Dicarbonyl-induced accelerated aging in vitro in human skin fibroblasts

Dicarbonyls glyoxal (GO) and methylglyoxal (MGO) produced during the autoxidation of reducing sugars are a source of macromolecular damage in cells. Since an accumulation of damaged macromolecules is a universal characteristic of aging, we have tested whether GO and MGO which cause oxidative damage to proteins and other macromolecules can bring about accelerated aging in normal human skin fibroblasts in vitro. A treatment of cells with 1.0 mM GO or 400 μM MGO leads to the appearance of senescent phenotype within 3 days, as judged by the following criteria: morphological phenotype, irreversible growth arrest and G₂ arrest, increased senescence-associated β-galactosidase (SABG) activity, increased H₂O₂ level, increased N^ξ-(carboxymethyl)-lysine (CML) protein level, and altered activities of superoxide dismutase and catalase antioxidant enzymes. This experimental model of accelerated cellular aging in vitro can be useful for studies on testing the effects of various physical, chemical and biological conditions, including natural and synthetic molecules, for the modulation of aging.     

Ploidy of human embryonic fibroblasts during in vitro aging

The DNA content of single nuclei in confluent cultures of aging human embryonic fibroblasts, TIG-3, was measured by means of flow cytofluorometry. In the early and late stages of their in vitro lifespan, they had considerable numbers of 4C, 8C and 16C nuclei. In the other period more than 95% of their nuclei were 2C nuclei. These results were confirmed by karyotype analysis. Presumably, the polyploid cells in young cell populations are removed due to their low growth potential and those in old cell populations are accumulated as a result of cellular aging. The saturation density of TIG-3 cells increased at the beginning of their lifespan and thereafter decreased. The rise in their saturation density seems to reflect cell selection advantageous for young diploid cells.     

The cellular aging of rat fibroblasts in vitro is a differentiation process

Cellular aging of diploid rat fibroblasts in vitro occurs as the result of a three-stage differentiation sequence, taking place in the morphologically recognizable differentiating cell compartment of the fibroblast stem cell system. Simultaneously with an age-dependent morphological differentiation there is an age-dependent biochemical differentiation of the fibroblasts. In cell strains with the genetic background Lewis, the cellular differentiation state, the major histocompatibility complex composition (H-1) and the H-1 complex-controlled expression of endogenous type C viruses determine the ultimate fate of the senescent cell, leading either to cellular degeneration or to cellular neoplastic transformation.     

Up-regulation of S100A8 and S100A9 protein in bronchial epithelial cells by lipopolysaccharide

Increased serum levels of the S100A8 (MRP-8) protein have been reported in inflammatory conditions including bacterial infection, arthritis, and cystic fibrosis (CF). This protein is expressed constitutively with S100A9 (MRP-14) in neutrophils and is regulated by inflammatory stimulants. It has been hypothesized that increased inflammatory response to persistent bacterial infection is a major feature of CF lung disease. Therefore, the authors wished to determine the involvement of these two proteins in the innate defense response of the bronchial epithelium to lipopolysaccharide (LPS). Human bronchial epithelial cells (16HBE14o-) and primary bronchial epithelial cells (NHBE) were grown at air-liquid interface (ALI) and stimulated for up to 96 hours with LPS from Pseudomonas aeruginosa. The 16HBE14o- cells responded to LPS with a 2.9-fold increase in S100A8 mRNA production after 12 hours. S100A9 mRNA production was increased by 1.8-fold after 12 hours and 2.9-fold after 24 hours. It was also found that the S100A8 and S100A9 proteins were increased in the secretions of the 16HBE14o- and NHBE cells after LPS stimulation. This finding suggests that S100A8 and S100A9 are involved in the innate defense of the bronchial epithelium.     

