Fibroblast cell lines from young adult mice of long-lived mutant strains are resistant to multiple forms of stress

Adam B. Salmon,1 Shin Murakami,2 Andrzej Bartke,3 John Kopchick,4 Kyoko Yasumura,5 and Richard A. Miller1,5,6

1Cellular and Molecular Biology Graduate Program, University of Michigan School of Medicine, Ann Arbor, Michigan; 2Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky; 3Departments of Internal Medicine and Physiology, Geriatrics Research, Southern Illinois University, School of Medicine, Springfield, Illinois; 4Edison Biotechnology Institute and Department of Biomedical Sciences, Ohio University, Athens, Ohio; 5Department of Pathology, University of Michigan School of Medicine; and 6University of Michigan Geriatrics Center and Institute of Gerontology and the Ann Arbor Department of Veterans Affairs Medical Center, Ann Arbor Michigan

Submitted 7 December 2004 ; accepted in final form 2 February 2005


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Previous studies have shown that dermal fibroblast cell lines derived from young adult mice of the long-lived Snell dwarf mutant stock are resistant, in vitro, to the cytotoxic effects of H2O2, cadmium, UV light, paraquat, and heat. We show here that similar resistance profiles are seen in fibroblast cells derived from a related mutant, the Ames dwarf mouse, and that cells from growth hormone receptor-null mice are resistant to H2O2, paraquat, and UV but not to cadmium. Resistance to UV light, cadmium, and H2O2 are similar in cells derived from 1-wk-old Snell dwarf or normal mice, and thus the resistance of cell lines derived from young adult donors reflects developmental processes, presumably hormone dependent, that take place in the first few months of life. The resistance of cells from Snell dwarf mice to these stresses does not reflect merely antioxidant defenses: dwarf-derived cells are also resistant to the DNA-alkylating agent methyl methanesulfonate. Furthermore, inhibitor studies show that fibroblast resistance to UV light is unaffected by the antioxidants ascorbic acid and N-acetyl-L-cysteine. These data suggest that postnatal exposure to altered levels of pituitary hormones leads to development of cellular resistance to oxidative and nonoxidative stressors, which are stable through many rounds of in vitro cell division and could contribute to the remarkable disease resistance of long-lived mutant mice.

oxidation; life span; Snell dwarf; aging; insulin-like growth factor I


THE ABILITY OF AN ORGANISM to respond to environmental and cytotoxic stresses may play an important role in regulation of the aging processes. Recent studies on the genetics of aging in the yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, and the fruit fly Drosophila melanogaster have shown a positive correlation between resistance to stress and extended longevity. Genetic manipulations and environmental selections that increase life span in these models tend to enhance resistance to such stressors as reactive oxygen species, UV light, heavy metals, and heat, suggesting that the ability to respond to stress may be an important determinant of life span (30, 31, 33, 45, 49, 58, 59, 62).

There is now a growing pool of evidence to suggest that stress resistance, at the cellular level, may also contribute to longevity in mammals (27, 32, 3739, 68). Many of these studies show that the animals are resistant to stressors in vivo, measured either through animal death or through modulation of stress response elements (27, 37, 39, 53, 54). However, homeostatic mechanisms within whole animals make results from such studies difficult to interpret. Recent studies have suggested that cells isolated from long-lived animals may retain properties of cellular resistance to stress in culture (27, 32, 39, 42). Such results suggest that studies of cellular resistance to stress may help to delineate the mechanisms that may regulate both stress resistance and the aging process.

