Mutagenicity of ultraviolet A radiation in the lacI transgene in Big Blue mouse embryonic fibroblasts

Sunlight ultraviolet A (UVA) irradiation has been implicated in the etiology of human skin cancer. A genotoxic mode of action for UVA radiation has been suggested that involves photosensitization reactions giving rise to promutagenic DNA lesions. We investigated the mutagenicity of UVA in the lacI transgene in Big Blue mouse embryonic fibroblasts. UVA irradiation of these cells at a physiologically relevant dose of 18J/cm(2) caused a 2.8-fold increase in the lacI mutant frequency relative to control, i.e., 12.12+/-1.84 versus 4.39+/-1.99 x 10(-5) (mean+/-S.D.). DNA sequencing analysis showed that of 100 UVA-induced mutant plaques and 54 spontaneously arisen control plaques, 97 and 51, respectively, contained a minimum of one mutation along the lacI transgene. The vast majority of both induced- and spontaneous mutations were single base substitutions, although less frequently, there were also single and multiple base deletions and insertions, and tandem base substitutions. Detailed mutation spectrometry analysis revealed that G:C-->T:A transversions, the signature mutations of oxidative DNA damage, were significantly induced by UVA irradiation (P<0.003). The absolute frequency of this type of mutations was 7.4-fold increased consequent to UVA irradiation as compared to control (3.38 versus 0.454 x 10(-5); P<0.00001). These findings are in complete agreement with those previously observed in the cII transgene of the same model system, and reaffirm the notion that intracellular photosensitization reactions causing promutagenic oxidative DNA damage are involved in UVA genotoxicity.     

Endogenous mitochondrial oxidative stress in MnSOD-deficient mouse embryonic fibroblasts promotes mitochondrial DNA glycation

The accumulation of somatic mutations in mitochondrial DNA (mtDNA) induced by reactive oxygen species (ROS) is regarded as a major contributor to aging and age-related degenerative diseases. ROS have also been shown to facilitate the formation of certain advanced glycation end-products (AGEs) in proteins and DNA and N(2)-carboxyethyl-2'-deoxyguanosine (CEdG) has been identified as a major DNA-bound AGE. Therefore, the influence of mitochondrial ROS on the glycation of mtDNA was investigated in primary embryonic fibroblasts derived from mutant mice (Sod2(-/+)) deficient in the mitochondrial antioxidant enzyme manganese superoxide dismutase. In Sod2(-/+) fibroblasts vs wild-type fibroblasts, the CEdG content of mtDNA was increased from 1.90 ± 1.39 to 17.14 ± 6.60 pg/μg DNA (p<0.001). On the other hand, the CEdG content of nuclear DNA did not differ between Sod2(+/+) and Sod2(-/+) cells. Similarly, cytosolic proteins did not show any difference in advanced glycation end-products or protein carbonyl contents between Sod2(+/+) and Sod2(-/+). Taken together, the data suggest that mitochondrial oxidative stress specifically promotes glycation of mtDNA and does not affect nuclear DNA or cytosolic proteins. Because DNA glycation can change DNA integrity and gene functions, glycation of mtDNA may play an important role in the decline of mitochondrial functions.     

N-WASP involvement in dorsal ruffle formation in mouse embryonic fibroblasts

The Wiskott-Aldrich syndrome protein (WASP) family activates the Arp2/3 complex leading to the formation of new actin filaments. Here, we study the involvement of Scar1, Scar2, N-WASP, and Arp2/3 complex in dorsal ruffle formation in mouse embryonic fibroblasts (MEFs). Using platelet-derived growth factor to stimulate circular dorsal ruffle assembly in primary E13 and immortalized E9 Scar1(+/+) and Scar1 null MEFs, we establish that Scar1 loss does not impair the formation of dorsal ruffles. Reduction of Scar2 protein levels via small interfering RNA (siRNA) also did not affect dorsal ruffle production. In contrast, wiskostatin, a chemical inhibitor of N-WASP, potently suppressed dorsal ruffle formation in a dose-dependent manner. Furthermore, N-WASP and Arp2 siRNA treatment significantly decreased the formation of dorsal ruffles in MEFs. In addition, the expression of an N-WASP truncation mutant that cannot bind Arp2/3 complex blocked the formation of these structures. Finally, N-WASP(-/-) fibroblast-like cells generated aberrant dorsal ruffles. These ruffles were highly unstable, severely depleted of Arp2/3 complex, and diminished in size. We hypothesize that N-WASP and Arp2/3 complex are part of a multiprotein assembly important for the generation of dorsal ruffles and that Scar1 and Scar2 are dispensable for this process.     

