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Fifty Years of Skin Aging

      In developed countries, interest in cutaneous aging is in large part the result of a progressive, dramatic rise over the past century in the absolute number and the proportion of the population who are elderly (
      • Smith E.S.
      • Fleischer A.B.
      • Feldman S.R.
      Demographics of aging and skin disease.
      ). The psychosocial as well as physiologic effects of skin aging on older individuals have created a demand for better understanding of the process and particularly for effective interventions.
      Skin aging is a complex process determined by the genetic endowment of the individual as well as by environmental factors. The appearance of old skin and the clinical consequences of skin aging have been well known for centuries, but only in the past 50 y have mechanisms and mediators been systematically pursued. Still, within this relatively short time there has been tremendous progress, a progress greatly enhanced by basic gerontologic research employing immunologic, biochemical, and particularly molecular biologic approaches (Figure 1, Figure 2).

      Keywords

      What is Aging?

      Aging is a process perhaps best defined as decreased maximal function and reserve capacity in all body organs, resulting in an increased likelihood of disease and death. Aging occurs at the level of individual cells and is regarded by many authorities as a cancer prevention mechanism (
      • Campisi J.
      Replicative senescence: an old lives' tale?.
      ), impeding the unregulated growth of cells whose DNA has been progressively damaged over the lifespan by internal and external mutagens.

      Photoaging Versus Intrinsic Aging

      Intrinsic skin aging is characterized primarily by functional alterations rather than by gross morphologic changes in the skin. Of equal or greater clinical importance is photoaging, the superposition of chronic sun damage on intrinsic aging. Photoaging is neither universal nor inevitable and is characterized by often striking morphologic and physiologic changes. Briefly, chronologically aged skin appears dry and pale with fine wrinkles, displaying a certain degree of laxity and a variety of benign neoplasms. In contrast, depending on the individual's skin type and degree of damage, photoaged skin may appear not only dry but also irregularly pigmented and (in darker-skinned persons) sallow, often displaying deep furrows in addition to fine wrinkling, or (in fair-skinned persons) severely atrophic, with multiple telangiectases and a variety of premalignant lesions such as actinic keratoses (reviewed in
      • Yaar M.
      • Gilchrest B.A.
      Skin aging: postulated mechanisms and consequent changes in structure and function.
      ).
      Histologically, the most consistent change of intrinsic cutaneous aging is flattening of the dermal-epidermal junction. Also, there is a progressive decrease in melanocyte and Langerhans cell density. The dermis displays loss of extracellular matrix and increased levels of collagen-degrading metalloproteinases (
      • Varani J.
      • Warner R.L.
      • Gharaee-Kermani M.
      • et al.
      Vitamin A antagonizes decreased cell growth and elevated collagen- degrading matrix metalloproteinases and stimulates collagen accumulation in naturally aged human skin.
      ), loss of fibroblasts and vascular network, and, in particular, loss of the capillary loops that occupy the dermal papillae (
      • Gilchrest B.A.
      • Stoff J.S.
      • Soter N.
      Chronologic aging alters the response to ultraviolet-induced inflammation in human skin.
      ). The characteristic histologic changes of aged skin are associated with decrements in signal transduction pathways that influence protein phosphorylation. Specifically, there is decreased activity of growth factor associated mitogen-activated protein kinases and increased activity of stress-associated kinases (p38 and c-jun-amino-terminal kinase) (
      • Chung J.H.
      • Kang S.
      • Varani J.
      • Lin J.
      • Fisher G.J.
      • Voorhees J.J.
      Decreased extracellular-signal-regulated kinase and increased stress-activated MAP kinase activities in aged human skin invivo.
      ). The main histologic change in photodamaged skin is dermal elastosis, the deposition of abnormal amorphous elastic material in the papillary dermis. Epidermal changes include variability in thickness accompanied by disorganized maturation and some cytologic atypia. Melanocytes are unevenly distributed in the basal layer, displaying areas of increased number of melanocytes and areas with reduced melanocytes. There is also a significant decrease in the number of Langerhans cells (
      • Gilchrest B.A.
      • Murphy G.F.
      • Soter N.A.
      Effect of chronologic aging and ultraviolet irradiation on Langerhans cells in human epidermis.
      ). Photodamaged skin frequently displays abundant inflammatory cells in the dermis, and collagen and elastic fibers show degenerative changes (
      • Lavker R.M.
      • Kligman A.M.
      Chronic heliodermatitis: a morphologic evaluation of chronic actinic dermal damage with emphasis on the role of mast cells.
      ).
      The term photoaging, coined by
      • Kligman L.H.
      Photoaging. Manifestations, prevention and treatment.
      , was a concept rediscovered in the twentieth century. In the late nineteenth century Unna and Dubreuilh, comparing the skin of farmers and sailors to that of indoor workers, recognized the devastating effects of solar irradiation on the appearance of facial skin. This insight was then lost until 1969, when Kligman published his landmark findings on the structural changes that occur in human skin as a result of sun damage, damage separable from the intrinsic aging process of the skin (
      • Kligman A.M.
      Early destructive effect of sunlight on human skin.
      ).
      • Lavker R.M.
      Structural alterations in exposed and unexposed aged skin.
      also described profound structural differences between sun-exposed and sun-protected skin.

