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Induction of Apoptosis in Human Replicative Senescent Fibroblasts
Experimental Cell Research 274, 92–99 (2002) doi:10.1006/excr.2001.5425, available online at http://www.idealibrary.com on
Induction of Apoptosis in Human Replicative Senescent Fibroblasts Vimaris DeJesus,* Ileana Rios,* ,† Claudette Davis,† ,‡ Yengsi Chen,† ,§ David Calhoun,† ,§ Zahra Zakeri,† ,‡ and Karen Hubbard* ,† ,1 *Department of Biology and §Department of Chemistry, City College of New York, 138th and Convent Avenue, New York, New York 10031; ‡Department of Biology, Queens College, Flushing, New York 11387; and †Graduate School and University Center of CUNY, New York, New York 10016
licative senescence is currently under investigation. Terminal exiting from the cell cycle may serve to function as a tumor suppressor mechanism [3]. One potential causal factor may be the attrition of telomeres [4]. Telomere length has been shown to decrease by many investigators both in vivo [5, 6] and, in cell culture, in vitro [4, 7]. This reduction may act as a DNA damage signal [8] and thus initiate senescence-associated pathways that ultimately result in the cessation of the cell cycle. Transforming events, such as SV40-mediated transformation and immortalization, allow cells to bypass replicative senescence [9]. Both normal and transformed cells have the ability to undergo apoptosis (programmed cell death). This becomes important in terms of a cell’s responsiveness to infection and the progression of tumor formation [10]. There are few studies that investigate the induction of apoptosis in senescent fibroblasts [11–13]. Senescent fibroblasts appear to respond to certain apoptotic signals, such as Fas L [11], but not to others, such as serum withdrawal [12]. It has been proposed that the forced reentry of senescent fibroblasts into the cell cycle may induce apoptosis [13]. Therefore, the failure to respond to certain apoptotic signals during aging may result in an accumulation of senescent cells and/or affect the tumor suppressor function of replicative senescence. Thus, it is important to understand the mechanisms whereby senescent cells can undergo programmed cell death. Senescent fibroblasts have large lysosomal bodies and a vacuolated cytoplasm [14 –16]. The enzyme activity for lysosomal cathepsin B is reduced in senescent cells due to the increased proteolysis of the singlechain form, whereas the less active two-chained form accumulates [17, 18]. The lysosomal enzyme -galactosidase increases with age in culture [19]. Taken together, it is possible that the morphological changes and the increases in -galactosidase activity suggest that lysosomal activation may potentially play a mechanistic role in apoptosis during replicative senescence. More importantly, there are several studies that dem-
Cellular senescence is an irreversible growth phase characteristic of normal cells. We have found that human senescent fibroblasts can be induced to undergo programmed cell death (apoptosis) by ceramide, TNF-␣, or okadaic acid. The most profound effects were induced by TNF-␣ and okadaic acid treatment. In the present study, we also evaluated the contribution of lysosomal activation as a possible mechanism underlying the induction of apoptosis. Four lysosomal enzyme activities were measured: -galactosidase, ␣-galactosidase A, -glucoronidase, and acid phosphatase. Using an in situ assay, we have found that the activity of -galactosidase, which is also a biochemical marker of senescence, is induced in young proliferating fibroblasts following exposure to all three apoptotic inducing agents. The other enzymes were not significantly induced in young fibroblasts following exposure to agents that induce apoptosis. During replicative senescence, three of the four lysosomal enzymes tested (-galactosidase, ␣-galactosidase A, and -glucoronidase) are constitutively expressed at high levels. TNF-␣ was the only agent that induced lysosomal activity in senescent fibroblasts, of which only ␣-galactosidase A activity was induced. Our studies show that senescent fibroblasts can be induced to undergo apoptosis in a signal-dependent manner. However, the lysosomal enzymes examined do not appear to be correlated with apoptotic induction. © 2002 Elsevier Science (USA)
Key Words: apoptosis; senescence; lysosomal; -galactosidase; aging; biomarker; human diploid fibroblasts.
INTRODUCTION
Replicative senescence is characterized as the terminal growth phase of normal diploid fibroblasts [1]. Although it is the culmination of numerous changes in gene expression [reviewed in 2], the causation of rep1 To whom correspondence and reprint requests should be addressed. Fax: 212.650.8585. E-mail: [email protected].
0014-4827/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
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FIG. 1. Induction of cell death in young proliferating and senescent fibroblasts. Young and senescent HS74 cells (see Material and Methods) were incubated with 10 M ceramide, 10 nM okadaic acid, or 10 ng/ml TNF-␣ or were untreated (control) for 24 h. A: The percentage viability determined by cell counts. Cell counts were measured for each condition and normalized to the appropriate growth status (either young or senescent) as a percentage of control values. The data for young cells are the average of seven individual experiments, in which the total number of cells ranged from 1.54 to 2.38 ⫻ 10 6. The data for senescent cells are the average of 9 individual experiments, in which the total number of cells ranged from 1.26 to 2.52 ⫻ 10 6. B: Cell death was measured as the percentage of cells excluding trypan blue cells. Cells floating in the medium were collected and analyzed together with attached cells. The data for young cells are the average of 10 individual experiments, in which the total number of cells analyzed for each experiment ranged from 1560 to 2200. The data for senescent cells are represented by the average of 9 individual experiments, in which the total number of cells ranged from 155 to 417.