Mitochondrial oxidative stress causes chromosomal instability of mouse embryonic fibroblasts

Reactive oxygen species are an inevitable by-product of mitochondrial respiration. It has been estimated that between 0.4 and 4% of molecular oxygen is converted to the radical superoxide (O2*-) and this level is significantly influenced by the functional status of the mitochondria. It is well established that exogenous oxidative stress and high doses of mitochondrial poisons such as paraquat and carbonyl cyanide 4 (trifluoromethoxy) phenylhydrazone (FCCP) can lead to genomic instability. In this report we show for the first time that endogenous mitochondrial oxidative stress in standard cell culture conditions results in nuclear genomic instability in primary mouse embryonic fibroblasts (MEFs). We show that lack of mitochondrial superoxide dismutase in MEFs leads to a severe increase of double strand breaks, end-to-end fusions, chromosomal translocations, and loss of cell viability and proliferative capacity. Our results predict that endogenous mitochondrial oxidative stress can induce genomic instability, and therefore may have a profound effect in cancer and aging.     

PTEN deletion leads to deregulation of antioxidants and increased oxidative damage in mouse embryonic fibroblasts

Despite the significance of oxidative damage in carcinogenesis, the molecular mechanisms that lead to increased susceptibility to oxidative stress are not well understood. We now report a link between loss of protection against oxidative damage and loss of function of PTEN, a highly mutated tumor suppressor gene in a variety of human tumors. Using two-dimensional gel electrophoresis, combined with Western and Northern blot analyses, we found that PTEN deficiency in mouse embryonic fibroblasts (MEFs) displays deregulated expression of several antioxidant enzymes, including peroxiredoxins 1, 2, 5, and 6 and Cu, Zn superoxide dismutase. In these Pten-deleted MEFs, the basal levels of reactive oxygen species (ROS) were increased, and both the basal level and the ROS-induced oxidative damage of DNA were increased, as evidenced by increased levels of hydrogen peroxide (H2O2), superoxide anion, 8-hydroxy-2'-deoxyguanosine, and DNA double-strand breaks. We further show that Pten deletion is correlated with resistance to H2O2-induced expression of several antioxidants. These findings suggest an essential role for PTEN in maintaining the normal redox state of mouse embryonic fibroblasts against oxidative damage. They also provide a molecular link between PTEN, whose inactivation is known to be involved in a variety of human tumors, and antioxidants, whose perturbation leads to oxidative damage of cells.     

DNA repair kinetics in SCID mice Sertoli cells and DNA-PKcs-deficient mouse embryonic fibroblasts