Our own prior investigations involved analysis of fibroblast cell lines derived from biopsies of the dermis of young adult mice of the Snell dwarf stock (42). Snell dwarf mice (dw/dw) have a mutation in the gene encoding a transcription factor, Pit-1, that results in abnormal pituitary development and an altered hormone profile featuring primary deficiencies in growth hormone (GH), thyroid-stimulating hormone (TSH), and prolactin and thus secondary deficiencies in insulin-like growth factor I (IGF-I) and thyroxine. Snell dwarf mice live 40% longer than littermate controls and show delays or deceleration of multiple aspects of aging, including arthritis (52), collagen cross-linking and T cell subset changes (19), and cancer (29), as well as delays in development of cataracts and glomerular pathology (65). Fibroblast cell lines derived from young adult Snell dwarf mice were found to be resistant to death induced by exposure to cadmium, hydrogen peroxide (H2O2), paraquat, UV light, and heat compared with fibroblasts from control mice of normal life span (42). These results thus suggested a parallelism with previous work in C. elegans and D. melanogaster, in which perturbations to the IGF/insulin-signaling pathway result in extended longevity and organismal resistance to stress (6, 15, 28, 33, 36, 43, 60, 62).

The previous experiments on fibroblasts from Snell dwarf mice suggest the hypothesis that cellular stress resistance may contribute to the disease resistance and longevity seen in vivo in mutant mice with alterations in the IGF-I pathway, such as the Ames dwarf mice (Prop-1df/df), and in mice deficient in the GH receptor (GHR-KO mice) (16, 17, 35). To test this idea, we have now evaluated stress resistance in cell lines derived from young adult mice of these long-lived models. In addition, we have conducted experiments to determine whether stress resistance is present in cells from neonatal mice or is acquired in the context of postnatal development and have tested whether stress resistance of Snell dwarf fibroblast lines is or is not due simply to resistance to oxidative damage.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Animal subjects. Snell dwarf animals (and heterozygote controls) were dw/dw mice bred as the progeny of (DW/J x C3H/HeJ)-dw/+ females and (DW/J x C3H/HeJ)F1-dw/dw males. These sires had been previously treated with GH and thyroxine to increase body size and fertility. Littermates with the (+/dw) genotype were used as controls. Tail skin biopsies were taken from male mice 4–5 days of age or from male mice 3–4 mo of age. Protocols were approved by the University Committee on the Use and Care of Animals.

Ames dwarf mice and GH receptor null or knockout (GHR-KO) mice (and littermate controls) were kindly provided by Dr. Andrzej Bartke at Southern Illinois University (Springfield, IL), from breeding stock originally generated by Dr. Kopchick's group at Ohio University. Tail skin biopsies from these mice were obtained from 3- to 6-mo-old males and sent overnight on ice in DMEM supplemented with 20% heat-inactivated fetal calf serum, antibiotics, fungizone, and 10 mM HEPES. Fibroblasts were prepared from the biopsy tissue as described in General procedure. Ames dwarf (df/df) and control (+/+ or +/df) mice were produced by crosses between df/+ parents or between fertile df/df males and df/+ females. These mice were produced in a random-bred, closed colony with a heterogeneous background, which has been maintained for more than 20 yr. GHR-KO mice and normal littermate controls were produced by mating heterozygous (+/–) carriers of the disrupted GHR/GHBP gene or homozygous knockout (–/–) males with (+/–) females. The genetic background of these animals is derived from 129/Ola embryonic stem cells and from BALB/c, C57BL/6, and C3H inbred strains.

(C57BL/6J x BALB/cJ)F1 hybrid (CB6F1) animals were obtained from the Jackson Laboratory (Bar Harbor, ME). Tail skin biopsies were taken from 3- to 4-mo-old males.

