The purification of plasma membranes from WI-38 fibroblasts: effects of ageing on their composition

A three-step method for the purification of plasma membranes from WI-38 fibroblasts was developed thus allowing the recovery of 36--44% of the plasma membrane. Except in the case of galactosyltransferase, the activity of the contaminating enzymes was very low. Morphological observations confirm the presence of a homogeneous population of vesicles. Preparations obtained from young and old cell cultures were compared for their enzymatic and protein contents. With ageing the activity of 5-nucleotidase significantly increases whereas that of alkaline phosphodiesterase I decreases. Out of the 26 components detected after sodium dodecyl sulphate polyacrylamide gel electrophoresis, four decreased but only one increased. Cellular ageing seems to fulfil a specific and localized effect on the plasma membrane.     

The purification of plasma membranes from WI-38 fibroblasts Effects of ageing on their composition

A three-step method for the purification of plasma membranes from WI-38 fibroblasts was developed thus allowing the recovery of 36–44% of the plasma membrane. Except in the case of galactosyltransferase, the activity of the contaminating enzymes was very low. Morphological observations confirm the presence of a homogeneous population of vesicles.Preparations obtained from young and old cell cultures were compared for their enzymatic and protein contents. With ageing the activity of 5′-nucleotidase significantly increases whereas that of alkaline phosphodiesterase I decreases. Out of the 26 components detected after sodium dodecyl sulphate polyacrylamide gel electrophoresis, four decreased but only one increased. Cellular ageing seems to fulfil a specific and localized effect on the plasma membrane.     

Circular dichroism and ethidium bromide binding studies of chromatin from WI-38 fibroblasts stimulated to proliferate.

Confluent monolayers of WI-38 diploid fibroblasts can be stimulated to proliferate by fresh serum. In the first 3 h after stimulation (that is, several hours before DNA replication) the chromatin of stimulated cells show structrual changes which include: (1) an increase in maximum positive ellipticity and a blue shift in the 250-300 nm region of circular dichroism spectra; and (2) an increase,in isolated chromatin, of the number of binding sites for the intercalating dye, ethidium bromide.The differences between chromtin of stimulated and chromatin of unstimulated cells are abolised when bother chromatins are treated with 0.25 M NaCL.     

DNA hypermethylation in sodium butyrate-treated WI-38 fibroblasts

Sodium butyrate is very often used to alter gene expression in cultured cells. In this study, we examined the effects of this compound on various cellular events in WI-38 human embryonic lung fibroblasts in culture. During a 16-20-h treatment at sodium butyrate concentrations of between 5 and 20 mM, no adverse effects on cell morphology were observed. However, cell division and DNA synthesis were reversibly inhibited, the latter by 85, 80, and 70% at sodium butyrate concentrations of 5, 10, and 20 mM, respectively. Although overall protein synthetic activity was not significantly affected, RNA synthesis decreased to 76% of the control values at a sodium butyrate concentration of 5 mM. Butyrate treatment also caused hypermethylation of DNA cytosines as determined by differential digestion by MspI/HpaII restriction endonucleases and by high performance liquid chromatography analysis of the DNA. The 5-methylcytosine content of the DNA in untreated WI-38 fibroblasts was 2.94 +/- 0.46% of total cytosine residues, while in cultures treated with 5, 10, and 20 mM sodium butyrate, these values were 5.76 +/- 0.28, 5.91 +/- 0.37, and 6.8 +/- 0.44%, respectively. An interesting feature is that this hypermethylation occurred in DNA which was synthesized in the presence of sodium butyrate (newly synthesized) as well as in DNA which had been synthesized before butyrate administration (pre-existing DNA). The hypermethylated state was conserved only in the former situation, since the methylcytosines were rapidly lost in the subsequent generation in the latter case. It would therefore appear that methylcytosines are maintained after cell replication only if they are generated on newly synthesized DNA.     

Subcellular fractionation of WI-38 fibroblasts: Compatison between young and old cells

WI-38 fibroblasts cultivated in vitro were homogenized and their subcellular organelles analysed by the techniques of differential centrifugation and isopycnic equilibration in density gradient. In these experiments, the assayed enzymes were known to be specifically associated with subcellular components in other cells types. In most cases, their behaviour and properties corresponded with observations made in earlier studies and we could consider them as being representative of the specific subcellular organelles.Some significant differences were observed between young and old fibroblasts. The specific activity of alkaline phosphodiesterase was lower in the old cells whereas for the other enzymes it was identical or higher, especially for the 5′-nucleotidase; also the particulate fractions obtained by differential centrifugation contained more material. After equilibration in density gradient, the average density of the 5′-nucleotidase, alkaline phosphodiesterase and $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-glucosaminidase}$ was less in the old than in the young cells, whereas that of the galactosyltransferase of Golgi apparatus was greater. For mitochondria, endolasmic reticulum and peroxisomes, the differences observed were small.     