Gerontomodulatory and youth-preserving effects of zeatin on human skin fibroblasts undergoing aging in vitro

Our studies have shown that zeatin, (6-[4-hydroxy-3-methyl-but-2-enylamino]adenine), a cytokinin plant growth factor, has gerontomodulatory, youth preserving and anti-aging effects on serially passaged human adult skin fibroblasts undergoing aging in vitro. There were no immediate negative or toxic effects in terms of cell attachment, cell proliferation, cell survival, cytoskeletal organization, and cellular growth by treatment with zeatin concentrations between 1 and 200 microM. During long-term treatment, cells could be maintained throughout their replicative lifespan in the presence of 40, 80, and 200 microM zeatin, but the optimal concentration of zeatin's anti-aging and youth preserving effects was found to be 80 microM. Life-long serial passaging of human skin fibroblasts in the presence of zeatin resulted in the prevention of cell enlargement, reduction of intracellular debris, prevention of actin polymerization, and enhancement of cellular ability to decompose hydrogen peroxide and to cope with ethanol and oxidative stresses. Most importantly, anti-aging and beneficial effects of zeatin were observed without any induction of additional cell proliferation or an increase in the maximum proliferative capacity, thus ruling out any potentially harmful and carcinogenic effects.     

Energetic stress induces premature aging of diploid human fibroblasts (Wi-38) in vitro

Oxidative phosphorylation is the main endogenous source for the generation of reactive oxygen species (ROS). In order to investigate the influence of enhanced ROS production on the in vitro senescence of Wi-38 fibroblasts, cells were cultivated in medium with elevated (hypertonic) NaCl concentrations. The number of active Na(+)/K(+)-ATPase molecules per cell was found to be increased. A rise in both respiration and glycolysis as evidenced by the increases in oxygen and glucose consumption and lactate production was revealed. Cells stayed alive in medium with NaCl concentrations of up to 0.30 M and could be adapted to growth under these hypertonic conditions (high-NaCl tolerant cells). These cells exhibited an increased cell size and protein content. A growing number of cells showed stress fibers and granulation. The proliferation rate and the maximum number of cumulative population doublings of these high-NaCl tolerant cultures were reduced and saturation density was decreased. Thus, these cells under energetic stress due to increased energy requirements for active ion transport expressed features typical for aging in vitro. We conclude therefore that energetic stress induces premature aging in human diploid fibroblasts.     

人高亲和力钠依赖二羧酸转运蛋白3对人成纤维细胞衰老进程的影响

目的 探讨人高亲和力钠依赖二羧酸转运蛋白3(hNaDC3)在人胚肺二倍体成纤维细胞(WI38)衰老中的作用。方法 通过构建逆转录病毒载体．使用含有hNaDC3基因的逆转录病毒液将hNaDC3基因导入WI-38中．并获得表达．观察其对WI-38细胞衰老的影响。结果 与对照细胞相比，hNaDC3基因导入后．WI-38细胞传代数减少10～13代，生长速率降低40％．细胞周期阻滞于G期，细胞形态呈衰老细胞样变化．衰老相关-β-半乳糖苷酶(SA-β-gal)染色阳性率上升-线粒体膜电位下降。结论 hNaDC3可能促进人WI-38衰老。     

Protein variations associated with in vitro aging of human fibroblasts and quantitative limits on the error catastrophe hypothesis

Two-dimensional electrophoresis was used to examine protein alterations associated with in vitro cellular aging. Patterns of cellular proteins from early and late passage human fibroblasts of two strains (normal and trisomy 21) were analyzed in silver-stained gels and autoradiograms with computerized microdensitometry. Four proteins were significantly altered in density in both cell strains. In late passage cells, these proteins were from 6 to 66% the density in early passage cells. The error catastrophe hypothesis predicts that random amino acid substitutions accumulate with cellular aging. No new proteins or satellite spots due to such substitutions, however, were detected in late passage cells. An upper bound of 2.5% was set by high resolution densitometry for the fraction of abnormal protein that could be present but undetected by these methods.     

Abrupt telomere shortening in normal human fibroblasts

Aging is one of the most basic properties of living organisms. Abundant evidence supports the idea that cell senescence underlies organismal aging in higher mammals. Therefore, examining the molecular mechanisms that control cell and replicative senescence is of great interest for biology and medicine. Several discoveries strongly support telomere shortening as the main molecular mechanism that limits the growth of normal cells. Although cultures gradually approach their growth limit, appearance of individual senescent cells is sudden and stochastic. A theoretical model of abrupt telomere shortening has been proposed in order to explain this phenomenon, but until now there was no reliable experimental evidence supporting this idea. Here, we have employed novel methodology to provide evidence for the generation of extrachromosomal circular telomeric DNA as a result of abrupt telomere shortening in normal human fibroblasts. This mechanism ensures heterogeneity in growth potential among individual cells, which is crucial for gradual progression of the aging process.     

