Identification of Recurrent Chromosome Breaks Underlying Structural Rearrangements in Mammary Cancer Cell Lines.

Cancer genomes are characterized by the accumulation of small-scale somatic mutations as well as large-scale chromosomal deletions, amplifications, and complex structural rearrangements. This characteristic is at least partially dependent on the ability of cancer cells to undergo recurrent chromosome breakage. In order to address the extent to which chromosomal structural rearrangement breakpoints correlate with recurrent DNA double-strand breaks (DSBs), we simultaneously mapped chromosome structural variation breakpoints (using whole-genome DNA-seq) and spontaneous DSB formation (using Break-seq) in the estrogen receptor (ER)-positive breast cancer cell line MCF-7 and a non-cancer control breast epithelium cell line MCF-10A. We identified concurrent DSBs and structural variation breakpoints almost exclusively in the pericentromeric region of chromosome 16q in MCF-7 cells. We fine-tuned the identification of copy number variation breakpoints on 16q. In addition, we detected recurrent DSBs that occurred in both MCF-7 and MCF-10A. We propose a model for DSB-driven chromosome rearrangements that lead to the translocation of 16q, likely with 10q, and the eventual 16q loss that does not involve the pericentromere of 16q. We present evidence from RNA-seq data that select genes, including SHCBP1, ORC6, and MYLK3, which are immediately downstream from the 16q pericentromere, show heightened expression in MCF-7 cell line compared to the control. Data published by The Cancer Genome Atlas show that all three genes have increased expression in breast tumor samples. We found that SHCBP1 and ORC6 are both strong poor prognosis and treatment outcome markers in the ER-positive breast cancer cohort. We suggest that these genes are potential oncogenes for breast cancer progression. The search for tumor suppressor loss that accompanies the 16q loss ought to be augmented by the identification of potential oncogenes that gained expression during chromosomal rearrangements.

Chromosome breaks generated by low doses of ionizing radiation in G2-phase are processed exclusively by gene conversion.

Four repair pathways process DNA double-strand breaks (DSBs). Among these pathways the homologous recombination repair (HRR) subpathway of gene conversion (GC) affords error-free processing, but functions only in S- and G2-phases of the cell cycle. Classical non-homologous end-joining (c-NHEJ) operates throughout the cell cycle, but causes small deletions and translocations. Similar deficiencies in exaggerated form, combined with reduced efficiency, are associated with alternative end-joining (alt-EJ). Finally, single-strand annealing (SSA) causes large deletions and possibly translocations. Thus, processing of a DSB by any pathway, except GC, poses significant risks to the genome, making the mechanisms navigating pathway-engagement critical to genome stability. Logically, the cell ought to attempt engagement of the pathway ensuring preservation of the genome, while accommodating necessities generated by the types of DSBs induced. Thereby, inception of DNA end-resection will be key determinant for GC, SSA and alt-EJ engagement. We reported that during G2-phase, where all pathways are active, GC engages in the processing of almost 50 % of DSBs, at low DSB-loads in the genome, and that this contribution rapidly drops to nearly zero with increasing DSB-loads. At the transition between these two extremes, SSA and alt-EJ compensate, but at extremely high DSB-loads resection-dependent pathways are suppressed and c-NHEJ remains mainly active. We inquired whether in this processing framework all DSBs have similar fates. Here, we analyze in G2-phase the processing of a subset of DSBs defined by their ability to break chromosomes. Our results reveal an absolute requirement for GC in the processing of chromatid breaks at doses in the range of 1 Gy. Defects in c-NHEJ delay significantly the inception of processing by GC, but leave processing kinetics unchanged. These results delineate the essential role of GC in chromatid break repair before mitosis and classify DSBs that underpin this breakage as the exclusive substrate of GC.

Topoisomerase I prevents transcription-replication conflicts at transcription termination sites.

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops, we mapped RNA:DNA hybrids, markers of replication fork stalling and DNA double-strand breaks along the human genome. This analysis indicates that transient replication fork pausing occurs at the transcription termination sites of highly expressed genes enriched in R-loops and prevents head-on conflicts with transcription, in a topoisomerase I-dependent manner.

Spatial Chromosome Folding and Active Transcription Drive DNA Fragility and Formation of Oncogenic MLL Translocations.

How spatial chromosome organization influences genome integrity is still poorly understood. Here, we show that DNA double-strand breaks (DSBs) mediated by topoisomerase 2 (TOP2) activities are enriched at chromatin loop anchors with high transcriptional activity. Recurrent DSBs occur at CCCTC-binding factor (CTCF) and cohesin-bound sites at the bases of chromatin loops, and their frequency positively correlates with transcriptional output and directionality. The physiological relevance of this preferential positioning is indicated by the finding that genes recurrently translocating to drive leukemias are highly transcribed and are enriched at loop anchors. These genes accumulate DSBs at recurrent hotspots that give rise to chromosomal fusions relying on the activity of both TOP2 isoforms and on transcriptional elongation. We propose that transcription and 3D chromosome folding jointly pose a threat to genomic stability and are key contributors to the occurrence of genome rearrangements that drive cancer.

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways.

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

The genetic landscape of gliomas arising after therapeutic radiation.

