Sulfate transport in human lung fibroblasts (IMR-90)

Sulfate transport in a fibroblast cell line derived from human lung (IMR-90) occurred mainly via high- and low-affinity, SITS-sensitive pathways and to a lesser extent by an SITS-insensitive mechanism. In low-ionic-strength media (sucrose substituted for salts) the apparent Km of the carrier-mediated sulfate influx was 1 mM. At 0.3 mM, the sulfate concentration normally found in human serum, the contribution of the SITS-insensitive pathway was negligible. In physiological salts solution, an SITS-sensitive, high-affinity (Km 34 +/- 14 microM) sulfate influx system was observed at extracellular sulfate concentrations less than 100 microM. Between 100 and 500 microM sulfate, the range normally found in human serum, sulfate influx occurred via an SITS-sensitive, low-affinity pathway and to a small extent by an SITS-insensitive mechanism. Extracellular chloride inhibited the influx and stimulated the efflux of sulfate. Bicarbonate and thiosulfate inhibited sulfate influx but had no effect on sulfate efflux. Phosphate, arsenate, or Na+ did not affect sulfate uptake. These results indicate that in human lung fibroblast IMR-90 cells sulfate is transported mainly via an SO4(2-)/Cl- exchange system independent of the phosphate or Na+ transport. Since sulfate concentration as high as 50 mM only slightly increased sulfate efflux, SO4(2-)/SO4(2-) exchange is probably a minor component of sulfate uptake.     

Degradation of proteins microinjected into IMR-90 human diploid fibroblasts

Erythrocyte ghosts loaded with 125I-labeled proteins were fused with confluent monolayers of IMR-90 fibroblasts using polyethylene glycol. Erythrocyte-mediated microinjection of 125I-proteins did not seriously perturb the metabolism of the recipient fibroblasts as assessed by measurements of rates of protein synthesis, rates of protein degradation, or rates of cellular growth after addition of fresh serum. A mixture of cytosolic proteins was degraded after microinjection according to expected characteristics established for catabolism of endogenous cytosolic proteins. Furthermore, withdrawal of serum, insulin, fibroblast growth factor, and dexamethasone from the culture medium increased the degradative rates of microinjected cytosolic proteins, and catabolism of long-lived proteins was preferentially enhanced with little or no effect on degradation of short-lived proteins. Six specific polypeptides were degraded after microinjection with markedly different half-lives ranging from 20 to 320 h. Degradative rates of certain purified proteins (but not others) were also increased in the absence of serum, insulin, fibroblast growth factor, and dexamethasone. The results suggest that erythrocyte- mediated microinjection is a valid approach for analysis of intracellular protein degradation. However, one potential limitation is that some microinjected proteins are structurally altered by the procedures required for labeling proteins to high specific radioactivities. Of the four purified proteins examined in this regard, only ribonuclease A consistently showed unaltered enzymatic activity and unaltered susceptibility to proteolytic attack in vitro after iodination.     

Regulation of intracellular protein degradation in IMR-90 human diploid fibroblasts

Human diploid fibroblasts (IMR-90) regulate their overall rates of proteolysis in response to the composition of the culture medium and the ambient temperature. The magnitude and, in some cases, the direction of the response depend on the half-lives of the cellular proteins that are radioactively labeled and the time chosen for measurements of protein degradation. Fetal calf serum, insulin, fibroblast growth factor, epidermal growth factor, and amino acids selectively regulate catabolism of long-lived proteins without affecting degradation of short-lived proteins. Fetal calf serum reduces degradative rates of long-lived proteins and is maximally effective at a concentration of 20%, but the effect of serum on proteolysis is evident only for the first 24 hr. Insulin inhibits degradation of long-lived proteins in the presence or absence of glucose and amino acids in the medium, but is maximally effective only at high concentrations (10(-5) M). Amino acid deprivation increases degradative rates of long-lived proteins for the first 6 hr, but then decreases their catabolism for the subsequent 20 hr. Lowered temperature is the only condition tested that significantly alters degradative rates of short-lived proteins. Although cells incubated at 27 degrees C have reduced rates of degradation for both short-lived and long-lived proteins compared to cells at 37 degrees C, lowered temperature reduces catabolism of long-lived proteins to a greater extent.     

The structure and amount of actin in IMR-90 fibroblasts.

