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PubMed1984

Histone acetylation and deacetylation in senescent human diploid fibroblasts.

We measured the kinetics of histone H4 hyperacetylation and kinetics of deacetylation for all core histones in young and senescent human diploid fibroblast-like cells. Both cell populations contained a fraction of histone H4 characterized by very rapid acetylation and deacetylation. The kinetics of these reactions were similar in young and senescent cells. The distribution of acetylated species of core histones was also similar in young and senescent human diploid fibroblast-like cells. These results indicated that previously-reported alterations in chromatin template activity in senescent cells were due to a mechanism other than histone acetylation.

Acetylation↗

PubMed1984

The effects of in vitro ageing on the exopeptidases of human diploid fibroblasts.

Six exopeptidases present in human diploid fibroblasts were identified by separation on polyacrylamide gel electrophoresis and their activity profiles against 17 dipeptides, two tripeptides and L-leucine-p-nitroanilide determined. No differences in relative activity or in the electrophoretic patterns of any of the six exopeptidases were detected with ageing. Aminoacylarylamidase activity assayed spectrophotometrically showed significantly increased activity in the middle age-group cells as opposed to the enzyme isolated from young and old cells. Heat-inactivation studies using the same substrate suggested the possibility of an increased proportion of heat-labile enzyme in the old cells but interpretation of the data was difficult because of the complex nature of the inactivation curves obtained. Overall, the results tended to refute the hypothesis that age-related changes in the free amino acid pool of human diploid fibroblasts were associated with significant alterations in the activities of cellular exopeptidases.

Cell Survival↗

PubMed1984

Human diploid fibroblasts with alterations in ribonucleotide reductase activity, deoxyribonucleotide pools and in vitro lifespan.

Three drug resistant human diploid fibroblast clones were isolated which contained elevated levels of ribonucleotide reductase activity when compared to wild type fibroblasts. The drug resistant cells do not appear to possess an enzyme with altered affinity for hydroxyurea. The increase in enzyme activity can entirely account for cellular drug resistance. In keeping with the observed changes in reductase activity in drug resistant fibroblasts, deoxyribonucleotide pools were also found to be altered. Most significantly, there was a 1.8-fold expansion of the dCTP pool size, suggesting that elevation in intracellular dCTP concentrations plays an important role in cellular resistance. Furthermore, the drug resistant fibroblasts exhibited substantial reductions in their replicative abilities, suggesting that the regulation of ribonucleotide reductase and the accompanying deoxyribonucleotide pools in human diploid cells is involved in aspects of cellular senescence.

Cell Survival↗

PubMed1985

Ribonucleotide reductase activity during the senescence of normal human diploid fibroblasts in culture.

Changes in ribonucleotide reductase occur during the senescence of normal human diploid fibroblasts in culture. Enzyme activity is significantly lower in cells and extracts at high mean population doublings (MPD) as compared to fibroblasts at low MPD. Although many of the kinetic properties of the enzyme remain unaltered in cells at high MPD, changes in the extent and kinetic mechanism of inhibition of CDP and ADP reductase activity by dATP, the overall negative effector of ribonucleotide reductase, were observed. These results and a previous observation that the four deoxyribonucleotide pools are markedly altered during in vitro senescence of human diploid fibroblasts, provide evidence for a link between ribonucleotide reduction, deoxyribonucleotide pools, and the establishment of the non-proliferative or senescent state.

Cell Division↗

PubMed1985

Reduction of filamin in late passage human diploid fibroblasts (IMR-90).

Progressive subcultivation of IMR-90 cells results in non-proliferative, heterogeneous cultures which may reflect aging of the diploid line (Hayflick, Exp. Cell Res., 37 (1965) 614). We have observed that late passage cells exhibit different rates of spreading and morphogenesis when compared to early passage groups, phenomena which we attribute to altered reassembly of the cytoskeleton in senescent cells (Kelley et al. Mech. Ageing Dev., 13 (1980) 127). To determine whether potential differences in cytoskeletal proteins develop with progressive subcultivation, early and late passage cultures were extracted with 0.5% Triton X-100 for 1 min followed by 1.0% sodium dodecyl sulfate (SDS) prior to separation and characterization of extracted proteins by electrophoresis on 7.5-15% gradient SDS gels. Extractions were made of both culture groups 3, 6 and 24 h after reseeding. Cytoskeletal ultrastructure at each stage of spreading was examined either in replicas of extracted cells or directly by scanning electron microscopy. Although considerable variation in cytoskeletal organization was observed, qualitative differences in gel banding patterns of actin, myosin and tubulin were not apparent at selected time points. However, late passage cells at 6 h and 24 h did not exhibit filamin associated with the Triton insoluble fraction as did early passage cells. Since it has been demonstrated that filamin is capable of cross-linking actin microfilaments into bundles or sheets, we suggest that it is a principal element for the variant cell shape and cytoskeletal morphology observed during altered spreading behavior of late passage human diploid fibroblasts.