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described by Hubbard and Ozer [21] in Dulbecco’s modified Eagle’s medium and Ham’s F10 medium in a 1:1 mixture supplemented with 10% fetal bovine serum. HS74 fibroblasts at population doubling 44 were used in all experiments and are considered comparable to young fibroblasts as determined by gene expression profiles previously performed [21]. Senescent HS74 in all experiments were at a population doubling of 62. Cell counts were quantified by a hemocytometer and viability by trypan blue exclusion was determined as previously described [22]. In situ -galactosidase assay. An in situ method developed by Dimri and others [19] was used to label -galactosidase activity in the cells. Cells were seeded at a density of 1 ⫻ 10 5 per 60-mm culture plates. After 24 h, the medium was exchanged for that containing 10 M C 6 ceramide, 10 nM okadaic acid, or 10 ng/ml TNF-␣ and incubation for 24 h followed. Dishes were then washed with 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 137 mM NaCL, and 8 mM Na 2 HPO 4 䡠 7 H 2 O, pH 7.0 (PBS), followed by fixation with 2% formaldehyde and 0.2% glutaraldehyde in PBS. -Galactosidase activity was detected by histochemical staining. Dishes were incubated with a staining solution (20 mg/ml X-Gal, 40 mM citric acid/ sodium phosphate buffer, pH.4.0, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 150 mM sodium chloride, and 2 mM magnesium chloride) for 12–16 h. Lysosomal enzyme activity analysis. Crude cell lysates were prepared by centrifugation of HS74 fibroblasts at 2000g for 10 min to obtain a cell pellet. The cell pellet was resuspended in 100 –120 l of lysis buffer which contained 0.1% Triton X-100, 10 mM sodium phosphate, pH 6.5, 0.02% sodium azide, and 0.05 mM phenylmethylsulfonyl fluoride. Complete lysis was achieved by three freeze– thaw cycles in dry ice–alcohol baths. The cell lysate was then centrifuged in an Eppendorf microcentrifuge for 5 min at 15,000g. The protein concentration of the various cell lysates was quantified by Bradford–Lowry protein assay (Bio-Rad) using bovine serum albumin as a standard for protein concentrations. -Galactosidase and acid phosphatase activities were measured spectrophotometrically as previously described by Meisler [23] and by a modification of Sleyester and Knook [24], respectively. ␣-Galactosidase A and -glucoronidase activities were measured by the fluorescence spectrophotometric procedure described by Copola et al. [25]. Apoptosis analysis. HS74 fibroblasts were plated on coverslips 24 h prior to incubation with 10 M C 6 ceramide, 10 ng/ml TNF-␣, or 10 nM okadaic acid. Cells were then incubated overnight (24 h) with each agent follow by fixation with 3% paraformaldehyde in PBS. The number of cells undergoing apoptosis was quantified using the TUNEL assay (TdT FrageL DNA fragmentation detection kit, Oncogene Research Products, CalBiochem) which labels chromosomal termini. A total of 254 young and 384 senescent cells were counted in at least three to four fields of cells.
RESULTS AND DISCUSSION
onstrate that lysosomal activation plays a significant role in cell death [20]. In the present study, we have examined the effects of several apoptotic signals during replicative senescence in human diploid fibroblasts. We also sought to determine the relationship between lysosomal activation and programmed cell death. In this regard, several lysosomal enzyme activities were measured under conditions that induced significant levels of apoptosis. MATERIALS AND METHODS Cell culture. The human fetal fibroblast cell strain HS74 was subcultured from early passage to terminal passage as previously
Cell Death Induction in Young and Senescent Fibroblasts The ability of senescent fibroblasts to respond to apoptotic signaling is poorly understood. Senescent cells are resistant to serum withdrawal [12] but have been shown more recently to be sensitive to Fas L [11]. In this study, we investigated other apoptotic inducing agents to further define the role of apoptosis during replicative senescence. Young and senescent fibroblasts were exposed to okadaic acid, TNF-␣, and ceramide at concentrations which have been shown to induce apoptosis in other systems [13, 26 –28]. Okadaic
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FIG. 2. Induction of -galactosidase activity in young and senescent human fibroblasts. Young and senescent HS74 cells (see Material and Methods) were incubated with 10 M ceramide, 10 nM okadaic acid, or 10 ng/ml TNF-␣ or were untreated (control) for 24 h and then stained for -galactosidase. -Galactosidase activity identified by in situ histochemical staining as described under Materials and Methods. The original magnification for young and senescent fibroblasts was 200X and 400X, respectively.
acid has been shown to induce cell death in senescent fibroblasts in an earlier report by Afshari et al. [13]. Ceramide was chosen since its levels increase in senescent fibroblasts [27] and can induce apoptosis in several systems [26]. TNF-␣ induces apoptosis via several mechanisms, including activation of TRADD/FADD
death adapter proteins and lysosomal sphingomyelinase activity (which generates ceramide) [26]. We found that all three apoptotic inducers resulted in cell death in both young and senescent fibroblasts. There was more induction of cell death by TNF-␣ and okadaic acid compared to ceramide in young fibroblasts; see
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FIG. 3. In situ analysis of apoptosis in young proliferating and senescent fibroblasts. Apoptotic cells were identified by in situ labeling of fragmented chromosomal termini using TUNEL assays. The original magnification for all conditions was 200X.
