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Inhibition of phosphatidylinostol 3-kinase uncouples H2O2-induced senescent phenotype and cell cycle arrest in normal human diploid fibroblasts
Experimental Cell Research 298 (2004) 188 – 196 www.elsevier.com/locate/yexcr
Inhibition of phosphatidylinostol 3-kinase uncouples H2O2-induced senescent phenotype and cell cycle arrest in normal human diploid fibroblasts Yong Wang, Aimin Meng, and Daohong Zhou * Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA Department of Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA Received 9 February 2004, revised version received 8 April 2004 Available online 10 May 2004
Abstract Exposure of WI38 human diploid fibroblasts (HDFs) to hydrogen peroxide (H2O2) induced premature senescence. The senescent HDFs were permanently arrested and exhibited a senescent phenotype including enlarged and flattened cell morphology and increased senescenceassociated h-galactosidase (SA-h-gal) activity. The induction of HDF senescence was associated with an activation of p53, increased expression of p21Cip1/WAF1, and hypophosphorylation of retinoblastoma protein (Rb), while no changes in the expression of p16Ink4a, p27Kip1, and p14Arf were observed. Exposure of WI38 cells to H2O2 also selectively activated phosphatidylinostol 3-kinase (PI3 kinase) and mitogenactivated protein kinase (MAPK) kinase (MEK), while no changes in p38 MAPK and Jun kinase (JNK) activities were observed. Selective inhibition of PI3 kinase activity with LY294002 abrogated H2O2-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-h-gal activity, but did not affect H2O2-induced cell cycle arrest. In contrast, selective inhibition of MEK and p38 MAPK with PD98059 and SB203580, respectively, produced no significant effect on H2O2-induced senescent phenotype and cell cycle arrest. These findings demonstrate that expression of the senescent phenotype can be uncoupled from cell cycle arrest in prematurely senescent cells induced by H2O2 and does not contribute to the maintenance of permanent cell cycle arrest. D 2004 Elsevier Inc. All rights reserved. Keywords: Human diploid fibroblasts; Hydrogen peroxide; Senescence; PI3 kinase
Introduction Premature senescence refers to a shortened intrinsic replicative life span in cells exposed to a stress [1,2]. This occurs when cells are exposed to an oxidative or genotoxic stress that causes DNA damage. It also happens when they are subjected to an oncogenic stress (such as ectopic overexpression of Ras or Raf by gene transfection) that results in aberrant regulation of cell proliferation. Many types of normal and tumor cells undergo premature senescence after exposure to H2O2, radiation, or a DNA damaging agent [1– 3]. Unlike replicatively senescent cells, prematurely senes-
* Corresponding author. Department of Pathology, Medical University of South Carolina, 165 Ashley Avenue, Suite 309, PO Box 250908, Charleston, SC 29425. Fax: +1-843-792-0368. E-mail address: [email protected] (D. Zhou). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.04.012
cent cells show no erosion in telomeres [1,2]. However, cells undergoing premature senescence are morphologically indistinguishable from replicatively senescent cells [1,2]. Prematurely senescent cells exhibit some of the same characteristics as replicatively senescent cells, including permanent cell cycle arrest, enlarged and flattened cell morphology, and increased senescence-associated h-galactosidase (SA-h-gal) activity [1,2]. It has been hypothesized that the induction of senescence acts as failsafe mechanism to prevent cancer by eliminating cells that are at risk of neoplastic transformation. Although senescent cells are permanently arrested, their cell size continuously enlarges. This paradoxical phenomenon suggests that cell proliferation (including cell cycling and division) and growth (in mass or size) are uncoupled in senescent cells, and these two processes may be regulated by distinct mechanisms. It has been well established that the cell cycle arrest in senescent cells is
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mediated by activation of p53 and induction of various cyclin-dependent kinase (CDK) inhibitors resulting from DNA damage or oncogenic stress [1,2]. However, the mechanisms that are responsible for the continuous cell growth and increase in SA-h-gal expression in senescent cells have not been well elucidated [4– 9]. Moreover, it remains to be determined (1) whether permanent cell cycle arrest and phenotypic changes in senescent cells are coupled or distinct processes and (2) whether these phenotypic changes contribute to the maintenance of permanent cell cycle arrest in senescent cells [4– 9]. Exposure of normal human diploid fibroblasts (HDFs) to H2O2 has been widely used as a model system to investigate the mechanisms of senescence induction [6,10,11]. Using this model system we examined the role of phosphatidylinostol 3-kinase (PI3 kinase) in H2O2-induced permanent cell cycle arrest and phenotypic changes in HDFs. This is because H2O2 stimulates PI3 kinase activity, and cell growth (in mass or size) is mainly regulated by the PI3 kinase pathway [12 – 14]. Our results showed that HDFs senesced after brief exposure to a sublethal dose of H2O2. The induction of HDF senescence was associated not only with a prolonged activation of the p53 – p21Cip1/WAF1 pathway but also with a sustained stimulation of PI3 kinase signaling. Inhibition of PI3 kinase activity with a selective PI3 kinase inhibitor abrogated H2O2-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-h-gal expression, but did not affect H2O2-induced cell cycle arrest. In contrast, selective inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) or p38 MAPK produced no significant effect on the H2O2-induced cell cycle arrest or senescent phenotype. These results suggest that activation of PI3 kinase is primarily responsible for mediating H2O2-induced phenotypic changes in senescent HDFs. The cell enlargement and increased expression of SA-h-gal are uncoupled from cell cycle arrest in senescent cells and do not contribute to the maintenance of permanent cell cycle arrest.
Materials and methods Reagents and antibodies Selective inhibitors for PI3 kinase (LY294002 and Wortmannin), MEK (PD98059), and p38 MAPK (SB203580) were purchased from Calbiochem (San Diego, CA). H2O2, 5-bromo-2V-deoxyuridine (BrdU), phenylmethylsulfonyl fluoride (PMSF), leupeptin, and aprotinin were obtained from Sigma (St. Louis, MO). Mouse monoclonal antibody against p21Cip1/WAF1 (SC-6246); rabbit polyclonal antibodies against p14Arf (SC-8340) and p27Kip1 (SC-528); and goat polyclonal antibodies against actin (SC-1615) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit polyclonal antibodies specific for extracel-
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lular-regulated kinase (ERK, or p44/42 MAPK) (9102), p53 (9282), Ser15 phosphorylated-p53 (p-p53) (9284), protein kinase B (PKB or Akt) (9272), Ser473 phosphorylated-PKB (p-PKB) (9271), p38 MAPK (9212), Thr180/Tyr182 phosphorylated-p38 MAPK (p-p38 MAPK) (9211), stress-activated protein kinase (SAPK)/Jun kinase (JNK) (9252), Thr183/Tyr185 phosphorylated-JNK (p-JNK) (9251), and Ser807/811 phosphorylated-retinoblastoma protein (pRb) (9308); and mouse monoclonal antibody against Thr202/ Tyr204 phosphorylated-ERK (p-ERK) (9106) were obtained from Cell Signaling Technology (Beverly, MA). Mouse anti-human p16Ink4a antibody (554,070) was obtained from BD PharMingen (San Diego, CA). Cell culture WI-38 cells (human embryonic lung-derived diploid fibroblasts) were originally obtained from ATCC. They were cultured in complete medium (Modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 Ag/ml streptomycin) in a humidified incubator at 37jC and 5% CO2. Cells at early passages (between 16 and 25 passages) were used in all experiments to avoid complications of replicative senescence as WI38 cells have a mean life span about 45 – 60 passages. For the induction of premature senescence, WI-38 cells at about 75% confluence were briefly exposed to 150 AM H2O2 (diluted in Modified Eagle’s medium supplemented with 10% FBS) for 2 h. The cells were washed twice with Modified Eagle’s medium (MEM) to remove H2O2 and recultured in fresh complete medium for 24 h and then subcultured for various durations as specified in individual experiments. Cell proliferation assay Cell proliferation and viability were assessed by the crystal violet-staining assay as described previously [15]. Briefly, WI-38 cells (2 104 cells/well) were seeded into wells of a 12-well plate and cultured in complete medium for 24 h and then pulse-exposed to 150 AM H2O2 as described above in the presence or absence of an inhibitor. After various days of culture, the cells were stained with 0.1% crystal violet for 30 min, washed with phosphatebuffered saline (PBS) extensively, and allowed to air dry. They were lysed in 0.5 ml of 10% acetic acid and the absorbance at 562 nM was measured using an automatic microtiter plate reader (Molecular Devices Corp., Sunnyvale, CA). Cell cycle analysis Cells were harvested from tissue culture by trypsinization, washed with cold PBS, and then fixed with ice-cold 70% ethanol at 4jC overnight. The cells were incubated in 1 ml of DNA staining solution (0.1 mM EDTA in PBS, pH
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7.4, 0.05 mg/ml RNase A, 50 Ag/ml propidium iodide) at room temperature for 30 min. The intensity of PI DNA staining was measured using a FACS Caliber flow cytometer equipped with CellQuest software, and the cell cycle distribution was analyzed using ModFit program (Becton Dickinson, San Jose, CA).
ferrocyanide, 5 mM potassium ferricyanide, 150 mM sodium chloride, and 2 mM magnesium chloride) at 37jC for 10 h. Senescent cells were identified as blue-stained cells by standard light microscopy, and a total of 1000 cells were counted in 20 random fields on a slide to determine the percentage of SA-h-gal positive cells.
