Dissimilar cyclic nucleotide phosphodiesterase activities in subcellular fractions from normal and SV40-transformed WI-38 fibroblasts.

Broken cell preparations of WI-38 and SV40-transformed WI-38 (VA13) fibroblasts were used to compare the cyclic nucleotide phosphodiesterase activities of the two cell strains. The bulk of the cAMP or cGMP phosphodiesterase activity of WI-38 and VA13 homogenates was found in the 100,000 x g fibroblast supernatant fractions. WI-38 and VA13 soluble phosphodiesterase activities showed anomalous kinetic behavior with either cAMP or cGMP as the substrate. At low substrate concentrations, e.g., 0.1 muM, WI-38 supernatant fractions hydrolyzed cGMP much more rapidly than cAMP. At high substrate concentrations, e.g., 100muM, the same enzyme preparations degraded cAMP more than twice as fast as cGMP. In contrast, VA13 soluble phosphodiesterase activity catalyzed the hydrolysis of a wide range of cAMP and cGMP concentrations at similar rates. Phosphodiesterase activity in WI-38 supernatant fractions was generally more sensitive than that of the comparable VA13 enzyme activity to inhibition by MIX and papaverine. The cAMP phosphodiesterase activity of both WI-38 and VA13 supernatant preparations was decreased by cGMP in a concentration-dependent manner. cAMP was an effective inhibitor of cGMP hydrolysis by VA13 soluble phosphodiesterase activity. Yet, the cGMP phosphodiesterase activity of WI-38 supernatant fractions was only slightly reduced in the presence of cAMP. DEAE-cellulose chromatography of WI-38 and VA13 supernatant preparations revealed two major peaks of phosphodiesterase activity for each cell type. WI-38 peak I showed much greater activity with 1muM cGMP than with 1muM cAMP and appeared to be composed of two different phosphodiesterase activities. WI-38 peak Ia included phosphodiesterase activity which could be stimulated by boiled, dialyzed fibroblast homogenates while WI-38 peak Ib coincided with column fractions which contained most of the cyclic GMP hydrolytic activity. VA13 peak I phosphodiesterase activity was eluted from DEAE cellulose columns at the same ionic strength as WI-38 peak Ia and hydrolyzed these two substrates at nearly identical rates. This enzyme activity was also increased in the presence of boiled, dialyzed fibroblast preparations. Peak II phosphodiesterase activities from both WI-38 and VA13 fibroblasts were relatively specific for cAMP as the substrate. Phosphodiesterase activity with the properties of WI-38 peak Ib was not isolated from VA13 supernatant fractions. These results suggested that the dissimilar patterns of cAMP accumulation in WI-38 and VA13 cultures may be at least partially related to different phosphodiesterase activities in the normal and the transformed fibroblasts.     

epsilon-(gamma-Glutamyl)lysine isopeptide bonds in normal and virus transformed human fibroblasts.

Normal human lung cells (WI-38) possessed 20–40 times more ε-(γ-glutamyl)lysine isopeptide bonds than Simian virus transformed counterparts, WI-38 VA13A and WI-38 VA13-2RA. Normal cells arrested in an essentially nonmitotic state had more isopeptide bonds than proliferating cells. Isopeptide content paralleled the transglutaminase activity of these cells. The results suggest that isopeptide crosslinks contribute to a cellular architecture conducive to a nonproliferating state.     

Prostaglandin synthetase activity and hormone responsiveness in normal and SV40 transformed WI-38 fibroblasts.