Fig. 1A for cell counts. However, all three agents induced significant cell death in senescent fibroblasts (Fig. 1A). When cell viability was analyzed by trypan blue exclusion (Fig. 1B), we found that the data for young cells differed from viability quantitated solely by cell counts. Cells analyzed for trypan blue analysis were primarily attached to the dish and, thus, may be an overestimation of the actual cell viability. The dis-
crepancy between the cell count data exhibiting at least 40% cell death for okadaic acid or TNF-␣ with that of trypan blue exclusion data (which indicates only 10% cell death) may be due to the lack of recovery of dead cells floating in the medium due to cell fragmentation. The induction of cell death in senescent cells determined by trypan blue exclusion mirrors cell count analysis (compare Figs. 1A and 1B). These re-
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FIG. 4. TUNEL analysis of DNA fragmentation in young proliferating and senescent fibroblasts. Young and senescent HS74 cells (see Material and Methods) were incubated with 10 M ceramide, 10 nM okadaic acid, or 10 ng/ml TNF-␣ or were untreated (control) for 24 h, stained for DNA fragmentation, and counted as described under Materials and Methods.
sults indicate that senescent cells may be more sensitive to a variety of apoptotic signals than young cells. The induction of cell death in senescent cells resulted in a marked alteration in morphology as shown in Fig. 2. In situ lysosomal -galactosidase activity is apparent and the morphological changes induced by all three agents in senescent cells compared to young cells illustrate the differences between young and senescent cells. Cells became larger and tended to round up following exposure to okadaic acid. TNF-␣ and ceramide did not produce significant morphological changes in young or senescent fibroblasts. In situ lysosomal -galactosidase activity increased in young cells following exposure to all three agents (Fig. 2). The in situ lysosomal -galactosidase activity in senescent fibroblasts was greatly increased compared to that of young fibroblasts with or without cell death inducers. Induction of Apoptosis in Young and Senescent Fibroblast To determine whether the morphological observation seen in Fig. 2 and the increased levels of cell death were indeed due to apoptosis, we performed TUNEL analysis which is used as an indicator of DNA fragmentation associated with programmed cell death. As expected, there were few TUNEL-positive young cells following exposure to the apoptotic inducers (Fig. 3) and any differences noted were not statistically significant. In contrast, in senescent cells, we observed extensive labeling of cells following exposure to all three agents. The actual level of induction was quantitated and is shown in Fig. 4. By this assay, okadaic acid and TNF-␣ but not ceramide resulted in significant induction of apoptosis. Although trypan blue data as well as cell counts (Figs. 1A and 1B) indicate that ceramide induces cell death in senescent cells, this cell death
may not result in the typical DNA fragmentation seen in many apoptotic systems because the level of TUNEL-positive cells was not significantly higher than that of control senescent cells (Fig. 4). Another measure of apoptosis is caspase activation. While Fas L induced caspase 3 activation in senescent cells [11], we have been unable to detect cleavage of caspase 3 or 8 in our studies (data not shown). It is not clear which caspase cascade signaling mechanism is important for our observations and needs further clarification. However, we have extended these analyses to induction of apoptosis in senescent WI-38 and IMR-90 cells (both are normal human diploid fibroblasts) and have found for both strains more TUNEL-positive cells in treated senescent fibroblasts than in young cells (unpublished observations). Lysosomal Enzyme Activity It has been previously shown by Dimri et al. [19] that senescent fibroblasts express high levels of -galactosidase activity in situ. Given the possible involvement of lysosomal activity in both cell death and cell senescence, we examined the induction of four lysosomal enzymes during cell death in young and old cells. We measured -galactosidase, ␣-galactosidase A, -glucoronidase, and acid phosphatase activity. To further explore whether constitutive levels of -galactosidase prime fibroblasts to undergo apoptosis, we investigated the effects of ceramide, TNF-␣, and okadaic acid on -galactosidase activity in young and senescent cells under conditions that produce significant levels of cell death. In situ analysis of -galactosidase activity indicated that all three inducers increase the expression of this enzyme in young fibroblasts (Fig. 2). Quantification of these in situ data showed a 1.6-, 2.3-, and 2-fold induction of -galactosidase in ceramide-, okadaic-acid-, and TNF-␣-treated young fibroblasts, respectively. However, we could not determine any quantitative difference for senescent fibroblast exclusively by in situ analysis because senescent fibroblasts normally express high levels of -galactosidase [19]. Since in situ evaluation of -galactosidase is qualitative rather than quantitative, we measured the total specific activity of -galactosidase in whole cell lysates following drug exposure. The in situ assay for -galactosidase (Fig. 2) takes place at pH 6.0 and may measure a different enzyme activity than that measured in the biochemical assay used for lysosomal enzymes at pH 4.5 (discussed in [19]). The results of our assays for four lysosomal enzymes in young and senescent cells in the presence and absence of inducers of apoptosis are shown in Fig. 5 and normalized in Table 1. First, a comparison of the enzyme levels in young and senescent cells indicates little or no difference for acid
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FIG. 5. Lysosomal activity in young and senescent human fibroblasts. Young and senescent HS74 cells (see Material and Methods) were incubated with 10 M ceramide, 10 nM okadaic acid, or 10 ng/ml TNF-␣ or were untreated (control) for 24 h, harvested for lysis, and assayed for -galactosidase (A), ␣-galactosidase (B), -glucoronidase (C), and acid phosphatase (D) activity as described under Materials and Methods.
