Changes in the cell surface of human diploid fibroblasts during cellular aging

The electrophoretic mobility of 13 human diploid cell strains, TIG-1, TIG-2, TIG-3, TIG-7, WI-38, IMR-90, MRC-5, MRC-9, TIG-1H, TIG-1L, TIG-2M, TIG-2B, and TIG-3S, which were established from different tissues of human embryos, was studied at different passages. The net negative surface charge of the cells was characteristic for each cell strain decreased significantly during the in vitro aging of the cells. The decrease in the net negative charge of the cells correlated well with the decrease in cell density throughout the life span of the cells. A strict linear correlation between the electrophoretic mobility and the number of cells harvested at each passage was obtained for all the human diploid cell strains. Moreover, almost of the same linear regression coefficient of the cells was obtained among these cell strains. Therefore, the net negative surface charge of human diploid cell strains could serve as a cell surface marker for in vitro cellular aging.     

Changes in the migratory ability of human lung and skin fibroblasts during in vitro aging and in vivo cellular senescence.

The migration of human lung and skin fibroblasts was determined during in vitro aging and in vivo cellular senescence by measuring their migration from the edge of a denuded area of a monolayer. The migration of human fetal lung fibroblasts (TIG-1 and TIG-3) decreased only very slightly with increasing passage, whereas the migration of human fetal skin fibroblasts (TIG-3S) declined gradually: the difference in cell migratory ability between early and late passages was significant (P less than 0.05). The migratory patterns of skin fibroblasts from adult and elderly donors were also similar to that of fetal skin fibroblasts. Next, the migratory abilities of fibroblast lines from adult and elderly donor groups were compared, using relatively early passaged cells. The migratory ability of the elderly-donor skin fibroblast lines was significantly lower (P less than 0.05) than that of the adult-donor skin fibroblast lines. Addition of suramin and monensin suppressed the migration of fibroblasts from fetal, adult and elderly donors, which implies that fibroblast migration is regulated by growth factors and matrix substances. The relationships between the age-dependent decline of migratory ability, growth factors and the extracellular matrix are discussed.     

Changes in cell-surface glycosaminoglycans in human diploid fibroblasts during in vitro aging

Changes in glycosaminoglycans during in vitro aging were investigated in human diploid fibroblasts. The cells were found to produce predominantly hyaluronate and smaller amounts of chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and heparan sulfate. Accumulation of heparan sulfate on the cell surface was notable during aging. Total glycosaminoglycan production in preconfluent culture did not change with population doubling level (PDL), while in confluent culture a decline in glycosaminoglycan production was observed. In contrast with this, heparan sulfate on the cell surface increased as a function of PDL in both confluent and preconfluent cultures. The distribution pattern of heparan sulfate in medium and cell surface indicated that the increase in heparan sulfate on the cell surface could be attributed to an increased accumulation on the cell surface, but not to an elevated production. Thus, we conclude that the increased accumulation of heparan sulfate on the cell surface might be involved in an age-related alteration in the cell membrane.     

The transition in frequency of Y-chromatin in males during early infancy

The frequency of Y-positive lymphocytes in blood smears at delivery and during early infancy was assessed to determine the exact time at which it is similar to the frequency in adults. It was found that at day 0 of life the frequency of Y-chromatin-positive cells was 26.6 ± 13.2% (mena ± SD); subsequently, it increased gradually to 41.2 ± 12.2 %, 49.7 ± 9.7 % and 55.4 ± 5.2 % at the 30th day, the end of the 3rd month and the end of the 4th month of life, respectively (Fig. 1). The frequency in adult males was 57.8 ± 5.8 %. This is the first time that the same infant population has been investigated in all the stages of a study.     

Changes in negative surface charge of human diploid fibroblasts, TIG-1, during in vitro aging

The electrophoretic mobility of human diploid fibroblasts, TIG-1, was studied at different passages. The net negative surface charge of the cells decreased from -1.658 +/- 0.108 micron/s/V/cm at an early passage (15 population doublings, PD) to -1.173 +/- 0.116 at the final passage (67 PD) in 1/15 M phosphate buffer supplemented with 5.4% glucose. The decrease was slow at 15-45 PD, but was rapid at 45-67 PD. The net negative surface charge of small cells in the late passage populations was not different from that of larger cells in this population, and was significantly lower than that of small cells in the middle passage populations. The distribution of the mobilities of cells in each passage was independent of the size of the individual cells, and the mean value was distinct for the passage number. The viability of the cells was retained during the assay of electrophoretic mobility under these conditions. These results indicate that the net negative surface charge of human diploid fibroblasts represents a cell surface maker for in vitro cellular age in the population.     

