Decrease In DNA Content Research Articles

Biochemical disorders induced by cytotoxic marine natural products in breast cancer cells as revealed by proton NMR spectroscopy-based metabolomics

Marine plants and animals are sources of a huge number of pharmacologically active compounds, some of which exhibit antineoplastic activity of clinical relevance. However the mechanism of action of marine natural products (MNPs) is poorly understood. In this study, proton NMR spectroscopy-based metabolomics was applied to unravel biochemical disorders induced in human MCF7 breast cancer cells by 3 lead candidate anticancer MNPs: ascididemin (Asc), lamellarin-D (Lam-D), and kahalalide F (KF). Asc, Lam-D, and KF provoked a severe decrease in DNA content in MCF7 cells after 24-h treatment. Asc and Lam-D provoked apoptosis, whereas KF induced non-apoptotic cell death. Metabolite profiling revealed major biochemical disorders following treatment. The response of MCF7 tumor cells to Asc involved the accumulation of citrate (×17 the control level, P < 0.001), testifying enzyme blockade in citrate metabolism, and the accumulation of gluconate (×9.8, P < 0.005), a metabolite never reported at such concentration in tumor cells, probably testifying glycolysis shutdown. The response to Lam-D involved the accumulation of aspartate (×7.2, P < 0.05), glutamate (×14.7, P < 0.05), and lactate (×2.3, P < 0.05), probably in relation with the targeting of the malate–aspartate shuttle, as discussed. The response to KF involved increased lipid accumulation (polyunsaturated fatty acids ×9.8, P < 0.05), and phospholipid and acetate derivative alterations. Altogether, this study demonstrates the potential of proton NMR spectroscopy-based metabolomics to help uncover metabolic targets and elucidate the mechanism of cytotoxicity of candidate antineoplastic MNPs.

Does prolactin induce apoptosis? Evidences in a prostate cancer in vitro model

Prolactin (PRL) regulates prostate growth and differentiation. Some studies have suggested that PRL has a pro-apoptotic effect on a myeloma cell line and in newt spermatogonia. The proliferative effect of PRL on prostate cancer cell lines is, however, a controversial area. On this account, we evaluated the effects of PRL on the prostate cancer cell lines LNCaP and PC3 apoptosis and proliferation. LNCaP and PC3 cells were exposed to increasing concentrations of PRL for 24, 48, 72 and 96 hours. Staining with propidium iodide (PI) and TUNEL assay followed by flow cytometry were used to detect apoptosis. LNCaP and PC3 proliferation was assessed by optical microscopy counting. PRL induced a dose-dependent decrease of DNA content and an increase of DNA fragmentation in LNCaP after 96 hours of incubation. These effects were observed with physiological concentrations of PRL and were counteracted by a prolactin receptor antagonist. On the other hand, PRL did not have any effect on DNA content or fragmentation in PC3 cells. No effect of PRL on LNCaP and PC3 proliferation was found. This study indicates that PRL induces apoptosis in the androgen-responsive cell line LNCaP, whereas no effect was observed in the androgen-insensitive PC3 cell line. These findings suggest that androgen responsiveness may be required for PRL to be effective on prostatic cells.

Developing an Articular Cartilage Decellularization Process Toward Facet Joint Cartilage Replacement

The facet joint has been identified as a significant source of morbidity in lower back pain. In general, treatments have focused on reducing the pain associated with facet joint osteoarthritis, and no treatments have targeted the development of a replacement tissue for arthritic facet articular cartilage. Therefore, the objective of this study was to develop a nonimmunogenic decellularized articular cartilage replacement tissue while maintaining functional properties similar to native facet cartilage tissue. In vitro testing was performed on bovine articular cartilage explants. The effects of 2% sodium dodecyl sulfate (SDS), a detergent used for cell and nuclear membrane solubilization, on cartilage cellularity, biochemical, and biomechanical properties, were examined. Compressive biomechanical properties were determined using creep indentation, and the tensile biomechanical properties were obtained with uniaxial tensile testing. Biochemical assessment involved determination of the DNA content, glycosaminoglycan (GAG) content, and collagen content. Histological examination included hematoxylin and eosin staining for tissue cellularity, as well as staining for collagen and GAG. Treatment with 2% SDS for 2 hours maintained the compressive and tensile biomechanical properties, as well as the GAG and collagen content while resulting in a decrease in cell nuclei and a 4% decrease in DNA content. Additionally, treatment for 8 hours resulted in complete histological decellularization and a 40% decrease in DNA content while maintaining collagen content and tensile properties. However, a significant decrease in compressive properties and GAG content was observed. Similar results were observed with 4 hours of treatment, although the decrease in DNA content was not as great as with 8 hours of treatment. Treatment with 2% SDS for 8 hours resulted in complete histological decellularization with decreased mechanical properties, whereas treatment for 2 hours maintained mechanical properties, but had a minimal effect on DNA content. Therefore, future studies must be performed to optimize a treatment for decellularization while maintaining mechanical properties close to those of facet joint cartilage. This study served as a step in creating a decellularized articular cartilage replacement tissue that could be used as a treatment for facet cartilage osteoarthritis.

