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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 60:695-701 (2005)
© 2005 The Gerontological Society of America

The Comet Assay Approach to Senescent Human Diploid Fibroblasts Identifies Different Phenotypes and Clarifies Relationships Among Nuclear Size, DNA Content, and DNA Damage

Alessandra Mocali1,, Lisa Giovannelli2, Piero Dolara2 and Francesco Paoletti1

Departments of 1 Experimental Pathology and Oncology
2 Preclinical and Clinical Pharmacology, University of Florence, Italy.

Address correspondence to Alessandra Mocali, Department of Experimental Pathology and Oncology, Viale G.B. Morgagni 50, 50134, Firenze, Italy. E-mail: amocali{at}unifi.it


   Abstract
Top
 Abstract
Materials and Methods
 Results and Discussion
 References
 
The comet assay methodology was used to monitor nuclear changes occurring in MRC5 human fibroblasts during transition from young to senescent cultures and to study heterogeneity of senescent populations. Nuclear morphology and size, DNA content per nucleus, and DNA damage (basal strand break, total damage, and oxidized base levels) were evaluated; moreover, visually identified large and small nuclei were analyzed separately and arranged in classes of increasing DNA damage. Oxidized base levels were definitely lower in young versus senescent fibroblasts of which, however, a significant proportion showed negligible DNA damage. Nuclear size enlargement accompanying senescence was almost equally influenced by cell ploidy increase and also by a chromatin decondensation process involving diploid cells. It is noteworthy that DNA damage in senescent fibroblasts correlated significantly to nuclear size, but not to DNA content. The comet assay allowed us to identify different senescent phenotypes and to investigate changes in nuclear features and/or DNA damage irrespective of time elapsed in culture.

NORMAL human diploid fibroblasts in culture undergo a phenomenon first described by Hayflick and Moorhead (1) and known as replicative senescence, i.e., the permanent proliferative arrest following a limited number of population doublings. Additional features of replicative senescent cells involve resistance to apoptosis and altered differentiation, a large flat morphology and decreased saturation density in culture, as well as the activation of neutral beta-galactosidase (2–4). Moreover, senescent human fibroblasts may remain metabolically active for a long time (1,5,6) by influencing the local tissue microenvironment (4). Replicative senescence has been proposed as a biological program aimed at inducing terminal growth arrest and then antagonizing neoplastic transformation as implied also by a number of selected molecular changes which are linked to the control of tumor-suppression mechanisms (7,8).

Although replicative block in human senescent cells has been reported to be induced primarily by the process of telomere shortening that occurs at each cell division (9,10), the activation of other pathways (11,12) might also be involved. In fact, certain types of nuclear DNA damage, including oxidative damage and double strand breaks (3,13–16) resulting from an altered balance between intensity of stress and efficiency of antioxidant cellular systems, may eventually lead to the acquisition of additional senescent features. This is especially important in vitro due to the fact that, under standard culture conditions, cells are usually exposed to an oxygen tension far exceeding that present in vivo. Other mechanisms which might be significant in senescence are: (a) nuclear somatic mutations involving Ras or Raf (17,18), (b) some forms of supraphysiological mitogenic signaling (19), (c) a progressive decline in mitochondrial functions (20), and (d) epigenetic alterations, errors in the accuracy of information transfer, and postsynthetic changes in proteins, as reviewed by Holliday (21). However, regardless of the mechanisms involved, replicative senescence develops as an intrinsically stochastic process (22) to eventually yield coexisting different cell phenotypes characterized by high structural, metabolic, and proliferative heterogeneity (2).

The aims of this work were to investigate the transition from young to senescent MRC5 fibroblasts and to possibly identify various phenotypes within heterogeneous senescent populations. The alkaline comet assay has been used as a flexible and fine methodology to measure the distribution of various types of DNA damage, nuclear morphology, and DNA content at the single cell level along with senescence. A novel approach—previously used by some of us (23) to analyze visually recognized polymorphonuclear and mononuclear white blood cells—was used herein to identify different fibroblast subpopulations. The combined "conventional and unconventional" use of the comet assay methodology helped us to rediscover some classic observations and provide new information on senescent fibroblasts.