Noncycling and terminally differentiated (TD) cells display differences in radiosensitivity and DNA damage response. Unlike other TD cells, Sertoli cells express a mixture of proliferation inducers and inhibitors in vivo and can reenter the cell cycle. Being in a G₁-like cell cycle stage, TD Sertoli cells are expected to repair DSBs by the error-prone nonhomologous end-joining pathway (NHEJ). Recently, we have provided evidence for the involvement of Ku-dependent NHEJ in protecting testis cells from DNA damage as indicated by persistent foci of the DNA double-strand break (DSB) repair proteins phospho-H2AX, 53BP1, and phospho-ATM in TD Sertoli cells of Ku70-deficient mice. Here, we analyzed the kinetics of 53BP1 foci induction and decay up to 12 h after 0.5 Gy gamma irradiation in DNA-PKcs-deficient (Prkdc ^scid ) and wild-type Sertoli cells. In nonirradiated mice and Prkdc ^scid Sertoli cells displayed persistent DSBs foci in around 12 % of cells and a fivefold increase in numbers of these DSB DNA damage-related foci relative to the wild type. In irradiated mice, Prkdc ^scid Sertoli cells showed elevated levels of DSB-indicating foci in 82 % of cells 12 h after ionizing radiation (IR) exposure, relative to 52 % of irradiated wild-type Sertoli cells. These data indicate that Sertoli cells respond to and repair IR-induced DSBs in vivo, with repair kinetics being slow in the wild type and inefficient in Prkdc ^scid . Applying the same dose of IR to Prdkc ^?/? and Ku ^?/? mouse embryonic fibroblast (MEF) cells revealed a delayed induction of 53BP1 DSB-indicating foci 5 min post-IR in Prdkc ^?/? cells. Inefficient DSB repair was evident 7 h post-IR in DNA-PKcs-deficient cells, but not in Ku ^?/? MEFs. Our data show that quiescent Sertoli cells repair genotoxic DSBs by DNA-PKcs-dependent NEHJ in vivo with a slower kinetics relative to somatic DNA-PKcs-deficient cells in vitro, while DNA-PKcs deficiency caused inefficient DSB repair at later time points post-IR in both conditions. These observations suggest that DNA-PKcs contributes to the fast and slow repair of DSBs by NHEJ.     

Quantification of oxidative stress in live mouse embryonic fibroblasts by monitoring the responses of polyubiquitin genes

Stress-regulated polyubiquitin genes in mammals are expected to be upregulated under oxidative stress conditions. In order to assess gene regulation via the conventional method, the isolation of RNA molecules or the transfection of reporter constructs into cells is frequently required. If the stress response within cells can be monitored in a reversible manner with minimal manipulation, the study of the stress response pathways will become much easier. Herein, we have developed a simple fluorescence plate reader-based assay to monitor the stress responses of polyubiquitin genes in mouse embryonic fibroblasts, in which one allele of the ubiquitin-coding region of the polyubiquitin gene Ubb or Ubc was replaced by the eGFP-puro cassette, thereby placing GFP expression under the control of the endogenous polyubiquitin gene promoter. Using this simple assay, we established that both mammalian polyubiquitin genes are upregulated upon oxidative stress with slightly higher responses from the Ubb promoter. The principal advantage of this assay is that it allows for the monitoring of stress responses of polyubiquitin genes without disrupting cellular growth; this assay can therefore be applied repeatedly to the same cells. Furthermore, by calculating the increase in fluorescence deriving from newly synthesized GFP upon stress, which can be regarded as a bona fide polyubiquitin gene stress response, we were able to determine and directly compare the concentrations of various oxidative stressors that induce the similar cellular stress levels. Therefore, this simple assay may also be employed in the screening of potentially toxic reagents that induce the stress response pathways.     

Modulation of exogenous and endogenous levels of thioredoxin in human skin fibroblasts prevents DNA damaging effect of ultraviolet A radiation

Thioredoxin (Trx) plays important biological roles both intra- and extracellularly via thiol redox control. We have previously demonstrated that Trx exhibited protective effects against UVA cytotoxicity in human skin fibroblasts. As an extension of the latter investigation, the present work is aimed at assessing ability of Trx to maintain genomic integrity in human skin fibroblasts upon exposure to UVA radiation. Indeed, UVA (320--380 nm) is mutagenic and induces genomic damage to skin cells. The alkaline comet assay was used in association with DNA repair enzyme including formamido pyrimidine glycosylase (Fpg) and endonuclease III (endo III) to estimate the amount of modified bases together with the level of strand breaks and alkali-labile sites. The HPLC-EC assay was applied to assess 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) levels and to permit the calibration of comet assay as previously described. We reported that overexpression of human Trx (transient transfection) as well as exogenous human recombinant Trx added to the culture medium, decreased the level of DNA damage in UVA irradiated cells. Interestingly, transfection appeared to prevent UVA-induced 8-oxodGuo (3.06 au per Joules.cm(-2) compared to 4.94 au per Joules.cm(-2) for nontransfected cells). Moreover, Trx accumulates into nuclei in transfected cells. This finding supports the notion that Trx is important for the maintenance of the integrity of genetic information. This work demonstrated that under conditions of UVA oxidative stress, Trx prevented the UVA-induced DNA damage.     

