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Originally published In Press as doi:10.1074/jbc.M501939200 on March 2, 2005

J. Biol. Chem., Vol. 280, Issue 18, 18033-18041, May 6, 2005
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Manganese Superoxide Dismutase Protects the Proliferative Capacity of Confluent Normal Human Fibroblasts*

Ehab H. Sarsour{ddagger}, Manjula Agarwal§, Tej K. Pandita§, Larry W. Oberley{ddagger}, and Prabhat C. Goswami{ddagger}

From the {ddagger}Free Radical and Radiation Biology Program, Department of Radiation Oncology, the University of Iowa, Iowa City, Iowa 52242 and the §Department of Radiation Oncology, Washington University School of Medicine, St. Louis, Missouri 63108

Received for publication, February 22, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We tested the hypothesis that manganese superoxide dismutase (MnSOD), an antioxidant enzyme, regulates the proliferative potential of confluent human fibroblasts. Normal human skin (AG01522) and lung (WI38, CCL-75) fibroblasts kept in confluence (>95% G0/G1) showed a significant decrease in their capacity to re-enter the proliferation cycle after 40-60 days. The inhibition of re-entry was accompanied with the age-dependent increase of p16 protein levels in the confluent culture. Adenoviral mediated overexpression of MnSOD during confluent growth suppressed p16, enhanced p21 protein accumulation, and protected fibroblasts against the loss of proliferation potential. Increases in p21 protein levels in MnSOD overexpressing confluent fibroblasts were independent of p53 protein levels. p53 protein levels did not change in control, replication-defective adenovirus containing an insertless vector (AdBgl II), or AdMnSOD-infected confluent cells cultured for 20 and 60 days. In addition, MnSOD-induced protection of the proliferation capacity of confluent fibroblasts was independent of their telomerase activity. However, telomerase-transformed fibroblasts showed increased MnSOD expression in confluent growth, maintaining their capacity to re-enter the proliferation cycle. Although inactivation of the retinoblastoma protein in cells subcultured from the 60-day confluent control, AdBgl II-, and AdMnSOD-infected fibroblasts was identical, only MnSOD-overexpressing cells showed a higher percentage of S-phase. These results support the hypothesis that a redox-sensitive checkpoint regulated the progression of fibroblasts from G0/G1 to S-phase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammalian cells, intracellular antioxidant enzymes include superoxide dismutase, catalase, and glutathione peroxidase. There are two intracellular forms of superoxide dismutase as follows: CuZnSOD,1 found in the cytoplasm and nucleus, and MnSOD, found in mitochondria (1, 2). Different isozymes of glutathione peroxidase are found in most subcellular compartments, whereas catalase is found primarily in peroxisomes and cytoplasm (1). Antioxidant enzymes neutralize reactive oxygen species (ROS) generated from the univalent reduction of oxygen by mitochondrial electron transport chains as well as biochemical reactions of oxygen-metabolizing enzymes (3-5). ROS, including superoxide, hydrogen peroxide, hydroxyl radical, singlet molecular oxygen, and organic hydroperoxides, are oxygen-containing molecules that have higher chemical reactivity than ground state molecular oxygen. ROS have traditionally been thought of as unwanted and toxic by-products of living in an aerobic environment (6, 7). In recent years, several studies suggest metabolic production of ROS is tightly regulated and serves a physiological function during mitogenic stimulation of cultured cells (6-11). It has been suggested that ROS operate as a key signaling process in the cascade of events leading to cell proliferation following stimulation with platelet-derived growth factor (9), epidermal growth factor (10), cytokines and antigen receptors (11), and oncogenic Ras (12). Although these studies provide evidence for a possible role of redox signaling during cellular proliferation, it is currently unknown whether antioxidant enzymes regulate progression through the cell cycle.

The cell cycle is a tightly regulated sequence of transitions from G0/G1 to S to G2 to M phases, which requires assembly and activation of phase-specific cyclin and cyclin-dependent kinase (Cdk) complexes (13-16). Cyclins D (D1, D2, and D3) and E in association with Cdks (Cdk2, Cdk4, and Cdk6) regulate progression from G1 to S. The enzymatic activity of cyclin/Cdk is regulated at three levels (15) as follows: cyclin binding and activation, subunit phosphorylation, and inhibition by one of the Cdk inhibitors (CIP/KIP family (p21, p27, and p57) and INK4 family (p15, p16, p18, and p19)). Active cyclin-CDK kinase complex partially phosphorylates the retinoblastoma (Rb) protein, which causes the release of the E2F family of proteins initiating transcription of E2F-mediated gene expression and entry into the S-phase (17). Whereas redox regulation of cell cycle regulatory proteins is not completely understood, other laboratories, including our own, have demonstrated that thiolantioxidants affect cyclin D1, p21, p27, and Rb phosphorylation (18-20). These results support the hypothesis that intracellular redox environment could regulate the proliferative capacity of normal fibroblasts in culture.

Normal human fibroblasts (NHF) have a finite proliferative life span in culture prior to undergoing irreversible growth arrest, commonly known as the "Hayflick Limit" (21). In recent years, the majority of studies favor the idea that the Hayflick Limit depends on the cumulative number of cell divisions rather than metabolic or calendar time. Therefore, a mitotic counting mechanism, "telomere attrition," has been hypothesized to be the molecular clock of cellular aging (22, 23). In addition to telomere attrition, two Cdk inhibitors, p16 and p21, mediate irreversible growth arrest in replicating cells (24-26). However, several other studies have shown confluent human fibroblasts kept for a prolonged period in culture lose their capacity to re-enter the proliferative cycle (27-29). More interestingly, increases in protein levels of both Cdk inhibitors p16 and p21 correlate with the inability of the confluent cultures to re-enter the proliferative cycle. These previous reports clearly suggest mechanisms other than cumulative cell division and teleomeric attrition could regulate the proliferative capacity of normal human fibroblasts. We therefore tested the hypothesis that MnSOD could regulate the proliferative capacity of confluent normal human diploid fibroblasts.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Human normal skin (NHF, AG01522) and lung (WI38, CCL-75) fibroblasts were obtained from Coriell Cell Repositories and the ATCC, respectively. The generation of telomerase-immortalized NHF (NHF + hTERT) has been described previously (30). All cell lines were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, supplemented with penicillin/streptomycin antibiotics. All experiments were performed using cells from passage 8.