Glycine decreases developmental damage induced by hyperglycaemia in mouse embryos

Hyperglycaemia induces neural tube defects and growth retardation in cultured mouse and rat embryos. In this study the possibility that glycine could prevent hyperglycaemia-induced embryopathy was researched. Early somite mouse embryos were cultured in normal medium, hyperglycaemic medium (50 mmol L(-1) glucose), or with glycine (1 mmol L(-1)) supplementation of normal and hyperglycaemic rat serum for 48 h. The embryo growth and differentiation were determined to estimate developmental and congenital malformations as well as lipid peroxidation levels. Adding glycine to the control culture medium did not affect embryonic development. Whereas the amino acid protected against telencephalon dysmorphogenesis, the decreased DNA content and number of somites, and the morphological score affectation induced by the hyperglycaemic medium, it had no preventive effect on the retarded differentiation of the otic system. Moreover, it prevented the high hyperglycaemia-induced lipoperoxidation levels of embryonic tissues. Embryos were partially protected from the hyperglycaemia-induced teratogenesis due to the antioxidative effect of glycine. As no other mechanisms related to the antiglycation or other protective effects of glycine were examined, the mechanism whereby it acted as an antiteratogenic agent needs further study.

Pharmacometabolomics of docetaxel-treated human MCF7 breast cancer cells provides evidence of varying cellular responses at high and low doses

There is growing evidence that docetaxel, a microtubule-targeting agent like the other taxane paclitaxel, induces dual cytotoxicity mechanism according to dose level. Postgenomics screening technologies are now more and more applied to the elucidation of drug response mechanisms. Proton nuclear magnetic resonance spectroscopy-based pharmacometabolomics was here applied to get further insight into the response of human MCF7 breast carcinoma cells to docetaxel at high (clinical, 5 microM) and low (1 nM) doses. The global response to both doses was evaluated by nuclear morphology and DNA content, the latter as an index of cell proliferation and DNA ploidy. High dose provoked long-lasting cell cycle arrest in mitosis during the first 48 h of exposure to treatment and severe decrease in DNA content followed by significant amount of cell death. In contrast, at low dose, no long-lasting cell cycle arrest was observed on micrographies, and DNA content was decreased but less than at high dose (P < 0.05), without significant cell death. This response was compared to biochemical alteration assessed by pharmacometabolomics. Thirty metabolites were identified and quantified. Metabolite profiling at clinical dose revealed time-dependent disorders in derivatives of glycolysis, lipid metabolism and glutathione metabolism. Comparison between high and low doses was performed at 72 h and showed common traits including the accumulation of cytidinediphosphocholine (x 5.0 and x 6.9, respectively, P < 0.03), the decrease in phosphatidylcholine (x 0.3 and x 0.2, respectively, P < 0.03), and gluthathione (x 0.6 and x 0.6, respectively, P < 0.03). Despite that, significant dose-dependent differences were found in 12 of 30 measured metabolites. Among them, the most discriminant metabolites were polyunsaturated fatty acids (ratio of high-to-low dose of 14.8, P < 0.05), glutamate, myoinositol, and homocysteine (ratio < 0.4, P < 0.05). In addition, the mechanism for glutathione decrease was different. At high dose, it resulted from extensive consumption with precursor starvation (glutamate: -89%, P < 0.05) and increased glutathione S-transferase activity (x 5, P < 0.01), whereas at low dose, it resulted from glutathione biosynthesis blockade with homocysteine accumulation (+144%, P < 0.03) and decreased glutathione S-transferase activity (-70%, P < 0.01). Altogether, this pharmacometabolomics analysis provides further evidence of the varying cellular responses at high and low doses of docetaxel in MCF7 breast cancer cells.

