pyrimidine dimers (CPD). The DDB2 proteo-probe was immobilized on agarose beads, and incubated with the DNA restriction fragments of a plasmid containing, or not, a unique lesion [(6-4)PP or CPD]. The average ratio of the amount of lesion-containing over lesion-free DNA fragments bound to the proteo-probe is shown (<i>n</i> = 3). Error bars: s.e.m.</p

The decrease of DDB2 proteo-probe and 6-4 PP signals over time are nearly identical.

<p>(<b>A</b>) Typical signals after UV damage observed <i>in situ</i> with the DDB2 proteo-probe, an anti-CPD antibody, or an anti-(6-4)PP antibody. Nuclei are delineated based on DAPI staining and using CellProfiler. (<b>B</b>) The DDB2 proteo-probe signal decreases exponentially with time. Average signal per nucleus normalized to signal at 5 minutes. Red dashed curve: one phase exponential decay fit calculated with a non-linear least square method (R² = 0.86). (<b>C</b>) The anti-(6-4)PP signal decreases exponentially with time. Average signal per nucleus normalized to signal at 5 minutes. Blue dashed curve: one phase exponential decay fit calculated with a non-linear least square method (R² = 0.83). (<b>D</b>) The anti-CPD signal remains constant over a two hour period. Average signal per nucleus normalized to signal at 5 minutes. Black dashed line: linear fit on the α-CPD signal (R² = 0.18). (<b>B</b>), (<b>C</b>), and (<b>D</b>): cells were irradiated with UV-C (10 J/m²). The average of three replicas is shown. Each replica represents an average of at least 60 cells. Error bars: s.e.m. (<b>E</b>) A single one phase exponential decay model summarizes the kinetic of (6-4)PPs removal <i>in situ</i>. The single model is based on the decay fits obtained with DDB2 proteo-probe and anti-(6-4)PP data. The grey band represents the area enclosing the true decay curve with 99% confidence. The dotted line indicates the predicted half-life (<i>t</i>_1/2) of (6-4)PPs <i>in situ</i> after UV irradiation.</p

Identification of a Synthetic Lethal Relationship between Nucleotide Excision Repair Deficiency and Irofulven Sensitivity in Urothelial Cancer

PURPOSE: Cisplatin-based chemotherapy is a first-line treatment for muscle-invasive and metastatic urothelial cancer. Approximately 10% of bladder urothelial tumors have a somatic missense mutation in the nucleotide excision repair (NER) gene, ERCC2, which confers increased sensitivity to cisplatin-based chemotherapy. However, a significant subset of patients is ineligible to receive cisplatin-based therapy due to medical contraindications, and no NER-targeted approaches are available for platinum-ineligible or platinum-refractory ERCC2-mutant cases. EXPERIMENTAL DESIGN: We used a series of NER-proficient and NER-deficient preclinical tumor models to test sensitivity to irofulven, an abandoned anticancer agent. In addition, we used available clinical and sequencing data from multiple urothelial tumor cohorts to develop and validate a composite mutational signature of ERCC2 deficiency and cisplatin sensitivity. RESULTS: We identified a novel synthetic lethal relationship between tumor NER deficiency and sensitivity to irofulven. Irofulven specifically targets cells with inactivation of the transcription-coupled NER (TC-NER) pathway and leads to robust responses in vitro and in vivo, including in models with acquired cisplatin resistance, while having minimal effect on cells with intact NER. We also found that a composite mutational signature of ERCC2 deficiency was strongly associated with cisplatin response in patients and was also associated with cisplatin and irofulven sensitivity in preclinical models. CONCLUSIONS: Tumor NER deficiency confers sensitivity to irofulven, a previously abandoned anticancer agent, with minimal activity in NER-proficient cells. A composite mutational signature of NER deficiency may be useful in identifying patients likely to respond to NER-targeting agents, including cisplatin and irofulven