      Functional changes

      Early studies attempting to define physiologic changes that accompany aging skin date back to the 1950s and perhaps earlier. Similar to studies examining morphologic and histologic changes in aged skin, however, these studies were confounded by not distinguishing between extrinsic and intrinsic aging processes. More interpretable studies measuring functional decrements that occur in non-sun-exposed skin (Table 1) date from the 1960s and 1970s.
      Despite relatively little change in gross and histologic cutaneous appearance, maximal function and reserve capacity deteriorates with aging. This compromised function is particularly evident after injury. Briefly, functional decrements that are the result of intrinsic aging include slow wound healing due to decreased keratinocyte and fibroblast proliferative capacity (
      • Gerstein A.D.
      • Phillips T.J.
      • Rogers G.S.
      • Gilchrest B.A.
      Wound healing and aging.
      ), reduced cytokine production (
      • Sunderkotter C.
      • Kalden H.
      • Luger T.A.
      Aging and the skin immune system.
      ), decreased fiber synthesis, and delayed recovery of barrier function after damage (
      • Elias P.M.
      • Ghadially R.
      The aged epidermal permeability barrier: basis for functional abnormalities.
      ). Also, the barrier to water loss is more easily disturbed in the elderly compared to young adults, and the time required to reconstitute competent stratum corneum is more than double in the elderly, in part because of decreased lipid synthetic capacity (
      • Ghadially R.
      • Brown B.E.
      • Sequeira-Martin S.M.
      • et al.
      The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model.
      ). Interestingly, female hormones were reported to significantly influence stratum corneum sphingolipid composition, suggesting that age-associated decreases in hormone levels may impact barrier function in the elderly (
      • Denda M.
      • Koyama J.
      • Hori J.
      • Horii I.
      • Takahashi M.
      • Hara M.
      • Tagami H.
      Age- and sex-dependent change in stratum corneum sphingolipids.
      ). Moreover, in women, both bone mass and skin collagen decline rapidly in the immediate postmenopausal years, suggesting that estrogens influence collagen synthesis and degradation in both sites (
      • Castelo-Branco C.
      • Pons F.
      • Gratacos E.
      • et al.
      Relationship between skin collagen and bone changes during aging.
      ).
      In combination with age-associated changes in B and T lymphocyte function (
      • Miller R.A.
      The aging immune system: primer and prospectus.
      ), decreased production of keratinocyte immune cytokines and decreased density of Langerhans cells lead to decreased cutaneous immune responsiveness with aging (
      • Sauder D.N.
      Effect of age on epidermal immune function.
      ). The decreased number of melanocytes is presumed to contribute to reduced protection against ultraviolet (UV) irradiation and to render the elderly more susceptible to UV-induced epidermal DNA damage (
      • Gilchrest B.A.
      ). Furthermore, DNA repair rate is decreased in the elderly, a loss shown to correlate inversely with mutation risk and skin cancer susceptibility (
      • Moriwaki S.
      • Ray S.
      • Tarone R.E.
      • Kraemer K.H.
      • Grossman L.
      The effect of donor age on the processing of UV-damaged DNA by cultured human cells: reduced DNA repair capacity and increased DNA mutability.
      ;
      • Goukassian D.A.
      • Bagheri S.
      • el-Keeb L.
      • Eller M.S.
      • Gilchrest B.A.
      DNA oligonucleotide treatment corrects the age-associated decline in DNA repair capacity.
      ). Indeed, skin cancer incidence increases exponentially with aging independent of insolation (reviewed in
      • Gilchrest B.A.
      ).
      An important endocrinologic function of human epidermis that declines with aging is vitamin D3 production. UV irradiation (< 320 nm) converts the epidermal precursor of vitamin D3, 7-dehydrocholesterol, to previtamin D3, which subsequently isomerizes to form vitamin D3. With aging, the levels of the epidermal precursor of vitamin D3 decrease, contributing to decreased vitamin D3 production in the elderly (
      • MacLaughlin J.
      • Holick M.F.
      Aging decreases the capacity of human skin to produce vitamin D3.
      ) and rendering them susceptible to vitamin D3 deficiency in the absence of regular sun exposure (
      • Gloth 3rd, F.M.
      • Gundberg C.M.
      • Hollis B.W.
      • Haddad Jr, J.G.
      • Tobin J.D.
      Vitamin D deficiency in homebound elderly persons.
      ).
      Changes in vessel wall architecture contribute to vascular fragility, a common clinical occurrence in the elderly. In addition, changes in dermal vascular bed and vascular responsiveness lead to compromised thermoregulation, predisposing the elderly to heat stroke or hypothermia (
      • Wagner J.A.
      • Robinson S.
      • Tzankoff S.P.
      • Marino R.P.
      Heat tolerance and acclimatization to work in the heat in relation to age.
      ;
      • Bruck K.
      Thermoregulation in man in relation to age.
      ). Also, the reduction of vascular network around the hair bulbs and eccrine, apocrine, and sebaceous glands probably contributes to their gradual age-associated atrophy and as a result decreased sweat and sebum production. Decrements in vascular network are probably also responsible for decreased integumental reactivity and attenuated inflammatory response to a variety of stimuli including injurious chemicals and UV irradiation. Changes in microvasculature may also diminish the rate at which substances are cleared from the dermis.
      Finally, decreased perception of light touch, vibratory and corneal sensation, ability to discriminate two points, and spatial acuity have been documented in the elderly (
      • Shimokata H.
      • Kuzuya F.
      Two-point discrimination test of the skin as an index of sensory aging.
      ;
      • Stevens J.C.
      • Patterson M.Q.
      Dimensions of spatial acuity in the touch sense: changes over the life span.
      ). Available data do not allow differentiation between aging changes in skin innervation and altered central perception of stimuli, and both phenomena probably contribute to the age-associated decline in processing outside stimuli.

      Insights into Cellular Gerontology (1950–2000)

      Early tissue culture systems

      Studies by Carrel et al at the beginning of the twentieth century appeared to suggest that cells, once separated from the organism, could be maintained for an unlimited length of time (
      • Carrel A.
      • Burrows M.T.
      Cultivation of adult tissues and organs outside of the body.
      ), although this immortality was eventually found to be an artifact of the then-crude cell culture methodologies. Half a century later, Hayflick (
      • Hayflick L.
      • Moorehead P.S.
      The serial cultivation of human diploid cell strains.
      ;
      • Hayflick L.
      The limited in vitro lifetime of human diploid cell strains.
      ;
      • Hayflick L.
      The cellular basis for biological aging.
      ) showed that normal human diploid fibroblasts derived from fetal lung proliferated in culture only for finite periods of time, laying the foundation for modern cellular gerontology. He convincingly demonstrated that human fibroblasts eventually enter a permanent state of proliferative refractoriness and that the number of divisions before attaining proliferative senescence is inversely proportional to donor age.
      Hayflick's seminal work was later expanded by other investigators throughout the world who demonstrated in a variety of cell types derived from different species, different tissues, and donors of different ages, that normal cells have limited proliferative lifespan. The exception to this rule appeared to be embryonic stem cells and germline cells (
      • Suda Y.
      • Suzuki M.
      • Ikawa Y.
      Mouse embryonic stem cells exhibit indefinite proliferative potential.
      ). It was proposed that before differentiation into somatic cells, stem cells or germ cells possess the machinery required for continuous proliferation, a machinery that is subsequently lost upon their differentiation.