Newcastle disease virus-induced cytopathic effect in infected cells is caused by apoptosis

The velogenic Newcastle disease virus (NDV) causes highly infectious and economically significant Newcastle disease (ND) in birds of various species. In cell culture NDV induces cytopathic effect (CPE) characterized by rounding, vacuolation, syncytia formation and cell death. Aside from cell to cell fusion caused by the F and HN glycoprotein of the virus molecular events leading to cell death are not known. In the current study, NDV-infected Vero cells, at 48 h p.i., showed nuclear condensation, cytoplasm blebbing, DNA fragmentation, and phosphatidylserine translocation to the cell surface. In addition, virus-infected cells demonstrated decreased DNA content and an increased Bax to Bcl-2 ratio, p53 level and caspase 3, 8, 9 expression compared to mock-infected cells. Based on these results, it was concluded that CPE in NDV-infected cells was caused by to the induction of apoptosis with the involvement of p53 and the Bax, dependent apoptotic pathways.

P.174 Conservative-surgical treatment of parodontitis

Augmentation of the craniofacial region is necessary for many aesthetic and reconstructive procedures. Tissue engineering offers a new option to supplement existing treatment regimens. In this procedure, materials composed of hydroxyapatite (HA), of synthetic or natural origin, are used as scaffolds. The aim of this study was to evaluate the effects of three HA materials on cultured human osteoblasts in vitro.Explant cultures of cells from human alveolar bone were established. Human osteoblasts were cultured on the surface of HA calcified from red algae (C GRAFT™/Algipore®), deproteinized bovine HA (Bio-Oss®) and bovine HA carrying the cell binding peptide P-15 (Pep Gen P-15™). Cultured cells were evaluated with respect to cell attachment, proliferation and differentiation. Cells were cultured for 6 and 21 days under osteogenic differentiation conditions, and tissue-culture polystyrene dishes were used as control. The ability of cells to proliferate and form extracellular matrix on these scaffolds was assessed by a DNA quantification assay, protein synthesis analysis and by scanning electron microscopical examination. Osteogenic differentiation was screened by the expression of alkaline phosphatase. The osteoblastic phenotype of the cells was monitored using mRNA levels of the bone-related proteins including osteocalcin, osteopontin and collagen Type I.We found that cells cultured on C GRAFT™/Algipore® and Pep Gen P-15™ showed a continuous increase in DNA content and protein synthesis. Cells cultured on Bio-Oss®showed a decrease in DNA content from Day 6 (P < 0.05) to Day 21 (P < 0.0001) and protein synthesis on Day 21 (P < 0.005). Alkaline phosphatase activity increased in cells grown on C GRAFT™/Algipore® and Pep Gen P-15™ in contrast to cells grown on Bio-Oss®, in which the lowest levels of activity could be observed on Day 21 (P < 0.05). Reverse transcriptase polymerase chain reaction analysis confirmed the osteoblastic phenotype of the cells grown on all three materials throughout the whole culture period.The results of our in vitro study show that the differences in metabolic activity of cells grown on HA materials are directly related to the substrate on which they are grown. They confirm the excellent properties of HA carrying the cell binding peptide P-15 and HA calcified from red algae as used in maxillofacial surgery procedures.

Induction of apoptosis in Vero cells by Newcastle disease virus requires viral replication, de-novo protein synthesis and caspase activation

Newcastle disease virus causes (NDV) apoptotic death of infected cells. In the present study, the stimulus that provoked the induction of apoptosis in infected cells was examined. Vero cells infected with NDV developed apoptosis as characterized by DNA fragmentation and decreased DNA content. In presence of ammonium chloride, infected cells did not show reduced DNA content indicating the requirement of virus entry for the induction of apoptosis. UV-inactivated NDV did not induce apoptosis in cells suggesting the need of virus replication. Although cycloheximide blocked NDV-induced apoptosis, actinomycin-D did not, suggesting that de-novo viral protein synthesis was critical for the induction of apoptosis. In addition, activation of caspases was also detected by flowcytometry, indirect fluorescent and colorimetric assays. Based on the results, it was concluded that NDV-induced apoptosis in Vero cells required virus replication, de-novo protein synthesis and caspase activation.

