ATMIN is required for the ATM-mediated signaling and recruitment of 53BP1 to DNA damage sites upon replication stress

Unresolved replication intermediates can block the progression of replication forks and become converted into DNA lesions, hence exacerbating genomic instability. The p53-binding protein 1 (53BP1) forms nuclear bodies at sites of unrepaired DNA lesions to shield these regions against erosion, in a manner dependent on the DNA damage kinase ATM. The molecular mechanism by which ATM is activated upon replicative stress to localize the 53BP1 protection complex is unknown. Here we show that the ATM-INteracting protein ATMIN (also known as ASCIZ) is partially required for 53BP1 localization upon replicative stress. Additionally, we demonstrate that ATM activation is impaired in cells lacking ATMIN and we define that ATMIN is required for initiating ATM signaling following replicative stress. Furthermore, loss of ATMIN leads to chromosomal segregation defects. Together these data reveal that chromatin integrity depends on ATMIN upon exposure to replication-induced stress.     

Changes of Saccharomyces cerevisiae cell membrane components and promotion to ethanol tolerance during the bioethanol fermentation

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane might provide main protection to tolerate accumulated ethanol, and S. cerevisiae cells might also remodel their membrane compositions or structure to try to adapt to or tolerate the ethanol stress. However, the exact changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation still remains poorly understood. This study was performed to clarify changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation. Both cell diameter and membrane integrity decreased as fermentation time lasting. Moreover, compared with cells at lag phase, cells at exponential and stationary phases had higher contents of ergosterol and oleic acid (C_18:1) but lower levels of hexadecanoic (C_16:0) and palmitelaidic (C_16:1) acids. Contents of most detected phospholipids presented an increase tendency during fermentation process. Increased contents of oleic acid and phospholipids containing unsaturated fatty acids might indicate enhanced cell membrane fluidity. Compared with cells at lag phase, cells at exponential and stationary phases had higher expressions of ACC1 and HFA1. However, OLE1 expression underwent an evident increase at exponential phase but a decrease at following stationary phase. These results indicated that during bioethanol fermentation process, yeast cells remodeled membrane and more changeable cell membrane contributed to acquiring higher ethanol tolerance of S. cerevisiae cells. These results highlighted our knowledge about relationship between the variation of cell membrane structure and compositions and ethanol tolerance, and would contribute to a better understanding of bioethanol fermentation process and construction of industrial ethanologenic strains with higher ethanol tolerance.     

Glycolysis-mediated control of blood-brain barrier development and function

The blood-brain barrier (BBB) consists of differentiated cells integrating in one ensemble to control transport processes between the central nervous system (CNS) and peripheral blood. Molecular organization of BBB affects the extracellular content and cell metabolism in the CNS. Developmental aspects of BBB attract much attention in recent years, and barriergenesis is currently recognized as a very important and complex mechanism of CNS development and maturation. Metabolic control of angiogenesis/barriergenesis may be provided by glucose utilization within the neurovascular unit (NVU). The role of glycolysis in the brain has been reconsidered recently, and it is recognized now not only as a process active in hypoxic conditions, but also as a mechanism affecting signal transduction, synaptic activity, and brain development. There is growing evidence that glycolysis-derived metabolites, particularly, lactate, affect barriergenesis and functioning of BBB. In the brain, lactate produced in astrocytes or endothelial cells can be transported to the extracellular space via monocarboxylate transporters (MCTs), and may act on the adjoining cells via specific lactate receptors. Astrocytes are one of the major sources of lactate production in the brain and significantly contribute to the regulation of BBB development and functioning. Active glycolysis in astrocytes is required for effective support of neuronal activity and angiogenesis, while endothelial cells regulate bioavailability of lactate for brain cells adjusting its bidirectional transport through the BBB. In this article, we review the current knowledge with regard to energy production in endothelial and astroglial cells within the NVU. In addition, we describe lactate-driven mechanisms and action of alternative products of glucose metabolism affecting BBB structural and functional integrity in developing and mature brain.     

Interaction between APC and Fen1 during breast carcinogenesis

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER – adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) – promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents.     

