Increased susceptibility of late passage human diploid fibroblasts to oxidative stress

Three strains of human diploid fibroblasts, TIG-3, TIG-7, and MRC-5, were serially cultivated. The susceptibility of early-passage and late-passage cells at 20-30 and 60-70 population doubling levels, respectively, to hydrogen peroxide, the superoxide radical (exposure to the hypoxanthine-xanthine oxidase system), or linoleic acid hydroperoxide was examined for lactate dehydrogenase release. The susceptibility of late-passage cells to such oxidative stress was considerably enhanced compared with early-passage cells. The concentration of reduced glutathione in late-passage cells was lower by 24-44% on a per-cell-number basis and by 86.0-94.5% on a per-protein-quantity basis than in early-passage cells. In addition, the activity of catalase in late-passage cells was lower by 19-46% compared with early-passage cells. There was, however, no difference between the mRNA levels of catalase in early-passage and late-passage cells. The activities and mRNA levels of copper/zinc superoxide dismutase, manganese superoxide dismutase, and glutathione peroxidase in late-passage cells were all higher than in early-passage cells. These results suggest that late-passage cells are more susceptible to oxidative stress than early-passage cells presumably because of decreases in cellular reduced glutathione concentration and catalase activity, and that their primary defense against oxidative stress is reduced glutathione.     

Effect of beta-carotene on cell cycle progression of human fibroblasts

The uptake of beta-carotene (BC) and its effect on the cell cycle progression of normal human fibroblasts in primary culture were investigated by using two different delivery methods: exposure to BC solubilized in the organic solvent tetrahydrofuran (THF) or to BC incorporated into dipalmitoylphosphatidylcholine (DPPC) liposomes. Cell cycle progression was evaluated by immunofluorescence detection and flow cytometric analysis of the proliferating cell nuclear antigen (PCNA). In contrast to THF, which induced a marked reduction in the number of cells in S phase and in the extent of PCNA immunolabeling, DPPC liposomes proved to be an effective delivery system that does not interfere with cell proliferation. Cellular uptake of 0.23 nmol/10(6) cells was found after 24 h incubation in BC-containing DPPC liposomes. This value increased to 1.2 nmol/10(6) cells after 72 h. After the first day of incubation, the number of cells in S phase was reduced by approximately 50%, with a consequent accumulation of cells in G1 phase. This effect was maintained up to 3 days incubation, with no detectable effects on cell viability. This cell cycle delay was found to be reversible, returning the percentage of cells in S phase to the control value 24 h after removal of BC from the medium. In order to determine whether the activity of BC could be attributed to the molecule itself or to its conversion into retinoids, the production of BC metabolites was assessed. Analysis of cellular levels of retinoids failed to demonstrate the presence of retinal, retinol, retinoic acid or retinyl esters during an incubation period of 6 days. These results suggest that in normal human fibroblasts, BC induces a cell cycle delay in the G1 phase and that this effect is independent of conversion to known retinoids.     

The recetor sites for complement (C3) on human diploid fibroblasts

Sheep red cells, sensitized with 19S fraction of antiserum and subsequently treated with mouse serum as the source of complement (EAC), interact with human diploid fibroblasts (WI-38 cells) and form "Rosettes". Under a scanning electron microscope, EAC have not attached directly to the cell surface of fibroblasts, but to the fine processes or microvilli of the latter, as if there were fine bridges between EAC and the surface of fibroblasts. On the other hand, the attachment of sheep red cells washed in PBS (E) or sensitized with 19S fraction of antiserum (EA) to WI-38 cells was not observed. The pretreatment of WI-38 cells with mouse serum did not inhibit the interaction of WI-38 cells and EAC. No phagocytosis of EAC by WI-38 cells was observed in the 2 hrs incubation of both cells. From these results it is suspected that the interaction of WI-38 cells and EAC is immune adherence, and that WI-38 cells have the receptor site for complement, especially for C3, on the surface of cell membrane.     

Altered protein metabolism in arrested populations of aging human diploid fibroblasts

Protein synthesis and turnover were measured in human diploid fibroblasts which were arrested in an essentially nonmitotic state by reducing the serum concentration in the incubation medium to 0.5%. Through the first 4 days of the arrested period both early and late passage cells lost about 20% of their cellular protein. There was a reduction in the rate of protein synthesis at both passage levels during this period, but there was no significant age-related difference in the synthetic rate or the rate of protein turnover. After day 4 both early and late passage cells maintained a constant protein content, but late passage cells did this while processing more protein through faster rates of both synthesis and turnover than did early passage cells. These results support those theories of cellular senescence which predict altered protein metabolism as a major consequence of the aging process.     

