Search

Mitochondria dynamics results from fission and fusion events that may be unbalanced in favor of mitochondrial fragmentation upon cell stress. During oxidative stress, microtubules are hyperacetylated in a mitochondria-dependent manner. In this study, we show that under stress conditions, most of the mitochondria form foci with microtubule domains that carry Drp1. We also demonstrate that stress-induced hyperacetylation of microtubules is required for the effective induction of Drp1 phosphorylation at 616Ser, in a kinesin-1- and c-Jun N-terminal kinase-dependent manner. Furthermore, hyperacetylation of microtubules contributes to the recruitment of total Drp1 to mitochondria to enhance fission. These results highlight a new way of interaction between microtubules and mitochondria dynamics.

Mitochondria respond to many cellular functions and act as central hubs in innate immunity against viruses. This response is notably due to their role in the activation of interferon (IFN) signaling pathways through the activity of MAVS (mitochondrial antiviral signaling protein) present at the mitochondrial surface. Here, we report that the BHRF1 protein, a BCL2 homolog encoded by Epstein-Barr virus (EBV), inhibits IFNB/IFN-β induction by targeting the mitochondria. Indeed, we have demonstrated that BHRF1 expression modifies mitochondrial dynamics and stimulates DNM1L/Drp1-mediated mitochondrial fission. Concomitantly, we have shown that BHRF1 is pro-autophagic because it stimulates the autophagic flux by interacting with BECN1/Beclin 1. In response to the BHRF1-induced mitochondrial fission and macroautophagy/autophagy stimulation, BHRF1 drives mitochondrial network reorganization to form juxtanuclear mitochondrial aggregates known as mito-aggresomes. Mitophagy is a cellular process, which can specifically sequester and degrade mitochondria. Our confocal studies uncovered that numerous mitochondria are present in autophagosomes and acidic compartments using BHRF1-expressing cells. Moreover, mito-aggresome formation allows the induction of mitophagy and the accumulation of PINK1 at the mitochondria. As BHRF1 modulates the mitochondrial fate, we explored the effect of BHRF1 on innate immunity and showed that BHRF1 expression could prevent IFNB induction. Indeed, BHRF1 inhibits the IFNB promoter activation and blocks the nuclear translocation of IRF3 (interferon regulatory factor 3). Thus, we concluded that BHRF1 can counteract innate immunity activation by inducing fission of the mitochondria to facilitate their sequestration in mitophagosomes for degradation. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; BCL2: BCL2 apoptosis regulator; CARD: caspase recruitment domain; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CI: compaction index; CQ: chloroquine; DAPI: 4ʹ,6-diamidino-2-phenylindole, dihydrochloride; DDX58/RIG-I: DExD/H-box helicase 58; DNM1L/Drp1: dynamin 1 like; EBSS: Earle’s balanced salt solution; EBV: Epstein-Barr virus; ER: endoplasmic reticulum; EV: empty vector; GFP: green fluorescent protein; HEK: human embryonic kidney; IFN: interferon; IgG: immunoglobulin G; IRF3: interferon regulatory factor 3; LDHA: lactate dehydrogenase A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MMP: mitochondrial membrane potential; MOM: mitochondrial outer membrane; PINK1: PTEN induced kinase 1; RFP: red fluorescent protein; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TOMM20: translocase of outer mitochondrial membrane 20; VDAC: voltage dependent anion channel.

Mitochondria are essential dynamic organelles that ordinarily balance between fragmentation and fusion. Under stress conditions, a shift toward fragmentation or hyper-fusion is observed as a pro-survival reaction. Fragmentation of mitochondria occurs within minutes or hours after the beginning of the stress and occurs in response to a large number of stress stimuli, including those triggered by environmental contaminants. In this study, we tested whether the change in the mitochondrial phenotype, from tubular to fragmented, could be used as a potential environmental stress biomarker in cells and compared this response with the standard MTT-based viability assay. Firstly, we show that mitochondrial fragmentation induced by selected stressors not only increases with concentrations, but also correlates positively with the cytotoxicity. Secondly, we found that the mitochondrial fragmentation that occurs in the first hour of stress correlated with the viability measured after a 24-h stress, allowing the establishment of a linear relation between mitochondrial fragmentation at 1 h and the predictable associated cytotoxicity of environmental contaminants alone or in mixture. In conclusion, we have succeeded in developing a model of predictable 24 h-cytotoxicity given mitochondrial fragmentation at 1 h with a set of chemicals. This model has been successful applied to three environmental toxicants and to a set of two chemical mixtures. We thus propose that mitochondrial fragmentation is a response that could be used as an early in vitro biomarker of environmental stress for toxicants alone or in mixture.