General procedure. Tail skin biopsies ~3–5 mm in length were obtained from the latter half of the intact tail of isoflurane-anesthetized mice after skin sterilization with 70% ethanol. Biopsies were further washed in 70% ethanol, placed in DMEM (high-glucose variant; GIBCO-Invitrogen, Carlsbad, CA), diced to <0.5 mm, and digested overnight with collagenase type II (400 U/ml, 1,000 U total per tail, GIBCO-Invitrogen) dissolved in DMEM supplemented with 20% heat-inactivated fetal bovine serum, antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin; Sigma, St. Louis, MO), and 0.25 µg/ml fungizone (Biowhittaker-Cambrex Life Sciences, Walkersville, MD) at 37°C in a humidified incubator with 5% CO2 in air. After collagenase treatment, cells were dislodged from digested tissue by repeated pipetting and passed through sterile nylon netting into sterile 14-ml centrifuge tubes (BD Dickenson, Bedford, MA). Samples were centrifuged for 5 min at 200 g, and collagenase solution was drawn off the cell pellet. Cells were resuspended in DMEM with 20% heat-inactivated fetal bovine serum, antibiotics, and fungizone. Approximately 2.5 x 105 cells in 3 ml of medium were seeded into tissue culture flasks of 25 cm2 surface area (Corning Costar, Corning, NY). After 3 days, ~2/3 total volume of medium was removed on day 3 and replaced with fresh DMEM with 20% heat-inactivated fetal bovine serum, antibiotics, and fungizone. Six or seven days after seeding, initial cultures (designated first-passage cells) were either 1) split six- or ninefold by volume to create second-passage cells, with twofold or threefold dilutions at each subsequent passage, or 2) split and seeded at a density of 1 x 105 cells/cm2 flask surface area at each passage into tissue culture flasks of 75 or 175 cm2 surface area (Corning Costar). Cells were split by first washing flasks with 1x phosphate-buffered saline solution (PBS: 8.8 g NaCl, 2.25 g Na2HPO4, and 0.26 g NaH2PO4/liter distilled H2O, pH 7.3) followed by incubation with ~3 ml of trypsin/100 cm2 surface area of flask 1x trypsin-EDTA (GIBCO-Invitrogen) for ~5 min at 37°C in a humidified incubator with 5% CO2 in air. Trypsin activity was inhibited with an equal volume of DMEM with 20% heat-inactivated fetal bovine serum, antibiotics, and fungizone. Subsequent passages were split at 6-day intervals with ~2/3 total volume of medium removed on day 3 and replaced with fresh DMEM with 20% heat-inactivated fetal bovine serum, antibiotics, and fungizone. At the end of the third passage (6 days after seeding), confluent cells were used for assessment of stress resistance.

Assessment of cytotoxicity after exposure to stress. Six days after seeding, third-passage cells were trypsinized as described. Cells were counted by hemocytometer and diluted to a concentration of 3 x 105/ml in DMEM with 20% fetal bovine serum, antibiotics, and fungizone and seeded into a 96-well tissue culture-treated microtiter plate at a volume of 100 µl/well. After an 18-h overnight incubation, cells were washed with 1x PBS and incubated in DMEM supplemented with 2% bovine serum albumin (BSA; Sigma), antibiotics, and fungizone for ~24 h. Previous work (42) has shown that this period of incubation in serum-starved conditions is critical for showing differences between mutant and control cells, because the presence of serum greatly increases stress resistance of cells from both kinds of mouse. Cells were then exposed to a range of doses of one of the cytotoxic stressors. For UV light testing, cells were washed and then irradiated with UV light (254 nm at 5.625 J·m–2·s–1) in 100 µl of Dulbecco's PBS (Biowhittaker-Cambrex Life Sciences). Cells were then incubated in DMEM supplemented with 2% BSA, antibiotics, and fungizone, and their survival was measured 18 h later by a test based on oxidative cleavage of the tetrazolium dye WST-1 (Roche Applied Science, Indianapolis, IN) to a formazan product, using the protocol suggested by the manufacturer. For assessment of resistance to H2O2, paraquat (methyl viologen), or cadmium (Sigma), the cells in the 96-well plates were incubated with a range of doses of stress agent for 6 h in DMEM. Cells were then washed and incubated with DMEM supplemented with 2% BSA, antibiotics, and fungizone, and survival was measured 18 h later by the WST-1 test. For assessment of resistance to methyl methanesulfonate (MMS), and actinomycin D (Sigma), cells in 96-well plates were incubated with a range of doses of each agent in DMEM for 24 h. Each agent was administered as a stock solution in dimethyl sulfoxide (DMSO, Sigma), and an equivalent level of DMSO (0.5% final concentration) was added to control cultures. Cells were then washed and incubated with DMEM supplemented with 2% BSA, antibiotics, and fungizone, and survival was measured 18 h later by the WST-1 test. All incubations were at 37°C in a humidified incubator with 5% CO2 in air.

