Jean Breton

Proteomic Screening of a Cell Line Model of Esophageal Carcinogenesis Identifies Cathepsin D and Aldo-Keto Reductase 1C2 and 1B10 Dysregulation in Barrett’s Esophagus and Esophageal Adenocarcinoma

Esophageal adenocarcinoma (EA) incidence is increasing rapidly and is associated with a poor prognosis. Identifying biomarkers of disease development and progression would be invaluable tools to inform clinical practice. Two-dimensional polyacrylamide gel electrophoresis was used to screen 10 esophageal cell lines representing distinct stages in the development of esophageal cancer. Thirty-three proteins were identified by MALDI-TOF-MS which demonstrated differences in expression across the cell lines. Western blotting and qRT-PCR confirmed increased cathepsin D and aldo-keto reductases 1C2 and 1B10 expression in metaplastic and dysplastic cell lines. Expression of these proteins was further assessed in esophageal epithelium from patients with nonerosive (NERD) and erosive gastro-esophageal reflux disease, Barretts esophagus (BE) and EA. When compared with normal epithelium of NERD patients, (i) cathepsin D mRNA levels demonstrated a stepwise increase in expression (p<0.05) in erosive, metaplastic and EA tissue; (ii) AKR1B10 expression increased (p<0.05) 3- and 9-fold in erosive and Barretts epithelium, respectively; and (iii) AKR1C2 levels increased (p<0.05) in erosive and Barretts epithelium, but were reduced (p<0.05) in EA. These proteins may contribute to disease development via effects on apoptosis, transport of bile acids and retinoid metabolism and should be considered as candidates for further mechanistic and clinical investigations.

A microarray to measure repair of damaged plasmids by cell lysates

DNA repair mechanisms constitute major defences against agents that cause cancer, degenerative disease and aging. Different repair systems cooperate to maintain the integrity of genetic information. Investigations of DNA repair involvement in human pathology require an efficient tool that takes into account the variety and complexity of repair systems. We have developed a highly sensitive damaged plasmid microarray to quantify cell lysate excision/synthesis (ES) capacities using small amounts of proteins. This microsystem is based on efficient immobilization and conservation on hydrogel coated glass slides of plasmid DNA damaged with a panel of genotoxic agents. Fluorescent signals are generated from incorporation of labelled dNTPs by DNA excision-repair synthesis mechanisms at plasmid sites. Highly precise DNA repair phenotypes i.e. simultaneous quantitative measures of ES capacities toward seven lesions repaired by distinct repair pathways, are obtained. Applied to the characterization of xeroderma pigmentosum (XP) cells at basal level and in response to a low dose of UVB irradiation, the assay showed the multifunctional role of different XP proteins in cell protection against all types of damage. On the other hand, measurement of the ES of peripheral blood mononuclear cells from six donors revealed significant diversity between individuals. Our results illustrate the power of such a parallelized approach with high potential for several applications including the discovery of new cancer biomarkers and the screening of chemical agents modulating DNA repair systems.

TOX4 and its binding partners recognize DNA adducts generated by platinum anticancer drugs.

Platinating agents are commonly prescribed anticancer drugs damaging DNA. Induced lesions are recognized by a wide range of proteins. These are involved in cellular mechanisms such as DNA repair, mediation of cytotoxicity or chromatin remodeling. They therefore constitute crucial actors to understand pharmacology of these drugs. To expand our knowledge about this subproteome, we developed a ligand fishing trap coupled to high throughput proteomic tools. This trap is made of damaged plasmids attached to magnetic beads, and was exposed to cell nuclear extracts. Retained proteins were identified by nanoHPLC coupled to tandem mass spectrometry. This approach allowed us to establish a list of 38 proteins interacting with DNA adducts generated by cisplatin, oxaliplatin and satraplatin. Some of them were already known interactome members like high mobility group protein 1 (HMGB1) or the human upstream binding factor (hUBF), but we also succeeded in identifying unexpected proteins such as TOX HMG box family member 4 (TOX4), phosphatase 1 nuclear targeting subunit (PNUTS), and WD repeat-containing protein 82 (WDR82), members of a recently discovered complex. Interaction between TOX4 and platinated DNA was subsequently validated by surface plasmon resonance imaging (SPRi). These interactions highlight new cellular responses to DNA damage induced by chemotherapeutic agents.

Measurement of 8-oxo-7,8-dihydro-2′-deoxyguanosine in peripheral blood mononuclear cells: Optimisation and application to samples from a case-control study on cancers of the oesophagus and cardia

8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is a widely used biomarker to evaluate the level of oxidative stress. This study describes in its first part the optimisation of our analytical procedure (HPLC/electrochemical detection). Particular care was exercised to avoid artefactual oxidation and in the precision of measurement, which was evaluated with blood bags from hemochromatosis patients. The best results were obtained with a DNA extraction step using the “chaotropic method” recommended by the European Standards Committee on Oxidative DNA Damage (ESCODD). Other approaches such as anion exchange columns gave ten times as much 8-oxodG as this method. Moreover, a complete DNA hydrolysis using five different enzymes allowed improved precision. The optimised protocol was applied to peripheral blood mononuclear cells (PBMC) sampled during a case-control study on cancers of the oesophagus and cardia. With 7.2±2.6 8-oxodG/106 2′-deoxyguanosines (2′-dG) (mean ± SD), patients (n=17) showed higher levels of 8-oxodG than controls (4.9±1.9 8-oxodG/106 2′-dG, n=43, Students t-test: p<0.001). This difference remained significant after technical (storage, sampling period, 2′-dG levels) and individual (age, sex, smoking, alcohol) confounding factors were taken into account (p<0.0001, Generalised Linear regression Model). To our knowledge, this is the first report to demonstrate an increase of 8-oxodG in PBMCs of patients suffering from a cancer of the upper digestive tract. This elevated level of DNA damage in patients can raise interesting issues: is oxidative stress the cause or the result of the pathology? Could this biomarker be used to evaluate chemoprevention trials concerning digestive tract cancers?