Double-labelling and peptide isolation have been used to examine the homology between the actin of IMR-90 human embryo fibroblasts and muscle actin. After separation of mixtures of [14C]carboxymethylated muscle actin and [3H]carboxymethylated proteins of IMR-90 cells of electrophoresis on sodium dodecyl sulphate-containing polyacrylamide gels, peptides were generated from the material co-migrating with actin by digestion with chymotrypsin. Peptides homologous with peptides accounting for Cys-217, Cys-256, Cys-284 and Cys-373 of muscle actin are present in this material, but no peptide homologous with a Cys-10-containing peptide was detected. From the amount of actin-derived peptides present, the actin content of IMR-90 fibroblasts was calculated to be 4.2% of the total protein of these cells.     

Cell-surface glycosaminoglycans: Turnover in cultured human embryo fibroblasts (IMR-90)

As constituents of both extracellular matrix and the cell surface, glycosaminoglycans are in a strategic position to influence several basic cell features. The localization and turnover of glycosaminoglycans was investigated in cultured normal human embryo fibroblasts of lung origin (IMR-90). Attention was directed particularly toward that compartment of the culture which could be released by gentle proteloysis (trypsin, 0.1 mg/ml, 15 min) and is considered to represent the cell surface. In the presence of Na₂SO₄, sulfated glycosaminoglycans (S-GAGs) of the cell surface were labeled rapidly, but within 30 min some ³⁵S-GAG appeared in the extracellular medium. The intracellular pool of S-GAGs labeled during a 10-min period was lost during the first hr of chase with a half-life of 18 min, compared with 16 hr for S-GAGs labeled over a 48-hr period. Pulse-labeled S-GAGs of the surface turned over with an initial half-life of 60 min, compared with 7 hr for surface material labeled over a 48-hr period. These rapid movements of the early chase period were followed by similar movement at a much slower rate. The results are consistent with a model in which most of the S-GAGs synthesized in the cell move rapidly to the surface. The surface GAGs are then released immediately to the medium or accumulate at the membrane to be shed more slowly at a later time or to be degraded. The S-GAG which left the cell layer most rapidly during chase was dermatan sulfate, while heparan sulfate made up an increasing percentage of the cell layer as chase progressed. These cultures produce a fibrillar matrix of fibronectin, but the kinetics of this study suggest that the S-GAGs of the surface are membrane-bound, and an extracellular glycosaminoglycan matrix does not form.     

Synthesis of cytoskeletal and contractile proteins by cultured IMR-90 fibroblasts

Models of the assembly of cytoskeletal and contractile proteins of eukaryotic cells require quantitative information about the rates of synthesis of individual component proteins. We applied the dual isotope technique of Clark and Zak (1981, J. Biol. Chem., 256:4863-4870) to measure the synthesis rates of cytoskeletal and contractile proteins in stationary and growing cultures of IMR-90 fibroblasts. Fibroblast proteins were labeled to equilibrium with [14C]leucine over several days, at the end of which there was a 4-h pulse with [3H]leucine. Fractional synthesis rates (percent per hour) were calculated from the 3H/14C ratio of cell protein extracts or protein purified by one- or two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of medium-free leucine. The average fractional synthesis rate for total, SDS- or urea-soluble; Triton-soluble; and cytoskeletal protein extracts in stationary cells each was approximately 4.0%/h. The range of values for the synthesis of individual proteins from total cell extracts or cytoskeletal extracts sliced from one-dimensional gels was similar, though this range was greater than that for major proteins of Triton-soluble protein extracts. Three specific cytoskeletal proteins--actin, vimentin, and tubulin--were synthesized at similar rates that were significantly slower than the average fractional synthesis rate for total protein. Myosin, on the other hand, was synthesized faster than average. Synthesis rates were the same for beta-and gamma-actin and polymerized (cytoskeletal extract) vs. Triton-soluble actin. The same was true for alpha- and beta-tubulin and two different forms of vimentin. Synthesis rates were uniformly higher in growing cells, though the same pattern of differential rates was observed as for stationary cells. Synthesis rates in growing cells were higher than the rate necessary to maintain the growth rate, even for those cytoskeletal proteins being synthesized slowly. Therefore, there appears to be some turnover of these cytoskeletal elements even during growth. We conclude that proteins in cytoskeletal extracts may have nonuniform rates of synthesis, but at least one important subclass of cytoskeletal proteins that comprise filament subunits have the same synthesis rates.     

