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Ⅰ. Introduction
A. Replicative senescenceReplicative cellular senescence entails an irreversible arrest of cell proliferation and altered cell function (Hayflick and Moorhead, 1961) primarily depending on the number of cell division, not on time to culture. Replicative senescence has been described in many cell types such as fibroblasts (Hayflick, 1992; Stein and Dulic, 1995; Seshadri and Campisi, 1990), keratinocytes (Bregegere, 2003), endothelial cells (Foreman and Tang, 2003), etc, and its characteristics depends on the cell type, the species, and age of donor (Stanulis-Praeger, 2001; Campisi et al., 1996). The phenotype of cellular senescence is generally characterized by a series of features, termed “biomarkers” (Dimri et al., 1995). The most obvious biomarker is growth arrest, i.e. cells stop dividing.
Most senescent cells are growth arrested in the transition from G1 phase to S phase of the cell cycle (Sherwood et al., 1988). The arrest is irreversible in the sense that growth factors cannot stimulate the cells to divide further (Cristofalo and Pignolo, 1993), even though senescent cells can remain metabolically active for long periods of time (Goldstein, 1990). Another important biomarker is cellular morphology. Senescent cells show characteristic changes such as enlarged and flattened cell shape, increased adhesion, and granularity (Chang, 1999; Judith, 1999). In addition, the enzyme β-galactosidase has an abnormal
behavior associated with senescent cells, which is termed senescence-associated β-galactosidase (SA β-gal) activity. β-galactosidase, a lysosomal hydrolase, is normally active at pH 4, but in senescent cells it becomes often active at pH 6. Early reports also showed that lysosomes increase in number and size in senescent cells (Robbins, 1970; Campisi, 2001), implying that SA β-gal appears to be a result of increased lysosomal activity (Brunk et al., 1973). Recent studies also suggest that during “in vitro” aging increased autophagy may be associated with an increase of lysosomal mass and SA β-gal activity (Kurz et al., 2000; Gerland et al., 2003).
B. Stress-induced senescence
Normally, cell culture conditions include 20% oxygen (O2) and these were the conditions initially used by Hayflick and Moonrhead and subsequent works. When HDF cells are cultured at 3 % O2, which is closer to physiological conditions, they appear normal growth rate. In contrast, different types of human cells cultured above 20 % display a reduced growth rate (horikoshi et al., 1986; Michiels et al., 1990; Horikoshi et al., 1991; Von Zglinicki et al., 1995). In normal human cells, O2 has been shown to accelerate growth arrest (Alaluf, 2000).If O2 is above 50 %, it becomes cytotoxic (Horikoshi et al., 1991). The subcytotoxic stress can accelerate the appearance of the senescent phenotype in
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cells. Therefore, it has been well accepted that, depending on the dose of stressor used, a cell population will respond in different ways. A high cytotoxic dosage of a stressor induces such an amount of damage that cellular biochemical activities decrease, often leading to cell death by necrosis. The level of damage sustained with cells determines the cellular fate to necrosis or apoptosis (Lemeasters et al., 1999; Nicotera et al., 1999). In order to destine to senescence, the cells require a precise subcytotoxic dose of O2. It has been often reported that, in addition to O2, several other stressors - ehanol, ionizing radiations, etc, can induce senescence in many types of proliferative cells such as lung and skin fibroblasts, endothelial cells, melanocytes, and retinal pigment epithelial cells (Ames et al., 1993; Finkel et al., 2000).
C. ROS and mitochondria
Cellular senescence has long been proposed to result from cumulative irreversible damages caused by toxic oxygen intermediates, so called reactive oxygen species (ROS) (Wallace, 1992; Boveris et al., 1972; Beckman and Ames, 1998, Harman, 1992). Although intracellular ROS production can be stimulated by activation of several enzymes such as NAD(P)H oxidase, β-oxidation, prostaglandin systhesis, cytochrome P450, mitochondria is targeted as one of the major sites (Boonstra and Post, 1997). A pivotal role of mitochodnria is energy
production through oxidative phosphorylation. Electrons from nicotinamide adenine dinucleotide (NADH) and flavine adenine dinucleotide (FADH2) flow down to O2 via the electron transport chain (ETC). The ETC is composed of four complexes within the inner mitochondrial membrane, NADH dehydrogenase (complexⅠ), succinate dehydrogenase (complexⅡ), cytochrome bc1 complex (complexⅢ), and cytochrome c oxidase (complexⅣ). In addition to electron transfer, Complex Ⅰ, Ⅲ and Ⅳ also pump protons across the inner mitochondrial membrane into the cytoplasm generating the proton gradient, the mitochondrial transmembrane potential (∆Ψm), which is finally harnessed by F0F1 ATP synthase for production of ATP (Wallace, 1992). The mitochondrial ETC continuously consumes more than 90 % of the oxygen taken up by the cell (Boveris et al., 1972). From this, about 1-5 % is converted into superoxide (O2.-) even during normal physiological state (Terrens and Boveris, 1980). Due to this reason, sustained production of the ROS through mitochondrial respiration, a major by-product of oxygen-derived reaction, is inevitable.
D. Mitochondrial DNA theory of aging
The identification of mitochondria as the major source of ROS production leads to the mitochondrial DNA hypothesis of aging (Harman, 1956; Miquel et
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al., 1980; Miquel, 1991). In contrast to other intracellular organelles,
mitochondria possess their own DNA, termed mitochondrial DNA (mtDNA). Moreover, mtDNA locates in the proximity to the ROS generation site within mitochondrial innermembrane and has naked structure without chromatin conformation, allowing itself highly vulnerable to oxidative damage (Miquel, 1991). For the proper mitochondrial respiration, it is quite important to coordinate well the expressions and structural arrangement of about 85 proteins, of which about 72 proteins encoded by nuclear DNA and 13 proteins by mitochondrial DNA (mtDNA). Since all the polypeptides encoded by mtDNA are involved in electron transport and ATP production, mtDNA damage by a certain oxidative damage will lead to a decrease in mtRNA and mitochondrial protein synthesis (Shoubridge, 2001; Triepels et al., 2001; Orth and Schapira, 2001). Thus, any damage of this DNA results in a loss of one or several proteins essential for electron transport or ATP systhesis. This would in turn lead to decreased energy production, which is in fact a hallmarker of many mitochondrial disorders. Also, the mitochondrial respiration is more prone to be damaged by ROS produced within their compartment, and consequently the damaged mitochondria release more ROS in a motion of vicious cycle, which has been a generally accepted theory of cellular aging (Fig. 1) (Boveris et al., 1999; Sohal et al., 1994; Lee and Wei, 2001; Raha and Robinson, 2000).
E. ROS and signaling-activated senescence
Although the generation of mitochondrial ROS has long been implicated in aging process, several recent studies have demonstrated that a moderate and sustained ROS produced by the activated stress signaling pathways, including Ras/MAPK (Lee et al., 1999), phosphoinositide 3-kinase (PI3K)/Akt (Collado et al., 2000), NF-kappaB (Poynter and Daynes, 1998; Supakar et al., 1995) and
p53 (Macip et al., 2003) and p16INK4a (Schmitt et al., 2000) activation, can also trigger the senescence program (Serrano and Bringold, 2000). Those stress signaling pathways can generally be activated by various environmental stimuli, including cytokines, UV irradiation, and chemotherapeutic agents (Finkel, 2000). Modulation of the pathways is not unique to oxidative stress, but also plays central roles in controlling normal growth through regulating metabolic signaling (Cooper et al., 1983; Whiteman, 2002). However, the mechanism of how the mitochondrial metabolic function is involved in the stress signaling-mediated senescence has not yet been studied.
F. TGF ββββ1 and senescence
Transforming growth factor β1 (TGF β1) is a multifunctional cytokine that controls proliferation, differentiation, migration, and apoptosis of various cell
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types (Massague, 1998; Dernck and Zhang, 2000; Alevizopoulos and Mermod, 1997; Lin and Chon, 1992; Nakamura et al., 1985). Especially, it is mainly known that TGF β1 is a potent growth inhibitor for epithelial cells to irreversibly arrest cells in the late G1 phase of cell cycle (Hocevar and Howe, 1998). TGF β1 treatment of mink lung epithelial cells prevents efficiently the phosphorylation of pRB as a result of complex regulation of activities of Cdk’s and their inhibitors (Laiho et al., 1990). The involvement of TGF β1 in aging and senescent process has recently been demonstrated by its direct induction of cellular senescence in a few cell lines, such as human diploid fibroblasts, mammary epithelial cells, and lung carcinoma cells (Frippiat et al., 2001;Koedon et al., 1995; Katrakura et al., 1999), in addition to its over-expression in aged animal (Zhao et al., 2002) and oxidatively stressed cells (Frippiat, 2001; Yoon, 2002). Although the potential role of p27, p15, and p21 (Katakura, 1999; Warner et al., 1999; Gong et al., 2003; Li et al., 1995) or overproduction of ROS (Sanchez et al., 1996; Herrera et al., Islam et al., 1997) has been alluded in the TGF β1-induced senescent arrest, the detailed underlying mechanisms remain unclear.
