Influence of Cardiac MR Imaging on DNA Double-Strand Breaks in Human Blood Lymphocytes.

PURPOSE: To evaluate the ability of magnetic resonance (MR) imaging to induce deoxyribonucleic acid (DNA) damage in patients who underwent cardiac MR imaging in daily routine by using γ-H2AX immunofluorescence microscopy. MATERIALS AND METHODS: This study complies with the Declaration of Helsinki and was performed according to local ethics committee approval. Informed patient consent was obtained. Blood samples from 45 patients (13 women, 32 men; mean age, 50.3 years [age range, 20-89 years]) were obtained before and after contrast agent-enhanced cardiac MR imaging. MR imaging-induced double-strand breaks (DSBs) were quantified in isolated blood lymphocytes by using immunofluorescence microscopy after staining the phosphorylated histone variant γ-H2AX. Twenty-nine patients were examined with a myocarditis protocol (group A), 10 patients with a stress-testing protocol (group B), and six patients with flow measurements and angiography (group C). Paired t test was performed to compare excess foci before and after MR imaging. RESULTS: The mean baseline DSB level before MR imaging and 5 minutes after MR imaging was, respectively, 0.116 DSB per cell ± 0.019 (standard deviation) and 0.117 DSB per cell ± 0.019 (P = .71). There was also no significant difference in DSBs in these subgroups (group A: DSB per cell before and after MR imaging, respectively, 0.114 and 0.114, P = .91; group B: DSB per cell before and after MR imaging, respectively, 0.123 and 0.124, P = .78; group C: DSB per cell before and after MR imaging, respectively, 0.114 and 0.115, P = .36). CONCLUSION: By using γ-H2AX immunofluorescence microscopy, no DNA DSBs were detected after cardiac MR imaging.

DeltaNp63 induces beta-catenin nuclear accumulation and signaling.

The P53 homolog p63 encodes multiple proteins with transactivating, apoptosis-inducing, and oncogenic activities. We showed that p63 is amplified and that DeltaNp63 isotypes are overexpressed in squamous cell carcinoma (SCC) and enhance oncogenic growth in vitro and in vivo. Moreover, p53 associated with DeltaNp63alpha and mediated its degradation. Here, we report that DeltaNp63 associates with the B56alpha regulatory subunit of protein phosphatase 2A (PP2A) and glycogen synthase kinase 3beta (GSK3beta), leading to a dramatic inhibition of PP2A-mediated GSK3beta reactivation. The inhibitory effect of DeltaNp63 on GSK3beta mediates a decrease in phosphorylation levels of beta-catenin, which induces intranuclear accumulation of beta-catenin and activates beta-catenin-dependent transcription. Our results suggest that DeltaNp63 isotypes act as positive regulators of the beta-catenin signaling pathway, providing a basis for their oncogenic properties.

Influence of risk-organ-based tube current modulation on CT-induced DNA double-strand breaks in a biological phantom model.

Techniques for dose reduction in computed tomography (CT) are receiving increasing attention. Lowering the tube current in front of the patient, known as risk-organ-based tube current modulation (RTM), represents a new approach. Physical dose parameters can determine the exposure but are not able to assess the biological-X-ray interactions. The purpose of this study was to establish a biological phantom model to evaluate the effect of RTM on X-ray-induced DNA double-strand breaks (DSBs). In breast phantoms and in the location of the spine in an Alderson phantom, isolated human blood lymphocytes were irradiated using a 128-slice CT scanner. A standard thoracic CT protocol (120 kV, 110 ref. mAs, anatomy-based tube current modulation, pitch 0.6, scan length 30 cm) with and without RTM was used. X-ray-induced DSBs were quantified in isolated blood lymphocytes using immunofluorescence microscopy after staining for the phosphorylated histone variant γ-H2AX. Using RTM, the resulting DNA damage reduction was 41% in superficial breast locations (P = 0.0001), 28% in middle breast locations (P = 0.0003) and 29% in lower breast locations (P = 0.0001), but we found a DNA damage increase of 36% in superficial spine locations (P = 0.0001) and of 26% in deep spine locations (P = 0.0001). In summary, we established a biological phantom model that is suitable for detecting DNA damage in distinct organs. In addition, we were able to show that, using RTM, X-ray-induced DNA damage in the breast can be significantly reduced; however, there is a significant increase in DSBs in the location of the spine.

Reduction of X-ray-induced DNA damage in normal human cells treated with the PrC-210 radioprotector.

