The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast

Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein, required for cellular DNA replication, repair, and recombination. In human cells, RPA is phosphorylated during the S and G2 phases of the cell cycle and also in response to ionizing or ultraviolet radiation. Saccharomyces cerevisiae exhibits a similar pattern of cell cycle-regulated RPA phosphorylation, and our studies indicate that the radiation-induced reactions occur in yeast as well. We have examined yeast RPA phosphorylation during the normal cell cycle and in response to environmental insult, and have demonstrated that the checkpoint gene MEC1 is required for the reaction under all conditions tested. Through examination of several checkpoint mutants, we have placed RPA phosphorylation in a novel pathway of the DNA damage response. MEC1 is similar in sequence to human ATM, the gene mutated in patients with ataxia-telangiectasia (A-T). A-T cells are deficient in multiple checkpoint pathways and are hypersensitive to killing by ionizing radiation. Because A-T cells exhibit a delay in ionizing radiation-induced RPA phosphorylation, our results indicate a functional similarity between MEC1 and ATM, and suggest that RPA phosphorylation is involved in a conserved eukaryotic DNA damage-response pathway defective in A-T.

DNA damage and p53-mediated cell cycle arrest: A reevaluation

Most mammalian cells exhibit transient delays in the G1 and G2 phases of the cell cycle after treatment with radiation or radiomimetic compounds. p53 is required for the arrest in G1, which provides time for DNA repair. Recently, a role of p53 in the G2/M transition has also been suggested. However, it has been reported that the presence of functional p53 does not always correlate with the induction of these checkpoints. To precisely assess the role of p53 in activating cell cycle checkpoints and in cell survival after radiation, we studied the response of two isogenic human fibrosarcoma cell lines differing in their p53 status (wild type or mutant). We found that when irradiated cells undergo a wild-type p53-dependent G1 arrest, they do not subsequently arrest in G2. Moreover, wild-type p53 cells irradiated past the G1 checkpoint arrest in G2 but do not delay in the subsequent G1 phase. Furthermore, in these cell lines, which do not undergo radiation-induced apoptosis, the wild-type p53 cell line exhibited a greater radioresistance in terms of clonogenic survival. These results suggest that the two checkpoints may be interrelated, perhaps through a control system that determines, depending on the extent of the damage, whether the cell needs to arrest cell cycle progression at the subsequent checkpoint for further repair. p53 could be a crucial component of this control system.

Enzyme plus light therapy to repair DNA damage in ultraviolet-B-irradiated human skin

Ultraviolet-B (UVB) (290–320 nm) radiation-induced cyclobutane pyrimidine dimers within the DNA of epidermal cells are detrimental to human health by causing mutations and immunosuppressive effects that presumably contribute to photocarcinogenesis. Conventional photoprotection by sunscreens is exclusively prophylactic in nature and of no value once DNA damage has occurred. In this paper, we have therefore assessed whether it is possible to repair UVB radiation-induced DNA damage through topical application of the DNA-repair enzyme photolyase, derived from Anacystis nidulans, that specifically converts cyclobutane dimers into their original DNA structure after exposure to photoreactivating light. When a dose of UVB radiation sufficient to induce erythema was administered to the skin of healthy subjects, significant numbers of dimers were formed within epidermal cells. Topical application of photolyase-containing liposomes to UVB-irradiated skin and subsequent exposure to photoreactivating light decreased the number of UVB radiation-induced dimers by 40–45%. No reduction was observed if the liposomes were not filled with photolyase or if photoreactivating exposure preceded the application of filled liposomes. The UVB dose administered resulted in suppression of intercellular adhesion molecule-1 (ICAM-1), a molecule required for immunity and inflammatory events in the epidermis. In addition, in subjects hypersensitive to nickel sulfate, elicitation of the hypersensitivity reaction in irradiated skin areas was prevented. Photolyase-induced dimer repair completely prevented these UVB radiation-induced immunosuppressive effects as well as erythema and sunburn-cell formation. These studies demonstrate that topical application of photolyase is effective in dimer reversal and thereby leads to immunoprotection.

Conversion of a radioresistant phenotype to a more sensitive one by disabling erbB receptor signaling in human cancer cells

Inhibition of cell growth and transformation can be achieved in transformed glial cells by disabling erbB receptor signaling. However, recent evidence indicates that the induction of apoptosis may underlie successful therapy of human cancers. In these studies, we examined whether disabling oncoproteins of the erbB receptor family would sensitize transformed human glial cells to the induction of genomic damage by γ-irradiation. Radioresistant human glioblastoma cells in which erbB receptor signaling was inhibited exhibited increased growth arrest and apoptosis in response to DNA damage. Apoptosis was observed after radiation in human glioma cells containing either a wild-type or mutated p53 gene product and suggested that both p53-dependent and -independent mechanisms may be responsible for the more radiosensitive phenotype. Because cells exhibiting increased radiation-induced apoptosis were also capable of growth arrest in serum-deprived conditions and in response to DNA damage, apoptotic cell death was not induced simply as a result of impaired growth arrest pathways. Notably, inhibition of erbB signaling was a more potent stimulus for the induction of apoptosis than prolonged serum deprivation. Proximal receptor interactions between erbB receptor members thus influence cell cycle checkpoint pathways activated in response to DNA damage. Disabling erbB receptors may improve the response to γ-irradiation and other cytotoxic therapies, and this approach suggests that present anticancer strategies could be optimized.

