TY - JOUR
AU - Wang,, Chau-Jong
AB - Abstract Nitrogen oxides (NOx) are important indoor and outdoor air pollutants. Many studies have indicated that NOx gas causes lung tissue damage by its oxidation properties and its free radicals. In a previous study we demonstrated that NOx gas induced proliferation of human lung fibroblast MRC-5 cells. In this study we show that NOx gas stimulates MRC-5 cell proliferation by Rb (rubidium) phosphorylation via activation of cyclin-cell division protein kinase (cdk) complexes. Western blot and immunoprecipitation data showed that NOx gas increased the expressions of cyclinA/cdk2, cyclinD1/cdk4, and cyclinE/cdk2 complexes in the cells at 9 h after treatment. The levels of phospho-Rb were also increased and cdk inhibitors (CKIs) p27 and p16 were apparently decreased. These data suggested that NOx gas stimulates cell-cycle progression by Rb phosphorylation via activation of cyclin-cdk complexes and inhibition of CKIs. In conclusion, the NOx-gas that induced lung fibroblast cell proliferation by stimulation of cell-cycle progression may contribute to lung fibrosis by NOx pollutants. gaseous nitrogen oxides, proliferation, cdk inhibitor (CKI), Rb phosphorylation, cell cycle progression Nitrogen oxide species (NOx) exist in the atmosphere as air pollutants and include emissions from motor vehicles, outdoor facilities for heating and power generation, and some indoor appliances, all of which burn fossil fuels (Leaderer, 1982; Samet et al., 1987). Another important source for personal exposure to NOx is cigarette smoke, which may contain nitric oxide (NO) at a concentration as high as 100 ppm (Norman and Keith, 1965). The biological effects of NO2 and NO have been reviewed in several papers (Nakajima et al., 1980; WHO, 1977). In addition, clinical studies, using various exposure conditions, have indicated that the mechanisms by which NOx causes lung tissue damage are based on the oxidation properties and the free-radical potentials of these gases (Morrow, 1984; Samet and Utell, 1990). In lung tissue remodeling, epithelial-mesenchymal fibroblast cell interactions play an important role in lung repair (Young and Adamson, 1993). Even though NOx-mediated cytotoxicity in airway epithelium has been shown, in a recent study, to indicate a dual role for NO through destruction of epithelium and stimulation for fibroblast activity (Romanska et al., 2000) after transplantation. Endogenous NO is produced by a family of enzymes called nitric-oxide synthesizers (NOS), which includes constitutive (eNOS and nNOS) and inducible (iNOS) isoforms that oxidize the guanido group of L-arginine (Moochhala and Rajnakova, 1999). Our recent studies using human lung fibroblast cells (MRC-5) demonstrate that the treatment of gaseous nitrogen oxide results in increased synthesis of inducible NO synthase (iNOS) (Hsieh et al., 2001). Many previous reports have investigated the role of iNOS in promoting cell proliferation (Asano et al., 1994; Guo et al., 1995; Robbins et al., 1994; Romanska et al., 2002), but the role of gaseous nitrogen oxide in inducing iNOS to promote cell cycle progression is unknown. Eukaryotic cells have developed precise and well-regulated mechanisms to control progression through the cell cycle (Pardee, 1989). Regulation of the vertebrate cell cycle requires the periodic formation, activation, and inactivation of unique protein kinase complexes that consist of cyclin (regulatory) and cyclin-dependent kinase (cdk; catalytic) subunits. The associations of cyclin D1 and cdk4, cyclin E, and cdk2, and cyclin A and cdk2 have also been shown to phosphorylate rubidium (Rb) in the G0/G1 and the G1/S-phase transitions of the cell cycle (Weinberg, 1995). Upon phosphorylation, pRb releases and activates a number of proteins such as the E2F