Aging Aging

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28 Cristofalo, Volker, and Allenskin fibroblasts with growth promoters can extend the proliferative life of culturesestablished from young donors but has negligible effects on cultures establishedfrom older donors (79). Aside from inherent species differences and theeffects of inbreeding that may influence these results, it is also apparent thatrodent skin is better protected from some types of environmental injury such aslight exposure. However, even in rodents, the relationship between donor ageand proliferative potential is not entirely clear. For example, an examination ofhamster skin fibroblast cultures established from the same donors at differentages reveals no age-associated changes in proliferative potential in animalsolder than 12 mo (78).To address these issues, we recently examined the proliferative potential of124 human fibroblast cell lines from the Baltimore Longitudinal Study of <strong>Aging</strong>(BLSA) (80). All of these cell lines were established from donors described ashealthy, at the time the biopsy was taken, using the criteria of the BLSA. Thisstudy revealed no significant change in proliferative potential of cell lines withdonor age, nor did we observe a significant difference between fetal and postnatallyderived cultures (80). Goldstein et al. (68) also reported that no relationshipbetween proliferative life-span and donor age could be found in healthydonors but did observe a relationship in diabetic donors. In addition, we performeda longitudinal study by determining the replicative life-span of celllines established from individuals sampled sequentially at different ages. As inthe case of the cross-sectional analysis, no relationship between donor age andreplicative potential was found in this longitudinal study. Indeed, cell linesestablished from individuals at older ages frequently exhibited a slightly greaterproliferative potential than the cell lines established from the same individualsat younger ages (80).1.2.1. Relationship of In Vitro and In Vivo ModelsOne of the underlying assumptions of in vitro aging models is that thechanges observed during proliferative senescence bear at least some homologyto those observed during aging in vivo. In fact, both similar (concordant) anddissimilar (discordant) changes have been observed between aging-associatedchanges observed in vivo and in vitro; these are summarized in Table 1.Although the results presented in Table 1 clearly demonstrate that somesimilarities do exist between aging in vivo and replicative senescence, itremains unclear whether these changes arise through a common mechanism orvia distinct pathways. As seen in Table 1, senescence in tissue culture andaging in the intact organism are not homologous. Others have noted that progressivemorphological changes begin to develop in diploid cell cultures shortlyafter they are established regardless of the donor age; no cells are found in vivo
Fibroblast Model for Cell Senescence Studies 29Table 1<strong>Aging</strong> in Cell Culture Replicative Senescence vs Donor AgeConcordant featuresDiscordant featuresCollagenase (↑) a (81,82) c-fos induction (↓) (20,83,84)Stromelysin (↑) (85) EPC-1 mRNA (↓) (86,87)PAI-1 (↑) (88,89) H-twist mRNA (↓) (90;unpublished)IGF-BP3 (↓) (91) G-6-PDH mRNA (=) b (54,92)TIMP-1 (↓) (85) Fibronectin (↑) (93)HSP 70 (↓) (94,95) ND-4 mRNA (↑) (96,97)Response to Ca 2+ (↓) (98,99) p21 mRNA (↑) (100,101)MnSOD mRNA (↑?) (102,103)β-Galactosidase (↑) (Cristofalo,unpublished)Chemiluminescence (↑) (54,96)H 2 O 2 Generation (↑) (54,96)Collagen a(1)I mRNA (↓) (100,104,105)Proliferative capacity (↓) (80)Saturation density (↓) (80)a Arrow indicates direction of change in replicative senescence.bIndicates no change.G-6-PDH, glucose-6-phosphate dehydrogenase; HSP 70, heat shock protein 70; IGF-BP3,insulin-like growth factor binding protein-3; PAI-1, plasmogen activator inhibitor-1; SOD,superoxide dismutase; TIMP-1, tissue inhibitor of metalloproteinase-1.at any age that exhibit the morphological phenotype of cells, in vitro, at the endof their replicative life-span (106).Rubin (76) suggests that the limited replicative life-span in vitro may be anartifact that reflects the failure of diploid cells to adapt to the trauma of dissociationand the radically foreign environment of cell culture. However, that hypothesisignores factors such as telomere shortening that appear to influenceproliferative life and that are not dependent on the culture environment. Presently,it is possible to state that the loss of proliferative potential in vitro doesnot directly reflect changes in replicative capacity that occur in vivo during agingand that changes in gene expression associated with replicative senescence arenot completely homologous with changes observed during aging in vivo.1.2.2. Relationship Between Senescence and DevelopmentOne view of the limited proliferative capacity of cells in culture is that itstems from the effects of the culture environment on the state of differentiationof the cells (107–113). Although the state of differentiation may change in
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Page 22 and 23: 24 Cristofalo, Volker, and Allenmen
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Page 28 and 29: 30 Cristofalo, Volker, and AllenTab
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Page 52 and 53: 54 Pawelecdiluted cells on an irrad
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Detection of Molecular Events 79Fig
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Detection of Molecular Events 814.
