Translating particulate hexavalent chromium-induced chromosome instability from human lung cells to experimental animals, human lung tumors and whale cells.

Lung cancer is a major human health problem. While smoking is the most well-known cause of lung cancer, people who never smoked develop the disease. Understanding how non-tobacco environmental carcinogens cause lung cancer is key to combating this disease. Hexavalent chromium [Cr(VI)] is a well-established human lung carcinogen, but its carcinogenic mechanism is uncertain. Chromosome instability (CIN) is a hallmark of lung cancer and a major factor in Cr(VI)-induced lung cancer. Studies in human lung cells show Cr(VI) induces DNA double strand breaks and suppresses homologous recombination (HR) repair by targeting RAD51, resulting in CIN. We translated these outcomes to rats, as this species develops Cr(VI)-induced lung tumors. We exposed 12-week-old Wistar rats to a single dose of zinc chromate for 24 hours or a weekly dose for 90 days via oropharyngeal aspiration. DNA double strand breaks and HR repair increased in a concentration-dependent manner in rat lungs after 24-hour Cr(VI) exposure. After 90-day exposure, DNA double strand breaks increased, but HR repair decreased. These effects were distinct in bronchioles but muted in alveoli, consistent with Cr(VI)-induced human lung tumors originating in bronchial epithelium. We translated these outcomes to Cr(VI)-associated human lung tumors. DNA double strand breaks significantly increased but RAD51 expression decreased in lung tumors; demonstrating Cr(VI)-induced DNA double strand breaks and HR inhibition persist in tumors. Long-lived whales can experience long-term exposure to environmental contaminants but have low cancer rates. We measured the ability of Cr(VI) to induce DNA double strand breaks, HR repair, and chromosome damage in bowhead whale lung cells. Cr(VI) induced DNA strand breaks in whale cells, but the HR repair response remained intact. Thus, whale cells are resistant to Cr(VI)-induced loss of HR repair with no apparent CIN. These results indicate significant differences in the response of human and bowhead whale lung cells to Cr(VI) exposure. Overall, our studies translate Cr(VI)-induced DNA double strand breaks and HR repair impacts to rat lung tissue, human lung tumors and whale lung cells. Cr(VI) induces DNA double strand breaks and inhibits HR repair in vivo, but does not cause HR repair failure and CIN in whale lung cells

DNA Double-Strand Break Repair: A Relentless Hunt Uncovers New Prey

A major pathway for repair of DNA double-strand breaks is nonhomologous end-joining (NHEJ). In this issue of Cell, Buck et al. (2006a) and Ahnesorg et al. (2006) report the discovery of a new NHEJ factor called Cernunnos-XLF. Both groups report that this protein is mutated in a rare inherited human syndrome characterized by severe immunodeficiency, developmental delay, and hypersensitivity to agents that cause DNA double-strand breaks

DNA Double-Strand Break Repair: A Relentless Hunt Uncovers New Prey

A major pathway for repair of DNA double-strand breaks is nonhomologous end-joining (NHEJ). In this issue of Cell, Buck et al. (2006a) and Ahnesorg et al. (2006) report the discovery of a new NHEJ factor called Cernunnos-XLF. Both groups report that this protein is mutated in a rare inherited human syndrome characterized by severe immunodeficiency, developmental delay, and hypersensitivity to agents that cause DNA double-strand breaks

DNA double-strand break induction by gamma radiation

Includes bibliographical references.Radiation is a powerful tool in fighting breast cancer. However, the mechanism of cell killing is not fully understood. It is therefore important to characterize radiation so that it may be used more effectively. In the present study the relationship between gamma radiation and DNA double strand break induction is investigated. DNA double strand breaks are the focus of this study because the biological consequences of DNA double-strand breaks are significant, and can result in cell-killing/ apotosis (Heilmann 1995). DNA double strand break induction is surveyed using a constant-field gel electrophoresis approach previously described by J. Heilmann (Heilmann 1995). A linear increase in DNA double-strand breaks is reported here which is consistent with other models of DNA double-strand break induction. This study is part of ongoing research involving the delivery of a cytotoxic dose of radiation to cancer cells.B.S. (Bachelor of Science

Extensive ssDNA end formation at DNA double-strand breaks in non-homologous end-joining deficient cells during the S phase