Chromatin-bound PCNA complex formation triggered by DNA damage occurs independent of the ATM gene product in human cells

Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases δ and ɛ, is involved in DNA replication as well as in diverse DNA repair pathways. In quiescent cells, UV light-induced bulky DNA damage triggers the transition of PCNA from a soluble to an insoluble chromatin-bound form, which is intimately associated with the repair synthesis by polymerases δ and ɛ. In this study, we investigated the efficiency of PCNA complex formation in response to ionizing radiation-induced DNA strand breaks in normal and radiation-sensitive Ataxia telangiectasia (AT) cells by immunofluorescence and western blot techniques. Exposure of normal cells to γ-rays rapidly triggered the formation of PCNA foci in a dose-dependent manner in the nuclei and the PCNA foci (40–45%) co-localized with sites of repair synthesis detected by bromodeoxyuridine labeling. The chromatin-bound PCNA gradually declined with increasing post-irradiation times and almost reached the level of unirradiated cells by 6 h. The PCNA foci formed after γ-irradiation was resistant to high salt extraction and the chromatin association of PCNA was lost after DNase I digestion. Interestingly, two radiosensitive primary fibroblast cell lines, derived from AT patients harboring homozygous mutations in the ATM gene, displayed an efficient PCNA redistribution after γ-irradiation. We also analyzed the PCNA complex induced by a radiomimetic agent, Bleomycin (BLM), which produces predominantly single- and double-strand DNA breaks. The efficiency and the time course of PCNA complex induced by BLM were identical in both normal and AT cells. Our study demonstrates for the first time that the ATM gene product is not required for PCNA complex assembly in response to DNA strand breaks. Additionally, we observed an increased interaction of PCNA with the Ku70 and Ku80 heterodimer after DNA damage, suggestive of a role for PCNA in the non-homologous end-joining repair pathway of DNA strand breaks.

Report to the director, National Cancer Institute, from the Occupational Cancer Task Force.

Mode of access: Internet.

Fallout control : final report /

"Contract AT(04-3)-115."

MMTV-trBrca 1 mice display strain-dependent abnormalities in vaginal development

The breast cancer susceptibility gene Brca1 encodes a large multi-functional protein which is implicated as a caretaker of the genome, through its role in regulation of DNA damage response pathways, including apoptosis. Here we show that in mice expressing a dominant-negative Brca1 transgene on a BALB/c background, vaginal entrance remodeling is inhibited, and that the incidence of this phenotype is increased on a p53 +/- genotype. Given that this developmental process is mediated primarily by apoptosis, we hypothesized that disruption of BRCA1 may confer a resistance to apoptosis in normal epithelial cells. Consistent with this, we show that expression of this transgene in vitro leads to resistance to ionizing radiation induced cell killing in mammary epithelial cells. This is the first time that BRCA1 has been implicated in an apoptosis-mediated normal developmental process.

Cell death mechanisms associated with G(2) radiosensitivity in patients with prostate cancer and benign prostatic hyperplasia

Cells respond to genotoxic insults such as ionizing radiation by halting in the G(2) phase of the cell cycle. Delayed cell death (mitotic death) can occur when the cell is released from G(2), and specific spindle defects form endopolyploid cells (endoreduplication/tetraploidy). Enhanced G(2) chromosomal radiosensitivity has been observed in many cancers and genomic instability syndromes, and it is manifested by radiation-induced chromatid aberrations observed in lymphocytes of patients. Here we compare the G(2) chromosomal radiosensitivity in prostate patients with benign prostatic hyperplasia (BPH) or prostate cancer with disease-free controls. We also investigated whether there is a correlation between G(2) chromosomal radiosensitivity and aneuploidy (tetraploidy and endoreduplication), which are indicative of mitotic cell death. The G(2) assay was carried out on all human blood samples. Metaphase analysis was conducted on the harvested chromosomes by counting the number of aberrations and the mitotic errors (endoreduplication/tetraploidy) separately per 100 metaphases. A total of 1/14 of the controls were radiosensitive in G(2) compared to 6/15 of the BPH patients and 15/17 of the prostate cancer patients. Radiation-induced mitotic inhibition was assessed to determine the efficacy of G(2) checkpoint control in the prostate patients. There was no significant correlation of G(2) radiosensitivity scores and mitotic inhibition in BPH patients (P = 0.057), in contrast to prostate cancer patients, who showed a small but significant positive correlation (P = 0.029). Furthermore, there was no significant correlation between G(2) radiosensitivity scores of BPH patients and endoreduplication/ tetraploidy (P = 0.136), which contrasted with an extremely significant correlation observed in prostate cancer patients (P < 0.0001). In conclusion, cells from prostate cancer patients show increased sensitivity to the induction of G(2) aberrations from ionizing radiation exposure but paradoxically show reduced mitotic indices and aneuploidy as a function of aberration frequency.