Interaction of lactoperoxidase with hydrogen peroxide. Formation of enzyme intermediates and generation of free radicals

Peroxidases belong to a group of enzymes which catalyze the oxidation of numerous organic and inorganic substrates by hydrogen peroxide. Most peroxidases, including lactoperoxidase (LPO), contain ferriprotoporphyrin IX as a prosthetic group. A characteristic feature of hemoprotein peroxidases is their ability to exist in various oxidation states. There are five known enzyme intermediates. In increasing order of their oxidative equivalents these are ferrous enzyme, ferric or native enzyme, Compound II, Compound I, and Compound III (sections 5, 7). They are readily distinguished from each other by their absorbance in the Soret region (380-450 nm) and visible range (450-650 nm). In the course of Compound III and Compound II conversion back to the native peroxidase, oxygen derived free radicals such as O2-, HO.2, and .OH are generated. Simultaneously the enzyme is irreversibly damaged. In the presence of an exogenous electron donor, such as iodide, the interconversion between the various oxidation states of the peroxidase is markedly affected. Compound II and/or Compound III formation is inhibited, depending on the H2O2 concentration. In addition, the enzyme is largely protected from irreversible inactivation. These effects of iodide are readily explained by 1) the two-electron oxidation of iodide to Iox by Compound I, which bypasses Compound II as an intermediate, and 2) the rapid oxidation of H2O2 to O2 by the oxidized species of iodide which prevents the generation of oxygen derived free radicals.     

Superoxide and hydrogen peroxide in oxygen damage.

Escherichia coli were damaged and killed by exposure to hyperbaric oxygen. Lethality was measured as the decrease in the number of colonies formed upon plating the exposed cells onto rich agar. Damage was assessed by plating onto both rich and minimal agar. Cells which gave rise to visible colonies on rich but not on minimal agar were considered to be damaged. That this differential colony count was largely due to reparable damage rather than to stable mutagenesis was shown by replica plating from the rich onto the minimal agar. Most of the cells which had been unable to grow when directly plated onto minimal agar regained this ability after growth upon rich agar. Repair of the damage imposed by exposure to oxygen was thus more readily accomplished on a nutritionally rich medium. The enzymes superoxide dismutase, catalase, and peroxidase appeared to protect against oxygen damage. It is thus likely that both O₂^? and H₂O₂ are important agents of oxygen toxicity. In accord with this conclusion were the observations that augmented intracellular levels of these enzymes correlated with increased resistance towards oxygen damage, whereas increased respiratory capacity correlated with increased sensitivity towards hyperbaric oxygen.     

Production of proteoglycans by human lung fibroblasts (IMR-90) maintained in a low concentration of serum.

Maintenance of fibroblasts in 0.5% serum results in viable but non-proliferative cells that may be analogous to fibroblasts in vivo. The synthesis of proteoglycans by human embryo lung fibroblasts in Eagle's minimal essential medium with 0.5% newborn-bovine serum or with 10% serum has been compared. A similar amount of [35S]sulphate-labelled glycosaminoglycan per cell was secreted by fibroblasts in 10% or 0.5% serum. 35SO42-incorporation into sulphated glycosaminoglycans was enhanced in 0.5% serum when expressed per mg of cell protein, but [3H]glucosamine incorporation was decreased. The charge density of these glycosaminoglycans was not changed as determined by ion-exchange chromatography. It was concluded that decreased protein/ cell resulted in an apparent increase in 35S-labelled glycosaminoglycan synthesis/mg of cell protein, whereas decreased uptake of [3H]glucosamine resulted in a decrease in their glucosamine labelling. The proteoglycans secreted by fibroblasts in 0.5% serum were similar in glycosaminoglycan composition, chain length and buoyant density to the dermatan sulphate proteoglycan, which is the major secreted component of cells in 10% serum. Larger heparan sulphate and chondroitin sulphate proteoglycans, which comprise about 40% of the total secreted proteoglycans of cultures in 10% serum, were greatly diminished in the medium of cultures in 0.5% serum. The proteoglycan profile of medium from density-inhibited cultures in 10% serum resembles that of proliferating cultures, indicating that lack of proliferation was not responsible for the alteration. The dermatan sulphate proteoglycan, participating in extracellular matrix structure, may be the primary tissue product of lung fibroblasts in vivo.     