   MATERIALS AND METHODS
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 Abstract
 Materials and Methods
Results and Discussion
 References
 
Cell Culture
MRC5 cells (human embryonic lung-derived diploid fibroblasts) were obtained from the National Institute on Aging, Aging Cell Repository (Camden, NJ), and were maintained in culture with high-glucose (4500 g/L) Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum at 37°C in a 5% humidified atmosphere. Confluent cultures were propagated by trypsinization, and the population doubling level (PDL) was calculated according to the equation: PDL = 3.32 x log N/No (where N and No were the recovered and seeded cell number, respectively). Experiments were performed with confluent young (PDL < 30), old (50 < PDL < 58), and senescent (PDL ≥ 60) noncycling MRC5 fibroblast cultures. Irrespective of the reached PDL, cultures were assumed to be senescent when cells could not complete one PDL after three consecutive weeks of refeeding with fresh complete medium.

Comet Assay
DNA damage measurements.-- The alkaline comet assay was used to measure the number of double- and single-strand breaks in DNA (24). A modification of the method was also used to measure specific oxidative damage on DNA bases (25). Aliquots of the cell suspension containing about 4 x 104 cells were pelleted by centrifugation at 250 x g at 4°C for 10 minutes and resuspended in 32 µl of low-melting-point agarose (Fisher Scientific, Leicestershire, U.K.). Cells were then spotted onto precoated microscope slides in duplicate (16 µl for each spot), covered with a round coverslip (13 mm diameter), and allowed to solidify at 4°C. Three different agarose spots were usually accommodated on each slide, which was then covered with a further 120 µl of low-melting-point agarose layered by means of a standard coverslip (24 x 60 mm) and allowed to solidify. All the experimental slides with the agarose-embedded cells were then subjected to a lysis step (1-hour incubation at 4°C in 1% N-lauroyl-sarcosine, 2.5 M NaCl, 100 mM Na2EDTA, 1% Triton X-100, 10% dimethyl sulfoxide). To analyze oxidized DNA bases, a bacterial repair enzyme, endonuclease III (ENDO III; provided by Dr. A. R. Collins, University of Oslo, Norway, upon isolation from Escherichia coli) was used to introduce breaks at pyrimidine oxidation sites. After completion of the lysis step, the slides were washed twice in enzyme buffer [40 mM HEPES-KOH (pH 8.0), 100 mM KCl, 0.5 mM EDTA, bovine serum albumin at 0.2 mg/ml], incubated at 37°C for 1 hour with 80 µl of ENDO III (diluted 1:1000 in enzyme-buffer), and sealed with a coverslip. After the enzyme incubation step, the slides were subjected to electrophoresis in an ice-cold electrophoresis chamber (model GNA-200; Pharmacia, Milan, Italy) containing alkaline running buffer (300 mM NaOH, 1 mM Na2EDTA) for 20 minutes to allow DNA unwinding. The electrophoresis was subsequently conducted for 20 minutes at 0.8 V/cm and 300 mA, then the slides were washed with neutralization buffer (40 mM Tris-HCl, pH 7.4), stained overnight with ethidium bromide, and analyzed on the following day. Microscopic analysis was carried out with the aid of a Labophot-2 microscope (Nikon, Tokyo, Japan) provided with epifluorescence and equipped with a rhodamine filter (excitation wavelength 546 nm; barrier 580 nm). The images of 50 randomly chosen nuclei per slide were captured and analyzed using custom-made imaging software coupled with a CCD camera (model C5985; Hamamatsu, Sunayama-Cho, Japan). The amount of damaged DNA migrated in the tail was expressed as percentage of total fluorescence for each nucleus (% DNA in the tail). This value was then averaged over the 50 nuclei measured per slide, and the duplicate values were further averaged. Total DNA damage resulted from the sum of the basal level of strand breaks plus the additional breaks introduced by ENDO III at oxidized pyrimidine sites. Thus, the net oxidative DNA damage (oxidized bases) was calculated as the difference between damage detected in the enzyme-treated slides and that detected in their corresponding controls (slides incubated with buffer only). The basal level of DNA strand breaks was obtained in the slides which had not undergone enzyme treatment.