Adenovirus Infections—Confluent cultures were infected with replication-deficient adenovirus containing cytomegalovirus promoter-driven human MnSOD cDNA containing the N-terminal mitochondrial localization signal (AdMnSOD). The MnSOD cDNA was inserted into the E1 region of an E1/partial E3-deleted replication-deficient adenoviral vector (31). A nonmodified vector (AdBgl II, see Refs. 32-34) was used as control for adenoviral infections. All adenovirus constructs were obtained from the University of Iowa DNA-vector core. Transduction efficiency was measured by flow cytometry assay of AdGFP-infected confluent cultures. MnSOD expression was measured by immunoblotting, and a gel electrophoresis-based assay was used to measure MnSOD enzyme activity (35).

Proliferative Capacity for Assay Confluent Cultures—Initially, one million cells per T-75 tissue culture flask were plated for each cell line and cultured in regular medium until confluence. Cells from replicate flasks were trypsinized and assayed for cell cycle phase distributions by flow cytometry measurements of DNA content and cell number. In general, cells reached confluence after 7 days of plating. Cells with less than 5% S-phase were considered a confluent culture; this was marked as day 1 of their experimental duration. Adenoviral infections were performed at day 1 of confluence using 30 m.o.i. of either AdMnSOD or AdBgl II. Alternatively, infections were performed in cultures kept in confluent growth for 50 days. In either protocol, cells were kept in confluent growth for a total of 60 days, and medium was replaced every 3 days. Control, AdBgl II-, and AdMnSOD-infected confluent cultures from 10, 20, 40, and 60 days of confluence were subcultured by trypsinization and re-plated at a lower cell density (1:5-fold dilution). Re-plated cells were allowed to grow for 48 h and pulse-labeled with bromodeoxyuridine (BrdUrd) for 30 min prior to harvesting by trypsinization. Ethanol-fixed cells were analyzed for cell cycle phase distributions by flow cytometry. Changes in the S-phase distributions were used to measure the proliferative capacity of the cells.

Cell population doubling times were calculated from growth curves. Re-plated cells were continued in culture and cell numbers counted up to 6 days. Cell population doubling time (Td) values were calculated from the exponential portion of the growth curve using the following equation: Td = 0.693t/ln(Nt/N0) where t is time in days, and Nt and N0 represent cell numbers at time t and the initial time, respectively.

Flow Cytometry Assays—Flow cytometry assays for measurements of cell cycle phase distributions were performed following a previously published protocol (18). Briefly, nuclei isolated from ethanol-fixed cells were incubated with anti-BrdUrd primary antibody (Immunocytometry Systems) followed by incubation with FITC-conjugated goat anti-mouse IgG. Nuclei were treated with RNase A (0.1 mg/ml) and counterstained with 20 µg/ml PI. FITC and PI fluorescence were analyzed on a FACScan flow cytometer (BD Biosciences). Data from a minimum of 20,000 nuclei were acquired and were processed using Cellquest Pro software (Immunocytometry Systems). The acquired data were analyzed as dual-parameter propidium iodide (PI) versus log-FITC histograms, and three compartments (BrdUrd-positive S-phase cells and BrdUrd-negative G1 and G2 phases) were identified. The number of cells in each compartment was calculated and expressed as percentage of total gated population.

For univariate flow cytometry assay of DNA content, ethanol-fixed cells were treated with RNase A for 30 min followed by staining with PI. PI-stained cells were analyzed by flow cytometry, and cell cycle phase distributions were calculated using Cellquest Pro and ModFit softwares (Verity Software House, Topsham, ME).

PI staining of unfixed cells was used to measure viability. Monolayer cultures were trypsinized and resuspended in 0.5 ml of phosphate-buffered saline containing PI prior to flow cytometry. The percentage of PI-positive (nonviable) and -negative (viable) cells was determined using WinMD software.

Immunogold Staining and Electron Microscopy—Control and adenovirus-infected cultures were collected by scraping monolayer cells 5 days post-infection and were fixed with a light fixative mixture of 4% paraformaldehyde and 0.05% glutaraldehyde. Immunogold staining was performed at the University of Iowa Central Microscopy Research Core Facility using MnSOD rabbit anti-human polyclonal antibodies and F(ab)2 goat anti-rabbit ultra-small gold-conjugated secondary antibodies.

Immunoblotting Assay—Equal amounts of total protein extracts were separated by 12.5 or 7% SDS-PAGE, electrotransferred onto a nitrocellulose membrane, and probed with primary antibodies against human MnSOD, CuZnSOD, p53, p21, and p16. MnSOD and CuZnSOD rabbit anti-human polyclonal antibodies were obtained from the Radiation and Free Radical Research Core Facility (the University of Iowa); p53 (Pharmingen), p21 (Upstate Biotechnology, Inc., Lake Placid, NY), p16 (Pharmingen), and actin (Chemicon International, Temecula, CA) antibodies were used according to the manufacturer's supplied protocol. Immunoreactive bands were detected by an ECL Plus kit from Amersham Biosciences. Bands were quantified with a computerized digital imaging system interfaced with Scion Image software (Scion Corp., Frederick, MD). The integrated density value was obtained by integrating the entire pixel values in the area of one band after correction for background. All blots were re-probed with antibody to actin, and actin protein levels were used for loading corrections. Blots are representative of at least two separate experiments.