phosphatase, but increasingly greater enzyme levels in senescent cells compared to young cells for -galactosidase, ␣-galactosidase A, and -glucoronidase, respectively (Table 1). These results indicate that -glucoronidase and ␣-galactosidase A are significantly elevated in senescent cells compared to young cells and provide unambiguous markers of senescence in this experimental system. The levels of -galactosidase are also elevated in senescent cells (Table 1) in agreement with our in situ results (Fig. 2) and previous reports in the literature (e.g., [29, 30]). However, the smaller change in enzyme levels compared to ␣-galactosidase A and -glucoronidase makes -galactosidase a less reliable biomarker of senescence compared to ␣-galactosidase A and -glucoronidase. Our findings are supported by recent observations showing that -galactosidase activity in situ is not spe-
cific to replicative senescence but is dependent on cell density. That is, -galactosidase increases in young fibroblasts that have been contact inhibited by saturation density [31]. Second, with regard to the effects of inducers of apoptosis on the enzyme levels in young and senescent cells, there was no statistically significant change except for a twofold induction of ␣-galactosidase A by TNF-␣ in senescent, but not in young cells. These results indicate that there is not a significant global correlation between induction of apoptosis and changes in levels of these four lysosomal enzymes using this experimental technique. In addition, our studies show that senescent cells can undergo apoptosis. However, increased lysosomal activities measured per se do not appear to play a role
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TABLE 1 Lysosomal Enzyme Levels in Young and Senescent HS74 Cells in the Presence and Absence of Inducers of Apoptosis a Acid phosphatase
-Galactosidase
␣-Galactosidase A
-Glucoronidase
Inducer
Young
Senescent
Young
Senescent
Young
Senescent
Young
Senescent
Control Ceramide Okadaic acid TNF-␣
1.0 0.9 1.3 1.1
1.2 0.9 1.0 2.1
1.0 1.3 3.3 1.7
3.1 2.7 1.7 2.5
1.0 1.1 1.3 1.2
5.4 5.1 5.9 12.0
1.0 1.2 0.9 1.5
15 13 11 11
a
The indicated lysosomal enzymes were assayed in young and senescent cells in the presence and absence of inducers of senescence. For each enzyme levels were normalized to young cells in the absence of inducer.
during apoptosis because none of the conditions employed correlated with significant levels of TUNELpositive cells, except notably for TNF-␣ treatment of senescent fibroblasts. In this case, TNF-␣ induced apoptosis and ␣-galactosidase in senescent cells and requires further study to delineate the role of lysosomal activation during TNF-␣ induction of apoptosis. Induction of apoptosis during senescence is very complex. The dogma has been that senescent fibroblasts were resistant to apoptosis and was based on the lack of induction of DNA fragmentation occurring following serum withdrawal [12]. There have only been a few informative studies that have evaluated the response of senescent cells to various apoptotic signaling pathways. Apoptosis can be induced in senescent fibroblasts by Fas L [11]. Our studies show that okadaic acid and TNF-␣ can induce apoptosis in senescent fibroblasts. TNF-␣ is generally not cytotoxic to cells in the absence of protein synthesis inhibition because it can also induce the expression of anti-apoptotic genes through the NF-B pathway [32]. However, TNF-␣ may induce apoptosis in senescent cells, albeit at a modest level, because of a decrease in the nuclear activity of NF-B in senescent fibroblasts. Just very recently, it has been demonstrated that senescent fibroblasts are resistant to apoptosis induced by p53dependent DNA damage, although still capable of responding to p53-independent apoptosis induced by genotoxic stress [33]. Thus, the ability of senescent cells to undergo apoptosis is signal dependent. The understanding of the mechanisms that provide apoptotic signals during replicative senescence is important for our understanding of normal cell turnover with age. It has been proposed that senescent cells may influence the microenvironment and integrity of surrounding tissue [3] and may also impact on the ability of the aged to respond to anticancer treatment [33]. This work was supported by NIH/RCMI Grant RR03060 (to KH and DC), NIH/MBRS Grant RR08168 (to KH and DC), and CUNY Collaborative Incentive Grant 991927 (to KH, DC, and ZZ).