Tetrodotoxin-sensitive sodium channels in normal human fibroblasts and normal human glia-like cells.

Tetrodotoxin-sensitive sodium channels are detectable in normal human fibroblasts and in "glia-like" cells at appreciable levels when compared to what is observed in established neuronal cell lines in culture. Two- to 3-fold stimulations of sodium influx are observed in the presence of 0.2 mM veratridine and scorpion venom at 0.1 mg/ml. Tetrodotoxin (2 microM) inhibits the observed stimulation of sodium influx. Previous work has indicated that these neurotoxins act on the voltage-sensitive sodium ionophore of excitable cells, and the presence of such channels in cells generally considered nonexcitable raises questions regarding both the uniqueness of this ionophore as a property of excitable cells and the origin of the cells generally described as fibroblasts.     

Anchorage-independent growth of normal human fibroblasts.

Normal human fibroblasts, considered to be entirely anchorage dependent for proliferation, have been grown in methylcellulose medium. The most important factor required for growth in suspension appears to be the use of high levels of serum and hydrocortisone. Newborn foreskin or fetal lung fibroblasts form colonies as large as 0.5 mm in diameter after 3 wk, with a colony-forming efficiency as high as 70%. Mouse 3T3 cells that do not form colonies in standard assays for anchorage-independent growth also grow under these conditions. Colony formation results after inoculation of as few as 100 cells per 60-mm dish, and metaphase cells have been visualized with a fluorescent DNA stain, showing that colony formation is due to division rather than aggregation. Fibroblasts recovered from suspension and grown as monolayers retain a diploid karyotype and normal shape, do not form tumors upon injection into nude mice, and become senescent. Thus, the trait of anchorage-independent growth in vitro is clearly possessed by normal human fibroblasts and can be expressed under the proper conditions.     

The effect of light on the aging of human diploid fibroblasts

Cultures of human embryonic diploid lung fibroblasts were observed to die within 2–3 days during continual exposure to light. This toxic effect was found to be due to the interaction of light with some of the components in Eagle's medium, particularly between amino acids and some vitamins. A study of the influence of light on the growth rate and aging diploid cells disclosed that a 2 hr daily exposure to a strong light source reduced the growth rate significantly.     

Matrix homeostasis in aging normal human ankle cartilage

OBJECTIVE: To study age-related (as opposed to arthritis-related) changes in collagen and proteoglycan turnover. METHODS: Macroscopically nondegenerate normal ankle cartilage obtained from 30 donors (ages 16-75 years) was processed for in situ hybridization to detect messenger RNA (mRNA) of type IIB collagen (CIIB); antibodies to the C-propeptide of type II collagen (CPII), to the type II collagen (CII) collagenase-generated cleavage neoepitope (Col2-3/4C(short)), and to the CII denaturation product (Col2-3/4m) were used for immunohistochemistry analysis and immunoassay. In addition, immunoblotting was used to detect the 4 collagenases. Assays were also performed to detect glycosaminoglycan (GAG) content and the 846 epitope of aggrecan. RESULTS: There were no significant changes in CII, GAG, and the content of the 846 epitope after the age of 30 years. Both mRNA for CIIB and the CPII were present in all zones, and CPII content did not change significantly with age. While the collagenase-cleaved CII showed a trend to increase with age, the denatured collagen did not. However, the molar ratio of cleaved versus denatured collagen was positively correlated with age. All 4 collagenases were detectable in the ankle cartilage but showed no identifiable changes in content with age. CONCLUSION: Synthesis and degradation of CII is associated with the pericellular matrix and is maintained at a steady state throughout life. The contents of CII and proteoglycan did not change. There was a significant reduction in the denaturation of CII with age, relative to collagenase-mediated cleavage. These observations reveal that, in aging of the intact ankle articular cartilage, there is no evidence of molecular degenerative changes of the kind observed in osteoarthritis, thereby distinguishing aging from the osteoarthritis process.     