Cell Survival↗

PubMed1985

Membrane glycoprotein changes during the senescence of normal human diploid fibroblasts in culture.

The plasma membrane is intimately involved in a variety of cellular phenomena and may play an important role in the aging of human cells in culture. Significant differences in human diploid fibroblast surface glycoproteins were observed with in vitro aging. Senescent cells bound more concanavalin A (Con A) than young cells and exhibited two distinct classes of binding sites. Cell surfaces of senescent cells incorporated less labelled mannose and more labelled fucose and glucosamine than young cell surfaces. Membranes prepared from older cells were also less effective than membrane preparations from young cells in incorporating mannose from GDP-mannose into a group of oligolipid intermediates, required for the synthesis of glycoproteins with asparagine-linked oligosaccharides. These results demonstrate novel quantitative changes in membrane structure and lectin reactivity in aging human diploid fibroblasts, which must reflect the fundamental physiological modifications in the cell that occur during senescence.

Cell Membrane↗

PubMed1987

Lack of recovery from oxygen-induced damage to colony formation and DNA synthesis in senescent human diploid fibroblasts.

The colony formation and DNA synthesis of cultured human fibroblasts were inhibited under hyperbaric oxygen. When maintained in a growth-arrested state for a few days after oxygen exposure, early passage diploid and SV-40-transformed cells repaired the oxygen-induced damage, while late passage diploid cells did not. In early passage cells to which cycloheximide, a protein synthesis inhibitor, was administered, no recovery from the damage to colony formation was found, indicating that de novo protein synthesis is necessary to the repair process. The repair function of the late passage cells against oxidative damage seems to be impaired. Oxidative stress may be involved in cellular ageing.

Cell Division↗

PubMed1988

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.

Cell Division↗

PubMed1993

Escape from in vitro aging in SV40 large T antigen-transformed human diploid cells: a key event responsible for immortalization occurs during crisis.

A human diploid fibroblast strain MRC-5 was transfected with a replication origin-defective early region of SV40 containing the gene of large T antigen, and 48 clones of T antigen-transformed MRC-5 were isolated. Although T antigen prolonged the lifespan of MRC-5, all the transformed clones were still mortal. From two of the transformed MRC-5 clones, eight independent immortalized clones were obtained in triple experiments of immortalization. The transformed phenotypes of immortalized clones were widely varied and did not always retain those of the parental pre-immortalized clones. The immortalization occurred at the frequency of about 1-3 x 10(-7)/cell. From cell number and population doubling time of the immortalized clones, the immortalization was estimated to occur in or just before the crisis of parental mortal cells. The decreases of modal chromosome numbers and the loss of chromosomes 5 and 10 were found to be common in three independent immortalized clones examined. Thus, the escape from the cellular aging process seemed to be caused by certain genetic events including the loss of chromosomes at the end of lifespan in T antigen-transformed human diploid cells.

Aneuploidy↗

PubMed1995

Relevance of oxidative stress to the limited replicative capacity of cultured human diploid cells: the limit of cumulative population doublings increases under low concentrations of oxygen and decreases in response to aminotriazole.

Human diploid cells, TIG-7, were serially cultivated under 1%, 5% or 21% (air) oxygen. The limit of their cumulative population doublings was extended by 16% or 10% under 1% or 5% oxygen, respectively, as compared with that under 21% oxygen. When TIG-7 cells were serially cultivated in the presence of 10 mM or 20 mM 3-amino-1H-1,2,4-triazole, an inhibitor of catalase scavenging hydrogen peroxide, the limit of their cumulative population doublings was shortened by 4.4% or 14.4%, respectively, as compared with untreated cells. In addition, long-term, rather than short-term, exposure of TIG-7 cells to aminotriazole retarded cell growth. Treatment with aminotriazole caused decreases not only in catalase activity but also in superoxide dismutase activity and reduced glutathione concentration, and an increase in glutathione peroxidase activity. These results suggest that the limit of the cumulative population doublings of human diploid cells is extended or shortened under decreasing or increasing oxidative stress, respectively. Oxidative stress may be relevant to replicative capacity, and a causative factor for oxidative stress may be hydrogen peroxide.

Amitrole↗

PubMed1989

Microencapsulation of human diploid fibroblasts in cationic polyacrylates.