family of transcription factors at the G1/S transition phase (Nevins et al., 1997; Wang et al., 1994), which in turn regulates the expression of several genes involved in DNA replication, such as dihydrofolate reductase, thymidine kinase, and DNA polymerase α (Izumi et al., 2000). Regulation of G1 cyclin-cdk activity is also dependent on cdk inhibitory proteins (CKIs), which can bind and inactivate cyclin-cdk complexes (Hunter, 1993; Hunter and Pines, 1994; Peters and Herskowitz, 1994). Several inhibitory proteins have been identified, including p27, p16, and p21, which have been reported to mediate G1 cell-cycle arrest (Hall et al., 1995). Our previous work on the action of NOx gas to induce human lung fibroblast cell MRC-5 proliferation demonstrated a relationship between iNOS expression and cell proliferation (Chou et al., 2002). In this current study, we clarify the effect of gaseous NOx on human lung fibroblast cell proliferation by cell cycle-regulatory proteins in order to explore the mechanism of gaseous NOx-mediated lung fibrosis. MATERIALS AND METHODS Chemicals. Gasous NO (10,000 ppm in N2) and N2 were obtained from Sanfa Co. (Hsingcha, Taiwan). Tris–HCl, EDTA, SDS, phenylmethylsulfonyl fluoride, bovine serum albumin (BSA), leupeptin, nonidet p-40, deoxycholic acid, sodium orthovanadate, and aprotinin were purchased from the Sigma Chemical Co. (St. Louis, MO). Protein assay kits were obtained from Bio-Rad Labs. (Hercules, CA). Eagle’s minimal essential medium (MEM), fetal calf serum, trypsin-EDTA, and penicillin, streptomycin, and neomysin mixture (PSN) were purchased from Gibco/BRL (Gaithersburg, MD). Polyclonal antibodies against cyclin A, cyclin B, cdk 2, and Rb were obtained from Transduction Lab (KY). Antibodies against cdk4 and E2F were obtained from Pharmingen (CA), and those against cyclin D1, cyclin E, cdk2, p27, p21, and p16 were from Santa Cruz (CA). Phospho-Rb was from Cell Signaling Tech (MA). Cell culture. Human lung fibroblast cells (MRC-5) were maintained as monolayers in MEM supplemented with 10% heat-inactivated fetal-calf serum and PSN (100 units/ml penicillin and 10 μg/ml streptomycin) at 37°C in a humidified atmosphere of 95% air/5% CO2. Treatment of gaseous NOx. A gaseous NOx-saturated buffer was prepared by bubbling through phosphate-buffered saline (PBS) with N2 gas for 30 min to deoxygenate the solution, and then bubbling with authentic NO gas (10,000 ppm) for another 30 min. The above-surface space was briefly spurted with N2. This saturated solution had an NO concentration of approximately 1.25 mM, as measured by the ISO-NO analyzer, and was used for cell treatment. MRC-5 cells seeded on 60-mm plastic dishes (5 × 106 cells/dish) were treated with NOx gas-saturated PBS at various dilutions for the indicated time points (0–24 h) (as a gas-liquid interface culture system). After exposure, the cells were harvested, and cell extracts were prepared for immunoblot and immunoprecipitation analysis. Preparation of cell extract and immunoblot analysis. To prepare whole-cell extract, cells were washed with PBS containing zinc ion (1 mM), and then suspended in a lysis buffer (50 mM Tris, 5 mM EDTA, 150 μM sodium chloride, 1% Nonidet P-40, 0.5% deoxycholic acid, 1 mM sodium orthovanadate, 81 μg/ml aprotinin, 170 μg/ml leupeptin, and 100 μg/ml phenylsulfonyl fluoride; pH7.5). After 30 min of mixing at 4°C, the mixture was centrifuged at 10,000 × g for 10 min, and the supernatant was collected as whole-cell extract. Protein content of the samples was determined with the Bio-Rad protein assay reagent using BSA as a standard. For Western-blotting analysis, whole-cell extracts (20 μg protein) from control and gaseous NOx-treated samples were resolved on 10% SDS–PAGE gels along with pre-stained protein molecular weight standards (Bio-Rad). The separated proteins were then blotted onto NC membrane (0.45 μm, Bio-Rad) and reacted with primary antibodies (against cyclin A, cyclin B, cyclin D1, cyclin E, cdc2, cdk2, cdk4, p27, p21, p16, Rb, phospho-Rb, E2F, and β-actin as internal control). After washing, the blots were incubated with peroxidase-conjugated goat anti-mouse antibody. Immunodetection was carried out using the ECL Western-blotting detection kit (Amersham Corp., U.K.). Relative protein expression levels were quantified by densitometric measurement of ECL reaction bands and normalized with values of β-actin. Immunoprecipitation. Cell lysates were prepared using lysis buffer: 50 mM Tris, 5 mM EDTA, 150 mM sodium chloride, 1% Nonidet P-40, 0.5% deoxycholic acid, 1 mM sodium orthovanadate, 81 μg/ml aprotinin, 170 μg/ml leupeptin, and 100 μg/ml phenylsulfonyl fluoride; pH7.5. 500 μg of protein from cell lysates was pre-cleared with protein A-Sepharose (Amersham Pharmacia Biotech), followed by immunoprecipitation using monoclonal anti-cdk2, -cdk4, and -E2F (Santa Cruz Biotech) antibodies. Immune complexes were harvested with protein A, and immunoprecipitated proteins were analyzed by SDS–PAGE, as above. Immunodetection was carried out using monoclonal anti-cdk2, -cdk4, -E2F, and -cyclin D1; polyclonal anti-cyclin A, -cyclin E, and -Rb antibodies. Assessment of cell viability. Cells were seeded at a density of 4 × 104 cells/well and exposed to NOx (1 μm) for various periods of time (0, 24, 48, and 72 h). Thereafter, the medium was removed and replaced with 3-(4, 5-dimethylthiazol-2-xl)-2,5-diphenyltetrazolium bromide [MTT, 0.1 mg/ml] for 4 h. The numbers of viable cells was directly proportional to the production of formazan, which was solubilized in isopropanol and measured spectrophotometrically at 563 nm (Mosmann, 1983). Cell cycle analysis. To analyze the cell cycle distribution, cells were treated with gaseous NOx-saturated buffer, and then trypsinized and resuspended in 70% ethanol. After incubation on ice for at least 1 h, the cells were resuspended in 1 ml of cell cycle assay buffer (0.38 mM sodium citrate, 0.5 mg/ml RNAse A, and 0.01 mg/ml propidium iodide) at a concentration of 5 × 105 cells/ml. Cell cycle analysis was carried out by use of a flow cytometer and ModFit LT 3.0 software (Verity Software, Topsham, ME). Statistical analysis. Results were reported as means ± SD, and statistical analysis was obtained using an unpaired t-test. A value of p < 0.05 was considered statistically significant. RESULTS Effect of NOx Gas on MRC-5 Cell Proliferation It was previously reported that NOx gas can bring about inducible nitric oxide synthesis (iNOS) expression (Hsieh, 2001), and that iNOS is involved in many kinds of cell proliferation (Asano et al., 1994; Guo et al., 1995; Robbins et al., 1994; Romanska et al., 2002). To prove that NOx gas can induce cell proliferation, human lung fibroblast MRC-5 cells were treated with NOx gas-saturated solution (1 μM) to test cell viability (Fig. 1A). The results show that living cell numbers were significantly larger than those of untreated samples, as measured by MTT assay at 24, 48, and 72 h. Further examination of the cell cycle distribution confirmed that NOx-induced MRC-5 cells entered the S phase, as revealed by the increased number of cells in the S phase at 12 h (19%) after the treatment, and these cells continued to increase at 24 h (25%, Fig. 1B). These data indicate that NOx could induce cell proliferation. Effect of NOx Gas on the Expression of Cyclins In mammalian cells, cyclins comprise an extensive family of proteins whose cell cycle-dependent synthesis is postulated to control multiple events during the cell cycle (Hunter and Pines, 1994). Hence, to investigate how NOx gas induced cell proliferation, caused by