Cell size determination
Western blot analysis
Forward scatter profiles were analyzed by live cell flow cytometry using a FACS Caliber flow cytometer (Becton Dickinson) to measure the size of WI38 cells with or without H2O2 treatment in the presence or absence of LY294002 as described above. The results of the analysis were expressed as mean FSC-H.
WI38 cells were lysed on ice in a lysis buffer (20 mM Tris – HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1.0% NP-40, 0.1 M NaF, 1 mM DTT, 1 mM PMSF, 1 mM NaVO4, 2 Ag/ml leupeptin and aprotinin) for 30 min, and cell extracts were collected after centrifugation of the cell lysates at 12,000 g for 10 min. The protein concentrations of these cell extracts were quantified using the Bio-Rad Dc protein assay kit (BioRad Laboratories, Hercules, CA). An equal amount of protein (40 Ag/lane) from each cell extract was resolved on a 10% SDS-PAGE gel. Proteins were blotted to an Immuno-Blot polyvinylidenediflouride (PVDF) membrane (Bio-Rad Laboratories) by electrophoresis. The membranes were blocked with TBS-T blocking buffer (5% nonfat milk in 25 mM Tris – HCl, pH 7.4, 3 mM KCl, 140 mM NaCl, and 0.05% Tween) and subsequently probed with a relevant primary antibody at a predetermined optimal concentration overnight at 4jC or for 2 h at room temperature. After extensive washing with TBS-T, the immunoblots were then incubated with an appropriate peroxidase-conjugated secondary antibody for 1 h at room temperature. After three washes with TBS-T, the immunoblots were detected using the ECL Western Blotting Detection Reagents (Amersham Pharmacia Biotech, Piscataway, NJ) and recorded by exposure of the immunoblots to an X-ray film (Pierce Biotech, Inc., Rockford, IL).
BrdU incorporation assay DNA synthesis was determined by measuring BrdU incorporation into DNA as previously described with some modifications [16]. Briefly, WI-38 cells were incubated in complete medium containing 10 AM BrdU (Sigma) in a 4well glass slide chamber (Nalge Nunc Inc., Naperville, IL) for 6 h. They were washed twice with PBS and fixed in 70% ethanol at 20jC for 30 min. After three washes with PBS, the cells were incubated in DNA denaturing solution (2N HCl/0.1% Triton X-100 in PBS) at 37jC for 30 min followed by a 10-min incubation in 0.1 M sodium borate (pH 8.5) at room temperature to denature DNA. After being washed with PBS and a 60-min incubation in 1% bovine serum albumin/PBS, the cells were incubated overnight at 4jC with 2 Ag/ml mouse anti-BrdU monoclonal antibody (clone BU-33, from Sigma) and then with Texas Red Dyeconjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) with extensive washing between each step. The nuclear DNA of the cells was counterstained with Hoechst 33342 (Molecular Probes, Eugene, OR). The cells were viewed and photographed using an Axioplan research microscope (Carl Zeiss Inc, Jena, Germany) equipped with a 100 W mercury light source. The images were captured with a Dage CCD100 integrating camera (Dage-MTI, Michigan, USA) and a Flashpoint 128 capture board (Integral Technologies, Indiana, USA). The captured images were processed using Image Pro Plus software (Media Cybernetics, Maryland, USA) and displayed with Adobe Photoshop V6.0. SA-b-gal assay SA-h-gal activity was determined using a SA-h-gal staining kit from Cell Signaling Technology according to the manufacturer’s instruction. Briefly, cells were fixed in 2% (v/v) formaldehyde and 0.2% glutaraldehyde and then incubated in SA-h-gal staining solution (1 mg/ml 5-bromo4-chloro-3-indolyl h-D-galactosidase, 40 mM citric acid, pH 6.0, 40 mM sodium phosphate, pH 6.0, 5 mM potassium
Immunofluorescence staining Activation and nuclear translocation of p53 was analyzed by immunofluorescence staining. Briefly, WI38 cells cultured in a 4-well glass slide chamber were fixed with ice-cold methanol for 10 min at 4jC and then permeabilized with 0.2% Triton X-100. After blocking with 5% normal goat serum, they were incubated with a rabbit polyclonal antibody against p-p53 (from Cell Signaling) overnight at 4jC and then with a rhodamine-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) at room temperature for 1 h after extensive washing between each step. The slides were washed three times with PBS and incubated with Hoechst 33342 (Molecular Probes) at room temperature for 3– 5 min to stain DNA. After a final washing with PBS, the slides were mounted using Gel/Mount (Biomeda Corp, Forster, CA). A Zeiss Axiophot fluorescence microscope coupled to a digital camera utilizing Adobe Photoshop software was used to view and acquire images as described above.
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Results H2O2 induces apoptosis and senescence in HDFs in a dose-dependent manner
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cell size (Fig. 1D). These observations confirm that exposure of WI38 fibroblasts to a sublethal concentration of H2O2 induces premature senescence [6,10,11,17]. H2O2 activates p53– p21Cip1/WAF1 pathway in HDFs
WI38 cells were exposed to increasing concentrations of H2O2. Two hours after the exposure, H2O2 was removed from the culture and the cells were washed and recultured in fresh medium for various times to determine oxidative stress-induced apoptosis and senescence. It was found that a brief exposure of WI38 cells to H2O2 for 2 h at a concentration of 200 AM or above caused a significant increase in apoptosis as the cells were detached and stained positive for annexin V (data not shown). In contrast, pulse exposure to H2O2 for the same duration (2 h) at a concentration of 150 AM or below induced minimal cell death (data not shown). However, the majority of the cells that survived the brief treatment with 150 AM H2O2 ceased to proliferate within 3 –5 days after H2O2 had been removed from the culture (Fig. 1A). These surviving cells became permanently arrested in the G1 phase of the cell cycle and failed to synthesize DNA as measured by BrdU incorporation assay (Figs. 1B – C). In addition, they exhibited phenotypic changes that resemble those observed in the cells undergoing replicative senescence. These changes include increased SA-h-gal activity, flattened cell morphology, and enlarged
To determine if H2O2 induces HDF senescence by activation of p53, WI38 cells were treated with 150 AM H2O2 for 0, 0.2, 0.5, 1, or 2 h and then the cells were lysed. In addition, groups of cells treated with H2O2 for 2 h were lysed after they were incubated in fresh complete medium for 4, 8, or 16 h or for 1, 2, 3, 5, 7, or 9 days after removal of H2O2. The phosphorylation of p53 and expression of p53 and p21Cip1/WAF1 were examined in these cell extracts. As shown in Figs. 2A – B, control WI38 cells expressed a substantial amount of p53 under basal conditions and its expression was slightly augmented after H2O2 treatment. However, the expression of p21Cip1/WAF1 was minimal and phosphorylation of p53 was undetectable in untreated WI38 cells. Shortly after H2O2 exposure, a significant increase in p53 phosphorylation was detected. The increase in p53 phosphorylation occurred within 0.2 h, peaked around 2 h, and remained elevated up to 9 days after H2O2 treatment. Immunofluorescence staining using a polyclonal antibody against phosphorylated-p53 revealed that the majority of the phosphorylated-p53 had translocated into nuclei (Fig. 2C).
Fig. 1. H2O2 induces premature senescence of WI-38 HDFs. (A) H2O2 inhibits cell proliferation. The growth curve of control or H2O2-treated WI-38 cells was determined at days 1, 3, or 5 after brief H2O2 treatment (2 h) by the crystal violet-staining assay. The data are presented as mean F SE of OD value at 562 nM (n = 3). The error bars for control cells are invisible because the values of SEM are too small. (B) H2O2 induces a G1 cell cycle arrest. A representative analysis of cell cycle distribution in control or day 5 post H2O2-treated cells is presented. (C) H2O2 inhibits DNA synthesis. Cells in the S phase of the cell cycle were detected by BrdU incorporation assay in control or in day 5 post H2O2-treated cells. (D) H2O2 increases SA-h-gal activity. Representative photomicrographs of SA-h-gal staining for control or day 7 post H2O2-treated cells are presented.