Mitochondria dynamics results from fission and fusion events that may be unbalanced in favor of mitochondrial fragmentation upon cell stress. During oxidative stress, microtubules are hyperacetylated in a mitochondria-dependent manner. In this study, we show that under stress conditions, most of the mitochondria form foci with microtubule domains that carry Drp1. We also demonstrate that stress-induced hyperacetylation of microtubules is required for the effective induction of Drp1 phosphorylation at

Microtubules are highly dynamic polymers of α/β tubulin heterodimers that play key roles in cell division and in organizing cell cytoplasm. Although they have been discovered more than two decades ago, tubulin post-translational modifications recently gained a new interest as their role was increasingly highlighted in neuron differentiation and neurodegenerative disorders. Here, we specifically focus on tubulin acetylation from its discovery to recent studies that provide new insights into how it is regulated in health and disease and how it impacts microtubule functions. Even though new mechanisms involving tubulin acetylation are regularly being uncovered, the molecular links between its location inside the microtubule lumen and its regulators and effectors is still poorly understood. This review highlights the emerging roles of tubulin acetylation in multiple cellular functions, ranging from cell motility, cell cycle progression or cell differentiation to intracellular trafficking and signalling. It also points out that tubulin acetylation should no longer be seen as a passive marker of microtubule stability, but as a broad regulator of microtubule functions.

The molecular mechanisms underlying microtubule participation in autophagy are not known. In this study, we show that starvation-induced autophagosome formation requires the most dynamic microtubule subset. Upon nutrient deprivation, labile microtubules specifically recruit markers of autophagosome formation like class III-phosphatidylinositol kinase, WIPI-1, the Atg12-Atg5 conjugate, and LC3-I, whereas mature autophagosomes may bind to stable microtubules. We further found that upon nutrient deprivation, tubulin acetylation increases both in labile and stable microtubules and is required to allow autophagy stimulation. Tubulin hyperacetylation on lysine 40 enhances kinesin-1 and JIP-1 recruitment on microtubules and allows JNK phosphorylation and activation. JNK, in turn, triggers the release of Beclin 1 from Bcl-2-Beclin 1 complexes and its recruitment on microtubules where it may initiate autophagosome formation. Finally, although kinesin-1 functions to carry autophagosomes in basal conditions, it is not involved in motoring autophagosomes after nutrient deprivation. Our results show that the dynamics of microtubules and tubulin post-translational modifications play a major role in the regulation of starvation-induced autophagy.

Involved in a wide range of cellular processes such as signal transduction, microtubules are highly dynamic polymers that accumulate various post-translational modifications including polyglutamylation, polyglycylation, carboxyterminal cleavage and acetylation, the functions of which just begin to be uncovered. The molecular chaperone Hsp90, which is essential for the folding and activity of numerous client proteins involved in cell proliferation and apoptosis, associates with the microtubule network but the effects of tubulin post-translational modifications on its microtubule binding has not yet been investigated. Herein, we show that both the constitutive (beta) and the inducible (alpha) Hsp90 isoforms bind to microtubules in a way that depends on the level of tubulin acetylation. Tubulin acetylation also stimulates the binding and the signaling function of at least two of its client proteins, the kinase Akt/PKB and the transcription factor p53. This study highlights the role of tubulin acetylation in modulating microtubule-based transport of Hsp90-chaperoned proteins and thus in regulating signaling dynamics in the cytoplasm.

Genotoxic agents produce numerous cellular responses that are principally dedicated to maintain or restore DNA integrity. In human cells, nucleotide excision repair (NER) is one of the major pathways for the repair of DNA damage such as ultraviolet (UV) radiation-induced lesions. Endocrine disrupting compounds are environmental contaminants that interfere with the function of the endocrine system. Among them, the natural estrogen 17beta-estradiol (E(2)) exhibits the most potent activity. Some proteins directly or indirectly involved in NER also fulfill other functions such as transcription, DNA damage checkpoints or cell cycle. Moreover, steroids such as E(2) are believed to interact with a large number of proteins including some involved in NER and DNA damage checkpoint control. We therefore investigated the potential modulation of genotoxic stress-cells responses by E(2) treatment. Estrogen receptor (ER)-positive human breast cancer cells were submitted to E(2) before and/or after UVB irradiation and thereafter the repair kinetics of UV-induced DNA damage were evaluated. We report here that the repair rate of UVB-induced DNA damage is enhanced when cells are submitted to an estrogenic stimulation. Moreover, our results suggest that this response could be mediated by cell cycle regulatory proteins in a p53-independent manner.

