J Appl Physiol 100: 988 –995, 2006.
First published October 27, 2005; doi:10.1152/japplphysiol.01086.2005.
Early loss of proliferative potential of human peritoneal mesothelial cells in
culture: the role of p16INK4a-mediated premature senescence
Krzysztof Ksia˛żek,1 Katarzyna Piwocka,2 Agnieszka Brzezińska,2 Ewa Sikora,2
Maciej Zabel,3 Andrzej Bre˛borowicz,1 Achim Jörres,4 and Janusz Witowski1,4
1
Department of Pathophysiology and 3Department of Histology and Embryology, University Medical School, Poznań; and
Laboratory for Molecular Bases of Aging, Nencki Institute of Experimental Biology, Warsaw, Poland; and 4Department of
Nephrology and Medical Intensive Care, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Berlin, Germany
2
Submitted 6 September 2005; accepted in final form 25 October 2005
cultures of human peritoneal
mesothelial cells (HPMC) have been increasingly used in
biomedical research. Availability of mesothelial cells in vitro
has enabled studies that shed new light on their function. These
studies changed a traditional and simplified view of the mesothelium as merely a lining of the coelomic cavity. The
mesothelium has been found to be a source of an extremely
effective phospholipid-based surfactant, which provides lubrication needed for smooth sliding of the viscera. The ability of
HPMC to generate fibrinolytic activity makes the peritoneum a
nonthrombogenic surface and offers further protection against
adhesion formation and fibrosis. By secreting cytokines in a
tightly regulated manner, the mesothelium controls the peritoneal response to injury and inflammation, and, by producing
mediators of extracellular matrix turnover, it plays an active
role in tissue repair. These newly discovered aspects of HPMC
function have received a lot of attention, as they appear to
bear clinical significance in various disciplines, including
surgery (peritonitis, adhesions), nephrology (peritoneal dialysis-related complications), gynecology (endometriosis),
and oncology (intraperitoneal metastases). New advances in
the biology of mesothelial cells have recently been extensively reviewed (26, 27).
Cultures of HPMC are typically established from small
pieces of omentum removed during abdominal surgery (as
described in detail in Refs. 40, 43). Less often, HPMC are
isolated from the specimens of the parietal peritoneum (19),
peritoneal lavage, or ascites fluid (48), and from peritoneal
dialysis effluent (12). Gentle enzymatic degradation of omentum produces a significant yield of HPMC; however, their
capacity to proliferate further in vitro seems to be rather
limited. HPMC have been found to grow effectively for just a
few passages (usually 4 –9), and the rapid increase in percentage of large senescent-like cells appears to occur from the third
to fourth passage onwards (personal observations and Refs. 10,
17, 32, 40). On the other hand, some studies have reported on
mesothelial cells undergoing senescence after as many as 50
population doublings (PDs) (11, 25, 42). Neither the reason for
these discrepancies nor the molecular basis underlying HPMC
senescence has been firmly established.
It is well recognized that, after a limited number of PDs,
normal diploid cells enter a state of senescence characterized
by irreversible growth arrest, enlarged and flattened morphology, and a significantly different profile of gene expression
(37). Senescence that occurs after a predetermined number of
cell divisions is commonly referred to as replicative senescence. It is believed to result from critical shortening and/or
uncapping of telomeres (reviewed in Ref. 3). This process is
mediated by p53 tumor suppressor protein and its downstream
effector, p21Cip1, which acts as an inhibitor of cyclin-dependent kinases (CDK). On the other hand, it appears that some
cell types may senesce rapidly in a telomere-independent
manner in response to inadequate culture conditions (33). This
stress-induced premature activation of the senescence program
is thought to be mediated by another CDK inhibitor, the
p16INK4a protein (22).
In the present study, we have attempted to characterize the
development of senescence in omentum-derived HPMC. We
demonstrate that, under typical culture conditions, HPMC
Address for reprint requests and other correspondence: Dr. J. Witowski,
Dept. of Pathophysiology, Univ. Medical School, Świe˛cickiego 6, 60 –781
Poznań, Poland (e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
cellular senescence; oxidative stress
OVER THE PAST TWENTY YEARS,
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8750-7587/06 $8.00 Copyright © 2006 the American Physiological Society
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Ksia˛żek, Krzysztof, Katarzyna Piwocka, Agnieszka Brzezińska,
Ewa Sikora, Maciej Zabel, Andrzej Bre˛borowicz, Achim Jörres, and
Janusz Witowski. Early loss of proliferative potential of human peritoneal mesothelial cells in culture: the role of p16INK4a-mediated premature
senescence. J Appl Physiol 100: 988 –995, 2006. First published October
27, 2005; doi:10.1152/japplphysiol.01086.2005.—Much has been
learned about the mechanisms underlying cellular senescence. The
pathways leading to senescence appear to vary, depending on the cell
type and cell culture conditions. In this respect, little is known about
senescence of human peritoneal mesothelial cells (HPMC). Previous
studies have significantly differed in the reported proliferative lifespan
of HPMC. Therefore, in the present study, we have examined how
HPMC enter state of senescence under conditions typically used for
HPMC culture. HPMC were isolated from omentum and grown into
senescence. The cultures were assessed for the growth rate, the
presence of senescence markers, activation of cell-cycle inhibitors,
and the oxidative stress. HPMC were found to reach, on average, six
population doublings before senescence. The terminal growth arrest
was associated with decreased expression of Ki67 antigen, increased
percentage of cells in the G1 phase, reduced early population doubling
level cDNA-1 mRNA expression, and the presence of senescenceassociated ␤-galactosidase. Compared with early-passage cells, the
late-passage HPMC exhibited increased expression of p16INK4a but
not of p21Cip1. In addition, these cells generated more reactive oxygen
species and displayed increased presence of oxidatively modified
DNA (8-hydroxy-2⬘-deoxyguanosine). These results demonstrate that
early onset of senescence in omentum-derived HPMC may be associated with oxidative stress-induced upregulation of p16INK4a.