Survival and mutagenesis of ultraviolet irradiated simian virus 40 in foetal human fibroblasts

Survival and mutagenesis of UV-irradiated, temperature-sensitive simian virus 40 mutants (SV40) have been studied after infection of human fibroblasts. Survival of the viral progeny obtained after 6,8 or 10 days at permissive temperature decrease as a function of the UV-dose delivered to the virus. In cels which have been pretreated with 10 Jm-2 of UV 24 hours before infection, progeny survival was increased as compared to survival in control cells. The reactivation factor varies from one to ten, depending on the number of lytic cycles carried out at permissive temperature. The level of mutation frequency, as measured by the reversion from a temperature sensitive growth phenotype towards a wild type phenotype, increases with the dose of UV-irradiation given to the virus. Moreover, the mutation frequency is increased in the viral progeny produced in UV-irradiated human cells. Similar experiments carried out with SV40-transformed human fibroblasts, which constitutively express SV40 T antigen, gave comparable results. These experiments show that, as in monkey cells, a new error-prone recovery pathway can be induced by pretreating human cells with UV-light before infection.     

DNA damage response activation in mouse embryonic fibroblasts undergoing replicative senescence and following spontaneous immortalization

Primary mouse embryonic fibroblasts (MEFs) are a popular tool for molecular and cell biology studies. However, when MEFs are grown in vitro under standard tissue culture conditions, they proliferate only for a limited number of population doublings (PD) and eventually undergo cellular senescence. Presently, the molecular mechanisms halting cell cycle progression and establishing cellular senescence under these conditions are unclear. Here, we show that a robust DNA damage response (DDR) is activated when MEFs undergo replicative cellular senescence. Senescent cells accumulate senescence-associated DDR foci (SDFs) containing the activated form of ATM, its phosphorylated substrates and γH2AX. In senescent MEFs, DDR markers do not preferentially accumulate at telomeres, the end of linear chromosomes. It has been observed that proliferation of MEFs is extended if they are cultured at low oxygen tension (3% O2). We observed that under these conditions, DDR is not observed and senescence is not established. Importantly, inactivation of ATM in senescent MEFs allows escape from senescence and progression through the S-phase. Therefore, MEFs undergoing cellular senescence arrest their proliferation due to the activation of a DNA damage checkpoint mediated by ATM kinase. Finally, we observed that spontaneously immortalized proliferating MEFs display markers of an activated DDR, indicating the presence of chromosomal DNA damage in these established cell lines.     

Iron, oxidative stress and the example of solar ultraviolet A radiation

Iron has outstanding biological importance as it is required for a wide variety of essential cellular processes and, as such, is a vital nutrient. The element holds this central position by virtue of its facile redox chemistry and the high affinity of both redox states (iron II and iron III) for oxygen. These same properties also render iron toxic when its redox-active chelatable 'labile' form exceeds the normal binding capacity of the cell. Indeed, in contrast to iron bound to proteins, the intracellular labile iron (LI) can be potentially toxic especially in the presence of reactive oxygen species (ROS), as it can lead to catalytic formation of oxygen-derived free radicals such as hydroxyl radical that ultimately overwhelm the cellular antioxidant defense mechanisms and lead to cell damage. While intracellular iron homeostasis and body iron balance are tightly regulated to minimise the presence of potentially toxic LI, under conditions of oxidative stress and certain pathologies, iron homeostasis is severely altered. This alteration manifests itself in several ways, one of which is an increase in the intracellular level of potentially harmful LI. For example acute exposure of skin cells to ultraviolet A (UVA, 320-400 nm), the oxidising component of sunlight provokes an immediate increase in the available pool of intracellular LI that appears to play a key role in the increased susceptibility of skin cells to UVA-mediated oxidative membrane damage and necrotic cell death. The main purpose of this overview is to bring together some of the new findings related to intracellular LI distribution and trafficking under physiological and patho-physiological conditions as well as to discuss mechanisms and consequences of oxidant-induced alterations in the intracellular pool of LI, as exemplified by UVA radiation.     