The aim of our study was to determine the protective efficacy of the PrC-210 aminothiol radioprotector against X-ray-induced DNA damage in normal human cells and to establish dose- and time-effect models for future PrC-210 use in humans. The PrC-210 structure has a branched structure which enables scavenging of reactive oxygen species (ROS) away from DNA. Normal human blood lymphocytes, fibroblasts and naked genomic DNA were exposed to PrC-210 seconds to hours prior to irradiation. Biological (γ-H2AX foci), chemical (8-oxo-deoxyguanosine) and physical (genomic DNA electrophoretic migration) DNA damage endpoints were scored to determine the ability of PrC-210 to suppress radiation-induced DNA damage. X-ray-induced γ-H2AX foci in blood lymphocytes were reduced by 80% after irradiation with 10, 50 and 100 mGy, and DNA double-strand breaks in fibroblasts were reduced by 60% after irradiation with 20 Gy. Additionally, we observed a reduction of 8-oxo-deoxyguanosine (an ROS-mediated, DNA damage marker) in human genomic DNA to background in a PrC-210 dose-dependent manner. PrC-210 also eliminated radiation-induced cell death in colony formation assays after irradiation with 1 Gy. The protective efficacy of PrC-210 in each of these assay systems supports its development as a radioprotector for humans in multiple radiation exposure settings.

Influence of Different Antioxidants on X-Ray Induced DNA Double-Strand Breaks (DSBs) Using γ-H2AX Immunofluorescence Microscopy in a Preliminary Study.

BACKGROUND: Radiation exposure occurs in X-ray guided interventional procedures or computed tomography (CT) and γ-H2AX-foci are recognized to represent DNA double-strand breaks (DSBs) as a biomarker for radiation induced damage. Antioxidants may reduce the induction of γ-H2AX-foci by binding free radicals. The aim of this study was to establish a dose-effect relationship and a time-effect relationship for the individual antioxidants on DSBs in human blood lymphocytes. MATERIALS AND METHODS: Blood samples from volunteers were irradiated with 10 mGy before and after pre-incubation with different antioxidants (zinc, trolox, lipoic acid, ß-carotene, selenium, vitamin E, vitamin C, N-acetyl-L-cysteine (NAC) and Q 10). Thereby, different pre-incubation times, concentrations and combinations of drugs were evaluated. For assessment of DSBs, lymphocytes were stained against the phosphorylated histone variant γ-H2AX. RESULTS: For zinc, trolox and lipoic acid regardless of concentration or pre-incubation time, no significant decrease of γ-H2AX-foci was found. However, ß-carotene (15%), selenium (14%), vitamin E (12%), vitamin C (25%), NAC (43%) and Q 10 (18%) led to a significant reduction of γ-H2AX-foci at a pre-incubation time of 1 hour. The combination of different antioxidants did not have an additive effect. CONCLUSION: Antioxidants administered prior to irradiation demonstrated the potential to reduce γ-H2AX-foci in blood lymphocytes.

PUMA in head and neck cancer.

Genetic alterations of p53, which monitors DNA damage and operates cellular checkpoints, is a major factor in the development of many types of cancer in human. PUMA, a direct mediator of p53-associated apoptosis, was recently identified. The PUMA gene was mapped to chromosomal arm 19q, a region frequently deleted in head/neck and lung cancers. We analyzed 30 primary tumors (15 head/neck and 15 lung) for loss of heterozygosity (LOH) at 19q using seven widely spaced microsatellite markers. LOH in at least one marker was present in 8 (56%) of the head/neck and 4 (26.6%) of the lung cancer samples. Overall, D19S408 and D19S412, showed the highest rates of allelic loss (23.3 and 16.6%, respectively). We then sequenced the entire coding region of the PUMA gene in all the 30 primary tumors and in 10 head/neck cancer cell lines. No mutations of PUMA were detected in any samples examined, regardless of the mutational status of the p53 gene. Forced expression of wild-type PUMA in JHU-012 and JHU-013 head/neck cancer cell lines significantly inhibited colony formation. Although PUMA suppresses tumor cell growth in head/neck cancer, it does not appear to be a direct target of inactivation in head and neck tumorigenesis.

X-ray induced DNA double-strand breaks in coronary CT angiography: comparison of sequential, low-pitch helical and high-pitch helical data acquisition.

