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PubMed1979

Differences between transfer RNA methylase activity in human diploid fibroblasts during in vitro and in vivo aging.

The change of the specific activity of S-adenosylmethionine: tRNA methyltransferase in cultures of human diploid fibroblasts at different passages has been measured and compared with that in the same type of cells derived from donors of different ages. Whereas the specific activity of tRNA methylase in the in vitro aged cells was found to decline gradually with increasing passage number of the culture, a different activity--age relationship was observed for this enzyme in cells derived from donors of different ages. The activity of tRNA methylase is high in the fetal cells and drops drastically in the "newborn" cells. After a further 10% decline, the activity of this enzyme reaches a steady low level in the postnatal cells from donors ranging in age from 3 months to 94 years. These findings cast doubt on the validity of the assumption that the results obtained from in vitro aging experiments reflect the biochemistry of aging in vivo. The "fetal" enzyme can methylate the "aged" tRNA but the "aged" enzyme cannot methylate the "fetal" tRNA. The fetal cells contain enzyme activities specific for the formation of m1A, m5C and m1G. These activities are low or deficient in "aged" cells. Control experiments showed that all of these results are due neither to the presence of inhibitor or stimulator in the extract nor to effects related to the population density, sex or growth rate of the culture.

Adult↗
PubMed1980

Commitment theory of cellular aging: possibility of an immortal diploid cell strain.

Calculations based on the commitment theory of cellular aging indicate that the mean number of uncommitted cells is much greater than unity in populations consisting of 10(10) or more diploid cells. Consequently the probability that all of the uncommitted cells will be lost by in vitro subcultivation is very small. It may therefore be possible to establish a cell population in the laboratory which is, for all practical purposes, immortal.

Cell Differentiation↗
PubMed1980

The sensitivity to hyperbaric oxygen of human diploid fibroblasts during ageing in vitro.

The effect of hyperbaric oxygen on human diploid fibroblasts, WI-38, at various passages was examined. Their colony-forming ability deteriorates on exposure to hyperbaric oxygen. The cellular injury is not due to the high pressure per se, but to oxygen toxicity, since compressed nitrogen has no effect on the cells. When the cells beyond mid-40 population doublings (lifespan completed more than about 75%) are exposed to 50 atm of oxygen, there is a great decrease in the efficiency of the colony formation. A double exposure to 75 atm of oxygen inhibits the growth of the late passage cells more strongly than that of the early passage cells. These results suggest that the late passage cells are more sensitive to oxygen than the early passage cells.

Cell Survival↗
PubMed1980

A low-density inoculation method for the serial subcultivation of human diploid fibroblasts: an efficient model system for the study of cellular ageing.

Human diploid fibroblasts, TIG-1, were serially subcultivated at low inoculation densities thus maintaining the cultures in log phase throughout their in vitro lifespan. Their lifespan was shortened in calendar time without decreasing the cumulative number of population doublings when compared with the lifespan of cells serially cultivated by a standard method of 1:4 splitting. The cytokinetic and morphological changes during their lifespan are quite similar to those of control cells. The frequency of trypsinization doess not influence the cumulative number of population doublings. The "low-denstiy inoculation method" of subcultivation is efficiently and economically advantageous in studying cellular ageing in vitro.

Cell Division↗
PubMed1980

Cell-surface changes accompanying aging in human diploid fibroblasts: effects of tissue, donor age and genotype.

The generality of age-related changes in concanavalin A (Con A)-mediated red blood cell (RBC) adsorption to human diploid fibroblasts was investigated on fibroblast-like cells from fetal lung, heart, liver, skin and muscle tissues. All the cells examined showed the continuous increase from early passages in RBC adsorption with the RBC coating method (in which Con A-coated RBCs are adsorbed to fibroblasts) and the incraease only at phase III with the fibroblast coating method (in which RBCs are adsorbed to Con A-coated fibroblasts). All of the four strains of lung fibroblasts gave nearly the same extent of the age-related change in RBC adsorption, when expressed as a function of percentage life span consumed, indicating that the change in RBC adsorption is independent of genetic heterogeneity and conditions of primary culture. Liver and heart fibroblasts also gave results similar to those of lung fibroblasts. However, skin and muscle fibroblasts were lower in their RBC adsorption capacity throughout the life span. The continuous age-related increase in RBC adsorption to these cells could be sensitized by using glutaraldehyde-prefixed RBCs, trypsinized RBCs or phytohemagglutinin P in place of Con A. The relevance of the phenotype of in vitro aging revealed by RBC adsorption to in vivo aging was also demonstrated on skin fibroblasts from different ages of donors using glutaraldehyde-prefixed RBCs. In addition, fibroblasts from patients with Werner's syndrome, an hereditary disease manifested by early and widespread degenerative changes, showed senescent phenotype in RBC adsorption even at early passages.