SENESCENCE OF PERITONEAL MESOTHELIAL CELLS
senesce fairly quickly in a process associated with increased
generation of reactive oxygen species (ROS) and increased
expression of p16INK4a.
MATERIALS AND METHODS
J Appl Physiol • VOL
detected with the use of a specific monoclonal antibody (Dako,
Glostrup, Denmark), diluted 1:100. For 8-hydroxy-2⬘-deoxyguanosine (8-OH-dG) staining, DNA was first denatured by exposure to 4 N HCl. After neutralization with 50 mM Trizma base, the
specimens were blocked with 10% BSA and incubated overnight at
4°C with monoclonal anti-8-OH-dG antibody (Trevigen, Gaithersburg, MD), diluted 1:300. After they were washed with PBS, cells
were treated with 0.3% H2O2 to quench endogenous peroxidase
activity. Bound antibodies were detected by immunoperoxidase staining using the EnVision⫹ System (Dako), as per manufacturer’s
instructions.
Morphometric evaluation. Morphometric analysis was performed
with cells growing in Lab-Tek Chamber Slides. The slides were
inspected initially at low magnification to choose for analysis the
areas free of any damage that could be occasionally caused by
washing or fixation. Then, two randomly selected areas on each slide
were assessed at higher magnification. By moving from left to right
and from top to bottom, 500 cells were counted, and the number of
positively stained cells was recorded. Total cell surface and nucleus
areas were assessed on digitalized images (Nikon Eclipse E-400,
Tokyo, Japan) with the use of Screen Measurement 4.21 software
(Laboratory Imaging, Prague, Czech Republic).
Detection of ROS. Generation of ROS was assessed by 2⬘,7⬘dichlorodihydrofluorescein diacetate (H2DCFDA) labeling. Briefly,
cells (105) were incubated with 5 ␮M H2DCFDA (Molecular Probes)
for 45 min at 37°C and then solubilized with the lysis buffer (Promega, Madison, WI). Fluorescence emitted by cell lysates was measured in a Wallac Victor2 spectrofluorometer (Perkin-Elmer, Turku,
Finland) using wavelengths of 485 and 535 nm for excitation and
emission, respectively. The data were expressed as relative light units
per 105 cells.
RT-PCR. Total RNA from HPMC cultures was extracted with the
RNA Isolator (Sigma-Genosys, Cambridge, UK), purified and reverse
transcribed into cDNA with random hexamer primers, as previously
described (44). PCR amplification was performed on the Mastercycler
Gradient 5331 thermocycler (Eppendorf, Hamburg, Germany) using
Platinium Taq DNA polymerase (Invitrogen). Specific oligonucleotide primer pairs were synthesized by TIB MolBiol SyntheseLabor
(Berlin, Germany). Primer sequences and PCR conditions were as
shown in Table 1.
Western blotting. Cells were scraped on ice; lysed in a buffer
containing 20 mM Tris 䡠 HCl at pH 7.3, 200 mM NaCl, 1 mM EDTA,
1% Triton X-100, 1 mM PMSF, and protease inhibitor cocktail
(Roche Diagnostics, Mannheim, Germany); and homogenized by
sonication (Bandelin, Berlin, Germany). Samples corresponding to
40-␮g protein were resolved by 15% SDS-PAGE and transferred to
Immobilon-P polyvinylidene difluoride membranes (Millipore, Eschborn, Germany).
The membranes were blocked with 5% nonfat powdered milk and
probed with antibodies against either p16INK4a or GAPDH (Santa
Cruz Biotechnology, Santa Cruz, CA). Bound antibodies were visualized following incubation with the peroxidase-labeled secondary
antibodies (Dako) and the exposure to chemiluminescence reagent
(ECL; Amersham Pharmacia Biotech, Castle Hill, Australia).
