Long INterspersed element-1 mobility as a sensor of environmental stresses.

Long INterspersed element (LINE-1, L1) retrotransposons are the most abundant transposable elements in the human genome, constituting approximately 17%. They move by a "copy-paste" mechanism, involving reverse transcription of an RNA intermediate and insertion of its cDNA copy at a new site in the genome. L1 retrotransposition (L1-RTP) can cause insertional mutations, alter gene expression, transduce exons, and induce epigenetic dysregulation. L1-RTP is generally repressed; however, a number of observations collected over about 15 years revealed that it can occur in response to environmental stresses. Moreover, emerging evidence indicates that L1-RTP can play a role in the onset of several neurological and oncological diseases in humans. In recent years, great attention has been paid to the exposome paradigm, which proposes that health effects of an environmental factor should be evaluated considering both cumulative environmental exposures and the endogenous processes resulting from the biological response. L1-RTP could be an endogenous process considered for this application. Here, we summarize the current understanding of environmental factors that can affect the retrotransposition of human L1 elements. Evidence indicates that L1-RTP alteration is triggered by numerous and various environmental stressors, such as chemical agents (heavy metals, carcinogens, oxidants, and drugs), physical agents (ionizing and non-ionizing radiations), and experiential factors (voluntary exercise, social isolation, maternal care, and environmental light/dark cycles). These data come from in vitro studies on cell lines and in vivo studies on transgenic animals: future investigations should be focused on physiologically relevant models to gain a better understanding of this topic.

Exposure to low-dose (56)Fe-ion radiation induces long-term epigenetic alterations in mouse bone marrow hematopoietic progenitor and stem cells.

There is an increasing need to better understand the long-term health effects of high-linear energy transfer (LET) radiation due to exposure during space missions, as well as its increasing use in clinical treatments. Previous studies have indicated that exposure to (56)Fe heavy ions increases the incidence of acute myeloid leukemia (AML) in mice but the underlying molecular mechanisms remain elusive. Epigenetic alterations play a role in radiation-induced genomic instability and the initiation and progression of AML. In this study, we assessed the effects of low-dose (56)Fe-ion irradiation on epigenetic alterations in bone marrow mononuclear cells (BM-MNCs) and hematopoietic progenitor and stem cells (HPSCs). Exposure to (56)Fe ions (600 MeV, 0.1, 0.2 and 0.4 Gy) resulted in significant epigenetic alterations involving methylation of DNA, the DNA methylation machinery and expression of repetitive elements. Four weeks after irradiation, these changes were primarily confined to HPSCs and were exhibited as dose-dependent hypermethylation of LINE1 and SINE B1 repetitive elements [4.2-fold increase in LINE1 (P < 0.001) and 7.6-fold increase in SINE B1 (P < 0.01) after exposure to 0.4 Gy; n = 5]. Epigenetic alterations were persistent and detectable for at least 22 weeks after exposure, when significant loss of global DNA hypomethylation (1.9-fold, P < 0.05), decreased expression of Dnmt1 (1.9-fold, P < 0.01), and increased expression of LINE1 and SINE B1 repetitive elements (2.8-fold, P < 0.001 for LINE1 and 1.9-fold, P < 0.05 for SINE B1; n = 5) were observed after exposure to 0.4 Gy. In contrast, exposure to (56)Fe ions did not result in accumulation of increased production of reactive oxygen species (ROS) and DNA damage, exhibited as DNA strand breaks. Furthermore, no significant alterations in cellular senescence and apoptosis were detected in HPSCs after exposure to (56)Fe-ion radiation. These findings suggest that epigenetic reprogramming is possibly involved in the development of radiation-induced genomic instability and thus, may have a causative role in the development of AML.

Shortwave UV-induced damage as part of the solar damage spectrum is not a major contributor to mitochondrial dysfunction.

Because of the absence of a nucleotide excision repair in mitochondria, ultraviolet (UV)-induced bulky mitochondrial DNA (mtDNA) lesions persist for several days before they would eventually be removed by mitophagy. Long persistence of this damage might disturb mitochondrial functions, thereby contributing to skin ageing. In this study, we examined the influence of shortwave UV-induced damage on mitochondrial parameters in normal human skin fibroblasts. We irradiated cells with either sun-simulating light (SSL) or with ultraviolet C to generate bulky DNA lesions. At equivalent antiproliferative doses, both irradiation regimes induced gene expression of mitochondrial transcription factor A (TFAM) and matrix metallopeptidase 1 (MMP-1). Only irradiation with SSL, however, caused significant changes in mtDNA copy number and a decrease in mitochondrial respiration. Our results indicate that shortwave UV-induced damage as part of the solar spectrum is not a major contributor to mitochondrial dysfunction.