G. Purpose of this study
involved in the senescent arrest induced by TGF β1 in Mv1Lu mink lung epithelial cells which is extremely sensitive to growth inhibition by TGF β1 (Ewen, 1993). And we demonstrate that TGF β1 induced senescence-associated G1 arrest in Mv1Lu lung epithelial cells cultured in the presence (higher than 5 %) of fetal bovine serum (FBS). During the arrest, mitochondrial respiratory function was disrupted through decreased complex IV activity, and ROS was persistently produced through the defective respiration without triggering apoptogenic signaling. Furthermore, we also showed that hepatocyte growth factor (HGF) could reverse the senescent arrest by protecting the mitochondrial function. These results show a potential intimate coupling between TGF β 1-induced signaling and modulation of mitochondrial metabolism, and imply that the maintenance of mitochondrial function is important to protect cells from the senescent arrest.
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ROS
mtDNA damage mRNA
ROS
ROS
electron transport
chain
polypeptides
loss of membrane potential, release of cytochrome C,
activation of caspase, apoptosis.
Fig. 1. mtDNA damage-induced vicious cycle of ROS generation Since mtDNA
encodes polypeptides involved in electron transport or ATP production, mtDNA damage will lead to a decrease in mitochondrial mRNA and mitochondrial protein synthesis. Loss of these mitochondrial proteins will lead to inhibition of electron transport, the generation of ROS and more mtDNA damage in a vicious feed-forward cascade. Eventually the level of damage is so severe that the mitochondria loss their ability to maintain the membrane potential. There is a subsequent loss of cytochrome c into the cytoplasm, which initiates apoptosis through the activation of caspases. This model also predicts that mitochondrial mRNA will decrease after ROS. Another prediction of this model is that any toxicant that inhibits the electron transport can lead to the generation of ROS and subsequent mtDNA damage.
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Ⅱ. Materials and Methods
A . Reagents and antibodies
Recombinant TGF β1 and HGF was obtained from R&D systems (Minneapolis, MN, U.S.A.). 5,5',6,6'-tetrachloro-1,1',3,3'-tetra-thylbenzimidazole
carbocyanide iodide (JC-1), 2’,7’-dichlorodihydrofluorescein diacetate (DCF-DA), and dihydrorhodamine 123 (DHR123) were purchased from Molecular Probe Corp. (Eugene, OR, U.S.A.). NADH, cytochrome c, N-acetyl cysteine (NAC), antimycin A, 2,4-dinitrophenol (DNP), tetramethyl-p-phenylene diamine (TMPD), oligomycin, and 5-bromo-4-chloro-3-indolyl-β -D-galactopyranoside (X-gal) were from Sigma-Aldrich (St. Louis, MO, U.S.A.). Trolox, 2-(12-hydroxydodeca-5, 10-diynyl)-3,5,6-tetrimethyl-p-benzoquinone (AA861) and Baicalein were purchased from BioMol Research Laboratory Inc. (Plymouth, PA, U.S.A.).
Antibody against cytochrome c (556433) was purchased from BD Pharmingen (San Diego, CA, U.S.A), and antibody against Fp protein of complex II (A11142) was from Molecular Probe Corp. PARP antibody was obtained from Zymed Laboratories Inc. (South San Francisco, U.S.A.), MnSOD antibody was from Stressgen (Victoria, Britich Columbia, Canada), and tubulin antibody (Ab-1) and Smac (Ab-3) antibody were from Oncogene (Boston, MA,
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U.S.A.). AIF antibody was purchased from SantaCruz (Santa Cruz, CA, USA). Antibodies against Prx I, II, and III were kindly provided by Dr. HZ Chae (Chonnam National University, Gwangju, Korea).
B. Cell culture, growth rates, and viability
Mv1Lu cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, MD, U.S.A.) containing 10 % fetal bovine serum (FBS) (Gibco BRL) in a 37 oC incubator with 5 % CO2 in air and treated with 2 ng/ml TGF β1, unless indicated otherwise. Cellular growth rates of TGF β1- and/or HGF-treated Mv1Lu cells were monitored by counting the viable cells. Briefly, 1 x 103 cells were seeded into 24-well plates, cultured in DMEM containing 10 % FBS for 12 h, and treated with TGF β1 (2 ng/ml) and/or HGF (20 ng/ml) for the indicated periods. The cells were continuously cultured by refreshing every three days with the media containing the same growth factors, if necessary. At the indicated times, cells were harvested by trypsinization and counted with a hemocytometer after staining with final 0.2 % (w/v) trypan blue (Gibco BRL) to exclude dead cells. To evaluate cellular viability, the population of trypan blue-stained cells were counted under the inverted microscope (Olympus, Tokyo, Japan) and represented as percent of total cells.
C. Cell cycle analysis
Cell cycle analysis was performed by propidium iodide (PI) staining according to the protocol provided with CycleTESTTM PLUS DNA reagent kit (Becton Dickinson, Ontario, Canada). Stained cells were analyzed using a FACS Vantage flow cytometer (Becton Dickinson) at an excitation wave length of 488 nm. The percentage of cell in each phase was based on 10,000 cells counted.
D. Senescence associated β-galactosidase Assay
Senescence-associated β-galactosidase (SA β-gal) was assayed at pH 6.0 as described by Dimri et al. (Dimri et al., 1995) with a slight modification (Yoon et al., 2002). Briefly, cells were washed twice with PBS, fixed to plates by 3 %
formaldehyde for 5 min, washed with PBS, and then incubated overnight in freshly prepared staining solution [40 mM citrate-phosphate buffer, pH 6.0, containing 1 mg/ml of 5-bromo-4-chloro-3-indolyl-β−D-galactopyranoside (X-gal), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl2]. The stain was visible 12 h – 18 h after incubation at 37 oC. The population of senescence-associated cells was obtained by counting the number of blue-stained cells per field (0.5 ㎝ X 0.5 ㎝) and expressing as a percentage of the total number of cells.
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E. Mitochondrial transmembrane potential (∆Ψm)
∆Ψm was estimated by staining cells with JC-1 (Molecular Probe) fluorescence dye. To assess the ∆Ψm changes of cells exposed to TGFβ1, cells were incubated for 1 h with 10 ug/ml JC-1, washed with PBS (137 mM NaCl, 2.7 mM KCl, 10mM phosphate buffer, pH 7.4), and finally visualized by Fluoromicroscope (Diaphot 300, Nikon, Japan). To quantitate the degree of disrupted ∆Ψm, cells stained with JC-1 were collected by trypsinization and resuspended in PBS, and green fluorescences of the cells were analyzed by flow cytometry (FACS Vantage, Becton Dickinson corp.). The intensity of the green fluorescence was considered as a degree of ∆Ψm disruption.
F. Determination of intracellular ROS level
To determine intracellular level of ROS, we used two different fluorogenic probes; DCFH-DA and DHR123 (Molecular probe). DCFH-DA can be deacetylated in cells, where it can react quantitatively with intracellular radicals, mainly H2O2, to be converted to its fluorescent product, 2’,7’-dichlorofluorescein (DCF), which is retained within the cells, thus providing an index of cytosolic ROS level.DHR123 can easily cross cell membranes and
react with ROS in mitochondria to generate positively charged rhodamine 123, allowing as a useful probe to measure mitochondrial ROS production such as H2O2 (Royall, 1993; Rothe, 1991). Briefly, cells were treated with TGF β1 for the indicated periods and further incubated in media containing 10 µM DCFH-DA or DHR123 for 15 min at 37℃. Stained cells were washed, resuspended in PBS, and analyzed by flow cytometry.
To measure intracellular ROS level within one hour after treatment with KCN, cells were suspended in media containing 10 µM DCFH-DA and then treated with various concentrations of KCN for the periods indicated. Maximum incubation time for DCFH-DA did not exceed one hour. After washing the cells with PBS, the fluorescence of the stained cells (1 x 104) was analyzed by flow cytometry.