Changes in NAD levels in human lymphocytes and fibroblasts during aging and in premature aging syndromes

NAD levels markedly increase upon mitogen stimulation of lymphocytes from young subjects. In contrast, lymphocytes from old subjects do not increase NAD levels upon stimulation. A survey of 35 individuals aged 18-79 years revealed a significant age-dependent decrease in the NAD response to mitogen stimulation. No significant differences were noted in lymphocytes from age-matched individuals with Down's syndrome or diabetes mellitus. On the other hand, cultured skin fibroblasts showed elevated NAD levels with age. However, this effect appears to be due to increased size of the cells since the NAD/protein ratio is unchanged. Skin fibroblasts from patients with progeria exhibit much higher levels of NAD and protein per cell than age-matched controls.     

In vitro aging of macaque adherent cells: similar pattern of cellular aging between human and macaque

To explore new models for human cellular aging as well as to evaluate aging of the macaques, profiles of cellular aging in macaques were studied. Adherent cells were obtained from five Japanese macaques (Macaca fuscata), 14 long-tailed macaques (Macaca fascicularis), two bonnet monkeys (Macaca radiata) and a rhesus monkey (Macaca mulatta). A total of 35 cultures were performed and cell morphology, doubling time, telomere length and telomerase activity were studied. They were classified into three groups; group I: cell strains with a definite replicative life-span (-41 PDLs) (presence of M1), group II: cell strains with a limited extension of replicative life-span (79-106 PDLs) with p53 mutation(s) (presence of M2), and group III: a cell strain with an indefinite replicative life-span (>150 PDLs) with characteristics of transformation. Except for the last group, telomerase activity was not observed. Macaque cells demonstrated three chronological patterns comprising both human and rodent patterns, however, presence of the two limits of proliferation in vitro grants macaque cells to be more appropriate than rodents in both studying human aging and oncogenesis.     

Morphological and morphometric characteristics of choroid plexus psammoma bodies during the human aging

Psammoma bodies are one of many choroid plexus aging changes which origin is still enigma for the scientists. During our investigation psammoma bodies were studied on 30 postmortem brains by light microscopy. They stained red with HE, and were PAS and AB PAS positive. The largest number of lamellas were stained blue with Mallory's connective tissue stain, except peripheral and next to the center lamella which stained red. During the aging, psammoma bodies became larger and more irregular, which was followed with group area and perimeter, single psammoma body average area and average perimeter, average diameter and contour index increase. Psammoma bodies mearged in the second and the third age group and mearging process led to larger and more irregular structures formation. The results of this investigation suggest that psammoma bodies are more frequent in choroid plexus of healthy older people and during the aging they obtain larger dimensions, more irregular contours, which is the result of their mutual mearging.     

Sequence of centromere separation of mitotic chromosomes during human cellular aging

The sequence of centromere separation of mitotic chromosomes derived from lymphocytes of 17 individuals of various ages was studied. A comparison of the mean CSI (centromeric separation index) values for individual chromosomes in the complement indicated a significant difference between the older and the younger subjects only with regard to the chromosome nos. 6, 11, and 19 in both sexes and the X chromosome exclusively in the female. With age, chromosome no. 6 in both males and females and the X chromosome in females showed premature centromeric separation whereas chromosome nos. 11 and 19 in both males and females displayed delayed centromeric separation. Our study, therefore, indicates that changes in the sequence of centromeric separation of certain chromosomes do occur in lymphocytes during chronological aging in humans.     

Changes in apoptosis and cell proliferation in human pineal gland during aging

Immunohistochemical analysis of the expression of proapoptotic protein P53 and proliferation protein Ki-67 in human pineal gland showed that in people over 60 years pinealocyte proliferation is virtually absent. Moreover, pinealocyte apoptosis is more pronounced in elderly, whereas in senile and long-living individuals its intensity decreases.     

Iron accumulation during cellular senescence in human fibroblasts in vitro

Iron accumulates as a function of age in several tissues in vivo and is associated with the pathology of numerous age-related diseases. The molecular basis of this change may be due to a loss of iron homeostasis at the cellular level. Therefore, changes in iron content in primary human fibroblast cells (IMR-90) were studied in vitro as a model of cellular senescence. Total iron content increased exponentially during cellular senescence, resulting in 10-fold higher levels of iron compared with young cells. Low-dose hydrogen peroxide (H2O2) induced early senescence in IMR-90s and concomitantly accelerated iron accumulation. Furthermore, senescence-related and H2O2-stimulated iron accumulation was attenuated by N-tert-butylhydroxylamine (NtBHA), a mitochondrial antioxidant that delays senescence in vitro. However, SV40-transformed, immortalized IMR-90s showed no time-dependent changes in metal content in culture or when treated with H2O2 and/or NtBHA. These data indicate that iron accumulation occurs during normal cellular senescence in vitro. This accumulation of iron may contribute to the increased oxidative stress and cellular dysfunction seen in senescent cells.     

