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.     

Effect of prolonged prostaglandin exposure on prostaglandin synthesis by human lung fibroblasts

Pretreatment of human lung fibroblasts with PGE₂ but not PGF_2α enhanced synthesis of prostaglandins (PGs). The effect of the pretreatment on PG synthesis was related to the concentration of PGE₂ that was added to the culture medium. Pretreatment with PGE₂ at 5 × 10⁻¹²M did not enhance PG synthesis whereas pretreatment with PGE₂ at 5 × 10⁻⁶M induced a maximal effect. Production of PGs was increased following 1 day of pretreatment with PGE₂ and was increased further following 3 days of pretreatment. The PGE₂ treated cells showed only a slight increase in the bradykinin-induced release of radioactivity from cells prelabeled with [³H]arachidonic acid but showed a dramatic increase in the bradykinin-induced synthesis of radio-labeled PGs. The conversion of free arachidonate to PGs in both intact cells and in a cell-free preparation was increased by PGE₂ pretreatment. The presence of cyclohexamide during the pretreatment did not inhibit the PGE₂-induced activation of PG synthesis. Taken together, the results indicate that pretreatment of cells with PGE₂ increased PG synthesis by augmenting the conversion of arachidonate to PGs.     

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.     

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.     

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.     

Inhibition by collagen chains of lysyl hydroxylase of chick embryo and of WI-38 human lung fibroblasts.

Lysyl hydroxylase from chick embryos was strongly inhibited by heat-denatured collagens from various vertebrate sources, and by separated a chains and β components of rat tail tendon collagen. The kinetics exhibited with this enzyme when heat-denatured calf or rabbit skin collagens was used showed a mixed type of inhibition. On the other hand, a preparation of homologous heat-denatured 4,5-³H-L-lysine-labeled collagen, in itself an extremely poor substrate for the hydroxylase, showed non-competitive inhibition with a K_i of about 8–9 μM. Finally, the lysyl hydroxylase preparations from WI-38 fetal human lung fibroblasts and from transformed WI-38 cells (WI-38 VA 13) were also inhibited by heat-denatured collagens or, where tested, by separated collagen chains.     

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.     

Prolonged prostaglandin E1 stimulation of cyclic AMP production in transformed and normal WI-38 fibroblasts

Summary Long-term (48-hr) incubations of either the fibroblast strain WI-48 or its SV40-transformed counterpart, WI-38-VA13-2RA, in growth medium containing 1 μm prostaglandin E₁ (PGE₁) resulted in a sustained production and release of cyclic AMP from the cells into the medium. Despite the steady production, intracellular levels of the nucleotide decreased, reaching steady-state values within 4 hr of the initial exposure to PGE₁. These values were maintained for the remainder of the 48-hr experimental period. The steadystate levels of intracellular cyclic AMP were higher than those observed in unstimulated cells, and cyclic AMP-dependent protein phosphokinase was in a highly activated state as compared to controls. Under these conditions little change in the growth or morphology of either the normal or transformed cells was observed. In contrast, inhibition of growth, apparent cell death, and unusual morphological changes were observed in both normal and transformed cells when high concentrations of either PGE₁ (10 μm) or the phosphodiesterase inhibitor 1-methyl, 3-isobutylxanthine (0.5mm to 2mm) were used, which was indicative of toxic effects of the drugs. It was concluded that cyclic AMP-mediated activation of protein phosphokinase does not completely inhibit growth in WI-38 cells or restore normal growth and morphology to the SV40-transformed cells. This work was supported by Grants AM 13904 and CA 21612 from the National Institutes of Health, Department of Health, Education and Welfare.     

Phosphatidic acid inhibition of PGE1-stimulated cAMP accumulation in WI-38 fibroblasts: similarities with carbachol inhibition

To test the hypothesis that phosphatidic acid (PhA) is involved in the carbachol inhibition of hormone stimulated accumulation of cAMP we observed the effects of PhA on PGE1-stimulation of cAMP in WI-38 fibroblasts. PhA inhibited PGE1-stimulated cAMP accumulation of WI-38 fibroblasts; maximum inhibition (approximately 50-80%) occurred at a PhA concentration of 1.0 microM and significant inhibition was observed with a concentration of 0.1 microM. The full effects of PhA were evident within 15 sec after the co-addition of PGE1 and PhA. Addition of PhA to cells which had been pre-stimulated with PGE1 resulted in the rapid decay of cAMP levels to a new steady state level with a t 1/2 of approximately 65 sec. The inhibition produced by PhA did not appear to be simply attributable to a depolarization or increased intracellular Ca2+, since addition of either KCl or the Ca2+ ionophore A23187 did not lower PGE1-stimulated cAMP accumulation. When intact cells were pretreated with PhA then lysed and adenylate cyclase immediately assayed, no detectable changes in broken cell adenylate cyclase activities were observed. Also, PhA added directly to adenylate cyclase assays at concentrations as high as 100 microM produced no detectable inhibition of the membrane fraction adenylate cyclase activities. Nonetheless, our results suggest that adenylate cyclase activity in intact cells may be directly affected by physiological levels of PhA . Further, the similarities of carbachol [Butcher, R. W., Journal of Cyclic Nucleotide Research, 4:411 (1978)] and PhA inhibition support the hypothesis that carbachol (acetylcholine) exerts its effect on adenylate cyclase through alterations of the plasma membrane phospholipid composition.     