G. Redox state and intracellular localization of Prx proteins
In order to analyze the redox status of Prx proteins, 2-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) and/or non-reducing sodium dodecyl sulfate (SDS)-PAGE followed by Western blot analyses were performed. For 2D-PAGE analysis, cells were rinsed three times with ice-cold PBS and lysed in lysis buffer (8 M urea, 4% CHAPS,and 40 mM Tris base) by sonication in a sonic bath three to fourtimes for 30 s. After removal of insoluble materials
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by centrifugation at 14,000 g for 30 min, cell lysates were mixed with 10 volumesof rehydration buffer (8 M urea, 2 % CHAPS, 0.5% immobilized pH gradient buffer, 20 mM DTT, and 0.005% bromophenol blue) and loadedonto immobilized pH gradient strips (pH 3-10, nonlinear). Isoelectricfocusing on an IPGPhor isoelectrofocusing unit (Amersham Pharmacia Biotech., Piscataway, NJ)and preparation (reduction and alkylation) of the immobilizedpH gradient strips for the second-dimensional SDS-PAGE were carriedout according to the procedures recommended by the manufacturer.SDS-PAGE was conducted on 12 % gel using an Amersham Biosciences SE 600 vertical unit. For non-reducing PAGE, cells were lysed in lysis buffer [50 mM Tris-Cl, pH 7.5, 0.1 M NaCl, 1mM EDTA, 1 % Triton X-100, 10 mg/ml N-ethylmaleimide, 10mg/ml each of aprotinin and leupeptin, and 1 mM phenylmethylsulfonylfluoride (PMSF)]. Ten µg of lysate was diluted in mercaptoethanol-free sample buffer (75 mM Tris-Cl, pH 6.8, 2 % SDS, 6.5 % glycerol, 0.1 % bromophenol blue) and electrophoresed onto SDS-polyacrylamide gel. Subsequently, immunoblot analyses were performed to evaluate redox status of Prx I, II, and III for both 2D-PAGE and non-reducing PAGE gels.
To analyze intracellular localization of Prx III protein, immunocytochemical analysis was applied. Cells seeded onto cover glass were incubated with MitoTracker Red (Molecular Probe) for 15 min and then fixed with a mixture of methanol and acetone (1:1). Fixed cells were washed with
PBS and permeabilized in PBS containing 0.075 % Triton X-100 for 5 min. Cells were blocked in PBS containing 1 % BSA for 1 h and probed with a 1:400 dilution of primary antibody against Prx III overnight at 4 ℃. After washing, the immune complexes were probed with 1:200 dilution of FITC-conjugated anti-rabbit antibody (Jackson Laboratory, Bar Harbor, ME). Immuno-stainined cells were then washed with PBS and mounted with glycerol. Images were obtained using a Confocal Laser Scanning Microscope (FLUOVIEW FV300, Olympus, Tokyo, Japan).
H. Mitochondrial fractionation and detection of mitochondrial protein release
Cells (5 x 106) were washed with PBS, harvested by trypsinization, and resuspended in medium A (250 mM sucrose, 0.1 mM EDTA, 2 mM HEPES, pH 7.4). The cell slurry was homogenized in a Dounce homogenizer (StedFastTM Stirrer, Fisher Scientific, Pittsburgh, PA, U.S.A.), followed by spin at 570 g for 10 min to remove nuclei and cell debris. The supernatant was further centrifuged at 14300 g for 10 min. The final supernatant (crude cytosolic fraction) and the pellet (mitochondrial fraction) were lysed in lysis buffer [50 mM Tris-Cl, pH 7.5, 0.1 M NaCl, 1mM EDTA, 1 % (w/v) Triton X-100, 10 mg/ml each of aprotinin and leupeptin, and 1 mM PMSF] before being subjected to Western blot analysis
17 for detection of cytochrome c, Smac, and AIF.
I. Endogenous cellular oxygen consumption
Endogenous cellular respiration was measured as described previously with slight modification (Villani and Attardi, 2001; Yoon et al., 2003). Briefly, exponentially growing Mv1Lu cells (5 x 106) were cultured in the absence or presence of TGF β1, washed with TD buffer (0.137 M NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 25 mM Tris-HCl, pH 7.4), and collected by trypsinization. After resuspension of the cells in the complete medium without phenol red, the cells were transferred to the chamber of Mitocell equipped with Clark oxygen electrode (782 Oxygen Meter, Strathkelvin Instrument, Glasgow, UK). Oxygen consumption rates were measured after adding 30 µM DNP to obtain maximum respiration rate and its specificity for mitochondrial respiration was confirmed by adding 10 mM KCN. Maximum cellular respiration rates are expressed as the ratio of DNP-uncoupled O2 consumption rate versus KCN-inhibited O2 consumption rate.
J. Assays for mitochondrial respiratory chain activities
lysate were measured as described previously with slight modification (Birch-Machin and Turnbull, 2001; Tzagoloff, 1999). Complex IV activity was determined as described with slight modification, by measuring the capacity to oxidize reduced cytochrome c at 550 nm. To prepare reduced cytochrome c, 1 mM of cytochrome c was reduced by adding a few crystals of sodium hydrosulfite (Na2S2O4) and incubated at 25 ℃ for 5 min, and then the reduced cytochrome c was purified by Sephadex G-50 chromatography. To determine complex IV activity, cells were lysed in PES buffer (50 mM Na/K phosphate, pH 7.4, 1 mM EDTA, 0.5 % sodium cholate) and further incubated with 1mg/ml of n- dodecylmaltoside for 20 min on ice. The initial rate to oxidize the reduced cytochrome c was monitored spectrophotometrically (Ultrospec 4300) at 550 nm in the reaction mixture (20 mM potassium phosphate, pH 7.4, 100 uM reduced cytochrome c) in the presence of 20 µM antimycin A. The reaction was started by the addition of the n-dodecylmaltoside-treated cell lysates (30 µg) and a rapid mix to detect the KCN sensitive initial rate. Complex IV activity was also determined by monitoring KCN-sensitive complex IV-dependent O2 consumption rate, which was obtained by measuring O2 consumption rate in the presence of 3 mM TMPD under the treatment of 30 µM DNP and 20 µM antimycin A and expressed as a percentage of TMPD-activated versus KCN-inhibited activity.
19 K. Western blot analysis
Cells were washed twice with PBS and lysed with lysis buffer (50 mM Tris-Cl, pH 7.5, 0.1 M NaCl, 1 mM EDTA, 1 % Triton X-100, 10 mg/ml each of aprotinin and leupeptin, and 1mM PMSF). A portion (40 ug) of lysate was electrophoresed on SDS-polyacrylamide gel. The proteins were electrically transferred onto nitrocellulose membrane (Protran, S&S Inc., NH) and blocked for 1hat room temperature with PBST (PBS containing 0.05 % Tween 20) containing 5 % (w/v) non-fat milk. Blots were subsequently incubatedin the blocking solution for overnight at 4 oC with 1 µg/ml of primary antibodies. Membranes were then washed twice with PBST, incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Amersham Pharmacia Biotech.) or anti-mousesecondary antibodies (Amersham Pharmacia Biotech.) for 2 hr at room temperature, and visualized by exposing to X-ray film after reacting with the enhanced chemiluminscencesystem (ECL, Amersham Pharmacia Biotech.) according to the manufacturer'sinstructions.
L. Determination of cellular ATP level
Cellular ATP levels were measured by the bioluminescence assay according to the protocol provided with an ATP Determination Kit (Molecular Probe Corp.,
Eugene, OR). Cell lysate (5 µg) was mixed in 100 ul of luciferase reagent, and luminescence was analyzed with 10 sec integration on luminometer (TD-20/20, Sunnyvale, CA). ATP concentration of the samples was determined from a calibration curve for known ATP concentration performed at the same time. ATP levels are expressed as pmole of ATP per µg of cellular lysate protein
M. Lactate dehydrogenase assay
Cell pellets were lysed in PES buffer (50 mM Na/K phosphate, pH 7.4, 1mM EDTA, 0.5 % sodium cholate), and a portion (25 µg) of lysate was used in total 150 µl of assay mixture. To determine LDH activities, initial rates to oxidize NADH were monitored at 340 nm with freshly prepared 2 mg/ml NADH solution in 0.1 M potassium phosphate buffer, pH 7.4, after adding pyruvate to 4 mM final concentration. The activity was determined by comparing values with a calibration curve for known concentration of standard LDH performed at the same time. The LDH activities were calculated as unit of LDH per ug of cellular lysate protein and expressed as percentage of control.
