Cancer Biology

8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is considered to be a premutagenic DNA lesion generated by 2'-deoxyguanosine (dG) oxidation due to reactive oxygen species (ROS). In recent years, the 8-oxodG distribution in human, mouse, and yeast genomes has been underlined using various next-generation sequencing (NGS)–based strategies. The present study reports the OxiDIP-Seq protocol, which combines specific 8-oxodG immuno-precipitation of single-stranded DNA with NGS, and the pipeline analysis that allows the genome-wide 8-oxodG distribution in mammalian cells. The development of this OxiDIP-Seq method increases knowledge on the oxidative DNA damage/repair field, providing a high-resolution map of 8-oxodG in human cells.

The main cellular pathways to repair DNA double-strand breaks (DSBs) and protect the integrity of the genome are homologous recombination (HR), non-homologous end-joining (NHEJ), and alternative end-joining (Alt-EJ). Polymerase theta-regulated Alt-EJ is an error-prone DSB repair pathway characterized by microhomology usage. Considering its importance in cancer treatment, technologies for detection of Alt-EJ in cancer cells may facilitate the study of the mechanisms of carcinogenesis and the development of new therapeutic targets. DSB reporter assay is the classical method for detecting Alt-EJ, which is primarily based on components of EJ2-puro cassette integration, I-SceI cleaving, and flow cytometry analysis. Here, we described an assay based on a modified I-Scel plasmid that can screen head and neck squamous cell carcinoma (HNSC) cells that were successfully transfected using selection medium with hygrovetine. We expect that this protocol will improve the fidelity and accuracy of reporter assays.

Graphical abstract:

Schematic overview of the workflow for establishment of Alt-EJ reporters.

Bromodomain-containing protein 4 (BRD4) is an acetyl-lysine reader protein and transcriptional regulator implicated in chromatin dynamics and cancer development. Several BRD4 isoforms have been detected in humans with the long isoform (BRD4-L, aa 1-1,362) playing a tumor-suppressive role and a major short isoform (BRD4-S, aa 1-722) having oncogenic activity in breast cancer development. In vivo demonstration of the opposing functions of BRD4 protein isoforms requires development of mouse models, particularly transgenic mice conditionally expressing human BRD4-L or BRD4-S, which can be selectively induced in different mouse tissues in a spatiotemporal-specific manner. Here, we detail the procedures used to genotype transgenic mouse strains developed to define the effects of conditional human BRD4 isoform expression on polyomavirus middle T antigen (PyMT)-induced mouse mammary tumor growth, and the key steps for Western blot detection of BRD4 protein isoforms in those tumors and in cultured cells. With this protocol as a guide, interpretation of BRD4 isoform functions becomes more feasible and expandable to various biological settings. Adequate tracking of BRD4 isoform distributions in vivo and in vitro is key to understanding their biological roles, as well as avoiding misinterpretation of their functions due to improper use of experimental procedures that fail to detect their spatial and temporal distributions.

Graphic abstract:

In the context of precision medicine, the identification of novel biomarkers for the diagnosis of disease, prognosis, predicting treatment outcome and monitoring of treatment success is of great importance. The analysis of methylated circulating-cell free DNA provides great promise to complement or replace genetic markers for these applications, but is associated with substantial challenges. This is particularly true for the detection of rare methylated DNA molecules in a limited amount of sample such as tumor released hypermethylated molecules in the background of DNA fragments from normal cells, especially lymphocytes.

Technologies for the sensitive detection of DNA methylation have been developed to enrich specifically methylated DNA or unmethylated DNA using among other methods: enzymatic digestion, methylation-specific PCR (often combined with TaqMan like oligonucleotide probes (MethyLight)) and co-amplification at lower denaturation temperature PCR (COLD-PCR).

E-ice-COLD-PCR (Enhanced-improved and complete enrichment-COLD-PCR) is a sensitive method that takes advantage of a Locked Nucleic Acid (LNA)-containing oligonucleotide probe to block specifically unmethylated CpG sites allowing the strong enrichment of low-abundant methylated CpG sites from a limited quantity of input. E-ice-COLD-PCRs are performed on bisulfite-converted DNA followed by Pyrosequencing analysis. The quantification of the initially present DNA methylation level is obtained using calibration curves of methylated and unmethylated DNA. The E-ice-COLD-PCR reactions can be multiplexed, allowing the analysis and quantification of the DNA methylation level of several target genes. In contrast to the above-mentioned assays, E-ice-COLD-PCR will also perform in the presence of frequently occurring heterogeneous DNA methylation patterns at the target sites. The presented protocol describes the development of an E-ice-COLD-PCR assay including assay design, optimization of E-ice-COLD-PCR conditions including annealing temperature, critical temperature and concentration of LNA blocker probe followed by Pyrosequencing analysis.

Human centromeres are composed of large tandem arrays of repetitive alpha satellite DNA, which are often sites of aberrant rearrangement in cancers (Mitelman et al., 1997; Padilla-Nash et al., 2001). To date, annotation of the human centromere repetitive sequences remains incomplete, greatly hindering in-depth functional studies of these regions essential for chromosome segregation. In order to monitor sister chromatid exchange happening at the centromere (C-SCE) due to recombination and mutagenic events, I have applied the Chromosome-Orientation Fluorescence in situ Hybridization (CO-FISH) technique to centromeres (Cen-CO-FISH) in human cells. This hybridization-based method involves (1) the incorporation of nucleotide analogs through a single round of replication, (2) enzymatic digestion of the newly synthesized DNA strand and (3) subsequent hybridization of single-stranded probes, in absence of a denaturation step. The resulting signal allows to differentially label each sister chromatid based on the 5’-3’ directionality of the DNA and to score aberrant staining patterns indicative of C-SCE. The Cen-CO-FISH method applied to human centromeres revealed that human centromeres indeed undergo recombination in cycling cells resulting in C-SCE, and centromere instability is enhanced in cancer cell lines and primary cells undergoing senescence (Giunta and Funabiki, 2017). Here, I present the detailed protocol of the preparation, experimental procedure and data acquisition for the Cen-CO-FISH method in human cells. It also includes a conceptual overview of the technique, with examples of representative images and scoring guidelines. The Cen-CO-FISH represents a valuable tool to facilitate exploration of centromere repeats.