Adult↗
PubMed1981

Loss of organized fibronectin matrix from the surface of aging diploid fibroblasts (IMR-90).

Indirect immunofluorescence was used to investigate the production of a fibrillar fibronectin matrix by human diploid fibroblasts (IMR-90) as cells progress through their in vitro lifespan. Early and mid-passage cultures displayed a prominent fibrillar reticulum over the cell layer which formed within 24 hours of seeding. Even sparsely seeded early-passage cells exhibited fibrils of fibronectin on external surfaces. In contrast, fibrillar fibronectin was reduced or absent on surfaces of late-passage cells. However, the larger, non-proliferating, late-passage cells were producing fibronectin, 1h determined by radioimmunoassay of the medium.

Aging↗
PubMed1981

Evidence for an increased level of DNA damage in high doubling level human diploid cells culture.

Excision repair after ultraviolet (UV) irradiation was estimated in human diploid fibroblast-like cells (HDF) by treating DNA with a crude extract from Micrococcus luteus that contained pyrimidine dimer-specific endonucleases. The assays were done before and after the cells had been arrested in an essentially nonmitotic state. When low and high population doubling level (PDL) cells were assayed under these conditions, no age-related difference in the number of UV-induced endonuclease sensitive sites was observed, but the removal of these sites was more rapid in high PDL arrested cells. There was a difference between the calculated number average molecular weights (Mn) of DNA from unirradiated low and high PDL arrested cells. The lower Mn of the DNA from high PDL cells indicated that other enzymes present in the M. luteus extract were acting upon non-UV-induced DNA distortions and that these were present to a greater extent in the chromatin associated regions of older cells. These results support the hypothesis that DNA damage accumulates as HDF progress through their in vitro life span.

Cell Division↗
PubMed1981

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.

Cell Division↗
PubMed1981

Effects of in vitro aging and cell growth on the viability and recovery of human diploid fibroblasts, TIG-1, after freezing and thawing.

The viability and the recovery (cell attachment to the dish) after thawing of human diploid fibroblasts (TIG-1) frozen by four different methods were studied at different passages. Improved results were observed in a medium of 30% fetal bovine serum plus 15% glycerol, compared with the conventional medium which contained 10% fetal bovine serum plus 10% glycerol. Centrifugation to remove glycerol immediately after thawing had a negative effect on the viability and recovery of cells. The recovery of cells after freezing and thawing showed a maximal value in the middle of phase II (PD 35) during the finite lifespan of the cell (average PD 67). This results indicates that the cells at early and late passages are sensitive to injury by freezing and thawing. The modified method yielded improved recovery, especially in the cells at early and late passages, except for the extremely senile stage. The recovery was also affected by the state of cell growth after inoculation.

Cell Division↗
PubMed1982

A long-lived human diploid fibroblast line for cellular aging studies: applications in cell hybridization.

We have isolated a diploid fibroblast culture from human fetal lung with an in vitro lifespan of about 100 population doublings. The culture grows very well at clonal densities and long-lived clones can be isolated for use in cellular aging studies. The longer in vitro lifespan of the culture has allowed us to isolate from it a clone, containing a dominant and recessive mutation, having significant remaining proliferative potential. The nature of the mutations will allow for hybrid selection, after fusion of the mutant clone with wild type human cells. The mass culture and clones derived from it provide a valuable resource for cell aging studies.

Aminopterin↗
PubMed1982

The effects of in vitro ageing on the composition of the intracellular free amino acid pool of human diploid fibroblasts.

The composition of the free amino acid pool of the human diploid fibroblast cell line, BCL-D1, was analysed by automated ion-exchange chromatography throughout the life-span of the culture. Significant age-related changes, independent of growth effects, were found with six essential amino acids: a decrease in tyrosine, phenylalanine, leucine, isoleucine and valine, and an increase in methionine. The nature of the changes were suggestive of a decline in the operation of the L-system of amino acid transport with in vitro age. However, it is also possible that age-related changes in the proportions of tRNA iso-acceptors and/or the availability and accuracy of tRNA synthetases were involved.

Amino Acids↗
PubMed1984

S-phase transit times as a function of age in human diploid fibroblasts.

The minimum time it takes a cell to pass completely through the S phase (MIN S) was examined in human diploid fibroblasts using a sequential [14C]thymidine, [3H]thymidine, [14C]thymidine labeling protocol. MIN S appeared to be around 6-8 h for both WI-38 and MRC-5 cells. In addition, MIN S did not increase in senescent cultures. Since damage to either DNA, its polymerases, or both would result in a reduction in the rate of DNA synthesis and a corresponding increase in MIN S, this suggests that in senescent cultures at least a portion of the cells contain DNA that is relatively undamaged and DNA polymerases that exhibit normal replicative kinetics.

