Altered Nuclear Morphology Research Articles

Antagonistic Relationship between Human Cytomegalovirus pUL27 and pUL97 Activities during Infection.

Human cytomegalovirus (HCMV) is a member of the betaherpesvirus family. During infection, an array of viral proteins manipulates the host cell cycle. We have previously shown that expression of HCMV pUL27 results in increased levels of the cyclin-dependent kinase (CDK) inhibitor p21(Cip1). In addition, pUL27 is necessary for the full antiviral activity of the pUL97 kinase inhibitor maribavir (MBV). The purpose of this study was to define the relationship between pUL27 and pUL97 and its role in MBV antiviral activity. We observed that expression of wild-type but not kinase-inactive pUL97 disrupted pUL27-dependent induction of p21(Cip1). Furthermore, pUL97 associated with and promoted the phosphorylation of pUL27. During infection, inhibition of the kinase resulted in elevated levels of p21(Cip1) in wild-type virus but not a pUL27-deficient virus. We manipulated the p21(Cip1) levels to evaluate the functional consequence to MBV. Overexpression of p21(Cip1) restored MBV activity against a pUL27-deficient virus, while disruption reduced activity against wild-type virus. We provide evidence that the functional target of p21(Cip1) in the context of MBV activity is CDK1. One CDK-like activity of pUL97 is to phosphorylate nuclear lamin A/C, resulting in altered nuclear morphology and increased viral egress. In the presence of MBV, we observed that infection using a pUL27-deficient virus still altered the nuclear morphology. This was prevented by the addition of a CDK inhibitor. Overall, our results demonstrate an antagonistic relationship between pUL27 and pUL97 activities centering on p21(Cip1) and support the idea that CDKs can complement some activities of pUL97. HCMV infection results in severe disease upon immunosuppression and is a leading cause of congenital birth defects. Effective antiviral compounds exist, yet they exhibit high levels of toxicity, are not approved for use during pregnancy, and can result in antiviral resistance. Our studies have uncovered new information regarding the antiviral efficacy of the HCMV pUL97 kinase inhibitor MBV as it relates to the complex interplay between pUL97 and a second HCMV protein, pUL27. We demonstrate that pUL97 functions antagonistically against pUL27 by phosphorylation-dependent inactivation of pUL27-mediated induction of p21(Cip1). In contrast, we provide evidence that p21(Cip1) functions to antagonize overlapping activities between pUL97 and cellular CDKs. In addition, these studies further support the notion that CDK inhibitors or p21(Cip1) activators might be useful in combination with MBV to effectively inhibit HCMV infections.

Acute effects of a prooxidant herbicide on the microalga Chlamydomonas reinhardtii: Screening cytotoxicity and genotoxicity endpoints.

Since recent evidence has demonstrated that many types of chemicals exhibit oxidative and/or genotoxic potential on living organisms, reactive oxygen species (ROS) formation and DNA damage are currently the best accepted paradigms to assess the potential hazardous biological effects of a wide range of contaminants. The goal of this study was to evaluate the sensitivity of different cytotoxicity and genotoxicity responses on the model microalga Chlamydomonas reinhardtii exposed to the prooxidant herbicide paraquat. In addition to the growth endpoint, cell viability, mitochondrial membrane potential and presence of reactive oxygen species (ROS) were assayed as potential markers of cytotoxicity using flow cytometry (FCM). To study the effects of paraquat on C. reinhardtii DNA, several genotoxicity approaches were implemented for the first time in an ecotoxicological study on microalgae. Oxidative DNA base damage was analysed by measuring the oxidative DNA lesion 8-OHdG by FCM. DNA fragmentation was analysed by different methods: comet assay, and cell cycle analysis by FCM, with a particular focus on the presence of subG1-nuclei. Finally, effects on morphology of nuclei were monitored through DAPI staining. The evaluation of these endpoints showed that several physiological and biochemical parameters reacted to oxidative stress disturbances with greater sensitivity than integrative parameters such as growth rates or cell viability. The experiments revealed concentration-dependent cytotoxicity (ROS formation, depolarization of mitochondrial membrane), genotoxicity (oxidative DNA damage, DNA strand breakage, alterations in nuclear morphology), and cell cycle disturbances (subG1-nuclei, decrease of 4N population) in paraquat-treated cells. Overall, the genotoxicity results indicate that the production of ROS caused by exposure to paraquat induces oxidative DNA damage followed by DNA single- and double-strand breaks and cell cycle alterations, possibly leading to apoptosis in C. reinhardtii cells. This is supported by the observation of typical hallmarks of apoptosis, such as mitochondrial membrane depolarization, alterations in nuclear morphology and subG1 nuclei in cells exposed to the highest assayed concentrations. To our knowledge, this is the first study that provides a comprehensive analysis of oxidative DNA base damage in unicellular algal cells exposed to a prooxidant pollutant, as well as of its possible relation with other physiological effects. These results reinforce the need for additional studies on the genotoxicity of environmental pollutants on ecologically relevant organisms such as microalgae that can provide a promising basis for the characterization of potential pollutant hazards in the aquatic environment.