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Fig. 2. H2O2 activates the p53 – p21Cip1/WAF1 pathway in WI38 HDFs. (A) WI38 cells were treated with H2O2 for 0 – 2 h or they were treated with H2O2 for 2 h and then incubated for 2 – 14 h after removal of H2O2. The expression of phosphorylated p53 (p-p53), p53, and p21Cip1/WAF1 was determined by Western blot analysis. (B) WI38 cells were treated with H2O2 for 2 h and the expression of phosphorylated p53 (p-p53) and p21Cip1/WAF1 in control (C) or H2O2-treated cells was determined at various times by Western blot analysis. (C) The phosphorylation and nuclear translocation of p53 induced by H2O2 were examined in control cells or 2-h H2O2-treated cells at 24 h post H2O2 treatment by immunofluorescence and Hoechst 33342 staining. Representative photomicrographs of the staining are shown. (D) WI38 cells were treated with H2O2 for 2 h and then incubated for 5 days after removal of H2O2. The expression of phosphorylated p53 (p-p53), p21Cip1/WAF1 (p21), p16Ink4a (p16), p27Kip1 (p27), p14Arf (p14), and phosphorylated Rb (p-Rb) in control (C) or in H2O2-treated cells (H2O2) was determined by Western blot analysis. Actin immunoblots serve as a control for equal protein loading.
The nuclear translocated p53 acted as a transcription factor that induces p21Cip1/WAF1 expression, as the expression of p21Cip1/WAF1 was increased in H2O2-treated cells at 8 h after the appearance of phosphorylated-p53 and remained augmented up to 9 days after H2O2 treatment (Figs. 2A –B). These results suggest that exposure of WI38 cells to a sublethal concentration of H2O2 activates p53, probably
via induction of oxidative DNA damage [6,11,18]. Activated p53 induces the expression of p21Cip1/WAF1 that in turn may mediate H2O2-induced cell cycle arrest by inhibiting several CDKs that phosphorylate Rb as reported previously (Fig. 2D) [1,2,6,11]. This suggestion is further supported by the finding that H2O2 treatment induced hypophosphorylation of Rb while having no significant
Fig. 3. H2O2 selectively induces PI3 kinase and MEK activation in HDFs. (A) WI38 cells were incubated in H2O2 for 0 – 2 h or they were treated with H2O2 for 2 h and then incubated for 4 – 16 h after removal of H2O2. The expression of phosphorylated PKB (p-PKB), PKB, phosphorylated ERK (p-ERK), ERK, phosphorylated p38 MAPK (p-p38), p38 MAPK (p38), phosphorylated JNK (p-JNK), and JNK was determined by Western blot analysis. (B) WI38 cells were treated with H2O2 for 2 h and then incubated for various times after removal of H2O2. The expression of phosphorylated PKB (p-PKB) and PKB in control (C) or H2O2-treated cells was determined by Western blot analysis. (C) Anisomycin-treated C-6 glioma cell extracts and UV-treated 293 cell extracts (Provided by Cell Signaling Technology) were used as a positive control for the Western blot analysis of phosphorylated p38 MAPK (p-p38) and phosphorylated JNK (p-JNK) of WI38 cells with (H2O2) or without H2O2 treatment (C). Actin immunoblots serve as a control for equal protein loading.
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effect on the expression of p16Ink4a, p27Kip1, and p14Arf (Fig. 2D). However, increase in p16Ink4a and p14Arf expression may occur at later time points after H2O2 treatment, which plays an important role in the maintenance of irreversible cell cycle arrest [1,2,6,11]. H2O2 selectively induces PI3 kinase and MEK activation in HDFs Exposure of cells to H2O2 also activates various kinases that may modify the cellular responses to H2O2-induced DNA damage and participate in the processes of senescence induction [19,20]. Therefore, we examined the effect of H2O2 exposure on the activities of PI3 kinase, MEK, p38 MAPK, and JNK by measuring the levels of phosphorylated
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PKB, ERK, p38 MAPK, and JNK. This is because activation of these kinases has been observed in certain types of cells after exposure to an oxidant and implicated in the induction of apoptosis and/or senescence by oxidative stress or oncogenic stimulation [2,11,19,20]. As shown in Figs. 3A – B, exposure of WI38 cells to 150 AM H2O2 resulted in an immediate activation of PI3 kinase as manifested by the increase in PKB phosphorylation. The activation of PI3 kinase peaked around 0.5– 1 h after exposure to H2O2 and lasted for more than 5 days. Activation of MEK by H2O2 was also indicated by the increased phosphorylation of ERK after PI3 kinase activation (Fig. 3A). However, the activity of MEK declined to the basal level at 16 h after H2O2 treatment (Fig. 3A). In contrast, no activation of p38 MAPK and JNK was detected in the cells treated with H2O2. The
Fig. 4. Selective inhibition of PI3 kinase uncouples H2O2-induced senescent phenotype and cell cycle arrest in HDFs. (A) Selective inhibition of PI3 kinase and MEK by LY294002 (LY) and PD98059, respectively. WI38 cells were pretreated with LY294002 (LY, 20 AM) or PD98059 (PD, 25 AM) for 30 min before 1 h incubation with H2O2. The cells pretreated with vehicle (0.5% DMSO) or the JNK inhibitor SB203580 (SB, 5 AM) were used as control. The expression of phosphorylated PKB (p-PKB), PKB, phosphorylated ERK (p-ERK), and ERK was determined by Western blot analysis. (B and C) Inhibition of PI3 kinase abrogated the H2O2-induced senescence phenotype. WI38 cells were pretreated with vehicle (0.5% DMSO), LY294002 (LY, 20 AM), PD98059 (PD, 25 AM), or SB203580 (SB, 5 AM) for 30 min before 2 h incubation with H2O2. After removal of H2O2, the cells were continuously cultured with these inhibitors for 5 days. The changes in SA-h-gal activity and cell morphology were determined by SA-h-gal staining and microscopy. Representative microscopic images are presented in B and the percentages of SA-h-gal positively stained cells were quantified and are presented in C as mean F SE (n = 4). (D and E) Inhibition of PI3 kinase failed to affect H2O2-induced cell cycle arrest. WI38 cells were pretreated with vehicle (0.5% DMSO), LY294002 (LY, 20 AM), PD98059 (PD, 25 AM), or SB203580 (SB, 5 AM) for 30 min before 2 h incubation with H2O2. After removal of H2O2, the cells were cultured with these inhibitors for 5 days and then released from inhibition by subculturing the cells in fresh complete medium without inhibitors. The growth curves of these cells were determined at day 1, 3, or 5 after the release by the crystal violet-staining assay and the data are presented in D as mean F SE of OD value at 562 nM (n = 3). The cells in the S phase of the cell cycle were determined by BrdU incorporation assay 5 days after the subculture and the data presented in E are mean percentage of BrdU positive cells F SE (n = 5). A, P < 0.01 vs. control; B, P < 0.01 vs. H2O2 + DMSO.
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inability to detect p38 MAPK and JNK activation in these H2O2-treated cells was not due to a lack of the expression of p38 MAPK and JNK in WI38 cells, nor did it result from a deficiency in the antibodies used in the assay to detect phosphorylated p38 MAPK and JNK since phosphorylated p38 MAPK and JNK were detected in two positive control cell extracts (Figs. 3A, C). These results suggest that exposure of WI38 cells to H2O2 selectively activates PI3 kinase/PKB and MEK with different kinetics. Selective inhibition of PI3 kinase uncouples H2O2-induced senescent phenotype and cell cycle arrest in HDFs To determine if activation of PI3 kinase and/or MEK contributes to H2O2-induced premature senescence in HDFs, PI3 kinase and MEK were selectively inhibited by their respective inhibitors LY294002 (LY, 20 AM) and PD98059 (PD, 25 AM) before a brief exposure of WI38 cells to H2O2 as described above [21,22]. These inhibitors were kept in the culture for 5 days after the removal of H2O2, since the pulse exposure of the cells to H2O2 caused a prolonged activation of PI3 kinase activity (Figs. 3A, B). As a control, the cells were pretreated with vehicle (0.5% DMSO) or the selective p38 MAPK inhibitor-SB203580 (SB, 5 AM) [23]. As shown in Fig. 4A, incubation of WI38 cells with LY and PD selectively inhibited H2O2-induced activation of PI3 kinase and MEK, respectively, whereas the activities of these kinases were not affected by SB. Interestingly, selective inhibition of PI3 kinase with LY abrogated H 2 O 2 -induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-h-gal activity, but had no significant effect on H2O2induced cell cycle arrest (Figs. 4B –E). In addition, forward scatter analysis by flow cytometry confirmed the inhibitory effect of LY on H2O2-induced increase in cell size, as the means (FSD) of FSC-H were 146.5 (F0.4), 161.6 (F0.3), and 102.5 (F6.1) for control, H2O2-treated, and H2O2 plus LY-treated cells, respectively. Similar results were also found in the cells treated with another PI3 kinase inhibitor Wortmannin (data not shown). In contrast, selective inhibition of MEK or p38 MAPK did not produce any significant effect on H2O2-induced senescent phenotype and cell cycle arrest (Figs. 4B –E). These results suggest that the senescent phenotype is uncoupled from cell cycle arrest in the senescent HDFs induced by H2O2. Activation of PI3 kinase is mainly responsible for mediating H2O2-induced phenotypic changes in senescent HDFs.