Purification and active site modification studies on glyoxalase I from monkey intestinal mucosa

Glyoxalase I ((R)-S-lactoylglutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) from monkey intestinal mucosa was purified to homogeneity. The purified enzyme had a molecular weight of 48,000, composed of two apparently identical subunits. Active-site modification was carried out on the purified enzyme in presence and absence of S-hexylglutathione, a reversible competitive inhibitor of glyoxalase I. Modification by tetranitromethane and N-acetylimidazole caused inactivation of the enzyme. Inactivation by N-acetylimidazole was reversible with hydroxylamine treatment, suggesting the importance of tyrosine residues for the activity of the enzyme. The enzyme was inactivated by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, 2,4,6-trinitrobenzenesulphonic acid, pyridoxal phosphate and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, indicating the importance of tryptophan, lysine and glutamic acid/aspartic acid residues for the activity of the enzyme. The enzyme was inactivated by diethyl pyrocarbonate and the activity was not restored by hydroxylamine treatment, suggesting that histidine residues may not be important for activity. Modification by N-ethylmaleimide and p-hydroxymercuribenzoate did not affect its activity, indicating that sulphydryl groups may not be important for activity. These studies indicated that the amino acids present in the active site of glyoxalase I from intestinal mucosa which may be important for activity are tyrosine, tryptophan, lysine and glutamic acid/aspartic acid residues.     

Thromboxane A2 biosynthesis in human lung fibroblasts WI-38.

A fibroblast of human lung origin (WI-38) synthesizes thromboxane A₂ from the prostaglandin endoperoxide PGH₂. Thromboxane A₂ synthesis was demonstrated by radio thin layer chromatography, gas chromatography/mass spectrometry, and by bioassay. This is the first demonstration of thromboxane A₂ biosynthesis in a homogeneous cell population other than the human platelet.     

Translational control of protein synthesis in stimulated WI-38 fibroblasts.

A cell-free protein synthesis system employing ribosomes from WI-38 human diploid fibroblasts was developed and its optimum MgC12 and KC1 levels and pH value found. The rate at which ribosomes are able to incorporate radioactive leucine into proteins ([14C]leucine incorporation/10 min/100 mug rRNA) and the number of growing peptide chains [3H]puromycinpeptides formed/100 mug rRNA) was determined. When confluent monolayers of WI-38 cells were stimulated to proliferate by serum, a transient increase in the rate of peptide elongation by ribosomes was observed at 60 min after stimulation. This increase was not affected by the presence of actinomycin D (10 mug/ml) in the stimulating medium. A change in the relative amount of certain ribosome-associated proteins accompanied the increased elongation rate of peptide growth. The alteration in associated proteins could not be accounted for by an increased synthesis of protein. Finally, the early activation of ribosomes in stimulated WI-38 cells appears to result from the removal of an inhibitor(s) of ribosome function.     

gamma-Glutamyl transpeptidase and glutathione in aging IMR-90 fibroblasts and in differentiating 3T3 L1 preadipocytes

γ-Glutamyl transpeptidase, glutathionase, and phosphate-independent glutaminase activities, and glutathione contents were measured in IMR-90 cells, human embryonic lung diploid fibroblasts, “aging” in culture, and in cultures of 3T3 L1 mouse preadipocytes differentiating to adipocytes. γ-Glutamyl transpeptidase, glutathionase, and phosphateindependent glutaminase activities in IMR-90 cells were about 8-, 8-, and 7-fold higher in “old” cells as compared to the activities in “young” cells. Total and reduced glutathione contents, expressed in relation to either cell number or DNA content, of IMR-90 cells were 3- and 12-fold higher in “young” cells (population doubling level 22) as compared to “old” cells (population doubling level 49). The ratio of reduced to oxidized glutathione was relatively constant in “young” cells over a range of population doubling levels 18–25; however, relative to the “young” cells, the ratio was decreased in “old” cells over a range of population doubling levels 49–55. γ-Glutamyl transpeptidase, glutathionase, and phosphate-independent glutaminase activities in 3T3 L1 cells were about 4-, 5-, and 5-fold higher in differentiating cells as compared to the activities in undifferentiating cells. Total glutathione contents in 3T3 L1 cells differentiating to adipocytes were similar to glutathione contents in undifferentiating cells. As the culture differentiated to the extent of 50%, almost all of the glutathione was present as oxidized glutathione. These results suggest that the several enzymatic activities and glutathione contents may be used as markers for aging or differentiation in cells. In the course of these studies, a sensitive fluorometric assay was developed, using l-γ-glutamyl-4-methoxy-2-naphthylamide as substrate.     