Radiotherapy improves survival for common childhood cancers such as medulloblastoma, leukemia, and germ cell tumors. Unfortunately, long-term survivors suffer sequelae that can include secondary neoplasia. Gliomas are common secondary neoplasms after cranial or craniospinal radiation, most often manifesting as high-grade astrocytomas with poor clinical outcomes. Here, we performed genetic profiling on a cohort of 12 gliomas arising after therapeutic radiation to determine their molecular pathogenesis and assess for differences in genomic signature compared to their spontaneous counterparts. We identified a high frequency of TP53 mutations, CDK4 amplification or CDKN2A homozygous deletion, and amplifications or rearrangements involving receptor tyrosine kinase and Ras-Raf-MAP kinase pathway genes including PDGFRA, MET, BRAF, and RRAS2. Notably, all tumors lacked alterations in IDH1, IDH2, H3F3A, HIST1H3B, HIST1H3C, TERT (including promoter region), and PTEN, which genetically define the major subtypes of diffuse gliomas in children and adults. All gliomas in this cohort had very low somatic mutation burden (less than three somatic single nucleotide variants or small indels per Mb). The ten high-grade gliomas demonstrated markedly aneuploid genomes, with significantly increased quantity of intrachromosomal copy number breakpoints and focal amplifications/homozygous deletions compared to spontaneous high-grade gliomas, likely as a result of DNA double-strand breaks induced by gamma radiation. Together, these findings demonstrate a distinct molecular pathogenesis of secondary gliomas arising after radiation therapy and identify a genomic signature that may aid in differentiating these tumors from their spontaneous counterparts.

Evaluation of Cytotoxic and Genotoxic Effects of Extremely Low-frequency Electromagnetic Field on Mesenchymal Stromal Cells.

BACKGROUND: Interest in the use of extremely low-frequency (ELF) electromagnetic field (EMF) for the treatment of pain and inflammation is increasing due to the ability of this promising therapy to compete with pharmaceuticals without the adverse effects caused by drugs. However, there continues to be concerns regarding cytotoxic and genotoxic effects that may occur as a result of exposure to EMF. OBJECTIVE: To investigate this concern, we tested the effect of our known therapeutic 5 Hz, 0.4 milliTesla (mT) EMF on a human mesenchymal stromal cell (hMSC) line to determine whether ELF-EMF exposure would cause cytotoxic or genotoxic effects. METHODS: Treated samples along with controls were exposed to 5 Hz, 0.4 mT ELF-EMF for 20 min/day, 3×/week for 2 weeks and then assayed for cell viability, proliferation rates, and chromosome breaks. RESULTS: Cytogenetic analysis of the viability and proliferation rates along with analysis of morphological genome stability showed no cytotoxicity, and no chromosome breaks per karyotype analysis-therefore no genotoxicity. CONCLUSION: Exposure to an ELF-EMF of 5 Hz, 0.4 mT for 20 min/day, 3×/week for 2 weeks does not cause cytotoxic or genotoxic effects in hMSCs.

Inhibitor of caspase-activated DNase expression enhances caspase-activated DNase expression and inhibits oxidative stress-induced chromosome breaks at the mixed lineage leukaemia gene in nasopharyngeal carcinoma cells.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is commonly found in Asia, especially among the Chinese ethnic group. Chromosome rearrangements are common among NPC patients. Although the mechanism underlying the chromosome rearrangements in NPC is unclear, various mechanisms including activation of caspase-activated DNase (CAD) were proposed to contribute to chromosome rearrangements in leukaemia. Activation of CAD can be initiated by multiple agents, including oxidative stress, which is well implicated in carcinogenesis. CAD is the main enzyme that causes DNA fragmentation during apoptosis, and CAD is also implicated in promoting cell differentiation. In view of the role of oxidative stress in carcinogenesis and CAD activation, and since CAD was suggested to contribute to chromosome rearrangement in leukaemia, we hypothesise that oxidative stress-induced CAD activation could be one of the mechanisms that leads to chromosome rearrangements in NPC. METHODS: SUNEI cells were treated with various concentrations of H2O2 for different period of time to ensure that cells undergo H2O2-induced MLL gene cleavage. Transfections with hCAD, mCAD, mutant hCAD, or cotransfection with hCAD and mICAD, and cotransfection with mutant hCAD and mICAD were performed. Gene expression was confirmed by Western blotting and MLL gene cleavage was assessed by inverse polymerase chain reaction (IPCR). RESULTS: Treatment with H2O2 clearly induces cleavages within the MLL gene which locates at 11q23, a common deletion site in NPC. In order to investigate the role of CAD, CAD was overexpressed in SUNE1 cells, but that did not result in significant changes in H2O2-induced MLL gene cleavage. This could be because CAD requires ICAD for proper folding. Indeed, by overexpressing ICAD alone or co-expressing ICAD with CAD, Western blotting showed that CAD was expressed. In addition, ICAD overexpression also suppressed H2O2-induced MLL gene cleavage, suggesting a possible role of CAD in initiating chromosome cleavage during oxidative stress. CONCLUSIONS: Oxidative stress mediated by H2O2 induces cleavage of the MLL gene, most likely via the caspase-activated DNase, CAD, and CAD expression requires ICAD. Since the MLL gene is located at 11q23, a common deletion site in NPC, thus stress-induced CAD activation may represent one of the mechanisms leading to chromosome rearrangement in NPC.