Discussion Pulse exposure of HDFs to a sublethal concentration of H2O2 induces permanent cell cycle arrest and phenotypic changes that mimic replicatively senescent cells [6,10,11, 18]. As reported previously, prematurely senescent HDFs induced by H2O2 exhibited an elevation in p53 activation
and p21Cip1/WAF1 expression, probably due to H2O2-induced oxidative DNA damage [6,11,18]. Increased levels of p21Cip1/WAF1 may mediate the initiation of H2O2-induced cell cycle arrest by inhibiting various CDKs that phosphorylate Rb to allow the G1 to S phase progression [1,2,6,11,18]. This hypothesis is supported by the finding that hypophosphorylation of Rb was only associated with an increased expression of p21Cip1/WAF1, while no changes in p16Ink4a, p27Kip1, and p14Arf expression were observed in WI38 cells after H2O2 treatment. These findings are in agreement with previous reports demonstrating that inactivation of p53 and/or Rb by ectopic transfection of E6 and/or E7 genes abrogated the cell cycle arrest induced by H2O2 [6,11,18], indicating that p53 and Rb control G1 cell cycle arrest in HDFs in response to H2O2-induced oxidative damage. However, p16Ink4a and/or p14Arf can be induced in prematurely or replicatively senescent HDFs at later time points after the senescent process is initiated. It has been suggested that p16Ink4a and/or p14Arf may play an important role in the maintenance of irreversible cell cycle arrest [1,2,6,11]. Exposure of HDFs to H2O2 also induces increased SAh-gal activity, flattened cell morphology, and enlarged cell size. The mechanisms by which H2O2 induces these phenotypic changes were poorly elucidated and the relationship between the senescent phenotype and permanent cell cycle arrest was highly controversial. It was reported that inactivation of Rb by ectopic transfection with the human papillomaviral (HPV) E7 (E7) gene prevented IMR-90 cells from H2O2-induced cell cycle arrest and cell enlargement but had no significant effect on the increase in SA-h-gal activity [5,6,18,24]. This suggests that the irreversible growth arrest induced by H2O2 exposure in HDFs may be coupled to the cell enlargement but not to the increased SAh-gal activity. However, uncoupling of cell cycle arrest and the senescent phenotype has been found in terminal-passage HPV E6 transfected cells or p21Cip1/WAF1 / HDFs, which exhibit a senescent phenotype but continuously proliferate [7,25]. Furthermore, cells ectopically transfected with p21Cip1/WAF1 or p27Kip1 underwent senescence without the appearance of senescent phenotype [4]. In contrast, treatment of replicatively senescent HDFs with nicotinamide reversed their senescent phenotype but did not affect their replicative capacity [8]. To further clarify the relationship between the senescent phenotype and permanent cell cycle arrest and to elucidate the mechanisms that may be responsible for the induction of senescent phenotype, we examined the role of PI3 kinase in H2O2-induced senescence in HDFs. This is because it has been well documented that exposure of various types of cells to H2O2 activates PI3 kinase via tyrosine phosphorylation of the p110 catalytic and/or p85 regulatory subunits by receptor-associated and/or Src family tyrosine kinases [12 – 14,26]. The phosphorylation of the p85 subunit contributes to the relocation of p85/p110 complex from the cytosol to the plasma membrane, thereby enabling PI3 kinase to
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phosphorylate its substrate and produce PI(3,4,5)P3 [12 – 14,26,27]. In turn, PI(3,4,5)P3 stimulates cell growth (in mass or size) by activating PI3 kinase down-stream targets, including PKB, ribosomal protein S6 kinase (S6K), and mammalian target of rapamycin (mTOR) [27]. Our study showed that WI38 cells treated with the selective PI3 kinase inhibitor LY but not with a selective MEK or p38 MAPK inhibitor exhibited normal cell morphology after exposure to H2O2. A similar morphological change in H2O2-treated or aged IMR-90 cells has been also reported in a recent study when PI3 kinase was similarly inhibited by LY but without the examination of SA-h-gal expression and cell cycle arrest [9]. Although the cells treated with LY failed to develop a senescent phenotype in response to H2O2 treatment, they remained arrested and unable to reenter the cell cycle and proliferate even after LY and H2O2 were removed from the culture. These findings suggest that the cell cycle arrest and senescent phenotype induced by H2O2 are distinct processes and the phenotypic changes in senescent cells do not contribute to the maintenance of the cell cycle arrest. In addition, our study demonstrates that the expression of senescent phenotype in H2O2-treated HDFs is under the regulation of PI3 kinase signaling, probably by activation of PKB and other PI3 kinase down-stream targets. However, the mechanism by which PI3 kinase regulates SA-h-gal expression has yet to be determined. It should be noted that while inhibiting PI3 kinase with LY that abrogated the cell enlargement induced by H2O2 in HDFs, it only partially inhibited H2O2-induced increase in SA-h-gal expression. This suggests that activation of PI3 kinase by H2O2 is partially responsible for the increase in SA-h-gal activity, and pathways other than PI3 kinase may be also involved in the upregulation of SA-h-gal by H2O2 in HDFs. The finding that inhibition of PI3 kinase suppresses the H2O2-induced senescent phenotype in HDFs indicates that activation of PI3 kinase contributes to H2O2-induced cellular senescence. The involvement of PI3 kinase in cellular senescence has been demonstrated by the studies using C. elegans [28,29]. It was reported that C. elegans with a mutated age-1 gene that encodes a homologue of mammalian PI3 kinase lived much longer that wild-type worms [29]. Treatment of adult C. elegans with LY resulted in a significant increase in their life span [28]. In contrast, it was reported that incubation of normal early passage HDFs or mouse embryo fibroblasts (MEFs) with LY alone without subsequent exposure to H2O2 actually induced senescence [30,31]. The induction of MEF senescence by LY is partially mediated by inhibition of PKB, activation of the forkhead protein AFX, and upregulation of p27Kip1 [30]. However, it remains to be determined whether the cells treated with LY alone enter a real senescent state or undergo quiescence or cell differentiation, as the ‘‘senescent’’ HDFs induced by LY reentered cell cycle and proliferated after they were released from LY inhibition [31]. In contrast, our study showed that the prematurely senescent HDFs induced by H2O2 and LY
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remained arrested after removal of H2O2 and LY. In addition, it has been shown that inhibition of PI3 kinase induces cellular differentiation [9,31 – 33]. Therefore, these conflicting findings suggest that PI3 kinase plays multiple roles in regulation of cellular senescence and differentiation in a manner depending upon whether cells are concurrently exposed to stress. Thus, additional studies are needed to further establish the function of PI3 kinase in cellular senescence under various physiological and pathological conditions.
