Apoptosis in human primary fibroblasts.
To examine whether cellular aging affects the ability of cells to undergo apoptosis in response to genotoxic stress, we chose WI-38 primary human fibroblasts. Since the detection of apoptosis in fibroblasts is controversial (3
), we first analyzed the abilities of various DNA-damaging agents to induce apoptosis in these cells.
Exponentially growing populations of young human fibroblasts were treated with the following DNA-damaging agents: actinomycin D, UV irradiation, etoposide, and low (0.5- to 2-μg/ml) and high (5- to 20-μg/ml) concentrations of cisplatin. Apoptosis was determined by the appearance of apoptotic features, such as DNA fragmentation, chromatin condensation, and cleavage of PARP.
DNA fragmentation was analyzed by the DNA ladder technique. It was previously shown that fibroblasts produce mostly DNA fragments of high molecular weight not followed by the internucleosomal DNA cleavage typical of hematopoietic cells (8
). Furthermore, it was suggested that the cleavage of DNA into high-molecular-weight, approximately 50- and 300-kb fragments is an essential early step in apoptosis for all cell types, in contrast to the later and nonessential internucleosomal DNA fragmentation (8
). Therefore, fibroblasts undergoing apoptosis yield a high-molecular-weight smear instead of the typical 180-bp DNA ladder. The optimal time and drug concentration for the induction of apoptosis were defined for every DNA-damaging agent (data not shown). Upon treatment of young cells with actinomycin D, UV irradiation, etoposide, and low and high concentrations of cisplatin, we detected the appearance of high-molecular-weight smears in a time-dependent manner (Fig. A). Different levels of DNA fragmentation were observed for the different agents.
FIG. 1 Apoptosis in young and old normal human fibroblasts induced by DNA-damaging agents. Young and old fibroblasts were treated with various DNA-damaging agents (actinomycin D, UV, etoposide, and low [1-μg/ml] and high [10-μg/ml] (more ...)
Chromatin condensation was analyzed by acridine orange DNA staining followed by FACS analysis. Figure B shows similar kinetics of apoptosis for young cells treated with actinomycin D, UV irradiation, and a low concentration of cisplatin. A peak of apoptosis (20 to 60%) was reached 48 h after treatment. The reduction in the percentage of apoptosis measured 72 h after treatment may be due to the disintegration of the apoptotic cells. The percentage of spontaneous apoptosis in untreated cells did not exceed 2%. Cells treated with high concentrations of cisplatin and etoposide exhibited different kinetics of apoptosis than cells treated with the other drugs. It should be noted that low and high concentrations of cisplatin display different patterns of apoptosis: a low concentration leads to a peak at 48 h, and a high concentration shows a linear increase up to 72 h. The apoptotic patterns obtained for the young cells by DNA condensation analysis measured by acridine orange staining followed by FACS are in good correlation with those obtained by the DNA fragmentation assay.
Finally, cleavage of PARP, which is indicative of apoptosis-induced caspase-3 and/or -8 activity, was analyzed by Western blotting with an anti-PARP antibody (Fig. C). A typical apoptotic pattern of the uncleaved form of PARP (113 kDa) with an increase in the level of the cleaved fragment of PARP (89 kDa) was detected between 24 and 36 h after all treatments. This occurred despite the differences in the kinetics of the drugs.
In conclusion, we have demonstrated by three different techniques that young human fibroblasts are able to undergo apoptosis in response to actinomycin D, UV irradiation, etoposide, and cisplatin.