Ligation-mediated PCR was employed to quantify cyclobutane pyrimidine dimer (CPD) formation at nucleotide resolution along exon 2 of the adenine phosphoribosyltransferase (aprt) locus in Chinese hamster ovary (CHO) cells following irradiation with either UVA (340-400 nm), UVB (295-320 nm), UVC (254 nm) or simulated sunlight (SSL; lambda > 295 nm). The resulting DNA damage spectrum for each wavelength region was then aligned with the corresponding mutational spectrum generated previously in the same genetic target. The DNA sequence specificities of CPD formation induced by UVC, UVB or SSL were very similar, i.e., in each case the overall relative proportion of this photoproduct forming at TT, TC, CT and CC sites was approximately 28, approximately 26, approximately 16 and approximately 30%, respectively. Furthermore, a clear correspondence was noted between the precise locations of CPD damage hotspots, and of 'UV signature' mutational hotspots consisting primarily of C-->T and CC-->TT transitions within pyrimidine runs. However, following UVA exposure, in strong contrast to the above situation for UVC, UVB or SSL, CPDs were generated much more frequently at TT sites than at TC, CT or CC sites (57% versus 18, 11 and 14%, respectively). This CPD deposition pattern correlates well with the strikingly high proportion of mutations recovered opposite TT dipyrimidines in UVA- irradiated CHO cells. Our results directly implicate the CPD as a major promutagenic DNA photoproduct induced specifically by UVA in rodent cells.

It has been shown that sewage effluent can discharge human hormones and pharmaceuticals, particularly estrogens and synthetic chemicals, which are able to disrupt animal and human endocrine systems into surface waters. Since many surface waters receive sewage effluent and are subsequently used to produce drinking water, it is of principal interest to assess their contamination level and thereby their possible public health and environmental impact. To date, no data concerning the occurrence of estrogens present in the French aquatic environment are available. We therefore developed and validated an analytical procedure, which allows simultaneous quantitative determination of three natural estrogens, 17β-estradiol, estriol, and estrone and one of the synthetic estrogens most widely used in contraception, ethinylestradiol, in water. Water samples are extracted using solid-phase extraction (SPE) and then separated by gas-chromatography coupled with mass spectrometric detection. Under our conditions, detection limits of estrogens reached the pg range of injected sample, i. e. less than 0.1 ng L–1. Conjugated estrogens were also investigated using the same procedure as described above but with a enzymatic hydrolysis preliminary step before extraction. The analysis of samples collected from four wastewater treatment plants and from surface water showed significant concentrations of estrogens ranging from 2 to 18 ng L–1 and from 0.5 to 3 ng L–1, respectively. Furthermore, no estrogen conjugated forms were detected in the water samples.

Beyond its presence in stable microtubules, tubulin acetylation can be boosted after UV exposure or after nutrient deprivation, but the mechanisms of microtubule hyperacetylation are still unknown. In this study, we show that this hyperacetylation is a common response to several cellular stresses that involves the stimulation of the major tubulin acetyltransferase MEC-17. We also demonstrate that the acetyltransferase p300 negatively regulates MEC-17 expression and is sequestered on microtubules upon stress. We further show that reactive oxygen species of mitochondrial origin are required for microtubule hyperacetylation by activating the AMP kinase, which in turn mediates MEC-17 phosphorylation upon stress. Finally, we show that preventing microtubule hyperacetylation by knocking down MEC-17 affects cell survival under stress conditions and starvation-induced autophagy, thereby pointing out the importance of this rapid modification as a broad cell response to stress.