Aging↗
PubMedDate not supplied

Induction of host DNA synthesis in senescent human diploid fibroblasts by infection with human cytomegalovirus.

A human diploid fibroblast strain, TIG -1, ceased to proliferate at about 60-62 population doubling level. The percentage of nuclei incorporating [3H]thymidine during 24-h culture in fresh medium containing 10% fetal bovine serum was less than 2% in the senescent cells used in this study. Infection of these cells with human cytomegalovirus (HCMV), strain AD-169, increased the percentage of [3H]thymidine-labeled cells by about ten-fold. Equilibrium density gradient centrifugation analysis of purified DNA from infected cells showed that cellular DNA synthesis was stimulated preceded by the viral DNA synthesis. Ultraviolet irradiation of HCMV reduced the ability to induce DNA synthesis. Equilibrium density gradient centrifugation analysis of DNA which was labeled with 5-bromodeoxyuridine indicated semiconservative replication rather than repair synthesis. These results suggested that in a considerable fraction of human senescent cells host DNA replication could be reinitiated by infection with HCMV, but not by the addition of fetal bovine serum.

Autoradiography↗
PubMed1986

Events blocked in prereplicative phase in senescent human diploid cells, TIG-1, following serum stimulation.

When senescent human diploid cells, TIG-1, were stimulated with serum at the end of their proliferative life span, such biochemical events as uptakes of 2-deoxyglucose and uridine, and expression of c-myc, were enhanced. However, RNA synthesis, polyamine accumulation, thymidine uptake and DNA synthesis were not enhanced at all. Protein synthesis increased only moderately as compared with that observed in younger cells. These results indicated that the events in prereplicative phase known to be independent on protein synthesis are induced in senescent cells after the stimulation with serum, whereas those required protein synthesis failed to increase to the same extent as seen in young cells.

Blood↗
PubMed1986

Failure in S6 protein phosphorylation by serum stimulation of senescent human diploid fibroblasts, TIG-1.

When quiescent young or senescent human diploid cells, TIG-1, were metabolically labeled with 32Pi and stimulated with 10% fetal bovine serum, the phosphorylation of ribosomal S6 protein was enhanced in young cells but not in senescent cells while that of some other proteins were increased in both cells. Inability to stimulate the phosphorylation of S6 protein in senescent cells after serum addition may be the primary cause of the failure of enhancement in protein synthesis followed by the block of prereplicative events dependent on protein synthesis and thus of the failure of cells to enter S phase. However, when the cell-free preparation from serum-stimulated senescent cells was incubated with [gamma-32P]ATP, S6-kinase activity was stimulated and S6 in ribosomal fraction was susceptible to phosphorylation as observed in young cells. Differences in S6 phosphorylation of senescent cells between in vivo and in vitro was discussed.

Blood↗
PubMed1987

The effect of mild hypothermia (34 degrees C) and mild hyperthermia (39 degrees C) on DNA damage, repair and aging of human diploid fibroblasts.

We used mild hypothermia (34 degrees C) and mild hyperthermia (39 degrees C) to examine aging at the cellular level in relation to DNA damage and repair. With the filter elution technique we monitored spontaneous single-strand breaks (SSBs) and double-strand breaks (DSBs) in DNA during in vitro aging at 34 degrees C, 37 degrees C and 39 degrees C of normal human diploid fibroblasts (HDF). DNA repair was assessed after ionizing and non-ionizing (ultraviolet) radiation of HDF at different population doubling levels (PDLs): the former was assayed by filter elution and the latter by unscheduled DNA synthesis. Survival was assessed by trypan blue dye exclusion and colony formation. Cells at 37 degrees C achieve a higher cumulative PDL (67 +/- 6) than cells at 39 degrees C (60 +/- 5) or at 34 degrees C (55 +/- 6). The level of spontaneous SSBs and DSBs, and radiosensitivity of DNA to either 6 Gy or 100 Gy gamma rays, do not change with in vitro age at any of the three temperatures. Repair of SSBs (induced by 6 Gy) and DSBs (induced by 100 Gy) does not change with in vitro age: rejoining is 86-104% complete by 60 min repair and generally does not differ across temperatures. Response to non-ionizing radiation (254 nm, 75, 150, 300 ergs/mm2) does not change with in vitro age at 37 degrees C or 39 degrees C, whereas excision repair increases with age at 34 degrees C even though cell survival does not. The results do not support the rate of living theory of aging (Pearl, R., The Rate of Living, University of London Press, London, 1928) as applied to temperature effects on HDF aging in vitro (as measured by proliferative lifespan) and on their response to radiation-induced DNA damage.

Cell Division↗