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Detection of Molecular Events 8310.
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86 Kirk and Millerexperiments on un
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88 Kirk and MillerFig. 1. (A) Analy
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94 Kirk and Miller4. Run samples on
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96 Kirk and Miller16. Lange-Carter,
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98 Engel, Adibzadeh, and PawelecPCR
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100 Engel, Adibzadeh, and Pawelec5.
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102 Engel, Adibzadeh, and PawelecTa
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Xenobiotic-Metabolizing Enzymes 119
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Assessing Antioxidant Status 1339As
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Assessing Antioxidant Status 137Tab
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Assessing Antioxidant Status 139add
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Assessing Antioxidant Status 14121.
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Comet Assay of DNA Damage 14310Meas
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Comet Assay of DNA Damage 145contro
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Comet Assay of DNA Damage 1476. Lys
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Comet Assay of DNA Damage 1494. Gen
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Comet Assay of DNA Damage 151anode
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160 van der Schanswithin 1 h, after
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162 van der Schans10. High-binding
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164 van der Schans3. Neutralize aft
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166 van der SchansThe calculations
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8-oxoguanine Levels in Nuclear DNA
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Mutation and the Aging Process 1791
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Mutation and the Aging Process 181m
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Mutation and the Aging Process 183b
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190 HouThe HPRT mutational spectrum
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Susceptibility of LDL to Oxidation
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Analysis of Pentosidine 20916Measur
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Analysis of Pentosidine 211Fig. 2.
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Analysis of Pentosidine 213Fig. 4.
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Analysis of Pentosidine 2152. Preci
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Analysis of Pentosidine 2177. Nagar
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222 Miquellipoperoxides and malonal
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224 Miquelat 4°C. Protect this sol
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226 Miquel3.1. Animal AnesthesiaMed
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228 Miquelsteps to carry out a conv
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230 Miquelsuspending it over the so
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232 Miquelcontrast, washing with bu
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234 MiquelFig. 3. Electron microsco
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Damage to Mitochondria 23718Causes
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Damage to Mitochondria 239Fig. 1. E
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Damage to Mitochondria 2412. To obt
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Damage to Mitochondria 243synthesis
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Mitochondrial DNA Mutations 24519An
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Mitochondrial DNA Mutations 247F.P.
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Mitochondrial DNA Mutations 249desi
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Mitochondrial DNA Mutations 2514. M
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Mitochondrial DNA Mutations 253Fig.
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Mitochondrial DNA Mutations 2593. P
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Mitochondrial DNA Mutations 261X-10
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Mitochondrial DNA Mutations 26315.
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Mitochondrial DNA Mutations 267Tabl
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Mitochondrial DNA Mutations 269muta
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Mitochondrial DNA Mutations 2733.2.
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Mitochondrial DNA Mutations 275that
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Mitochondrial DNA Mutations 27711.
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282 BeckmanWhatever the mechanism e
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284 BeckmanT-cell adherence to vasc
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286 BeckmanT3 hybridoma cells satis
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NK Cell Function in Aging 31123Age-
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NK Cell Function in Aging 3135. Pet
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NK Cell Function in Aging 31518. Io
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NK Cell Function in Aging 3173.3. C
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NK Cell Function in Aging 3199mL of
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Immunogenetics and Life-Span 32124I
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Immunogenetics and Life-Span 349to
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Genetically Engineered Mice 36126Th
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Aging Aging

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