Statistical analysis. The means of data measured on an interval
scale were compared with the Wilcoxon signed ranks test for nonparametric paired data using GraphPad Prism 4.00 software (GraphPad Software, San Diego, CA). The differences between proportions
recorded on a nominal scale were analyzed with McNemar’s test for
nonparametric paired data by using Analyse-It 1.71 software (Analyse-It Software, Leeds, UK). Correlations were assessed with the
nonparametric Spearman correlation test. Results are expressed as
means ⫾ SD. Differences with a P value ⬍ 0.05 were considered to
be statistically significant.
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Materials. Unless indicated otherwise, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO). All tissue culture plastics were from Nunc (Roskilde, Denmark).
Isolation and culture of HPMC. HPMC were isolated by enzymatic
disaggregation of omentum, as described in detail elsewhere (32, 40,
43). Briefly, the specimens of omentum were obtained from consenting patients undergoing elective abdominal surgery. The tissue was
incubated in a solution of 0.05% trypsin and 0.02% EDTA for 30 min
at 37°C with gentle shaking. The cells obtained were washed and
propagated in Earle’s buffered M199 culture medium supplemented
with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100
␮g/ml), hydrocortisone (0.4 ␮g/ml), and 10% vol/vol FCS (Gibco,
Invitrogen, Karlsruhe, Germany). The cultures were maintained at
37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were
identified as pure mesothelial by their typical cobblestone appearance
at confluence and uniform positive staining for cytokeratins (32, 40)
and HBME-1 antigen (48). All cultures were established from individuals with no evidence of peritonitis and no overt diabetes, uremia,
and peritoneal malignancy. The age of the donors (7 men and 17
women) ranged from 24 to 77 yr.
Proliferation studies. Primary HPMC were grown to confluence
and then serially passaged at 3-day intervals using a fixed seeding
density of 3 ⫻ 104 cells/cm2. The passages were carried out up to the
point at which the number of cells harvested fell below the number of
cells plated. The plating density and the time frame of passages
chosen were optimized in preliminary experiments. For comparison,
in some experiments, HPMC were passaged not at fixed time intervals
but only when confluent, and not with the same seeding density but at
a split ratio of 1:4. Cells were counted using the Bürker chamber, and
the number of PDs was calculated as follows: PD ⫽ log2(Ct/Co),
where Co is the number of cells inoculated and Ct is the number of
cells harvested. HPMC derived from the primary culture were considered to be at PD ⫽ 0. At subsequent passages, no correction was
made for cells that failed to attach or reinitiate growth. Because cell
lines derived from different donors exhibited large variability in their
proliferative capacity and reached different numbers of passages, the
comparisons were made between cells at their first and last passages,
further referred to as “early” and “late,” respectively.
To ensure that late-passage cells ultimately lost their ability to
divide, few representative cultures were maintained for 2 mo further.
During this period, the cultures were regularly fed and repassaged at
weekly intervals, i.e., cells were harvested, counted, and, regardless of
the yield, all transferred to new flasks.
Measurement of DNA content by flow cytometry. Cells were harvested with trypsin-EDTA solution and fixed in ice-cold 70% ethanol
overnight at ⫺20°C. After they were washed with PBS, cells were
resuspended in 0.1 M sodium citrate, pH 7.8, for 1 min, and incubated
for 30 min in PBS containing 5 mg/ml of propidium iodide (Molecular
Probes, Eugene, OR) and 0.1 mg/ml of RNase A. For each condition,
one million cells were analyzed by using a FACSCalibur flow cytometer with Cell-Quest software (Becton-Dickinson, Warsaw, Poland).
Detection of senescence-associated ␤-galactosidase. Senescenceassociated ␤-galactosidase (SA-␤-Gal) was detected, according to
Dimri et al. (14). Briefly, HPMC were grown on Lab-Tek Chamber
Slides (Nunc, Roskilde, Denmark), fixed with 3% formaldehyde,
washed, and exposed for 2 h at 37°C to a solution containing 1 mg/ml
5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside, 5 mM potassium
ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM
MgCl2, and 40 mM citric acid, pH 6.0.
Immunocytochemistry. For immunostaining, HPMC were cultured
in Lab-Tek Chamber Slides and fixed with 70% ethanol. Ki67 was
989
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SENESCENCE OF PERITONEAL MESOTHELIAL CELLS
Table 1. Primer sequences and PCR conditions
Sequence
␤-actin
F: 5⬘-ATCCCCCAAAGTTCACAA-3⬘
R: 5⬘-CTGGGCCATTCTCCTTAG-3⬘
p16INK4a
F: 5⬘-CAGCATGGAGCCTTCGGCTGAC-3⬘
R: 5⬘AGCGTGCCATGGACGCGCGCCGAC-3⬘
EPC-1
F: 5⬘-GGAGCGGAGCAGCGAACAGAA-3⬘
R: 5⬘-TGCGCCACACCGAGAAGGAGA-3⬘
p21Cip1
F: 5⬘-CCTCTTCGGCCCGGTGGAC-3⬘
R: 5⬘-CCGTTTTCGACCCTGAGAG-3⬘
Product Size, bp
PCR Cycles
Annealing Temperature, °C
Reference No.
147
30
55
31
379
30
55
13
353
33
65
29
369
35
64
41
EPC-1, early population doubling level cDNA-1; F, forward; R, reverse.