Alcohol exposure alters cell cycle and apoptotic events during early neurulation

Fetal alcohol exposure causes growth deficits, microencephaly, and neurological abnormalities. Although the effects of alcohol on developmental delay and growth-related deficits have been hypothesized, little is understood about how alcohol alters, in particular, the cyclin pathway within the cell cycle, which is critical to proliferation and apoptotic control. In this study, we examined cell cycle proteins pertinent to the G1-S phase transition and apoptosis, to determine if cell cycle misregulation can be attributed to apoptotic induction and growth defects. We examined cell cycle regulation during G1 and S-phase, and DNA fragmentation damage, using E14 dorsal root ganglia neural stem cells (DRG-NC), and cultured mouse embryos exposed to 200 and 400 mg/dl ethanol. Alcohol-exposed DRG-NC demonstrated a dose-dependent increase in cells expressing increased cyclin D1 protein, and increased DNA fragmentation. Western blot analysis, using embryos, demonstrated an overexpression of cyclin D1, D2, and E2F1, key G1 to S-phase cell cycle regulatory components, and increases in p53, linking the cell cycle and apoptotic pathways. Bromodeoxyuridine incorporation indicated reduced DNA synthesis and growth in several embryonic regions. Propidium iodide staining demonstrated decreases in DNA content and increases in DNA fragmentation in several embryonic tissues. This study indicated that retarded growth of DRG-NC and embryos, induced by alcohol, is associated with altered expression of cell cycle and apoptotic proteins and concurrent inhibition of proliferation and increased DNA fragmentation. We suggest that alcohol induces an increase in cyclin D1 expression, premature S-phase entry, and disjointed DNA synthesis with increased apoptosis.

Skeletal muscle is enriched in hematopoietic stem cells and not inflammatory cells in cachectic mice

Objective: Cachexia, a debilitating syndrome characterized by skeletal muscle wasting, is associated to many chronic diseases and diminishes the quality of life and survival of patients. Tumor-derived factors and proinflammatory cytokines, including TNF-alpha, IL-6 and IL-1beta, mediate cachexia. In response to elevated cytokine levels, increased proteasome-mediated proteolysis and auto-phagocytosis result in muscle wasting. The histologic features of muscle cachexia are not fully elucidated. Therefore, we analysed alterations of different cell populations in cachectic muscle.Methods: By immunohistochemical and cytological approaches, we characterized changes in the abundance of cellular populations in the musculature of a murine model of cancer cachexia (C26-bearing mice).Results: Cachectic muscle displayed a decreased DNA content proportional to muscle mass wastage. A decrease in the number of nuclei occurred in the muscular but not in the stromal compartment. Cachectic muscle showed: mild modulation of myeloperoxidase activity, a neutrophil marker; reduction of macrophages in the endomysium; decrease in CD3+ lymphocyte number. Conversely, a statistically significant enrichment in Sca-1+ CD45+ hematopoietic stem cells (HSCs) occurred in cachectic muscle.Discussion: The elevated levels of cytokines which characterize cachexia may represent a trigger for inflammatory cell activation. However, we find that in cachexia, inflammatory cells in muscle are not increased while muscle tissue nuclei decline. Our data suggest that the inflammatory cell-mediated stress is not an etiologic component of muscle wasting in cachexia. The relative increase in HSCs in cachectic skeletal muscle suggests an attempt to maintain muscle homeostasis by recruitment and/or activation of stem cells.

HN protein of Newcastle disease virus causes apoptosis in chicken embryo fibroblast cells

Newcastle disease virus (NDV), an avian paramyxovirus, induces apoptosis in chicken embryo fibroblast (CEF) cells. In the present investigation, the ability of haemagglutinin-neuraminidase (HN) protein of NDV to cause apoptosis in CEF cells was examined. The results revealed that cells expressing the HN protein demonstrated decreased DNA content, phosphatidylserine exposure and increased cytoplasmic vacuolation. Up-regulation of caspase-1, -9, -8, -3, loss of mitochondrial transmembrane potential and an increase in oxidative stress were also observed in cells expressing the HN protein. Based on the above results it can be concluded that HN protein of NDV causes apoptosis in CEF cells.