Involvement of novel autophosphorylation sites in ATM activation

ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.

Inhibition of transforming growth factor-beta 1 signaling attenuates ataxia telanglectasia mutated activity in response to genotoxic stress

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor beta (TGF beta)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGF beta inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf beta 1 nail murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGF beta type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced gamma H2AX radiation-induced foci; and increased radiosensitivity compared with TGF beta competent cells. We determined that loss of TGF beta signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF beta restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgf beta 1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF beta may be used to advantage in cancer therapy.

An exploratory study to determine applicability of nano-hardness and micro-compression measurements for yield stress estimation

The superior properties of ferritic/martensitic steels in a radiation environment (low swelling, low activation under irradiation and good corrosion resistance) make them good candidates for structural parts in future reactors and spallation sources. While it cannot substitute for true reactor experiments, irradiation by charged particles from accelerators can reduce the number of reactor experiments and support fundamental research for a better understanding of radiation effects in materials. Based on the nature of low energy accelerator experiments, only a small volume of material can be uniformly irradiated. Micro and nanoscale post irradiation tests thus have to be performed. We show here that nanoindentation and micro-compression testing on T91 and HT-9 stainless steel before and after ion irradiation are useful methods to evaluate the radiation induced hardening.

Size dependent enhancement of helium ion irradiation tolerance in sputtered Cu/V nanolaminates

We have investigated the evolution of radiation damage and changes in hardness of sputter-deposited Cu/V nanolaminates upon room temperature helium ion irradiation. As the individual layer thickness decreases from 200 to 5 nm, helium bubble density and radiation hardening both decrease. The magnitude of radiation hardening becomes negligible for individual layer thickness of 2.5 nm or less. These observations indicate that nearly immiscible Cu/V interface can effectively absorb radiation-induced point defects and reduce their concentrations.

Towards standardization of UV eye protection:what can be learned from photodermatology?

While knowledge about standardization of skin protection against ultraviolet radiation (UVR) has progressed over the past few decades, there is no uniform and generally accepted standardized measurement for UV eye protection. The literature provides solid evidence that UV can induce considerable damage to structures of the eye. As well as damaging the eyelids and periorbital skin, chronic UV exposure may also affect the conjunctiva and lens. Clinically, this damage can manifest as skin cancer and premature skin ageing as well as the development of pterygia and premature cortical cataracts. Modern eye protection, used daily, offers the opportunity to prevent these adverse sequelae of lifelong UV exposure. A standardized, reliable and comprehensive label for consumers and professionals is currently lacking. In this review we (i) summarize the existing literature about UV radiation-induced damage to the eye and surrounding skin; (ii) review the recent technological advances in UV protection by means of lenses; (iii) review the definition of the Eye-Sun Protection Factor (E-SPF®), which describes the intrinsic UV protection properties of lenses and lens coating materials based on their capacity to absorb or reflect UV radiation; and (iv) propose a strategy for establishing the biological relevance of the E-SPF. © 2013 John Wiley & Sons A/S.

Quantifying uncertainty in citizen weather data

The sheer volume of citizen weather data collected and uploaded to online data hubs is immense. However as with any citizen data it is difficult to assess the accuracy of the measurements. Within this project we quantify just how much data is available, where it comes from, the frequency at which it is collected, and the types of automatic weather stations being used. We also list the numerous possible sources of error and uncertainty within citizen weather observations before showing evidence of such effects in real data. A thorough intercomparison field study was conducted, testing popular models of citizen weather stations. From this study we were able to parameterise key sources of bias. Most significantly the project develops a complete quality control system through which citizen air temperature observations can be passed. The structure of this system was heavily informed by the results of the field study. Using a Bayesian framework the system learns and updates its estimates of the calibration and radiation-induced biases inherent to each station. We then show the benefit of correcting for these learnt biases over using the original uncorrected data. The system also attaches an uncertainty estimate to each observation, which would provide real world applications that choose to incorporate such observations with a measure on which they may base their confidence in the data. The system relies on interpolated temperature and radiation observations from neighbouring professional weather stations for which a Bayesian regression model is used. We recognise some of the assumptions and flaws of the developed system and suggest further work that needs to be done to bring it to an operational setting. Such a system will hopefully allow applications to leverage the additional value citizen weather data brings to longstanding professional observing networks.