DNA signals for G2 checkpoint response in diploid human fibroblasts

Mammalian eggs are ovulated in metaphase II of meiosis, in a state characterized by high levels of cyclin B and of active maturation promoting factor (MPF). This arrest is mediated by an activity referred to as cytostatic factor (CSF) which prevents the degradation of cyclin. Fertilization triggers a train of Ca2+ spikes which is responsible for the decrease in activity of both MPF and CSF. The decline in MPF however much precedes that in CSF. Experimental observations on mammalian eggs indicate that the kinetics of cell cycle resumption much depends on the temporal pattern of the repetitive Ca2+ spikes. Here, we propose a theoretical model which accounts for Ca(2+)-induced relief from metaphase II arrest in mammalian eggs. The model is based on the fact that Ca2+/calmodulin kinase II (CaMKII) activation is the primary event leading to inactivation of both CSF and MPF. To account for experimental observations, it has to be assumed that CaMKII activation affects the level of the active form of the anaphase promoting complex (APC), which initiates the degradation of cyclin, through two pathways characterized by different time scales. Thus, we hypothesize that CaMKII activation by Ca2+ leads to the transformation of a mediator protein from a form which stimulates the inactivation of the APC into a form which gradually and indirectly induces the deactivation of CSF. In consequence, a sufficient number of Ca2+ spikes first triggers the decrease of MPF, thus allowing the egg to enter in interphase, and later that of CSF. Finally, when CSF is low and when Ca2+ oscillations have stopped, the level of MPF can increase again, a phenomenon that would correspond to the first mitosis. This model also accounts for the observed dependence of the time of entry in interphase (marked by the appearance of the pronuclei) on the frequency of Ca2+ spikes, as well as for the possible entry in metaphase III arrest, a pathological state of the egg which results from an insufficient activation by Ca2+. This study provides some theoretical prediction as to the time of the first mitosis as a function of the temporal pattern of Ca2+ oscillations.     

X-ray-induced mutation to 6-thioguanine resistance in cultured human diploid fibroblasts

X-ray induced mutation to 6-thioguanine (6TG)-resistance was studied in early passage cultures of human diploid fibroblasts. The appearance of phenotypic induced mutants in irradiated cell populations was linearly related to the number of post-irradiation cell doublings and to the duration of the growth period prior to mutant selection; the maximum yield of X-ray induced mutants was observed when cells surviving radiation had completed 3--4 douplings (6--7 days growth) in non-selective medium. The maximum induced mutation frequency was linearly related to X-ray dose and the mutation rate was estimated to be 3.1-10(-7) mutations per viable cell per rad. The data obtained for X-ray induced mutations in cultured human diploid fibroblasts were compared with (a) similar experimental data obtained with established cell cultures and (b) with theoretical predictions of X-ray mutation rates in human germ cells.     

The effect of high vacuum on the low cycle fatigue law

Push-pull fatigue tests have been conducted on several materials at various frequencies and temperatures in air and high vacuum (10⁻⁸ torr) and the fatigue life determined in terms of the cyclic plastic strain. In contrast to a changing exponent of the Coffin-Manson law with increasing temperature in air, in high vacuum this exponent is found to remain nearly constant at a value of about 0.5. Further, the temperature sensitivity of this exponent and of life at a specific plastic strain range in high vacuum is slight. Pronounced plastic instability (specimen shortening and fattening) was observed for the ductile metals investigated and crack nucleation was retarded. In all cases crack propagation was transgranular in vacuum. It is concluded that for the materials, temperature, and frequencies investigated, the degradation of fatigue life at elevated temperature is due to environmental enhancement of intergranular fracture. Materials investigated include A286 at room temperature and 593°C, nickel A at 550°C, 304 stainless steel at 816°C and 7075T6 aluminum alloy.     

Characterization of fractionated human diploid fibroblast cell populations

In a series of 1334 consecutive pneumoencephalographies in children 0-15 years, there were 2 deaths, 3 severe apneas and 1 pneumomediastinum. Respiratory insufficiency, due to contention of the patient with resulting impairment of the motion of thoracic muscles was an important mechanism and pneumoencephalography is contra-indicated in children with respiratory difficulties prior to the examination. In 90 consecutive patients submitted to pneymoencephalography CSF changes and fundus oculi abnormalities were studied prospectively. Aseptic meningitis of a mild degree is common following the examination, whereas significant meningeal haemorrhage is rare. Haemorrhages in the fundi were noted in 10% of the patients. The general tolerance of the examination was good.     

