Glucocorticoid-induced modulation of insulin-stimulated DNA synthesis: differential responsiveness in cell cultures derived from donors of different ages

The replicative ability of variously 'aged' cell cultures, their insulin binding and biological responsiveness under control and glucocorticoid (i.e. hydrocortisone) amplified conditions have been studied in human fibroblast cultures. Insulin stimulation of DNA synthesis in early and late passage cultures and in cultures from young and old donors showed no age-related difference in insulin responsiveness. Hydrocortisone amplification of insulin-stimulated DNA synthesis in early and late passage cells expressed no age-related differences. Hydrocortisone affected basal DNA synthesis in cultures from in vivo young and old donors differently. Additionally, hydrocortisone amplified insulin-stimulated DNA synthesis in young donor cell cultures was observed to be higher than in old donor cell cultures. Specific 125I-insulin binding was increased by hydrocortisone in both early and late passage cultures and in cultures from young and old donors but no age-related differences in 125I-insulin binding were observed in the presence or absence of hydrocortisone. The data suggest that an age-related loss of an insulin postreceptor interaction during hydrocortisone amplification of insulin-stimulated DNA synthesis is being expressed in the cultures from old donors.     

Colony size distributions as a measure of in vivo and in vitro aging.

Individual human diploid cells plated at low cell density and incubated for 2 weeks develop into colonies ranging in size from one cell to several thousand cells. The resultant colony size distribution is an accurate indicator of the number of subsequent in vitro population doublings that can be attained by the parent culture. This relationship holds for both human fetal lung and adult skin fibroblast cultures. In addition, the colony size distributions obtained from fetal, young adult, and old adult human cell cultures at the same low level of in vitro passage are indicative of the in vivo age of the level of in vitro passage are indicative of the in vivo age of the cell culture donor. Cell cultures of fetal origin give rise to the highest percentage of colonies with significant proliferative abilities, whereas cultures from old adults give rise to the lowest percentage of large colonies. Therefore, colony size distributions appear to be good indicators of both in vitro and in vivo human cellular aging.     

Collagenase production by early and late passage cultures of human fibroblasts

Comparison of the proteins secreted by early and late passage cell cultures of human fibroblasts revealed a high level of immunoreactive collagenase (Mr = 55,000 Da and 58,000 aDa) in the late passage cell culture conditioned medium. Both molecular weight species reacted with a monoclonal anticollagenase antibody and were apparently glycosylation varients of the same protein. The question of whether the apparent age-dependent differences in collagenase synthesis reflected changes in protein synthesis or secretion was addressed by assaying immunoreactive collagenase and collagenase mRNA. Immunofluorescence microscopy of cellular collagenase revealed that the percentage of collagenase positive cells ranged from 1 to 6% (early passage) to 35 to 46% (late passage) indicating that the late passage cells had higher basal levels of collagenase synthesis. Later passage cultures also secreted higher levels of immunoprecipitable collagenase into the culture medium and Northern analysis established that the basal levels of collagenase mRNA was also 10 times greater in late passage cells. High basal levels of collagenase were also observed in fibroblasts cultured from an in vivo aged donor and from donors with Werner's syndrome. Collagenase production was induced in both early and late passage cell cultures by exposure to fibroblast extracellular matrix, fibroblast conditioned media, polypeptide growth factors, or phorbol esters. The induced levels were always greater in the late passage cell cultures than in the early passage cell cultures.     

Changes in cell cultures of bovine corneal endothelium cells as related to donor age and number of passages in vitro

Aging changes of the acidic glycosaminoglycans (aGAG) were investigated in bovine corneal endothelium cells grown in vitro, using histochemical methods and the determination of hexosamine and uronic acid. The time interval between trypsination and confluency of the monolayer and the cell counts at different time intervals of the same passage number served as parameters for the proliferation capacity of the cultures. A cell line, derived from the eye of a 1,5 year old cattle showed a low proliferation capacity in the first 9 passages, which increased until the 15th passage, to decrease thereafter until the 29th passage. In cells from eyes aged 11 and 12 years respectively the initial proliferation capacity was very low to reach the same values as in cultures of the young eye at the 10th passage but remained constant thereafter. Chromosome counts showed an increased number of hyperploids in late passages of cells from the aging eye with counts around 4n-8n increased to 17% as compared to 5% in the cells derived from the young eye. The cell densities were increased in cultures of high passage numbers. Histochemically the aGAG were shown in the cell nucleus, and the cytoplasm as well as in the intercellular space. In the nuclei of cells from aging eyes and in late passage cells of the young eye they showed an increased resistance against resting hyaluronidase. The hexosamine and uronic acid values were high in early passages of cultures from the young eye and decreased in late passages of the young eye as well as in early passages of cells from the aging eyes. The changes in the aGAG as well as the changes in the cell proliferation are similar comparing early passage cells of aging eyes to late passage cells of the young eyes. But the aGAG changes are more pronounced in cells of eyes aged in vivo. In the eye the proliferation of the corneal endothelium cells is inhibited. As such these cells provide a useful model to comparing in vivo-aging changes of resting cells to changes of the same cells proliferating in vitro.     