Acknowledgments The authors thank Drs. Stephen Brown, Bradley Schulte, and Cynthia Wright for critically reviewing the manuscript. This study was supported in part by grants from the National Institutes of Health (R01-CA78688 and R01-CA86688) to Dr. Daohong Zhou. References [1] R. Marcotte, E. Wang, Replicative senescence revisited, J. Gerontol., A Biol. Sci. Med. Sci. 57 (2002) B257 – B269. [2] M. Serrano, M.A. Blasco, Putting the stress on senescence, Curr. Opin. Cell Biol. 13 (2001) 748 – 753. [3] I.B. Roninson, Tumor cell senescence in cancer treatment, Cancer Res. 63 (2003) 2705 – 2715. [4] K. Alexander, P.W. Hinds, Requirement for p27(KIP1) in retinoblastoma protein-mediated senescence, Mol. Cell. Biol. 21 (2001) 3616 – 3631. [5] Q.M. Chen, V.C. Tu, J. Liu, Measurements of hydrogen peroxide induced premature senescence: senescence-associated beta-galactosidase and DNA synthesis index in human diploid fibroblasts with down-regulated p53 or Rb, Biogerontology 1 (2000) 335 – 339. [6] Q.M. Chen, Replicative senescence and oxidant-induced premature senescence. Beyond the control of cell cycle checkpoints, Ann. N.Y. Acad. Sci. 908 (2000) 111 – 125. [7] V. Dulic, G.E. Beney, G. Frebourg, L.F. Drullinger, G.H. Stein, Uncoupling between phenotypic senescence and cell cycle arrest in aging p21-deficient fibroblasts, Mol. Cell. Biol. 20 (2000) 6741 – 6754. [8] K. Matuoka, K.Y. Chen, T. Takenawa, Rapid reversion of aging phenotypes by nicotinamide through possible modulation of histone acetylation, Cell. Mol. Life Sci. 58 (2001) 2108 – 2116. [9] K. Matuoka, K.Y. Chen, T. Takenawa, A positive role of phosphatidylinositol 3-kinase in aging phenotype expression in cultured human diploid fibroblasts, Arch. Gerontol. Geriatr. 36 (2003) 203 – 219. [10] Q. Chen, B.N. Ames, Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 4130 – 4134. [11] O. Toussaint, E.E. Medrano, T. von Zglinicki, Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes, Exp. Gerontol. 35 (2000) 927 – 945. [12] S. Qin, P.B. Chock, Implication of phosphatidylinositol 3-kinase membrane recruitment in hydrogen peroxide-induced activation of PI3K and Akt, Biochemistry 42 (2003) 2995 – 3003. [13] M. Shaw, P. Cohen, D.R. Alessi, The activation of protein kinase B by H2O2 or heat shock is mediated by phosphoinositide 3-kinase and not by mitogen-activated protein kinase-activated protein kinase-2, Biochem. J. 336 (Pt. 1) (1998) 241 – 246.
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Inhibition of phosphatidylinostol 3-kinase uncouples H2O2-induced senescent phenotype and cell cycle arrest in normal human diploid fibroblasts Yong Wang, Aimin Meng, and Daohong Zhou * Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA Department of Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29425, USA Received 9 February 2004, revised version received 8 April 2004 Available online 10 May 2004
Abstract Exposure of WI38 human diploid fibroblasts (HDFs) to hydrogen peroxide (H2O2) induced premature senescence. The senescent HDFs were permanently arrested and exhibited a senescent phenotype including enlarged and flattened cell morphology and increased senescenceassociated h-galactosidase (SA-h-gal) activity. The induction of HDF senescence was associated with an activation of p53, increased expression of p21Cip1/WAF1, and hypophosphorylation of retinoblastoma protein (Rb), while no changes in the expression of p16Ink4a, p27Kip1, and p14Arf were observed. Exposure of WI38 cells to H2O2 also selectively activated phosphatidylinostol 3-kinase (PI3 kinase) and mitogenactivated protein kinase (MAPK) kinase (MEK), while no changes in p38 MAPK and Jun kinase (JNK) activities were observed. Selective inhibition of PI3 kinase activity with LY294002 abrogated H2O2-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-h-gal activity, but did not affect H2O2-induced cell cycle arrest. In contrast, selective inhibition of MEK and p38 MAPK with PD98059 and SB203580, respectively, produced no significant effect on H2O2-induced senescent phenotype and cell cycle arrest. These findings demonstrate that expression of the senescent phenotype can be uncoupled from cell cycle arrest in prematurely senescent cells induced by H2O2 and does not contribute to the maintenance of permanent cell cycle arrest. D 2004 Elsevier Inc. All rights reserved. Keywords: Human diploid fibroblasts; Hydrogen peroxide; Senescence; PI3 kinase
Introduction Premature senescence refers to a shortened intrinsic replicative life span in cells exposed to a stress [1,2]. This occurs when cells are exposed to an oxidative or genotoxic stress that causes DNA damage. It also happens when they are subjected to an oncogenic stress (such as ectopic overexpression of Ras or Raf by gene transfection) that results in aberrant regulation of cell proliferation. Many types of normal and tumor cells undergo premature senescence after exposure to H2O2, radiation, or a DNA damaging agent [1– 3]. Unlike replicatively senescent cells, prematurely senes-
* Corresponding author. Department of Pathology, Medical University of South Carolina, 165 Ashley Avenue, Suite 309, PO Box 250908, Charleston, SC 29425. Fax: +1-843-792-0368. E-mail address: [email protected] (D. Zhou). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.04.012
cent cells show no erosion in telomeres [1,2]. However, cells undergoing premature senescence are morphologically indistinguishable from replicatively senescent cells [1,2]. Prematurely senescent cells exhibit some of the same characteristics as replicatively senescent cells, including permanent cell cycle arrest, enlarged and flattened cell morphology, and increased senescence-associated h-galactosidase (SA-h-gal) activity [1,2]. It has been hypothesized that the induction of senescence acts as failsafe mechanism to prevent cancer by eliminating cells that are at risk of neoplastic transformation. Although senescent cells are permanently arrested, their cell size continuously enlarges. This paradoxical phenomenon suggests that cell proliferation (including cell cycling and division) and growth (in mass or size) are uncoupled in senescent cells, and these two processes may be regulated by distinct mechanisms. It has been well established that the cell cycle arrest in senescent cells is
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mediated by activation of p53 and induction of various cyclin-dependent kinase (CDK) inhibitors resulting from DNA damage or oncogenic stress [1,2]. However, the mechanisms that are responsible for the continuous cell growth and increase in SA-h-gal expression in senescent cells have not been well elucidated [4– 9]. Moreover, it remains to be determined (1) whether permanent cell cycle arrest and phenotypic changes in senescent cells are coupled or distinct processes and (2) whether these phenotypic changes contribute to the maintenance of permanent cell cycle arrest in senescent cells [4– 9]. Exposure of normal human diploid fibroblasts (HDFs) to H2O2 has been widely used as a model system to investigate the mechanisms of senescence induction [6,10,11]. Using this model system we examined the role of phosphatidylinostol 3-kinase (PI3 kinase) in H2O2-induced permanent cell cycle arrest and phenotypic changes in HDFs. This is because H2O2 stimulates PI3 kinase activity, and cell growth (in mass or size) is mainly regulated by the PI3 kinase pathway [12 – 14]. Our results showed that HDFs senesced after brief exposure to a sublethal dose of H2O2. The induction of HDF senescence was associated not only with a prolonged activation of the p53 – p21Cip1/WAF1 pathway but also with a sustained stimulation of PI3 kinase signaling. Inhibition of PI3 kinase activity with a selective PI3 kinase inhibitor abrogated H2O2-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-h-gal expression, but did not affect H2O2-induced cell cycle arrest. In contrast, selective inhibition of mitogen-activated protein kinase (MAPK) kinase (MEK) or p38 MAPK produced no significant effect on the H2O2-induced cell cycle arrest or senescent phenotype. These results suggest that activation of PI3 kinase is primarily responsible for mediating H2O2-induced phenotypic changes in senescent HDFs. The cell enlargement and increased expression of SA-h-gal are uncoupled from cell cycle arrest in senescent cells and do not contribute to the maintenance of permanent cell cycle arrest.
Materials and methods Reagents and antibodies Selective inhibitors for PI3 kinase (LY294002 and Wortmannin), MEK (PD98059), and p38 MAPK (SB203580) were purchased from Calbiochem (San Diego, CA). H2O2, 5-bromo-2V-deoxyuridine (BrdU), phenylmethylsulfonyl fluoride (PMSF), leupeptin, and aprotinin were obtained from Sigma (St. Louis, MO). Mouse monoclonal antibody against p21Cip1/WAF1 (SC-6246); rabbit polyclonal antibodies against p14Arf (SC-8340) and p27Kip1 (SC-528); and goat polyclonal antibodies against actin (SC-1615) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit polyclonal antibodies specific for extracel-
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lular-regulated kinase (ERK, or p44/42 MAPK) (9102), p53 (9282), Ser15 phosphorylated-p53 (p-p53) (9284), protein kinase B (PKB or Akt) (9272), Ser473 phosphorylated-PKB (p-PKB) (9271), p38 MAPK (9212), Thr180/Tyr182 phosphorylated-p38 MAPK (p-p38 MAPK) (9211), stress-activated protein kinase (SAPK)/Jun kinase (JNK) (9252), Thr183/Tyr185 phosphorylated-JNK (p-JNK) (9251), and Ser807/811 phosphorylated-retinoblastoma protein (pRb) (9308); and mouse monoclonal antibody against Thr202/ Tyr204 phosphorylated-ERK (p-ERK) (9106) were obtained from Cell Signaling Technology (Beverly, MA). Mouse anti-human p16Ink4a antibody (554,070) was obtained from BD PharMingen (San Diego, CA). Cell culture WI-38 cells (human embryonic lung-derived diploid fibroblasts) were originally obtained from ATCC. They were cultured in complete medium (Modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 Ag/ml streptomycin) in a humidified incubator at 37jC and 5% CO2. Cells at early passages (between 16 and 25 passages) were used in all experiments to avoid complications of replicative senescence as WI38 cells have a mean life span about 45 – 60 passages. For the induction of premature senescence, WI-38 cells at about 75% confluence were briefly exposed to 150 AM H2O2 (diluted in Modified Eagle’s medium supplemented with 10% FBS) for 2 h. The cells were washed twice with Modified Eagle’s medium (MEM) to remove H2O2 and recultured in fresh complete medium for 24 h and then subcultured for various durations as specified in individual experiments. Cell proliferation assay Cell proliferation and viability were assessed by the crystal violet-staining assay as described previously [15]. Briefly, WI-38 cells (2 104 cells/well) were seeded into wells of a 12-well plate and cultured in complete medium for 24 h and then pulse-exposed to 150 AM H2O2 as described above in the presence or absence of an inhibitor. After various days of culture, the cells were stained with 0.1% crystal violet for 30 min, washed with phosphatebuffered saline (PBS) extensively, and allowed to air dry. They were lysed in 0.5 ml of 10% acetic acid and the absorbance at 562 nM was measured using an automatic microtiter plate reader (Molecular Devices Corp., Sunnyvale, CA). Cell cycle analysis Cells were harvested from tissue culture by trypsinization, washed with cold PBS, and then fixed with ice-cold 70% ethanol at 4jC overnight. The cells were incubated in 1 ml of DNA staining solution (0.1 mM EDTA in PBS, pH
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7.4, 0.05 mg/ml RNase A, 50 Ag/ml propidium iodide) at room temperature for 30 min. The intensity of PI DNA staining was measured using a FACS Caliber flow cytometer equipped with CellQuest software, and the cell cycle distribution was analyzed using ModFit program (Becton Dickinson, San Jose, CA).