gamma-Glutamyl transpeptidase: relation to immunoglobulin A and free secretory component.

The experiments reported show that bovine γ-glutamyl transpeptidase can be separated from free secretory component. An ion-exchange Chromatographic procedure was developed to analyze the incubation mixtures of the enzyme with glutathione or S-(2-acetamido)-glutathione and glycylglycine. Using this system or the γ-glutamyl p-nitroanilide assay, no significant transpeptidase activity could be detected in the free secretory component-containing fractions of DEAE-cellulose chromatography. Gel filtration on Biogel A-5M showed that the bovine whey transpeptidase chromatographed in the void volume suggesting an aggregate of a minimum molecular weight of about 5 × 10⁶. The transpeptidase could be separated from all immunoglobulins in bovine whey and human colostrum by a combination of agarose gel filtration and immunoadsorption. Concentrated samples of human and sheep saliva showed normal amounts of secretory component, but no detectable γ-glutamyl transpeptidase activity. These experiments show that (1) the transpeptidase and secretory component are two different proteins, and (2) the transpeptidase is present in bovine and human milk as a high molecular weight aggregate which does not include any of the immunoglobulins.     

Effects of chondroitin-4-sulphate on survival, sizes and ultrastructures of cultured WI-38 fibroblasts and fibroblasts from progeria patients

WI-38 fibroblasts from 'normal' individuals and skin fibroblasts from patients displaying classical symptoms of progeria (accelerated aging) were maintained in tissue culture with and without periodic supplementation of 0.25 mg/ml of chondroitin-4-sulphate (C-4-S) during the ultimate phase of slowed division (phase 3). When C-4-S was not present in the culture medium, cell counts (not necessarily indicative of relative rates of cell division) and mean cell volumes were lower, and intracellular aberrations were higher, in both types of fibroblasts (normal and progeria) at, and even before, the 47th-50th and 13th-16th passages, respectively. The authors emphasise effects of C-4-S in preserving normal ultrastructure, pointing out that C-4-S does not fulfil the criteria of a mitogen and that any possible increases in rates of mitosis following supplementation with this substance are probably secondary to an enhanced metabolic environment.     

Effects of prolonged quiescence on neuclei and chromatin of WI-38 fibroblasts.

DNA synthesis and cell division are markedly reduced in confluent mono-layers of WI-38 diploid fibroblasts, but resume again if the depleted medium is replaced by fresh medium containing 10% fetal calf serum. If the cells are kept quiescent for prolonged periods of time after confluence (1 or 2 weeks), the fraction of cells that can be stimulated to proliferate by fresh serum decreases and the length of the prereplicative phase increases. The template activity of isolated nuclei decreases with increasing time of quiescence, and parallel changes occur in chromatin as evidenced by circular dichroism spectra and capacity to bind the intercalating dye, ethidium bromide. When WI-38 cells are stimulated to proliferate after prolonged quiescence, the increase in template activity of nuclei is delayed by several hours in comparison to cells stimulated after short periods of quiescence. Two distinct steps, both requiring serum, can be identified in the prereplicative phase of cells stimulated to proliferative after prolonged quiescence. We interpret the results as indicating that, during prolonged quiescence, WI-38 fibroblasts go into a deeper GO state from which they can be rescued only after prolonged stimulation. In this respect, prolonged quiescence may bear some resemblance to the process of aging.     

Resticted growth of human cytomegalovirus in UV-irradiated WI-38 human fibroblasts.

The growth of human CMV was inhibited by uv irradiation of cells prior to infection or during the 48-hr latent period of virus replication but not after virus synthesis began. The duration of uv exposure sufficient to inhibit CMV replication was insufficient to inhibit replication of Herpes simplex and did not prevent uninfected cells from dividing normally. The effect of uv irradiation on CMV replication may have been mediated through prevention of the virus on host cell RNA(s) synthesis.     