Next, we analyzed the induction of apoptosis in old WI-38 human fibroblasts with the same set of DNA-damaging agents used for young cells. Apoptosis was determined by the DNA ladder and acridine orange techniques under the same conditions as for the young fibroblasts. According to the DNA ladder patterns obtained, it appears that young and old cells exhibit comparable fragmentation smears in response to high concentrations of cisplatin and etoposide (Fig. A). However, following treatment with actinomycin D, UV irradiation, or a low concentration of cisplatin, old cells show a much lower level of apoptosis than young cells. This difference is more pronounced in the acridine orange analysis. In this case, no significant levels of apoptosis are detected in old cells in response actinomycin D, UV irradiation, or a low concentration of cisplatin, whereas high levels of apoptosis are evident in young cells (Fig. B). In contrast, similar patterns of apoptosis were seen in young and old cells in response to etoposide. Treatment of old cells with a high concentration of cisplatin resulted in a low level of apoptosis at 36 h, followed by a marked increase at 48 h after treatment. By 72 h, the level of apoptosis in old cells was high and comparable to that of young cells.
To summarize, we found that the treatments with etoposide and high concentrations of cisplatin induced similar levels of apoptosis in both young and old cells whereas actinomycin D, UV irradiation, and a low concentration of cisplatin induced high levels of apoptosis in young cells and much lower levels of apoptosis in old cells. This possibly means that cellular response pathways induced by etoposide and high concentrations of cisplatin remain unchanged when cells enter senescence, while response pathways induced by actinomycin D, UV irradiation, and a low concentration of cisplatin are altered in senescent cells.
Human fibroblasts undergo p53-dependent or p53-independent apoptosis.
We were interested in studying whether there is a correlation between the differential apoptotic responses of the young and old cells and the tumor suppressor protein p53. Apoptosis is known to occur via p53-dependent and p53-independent pathways, depending on the treatment applied and the cell type. We first investigated the involvement of p53 in the induction of apoptosis by genotoxic stress in young human fibroblasts. Upon treatment of young fibroblasts with actinomycin D, cisplatin, and UV, we detected an increase in p53 levels by Western blotting with DO-1 anti-p53 antibodies (Fig. A). However, etoposide failed to induce accumulation of p53 (Fig. A), which suggests that etoposide, in contrast to actinomycin D, cisplatin, and UV, is unable to stabilize p53. In order to test whether stabilization of p53 is accompanied by its activation, we analyzed the induction of the p53 downstream gene Mdm2 upon genotoxic stress. Induction of Mdm2 was monitored by Western blotting with the anti-Mdm2 antibody (data not shown). We observed strong correlation between accumulation of p53 and induction of the Mdm2 gene in the cells treated with actinomycin D, UV, and low concentrations of cisplatin. Even though fibroblasts treated with high concentrations of cisplatin exhibit high levels of p53, we did not detect any induction of the Mdm2 gene. Etoposide-treated cells, which were found not to accumulate p53 (see above), were unable to induce Mdm2 in response to stress.
FIG. 2 Role of p53 in the induction of apoptosis in young and old human fibroblasts. (A) Accumulation of p53 in young and old fibroblasts upon treatment with various DNA-damaging agents. After 0, 5, 12, 24, 36, and 48 h of induction, all detached and adherent (more ...)