Astrocyte activation and neurotoxicity: A study in different rat brain regions and in rat C6 astroglial cells

The present study was conducted to investigate the effect of rotenone on astrocytes activation, their viability and its effect on neuronal death in different brain regions. Rotenone was injected in rat brain by intracerebroventricularly (bilateral) route at dose of 6μg and 12μg. In vitro C6 cells were treated with rotenone at concentration of 0.1, 0.25, 0.5, 1 and 2μM. Rotenone administration to rat brain caused significant astrocytes activation in frontal cortex, cerebellum, cerebellar nucleus, substantia nigra, hypothalamus and hippocampus regions of the rat brain. Rotenone administration also led to significant degeneration of cells in all the studied regions along with altered nuclear morphology assessed by hematoxylin–eosin and cresyl violet staining. Histological staining showed the significantly decreased number of cells in all the studied regions except cerebellar nucleus in dose and time dependant manner. Rotenone administration in the rat brain also caused significant decrease in glutathione levels and augmented nitrite levels. In vitro treatment of rotenone to astrocytic C6 cells caused significantly increased expression of glial fibrillar acidic protein (GFAP) and decreased viability in dose and time dependent manner. Rotenone treatment to C6 cells exhibited significant generation of reactive oxygen species, augmented nitrite level, impaired mitochondrial activity, apoptotic chromatin condensation and DNA damage in comparison to control cells. Findings showed that oxidative stress play a considerable role in rotenone induced astrocyte death that was attenuated with co-treatment of antioxidant melatonin. In conclusion, results showed that rotenone caused significant astrocytes activation, altered nuclear morphology, biochemical alteration and apoptotic cell death in different rat brain regions. In vitro observations in C6 cells showed that rotenone treatment exhibited oxidative stress mediated apoptotic cell death, which was attenuated with co treatment of melatonin.

Morphological Changes in Amphibian and Fish Cell Lines Infected with Andrias davidianus Ranavirus

Andrias davidianus ranavirus (ADRV) is an emerging viral pathogen that causes severe disease in Chinese giant salamanders, the largest extant amphibian in the world. A fish cell line, Epithelioma papulosum cyprinid (EPC), and a new amphibian cell line, Chinese giant salamander spleen cell (GSSC), were infected with ADRV and observed by light and electron microscopy. The morphological changes in these two cell lines infected with ADRV were compared. Cytopathic effect (CPE) began with rounding of the cells, progressing to cell detachment in the cell monolayer, followed by cell lysis. Significant CPE was visualized as early as 24 h post infection (hpi) in EPC cells and at 36 hpi in GSSC cells. Microscopical examination showed clear and significant CPE in EPC cells, while less extensive and irregular CPE with some adherent cells remaining was observed in GSSC cells. Following ADRV infection, CPE became more extensive. Transmission electron micrographs showed many virus particles around cytoplasmic vacuoles, formed as crystalline arrays or scattered in the cytoplasm of infected cells. Infected cells showed alteration in nuclear morphology, with condensed and marginalized nuclear chromatin on the inner aspect of the nuclear membrane and formation of a cytoplasmic viromatrix adjacent to the nucleus in both cell lines. Some virus particles were also detected in the nucleus of infected GSSC cells. Both cell lines are able to support replication of ADRV and can therefore be used to investigate amphibian ranaviruses.

