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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 56:B8-B20 (2001)
© 2001 The Gerontological Society of America

Human Diploid Fibroblasts That Undergo a Senescent-like Differentiation Have Elevated Ceramide and Diacylglycerol

Carol J. Millera and Gretchen H. Steina

a Molecular, Cellular, and Developmental Biology Department, University of Colorado at Boulder

Gretchen H. Stein, University of Colorado, Boulder, MCDB, Campus Box 347, Boulder, CO 80309-0347 E-mail: stein{at}boulder.colorado.edu.

Decision Editor: Jay Roberts, PhD


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Senescent human diploid fibroblasts (HDF) have elevated levels of ceramide and diacylglycerol (DAG) compared with young HDF. DNA fragmentation analysis demonstrated the increased ceramide in senescent HDF was not associated with apoptosis, whereas in young HDF, exogenous ceramide induced apoptosis. In young HDF treated with both exogenous ceramide and DAG, less DNA fragmentation was observed. Thus, elevated DAG levels in senescent HDF might protect against ceramide-induced apoptosis. To determine which characteristics of senescent HDF (aging per se, cell cycle arrest, elevated p21Sdi1,Waf1,Cip1, and senescent-like differentiation) might influence ceramide and DAG, we examined transformed or mitomycin C–treated HDF that shared some of these properties with senescent HDF. The elevation of ceramide and DAG did not depend on aging per se, cell cycle arrest, or elevation of p21. Rather, ceramide and DAG may be elevated as part of a program of differentiation that is induced by either aging or DNA damage.

SENESCENT human diploid fibroblasts (HDF) fail to initiate DNA synthesis in response to mitogenic stimulation, and, consequently, the cells are arrested with G1-phase DNA contents. In addition, senescent HDF demonstrate morphological differentiation by becoming enlarged and flattened with an increased cell volume, and functional differentiation by changing their role in the production and maintenance of the extracellular matrix (1)(2). For example, senescent HDF have decreased expression of several extracellular matrix components (collagen I-1{alpha}, collagen III-1{alpha}, and elastin) and increased expression of collagenase and stromelysin, two enzymes that serve to breakdown the extracellular matrix (3)(4). In this differentiated state, senescent HDF express a neutral senescence-associated ß-galactosidase activity (SA-ß-gal), which is used as a marker of senescence (5). Although much is known about the immediate mechanism for the cell cycle arrest (failure to phosphorylate the retinoblastoma protein [pRb] owing primarily to increases in the p21Sdi1,Cip1,Waf1 and p16Ink4a inhibitors of the cyclin-dependent kinases [(2),(6)(7)(8)(9)]), little is known about the molecules that regulate both these changes in cell cycle molecules and the expression of the senescence-associated differentiation.

Recently, ceramide, a sphingolipid second-messenger molecule, has been implicated as having a role in senescence, because endogenous ceramide levels are elevated fourfold in senescent HDF in comparison with quiescent HDF (10). Consistent with this, young cells exposed to a soluble ceramide analog acquired these senescent characteristics: activator protein 1 (AP-1) transcription factor activity was inhibited, pRb was dephosphorylated, and DNA synthesis was inhibited (10). In addition, ceramide has been shown to inhibit the production of diacylglycerol (DAG) from phosphatidylcholine in vitro (11), which mimics another characteristic of senescent cells. Taken together, these data suggest that ceramide could play an important role in the mechanism for senescence in HDF.

Elevated ceramide is not a unique feature of senescence, for it is also seen in a wide variety of cells when they are induced to undergo apoptosis (12)(13). This raised two questions about the high levels of ceramide in senescent HDF. First, is the elevation of ceramide in senescent HDF associated with an increased amount of apoptosis in these cells? Although senescent HDF populations clearly survive for long periods of time, Pignolo and colleagues (14) found that approximately 50% of senescent WI38 HDF died in the early stage of senescence. Furthermore, exposure of young HDF to increasing amounts of exogenous ceramide analog induces apoptosis in these cells (10). Thus, the second question raised by these results is how do senescent HDF survive a fourfold increase in endogenous ceramide? Studies on MOLT-4 human leukemia cells induced to apoptose by exogenous ceramide analogs suggested that concomitant increases in DAG provide a protective effect, resulting in cell cycle arrest rather than apoptosis (15). To answer these questions, we compared the amount of ceramide, DAG, and DNA fragmentation (a measure of apoptosis) in young, quiescent HDF and senescent HDF and investigated the effects of exogenous ceramide and DAG analogs on apoptosis in young HDF.