Subdivision of DNA damage in classes.-- To calculate the distribution of total DNA damage within each sample, we classified the nuclei into five categories with increasing tail migration (class 1: 0%–5%; class 2: 5.1%–17%; class 3: 17.1%–35%; class 4: 35.1%–60%; class 5: 60.1%–100%), as proposed by Giovannelli and colleagues (26). The percentage of frequency distribution into the five categories was then calculated for each duplicate and averaged within experimental groups.

DNA amount measurements.-- Total DNA amount per nucleus was also measured as total ethidium bromide fluorescence with the comet assay software to obtain bivariate distributions, as previously described by Olive and Banath (27). The DNA amount was expressed as pixels x 105, and nuclei were also grouped into intervals of 1 x 105 pixels.

Differential analyses in senescent small and large nuclei.-- Senescent MRC5 cultures were subjected to a differential analysis of DNA damage: Images of small and very large nuclei (as exemplified in Figure 2A) were acquired separately on the basis of visual recognition by the operator. DNA content and DNA damage in the two subpopulations were evaluated by the comet assay as reported above and used for comparative analyses.



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  		Figure 2. Changes in nuclear morphology and DNA content profiles of young to senescent MRC5 fibroblasts. A, Photomicrographs showing examples of nuclei in a comet assay slide from either young [population doubling level (PDL) = 28], presenescent (PDL = 55), or senescent (PDL = 62) noncycling fibroblasts. Cultures were assumed to be senescent when cells could not complete one PDL after three consecutive weeks of maintenance in culture. Pictures were taken with a Labophot-2 microscope provided with epifluorescence and equipped with a rhodamine filter (excitation wavelength: 546 nm; barrier: 580 nm) at x40 magnification. B, Frequency distribution of DNA content in young and senescent MRC5 cells. The DNA content per nucleus was measured by the comet assay-dedicated software as the total ethidium bromide fluorescence, and is reported as increasing intervals of 1 x 105 pixels. The mean percentage frequency of nuclei distribution in DNA content intervals for young and senescent cultures is shown

 
Statistical Methods
The results were expressed as means ± SEM (standard error of the mean). Differences between cell groups were assessed using Student's t test. Correlations were performed using linear regression analysis, and the significance level was considered to be p <.05.


   RESULTS AND DISCUSSION
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 Abstract
 Materials and Methods
 Results and Discussion
References
 
DNA Damage in Either Young or Senescent MRC5 Fibroblasts
The alkaline comet assay was used to monitor DNA damage in noncycling MRC5 fibroblasts from either young (PDL < 30) or senescent (PDL ≥ 60) cultures (Figure 1A). Levels of either basal strand breaks assessed prior to any treatment or, instead, the total DNA damage obtained following ENDO III treatment of samples were measured and expressed as percentages of DNA migrating in the tail of the comet. The relative content of oxidative bases in cell populations was derived, eventually, from the subtraction of basal strand breaks from total DNA damage. Young and senescent fibroblasts showed comparable amounts of basal DNA breaks in keeping with our previous data on MRC5 (28) and dermal (29) fibroblasts which were examined at low and high PDL for DNA integrity by DNA precipitation assay (30). Instead, levels of total DNA damage were considerably higher in senescent versus young fibroblasts (p =.054) as the result of significant variations in the amount of oxidized bases that accounted for about 12% and 21% of DNA in the tail of young and senescent nuclei, respectively (p =.017). These data confirmed previous reports (13,14) describing increased levels of oxidized bases along with senescence in cultured human diploid fibroblasts.