We next examined whether the increase of p53 protein is associated with the induction of apoptosis in fibroblasts. For this purpose, we used functional depletion of p53 by a dominant-negative fragment and the viral E6 protein. The minimal requirement for the dominant-negative function of mutant p53 is its C terminus from amino acids 302 to 390. Transient expression of this minimal dominant-negative p53 fragment, designated DD, leads to strong functional inactivation of endogenous p53 (53
). To exclude the possible gain-of-function effect of the DD fragment on suppression of apoptosis, we used a second method of p53 inactivation. To this end, we depleted p53 by the transient expression of the human papillomavirus type 16 protein E6, which binds to p53 and promotes its rapid proteolysis (21
Young fibroblasts were transiently transfected with plasmids harboring the dominant-negative DD or E6 under the strong constitutive CMV and simian virus 40 promoters, respectively. Empty vectors were used as a control. Inactivation of p53 was confirmed by cotransfection of DD or E6 with the plasmids carrying the reporter (luciferase) gene under a p53-inducible RGC or Bax promoter. In the cells transfected with the plasmids harboring DD or E6, the level of luciferase expression was strongly reduced compared to that in control cells transfected with empty vectors (data not shown). The efficiency of transfection was ~15%, as determined by cotransfection with the plasmid containing GFP under the CMV promoter followed by FACS analysis. We enriched the population for up to 80% of transfected cells using the magnetic cell sorter (see Materials and Methods). After recovery from transfection and cell sorting, the cells were treated with actinomycin D, UV irradiation, etoposide, and low and high concentrations of cisplatin, and apoptosis was analyzed by the more quantitative acridine orange method. The functional depletion of p53 in fibroblasts markedly decreased their ability to undergo apoptosis in response to actinomycin D, UV irradiation, or a low concentration of cisplatin (Fig. B). However, apoptosis induced by etoposide or a high concentration of cisplatin was not affected. Functional depletion of p53 by the dominant-negative DD fragment and E6 gave similar results, indicating that only inactivation of p53 is responsible for the observed effect rather than other effects of the DD or E6 protein. These results indicate that young WI-38 human fibroblasts undergo p53-dependent apoptosis in response to treatment with actinomycin D, UV irradiation, and a low concentration of cisplatin. In contrast, etoposide and a high concentration of cisplatin induce p53-independent apoptosis.
As described above, old WI-38 human fibroblasts predominantly underwent apoptosis in response to high concentrations of cisplatin and etoposide and to a much lower extent in response to actinomycin D, UV irradiation, and a low concentration of cisplatin. In light of the observations that actinomycin D, UV, and a low concentration of cisplatin induced p53-dependent apoptosis in young fibroblasts, it appears that old fibroblasts are able to undergo p53-independent apoptosis but not p53-dependent apoptosis.
Old human fibroblasts are unable to stabilize p53 in response to genotoxic stress.
We have found that, unlike young cells, old fibroblasts exhibit negligible levels of p53-dependent apoptosis in response to genotoxic stress. It is well accepted that p53-dependent apoptosis induced by genotoxic stress requires p53 protein stabilization. Hence, we analyzed the stabilization of p53 in old fibroblasts after treatment with actinomycin D, UV irradiation, etoposide, and low and high concentrations of cisplatin, using Western blotting with the DO-1 anti-p53 antibody (Fig. A). We detected very low or no stabilization of the p53 protein in old cells compared to that in young cells. However, the basal levels of p53 in young and old fibroblasts were comparable (Fig. A), as shown previously (1
). This suggests that the reduction in p53-dependent apoptosis in old fibroblasts may be due to their inability to stabilize p53.
Old human fibroblasts that are unable to enter p53-dependent apoptosis undergo necrosis.
The above-mentioned results showed that old human fibroblasts are unable to undergo p53-dependent apoptosis in response to DNA damage. A question arises as to the fate of those cells with damaged DNA. During preliminary microscopic examination, we observed similar death rates in the young and old cells treated with all the tested DNA-damaging agents (data not shown). Hence, we suspected that necrosis was responsible for the death of DNA-damaged old cells. Release of the cellular matrix from the cell and the appearance of “empty” ghost cells, consisting of only cell membranes, are typical necrotic features (20
). As mentioned previously, acridine orange staining followed by FACS analysis was effective in differentiating between viable and apoptotic WI-38 fibroblasts. Acridine orange binds to DNA and hence can detect sub-G1
DNA content. Moreover, it is dichromatic and undergoes a shift from green to red fluorescence when it binds to the condensed DNA typical of apoptosis. Therefore, it allows the detection of apoptosis even in cells that do not exhibit a sub-G1
content, like fibroblasts. Figure C shows an example of the fluorescence shift of acridine orange due to apoptosis induced by actinomycin D treatment.
