Transformed WI-38 Cells Research Articles

Levels of reactive oxygen species and primary antioxidant enzymes in WI38 versus transformed WI38 cells following bleomcyin treatment

Bleomycin (BLM) is an anticancer drug that generates reactive oxygen species (ROS) after interacting with iron and oxygen. We hypothesized that BLM could cause a different status of oxidative stress in normal versus tumor cells due to possible altered redox status and gene expression in cells following transformation. In this study, the extent of cytotoxicity, levels of ROS, and activities of antioxidant enzymes were compared between normal WI38 cells and SV40-transformed WI38 (VA13) cells following BLM treatment. Basal activities of MnSOD and catalase were lower in VA13 cells and basal ROS levels were higher in VA13 cells. Although BLM caused greater growth inhibition and apoptosis in VA13 cells, it increased ROS levels at an earlier time point in WI38 cells. Moreover, BLM treatment (100 μg/ml) had no effect on the activities of MnSOD, CuZnSOD, and catalase, but increased the activities of glutathione peroxidase (GPX) in WI38 cells after a 48-h treatment and in VA13 cells after a 24- and 48-h treatment. Northern blot analysis indicated that the increase in GPX activities was due to increased transcript levels of GPX1 but not GPX4 in both cells. Our results indicate selective induction of the GPX1 gene by BLM and different redox responses to BLM between WI38 and VA13 cells.

Effects of oxygen on the antioxidant responses of normal and transformed cells

Basal antioxidant defense levels are often aberrant in tumor cells; however, less attention has been given to differences in the way that normal and transformed cells respond to changes in oxidative stress. This study evaluated differences in the responses of various normal and transformed cell lines to different oxygen tensions. Exposure to hyperoxia generally failed to induce either the activity of GSH peroxidase (GPx) or the manganese-containing form of superoxide dismutase (MnSOD) after 48 h, although at 605 mm Hg oxygen, small inductions of MnSOD activity were observed in adult lung fibroblasts and amelanotic melanoma. Exposure to 605 mm Hg O 2 for 48 h was inhibitory to GPx activity. MnSOD activity was strongly induced in virally transformed WI-38 cells by treatment with the herbicide paraquat or inhibition of GSH synthesis with BSO. In normal cells GSH concentration was proportional to ambient oxygen tension. Tumor cells exhibited greater GSH concentrations at low oxygen tensions than normal cells but were unable to increase GSH in response to elevation of oxygen tension. These results reveal differences in tumor and normal cell responses to changes in ambient oxygen tension and show that MnSOD activity is inducible when an appropriate stimulus is applied.

Resveratrol analog, 3,4,5,4'-tetrahydroxystilbene, differentially induces pro-apoptotic p53/Bax gene expression and inhibits the growth of transformed cells but not their normal counterparts.

Resveratrol, a trihydroxystilbene found in grapes and other plants, has been shown to be active in inhibiting multistage carcinogenesis. Using resveratrol as a prototype, we have synthesized a number of polyhydroxy- and polymethoxy-stilbenes and tested their anti-proliferative effect in normal and transformed human cells. Here we show that one of the resveratrol analogs, 3,4,5,4'-tetrahydroxystilbene (R-4), specifically inhibited the growth of SV40 virally transformed WI38 cells (WI38VA) at 10 microM, but had no effect on normal WI38 cells at even higher concentrations. R-4 also prominently induced apoptosis in WI38VA cells, but not in WI38 cells. RNase protection assay showed that R-4 significantly induced the expression of p53, GADD45 and Bax genes and concomitantly suppressed the expression of bcl-2 gene in WI38VA, but not in WI38 cells. A large increase in p53 DNA binding activity and the presence of p53 in the Bax promoter binding complex suggested that p53 was responsible for the Bax gene expression induced by R-4 in transformed cells. Within 4 h of treatment with R-4, the Bax to bcl-2 protein ratio in WI38 and WI38VA cells was, respectively, 0.1 and 105, a difference of three orders of magnitude. While R-4 prominently induced the p53/Bax pro-apoptotic genes, it also concomitantly suppressed the expression of Cox-2 in WI38VA cells. Taken together, our study suggests that the induction of p53 gene by R-4 in transformed cells may play a key role in the differential growth inhibition and apoptosis of transformed cells.

