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PubMed2005

ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation.

Pollen exposure induces allergic airway inflammation in sensitized subjects. The role of antigenic pollen proteins in the induction of allergic airway inflammation is well characterized, but the contribution of other constituents in pollen grains to this process is unknown. Here we show that pollen grains and their extracts contain intrinsic NADPH oxidases. The pollen NADPH oxidases rapidly increased the levels of ROS in lung epithelium as well as the amount of oxidized glutathione (GSSG) and 4-hydroxynonenal (4-HNE) in airway-lining fluid. These oxidases, as well as products of oxidative stress (such as GSSG and 4-HNE) generated by these enzymes, induced neutrophil recruitment to the airways independent of the adaptive immune response. Removal of pollen NADPH oxidase activity from the challenge material reduced antigen-induced allergic airway inflammation, the number of mucin-containing cells in airway epithelium, and antigen-specific IgE levels in sensitized mice. Furthermore, challenge with Amb a 1, the major antigen in ragweed pollen extract that does not possess NADPH oxidase activity, induced low-grade allergic airway inflammation. Addition of GSSG or 4-HNE to Amb a 1 challenge material boosted allergic airway inflammation. We propose that oxidative stress generated by pollen NADPH oxidases (signal 1) augments allergic airway inflammation induced by pollen antigen (signal 2).

Aldehydes↗

PubMed2005

Lysosomal ROS formation.

Ubiquinone is inhomogenously distributed in subcellular biomembranes. Apart from mitochondria, where ubiquinone has bioenergetic and pathophysiological functions, unusually high levels of ubiquinone have also been reported in Golgi vesicles and lysosomes. In lysosomes, the interior differs from other organelles in its low pH value which is important to ensure optimal activity of hydrolytic enzymes. Since redox-cycling of ubiquinone is associated with the acceptance and release of protons, we assumed that ubiquinone is part of a redox chain contributing to unilateral proton distribution. A similar function of ubiquinone was earlier suggested by Crane to operate in Golgi vesicles. Support for the involvement of ubiquinone in a presumed couple of redox carriers came from our observation that almost 70% of total lysosomal ubiquinone was in the divalently reduced state. Further reduction was seen in the presence of external NADH. Analysis of the components involved in the transfer of reducing equivalents from cytosolic NADH to ubiquinone revealed the existence of an FAD-containing NADH dehydrogenase. The latter was found to reduce ubiquinone by means of a b-type cytochrome. Proton translocation into the interior was linked to the activity of the novel lysosomal redox chain. Oxygen was found to be the terminal electron acceptor, thereby also regulating acidification of the lysosomal matrix. In contrast to mitochondrial respiration, oxygen was only trivalently reduced giving rise to the release of HO radicals. The role of this novel proton-pumping redox chain and the significance of the associated ROS formation has to be elucidated.

Animals↗

PubMed1984

The effect of dexamethasone on parathyroid hormone stimulation of adenylate cyclase in ROS 17/2.8 cells.

Treatment of ROS 17/2.8 osteosarcoma-derived cells with dexamethasone potentiates the PTH stimulation of adenylate cyclase in these cells, yielding a detectable response to as little as 10 pM PTH. Isoproterenol stimulation was also enhanced. The dexamethasone effect is first apparent at 12 h and increases with time of treatment. The apparent EC50 for dexamethasone is 3 nM. Hydrocortisone and corticosterone act similarly to dexamethasone, but require 30-fold higher concentrations. Dexamethasone treatment produces no change in high affinity phosphodiesterase activity. Glucocorticoid-potentiating effects are much more pronounced in whole cells than in broken cells and do not influence forskolin stimulation. Particulate fractions of dexamethasone-treated cells have higher adenylate cyclase specific activity, but are stimulated by guanyl-5'-yl imidodiphosphate to the same extent as control cells. These findings suggest that the glucocorticoids potentiate hormone responsiveness through promotion of hormone receptor-adenylate cyclase coupling by a mechanism dependent on cellular integrity.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1989

Inhibitory effects of tumor necrosis factor-alpha and interferon-gamma on deoxyribonucleic acid and collagen synthesis by rat osteosarcoma cells (ROS 17/2.8).

