Inflammation-Induced Cell Proliferation Potentiates DNA Damage-Induced Mutations In Vivo

Mutations are a critical driver of cancer initiation. While extensive studies have focused on exposure-induced mutations, few studies have explored the importance of tissue physiology as a modulator of mutation susceptibility in vivo. Of particular interest is inflammation, a known cancer risk factor relevant to chronic inflammatory diseases and pathogen-induced inflammation. Here, we used the fluorescent yellow direct repeat (FYDR) mice that harbor a reporter to detect misalignments during homologous recombination (HR), an important class of mutations. FYDR mice were exposed to cerulein, a potent inducer of pancreatic inflammation. We show that inflammation induces DSBs (γH2AX foci) and that several days later there is an increase in cell proliferation. While isolated bouts of inflammation did not induce HR, overlap between inflammation-induced DNA damage and inflammation-induced cell proliferation induced HR significantly. To study exogenously-induced DNA damage, animals were exposed to methylnitrosourea, a model alkylating agent that creates DNA lesions relevant to both environmental exposures and cancer chemotherapy. We found that exposure to alkylation damage induces HR, and importantly, that inflammation-induced cell proliferation and alkylation induce HR in a synergistic fashion. Taken together, these results show that, during an acute bout of inflammation, there is a kinetic barrier separating DNA damage from cell proliferation that protects against mutations, and that inflammation-induced cell proliferation greatly potentiates exposure-induced mutations. These studies demonstrate a fundamental mechanism by which inflammation can act synergistically with DNA damage to induce mutations that drive cancer and cancer recurrence.Austrian Academy of Sciences (APART Fellowship)Singapore-MIT Alliance for Research and TechnologySingapore. National Research FoundationNational Institutes of Health (U.S.) (NIH R33-CA112151)National Institutes of Health (U.S.) (grant R01-CA079827

Are Cyberchondria and Intolerance of Uncertainty Related to Smartphone Addiction?

Smartphones are a medium for performing online activities, and one such activity could be the compulsive online health information search — cyberchondria. This study aimed to test whether cyberchondria and intolerance of uncertainty (IU) positively predict smartphone addiction (SA), adjusted for age, gender, daily use duration, the reason for using smartphones, and symptoms of anxiety and depression. The sample consisted of 471 adults (55.2% women) from the general population without chronic diseases ( M age = 38.67). Regression analysis showed that IU was a positive predictor of SA ( β = .17, p < .001), as well as cyberchondria ( β = .14, p < .001), which had a unique contribution to the explanation of SA, relative to IU. Other significant predictors were average daily smartphone use and entertainment use, the latter being the strongest predictor in the model. These results revealed cyberchondria as a unique predictor of SA

No direct effect of the -521 C/T polymorphism in the human dopamine D4 receptor gene promoter on transcriptional activity

BACKGROUND: The human dopamine D4 receptor (DRD4) gene has been studied extensively as a candidate gene for certain psychological traits and several behavioural and psychiatric disorders. Both the 5' regulatory region and the coding sequence contain a number of polymorphisms. The promoter variants have received particular attention in the past few years due to their possible role in the regulation of gene transcription. Previously, the -521C/T SNP was shown to influence promoter activity. The aim of this study is to perform an in-depth analysis of this effect in the context of various neural cell lines. RESULTS: Endogenous mRNA expression of the DRD4 gene was demonstrated in two neuroblastoma (SK-N-F1, IMR32) and one retinoblastoma cell line (Y79) by RT-PCR. In addition, very low DRD4 mRNA levels were also detected in HeLa cells. The transcriptional activity of a series of 5' promoter deletion mutants was determined by transient transfection of luciferase reporter constructs. The activity profile of these promoter fragments was similar in each of the cell lines tested. The highest luciferase reporter activity was obtained with a construct containing promoter sequences between nucleotides -668 to -389, while a putative silencer region was localised spanning from nucleotide -1571 to -800. Surprisingly, the -521 C/T polymorphism had no significant effect on transcriptional activity of the reporter construct with the highest activity (-668 to -389) in any of the three cell lines tested. CONCLUSION: Our results do not confirm previous data assigning different transcriptional activities to the -521 C/T alleles of the human DRD4 promoter. Furthermore, these findings highlight the need for further characterization of the 5' regulatory region of the DRD4 gene and identification of additional functional promoter polymorphic sites, especially in the context of haplotype

Rosa26-GFP Direct Repeat (RaDR-GFP) Mice Reveal Tissue- and Age-Dependence of Homologous Recombination in Mammals In Vivo

Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals.National Institutes of Health (U.S.) (Program Project Grant P01-CA026731)National Institutes of Health (U.S.) (R33-CA112151)National Institute of Environmental Health Sciences (P30-ES002109)Singapore-MIT Alliance for Research and Technology CenterNational Institutes of Health (U.S.) (P41-EB015871)National Cancer Institute (U.S.) (P30-CA014051

Gender Moderates the Partial Mediation of Impulsivity in the Relationship Between Psychiatric Distress and Problematic Online Gaming: Online Survey