Prostaglandin (PG) synthetase activity and selective hormone responsiveness were examined in normal and SV40 transformed WI-38 fibroblasts (VA13-2RA). The transformed VA13-2RA cells have significantly reduced rates of PGE1, PGE2, PGF1alpha and PGF2alpha synthesis as compared to the normal WI-38 fibroblast. The transformed cell in contrast to the normal cell hyperresponds to stimulation by L-epinephrine (10 muM) and PGE1 (8.5 muM) but is unresponsive to bradykinin (BK) as measured by the accumulation of intracellular cyclic AMP. Indomethacin treatment does not significantly alter the PGE1 and L-epinephrine (EPI) responsiveness of the normal WI-38 fibroblast, however it abolishes the BK response in these cells. These results provide further evidence for the dependency of cell activation by bradykinin on the PG synthetase system. No experimental data was found to support the role of PGs as negative regulators of PGE1 and EPI responsiveness in the WI-38 fibroblast. Using the VA13-2RA cells, limited attempts to recover PG synthetase activity comparable to that found in normal WI-38 cells were unsuccessful. The VA13-2RA cell and its normal counterpart represent models for investigating the role of PGs in cell function and the mechanism of BK activation and its effect on cell metabolism.     

Hypothesis: cyclic AMP turnover in S49 cells

The fractional turnover constant (ke) of cAMP in S49 lymphoma cells stimulated by epinephrine was essentially identical to the decay constant for cAMP in these cells. This was in sharp distinction to the situation in the human diploid lung fibroblast WI-38, in which ke was much lower in hormone stimulated cells. In this study we report a new method for the determination of cAMP turnover. The method was based on the assumption that for tritium label to be incorporated into cAMP on treatment of cells with [3H]-adenine, the label must first pass through ATP. An equation relating the rate of change in cAMP specific radioactivity with cAMP and ATP specific radioactivities was thereby determined. The equation was expressed in a form that gave a linear graphical plot with the fractional turnover constant as the slope of the line.     

Substrate mechanics and cell spreading

Cell spreading and cell locomotion arise from forces exerted by actin microfilaments upon the substratum. Using modified protein films at fluorocarbon oil--water interfaces as substrates, we have measured some minimal mechanical properties required of these films to support cell spreading forces in vitro. For murine 3T3-L1 fibroblasts, complete cell spreading was obtained when the films exceeded surface shear moduli and surface fracture points of 15 and 5 dyne/cm, respectively. The human WI-38 fibroblast required more robust films than did its transformed counterpart (WI-38/VA 13) in order to achieve equivalent spreading. These results are of significance in understanding the metastatic capabilities of cancer cells.     

Changes in enzymic activity during cultivation of human cells in vitro

The composition of chromatin, its template activity and the activity of certain chromatin-associated enzymes, including DNA polymerase (DP) and soluble RNase, DNase, DP and seryl tRNA synthetase, were examined in early and late passage of WI-38 cells and of WI-38VA13 cells.No significant changes in soluble RNase, DNase, seryl tRNA synthetase or soluble and chromatin-associated DP were found with increasing passage of WI-38 cells. The activity of seryl tRNA synthetase and DP in WI38VA13 cells was, however, significantly higher than WI-38 cells in all passages. A decline in RNA synthesizing activity of chromatin, an increase in the proportion of RNA and histone in chromatin, as well as an increase in the activities of ‘chromatin-associated enzymes’ (RNase, DNase, protease, nucleoside triphosphatase, DPN pyrophosphorylase) were noted in WI-38 cells with increasing passages. Although RNA synthesizing activity of chromatin from WI38VA13 cells was lower than that from WI-38 cells, the former also were much lower in ‘chromatin-associated enzymes’. An increase of chromatin-associated enzymes responsible for RNA, DNA and protein degradation in WI-38 cells in successive passages, and a much lower activity of these enzymes in WI-38VA13 cells (which have an indefinite doubling potential in vitro) suggests that an elevation in the activity of these enzymes, which would seriously interfere with the chromatin function, could result in ‘aging’ of WI-38 cells.     