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Received June 20, 2001 Revised version received October 29, 2001 Published online January 22, 2002
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Induction of Apoptosis in Human Replicative Senescent Fibroblasts Vimaris DeJesus,* Ileana Rios,* ,† Claudette Davis,† ,‡ Yengsi Chen,† ,§ David Calhoun,† ,§ Zahra Zakeri,† ,‡ and Karen Hubbard* ,† ,1 *Department of Biology and §Department of Chemistry, City College of New York, 138th and Convent Avenue, New York, New York 10031; ‡Department of Biology, Queens College, Flushing, New York 11387; and †Graduate School and University Center of CUNY, New York, New York 10016
licative senescence is currently under investigation. Terminal exiting from the cell cycle may serve to function as a tumor suppressor mechanism [3]. One potential causal factor may be the attrition of telomeres [4]. Telomere length has been shown to decrease by many investigators both in vivo [5, 6] and, in cell culture, in vitro [4, 7]. This reduction may act as a DNA damage signal [8] and thus initiate senescence-associated pathways that ultimately result in the cessation of the cell cycle. Transforming events, such as SV40-mediated transformation and immortalization, allow cells to bypass replicative senescence [9]. Both normal and transformed cells have the ability to undergo apoptosis (programmed cell death). This becomes important in terms of a cell’s responsiveness to infection and the progression of tumor formation [10]. There are few studies that investigate the induction of apoptosis in senescent fibroblasts [11–13]. Senescent fibroblasts appear to respond to certain apoptotic signals, such as Fas L [11], but not to others, such as serum withdrawal [12]. It has been proposed that the forced reentry of senescent fibroblasts into the cell cycle may induce apoptosis [13]. Therefore, the failure to respond to certain apoptotic signals during aging may result in an accumulation of senescent cells and/or affect the tumor suppressor function of replicative senescence. Thus, it is important to understand the mechanisms whereby senescent cells can undergo programmed cell death. Senescent fibroblasts have large lysosomal bodies and a vacuolated cytoplasm [14 –16]. The enzyme activity for lysosomal cathepsin B is reduced in senescent cells due to the increased proteolysis of the singlechain form, whereas the less active two-chained form accumulates [17, 18]. The lysosomal enzyme -galactosidase increases with age in culture [19]. Taken together, it is possible that the morphological changes and the increases in -galactosidase activity suggest that lysosomal activation may potentially play a mechanistic role in apoptosis during replicative senescence. More importantly, there are several studies that dem-
Cellular senescence is an irreversible growth phase characteristic of normal cells. We have found that human senescent fibroblasts can be induced to undergo programmed cell death (apoptosis) by ceramide, TNF-␣, or okadaic acid. The most profound effects were induced by TNF-␣ and okadaic acid treatment. In the present study, we also evaluated the contribution of lysosomal activation as a possible mechanism underlying the induction of apoptosis. Four lysosomal enzyme activities were measured: -galactosidase, ␣-galactosidase A, -glucoronidase, and acid phosphatase. Using an in situ assay, we have found that the activity of -galactosidase, which is also a biochemical marker of senescence, is induced in young proliferating fibroblasts following exposure to all three apoptotic inducing agents. The other enzymes were not significantly induced in young fibroblasts following exposure to agents that induce apoptosis. During replicative senescence, three of the four lysosomal enzymes tested (-galactosidase, ␣-galactosidase A, and -glucoronidase) are constitutively expressed at high levels. TNF-␣ was the only agent that induced lysosomal activity in senescent fibroblasts, of which only ␣-galactosidase A activity was induced. Our studies show that senescent fibroblasts can be induced to undergo apoptosis in a signal-dependent manner. However, the lysosomal enzymes examined do not appear to be correlated with apoptotic induction. © 2002 Elsevier Science (USA)
Key Words: apoptosis; senescence; lysosomal; -galactosidase; aging; biomarker; human diploid fibroblasts.
INTRODUCTION
Replicative senescence is characterized as the terminal growth phase of normal diploid fibroblasts [1]. Although it is the culmination of numerous changes in gene expression [reviewed in 2], the causation of rep1 To whom correspondence and reprint requests should be addressed. Fax: 212.650.8585. E-mail: [email protected].
0014-4827/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
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FIG. 1. Induction of cell death in young proliferating and senescent fibroblasts. Young and senescent HS74 cells (see Material and Methods) were incubated with 10 M ceramide, 10 nM okadaic acid, or 10 ng/ml TNF-␣ or were untreated (control) for 24 h. A: The percentage viability determined by cell counts. Cell counts were measured for each condition and normalized to the appropriate growth status (either young or senescent) as a percentage of control values. The data for young cells are the average of seven individual experiments, in which the total number of cells ranged from 1.54 to 2.38 ⫻ 10 6. The data for senescent cells are the average of 9 individual experiments, in which the total number of cells ranged from 1.26 to 2.52 ⫻ 10 6. B: Cell death was measured as the percentage of cells excluding trypan blue cells. Cells floating in the medium were collected and analyzed together with attached cells. The data for young cells are the average of 10 individual experiments, in which the total number of cells analyzed for each experiment ranged from 1560 to 2200. The data for senescent cells are represented by the average of 9 individual experiments, in which the total number of cells ranged from 155 to 417.