activation of cyclins, MRC-5 cells were treated with NOx gas-saturated solution (1 μM) for the indicated times, and the resulting levels of cyclin A, cyclin D1, and cyclin E were measured. As shown in Figure 2A, the expression of all cyclins was increased in response to NOx treatment in a time-dependent manner, especially in cell cycles G1-to-S phases, cyclins (cyclin A, cyclin D1, cyclin E) were increased with maximal induction folds of 3.5, 6, and 5.5 at 9 h. Effect Of NOx Gas on Expression of Cdks Cyclin-dependent kinases (cdks) play a critical role in the commitment of a cell to proliferate (Pardee, 1989). Cdks are activated by their association with regulatory subunits known as cyclins. To confirm that NOx gas induces cyclin activation and also activates cdks, MRC-5 cells were treated with NOx gas-saturated solution (1 μM) for the indicated times. Western-blot data showed that the protein level of cdk2 and cdk4 increased at 9 h (maximal inductions of 3- and 2.8-fold respectively) (Fig. 2B). The data in Figure 2 demonstrate that NOx gas stimulates cell-cycle progression from G1 to S phase by activation of cyclins and cdks at 9 h. Effect of NOx Gas Enhances Cyclin/Cdk Association Cell cycle transition from G1 to S requires the temporal activation of cyclin D1-cdk4, cyclin E-cdk2, and cyclin A-cdk2 (Weinberg, 1995). To investigate how NOx gas induces cyclin (Fig. 2A) and cdk (Fig. 2B) activation and how it can promote cell-cycle progression, we used immunoprecipitation to ensure that cyclin-cdk complexes were activated to promote cell cycles. As shown in Figure 3, cyclin D1-cdk4 and cyclin E-cdk2, which control the progression through the G1-phase, increased induction by 2.63- and 2.05-fold at 9 h. Cyclin A-cdk2 induced cell cycles from the S phase to initiation of DNA synthesis, and this was increased by 3.55-fold at 9 h. This result implies that the NOx gas activation of cyclin D1-cdk4, cyclin E-cdk2, and cyclin A-cdk2 complexes did promote cell-cycle transition from G1 to the S phase. Effect of CKI on NOx Gas Activates Cell-Cycle Progression Regulation of G1 cyclin-cdk activity is also dependent on cdk inhibitory proteins (CKIs), which can bind to and inactivate cyclin-cdk complexes (Hunter, 1993; Hunter and Pines, 1994; Peters and Herskowitz, 1994). Several inhibitory proteins have been identified, including p27, p16, and p21, which have been reported to mediate G1 cell-cycle arrest (Hall et al., 1995). To verify that NOx gas induces G1 cyclin-cdk activity, dependent on inhibition of CKIs, MRC-5 cells were treated with NOx gas-saturated solution (1 μM) for the indicated times. The protein levels of p27, p16, and p21 were measured. We found that expression of p27 and p16 were significantly reduced, as shown in Figure 4, and that there was no significant alternation in p21. Effect of NOx Gas on the Expression of Rb-Phosphorylation (pRb) Cyclin D1-cdk4 plays a major role in the initiation of the cell cycle, passage through the restriction point (G0), and entry into the S phase. The only known target of active cyclin D1-cdk4 is the retinoblastoma tumor-suppressor protein (pRb); however, other cyclin-dependent kinases, such as cyclin E-cdk2, and cyclin A-cdk2 have also been shown to phosphorylate pRb in the G1-phase and the G1/S transition of the cell cycle (Wang et al., 1994). Cyclin-cdk complexes phosphorylate the retinoblastoma gene product (Rb), releasing E2F from its sequestration by Rb and allowing E2F to transactivate genes essential for the S phase (Nevins et al., 1997). Demonstrating that the NOx gas induced cyclin-cdk-complexes association could phosphorylate the Rb, we found that Rb phosphorylation was significantly increased after 9 h (Fig. 5). At the same time, we also found that Rb/E2F association was markedly reduced at 9 h (Fig. 6). These