Background: Research has shown that some individuals can develop problematic patterns of online gaming, leading to significant psychological and interpersonal problems. Psychiatric distress and impulsivity have been suggested to contribute to problematic online gaming (POG).Objective: This study aimed to investigate the potential mediating or moderating mechanisms of impulsivity and gender-related differences in possible associations between psychiatric distress and POG.Methods: A total of 596 matched female and male participants, ranging in age from 14 to 38 years (mean 21.4, SD 4.5), were chosen from a large cross-sectional, nationwide Hungarian online gaming sample. Participants completed online questionnaires about self-reported impulsivity, psychiatric distress, and POG.Results: Psychiatric distress directly predicted POG, and impulsivity partially mediated the relationship between psychiatric distress and POG. However, this mediation effect was found only for the impatience factor of impulsivity. Impulsivity did not moderate the relationship between psychiatric distress and POG. A moderating effect of gender was not found in the direct relationship between psychiatric distress and POG. However, a moderated mediation analysis revealed that impatience mediated the association between psychiatric distress and POG in males, whereas the indirect effect of impatience was not significant in females.Conclusions: The results of this work highlight gender-related difference among online gamers in the mediation effect of impulsivity between psychiatric distress and POG and provide novel insights regarding clinical implications for preventing or treating POG

Overlapping bouts of inflammation induce more DSBs than independent bouts of inflammation.

<p>Inflammation, cell proliferation and γH2AX foci formation were quantified in pancreas sections from mice treated with independent bouts of inflammation (blue bars) and with overlapping bouts of inflammation (purple bars). (<b>A,B</b>) Cerulein induces inflammation in both independent (n = 7) and overlapping (n = 8) treatment regimens. Severity of inflammation in control and cerulein-treated mice was quantified by a trained pathologist. (<b>C, D</b>) Quantification of nuclei positive for the proliferation marker Ki-67 shows a moderate increase in independent bouts of inflammation (n = 7), and a large increase in overlapping bouts of inflammation (n = 8). (<b>E,F</b>) Quantification of nuclei positive for the DSB marker γH2AX (nuclei with >5 foci) shows a moderate increase in independent bouts of inflammation (n = 3), and a large increase in overlapping bouts of inflammation (n = 3). Data are mean ± SEM. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004901#sec004" target="_blank"><i>Methods</i></a> for detailed pathological scoring criteria. Statistical testing could not be performed in groups containing only zero values. * <i>P</i> < 0.05; ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 (Student’s <i>t</i>-test).</p

Independent and overlapping bouts of pancreatic inflammation.

<p>(<b>A</b>) For independent bouts of inflammation, three acute cerulein pancreatitis events were induced two weeks apart, and inflammation and proliferation were assessed at the second (analysis time A) and third (analysis time C) bout of inflammation. HR was quantified 10 to 15 days after the last pancreatitis event. (<b>B</b>) For overlapping bouts of inflammation, three acute cerulein pancreatitis events were induced on days 1, 4 and 9. Inflammation and proliferation were assessed at the second (analysis time B) and third (analysis time D) bout of inflammation. HR was quantified 10 to 15 days after the last pancreatitis event. (<b>C</b>) Pancreas section from a control mouse shows healthy tissue. (<b>D,E</b>) Treatment with cerulein (both independent and overlapping) results in edema and an inflammatory infiltrate chiefly of neutrophils, indicating acute inflammation. (<b>F</b>) Ki-67 immunohistochemistry shows low levels of baseline proliferation in control pancreata. (<b>G</b>) Cell proliferation remains low in the pancreas during acute inflammation. (<b>H</b>) During regeneration from acute inflammation, Ki-67 positive nuclei appear, indicating regenerative proliferation. (<b>I</b>) Immunohistochemical detection of γH2AX phosphorylation in pancreas sections show low levels of DSBs in healthy pancreata. (<b>J</b>) During independent bouts of inflammation, nuclei with γH2AX foci (arrowhead) become apparent. (<b>K</b>) During overlapping bouts of inflammation, more γH2AX positive nuclei are visible. (<b>C</b>-<b>E</b>) Original magnification, ×10. Scale bar = 200 μm. (<b>F</b>-<b>H</b>) Original magnification, ×20. Scale bar = 100 μm. (<b>I</b>-<b>K</b>) Original magnification, ×40.</p

The FYDR mouse detects HR-derived sequence rearrangements <i>in situ</i> in intact tissue.

<p>(<b>A</b>) Schematic of the reconstitution of full-length EYFP coding sequence from two truncated copies through replication fork restart by HR. Note that the appearance of fluorescent signal indicates the gain of one repeat unit (a duplication). Arrows represent expression constructs. EYFP coding sequences are in yellow, promoter and polyadenylation signal sequences are in white, and deleted sequences are in black. Drawing is not to scale. (<b>B</b>) Representative image of a FYDR pancreas showing fluorescent foci detectable <i>in situ</i> in intact tissue. Freshly harvested, unfixed whole pancreas was counterstained with Hoechst, compressed to 0.5 mm and imaged under an epifluorescent microscope. Fluorescence is pseudocolored. Original magnification, ×1. Scale bar = 1 cm. (<b>C</b>) Cluster of recombinant cells at ×60 original magnification. Fluorescence is pseudocolored. (<b>D</b>) A recombinant pancreatic acinar cell identified by the overlay of EYFP fluorescence and H&E staining. Fluorescence is pseudocolored. Original magnification, ×40. (<b>E</b>) The model alkylating agent MNU induces HR in the pancreas. Mice received 25 mg/kg MNU i.p., and HR was evaluated 3 to 5 weeks after treatment. Frequencies of recombinant foci per cm² tissue area are significantly greater in MNU-treated mice (n = 15) than in control mice (n = 16). Boxes show 25^th and 75^th percentiles, medians are indicated by horizontal lines. * <i>P</i> < 0.05 (Mann–Whitney <i>U</i>-test).</p