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described by Hubbard and Ozer [21] in Dulbecco’s modified Eagle’s medium and Ham’s F10 medium in a 1:1 mixture supplemented with 10% fetal bovine serum. HS74 fibroblasts at population doubling 44 were used in all experiments and are considered comparable to young fibroblasts as determined by gene expression profiles previously performed [21]. Senescent HS74 in all experiments were at a population doubling of 62. Cell counts were quantified by a hemocytometer and viability by trypan blue exclusion was determined as previously described [22]. In situ -galactosidase assay. An in situ method developed by Dimri and others [19] was used to label -galactosidase activity in the cells. Cells were seeded at a density of 1 ⫻ 10 5 per 60-mm culture plates. After 24 h, the medium was exchanged for that containing 10 M C 6 ceramide, 10 nM okadaic acid, or 10 ng/ml TNF-␣ and incubation for 24 h followed. Dishes were then washed with 2.7 mM KCl, 1.5 mM KH 2 PO 4 , 137 mM NaCL, and 8 mM Na 2 HPO 4 䡠 7 H 2 O, pH 7.0 (PBS), followed by fixation with 2% formaldehyde and 0.2% glutaraldehyde in PBS. -Galactosidase activity was detected by histochemical staining. Dishes were incubated with a staining solution (20 mg/ml X-Gal, 40 mM citric acid/ sodium phosphate buffer, pH.4.0, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 150 mM sodium chloride, and 2 mM magnesium chloride) for 12–16 h. Lysosomal enzyme activity analysis. Crude cell lysates were prepared by centrifugation of HS74 fibroblasts at 2000g for 10 min to obtain a cell pellet. The cell pellet was resuspended in 100 –120 l of lysis buffer which contained 0.1% Triton X-100, 10 mM sodium phosphate, pH 6.5, 0.02% sodium azide, and 0.05 mM phenylmethylsulfonyl fluoride. Complete lysis was achieved by three freeze– thaw cycles in dry ice–alcohol baths. The cell lysate was then centrifuged in an Eppendorf microcentrifuge for 5 min at 15,000g. The protein concentration of the various cell lysates was quantified by Bradford–Lowry protein assay (Bio-Rad) using bovine serum albumin as a standard for protein concentrations. -Galactosidase and acid phosphatase activities were measured spectrophotometrically as previously described by Meisler [23] and by a modification of Sleyester and Knook [24], respectively. ␣-Galactosidase A and -glucoronidase activities were measured by the fluorescence spectrophotometric procedure described by Copola et al. [25]. Apoptosis analysis. HS74 fibroblasts were plated on coverslips 24 h prior to incubation with 10 M C 6 ceramide, 10 ng/ml TNF-␣, or 10 nM okadaic acid. Cells were then incubated overnight (24 h) with each agent follow by fixation with 3% paraformaldehyde in PBS. The number of cells undergoing apoptosis was quantified using the TUNEL assay (TdT FrageL DNA fragmentation detection kit, Oncogene Research Products, CalBiochem) which labels chromosomal termini. A total of 254 young and 384 senescent cells were counted in at least three to four fields of cells.
RESULTS AND DISCUSSION
onstrate that lysosomal activation plays a significant role in cell death [20]. In the present study, we have examined the effects of several apoptotic signals during replicative senescence in human diploid fibroblasts. We also sought to determine the relationship between lysosomal activation and programmed cell death. In this regard, several lysosomal enzyme activities were measured under conditions that induced significant levels of apoptosis. MATERIALS AND METHODS Cell culture. The human fetal fibroblast cell strain HS74 was subcultured from early passage to terminal passage as previously
Cell Death Induction in Young and Senescent Fibroblasts The ability of senescent fibroblasts to respond to apoptotic signaling is poorly understood. Senescent cells are resistant to serum withdrawal [12] but have been shown more recently to be sensitive to Fas L [11]. In this study, we investigated other apoptotic inducing agents to further define the role of apoptosis during replicative senescence. Young and senescent fibroblasts were exposed to okadaic acid, TNF-␣, and ceramide at concentrations which have been shown to induce apoptosis in other systems [13, 26 –28]. Okadaic
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FIG. 2. Induction of -galactosidase activity in young and senescent human fibroblasts. Young and senescent HS74 cells (see Material and Methods) were incubated with 10 M ceramide, 10 nM okadaic acid, or 10 ng/ml TNF-␣ or were untreated (control) for 24 h and then stained for -galactosidase. -Galactosidase activity identified by in situ histochemical staining as described under Materials and Methods. The original magnification for young and senescent fibroblasts was 200X and 400X, respectively.
acid has been shown to induce cell death in senescent fibroblasts in an earlier report by Afshari et al. [13]. Ceramide was chosen since its levels increase in senescent fibroblasts [27] and can induce apoptosis in several systems [26]. TNF-␣ induces apoptosis via several mechanisms, including activation of TRADD/FADD
death adapter proteins and lysosomal sphingomyelinase activity (which generates ceramide) [26]. We found that all three apoptotic inducers resulted in cell death in both young and senescent fibroblasts. There was more induction of cell death by TNF-␣ and okadaic acid compared to ceramide in young fibroblasts; see
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FIG. 3. In situ analysis of apoptosis in young proliferating and senescent fibroblasts. Apoptotic cells were identified by in situ labeling of fragmented chromosomal termini using TUNEL assays. The original magnification for all conditions was 200X.