Carboxylic group-induced synthesis and characterization of selenium nanoparticles and its anti-tumor potential on Dalton's lymphoma cells

Carboxylic group-induced synthesis of selenium nanoparticles (SeNPs) was achieved using sodium selenosulphate as a precursor. The particles were stabilized and capped with 0.01% polyvinyl alcohol under ambient conditions. This is a simple and easy method of producing SeNPs in a size range from 35 to 105nm. The synthesized SeNPs were purified by centrifugation at 11,500×g for 20min and characterized by UV–visible spectroscopy, FTIR spectroscopy, XRD, DSC and TEM. It was observed that the synthesized SeNPs showed differences in their absorption spectra, phase composition and crystal structure, thermodynamic behaviour, size and shape. Further, to confirm anti-tumour potential of the synthesized SeNPs induced by the carboxylic group of acetic acid, pyruvic acid and benzoic acid, cell viability assay, nuclear morphology testing and DNA fragmentation assay were carried out using Dalton's lymphoma (DL) cells. DL cells treated with the SeNPs showed reduced cell viability, altered nuclear morphology, typical apoptotic DNA ladder and apoptosis. Therefore, these SeNPs may have therapeutic relevance to treat this type of cancer.

Morin, a dietary flavonoid, exhibits anti-fibrotic effect and induces apoptosis of activated hepatic stellate cells by suppressing canonical NF-κB signaling.

In experimental liver fibrosis, activated hepatic stellate cells (HSCs) play a central role and thus, induction of apoptosis of activated HSCs is a promising therapeutic strategy for liver fibrosis. The present study was designed to elucidate the molecular mechanisms of the pro-apoptotic effects of morin, a dietary flavonoid, invitro and invivo. Culture-activated human HSCs (LX-2 cells) were treated with morin (50μM) for 24 and 48h, and the mechanism of cell death induced by morin was evaluated. Also, the anti-fibrotic and pro-apoptotic effect of morin in diethylnitrosamine (DEN)-induced fibrotic rats were determined. Morin induced apoptosis in cultured LX-2 cells by preventing the nuclear translocation of nuclear factor-κBp65 (NF-κBp65) by inhibiting NF-κB activation via inhibition of IκBα degradation and thereby suppressing anti-apoptotic proteins and activating caspases. In fibrotic rats, morin treatment resulted in inhibition of canonical NF-κB signaling and induction of apoptosis, mainly by downregulating Bcl-2, upregulating Bax and cyt c and by activation of caspase-9 and caspase-3. Translocation of phosphatidylserine to the outer membrane, altered nuclear morphology and DNA fragmentation confirmed the induction of apoptosis by morin. Overall, morin treatment ameliorated experimental liver fibrosis, most likely through induction of apoptosis by inhibiting canonical NF-κB signaling in activated HSCs. It is therefore postulated that morin is a potential therapeutic candidate for liver fibrosis.

The Antitumor Activity of Meconopsis Horridula Hook, a Traditional Tibetan Medical Plant, in Murine Leukemia L1210 Cells

Background: Meconopsis horridula Hook (M. horridula) has been used as a traditional Tibetan medicine to relieve heat and pain as well as mobilize static blood, and it is recognized as a good treatment for bruises. This study is the first trial to evaluate the tumor inhibitory activity of M. horridula extract and its underlying mechanism in the hope of providing evidence to support the anticancer function of M. horridula. Methods and Results: M. horridula extract was cytotoxic to L1210 cells in a dose- and time-dependent manner. SEM (scanning electron microscope) observation revealed obvious morphological changes in L1210 cells after M. horridula treatment. Flow cytometry analysis demonstrated that the extract dose-dependently induced early apoptosis. Additional apoptosis parameters, such as alterations in nuclear morphology and DNA damage, were also observed. Furthermore, M. horridula treatment induced G2/M arrest. M. horridula treatment significantly increased reactive oxygen species (ROS) production, suggesting that ROS are a key factor in M. horridula-induced apoptosis. Volatile constituent detection found 15 abundant chemicals in M. horridula, which may contribute to its anticancer effect. Conclusion: In conclusion, M. horridula extract induced L1210 cell apoptosis and inhibited proliferation through G2/M phase arrest, and ROS were involved in the process.

Volume regulation and shape bifurcation in the cell nucleus.

Alterations in nuclear morphology are closely associated with essential cell functions, such as cell motility and polarization, and correlate with a wide range of human diseases, including cancer, muscular dystrophy, dilated cardiomyopathy and progeria. However, the mechanics and forces that shape the nucleus are not well understood. Here, we demonstrate that when an adherent cell is detached from its substratum, the nucleus undergoes a large volumetric reduction accompanied by a morphological transition from an almost smooth to a heavily folded surface. We develop a mathematical model that systematically analyzes the evolution of nuclear shape and volume. The analysis suggests that the pressure difference across the nuclear envelope, which is influenced by changes in cell volume and regulated by microtubules and actin filaments, is a major factor determining nuclear morphology. Our results show that physical and chemical properties of the extracellular microenvironment directly influence nuclear morphology and suggest that there is a direct link between the environment and gene regulation.