Acute high-dose X-radiation-induced genomic changes in A549 cells.

Accidents with ionizing radiation often involve single, acute high-dose exposures that can lead to acute radiation syndrome and late effects such as carcinogenesis. To study such effects at the cellular level, we investigated acute ionizing radiation-induced chromosomal aberrations in A549 adenocarcinoma cells at the genome-wide level by exposing the cells to an acute dose of 6 Gy 240 kV X rays. One sham-irradiated clone and four surviving irradiated clones were recovered by minimal dilution and further expanded and analyzed by chromosome painting and tiling-path array CGH, with the nonirradiated clone 0 serving as the control. Acute X-ray exposure induced specific translocations and changes in modal chromosome number in the four irradiated clones. Array CGH disclosed unique and recurrent genomic changes, predominantly losses, and revealed that the fragile sites FRA3B and FRA16D were preferential regions of genomic alterations in all irradiated clones, which is likely related to radioresistant S-phase progression and genomic stress. Furthermore, clone 4 displayed an increased radiosensitivity at doses >5 Gy. Pairwise comparisons of the gene expression patterns of all irradiated clones to the sham-irradiated clone 0 revealed an enrichment of the Gene Ontology term "M Phase" (P = 6.2 × 10(-7)) in the set of differentially expressed genes of clone 4 but not in those of clones 1-3. Ionizing radiation-induced genomic changes and fragile site expression highlight the capacity of a single acute radiation exposure to affect the genome of exposed cells by inflicting genomic stress.

Elevated mitochondrial DNA copy number and POL-γ expression but decreased expression of TFAM in murine intestine following therapeutic dose irradiation.

Mitochondria play pivotal roles in cellular handling of oxygen and in apoptosis, the ordered suicide response of cells to irradiation. The involvement of expression products from the 16.5 kb human mitochondrial genome in these activities has been studied widely. However, little is known about effects of irradiation on mammalian mitochondrial DNA (mtDNA). The relative lack of mtDNA repair mechanisms compared with nuclear DNA (nDNA) predicts particular vulnerability to irradiation. Using a technique developed to ascertain mtDNA:nDNA ratios, we previously showed that this ratio increases dramatically in murine small bowel within 48 hours following whole body irradiation. We now report that those levels continue to rise for four days and remain elevated at close to that level beyond 30 days after 5 Gy of irradiation.We further demonstrate that levels of the mtDNA-specific DNA polymerase-γ (Pol-γ ) also show a sharp and sustained increase during this time course after a 2-Gy dose. Paradoxically, transcription factor A (TFAM), exhibited the directly opposite response.

Independent effects of leaf growth and light on the development of the plastid and its DNA content in Zea species.

In maize (Zea mays L.), chloroplast development progresses from the basal meristem to the mature leaf tip, and light is required for maturation to photosynthetic competence. During chloroplast greening, it was found that chloroplast DNA (cpDNA) is extensively degraded, falling to undetectable levels in many individual chloroplasts for three maize cultivars, as well as Zea mexicana (the ancestor of cultivated maize) and the perennial species Zea diploperennis. In dark-grown maize seedlings, the proplastid-to-etioplast transition is characterized by plastid enlargement, cpDNA replication, and the retention of high levels of cpDNA. When dark-grown seedlings are transferred to white light, the DNA content per plastid increases slightly during the first 4 h of illumination and then declines rapidly to a minimum at 24 h during the etioplast-to-chloroplast transition. Plastid autofluorescence (from chlorophyll) continues to increase as cpDNA declines, whereas plastid size remains constant. It is concluded that the increase in cpDNA that accompanies plastid enlargement is a consequence of cell and leaf growth, rather than illumination, whereas light stimulates photosynthetic capacity and cpDNA instability. When cpDNA from total tissue was monitored by blot hybridization and real-time quantitative PCR, no decline following transfer from dark to light was observed. The lack of agreement between DNA per plastid and cpDNA per cell may be attributed to nupts (nuclear sequences of plastid origin).

Alteration of mtDNA copy number, mitochondrial gene expression and extracellular DNA content in mice after irradiation at lethal dose.