The carbohydrate structure of human fibronectins: A comparison between normal embryonic lung fibroblasts WI-38 and the SV40 virus transformed cell line VA13

The carbohydrates of human fibronectin released from non-transformed human fibroblasts WI-38 have been compared with those of fibronectin released from SV40 virus transformed WI-38/VA13 cells and those of fibronectin prepared from human plasma. The majority of the bi-antennary glycopeptides of fibronectin released from WI-38 fibroblasts was not sialylated at the terminal galactosyl residues, but was fucosylated at the coreN-acetylglucosaminyl residue directly linked to a peptide (structure A, below). Most of the minor sialylation detected was linked 2–3 to galactose. In contrast, the majority of the bi-antennary glycopeptides released from the transformed VA13 cells was highly sialylated at the terminal galactosyl residues with both 2–3 and 2–6 linkages, but was only partially fucosylated at the coreN-acetylglucosaminyl residue (structure B, below). This structure was similar to that of the bi-antennary glycopeptide of human plasma fibronectin which was, however, predominantly sialylated with an 2–6 linkage (structure C, below). These human fibronectins, regardless of their source, lack a high molecular weight lactosaminoglycan structure.In addition to the differences in bi-antennary structure described above, the quantity of tri- to tetra-antennary glycopeptides of fibronectin released from VA13 transformed cells was found to be much greater than the quantity of these glycopeptides of fibronectin released from normal WI-38 fibroblasts. Furthermore, there was a relatively small quantity of a glycopeptide fraction having a smaller molecular weight that did not bind to Con A-Sepharose and was separated on a Bio-Gel P-4 column. The presence of this fraction was characteristic for fibronectin released from transformed VA13 cells, and the fraction was absent in fibronectin from normal fibroblasts.     

Effect of vitamin A on epithelial morphogenesis in vitro

Quiescent confluent monolayers of WI-38 human diploid fibroblasts and of 3T6 mouse fibroblasts were stimulated to proliferate by nutritional changes. WI-38 cells had a stringent requirement for serum factor(s) but 3T6 did not require serum in order to proliferate again. In both cell lines there was an early increase in the synthesis of non-histone chromosomal proteins shortly after stimulation of cellular proliferation and this increase was linearly correlated to the number of cells entering the S phase several hours later. Only WI-38 diploid fibroblasts, however, showed an early increase in chromatin template activity 1 h after stimulation of cellular proliferation, while chromatin template activity in 3T6 cells remained unchanged. It is suggested that the activation of gene function represents a critical step for the passage of WI-38 cells in the G0 resting phase to the G1 phase of the cell cycle. It is also suggested that 3T6 cells are unable to enter or stay in a G0 phase but can be arrested predominantly in the G1 phase by nutritional deficit, probably amino acid starvation.     

The escape of cyclic AMP from human diploid fibroblasts: general properties.

A variety of human diploid fibroblasts released large amounts of cAMP to the medium in a time-dependent fashion concomitant with stimulation of the cells by agonists of the adenylate cyclase. In WI-38 cells increased medium cAMP levels were detectable as quickly as increased cellular levels. Escape was not secondary to serum deprivation nor cell injury. It occurred in defined media, and was pH and temperature dependent. Elevated rates of escape were maintained for up to 24 hours after stimulation. A variety of PDE inhibitors reduced the rate of escape. A rough proportionality existed between the potencies of the compounds as potentiators of PGE1 increased cellular cAMP levels on the one hand and as inhibitors of escape on the other. In the case of IBMX, the inhibition of escape was transient, the most pronounced effect being during the first 5 minutes of incubation. In addition, a variety of compounds without significant acute effects on cellular cAMP levels inhibited escape.     

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.     

Subcellular localization of acid carboxypeptidase in rat liver and human fibroblasts.

The subcellular distribution of acid carboxypeptidase was investigated in rat liver, normal human skin (CRL 1501) and lung (WI-38) fibroblasts, galactosialidosis skin fibroblasts (GM 00806) and transformed lung fibroblasts (WI-38 VA 13). Results of differential and isopycnic centrifugations and osmotic activation experiments clearly indicate that the enzyme is located in lysosomes, in agreement with observations suggesting that carboxypeptidase is the protective protein of the 'Galjaard complex' which is defective in galactosialidosis.     