Endogenous ceramide is also elevated in other types of cell cycle arrest that do not involve aging. For example, a high amount of ceramide is produced in MOLT-4 cells induced to arrest by serum deprivation (15). Likewise, ceramide is elevated in HL-60 cells when they are induced to terminally differentiate (16). Because the senescent phenotype includes aging, irreversible cell cycle arrest, differentiation, and possibly apoptosis, we analyzed a variety of HDF with subsets of these properties to determine how elevated cer-amide and DAG relate to each of these aspects of senescence.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Cell Culture
IMR90 human diploid lung fibroblasts and their precrisis SV40-transformed derivative (90VA-PC) were obtained from the Coriell Institute for Medical Research (Camden, NJ). IMR90 expressing the human papilloma virus 16 (HPV16) E6 oncogene (E6 cells) (17) were a gift from J. Shay and W.E. Wright (University of Texas Southwestern Medical Center, Dallas, TX). Cells were routinely cultured in 150-mm diameter tissue culture plates in EF medium (1:1, EMEM:F12) with either 10% fetal bovine serum (Irvine Scientific, Santa Ana, CA) or 5% fetal bovine serum (Irvine) and 5% newborn bovine serum (Irvine or Gemini Bioproducts, Calabasas, CA) (18). Young IMR90 (24–33 population doublings [PD]) were harvested 3 days after seeding (replicating), 12 to 14 days after seeding (quiescent), or 4 days after feeding a confluent culture serum-free medium (serum-deprived quiescent). Senescent IMR90 (74–86 PD) were harvested either 12 to 19 days after seeding (with weekly feeding) or after an additional 4 days in serum-free medium (serum-deprived senescent). Young 90VA-PC cells (60–65 PD) were cultured similarly to young IMR90 and harvested 3 days after seeding. Old 90VA-PC cells (88–89 PD) were subcultivated every 7 days until the cultures began to enter crisis, as determined by decreasing cell number, and harvested 3 days after seeding. Middle-aged E6 cells (43–54 PD) were cultured similarly to young IMR90 and harvested after 12 days, with a feeding at 7 days. Senescent E6 cells (78–85 PD) were cultured like senescent IMR90; however, when these cells could no longer achieve a net gain in cell number, their labeling index was still 30% or more. With weekly feedings for 2 months, these cells senesced and were then harvested.

Mitomycin C Treatment
Mitomycin C (MMC; 0.5 µg/ml; Sigma, St. Louis, MO) was added to the medium of quiescent middle-aged IMR90 cells (45 PD) and middle-aged (43 or 49.5 PD) E6 cells for 48 hours, whereupon the cells were rinsed with 1x phosphate buffered saline (PBS), subcultured at a density of 3.3 to 6.6 x 103 cells/cm2, fed on Day 7, and harvested on Day 14 for analysis. Old IMR90 (63.5 or 65 PD) were treated with MMC for 48 hours beginning 1 day after subcultivation, whereupon the cells were rinsed, refed, and harvested, as described previously, without subculturing.

Labeling Indices
When cells were harvested for experiments, sister plates were labeled with 1-µCi/ml 3H-thymidine for 24 hours with and without serum stimulation, then cells were cytospun, fixed, and autoradiographed as previously described (19). The percentages of labeled nuclei were as follows: replicating IMR90, >=50%; density-inhibited quiescent IMR90 or E6 cells, <=10%; serum-deprived quiescent IMR90, <=5%; senescent IMR90, <=5%; and senescent E6 cells, <=10%.

Cell Volume Determination
Relative cell volume was calculated from the volume of the pellet formed by 9 x 106 freshly trypsinized cells centrifuged at 1100 x g for 5 minutes. The results were consistent with cell volume calculated on the basis of cell diameter in a hemacytometer.

Senescence-Associated ß-Galactosidase Assay
Our study followed the procedure of Dimri and colleagues (5).

Preparation of Crude Lipid Extracts
Crude lipid extracts were prepared as described by Bligh and Dyer (20), with modifications. Briefly, the cells in 150-mm plates were placed on ice, rinsed with 5 ml of cold 1x PBS, scraped off the plates in 3.2 ml cold methanol, and transferred to a test tube on ice. Each plate was rinsed with 3.2 ml of cold methanol, which was added to the tube for a final yield of cells in 6.4 ml of cold methanol. Following the addition of 3.2 ml of chloroform, the mixture was shaken, and 1.0 mol/l NaCl was added just until a biphasic solution occurred, and then methanol was added to regain a monophase, thereby obtaining a 2:1:0.8 ratio of methanol to chloroform to NaCl. The sample was centrifuged at 500 x g at room temperature and the supernatant was removed to another tube. The pellet was resuspended and re-extracted with 1 ml of chloroform and 2 ml of methanol for 5 minutes, pelleted at 500 x g and the supernatant was added to the first supernatant. The phases were separated by addition of 4.2 ml of chloroform and 4.2 ml of 1.0 mol/l NaCl. Following centrifugation at 500 x g, the precipitate at the interface was removed with a drawn-out Pasteur pipette, and the aqueous phase was removed. The organic phase was rinsed with 2 ml of a one-to-one mixture of methanol to NaCl (1.0 mol/l), centrifuged at 500 x g, and the aqueous phase was removed again. The organic phase samples were stored under nitrogen at –70°C for 1 to 2 days until aliquots were prepared for the DAG kinase assay.