FIG. 3 Detection of necrotic and apoptotic cells by acridine orange staining followed by FACS analysis. (A) Density plot of normal cell cycle distribution of untreated fibroblasts. Cell populations at the G1, S, and G2 stages of the cell cycle are indicated. (more ...)
To examine whether the acridine orange technique can also detect necrotic cells as a population separate from viable and apoptotic cells, we treated viable cells with DNase and RNase to produce the empty WI-38 cells typical of necrosis and assayed them by acridine orange. We obtained a clearly segregated population, with a fluorescence lower and greener than that of viable cells (Fig. B), which is probably the result of acridine orange staining of membrane-bound glycosaminoglycans and proteoglycans, as shown previously (13
). We therefore designated this population necrotic cells.
Old and young human fibroblasts treated with actinomycin D, UV irradiation, etoposide, and low and high concentrations of cisplatin were stained with acridine orange and analyzed by FACS. The accumulation of necrotic and/or apoptotic cells was observed following all treatments (Fig. ). Strikingly, in response to actinomycin D, UV irradiation, and a low concentration of cisplatin, old fibroblasts exhibited a population that corresponded exactly to the population defined above as necrotic (compare Fig. B and D). Under the same conditions, young cells underwent apoptosis (Fig. C). A time-dependent increase in necrotic cells can be seen in Fig. , following treatment with actinomycin D, UV irradiation, and a low concentration of cisplatin. No significant levels of apoptosis were detected in the old cells in any of these treatments. Treatment of young and old cells with a high concentration of cisplatin and etoposide induced apoptosis to similar extents at most time points used, with no significant levels of necrosis.
FIG. 4 Induction of apoptosis and necrosis in young and old human fibroblasts by various DNA-damaging agents. After 36, 48, and 72 h of treatment, the cells were stained with acridine orange and analyzed by FACS (see Materials and Methods), which allowed the (more ...)
To further confirm that cell death in senescent cells occurred by necrosis, we used a different method for detection of necrosis which is based on the release of DNA from necrotic cells into the medium. Following treatment of senescent cells with actinomycin D, UV irradiation, and a low concentration of cisplatin, an increase in the DNA level detected in the medium was observed relative to that of untreated cells (Fig. ). We have not detected any significant levels of DNA release for young cells. The DNA release increased in a time-dependent manner in all cases. This indicates that old fibroblasts treated with actinomycin D, UV irradiation, and a low concentration of cisplatin undergo necrosis in response to the treatments. The kinetics and relative extent of necrosis detected by DNA release were similar to those detected by acridine orange. However, in all the treatments the percentage of necrotic cells detected by DNA release was twice as small as that with acridine orange. This can be explained by the fact that free DNA released into the medium undergoes rapid degradation, while empty cells (membrane vesicles) detected by acridine orange are much more stable.
FIG. 5 Induction of necrosis in old human fibroblasts by actinomycin D, UV, and low concentration of cisplatin. Necrosis was analyzed by the release of DNA from necrotic cells into the medium. Total DNA of the old fibroblasts was metabolically labeled with BrdU (more ...)
These findings suggest that towards senescence, human fibroblasts change their death pathway from p53-dependent apoptosis to necrosis in response to genotoxic stress. DNA-damaging agents that cause p53-dependent apoptosis in young cells induce necrosis in old cells.
Stabilization of p53 in old human fibroblasts restores their ability to undergo apoptosis.
To examine whether the reduction in p53-dependent apoptosis in old fibroblasts is due to their inability to stabilize p53, we used two approaches. One was aimed at stabilizing p53 in old cells by the inhibition of its proteolysis, and the other approach was to exogenously express the p53 protein by transient transfection of wild-type p53.
(i) Stabilization of p53 by proteasome inhibitors.