The Influence of Temperature, Light and Cytokinins on DNA Content of Musa acuminata (AAA Group) 'Kluai hom thong' and Musa balbisiana (BBB Group) 'Kluai hin'

The changes in DNA content of triploid bananas Musa acuminata (AAA group) 'Kluai Hom Thong' and Musa balbisiana (BBB group) 'Kluai Hin' under different environmental conditions were investigated. The results revealed showed that different temperatures and light regimes had no effects on DNA content of both bananas cultivated on MS (Murashige and Skoog, 1962) medium supplemented with 5 mg/l BA (N6-benzyladenine) and 15% coconut water. However, they had some influences on morphological characteristics. On the other hand, both bananas cultivated on MS medium containing different concentrations of TDZ (N-phenyl-1, 2, 3 thidiazol-5-ylurea; thidiazuron) and BA, differed in their DNA contents. Increase in TDZ and BA concentrations resulted in the decrease of DNA content in 'Kluai Hom Thong', while the DNA content of 'Kluai Hin' increased.

Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry

The analysis of changes in mitochondrial membrane potential (MMP) that can occur during apoptosis provides precious information on the mechanisms and pathways of cell death. For many years, the metachromatic fluorochrome JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide) was used for this purpose. Thanks to new dyes and to the technical improvements recently adopted in several flow cytometers, it is now possible to investigate, along with MMP, a variety of other parameters. Using three sources of excitation and polychromatic flow cytometry, we have developed a protocol that can be applied to cells undergoing apoptosis. In the model of U937 cells incubated with the chemopreventive agent quercetin (3,3',4',5,7-pentahydroxyflavone), we describe the detection at the single cell level of changes in MMP (by JC-1), early apoptosis (exposition of phosphatidylserine on the plasma membrane detected by annexin-V), late apoptosis and secondary necrosis (decreased DNA content by Hoechst 33342 and permeability of the plasma membrane to propidium iodide). The procedure can be completed in less than 2 h.

Effect of aquatic extracts of oleander and Melia azedarach on the nucleic acid content of trichomonas vaginalis in vivo

This study was conducted to elucidate the effect of aqueous leaf extractof Nerium oleander and fruit extract of Melia azedarach on nucleic acidscontent and activity of some enzymes in Trichomonas vagina/is whichgrowth in Diamond's medium tryptone, yeast and maltose (TYM) media.Results indicated that the exposure of T. vagina/is to IC50concentration of N. oleander leaves and M azedarach fruits extractsshowed asignificant decrease in DNA content by 31.5% and 38.2%,respectively, while the RNA content decreased by 28.3% and 50.7%,respectively. Furthermore the totel.l. nucleic acids content decreased by27.9% and 48.1% , respectively, compared with the untreatedtrichomonads with plant extracts after 72 hours of growth.The results of this study also indicated that T. vagina/is treated withN. oleander leaves and M azedarach fruits extracts showed inhibed in theactivity of thymidine phosphorylase enzyme at rate 32.7% and 69.2%respectively compared with untreated trichomonads with plant extractsafter 72 hours of growth.From the results of this study, 1t is obvious that the above mentionedextracts possess an inhibitory effect on nucleic acids and on the activityof the enzyme Thymidine phosphorylase.

SpxB Is a Suicide Gene ofStreptococcus pneumoniaeand Confers a Selective Advantage in an In Vivo Competitive Colonization Model