BACKGROUND: Aim of this study was to compare DNA double-strand breaks (DSBs) in blood lymphocytes of patients undergoing high-pitch helical, low-pitch helical and sequential coronary CT angiography. METHODS AND RESULTS: 66 patients were examined with various scan protocols and modes (low-pitch helical scan: 100-120 kV, 320-438 mAs/rot, pitch 0.18-0.39, with or without ECG-pulsing, n=35; prospectively ECG-triggered high-pitch helical scan: 100-120 kV, 320-456 mAs/rotation, pitch 3.2-3.4, n=19; prospectively ECG-triggered sequential scan: 100-120 kV, 150-300 mAs or 320-370 mAs/rotation, n=12) either using a 64-slice or 128-slice dual-source CT or a 128-slice single source CT scanner. Blood samples were obtained before and 30 min after CT and DSBs were analyzed in isolated lymphocytes using γ-H2AX immunofluorescence microscopy. A significant increase of DSBs was measurable 30 min after CTA (range 0.01-0.71/cell). CT induced DSBs showed a significant correlation with the estimated effective dose (ρ=0.90, p<0.00001). Both prospectively ECG-triggered sequential (0.10 DSBs/cell, 176 mGy cm, p<0.00001) and high-pitch helical scan protocols (0.03 DSBs/cell, 109 mGy cm, p<0.00001) led to a significant reduction of median DLP and DSB levels compared to low-pitch helical scans (0.34 DSBs/cell, 828 mGy cm). A reduction of the tube voltage resulted in significantly lower whereas additional calcium scoring resulted in elevated DLP and DNA damages (p<0.05 each). CONCLUSION: In coronary CTA, data acquisition protocols have a significant influence on the X-ray induced DSB levels. Using γ-H2AX immunofluorescence microscopy different scan modes in different CT generations can be compared concerning their biological impact.

The type 2C phosphatase Wip1: an oncogenic regulator of tumor suppressor and DNA damage response pathways.

The Wild-type p53-induced phosphatase 1, Wip1 (or PPM1D), is unusual in that it is a serine/threonine phosphatase with oncogenic activity. A member of the type 2C phosphatases (PP2Cdelta), Wip1 has been shown to be amplified and overexpressed in multiple human cancer types, including breast and ovarian carcinomas. In rodent primary fibroblast transformation assays, Wip1 cooperates with known oncogenes to induce transformed foci. The recent identification of target proteins that are dephosphorylated by Wip1 has provided mechanistic insights into its oncogenic functions. Wip1 acts as a homeostatic regulator of the DNA damage response by dephosphorylating proteins that are substrates of both ATM and ATR, important DNA damage sensor kinases. Wip1 also suppresses the activity of multiple tumor suppressors, including p53, ATM, p16(INK4a) and ARF. We present evidence that the suppression of p53, p38 MAP kinase, and ATM/ATR signaling pathways by Wip1 are important components of its oncogenicity when it is amplified and overexpressed in human cancers.

TAp63gamma regulates hOGG1 and repair of oxidative damage in cancer cell lines.

We showed that TAp63gamma regulates hOGG1. Using chromatin immunoprecipitation (ChIP), we found that TAp63gamma binds to the hOGG1 promoter. Reintroduction of wild-type TAp63gamma into HEK 293 cells, induced transcription of hOGG1 promoter, leading to increase in RNA and protein. Using RNAi studies, we observed that TAp63gamma-RNAi resulted in reduced hOGG1 RNA and protein in HeLa cells. This decrease in hOGG1 expression was associated with reduced cell viability upon oxidative damage. Taken together, our results indicate that hOGG1 is a direct target of TAp63gamma, suggesting a role for TAp63gamma in oxidative damage and repair.

DeltaNp63alpha overexpression induces downregulation of Sirt1 and an accelerated aging phenotype in the mouse.

p63 is highly expressed in the skin and appears to be an early marker of keratinocyte differentiation. To examine the role of p63 in vivo, we generated transgenic mice that overexpress deltaNp63alpha in the skin. These mice exhibited an accelerated aging phenotype in the skin characterized by striking wound healing defects, decreased skin thickness, decreased subcutaneous fat tissue, hair loss, and decreased cell proliferation. The accelerated skin aging was accompanied by a dramatic decrease in longevity of the mice. We found that aging in deltaNp63alpha transgenic mice and other mouse models correlated with levels of Sirt1, a mammalian SIR2 orthologue thought to extend the lifespan in lower species. Moreover, increased deltaNp63alpha expression induced cellular senescence that was rescued by Sirt1. Our data suggest that deltaNp63alpha levels may affect aging in mammals, at least in part, through regulation of Sirt1.