Differential regulation of collagenase and stromelysin mRNA in late passage cultures of human fibroblasts

Nontransformed human fibroblast cell cultures have been extensively studied as an in vitro model for cellular senescence. Recently there has been considerable interest in using the human fibroblast in the identification of genes relevant to the process of replicative senescence. We demonstrated that in comparison with early passage cultures the expression of collagenase and stromelysin mRNAs and proteins was increased > 8 x in late passage cultures of human fibroblasts and, in addition, expression of Il-1 alpha, a cytokine that regulates collagenase and stromelysin expression, was also significantly increased in late passage cell cultures. These findings suggested the hypothesis that constitutive Il-1 alpha expression in late passage cells may coordinately regulate the age-associated increase in the expression of collagenase and stromelysin. To test this hypothesis we examined the effects of long-term Il-1 alpha treatment, serum starvation, and cycloheximide inhibition on collagenase and stromelysin mRNA levels in early and late passage human fibroblast cell cultures. Here we report that in late passage cell cultures, collagenase and stromelysin mRNAs respond differentially to Il-1 alpha, serum starvation, and cycloheximide addition. Continuous exposure to Il-1 alpha reduced the half-life of stromelysin mRNA but had little effect on the half-life of collagenase mRNA. In contrast to stromelysin, the collagenase mRNA level is dependent on serum factors. Collagenase is induced during recovery from cycloheximide inhibition, but stromelysin expression is not affected. These results establish that collagenase and stromelysin mRNAs are differentially regulated in both early and late passage human fibroblasts and suggest that the mechanisms responsible for the age-associated increase in the two mRNAs are different. In addition, these studies support the conclusion that continuous long-term exposure to Il-1 alpha, a condition that is characteristic of late passage cells, is not the factor responsible for the high levels of collagenase expression, but may be critical for stromelysin expression.     

Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins

Natural background activity and food chain transfer of the uranium decay products, 210Po and 210Pb, were examined in the lichen-caribou-wolf food chain at two locations in the Northwest Territories of Canada. 210Po and 210Pb activities in lichens differed with species and location. Both 210Po and 210Pb were markedly higher in caribou bone than in wolf bone. 210Po activities in liver, kidney, and muscle were similar in both species. Caribou fetuses had lower activities of 210Po but higher activities of 210Pb than maternal muscle and placenta, suggesting greater placental transport of 210Pb than 210Po. Concentration ratios (CR = Bq kg-1 in consumer/Bq kg-1 in its food source) and ff values (ff in d kg-1 = Bq kg-1 in muscle/Bq d-1 ingested) showed that wolves retain more 210Po and less 210Pb from their diet than do caribou. 210Po CRs averaged 0.38 for caribou/lichens, 0.26 for caribou/rumen contents, and 0.40 for wolves/caribou. 210Pb CRs averaged 0.36 for caribou/lichens, 0.57 for caribou/rumen contents, and 0.13 for wolves/caribou.     

Defects in signal transduction during replicative senescence of diploid human fibroblasts in vitro

Decompression of the suprascapular nerve through the posterior approach minimizes muscular damage and postoperative scar. The difficulty with this approach is that the depth of the exposure makes operating around the delicate structures of the suprascapular artery and nerve challenging. Spine surgery instrumentation is very helpful in circumventing this problem. Once exposure is achieved, a nerve root retractor is used to retract the suprascapular artery and vein. A number 2 Woody Woodson elevator is used to protect the suprascapular nerve. A number 1 or 2 Kerrison rongeur is then used to resect the suprascapular ligament. The Kerrison rongeur is a particularly useful instrument if an ossified ligament is encountered.     

Carcinogens stimulate intrachromosomal homologous recombination at an endogenous locus in human diploid fibroblasts