The formation of cyclobutane pyrimidine dimers (CPD) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) was investigated in Chinese hamster ovary cells upon exposure to either UVC, UVB, UVA or simulated sunlight (SSL). Two cell lines were used, namely AT3-2 and UVL9, the latter being deficient in nucleotide excision repair and consequently UV sensitive. For all types of radiation, including UVA, CPD were found to be the predominant lesions quantitatively. At the biologically relevant doses used, UVC, UVB and SSL irradiation yielded 8-oxodGuo at a rather low level, whereas UVA radiation produced relatively higher amounts. The formation of CPD was 10(2) and 10(5) more effective upon UVC than UVB and UVA exposure. These yields of formation followed DNA absorption, even in the UVA range. The calculated relative spectral effectiveness in the production of the two lesions showed that efficient induction of 8-oxodGuo upon UVA irradiation was shifted toward longer wavelengths, in comparison with those for CPD formation, in agreement with a photosensitization mechanism. In addition, after exposure to SSL, about 19% and 20% of 8-oxodGuo were produced between 290-320 nm and 320-340 nm, respectively, whereas CPD were essentially (90%) induced in the UVB region. However, the ratio of CPD to 8-oxodGuo greatly differed from one source of light to the other: it was over 100 for UVB but only a few units for UVA source. The extent of 8-oxodGuo and CPD was also compared to the lethality for the different types of radiation. The involvement of 8-oxodGuo in cell killing by solar UV radiation was clearly ruled out. In addition, our previously reported mutation spectra demonstrated that the contribution of 8-oxodGuo in the overall solar UV mutagenic process is very minor.

As modern day society takes an increasing interest in outdoor activities, its exposure to sunlight has never been greater. As a consequence, countries throughout the world are experiencing a dramatic increase in the incidences of skin carcinomas and melanomas. From DNA photolesions to mutations, skin cancer and cell death provides an authoritative source of information for photobiologists interested in the series of genetic events that occur in the skin, and eventually lead to cancer. With contributions from eminent scientists in the field, this book includes the latest information on DNA photolesions and repair, as well as the key mechanisms of solar UV in skin cancer initiation and development. Significant information relating to UV-induced photolesions and mechanisms of skin tumour occurrence is also included. By providing the basic phenomena underlying the science and an overview of the biological events that take place when cells are exposed to solar UV radiation, From DNA photolesions to mutations, skin cancer and cell death is suitable to all researchers interested in the process of photocarcinogenesis.

Human populations and wildlife are exposed to mixtures of both anthropogenic and natural chemicals. Some of these compounds are known to interact principally with the endocrine function, whereas others act mainly on genomic DNA. Given this evidence, we wanted to address the question of whether concomitant exposure of such chemicals was able to interact at the cellular level. We have previously shown that 17β-Estradiol (E 2 ) modulates the DNA repair capacity of cells. In this work, we wanted to examine if other xenoestrogens (i.e. industrial compounds, pesticides or pharmaceuticals) were able to interact with the UVB-induced cellular response as E 2 does. Here, we show that xenoestrogens modulate the capacity of cells to repair their DNA damage according to the type of compounds. For example, the oral contraceptive 17α-Ethinylestradiol down-regulated the repair of UVB-induced DNA damage whereas the UV filter Eusolex 6007 up-regulated this pathway. The notion that xenoestrogens could interact with a genotoxic stress is reinforced by the modulation of the estrogens-dependent luciferase reporter gene expression when cells are UVB-irradiated. Finally, these observations suggested the potential role of xenoestrogens in carcinogenesis by their capacity to modulate cells responses to genotoxic stress.

For many years, surface waters have been shown to be contaminated by endocrine-disrupting compounds (EDCs), which can cause adverse effects on human and wildlife growth, development, and reproduction. It is therefore of primary importance to determine if drinking water could be contaminated by EDCs when produced from polluted surface waters. It is also essential to determine if disinfection by-products can account for estrogenic activity in treated waters. The estrogenic potential of river and treated waters was investigated using an in vivo assay. Adult male zebrafish were placed in three drinking water treatment plants (DWTPs) in the Paris area and exposed for 1 month to the two types of waters. After exposure, vitellogenin (VTG) was measured in the plasma of fish using a competitive ELISA. In addition, an in vitro assay (MELN cells) was used to assess the estrogenic potential of 10 major chlorination by-products. No significant induction of VTG was observed in fish exposed to river or treated waters. Among the 10 chlorination by-products tested, only 2-chlorophenol was found to be weakly estrogenic at concentrations up to 1 mg/L. Therefore, the risk for the three DWTPs studied to produce drinking water with significant level of estrogenic substances appears to be low.