Assay for DAG and Ceramide
Based on cell counts done on sister plates at the time of harvest, the concentration of each crude lipid extract was normalized to the number of cells harvested. Triplicate samples representing 7.5 x 104 – 1.5 x 105 cells were prepared (in 2-ml polypropylene test tubes with O-ring caps) for senescent and MMC-treated cells, which have increased cell size, whereas all other samples were aliquoted on the basis of 3 x 105 cells per aliquot. These aliquots were dried without heating in a speed vacuum system, stored at –20°C, and assayed for DAG and ceramide with a DAG kinase kit (Amersham, Piscataway, NJ) based on the protocol of Preiss and colleagues (21). This assay system adds radiolabeled phosphate to both DAG and ceramide, yielding 32P-labeled phosphatidic acid and ceramide-1-phosphate, respectively. The reaction products were separated by thin-layer chromatography on silica gel plates (E. Merck, Darmstadt, Germany) in a solution of chloroform, methanol, acetone, acetic acid, and water (10:4:3:2:1) and quantitated by an AMBIS radioanalytic scanner (AMBIS, San Diego, CA). The counts per minute (cpm) obtained for the corresponding DAG and ceramide spots were averaged. The amount of DAG and ceramide in each sample was normalized to total phospholipid phosphate for that sample (i.e., normalized value = [average cpm/nmol of total phospholipid phosphate]). Using these normalized values, experimental samples were then compared with young or untreated controls to derive the relative levels of DAG and ceramide (i.e., relative level = normalizedexperimental/normalizedcontrol). Then, those relative levels of ceramide and DAG were averaged, and those averages were reported in the figures. The standard error of the mean (SEM) reported reflects the SEM for the average relative level (i.e., SEM = standard deviation/square root of number of experiments [N]).

Phospholipid Phosphate Determination
Phospholipid phosphate, a measure of total phospholipid, was analyzed by the method of Rouser (22), as modified by Kano-Sueoka (personal communication). Aliquots of crude lipid extract equal to 1 x 104 – 2 x 105 cells were prepared in triplicate in disposable glass tubes and allowed to air dry. Briefly, 0.1 ml of 70% perchloric acid was added to the dried lipids and incubated at 100°C for 1 hour in a glycerol-filled heat block, with a glass marble covering each tube; 0.7 ml dH2O, 0.1 ml of 2.5% ammonium molybdate, and 0.1 ml of 10% ascorbic acid were added and incubated for 5 minutes in a boiling water bath, and the absorbance of each sample was read in a spectrophotometer at 820 nm. Two standard curves using 1.3 to 13 nmol of Phosphorous Standard Solution (Sigma) or bovine brain phosphatidylcholine (Sigma) were used to determine the amount of phospholipid phosphate in each sample.

DNA Fragmentation Assay
Aliquots of 2 or 3 million cells were prepared in triplicate from sister plates to the ones harvested for ceramide and DAG measurements, and the amount of DNA fragmentation was determined by the method of Sellins and Cohen (23), which separates the fragmented DNA from the chromatin by centrifugation. The percentage of DNA fragmentation = DNA in the supernatant/(DNA in the supernatant + DNA in the chromatin pellet).

3H-Thymidine-Labeled DNA Fragmentation Assay
The amount of DNA fragmentation in analog-treated cells (1–2 million cells) was determined by the methods of Venable and colleagues (10) and Sellins and Cohen (23), with some modifications. Briefly, young IMR90, seeded in duplicate in 100-mm plates at 4 x 105 cells per plate, were labeled with 3H-thymidine (0.4 µCi/ml) for the interval 48 to 72 hours after seeding. Following two rinses with 1x PBS, the cells were fed with fresh medium supplemented with either C6-ceramide, dioctanoylglycerol, or the ethanol vehicle used to dissolve these lipids. After 24 hours, the medium was aspirated from the dishes and centrifuged (100 x g, room temperature) to harvest floating cells, while the adherent cells were harvested by trypsinization and centrifugation at 100 x g at room temperature. Before this spin, a 100-µl aliquot of the trypsinized cells (3 ml in total) was analyzed for percentage of dead cells by trypan blue exclusion. The two cell pellets were combined, as were the two supernatants (yielding the plate supernatant). The cells were lysed in 0.5 ml of 10 mmol/l Tris-HCl, pH 7.4, and 1 mmol/l ethylenediaminetetraacetic acid (EDTA), 0.2% Triton X-100 for 10 minutes at room temperature, and microcentrifuged for 10 minutes at 4°C. The pellet supernatant was saved, and the pellet was resuspended in 0.5 ml of 10 mmol/l Tris-HCl, pH 7.4, and 1 mmol/l EDTA. The amount of radioactivity in the plate supernatant, the pellet supernatant, and the pellet solution was determined by scintillation counting to yield the percentage of DNA fragmentation: cpm in the two supernatant fractions/(cpm in the two supernatant fractions + cpm pellet fraction).

Determination of Percentage of Dead Cells
As mentioned previously, after trypsinization, a 100 µl aliquot of the analog-treated cells was transferred to an Eppendorf tube and used for the determination of percentage of dead cells by trypan blue stain exclusion. These cells were diluted (1:10) in PBS and stained with trypan blue stain (Flow Laboratories, McClean, VA) for 2 minutes. The cells were counted with the use of a hemacytometer. The percentage of dead cells was calculated as (blue cells/total cells) x 100%. Between 160 to 400 dead cells (trypan blue stained) were detected in the approximately 1800 cells analyzed per condition.

Statistical Analysis
Data for the 3H-thymidine–labeled DNA fragmentation assay and the percentage of dead cells (C6-ceramide and dioctanoylglycerol treatment experiments) were analyzed with a two-way analysis of variance. Data for ceramide and DAG levels were analyzed with a one-tailed t test, comparing the means of two samples. A p value <=.05 was considered statistically significant.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Ceramide and DAG Measurements in Senescent IMR90 Cells
Senescent IMR90 have an average of 4.0 and 4.4 times as much ceramide relative to total phospholipid as do young IMR90 that were made quiescent by serum deprivation or high cell density, respectively (Fig. 1). The difference is significant for both serum-deprived (p = .029) and non-serum-deprived (p = .017) senescent cells. In addition, the amount of ceramide present in a population of young replicating IMR90 is intermediate between that of senescent and quiescent cells (Fig. 1). For replicating cells compared with quiescent cells, the difference is significant (p = .018). These data imply that cell cycle arrest per se does not account for the elevated ceramide in senescent cells, because the cell cycle arrest in young cells is actually associated with a reduced amount of ceramide. Although Venable and colleagues (10) did not see a substantial difference in the amount of ceramide in exponentially growing WI38 cells and their quiescent counterparts, they found a similar fourfold increase in ceramide in senescent versus quiescent WI38. Thus, both sets of data support the conclusion that cell cycle arrest per se does not account for the elevation of ceramide in senescent human fibroblasts.