MnSOD Enzyme Activity Assay—Fifty micrograms of total protein extracts were separated by native polyacrylamide gel electrophoresis following a protocol published previously (35). Gels were stained with nitro blue tetrazolium and riboflavin-TEMED solution for 20 min at room temperature. CuZnSOD and MnSOD were differentiated by the addition of sodium cyanide in the staining solution (36). The bands were visualized and quantified with a computerized digital imaging system interfaced with AlphaImager 2000 software (Alpha Innotech., San Leandro, CA). Results are representative of two or more experiments.

Telomerase Activity Assay—Cells were resuspended in lysis buffer (0.01% Nonidet P-40, 10 mM Tris, pH 7.5, 50 mM KCl, 5 mM MgCl2, 2 mM dithiothreitol, 20% glycerol, and protease inhibitors). Lysates were centrifuged for 30 min at 16,000 x g, and supernatants were used for immunoprecipitation. The hTERT proteins were immunoprecipitated with anti-hTERT antibodies and protein A/G-Sepharose beads. Telomerase activity in the immunoprecipitates was determined using the telomerase PCR enzyme-linked immunosorbent assay kit (Roche Applied Science) following a protocol published previously (30). Telomerase activity in HeLa cells was used as positive control for the assay. Protein extracts denatured at 95 °C for 5 min were included as negative control. Changes in telomerase activities were calculated relative to the negative control.

Statistical Analysis—Statistical analysis was done using the one- and two-way analysis of variance with Tukey's honestly significant difference test. Homogeneity of variance was assumed with 95% confidence interval level. Results with p < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MnSOD Overexpression Protects the Capacity of Confluent Normal Fibroblasts to Re-enter the Proliferation Cycle—We have used adenovirus-mediated gene transfer methodology to determine whether the MnSOD antioxidant enzyme protects the proliferative capacity of normal human fibroblasts. Confluent cultures of NHF (>95% G0/G1 cells, based on DNA content as determined by flow cytometry) were infected with 10-100 m.o.i. of AdMnSOD or AdBgl II and harvested for MnSOD protein and enzyme activity measurements beginning at 48 h post-infection (Fig. 1). MnSOD protein levels increased in control cells 7 days post-plating compared with 48 h post-plating (Fig. 1A). This result is consistent with an earlier report in the literature demonstrating increased MnSOD protein and activity in confluent growth-arrested cells compared with exponential cultures (37). Both 10 and 30 m.o.i. AdMnSOD-infected cells showed a dose-dependent increase in MnSOD protein and enzyme activity at 48 h post-infection, which remained elevated even after 7 days of confluent growth (Fig. 1, A and B). Cells infected with 100 m.o.i. showed a decrease in MnSOD protein levels after 7 days of confluent growth (Fig. 1A). This decrease in MnSOD protein was associated with a decrease in cell viability as measured by PI staining of unfixed cells and flow cytometry (data not shown), suggesting this higher dose could result in toxicity. Because the majority of our experiments extended up to 60 days, we determined whether ectopic expression of MnSOD continued for this prolonged period. Confluent cultures were infected with 30 m.o.i. of AdBgl II or AdMnSOD and kept in confluent cultures for 60 days with regular changes in growth medium. The basal levels of MnSOD protein and enzyme activity in control and AdBgl II-infected cells showed marginal changes at 60 days of confluence compared with 20-day confluent cultures (Fig. 1C). However, AdMnSOD-infected cells continuously maintained 2-4-fold higher levels of MnSOD expression throughout the 60 days of confluent growth (Fig. 1C). Similar results were also obtained for AdMnSOD-infected confluent WI38 fibroblasts held in confluence for 60 days (Fig. 5C). Furthermore, the duration of confluent growth did not show any significant changes in CuZnSOD protein levels in control, AdBgl II, and AdMnSOD-infected cells (Fig. 1C).



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  		FIG. 1.
Expression of MnSOD antioxidant enzyme in confluent NHF. Confluent NHF were infected (10-30 m.o.i.) with AdMnSOD or AdBgl II and harvested at 48 h and 7 days (d) post-infection for immunoblotting of MnSOD protein levels (A), gel electrophoresis-based MnSOD enzyme activity assay (B), and MnSOD protein levels and enzyme activities in 30 m.o.i. AdMnSOD- or AdBgl II-infected NHF kept in confluent growth for 20, 40, and 60 days (C). Blots were re-hybridized for CuZnSOD and actin protein levels.

 



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  		FIG. 2.
MnSOD localization and transduction efficiency. A, electron microscopy visualization of mitochondria localized MnSOD. Confluent NHF infected with AdBgl II and AdMnSOD were scrape-harvested 7 days post-infection, fixed, and immunostained with primary antibodies to MnSOD and immunogold-conjugated secondary antibody. Negative pictures were recorded at x30,000 magnification; bars represent 200 nm; insets represent sections of mitochondria showing immunogold-positive MnSOD protein within the mitochondria. B, flow cytometry assay for transduction efficiency. Control and AdGFP-infected (30 and 100 m.o.i.) confluent NHFs were kept in culture for 7 days, trypsinized, and resuspended in 500 µl of phosphate-buffered saline. GFP fluorescence was measured by flow cytometry, and GFP-positive and -negative cell populations were analyzed by WINMDI software, FITC versus forward scatter.

 
Next, we determined if overexpression of MnSOD in AdMnSOD-infected cells caused MnSOD to be localized to the mitochondria. Immunologic detection of MnSOD using primary antibodies to MnSOD and ultra-small gold-conjugated secondary antibodies showed overexpression of MnSOD increased MnSOD-immunoreactive protein levels in the mitochondria of both 30 and 100 m.o.i. AdMnSOD-infected cells (Fig. 2A). Flow cytometry measurement of GFP fluorescence in AdGFP-infected cells was performed to determine transduction efficiency. Confluent cultures infected with 30 m.o.i. AdGFP showed ~80% GFP-positive cells, and the percentage of GFP-positive cells increased to ~96% in 100 m.o.i. infected cells (Fig. 2B). These results suggest the majority of cells express the transgene, and MnSOD protein levels were higher in the mitochondria of AdMnSOD-infected cells compared with controls.