Calculation of LD50 and statistical analysis. At each dose of chemical stressor, mean survival was calculated for triplicate wells for each cell line. The LD50, i.e., dose of stress agent that led to survival of 50% of the cells, was then calculated using probit analysis as implemented in NCSS software (NCSS, Kaysville, UT). For this analysis, extremely low doses of stress agents that caused no cell death in fibroblasts from normal animals, as measured by WST1 assay, were removed from all data sets. Differences between groups in mean LD50 levels were evaluated by paired t-test, by t-test, or by ANOVA, depending on the experimental design, with each day's work containing equal numbers of cultures from mutant and control or equal numbers of cultures tested with each treatment for antioxidant studies.

Antioxidant treatment. Tests for antioxidant effects were conducted using the method described above for stress exposure, except that ascorbic acid (Sigma) or N-acetyl-L-cysteine (NAC; Sigma) was added during the 24-h period before exposure to H2O2, cadmium, paraquat, or UV light. Ascorbic acid was administered as a stock solution in DMSO, and an equivalent level of DMSO (0.5% final concentration) was added to control cultures. Final medium and antioxidant solutions were brought to a pH of 7.2 by addition of NaOH (Sigma). Ascorbate or NAC remained present during the exposure to the stress agent, after which cells were washed, incubated for 18 h in DMEM with BSA, and tested for survival using the WST-1 procedure.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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To determine whether the association between life span and cellular resistance to stress extends to mouse mutant models of extended longevity other than Snell dwarf mice, we used a stress assay to test fibroblasts from Ames dwarf mice and GHR-KO mice. The results (Fig. 1) revealed that fibroblasts grown from homozygous Ames dwarf mice (df/df) are significantly more resistant to UV light, H2O2, and cadmium than fibroblasts derived from normal littermate controls. The effect sizes range from 43% (UV) to 95% (cadmium), and all results were significant by paired t-test at P < 0.05. The data also suggest that fibroblasts from Ames dwarf mice may be slightly more resistant to paraquat, although not significantly, in a study with a sample size of six per group.



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Fig. 1. Skin-derived primary fibroblasts from Ames dwarf mice are resistant to multiple forms of stress compared with control. Each symbol in the figure represents fibroblasts isolated from an individual mouse of the indicated genotype; the horizontal line indicates the mean value for each group. Percent increases in LD50 and P values for paired t-test were as follows: A: UV light (43%; P = 0.017, n = 12); B: H2O2 (79%; P = 0.002, n = 12); C: cadmium (95%; P = 0.021, n = 12); D: paraquat (25%; P = 0.141, n = 6).

 
Fibroblasts from mice lacking GH receptor (GHR-KO) were significantly more resistant to H2O2, UV light, and paraquat than fibroblasts from littermate control mice (Fig. 2). Effect sizes ranged from 47% (paraquat) to 194% (UV light) and were all significant by paired t-test at P <0.05. There was, however, no evidence for cadmium resistance in fibroblasts from GHR-KO mice.



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Fig. 2. Skin-derived primary fibroblasts from growth hormone receptor knockout (GHR-KO) mice are resistant to UV light, H2O2, and paraquat, but not to cadmium. Each symbol in the figure represents fibroblasts isolated from an individual mouse of the indicated genotype; the horizontal line indicates the mean value for each group. Percent increases in LD50 and P values for paired t-test were as follows: A: UV light (194%; P = 0.049; n = 12); B: H2O2 (108%; P = 0.002; n = 12); C: cadmium (7%; P = 0.36; n = 12); D: paraquat (47%; P = 0.016; n = 6).