      Cellular and molecular studies of skin aging

      Until the mid-1970s virtually all in vitro aging studies were performed using the system popularized by
      • Hayflick L.
      The limited in vitro lifetime of human diploid cell strains.
      ;
      • Hayflick L.
      The cellular basis for biological aging.
      . In this system, fetal lung fibroblasts are serially passaged under well-defined culture conditions. Early passage “young” cells are compared to late passage “old” cells. Work by
      • Schneider E.L.
      • Mitsui Y.
      The relationship between in vitro cellular aging and in vivo human age.
      , however, identified inconsistencies between the Hayflick model for cell aging and a newer model comparing early passage dermal fibroblasts from young adults versus old adults. In this newer model, cells are permitted to age in the organism and hence better reflect the in vivo aging process. Furthermore, this newer system allowed comparison between chronologic cutaneous aging and photoaging simply by comparing the behavior of cells obtained from photodamaged skin to that of cells obtained from sun-protected skin of the same individual (
      • Gilchrest B.A.
      Relationship between actinic damage and chronologic aging in keratinocyte cultures of human skin.
      ;
      • Gilchrest B.A.
      Prior chronic sun exposure decreases the lifespan of human skin fibroblasts in vitro.
      ). Moreover, it allowed studies of cell types other than fibroblasts, such as keratinocytes and melanocytes, allowing for generalization of the conclusions and more confidence in the results (
      • Rheinwald J.G.
      • Green H.
      Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.
      ;
      • Gilchrest B.A.
      In vitro assessment of keratinocyte aging.
      ;
      • Gilchrest B.A.
      • Vrabel M.A.
      • Flynn E.
      • Szabo G.
      Selective cultivation of human melanocytes from newborn and adult epidermis.
      ;
      • Medrano E.E.
      • Yang F.
      • Boissy R.
      • et al.
      Terminal differentiation and senescence in the human melanocyte: repression of tyrosine-phosphorylation of the extracellular signal-regulated kinase 2 selectively defines the two phenotypes.
      ).
      As in the Hayflick system, it was found that there is an inverse relationship between donor age and the number of population doublings achieved by cultured cells (
      • Martin G.M.
      • Sprague C.A.
      • Epstein C.J.
      Replicative life-span of cultivated human cells. Effects of donor's age, tissue, and genotype.
      ;
      • Schneider E.L.
      • Mitsui Y.
      The relationship between in vitro cellular aging and in vivo human age.
      ). Subsequent publications established that dermal fibroblasts derived from patients with diseases of premature aging, such as progeria, Werner's syndrome, and diabetes, have shorter culture lifespans (fewer cumulative population doublings, CPD) than do cells derived from age-matched controls (reviewed in
      • Stanulis-Praeger B.
      In vitro studies of aging.
      ). Additional evidence of the relevance of culture lifespan to physiologic age was the observation that both keratinocytes and fibroblasts derived from habitually sun-exposed (photoaged) skin undergo fewer CPD prior to senescence than do the same cells derived from sun-protected sites of the same individuals (
      • Gilchrest B.A.
      Relationship between actinic damage and chronologic aging in keratinocyte cultures of human skin.
      ;
      • Gilchrest B.A.
      Prior chronic sun exposure decreases the lifespan of human skin fibroblasts in vitro.
      ). The mechanism(s) of cellular aging and irreversible growth arrest remained unknown, however.
      Keratinocytes, fibroblasts, and melanocytes also display a decreased short-term response to mitogenic stimuli with increasing donor age (
      • Gilchrest B.A.
      In vitro assessment of keratinocyte aging.
      ;
      • Plisko A.
      • Gilchrest B.A.
      Growth factor responsiveness of cultured human fibroblasts declines with age.
      ). At least in the case of normal human fibroblasts there is age-associated decreased responsiveness to epidermal growth factor that can be ascribed to decreased number and density of receptors, as well as to decreased ligand binding, receptor autophosphorylation, and internalization (
      • Reenstra W.R.
      • Yaar M.
      • Gilchrest B.A.
      Effect of donor age on epidermal growth factor processing in man.
      ;
      • Reenstra W.R.
      • Yaar M.
      • Gilchrest B.A.
      Aging affects epidermal growth factor receptor phosphorylation and traffic kinetics.
      ). Keratinocytes derived from adult versus newborn donors also produce significantly less of the immunomodulatory cytokine now termed interleukin-1 (
      • Sauder D.N.
      • Stanulis-Praeger B.M.
      • Gilchrest B.A.
      Autocrine growth stimulation of human keratinocytes by epidermal cell-derived thymocyte-activating factor: implications for skin aging.
      ). Such findings suggest several mechanisms by which aging can compromise skin function.