Human diploid fibroblasts and Chinese hamster ovary cells were encapsulated in several copolymers of dimethylaminoethyl methacrylate with methacrylic acid and/or methyl methacrylate. Copolymers containing 16 to 25% dimethylaminoethyl methacrylate and less than or equal to 2.2% methacrylic acid (based on monomer mol%) supported human diploid fibroblast growth when the polymer was cast as a film on glass or polystyrene. The cells survived encapsulation and grew, but growth was only observed in those capsules which appeared to be flawed; the flaws were detected as an early loss of fluorescence, due to leakage of the FITC-dextran added as a marker to the encapsulated cell suspension. Presumably the capsule wall had too low a permeability to allow for unrestricted growth. Chinese hamster ovary cells behaved similarly in dimethylaminoethyl methacrylate/methyl methacrylate capsules. Increasing the water content, by addition of methacrylic acid, did not improve matters, since these materials were not as good a substrate for cell growth as the others. Preparing materials that are sufficiently permeable, with low toxicity and high processability and which support the growth of anchorage-dependent cells is difficult, yet it remains an appropriate goal for further study.

Acrylates↗

PubMed1994

Diploid and hyperdiploid rat Schwann cell strains displaying negative autoregulation of growth in vitro and myelin sheath-formation in vivo.

Neonatal rat Schwann cells were cultured for several months with intermittent exposure to the mitogen, cholera toxin, and infrequent passaging to avoid premature transformation. A cell line SCL4.1/F7 was derived following the cloning of one of these long-term cultures by limiting dilution in liquid medium to select for cells capable of continuous proliferation in the absence of mitogen. F7 cells have been passaged 40 times (80-120 generations) over 14 months. Two substrains were identified at passage 20, one of which ,s diploid and the other which has trisomy 7 (t7). The cell line displays a characteristic flattened or crescent-shaped morphology, substratum adhesion which is calcium-dependent in the millimolar range, and pronounced contact-inhibition of growth. Confluent or subconfluent cultures readily cease proliferation and change to a differentiated (stellate/bipolar) morphology through the mediation of an autocrine growth-inhibitory factor. F7 cells grafted into the site of a crush injury in adult rat sciatic nerves remained viable and myelinated host axons. F7 is the first clonally derived diploid immortal Schwann cell line to have been published and should provide a suitable tool for the study of the biochemical and cellular basis of sheath cell-neuron interactions, myelin stabilization in peripheral nerve and Schwann cell growth autoregulation.

Animals↗

PubMed1978

Quantification of chemical mutagenesis in diploid human fibroblasts: induction of azaguanine-resistant mutants by N-methyl-N'-nitro-N-nitrosoguanidine.

The toxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on cultured diploid human fibroblasts were studied. When 10(5) cells per 60 mm dish were exposed to MNNG for 4 h in Ham's medium F10 containing 0.02 M HEPES buffer at pH 6.8, MNNG concentrations of less than 1 X 10(-6) M resulted in cell survivals near 100%, while the average survival was less than one percent at concentrations greater than 5 X 10(-6) M. After treatment with MNNG, cells were subjected to selection using optimal conditions for the detection of diploid human fibroblasts that are resistant to the guanine-analogs AG and TG because they contain altered or deficient HPRT. The induced mutant frequency was maximized by allowing a 5 to 7 day post-treatment interval for the expression of the mutant phenotype and by replating the cells at the beginning of selection at a population density of less than 450 cells per cm2. Careful attention was given to counting statistically adequate numbers of mutants and to accurately determining cell survival and replating cloning efficiencies. Independent dose-response experiments gave induced mutant frequencies as high as 7.0 X 10(-4) to 8.8 X 10(-4) mutants per viable cell at about 5% survival, compared to a spontaneous mutation rate of 3.7 X 10(-6) to 7.2 X 10(-6) mutants per cell generation. The AGr mutants observed after treatment with MNNG were phenotypically stable and closely resembled prototype AGr cultures derived from humans who have inherited mutant X-chromosomal alleles for HPRT.

Azaguanine↗

PubMed1985

Growth of diploid cells from breast cancers.

Cell cultures were derived from normal and cancerous breast tissues and from metastases by methods that selected for relatively adherent epithelial aggregates. Karyotypic analyses of first or second passage cultures yielded predominantly normal diploid cells. Nonclonal aberrations were more common in tumor-derived than in normal cultures. Three of the cultures that originated from metastases were characterized by abnormal clones. These results support observations based on DNA content, which indicate that a considerable fraction of breast cancers are composed predominantly of diploid cells. They differ greatly from chromosomal findings in long-term cultures of tumor effusions and thus emphasize the karyotypic diversity that can be found in tumors from a single tissue of origin--the breast.

Adolescent↗

PubMed1990

A case of malignant mastocytosis with near-haploid, near-diploid, and polyploid cells in the bone marrow.