 
Newborn mice of the Snell and Ames genotype and GHR-KO mice are indistinguishable in size from control littermates (35, 69) but begin to show growth retardation in the first week of life as a consequence of their pituitary hormone deficiencies (Ames, Snell) or diminished response to GH (GHR-KO). To determine whether differential resistance to stress was present in neonates or, alternately, developed gradually after birth, we tested cell lines derived from tail biopsies from newborn (4- to 5-day-old) Snell dwarfs and controls in parallel with lines taken from separate mice that were young adults (3–4 mo old). The results are shown in Table 1. Consistent with previous findings, cells derived from young adult Snell dwarf mice show significantly greater resistance to UV light, H2O2, and cadmium. In contrast, fibroblasts from neonatal Snell dwarfs are not more resistant than control lines to UV light or cadmium. Fibroblasts from neonatal dwarf mice are significantly more resistant to H2O2 than controls, but the effect size (32%) is substantially less than that seen for lines derived from young adult mice (61%). These data suggest that development of stress resistance depends on developmental changes, presumably hormone dependent, that occur in the first few months of postnatal life.


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Table 1. Stress resistance of fibroblasts from normal and Snell dwarf mice of different ages

 
The resistance of Snell dwarf cell lines to multiple forms of stress is consistent with either of two models. It may be that each of the test agents injures cells principally via oxidation damage, to which dwarf-derived cells are particularly resistant. Alternately, dwarf fibroblast lines may be resistant to multiple forms of injury, only some of which involve oxidation damage. To begin to discriminate between these ideas, we evaluated cell injury induced by H2O2, cadmium, paraquat, and UV light in the presence of ascorbic acid (vitamin C) or NAC. Results are shown in Table 2 for ascorbic acid and in Table 3 for NAC. In each case, the cells were incubated with the antioxidant both for 24 h before stress exposure (during the period of serum deprivation) and then also during exposure to the stress. As expected, ascorbic acid protected against H2O2-mediated cell death (F = 10.1, P < 0.001) and also against paraquat (F = 8.8, P < 0.001), which is thought to act by production of intracellular free radicals (13). The greater degree of protection against H2O2 may reflect inactivation of H2O2 before its entry into the fibroblast cytoplasm. Ascorbic acid treatment also gave significant protection from cadmium-mediated cell death (F = 5.8, P = 0.008), albeit to a lower degree, consistent with previous reports that cadmium-mediated cytotoxicity involves, at least in part, oxidative damage (56). Ascorbic acid did not, however, provide any protection from cell death caused by UV light irradiation (Table 2, F = 1.2, P = 0.321). Table 3 shows similar results with an alternate antioxidant, NAC, that provided significant protection against cell injury produced by H2O2 (F = 17.3, P < 0.001) and cadmium (F = 5.1, P = 0.015), consistent with the ascorbate results. The high level of protection against cadmium may be an artifact related to the chelation of cadmium ions by NAC (66). NAC provided protection from paraquat-induced cell death, although these data did not reach significance (F = 0.8, P = 0.472). Once again, though, cytotoxicity induced by UV light was not altered by the presence of an antioxidant (F = 0.2, P = 0.832), suggesting that the protective effect seen in Snell dwarf cell lines was not restricted to agents that induce oxidative damage alone.


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Table 2. Stress resistance of fibroblasts exposed to ascorbic acid (vitamin C)

 

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Table 3. Stress resistance of fibroblasts exposed to NAC

 
To determine whether fibroblasts from long-lived mice are resistant to other forms of cell death that do not involve direct oxidative damage, we tested the effect of MMS and actinomycin D on fibroblasts from Snell dwarf mice and normal littermate controls. MMS is a monofunctional DNA-alkylating agent and a known carcinogen (8, 34) and primarily methylates DNA on N7-deoxyguanine and N3-deoxyadenine (47). Although the N7-methylguanine adduct may be nontoxic and nonmutagenic, N3-methyladenine is a lesion that inhibits DNA synthesis and needs to be actively repaired to prevent cell death (8, 9). Actinomycin D is an antineoplastic antibiotic that inhibits cell proliferation by forming stable complexes with DNA and blocking the movement of RNA polymerase, interfering with RNA synthesis and inducing apoptosis (22). Both agents induced dose-dependent cell death in dermal fibroblast cell lines from both genotypes as measured by WST1. In cultures isolated from 12 Snell dwarf animals and 11 normal littermates, dwarf fibroblasts were significantly more resistant to cell death induced by MMS (Fig. 3A). There was, however, no difference in resistance of these cultures to cell death induced by actinomycin D (Fig. 3B).