BACKGROUND: Efficient and correct repair of DNA damage, especially DNA double-strand breaks, is critical for cellular survival. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks in mammalian cells. The ability of other repair pathways, such as homologous recombination, to compensate for loss of NHEJ and the ways in which contributions of different pathways are regulated are far from fully understood. RESULTS: In this report we demonstrate that long single-stranded DNA (ssDNA) ends are formed at radiation-induced DNA double-strand breaks in NHEJ deficient cells. At repair times ≥ 1 h, processing of unrejoined DNA double-strand breaks generated extensive ssDNA at the DNA ends in cells lacking the NHEJ protein complexes DNA-dependent protein kinase (DNA-PK) or DNA Ligase IV/XRCC4. The ssDNA formation was cell cycle dependent, since no ssDNA ends were observed in G(1)-synchronized NHEJ deficient cells. Furthermore, in wild type cells irradiated in the presence of DNA-PKcs (catalytic subunit of DNA-PK) inhibitors, or in DNA-PKcs deficient cells complemented with DNA-PKcs mutated in six autophosphorylation sites (ABCDE), no ssDNA was formed. The ssDNA generation also greatly influences DNA double-strand break quantification by pulsed-field gel electrophoresis, resulting in overestimation of the DNA double-strand break repair capability in NHEJ deficient cells when standard protocols for preparing naked DNA (i. e., lysis at 50°C) are used. CONCLUSION: We provide evidence that DNA Ligase IV/XRCC4 recruitment by DNA-PK to DNA double-strand breaks prevents the formation of long ssDNA ends at double-strand breaks during the S phase, indicating that NHEJ components may downregulate an alternative repair process where ssDNA ends are required

DNA Double-Strand Breaks Come into Focus

The Mre11-Rad50-Nbs1 (MRN) complex senses DNA double-strand breaks and recruits different repair pathway and checkpoint proteins to break foci. Two new studies (Williams et al., 2009; Lloyd et al., 2009) identify Nbs1 as a key factor in this process and reveal how an N-terminal protein recruitment module in Nbs1 binds to different response factors through shared phosphopeptide motifs

Early neuronal accumulation of DNA double strand breaks in Alzheimer's disease.

The maintenance of genomic integrity is essential for normal cellular functions. However, it is difficult to maintain over a lifetime in postmitotic cells such as neurons, in which DNA damage increases with age and is exacerbated by multiple neurological disorders, including Alzheimer's disease (AD). Here we used immunohistochemical staining to detect DNA double strand breaks (DSBs), the most severe form of DNA damage, in postmortem brain tissues from patients with mild cognitive impairment (MCI) or AD and from cognitively unimpaired controls. Immunostaining for γH2AX-a post-translational histone modification that is widely used as a marker of DSBs-revealed increased proportions of γH2AX-labeled neurons and astrocytes in the hippocampus and frontal cortex of MCI and AD patients, as compared to age-matched controls. In contrast to the focal pattern associated with DSBs, some neurons and glia in humans and mice showed diffuse pan-nuclear patterns of γH2AX immunoreactivity. In mouse brains and primary neuronal cultures, such pan-nuclear γH2AX labeling could be elicited by increasing neuronal activity. To assess whether pan-nuclear γH2AX represents DSBs, we used a recently developed technology, DNA damage in situ ligation followed by proximity ligation assay, to detect close associations between γH2AX sites and free DSB ends. This assay revealed no evidence of DSBs in neurons or astrocytes with prominent pan-nuclear γH2AX labeling. These findings suggest that focal, but not pan-nuclear, increases in γH2AX immunoreactivity are associated with DSBs in brain tissue and that these distinct patterns of γH2AX formation may have different causes and consequences. We conclude that AD is associated with an accumulation of DSBs in vulnerable neuronal and glial cell populations from early stages onward. Because of the severe adverse effects this type of DNA damage can have on gene expression, chromatin stability and cellular functions, DSBs could be an important causal driver of neurodegeneration and cognitive decline in this disease

Repair of DNA double-strand breaks in heterochromatin

DNA double-strand breaks (DSBs) are among the most damaging lesions in DNA, since, if not identified and repaired, they can lead to insertions, deletions or chromosomal rearrangements. DSBs can be in the form of simple or complex breaks, and may be repaired by one of a number of processes, the nature of which depends on the complexity of the break or the position of the break within the chromatin. In eukaryotic cells, nuclear DNA is maintained as either euchromatin (EC) which is loosely packed, or in a denser form, much of which is heterochromatin (HC). Due to the less accessible nature of the DNA in HC as compared to that in EC, repair of damage in HC is not as straightforward as repair in EC. Here we review the literature on how cells deal with DSBs in HC