Norepinephrine stimulation of alpha1D-adrenoceptor promotes proliferation of pulmonary artery smooth muscle cells via ERK-1/2 signaling

It has been shown that the sympathetic nervous system is activated in pulmonary arterial hypertension (PAH). Norepinephrine (NE) levels are increased by chemoreflex-dependent sympathetic overactivation and involved in pulmonary vascular remodeling. However, the underlying mechanisms of the remodeling induced by NE are poorly understood. In this study, we found that, in vivo, the expression of tyrosine hydroxylase and the concentration of plasma NE were increased in PAH rats compared with normal rats. Increases in ventricular hypertrophy and medial width of the pulmonary arteries were reversed by prazosin, α₁-adrenoceptor (α₁-AR) antagonists, in PAH rats. Elevated expression of α_1D-AR was detected in PAH rats. In addition, prazosin reduced the increasing expression of PCNA, CyclinA and CyclinE induced by hypoxia. In vitro, MTT assay, flow cytometry, Western blotting and immunofluorescence were performed to investigate the effects of NE on proliferation of pulmonary artery smooth muscle cells (PASMCs). We revealed that NE promoted PASMCs viability, increased the expression of PCNA, CyclinA and CyclinE, made more cells from G₀/G₁ phase to G₂/M + S phase and enhanced the microtubule formation. Above NE-induced changes could be suppressed by BMY 7378, an inhibitor of α_1D-AR. Furthermore, ERK-1/2 pathway was activated by NE. U0126, a specific inhibitor for ERK-1/2, attenuated the NE-induced proliferation of PASMCs under normoxia and hypoxia. Taken together, our results suggest that NE which stimulates α_1D-AR promotes proliferation of PASMCs and the effect is, at least in part, mediated via the ERK-1/2 pathway.     

LAMMER kinase contributes to genome stability in Ustilago maydis

Here we report identification of the lkh1 gene encoding a LAMMER kinase homolog (Lkh1) from a screen for DNA repair-deficient mutants in Ustilago maydis. The mutant allele isolated results from a mutation at glutamine codon 488 to a stop codon that would be predicted to lead to truncation of the carboxy-terminal kinase domain of the protein. This mutant (lkh1^Q488*) is highly sensitive to ultraviolet light, methyl methanesulfonate, and hydroxyurea. In contrast, a null mutant (lkh1Δ) deleted of the entire lkh1 gene has a less severe phenotype. No epistasis was observed when an lkh1^Q488* rad51Δ double mutant was tested for genotoxin sensitivity. However, overexpressing the gene for Rad51, its regulator Brh2, or the Brh2 regulator Dss1 partially restored genotoxin resistance of the lkh1Δ and lkh1^Q488* mutants. Deletion of lkh1 in a chk1Δ mutant enabled these double mutant cells to continue to cycle when challenged with hydroxyurea. lkh1Δ and lkh1^Q488* mutants were able to complete the meiotic process but exhibited reduced heteroallelic recombination and aberrant chromosome segregation. The observations suggest that Lkh1 serves in some aspect of cell cycle regulation after DNA damage or replication stress and that it also contributes to proper chromosome segregation in meiosis.     

Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway

Prostate cancer (CaP) is the second leading malignancy in men. The role of epithelial cell adhesion molecule (EpCAM), also known as CD326, in CaP progression and therapeutic resistance is still uncertain. Here, we aimed to investigate the roles of EpCAM in CaP metastasis and chemo/radioresistance. Expression of EpCAM in CaP cell lines and human CaP tissues was assessed using immunofluorescence and immunohistochemistry, respectively. EpCAM was knocked down (KD) in PC-3, DU145 and LNCaP-C4-2B cells using small interfering RNA (siRNA), and KD results were confirmed by confocal microscope, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The invasive potential was assessed using a matrigel chamber assay. Tumorigenesis potential was measured by a sphere formation assay. Chemo-/radiosensitivity were measured using a colony formation assay. Over-expression of EpCAM was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of EpCAM suppressed CaP proliferation and invasive ability, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated E-cadherin, p-Akt, p-mTOR, p-4EBP1 and p-S6K expression in CaP cells. Our findings suggest that EpCAM plays an important role in CaP proliferation, invasion, metastasis and chemo-/radioresistance associated with the activation of the PI3K/Akt/mTOR signaling pathway and is a novel therapeutic target to sensitize CaP cells to chemo-/radiotherapy.     