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Fig. 3. Skin-derived primary fibroblasts from Snell dwarf mice are resistant to cell death induced by methyl methanesulfonate (MMS) but not to cell death induced by actinomycin D. Each symbol in the figure represents fibroblasts isolated from an individual mouse of the indicated genotype; the horizontal line indicates the mean value for each group. Percent increases in LD50 and P values for t-test were as follows: A: MMS (60%; P = 0.017, n = 12 dw/dw, 11 dw/+); B: actinomycin D (–10%; P = 0.43, n = 12 dw/dw, 11 dw/+).

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Our previous work (42) has shown that fibroblasts isolated from long-lived Snell dwarf mice are more resistant to multiple cytotoxic stressors than are fibroblasts from control mice of normal life span. The results presented here replicate the earlier data and also show that stress resistance is seen in fibroblasts from phenotypically similar mouse mutants with extended longevity, i.e, in Ames dwarf and GHR-KO mice. We also show that this stress resistance is not present at birth but is acquired in the first few months of life and that resistance is seen not just to oxidative insults but also to agents like UV light and MMS, which interfere with cellular functions principally through other means.

Our results extend the evidence for the hypothesis that mutations that extend life span may do so by increasing the resistance of cells to stress. The Ames dwarf and the Snell dwarf have different mutations within a shared pathway critical to development of the embryonic pituitary gland. As a result, mice with these mutations share an altered hormonal profile that features declines in GH, TSH, prolactin, IGF-I, and thyroxine. The similarities in stress resistance profiles of fibroblasts from Snell and Ames dwarf mice not only confirm our earlier findings in the Snell mutant but also show that the association of cellular resistance to organismal longevity is replicable on different genetic backgrounds and on mice raised in different vivaria. GHR-KO mice differ in their hormonal profile from Ames and Snell dwarf mice; the GHR-KO mice have high levels of GH, modest declines in thyroid hormones, and very low IGF-I levels (16, 17, 35). Thus the observation that resistance to H2O2, paraquat, and UV light is seen in cells from the GHR-KO mice indicates that IGF-I deficits may underlie fibroblast cell resistance to these three agents. The absence of evidence for cadmium resistance in GHR-KO cell lines suggests that resistance to this agent may depend on hormonal, or perhaps other, factors that differ between the pituitary mutants and the GHR-KO model.

Damage caused by reactive oxygen species, accruing over a lifetime, has been postulated to be an important determinant of life span both in normal individuals (23, 24) and in animal models that show extended longevity (3, 5, 26). This hypothesis is supported by evidence that levels of antioxidants are higher in tissues of Ames dwarf mice (11, 48), although results from GHR-KO mice are less clear (25). To a limited extent, our results are consistent with this idea by showing that fibroblasts from Ames dwarf, Snell dwarf, and GHR-KO mice are relatively resistant to damage induced by H2O2 or paraquat. However, the results with ascorbate and NAC inhibition, as well as the MMS data, clearly indicate that resistance of the fibroblasts cannot be attributed solely to differential vulnerability to reactive oxygen species. In our assay, the antioxidants ascorbic acid and NAC provide no protection from UV light, to which cells from all three mutant mice are resistant. In addition, Snell dwarf-derived cell lines are resistant to MMS, whose injury reflects DNA alkylation (8, 34). These data suggest that fibroblasts from dwarf mice are resistant to multiple forms of internal damage, only some of which are mediated by reactive oxygen species. One potential complication of our design is that our standard culture conditions expose the cells to atmospheric oxygen levels, ~20% in air, that are higher than typical tissue oxygen levels (63) and could potentially induce either oxidative damage, or compensatory responses to damage, that would not have been seen in cells grown under lower oxygen tension. Atmospheric levels of oxygen have previously been shown to induce cellular damage, particularly to DNA (14, 46). To address this issue, studies are being undertaken in our laboratory using fibroblasts from Snell dwarf mice and normal littermate controls grown in culture conditions using the standard 20% oxygen in air or using 3% oxygen in air, which may better represent physiological levels.