Changes in membrane transport function in G0 and G1 cells

Confluent quiescent monolayers of aneuploid and euploid cells in culture can be stimulated to proliferate by appropriate nutritional changes. In confluent monolayers of WI-38 human diploid fibroblasts the uptake of cycloleucine is increased three hours after these cells are stimulated to proliferate by a change of medium plus 10% serum. No changes in the uptake of cycloleucine are observed in logarithmically-growing WI-38 cells exposed to fresh medium plus 10% serum, or in WI-38 confluent monolayers in which the conditioned medium has been replaced by fresh medium with 0.3% serum (a change that does not cause stimulation of cellular proliferation in WI-38 cells). In 3T6 cells in the stationary phase stimulated to proliferate by nutritional changes, there is a prompt increase in the uptake of cycloleucine, within one hour after stimulation of cell proliferation. Similar results were obtained with stationary 2RA cells which are SV-40 transformed WI-38 fibroblasts. In addition, chromatin template activity which is known to increase in the early stages after stimulation of confluent WI-38 cells, was unchanged in confluent 3T6 or 2RA cells stimulated to proliferate. These results show that at least two of the very early biochemical events occurring in response to stimulation of cell proliferation are different in WI-38 diploid cells and in aneuploid 2RA or 3T6 cells. It is proposed that WI-38 cells in the stationary phase are arrested in the G₀ phase of the cell cycle, while 2RA and 3T6 cells are arrested in the G₁ phase.     

Decreased cAMP levels in human diploid cells exposed to cholinergic stimuli.

Carbachol at a concentration of 1 micron caused very significant decreases in PGE1 stimulated cAMP levels in three human diploid lung fibroblasts, WI-38, MRC-5, and IMR-90. Detailed studies with WI-38 cells showed that carbachol acted at low concentrations and very rapidly, cAMP levels being reduced by 50% in about 1 minute. The effect was moderately reduced but still very significant in Ca++ free medium containing 10(-5)M EGTA. Acetylcholine (in the presence of physostigmine), pilocarpine, and muscarine lowered cAMP levels at concentrations of 1 or 10 micron. The effects of carbachol were exquisitely sensitive to atropine but were unaffected by hexomethonium or d-tubocurarine. These data suggested that the cholinergic response of WI-38 cells was of the muscarinic type.     

Induction of cellular transglutaminase biosynthesis by sodium butyrate

Cellular transglutaminase activity was induced in simian virus-transformed human embryonic lung fibroblasts (WI-38 VA13A) by sodium butyrate. The level of enzyme activity approached a maximum by 6 days; 9–11-fold higher in the presence of sodium butyrate (1 mM) than in its absence. The observed increases in cellular transglutaminase activity could be entirely accounted for by equivalent increases in the levels of enzyme protein measured by inhibition enzyme-linked immunosorbent assay. Sodium butyrate also increased the rate of enzyme synthesis, but had no effect on the rate of cellular transglutaminase degradation. The increase in the rate of enzyme synthesis was matched by an increased level of translatable transglutaminase mRNA as measured in a cell-free translation system. Our results suggest that sodium butyrate regulates cellular transglutaminase at the pretranslational level.     

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.     

beta-Adrenergic receptors and cyclic AMP responses to epinephrine in cultured human fibroblasts at various population densities

The beta-adrenergic receptors of the intact human lung diploid fibroblast line Wl-38 and an SV-40 transformed clone of Wl-38, Wl-38-VA-13-2RA (VA13), were estimated in experiments utilizing the beta-adrenergic ligand, 125l-hydroxybenzylpindolol (125IHYP). When specific 125IHYP binding was measured in cells grown to relatively low population densities (0.15x10(6)cells/35mm dish), both Wl-38 and VA13 cells had approximately 40,000 beta-adrenergic receptors per cell. Wl-38 cells, when cultured to a high population density (0.5x10(6) cells/35/mm dish) had clearly diminished numbers of beta-adrenergic receptors and greatly decreased cAMP responses to epinephrine stimulation. On the other hand, in VA13 cells, neither the receptor number nor the beta-adrenergic response was affected by cell population density. In Wl-38 cells, the diminished cAMP response to epinephrine paralleled the decrease in number of beta-adrenergic receptors. Prostaglandin E1 (PGE1) stimulation of cAMP levels was unaffected by cell population density in either Wl-38 or VA13 cells. Thus, increased cell population density, perhaps related to density dependent inhibition of growth, caused a specific diminution in 125IHYP binding concomitant with decreased cAMP responses to epinephrine.     