Síndrome de Progeria de Hutchinson-Gilford. Causas, investigación y tratamientos farmacológicos

Hutchinson-Gilford progeria syndrome (HGPS) also known as childhood progeria is a rare genetic disease characterized by accelerated aging beginning in early childhood. The phenotypic features of this syndrome are caused by alterations in the lamin A protein, fibrillar component of the nuclear lamina which maintain the structure of the nuclear envelope and participates in organization chromatin. Children with progeria have a point mutation in the LMNA gene which leads to the production of a permanently farnesylated mutant lamin A called progerin, that contribute to premature aging. In normal protein, this farnesyl group is removed, but this step does not take place in progeria and the progerin remain attached to the inner nuclear membrane, causing alterations in nuclear morphology. The use of several drugs that inhibit the farnesylation of progerin has been proposed as a promising therapeutic approach to reverse the adverse effects of progerin synthesis. Some studies have determined that progerin is also produced in normal individuals and increase with age, suggesting that these studies of progeria can shed light on the normal process of aging. In this paper the main aspects of HGPS such signs and symptoms, genetic basis, and how it has driven the discovery of strategies for treating the disease will be discussed.

Caspase-activated DNase Is Necessary and Sufficient for Oligonucleosomal DNA Breakdown, but Not for Chromatin Disassembly during Caspase-dependent Apoptosis of LN-18 Glioblastoma Cells

Caspase-dependent apoptosis is a controlled type of cell death characterized by oligonucleosomal DNA breakdown and major nuclear morphological alterations. Other kinds of cell death do not share these highly distinctive traits because caspase-activated DNase (DFF40/CAD) remains inactive. Here, we report that human glioblastoma multiforme-derived LN-18 cells do not hydrolyze DNA into oligonucleosomal fragments after apoptotic insult. Furthermore, their chromatin remains packaged into a single mass, with no signs of nuclear fragmentation. However, ultrastructural analysis reveals that nuclear disassembly occurs, although compacted chromatin does not localize into apoptotic nuclear bodies. Caspases become properly activated, and ICAD, the inhibitor of DFF40/CAD, is correctly processed. Using cell-free in vitro assays, we show that chromatin from isolated nuclei of LN-18 cells is suitable for hydrolysis into oligonuclesomal fragments by staurosporine-pretreated SH-SY5Y cytoplasms. However, staurosporine-pretreated LN-18 cytoplasms do not induce DNA laddering in isolated nuclei from either LN-18 or SH-SY5Y cells because LN-18 cells express lower amounts of DFF40/CAD. DFF40/CAD overexpression makes LN-18 cells fully competent to degrade their DNA into oligonucleosome-sized fragments, and yet they remain unable to arrange their chromatin into nuclear clumps after apoptotic insult. Indeed, isolated nuclei from LN-18 cells were resistant to undergoing apoptotic nuclear morphology in vitro. The use of LN-18 cells has uncovered a previously unsuspected cellular model, whereby a caspase-dependent chromatin package is DFF40/CAD-independent, and DFF40/CAD-mediated double-strand DNA fragmentation does not warrant the distribution of the chromatin into apoptotic nuclear bodies. The studies highlight a not-yet reported DFF40/CAD-independent mechanism driving conformational nuclear changes during caspase-dependent cell death.

Synergistic anticancer activity of valproate combined with nicotinamide enhances anti-proliferation response and apoptosis in MIAPaca2 cells