ferrocyanide, 5 mM potassium ferricyanide, 150 mM sodium chloride, and 2 mM magnesium chloride) at 37jC for 10 h. Senescent cells were identified as blue-stained cells by standard light microscopy, and a total of 1000 cells were counted in 20 random fields on a slide to determine the percentage of SA-h-gal positive cells.
Cell size determination
Western blot analysis
Forward scatter profiles were analyzed by live cell flow cytometry using a FACS Caliber flow cytometer (Becton Dickinson) to measure the size of WI38 cells with or without H2O2 treatment in the presence or absence of LY294002 as described above. The results of the analysis were expressed as mean FSC-H.
WI38 cells were lysed on ice in a lysis buffer (20 mM Tris – HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1.0% NP-40, 0.1 M NaF, 1 mM DTT, 1 mM PMSF, 1 mM NaVO4, 2 Ag/ml leupeptin and aprotinin) for 30 min, and cell extracts were collected after centrifugation of the cell lysates at 12,000 g for 10 min. The protein concentrations of these cell extracts were quantified using the Bio-Rad Dc protein assay kit (BioRad Laboratories, Hercules, CA). An equal amount of protein (40 Ag/lane) from each cell extract was resolved on a 10% SDS-PAGE gel. Proteins were blotted to an Immuno-Blot polyvinylidenediflouride (PVDF) membrane (Bio-Rad Laboratories) by electrophoresis. The membranes were blocked with TBS-T blocking buffer (5% nonfat milk in 25 mM Tris – HCl, pH 7.4, 3 mM KCl, 140 mM NaCl, and 0.05% Tween) and subsequently probed with a relevant primary antibody at a predetermined optimal concentration overnight at 4jC or for 2 h at room temperature. After extensive washing with TBS-T, the immunoblots were then incubated with an appropriate peroxidase-conjugated secondary antibody for 1 h at room temperature. After three washes with TBS-T, the immunoblots were detected using the ECL Western Blotting Detection Reagents (Amersham Pharmacia Biotech, Piscataway, NJ) and recorded by exposure of the immunoblots to an X-ray film (Pierce Biotech, Inc., Rockford, IL).
BrdU incorporation assay DNA synthesis was determined by measuring BrdU incorporation into DNA as previously described with some modifications [16]. Briefly, WI-38 cells were incubated in complete medium containing 10 AM BrdU (Sigma) in a 4well glass slide chamber (Nalge Nunc Inc., Naperville, IL) for 6 h. They were washed twice with PBS and fixed in 70% ethanol at 20jC for 30 min. After three washes with PBS, the cells were incubated in DNA denaturing solution (2N HCl/0.1% Triton X-100 in PBS) at 37jC for 30 min followed by a 10-min incubation in 0.1 M sodium borate (pH 8.5) at room temperature to denature DNA. After being washed with PBS and a 60-min incubation in 1% bovine serum albumin/PBS, the cells were incubated overnight at 4jC with 2 Ag/ml mouse anti-BrdU monoclonal antibody (clone BU-33, from Sigma) and then with Texas Red Dyeconjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) with extensive washing between each step. The nuclear DNA of the cells was counterstained with Hoechst 33342 (Molecular Probes, Eugene, OR). The cells were viewed and photographed using an Axioplan research microscope (Carl Zeiss Inc, Jena, Germany) equipped with a 100 W mercury light source. The images were captured with a Dage CCD100 integrating camera (Dage-MTI, Michigan, USA) and a Flashpoint 128 capture board (Integral Technologies, Indiana, USA). The captured images were processed using Image Pro Plus software (Media Cybernetics, Maryland, USA) and displayed with Adobe Photoshop V6.0. SA-b-gal assay SA-h-gal activity was determined using a SA-h-gal staining kit from Cell Signaling Technology according to the manufacturer’s instruction. Briefly, cells were fixed in 2% (v/v) formaldehyde and 0.2% glutaraldehyde and then incubated in SA-h-gal staining solution (1 mg/ml 5-bromo4-chloro-3-indolyl h-D-galactosidase, 40 mM citric acid, pH 6.0, 40 mM sodium phosphate, pH 6.0, 5 mM potassium
Immunofluorescence staining Activation and nuclear translocation of p53 was analyzed by immunofluorescence staining. Briefly, WI38 cells cultured in a 4-well glass slide chamber were fixed with ice-cold methanol for 10 min at 4jC and then permeabilized with 0.2% Triton X-100. After blocking with 5% normal goat serum, they were incubated with a rabbit polyclonal antibody against p-p53 (from Cell Signaling) overnight at 4jC and then with a rhodamine-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) at room temperature for 1 h after extensive washing between each step. The slides were washed three times with PBS and incubated with Hoechst 33342 (Molecular Probes) at room temperature for 3– 5 min to stain DNA. After a final washing with PBS, the slides were mounted using Gel/Mount (Biomeda Corp, Forster, CA). A Zeiss Axiophot fluorescence microscope coupled to a digital camera utilizing Adobe Photoshop software was used to view and acquire images as described above.
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Results H2O2 induces apoptosis and senescence in HDFs in a dose-dependent manner
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cell size (Fig. 1D). These observations confirm that exposure of WI38 fibroblasts to a sublethal concentration of H2O2 induces premature senescence [6,10,11,17]. H2O2 activates p53– p21Cip1/WAF1 pathway in HDFs
WI38 cells were exposed to increasing concentrations of H2O2. Two hours after the exposure, H2O2 was removed from the culture and the cells were washed and recultured in fresh medium for various times to determine oxidative stress-induced apoptosis and senescence. It was found that a brief exposure of WI38 cells to H2O2 for 2 h at a concentration of 200 AM or above caused a significant increase in apoptosis as the cells were detached and stained positive for annexin V (data not shown). In contrast, pulse exposure to H2O2 for the same duration (2 h) at a concentration of 150 AM or below induced minimal cell death (data not shown). However, the majority of the cells that survived the brief treatment with 150 AM H2O2 ceased to proliferate within 3 –5 days after H2O2 had been removed from the culture (Fig. 1A). These surviving cells became permanently arrested in the G1 phase of the cell cycle and failed to synthesize DNA as measured by BrdU incorporation assay (Figs. 1B – C). In addition, they exhibited phenotypic changes that resemble those observed in the cells undergoing replicative senescence. These changes include increased SA-h-gal activity, flattened cell morphology, and enlarged
To determine if H2O2 induces HDF senescence by activation of p53, WI38 cells were treated with 150 AM H2O2 for 0, 0.2, 0.5, 1, or 2 h and then the cells were lysed. In addition, groups of cells treated with H2O2 for 2 h were lysed after they were incubated in fresh complete medium for 4, 8, or 16 h or for 1, 2, 3, 5, 7, or 9 days after removal of H2O2. The phosphorylation of p53 and expression of p53 and p21Cip1/WAF1 were examined in these cell extracts. As shown in Figs. 2A – B, control WI38 cells expressed a substantial amount of p53 under basal conditions and its expression was slightly augmented after H2O2 treatment. However, the expression of p21Cip1/WAF1 was minimal and phosphorylation of p53 was undetectable in untreated WI38 cells. Shortly after H2O2 exposure, a significant increase in p53 phosphorylation was detected. The increase in p53 phosphorylation occurred within 0.2 h, peaked around 2 h, and remained elevated up to 9 days after H2O2 treatment. Immunofluorescence staining using a polyclonal antibody against phosphorylated-p53 revealed that the majority of the phosphorylated-p53 had translocated into nuclei (Fig. 2C).