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Fig. 1. Proliferative capacity of human peritoneal mesothelial cells (HPMC). A: cells
were cultured as described in MATERIALS AND
METHODS and photographed at 3-day intervals
at consecutive passages (magnification ⫻40).
B: the number of population doublings (PDs)
reached by HPMC isolated from 24 different
donors. The horizontal bar represents a median value. C: correlation between the number of population doublings achieved in vitro
and the age of the donors (n ⫽ 24).
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RESULTS
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Proliferative capacity of HPMC. Serial passages of HPMC
led to rapid exhaustion of their proliferative capacity. During
the first few (1–3) passages, the seeding density of 3 ⫻ 104
cells/cm2 sufficed for HPMC to easily reach a monolayer
within 3 days. However, this ability gradually declined, and the
next subcultures assessed at the same intervals displayed progressively dwindling cell density (Fig. 1A). The calculated
number of PDs was (median and range) six (3–10) (n ⫽ 24)
(Fig. 1B). While all cell lines could be passaged at least five
times, one-half of the cultures expired at their seventh or eighth
passages, and only 13% of cultures reached a maximum of 10
passages. Even if late-passage cultures were regularly fed and
maintained in culture for 2 additional mo, the number of cells
did not increase any further. Comparison of randomly selected
four cell lines revealed that the number of PDs achieved did not
differ significantly from that recorded in cultures first grown to
confluence and then passaged at a split ratio of 1:4. Also, these
cultures never exceeded 10 PDs. There was a borderline
negative correlation between the number of PDs achieved by
HPMC in vitro and the age of the donor from whom the culture
was established (Fig. 1C).
Analysis of DNA content by flow cytometry showed an
increased percentage of late-passage cells arrested in the G1
phase of the cell cycle with a concomitant reduction of cells in
S and G2/M phases (Fig. 2A). Terminal growth arrest of these
cells was also confirmed by a decrease in the expression of
proliferation-related antigen Ki67 (Fig. 2B).
Replicative senescence markers. The EPC-1 (early PD level
cDNA-1) gene is thought to be a marker of senescence, as its
expression decreases with replicative age of fibroblasts in
culture (29, 30). We found that, in HPMC, the expression of
EPC-1 mRNA in late-passage cells was significantly reduced
compared with early-passage cells (Fig. 3A). Another commonly used senescence marker is SA-␤-Gal (14). While the
presence of SA-␤-Gal was only sporadic in early-passage
cultures, 78% (SD 13) of late-passage cells stained positively
for SA-␤-Gal (Fig. 3B). Furthermore, senescent cells are
known to undergo a substantial change in morphology. We
found that early-passage HPMC cells displayed typical homogenous epithelial-like cobblestone appearance, but late-passage
cells acquired greatly enlarged, irregular, and vacuolated morphology (Figs. 1A and 3B). Morphometric analysis of eight
representative cell lines revealed that the average cell size
increased from 381 ␮m2 (SD 56) in early-passage cells to
2,032 ␮m2 (SD 313) in late-passage HPMC (n ⫽ 8, P ⬍
0.008). Size of the nucleus increased from 113 ␮m2 (SD 12)
to 156 ␮m2 (SD 24) (n ⫽ 8, P ⬍ 0.008) so that the ratio of
cytoplasm to nucleus area rose from 2.4 (SD 0.3) to 12.3
(SD 4.0) for early- and late-passage HPMC, respectively.
Expression of p16INK4a and p21Cip1. HPMC were analyzed
for the expression of CDK inhibitors that modulate different
pathways leading to senescence. The expression of p21Cip1
mRNA did not differ significantly between early- and latepassage HPMC (Fig. 4A). In the same cell lines, however,
late-passage cells consistently exhibited increased expression
of p16INK4a seen at both the mRNA and protein level (Fig. 4,
B and C).
ROS production and oxidative DNA damage. Oxidative
stress is believed to be associated with (and most likely
Fig. 2. Cessation of HPMC proliferation. A: analysis of DNA content by flow
cytometry in early- and late-passage HPMC. The histograms represent results
obtained from a representative cell line with relative numbers of cells in
sub-G1, G1, and S-G2/M phases indicated. B: percentage of cells expressing
proliferation-related antigen Ki67 in early- and late-passage HPMC. Data were
obtained from 17 separate cell lines. ⫹ve, Positive.
contribute to) cellular senescence (5). The production of ROS
by late-passage HPMC increased almost fourfold compared
with early-passage cells (Fig. 5A). Nucleic acids are one of the
main targets of ROS, and accumulation of 8-OH-dG adducts is
viewed as a marker of oxidative DNA damage (45). We
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Fig. 3. Replicative senescence markers in HPMC. A: expression of early population doubling level cDNA-1
(EPC-1) mRNA in early- and late-passage HPMC, as
assessed by RT-PCR. Results are of 1 representative experiment of 6 performed with cells from different donors.