      Gene expression and aging

      With the advent of recombinant DNA technology, there has been an explosion of data regarding specific mRNA transcripts whose expression changes with age, both in early versus late passage cells and in cells derived from young versus old donors. Although the expression of many genes does not change, some transcript levels decrease with age, and some even increase.
      Of relevance to cutaneous biology, the expression of extracellular matrix proteins and enzymes involved in their degradation appear to be affected with aging. Both in the early/late passage senescent model and the young/old donor model, fibronectin expression increases with age (
      • Smith J.R.
      • Pereira-Smith O.M.
      Altered gene expression during cellular aging.
      ;
      • Kumazaki T.
      • Kobayashi M.
      • Mitsui Y.
      Enhanced expression of fibronectin during in vivo cellular aging of human vascular endothelial cells and skin fibroblasts.
      ). Also, the expression of certain metalloproteinases, collagenase and stromelysin, increases as a function of serial passage (
      • Millis A.J.
      • Hoyle M.
      • McCue H.M.
      • Martini H.
      Differential expression of metalloproteinase and tissue inhibitor of metalloproteinase genes in aged human fibroblasts.
      ;
      • Zeng G.
      • Millis A.J.
      Differential regulation of collagenase and stromelysin mRNA in late passage cultures of human fibroblasts.
      ;
      • Khorramizadeh M.R.
      • Tredget E.E.
      • Telasky C.
      • Shen Q.
      • Ghahary A.
      Aging differentially modulates the expression of collagen and collagenase in dermal fibroblasts.
      ) and donor age (
      • Burke E.M.
      • Horton W.E.
      • Pearson J.D.
      • Crow M.T.
      • Martin G.R.
      Altered transcriptional regulation of human interstitial collagenase in cultured skin fibroblasts from older donors.
      ), whereas the level of the tissue inhibitor of metallo-proteinases (TIMP-1) decreases (
      • Burke E.M.
      • Horton W.E.
      • Pearson J.D.
      • Crow M.T.
      • Martin G.R.
      Altered transcriptional regulation of human interstitial collagenase in cultured skin fibroblasts from older donors.
      ;
      • Khorramizadeh M.R.
      • Tredget E.E.
      • Telasky C.
      • Shen Q.
      • Ghahary A.
      Aging differentially modulates the expression of collagen and collagenase in dermal fibroblasts.
      ). These changes are consistent with the age-associated decrease in dermal thickness (reviewed in
      • Yaar M.
      • Gilchrest B.A.
      Aging of skin.
      ), presumed to reflect a shift in the balance between synthesis and degradation of collagen.
      With regard to genes that participate in cell cycle progression, a coordinated expression of genes that encode nuclear transcription factors, cyclins, and cyclin-dependent kinases (Cdk) takes place during the G1 phase of the cell cycle, and this program is dysregulated during aging and senescence (reviewed in
      • Stein G.H.
      • Dulic V.
      Molecular mechanisms for the senescent cell cycle arrest.
      ). In response to serum stimulation, senescent cells fail to induce some of the genes that are induced in early G1 (immediate early genes), including c-fos that encodes a component of the AP-1 transcription factor (
      • Seshadri T.
      • Campisi J.
      Repression of c-fos transcription and an altered genetic program in senescent human fibroblasts.
      ), and Id1H and Id2H, encoding helix-loop-helix proteins that are thought to act as inhibitors of differentiation (
      • Hara E.
      • Yamaguchi T.
      • Nojima H.
      • Ide T.
      • Campisi J.
      • Okayama H.
      • Oda K.
      Id-related genes encoding helix-loop-helix proteins are required for G1 progression and are repressed in senescent human fibroblasts.
      ). Senescent cells fail to express many genes that are expressed in late G1, including cyclin A, p34 kinase, and the E2F family of transcription factors that induce the expression of genes required for cell proliferation (
      • Stein G.H.
      • Drullinger L.F.
      • Robetorye R.S.
      • Pereira-Smith O.M.
      • Smith J.R.
      Senescent cells fail to express cdc2, cycA, and cycB in response to mitogen stimulation.
      ;
      • Dimri G.P.
      • Hara E.
      • Campisi J.
      Regulation of two E2F-related genes in presenescent and senescent human fibroblasts.
      ). Senescent cells also fail to express several genes whose protein products are required for DNA synthesis, including dihydrofolate reductase, PCNA, and histones. Senescence does not affect the expression of genes encoding Cdk4/6 and Cdk2, c-jun, the other component of the AP-1 transcription factor, c-myc, and c-H-ras. Furthermore, certain genes like cyclin D1 and cyclin E are over-expressed in serum-stimulated senescent cells. These studies suggest that coordinated expression of an entire array of genes is required to enable the cell to progress through the G1 phase of the cell cycle, and that this coordination is compromised during aging.
      The activities of other nuclear proteins that play a crucial role in cell cycle progression are not regulated at the transcription level but rather by the phosphorylated state of the protein. For example, phosphorylation of the retinoblastoma protein (Rb) is a critical event required for cell cycle progression through G1, as phosphorylated Rb releases factors like E2F that are otherwise bound to the hypophosphorylated form of Rb and hence lack transcriptional activity (reviewed in
      • Yaar M.
      Oncogenes and tumor suppressor genes.
      ). In contrast with quiescent cells, senescent cells fail to phosphorylate Rb despite serum stimulation, consistent with their inability to progress through G1 into S (
      • Stein G.H.
      • Beeson M.
      • Gordon L.
      Failure to phosphorylate the retinoblastoma gene product in senescent human fibroblasts.
      ).
      Direct inhibition of cell cycle progression proteins offers another mechanism of regulation. Notably, p21 ubiquitously inhibits cyclin-Cdk complexes (
      • Xiong Y.
      • Hannon G.J.
      • Zhang H.
      • Casso D.
      • Kobayashi R.
      • Beach D.
      p21 is a universal inhibitor of cyclin kinases.
      ;
      • Zhang H.
      • Hannon G.J.
      • Beach D.
      p21-containing cyclin kinases exist in both active and inactive states.
      ). In fibroblasts, p21 transcripts increase gradually with increasing CPD and reach their highest level in early senescence (
      • Noda A.
      • Ning Y.
      • Venable S.F.
      • Pereira-Smith O.M.
      • Smith J.R.
      Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen.
      ). The levels of p21 transcripts and protein decrease to normal levels, however, within 2 mo of attaining senescence. In contrast, the expression of p16, another Cdk inhibitor, does not change with fibroblast CPD level but increases sharply during senescence and appears to remain elevated (
      • Alcorta D.A.
      • Xiong Y.
      • Phelps D.
      • Hannon G.
      • Beach D.
      • Barrett J.C.
      Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts.
      ;
      • Hara E.
      • Smith R.
      • Parry D.
      • Tahara H.
      • Stone S.
      • Peters G.
      Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence.
      ). These studies suggest that the persistent elevation of p21 is essential for entry into senescence, whereas p16 elevation is required to maintain the senescent phenotype.

      Oxidative stress and aging

      Oxidative stress is considered to be a major contributor to the process of aging (reviewed in
      • Harman D.
      Aging: overview.
      ). Oxygen, required for survival of aerobic organisms, readily accepts single electron transfers, generating reactive oxygen species (ROS) such as ·O-2, H2O2, and ·OH that can damage biologic molecules (
      • Davies K.J.
      Oxidative stress: the paradox of aerobic life.
      ). Such damage continuously occurs throughout the lifespan of the organism and contributes to membrane peroxidation, DNA base alterations, single-strand DNA breaks, sister chromatin exchange, DNA-protein cross-links, carbonyl modifications, and loss of sulfhydryl groups in proteins (
      • Stadtman E.R.
      Protein oxidation and aging.
      ). Oxidative stress also hastens replicative senescence of cultured fibroblasts (
      • Chen Q.M.
      • Liu J.
      • Merrett J.B.
      Apoptosis or senescence-like growth arrest: influence of cell-cycle position, p53, 21 and bax in H2O2 response of normal human fibroblasts.
      ;
      • Dumont P.
      • Burton M.
      • Chen Q.M.
      • et al.
      Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast.
      ). Although cellular and mitochondrial defense mechanisms including the antioxidant enzymes glutathione peroxidase, glutathione reductase, and superoxide dismutases have evolved to quench ROS, these antioxidant defense systems are not fully efficient, and hence throughout life cells accumulate molecular oxidative damage, sometimes leading to apoptotic cell death (
      • Stadtman E.R.
      Protein oxidation and aging.
      ;
      • Ames B.N.
      • Shigenaga M.K.
      • Hagen T.M.
      Oxidants, antioxidants, and the degenerative diseases of aging.
      ). Several studies show that there is an age-associated increase in both ROS generation (
      • Sohal R.S.
      • Brunk U.T.
      Mitochondrial production of pro-oxidants and cellular senescence.
      ) and the level of oxidatively damaged proteins (
      • Stadtman E.R.
      Protein oxidation and aging.
      ) and DNA (
      • Ames B.N.
      • Shigenaga M.K.
      • Hagen T.M.
      Oxidants, antioxidants, and the degenerative diseases of aging.
      ;
      • Agarwal S.
      • Sohal R.S.
      DNA oxidative damage and life expectancy in houseflies.
      ). Caloric restriction extends the lifespan in all species examined, including fish, spiders, rats, mice, and primates (
      • Sohal R.S.
      • Weindruch R.
      Oxidative stress, caloric restriction, and aging.
      ). It was speculated that caloric restriction decreases the metabolic rate of the organism, reduces oxidative stress, and hence contributes to lifespan extension. Recent findings in Saccharomyces cerevisiae, however, show that caloric restriction increased the yeast's respiration rate implying that, as ROS are a by-product of respiration, lifespan extension is not the result of decreased ROS but may be the outcome of improved ability to detoxify these free radicals (
      • Lin S.J.
      • Kaeberlein M.
      • Andalis A.A.
      • et al.
      Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration.
      ).