Fig. 1A for cell counts. However, all three agents induced significant cell death in senescent fibroblasts (Fig. 1A). When cell viability was analyzed by trypan blue exclusion (Fig. 1B), we found that the data for young cells differed from viability quantitated solely by cell counts. Cells analyzed for trypan blue analysis were primarily attached to the dish and, thus, may be an overestimation of the actual cell viability. The dis-
crepancy between the cell count data exhibiting at least 40% cell death for okadaic acid or TNF-␣ with that of trypan blue exclusion data (which indicates only 10% cell death) may be due to the lack of recovery of dead cells floating in the medium due to cell fragmentation. The induction of cell death in senescent cells determined by trypan blue exclusion mirrors cell count analysis (compare Figs. 1A and 1B). These re-
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FIG. 4. TUNEL analysis of DNA fragmentation in young proliferating and senescent fibroblasts. Young and senescent HS74 cells (see Material and Methods) were incubated with 10 M ceramide, 10 nM okadaic acid, or 10 ng/ml TNF-␣ or were untreated (control) for 24 h, stained for DNA fragmentation, and counted as described under Materials and Methods.
sults indicate that senescent cells may be more sensitive to a variety of apoptotic signals than young cells. The induction of cell death in senescent cells resulted in a marked alteration in morphology as shown in Fig. 2. In situ lysosomal -galactosidase activity is apparent and the morphological changes induced by all three agents in senescent cells compared to young cells illustrate the differences between young and senescent cells. Cells became larger and tended to round up following exposure to okadaic acid. TNF-␣ and ceramide did not produce significant morphological changes in young or senescent fibroblasts. In situ lysosomal -galactosidase activity increased in young cells following exposure to all three agents (Fig. 2). The in situ lysosomal -galactosidase activity in senescent fibroblasts was greatly increased compared to that of young fibroblasts with or without cell death inducers. Induction of Apoptosis in Young and Senescent Fibroblast To determine whether the morphological observation seen in Fig. 2 and the increased levels of cell death were indeed due to apoptosis, we performed TUNEL analysis which is used as an indicator of DNA fragmentation associated with programmed cell death. As expected, there were few TUNEL-positive young cells following exposure to the apoptotic inducers (Fig. 3) and any differences noted were not statistically significant. In contrast, in senescent cells, we observed extensive labeling of cells following exposure to all three agents. The actual level of induction was quantitated and is shown in Fig. 4. By this assay, okadaic acid and TNF-␣ but not ceramide resulted in significant induction of apoptosis. Although trypan blue data as well as cell counts (Figs. 1A and 1B) indicate that ceramide induces cell death in senescent cells, this cell death
may not result in the typical DNA fragmentation seen in many apoptotic systems because the level of TUNEL-positive cells was not significantly higher than that of control senescent cells (Fig. 4). Another measure of apoptosis is caspase activation. While Fas L induced caspase 3 activation in senescent cells [11], we have been unable to detect cleavage of caspase 3 or 8 in our studies (data not shown). It is not clear which caspase cascade signaling mechanism is important for our observations and needs further clarification. However, we have extended these analyses to induction of apoptosis in senescent WI-38 and IMR-90 cells (both are normal human diploid fibroblasts) and have found for both strains more TUNEL-positive cells in treated senescent fibroblasts than in young cells (unpublished observations). Lysosomal Enzyme Activity It has been previously shown by Dimri et al. [19] that senescent fibroblasts express high levels of -galactosidase activity in situ. Given the possible involvement of lysosomal activity in both cell death and cell senescence, we examined the induction of four lysosomal enzymes during cell death in young and old cells. We measured -galactosidase, ␣-galactosidase A, -glucoronidase, and acid phosphatase activity. To further explore whether constitutive levels of -galactosidase prime fibroblasts to undergo apoptosis, we investigated the effects of ceramide, TNF-␣, and okadaic acid on -galactosidase activity in young and senescent cells under conditions that produce significant levels of cell death. In situ analysis of -galactosidase activity indicated that all three inducers increase the expression of this enzyme in young fibroblasts (Fig. 2). Quantification of these in situ data showed a 1.6-, 2.3-, and 2-fold induction of -galactosidase in ceramide-, okadaic-acid-, and TNF-␣-treated young fibroblasts, respectively. However, we could not determine any quantitative difference for senescent fibroblast exclusively by in situ analysis because senescent fibroblasts normally express high levels of -galactosidase [19]. Since in situ evaluation of -galactosidase is qualitative rather than quantitative, we measured the total specific activity of -galactosidase in whole cell lysates following drug exposure. The in situ assay for -galactosidase (Fig. 2) takes place at pH 6.0 and may measure a different enzyme activity than that measured in the biochemical assay used for lysosomal enzymes at pH 4.5 (discussed in [19]). The results of our assays for four lysosomal enzymes in young and senescent cells in the presence and absence of inducers of apoptosis are shown in Fig. 5 and normalized in Table 1. First, a comparison of the enzyme levels in young and senescent cells indicates little or no difference for acid
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FIG. 5. Lysosomal activity in young and senescent human fibroblasts. Young and senescent HS74 cells (see Material and Methods) were incubated with 10 M ceramide, 10 nM okadaic acid, or 10 ng/ml TNF-␣ or were untreated (control) for 24 h, harvested for lysis, and assayed for -galactosidase (A), ␣-galactosidase (B), -glucoronidase (C), and acid phosphatase (D) activity as described under Materials and Methods.