Histone deacetylase is strongly associated with epigenetic regulation and carcinogenesis, and its inhibitors can induce cell cycle arrest and apoptosis of the cancer cells. In this study we aimed to examine the antiproliferative effects a combination of the valproate with nicotinamide in MIAPaca2 cell line. We revealed that valproate acted in a synergistic/additive with nicotinamide to inhibit the proliferation and induction of apoptosis in MIAPaca2 cancer cell line. MIAPaca2 was treated with various concentrations of valproate. The MTT assay and colony formation in soft agar indicated that valproate at 0.5 mM, when used alone weakly, suppressed proliferation of cells (37 ± 3.02%) whereas the combination treatment of valproate + nicotinamide significantly suppressed cell proliferation (58 ± 3.5%). The effect of nicotinamide at 25 mM on cell proliferation and cell colonization induced 50% apoptosis of MIAPaca2 cells. To identify the anti-proliferation and apoptotic effects of valproate and nicotinamide we performed flow cytometric and microscopic analyses. The results indicated significant apoptosis induction and nuclear morphological alterations greater than when valproate was used alone. Furthermore, western blot analyses was performed to study the role of acetyl-histone H3 levels, and quantitative RNA expression analyses were performed on expression of thrombospondin (TSP) and maspin genes in MIAPaca2. We found that the combination treatment of valproate + nicotinamide enhanced the expression of maspin and TSP genes and the biological response of the cell line was correlated with the increase of histone H3 acetylation after nicotinamide and valproate application. Together our findings indicate that valproate which act as inhibitor of cell proliferation and inducer of apoptosis in human cancer MIAPaca2 cells when used in combination with nicotinamide makes it a potentially good candidate for new anticancer drug development.

The enhanced apoptosis and antiproliferative response to combined treatment with valproate and nicotinamide in MCF-7 breast cancer cells

Acetylation of histone is a major player in epigenetic modifications, resulting in open chromatin structures and, hence, permissive conditions for transcription-factor recruitment to the promoters, followed by initiation of transcription. Histone deacetylase inhibitors arrest cancer cell growth and cause apoptosis with low toxicity thereby constituting a promising treatment for cancer. In this study, we examined the antiproliferative effects of valproate with a combination of nicotinamide in the MCF-7 cell line. MCF-7 was treated with various concentrations of valproate. The MTT assay showed that the viability of MCF-7 cells was inhibited and the cell activity was decreased. Viability percent of valproate and nicotinamide combined treatment cells (28 ± 2) was 1.78 times increased compared with the valproate-alone (0.5mM) treated cells (50 ± 2). Colony formation in soft agar indicated that valproate at 0.3mM, when used alone, weakly suppressed proliferation of cells (82 ± 3) and the combination treatment of valproate + nicotinamide strongly suppressed cell proliferation (51 ± 3). The flow cytometric and microscopic analyses of HDACI combined with treated cells indicated strong apoptosis induction and nuclear morphological alterations greater than those of valproate alone. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed the efficiency of the HDAC inhibitor combination, revealing the effectively upregulated p16 and p21. Furthermore, to investigate the role of acetyl-histone H3 levels, western blot analyses have been performed and high levels of acetylated histone H3 were detected in valproate- and nicotinamide-treated cells. These results suggest that the combination treatment of valproate with nicotinamide exerts significant antitumor activity and could be a promising therapeutic candidate to treat human breast cancer.

Effect of morphological preservation artifacts on the quality of immunohistochemical staining for ER and Her-2

Introduction: The most commonly encountered morphological preservation artifacts in the histological assessment of breast cancer include retraction of tumour cell clusters from the stroma, cytoplasmic retraction and alterations in nuclear morphology. This study intended to assess how these artifacts affect the quality of immunohistochemical (IHC) staining for ER and Her-2. Methods: For this retrospective study, 120 cases of consecutive breast cancers reported by the 2nd and 3rd authors during 2009 to 2010 were included. Haemotoxylin and Eosin stained sections of each breast cancer were assessed to obtain a consensus score for the degree of morphological preservation artifacts present. For each artifact (Retraction of tumour cell clusters, cytoplasmic retraction and alterations in nuclear morphology) a consensus score was given (0, 1, 2, 3) depending on the percentage of cell clusters/cells affected. Each IHC slide was assessed to obtain a consensus score for the quality of IHC staining for ER and Her-2. Parameters measured were uniformity of staining (0, 1), background staining (0, 1, 2), and adequacy of counterstaining (0, 1). Results: The study included a total of 120 consecutive breast cancer cases. There was no statistically significant relationship between the consensus score for the commonly encountered morphological preservation artifacts and the quality of IHC staining of ER (p = 0.44) and Her-2 (p = 0.51). Conclusions: Commonly encountered morphological preservation artifacts have no significant effect on the quality of IHC staining for ER and Her-2 measured in terms of uniformity of staining, background staining and adequacy of counterstaining. Galle Medical Journal, Vol 18: No. 2, September 2013;Page 8-12 DOI: http://dx.doi.org/10.4038/gmj.v18i2.6193

Promising Role of Exfoliative Cytology in the Evaluation of Glycaemic Status of Type II Diabetics: A Pilot Study.