The proliferative capacity of the confluent fibroblasts was measured by subculturing confluent cultures at a lower cell density and monitoring BrdUrd incorporation 48 h after replating. Confluent cultures of NHF were subcultured at 10, 30, 40, and 60 days of confluent growth and re-plated at a lower cell density. Forty eight hours later, cells were pulse-labeled with BrdUrd and ethanol-fixed for flow cytometry measurements of cell cycle phase distributions. Results presented in Table I show more than 95% G0/G1 cells in each treatment group, suggesting the majority of cells were growth-arrested prior to subculturing at both 20 and 60 days of confluence. However, cell cycle phase distributions following 48 h of replating were different among the three treatment groups. Fig. 3A shows representative BrdUrd-PI bivariate histograms of cell cycle phase distributions at 48 h post-plating of cells subcultured from 10-day (upper panel) and 60-day (lower panel) confluent cultures. NHF subcultured from 10- and 30-day confluent cultures showed ~20-25% S-phase cells in control, AdBgl II-, and AdMnSOD-infected cells (Fig. 3B). However, control and AdBgl II-infected NHF subcultured from confluent cultures held in confluence for 40-60 days showed a significant inhibition in re-entry to the proliferation cycle: 10-15% in control and 1-3% S-phase in AdBgl II-infected cells (Fig. 3B and Table II). In contrast, cells subcultured from AdMnSOD-infected confluent NHF showed a consistently higher percentage of S-phase cells, even after 60 days of confluent growth, ~20% in AdMnSOD-infected cells compared with 2% S-phase cells in AdBgl II-infected cells (p < 0.05; Table II).



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  		FIG. 3.
MnSOD overexpression protects G0/G1 capacity of the cells to re-enter S-phase. Confluent NHF were infected with 30 m.o.i. of AdBgl II or AdMnSOD. Cells were kept in confluent growth with regular change in growth medium and subcultured by re-plating at lower cell densities beginning at 10 days of confluent growth. Re-plated cells were cultured for additional 48 h and pulse-labeled with BrdUrd prior to harvesting. Cell cycle phase distributions were analyzed by flow cytometry following a protocol published previously (18). A, representative BrdUrd versus PI bi-variate histograms of cell cycle phase distributions in cells subcultured from 10 and 60 days of confluent growth; S-phase distributions in NHF (B) and WI38 (C) were calculated using Cellquest software n = 5; *, p < 0.05. All cultures had >95% G1 cells (based on PI-staining and flow cytometry measurements of DNA content) prior to subculturing (Table I).

 


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  		TABLE I
Cell cycle phase distributions in confluent NHFs The flow cytometric measurement of DNA content is n = 3, p > 0.05; average ± S.D.

 


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  		TABLE II
Flow cytometry measurements of cell cycle phase distributions at 48 h post-plating of cells kept in confluent cultures for different days

 
MnSOD overexpression also prolonged the capacity of confluent normal human lung fibroblasts (WI38) to re-enter the cell cycle (Fig. 3C). Similar to NHF, control and AdBgl II-infected confluent WI38 fibroblasts lost their proliferation potential after 30-40 days in culture: 25% S-phase in cells subcultured from 10 days of confluent culture were compared with 4% S-phase in cells subcultured from 40 to 60 days of confluent culture. In contrast, the percentage of S-phase cells in MnSOD-overexpressing WI38 fibroblasts remained essentially unaltered in cells subcultured from 60 days of confluent culture compared with cells subcultured from 10 days of confluent culture. These results clearly demonstrate a finite duration of the proliferative potential of human fibroblasts kept in confluent growth and that MnSOD protects the proliferative capacity of confluent normal human fibroblasts.

MnSOD-dependent prolongation of NHF proliferation potential was also apparent from preservation of NHF growth characteristics, measured by counting cell numbers up to 6 days after subculturing (Fig. 4A). The increase in cell numbers in cultures subcultured from 10-day confluent cultures did not show any significant difference among control, AdBgl II-, and AdMnSOD-infected cells (Fig. 4A, left panel). However, cell growth in cultures subcultured from 60-day confluent control and AdBgl II-infected NHF was significantly delayed (Fig. 4A, right panel). These observations support the results from Fig. 3 and clearly demonstrate that NHF kept in confluent culture gradually lose their capacity to re-enter the cell cycle. Most interestingly, MnSOD overexpression protected the capacity of confluent NHF to re-enter the proliferation cycle, even after 60 days in confluence.

Furthermore, subsequent to re-entry, the cell number continued to increase during 6 days of post-subculturing (Fig. 4A, right panel). To determine whether the inhibition in cell growth following re-plating of 60-day confluent cultures could be due to cell death and/or cell cycle arrest, DNA content was assayed at 2, 4, and 6 days post-plating. Ethanol-fixed cells were stained with PI, and DNA content was analyzed by flow cytometry. Results from these experiments showed a significantly higher percentage of cells with sub-G0/G1 DNA content in control and AdBgl II-infected cells compared with AdMnSOD-infected cells at 4 and 6 days post-plating (Fig. 4, B and C). The increase in percentage of cells with sub G0/G1 DNA content corresponded to a decrease in cell number in control and AdBgl II-infected cells, suggesting the decrease could be due to cell death. Most interestingly, whereas the cell number at 2-day post-plating did show a decrease in control and AdBgl II compared with AdMnSOD-infected cells, there were no changes in cell population with sub-G0/G1 DNA content. This difference in cell numbers at 2 days post-plating could be due to cell cycle arrest.