Changes in mitochondrial DNA during aging

Mitochondrial DNA (mtDNA) was isolated from liver mitochondria of rats between 2 and 24 months of age. The mtDNA was purified by cesium chloride—ethidium bromide isopycnic density gradient centrifugation. In the gradients, in addition to the two expected bands of ethidium-DNA complex, there was observed a third, more dense band ($\text{d = 1.69}\text{g}\text{cm}{}^3$). This novel band, rarely observed in preparations from younger animals, was present in most preparations from older animals. The latter was characterized using the diphenylamine assay(s) and ascertained to contain DNA and carbohydrate components. Agarose gel electrophoresis revealed the DNA of the novel band to have a migration identical to form I mtDNA. Digestion of the novel band with the restriction endonuclease Bam HI yielded products identical to those obtained upon treatment of form I mtDNA with Bam HI. The observation of mtDNA at a density of $\text{1.69 g}\text{cm}{}^3$ indicates the presence, predominantly in older animals, of a subclass of mtDNA molecules with altered ethidium binding properties. The significance of this mtDNA and its position in the gradient is unclear at this time.     

Changes of the basement membrane and type IV collagen in human skin during aging

Objectives: To determine the relationship between skin collagen IV and basement membrane changes during aging, a total of 35 women who had been admitted for surgery, were studied. Methods: Subjects were arranged into six age-groups (from 35 to 60 years). Skin biopsies were performed in all patients and the samples were taken from a site 6 cm above the pubic symphysis. The collagen IV content and the epithelial basement membrane were analyzed by using immunohistochemical, transmission electron microscopy and computer-assisted image analysis methods. The skin collagen IV content was measured by an image analysis program and expressed in arbitrary units of integrate optical density, and, the basement membrane thickness was expressed in nanometers. Results: Type IV collagen content decreased with age after 35 years (r = −0.9561). The epithelial basement membrane thickness increased significantly with age (r = 0.98192; P < 0.01) and there is an inverse correlation between these two parameters (r = −0.990502). Conclusions: Although type IV collagen is a basement membrane component and declines with aging, the total thickness of this membrane increases, which suggests a reduction in tissue turnover.     

Influence of aging on human sound localization

Errors in sound localization, associated with age-related changes in peripheral and central auditory function, can pose threats to self and others in a commonly encountered environment such as a busy traffic intersection. This study aimed to quantify the accuracy and precision (repeatability) of free-field human sound localization as a function of advancing age. Head-fixed young, middle-aged, and elderly listeners localized band-passed targets using visually guided manual laser pointing in a darkened room. Targets were presented in the frontal field by a robotically controlled loudspeaker assembly hidden behind a screen. Broadband targets (0.1-20 kHz) activated all auditory spatial channels, whereas low-pass and high-pass targets selectively isolated interaural time and intensity difference cues (ITDs and IIDs) for azimuth and high-frequency spectral cues for elevation. In addition, to assess the upper frequency limit of ITD utilization across age groups more thoroughly, narrowband targets were presented at 250-Hz intervals from 250 Hz up to ～ 2 kHz. Young subjects generally showed horizontal overestimation (overshoot) and vertical underestimation (undershoot) of auditory target location, and this effect varied with frequency band. Accuracy and/or precision worsened in older individuals for broadband, high-pass, and low-pass targets, reflective of peripheral but also central auditory aging. In addition, compared with young adults, middle-aged, and elderly listeners showed pronounced horizontal localization deficiencies (imprecision) for narrowband targets within 1,250-1,575 Hz, congruent with age-related central decline in auditory temporal processing. Findings underscore the distinct neural processing of the auditory spatial cues in sound localization and their selective deterioration with advancing age.     

Antigenic variation during cellular aging in Paramecium tetraurelia

Clones of Paramecium tetraurelia undergoing cellular aging were exposed to two antisera, anti-32 and anti-25, raised against surface antigen isolated from cells grown at 32 degrees C or 25 degrees C. When young clones were exposed to the two antisera about 50% were immobilized by anti-32 while others were not immobilized by either antisera. These clones were grouped as group A or B depending upon their sensitivity or nonsensitivity to the two antisera. Young cells of group A were immobilized by anti-32 and during aging these cells either maintained the same antigenic type or transformed to another. Young group B clones were not immobilized by either antisera but gradually became sensitive to anti-32. Some clones from both groups showed sensitivity to both antisera. The possible mechanism of antigenic variation during fission age is discussed.     