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  		Figure 1. Senescent IMR90 cells have elevated basal amounts of ceramide and diacylglycerol (DAG). Relative levels of ceramide (shown in A) and DAG (shown in B) in quiescent (q), senescent (s), and replicating (r) IMR90 of serum-deprived (sd) or non-serum-deprived (non-sd) cells. The ceramide and DAG levels were normalized to total phospholipid phosphate and then normalized to the levels in the quiescent IMR90 of each experiment, which were set equal to 1. The data represent the average of four-to-ten triplicate measurements ± SEM. p values relative to qIMR90: *p <= .05; ***p <= .001.

 
Senescent HDF also differ from young HDF in cell size, with newly senescent cells being two- to threefold larger than quiescent cells (1). We have presented our data as the amount of ceramide relative to total phospholipid, which generally correlates with plasma membrane size. The differences that we report between senescent cells and young cells would be even greater if we expressed them on a per cell basis. Because it is not known whether the concentration or the absolute amount of ceramide is most important for its effects on cellular processes, we chose the more conservative method of analysis.

Because increased amounts of ceramide have been linked to apoptosis in several cell systems, we investigated whether apoptosis could account for the elevated ceramide in senescent HDF. Although apoptosis is not generally thought of as a significant feature of normal cell senescence, Pignolo and colleagues (14) found that, on average, half of the WI38 cells in a senescing population die. This prompted us to investigate the level of apoptosis in our cultures. We analyzed DNA fragmentation as a measure of apoptosis and found that senescent IMR90 cells had only a small increase in DNA fragmentation, even though they consistently demonstrated a large increase in ceramide (Table 1 ). For experiments 21 and 25, the difference in DNA fragmentation is not significant (p = .26) and (p = .07), respectively. In experiment 23, the difference for serum-deprived cells is significant (p = .039) and for non-serum-deprived cells is very significant (p = .004). The difference in ceramide is very significant or highly significant for all cells (serum-deprived cells, experiment 21 [ p = .009] and experiment 23 [ p = .00085], and non-serum-deprived cells, experiment 23 [ p = 5.6 x 10-6] and experiment 25 [ p = .004]). These data suggest that the increase in ceramide in senescent HDF is not related to apoptosis.


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  		Table 1. Amounts of DNA Fragmentation and Ceramide in IMR90 Cells

 
The basal amount of DAG is also elevated in senescent HDF, albeit to a lesser extent than ceramide (Fig. 1) (10). Senescent IMR90 have approximately twice as much DAG as serum-deprived and non-serum-deprived young quiescent IMR90. The difference is significant (p = .032) for serum-deprived cells, but not significant (p = .098) for non-serum-deprived cells. On the other hand, young replicating cells had more DAG than either senescent cells or quiescent cells (Fig. 1), which is consistent with previous reports that DAG is usually elevated in cells that are proliferating (24). The replicating cells have a significantly different amount of DAG (p = 6.7 x 10-7) compared with quiescent cells. Our results with senescent HDF are consistent with the hypothesis that cells with high ceramide may be protected from apoptosis by a concomitant increase in DAG.

Treatment of Young IMR90 With C6-Ceramide and Dioctanoylglycerol
As a further test of the hypothesis that elevation of DAG might be important to protect senescent HDF from ceramide-induced apoptosis, we treated young IMR90 with C6-ceramide and/or a DAG analog (dioctanoylglycerol) and monitored the induction of apoptosis by changes in the amount of DNA fragmentation and cell death. Previous studies had shown that in young WI38 HDF treated with C6-ceramide, cell proliferation was inhibited, pRb was dephosphorylated, and AP-1 activity was inhibited, suggestive of a senescent cell cycle arrest; however, both the amount of DNA fragmentation and cell death were noticeably increased, which is different from the higher viability of normal senescent cells (10). Our results indicate that treatment of young IMR90 with 15 µmol/l C6-ceramide increased the DNA fragmentation (up to 2.7-fold), whereas treatment with dioctanoylglycerol alone (up to 90 µmol/l) had little effect (Fig. 2). Although the effect of C6-ceramide on DNA fragmentation is highly significant (F = 157.5, p = .0001), the effect of dioctanoylglycerol on DNA fragmentation is not significant (F = 2.2, p = .171). When the cells were treated with both C6-ceramide and dioctanoylglycerol, the amount of DNA fragmentation significantly decreased compared with the C6-ceramide-treated cells. The data showed a significant interaction between dioctanoylglycerol and C6-ceramide (F = 7.27, p = .02). Although analysis of cell death by trypan blue exclusion also suggested a protective effect, these changes were not statistically significant (F = 0.75, p = .51). The result of the percentage of dead cells may reflect the variability in this method, whereas the DNA fragmentation method showed less variability. Thus, based on the DNA fragmentation results, these analog experiments in IMR90 HDF further support the hypothesis that elevated endogenous DAG levels may serve to protect senescent HDF from ceramide-induced apoptosis.