The degree of metabolic control in diabetes mellitus influences the susceptibility of patients to oral diseases. It is mandatory to regularly monitor glycaemic status, however invasive methods may be contraindicated or intolerable to diabetic individuals. Thus, cytology, being a simple, non-invasive and rapid procedure, is a promising protocol for assessing diabetic status and assisting in management. To assess the number of PAS positive glycogen containing cells and associated cellular changes in buccal smears of type II diabetics and correlate the findings with their serum glucose levels. The study was conducted at the out patient Department of Oral and Maxillofacial Pathology, KLES Institute of Dental Sciences, Belgaum. Fifty known cases of type II diabetes mellitus and 50 healthy individuals were included in the study. Fasting blood glucose levels were estimated and buccal smears stained with Periodic Acid Schiff stain. The observed cellular changes were correlated with the glycaemic status of each patient. Statistical evaluations such as Student's t test (P<0.01-very significant; P<0.001-highly significant), correlation-coefficient and probability values were computed. Smears of diabetic patients depicted an increase in the number of PAS positive cells in significant correlation to their glycaemic status. Cellular and nuclear morphological alterations were also found in squames of diabetic individuals. Cytological findings and clinical observations, suggest a correlation between the extent of these changes and clinical parameters like glycaemic control. Further studies in this aspect can help in improving the reliability of oral cytology as a diagnostic tool in diabetes.

Lamin A/C Depletion Enhances DNA Damage-Induced Stalled Replication Fork Arrest

The human LMNA gene encodes the essential nuclear envelope proteins lamin A and C (lamin A/C). Mutations in LMNA result in altered nuclear morphology, but how this impacts the mechanisms that maintain genomic stability is unclear. Here, we report that lamin A/C-deficient cells have a normal response to ionizing radiation but are sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with ICL agents (cisplatin, camptothecin, and mitomycin), lamin A/C-deficient cells displayed normal γ-H2AX focus formation but a higher frequency of cells with delayed γ-H2AX removal, decreased recruitment of the FANCD2 repair factor, and a higher frequency of chromosome aberrations. Similarly, following hydroxyurea-induced replication stress, lamin A/C-deficient cells had an increased frequency of cells with delayed disappearance of γ-H2AX foci and defective repair factor recruitment (Mre11, CtIP, Rad51, RPA, and FANCD2). Replicative stress also resulted in a higher frequency of chromosomal aberrations as well as defective replication restart. Taken together, the data can be interpreted to suggest that lamin A/C has a role in the restart of stalled replication forks, a prerequisite for initiation of DNA damage repair by the homologous recombination pathway, which is intact in lamin A/C-deficient cells. We propose that lamin A/C is required for maintaining genomic stability following replication fork stalling, induced by either ICL damage or replicative stress, in order to facilitate fork regression prior to DNA damage repair.

Enhanced levels of double-strand DNA break repair proteins protect ovarian cancer cells against genotoxic stress-induced apoptosis

BackgroundEarlier, proteomic profiling of a Serous Ovarian Carcinoma (SeOvCa) progression model in our lab had identified significantly enriched expression of three double-strand break (DSB) -repair proteins viz. RAD50, NPM1, and XRCC5 in transformed cells over pre-transformed, non-tumorigenic cells. Analysis of the functional relevance of enhanced levels of these proteins was explored in transformed ovarian cancer cells.MethodsExpression profiling, validation and quantitation of the DSB-repair proteins at the transcriptional and protein levels were carried out. Further analyses included identification of their localization, distribution and modulation on exposure to Estradiol (E2) and cisplatin. Effects on silencing of each of these under conditions of genomic-stress were studied with respect to apoptosis, alterations in nuclear morphology and DNA fragmentation; besides profiling known mitotic and spindle check-point markers in DSB-repair.ResultsWe identified that levels of these DSB-repair proteins were elevated not only in our model, but generally in cancer and are specifically triggered in response to genotoxic stress. Silencing of their expression led to aberrant DSB repair and consequently, p53/p21 mediated apoptosis. Further compromised functionality generated genomic instability.ConclusionsPresent study elucidates a functional relevance of NPM1, RAD50 and XRCC5 DSB-repair proteins towards ensuring survival and evasion of apoptosis during ovarian transformation, emphasizing their contribution and association with disease progression in high-grade SeOvCa.