phosphatase, but increasingly greater enzyme levels in senescent cells compared to young cells for -galactosidase, ␣-galactosidase A, and -glucoronidase, respectively (Table 1). These results indicate that -glucoronidase and ␣-galactosidase A are significantly elevated in senescent cells compared to young cells and provide unambiguous markers of senescence in this experimental system. The levels of -galactosidase are also elevated in senescent cells (Table 1) in agreement with our in situ results (Fig. 2) and previous reports in the literature (e.g., [29, 30]). However, the smaller change in enzyme levels compared to ␣-galactosidase A and -glucoronidase makes -galactosidase a less reliable biomarker of senescence compared to ␣-galactosidase A and -glucoronidase. Our findings are supported by recent observations showing that -galactosidase activity in situ is not spe-
cific to replicative senescence but is dependent on cell density. That is, -galactosidase increases in young fibroblasts that have been contact inhibited by saturation density [31]. Second, with regard to the effects of inducers of apoptosis on the enzyme levels in young and senescent cells, there was no statistically significant change except for a twofold induction of ␣-galactosidase A by TNF-␣ in senescent, but not in young cells. These results indicate that there is not a significant global correlation between induction of apoptosis and changes in levels of these four lysosomal enzymes using this experimental technique. In addition, our studies show that senescent cells can undergo apoptosis. However, increased lysosomal activities measured per se do not appear to play a role
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TABLE 1 Lysosomal Enzyme Levels in Young and Senescent HS74 Cells in the Presence and Absence of Inducers of Apoptosis a Acid phosphatase
-Galactosidase
␣-Galactosidase A
-Glucoronidase
Inducer
Young
Senescent
Young
Senescent
Young
Senescent
Young
Senescent
Control Ceramide Okadaic acid TNF-␣
1.0 0.9 1.3 1.1
1.2 0.9 1.0 2.1
1.0 1.3 3.3 1.7
3.1 2.7 1.7 2.5
1.0 1.1 1.3 1.2
5.4 5.1 5.9 12.0
1.0 1.2 0.9 1.5
15 13 11 11
a
The indicated lysosomal enzymes were assayed in young and senescent cells in the presence and absence of inducers of senescence. For each enzyme levels were normalized to young cells in the absence of inducer.
during apoptosis because none of the conditions employed correlated with significant levels of TUNELpositive cells, except notably for TNF-␣ treatment of senescent fibroblasts. In this case, TNF-␣ induced apoptosis and ␣-galactosidase in senescent cells and requires further study to delineate the role of lysosomal activation during TNF-␣ induction of apoptosis. Induction of apoptosis during senescence is very complex. The dogma has been that senescent fibroblasts were resistant to apoptosis and was based on the lack of induction of DNA fragmentation occurring following serum withdrawal [12]. There have only been a few informative studies that have evaluated the response of senescent cells to various apoptotic signaling pathways. Apoptosis can be induced in senescent fibroblasts by Fas L [11]. Our studies show that okadaic acid and TNF-␣ can induce apoptosis in senescent fibroblasts. TNF-␣ is generally not cytotoxic to cells in the absence of protein synthesis inhibition because it can also induce the expression of anti-apoptotic genes through the NF-B pathway [32]. However, TNF-␣ may induce apoptosis in senescent cells, albeit at a modest level, because of a decrease in the nuclear activity of NF-B in senescent fibroblasts. Just very recently, it has been demonstrated that senescent fibroblasts are resistant to apoptosis induced by p53dependent DNA damage, although still capable of responding to p53-independent apoptosis induced by genotoxic stress [33]. Thus, the ability of senescent cells to undergo apoptosis is signal dependent. The understanding of the mechanisms that provide apoptotic signals during replicative senescence is important for our understanding of normal cell turnover with age. It has been proposed that senescent cells may influence the microenvironment and integrity of surrounding tissue [3] and may also impact on the ability of the aged to respond to anticancer treatment [33]. This work was supported by NIH/RCMI Grant RR03060 (to KH and DC), NIH/MBRS Grant RR08168 (to KH and DC), and CUNY Collaborative Incentive Grant 991927 (to KH, DC, and ZZ).
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Received June 20, 2001 Revised version received October 29, 2001 Published online January 22, 2002
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