Protein synthetic errors do not increase during aging of cultured human fibroblasts.

To test the error catastrophe theory of aging we determined the error frequency of protein synthesis in several strains of cultured human fibroblasts at early and late passage. Error rates were calculated from analysis of native and substituted actins on two-dimensional gels of cellular proteins after induction of mistranslation by histidine starvation in the presence of histidinol. Early-passage cells from fetal, young, and old donors and cells from subjects with the Hutchinson-Gilford and Werner syndromes of accelerated aging had similar error frequencies. Late-passage cells from fetal, young, and old normal donors had similar or lower error frequencies than corresponding early-passage cells. No correlation was observed between error frequency, donor age, or maximal life span in vitro. We also examined an immortal cell line, simian virus 40-transformed W138 fibroblasts. These cells had a significantly elevated rate of mistranslation (2.8 +/- 0.2 x 10(-4))(+/- SEM) compared to their untransformed counterpart WI38 (0.6 +/- 0.1 X 10(-4)) or all diploid cells combined (1.1 +/- 0.1 x 10(-4)). Taken together, the data fail to support the error catastrophe theory 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.     

Ganglioside changes during cell aging in human diploid fibroblast tig-1

Gangliosides of human diploid fibroblast, TIG-1, derived from fetal lung were analyzed during cell aging. The major gangliosides of TIG-1 cells were G_M3 and G_D3. During cell aging, G_M3 and other gangliosides were decreased or below detection level. Lipid-bound sialic acid per mg protein was 2μg at the early, middle and late passages, but between the middle and late passage it was twofold higher than the other passages.     

Mechanism of enhanced cyclic AMP stimulation by isoproterenol in aged human fibroblasts.

Human diploid lung fibroblasts (IMR-90) were used to investigate the reported increase in beta-adrenergic-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) levels in fibroblasts aged in culture. Under basal conditions cellular cAMP was 34.2 +/- 5.6 and 38.4 +/- 9.1 pmol/mg protein in early (PDL 22-24) and late (PDL 47-52) passage fibroblasts, respectively. Net release of cAMP from fibroblasts was 67.8 +/- 8.6 and 18.5 +/- 7.0 pmol/30 min/mg protein in early and late passage cultures, respectively. In confluent, early passage fibroblasts, cellular cAMP and net release of cAMP increased by 2.7-fold and 3.8-fold, respectively, after a 30 min incubation in 2 microM isoproterenol. In confluent late passage fibroblasts, isoproterenol incubation increased cellular cAMP and net release of cAMP by 7.8-fold and 26.1-fold, respectively. Adenosine failed to inhibit isoproterenol-induced stimulation of cAMP in early or late passage fibroblasts. There was no passage-related difference in basal, isoproterenol, or forskolin-stimulated adenylyl cyclase activity in crude fibroblast membrane preparations. The activity of cAMP-phosphodiesterase in sonicates of early and late passage IMR-90 was 9.61 +/- 1.15 and 5.81 +/- 1.11 pmol/min/mg protein respectively. Measurements of cAMP in subconfluent early passage fibroblasts indicated that mechanisms related to the reduced cell density in confluent late passage IMR-90 may, in part, account for the enhanced isoproterenol-induced cAMP levels observed in these cultures. The results suggest that the remainder of the enhanced cAMP response to isoproterenol of in vitro aged fibroblasts may be due to a lower cAMP phosphodiesterase activity in these cells.     