Nuclear Morphometric Analysis of Leydig Cells of Male Pubertal Rats Exposed In Utero to Di(n-butyl) Phthalate.

We recently reported that prenatal rat exposure to di(n-butyl) phthalate (DBP) induced Leydig cell (LC) hyperplasia after nine weeks (wks) of age, yet the number of LCs was similar to that of the vehicle group until seven weeks. Nuclear pleomorphism of hyperplastic LCs is common and is considered to be continuous progressive degeneration. Thus, computer-assisted image cell nuclear analysis of LCs was performed on 5- and 7-wk-old Sprague-Dawley (SD) rats whose dams had been administered DBP (i.g.) at 100 mg/kg/day or vehicle (corn oil) on gestation day 12 to 21. The results of the 5-wk-old DBP group were similar to those of the vehicle group; LC nuclei of the 7-wk-old DBP group showed normal ploidy and similar amounts of DNA. However, the size, elongation and peripheral chromatin aggregation parameters were significantly higher, and the reticular chromatin distribution and isolated chromatin aggregation parameters were significantly lower compared with the vehicle group. The present study quantitatively demonstrated nuclear morphological alterations in rat LCs at 7 wks old (puberty) due to the prenatal DBP administration before apparent LC hyperplasia developed.

Inducción de micronúcleos y otras anormalidades nucleares en Astyanax gr. bimaculatus (Pisces: Characidae) expuestas a fenantreno

Título en ingles: Induction of micronuclei and other nuclear abnormalities in Astyanax gr. bimaculatus (Pisces: Characidae) exposed to phenanthreneTítulo en portugués: Indução de micronúcleos e de outras anormalidades nucleares em Astyanax gr. bimaculatus (Pisces: Characidae) expostos a fenantreno Resumen: El fenantreno es un hidrocarburo policíclico aromático (HPA) asociado a la actividad petrolera, altamente lipofílico, con amplia distribución y con la propiedad de persistir en el ambiente por tiempos prolongados. A pesar de su impacto, el efecto de los HPA ha sido poco estudiado en peces tropicales; por tanto, el objetivo de este estudio fue determinar la presencia de micronúcleos y otras anormalidades nucleares en eritrocitos de sangre periférica de Astyanax gr. bimaculatus expuestas a fenantreno. Los peces (n=9) fueron inyectados intraperitonealmente con 0.1, 1 y 10 µg/g de PV de fenantreno (PHE, Sigma Aldrich, pureza 98%) disuelto en aceite vegetal, un control positivo inyectados con ciclofosfamida y un control negativo con aceite vegetal. Se colectaron muestras de sangre periférica a las 0 y 96 horas de la inyección. A las 96 horas, las dosis más altas de fenantreno mostraron efectos genotóxicos sobre los eritrocitos de Astyanax gr. bimaculatus, evidenciados por la formación de micronúcleos y alteraciones morfológicas nucleares. Estos resultados sugieren que Astyanax gr. bimaculatus, especie íctica dulceacuícola nativa, tiene gran potencial para ser utilizada como bioindicador de genotoxicidad causada por hidrocarburos policíclicos aromáticos.Palabras clave: Aberración cromosómica; bioensayo; genotóxico; hidrocarburos aromáticos policíclicos; toxicidad.Abstract: Phenanthrene is a polycyclic aromatic hydrocarbon (PAH) associated with the oil industry, highly lipophilic, ubiquitous and with the property to persist in the environment for longer periods. Despite its impact, few studies has been carry out in tropical fish evaluating the PAH effect, therefore, the aim of this study was to determine the presence of micronucleus and other nuclear abnormalities in peripheral blood erythrocytes in Astyanax gr. bimaculatus exposed to phenantrene. Fish (n=9) were injected intraperitoneally with 0.1, 1 and 10 µg/g BW of phenantrene solution. Cicliphosphamide and vegetable oil were used as a positive and negative control, respectively. Peripheral blood samples were collected at 0 and 96 hours. At 96 h exposure, the two higher phenantrene concentrations showed genotoxic effect on Astyanax gr. bimaculatus erythrocytes caused by the micronucleus induction and other nuclear morphological alterations. These results suggest that Astyanax gr. bimaculatus, a native freshwater fish, has a great potential as a bioindicator of polycyclic aromatic hydrocarbons genotoxicity.Key words: Chromosomal aberration; bioassays; genotoxic; polycyclic aromatic hydrocarbons; toxicity.Resumo: Fenantreno é um hidrocarboneto aromático policíclico (HAP) associado com a indústria do petróleo, altamente lipofílico, com ampla distribuição e propriedade de persistirem no ambiente por longos períodos. No entanto seu impacto, o efeito dos HPA há sido pouco estudado em peixes tropicais, por tanto, o objetivo deste estudo foi determinar a presença dos micronúcleos e outras anormalidades nucleares em eritrócitos de sangue periférico do Astyanax gr. Bimaculatus expostas a fenantreno. Os peixes (n = 9) foram injetados intraperitonealmente com 0.1, 1 e 10 µg/g de fenantreno (PHE, Sigma Aldrich, 98% de pureza), dissolvido em óleo vegetal, um controle positivo injetado com ciclofosfamida e um controlo negativo, injetado com o óleo vegetal. Amostras de sangue periférico foram coletadas as 0 e 96 horas de exposição. Às 96 horas, as doses mais altas de fenantreno mostraram efeitos genotóxicos sobre os eritrócitos de Astyanax gr. bimaculatus, evidenciados por a formação de micronúcleos e outras alterações morfológicas nucleares. Baseados nos resultados obtidos pode-se inferir que Astyanax gr. bimaculatus, peixe nativo de água doce, tem grande potencial para ser utilizado como um bioindicador de genotoxicidade, causada por hidrocarbonetos aromáticos policíclicos.Palavras chave: Aberração cromossômica; hidrocarbonetos aromáticos policíclicos; genotóxicos; toxicidade.