B: the presence of senescence-associated ␤-galactosidase in
early- and late-passage cells in a representative HPMC
culture (magnification ⫻100, note the difference in cell size
and shape). C: percentage of cell staining positive for
senescence-associated ␤-galactosidase. Data were obtained
from cultures established from 16 separate donors.
observed that the presence of 8-OH-dG rose from 11% (SD 8)
in early-passage cells to 65% (SD 24) in late-passage HPMC
(Fig. 5B).
DISCUSSION
Previous studies with omentum-derived HPMC commented
on early cessation of cell growth and rapid appearance of
J Appl Physiol • VOL
enlarged vacuolated cells (10, 17, 32, 40). With the present
study, we confirm these observations and show that the loss of
proliferative capacity by HPMC is associated with the development of other features of a senescent phenotype. They
include enlarged morphology, decreased expression of Ki67
antigen, increased percentage of cells arrested in the G1 phase
of the cell cycle, and the presence of SA-␤-Gal, a commonly
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SENESCENCE OF PERITONEAL MESOTHELIAL CELLS
993
One may argue that HPMC cultures could have reached
more PD, if given more time between passages rather than
being split every 3 days. Therefore, we assessed the growth of
HPMC that, at each passage, were allowed to grow until
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Fig. 4. Expression of p21Cip1 and p16INK4a in early- and late-passage HPMC.
Total RNA and protein were obtained from HPMC and analyzed by RT-PCR
and Western blotting, respectively, as described in MATERIALS AND METHODS.
Bands corresponding to target molecules were compared with those for ␤-actin
(RT-PCR) and GAPDH (Western blotting). Results are of representative
experiments from either 2 (Western blotting) or 6 (RT-PCR) performed. A:
p21Cip1 mRNA expression. B: p16INK4a mRNA expression. C: p16INK4a protein (Western blot) expression.
used marker of senescence in vitro (14). In addition, we
detected reduced expression of EPC-1 mRNA in late-passage
HPMC. This effect was previously described in senescent
human fibroblasts (29). It is believed that the EPC-1 transcript
is present in early-passage cell growth arrested due to densitydependent contact inhibition but disappears from terminally
growth-arrested senescent cells (15).
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Fig. 5. Reactive oxygen species (ROS) generation and oxidative DNA damage
in early- and late-passage HPMC. A: quantification of ROS by 2⬘,7⬘-dichlorodihydrofluorescein diacetate labeling. Emitted fluorescence was expressed as
relative light units per 105 cells. Results were obtained from 15 separate cell
lines. B: immunocytochemical detection of 8-hydroxy-2⬘-deoxyguanosine (8OH-dG). The number of cells positive for 8-OH-dG was expressed as a
percentage of 500 cells counted. Data are from 14 experiments with cells from
different donors.
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SENESCENCE OF PERITONEAL MESOTHELIAL CELLS
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lated to the activation of p53 and p21Cip1 (16, 21, 39). We
did not find obvious differences between early- and latepassage cells in p21Cip1 mRNA expression. This fact, together with a low number of PD (and hence probably only
minor telomere shortening) suggests that HPMC under conditions employed here senesce predominantly as a result of
“culture shock” (22, 33).
Oxidative stress is viewed as one of the leading causes that
limit long-term cell growth in culture. It has been demonstrated
that mouse embryo fibroblasts can achieve up to 60 doublings
when cultured in low- (⬃3%) oxygen milieu, but senesce after
8 –10 divisions when maintained in standard (⬃20%) oxygen
tension (28). Cristofalo’s group (1) has demonstrated that
enhanced production of ROS in senescent fibroblasts is associated with increased activity of cytochrome-c oxidase and
NADH dehydrogenase, the enzymes that regulate the rate of
electron flow through the electron transport chain. In this study,
we found that the level of ROS generated by late-passage
HPMC was remarkably higher compared with early-passage
cells. Moreover, we detected increased staining of senescent
HPMC for 8-OH-dG, the most abundant product of DNA
oxidation. Our findings are consistent with previous reports
showing increased levels of 8-OH-dG in senescent human
fibroblasts (20, 23, 45). Importantly, the accumulation of oxidative DNA damage has been linked to the short proliferative
lifespan of mouse fibroblasts (7). In addition, several studies
have shown that oxidative stress may directly induce p16INK4adependent growth inhibition (8). It is, therefore, possible that
the premature senescence of HPMC observed in the present
study was largely related to oxidative stress. The impact of
ROS on the peritoneal mesothelium in vivo may particularly
affect patients treated with peritoneal dialysis for kidney failure. These patients are exposed to both uremic environment
and excessive load of glucose from dialysis fluids and, in
addition, are at risk of peritonitis. All of these situations are
associated with increased oxidative stress (4, 18, 24).
Furthermore, many in vitro studies with the use of HPMC
have frequently recorded huge standard deviations in the parameters investigated. In view of our data, it is quite possible
that this effect was partly related to the different proportions of
senescent cells in the populations examined. Therefore, it may
be advisable to standardize future studies by including senescence markers in the experimental protocols.