Glucocorticoid receptors in WI-38 fibroblasts: characterization and changes with population doubling in culture.

A high-affinity dexamethasone binding macromolecule was identified in WI-38 human fetal lung fibroblasts. High specificity of binding for glucocorticoids was shown by competition studies in which binding of dexamethasone was inhibited by cortisol and corticosterone but not by testosterone or 17 beta-estradiol. WI-38 cells exposed to [3H]dexamethasone at 30 degrees C were able to transfer the 3H-labeled steroid-receptor complex to the nuclear materal. A reduction of 30--50% was observed in the number of [3H]dexamethasone-receptor binding sites per cell as well as in the nuclear fraction of the cells as a function of age (passage levels 27 and 54). However, in the same cells no significant changes in affinity of receptor for [3H]dexamethasone as a function of the two passage levels were detected.     

Glucocorticoid receptors in WI-38 fibroblasts Characterization and changes with population doubling in culture

A high-affinity dexamethasone binding macromolecule was identified in WI-38 human fetal lung fibroblasts. High specificity of binding for glucocorticoidc was shown by competition studies in which binding of dexamethasone was inhibited by cortisol and corticosterone but not by testosterone of 17β-estradiol. WI-38 cells exposed to [³H]dexamethasone at 30°C were able to transfer the ³H-labeled steroid-receptor complex to the nuclear material. A reduction of 30–50% was observed in the number of [³H]dexamethasone-receptor binding sites per cell as well as in the nuclear fraction of the cells as a function of age (passage levels 27 and 54). However, in the same cells no significant changes in affinity of receptor for [³H]dexamethasone as a function of the two passage levels were detected.     

Synthesis of proline and hydroxyproline in human lung (WI-38) fibroblasts.

Human lung fibroblasts (WI-38) in late exponential phase of growth, in stationary phase after confluency was reached, and at high or low number of population doublings were used to investigate the synthesis of proline and hydroxyproline from glutamate or arginine. Glutamate was from two to five times as effective a precursor as arginine; glutamine did not seem to be involved in these metabolic pathways. Accumulation of protein-bound hydroxyproline in cell layers was observed only after confluency. Confluent cells synthesized more proline from glutamate than did cells in late exponential growth. Conversion of glutamate into intracellular free proline was conducted also to a greater extent in confluent cells at a high number of population doublings. Conversion of glutamate into proline or hydroxyproline in cell-layer protein was not affected significantly by the number of population doublings. Less total protein as well as less hydroxyproline accumulated with cells at a high number of population doublings.     

Glycosylases that excise modified DNA pyrimidines in young and senescent human WI-38 fibroblasts

Cellular DNA is continuously subject to damages by both endogenous and exogenous oxidizing agents. Excision repair in human cells is initiated by DNA glycosylases which remove oxidized bases from DNA. 5-Hydroxymethyluracil-DNA glycosylase excises 5-hydroxymethyluracil from DNA. A different enzyme has glycosylic activity against many ring-saturated DNA pyrimidines. Levels of these enzymes were examined in WI-38 fibroblasts of different culture ages. All glycosylases were assayed by measurements of direct release of modified free bases from their respective DNA substrates. Levels of 5-hydroxymethyluracil-DNA glycosylase were reduced in aging cells. Specific activities of the glycosylase that releases ring-saturated pyrimidines and of uracil-DNA glycosylase were not substantially altered in senescent cells. Therefore, although aging cells might have reduced excision of DNA 5-hydroxymethyluracil, there is no overall age-dependent decrease of DNA glycosylase activities.     

TPA-induction of c-fos mRNA during the cell cycle of WI-38 human fibroblasts

A brief exposure of quiescent (Go) WI-38 human fibroblasts to the tumor promoter TPA results in an increase in the mRNA levels of c-fos protooncogene. The same effect is produced by exposing to TPA human diploid fibroblasts WI38 synchronized in S phase by treatment with 2.5 mM hydroxyurea. Induction of c-fos mRNA in response to TPA occurs also during the progression of synchronized WI38 throughout the second and third cell cycle, but it is not associated with measurable changes in the cell cycle progression of these cells. These findings suggest that TPA induction of c-fos mRNA levels in proliferating cells is a stimulus specific rather than a function specific event.