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  		Figure 1. Evaluation of DNA damage in young (population doubling level <30) and senescent (population doubling level ≥60) cultures of MRC5 fibroblasts by single cell comet assay. A, Total DNA damage (sum of breaks and oxidized bases), basal level of DNA damage (breaks), and the net oxidative DNA damage (ox. bases) resulting from the difference between damage detected after and before ENDO III addition, were measured and expressed as percentages of DNA migrated in the tail of the comet. Values were the mean ± standard error of the mean of four separate experiments; *p <.05 senescent vs young cultures. B, Frequency distribution of nuclei in five different classes of increasing total DNA damage (class 1: 0%–5%; class 2: 5.1%–17%; class 3: 17.1%–35%; class 4: 35.1%–60%; class 5: 60.1%–100% of DNA in the tail of the comet; see Materials and Methods). The mean percentage values for young and senescent cultures from a typical experiment of three were reported

 
The DNA damage reported above as the mean of several observations (100 nuclei for each sample) could lead to overlooking the range of variations occurring within a given population and, especially, in nuclei from senescent cultures. For a more accurate analysis of data, the latter have been collected into five arbitrarily established classes of increasing total DNA damage which were, namely, class 1: 0%–5%; class 2: 5.1%–17%; class 3: 17.1%–35%; class 4: 35.1%–60%; and class 5: 60.1%–100% of DNA in the tail. The use of classes of DNA damage (subsequently indicated as "discontinuous" comet assay approach) was originally developed to measure the effect of genotoxic agents (31) and is used herein to compare the distribution frequency of spontaneously damaged nuclei in young and senescent MRC5 cultures (Figure 1B). Approximately 20%–25% of nuclei of both young and senescent fibroblasts were included in class 1 of damage, but the frequencies in other classes diverged significantly between the two populations. The bulk of young nuclei presented low levels of damage and were mainly gathered (>60%) within classes 2 and 3, whereas highly damaged nuclei in classes 4 and 5 accounted for approximately 15% of the whole population. In contrast, senescent cultures were characterized by decreased frequency of moderately damaged nuclei in classes 2 and 3, whereas there was a striking increase in severely damaged nuclei so as to include >40% of senescent cells in classes 4 and 5. Overall, nuclei of senescent MRC5 populations were spread over the five classes of DNA damage; it is noteworthy that class 1 contained a significant proportion (approximately 20%) of very well preserved nuclei which would have been overlooked by using the "average" rather than "discontinuous" data analysis.

Changes in Nuclear Morphology and DNA Content During the Transition From Young to Senescent Fibroblast Cultures
Along with the determination of basal DNA damage in noncycling young, presenescent, and senescent MRC5 fibroblast cultures, marked differences in nuclear size among the three cell populations were observed (Figure 2A). Young MRC5 cultures presented nuclei that were all relatively small in size and with a fairly regular round morphology. Significant changes in nuclear shape were seen in presenescent cultures, showing the coexistence of both small and large nuclei, whereas in senescent cultures nuclei were mainly very large in size and characterized by an irregular distribution of fluorescence. Nuclear size increase was reported to occur also in senescent human WI38 fibroblasts [(32) and cited in (2)] and correlated to a decreased replicative capability that characterizes populations with high PDLs. Changes in fluorescence intensity and distribution observed in senescent nuclei might suggest (a) alterations in chromatin condensation status (33,34) capable of modifying accessibility of template regions and (b) possibly, decreasing rates of transcription, as reviewed by Cristofalo and Pignolo (2). However, differences in nuclear morphology between young and senescent MRC5 cultures could not be ascribed to the amount of basal DNA breaks as these were virtually identical (around 10%) in the two populations (see Figure 1A). Alternative hypotheses to explain senescence-related enlargement of nuclei pointed to an increase in either (a) the amount of DNA per cell and/or (b) the level of oxidized bases that may lead to alterations in chromatin assembling. To explore the first issue, DNA content per nucleus was measured as total ethidium bromide fluorescence by the comet assay according to Olive and Banath (27), showing differences between young and senescent MRC5 noncycling cultures (Figure 2B). Nuclei from young cultures were mainly grouped within a single peak with a maximum of fluorescence around 2.5 x 105 pixels and assumed to be equivalent to a DNA content of 2n. In senescent cultures, a significant portion of nuclei (over 40%) exhibited DNA fluorescence values >2.5 x 105 pixels with two distinct peaks around 4 x 105 and 6 x 105 pixels to indicate nuclear DNA content ≥4n; these results were in keeping with other reports on fibroblast cell lines WI38 (3,35,36) and MRC5 (37) showing that DNA content increased progressively along with senescence. Nevertheless, it was worth noting that approximately half of senescent nuclei had a diploid DNA content, as also reported in MRC5 cells by metaphase chromosome counting (37). Therefore, the nuclear enlargement observed in senescent MRC5 fibroblast cultures cannot be ascribed solely to increase in cell ploidy but also to a process of chromatin decondensation occurring in a large proportion of senescent diploid cells.