High steady-state transcriptional activity is essential for normal mitochondrial function. The requisite transcription rate is satisfied in part by high copy number of mitochondrial DNA (mtDNA). In the present study, we analyze mtDNA copy number by real-time PCR in nucleated blood cells from control mice and mice exposed to 1- or 10-Gy X-radiation. Transcription of the oxidative phosphorylation-associated genes cytb, atp6, nd4, nd2 and d-loop region was monitored in these nucleated blood cells similarly by real-time PCR. We observed a 50% decrease in the ratio of mitochondrial to nuclear DNA (mtDNA/nDNA) in blood cells, while the mtDNA/nDNA ratio in serum increased. After a lethal 10-Gy dose of X-irradiation, we observed an 80% decrease in the number of circulating lymphocytes. In response to a 10-Gy radiation dose, we observed transiently increased mtDNA/nDNA ratio and transcription within the initial 5 h post-treatment. At 24-72 h, the mtDNA/nDNA ratio in surviving cells was reduced to the level observed in blood cells irradiated with 1 Gy. We observed a decrease in the serum mtDNA/nDNA ratio due to an increase in nDNA content rather than a decrease in mtDNA. Taken together, results presented herein suggest that the mtDNA/nDNA ratio may be of clinical value potentially as a diagnostic tool, particularly in oncology patients undergoing radiation therapy.

Upregulation of FOXM1 induces genomic instability in human epidermal keratinocytes.

BACKGROUND: The human cell cycle transcription factor FOXM1 is known to play a key role in regulating timely mitotic progression and accurate chromosomal segregation during cell division. Deregulation of FOXM1 has been linked to a majority of human cancers. We previously showed that FOXM1 was upregulated in basal cell carcinoma and recently reported that upregulation of FOXM1 precedes malignancy in a number of solid human cancer types including oral, oesophagus, lung, breast, kidney, bladder and uterus. This indicates that upregulation of FOXM1 may be an early molecular signal required for aberrant cell cycle and cancer initiation. RESULTS: The present study investigated the putative early mechanism of UVB and FOXM1 in skin cancer initiation. We have demonstrated that UVB dose-dependently increased FOXM1 protein levels through protein stabilisation and accumulation rather than de novo mRNA expression in human epidermal keratinocytes. FOXM1 upregulation in primary human keratinocytes triggered pro-apoptotic/DNA-damage checkpoint response genes such as p21, p38 MAPK, p53 and PARP, however, without causing significant cell cycle arrest or cell death. Using a high-resolution Affymetrix genome-wide single nucleotide polymorphism (SNP) mapping technique, we provided the evidence that FOXM1 upregulation in epidermal keratinocytes is sufficient to induce genomic instability, in the form of loss of heterozygosity (LOH) and copy number variations (CNV). FOXM1-induced genomic instability was significantly enhanced and accumulated with increasing cell passage and this instability was increased even further upon exposure to UVB resulting in whole chromosomal gain (7p21.3-7q36.3) and segmental LOH (6q25.1-6q25.3). CONCLUSION: We hypothesise that prolonged and repeated UVB exposure selects for skin cells bearing stable FOXM1 protein causes aberrant cell cycle checkpoint thereby allowing ectopic cell cycle entry and subsequent genomic instability. The aberrant upregulation of FOXM1 serves as a 'first hit' where cells acquire genomic instability which in turn predisposes cells to a 'second hit' whereby DNA-damage checkpoint response (eg. p53 or p16) is abolished to allow damaged cells to proliferate and accumulate genetic aberrations/mutations required for cancer initiation.

Exposure to 1800 MHz radiofrequency radiation induces oxidative damage to mitochondrial DNA in primary cultured neurons.

Increasing evidence indicates that oxidative stress may be involved in the adverse effects of radiofrequency (RF) radiation on the brain. Because mitochondrial DNA (mtDNA) defects are closely associated with various nervous system diseases and mtDNA is particularly susceptible to oxidative stress, the purpose of this study was to determine whether radiofrequency radiation can cause oxidative damage to mtDNA. In this study, we exposed primary cultured cortical neurons to pulsed RF electromagnetic fields at a frequency of 1800 MHz modulated by 217 Hz at an average special absorption rate (SAR) of 2 W/kg. At 24 h after exposure, we found that RF radiation induced a significant increase in the levels of 8-hydroxyguanine (8-OHdG), a common biomarker of DNA oxidative damage, in the mitochondria of neurons. Concomitant with this finding, the copy number of mtDNA and the levels of mitochondrial RNA (mtRNA) transcripts showed an obvious reduction after RF exposure. Each of these mtDNA disturbances could be reversed by pretreatment with melatonin, which is known to be an efficient antioxidant in the brain. Together, these results suggested that 1800 MHz RF radiation could cause oxidative damage to mtDNA in primary cultured neurons. Oxidative damage to mtDNA may account for the neurotoxicity of RF radiation in the brain.

Modulation of ionizing radiation-induced apoptosis by bantam microRNA in Drosophila.