N. Electron microscopy
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2 % glutaraldehyde, 2 mM calcium chloride, 100 mM cacodylate buffer, pH 7.4) for 2 hr, washed with cacodylate buffer, and post-fixed in 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1 hr. Cells were then stained en bloc in 0.5% uranyl acetate, dehydrated through a graded ethanol series and
embedded in Poly/Bed 812 resin (Pelco, CA, U.S.A.). Cells were sectioned using Reichert Jung Ultracut S (Leica, Cambridge, UK). After staining cells with uranyl acetate and lead citrate, cells were observed and photographed under transmission electron microscope (Zeiss EM 902A, Leo, Oberkohen, Germany).
O. Statistical analysis
All bars represent the mean and standard deviations of determinants. Experiments were repeated for more than 2 times. Statistical analysis was done with Student’s t-test.
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Ⅲ. Results
A. TGF ββββ1-induced senescence-associated G1 arrest of Mv1Lu cells depends on the presence of fetal bovine serum
Mv1Lu cells exhibit one of the best-characterized antimitogenic responses to TGF β1. It has generally been accepted that the growth of Mv1Lu cells is prone to be arrested and finally committed to apoptosis when exposed to TGF β1 in serum-free media (Parekh et al., 1998), nevertheless, the growth arrest by TGF β1 has also been demonstrated even in the presence of 10% FBS (Laiho et al., 1990; Taipale and Keski-Oja, 1996). In order to ascertain the effect of TGF β1 on cellular destination of Mv1Lu cells in the presence of 10 % FBS, we continuously cultured the cells in the medium containing TGF β1 (2 ng/ml) and 10 % FBS. As expected, we could observe delayed growth rate with G1 arrest (Fig. 2, A and B) in accordance with the studies previously described. However, we also observed progressive change of cellular morphology from the second day to enlarged and flattened form (Fig. 2, C), which is one of the typical characteristics of cellular senescence. By confirming appearance of increase of cellular granularity (Fig. 3) and SA β-gal activity (Fig. 4), generally accepted senescent markers, the arrest was proved to be associated with cellular senescence. The senescence like phenotype by TGF β1 was developed in dose-
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and time-dependent manners (Fig. 4). Forthemore, these senescence-like phenotypes were not found in R1B cells, Type Ⅰ receptor null Mv1Lu mutant cells (Fig. 5), clearly suggesting that these changes are acquired through the TGFβ receptor signaling.
To assess the effect of FBS on the growth arrest induced by TGF β1, TGF β1 was applied in the presence of various concentrations of FBS. TGF β1 induced most of the cells to apoptosis with serum concentration lower than 2 %, evidenced by trypan blue-stained dead cells, sub G1 population after PI staining, and even by cleavage of poly (ADP-ribose) polymerase (PARP). However, when treated with TGF β1 in media containing higher than 2 % FBS concentration, the apoptotic dead cells gradually decreased (Fig. 6), implying that the senescent arrest of Mv1Lu cells by TGF β1 needs the help of serum to protect from triggering its apoptotic signal pathway.
(A) N u m b e r o f c e lls N u m b e r o f c e lls N u m b e r o f c e lls N u m b e r o f c e lls 103 104 105 106 0 00
0 1111 3333 5555 7777 (days)(days)(days)(days)
TGF TGF TGF TGF ββββ1111 (2ng/ml) (2ng/ml)(2ng/ml) (2ng/ml) CON CON CON CON N u m b e r o f c e lls N u m b e r o f c e lls N u m b e r o f c e lls N u m b e r o f c e lls 103 104 105 106 0 00
0 1111 3333 5555 7777 (days)(days)(days)(days)
103 104 105 106 103 104 105 106 0 00
0 1111 3333 5555 7777 (days)(days)(days)(days)
TGF TGF TGF TGF ββββ1111 (2ng/ml) (2ng/ml)(2ng/ml) (2ng/ml) CON CON CON CON G1 63.4% S 20.1% G2/M 13.8% G1 79.9% S 12.2% G2/M 6.9% CON CON CON CON TGF TGF TGF TGF ββββ1111 3days 3days3days 3days (B) CON TGF β1 (2ng/ml) 3day (C)
Fig. 2. TGF ββββ1 induced G1 arrest and morphological change of Mv1Lu cells.
(A) Exponentially growing Mv1Lu cells (1 x 103) were seeded in 24-well plates and cultured in DMEM containing 10% FBS and were treated with (■) or without (◆) TGF β1 for the periods indicated. At the indicated times, cells were harvested by trypsinization and counted by excluding dead cells with trypan blue staining. Average counts and S.D. from four independent experiments are presented. (B), (C) Mv1Lu cells were cultured in DMEM medium containing 10 % FBS and 2 ng/ml TGF β1 for three days. Cell cycle patterns were examined by staining with PI for flow cytometric analysis. Cellular morphology was visualized by inverted microscope.
25 0 10 20 30 40 50 60 70 0 1 2 3 4 (days) G ra n u la ri ty i n c re a s e (R 1 % o f to ta l) 0 10 20 30 40 50 60 70 0 1 2 3 4 (days) G ra n u la ri ty i n c re a s e (R 1 % o f to ta l) (A) (B) C 1d 2d 4d 3d R1 R1 R1 R1 R1 C 1d 2d 4d 3d R1 R1 R1 R1 R1
Fig. 3. Analysis of increase in TGF ββββ1 induced cellular granularity. Mv1Lu cells
were treated with 2 ng/ml TGF β1 in DMEM containing 10 % FBS for the indicated times. Then cells were analyzed for cellular granularity by flow cytometric analysis (A). Percentages of R1 region are presented by means and S.D. (B).
(X200)
(X200) (X100)(X100)
con
concon
con 3 days3 days3 days3 days 5 days5 days5 days5 days 5 days5 days5 days5 days (A)
(B)
Fig. 4. TGF ββββ1 induced SA ββββ-gal activity in dose- and time-dependent manner in
Mv1Lu cells. Mv1Lu cells treated for the indicated time in the presence of 1, 2 and 4
ng/ml TGF β1 were stained for SA β-gal activities (A). Strongly stained cells for SA β -gal activity were counted and expressed as the percentage of total cells. Averages ± SD of the percentage are presented (B).
1 11 1 0 2.110 2.110 2.11±0 2.11±±±0.16 0.16 0.16 0.16 2.122.122.122.12±±0.24±±0.240.240.24 2.852.852.852.85±±±±0.500.500.500.50 3 33 3 0 18.020 18.020 18.02±0 18.02±±±6.53 6.53 6.53 6.53 33.2833.28±33.2833.28±3.52±±3.523.523.52 44.2544.2544.2544.25±±±4.32±4.324.324.32 5 55 5 0.580.58±0.580.58±±±0.15 62.530.15 62.53±0.15 62.530.15 62.53±±12.61 ±12.61 12.61 12.61 79.34±79.3479.3479.34±±8.30±8.308.308.30 86.27±86.2786.2786.27±±±3.003.003.003.00 7 7 7 7 0.870.87±0.870.87±±±0.25 86.540.25 86.54±0.25 86.540.25 86.54±±2.93 ±2.93 2.93 2.93 86.01±86.0186.0186.01±±±4.07 4.07 4.07 4.07 87.14±87.1487.1487.14±±±3.173.173.173.17 C C C C 1 1 ng1 1 ngng/mlng/ml/ml/ml 2 2 2 2 ngng/mlngng/ml/ml/ml 4 4 4 4 ngngngng/ml /ml /ml /ml days days days days
(stained cells/total cells, %) (stained cells/total cells, %) (stained cells/total cells, %) (stained cells/total cells, %)
TGF TGF TGF TGF----ββββ1111 1 11 1 0 2.110 2.110 2.11±0 2.11±±±0.16 0.16 0.16 0.16 2.122.122.122.12±±0.24±±0.240.240.24 2.852.852.852.85±±±±0.500.500.500.50 3 33 3 0 18.020 18.020 18.02±0 18.02±±±6.53 6.53 6.53 6.53 33.2833.28±33.2833.28±3.52±±3.523.523.52 44.2544.2544.2544.25±±±4.32±4.324.324.32 5 55 5 0.580.58±0.580.58±±±0.15 62.530.15 62.53±0.15 62.530.15 62.53±±12.61 ±12.61 12.61 12.61 79.34±79.3479.3479.34±±8.30±8.308.308.30 86.27±86.2786.2786.27±±±3.003.003.003.00 7 7 7 7 0.870.87±0.870.87±±±0.25 86.540.25 86.54±0.25 86.540.25 86.54±±2.93 ±2.93 2.93 2.93 86.01±86.0186.0186.01±±±4.07 4.07 4.07 4.07 87.14±87.1487.1487.14±±±3.173.173.173.17 C C C C 1 1 ng1 1 ngng/mlng/ml/ml/ml 2 2 2 2 ngng/mlngng/ml/ml/ml 4 4 4 4 ngngngng/ml /ml /ml /ml days days days days 1 11 1 0 2.110 2.110 2.11±0 2.11±±±0.16 0.16 0.16 0.16 2.122.122.122.12±±0.24±±0.240.240.24 2.852.852.852.85±±±±0.500.500.500.50 3 33 3 0 18.020 18.020 18.02±0 18.02±±±6.53 6.53 6.53 6.53 33.2833.28±33.2833.28±3.52±±3.523.523.52 44.2544.2544.2544.25±±±4.32±4.324.324.32 5 55 5 0.580.58±0.580.58±±±0.15 62.530.15 62.53±0.15 62.530.15 62.53±±12.61 ±12.61 12.61 12.61 79.34±79.3479.3479.34±±8.30±8.308.308.30 86.27±86.2786.2786.27±±±3.003.003.003.00 7 7 7 7 0.870.87±0.870.87±±±0.25 86.540.25 86.54±0.25 86.540.25 86.54±±2.93 ±2.93 2.93 2.93 86.01±86.0186.0186.01±±±4.07 4.07 4.07 4.07 87.14±87.1487.1487.14±±±3.173.173.173.17 C C C C 1 1 ng1 1 ngng/mlng/ml/ml/ml 2 2 2 2 ngng/mlngng/ml/ml/ml 4 4 4 4 ngngngng/ml /ml /ml /ml days days days days
(stained cells/total cells, %) (stained cells/total cells, %) (stained cells/total cells, %) (stained cells/total cells, %)
TGF TGF TGF TGF----ββββ1111
27
CON
TGF
β
1(2ng/ml) 3days
Fig. 5. TGF ββββ1 doesn’t induce senescent phenotypes in R1B cells R1B cells were
cultured in DMEM medium containing 10 % FBS and 2 ng/ml TGF β1 for three days. Cells were stained for SA β-gal activity.