A marked increase of fucosylation of glycoproteins in IMR-90 human diploid fibroblasts during senescence in vitro

Possible changes of glycoproteins in IMR-90 human embryonic lung fibroblasts during senescence in vitro were studied by the metabolic labeling technique using radioactive precursors for carbohydrate moieties of glycoproteins. IMR-90 fibroblasts at three different population doubling level (PDL) were incubated with [3H]fucose and [3H]glucosamine for various periods of time. The radioactively labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. The results indicated a marked increase, by more than eight-fold on per mg protein basis, of labeling by [3H]fucose in old IMR-90 fibroblasts (PDL = 45) as compared to young (PDL = 22) and middle-age (PDL = 30) IMR-90 fibroblasts. In contrast, no significant difference in [3H]glucosamine labeling was observed in young and old IMR-90 cells.     

Regulation of ornithine decarboxylase and other cell cycle-dependent genes during senescence of IMR-90 human diploid fibroblasts

Aging of IMR-90 human diploid fibroblasts in vitro is accompanied by significant changes of polyamine metabolism, most notably, a 5-fold decrease of serum-induced activity of ornithine decarboxylase, the key enzyme in the biosynthesis of polyamines (Chen, K. Y., Chang, Z. F., and Liu, A. Y.-C. (1986) J. Cell. Physiol. 129, 142-146). In this paper, we employed Northern blot hybridization and affinity radiolabeling techniques to investigate the molecular basis of this age-associated change of ornithine decarboxylase activity. Since the induction of ornithine decarboxylase by serum is a mid-G1 event, we also examined expressions of other cell cycle-dependent genes that are induced before and after the mid-G1 phase to determine if their expressions may also be age-dependent. Our results demonstrated a 3-fold decrease of the amount of active ornithine decarboxylase molecules that can be labeled by alpha-difluoromethyl[3H]ornithine in senescent IMR-90 cells (population doubling level (PDL) = 52) as compared to young cells (PDL = 22). However, the levels and kinetics of induction of ornithine decarboxylase mRNA in both young and senescent IMR-90 cells were found to be identical throughout a 24-h time period after serum stimulation. The time course and the magnitude of the expression of c-myc, an early G1 gene, were quite similar in young and senescent IMR-90 cells and appeared to be PDL-independent. In contrast, the expression of thymidine kinase, a late G1/S gene, was significantly reduced in senescent IMR-90 cells. Levels of thymidine kinase mRNA and thymidine kinase activity in senescent IMR-90 cells were 6- and 8-fold less than those in young cells, respectively. Based on these data, we proposed that impairment of cell cycling in senescent IMR-90 cells may occur at the late G1/S phase and that decreases of ornithine decarboxylase activity and putrescine accumulation during cell senescence may contribute to this impairment.     

Changes of serum-induced ornithine decarboxylase activity and putrescine content during aging of IMR-90 human diploid fibroblasts

The roles of ornithine decarboxylase (ODC, EC 4.1.1.17) and polyamines in cellular aging were investigated by examining serum-induced changes of these parameters in quiescent IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL) and in human progeria fibroblasts. Serum stimulation caused increases of ODC and DNA synthesis in IMR-90 human diploid fibroblasts, with maximal values occurring, respectively, 10 hr and 22 hr after serum stimulation. Both serum-induced ODC activity and DNA synthesis in IMR-90 cells were found to be inversely related to their PDL. Maximal ODC activity and DNA synthesis in young cells (PDL = approximately 18-22) were, respectively, five-fold and six-fold greater than that in old cells (PDL = approximately 50-55), which in turn were comparable or slightly higher than that in progeria fibroblasts. Polyamine contents (putrescine, spermidine, and spermine) in quiescent IMR-90 cells did not show significant PDL-dependency. The putrescine and spermine contents in quiescent progeria cells were comparable to those in quiescent IMR-90 cells. The spermidine content in quiescent progeria cells, however, was extremely low, less than half of that in quiescent IMR-90 cells. Serum stimulation caused a marked increase in putrescine content in young cells but not in old cells or in progeria cells. The spermidine and the spermine content in IMR-90 cells, either young or old, and in progeria cells did not change significantly after serum stimulation. Our study indicated that aging of IMR-90 human diploid fibroblasts was accompanied by specific changes of polyamine metabolism, namely, the serum-induced ODC activity and putrescine accumulation. These changes were also observed in progeria fibroblasts derived from patients with Hutchinson-Gilford syndrome.     