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  		Figure 2. Death occurs in IMR90 cells treated with ceramide analog, but is decreased with concomitant treatment with diacylglycerol (DAG) analog. Percentage of DNA fragmentation of total DNA and the percentage of dead versus total cells of 3H-thymidine prelabeled young IMR90 untreated or treated with various concentrations of C6-ceramide and/or dioctanoylglycerol. The data shown are the average ± SD of one experiment performed in duplicate and are representative of two experiments. *p <= .05, relative to 15 µmol/l C6-ceramide and 0 µmol/l dioctanoylglycerol; ***p <= .001, relative to 0 µmol/l C6-ceramide and 0 µmol/l dioctanoylglycerol.

 
Aging and Senescent-like Differentiation of 90VA-PC Cells
Although cell cycle arrest per se does not result in an elevated amount of ceramide, we wished to investigate whether the cell cycle arrest at the end of the life span is a prerequisite for the age-related increase in ceramide, or whether aging without arrest can also lead to elevated ceramide. For this purpose, we chose to study 90VA-PC cells, which are precrisis IMR90 cells transformed with SV40, because they have a finite proliferative life span but do not enter a senescent arrest state; rather, they die in crisis at the end of their life span. Because a SV40 T antigen can bind to pRb and p53, rendering these proteins nonfunctional, 90VA-PC cells cannot arrest at quiescence or senescence. Even though precrisis SV40-transformed HDF do not achieve a senescent cell cycle arrest, they demonstrate a number of other age-related changes similar to wild-type HDF (e.g., their growth rate slows down over their life span, their requirement for serum increases, their telomeres shorten, their p21 expression increases, and their cyclin A and cyclin B expression decreases) (25)(26)(27). We found that old 90VA-PC cells also show signs of the senescent-like differentiation; that is, their average cell volume is increased approximately fivefold (data not shown), their morphology is enlarged and flattened, resembling senescent IMR90, (Fig. 3), and the fraction of cells positive for SA-ß-gal activity is elevated from 2% in a young population to 12% ± 5% in old populations. Furthermore, Dimri and colleagues (5) have found that when SV40-transformed HDF are in full crisis, 95% of the cells are positive for SA-ß-gal. Taken together, these data strongly suggest that the aging process occurs in precrisis SV40-transformed HDF independent of the effects of irreversible cell cycle arrest.



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  		Figure 3. With age, the cellular morphology of 90VA-PC cells enlarged and flattened as in normal IMR90. Fixed cells were stained with Pierce Gelcode protein stain. A, Young IMR90, B, senescent IMR90, C, young 90VA-PC, and D, old 90VA-PC cells.

 
Ceramide and DAG Levels in 90VA-PC Cells
To investigate the age-related changes in ceramide and DAG in 90VA-PC, without involving the massive death of these cells at crisis, we examined old 90VA-PC cells that were at the beginning of crisis, and we eliminated the minority of dying cells in young and old cultures by harvesting only the cells that remained attached to the tissue culture plate after rinsing. Because 90VA-PC cells cannot arrest at either quiescence or senescence, they were harvested 3 days after subcultivation for comparison with replicating cultures of normal IMR90 HDF. Analysis of DNA fragmentation showed that old 90VA-PC cells harvested in this way had the same low level of apoptosis as comparably treated young 90VA-PC cells and young IMR90 cells (Table 2 ). The percentage of DNA fragmentation was not significantly different for old 90VA-PC compared with young 90VA-PC (p = .15, p = .059, and p = .19 for experiments 24, 25, and 26, respectively). Thus, this protocol was used to study age-related changes in 90VA-PC cells, independent of apoptosis at the crisis phase.


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  		Table 2. DNA Fragmentation in IMR90 and 90VA-PC Cells

 
Old 90VA-PC cells at the beginning of crisis had a 3.3-fold increase in ceramide, compared with young 90VA-PC that were 20 to 30 PD before crisis, and a 2.0-fold increase compared with young, replicating IMR90 (Fig. 4). This increase in ceramide is significant for old 90VA-PC compared with young 90VA-PC cells (p = .032) and for old 90VA-PC compared with young, replicating IMR90 (p = .04). The replicating IMR90, young 90VA-PC, and old 90VA-PC cells all had high percentages of labeled nuclei, indicating that all of these cultures were in a comparable replicative state (Fig. 4). Thus, 90VA-PC cells that were aged, but not arrested or dying, had an increased amount of ceramide compared with young 90VA-PC cells.



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  		Figure 4. Aged, but not arrested, 90VA-PC cells have increased ceramide. A, Ceramide and B, diacylglycerol (DAG) amounts in qIMR90 at 12 days after subculturing, rIMR90 at 3 days, young (y) 90VA-PC at 3 days, and old 90VA-PC at 3 days relative to the y90VA-PC cells. Most of the 90VA-PC cells in a population are replicating, so they were compared with replicating normal cells. The qIMR90 are included to show how 90VA-PC cells relate to normal quiescent cells. The ceramide and DAG levels were normalized to total phospholipid phosphate and then normalized to the levels in the young 90VA-PC, which were set equal to 1. The data represent the average of two or three triplicate measurements ± SEM. C, Percentage of labeled nuclei of unstimulated qIMR90, rIMR90, y90VA-PC, and old 90VA-PC cells from a representative experiment. *p <= .05, relative to y90VA-PC.