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  		FIG. 4.
MnSOD overexpression protects the proliferative capacity of confluent normal human fibroblasts. A, growth characteristics of control, 30 m.o.i. AdBgl II-, and AdMnSOD-infected NHF subcultured from cultures kept at confluence for 10 (left panel) and 60 (right panel) days; n = 3; *, p < 0.05. Cells from replicate plates were harvested by trypsinization and fixed in ethanol. Ethanol-fixed cells were treated with RNase A and stained with PI, and DNA content was analyzed by flow cytometry. Representative histograms are shown in B, and the fraction of cells with sub-G0/G1 DNA content is shown in C; an arrow indicates the sub-G0/G1 peak.

 
MnSOD Overexpression Suppresses Accumulation of p16 and Enhances p21 Cyclin-dependent Kinase Inhibitor Protein Levels during Confluent Growth of Fibroblasts—Because changes in p16 and p21 protein levels were associated with irreversible growth arrest of the replicating fibroblasts, we determined if similar changes were also associated with the loss of the proliferative capacity of confluent fibroblasts. Results presented in Fig. 5 demonstrate increased p16 protein levels in control and AdBgl II-infected confluent cultures at 60 days post-confluence both for NHF (Fig. 5A) and WI38 (Fig. 5C) cultures. In contrast, MnSOD overexpression suppressed p16 protein accumulation in both cell lines. This MnSOD-induced decrease in p16 protein levels correlated with the protection of the proliferative capacity. Most interestingly, whereas MnSOD overexpression suppressed p16 accumulation in cultures held at confluence for 60 days, the same treatment caused an increase in p21 protein levels both in NHF and WI38 fibroblasts (Fig. 5). The increase in p21 protein levels was independent of changes in p53 protein levels. Results showed no change in p53 protein levels in confluent cultures of control, AdBgl II-, and AdMnSOD-infected cells at 20 and 60 days of post-confluence (Fig. 5B). This suggests the MnSOD-mediated protection of proliferative capacity could be mediated via MnSOD-dependent regulation of the Cdk inhibitors, p16 and p21.



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  		FIG. 5.
MnSOD overexpression suppressed p16 and enhanced p21 Cdk inhibitors in confluent normal human skin and lung fibroblasts. Immunoblotting assay of cyclin-dependent kinase inhibitors (p16 and p21), protein levels in control, 30 m.o.i. AdBgl II, and AdMnSOD-infected confluent cultures are shown. A, NHF, 20 and 60 days post-infection; C, WI38, 60-days post-infection. B, immunoblot assay of p53 protein levels in 20- and 60-day confluent control and, AdBgl II-, and AdMnSOD-infected NHFs. Actin protein levels were used for comparison of results.

 
We then determined whether MnSOD overexpression in cells that have already accumulated p16 protein could reverse the inhibition in cell cycle re-entry. NHF kept in confluence for 1 and 50 days were infected with 30 m.o.i. AdMnSOD or AdBgl II and harvested after 60 days of initial confluent growth, i.e. confluent cultures infected on day 1 of confluence were kept in confluent growth for 59 days, whereas cells infected at 50 days of confluent growth were kept in confluence for an additional 10 days. The results presented in Fig. 6 show that whereas AdMnSOD infection on day 1 of confluence suppresses p16 accumulation, MnSOD overexpression after 50 days of confluence had no significant effect on p16 protein levels (Fig. 6A, lanes 4 and 7). Therefore, confluent fibroblasts infected with AdMnSOD after 50 days of confluence showed no significant difference in S-phase distributions 48 h after subculture compared with control and AdBgl II-infected cells (Fig. 6B, p > 0.05). Thus, whereas MnSOD overexpression prior to increases in p16 protein levels can suppress p16 accumulation, MnSOD overexpression after p16 protein accumulation was ineffective in suppressing p16 protein accumulation.



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  		FIG. 6.
MnSOD overexpression after p16 accumulation failed to reverse the inhibition in NHF re-entry into the proliferation cycle. Confluent NHF held at confluence for 50 days were infected with 50 m.o.i. of AdMnSOD or AdBgl II. Cells were continued in confluent culture for 10 days post-infection and harvested for immunoblot analysis (A, lanes 2-4). Cells from replicate plates were trypsinized and re-plated at a lower cell density. Forty eight hours post-plating, cells were pulse-labeled with BrdUrd and assayed for S-phase distribution by flow cytometry (B), n = 3, p > 0.05. Lanes 5-7 in A represent proteins extracted from cells that were infected at day 1 of confluent growth and held at confluence for 60 days. Lane 1 represents cultures held at confluence for 50 days prior to the adenovirus infection.

 
Because telomerase activity has been shown to regulate the replicative capacity of the exponential cultures, we determined if this MnSOD-dependent protection could be due to changes in telomerase activity. Control, AdMnSOD-, and AdBgl II-infected confluent NHF kept in cultures for 60 days were assayed for telomerase activity (Fig. 7). These results showed no significant difference in telomerase activity among control, AdBgl II-, and AdMnSOD-infected cells. These results suggest the MnSOD-dependent protection of the proliferation potential for the confluent fibroblasts is independent of telomerase activity.

Increased MnSOD Enzyme Activity in Telomerase-transformed Normal Human Fibroblasts Protected the Capacity of Confluent Cells to Re-enter the Proliferation Cycle—The relationship between MnSOD antioxidant enzyme activity in confluent cultures and protection of the proliferation capacity for the NHF was further tested in telomerase-immortalized NHF. Telomerase immortalization of NHF has been shown previously to increase MnSOD mRNA levels 6-7-fold (30). Initially, MnSOD protein levels and enzyme activity were measured in exponential growth and cell transiting to confluent growth stage (Fig. 8A). Both NHF and telomerase immortalized NHF (NHF + hTERT) showed comparable MnSOD protein and enzyme activity during exponential growth (20-30% S-phase, based on flow cytometry measurement of DNA content). Although MnSOD protein levels and enzyme activity increased in both cell lines during the transit of the cells to confluent growth stage (<10% S-phase), NHF + hTERT showed higher levels compared with NHF (Fig. 8A, lanes 3 and 6). MnSOD protein levels continued to increase in confluent NHF + hTERT cultures at 10, 30, and 60 days of confluent growth (Fig. 8B).



