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J Appl Physiol 100: 988-995, 2006. First published October 27, 2005; doi:10.1152/japplphysiol.01086.2005
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Early loss of proliferative potential of human peritoneal mesothelial cells in culture: the role of p16INK4a-mediated premature senescence

Krzysztof Ksiazek,1 Katarzyna Piwocka,2 Agnieszka Brzezinska,2 Ewa Sikora,2 Maciej Zabel,3 Andrzej Breborowicz,1 Achim Jörres,4 and Janusz Witowski1,4

1Department of Pathophysiology and 3Department of Histology and Embryology, University Medical School, Poznan; and 2Laboratory 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

Submitted 6 September 2005 ; accepted in final form 25 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 senescence-associated beta-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.

cellular senescence; oxidative stress

OVER THE PAST TWENTY YEARS, 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 senesce fairly quickly in a process associated with increased generation of reactive oxygen species (ROS) and increased expression of p16INK4a.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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 µg/ml), hydrocortisone (0.4 µg/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 x 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 beta-galactosidase.   Senescence-associated beta-galactosidase (SA-beta-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-beta-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 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 µM 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.


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  		Table 1. Primer sequences and PCR conditions

 
Western blotting.   Cells were scraped on ice; lysed in a buffer containing 20 mM Tris·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-µg 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.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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 x 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).


Figure 1
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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 x40). 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).

 
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).


Figure 2
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  		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.

 
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-beta-Gal (14). While the presence of SA-beta-Gal was only sporadic in early-passage cultures, 78% (SD 13) of late-passage cells stained positively for SA-beta-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 µm2 (SD 56) in early-passage cells to 2,032 µm2 (SD 313) in late-passage HPMC (n = 8, P < 0.008). Size of the nucleus increased from 113 µm2 (SD 12) to 156 µm2 (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.


Figure 3
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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 beta-galactosidase in early- and late-passage cells in a representative HPMC culture (magnification x100, note the difference in cell size and shape). C: percentage of cell staining positive for senescence-associated beta-galactosidase. Data were obtained from cultures established from 16 separate donors.

 
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 late-passage 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).


Figure 4
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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 beta-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.

 
ROS production and oxidative DNA damage.   Oxidative stress is believed to be associated with (and most likely 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 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).


Figure 5
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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 (8-OH-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.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies with omentum-derived HPMC commented on early cessation of cell growth and rapid appearance of 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-beta-Gal, a commonly 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 density-dependent contact inhibition but disappears from terminally growth-arrested senescent cells (15).

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 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 p16INK4a-independent 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 related to the activation of p53 and p21Cip1 (16, 21, 39). We did not find obvious differences between early- and late-passage 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 p16INK4a-dependent 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.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Dr. J. Witowski, Dept. of Pathophysiology, Univ. Medical School, Swiecickiego 6, 60–781 Poznan, Poland (e-mail: jwitow{at}amp.edu.pl)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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