 
Although both 90VA-PC and IMR90 showed an age-related increase in ceramide, the effect of aging on DAG levels in 90VA-PC is ambiguous because only replicative cells, which have elevated DAG, can be analyzed. Our data indicate that old 90VA-PC cells have nearly the same amount of DAG as young 90VA-PC and IMR90 (Fig. 4). The differences are not significant for young and old 90VA-PC cells (p = .44) nor for young, replicating IMR90 and old 90VA-PC (p = .26). Furthermore, all of these replicating cultures had a greater increase in DAG compared with quiescent IMR90 than did senescent IMR90 (cf, Fig. 4 and Fig. 1). Hence, if aging causes a modest increase in DAG (like the twofold increase seen in senescent IMR90), then this change could be masked in old 90VA-PC cells because they have a replicating level of DAG.

Ceramide and DAG Levels in E6 Cells
Because 90VA-PC cells, which lack both functional pRb and p53, cannot be studied in an arrested state, we investigated whether ceramide and/or DAG are increased in senescent cells that lack p53 activity, but have functional pRb. For this purpose, we analyzed IMR90-E6 cells, which contain a low amount of p53 owing to the presence of the HPV16 E6 oncogene, which binds to p53 and promotes its degradation (28). As a consequence of their deficiency in p53, young E6 cells also have a greatly reduced amount of p21, which is positively transactivated by p53 (29). Surprisingly, we have found that p21 remains low (10%–20% of wild-type levels) throughout the life span of E6 cells, yet these cells eventually become senescent after an extended life span (i.e., they are irreversibly arrested, senescent-like in appearance, and express high levels of SA-ß-gal activity) (30). Hence, we analyzed senescent E6 cells (Fig. 5) for their ceramide and DAG content. These senescent E6 cells had 6.8 times as much ceramide and 3.6 times as much DAG as middle-aged, quiescent E6 cells, which were fairly comparable to control populations of IMR90 (Fig. 5,Fig. 5). These increases are significant for both ceramide (p = .039) and DAG (p = .04). The amount of DNA fragmentation in the E6 populations was the same, regardless of age, 6.0% for middle-aged and 5.7% for senescent cells (p = .22), and in line with previous measurements of IMR90 cells. Thus, these data indicate that both ceramide and DAG are elevated in viable cells that are able to arrest at the end of the life span, regardless of whether those cells are deficient in p53 and p21.



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  		Figure 5. Ceramide and diacylglycerol (DAG) are elevated in senescent E6 cells. A, Percentage of labeled nuclei of unstimulated (unstim) and serum-stimulated (stim) young (y) IMR90, middle-aged (m-a) IMR90, m-a E6, and senescent (sen) E6 from a representative experiment. Levels of ceramide (shown in B) and DAG (shown in C) in yIMR90, m-a IMR90, m-a E6, and sen E6 relative to the levels in the m-a E6. Both young and middle-aged IMR90 were included to show how E6 cells compare with normal cells. The ceramide and DAG levels were normalized to total phospholipid phosphate and then normalized to the levels in the m-a E6, which were set equal to 1. These data represent the average of three or five triplicate measurements ± SEM. *p <= .05, relative to m-a E6.

 
Ceramide and DAG Levels in MMC-treated Cells
Although cessation of proliferation due to aging is the defining characteristic of senescent cells, senescent HDF are also clearly differentiated relative to young cells. Consequently, as part of our strategy to elucidate which aspects of the multifactorial senescent phenotype are associated with elevated ceramide and DAG, we have investigated whether ceramide and/or DAG are elevated in HDF that exhibit a senescent-like differentiation, but are not aged.

Previous experiments have shown that MMC treatment induces young HDF to become irreversibly arrested and differentiated into senescent-like cells based on their morphology, overall protein expression patterns, ability to inhibit DNA synthesis in heterodikaryons formed with replicating HDF, and expression of SA-ß-gal activity (31)(32)(33). We treated quiescent, middle-aged IMR90 cells with MMC for 2 days and allowed them to arrest and differentiate for 2 weeks. After the 2-week period, their percentage of labeled nuclei was <1% (Fig. 6) and they had a senescent-like morphology (Fig. 7). Ceramide was elevated 4.0-fold and DAG was elevated 2.3-fold in these MMC-treated IMR90 cells (Fig. 6,Fig. 6). The elevations in both lipids are highly significant, (p = .00011) for ceramide and (p = 2.3 x 10-5) for DAG. Because MMC causes DNA damage by crosslinking the DNA (34), it was not surprising to find a modest, but not significant, increase in DNA fragmentation from 4.3% in the control to 7.7% (p = .076) in the treated cells, but the increase in ceramide was significantly greater. These data indicate that ceramide and DAG are increased in non-aged cells when they are induced to arrest and differentiate like senescent cells.



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  		Figure 6. Mitomycin C (MMC) treatment arrests middle-aged IMR90 resulting in elevated ceramide and diacylglycerol (DAG). A, Percentage of labeled nuclei of unstimulated (unstim) and serum-stimulated (stim) control and MMC-treated IMR90. The data are the average of two experiments. Amounts of ceramide (shown in B) and DAG (shown in C) in MMC-treated IMR90 relative to control IMR90. Each lipid was normalized to the total phospholipid phosphate and then normalized to the control, which was set equal to 1. The data are the average ± SEM of two experiments performed in triplicate. ***p <= .001, relative to IMR90 control.