The human bacterial pathogen Streptococcus pneumoniae dies spontaneously upon reaching stationary phase. The extent of S. pneumoniae death at stationary phase is unusual in bacteria and has been conventionally attributed to autolysis by the LytA amidase. In this study, we show that spontaneous pneumococcal death is due to hydrogen peroxide (H(2)O(2)), not LytA, and that the gene responsible for H(2)O(2) production (spxB) also confers a survival advantage in colonization. Survival of S. pneumoniae in stationary phase was significantly prolonged by eliminating H(2)O(2) in any of three ways: chemically by supplementing the media with catalase, metabolically by growing the bacteria under anaerobic conditions, or genetically by constructing DeltaspxB mutants that do not produce H(2)O(2). Likewise, addition of H(2)O(2) to exponentially growing S. pneumoniae resulted in a death rate similar to that of cells in stationary phase. While DeltalytA mutants did not lyse at stationary phase, they died at a rate similar to that of the wild-type strain. Furthermore, we show that the death process induced by H(2)O(2) has features of apoptosis, as evidenced by increased annexin V staining, decreased DNA content, and appearance as assessed by transmission electron microscopy. Finally, in an in vivo rat model of competitive colonization, the presence of spxB conferred a selective advantage over the DeltaspxB mutant, suggesting an explanation for the persistence of this gene. We conclude that a suicide gene of pneumococcus is spxB, which induces an apoptosis-like death in pneumococci and confers a selective advantage in nasopharyngeal cocolonization.

A naturally derived, cytocompatible, and architecturally optimized scaffold for tendon and ligament regeneration

Tissue-engineered tendon scaffolds have the potential to significantly improve the treatment of tendon and ligament injuries, especially those associated with tumors, trauma, and congenital deficiencies where autograft or allograft tissue might not be available in sufficient quantity for reconstruction. In this study, a tendon scaffold was produced that: (1) has decreased/absent cellular material histologically, as well as significantly decreased DNA content in comparison with the material it is derived from—fresh-frozen flexor digitorum profundus tendon; (2) is cytocompatible in vitro; (3) has been modified to produce increased pore size and porosity; (4) retains 76–78% of the tensile properties of the material it is derived from; (5) is readily infiltrated by fibroblast-like, mononuclear host cells; and (6) does not exhibit a host-cell-mediated foreign-body immune response after implantation in vivo.

Apoptosis of lens epithelial cell induced by curcumin and its mechanism

To investigate the effects of natural drug curcumin (Cur) on apoptosis of lens epithelial cell (LEC) in vitro and its mechanism. The bovine LEC were cultured with Cur, the ultrastructure changes were observed under transmission electron microscope (TEM), the DNA content and mitochondrial transmembrane potential (DeltaPsim) changes were studied by flow cytometry (FCM). The typical morphological changes of LEC apoptosis in Cur group detected by TEM included chromatin condensation and aggregation at the periphery of the nucleons and nuclear fragmentation. The DNA content of LEC in Cur group decreased time-dependently. The DNA content was significantly lower than that of the control group (P < 0.01). The DeltaPsim of LEC in Cur group was decreased, appeared in early stage (8 hours) and reached the maximum after 72 hours. The difference of DeltaPsim of LEC between Cur group and the control group was significant (P < 0.01). Cur can remarkably induce apoptosis of LEC in vitro. Cur induced LEC apoptosis is caused by decrease of DNA content in LEC nucleus. Collapse of DeltaPsim in cytoplasm induced by Cur results in the irreversible apoptosis process of LEC. This is the early event of LEC apoptosis. LEC apoptosis induced by Cur may pass through two pathways: nuclear pathway and cytoplasmic pathway. The apoptosis of LEC induced by Cur may be the cellular and molecular mechanisms of reducing lens posterior capsular opacification by Cur. Cur may become an effective and low toxic medication for the prevention and treatment of after-cataract.

Prolactin inhibits cell loss and decreases matrix metalloproteinase expression in the involuting mouse mammary gland but fails to prevent cell loss in the mammary glands of mice expressing IGFBP-5 as a mammary transgene

Insulin-like growth factor-binding protein 5 (IGFBP-5) mediates involution of the mammary gland. The decrease in DNA content and mammary gland weight which accompanies involution was inhibited by prolactin (PRL) in wild-type but not transgenic mice expressing IGFBP-5. Phospho-STAT5 protein levels were significantly lower in IGFBP-5 transgenic mice during lactation suggesting that IGFBP-5 antagonises PRL signalling in the mammary epithelium. In contrast, phospho-STAT3 levels increased during involution to a similar extent in both wild-type and transgenic mice and were unaffected by PRL. PRL inhibited gene expression of matrix metalloproteinases (MMPs) 3 and 12 but not tissue plasminogen activator or plasmin in wild-type and transgenic animals. The effects of PRL on MMPs appear to be indirect since PRL failed to inhibit MMP-3, -7 or -12 expression in HC-11 cells or in a co-transfection including an activated PRL receptor, STAT5 and a MMP-3-luciferase reporter gene. PRL is a potent inhibitor, both of cell death, an effect which is suppressed by IGFBP-5, and of MMP expression, which is independent of the actions of IGFBP-5.