Wastewater treatment plants (WWTPs) receive a large spectrum of endocrine disrupting compounds (EDCs) that are partially eliminated during treatment processes and discharged into rivers. Given the lack of information in France about river contamination by EDCs, we chose to examine estrogenic potential of WWTP influents, effluents and receiving waters in Paris and its suburbs. Water samples were analyzed using gas chromatography coupled with mass spectrometry for quantifying natural and synthetic estrogens combined with an in vitro estrogenicity bioassay associated to a high pressure liquid chromatography fractionation. The four estrogens investigated, Estrone (E1), 17beta-Estradiol (E2), Estriol (E3) and 17alpha-Ethinylestradiol (EE2) were found in all WWTP and river samples at concentrations ranging from 2.7 to 17.6 ng/l and 1.0 to 3.2 ng/l, respectively. The synthetic estrogen EE2 seems more resistant to biodegradation in WWTPs and thus accounted for 35-50% of the estimated estrogenic activity in rivers. However, fractionation of samples and differences between concentrations of E1, E2, E3 and EE2 and the estrogenic activity measured by the in vitro bioassay suggested a complexity of mechanisms underlying the biological response that could not be attributed only to the investigated molecules.

In order to better understand the relative contribution of the different UV components of sunlight to solar mutagenesis, the distribution of the bipyrimidine photolesions, cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts ((6-4)PP), and their Dewar valence photoisomers (DewarPP) was examined in Chinese hamster ovary cells irradiated with UVC, UVB, or UVA radiation or simulated sunlight. The absolute amount of each type of photoproduct was measured by using a calibrated and sensitive immuno-dot-blot assay. As already established for UVC and UVB, we report the production of CPD by UVA radiation, at a yield in accordance with the DNA absorption spectrum. At biologically relevant doses, DewarPP were more efficiently produced by simulated solar light than by UVB (ratios of DewarPP to (6-4)PP of 1:3 and 1:8, respectively), but were detected neither after UVA nor after UVC radiation. The comparative rates of formation for CPD, (6-4)PP and DewarPP are 1:0.25 for UVC, 1:0. 12:0.014 for UVB, and 1:0.18:0.06 for simulated sunlight. The repair rates of these photoproducts were also studied in nucleotide excision repair-proficient cells irradiated with UVB, UVA radiation, or simulated sunlight. Interestingly, DewarPP were eliminated slowly, inefficiently, and at the same rate as CPD. In contrast, removal of (6-4)PP photoproducts was rapid and completed 24 h after exposure. Altogether, our results indicate that, in addition to CPD and (6-4)PP, DewarPP may play a role in solar cytotoxicity and mutagenesis.

DNA damage profiles have been established in plasmid DNA using purified DNA repair enzymes and a plasmid relaxation assay, following exposure to UVC, UVB, UVA or simulated sunlight (SSL). Cyclobutane pyrimidine dimers (CPDs) are revealed as T4 endonuclease V-sensitive sites, oxidation products at purine and pyrimidine as Fpg- and Nth-sensitive sites, and abasic sites are detected by Nfo protein from Escherichia coli. CPDs are readily detected after UVA exposure, though produced 103 and 105 times less efficiently than by UVB or UVC, respectively. We demonstrate that CPDs are induced by UVA radiation and not by contaminating UVB wavelengths. Furthermore, they are produced at doses compatible with human exposure and are likely to contribute to the mutagenic specificity of UVA [E. Sage et al., Proc. Natl. Acad. Sci. USA 93 (1996) 176–180]. Oxidative damage is induced with a linear dose dependence, for each region of the solar spectrum, with the exception of oxidized pyrimidine and abasic sites, which are not detectable after UVB irradiation. The distribution of the different classes of photolesions varies markedly, depending on wavelengths. However, the unexpectedly high yield of oxidative lesions, as compared to CPDs, by UVA and SSL led us to investigate their production mechanism. An artificial formation of hydroxyl radicals is observed, which depends on the material of the sample holder used for UVA irradiation and is specific for long UV wavelengths. Our study sheds light on a possible artefact in the production of oxidative damage by UVA radiation. Meanwhile, after eliminating some potential sources of the artefact ratios of CPDs to oxidized purine of three and five upon irradiation with UVA and SSL, respectively, are still observed, whereas these ratios are about 140 and 200 after UVC and UVB irradiation.