Relationship Between Nuclear Morphology and DNA Damage in Senescent MRC5 Cultures
The above-mentioned conclusions prompted us to verify the second hypothesis, i.e., whether DNA damage could contribute to morphological heterogeneity of senescent nuclei. To this aim, small and large nuclei of the same senescent culture were analyzed separately on the basis of visual recognition by the operator and examined as two different populations. This comparison was intended, therefore, to provide a qualitative analysis of DNA damage in large versus small nuclei which represented the major and minor portion of senescent fibroblast populations, respectively. Small nuclei appeared to be significantly less damaged than large senescent nuclei (Figure 3A). However, the most striking differences (p =.008) between small and large nuclei involved levels of (a) total DNA damage, with 15% and 35% mean percentages of DNA migrating in the tail, respectively, and (b) oxidized bases that were approximately fivefold lower in small than in large senescent nuclei. These data suggested that, within senescent cultures, small and large nuclei represented two distinct subpopulations and pointed to a direct correlation existing between nuclear size and extent of oxidative DNA damage. In past years, these two parameters were often considered in regard to their influence on the decline in cell proliferative activity, whereas much less was known of their mutual relationships in senescence. Actually, a less condensed status of chromatin within senescent nuclei has been hypothesized to favor oxidative damage of DNA and increase levels of 8-hydroxy-2'deoxyguanosine (13). Moreover, our results allowed us to compare the DNA integrity in a small nuclei subpopulation from senescent cultures and that in the bulk of small nuclei from young cultures (Figure 3A, dashed line). Total DNA damage appeared to be lower in small senescent versus small young nuclei (p =.054); this discrepancy, however, was due to a significant decrease (p =.025) in the amount of oxidized bases of senescent small nuclei representing a subset of cells that, although very limited in number, were qualitatively important because of their distinctive resistance to oxidative damage.



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  		Figure 3. Levels of DNA damage in nuclei of senescent MRC5 cultures. A, Mean levels of DNA damage (total, breaks, and ox. bases) were measured after visual separation of nuclei according to size into small and large groups (100 nuclei per group). DNA damage was expressed as the percentage of DNA migrated in the tail of the comet. Average values of young MRC5 cultures (from results of
Figure 1A) were also reported as dashed lines for comparison. Values were the mean ± standard error of the mean of four separate experiments. *p <.05, **p <.01 large vs small nuclei; #p <.05 small nuclei vs young cultures. B, Frequency distribution of nuclei in classes of total DNA damage. The mean percentage frequency of nuclei in each class of damage (from class 1 to 5, as reported in
Figure 1 legend) for either small or large senescent nuclei is shown. Results were from a typical experiment of three

 
A further level of complexity within senescent cell subpopulations was disclosed by the analysis of distribution of total DNA damage in classes as above. Two very distinct profiles were obtained (Figure 3B): Small senescent nuclei were confined mainly (approximately 60%) to classes 1 and 2 of DNA damage, with virtually no cells in class 5. Conversely, large senescent nuclei distribution shifted toward higher classes of damage, with >55% of nuclei in classes 4 and 5, in both of which broken DNA accounted for approximately 35%–100% of the total. It is interesting, however, that approximately 25% of large nuclei were in class 1 of damage (i.e., negligible or null DNA damage); this finding indicated that chromatin decondensation might actually render nuclei more susceptible to oxidation but, conceivably, it was not the result of DNA damage. Overall, in senescent cultures there was a significant portion of cells which, regardless of their small or large nuclear size, presented exceedingly low levels of DNA damage. These subsets of "undamaged" senescent cells showing no gross DNA injury seemed to have lost their proliferative capability for reasons that might reflect only a few modifications involving either expression of specific target genes or cell response to extranuclear events (2,3).