Tumor necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) have potent effects on bone resorption and collagen synthesis in cultured rat long bones. Since the effects of TNF alpha and IFN gamma may result from interaction with multiple cell types, we studied the effects of these cytokines on the synthesis of DNA and collagen in one cell type with osteoblast phenotype, cloned rat osteosarcoma cells (ROS 17/2.8). Recombinant human TNF alpha did not affect DNA synthesis after 48 h with concentrations of 10(-11)-10(-8) M and inhibited DNA synthesis slightly at 10(-6) M. Recombinant rat IFN gamma (5-500 U/ml) caused a dose-dependent inhibition of DNA synthesis. Coincubation with TNF alpha and IFN gamma inhibited DNA synthesis more than maximal doses of either cytokine alone. This enhanced inhibitory effect was due to the induction of a response to TNF alpha by IFN gamma, since preexposure of cells to IFN gamma for 24 h, followed by incubation with TNF alpha alone for an additional 48 h, also resulted in increased inhibition of DNA synthesis. Preexposure to TNF alpha for 24 h, followed by IFN gamma alone, did not increase the inhibition of DNA synthesis. Incubation with either IFN gamma (5-500 U/ml) or TNF alpha (10(-10)-10(-6) M) inhibited the incorporation of [3H]proline into collagen. Coincubation with intermediate concentrations of both cytokines resulted in an inhibitory effect greater than that produced by maximal concentrations of either alone. The results indicate that 1) IFN gamma and TNF alpha have direct actions on osteoblast-like cells in vitro; 2) IFN gamma modulates the DNA response to TNF alpha; and 3) the greater responses to combined cytokines than to high doses of either alone suggest that these cytokines act, at least in part, through different pathways.

Animals↗

PubMed1990

Treatment of bone-derived ROS 17/2.8 cells with dexamethasone and pertussis toxin enables detection of partial agonist activity for parathyroid hormone antagonists.

In the design and biological evaluation of PTH antagonists, certain analogs, although antagonists in vitro, possess partial agonist properties in vivo that preclude their utility as antagonists. In an effort to identify weak agonism of PTH analogs, an attempt was made to enhance the responsiveness of the widely employed rat osteosarcoma (ROS 17/2.8) cell adenylate cyclase assay. Because responsiveness to PTH in these cells is enhanced upon treatment with dexamethasone (dex) or pertussis toxin (PT), we have evaluated their use to aid in detection of partial agonism for PTH and PTH-related protein (PTHrP) antagonist analogs. Treatment of cells with dex alone (30 nM for 3 days) or with PT alone (40 ng/ml for 1 day) increased basal adenylate cyclase activity by 27%. However, combination of the dex and PT treatments increased basal cAMP production 70%. The in vivo partial agonist [Nle8,18,Tyr34]bPTH(3-34)NH2 increased cAMP production 3-fold over basal levels in untreated cells, nearly 5-fold in PT-treated cells, 8-fold in cells treated with dex, and 10-fold in cells treated with dex plus PT. Similar results were obtained with PTHrP(7-34)NH2: the 6-fold stimulation observed in control cells was converted to 14-fold in cells treated with dex plus PT. Agonist activity undetectable in the conventional assay was observed in the dex plus PT system: [Tyr34]- and [D-Trp12,Tyr34]bPTH(7-34)NH2, which exhibit no agonist activity under control conditions, stimulated cAMP production 2.6- and 2.1-fold, respectively, under dex plus PT treatment. In contrast, the antagonist analogs [Asn10,Leu11]- and [Leu11,D-Trp12]PTHrP(7-34)NH2, hybrid peptides of PTH and PTHrP, had no agonist activity under any conditions. Because of increased responsiveness, this assay should occupy an important step in the pathway for evaluation of PTH antagonists and permit identification of weak partial agonist activity before extensive in vivo testing.