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  		FIG. 7.
MnSOD overexpression in confluent NHF cultures did not affect telomerase activity. Relative telomerase activity was measured by an in vitro PCR-based enzyme-linked immunosorbent assay; confluent cultures of NHF were infected with 30 m.o.i. of AdBgl II or AdMnSOD and continued in confluent cultures for 60 days. Telomerase assay was performed following a protocol published previously (30). n = 3, p > 0.05.

 
Increased MnSOD protein levels in confluent NHF + hTERT protected cellular capacity to re-enter the proliferation cycle. The results demonstrated that whereas the percentage of S-phase upon re-entry decreased in NHF subcultured from 60 days of confluent growth (<5% compared with 10% S-phase in cells subcultured from 20 days of confluent growth; Fig. 8D), the S-phase percentage remained high (>30%; Table II) in NHF + hTERT subcultured from either 20 or 60 days of confluent growth. Because both cell lines had >95% G0/G1 cells prior to subculturing, and only NHF + hTERT maintained high S-phase upon re-entry to the proliferation cycle, these results further show increased MnSOD levels in confluent cultures maintain reproductive integrity of the fibroblasts. More interestingly, the hTERT-induced increase in MnSOD protein levels in confluent cultures was associated with inhibition in age-dependent increase in p16 protein accumulation of the confluent fibroblasts (Fig. 8B, lanes 3 and 6). Increased MnSOD protein levels were also associated with enhanced p21 protein accumulation. Suppression of p16 accumulation in confluent NHF + hTERT fibroblasts facilitates the entry of the cells into the proliferation cycle (Fig. 8, C and D).

MnSOD Overexpression in Confluent Fibroblasts Did Not Affect Phosphorylation of Rb Upon Re-entry to the Proliferation Cycle—Because hyperphosphorylation of Rb accompanies the entry of the cells from the G1 to S-phase, we determined whether the inability of 60-day confluent cultures to enter the S-phase could be due to the differences in Rb phosphorylation. Sixty-day confluent cultures of control, AdBgl II-, and AdMnSOD-infected NHFs were subcultured and re-plated at a lower cell density. Re-plated cells were harvested 48 h later and analyzed for p16, p21, and Rb phosphorylation by immunoblotting. In 60-day confluent cells prior to subculturing, Rb was hypophosphorylated in control, AdBgl II-, and AdMnSOD-infected cells (Fig. 9, lanes 1-3). As shown before (Fig. 5), p16 protein levels in confluent fibroblasts were higher in control and AdBgl II-infected cells compared with AdMnSOD-infected cells, and overexpression of MnSOD enhanced p21 protein levels. Although p16 protein levels decreased substantially following re-entry into the proliferation cycle, AdMnSOD-infected cells still showed higher p21 protein levels compared with control and AdBgl II-infected fibroblasts (Fig. 9, lanes 4-6). Most interestingly, inactivation of Rb following re-entry into the proliferation cycle was essentially similar in control, AdBgl II-, and AdMnSOD-infected cells (Fig. 9, lanes 4-6). Although Rb is hyperphosphorylated in all three experimental conditions, only MnSOD-overexpressing cells entered S-phase (Fig. 3A) and continued to proliferate as evidenced from the increases in cell numbers (Fig. 4A).



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  		FIG. 8.
Telomerase-immortalized normal human fibroblasts showed increased MnSOD protein levels and enzyme activity during confluence, suppressed p16 and enhanced p21 protein levels, and maintained capacity of the confluent fibroblasts to re-enter the proliferation cycle. NHF and NHF immortalized with hTERT (NHF + hTERT) were harvested from exponential (lanes 1, 2, 4, and 5) and transit to confluent growth (lanes 3 and 6) (A), and 10, 30, and 60 days of confluent growth for immunoblotting analysis of MnSOD, p21, and p16 protein levels (B). Cells from 20, 30, 40, 50, and 60 days confluent growth were trypsinized and re-plated at lower cell density. Re-plated cells were cultured for additional 48 h and pulse-labeled with BrdUrd prior to harvesting. C, representative BrdUrd versus PI bi-variate histograms of cell cycle phase distributions in cells subcultured from 20 and 60 days confluent cultures. D, S-phase distributions were calculated using Cellquest software. n = 3, * p < 0.05 at all time points for NHF compared with NHF + hTERT.

 



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  		FIG. 9.
MnSOD overexpression-induced protection of the proliferation capacity of NHF appears to be independent of Rb phosphorylation status. Confluent NHF infected with 30 m.o.i. AdBgl II or AdMnSOD were kept in confluence for 60 days and subcultured by re-plating at a lower cell density. Cells were collected prior to re-plating (lanes 1-3) and 48 h after subculturing (lanes 4-6) for immunoblot analysis of p16, p21, and Rb. Actin protein levels were used for comparison of results. Shown are blots representative of three different experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Human somatic cells are known to have a finite number of proliferative life spans prior to irreversible growth arrest. This phenomenon, commonly known as the Hayflick Limit (21) or replicative life span, is measured by counting the number of times a cell population undergoes cell division and doubles in size. Although this concept is still considered a fundamental characteristic of cellular aging, another characteristic of aging appears to be the maintenance of the reproductive integrity of G0/G1 quiescent cells, also known as chronological life span. Several earlier studies report human fibroblasts kept in confluent culture in vitro for a long period of time have a reduced replicative life span (27-29). These observations are further supported from findings that fibroblasts cultured from skins of older individuals undergo fewer cycles of cell division compared with fibroblasts isolated from younger individuals (38). Consistent with these observations, our results show normal human diploid fibroblasts kept in confluent cultures for a prolonged period lose their capacity to proliferate upon re-entry to the cell cycle.