Cloning of differentially expressed genes in skin fibroblasts from centenarians

Normal human fibroblasts undergoing serial passaging have been extensively used to identify genes linked with aging. Most of the isolated genes relate to growth retardation signals and the failure of homeostasis that accompanies aging and senescence. In contrast, there is still limited knowledge regarding the nature of the genes that influence positively the rate of aging and longevity. Healthy centenarians represent the best example of successful aging and longevity. Studies using samples from these individuals have proved very valuable for identifying a variety of factors that contribute to successful aging. The aim of the current work was to take advantage of skin fibroblast cultures established from healthy donors including centenarians in order to clone differentially expressed genes in centenarians. First, we demonstrate that centenarian derived cultures follow the typical Hayflick curve and they enter senescence after serial passaging. Application of differential screening techniques in minimally passaged cultures of four control donors of different ages (18–80 years old) and four centenarians has resulted in the cloning of six differentially expressed genes in centenarians. Four of the cloned genes, namely adlican, KBL, EST 38 and EST 39, were over-expressed in centenarians, while VDUP1 and OCIF were down-regulated in the same samples. We have also compared the expression levels of two representative cloned genes in cultures of human embryonic and adult fibroblasts to establish potential links with replicative senescence. Interestingly, VDUP1 was found over-expressed in late passage cells, while EST 39 was down-regulated in the same cultures. Thus our work demonstrates that a combination of the use of both biopsies derived cells and classical in vitro cells passaging will facilitate the better understanding of the biology of aging and longevity.Normal human fibroblasts undergoing serial passaging have been extensively used to identify genes linked with aging. Most of the isolated genes relate to growth retardation signals and the failure of homeostasis that accompanies aging and senescence. In contrast, there is still limited knowledge regarding the nature of the genes that influence positively the rate of aging and longevity. Healthy centenarians represent the best example of successful aging and longevity. Studies using samples from these individuals have proved very valuable for identifying a variety of factors that contribute to successful aging. The aim of the current work was to take advantage of skin fibroblast cultures established from healthy donors including centenarians in order to clone differentially expressed genes in centenarians. First, we demonstrate that centenarian derived cultures follow the typical Hayflick curve and they enter senescence after serial passaging. Application of differential screening techniques in minimally passaged cultures of four control donors of different ages (18–l80 years old) and four centenarians has resulted in the cloning of six differentially expressed genes in centenarians. Four of the cloned genes, namely adlican, KBL, EST 38 and EST 39, were over-expressed in centenarians, while VDUP1 and OCIF were down-regulated in the same samples. We have also compared the expression levels of two representative cloned genes in cultures of human embryonic and adult fibroblasts to establish potential links with replicative senescence. Interestingly, VDUP1 was found over-expressed in late passage cells, while EST 39 was down-regulated in the same cultures. Thus our work demonstrates that a combination of the use of both biopsies derived cells and classical in vitro cells passaging will facilitate the better understanding of the biology of aging and longevity.     

Effects of serially passaged fibroblasts on dermal and epidermal morphogenesis in human skin equivalents

Serial passaging has a profound effect on primary cells. Since serially passaged cells show signs of cellular aging, serial passaging is used as an in vitro model of aging. To relate the effect of in vitro aging more to in vivo aging, we generated human skin equivalents (HSEs). We investigated if HSEs generated with late passage fibroblasts show characteristics of aged skin when compared with HSEs generated with early passage fibroblasts. Late passage fibroblasts had enlarged cell bodies and were more often positive for myofibroblast marker α-smooth muscle actin, senescence associated β-galactosidase and p16 compared with early passage fibroblasts. Skin equivalents generated with late passage fibroblasts had a thinner dermis, which could partly be explained by increased matrix metalloproteinase-1 secretion. In equivalents generated with late passage fibroblasts epidermal expression of keratin 6 was increased, and of keratin 10 slightly decreased. However, epidermal proliferation, epidermal thickness and basement membrane formation were not affected. In conclusion, compared with HSEs generated with early passage fibroblasts, HSEs generated with late passage fibroblasts showed changes in the dermis, but no or minimal changes in the basement membrane and the epidermis.     

Absence of three secreted proteins and presence of a 57-kDa protein related to irreversible arrest of cell growth.

Two subsets of proteins from the conditioned medium of normal human diploid fibroblasts were detected by NaDodSO4/polyacrylamide gel electrophoresis analysis; the presence of these subset is related to the nonproliferative state of in vitro aged cells. One subset consists of three proteins (molecular mass from 80 to 87 kDa) secreted by replicating cells in sparse cultures, as well as by quiescent cells in confluent or serum-starved cultures of young fibroblasts. These proteins disappear from the medium when cultures reach the state of senescence. The other subset consists of a single protein of 57 kDa, detected only in conditioned medium of senescent fibroblast cultures. The results suggest that when human fibroblasts reach senescence, they secrete the 57-kDa protein and concomitantly stop secreting the other three proteins normally found in the culture medium of young fibroblasts. Therefore, the alternative secretion of these protein subsets could specifically signify irreversible arrest of cell growth during in vitro cellular aging.     

T-lymphocyte long-term cultures have a constant histone variant pattern during aging

Human peripheral long-term T-lymphocyte cell cultures show some characteristics similar to those of fibroblast cell lines, the latter of which have been used as in vitro systems for cellular aging studies for many years. Both show a limited in vitro life span, as well as a progressive prolongation of their cell cycle with increasing age. However, whereas T-cell cultures die from apoptosis at the end of their proliferative capacity, fibroblasts can be maintained for long periods of time in stationary cultures as postmitotic senescent cells. Previous studies analyzing the histone variant pattern of a human lung embryonic fibroblast cell line have shown that this pattern changes as a function of cumulative population doublings in a manner not unlike that found in terminally differentiating systems. In the present study the histone variant composition of long-term T-cell cultures was analyzed as a function of population doublings and compared to a human diploid fibroblast system. The results from this study provide a distinction at the molecular level among these two in vitro aging model systems, because it was found that long-term T-cell cultures show a constant histone variant constitution throughout their in vitro life, dissimilar to previous findings using the fibroblast cell system.     