Lysosomal enzyme activities and RNA turnover rates in growing and nongrowing WI-38 and HeLa cells

Activities of three lysosomal enzymes--acid RNase. N-acetyl-beta-D-glucosaminidase and acid phosphatase--were determined during the growth cycles of WI-38 and HeLa cells, as well as in radiation-arrested WI-38 cells. In confluent and growth-arrested cultures of WI-38 cells, the lysosomal RNase increased six- to sevenfold; glucosaminidase, four- to fivefold; and phosphatase, two- to threefold. In HeLa cells, the lysosomal enzymes also increased in confluent cultures, but less than twofold; and the RNase level increased only transiently. In both WI-38 and HeLa cells, the rate of RNA breakdown also increased as cultures approached confluency. The rate of turnover of RNA, like the level of acid RNase, was higher in WI-38 cells than in HeLa cells (4 d half-life compared to 8 d). The increase in acid RNase could be prevented by incubation of cells in NH4Cl, but the rate of turnover in the presence of NH4Cl increased just as much when cells became confluent or stopped growth. The content of acid RNase could be changed more than 10-fold without altering the rate of RNA turnover. It is suggested that the increase in enzyme level is more important for possible autophagy or increased digestion of engulfed RNA, rather than for normal RNA turnover, when growth stops.     

Properties of the genome in normal and SV-40 transformed WI-38 human diploid fibroblasts. III. Turnover of nonhisone chromosomal proteins and their phosphate groups.

The turnover of nonhistone chromosomal proteins and their phosphate groups was compared in normal and in SV-40 virus transformed WI-38 human diploid fibroblasts. Cells were pulse labelled with tryptophan-³H and ³²P for 30 minutes and the specific activities of tryptophan-³H and ³²P in the various molecular weight classes of nonhistone chromosomal proteins were determined during the first four hours following termination of labelling. While a rapid turnover of high molecular weight nonhistone polypeptides (142, 000 to 200, 000 Daltons) is evident after one hour in SV_40 transformed cells, the specific activities of these nonhistone chromosomal polypeptides are not significantly decreased in normal cells. In contrast, a rapid turnover of low molecular weight (30, 000 to 51, 000 Daltons) nonhistone chromosomal proteins occurs during the first hour in normal WI-38 cells with no corresponding decrease in the specific activities of these proteins in SV-40 transformed cells. There is no apparent net turnover of phosphate groups on nonhistone chromosomal proteins in either normal or SV-40 transformed cells four hours following pulse labelling. Rather, during the first four hours significant fluctuations are observed in the ³²P specific activities of defined molecular weight fractions. Taken together with previous reports of differences in the composition, synthesis and phosphorylation of nonhistone chromosomal proteins in normal and SV-40 transformed human diploid cells the present results further indicate the complex nature of the alterations in these proteins which accompany viral transformation.     

Hyperoxic-induced alterations in lung cell structure and function. Effects on cellular cyclic AMP content and comparison to alterations produced by exogenous cyclic AMP

Hyperoxic exposure in vitro of two lung-derived cell types (the epithelial-derived L2 cells and WI-38 fibroblasts) inhibits cellular replication, produces striking morphologic changes and may result in cell death; these effects have been observed consistently in other cell types. Hyperoxic exposure of L2 cells is associated with an increase in cellular cyclic AMP content (cellular cyclic AMP content 454 +/- 115 fmol/micrograms DNA in cells exposed to pO2 677 Torr for 96 h compared to 136 +/- 17 fmol/microgram DNA in air-grown cells). Hyperoxic exposure of WI-38 fibroblasts is not associated with increased cyclic AMP content. Although cultivation of L2 cells in the presence of exogenous dibutyryl cyclic AMP does inhibit replication and produce morphologic alterations, similar effects are produced by sodium butyrate alone. Hyperoxic exposure alters cyclic AMP metabolism in some cell types, but the structural and functional alterations observed in L2 cells and WI-38 fibroblasts following hyperoxic exposure are not produced by changes in cellular cyclic AMP content.