results demonstrate that NOx gas-induced cyclin-cdk complexes could phosphorylate Rb, thus allowing the release of E2F from Rb to promote cell-cycle transition from the G1 to the S phase. DISCUSSION In this study, we demonstrate that gaseous nitrogen oxides stimulate human lung fibroblast cell proliferation by activation of cell-cycle regulators. As summarized in Figure 7, the mechanisms of NOx mediated human lung fibroblast cell proliferation involved NOx inhibition of CKI proteins p27 and p21, and then activated cyclin D1-cdk4 and cyclin E-cdk2 complexes. These Cyclin-cdk complexes phosphorylate the Rb, releasing E2F from its sequestration by Rb to promote cell-cycle transition from the G1 to the S phase. NOx is present in the gaseous phase of smoke and air pollution at high concentrations (Church and Pryor, 1985; Last et al., 1994; Mohsenin, 1994). For example, freshly generated cigarette smoke contains up to 600 μg of NO per cigarette in the gaseous phase (Brunnemann and Hoffmann, 1982). Cigarette smoking is a risk factor for idiopathic pulmonary fibrosis (IPF) (Baumgartner et al., 1997). The reactive oxygen and nitrogen species could initiate inflammation and NO production that might eventually lead to pulmonary fibrosis (Chan et al., 2001; MacNee and Rahman, 1995; Saleh et al., 1997). Earlier studies have shown that environmental solid pollutants such as asbestos (Chao et al., 1996; Tanaka et al., 1998), induced iNOS in the lung. Some inflammatory mediators could induce iNOS expression in rat lung fibrosis, which might lead to their proliferation (Gansauge et al., 1997). Both endogenous and exogenous NO stimulate proliferation of human lung fibroblast cells (Gansauge et al., 1997; Romanska et al., 2002). In a previous study, we demonstrated that gaseous NOx could activate iNOS in human lung cells (Hsieh et al., 2001). Gaseous NOx was able to induce the expression of iNOS, which might initiate a secondary NO production and manipulate long-term effects in MRC-5 cells. Therefore, gaseous NOx-induced proliferation of MRC-5 cells may be mediated directly (that is, it is a primary effect of NOx) and indirectly (via the induction of iNOS) by the production of endogenous NO, and both sources could contribute to the process of lung fibrosis. In addition, interaction between airway epithelial cells and mesenchymal fibroblast cells has been investigated in human and animal studies of lung development, injury, and repair. (Sanders, 1988; Young and Adamson, 1993). After lung transplantation, iNOS immunoreactivity could be seen in damaged bronchiolar epithelium that initiated mesenchymal fibroblast cell proliferation (Mason et al., 1998). It is possible that, after transplantation, the injured epithelial and the destroyed basement membrane could directly expose fibroblasts to the injured epithelial cells, and induce tissue repairing by activation of inflammatory cytokines and growth factors that can stimulate fibroblast proliferation (Nakamura et al., 1995; Saleh et al., 1997; Young and Adamson, 1993). Therefore, NO derived from epithelial and inflammatory cells maybe a key mediator of fibroblast activation. This hypothesis is supported by the finding of this present study, using an in vitro treatment of gaseous NOx to induce the proliferation of human lung fibroblast cells. Pulmonary fibrosis includes an inflammatory constituent that implicates iNOS expression in the fibrotic lung (MacNee and Rahman, 1995; Saleh et al., 1997). Gaseous NOx-induced iNOS expression should occur in response to lung fibrosis (Paredi et al., 1999). Thus, iNOS activation and NO release may be involved in the proliferative response of fibroblasts to NOx gas exposure. It has been proven that NO synthesis activity can regulate NF-κB (Marshall and Stamler, 1999). NF-κB functions