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.     

MicroRNA-663 induction upon oxidative stress in cultured human fibroblasts depends on the chronological age of the donor

MicroRNAs, regulators of messenger RNA translation, have been observed to influence many physiological processes, amongst them the process of aging. Higher levels of microRNA-663 (miR-663) have previously been observed in human dermal fibroblasts subject to both replicative and stress-induced senescence compared to early passage cells. Also, higher levels of miR-663 have been found in memory T-cells and in human fibroblasts derived from older donors compared to younger donors. In previous studies we observed that dermal fibroblasts from donors of different chronological and biological age respond differentially to oxidative stress measured by markers of cellular senescence and apoptosis. In the present study we set out to study the association between miR-663 levels and chronological and biological age. Therefore we tested in a total of 92 human dermal fibroblast strains whether the levels of miR-663 in non-stressed and stressed conditions (fibroblasts were treated with 0.6 μM rotenone in stressed conditions) were different in young, middle aged and old donors and whether they were different in middle aged donors dependent on their biological age, as indicated by the propensity for familial longevity. In non-stressed conditions the level of miR-663 did not differ between donors of different age categories and was not dependent on biological age. Levels of miR-663 did not differ dependent on biological age in stressed conditions either. However, for different age categories the level of miR-663 in stressed conditions did differ: the level of miR-663 was higher at higher age categories. Also, the ratio of miR-663 induction upon stress was significantly higher in donors from older age categories. In conclusion, we present evidence for an association of miR-663 upon stress and chronological age.     

Decreased fidelity of DNA polymerase activity isolated from aging human fibroblasts.

DNA polymerase (deoxynucleosidetriphosphate: DNA nucleotidyltransferase, EC 2.7.7.7 or DNA nucleotidyltransferase) activity, isolated from late and early passage cells of the diploid human fibroblast line, MRC-5, was compared. The level of activity dropped with increasing passage. In addition, when the fidelity of polymerization was monitored with four synthetic templates under a variety of conditions, it was observed that the enzyme from late passage cells was more error-prone. The possible relation of these observations to "senescence" of the fibroblasts is discussed.     

Altered sensitivity of protein synthesis to paromomycin in extracts from aging human diploid fibroblasts

Age-related differences in the effects of paromomycin (Pm) on protein synthesis have been investigated in translation reactions with extracts derived from young and old human diploid fibroblasts. Translation products from reactions directed by endogenous or exogenous mRNA were analyzed by polyacrylamide gel electrophoresis and fluorography. The exogenous mRNA lacked codons for cysteine, and therefore cysteine incorporation into translation products represented translational error. This laboratory has previously used this assay to show that the basal translational error level in the absence of Pm increases in extracts from old fibroblasts. In this report, Pm stimulated the misincorporation of cysteine by 6-7 fold over cysteine misincorporation levels in the absence of Pm. This degree of Pm stimulation was similar in extracts from young and old fibroblasts. However, other results showed quantitative differences in the responses to Pm between young and old cell extracts. Old cell extracts were less sensitive to the stimulation of the rate of protein synthesis, and more sensitive to the inhibition of protein synthesis, by Pm. It is proposed that aging human diploid fibroblasts contain altered ribosomes which react differently with Pm.     

Age-related accumulation of a novel CD44 + CD25lowgammadelta T-cell population in hematopoietic organs of the mouse

We discovered a novel population of gammadelta T cells in the mouse that accumulates with age in hematopoietic organs, but not in epithelia. These cells are CD25low (an unusual phenotype for gammadelta T cells in the mouse); express higher levels of TCRgammadelta and CD44 than do CD25- gammadelta T cells; mainly express Vgamma2, Vgamma3, and Vgamma4 chains; and are largely quiescent. A very similar cell population appears in the late stages of fetal thymus organ cultures, suggesting that the accumulation of CD44 + CD25lowTCRgammadelta + cells is a response to stress induced by aging in vivo or by culture in vitro. The precursors of CD44 + CD25lowTCRgammadelta + cells are generated during fetal or very young adult life, as this population was undetectable in aged recipients of bone marrow from old or young donors. CD44 + CD25lowTCRgammadelta + cells may be a biomarker of aging, but could also play a role in the inflammatory changes that accompany aging.