Age-associated decrease in proteasome content and activities in human dermal fibroblasts: restoration of normal level of proteasome subunits reduces aging markers in fibroblasts from elderly persons

We measured proteasome activities and the levels of proteasome subunits in dermal fibroblasts from individuals aged 20-82 years. Proteasome activities changed with age in a biphasic manner, decreasing significantly up to 50 years of age and showing no significant change between 50 and 78 years of age. Similarly, proteasome activities in replicatively senescent dermal fibroblasts showed a passage-dependent biphasic change. We confirmed that the decreases in proteasome activities were accompanied by the accumulation of oxidized and ubiquitinated proteins. The decline in proteasome activities in aging fibroblasts was associated with a decrease in the expression of proteasome subunits. We found that the restoration of the normal level of proteasome catalytic subunits, using a lentivirus gene-delivery system, decreased the severity of the aging markers in dermal fibroblasts from elderly donors. These findings suggest that proteasome malfunction may contribute to the aging process in human skin and that the maintenance of normal proteasome activities could delay skin aging.     

A practical preservation method for human diploid fibroblasts in aging research

A practical method for long-term storage of human diploid fibroblasts was investigated. The conditions of increased cell attachment (recovery) and/or viability are as follows; freezing 1?10 × 10⁶ cells/ampoule at a rate of 1°C/min to ?35°C with the culture medium containing 30% FBS and 15% glycerol, thawing the contents rapidly by agitation in a 40°C water bath, and diluting 5-fold by the addition of the growth medium at 15 min after thawing. Consequently, this recommended method shows 78.8% recovery and 90.8% viability, in contrast to 32.9% recovery (82.5% viability) obtained in the conventional method. On the other hand, cells frozen with DMSO as an additive showed 52.9% recovery and 85.9% viability. The preservation of human diploid fibroblasts with glycerol was accordingly preferred to a method with DMSO. This practical method is useful for long-term storage of not only young cells but aged cells for the research on cellular aging.     

Characteristics of cultured lung fibroblasts from bleomycin-treated rats. Comparisons with in vitro exposed normal fibroblasts

Bleomycin is an antineoplastic agent that causes pulmonary fibrosis. This report describes fibroblasts cultured from lung tissue explants of in vivo bleomycin-exposed and control rats. Proliferation of cultured fibroblasts from bleomycin-exposed lungs was decreased compared with that from control cultures, both in rate of growth and maximal cell yield. The diminished proliferative capacity of these fibroblasts from exposed lungs persisted over 9 wk of serial subcultivation. Fibroblast cultures from exposed lungs typically had a greater proportion of large cells exhibiting extensive cytoskeletal structures than did control cultures. Comparisons of these fibroblasts with normal fibroblasts exposed in vitro to bleomycin showed similarities in size, morphologic features, and proliferation.     

Dry mass and area changes in the GM-10 skin fibroblasts during aging in vitro

Normal, human diploid skin fibroblasts (GM-10) were grown in culture and the changes in the mean cellular, nuclear and nucleolar dry mass and area were monitored simultaneously at regular intervals until the last passage. The results which were analyzed statistically showed that all measures increased during aging in vitro with a most striking surge for all values from passage 29 to 31. The area measures of the cell, nucleus and nucleosis were well correlated with the measures of dry mass for the same structures. The results are discussed and possible reasons for the findings are offered.     

Decreased accuracy of protein synthesis in extracts from aging human diploid fibroblasts

The accuracy of protein synthesis has been measured in extracts from human diploid fibroblasts of different ages. Extracts were supplied with purified mRNA for the coat protein of the cowpea variant of tobacco mosaic virus (CcTMV), which lacks codons for cysteine and methionine. The presence of 35S-cysteine in CcTMV coat protein synthesized during translation reactions therefore represents translational error. Translation reactions were performed with extracts from young fibroblasts (less than 50% of life span completed) and old fibroblasts (more than 90% of life span completed), and the translation products were purified by immunoprecipitation and analyzed by polyacrylamide gel electrophoresis. The error frequency increased from 4.2 x 10(-5) cysteines/amino acid in young cell extracts to 2.9 x 10(-4) cysteines/amino acid in old cell extracts. Cysteine incorporation was not due to nonspecific binding, and could be increased approximately sixfold by the addition of the misreading antibiotic, paromomycin. It is concluded that translational accuracy is not stable during aging in vitro, and it is proposed that this decrease in the fidelity of information transfer could be responsible for the variety of changes observed in aging cultured human cells.