Mitotic recombination is believed to play an important role in the development of many cancers. An improved system has been developed to detect reversion of an intragenic DNA duplication, as a model for intrachromosomal homologous recombination. The 'LNtd' strain of human fibroblasts, derived from a Lesch-Nyhan donor, produces no detectable hypoxanthine phosphoribosyltransferase (HPRT) activity due to a 13.7-kilobase-pair DNA insertion duplicating exons 2 and 3 of the HPRT locus. These cells are therefore sensitive to selection in HAT medium, against cells lacking functional HPRT enzyme. Clonal reversion to HAT resistance occurs spontaneously at 1-3 x 10(-5)/cell/generation, and can be induced by brief exposure to a variety of carcinogenic agents. Six known carcinogens, including two (diethylstilbestrol and nickel chloride) which were non-mutagenic in Salmonella by Ames HIS-reversion tests, showed dose-dependent induction of LNtd reversion by a maximum of 2.4- to > 11-fold over controls (each p < 0.01). In contrast, 5 non-carcinogenic agents, including two 'Ames-positive' chemicals, sodium azide and 8-hydroxyquinoline, evoked no more than a 1.7-fold increase in reversion (not significant). The molecular events associated with reversion to HAT-resistance were characterized, relative to the parental strain, in HATR clones derived from either untreated or carcinogen-treated cells. Both the intron-3:intron-1 junction situated between the duplicated HPRT segments in LNtd cells (amplified by polymerase chain reaction), and a restriction fragment corresponding to the duplicated HPRT DNA (assessed by Southern-blot hybridization), were lost from the majority of HATR revertant clones, whether they arose spontaneously or following exposure to Cr(VI) or ultraviolet light. These results imply that HATR reversion is induced in LNtd cells by carcinogenic treatments, through a mechanism consistent with homologous recombination, and is highly concordant with induction of in vivo carcinogenesis by the same agents.     

Regulation of growth of human diploid fibroblasts by factors elaborated by activated lymphoid cells

Fibroblasts growth synthesis activities appear to be under exquisite control. This control is mediated in part by substances present in blood plasma or released by other cells. We have studied the role of peripheral blood mononuclear cells (PBM) activated with phytohemagglutinin-P (PHA) on DNA synthesis, proliferation, and the cell cycle of human diploid fibroblasts. Culture medium from activated but not from unactivated PBM cultures inhibited fibroblast DNA synthesis and growth in a dose-dependent manner. The activity, which was designated as lymphocyte factor (LF), was very potent; it inhibited 50% of the DNA synthesis and cell growth at a dilution of 1:160. It has a molecular weight between 50,000 and 100,000 daltons and it is destroyed by trypsin digestion or by heating at 80 degrees C for 30 minutes. The activity was not due to the presence of prostaglandin. Furthermore, using immunoprecipitation and affinity chromatography, it was shown conclusively to to be distinctly different from alpha lymphotoxin (alpha-LT). It was not cytotoxic, as shown by the 51chromium release technique. Using flow microfluorimetry it was shown that the activity regulates fibroblast growth by preventing quiescent cells in the G0 or G1 stage of the cell cycle from entering the S phase. Cells already in S at the time of exposure complete DNA synthesis but cannot divide, and they accumulate in G2. The activity also has marked effects on protein synthesis. Activated mononuclear cells may play a major role in regulating fibroblast growth and synthesis in normally healing wounds and in acute and chronic inflammatory processes.     

Relationships between p53 induction, cell cycle arrest and survival of normal human fibroblasts following DNA damage

It is now well established that in response to genotoxic stresses mammalian cells show an increased p53 protein levels and undergo cell cycle arrest at G1/S and G2/M checkpoints. But, the consequences of these cell cycle arrests on cell survival are not yet elucidated. In this study, we have analysed the relationships between p53 protein induction, cell cycle arrest and cell survival following exposure of normal human fibroblasts (NHFs) to various genotoxic agents such as cisplatin, UV radiation and gamma radiation. p53 protein accumulation and G2/M arrest arose at the same time following exposure to DNA damaging agents, suggesting that p53 is responsible for the G2/M block. However, following inhibition of p53 induction by an antisense oligonucleotide, this G2/M arrest is even more important and correlates with an enhanced sensitivity of NHFs to UV radiation. In addition, there appears to be a threshold in the response of NHFs to DNA damaging agents, p53 induction and cell cycle arrest being observed only with lethal UV doses. We show that: 1) there appears to be a threshold in the cellular response to genotoxic agents, below which neither p53 induction, nor cell cycle arrest, nor cell survival alteration occur and beyond which p53 induction is accompanied by cell cycle arrest and decreased cell survival; 2) although there is a tight temporal relationship, the onset of which depends of the DNA damaging agent used, between the start of p53 induction and the occurrence of G2/M arrest, this latter is independent of p53; 3) p53 inhibition enhances NHFs' sensitivity to DNA damaging agents, the extent of the G2/M arrest correlating with decreased cell survival. Finally, the lack of obligatory correlation between p53 inactivation, apoptosis and radio- or chemoresistance is discussed.     