The complexity of DNA double strand break is a crucial factor for activating ATR signaling pathway for G2/M checkpoint regulation regardless of ATM function

DNA double strand break (DSB) repair pathway choice following ionizing radiation (IR) is currently an appealing research topic, which is still largely unclear. Our recent paper indicated that the complexity of DSBs is a critical factor that enhances DNA end resection. It has been well accepted that the RPA-coated single strand DNA produced by resection is a signaling structure for ATR activation. Therefore, taking advantage of high linear energy transfer (LET) radiation to effectively produce complex DSBs, we investigated how the complexity of DSB influences the function of ATR pathway on the G2/M checkpoint regulation. Human skin fibroblast cells with or without ATM were irradiated with X rays or heavy ion particles, and dual-parameter flow cytometry was used to quantitatively assess the mitotic entry at early period post radiation by detecting the cells positive for phosphor histone H3. In ATM-deficient cells, ATR pathway played a pivotal role and functioned in a dose- and LET-dependent way to regulate the early G2/M arrest even as low as 0.2 Gy for heavy ion radiation, which indicated that ATR pathway could be rapidly activated and functioned in an ATM-independent, but DSB complexity-dependent manner following exposure to IR. Furthermore, ATR pathway also functioned more efficiently in ATM-proficient cells to block G2 to M transition at early period of particle radiation exposure. Accordingly, in contrast to ATM inhibitor, ATR inhibitor had a more effective radiosensitizing effect on survival fraction following heavy ion beams as compared with X ray radiation. Taken together, our results reveal that the complexity of DSBs is a crucial factor for the activation of ATR pathway for G2/M checkpoint regulation, and ATM-dependent end resection is not essential for the activation.     

Vimentin-mediated regulation of cell motility through modulation of beta4 integrin protein levels in oral tumor derived cells

Vimentin expression correlates well with migratory and invasive potential of the carcinoma cells. The molecular mechanism by which vimentin regulates cell motility is not yet clear. Here, we addressed this issue by depleting vimentin in oral squamous cell carcinoma derived cell line. Vimentin knockdown cells showed enhanced adhesion and spreading to laminin-5. However, we found that they were less invasive as compared to the vector control cells. In addition, signaling associated with adhesion behavior of the cell was increased in vimentin knockdown clones. These findings suggest that the normal function of β4 integrin as mechanical adhesive device is enhanced upon vimentin downregulation. As a proof of principle, the compromised invasive potential of vimentin depleted cells could be rescued upon blocking with β4 integrin adhesion-blocking (ASC-8) antibody or downregulation of β4 integrin in vimentin knockdown background. Interestingly, plectin which associates with α6β4 integrin in the hemidesmosomes, was also found to be upregulated in vimentin knockdown clones. Furthermore, experiments on lysosome and proteasome inhibition revealed that perhaps vimentin regulates the turnover of β4 integrin and plectin. Moreover, an inverse association was observed between vimentin expression and β4 integrin in oral squamous cell carcinoma (OSCC). Collectively, our results show a novel role of vimentin in modulating cell motility by destabilizing β4 integrin-mediated adhesive interactions. Further, vimentin-β4 integrin together may prove to be useful markers for prognostication of human oral cancer.     

Tebufenozide disrupts ovarian development and function in silkmoths

Adult development and production of up to 400 eggs within the pupal case of female silkmoths are both dependent on 20-hydroxyecdysone (20E), the steroid hormone of insects. When adult development was initiated with tebufenozide, the non-steroidal ecdysteroid agonist, instead of 20E, full development of all epidermal tissues like the wing was witnessed, but ovarian growth and egg formation was minimal. Administration of tebufenozide to female pharate adults caused disruption of the follicular epithelium, produced nurse cell damage, and inhibited oogenesis. Reduced ability to synthesize RNA and protein accompanied these tebufenozide induced morphological disturbances of the follicles. In vivo accumulation of vitellogenin (Vg) from the hemolymph was reduced in tebufenozide treated female ovaries as well as their ability to accumulate Vg in vitro. Determination of protein staining intensity and antibody reactivity of Vg pointed out that hemolymph Vg level remained fairly constant all through adult development whether induced by 20E or tebufenozide. Measurement of hemolymph volumes and hemolymph Vg levels of control and experimental animals allowed us to conclude that egg development involves the uptake of all the hemolymph proteins and not Vg alone. The loss of hemolymph that accompanies egg maturation was considerably reduced in tebufenozide initiated female pharate adults. 20E could not overcome ovarian growth inhibitory effects of tebufenozide. Dual mechanisms, one involving ecdysteroid antagonist action at the beginning of development, and the other unrelated to that function during heightened egg formation, are needed explain the biphasic inhibitory actions of tebufenozide on silkmoth ovaries.     