      The role of telomeres in cellular aging

      The Hayflick limit, the finite “lifespan” or number of CPD for cultured human cells (
      • Hayflick L.
      • Moorehead P.S.
      The serial cultivation of human diploid cell strains.
      ;
      • Hayflick L.
      The limited in vitro lifetime of human diploid cell strains.
      ;
      • Hayflick L.
      The cellular basis for biological aging.
      ), implies that aging occurs at the cellular level and is the consequence at least in part of a genetic program. Subsequently, strong correlations were noted between DNA repair capacity and average lifespan in a large number of diverse species (
      • Hart R.W.
      • Setlow R.B.
      Correlation between deoxyribonucleic acid excision-repair and life-span in a number of mammalian species.
      ) and between metabolic rate and average lifespan (
      • Sohal R.S.
      • Allen R.G.
      Relationship between metabolic rate, free radicals, differentiation and aging: a unified theory.
      ). These findings suggested that aging was also in part the consequence of “wear and tear” from both internal and external environmental insults, with aging rate determined by the balance between incurred damage and its repair.
      The first major advance in understanding the mechanism of cellular aging was the demonstration that critical shortening of telomeres underlies proliferative senescence of cultured human cells. First described in eukaryotic cells by
      • Greider C.W.
      • Blackburn E.W.
      Identification of a specific telomere terminal transferase activity in Tetrahymena extracts.
      , telomeres are tandem repeats of short base sequences, …TTAGGG… in mammals, at the end of each chromosome, that are required for chromosome stability.
      Harley et al (1990) observed that increasing CPD of normal human fibroblasts is associated with shortening of their telomeres. Further work established that telomere length of cells obtained directly from tissues in vivo is inversely related to the individual's physiologic age, being shorter in cells derived from older versus younger adults (
      • Harley C.B.
      • Futcher A.B.
      • Greider C.W.
      Telomeres shorten during ageing of human fibroblasts.
      ;
      • Allsopp R.C.
      • Vaziri H.
      • Patterson C.
      • et al.
      Telomere length predicts replicative capacity of human fibroblasts.
      ) and in cells from patients with premature aging syndromes like Werner's and progeria versus age-matched controls (
      • Allsopp R.C.
      • Vaziri H.
      • Patterson C.
      • et al.
      Telomere length predicts replicative capacity of human fibroblasts.
      ;
      • Schulz V.P.
      • Zakian V.A.
      • Ogburn C.E.
      • McKay J.
      • Jarzebowicz A.A.
      • Edland S.D.
      • Martin G.M.
      Accelerated loss of telomeric repeats may not explain accelerated replicative decline of Werner syndrome cells.
      ).
      Interest in the biologic role of telomeres was heightened by the observation (reviewed in
      • Harley C.B.
      • Kim N.W.
      • Prowse K.R.
      • et al.
      Telomerase, cell immortality, and cancer.
      ;
      • Harley C.B.
      • Kim N.W.
      Telomerase and cancer.
      ) that almost all human malignant cells express the reverse transcriptase component of telomerase (hTERT) responsible for maintaining telomere length in germline cells but not normally considered to be expressed in somatic cells (reviewed in
      • Harley C.B.
      Telomere loss: mitotic clock or genetic time bomb?.
      ). This finding suggested that prevention of progressive telomere shortening with each round of cell division was necessary for dysregulated cells to divide indefinitely and hence become sufficiently numerous to harm the host. This finding was expanded by the report that transfecting normal human cells (fibroblasts and retinal pigment epithelial cells) with hTERT appeared to immortalize them, increasing their in vitro lifespan at least several fold (
      • Jiang X.R.
      • Jimenez G.
      • Chang E.
      • et al.
      Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype.
      ). Further studies determined that in other cell types, such as keratinocytes, immortalization requires ablation of the Rb/p16 protein pathway, in addition to expression of telomerase activity (
      • Kiyono T.
      • Foster S.A.
      • Koop J.I.
      • McDougall J.K.
      • Galloway D.A.
      • Klingelhutz A.J.
      Both Rb/p16INK4a inactivation and telomerase activity are required to immortalize human epithelial cells.
      ). Importantly, hTERT expression did not alter the cells' behavior, only their proliferative capacity (
      • Jiang X.R.
      • Jimenez G.
      • Chang E.
      • et al.
      Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype.
      ). Transfected cells remained appropriately responsive to environmental signals and did not display characteristics of transformed cells, confirming the prediction that unlimited growth potential is necessary but not sufficient for malignant conversion.
      Interestingly, although the current dogma states that normal somatic cells do not express telomerase, telomerase activity is found in vitro in normal epidermal keratinocytes (
      • Yasumoto S.
      • Kunimura C.
      • Kikuchi K.
      • et al.
      Telomerase activity in normal human epithelial cells.
      ). Telomerase activity is also detected in vivo in normal human epidermis, primarily in the proliferative basal layer, but not in the dermal compartment of the skin or in cultured fibroblasts (
      • Harle-Bachor C.
      • Boukamp P.
      Telomerase activity in the regenerative basal layer of the epidermis in human skin and in immortal and carcinoma-derived skin keratinocytes.
      ). During the growth phase of human hair follicles, telomerase activity is detected in the bulb, the portion of the hair follicle that contains rapidly multiplying cells, and its activity in this part of the hair follicle is higher than its activity in hair follicle stem cells located in the bulge area (
      • Ramirez R.D.
      • Wright W.E.
      • Shay J.W.
      • Taylor R.S.
      Telomerase activity concentrates in the mitotically active segments of human hair follicles.
      ). These findings suggest that telomerase expression may not be restricted to stem cells, germline cells, and malignant cells although its role in normal tissues remains to be elucidated.
      In tandem with work elucidating the role of telomeres and telomerase in aging and malignancy,
      • Yu C.E.
      • Oshima J.
      • Fu Y.H.
      • et al.
      Positional cloning of the Werner's syndrome gene.
      identified the gene defective in Werner's syndrome, a recessively inherited classic premature aging syndrome in which development is relatively normal but many features of aging, including malignancies, become apparent in early to mid-adulthood and death usually occurs in the 40s or 50s. Although the precise in vivo function of the Werner's syndrome gene product is largely unknown, it is a DNA helicase/exonuclease and is thought to play a role in DNA replication and recombination during cell division and DNA repair (
      • Gray M.D.
      • Shen J.C.
      • Kamath-Loeb A.S.
      • et al.
      The Werner syndrome protein is a DNA helicase.
      ;
      • Huang S.
      • Li B.
      • Gray M.D.
      • Oshima J.
      • Mian I.S.
      • Campisi J.
      The premature ageing syndrome protein, WRN, is a 3′ 5′ exonuclease.
      ;
      • Shen J.C.
      • Gray M.D.
      • Oshima J.
      • Kamath-Loeb A.S.
      • Fry M.
      • Loeb L.A.
      Werner syndrome protein. I. DNA helicase and dna exonuclease reside on the same polypeptide.
      ;
      • Fry M.
      • Loeb L.A.
      Human Werner syndrome DNA helicase unwinds tetrahelical structures of the fragile X syndrome repeat sequence d (CGG) n.
      ). The Werner's syndrome protein in particular unwinds G-rich DNA structures (
      • Wyllie F.S.
      • Jones C.J.