in controlling cell growth by regulation of cyclin D1 expression and G0/G1-to-S phase transition (Hinz et al., 1999). Hence, our previous data demonstrating that gaseous NOx could activate NF-κB (Chou et al., 2002) might also relate to the regulation of NOx gas-induced cell cycle progression by Rb phosphorylation via activation of cyclin/cdk complexes. In conclusion, our previous results demonstrated that gaseous NOx stimulated proliferation via direct and indirect activation of MEKK1, JNK, and p38 signaling pathways (Chou et al., 2002). In this current study, NOx-saturated solution induced cell proliferation through inhibition of cdk inhibitory proteins p27 and p21, and then activated cyclin D1-cdk4 and cyclin E-cdk2 complexes to induce Rb phosphorylation and to promote cell-cycle transition from the G1 to the S phase. Therefore, the results of this study support the hypothesis that NOx and NO may act as profibrotic mediators, thus contributing to the pathogenesis of IPF. FIG. 1. Open in new tabDownload slide Effect of gaseous NOx on the cell viability and the cell cycle progression of MRC-5 cells: Cultured cells were treated with NO gas-saturated solution (1 μM NOx for final concentration) for the indicated times. (A) Cell viability was analyzed by MTT assay. (B) Cell cycle was analyzed by flow cytometry, and the percentage of cells distributed in S phase was compared to the untreated controls. Values are presented as mean ± SD of four independent experiments; *p < 0.05; **p < 0.005. FIG. 1. Open in new tabDownload slide Effect of gaseous NOx on the cell viability and the cell cycle progression of MRC-5 cells: Cultured cells were treated with NO gas-saturated solution (1 μM NOx for final concentration) for the indicated times. (A) Cell viability was analyzed by MTT assay. (B) Cell cycle was analyzed by flow cytometry, and the percentage of cells distributed in S phase was compared to the untreated controls. Values are presented as mean ± SD of four independent experiments; *p < 0.05; **p < 0.005. FIG. 2. Open in new tabDownload slide Time course of gaseous NOx treatment on the protein expression of cyclins (A) and cdks (B) in MRC-5 cells: Cell lysates were prepared from MRC-5 fibroblasts at the times indicated following treatment with NOx gas-saturated solution (1 μM NOx for final concentration). The protein levels of cyclin A, D1, E, and cdk2 and cdk4 were analyzed by Western blotting. The quantitative data were presented as means ± SD of three repeats from one independent study. *p < 0.05; **p < 0.005, compared with 0-h control cells. N is normal control; NOx is MRC-5 cells treated with NOx gas-saturated solution. FIG. 2. Open in new tabDownload slide Time course of gaseous NOx treatment on the protein expression of cyclins (A) and cdks (B) in MRC-5 cells: Cell lysates were prepared from MRC-5 fibroblasts at the times indicated following treatment with NOx gas-saturated solution (1 μM NOx for final concentration). The protein levels of cyclin A, D1, E, and cdk2 and cdk4 were analyzed by Western blotting. The quantitative data were presented as means ± SD of three repeats from one independent study. *p < 0.05; **p < 0.005, compared with 0-h control cells. N is normal control; NOx is MRC-5 cells treated with NOx gas-saturated solution. FIG. 3. Open in new tabDownload slide Analysis of cyclin-cdk complexes in MRC-5 fibroblasts following gaseous NOx treatment: Cell extracts prepared from MRC-5 fibroblasts at the times indicated following treatment with NO gas-saturated solution (1 μM NOx for final concentration) were immunoprecipitated with cdk2 (A and B) and cdk4 (C). The precipitated complexes were examined for immunoblotting using cyclin A (A), cyclin E (B), and cyclin D1 (C) antibodies. FIG. 3. Open in new tabDownload slide Analysis of cyclin-cdk