In conclusion, our results demonstrate that the proliferative
lifespan of HPMC derived from apparently normal omentum
and cultured under conditions commonly used by many laboratories is severely limited. This process of premature senescence is likely to be a result of oxidative stress-induced
activation of p16INK4a.
REFERENCES
1. Allen RG, Tresini M, Keogh BP, Doggett DL, and Cristofalo VJ.
Differences in electron transport potential, antioxidant defenses, and
oxidant generation in young and senescent fetal lung fibroblasts (WI-38).
J Cell Physiol 180: 114 –122, 1999.
2. Beausejour CM, Krtolica A, Galimi F, Narita M, Lowe SW, Yaswen
P, and Campisi J. Reversal of human cellular senescence: roles of the p53
and p16 pathways. EMBO J 22: 4212– 4222, 2003.
3. Ben Porath I and Weinberg RA. The signals and pathways activating
cellular senescence. Int J Biochem Cell Biol 37: 961–976, 2005.
4. Billing AG, Jochum M, Frohlich D, Cheronis JC, and Fritz H.
Oxidative autoaggression by phagocytes in human peritonitis. Eur J Clin
Invest 27: 1030 –1037, 1997.
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confluence. Despite prolonged incubation and repeated feeding, the number of PD did not increase.
It is possible that the calculated number of PD may be
underestimated to some extent, because we could not accurately control early stages of HPMC proliferation immediately
after the isolation. However, it is rather unlikely that, even with
this correction, the PD values would change dramatically.
Therefore, they stay in sharp contrast to those of 42–55
reported by Thomas et al. (42) and the group of Rheinwald (11,
25, 46). In addition, these authors passaged mesothelial cells
using seeding densities as low as 400/cm2 (25) or 6,000/cm2
(42). Both in our and other authors’ experience (32), seeding
densities of this magnitude were usually too low for the cells to
effectively resume proliferation. The possible explanation for
this discrepancy could be that these studies (11, 42, 46) were
performed on a mesothelial cell line, AG07086A (LP9), established from ascites fluid associated with ovarian malignancy.
In this respect, Zhang et al. (48) compared growth patterns of
HPMC isolated from patients with or without advanced ovarian
cancer and observed accelerated growth of mesothelial cells
obtained from patients with ovarian cancer in advanced stages.
Several studies with different cell types reported an inverse
relationship between the proliferative lifespan of cells in culture and the age of the donor from whom the cells were isolated
(reviewed in Ref. 47). We observed a similar tendency in our
study. However, these results should be interpreted with caution, given that, as mentioned above, we could only approximate the number of cumulative PD, and the age spectrum of
our donors was relatively narrow. In this respect, there are
some doubts about the immediate significance of cell replicative lifespan in vitro in relation to organismal ageing in vivo
(thoroughly discussed in Ref. 36).
It is now believed that the rapid onset of senescence in some
cell types may be caused by a hostile culture environment. The
multiple stresses of cell culture include high oxygen tension,
lack of interactions with neighboring cells, growth on plastic
surfaces, and growth factor and nutrient deficiencies (3, 38).
This pathway of stress-induced senescence is thought to be
mediated by p16INK4a (33). Increased expression of p16INK4a
was seen in senescent cells suffering from DNA damage (35)
and oxidative stress (9). The key role of p16INK4a in this
response was confirmed when keratinocytes grown on fibroblast feeder layers (instead of plastic dishes) displayed reduced
expression of p16INK4a and a concomitant increase in replicative lifespan (33). Conversely, it has been demonstrated that BJ
foreskin fibroblasts, which typically senesce in a p16INK4aindependent manner, rapidly accumulate p16INK4a and arrest
growth when placed under stressful serum-deprived conditions
(33). Therefore, increased p16INK4a may serve as a marker of
the environmental trauma. We found that senescence of HPMC
was associated with increased expression of p16INK4a, at both
the mRNA and protein level. In contrast, Rheinwald et al. (34)
showed that the abolition of p16INK4a control did not improve
proliferative capacity of peritoneal mesothelial cell line HM3.
Because, however, cell strains appear to vary in their ability to
upregulate p16INK4a at senescence (2, 6), and HM3 cells were
cultured under different conditions, we are currently investigating how various elements of the culture environment affect
the expression of p16INK4a in HPMC.
In contrast to stress-induced premature senescence, the
classic, telomere-dependent pathway of senescence is re-
SENESCENCE OF PERITONEAL MESOTHELIAL CELLS
J Appl Physiol • VOL
28. Parrinello S, Samper E, Krtolica A, Goldstein J, Melov S, and
Campisi J. Oxygen sensitivity severely limits the replicative lifespan of
murine fibroblasts. Nat Cell Biol 5: 741–747, 2003.
29. Pignolo RJ, Cristofalo VJ, and Rotenberg MO. Senescent WI-38 cells
fail to express EPC-1, a gene induced in young cells upon entry into the
G0 state. J Biol Chem 268: 8949 – 8957, 1993.