Adenylate Cyclase Toxin↗

PubMed1991

Tumor necrosis factor-alpha inhibits 1,25-dihydroxyvitamin D3-stimulated bone Gla protein synthesis in rat osteosarcoma cells (ROS 17/2.8) by a pretranslational mechanism.

Tumor necrosis factor-alpha (TNF alpha), a 17,000 mol wt protein, mediates a variety of immunological and inflammatory events. TNF alpha is a potent inhibitor of bone collagen synthesis and stimulator of osteoclastic bone resorption, the net effect of which is to cause bone loss. We have previously reported that TNF alpha inhibits the synthesis of collagen by osteoblastic cells in culture out of proportion to effects on total protein synthesis, suggesting that inhibition of bone formation by TNF alpha may be due to selective inhibition of matrix protein synthesis. To further test this hypothesis and to evaluate the mechanism of TNF alpha action, we studied the effect of TNF alpha on synthesis of the osteoblast-specific bone Gla protein (BGP) by ROS 17/2.8 cells, which have the osteoblast phenotype. Cells were cultured with 10 nM 1,25-dihydroxyvitamin D3 to stimulate BGP secretion, followed by the addition of TNF alpha (1-100 ng/ml) in 1,25-dihydroxyvitamin D3-containing medium. TNF alpha (10 ng/ml) inhibited BGP secretion to 42 +/- 5%, 19 +/- 10%, and 15 +/- 3% of control values after 24, 48, and 72 h of treatment. After 48 h, inhibition of BGP secretion was observed with 2 ng/ml TNF alpha and was maximum with 100 ng/ml. To determine the effect of TNF alpha on total protein synthesis, cells were pulse labeled with [14C]leucine during the last 4 h of TNF alpha treatment, and incorporation of radioactivity into trichloroacetic acid-precipitable protein in cell layer and medium was determined. The TNF alpha inhibition of BGP secretion was independent of changes in [14C]leucine incorporation, suggesting that TNF alpha did not have a general inhibitory effect on total protein synthesis. Cell number was not affected by TNF alpha. Northern analysis of steady state BGP mRNA revealed a dose-dependent decrease in the BGP/cyclophilin mRNA hybridization signal intensity after 24 h of treatment. The maximum inhibitory effect was 41 +/- 5% of the control value with 100 ng/ml TNF alpha. The effect of TNF alpha on steady state BGP mRNA levels was not prevented by treatment of cells with cycloheximide, suggesting that TNF-induced new protein synthesis was not required for TNF alpha action. These results suggest that the mechanism of TNF alpha inhibition of BGP synthesis includes a pretranslational site and support the hypothesis that TNF alpha inhibits bone formation by a selective inhibition of matrix protein production.

Animals↗

PubMed1992

Evidence that estrogens modulate activity and increase the number of 1,25-dihydroxyvitamin D receptors in osteoblast-like cells (ROS 17/2.8).

A detailed understanding of the mechanism of action of estrogen on bones is still lacking. The present study was designed to examine possible modulation by 17 beta-estradiol (E2) on the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and on vitamin D receptors (VDR) in the ROS 17/2.8 osteoblast-like cell line. Cells were grown in phenol-red free medium supplemented with charcoal-stripped fetal calf serum (FCS). Total cellular VDR were measured in cell homogenates after extraction with a KCl hypertonic buffer. VDR-binding capacity doubled in the presence of 10 nM E2 (16.2 +/- 2.3 vs. 7.0 +/- 1.3 fmol/mg protein, respectively; P less than 0.01), while the Kd for 1,25-(OH)2D3 did not change (approximately 0.1 nM). Tamoxifen alone had no effect on VDR, while it completely abolished the E2-induced increase in VDR, indicating that the effect was specific for E2 and estrogen receptor mediated. 1,25-(OH)2D3 inhibited cell proliferation, determined by [3H] thymidine incorporation to DNA, in a dose-dependent fashion between 0.01-100 nM. The inhibitory effect of 1,25-(OH)2D3 on cell proliferation was significantly augmented in the presence of E2 (10 nM). 1,25-(OH)2D3 increased osteocalcin secretion to the medium by the cells in a dose-dependent fashion between 0.01-100 nM. In the presence of E2 (10 nM), maximal osteocalcin secretion in response to 1,25-(OH)2D3 was 3.5-fold higher than that in response to 1,25-(OH)2D3 alone. These results indicate that E2 modulates 1,25-(OH)2D3 activity in osteoblast-like cells, and that this effect can be attributed to an increase in VDR.