Fig. 1. H2O2 induces premature senescence of WI-38 HDFs. (A) H2O2 inhibits cell proliferation. The growth curve of control or H2O2-treated WI-38 cells was determined at days 1, 3, or 5 after brief H2O2 treatment (2 h) by the crystal violet-staining assay. The data are presented as mean F SE of OD value at 562 nM (n = 3). The error bars for control cells are invisible because the values of SEM are too small. (B) H2O2 induces a G1 cell cycle arrest. A representative analysis of cell cycle distribution in control or day 5 post H2O2-treated cells is presented. (C) H2O2 inhibits DNA synthesis. Cells in the S phase of the cell cycle were detected by BrdU incorporation assay in control or in day 5 post H2O2-treated cells. (D) H2O2 increases SA-h-gal activity. Representative photomicrographs of SA-h-gal staining for control or day 7 post H2O2-treated cells are presented.
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Fig. 2. H2O2 activates the p53 – p21Cip1/WAF1 pathway in WI38 HDFs. (A) WI38 cells were treated with H2O2 for 0 – 2 h or they were treated with H2O2 for 2 h and then incubated for 2 – 14 h after removal of H2O2. The expression of phosphorylated p53 (p-p53), p53, and p21Cip1/WAF1 was determined by Western blot analysis. (B) WI38 cells were treated with H2O2 for 2 h and the expression of phosphorylated p53 (p-p53) and p21Cip1/WAF1 in control (C) or H2O2-treated cells was determined at various times by Western blot analysis. (C) The phosphorylation and nuclear translocation of p53 induced by H2O2 were examined in control cells or 2-h H2O2-treated cells at 24 h post H2O2 treatment by immunofluorescence and Hoechst 33342 staining. Representative photomicrographs of the staining are shown. (D) WI38 cells were treated with H2O2 for 2 h and then incubated for 5 days after removal of H2O2. The expression of phosphorylated p53 (p-p53), p21Cip1/WAF1 (p21), p16Ink4a (p16), p27Kip1 (p27), p14Arf (p14), and phosphorylated Rb (p-Rb) in control (C) or in H2O2-treated cells (H2O2) was determined by Western blot analysis. Actin immunoblots serve as a control for equal protein loading.
The nuclear translocated p53 acted as a transcription factor that induces p21Cip1/WAF1 expression, as the expression of p21Cip1/WAF1 was increased in H2O2-treated cells at 8 h after the appearance of phosphorylated-p53 and remained augmented up to 9 days after H2O2 treatment (Figs. 2A –B). These results suggest that exposure of WI38 cells to a sublethal concentration of H2O2 activates p53, probably
via induction of oxidative DNA damage [6,11,18]. Activated p53 induces the expression of p21Cip1/WAF1 that in turn may mediate H2O2-induced cell cycle arrest by inhibiting several CDKs that phosphorylate Rb as reported previously (Fig. 2D) [1,2,6,11]. This suggestion is further supported by the finding that H2O2 treatment induced hypophosphorylation of Rb while having no significant
Fig. 3. H2O2 selectively induces PI3 kinase and MEK activation in HDFs. (A) WI38 cells were incubated in H2O2 for 0 – 2 h or they were treated with H2O2 for 2 h and then incubated for 4 – 16 h after removal of H2O2. The expression of phosphorylated PKB (p-PKB), PKB, phosphorylated ERK (p-ERK), ERK, phosphorylated p38 MAPK (p-p38), p38 MAPK (p38), phosphorylated JNK (p-JNK), and JNK was determined by Western blot analysis. (B) WI38 cells were treated with H2O2 for 2 h and then incubated for various times after removal of H2O2. The expression of phosphorylated PKB (p-PKB) and PKB in control (C) or H2O2-treated cells was determined by Western blot analysis. (C) Anisomycin-treated C-6 glioma cell extracts and UV-treated 293 cell extracts (Provided by Cell Signaling Technology) were used as a positive control for the Western blot analysis of phosphorylated p38 MAPK (p-p38) and phosphorylated JNK (p-JNK) of WI38 cells with (H2O2) or without H2O2 treatment (C). Actin immunoblots serve as a control for equal protein loading.
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effect on the expression of p16Ink4a, p27Kip1, and p14Arf (Fig. 2D). However, increase in p16Ink4a and p14Arf expression may occur at later time points after H2O2 treatment, which plays an important role in the maintenance of irreversible cell cycle arrest [1,2,6,11]. H2O2 selectively induces PI3 kinase and MEK activation in HDFs Exposure of cells to H2O2 also activates various kinases that may modify the cellular responses to H2O2-induced DNA damage and participate in the processes of senescence induction [19,20]. Therefore, we examined the effect of H2O2 exposure on the activities of PI3 kinase, MEK, p38 MAPK, and JNK by measuring the levels of phosphorylated
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PKB, ERK, p38 MAPK, and JNK. This is because activation of these kinases has been observed in certain types of cells after exposure to an oxidant and implicated in the induction of apoptosis and/or senescence by oxidative stress or oncogenic stimulation [2,11,19,20]. As shown in Figs. 3A – B, exposure of WI38 cells to 150 AM H2O2 resulted in an immediate activation of PI3 kinase as manifested by the increase in PKB phosphorylation. The activation of PI3 kinase peaked around 0.5– 1 h after exposure to H2O2 and lasted for more than 5 days. Activation of MEK by H2O2 was also indicated by the increased phosphorylation of ERK after PI3 kinase activation (Fig. 3A). However, the activity of MEK declined to the basal level at 16 h after H2O2 treatment (Fig. 3A). In contrast, no activation of p38 MAPK and JNK was detected in the cells treated with H2O2. The
Fig. 4. Selective inhibition of PI3 kinase uncouples H2O2-induced senescent phenotype and cell cycle arrest in HDFs. (A) Selective inhibition of PI3 kinase and MEK by LY294002 (LY) and PD98059, respectively. WI38 cells were pretreated with LY294002 (LY, 20 AM) or PD98059 (PD, 25 AM) for 30 min before 1 h incubation with H2O2. The cells pretreated with vehicle (0.5% DMSO) or the JNK inhibitor SB203580 (SB, 5 AM) were used as control. The expression of phosphorylated PKB (p-PKB), PKB, phosphorylated ERK (p-ERK), and ERK was determined by Western blot analysis. (B and C) Inhibition of PI3 kinase abrogated the H2O2-induced senescence phenotype. WI38 cells were pretreated with vehicle (0.5% DMSO), LY294002 (LY, 20 AM), PD98059 (PD, 25 AM), or SB203580 (SB, 5 AM) for 30 min before 2 h incubation with H2O2. After removal of H2O2, the cells were continuously cultured with these inhibitors for 5 days. The changes in SA-h-gal activity and cell morphology were determined by SA-h-gal staining and microscopy. Representative microscopic images are presented in B and the percentages of SA-h-gal positively stained cells were quantified and are presented in C as mean F SE (n = 4). (D and E) Inhibition of PI3 kinase failed to affect H2O2-induced cell cycle arrest. WI38 cells were pretreated with vehicle (0.5% DMSO), LY294002 (LY, 20 AM), PD98059 (PD, 25 AM), or SB203580 (SB, 5 AM) for 30 min before 2 h incubation with H2O2. After removal of H2O2, the cells were cultured with these inhibitors for 5 days and then released from inhibition by subculturing the cells in fresh complete medium without inhibitors. The growth curves of these cells were determined at day 1, 3, or 5 after the release by the crystal violet-staining assay and the data are presented in D as mean F SE of OD value at 562 nM (n = 3). The cells in the S phase of the cell cycle were determined by BrdU incorporation assay 5 days after the subculture and the data presented in E are mean percentage of BrdU positive cells F SE (n = 5). A, P < 0.01 vs. control; B, P < 0.01 vs. H2O2 + DMSO.