30. Pignolo RJ, Rotenberg MO, and Cristofalo VJ. Analysis of EPC-1
growth state-dependent expression, specificity, and conservation of related
sequences. J Cell Physiol 162: 110 –118, 1995.
31. Ponte P, Ng SY, Engel J, Gunning P, and Kedes L. Evolutionary
conservation in the untranslated regions of actin mRNAs: DNA sequence
of a human beta-actin cDNA. Nucleic Acids Res 12: 1687–1696, 1984.
32. Pronk A, Leguit P, Hoynck van Papendrecht AA, Hagelen E, van
Vroonhoven TJ, and Verbrugh HA. A cobblestone cell isolated from the
human omentum: the mesothelial cell; isolation, identification, and growth
characteristics. In Vitro Cell Dev Biol 29A: 127–134, 1993.
33. Ramirez RD, Morales CP, Herbert BS, Rohde JM, Passons C, Shay
JW, and Wright WE. Putative telomere-independent mechanisms of
replicative aging reflect inadequate growth conditions. Genes Dev 15:
398 – 403, 2001.
34. Rheinwald JG, Hahn WC, Ramsey MR, Wu JY, Guo Z, Tsao H, De
Luca M, Catricala C, and O’Toole KM. A two-stage, p16(INK4A)- and
p53-dependent keratinocyte senescence mechanism that limits replicative
potential independent of telomere status. Mol Cell Biol 22: 5157–5172,
2002.
35. Robles SJ and Adami GR. Agents that cause DNA double strand breaks
lead to p16INK4a enrichment and the premature senescence of normal
fibroblasts. Oncogene 16: 1113–1123, 1998.
36. Rubin H. The disparity between human cell senescence in vitro and
lifelong replication in vivo. Nat Biotechnol 20: 675– 681, 2002.
37. Shelton DN, Chang E, Whittier PS, Choi D, and Funk WD. Microarray
analysis of replicative senescence. Curr Biol 9: 939 –945, 1999.
38. Sherr CJ and DePinho RA. Cellular senescence: mitotic clock or culture
shock? Cell 102: 407– 410, 2000.
39. Stein GH, Drullinger LF, Soulard A, and Dulic V. Differential roles for
cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of
senescence and differentiation in human fibroblasts. Mol Cell Biol 19:
2109 –2117, 1999.
40. Stylianou E, Jenner LA, Davies M, Coles GA, and Williams JD.
Isolation, culture and characterization of human peritoneal mesothelial
cells. Kidney Int 37: 1563–1570, 1990.
41. Taniguchi T, Endo H, Chikatsu N, Uchimaru K, Asano S, Fujita T,
Nakahata T, and Motokura T. Expression of p21(Cip1/Waf1/Sdi1) and
p27(Kip1) cyclin-dependent kinase inhibitors during human hematopoiesis. Blood 93: 4167– 4178, 1999.
42. Thomas E, al Baker E, Dropcova S, Denyer S, Ostad N, Lloyd A, Kill
IR, and Faragher RG. Different kinetics of senescence in human fibroblasts and peritoneal mesothelial cells. Exp Cell Res 236: 355–358, 1997.
43. Van Bronswijk H, Verbrugh HA, Bos HJ, Heezius EC, Oe PL, van der
MJ, and Verhoef J. Cytotoxic effects of commercial continuous ambulatory peritoneal dialysis (CAPD) fluids and of bacterial exoproducts on
human mesothelial cells in vitro. Perit Dial Int 9: 197–202, 1989.
44. Witowski J, Jörres A, Coles GA, Williams JD, and Topley N. Superinduction of IL-6 synthesis in human peritoneal mesothelial cells is related
to the induction and stabilization of IL-6 mRNA. Kidney Int 50: 1212–
1223, 1996.
45. Wolf FI, Torsello A, Covacci V, Fasanella S, Montanari M, Boninsegna A, and Cittadini A. Oxidative DNA damage as a marker of aging
in WI-38 human fibroblasts. Exp Gerontol 37: 647– 656, 2002.
46. Wu YJ, Parker LM, Binder NE, Beckett MA, Sinard JH, Griffiths CT,
and Rheinwald JG. The mesothelial keratins: a new family of cytoskeletal proteins identified in cultured mesothelial cells and nonkeratinizing
epithelia. Cell 31: 693–703, 1982.
47. Yegorov YE and Zelenin AV. Duration of senescent cell survival in vitro
as a characteristic of organism longevity, an addition to the proliferative
potential of fibroblasts. FEBS Lett 541: 6 –10, 2003.
48. Zhang XY, Pettengell R, Nasiri N, Kalia V, Dalgleish AG, and Barton
DP. Characteristics and growth patterns of human peritoneal mesothelial
cells: comparison between advanced epithelial ovarian cancer and nonovarian cancer sources. J Soc Gynecol Investig 6: 333–340, 1999.
100 • MARCH 2006 •
www.jap.org
Downloaded from http://jap.physiology.org/ by 10.220.32.247 on June 14, 2017
5. Bokov A, Chaudhuri A, and Richardson A. The role of oxidative
damage and stress in aging. Mech Ageing Dev 125: 811– 826, 2004.