NBT-II cell locomotion is modulated by restricting the size of focal contacts and is improved through EGF and ROCK signaling

Focal contacts, large macromolecular complexes that link the extracellular matrix and the internal cell cytoskeleton, are thought to govern cell locomotion. However, the maturation process through which focal contacts control the cellular migratory machinery by changes in size and molecular composition remain unclear. Here, we fabricated cell growth substrates that contained linear ECM strips of micron- or submicron-width in order to limit the enlargement of focal contacts. We found that NBT-II cells plated on the submicron substrate possessed smaller focal complexes that exhibited a highly dynamic turnover. These cells possessed various leading edges at multiple sites of the cell periphery, which prevented the cell from advancing. In contrast, cells grown on the micron-width substrate possessed large and stable focal adhesions. Most of these cells were elongated bipolar cells that were tethered at both ends and were immobile. Further, EGF and ROCK signaling pathways can modulate the cellular migratory responses according to the substrate guidance. On the submicron-width substrate, EGF treatment increased the focal contact size and the contractile force, causing these cells to develop one leading edge and migrate along the submicron-sized ECM paths. In contrast, inhibition of ROCK signaling decreased the focal contact size for cells plated on the micron substrate. These cells became less tethered and were able to migrate along or even across the micron-sized ECM paths. Our results indicate that formation and maturation of focal contacts is controlled by both ECM cues and intracellular signaling and they play a central role in directed cell motion.     

Seasonal patterns of microcystin-producing and non-producing Planktothrix rubescens genotypes in a deep pre-alpine lake

The filamentous cyanobacterium Planktothrix rubescens produces secondary metabolites called microcystins (MC) that are potent toxins for most eukaryotes, including zooplankton grazers, cattle and humans. P. rubescens occurs in many deep and thermally stratified lakes throughout Europe. In Lake Zurich (Switzerland), it re-appeared in the 1970s concomitant with decreasing eutrophication. Since then, P. rubescens has become the dominant species in this major drinking water reservoir, where it forms massive metalimnetic blooms during late summer. These cyanobacteria harbor subpopulations of non-MC producers, but little is known about the environmental factors affecting the success of such genotypes. The non-MC-producing subpopulation of P. rubescens was studied using a quantitative real-time PCR (qPCR) assay on the MC synthetase (mcy) gene cluster that targets a deletion on the mcyH and mcyA genes, which inactivates MC biosynthesis. Two complementary qPCR assays were used to assess the total population abundance (based on the 16S rDNA gene) and the mcy gene copy number (based on a conserved region in the adenylation domain of the mcyB gene). The objective was to evaluate the seasonal patterns of the share of non-MC-producing filaments in the total P. rubescens population. The mcyHA mutants were present in low proportions (up to 14%) throughout the year. Their highest relative abundances occurred during the winter mixis, when total concentrations of P. rubescens were minimal. The MC deficient mutants seemed to better survive in sparse populations, possibly because of lower grazing pressure and a consequently reduced need for MC-mediated protection. Alternatively, the mutants might cope better with the sub-optimal, stressful pressure and light conditions during the winter mixis. Altogether, our results suggest that subtle trade-offs might seasonally determine the proportions of non-MC producers within P. rubescens populations.     

Stochastic exposure to sub-lethal high temperature enhances exopolysaccharides (EPS) excretion and improves Bifidobacterium bifidum cell survival to freeze–drying

Exposure of microbial cells to sub-lethal stresses is known to increase cell robustness. In this work, a two-compartment bioreactor in which microbial cells are stochastically exposed to sub-lethal temperature stresses has been used in order to investigate the response of the stress sensitive Bifidobacterium bifidum THT 0101 to downstream processing operations. A stochastic model validated by residence time distribution experiments has shown that in the heat-shock configuration, a two-compartment bioreactor (TCB) allows the exposure of microbial cells to sub-lethal temperature of 42 °C for a duration comprised between 100 and 300 s. This exposure resulted in a significant increase of cell resistance to freeze–drying by comparison with cells cultivated in conventional bioreactors or in the TCB in the cold shock mode (CS-TCB). The mechanism behind this robustness seems to be related with the coating of microbial cells with exopolysaccharide (EPS), as assessed by the change of the zeta potential and the presence of higher EPS concentration after heat shock. Conditioning of Bifidobacteria on the basis of the heat shock technique is interesting from the practical and economical point of view since this strategy can be directly implemented in the bioreactor during stationary phase preceding cell recovery and freeze–drying.     