Animals↗

PubMed1992

Estrogens modulate the responsiveness of osteoblast-like cells (ROS 17/2.8) stably transfected with estrogen receptor.

Recent studies have demonstrated the presence of estrogen receptor (ER) in both normal human osteoblast-like and osteoblast-like osteosarcoma cells. The number of ER in cultured osteoblastic cells is very low (200-500 sites/cell). This has complicated characterization of the biological role of estrogens in bone cells. To study the responsiveness of bone cells to estrogens, we established osteoblast-like cell lines expressing higher ER levels. ROS 17/2.8, an osteoblastic cell line, was stably transfected with the cDNA encoding for the mouse ER. After a selection period, positive clones were isolated and evaluated for the presence of ER by both Northern blot analysis and ligand binding assays. Using these techniques, we detected a significant increase in the level of both ER transcript and binding compared to that in wild-type cells. The levels of expressed ER protein were similar to those reported in normal human osteoblast-like cells in primary culture (approximately 2000 sites/cell). To test whether the exogenously inserted ER was responsive, both wild-type and ER stably transfected cells were transiently transfected with a reporter construct containing an estrogen-responsive element linked to a truncated thymidine kinase promoter and a chloramphenicol acetyltransferase (CAT) reporter gene. Exposure of the cells to increased concentrations of estradiol induced a slight increase in CAT activity in wild-type cells (approximately 1.5-fold) at maximal stimulation; however, it provoked a clear concentration-dependent increase in CAT activity in the ER stably transfected cells, with a maximal stimulation of approximately 10-fold. This event was receptor mediated, since ICI 164,384, an ER antagonist, blocked the enhancement of estradiol-induced CAT activity, and it was specific, since other steroid hormones did not stimulate CAT activity. Finally, we evaluated the ability of ER to modulate an endogenous estrogen-responsive gene by measuring the activity of the enzyme alkaline phosphatase. In addition, diethylstilbestrol, a synthetic estrogen agonist, increased the activity of both the CAT reporter gene and the endogenous alkaline phosphatase enzyme. In summary, we have established osteoblast-like cells expressing high levels of an exogenously inserted ER, which has characteristics similar to those of the endogenous ER in terms of its Kd. Finally, the exogenous ER regulates both exogenously inserted construct (VITERECAT) and endogenous properties of the cells (enzymatic activity and proliferation).

Alkaline Phosphatase↗

PubMed1994

Parathyroid-responsive modifications in the nuclear matrix of ROS 17/2.8 rat osteosarcoma cells.

PTH is a mediator of skeletal development and remodeling that influences gene expression in osteoblastic cells. It is well established that PTH modulates the activity of membrane-associated second messenger signal transduction pathways. In these studies we have addressed the potential contribution of components of cell structure to the integration of PTH-related regulatory signals that influence the expression of bone cell genes. Chronic treatment of ROS 17/2.8 rat osteosarcoma cells with PTH is accompanied by changes in gene expression that are at least in part transcriptionally controlled. To explore the involvement of nuclear architecture in PTH-responsive modifications in gene expression, we investigated changes in the nuclear matrix after PTH treatment. Consistent with a role for the nuclear matrix in determining spatial organization and topology of chromatin as well as in the localization and targeting of transcription factors, we observed PTH-associated changes in a 200-kilodalton nuclear matrix protein in response to PTH. A significant down-regulation of synthesis was observed when nuclear matrix proteins were resolved electrophoretically in two-dimensional gels. This protein was restricted to the nuclear matrix and was not detected in the chromatin or cytoskeletal cellular fractions. These alterations in nuclear matrix proteins that occur after PTH treatment in osteosarcoma cells were phenotype related. They did not occur in UMR-106 POL or H4 hepatoma cells. Our findings support a role for the nuclear matrix in transducing PTH-mediated regulatory signals to facilitate the extent to which genes in osteoblasts are transcribed.