α αα
α--tubulin--tubulintubulintubulin
(FBS, %) 0.5 1 2 5 10 0.5 1 2 5 10 0.5 1 2 5 10 0.5 1 2 5 10 PARP PARP PARP PARP 0 10 20 30 0.5 1 2 5 10 c e lls i n s u b -G 1 (% o f to ta l c e lls ) (FBS, %) 0 10 20 30 40 50 60 70
0.5
0.5
0.5
0.5
1111
2222
5555
10
10 (FBS, %)
10
10
(FBS, %)
(FBS, %)
(FBS, %)
S ta in e d c e ll s ( % o f to ta l c e ll s ) S ta in e d c e ll s ( % o f to ta l c e ll s ) S ta in e d c e ll s ( % o f to ta l c e ll s ) S ta in e d c e ll s ( % o f to ta l c e ll s ) (A) (B) (C)Fig. 6. TGF ββββ1-induced senescence-like arrest of Mv1Lu cells depends on the
presence of fetal bovine serum. (A-C) After Mv1Lu cells were treated with 2 ng/ml
TGF β1 in DMEM containing various concentrations (0.2 – 10 %) of FBS for 3 days, trypan blue-stained dead cells were counted (A) and proportion of cells in sub G1 phase was analyzed by flow cytometric analysis after PI staining (C). Average and S.D. from four independent experiments are presented. (B) PARP expression level and cleavage pattern were examined by Western blot analysis.
29
B. Prolonged ROS generation and mitochondrial membrane potential (MMP) disruption during TGF ββββ1-induced senescence-like growth arrest
It has long been hypothesized that mitochondrial defect and its resultant oxidative stress play a key role in the cellular aging process (Sastre et al., 1999). Therefore, we questioned whether and how mitochondrial defect and ROS generation were involved in the senescence like arrest induced by TGF β1. First, we monitored mitochondrial membrane potential (∆Ψm) by staining the cells with JC-1 fluorescence dye. With increase of exposure time to TGF β1, green fluorescence-stained cells, which represented the cells harboring inactive mitochondria with low ∆Ψm, gradually increased and the degree of ∆Ψm disruption reached maximum on day 3 to 4 (Fig. 7). When monitored with DCF fluorescence (Fig. 8, A and B), intracellular ROS level under the same condition also persistently rose until the fourth day and sustained thereafter. The ROS increase was also observed with DHR123, a dye known to capture mitochondrial ROS, implying that possibility of the ROS was generated from mitochondria (Fig. 8, C). We also found that the senescence like growth arrest was also developed by TGF-β1 in WI 26 cells, an immortalized human lung epithelial cell line (Fig. 9). Under the same condition, the ∆Ψm disruption and ROS increase were also accompanied (Fig. 10), implying that the ∆Ψm collapse and ROS production by TGF β1 might not be a result from specific cell line.
(B) (196.1) (274.7) (258.8) (365.7) (352.5) con 1d 2d 3d 4d (mean value) (196.1) (274.7) (258.8) (365.7) (352.5) con 1d 2d 3d 4d (mean value) Con T1d T3d T5d Con T1d T3d T5d (A) JCJCJCJC ----1 g re e n f lu o re s c e n c e 1 g re e n f lu o re s c e n c e 1 g re e n f lu o re s c e n c e 1 g re e n f lu o re s c e n c e (a rb it ra ry u n it , m e a n v a lu e ) (C) 0 100 200 300 400 500 600 700 C 1 2 3 4 5 * ** ** ** *, p<0.05 **, p<0.01 (days)
Fig. 7. TGF ββββ1 induced disruption of mitochondrial membrane potential. Mv1Lu
cells were cultured in DMEM containing 10 % FBS and were treated with 2 ng/ml TGF β1 for the periods indicated. Mitochondrial membrane potential was visualized by staining cells with JC-1 fluorescence dye. Red fluorescence indicates active mitochondria with high ∆Ψm and green for inactive mitochondria (A). The extent of
∆Ψm disruption was quantitated by counting the cells fluoresced green (cells harboring inactive mitochondria) using flow cytometry after staining with JC-1 fluorescence dye (B). Representative pattern of more than three independent experiments is shown. Mean values and S.D. of the arbitrary units of the green fluorescence of are presented (C).
31 con 3d con 3d D H R 1 2 3 f lu o re s c e n c e D H R 1 2 3 f lu o re s c e n c e D H R 1 2 3 f lu o re s c e n c e D H R 1 2 3 f lu o re s c e n c e (a rb it ra ry u n it , m e a n va lu e ) 0 40 80 120 160 200 0 00 0 1111 2222 3333 4444 5555 (days) (days) (days) (days) D H R 1 2 3 f lu o re s c e n c e D H R 1 2 3 f lu o re s c e n c e D H R 1 2 3 f lu o re s c e n c e D H R 1 2 3 f lu o re s c e n c e (a rb it ra ry u n it , m e a n va lu e ) 0 40 80 120 160 200 0 00 0 1111 2222 3333 4444 5555 (days) (days) (days) (days)
(A)
D C F f lu o re s c e n c e (a rb it ra ry u n it , m e a n v a lu e ) (days) (days)(days) (days) 0 50 100 150 200 250 300 350 0 00 0 0.50.50.50.5 1111 2222 3333 4444(B)
* *
*
*
*, p < 0.01 D C F f lu o re s c e n c e (a rb it ra ry u n it , m e a n v a lu e ) (days) (days)(days) (days) 0 50 100 150 200 250 300 350 0 00 0 0.50.50.50.5 1111 2222 3333 4444(B)
* *
*
*
*, p < 0.01(C)
Fig. 8. Prolonged ROS generation during TGF ββββ1-induced senescence like growth
arrest. Mv1Lu cells were exposed to 2 ng/ml TGF β1 for the periods indicated.
Intracellular ROS levels were estimated by comparing the fluorescence using flow cytometry after staining the cells with DCFH-DA fluorescence dye. Representative patterns of flow cytometric analysis (A) and mean values (B) are presented. The ROS levels were also detected with DHR123 fluorescence dye with the same method (C).
103 104 105 106 1 3 5 7 (days) 103 104 105 106 1 3 5 7 (days) N u m b e r o f c e lls N u m b e r o f c e lls N u m b e r o f c e lls N u m b e r o f c e lls TGF TGF TGF TGF----ββββ1111 CON CON CON CON con con con con 3 days 3 days3 days
3 days 5 days5 days5 days5 days
(A)
(B)
Fig. 9. TGF ββββ1 induced senescent phenotypes in WI 26 cells. WI 26 human lung type
I epithelial cells (1 x 103) were seeded in 24-well plates and cultured in DMEM containing 0.5 % FBS, and were treated with (■) or without (◆) TGF β1 for the indicated periods. (A) Cellular growth rates were obtained by counting live cells after staining cells with trypan blue. Average counts and S.D. from four independent experiments are presented. (B) Cells were stained for SA β-gal activities.