Adipocyte Apoptosis After Burn Injury Is Associated With Altered Fat Metabolism

Burn injury often is associated with the abnormal lipid metabolism, including hyperlipidemia, desensitization to lipolytic responses to catecholamines, and reduction in the size of the white adipose tissue. Understanding the biological mechanisms for the decrease in fat mass despite desensitization to catecholamines is important both for the study of lipid metabolism and for the study of its relationship to concomitant insulin resistance. Using epididymal adipose tissue from adult male Sprague-Dawley rats after burn injury (n = 102) or sham-burn injury (n = 102), we tested the hypothesis that a whole-body burn injury causes apoptosis in that tissue. At 1, 3, and 7 days after 40% to 50% body burn injury to the rat, epidydimal adipose tissue was harvested and studied for apoptotic changes and lipolytic properties. For apoptosis, paraformaldehyde-fixed tissue sections were analyzed by in situ TdT-mediated dUTP-X nick-end labeling (TUNEL) staining, and tissue homogenates were also analyzed for DNA fragmentation by enzyme-linked immunoassay and ligation-mediated polymerase chain reaction ladder assay. Isolated adipocytes were stimulated with isoprotenerol, and glycerol production was measured as a reflector of effectiveness of lipolysis. Epididymal adipose tissue showed increased apoptosis manifested by the positive TUNEL staining and increased DNA fragmentation by enzyme-linked immunoassay at day 3 and 7 after burn injury. The DNA fragmentation was confirmed further by the ligation-mediated polymerase chain reaction ladder assay. This elevated DNA fragmentation persisted in the burned animals from day 3 until day 7 after burn injury, the end of observation period. Increase in apoptosis was correlated with decrease in DNA content and tissue weight in the epidydimis. At the functional level, a significant decrease in isoproterenol-induced lipolytic activity (glycerol production) was observed to almost 50% of control level at day 3 and 7 but was not decreased at day 1. Apoptosis of adipocytes may play a role in the altered lipid metabolism, including hyperlipidemia observed in burned subjects.

Nitric oxide and peroxynitrite induce cellular death in bovine chromaffin cells: Evidence for a mixed necrotic and apoptotic mechanism with caspases activation

Treatment of chromaffin cells with nitric oxide (NO) donors (SNP and SNAP) and peroxynitrite produces a time- and dose-dependent necrotic and apoptotic cell death. Necrotic cell death was characterized by both an increase in lactate dehydrogenase and ATP release and changes in nuclei and cell morphology (as seen with fluorescence microscopy analysis with propidium iodide and Hoechst 33342). Apoptotic cell death was characterized by nuclear fragmentation and presence of apoptotic cell bodies, by a decrease in DNA content, and by an increase in DNA fragmentation. Treatment of chromaffin cells with lipopolysaccharide (LPS) or cytokines (interferon-gamma, tumor necrosis factor-alpha) resulted only in apoptotic cell death. Apoptotic effects of NO-inducing compounds were specifically reversed, depending on the stimuli, by the NO scavenger carboxy-PTIO (CPTio) or by the NOS inhibitors L-NMA and thiocitrulline. NO-induced apoptotic death in chromaffin cells was concomitant to a cell cycle arrest in G0G1 phase and a decrease in the number of chromaffin cells in the G2M and S phases of cell cycle. All NO-producing compounds were able to induce activation of caspase 3 and cytochrome c release, and specific inhibitors of caspase 3 and 9, such as Ac-DEVD-CHO (CPP32) and Ac-Z-LEHD-FMK, respectively, prevented NO-induced apoptosis in chromaffin cells. These results suggest that chromaffin cells could be good models for investigating the molecular basis of degeneration in diseases showing death of catecholaminergic neurons, phenomenon in which NO plays an important role.