Degradation of p53 is mediated by the ubiquitin-proteasome pathway (40
). In order to induce the accumulation of endogenous p53 in old fibroblasts, we used the proteasome inhibitors MG-115, MG-132, and PSI (proteasome inhibitor I), which were shown to stabilize p53 (11
). Importantly, it was demonstrated that apoptosis of immortalized cells induced by MG-115 and PSI is p53 dependent, suggesting that stabilization of p53 plays a key role in apoptosis induced by proteasome inhibitors (39
Young and old human fibroblasts were treated with proteasome inhibitors, and the accumulation of p53 was analyzed up to 48 h after the treatment. All the tested proteasome inhibitors induced rapid and strong accumulation of p53 in young and old fibroblasts (Fig. A). These results clearly demonstrate that even though p53 in the old fibroblasts is not stabilized in response to genotoxic stress, it could be stabilized by proteasome inhibitors. Furthermore, the kinetics of p53 accumulation confirmed previous observations that the rates of synthesis of p53 in young and old cells are similar (1
FIG. 6 Stabilization of p53 and induction of apoptosis in young and old fibroblasts treated with proteasome inhibitors. Young and old fibroblasts were treated with the following proteasome inhibitors: MG-115 (30 μM), MG-132 (10 μM), and PSI (30 (more ...)
In order to examine the induction of apoptosis in the cells treated by proteasome inhibitors, the cells were analyzed by acridine orange DNA staining. Rapid and strong induction of apoptosis was observed in both young and old fibroblasts treated with MG-115, MG-132, and PSI (Fig. B). It should be noted that up to 24 h following this treatment, the level of apoptosis in old cells was lower than that in young cells. However, both young and old cells reached ~100% apoptosis 48 h after treatment. The fact that the proteasome inhibitors that are associated with p53 stabilization were able to induce apoptosis in old cells suggests that induction of apoptosis under these conditions was p53 dependent. Apoptosis induced by proteasome inhibitors was higher than apoptosis induced by other treatments. This can be explained by the fact that proteasome inhibitors lead to the accumulation of other regulatory molecules in addition to p53. However, it has been shown that modulation of p53 turnover is a key event in apoptosis induced by proteasome inhibitors (39
(ii) Exogenous expression of wild-type p53.
To confirm that the observed apoptosis induced by the proteasome inhibitors is indeed due to an increase in p53 levels rather than stabilization of other factors, we tested whether overexpression of exogenous p53 would force old cells to enter apoptosis instead of undergoing necrosis. To this end, old human fibroblasts were transfected with a plasmid carrying wild-type p53 under the CMV promoter. The efficiency of transfection was ~15%, as monitored by cotransfection with a GFP-harboring plasmid. The population of transfected cells was enriched up to 80% by magnetic cell sorting (see Materials and Methods). The exogenous p53 was highly expressed in transfected old fibroblasts within 48 h after transfection, as shown by Western blotting (Fig. A). This high level of p53 in transfected cells induced massive apoptosis even without genotoxic stress (Fig. B). Similar massive apoptosis was observed with p53-transfected old fibroblasts that were treated with actinomycin D, UV irradiation, and a low concentration of cisplatin. This may indicate that following exogenous expression of p53, the cells reached a maximum level of p53-dependent apoptosis that could not be further increased by drug treatment. Importantly, the level of DNA damage-induced necrosis was significantly reduced by overexpression of p53 in the old cells in comparison to that observed for the old cells transfected with the control vector. Therefore, by overexpression of exogenous p53, we were able to override senescence-related changes in the old cells and switch them back from the necrotic to the apoptotic pathway of cell death.
FIG. 7 Transient expression of p53 in old fibroblasts restores their ability to undergo apoptosis and inhibits their ability to undergo necrosis. Old fibroblasts were transiently transfected with a plasmid harboring the wild-type p53 gene under the CMV promoter (more ...)
Taken together, these results demonstrate that the cellular milieu of old fibroblasts permits the expression of high levels of p53 sufficient for apoptosis. Furthermore, the apoptotic machinery downstream of p53 is fully functional in old cells.