33 CON CON CON CON T3 T3 T3 T3 (195.9) (267.1) (mean value) ROS ROS ROS ROS (290.5) (371.2) (mean value) MMP MMPMMP MMP (A) (B)
Fig. 10. TGF ββββ1 induced increased in intracellular ROS level and ∆Ψm disruption
in WI 26 cells. WI 26 human lung type I epithelial cells (1 x 103) were seeded in
24-well plates and cultured in DMEM containing 0.5 % FBS. Intracellular ROS level (A) and ∆Ψm disruption (B) were analyzed without (con) or with treatment of TGF β1 for 3 days (T3).
C. The increased ROS level resulted from defective mitochondrial respiration
To explore the relationship between the ROS production and ∆Ψm disruption, we first examine the effect of antioxidants, N-acetyl cysteine (NAC) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), on TGF β 1-induced ROS overproduction and ∆Ψm disruption. Pretreatment of the cells for 3 hour with either NAC or Trolox could sufficiently eluminate the ROS generated by TGF β1, but not ameliorate the disrupted ∆Ψm (Fig. 11 and 12), implying that the ROS generation and ∆Ψm disruption were independent from each other or the ROS might be generated by defective mitochondrial function. Since mitochondrial respiratory complexes have long been considered as one of the main sources of ROS generation (Islam et al., 1997; Ewen et al., 1993), to assess the hypothesis, we employed specific inhibitors of mitochondrial oxidative phosphorylation. When 2mM KCN (a complex IV inhibitor) or 2 µM oligomycin (a complex V inhibitor) was applied to the cells for 5 hours which were exposed to TGF β1 for 3days, ROS generation was completely blocked (Fig. 13, A). On the other hand, the TGF β1-induced ROS generation was not blocked by DPI (an inhibitor of NAD(P)H oxidase) and only slightly reduced by AA 861 (Lipoxygenase 5 inhibitor) and Baicalein (Lipoxygenase 5 and 12 inhibitor) (Fig. 13, B), leading to conclusion that the continually overproduced
35
ROS by TGF β1 was mainly derived from mitochondrial respiratory chain. To further elucidate whether TGF β1 really affected mitochondrial respiration, we monitored cellular O2 consumption rate. As shown in Fig. 15, DNP-uncoupled cellular respiration rate decreased upon treatment of TGF β1 in a time-dependent manner and coupled-respiration rate was also decreased (data not shown), suggesting that the late prolonged ROS generation by TGF β1 results from the defect of mitochondrial respiration.
In a few recent reports, TGF β1 has already been known to induce ROS generation within 18 h after treatment. The ROS production was associated with NAD(P)H oxidase activity and accompanied by apoptotic process of fibroblasts. We therefore tested the overall pattern of ROS generation by TGF β1 in our model system. We also could detect early transient ROS production within 12 h and thereafter prolonged ROS generation (Fig. 14, A). The early ROS could partially be removed by treatment of DPI, but not by AA 861 or Baicalein (Fig. 14, B), implying that it might be generated through NAD(P)H oxidase and other hither-to-unidentified oxygen-derived reaction.
Next, we examined whether intra-mitochondrial redox state was changed by the ROS produced from mitochondria. As shown in Fig. 17 A, the expression level of MnSOD, famed mitochondrial antioxidant system, was not changed, whereas Prx III which is exclusively expressed within mitochondria was gradually oxidized and shifted to monomer form (Fig. 16, B-D), supporting that
the mitochondria are situated under oxidative stress due to the elevated ROS production from respiration.
It has been known that disruption of mitochondrial membrane potential and its respiratory function is critical for release of mitochondrial apoptogenic proteins. However, the mitochondrial respiratory defects and consequent ∆Ψm collapse by TGF β1, no mitochondrial apoptogenic molecules such as cytochrome C, Smac, and AIF were released.
37
(A)
(362.28 ±64.68) (723.82 ± 137.88) (386.26 ± 78.02)CON
TGF-
ββββ
1
(3day)
NAC
(5mM)/
TGF-
ββββ
1
(363.88 ±45.43) (258.02 ±3.29) (379.75 ±39.33(mean value) (mean value)
MMP
ROS
DCF staining
DCF staining
DCF staining
DCF staining
JC
JC
JC
JC-
-
-
-1 staining
1 staining
1 staining
1 staining
(B)
Fig. 11. Effect of antioxidants (NAC) on ROS level and mitochondrial membrane potential. Mv1Lu cells were pretreated with NAC (5 mM) for 3 hr before exposure to 2
ng/ml TGF β1. After 3 day-exposure, ROS level (A) and the extent of ∆Ψm disruption (B) were analyzed by staining with DCFH-DA and JC-1 fluorescence dye, respectively. Average + SD of mean values of the fluorescence intensity for ROS level and ∆Ψm disruption are presented
Trolox Trolox Trolox Trolox: : : : ---- ---- ++++ TGF TGF TGF TGF ββββ1: 1: 1: 1: ---- + ++ ++ ++ + 0 20 40 60 80 100 120 140 JCJCJCJC ----1 gr ee n flu or es ce nc e 1 gr ee n flu or es ce nc e 1 gr ee n flu or es ce nc e 1 gr ee n flu or es ce nc e (a rb itr ar y un it, m ea n va lu e) Trolox TroloxTrolox Trolox: : : : ---- ---- ++++ TGF TGF TGF TGF ββββ1: 1: 1: 1: ---- + ++ ++ ++ + 0 40 80 120 160 200 D C F flu or es ce nc e D C F flu or es ce nc e D C F flu or es ce nc e D C F flu or es ce nc e (a rb itr ar y un it, m ea n va lu e) (A) (B)
Fig. 12. Effect of antioxidants (Trolox) on mitochondrial membrane potential.
Mv1Lu cells were pretreated with Trolox (10 mM) for 3 hr before exposure to 2 ng/ml TGF β1. After 3 day-exposure, ROS level (A) and the extent of ∆Ψm disruption (B) were analyzed by staining with DCFH-DA and JC-1 fluorescence dye, respectively. Average and SD of mean values of the fluorescence intensity for ROS level (A) and
39 KCN : KCN : KCN : KCN : ---- ---- + + -+ + + + + + --- --- -TGF TGFTGF
TGF----ββββ1(3day) : 1(3day) : 1(3day) : 1(3day) : ---- + -+ + + --- + -+ + + --- ++++ oligomycin oligomycin oligomycin oligomycin : : : -: --- ---- ---- ---- + ++ ++ ++ + D C F f lu o re s c e n c e D C F f lu o re s c e n c e D C F f lu o re s c e n c e D C F f lu o re s c e n c e (a rb it ra ry u n it , m e a n v a lu e ) 0 50 100 150 200 250 TGF TGFTGF
TGF----ββββ1: 1: 1: 1: ---- ---- ---- ---- + + + + (3day)+ + + + (3day)+ + + + (3day)+ + + + (3day) DPI: DPI: DPI: DPI: ---- + + + + ---- ---- ---- + + + + ---- --- -AA861: AA861: AA861: AA861: ---- ---- + -+ + + --- ---- ---- + + + + --- -Baicalein Baicalein Baicalein Baicalein: : : : ---- ---- ---- + -+ + + --- ---- ---- ++++ 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 D C F f lu o re sc e n c e D C F f lu o re sc e n c e D C F f lu o re sc e n c e D C F f lu o re sc e n c e (a rb it ra ry u ni t, m e a n va lu e ) (A) (B)
Fig. 13. The prolonged ROS generation was originated from defective
mitochondrial function. (A) TGF β1-treated cells for 3 days were further incubated
with KCN (2 mM) or oligomycin (2 µM) for 5 hr, and then ROS levels were measured using DCFH-DA staining. (B) TGF β1-treated cells for 3 days were further incubated with DPI (5 µM), AA861 (10 µM) and Baicalein (10 µM) for 1 hr and ROS levels were measured using DCFH-DA staining. Average and S.D. of mean values of fluorescence intensity for ROS level are presented.