In vitro modulation of cellular localization of milk fat globule membrane antigens in human breast carcinomas

Alterations in the cellular localization of cell surface components such as the milk fat globule membrane are a common feature of breast carcinomas and relate to the differentiation of a tumour. This study has examined the potential modulation of such components. A group of carcinomas were cultured with and without insulin and/or hydrocortisone and the site of staining for milk fat globule membrane, as detected by the antibodies HMFG 1, HMFG 2, and NCRC 11, was assessed using light microscopic and electron microscopic immunohistochemistry. Modulation of localization, with a shift from cytoplasmic vesicle labelling to submembraneous vesicles/cell surface labelling and intracytoplasmic luminal labelling, was observed in 5 of 14 moderately differentiated and 8 of 11 poorly differentiated carcinomas. Two well differentiated carcinomas continued to show peripheral labelling; three poorly differentiated carcinomas showed no change from cytoplasmic labelling only; and the other carcinomas exhibited heterogeneous localization, making any change difficult to assess. Insulin was required for any change to be observed and it is suggested that this has an effect on the mechanisms for intracellular transport of membrane and secretory proteins.     

Changes in biological characteristics during the cellular aging of ligament fibroblasts derived from patients with prolapsus uteri

Prolapsus uteri in pelvic support disorders are common in elderly women. The etiology is unclear and more likely to be multifactorial. We examined changes in biological characteristics and responsiveness to growth factors during the in vitro cellular aging of cardinal ligamental fibroblasts derived from patients with prolapsus uteri (HPLiF), and compared them with those of cells from age-matched control subjects (HCLiF). HPLiF and HCLiF had almost the same in vitro life span and the age-related patterns of biological parameters were essentially the same. However, the saturation density was significantly higher in HPLiF than in HCLiF. Furthermore, the high proliferative activity of HPLiF to serum mitogens, especially to platelet-derived growth factor, was retained throughout the in vitro life span. p53 protein levels in HPLiF increased at late passages, but were significantly less than in aged HCLiF. These results indicate that the higher proliferative activity in prolapsus fibroblasts may result from the decreased expression of p53 protein and may lead to a decrease in the synthesis and deposition of extracellular matrix components. These results support the hypothesis that functional alterations in ligament fibroblasts are involved in the mechanism of the development of prolapsus uteri.     

Changes in the higher order organization of DNA during aging of human fibroblast-like cells

Nucleosome spacing (DNA repeat length) was determined in human diploid fibroblast-like cells (HDF) of different in vitro ages following the electrophoretic separation of micrococcal nuclease digestion products. The results indicate that a heterogeneity of DNA repeat lengths is present in HDF of all in vitro ages. In older cells the organization of part of the DNA is conserved, but a greater proportion of shorter repeats is evident. The shorter repeat lengths are not due to nucleosome sliding, but result from the presence of shorter linker regions which are reduced by as much as 25% in part of the chromatin of high PDL cells.     

In vitro investigations of interphase and metaphase argyrophilic nucleolar organizer regions and cellular proliferation in the human urothelial cancer cell line HOK-1

Detailed investigation of cell growth and nucleolar organizer region associated argyrophilic proteins (Ag-NORs) is necessary to asses a possible impact of Ag-NOR quantification on the diagnosis and prognosis of tumours. In this study, cellular proliferation of the transitional-cell carcinoma cell line HOK-1 was modulated over a period of 11 days by starvation and subsequent medium addition. Proliferation was determined daily by DNA flow cytometric estimation of S-phase fraction (SPF) and mitotic index (MI) calculation. The number and area of interphase Ag-NORs were quantified by automated image analysis daily and the number of Ag-NOR bearing chromosomes in metaphase was counted. In interphase nuclei, Ag-NOR area showed a highly significant correlation with SPF (p<0.0001) whereas interphase Ag-NOR number showed significant correlation with MI (p<0.05). A positive relationship between the number of Ag-NOR bearing chromosomes in metaphases and cellular proliferation was also observed. There is variability in Ag-NOR quantity during interphase and metaphase depending on growth conditions in vitro. Correlations of the number of interphase Ag-NORs with the MI on one hand and Ag-NOR area with SPF on the other provide further evidence that distribution and quantity of Ag-NORs are strongly influenced by the cell cycle phase within the structural-functional unit of the nucleolus.     

Changes in the ultrastructure of axospike contacts in human brain during normal aging and in vascular disorders

Light spikes, the majority of which are electron-transparent, are the main structural unit of the axospike contacts in normal brain of aged people (73 and 83 years). Dark spikes and spikes with moderate osmiophilia predominate in the brain of people with vascular disorders at the age of 70–73 years. A “synaptic block”, which was morphologically represented by presynaptic terminals with densely packed synaptic vesicles, is revealed in the brain of aged people with vascular disorders. The role of hypoxia in the reorganization of axospike synapes is discussed. It is hypothesized that changes in the structure of axospike contacts in normal aging and vascular disorders are associated with modifications in the ultrastructure of spikes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 7, pp. 15–19, July, 1998