The induction of DNA strand breaks in normal human skin fibroblasts exposed to solar ultraviolet radiation

The levels of apparent DNA single-strand breaks (ssb) were measured, following a 0-20 h incubation of normal human skin fibroblasts exposed to the solar uv wavelengths produced by a fluorescent sunlamp. The ssb were determined using the alkaline elution assay, which was performed either without proteinase K (proK) or in its presence, so as to eliminate any DNA-protein crosslinks that may be present in the cells. Cells were irradiated with either 3 kJ/m2 of sunlamp uv greater than 295 nm, 150 kJ/m2 of sunlamp uv greater than 315 nm, or 150 kJ/m2 of sunlamp uv greater than 320 nm. These treatments resulted in the production of 5-10 ssb/10(10) Da. For the two shorter wavelength irradiations, the levels of ssb decreased rapidly upon incubation of the cells. However, when the elutions were performed using proK, the number of ssb increased about twofold following a 2-4 h incubation. In contrast, the levels of ssb decreased in the sunlamp uv greater than 320 nm irradiated cells for elutions performed with or without proK. These results suggest that under certain irradiation conditions, ssb are formed in cells upon incubation, which are hidden by the crosslinking of protein to DNA.     

Baicalin protects human skin fibroblasts from ultraviolet A radiation-induced oxidative damage and apoptosis

Reactive oxygen species (ROS) are an important factor in the development of skin photodamage after ultraviolet A (UVA) radiation. A flavonoid antioxidant, baicalin, can selectively neutralize super-oxide anion (O₂^?) while having no significant effect on ^?OH. Fibroblasts are a key component of skin dermis. In the present study, we investigated the protective effects of baicalin on human skin fibroblasts (HSFs) under UVA induced oxidative stress. Fluorescence microscopy and flow cytometry were used to assay the intracellular O₂^?, NO, ROS concentrations and the mitochondrial membrane potential. Cell viability was determined using the Cell Counting Kit-8 (CCK-8). The concentrations of cellular MDA, SOD, GSH, T-AOC, and 8-oxo-dG were also measured. Cellular apoptosis was measured by flow cytometry and caspase-3 detection. The results revealed that UVA radiation could cause oxidative stress and apoptosis in HSFs. Interestingly, the use of baicalin after UVA radiation signi?cantly reduced the level of intracellular O₂^?, NO, and ROS, stabilized the mitochondrial membrane potential, and attenuated production of MDA and 8-oxo-dG. These ef?ciently enhanced the antioxidative defense system and protected the HSFs from subsequent oxidative stress damage and apoptosis. In other words, baicalin decreased the excessive generation of intracellular ROS and NO, and elevated the cellular antioxidative defense, which eventually mitigate the UVA-induced apoptosis. Based on our results, baicalin may have applications in the treatment of skin photodamage caused by UVA irradiation.     

A role of oxidative DNA sugar damage in mutagenesis by neocarzinostatin and bleomycin

The anti-tumor antibiotics neocarzinostatin and bleomycin specifically oxidize deoxyribose in DNA at the C-5' and C-4' positions, respectively. The resulting DNA lesions include strand breaks and apyrimidinic sites. Both agents are broad specificity mutagens, inducing, in various systems, base substitutions, frameshifts and deletions. Sequencing studies in bacterial systems have suggested that the base substitutions may result primarily from replicative bypass of the oxidized apyrimidinic sites.     

DNA damage caused by ionizing radiation in embryonic diploid fibroblasts WI-38 induces both apoptosis and senescence

Cellular response to ionizing radiation-induced damage depends on the cell type and the ability to repair DNA damage. Some types of cells undergo apoptosis, whereas others induce a permanent cell cycle arrest and do not proliferate. Our study demonstrates two types of response of embryonic diploid fibroblasts WI-38 to ionizing radiation. In the WI-38 cells p53 is activated, protein p21 increases, but the cells are arrested in G2 phase of cell cycle. Some of the cells die by apoptosis, but in remaining viable cells p16 increases, senescence associated DNA-damage foci occur, and senescence-associated beta-galactosidase activity increases, which indicate stress-induced premature senescence.