TGF TGFTGF TGF----ββββ1: 1: 1: 1: ---- + + + + -+ + + + + + + + + + + + --- ---- ---- (4hr) (4hr) (4hr) (4hr) DPI: DPI: DPI: DPI: ---- ---- + + + + ---- ---- + + + + ---- --- -AA861: AA861: AA861: AA861: ---- ---- ---- + + -+ + --- ---- + + + + --- -Baicalein Baicalein Baicalein Baicalein: : : -: --- ---- ---- ---- + + + + ---- ---- ++++ 0 40 80 120 160 200 D C F f lu o re s c e n c e D C F f lu o re s c e n c e D C F f lu o re s c e n c e D C F f lu o re s c e n c e (a rb it ra ry u n it , m e a n v a lu e ) D C F f lu o re s c e n c e D C F f lu o re s c e n c e D C F f lu o re s c e n c e D C F f lu o re s c e n c e (a rb it ra ry u n it , m e a n v a lu e ) (hr) (hr) (hr) (hr) 0 50 100 150 200 250 0 1 4 6 12 24 48 0 1 4 6 12 24 48 0 1 4 6 12 24 48 0 1 4 6 12 24 48 72 9672 9672 9672 96 (A) (B)
Fig. 14. Overall pattern of ROS generation during TGF ββββ1-induced
senescence-like growth arrest. Mv1Lu cells were cultured in DMEM medium containing 10%
FBS and were treated with 2 ng/ml TGF-β1 for the indicated periods. Intracellular ROS level was detected with DCFH-DA fluorescence dye. Average and S.D. from three independent experiments are presented (A). To identify the involvement of NADPH oxidase and lipoxygenase in the ROS generation, DPI (5 µM), AA861 (10 µM) and Baicalein (10 µM) were applied for 30 min before TGF-β1 treatment, and then cells were stained with DCFH-DA. fluorescence dye (B).
41 CON CON CON CON T3 T3 T3 T3 0 5 10 15 20 25 30 35 0 0 0 0 12121212 24242424 48484848 72727272 (hr)(hr)(hr)(hr) OOOO2222 c o n s u m p ti o n r a te c o n s u m p ti o n r a te c o n s u m p ti o n r a te c o n s u m p ti o n r a te (O 2 u m o l/ L /m in ) DNP DNP DNP
DNP CellCellCellCell KCNKCNKCNKCN
0 1 2 3 4 5 6 7(min) 300 250 200 150 100 50 0 OOOO2222 (((( µµµµ m o l/ L ) m o l/ L ) m o l/ L ) m o l/ L )
Fig. 15. The prolonged ROS generation was originated from defective
mitochondrial respiration. After exposure to TGF β1 (2 ng/ml) for the indicated times,
maximum cellular respiration rates were determined as a DNP-coupled versus KCN-inhibited O2 consumption rate, and expressed as percentage of control (inset) as described in “Experimental Procedures”.
Prx Prx Prx PrxⅢⅢⅢⅢ complex II ( complex II ( complex II ( complex II (FpFpFp))))Fp 0 1 2 3 0 1 2 3 (days) mt mt mt
mt cytcytcytcyt
dimer dimerdimer dimer monomer monomermonomer monomer Prx PrxPrx
Prx IIIIIIIIIIII MitoMitoMitoMito MergeMergeMergeMerge
0 1 2 3 (days) VDAC Mn-SOD mt mt mt mt C C C C 1d 1d1d 1d 2d 2d2d 2d 3d 3d3d 3d 3d 3d 3d 3d pH=3 pH=10 PrxⅠⅠⅠⅠ PrxⅢⅢⅢⅢ PrxⅡⅡⅡⅡ PrxⅢⅢⅢⅢ oxi oxioxi
oxi redredredred
(A) (D)
(B)
(C)
Fig. 16. Oxidation of mitochondrial peroxiredoxin III. (A) Expression level of
Mn-SOD is analyzed by Western blot analysis. VDAC was used as a control for fractionated mitochondrial proteins. (B) Western blot analysis for Prx III after non-reducing PAGE of mitochondrial and cytosolic fractions of TGF β1-treated cells. Antibody against Fp subunit of complex II was used to identify the mitochondrial fractions. (C) Intracellular localization of Prx III within Mv1Lu cells was analyzed by immunocytochemistry (Prx III). Mitochondria were visualized by pre-staining with MitoTracker-red (Mito). (D) Redox patterns of Prx proteins were analyzed by Western blot analysis after 2D-PAGE using a mixtures of antibodies against Prx I (open arrow), Prx II (open arrowhead), and Prx III (arrow, reduced form; arrowhead, oxidized form). Western blot analysis with Prx III antibody only is shown in the bottom panel to identify Prx III.
43
0 1 2 3 0 1 2 3 0 1 2 3
0 1 2 3 0 1 2 3 (days)0 1 2 3 (days)0 1 2 3 (days)0 1 2 3 (days)
αααα
-
-
-
-tubulin
tubulin
tubulin
tubulin
complex
complex
complex
complexⅡ
Ⅱ
Ⅱ
Ⅱ(Fp
(Fp
(Fp
(Fp))))
Cyt.
Cyt.
Cyt.
Cyt.
c
c
c
c
14.3- 45- 66-Mitochondria Mitochondria MitochondriaMitochondria CytosolCytosolCytosolCytosol
AIF
AIF
AIF
AIF
50-Smac
Smac
Smac
Smac
30-FP
FP
FP
FP
66-0 1 2 3
0 1 2 3 (days)
αααα
-
-tubulin
-
-
tubulin
tubulin
tubulin
45-Mitochondria Mitochondria Mitochondria
Mitochondria CytosolCytosolCytosolCytosol 0 1 2 3
0 1 2 3 0 1 2 3
0 1 2 3 0 1 2 3 (days)0 1 2 3 (days)0 1 2 3 (days)0 1 2 3 (days)
αααα
-
-
-
-tubulin
tubulin
tubulin
tubulin
complex
complex
complex
complexⅡ
Ⅱ
Ⅱ
Ⅱ(Fp
(Fp
(Fp
(Fp))))
Cyt.
Cyt.
Cyt.
Cyt.
c
c
c
c
14.3- 45- 66-Mitochondria Mitochondria MitochondriaMitochondria CytosolCytosolCytosolCytosol
AIF
AIF
AIF
AIF
50-Smac
Smac
Smac
Smac
30-FP
FP
FP
FP
66-0 1 2 3
0 1 2 3 (days)
αααα
-
-tubulin
-
-
tubulin
tubulin
tubulin
45-Mitochondria Mitochondria Mitochondria
Mitochondria CytosolCytosolCytosolCytosol
Fig. 17. The levels of mitochondrial apoptogenic proteins in mitochondrial
and cytosolic fractions. TGF β1 treated Mv1Lu cells harvested and mitochondria fractionated. Antibodies against Fp subunit of complex II and tubulin were used to identify the mitochondrial and cytosolic fractions. Slowly migrated bands (*) of the α-tubulin blot are the traces of complex II bands.
D. The late prolonged ROS production was associated with decreased complex ⅣⅣⅣⅣ activity.
Then, how could mitochondrial respiration be decreased? In order to answer this question, we monitored the activities of mitochondrial respiratory chain complexes. There were no significant changes observed in the complex I/III-coupled activity (NADH-cytochrome c reductase activity) and complex II/III-coupled activity (succinate-cytochrome c reductase), implying that all the complex I, II, and III activities are intact (Fig. 18). However, when exposed to TGF β1, the complex IV activity was significantly decreased from 24 hr, estimated by its oxidizing capacity of reduced cytochrome c spectrophotometrically (Fig. 19, A). And complex IV activity was further analyzed by O2 consumption rate under the condition that the complex III was inhibited by antimycin A and endogenous cytochrome c was reduced by TMPD (Fig. 19, B). The O2 consumption rate by the complex IV was clearly decreased from 12hr, suggesting that the defective mitochondrial respiration might have been primarily due to the decreased complex IV activity.
Employing KCN, a complex IV activity inhibitor, we next addressed whether the decrease of the complex IV activity could be the cause of the ROS production. With the concentrations of KCN (Fig. 20, A), at which cellular respiration was partially or completely inhibited, significant generation of
45
intracellular ROS was observed (Fig. 20, B). Furthermore, transient treatment of Mv1Lu cells with H2O2 (0.8 or 1mM) for 2 hr was enough to destine the cells to senescence like arrest (Fig. 21), demonstrating that Mv1Lu cells exposed to increased intracellular ROS level caused by TGF β1 were susceptible to develop senescence like growth arrest .
Finally, we analyzed that removal of ROS generated could reverse the acquisition of senescent phenotypes. As shown in Fig. 22, the cellular granularity decreased significantly, strongly implying the critical role of ROS in
140 0 20 40 60 80 100 120 C 0.5 1 2 3 (days) C o m p le x I-III c o u p le d a ct iv it y (% o f c on tr ol ) 0 20 40 60 80 100 120 140 C 0.5 1 2 3 (days) C o m p lex II -I II co u p le d act iv it y (% o f c ont rol ) (A) (B)
Fig. 18. Effects of TGF ββββ1 on mitochondrial respiratory complex activity. (A)
Complex I-III coupled activity was measured by spectrometric monitoring of the activity to reduce cytochrome c after adding NADH. (B) Complex II-III coupled activity was spectrometrically monitored by the activity to reduce cytochrome c in the presence of excessive succinate.