Radiation-mediated formation of complex damage to DNA: a chemical aspect overview.

During the last three decades, a considerable amount of work has been undertaken to determine the nature, the mechanism of formation and the biological consequences of radiation-induced DNA lesions. Most of the information was obtained via the development of chemical approaches, including theoretical, analytical and organic synthesis methods. Since it is not possible to present all the results obtained in this review article, we will focus on recent data dealing with the formation of complex DNA lesions produced by a single oxidation event, as these lesions may play a significant role in cellular responses to ionizing radiation and also to other sources of oxidative stress. Through the description of specific results, the contribution of different chemical disciplines in the assessment of the structure, the identification of the mechanism of formation and the biological impacts in terms of repair and mutagenicity of these complex radiation-induced DNA lesions will be highlighted.

DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation

Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis.

Tools and strategies for DNA damage interactome analysis

DNA is the target of multiple endogenous and exogenous agents generating chemical lesions on the double helix. Cellular DNA damage response pathways rely on a myriad of proteins interacting with DNA alterations. The cartography of this interactome currently includes well known actors of chromatin remodelling, DNA repair or proteins hijacked from their natural functions such as transcription factors. In order to go further into the characterisation of these protein networks, proteomics-based methods began to be used in the early 2000s. The strategies are diverse and include mainly (i) damaged DNA molecules used as targets on protein microarrays, (ii) damaged DNA probes used to trap within complex cellular extracts proteins that are then separated and identified by proteomics, (iii) identification of chromatin- bound proteins after a genotoxic stress, or (iv) identification of proteins associated with other proteins already known to be part of DNA damage interactome. All these approaches have already been performed to find new proteins recognizing oxidised bases, abasic sites, strand breaks or crosslinks generated by anticancer drugs such as nitrogen mustards and platinating agents. Identified interactions are generally confirmed using complementary methods such as electromobility shift assays or surface plasmon resonance. These strategies allowed, for example, demonstration of interactions between cisplatin-DNA crosslinks and PARP-1 or the protein complex PTW/PP. The next challenging step will be to understand the biological repercussions of these newly identified interactions which may help to unravel new mechanisms involved in genetic toxicology, discover new cellular responses to anticancer drugs or identify new biomarkers and therapeutic targets.

Specificity of TP53 mutation screening methods in cancerous tissues

Dear Sir, Yamanoshita et al. recently published a TP53 mutation pattern obtained by analysing oesophageal cancerous tissues from 207 Chinese patients. With great interest, we read their article, which was mainly dedicated to the comparison of 2 mutation screening approaches: single-strand conformation polymorphism (SSCP) and denaturing high-performance liquid chromatography (DHPLC). Reliable TP53 mutations screening and identification methods are needed in numerous fields of cancer research. In molecular epidemiology and environmental toxicology, the establishment of mutational patterns provides interesting clues in the involvement of cancer risk factors. Two of the most famous examples are tandem CC to TT substitutions related to UV radiations in skin cancers and G to T transversions at codon 249 in aflatoxin B1-related liver cancers. Another application is the use of TP53 mutations as a tool to improve diagnosis, prognosis or prediction of response to therapy. In these areas, the precise determinations of mutation type and localisation inside the gene seem to bring more relevant information than a simple immunohistochemical determination of the p53 protein overexpression. Finally, the identification of TP53 mutations could be of great value if new therapies aiming to restore the protein p53 functionality confirm their promising preliminary results in the future. For all these reasons, many approaches are optimised to improve the detection and the following identification of TP53 mutations in human tissues. These last few years, the most popular of them have been SSCP, functional assay for the separation of alleles in yeast (FASAY), DNA microarrays dedicated to TP53 analysis, restriction methods and techniques based on double-stranded DNA denaturation such as DHPLC, denaturing gradient gel electrophoresis (DGGE) or temporal temperature gradient gel electrophoresis (TTGE). DHPLC has already been used to detect TP53 alterations in different cancerous tissues including oesophagus, stomach, ovary, blood cells, bladder or conjunctiva. As mentionned by Yamanoshita et al., this technique is sensitive, easy to use (in comparison to electrophoretic methods) and offers an interesting throughput. However, the authors reported a disappointing specificity with 50% of falsepositives and considered this performance as ‘‘not yet understood.’’ As this issue is essential to obtain reliable results, we would like to provide here some clues to explain at least one part of this lack of specificity and some practical solutions for clinicians and researchers beginning to screen TP53 mutations. Indeed, we will demonstrate that some false-positives attributed to a lack of DHPLC specificity could in fact constitute genuine mutated DNA fragments. The inclusion of these additional positive results in tumor series could improve the sensitivity of the whole procedure (screening 1 sequencing) and increase mutation rates found in studies similar to that of Yamanoshita et al. These comments are based on our own experience and on technical information collected in other laboratories and published studies. DHPLC is indeed likely to give false-positives. In these cases, additional peaks or irregularities of the baseline can result from degradation products, aspecific DNA amplification or from mutations generated during the polymerase chain reaction (PCR). Provisions against these potential artefacts must be taken: (i) systematic use of well-characterized positive and negative DNA controls for each fragment; (ii) replication of any doubtful