Chromosome analysis in bone marrow cells of a 41-year-old woman with malignant mastocytosis (MM) revealed near-haploid (59%), near-diploid (36%), and polyploid (5%) cells. Near-haploid cells had a mode of 25 chromosomes with variable disomy and nullisomy of particular chromosomes. Disomy for chromosome 19 and chromosome 20 were most common. A 17p+ marker was observed in all cells. Other structural chromosomal changes were dicentrics, ring chromosomes, translocations, and double minutes. The modal chromosome number in near-diploid cells was 46 with a range of 41-52. The majority of cells with 46 chromosomes were pseudodiploid and only a few had a normal female karyotype. As in near-haploid cells, various structural chromosomal changes were found. The 17p+ marker was also present in occasional cells suggesting a relationship between the two tumor cell populations. Chromosome numbers in polyploid cells ranged from 70 to about 600. Identical copies of dicentrics or markers were identified in some cells.

Adult↗

PubMed1984

Cytotoxicity, chromosome aberrations and unscheduled DNA synthesis in cultured human diploid fibroblasts induced by sodium fluoride.

The effects of exposure of cultured human diploid fibroblasts (JHU-1 cells) to sodium fluoride have been studied with respect to cytotoxicity and induction of chromosome aberrations and unscheduled DNA synthesis (UDS) Cytotoxicity of NaF on JHU-1 cells, as determined by a decrease in colony-forming ability, linearly increased with increasing dose of NaF (50-150 micrograms/ml) or exposure time (1-24 h). Treatment of the cells with 50 micrograms/ml NaF for 24 h resulted in a lethality (approximately 70%) similar to that obtained with 100 micrograms/ml for 12 h. A linear increase in cytotoxicity was observed as a fraction of the product of NaF treatment time and dose. JHU-1 cells treated with 20-50 micrograms/ml NaF for 12 or 24 h were analyzed for chromosome aberrations. A significant increase in the frequency of chromosome aberrations at the chromatid level was observed in treated cells in a dose-dependent manner. For detection of UDS, confluent JHU-1 cells were cultured with medium containing low serum and then exposed to NaF in the presence of 10 mM hydroxyurea. Treatment with 100-400 micrograms NaF/ml for 4-24 h reproducibly elicited UDS in a dose-related fashion as determined by direct scintillation counting of [3H]thymidine incorporated into DNA during repair synthesis. These results suggest that NaF causes DNA damage in human diploid fibroblasts in culture.

Cell Survival↗

PubMed1990

Induction of anchorage-independent growth in human diploid fibroblasts by the cyclopenta-polycyclic aromatic hydrocarbon, benz[l]aceanthrylene.

Cyclopenta-fused polycyclic aromatic hydrocarbons are a class of environmental PAH that have been recently identified. Many of these chemicals have been found to be more active than benzo[a]pyrene in tests for genetic toxicity using bacterial and rodent cells. Benz[l]aceanthrylene, a cyclopenta-polycyclic aromatic hydrocarbon related to benz[a]anthracene, and benzo[a]pyrene were compared for their activity to induce cytotoxicity and anchorage-independent growth with normal human diploid fibroblasts. Both benz[l]aceanthrylene and benzo[a]pyrene were relatively non-cytotoxic to normal human diploid fibroblasts. However, benz[l]aceanthrylene was twice as active compared to benzo[a]pyrene over the concentration range examined as an inducer of anchorage-independent growth. The ability of benz[l]aceanthrylene to induce anchorage-independent colony growth in normal human cells, in combination with its demonstrated ability as a mouse-skin tumorigen, suggests this PAH to be a potential multi-species carcinogen.

Benz(a)Anthracenes↗

PubMed1986

A sensitive, enzymatic assay for the detection of closely opposed cyclobutyl pyrimidine dimers induced in human diploid fibroblasts.

A sensitive, enzymatic assay has been developed for the detection of closely opposed cyclobutyl pyrimidine dimers induced in UV-irradiated human diploid fibroblasts. In this assay closely opposed dimers are detected as bifilar enzyme-sensitive sites. Single-strand incisions are made at the positions of individual pyrimidine dimers through the action of M. luteus pyrimidine dimer-DNA glycosylase. Incisions at closely opposed dimers, effectively expressed as double-strand breaks, are quantified from the resulting reduction in DNA double-strand molecular weight as determined by velocity sedimentation through neutral sucrose density gradients. The stability of the bacteriophage lambda cos site under our reaction conditions indicates that opposed incisions must be relatively close to be expressed as a double-strand break. The dose response for the induction of bifilar enzyme-sensitive sites in mammalian cells was found to be complex but can be approximated by a function that increases as the 1.2-1.4 power of UV dose. The frequency of bifilar enzyme-sensitive sites observed decreased during postirradiation incubation of excision-repair-proficient human diploid fibroblasts with less than 20% still detectable at 24 h after irradiation with 5 J/m2 (254 nm). By comparison, over 80% of the bifilar enzyme-sensitive sites induced in fibroblasts assigned to xeroderma pigmentosum complementation group A remained detectable 24 h after irradiation. The implications of these results for models addressing the induction and repair of closely opposed pyrimidine dimers are discussed.

Cells, Cultured↗