The replicative life span of in vitro cell culture and in vivo model organisms (yeast, Drosophila, and Caenorhabditis elegans) is believed to be regulated by oxidative stress, successive erosion of telomere length during replication, and cell cycle checkpoint pathways (6-17). Life span extensions in transgenic flies overexpressing superoxide dismutase antioxidant enzymes support the idea that antioxidants could influence the longevity of an organism (39-42). Transgenic flies overexpressing the cytosolic antioxidant enzyme, copper and zinc-containing superoxide dismutase (CuZnSOD), showed a 33-48% increase in life span (40-42). Overexpression of the mitochondrial antioxidant enzyme, MnSOD, demonstrated a dose-dependent increase in life span (42). Consistent with oxidative stress-mediated regulation of life span, cells cultured in vitro under chronic hypoxia (3% oxygen partial pressure) showed an increase in replicative life span (43, 44). In yeast, MnSOD overexpression shortened the replicative life span but prolonged the chronological life span (39, 45), suggesting antioxidant enzymes could regulate different pathways in replicative versus chronological life span. Our results show constitutive overexpression of MnSOD antioxidant enzyme during confluent growth protected normal human diploid fibroblasts from time-dependent loss of proliferative capacity (Fig. 3). These results support the hypothesis that mitochondrial generated ROS might influence the proliferative potential of quiescent fibroblasts under in vitro cell culture conditions.

Although MnSOD-dependent regulatory pathways are not clearly defined, a recent study reports overexpression of extracellular superoxide dismutase in human fibroblasts slows telomere shortening (46). Alternatively, confluent growth-arrested human fibroblasts stably overexpressing human telomerase enzyme enhanced MnSOD expression 6-7-fold (30). The successive decrease in telomere length during cell division, also known as the "end replication problem," has been attributed to the inability of the DNA replication machinery to replicate completely the very end of the lagging strand (47). Whereas telomeric attrition requires S-phase and subsequent cell divisions during the replicative life span, results presented in Fig. 7 suggest the same pathway might not influence the proliferative capacity for confluent fibroblasts. Because a change in telomerase activity is routinely used as an indicator of telomeric attrition, telomerase activity was measured at the end of a 60-day confluent growth in control, AdBgl II-, and AdMnSOD-infected fibroblasts. Results presented in Fig. 7 display no significant difference in telomerase activity among control, AdBgl II-, and AdMnSOD-infected cells. This is also consistent with the observation that ~100% of the cells were in G1 phase (Table I), and absence of a cycling population is expected to have very little effect on telomerase activity and subsequently telomere length. The idea that telomeric attrition requires extensive cell division rather than the calendar time has also been supported from a number of reports in the literature (27, 28, 48). Allsopp et al. (48) and Sitte et al. (28) both reported no significant level of telomere shortening in mid-life span fibroblasts kept in confluent culture for 7 and 10 weeks, respectively. A more recent report from Munro et al. (27) showed very little telomere attrition in human fibroblasts kept in culture for 10 months with intermittent confluence. These results further suggest that telomere attrition is exclusively a mitotic counting mechanism in replicating human fibroblasts.

Although telomere attrition might not be a significant event during confluence, fibroblasts immortalized with hTERT showed a gradual increase in MnSOD protein levels and activity during confluence compared with control (Fig. 8). Considering constitutive expression of hTERT in these stable transfectants, it is not clear why hTERT overexpression selectively activates MnSOD expression in confluent versus exponential (20-30% S-phase) cells (Fig. 8, A and B). Most interestingly, increased levels of MnSOD in confluent hTERT-overexpressing fibroblasts (NHF + hTERT) also showed a higher percentage of S-phase upon re-entry into the proliferation cycle (Fig. 8, C and D, and Table II). Thus, increased MnSOD protein levels and enzyme activity in confluent fibroblasts, either due to AdMnSOD infection or hTERT overexpression, appear to maintain the proliferative potential of these cells upon re-entry to the cell cycle. These results, as well as those of others (27, 28), suggest a large number of confluent cells undergo telomere-independent irreversible growth arrest (measured by their inability to re-enter the proliferation cycle), and only a small subset of confluent cells retain their reproductive integrity to repopulate.

Re-entry and progression through the cell cycle is a tightly regulated sequence of transitions from G0/G1 to S to G2 to M phases involving the sequential activation of cyclin-dependent kinase (Cdk) activities that are regulated positively by cyclins and negatively by Cdk inhibitors. Increases in both p16 and p21 Cdk inhibitors during an irreversible growth arrest are generally thought to be associated with accumulation of hypophosphorylated Rb (24). Hypophosphorylated Rb is known to sequester members of the E2F transcription factor family, and the unavailability of E2F inhibits progression through the cell cycle. Consistent with these reports, our results also demonstrated hypophosphorylated Rb during the confluent growth of the fibroblasts in control, AdBgl II-, and AdMnSOD-infected cells (Fig. 9), suggesting overexpression of MnSOD in confluent cells does not affect Rb phosphorylation. However, MnSOD overexpression did alter p16 and p21 protein accumulation patterns during confluence. Although an age-dependent increase in p16 protein accumulation in confluent fibroblasts is associated with a cellular inability of the cells to re-enter the cell cycle, MnSOD-dependent suppression in p16 protein accumulation maintained the capacity of the confluent cells to re-enter the proliferation cycle (Figs. 5 and 8 and Table II). Because p16 is a specific inhibitor of the cyclin D1-Cdk4, 6 kinase complex and Rb is one of the substrates for this kinase activity, suppression of p16 would be expected to facilitate Rb phosphorylation upon re-entry to the proliferation cycle. Indeed, MnSOD overexpression-induced suppression of p16 in the confluent fibroblasts is associated with Rb hyperphosphorylation upon re-entry to the proliferation cycle and the subsequent entry of the fibroblasts into S-phase (Fig. 9, lane 6, and Figs. 3 and 8). However, the Rb phosphorylation pattern in MnSOD-overexpressing cells following re-entry was not different compared with control and AdBgl II-infected cells (Fig. 9, lanes 4-6), yet S-phase distributions were much higher in MnSOD-overexpressing cells. These results suggest the MnSOD-induced decrease in p16 protein accumulation in confluent cells might have a different end point compared with the role of p16 in Rb phosphorylation in proliferating cells. Furthermore, an earlier report of p16 accumulation as a function of replicative senescence in mouse fibroblasts and p16 accumulation being independent of Rb gene status (26) further supports the idea that p16 could have a differential regulatory role in confluent versus exponential growth phase. Alternatively, MnSOD overexpression in confluent fibroblasts could preserve the cell cycle re-entry pathways that are Rb-independent. Furthermore, because p16 protein levels decreased upon re-entry in control, AdBgl II-, and AdMnSOD-infected cells (Fig. 9), with only AdMnSOD-infected cells continuing to cycle upon re-entry, the regulatory role of p16 during confluency is more critical in maintaining the reproductive integrity of confluent cultures. Consistent with this hypothesis, overexpression of MnSOD failed to protect the loss of proliferative capacity in confluent fibroblasts after p16 accumulated (Fig. 5).