Effect of 3-aminotriazole on anchorage independence and mutagenicity in cadmium- and lead-treated diploid human fibroblasts

Mobilization of intracellular Ca2+ is a critical cellular response to lysophosphatidic acid (LPA) in many cell types. Recent identification of endothelial differentiation gene (Edg) 2 and Edg4 as subtypes of G protein-coupled receptors for LPA allowed examination of the Ca2+ mobilization mediated specifically by each subtype. To reduce endogenous background levels while enhancing recombinant receptor-specific signals, the aequorin luminescence method was used to quantify cytoplasmic Ca2+ levels. In TAg-Jurkat T cells transiently co-transfected with apoaequorin and human Edg2 or Edg4 cDNA, LPA dose-dependently increased light emission triggered by increased Ca2+ bound to aequorin. N-Palmitoyl-L-serine-phosphoric acid and N-palmitoyl-L-tyrosine-phosphoric acid, which had been previously shown to be antagonists for Xenopus laevis LPA receptors, did not antagonize the Ca2+-mobilizing effects of Edg2 and Edg4. Surprisingly, they acted as agonists or partial agonists for Edg2 and Edg4. The Ca2+ mobilization by Edg2 and Edg4 was further characterized in stable transfectants of rat HTC4 hepatoma cells. By using the fura-2 fluorescence method, a difference in the kinetics of Ca2+ flux with Edg2 and Edg4 was observed. With Edg2, but not Edg4, the initial increase in the Ca2+ concentration was followed by a sustained influx of extracellular Ca2+. The coincident production of inositol phosphates and the inhibition of Ca2+ mobilization by the phospholipase C inhibitor U73122 strongly suggested that Edg2 and Edg4 mobilize Ca2+ through inositol trisphosphate generated by phospholipase C activation. Pertussis toxin almost completely blocked LPA-induced Ca2+ mobilization by Edg2 but only partially blocked that by Edg4, which suggests that Edg2 transduces Ca2+ mobilization largely through pertussis toxin-sensitive Gi proteins, whereas Edg4 requires both Gi and Gq.     

The high temperature low cycle fatigue behavior of the nickel base alloy IN-617

Fully reversed strain controlled LCF tests were performed on 4.5 mm thick sheet specimens IN-617 at 1033 K and 1144 K in air. The strain-life and cyclic stress-strain data were analyzed parametrically. While the inelastic strain amplitude-life relationships are similar at the two temperatures, the softer cyclic stress-strain relationship observed at 1144 K produces inferior fatigue resistance to that at 1033 K when comparisons are based on stress or total strain amplitude. Transmission electron microscope observations suggest that grain boundary sliding was the primary mechanism of deformation at 1144 K while at 1033 K intragranular slip produced a dense dislocation substructure which was stabilized by fine scale precipitation of M₂₃C₆. At 1144 K, grain boundary migration occurred within the specimen interiors, producing a cellular precipitation of M₂₃C₆ as well as the intragranular M₂₃C₆ observed at 1033 K. Cellular precipitation was not observed in the near surface regions. This is attributed to oxygen penetration along grain boundaries. Comparison of the data with mean stress-HCF data indicates that the mean load significantly reduces fatigue resistance at both temperatures.     

Levels of DNA polymerases alpha, beta, and gamma in control and repair-deficient human diploid fibroblasts 1

The activities of DNA polymerases alpha, beta, and gamma were determined in control and repair-deficient human fibroblasts (xeroderma pigmentosum complementation groups A, C, and D; Fanconi's Anemia; and Bloom's syndrome). Assays were done on 103,000XG supernatants which had been chromatographed on DEAE cellulose to remove nucleic acids and on fractions containing polymerase activities which had been separated from one another on a second DEAE cellulose column. All repair-deficient cell types contained all three DNA polymerase activities. Caffeine, which has been observed to inhibit some DNA-repair processes in intact cells, had no effect on DNA polymerase activities from XP-A, XP-C, XP-D or XP-variant cells. These data indicate that all three polymerases are present in cells which have reduced or absent repair functions and that the caffeine effects observed in living cells are probably not due to the direct action of caffeine on DNA polymerases.     

Photosensitization of human diploid cell cultures by intracellular flavins and protection by antioxidants

Semicarbazide hydrochloride (0.1 M in glass-distilled water), on injection, showed mutagenic action on the spermatocyte chromosomes of the grasshopper, Spathosternum prasiniferum. Aberrations such as chromatid and chromosome breaks, translocations, fragments and bridges were encountered. The sex chromosome and the long autosomes were affected. Semicarbazide, perhaps, reacts with DNA and the chromosome in a way similar to that of hydroxylamine and hydrazines.