 


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  		Figure 7. Mitomycin C (MMC) treatment results in the same morphological changes as aging (i.e., cellular enlargement and flattening). Photographs of live cells: A, replicating IMR90 3 days after subculturing; B, senescent IMR90 12 days after subculturing; C, quiescent IMR90 14 days after subculturing; D, IMR90 14 days after MMC treatment; E, quiescent E6 14 days after subculturing; and F, E6 14 days after MMC treatment.

 
Since MMC causes DNA damage and p53 mediates a number of responses to DNA damage, we investigated whether the MMC-induced elevation of ceramide and DAG depends on p53. MMC-treated E6 cells become enlarged, morphologically senescent-like (Fig. 7,Fig. 7), and express the SA-ß-gal marker (30). We examined E6 cells 2 weeks after MMC treatment, at which time the MMC-treated cells were almost all arrested (Fig. 8). Both ceramide and DAG were increased in MMC-treated E6 relative to the untreated E6 control, 5.9- and 3.9-fold, respectively, and to the untreated young IMR90 control, 11.3- and 3.5-fold, respectively (Fig. 8,Fig. 8). For both ceramide and DAG in MMC-treated E6 cells versus the control E6 cells, the differences were significant (p = .013) and (p = .023), respectively. Thus, even in p53-deficient, non-aged cells, MMC induces increased amounts of ceramide, and DAG. Taken together, our studies on MMC-treated IMR90 and E6 cells indicate that ceramide and DAG are elevated in cells that are arrested and differentiated like senescent cells, but are not aged.



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  		Figure 8. Mitomycin C (MMC)–treated E6 cells have elevated ceramide and diacylglycerol (DAG). A, Percentage of labeled nuclei of unstimulated (unstim) and serum-stimulated (stim) control IMR90, untreated E6, and MMC-treated E6. Amounts of ceramide (shown in B) and DAG (shown in C) in untreated and MMC-treated E6 relative to control IMR90. Each lipid was normalized to the total phospholipid phosphate and then normalized to the control, which was set equal to 1. The data are the average ± SEM of two experiments performed in triplicate. ***p <= .05, relative to E6.

 

   Discussion
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Using IMR90 HDF for our studies, we confirmed the approximately fourfold increase in ceramide in senescent HDF versus young quiescent HDF and showed that this occurred without increased apoptosis in the senescent populations. We tested the hypothesis that senescent HDF might be protected from ceramide-induced apoptosis by a concomitant elevation of DAG, as had been seen in MOLT-4 cells. Our data supported this hypothesis, as we found that the basal amount of DAG was elevated in senescent HDF, as well as in all the other arrested cells that had high ceramide (i.e., senescent E6 cells and both HDF and E6 cells treated with MMC). In addition, we found that when young HDF were induced to undergo apoptosis by treatment with a soluble ceramide analog, this effect was partially mitigated by treating the cells concurrently with a DAG analog.

Our analysis of ceramide and DAG amounts in young versus senescent HDF revealed that young proliferating IMR90 had elevated ceramide in comparison with quiescent cells, albeit to a lesser degree than was seen in senescent cells. These results were unexpected because previous studies of WI38 cells indicated that ceramide was present at comparable levels in proliferating and quiescent young cells. Other proliferation-associated molecules, such as cyclin D1, cyclin E (35), and DAG (11), are also elevated in senescent IMR90. These data suggest the interesting possibility that some proliferation-associated molecules accumulate in senescent IMR90 because these molecules are produced in response to mitogenic stimulation, yet are not utilized or downregulated again because the cells are unable to progress through the cell cycle.

To investigate which aspects of the senescent HDF phenotype might be mediated by increased ceramide, we analyzed ceramide, DAG, and DNA fragmentation in cells that were able to express only a subset of the characteristics of senescent HDF. These results are summarized in Table 3 . Our studies of old precrisis 90VA-PC cells show that HDF that are aged, but not able to achieve a senescent cell cycle arrest, nevertheless have elevated ceramide. Although old 90VA-PC cells displayed low levels of DNA fragmentation and high labeling indices like the young cells, they had three times as much ceramide and also displayed some features of the senescent-like differentiation (i.e., they were enlarged, flattened, and had increased SA-ß-gal activity). These results imply that the increase in ceramide in senescent HDF does not depend on the cell cycle arrest, but may be associated with aging per se and/or the senescence-associated differentiation.


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  		Table 3. Ceramide and Cellular Phenotype

 
An increased amount of ceramide is also present in young cells that are induced to become irreversibly arrested following treatment with MMC. Previous studies have shown that when young HDF are treated with appropriate doses of DNA-damaging agents, such as MMC, ultraviolet radiation, ionizing radiation, and hydrogen peroxide, they became permanently arrested and also differentiated into senescent-like cells (31)(32)(36)(37). Taken together, these data show that aging per se is not necessary to achieve an elevation of ceramide in association with a senescent-like arrest and differentiation.

Transient increases in ceramide have been shown in other models of terminal differentiation such as vitamin D-induced differentiation of HL60 (38). However, in our experiments, ceramide was elevated 2 weeks after MMC treatment of IMR90, suggesting that it is a long-term change in these cells. Thus, our data suggest that both aging and exogenous DNA damage induce HDF to arrest and differentiate by a mechanism that involves a long-term increase in ceramide.