      • Skinner J.W.
      • et al.
      Telomerase prevents the accelerated cell aging of Werner syndrome fibroblasts.
      ;
      • Brosh Jr, R.M.
      • Majumdar A.
      • Desai S.
      • Hickson I.D.
      • Bohr V.A.
      • Seidman M.M.
      Unwinding of a DNA triple helix by the Werner and Bloom syndrome helicases.
      ) and has recently been implicated in telomere maintenance (
      • Hisama F.M.
      • Chen Y.H.
      • Meyn M.S.
      • Oshima J.
      • Weissman S.M.
      WRN or telomerase constructs reverse 4-nitroquinoline 1-oxide sensitivity in transformed Werner syndrome fibroblasts.
      ;
      • Ouellette M.M.
      • McDaniel L.D.
      • Wright W.E.
      • Shay J.W.
      • Schultz R.A.
      The establishment of telomerase immortalized cell lines representing human chromosome instability syndromes.
      ). Recently, it was reported that patients with dyskeratosis congenita, a second condition characterized by premature graying of hair and cancer predisposition, have markedly shorter telomeres than normal individuals (
      • Vulliamy T.J.
      • Knight S.W.
      • Mason P.J.
      • Dokal I.
      Very short telomeres in the peripheral blood of patients with X-linked and autosomal dyskeratosis congenita.
      ). Finally, in human skin specimens derived from donors of different ages, an inverse relationship was found between telomere length and donor age (
      • Friedrich U.
      • Griese E.
      • Schwab M.
      • Fritz P.
      • Thon K.
      • Klotz U.
      Telomere length in different tissues of elderly patients.
      ).
      Throughout the 1990s, data implying interrelatedness of telomeres, aging, cancer, DNA damage, and DNA damage responses continued to accumulate. Most compelling were a series of reports demonstrating substantial overlap in the proteins mediating proliferative senescence, reversible cell cycle arrest, apoptosis, and DNA repair. For example, the p53 tumor suppressor protein and transcription factor, first identified in the late 1970s as a cellular protein that binds the SV40 large T antigen (
      • Lane D.P.
      • Crawford L.V.
      T antigen is bound to a host protein in SV40-transformed cells.
      ;
      • Lane D.P.
      • Crawford L.V.
      The complex between simian virus 40 T antigen and a specific host protein.
      ;
      • Crawford L.V.
      • Lane D.P.
      • Denhardt D.T.
      • Harlow E.E.
      • Nicklin P.M.
      • Osborn K.
      • Pim D.C.
      Characterization of the complex between SV40 large T antigen and the 53K host protein in transformed mouse cells.
      ), was found to be upregulated immediately before cells entered the senescent state (reviewed in
      • Stein G.H.
      • Dulic V.
      Molecular mechanisms for the senescent cell cycle arrest.
      ). The p53-regulated protein p21, centrally involved in DNA damage responses (
      • Stein G.H.
      • Dulic V.
      Molecular mechanisms for the senescent cell cycle arrest.
      ), was independently identified as playing a major role in proliferative senescence in the absence of DNA damage (
      • Noda A.
      • Ning Y.
      • Venable S.F.
      • Pereira-Smith O.M.
      • Smith J.R.
      Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen.
      ), leading to its alternative designation as the senescent cell-derived (growth) inhibitor or SDI-1. Furthermore, it was noted that over-expression of proto-oncogenes such as ras and raf may induce proliferative senescence rather than increase cellular proliferation (
      • Serrano M.
      • Lin A.W.
      • McCurrach M.E.
      • Beach D.
      • Lowe S.W.
      Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a.
      ;
      • Zhu J.
      • Woods D.
      • McMahon M.
      • Bishop J.M.
      Senescence of human fibroblasts induced by oncogenic Raf.
      ). Interestingly, E2F1, a transcription factor that regulates cell cycle progression, can induce senescence in human fibroblasts when over-expressed in the presence of p53 (
      • Dimri G.P.
      • Itahana K.
      • Acosta M.
      • Campisi J.
      Regulation of a senescence checkpoint response by the E2F1 transcription factor and p14 (ARF) tumor suppressor.
      ). In addition, E2F1 together with p53 can induce apoptosis (
      • Wu X.
      • Levine A.J.
      p53 and E2F-1 cooperate to mediate apoptosis.
      ;
      • DeGregori J.
      • Kowalik T.
      • Nevins J.R.
      Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes.
      ), suggesting that, depending on the cell type and specific experimental conditions, certain proteins may induce apparently the antithecal cellular behaviors of growth versus proliferative senescence or programmed cell death. These combined observations have led to the concept that aging, or at least proliferative senescence, is a fundamental cancer prevention mechanism in multicellular organisms, employing many of the same gene products as DNA repair (
      • Campisi J.
      Replicative senescence: an old lives' tale?.
      ). Interesting in vivo observation consistent with this concept is the report that mice with amplified p53 activity as a result of a mutated p53 gene display signs of premature aging including decreased lifespan, premature hair graying, and cutaneous atrophy, as well as osteoporosis and internal organ atrophy (
      • Tyner S.D.
      • Venkatachalam S.
      • Choi J.
      • et al.
      p53 mutant mice that display early ageing-associated phenotypes.
      ), in addition to the anticipated decrease in cancer incidence.
      How or why telomere shortening might induce genes also involved in DNA damage responses is at present the subject of intense investigation. The de Lange group has reported that disrupting the telomere loop structure by ectopic expression of a dominant negative form of the telomeric repeat binding factor 2 (TRF2DN) induces p53 and, in at least one cell type, the classic DNA damage response of apoptosis (
      • Karlseder J.
      • Broccoli D.
      • Dai Y.
      • de Hardy S.
      • Lange T.
      p53 and ATM-dependent apoptosis induced by telomeres lacking TRF2.
      ), and in another cell type a senescent phenotype (
      • Van Steensel B.
      • de Smogorzewska A.
      • Lange T.
      TRF2 protects human telomeres from end-to-end fusions.
      ). Eller has observed that supplementing cultured cells with oligonucleotides substantially or completely homologous to the telomere 3′ overhang sequence TTAGGG also induces and activates p53 and generally results in cell behaviors observed after DNA damage (
      • Eller M.S.
      • Puri N.
      • Hadshiew I.M.
      • Venna S.S.
      • Gilchrest B.A.
      Induction of apoptosis by telomere 3′ overhang-specific DNA.
      ;
      • Eller M.S.
      • Li G.Z.
      • Firoozabadi R.
      • Puri N.
      • Gilchrest B.A.
      Induction of p95/NBS1-mediated S-phase checkpoint by telomere 3′ overhang specific DNA.
      ). In combination, these findings suggest that an as yet unidentified nuclear sensor of this single stranded DNA sequence, which is normally concealed in the telomere loop, may be responsible for initiating these molecular pathways. Telomere loop disruption may occur after acute DNA damage, TRF2DN expression, or critical telomere shortening, all leading to exposure of the 3′ overhang (Li et al, in press) (Figure 3). Furthermore, this model provides a molecular connection between photoaging (UV-induced DNA damage) and chronologic aging (telomere shortening through replication), both pathways leading to telomere destabilization and exposure of the 3′ overhang. Of interest, in fibroblasts these oligomers and telomere loop disruption also induce the same gene products as serial passage and critical telomere shortening, leading to a senescent phenotype, allowing for the speculation that critically short telomeres may be stochastically unstable and hence prone to disruption of the loop structure with consequent exposure of the TTAGGG signal (
      • Eller M.S.
      • Puri N.
      • Hadshiew I.M.