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inability to detect p38 MAPK and JNK activation in these H2O2-treated cells was not due to a lack of the expression of p38 MAPK and JNK in WI38 cells, nor did it result from a deficiency in the antibodies used in the assay to detect phosphorylated p38 MAPK and JNK since phosphorylated p38 MAPK and JNK were detected in two positive control cell extracts (Figs. 3A, C). These results suggest that exposure of WI38 cells to H2O2 selectively activates PI3 kinase/PKB and MEK with different kinetics. Selective inhibition of PI3 kinase uncouples H2O2-induced senescent phenotype and cell cycle arrest in HDFs To determine if activation of PI3 kinase and/or MEK contributes to H2O2-induced premature senescence in HDFs, PI3 kinase and MEK were selectively inhibited by their respective inhibitors LY294002 (LY, 20 AM) and PD98059 (PD, 25 AM) before a brief exposure of WI38 cells to H2O2 as described above [21,22]. These inhibitors were kept in the culture for 5 days after the removal of H2O2, since the pulse exposure of the cells to H2O2 caused a prolonged activation of PI3 kinase activity (Figs. 3A, B). As a control, the cells were pretreated with vehicle (0.5% DMSO) or the selective p38 MAPK inhibitor-SB203580 (SB, 5 AM) [23]. As shown in Fig. 4A, incubation of WI38 cells with LY and PD selectively inhibited H2O2-induced activation of PI3 kinase and MEK, respectively, whereas the activities of these kinases were not affected by SB. Interestingly, selective inhibition of PI3 kinase with LY abrogated H 2 O 2 -induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-h-gal activity, but had no significant effect on H2O2induced cell cycle arrest (Figs. 4B –E). In addition, forward scatter analysis by flow cytometry confirmed the inhibitory effect of LY on H2O2-induced increase in cell size, as the means (FSD) of FSC-H were 146.5 (F0.4), 161.6 (F0.3), and 102.5 (F6.1) for control, H2O2-treated, and H2O2 plus LY-treated cells, respectively. Similar results were also found in the cells treated with another PI3 kinase inhibitor Wortmannin (data not shown). In contrast, selective inhibition of MEK or p38 MAPK did not produce any significant effect on H2O2-induced senescent phenotype and cell cycle arrest (Figs. 4B –E). These results suggest that the senescent phenotype is uncoupled from cell cycle arrest in the senescent HDFs induced by H2O2. Activation of PI3 kinase is mainly responsible for mediating H2O2-induced phenotypic changes in senescent HDFs.
Discussion Pulse exposure of HDFs to a sublethal concentration of H2O2 induces permanent cell cycle arrest and phenotypic changes that mimic replicatively senescent cells [6,10,11, 18]. As reported previously, prematurely senescent HDFs induced by H2O2 exhibited an elevation in p53 activation
and p21Cip1/WAF1 expression, probably due to H2O2-induced oxidative DNA damage [6,11,18]. Increased levels of p21Cip1/WAF1 may mediate the initiation of H2O2-induced cell cycle arrest by inhibiting various CDKs that phosphorylate Rb to allow the G1 to S phase progression [1,2,6,11,18]. This hypothesis is supported by the finding that hypophosphorylation of Rb was only associated with an increased expression of p21Cip1/WAF1, while no changes in p16Ink4a, p27Kip1, and p14Arf expression were observed in WI38 cells after H2O2 treatment. These findings are in agreement with previous reports demonstrating that inactivation of p53 and/or Rb by ectopic transfection of E6 and/or E7 genes abrogated the cell cycle arrest induced by H2O2 [6,11,18], indicating that p53 and Rb control G1 cell cycle arrest in HDFs in response to H2O2-induced oxidative damage. However, p16Ink4a and/or p14Arf can be induced in prematurely or replicatively senescent HDFs at later time points after the senescent process is initiated. It has been suggested that p16Ink4a and/or p14Arf may play an important role in the maintenance of irreversible cell cycle arrest [1,2,6,11]. Exposure of HDFs to H2O2 also induces increased SAh-gal activity, flattened cell morphology, and enlarged cell size. The mechanisms by which H2O2 induces these phenotypic changes were poorly elucidated and the relationship between the senescent phenotype and permanent cell cycle arrest was highly controversial. It was reported that inactivation of Rb by ectopic transfection with the human papillomaviral (HPV) E7 (E7) gene prevented IMR-90 cells from H2O2-induced cell cycle arrest and cell enlargement but had no significant effect on the increase in SA-h-gal activity [5,6,18,24]. This suggests that the irreversible growth arrest induced by H2O2 exposure in HDFs may be coupled to the cell enlargement but not to the increased SAh-gal activity. However, uncoupling of cell cycle arrest and the senescent phenotype has been found in terminal-passage HPV E6 transfected cells or p21Cip1/WAF1 / HDFs, which exhibit a senescent phenotype but continuously proliferate [7,25]. Furthermore, cells ectopically transfected with p21Cip1/WAF1 or p27Kip1 underwent senescence without the appearance of senescent phenotype [4]. In contrast, treatment of replicatively senescent HDFs with nicotinamide reversed their senescent phenotype but did not affect their replicative capacity [8]. To further clarify the relationship between the senescent phenotype and permanent cell cycle arrest and to elucidate the mechanisms that may be responsible for the induction of senescent phenotype, we examined the role of PI3 kinase in H2O2-induced senescence in HDFs. This is because it has been well documented that exposure of various types of cells to H2O2 activates PI3 kinase via tyrosine phosphorylation of the p110 catalytic and/or p85 regulatory subunits by receptor-associated and/or Src family tyrosine kinases [12 – 14,26]. The phosphorylation of the p85 subunit contributes to the relocation of p85/p110 complex from the cytosol to the plasma membrane, thereby enabling PI3 kinase to
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phosphorylate its substrate and produce PI(3,4,5)P3 [12 – 14,26,27]. In turn, PI(3,4,5)P3 stimulates cell growth (in mass or size) by activating PI3 kinase down-stream targets, including PKB, ribosomal protein S6 kinase (S6K), and mammalian target of rapamycin (mTOR) [27]. Our study showed that WI38 cells treated with the selective PI3 kinase inhibitor LY but not with a selective MEK or p38 MAPK inhibitor exhibited normal cell morphology after exposure to H2O2. A similar morphological change in H2O2-treated or aged IMR-90 cells has been also reported in a recent study when PI3 kinase was similarly inhibited by LY but without the examination of SA-h-gal expression and cell cycle arrest [9]. Although the cells treated with LY failed to develop a senescent phenotype in response to H2O2 treatment, they remained arrested and unable to reenter the cell cycle and proliferate even after LY and H2O2 were removed from the culture. These findings suggest that the cell cycle arrest and senescent phenotype induced by H2O2 are distinct processes and the phenotypic changes in senescent cells do not contribute to the maintenance of the cell cycle arrest. In addition, our study demonstrates that the expression of senescent phenotype in H2O2-treated HDFs is under the regulation of PI3 kinase signaling, probably by activation of PKB and other PI3 kinase down-stream targets. However, the mechanism by which PI3 kinase regulates SA-h-gal expression has yet to be determined. It should be noted that while inhibiting PI3 kinase with LY that abrogated the cell enlargement induced by H2O2 in HDFs, it only partially inhibited H2O2-induced increase in SA-h-gal expression. This suggests that activation of PI3 kinase by H2O2 is partially responsible for the increase in SA-h-gal activity, and pathways other than PI3 kinase may be also involved in the upregulation of SA-h-gal by H2O2 in HDFs. The finding that inhibition of PI3 kinase suppresses the H2O2-induced senescent phenotype in HDFs indicates that activation of PI3 kinase contributes to H2O2-induced cellular senescence. The involvement of PI3 kinase in cellular senescence has been demonstrated by the studies using C. elegans [28,29]. It was reported that C. elegans with a mutated age-1 gene that encodes a homologue of mammalian PI3 kinase lived much longer that wild-type worms [29]. Treatment of adult C. elegans with LY resulted in a significant increase in their life span [28]. In contrast, it was reported that incubation of normal early passage HDFs or mouse embryo fibroblasts (MEFs) with LY alone without subsequent exposure to H2O2 actually induced senescence [30,31]. The induction of MEF senescence by LY is partially mediated by inhibition of PKB, activation of the forkhead protein AFX, and upregulation of p27Kip1 [30]. However, it remains to be determined whether the cells treated with LY alone enter a real senescent state or undergo quiescence or cell differentiation, as the ‘‘senescent’’ HDFs induced by LY reentered cell cycle and proliferated after they were released from LY inhibition [31]. In contrast, our study showed that the prematurely senescent HDFs induced by H2O2 and LY
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remained arrested after removal of H2O2 and LY. In addition, it has been shown that inhibition of PI3 kinase induces cellular differentiation [9,31 – 33]. Therefore, these conflicting findings suggest that PI3 kinase plays multiple roles in regulation of cellular senescence and differentiation in a manner depending upon whether cells are concurrently exposed to stress. Thus, additional studies are needed to further establish the function of PI3 kinase in cellular senescence under various physiological and pathological conditions.
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