Animals↗

PubMed2000

Culture in vector-averaged gravity under clinostat rotation results in apoptosis of osteoblastic ROS 17/2.8 cells.

Space flight experiments and studies carried out in altered gravity environments have revealed that exposure to altered gravity conditions results in (mal)adaptation of cellular function. In the present study, we used a clinostat to generate a vector-averaged gravity environment. We then evaluated the responses of osteoblast-like ROS 17/2.8 cells subsequent to rotation at 50 revolutions per minute (rpm) for 6-24 h. We found that the cells started to detach from the substrate between 12 h and 24 h of rotation in clinostat but not in stationary cultures or after horizontal rotation (the latter serving as a motion control for turbulence, shear forces, and vibrations). At 24 h, 35% of clinorotated cells had detached and the cells underwent apoptotic death as evidenced by DNA fragmentation analysis, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining, and flow cytometry with Annexin V staining. The apoptotic death was associated with perinuclear distribution of cell-surface integrin beta1 and disorganization of actin cytoskeleton. These results suggest that vector-averaged gravity causes apoptosis of osteoblasts by altering the organization of the cytoskeleton. We hypothesize that apoptotic death of osteoblasts might play an important role in the pathogenesis of osteoporotic bone loss as observed in actual space flights.

Actins↗

PubMedDate not supplied

Delayed reproductive death and ROS levels in the progeny of irradiated melanoma cells.

The cell death and survival of proliferating (clonogenic) cells were investigated in two human melanoma cell lines to assess the optimal conditions for preparation of apoptotic bodies from melanoma cells. After 50 J/m2 UVB+UVC the maximal levels of apoptotic cells assayed by Trypan blue staining, nucleosomal DNA fragmentation, MTT, and TUNEL tests were observed within 2-3 d of radiation. In 100 Gy gamma-irradiated cultures these apoptosis indicators were delayed for up to 3 weeks. In addition, clonogenic cells were observed only in exponentially growing cultures irradiated with UV at high cell density but not in gamma-irradiated cultures. The response of melanoma cultures after high UV radiation doses contrasted to the response in lethally gamma-irradiated cultures. UV-irradiated melanoma cultures were recovered within two weeks. Most of the clonogenic cells in the recovered colonies contained micronuclei. ROS levels determined by DCF fluorescence and a modified MTT test were also normalized obviously due to the extensive antioxidant defense system of melanoma cells. UV radiation of tumor cells might be the preferential method for preparation of apoptotic bodies. The presence of clonogenic cells in the suspension of apoptotic bodies from melanoma cells used for pulsing of dendritic cells with tumor antigens might compromise this protocol for preparation of cell vaccines.

Apoptosis↗

PubMed1993

Individual and combined effects of intact PTH, amino-terminal, and a series of truncated carboxyl-terminal PTH fragments on alkaline phosphatase activity in dexamethasone-treated rat osteoblastic osteosarcoma cells, ROS 17/2.8.