6. Brookes S, Rowe J, Gutierrez DA, Bond J, and Peters G. Contribution
of p16(INK4a) to replicative senescence of human fibroblasts. Exp Cell
Res 298: 549 –559, 2004.
7. Busuttil RA, Rubio M, Dolle ME, Campisi J, and Vijg J. Oxygen
accelerates the accumulation of mutations during the senescence and
immortalization of murine cells in culture. Aging Cell 2: 287–294, 2003.
8. Chen JH, Stoeber K, Kingsbury S, Ozanne SE, Williams GH, and
Hales CN. Loss of proliferative capacity and induction of senescence in
oxidatively stressed human fibroblasts. J Biol Chem 279: 49439 – 49446,
2004.
9. Chen QM. Replicative senescence and oxidant-induced premature senescence. Beyond the control of cell cycle checkpoints. Ann N Y Acad Sci
908: 111–125, 2000.
10. Chung-Welch N, Patton WF, Shepro D, and Cambria RP. Two-stage
isolation procedure for obtaining homogenous populations of microvascular endothelial and mesothelial cells from human omentum. Microvasc
Res 54: 121–134, 1997.
11. Connell ND and Rheinwald JG. Regulation of the cytoskeleton in
mesothelial cells: reversible loss of keratin and increase in vimentin during
rapid growth in culture. Cell 34: 245–253, 1983.
12. Diaz C, Selgas R, Castro MA, Bajo MA, Fernandez DC, Molina S,
Jimenez C, Ortiz A, and Vara F. Ex vivo proliferation of mesothelial
cells directly obtained from peritoneal effluent: its relationship with
peritoneal antecedents and functional parameters. Adv Perit Dial 14:
19 –24, 1998.
13. Dimri GP, Itahana K, Acosta M, and Campisi J. Regulation of a
senescence checkpoint response by the E2F1 transcription factor and
p14(ARF) tumor suppressor. Mol Cell Biol 20: 273–285, 2000.
14. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano
EE, Linskens M, Rubelj I, Pereira-Smith O, Peacocke M, and Campisi
J. A biomarker that identifies senescent human cells in culture and in
aging skin in vivo. Proc Natl Acad Sci USA 92: 9363–9367, 1995.
15. DiPaolo BR, Pignolo RJ, and Cristofalo VJ. Identification of proteins
differentially expressed in quiescent and proliferatively senescent fibroblast cultures. Exp Cell Res 220: 178 –185, 1995.
16. Dulic V, Beney GE, Frebourg G, Drullinger LF, and Stein GH.
Uncoupling between phenotypic senescence and cell cycle arrest in aging
p21-deficient fibroblasts. Mol Cell Biol 20: 6741– 6754, 2000.
17. Fischereder M, Luckow B, Sitter T, Schroppel B, Banas B, and
Schlondorff D. Immortalization and characterization of human peritoneal
mesothelial cells. Kidney Int 51: 2006 –2012, 1997.
18. Floccari F, Aloisi C, Crasci E, Sofi T, Campo S, Tripodo D, Criseo M,
Frisina N, and Buemi M. Oxidative stress and uremia. Med Res Rev 25:
473– 486, 2005.
19. Hjelle JT, Golinska BT, Waters DC, Steidley KR, McCarroll DR, and
Dobbie JW. Isolation and propagation in vitro of peritoneal mesothelial
cells. Perit Dial Int 9: 341–347, 1989.
20. Homma Y, Tsunoda M, and Kasai H. Evidence for the accumulation of
oxidative stress during cellular ageing of human diploid fibroblasts.
Biochem Biophys Res Commun 203: 1063–1068, 1994.
21. Itahana K, Dimri G, and Campisi J. Regulation of cellular senescence
by p53. Eur J Biochem 268: 2784 –2791, 2001.
22. Jacobs JJ and de Lange T. Significant role for p16INK4a in p53independent telomere-directed senescence. Curr Biol 14: 2302–2308,
2004.
23. Kaneko T, Tahara S, Taguchi T, and Kondo H. Accumulation of
oxidative DNA damage, 8-oxo-2⬘-deoxyguanosine, and change of repair
systems during in vitro cellular aging of cultured human skin fibroblasts.
Mutat Res 487: 19 –30, 2001.
24. Loots MA, Lamme EN, Mekkes JR, Bos JD, and Middelkoop E.
Cultured fibroblasts from chronic diabetic wounds on the lower extremity
(non-insulin-dependent diabetes mellitus) show disturbed proliferation.
Arch Dermatol Res 291: 93–99, 1999.
25. Murphy JE and Rheinwald JG. Intraperitoneal injection of genetically
modified, human mesothelial cells for systemic gene therapy. Hum Gene
Ther 8: 1867–1879, 1997.
26. Mutsaers SE. Mesothelial cells: their structure, function and role in
serosal repair. Respirology 7: 171–191, 2002.
27. Mutsaers SE. The mesothelial cell. Int J Biochem Cell Biol 36: 9 –16,
2004.
995