Because senescent IMR90, old 90VA-PC, and MMC-treated IMR90 share the characteristic of high ceramide, but are neither uniformly aged nor uniformly arrested, we sought other characteristics that these three types of cells might have in common. Two additional characteristics that they share are elevated p21 and the senescent-like differentiation (7)(27)(30) (Table 3 ). However, IMR90-E6 cells, which are deficient in p53 and p21 throughout their life span (30), have high ceramide both at senescence and after MMC treatment. Thus, elevated ceramide occurs independently of elevated p21.

Among the characteristics of senescent HDF that we studied, only the senescent-like differentiation is a common characteristic among all the cells that had elevated ceramide, suggesting that perhaps ceramide is elevated as part of a putative differentiation program that is turned on in senescent cells. Alternatively, ceramide itself may be upstream of the putative differentiation program. In addition, even though our results with 90VA-PC cells imply that a senescent-like cell cycle arrest is not necessary for the elevation of ceramide, it is possible that ceramide contributes to the senescent cell cycle arrest in cells whose pRb is not inactivated by binding to SV40 T antigen.

In addition to ceramide, DAG may also play a role in differentiation. For example, DAG is a known activator of several protein kinase C (PKC) isozymes, which have been implicated in differentiation in several cell culture systems (HL-60 and U-937 human leukemia cells, B and T lymphocytes, keratinocytes, neuronal cells, and neuroblastoma cells) (39). One way PKC could promote differentiation is by promoting the failure of pRb phosphorylation. In various cell types, activation of PKC has been shown to lead to increased p53, p21, p27, and decreased Cdk-activating kinase (CAK) activity and cyclin–Cdk 2 kinase activity (40). All of these changes can contribute to a lack of pRb phosphorylation. Indeed, in IMR90 cells, activation of PKC by phorbol-12, 13-dibutyrate 5 hours after serum stimulation results in complete inhibition of Cdk 2 activity and DNA synthesis (41). These cells had decreased CAK and CAK-mediated phosphorylation of Cdk 2. It is important to note that PKC has this inhibitory effect when it is activated late in G1, whereas activation of PKC (and DAG), which normally occurs shortly after serum stimulation, promotes proliferation. This suggests that DAG present at different points in G1 may promote alternative outcomes. Thus, it is possible that the overall elevation of DAG in senescent IMR90, senescent E6, and MMC-treated IMR90 and E6 could play a role in the differentiated phenotype of these cells via the activity of PKC and signaling molecules downstream of PKC.

The results obtained with E6 cells are particularly interesting because the ability of these cells to become senescent without the contribution of a high amount of p21 suggests that there are multiple effectors of the senescent cell cycle arrest. In normal senescent HDF, pRb fails to be phosphorylated following serum stimulation, primarily because of p21- and p16-mediated inactivation of the G1 cyclin–cyclin-dependent kinase complexes that act as pRb kinases during the G1 phase (Fig. 9). Because ceramide activates a protein phosphatase (CAPP), which is thought to dephosphorylate pRb (42), we suggest that in E6 cells where p21 is low, a less efficient cell cycle arrest might be mediated by the combined effect of p16 and CAPP. This could explain the cell cycle arrest of both senescent E6 and MMC-treated E6 cells. If CAPP activation can lead to pRb dephosphorylation, then this should also occur in senescent and MMC-treated HDF, even though it may be overshadowed by the effects of a high amount of p21. Interestingly, treatment of newly senescent HDF with phosphatase inhibitors allowed 20% of these cells to enter DNA synthesis again, suggesting that phosphatase activity does play a role in the senescent cell cycle arrest (43). Ceramide-mediated activation of CAPP could also be important for maintenance of the senescent cell cycle arrest in normal HDF because the amount of p21 reaches a peak at, or just before, replicative senescence is achieved and then declines (2)(9). Therefore, it is possible that additional mechanisms, such as the activation of CAPP, play a role in maintenance of the senescent cell cycle arrest.



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  		Figure 9. Model for the phosphorylation and dephosphorylation of the retinoblastoma protein (pRb). The cyclin-Cdk complexes phosphorylate pRb, depending on complex formation and the presence of p21 or p16. The phosphorylation of pRb leads to DNA synthesis. We propose that ceramide-activated protein phosphatase (CAPP) dephosphorylates pRb resulting in cell cycle arrest. Cyc D = cyclin D; Cyc E = cyclin E; Cdk = cyclin-dependent kinase; pRb-P = phosphorylated pRb.

 


   Acknowledgments
 
We thank Dr. Tami Kano-Sueoka, Dr. Anita Miller, and Linda Drullinger from the Molecular, Cellular, and Developmental Biology Department, University of Colorado, Boulder, and Dr. Laurie Goodman from Genome Research, Cold Spring Harbor Laboratory Press. We thank Diane Lorenz and Chris Dagsgaard for technical assistance with figures. We thank Dr. Beth Bennett from the Institute for Behavioral Genetics, University of Colorado, Boulder, for assistance with statistical analyses. This research was partially supported by fellowships from the Glenn Foundation for Medical Research (CJM) and by Public Health Service Grant AG00947 from the National Institute of Aging (GHS).

Received November 26, 1999

Accepted June 12, 2000


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 Results
 Discussion
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