The individual and combined effects of intact PTH, amino-terminal, and a series of truncated carboxyl-terminal PTH fragments on alkaline phosphatase activity were examined in dexamethasone-treated rat osteoblastic osteosarcoma cells ROS 17/2.8. Dexamethasone-induced alkaline phosphatase activity was inhibited not only by hPTH(1-84) and amino-terminal PTH fragment hPTH(1-34), but also by carboxyl-terminal PTH fragment hPTH(69-84) in a dose-related fashion. At 10(-7) mol/l, hPTH(1-84) completely abolished dexamethasone-induced alkaline phosphatase activity, while hPTH(1-34) and hPTH(69-84) reduced alkaline phosphatase activity to 0.16 +/- 0.02 and 0.80 +/- 0.03 fold, respectively, of the control value obtained in the absence of PTH peptides. The combination of hPTH(1-34) and hPTH(69-84) resulted in reduction of alkaline phosphatase activity to the level obtained by hPTH(1-84). The shorter carboxyl-terminal PTH fragment hPTH(71-84) did not affect alkaline phosphatase activity or modulate the action of hPTH(1-34). The longer carboxyl-terminal PTH fragment hPTH(53-84) stimulated alkaline phosphatase activity up to 1.23 +/- 0.03 fold and partially blunted the inhibitory effect of hPTH(1-34) on alkaline phosphatase activity. These findings suggest that carboxyl-terminal PTH fragments could exert diverse effects on the target cells, depending on the length of deletion of amino-terminal amino acids of PTH molecule, and interact with amino-terminal PTH fragment. The two amino-terminal amino acids of hPTH(69-84) and the 53-68 portion of hPTH(53-84) might be responsible for the respective inhibitory and stimulatory effects of the peptides on alkaline phosphatase activity.

Alkaline Phosphatase↗

PubMed2005

Could MYC induction of mitochondrial biogenesis be linked to ROS production and genomic instability?

MYC has been linked separately to genomic instability and to reactive oxygen species production, but the connection between these two processes has not been firmly established. Our recent study along with previous reports demonstrate that Myc induces nuclear encoded mitochondrial gene expression and mitochondrial biogenesis, thereby directly associating Myc's transcriptional properties, to the production of mitochondrial ROS and the promotion of genomic oxidative damage and genomic instability.

Animals↗

PubMed2000

Extracellular presence of reactive oxygen species (ROS) in fresh and frozen-thawed canine semen and their effects on some semen parameters.

The objective of this study was to determine the presence of reactive oxygen species (ROS), especially superoxide anion (O2-), in fresh and frozen-thawed canine semen, its effect on other seminal parameters and whether the freezing-thawing process influences the O2- production rate. A total of 56 mature male domestic dogs were used in this study. Their semen was examined before and after freezing-thawing process. Sperm motility, hypo-osmotic swelling, sperm morphology and O2- extracellular production were measured. The dogs were divided in two groups, with the criterion of the absence or presence of O2- in fresh semen (groups A and B, respectively). Values of group A were compared with the respective values of group B. A significant decrease in motility and percentage of swollen spermatozoa and an increase in morphological abnormalities and O2- production in group B were found in comparison with group A after freezing-thawing process (p < 0.05). It seems that the freezing-thawing process leads to an increase of the O2- production. However, it remains to be documented if the increased concentrations of O2- in canine semen are responsible for the alterations in the seminal parameters observed in this study.

Animals↗

PubMedDate not supplied

Control of mitochondrial membrane potential and ROS formation by reversible phosphorylation of cytochrome c oxidase.

Phosphorylation of isolated cytochrome c oxidase from bovine kidney and heart, and of the reconstituted heart enzyme, with protein kinase A, cAMP and ATP turns on the allosteric ATP-inhibition at high ATP/ADP ratios. Also incubation of isolated bovine liver mitochondria only with cAMP andATP turns on, and subsequent incubation with Ca2+ turns off the allosteric ATP-inhibition of cytochrome c oxidase. In the bovine heart enzyme occur only three consensus sequences for cAMP-dependent phosphorylation (in subunits I, III and Vb). The evolutionary conservation of RRYS441 at the cytosolic side of subunit I, together with the above results, suggest that phosphorylation of Ser441 turns on the allosteric ATP-inhibition of cytochrome c oxidase. The results support the 'molecular-physiological hypothesis' [29], which proposes a low mitochondrial membrane potential through the allosteric ATP-inhibition. A hormone- or agonist-stimulated increase of cellular [Ca2+] is suggested to activate a mitochondrial protein phosphatase which dephosphorylates cytochrome c oxidase, turns off the allosteric ATP-inhibition and results in increase of mitochondrial membrane potential and ROS formation.