Most interestingly, whereas MnSOD overexpression suppressed p16 protein accumulation in confluent fibroblasts, the same treatment enhanced p21 protein levels (Fig. 5). MnSOD-dependent accumulation of p21 protein levels was independent of p53 protein levels (Fig. 5B). Whereas p16 is elevated at the end of the replicative life span and drives cells to an irreversible growth arrest, increases in p21 protein levels are biphasic, and the inhibitory effect of p21 on cell growth is a reversible process. Recent discoveries suggest p21 has multiple functions, including assembling cyclin-Cdk kinase complexes, inhibiting cyclin/Cdk kinase activity, binding to proliferating cell nuclear antigen and regulation of DNA synthesis, association with ASK1 and possible regulation of cell death pathways, and binding to the Rho protein and subsequent effects on integrin-signaling pathways (49). In addition, p21 has been shown to maintain hematopoietic stem cell quiescence, and consequently transplanted bone marrow from p21 knock out mice succumbed to hematopoietic failure (50). Furthermore, antisense p21 is known to release human mesenchymal cells from G0 (51). These reports support a regulatory role of p21 in maintaining quiescence and protecting the replicative integrity of proliferation-competent cells. It is currently unknown if any or all of these p21 functions are sensitive to the intracellular redox environment. However, our results suggest that MnSOD-induced protection of the reproductive integrity of the confluent fibroblasts could be mediated via maintaining a higher p21 protein level (Fig. 5). It is possible that MnSOD-induced accumulation of p21 protein levels would protect cyclin E(A)-Cdk 2 kinase complexes, which upon subculturing would facilitate a Rb phosphorylation-independent entry into S-phase. Such an assumption is supported by previous studies in the literature demonstrating cyclin E-dependent and Rb phosphorylation-independent entry into S-phase (24, 52). Nonetheless, the observation that MnSOD induces p21 protein levels might be of broad interest because of the possibility that antioxidants, in particular MnSOD, could have a protective role in maintaining the reproductive integrity of a variety of proliferation-competent quiescent cells, including stem cells.



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  		FIG. 10.
A schematic illustration of MnSOD-induced balance between p16 and p21 Cdk inhibitors, and protection of proliferative capacity of confluent fibroblasts.

 
In summary, our results demonstrate that a balance between two critical Cdk inhibitors, p16 and p21, could mediate an MnSOD-induced protection of the proliferation capacity of the confluent fibroblasts (Fig. 10). Although a majority of the aging research in mammalian cells focuses on understanding of the replicative life span of somatic cells, very few studies are centered on investigating the proliferative potential of quiescent cells, also known as the chronological life span. Because longevity of an organism depends on proliferative capacities of cycling and replicative-competent noncycling cells, a detailed understanding of the mechanisms regulating the chronological life span becomes highly essential. Our results support the hypothesis that two separate but interdependent pathways could regulate cellular longevity, a redox-sensitive checkpoint regulating transition from G0/G1 to S-phase followed by telomeric attrition controlling the "mitotic clock."


   FOOTNOTES
 
* This work was supported by American Cancer Society Grant IRG77-004-25 (to P. C. G.) and National Institutes of Health Grants 5PO1 CA66081 (to L. W. O.) and NS34736 (to T. K. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Free Radical and Radiation Biology Program, Dept. of Radiation Oncology, B180 Medical Laboratories, University of Iowa, Iowa City, IA 52242. Tel.: 319-384-4466; Fax: 319-335-8039; E-mail: prabhat-goswami{at}uiowa.edu.

1 The abbreviations used are: CuZnSOD, copper and zinc-containing superoxide dismutase; AdMnSOD, replication-defective adenovirus containing cytomegalovirus promoter-driven human Mn-superoxide dismutase antioxidant enzyme cDNA; AdBgl II, replication-defective adenovirus containing an insertless vector; BrdUrd, bromodeoxyuridine; FITC, fluorescence isothiocyanate; hTERT, human telomerase reverse transcriptase; m.o.i., multiplicity of infection; NHF, normal human fibroblasts; PI, propidium iodide; redox, reduction and oxidation; ROS, reactive oxygen species; Rb, retinoblastoma; GFP, green fluorescent protein; TEMED, N,N,N',N'-tetramethylethylenediamine; Cdk, cyclin-dependent kinase. Back


   ACKNOWLEDGMENTS
 
We thank Amanda Kalen, Yuping Zhang, and Sarita G. Menon for their technical assistance and Kellie Bodeker for valuable editorial assistance.



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