      • Venna S.S.
      • Gilchrest B.A.
      Induction of apoptosis by telomere 3′ overhang-specific DNA.
      ;
      • Eller M.S.
      • Li G.Z.
      • Firoozabadi R.
      • Puri N.
      • Gilchrest B.A.
      Induction of p95/NBS1-mediated S-phase checkpoint by telomere 3′ overhang specific DNA.
      )
      Figure thumbnail gr1
      Figure 1Timeline for skin aging research. In the 1960s Kligman published his findings on structural changes in human skin as a result of sun damage, distinguishing between chronologic aging and photoaging. A succession of reports have later described and detailed the structural and functional changes that accompany chronologic aging and contrasted them with photoaging. In the mid-1960s and through the mid-1980s, the structural and functional changes that accompany chronologic aging and photoaging were described. With the advent of molecular biology methodologies in the 1970s, numerous studies investigated changes in gene expression that are the result of aging, with interesting observations on age-associated alterations in the expression of genes encoding extracellular matrix proteins and enzymes involved in their degradation. Also, changes in the expression of genes whose protein products participate in cell cycle regulation were well documented. In the 1980s and 1990s, with the projection for a dramatic increase in the number of elderly people in the population, numerous studies focused on possible interventions in the aging process. Caloric restriction is probably the most convincing approach to date to slow aging in general, and topical retinoic acid the innovative approach to reverse cutaneous photoaging. Later in the 1990s, Voorhees and colleagues reported the molecular basis of acute photodamage responses. The pathways by which retinoids antagonize the response were demonstrated as well. During this period molecular mechanisms that underlie the aging/photoaging process were described as well.
      Figure thumbnail gr2
      Figure 2Timeline for cellular aging research. In the early 1960s Hayflick was the first to show that normal human diploid fibroblasts derived from fetal lung have a finite proliferative capacity. His studies were later confirmed and expanded by many investigators who demonstrated that all normal cells have limited proliferative lifespan. In the late 1970s correlations have been demonstrated between species lifespan, which is relatively constant among individuals of the same species, and the ability to repair DNA damage. Although first described in the mid-1980s, major interest in telomeres as the molecular basis for aging began in the 1990s with the observation that telomeres shorten during cellular lifespan, that immortal cells that have telomerase activity have increased in vitro lifespan, and that cells derived from individuals with progeroid syndromes display accelerated telomere shortening, suggesting that telomeres represent a biologic “clock” that determines the lifespan of the cell. Very recently, it was demonstrated that genes positioned near a telomere could be silenced as telomeres become shorter. Although the story is still evolving, it is possible that in the future studies will identify those pathways that are activated or silenced by critically short telomeres to provide a better understanding of the complex universal process of aging.
      Figure thumbnail gr3
      Figure 3Proposed mechanism for induction of senescence, cell cycle arrest, differentiation, or apoptosis by exposure of the single-stranded telomere DNA sequence. The 3′ telomere overhang is normally sequestered within a loop structure stabilized by TRF2. Destabilization of this loop structure by DNA damage due to UV irradiation or chemical adducts, expression of TRF2DN, or gradual erosion during aging is hypothesized to expose this single-stranded DNA (repeats of TTAGGG). Displacement of the TRF2 protein as shown in the figure might or might not accompany loop disruption under physiologic conditions. This single-stranded DNA is then detected by an as yet unidentified sensing mechanism. Interaction of this sensor with the 3′ overhang initiates a cascade of events. Depending on cell type and/or intensity and duration of the signal, these events might lead to cell cycle arrest, the eventual induction of senescence, differentiation, or apoptosis. In the present experiments, we hypothesize that DNA oligonucleotides homologous to the overhang sequence are recognized by the same sensing mechanism, triggering the cascade in the absence of telomere disruption.
      One apparent anomaly in the accumulating evidence that aging results in some way from telomere shortening was the fact that the telomeres of mice, a widely used animal model with a relatively short lifespan (approximately 2–4 y), are far longer than human telomeres and do not shorten appreciably (as a percentage of initial length) over the murine lifespan (
      • Kipling D.
      • Cooke H.J.
      Hypervariable ultra-long telomeres in mice.
      ;
      • Starling J.A.
      • Maule J.
      • Hastie N.D.
      • Allshire R.C.
      Extensive telomere repeat arrays in mouse are hypervariable.
      ). To further investigate this apparent exception to the rule, Greider, DePinho and colleagues created telomerase knock-out mice and observed the animals through multiple in-bred generations (
      • Blasco M.A.
      • Lee H.W.
      • Hande M.P.
      • Samper E.
      • Lansdorp P.M.
      • DePinho R.A.
      • Greider C.W.
      Telomere shortening and tumor formation by mouse cells lacking telomerase RNA.
      ;
      • Rudolph K.L.
      • Chang S.
      • Lee H.W.
      • Blasco M.
      • Gottlieb G.J.
      • Greider C.
      • DePinho R.A.
      Longevity, stress response, and cancer in aging telomerase-deficient mice.
      ). The first and second generation animals (G1, G2) appeared completely normal, but the G3-G6 mice developed gray hair and hair loss as early as 6 mo of age and delayed wound healing or chronic ulcers at 15–18 mo of age (
      • Rudolph K.L.
      • Chang S.
      • Lee H.W.
      • Blasco M.
      • Gottlieb G.J.
      • Greider C.
      • DePinho R.A.
      Longevity, stress response, and cancer in aging telomerase-deficient mice.
      ). G4-G6 mice also developed spontaneous tumors at a younger age and had shortened lifespans, phenomena suggestive of premature aging. Although later generations could not be studied because the G6 mice were infertile, telomerase null mice displayed a 4–5 kb decrease in telomere length per generation. These findings strongly suggest that, whereas lifespan in mice must normally be regulated differently than in man, critical telomere shortening, if achieved experimentally over several generations by inactivating telo-merase in germline cells, ultimately has the same consequences as in human cells.
      Caloric restriction, defined as reduction by more than half an animal's ad libitum food intake, had been observed to increase lifespan and delay onset of age-associated diseases in many species, including mammals (reviewed in
      • Lane M.A.
      • Ingram D.K.
      • Roth G.S.
      Nutritional modulation of aging in nonhuman primates.
      ). Although the mechanism of this effect is poorly understood, caloric restriction was widely presumed to act through reduction in oxidative damage that is secondary to ROS generated during cellular metabolism (
      • Lass A.
      • Sohal B.H.
      • Weindruch R.
      • Forster M.J.
      • Sohal R.S.
      Caloric restriction prevents age-associated accrual of oxidative damage to mouse skeletal muscle mitochondria.
      ;
      • Lee C.K.
      • Klopp R.G.
      • Weindruch R.
      • Prolla T.A.
      Gene expression profile of aging and its retardation by caloric restriction.
      ;
      • Zainal T.A.
      • Oberley T.D.
      • Allison D.B.
      • Szweda L.I.
      • Weindruch R.
      Caloric restriction of rhesus monkeys lowers oxidative damage in skeletal muscle.
      ). Employing a yeast model,