A filtering strategy identifies FOXQ1 as a potential effector of lamin A dysfunction.

Small increases in the expression of wild-type prelamin A are sufficient to recapitulate the reduced cell proliferation and altered nuclear membrane morphology observed in cells expressing progerin, the mutant lamin A associated with progeria. We hypothesized that the manifestation of these phenotypes in cells expressing elevated levels of wild-type prelamin A or progerin is caused by the same molecular effectors, which play a central role in the onset of the progeroid phenotype. To experimentally test this hypothesis, we compared the transcriptomes of isogenic diploid fibroblasts expressing progerin or elevated levels of wild-type prelamin A with that of wild-type fibroblasts. We subsequently used the reversion towards normal of two phenotypes, reduced cell growth and dismorphic nuclei, by treatment with farnesyltransferase inhibitor (FTI) or overexpression of ZMPSTE24, as a filtering strategy to identify genes linked to the onset of these two phenotypes. Through this analysis we identified the gene encoding for the transcription factor FOXQ1, as a gene whose expression is induced in both cells expressing progerin and elevated levels of wild-type prelamin A, and subsequently reduced in both cell types upon conditions that ameliorate the phenotypes. We overexpressed FOXQ1 in normal fibroblasts and demonstrated that increased levels of this factor lead to the development of both features that were used in the filtering strategy. These findings suggest a potential link between this transcription factor and cell dysfunction induced by altered prelamin A metabolism.

Astrocyte Activation: A Key Step in Rotenone Induced Cytotoxicity and DNA Damage

Astrocytes are the most abundant glial cells, which provide metabolic support for neurons. Rotenone is a botanical pesticide of natural origin, known to exhibit neurotoxic potential via inhibition of mitochondrial complex-I. This study was carried out to explore the effect of rotenone on C6 cells. The cell line C6 derived from rat glioma cells represents astrocyte-like cell. C6 cells were treated with rotenone (0.1, 1 and 10 μM) for 4 h. The effect of rotenone was studied on cell survival (MTT reduction and PI uptake); free radicals (ROS and RNS) and DNA damage (comet assay and Hoechst staining). The glial cell activation and apoptotic cell death was evaluated by expression of Glial fibrillary acidic protein (GFAP) and caspase-3 respectively. The treatment with rotenone resulted in decreased cell survival and increased free radical generation. Altered nuclear morphology and DNA damage were evident following rotenone treatment in Hoechst staining and Comet assay. Rotenone elevated expression of GFAP and caspase-3 that indicates glial cell activation and apoptosis, respectively. We further studied the effect of melatonin, an antioxidant, on the observed toxic effects. Co-incubation of antioxidant, melatonin (300 μM), significantly suppressed rotenone induced above-mentioned effects in C6 cells. Inhibitory effects of melatonin suggest that free radicals play a major role in rotenone induced astrocyte activation and cellular toxicity leading to apoptosis of astroglial cells.