Stimulation of DNA synthesis in human fibroblasts by thrombin.

Earlier work showed that thrombin stimulates proliferation of human fibroblasts in serumfree medium. This work demonstrates (1) that thrombin has to be prensent during most or all of the G1 period to ensure maximal DNA synthesis, (2) that DNA synthesis increases about three hours later after thrombin than after serum treatment, (3) that both thrombin and serum activate transport of uridine, D--2-deoxy-glucose and putrescine, (4) that thrombin is able to increase 3H-thymidine incorporation also in SV40 transformed human fibroblasts, in HeLa cells and in two continuous monkey cell lines.     

Prolonged prostaglandin E1 stimulation of cyclic AMP production in transformed and normal WI-38 fibroblasts.

Long-term (48-hr) incubations of either the fibroblast strain WI-38 or its SV40-transformed counterpart, WI-38-VA13-2RA, in growth medium containing 1 micron prostaglandin E1 (PGE1) resulted in a sustained production and release of cyclic AMP from the cells into the medium. Despite the steady production, intracellular levels of the nucleotide decreased, reaching steady-state values within 4 hr of the initial exposure to PGE1. These values were maintained for the remainder of the 48-hr experimental period. The steady-state levels of intracellular cyclic AMP were higher than those observed in unstimulated cells, and cyclic AMP-dependent protein phosphokinase was in a highly activated state as compared to controls. Under these conditions little change in the growth or morphology of either the normal or transformed cells was observed. In contrast, inhibition of growth, apparent cell death, and unusual morphological changes were observed in both normal and transformed cells when high concentrations of either PGE1 (10 micron) or the phosphodiesterase inhibitor 1-methyl, 3-isobutylxanthine (0.5 mM to 2 mM) were used, which was indicative of toxic effects of the drugs. It was concluded that cyclic AMP-mediated activation of protein phosphokinase does not completely inhibit growth in WI-38 cells or restore normal growth and morphology to the SV40-transformed cells.     

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.     

Partial characterization of a mitogenic factor with somatomedin-like activity produced by culture WI-38 human fibroblasts

Conditioned serum-free medium (CSFM) obtained from WI-38 human fibroblasts was found to contain a mitogenic factor(s) with somatomedin (SM)-like activity. Treatment of the cells with cycloheximide eliminated the SM-like activity in CSFM, suggesting that these cells produce and release the activity. Gel filtration revealed that the fibroblast SM-like activity (FSLA) had a molecular size near 45,000. Isoelectric focusing of this FSLA yielded 2 bands of SM activity with pIs of 4.7 and 6.1, and corresponding molecular sizes of ～29,000 and 16,500, respectively. The FSLA obtained by gel filtration revealed parallel dose response curves with a basic SM in a SM radioreceptor and radioimmunoassay and stimulated: (1) ³⁵So₄ uptake by hypophysectomized rat cartilage; (2) (U-¹⁴C) glucose oxidation is isolated rat adipocytes; and (3) (³H) thymidine uptake and cell division in these same WI-38 fibroblasts. Out studies indicate that this FSLA and basic SM are similar but not identical.     

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.     

Loss of DLK expression in WI-38 human diploid fibroblasts induces a senescent-like proliferation arrest

DLK, a serine/threonine kinase that functions as an upstream activator of the mitogen-activated protein kinase (MAPK) pathways, has been shown to play a role in development, cell differentiation, apoptosis and neuronal response to injury. Interestingly, recent studies have shown that DLK may also be required for cell proliferation, although little is known about its specific functions. To start addressing this issue, we studied how DLK expression is modulated during cell cycle progression and what effect DLK depletion has on cell proliferation in WI-38 fibroblasts. Our results indicate that DLK protein levels are low in serum-starved cells, but that serum addition markedly stimulated it. Moreover, RNA interference experiments demonstrate that DLK is required for ERK activity, expression of the cell cycle regulator cyclin D1 and proliferation of WI-38 cells. DLK-depleted cells also show a senescent phenotype as revealed by senescence-associated galactosidase activity and up-regulation of the senescence pathway proteins p53 and p21. Consistent with a role for p53 in this response, inhibition of p53 expression by RNA interference significantly alleviated senescence induced by DLK knockdown. Together, these findings indicate that DLK participates in cell proliferation and/or survival, at least in part, by modulating the expression of cell cycle regulatory proteins.