47
(B)
0 20 40 60 80 100 120 OOOO2222 c o n su m p ti o n r a te c o n su m p ti o n r a te c o n su m p ti o n r a te c o n su m p ti o n r a te (O 2 u m o l/ L /m in ) C C C C 0.50.50.50.5 1111 2222 33 (days)33 0 20 40 60 80 100 120 OOOO2222 c o n su m p ti o n r a te c o n su m p ti o n r a te c o n su m p ti o n r a te c o n su m p ti o n r a te (O 2 u m o l/ L /m in ) C C C C 0.50.50.50.5 1111 2222 33 (days)33 0.0 0.5 1.0 1.5 2.0 2.5 3.0 p m o l c yt .C /m in /u g p ro te in C 0.5 1 2 3 (days)(A)
* ** **
* *, p< 0.01*, p< 0.05Fig. 19. Specific inhibition of complex IV activity Complex IV activity was
determined by spectrophotometric measurement of the ability to oxidize the reduced cytochrome c (A) and by monitoring complex IV-dependent O2 consumption rate in the presence of TMPD (B) after blocking complex III with antimycin A in both assays, as described in “Materials and Methods.”
0 100 200 300 400 500 600 700 0 5 30 60 0.8mM 1.5mM 3mM D C F f lu o re s c e n c e (A rb it ra ry u n it , m e a n v a lu e ) (min) 0 100 200 300 400 500 600 700 0 5 30 60 0.8mM 1.5mM 3mM D C F f lu o re s c e n c e (A rb it ra ry u n it , m e a n v a lu e ) (min) 0 20 40 60 80 100 120 C 0.8 1.5 2 KCN (mM)
C
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(% o f co nt ro l) (A) (B)Fig. 20. Enhanced generation of intracellular ROS level by inhibiting complex IV
activity with KCN. (A) DNP-uncoupled cellular O2 consumption rates of Mv1Lu cells
were examined in the presence of various concentrations of KCN to optimize the KCN concentration required for complex IV inhibition. (B) Cells pre-incubated with DCFH-DA (10 µM) were treated with 0.8 (◆), 1.5 (■) and 3 mM () KCN for the indicated
periods. The intracellular ROS level was quantitated by flow cytometric analysis after washing the DCF-stained cells with PBS.
49
CON
CONCON
CON HHHH2222OOOO2,2,2,2,(1mM)+Rel 3day(1mM)+Rel 3day(1mM)+Rel 3day(1mM)+Rel 3day
Fig. 21. Acquisition of senescent phenotypes by H2O2 in Mv1Lu cells. Mv1Lu cells
were cultured in DMEM containing 10 % FBS, treated with H2O2 (1 mM) for 2 hr, then replaced with fresh medium without H2O2, and further incubated for another 3 days after release. Then cells were stained for SA β-gal activities.
C NAC NACT T3 R1 R1 R1 R1 (A) 0 10 20 30 40 50 60 C T NACT NAC
*
* : p < 0.05 G ra n u la ri ty i n c re a s e ( R 1 % o f to ta l)(B)
Fig. 22. Partial reversal of senescent phenotype by pre-treatment of NAC. Mv1Lu
cells were pretreated with NAC (5 mM) for 3 hr before exposure to 2 ng/ml TGF β1. After 3 days, cellular granularity was analysed with side-scatter analysis by flow cytometer (A). Value of R1 region was represented with mean and S.D. (B).
51
E. HGF reversed TGF ββββ1-induced senescent arrest by protecting mitochondrial function
Recently, several reports described the reciprocal effect of HGF on TGF β 1-induced growth arrest of epithelial cells (Tsubari et al., 1999), however, its effect on senescence like arrest has not well been documented. In our study, simultaneous treatment of HGF together with TGF β1 could clearly reverse the senescence like phenotypes including decrease of growth rate, G1 arrest, and appearance of SA β-gal activity in the presence of 10 % FBS (Fig. 23 and Fig. 24). As shown in Fig. 24, clear reversal was observed even with transient 3 h-pretreatment of HGF followed by wash with 2 M NaCl, implying that primary gene products induced by HGF might block the senescent arrest by TGF β1. Eventually, we examined the effect of HGF on mitochondrial function. As shown in Fig. 25, HGF maintained ∆Ψm, and prevented the mitochondrial ROS overproduction. And HGF also protected mitochondrial respiratory function from TGF β1-induced damage (Fig. 26).
Control ControlControl Control TGF TGFTGF TGF----β1 (β1 (β1 (β1 (2ng/ml)2ng/ml)2ng/ml)2ng/ml) T2+H (20ng/ml) T2+H (20ng/ml)T2+H (20ng/ml) T2+H (20ng/ml) H20 ( H20 ( H20 ( H20 (ngngngng/ml)/ml)/ml)/ml) Control ControlControl Control TGF TGFTGF TGF----β1 (β1 (β1 (β1 (2ng/ml)2ng/ml)2ng/ml)2ng/ml) T2+H (20ng/ml) T2+H (20ng/ml)T2+H (20ng/ml) T2+H (20ng/ml) H20 ( H20 ( H20 ( H20 (ngngngng/ml)/ml)/ml)/ml) 103 104 105 106 107 0 1 3 5 7 (days) N um be r of c el ls C C C C 55.88 55.88 55.88 55.88 24.20 12.21 24.20 12.21 24.20 12.21 24.20 12.21 T2 T2 T2 T2 ngngngng/ml/ml/ml/ml 64.06 64.06 64.06 64.06 12.99 5.6612.99 5.6612.99 5.6612.99 5.66 H20 H20 H20 H20 ngngngng/ml/ml/ml/ml 58.4458.4458.4458.44 23.17 10.6623.17 10.6623.17 10.6623.17 10.66 T2+H20 T2+H20 T2+H20 T2+H20 ngngngng/ml/ml/ml/ml 56.0856.0856.0856.08 24.36 7.4524.36 7.4524.36 7.4524.36 7.45 G1 S G2/M G1 S G2/M G1 S G2/M G1 S G2/M (3 days) (A) (B)
Fig. 23. HGF reversed the TGF β1β1β1β1-induced growth arrest. (A) Mv1Lu cells were
treated with 2 ng/ml TGF β1 in the absence or presence of 20 ng/ml HGF for the indicated periods. After the indicated periods, cellular growth rate was examined by counting live cells after staining them with trypan blue. (B) Cells cycle patterns were examined by staining PI for flow cytometric analysis. [C, untreated control cells; T, cells treated with TGF β1 (2 ng/ml) alone; H, cells with HGF (20 ng/ml) alone; T+H, cells co-treated with TGF β1 (2 ng/ml) and HGF (20 ng/ml) for 3 days].
53 TGF TGF TGF TGF ββββ1: 1: 1: 1: ---- + + + + + + + + + + + + --- -HGF: HGF: HGF: HGF: ---- ---- + + ++ + ++ + ++ + + N u m b e r o f N u m b e r o f N u m b e r o f N u m b e r o f ββββ ----g a l s ta in e d c e lls g a l s ta in e d c e lls g a l s ta in e d c e lls g a l s ta in e d c e lls (% o f to ta l c e lls ) (% o f to ta l c e lls ) (% o f to ta l c e lls ) (% o f to ta l c e lls ) 0 10 20 30 40 50 60 co co co co prepreprepre
C e ll n u m b e r C e ll n u m b e r C e ll n u m b e r C e ll n u m b e r TGF TGF TGF TGF ββββ1: 1: -1: 1: --- + + + + + + -+ + + + + + -- -HGF: HGF: HGF: HGF: ---- ---- + + ++ + ++ + ++ + + 103 104 105 106 co co co co prepreprepre
(B)
(A)
Fig. 24. HGF reversed the TGF β1β1β1β1-induced senescence like growth arrest. (A)
Mv1Lu cells were treated with 2 ng/ml TGF β1 plus HGF for 3 days (co) or with HGF alone for 3 hr, washed with 2 M NaCl, and then treated with TGF β1 for 3 days (pre). Numbers of total live cells (A) and SA β-gal stained cells (B) were counted. [C, untreated control cells; T, cells treated with TGF β1 (2 ng/ml) alone; H, cells with HGF (20 ng/ml) alone; T+H, cells co-treated with TGF β1 (2 ng/ml) and HGF (20 ng/ml) for 3 days].