Relationship Between DNA Content and Damage in Senescent MRC5 Cultures
We provided evidence that an increase in nuclear size of senescent MRC5 fibroblasts was closely related to the extent of DNA damage and, especially, levels of oxidized bases (see Figure 3A). However, it was also important to assess whether the higher frequency of polyploid cells observed in senescent cultures might be a consequence of damage. To answer this question, DNA content was measured in large and small nuclei from 3-week cultures of subconfluent senescent fibroblasts, and results were plotted against total DNA damage expressed as a percentage of DNA in the tail (Figure 4). Small nuclei (filled squares) with a mean fluorescence value of approximately 2.5 x 105 pixels corresponding to a DNA content of 2n exhibited from low to moderate levels of DNA damage, although some nuclei with >50% DNA in the tail were also identified. For the majority of large nuclei (empty squares)—yielding fluorescence values >4 x 105 pixels to reaffirm the marked increase in ploidy along with MRC5 fibroblast senescence—there was a broad distribution of damage within the range of 0%–80%. A similarly wide extent of damage was seen in a small subset of large nuclei with a DNA content close to diploid. Linear regression analysis of data indicated no correlation between total DNA damage and DNA content per nucleus in either small or large senescent nuclei. This result implies that polyploidy and nuclear shape are not strictly related in senescent nuclei, which can undergo large variations in size, morphology, and chromatin asset which are virtually independent of their DNA content. Paradoxically, therefore, a biochemical parameter such as DNA content appeared to be much less informative than did morphological analysis of nuclei by comet assay to gain insight on the relationships among nuclear size, levels of DNA damage, and chromatin condensation status in cultured human fibroblasts.



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  		Figure 4. Total DNA damage and DNA amount in small ({block}) and large ({square}) nuclei of senescent MRC5 fibroblasts. DNA content per nucleus (100 nuclei for each group) was measured by the comet assay-dedicated software as the total ethidium bromide fluorescence, and is reported as pixels x 105. Values of total DNA damage, expressed as the percentage of DNA in the tail, were crossed with DNA content of each single nucleus. No significant correlation between DNA damage and content was observed for either small or large nuclei by linear regression analyses

 
Final Remarks
The present results confirm the increase in nuclear heterogeneity in MRC5 fibroblasts approaching their replicative senescence. Different phenotypes have been identified in senescent cultures by using the comet assay methodology, which has proved to be an especially flexible and efficient tool for assessing changes in nuclear size, DNA content, and morphology as well as for measuring various types and extent of DNA damage. Analyses carried out at the single cell level and the evaluation of different classes of DNA damage helped us to (a) characterize more deeply the several subsets of cells occurring in senescent cultures, (b) clarify significant relationships among the aforementioned changes in nuclear features and, eventually, (c) hypothesize whether these changes might influence or be influenced by the replicative senescence process. The comet assay approach used herein for senescent fibroblasts might also be used generally for in vitro studies to provide detailed information on changes in nuclear status of cultured cells over time and/or treatments.


   Acknowledgments
 
This work was supported by grants from the Ministero dell'Istruzione, dell'Università e della Ricerca, Italy (ex 60%, Progetti di Rilevante Interesse Nazionale 1999 and 2001), from the Regione Toscana-Ministero della Salute (project 2001–2004), and from European Union grants QLKI-1999-00346, QLRT-1999-00505, and QLK1-CT-1999-00568. We thank Dr. Andrew Collins of the Institute for Nutrition Research, University of Oslo, Norway, for providing the enzyme ENDO III.


   Footnotes
 
Decision Editor: James R. Smith, PhD

Received October 27, 2004

Accepted January 12, 2005


   References
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 Abstract
 Materials and Methods
 Results and Discussion
 References
 

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