Home environment and cord blood levels of lead,arsenic, and zinc on neurodevelopment of 24 months children living in Chitwan Valley,Nepal

In a birth cohort living in Chitwan Valley, lowland Nepal, we have previously reported inverse associations between in utero levels of lead (Pb), arsenic (As) and neurodevelopment at birth measured by the Brazelton Neonatal Behavioral Assessment Scale, third edition (NBAS III). In the present paper, a follow-up of the same cohort was made on 24-month-old infants regarding the neurodevelopmental effects of these metals, taking the postnatal environment into account. In total, the same100 mother-infant pairs as the previous study, whose Pb, As, and Zn concentrations in cord blood were known, were recruited. Postnatal raising environment was evaluated using the Home Observation for Measurement of Environment (HOME) scale. Neurodevelopment of children at 24 months of age (n = 74) was assessed using the Bayley Scale of Infant Development, Second Edition (BSID II). Multivariable regression adjusting for covariates was performed to determine the associations of in utero levels of toxic and essential elements and the home environment with neurodevelopment scores. Unlike the NBAS III conducted for newborns, none of the BSID II cluster scores in 24-month-old infants were associated with cord blood levels of Pb, As, and Zn. The total HOME score was positively associated with the mental development scale (MDI) score (coefficient = 0.67, at 95% CI = 0.03 to 1.31). In this cohort, a detrimental effect of in utero Pb and As on neurodevelopmental indicators observed at birth disappeared at 24 months, while an association between neurodevelopment and home environment continued.     

The relationships between XPC binding to conformationally diverse DNA adducts and their excision by the human NER system: Is there a correlation?

The first eukaryotic NER factor that recognizes NER substrates is the heterodimeric XPC-RAD23B protein. The currently accepted hypothesis is that this protein recognizes the distortions/destabilization caused by DNA lesions rather than the lesions themselves. The resulting XPC-RAD23B–DNA complexes serve as scaffolds for the recruitment of subsequent NER factors that lead to the excision of the oligonucleotide sequences containing the lesions. Based on several well-known examples of DNA lesions like the UV radiation-induced CPD and 6–4 photodimers, as well as cisplatin-derived intrastrand cross-linked lesions, it is generally believed that the differences in excision activities in human cell extracts is correlated with the binding affinities of XPC-RAD23B to these DNA lesions. However, using electrophoretic mobility shift assays, we have found that XPC-RAD23B binding affinities of certain bulky lesions derived from metabolically activated polycyclic aromatic hydrocarbon compounds such as benzo[a]pyrene and dibenzo[a,l]pyrene, are not directly, or necessarily correlated with NER excision activities observed in cell-free extracts. These findings point to features of XPC-RAD23B–bulky DNA adduct complexes that may involve the formation of NER-productive or unproductive forms of binding that depend on the structural and stereochemical properties of the DNA adducts studied. The pronounced differences in NER cleavage efficiencies observed in cell-free extracts may be due to differences in the successful recruitment of subsequent NER factors by the XPC-RAD23B–DNA adduct complexes, and/or in the verification step. These phenomena appear to depend on the structural and conformational properties of the class of bulky DNA adducts studied.     

Inhibition of vascular smooth muscle cell calcification by vasorin through interference with TGFβ1 signaling

Elevated transforming growth factor β1 (TGFβ1) levels are frequently observed in chronic kidney disease (CKD) patients. TGFβ1 contributes to development of medial vascular calcification during hyperphosphatemia, a pathological process promoted by osteo−/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Vasorin is a transmembrane glycoprotein highly expressed in VSMCs, which is able to bind TGFβ to inhibit TGFβ signaling. Thus, the present study explored the effects of vasorin on osteo−/chondrogenic transdifferentiation and calcification of VSMCs. Primary human aortic smooth muscle cells (HAoSMCs) were treated with recombinant human TGFβ1 or β-glycerophosphate without or with recombinant human vasorin or vasorin gene silencing by siRNA. As a result, TGFβ1 down-regulated vasorin mRNA expression in HAoSMCs. Vasorin supplementation inhibited TGFβ1-induced pathway activation, SMAD2 phosphorylation and downstream target genes expression in HAoSMCs. Furthermore, treatment with exogenous vasorin blunted, while vasorin knockdown augmented TGFβ1-induced osteo−/chondrogenic transdifferentiation of HAoSMCs. In addition, phosphate down-regulated vasorin mRNA expression in HAoSMCs. Phosphate-induced TGFβ1 expression was not affected by addition of exogenous vasorin. Nonetheless, the phosphate-induced TGFβ1 signaling, osteo−/chondrogenic transdifferentiation and calcification of HAoSMCs were all blunted by vasorin. Conversely, silencing of vasorin aggravated osteoinduction in HAoSMCs during high phosphate conditions. Aortic vasorin expression was reduced in the hyperphosphatemic klotho-hypomorphic mouse model of CKD-related vascular calcification. In conclusion, vasorin, which suppresses TGFβ1 signaling and protects against osteo−/chondrogenic transdifferentiation and calcification of VSMCs, is reduced by pro-calcifying conditions. Thus, vasorin is a novel key regulator of VSMC calcification and may represent a potential therapeutic target for vascular calcification during CKD.