The involvement of IGF-I in our stress resistance system is likely to be indirect. Although IGF-I levels in culture do modulate cell proliferation and stress resistance (10, 21, 40, 57), our stress assays are conducted on cell lines that have been grown in defined culture medium, containing serum, for at least three passages (about 3 wk in vitro) before the withdrawal of serum and exposure to stress. Thus it seems likely that the stress resistance phenotypes are regulated by developmental differences in exposure to hormones, presumably including IGF-I, during the development of the mouse prior to tissue biopsy (7). The epigenetic changes induced in vivo might then be retained in cells isolated from these mutant mice.

Our data showing that cell lines derived from mice 4–5 days of age show no genotypic effect on resistance to UV light or cadmium and only a small difference in H2O2 resistance are consistent with this interpretation. The prenatal environments for both Snell dwarf and littermate control mice are identical, as both develop in the same uterus with hormones and nutrients provided through the maternal circulation. Snell dwarf and normal littermates are indistinguishable in size at birth, but thereafter Snell dwarf mice grow more slowly than normal mice (35). Similarly in Drosophila, alterations in early-life growth due to differences in hormonal signaling, either through genetic mutation or experimental manipulations, tend to be correlated with extended life span and increased resistance to stress (2, 12, 15, 20).

Our data suggest that cells from long-lived mutant mice may be more resistant to direct DNA damage, whether induced directly by UV light and MMS or perhaps indirectly by exposure to H2O2, paraquat, and cadmium. Such resistance might also contribute to the relative delay in cancer incidence rates in these animals (29). It will be interesting to examine levels of specific forms of DNA damage and repair systems in fibroblasts from long-lived mice and in cells within the intact animal. More work is also needed to define the relationship between cellular stress resistance and proteins such as p53 and mammalian Sir2 homologs that participate in DNA damage response pathways and to proteins that control stress-induced changes in cell cycle regulation, such as Foxo (41, 51, 61, 64, 67).

We should also mention the possibility that fibroblasts isolated from dermis may represent a heterogeneous collection of cells with overlapping properties (55) and that these populations may differ between cells from dw/dw and dw/+ mice. It will take additional studies to determine whether the differences seen between fibroblasts of dwarf and control mice represent changes in all fibroblasts isolated for each sample, or are a result of changes in the relative proportion of cells with stress-resistant properties (4, 18).

Dermal fibroblasts provide convenient in vitro models for cellular and molecular studies but are unlikely to regulate aging and late-life pathology in mammals. It will thus be useful to measure cellular stress resistance in vivo, using systems that evaluate liver function in response to an inducer of oxidative stress such as acetaminophen (1) or the resistance to kainic acid-induced lesions and excitotoxic neurodegeneration of cells within the hippocampus (50). Studies of fibroblasts from long-lived rodents may also provide new insights into the possible role of stress resistance in modulating longevity among closely related species (32, 44).


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 MATERIALS AND METHODS
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This work was supported by National Institutes of Health Grants AG-19989, AG-08808, AG-13283, AG-Z3122, and T32-GM-07315.


    ACKNOWLEDGMENTS
 
We thank Maggie Vergara and Ray Krzesicki for expert technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Miller, Univ. of Michigan Medical School, 1500 E. Medical Center Dr., 5316 CCGC 0940, Ann Arbor MI 48105-0940 (e-mail: millerr{at}umich.edu)

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
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 MATERIALS AND METHODS
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 DISCUSSION
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 REFERENCES
 

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