The effect of canavanine on protein synthesis and protein degradation in IMR-90 fibroblasts

Proteins of IMR-90 fibroblasts incorporating [³⁵S]methionine during a 1 h labelling period in the presence of the arginine analogue canavanine were degraded twice as rapidly in the cells as were proteins similarly made in the presence of arginine. Using both isoelectric focusing and SDS-polyacrylamide gel electrophoretic analyses, the banding patterns of proteins labelled in the presence of canavanine and arginine were found to differ. This banding difference was detected as early as 15 min after canavanine treatment. With the exception of one minor band in isoelectric focusing gel, the relative intensity of labelled protein bands for the control samples remained unchanged during the 2 h period of protein degradation being investigated. This was also true for the proteins labelled in the presence of canavanine, despite the increase in their rate of degradation. Banding difference between canavanine and arginine treatment was also detected in an in vitro reticulocyte lysate translation system dependent on fibroblast mRNA. Proteins labelled in the presence of a different analogue, p-fluorophenylalanine instead of phenylalanine, however, had similar banding patterns as the control both in the lysate system and in intact cells.     

K12-biotinylated histone H4 is enriched in telomeric repeats from human lung IMR-90 fibroblasts

Covalent modifications of histones play a role in regulating telomere attrition and cellular senescence. Biotinylation of lysine (K) residues in histones, mediated by holocarboxylase synthetase (HCS), is a novel diet-dependent mechanism to regulate chromatin structure and gene expression. We have previously shown that biotinylation of K12 in histone H4 (H4K12bio) is a marker for heterochromatin and is enriched in pericentromeric alpha satellite repeats. Here, we hypothesized that H4K12bio is also enriched in telomeres. We used human IMR-90 lung fibroblasts and immortalized IMR-90 cells overexpressing human telomerase (hTERT) in order to examine histone biotinylation in young and senescent cells. Our studies suggest that one out of three histone H4 molecules in telomeres is biotinylated at K12 in hTERT cells. The abundance of H4K12bio in telomeres decreased by 42% during telomere attrition in senescent IMR-90 cells; overexpression of telomerase prevented the loss of H4K12bio. Possible confounders such as decreased expression of HCS and biotin transporters were formally excluded in this study. Collectively, these data suggest that H4K12bio is enriched in telomeric repeats and represents a novel epigenetic mark for cell senescence.     

The biological activity of hydrogen peroxide. I. Induction of chromosome-type aberrations susceptible to inhibition by scavengers of hydroxyl radicals in human embryonic fibroblasts

The cytogenetic effect of hydrogen peroxide (H2O2) was investigated in human embryonic fibroblasts. Chromosome-type aberrations were found together with chromatid-type aberrations in metaphase cells harvested 24 h after a single 10-min treatment with 10(-5)-10(-3) M H2O2 in 0.9% NaCl solution. The chromosome-type aberrations were observed to be predominantly dicentrics and deletions. Both types of aberration showed a dose-response relationship to the dose of H2O2 over the range of 10(-5)-1.5 X 10(-4) M H2O2. The intercellular distribution of dicentrics showed a Poisson distribution. Centric and acentric rings and abnormal monocentrics were a minor fraction of the chromosome-type aberrations. The chromatid-type aberrations observed, such as breaks, exchanges and gaps, showed no dose-response relationship. The frequency of isochromatid breaks was higher than that of chromatid breaks and approximately 70% of the isochromatid breaks were found in the centromeric or pericentromeric region. The intercellular distribution of chromatid exchanges showed an over-dispersed distribution. The generation of aberrations by H2O2 was effectively suppressed by catalase and several scavengers of hydroxyl radicals (.OH) such as ethanol, dimethyl sulfoxide (DMSO) and mannitol. This result suggest that .OH plays an essential role in the generation of the chromosome aberrations by H2O2.     

Oxygen and hydrogen peroxide enhance light-induced carotenoid synthesis in Neurospora crassa

Previously, we found that intracellular reactive oxygen species (ROS) affect photomorphogenesis in Neurospora crassa. In this study, we investigated the physiological roles of ROS in the response to light and found that the exposure of mycelia to air was important for the light-induced carotenogenesis. Mycelia treated with a high concentration of O(2) gas and H(2)O(2) to release ROS showed an enhancement of light-induced carotenoid accumulation and the expression of gene related to light-inducible carotenogenesis. These results suggested that stimuli caused by the exposure of the mycelia to air containing O(2) gas triggered the light-induced carotenoid synthesis.