      Li GZ, Eller MS, Firoozabadi R, Gilchrist BA: Evidence that Exposure of the Felomere 30 Ovehang Sequence Induces Senescence. Proc Natl Acad Sci, in press

      showed that lifespan extension by caloric restriction requires the presence of Sir2, an rDNA silencing protein, and nicotinamide adenine dinucleotide (NAD) that is necessary for Sir2 function. Lifespan extension of the yeast as a result of Sir2 activation is most probably due to decreased rDNA recombination and hence decreased senescence inducing extrachromosomal rDNA circles (
      • Sinclair D.A.
      • Guarente L.
      Extrachromosomal rDNA circles–a cause of aging in yeast.
      ). Also, as noted above, caloric restriction in this model increased the yeast respiration rate, suggesting that mechanism(s) other than decreased ROS production give rise to extended lifespan in this model (
      • Lin S.J.
      • Kaeberlein M.
      • Andalis A.A.
      • et al.
      Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration.
      ). It is possible that Sir2 encodes proteins that have the capacity to quench ROS. Such proteins have not been identified to date, however. Although the extrapolation to higher organisms is not intuitively obvious, this work suggests molecular links between aging, oxidative metabolism, and genomic instability that might well apply to mammalian cells as well as to yeast. Very recently, using human HeLa cells, Wright, Shay, and colleagues provided compelling evidence that genes positioned near a telomere can be silenced, demonstrating a 10-fold variation in expression of a reporter gene depending on both its proximity to the telomere and telomere length (
      • Baur J.A.
      • Zou Y.
      • Shay J.W.
      • Wright W.E.
      Telomere position effect in human cells.
      ). These experiments offer an explanation for the selectively altered gene expression patterns observed in late passage or old donor cells and again tie telomere length to the aging phenotype, with the caveat that aging is a complex process that occurs in nondividing cells as well as in proliferative ones and hence may be incompletely modeled by cultured cells (
      • Rubin H.
      Cell aging in vivo and in vitro.
      ).

      Summary and Conclusions

      Skin aging has been well characterized clinically, histologically, and functionally. As in other organ systems, skin aging has been shown to have an intrinsic component and a variable environmental component termed photoaging. Since Hayflick's in vitro aging studies in the early 1960s, there has been an explosion of information regarding molecular mechanisms that regulate these aging processes. Aging is now conceptualized as a cellular safeguard against malignant transformation and aging rate as a balance between the rate at which DNA damage is incurred and repaired, with many of the same gene products mediating both DNA damage responses and proliferative senescence. Telomeres appear to play a central role. These observations suggest that, in skin as in all tissues, a common signaling pathway is activated both by critical telomere shortening after multiple rounds of cell division and by repeated environmentally induced DNA damage to yield the aging phenotype.

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