Allosteric Regulation↗

PubMed1999

Culture in vector-averaged gravity environment in a clinostat results in detachment of osteoblastic ROS 17/2.8 cells.

Studies carried out in space flights and in altered gravitational environments have shown that exposure to altered gravity conditions results in alterations in cellular structure and function. In the present study, we used a clinostat to generate a vector-averaged gravity environment, and evaluated the responses of osteoblast-like ROS 17/2.8 cells subsequent to rotation at 50 r.p.m from 24 to 72 hr. We found that the cells started to detach during the first 24 hr of culture in clinostat, but not in stationary and horizontal rotation (the latter serving as a control for turbulence, shear forces and vibrations). At 24 hr, there was a significant decrease in the number of adherent cells under clino-rotation (2.75 +/- 0.5 x 10(5) in stationary culture versus 2.02 +/- 0.27 x 10(5) under clino-rotation), and 19.8% of adherent cells were trypan-blue positive when cultured in 2% fetal bovine serum. All the detached cells were trypan-blue positive. At 72 hr, the cells became confluent in all three groups. These results suggest that vector-averaged gravity could cause the death of osteoblasts during the first 24 hr of clino-rotation. We hypothesize that this cell death might play a role in the pathogenesis of osteoporotic bone loss as observed in actual space flight.

Animals↗

PubMed2002

[In vitro study of the apoptosis effect of DNR on HL-60 cells and its relationship with ROS, CER and NF-kappaB].

OBJECTIVE: To study the effect of DNR on HL-60 cells apoptosis in vitro and the related mechanism. METHODS: The apoptosis of HL-60 was observed by microscope, flow cytometry (FCM) and DNA electrophoresis and various apoptosis-associated proteins expression by immunocytochemistry (IC) and FCM assays; the changes of apoptosis in HL-60 cells treated with DNR or suppressors PDTC or FB1 were also observed. RESULTS: When treated with 0.2 approximately 2.0 micro mol/L DNR, the percentage of apoptotic HL-60 cells increased with the dose increasing and the time extending, and the typical apoptotic cells and the appearance of apoptotic DNA ladder were observed. It was shown that after treatment with 1 micro mol/L DNR, the fluorescence intensity index (FI) of both bcl-2 and c-myc in HL-60 cells decreased, the FI of Bax, caspase-3 increased at 2 h, but decreased at 5 h, the FI of NF-kappaB increased. After adding PDTC, the apoptosis percentage of HL-60 cells decreased, but FB1 didn't present these effect. CONCLUSION: It suggested from the results that at certain concentration, DNR can induce the apoptosis of HL-60 cells in vitro. The mechanism was supposed by suppressing the expression of bcl-2 and c-myc and activating the expression of Bax and caspase-3, NF-kappaB and ROS had the marked correlation with the apoptosis process, but the ceramide synthase wasn't associated with it.

Apoptosis↗

PubMed2000

Rotation in clinostat results in apoptosis of osteoblastic ROS 17/2.8 cells.

Clinostat is an effective, ground-based tool which can be used to verify data from space flight, and to test hypotheses and experimental conditions for eventual space flights. Rotation in clinostat appears to mimic the microgravity environment by nulling the gravitational vector by continuous averaging. In the present study, we exposed osteoblast-like ROS 17/2.8 cells to a vector-averaged gravity environment in a clinostat and found that the cells undergo apoptotic death during the first 24 hr of clino-rotation. We suggest that apoptosis might be one of the mechanisms for reduced bone formation as observed in actual space flights.

Apoptosis↗