In Drosophila, heterozygosity in the pro-apoptotic gene hid significantly reduces apoptosis that is induced by ionizing radiation (IR). Therefore, mechanisms that regulate Hid levels can potentially contribute to life-or-death decision of an irradiated cell. 3'UTR of hid mRNA contains 5 potential binding sites for bantam microRNA. Ectopic expression of ban attenuated apoptosis that results from ectopic expression of hid but the significance of this regulation under physiological conditions remained to be investigated. We report here that ban is needed to limit IR-induced apoptosis in larval imaginal discs. Using tubulin-EGFP ban sensors with ban consensus sequences in the 3'UTR, we find that EGFP decreases following IR, indicating that IR activates ban. Likewise, a tubulin-EGFP reporter with hid-3'UTR is repressed in irradiated discs and this repression requires ban consensus sites in the hid 3'UTR. ban mutant larvae show increased sensitivity to killing by IR, which is suppressed by a mutation in hid. These results can fit into a model in which IR activates ban and ban represses hid to limit IR-induced apoptosis. miRNAs have been shown previously to be induced by radiation but this is the first report that a miRNA is functionally important for radiation responses.

DNA copy-number instability in low-dose gamma-irradiated TK6 lymphoblastoid clones.

Genomic instability that might occur early during low-dose, fractionated radiation exposures may be traceable in radiogenic compared to spontaneous cancers. Using a human 18K cDNA microarray-based comparative genome hybridization protocol, we measured changes in DNA copy number at over 14,000 loci in nine low-dose (137)Cs gamma-irradiated (acute exposure to 10 cGy/day x 21 days) and nine unirradiated TK6 clones and estimated locus-specific copy-number differences between them. Radiation induced copy-number hypervariability at thousands of loci across all chromosomes, with a sevenfold increase in low-level, randomly positioned DNA gains. Recurrent gains at 40 loci occurred among irradiated clones and were distributed nonrandomly across the genome, with the highest densities in 3q, 13q and 20q at sites that were hypodiploid without irradiation. Another nonrandomly distributed set of 94 loci exhibited relative recurrent gains from a hypodiploid state to a diploid state, suggesting hemizygous-to-homozygous transitions. Frequently recurring losses at 57 loci were concentrated on the single X-chromosome but were sparsely distributed at 0-2 loci per autosome. These results suggest induced mitotic homologous recombination as a possible mechanism of low-dose radiation-induced genomic instability. Genomic instability induced in TK6 cells resembled that seen in radiogenic tumors and suggests a way that radiation could induce genomic instability in preneoplastic cells.

Tandem duplication and copy number polymorphism of the SRY gene in patients with sex chromosome anomalies and males exposed to natural background radiation.

Mutations in the SRY gene encompassing the HMG box have been well characterized in gonadal dysgenesis, male infertility and other types of sex chromosome related anomalies (SCRA). However, no information is available on copy number status of this gene under such abnormal conditions. Employing 'Taqman Probe Assay' specific to the SRY gene, we screened 16 DNA samples from patients with SCRA and 36 samples from males exposed to high levels of natural background radiation (HNBR). Patients with SCRA showed 2-16 copies of the SRY gene of which, one, Oxen (49, XYYYY) had eight copies with sequences different from one another. Of the 36 HNBR samples, 12 had one copy whereas 24 harboured 2-8 copies of the SRY gene. A HNBR male 33F had one normal and one mutated copy of this gene. Analysis of 25 DNA samples from blood and semen of normal males showed only one copy of this gene. Despite multiple copies in affected males, fluorescence in-situ hybridization (FISH) with SRY probe detected a single signal on the Y chromosome in HNBR males suggesting its possible localized tandem duplication. Copy number status of the other Y-linked loci is envisaged to augment DNA diagnostics facilitating genetic counselling to affected patients.

The increase in mitochondrial DNA copy number in the tissues of gamma-irradiated mice.

Changes in the number of mitochondrial DNA (mtDNA) copies in the brain and spleen tissues of gamma-irradiated (3 Gy) mice were studied by comparative analysis of the long-extension PCR products of mtDNA (15.9 kb) and a fragment of the cluster nuclear beta-globin gene (8.7 kb) amplified simultaneously in one and the same test-tube within total DNA. The analysis showed that, compared to the nuclear beta-globin gene, an increase in mtDNA copy number (polyploidization) took place in the brain and spleen cells of mice exposed to gamma-radiation. This data led to the suggestion that the major mechanism for maintenance of the mitochondrial genome, which is constantly damaged by endogenous ROS and easily affected by ionizing radiation or other exogenous factors, is the induction of synthesis of new mtDNA copies on intact or little affected mtDNA templates because the repair systems in the mitochondria function at a low level of efficiency.