complexes in MRC-5 fibroblasts following gaseous NOx treatment: Cell extracts prepared from MRC-5 fibroblasts at the times indicated following treatment with NO gas-saturated solution (1 μM NOx for final concentration) were immunoprecipitated with cdk2 (A and B) and cdk4 (C). The precipitated complexes were examined for immunoblotting using cyclin A (A), cyclin E (B), and cyclin D1 (C) antibodies. FIG. 4. Open in new tabDownload slide Expression of cell-cycle inhibitors in MRC-5 fibroblasts following gaseous NOx treatment. Cell lysates were prepared from MRC-5 fibroblasts at the times indicated following treatment with NO gas-saturated solution (1 μM NOx for final concentration(. The expression of p27, p21, and p16 were analyzed by Western blotting. The quantitative data were presented as means ± SD of three repeats from one independent study; *p < 0.05; **p < 0.005, compared with 0-h control cells. FIG. 4. Open in new tabDownload slide Expression of cell-cycle inhibitors in MRC-5 fibroblasts following gaseous NOx treatment. Cell lysates were prepared from MRC-5 fibroblasts at the times indicated following treatment with NO gas-saturated solution (1 μM NOx for final concentration(. The expression of p27, p21, and p16 were analyzed by Western blotting. The quantitative data were presented as means ± SD of three repeats from one independent study; *p < 0.05; **p < 0.005, compared with 0-h control cells. FIG. 5. Open in new tabDownload slide Expression of Rb and Rb phosphorylation in MRC-5 fibroblasts following gaseous NOx treatment: Cell lysates were prepared from MRC-5 fibroblasts at the times indicated, following treatment with NO gas-saturated solution (1 μM NOx for final concentration). The expression of Rb and phosphor-Rb were analyzed by Western blotting. The quantitative data were presented as means ± SD of three repeats from one independent study; *p < 0.05; **p < 0.005, compared with 0-h control cells. FIG. 5. Open in new tabDownload slide Expression of Rb and Rb phosphorylation in MRC-5 fibroblasts following gaseous NOx treatment: Cell lysates were prepared from MRC-5 fibroblasts at the times indicated, following treatment with NO gas-saturated solution (1 μM NOx for final concentration). The expression of Rb and phosphor-Rb were analyzed by Western blotting. The quantitative data were presented as means ± SD of three repeats from one independent study; *p < 0.05; **p < 0.005, compared with 0-h control cells. FIG. 6. Open in new tabDownload slide Analysis of Rb-E2F complexes in MRC-5 fibroblasts following gaseous NOx treatment: Cell extracts prepared from MRC-5 fibroblasts at the times indicated following treatment with NO gas-saturated solution (1 μM NOx for final concentration) were immunoprecipitated with E2F. The precipitated complexes were examined for immunoblotting using Rb antibody. The quantitative data were presented as means ± SD of three repeats from one independent study; *p < 0.05; **p < 0.005, compared with 0-h control cells. FIG. 6. Open in new tabDownload slide Analysis of Rb-E2F complexes in MRC-5 fibroblasts following gaseous NOx treatment: Cell extracts prepared from MRC-5 fibroblasts at the times indicated following treatment with NO gas-saturated solution (1 μM NOx for final concentration) were immunoprecipitated with E2F. The precipitated complexes were examined for immunoblotting using Rb antibody. 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TI - Gaseous Nitrogen Oxides Stimulate Cell Cycle Progression by Rubidium Phosphorylation via Activation of Cyclins/Cdks
JO - Toxicological Sciences
DO - 10.1093/toxsci/kfg221
DA - 2003-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/gaseous-nitrogen-oxides-stimulate-cell-cycle-progression-by-rubidium-8hdVGEDqyw
SP - 83
EP - 90
VL - 76
IS - 1
DP - DeepDyve
ER -