Cell cycle regulated expression of nucleolar antigen P120 in normal and transformed human fibroblasts

Normal and SV40 virus-transformed WI-38 human lung fibroblasts were serum starved and refed, or synchronized by double thymidine block and released from the block. At different time points in the cell cycle, steady state levels of P120 mRNA and P120 protein content of the cells were determined by densitometric scans of Northern and Western blots. At the same time points, [3H]thymidine uptake was measured and flow cytometric analysis performed for DNA content and P120 antigen staining. Levels of P120 protein and P120 mRNA were approximately 4 times greater in non-synchronous, exponentially growing transformed cells than in similarly growing normal cells. Early G1-phase cells, synchronized either with serum deprivation or with metabolic block, contained only a trace amount of P120 protein and mRNA. The P120 gene was transcribed early in G1 and P120 protein synthesis initiated in middle G1. A dramatic increase of P120 protein level occurred in S-phase with a corresponding mRNA peak preceding the P120 protein peak. These results indicate that P120 is overexpressed in transformed WI-38 cells and that P120 is temporally regulated during the cell cycle of both transformed and normal fibroblasts. The dramatic increase in P120 protein expression at the G1 to S boundary suggests that P120 may play a role in the regulation of cell cycle and increased nucleolar activity that is associated with cell proliferation.

Inhibition by selenium of DNA and RNA synthesis in normal and malignant human cells in vitro

Several studies have demonstrated differences between normal and malignant cells in their sensitivity to various effects of selenite. We have compared the effect of selenite on DNA and RNA synthesis in two pairs of normal and malignant human cell lines. One pair of cells, CCL-210 (normal lung fibroblasts) and A549 (lung adenocarcinoma cells), exhibited a large difference in their sensitivity to selenite but no significant difference in their sensitivity to selenodiglutathione. They also had a large difference in the level of intracellular sulfhydryl (SH) compounds. In contrast the other pair of cells, WI-38 (normal fetal lung fibroblasts) and WI-38VA (SV-40 transformed WI-38 cells) both had low levels of intracellular SH compounds and exhibited similar (low) sensitivity to selenite. Our results indicate that differences between normal and malignant cells in their sensitivity to selenite could be due to a difference in the reaction of selenite with intracellular sulfhydryl compounds to form selenotrisulfides.

Filopodia number increases with age and quiescence in populations of normal WI-38 cells, and is correlated with drug-induced changes in proliferation in both normal and transformed populations

Filopodia in log and stationary phase populations of human fetal lung fibroblasts (WI-38) at low and high population doubling levels (PDLs), and of SV40 transformed WI-38 cells (VA13A), were observed and counted under different conditions of in vitro growth by scanning electron microscopy. Cells from old non-vigorously growing WI-38 populations (those at a high PDL) had more filopodia than younger populations (those at a lower PDL) at all times after seeding, and for any given population stationary phase cells (those entering, or in, quiescence), had more than log phase cells. Hydrocortisone (HC, 14 μM), which stimulates proliferation and increases life span of WI-38 cells, was associated with a marked decrease in filopodia. Conversely, retinoic acid (RA, 10 μM), which inhibits growth and decreases life span of WI-38 cells, was associated with an increase in filopodia. Since old cell populations have lower saturation densities than young, it is suggested that cell contact signaling growth cessation in these populations may be mediated by filopodia. The HC-associated decrease in filopodia may thus be possibly interpreted as a decrease in filopodia-mediated “density dependent inhibition,” and the increase in filopodia with RA as a possible increase in this “inhibition.” Both HC and RA inhibit growth and are associated with an increase in filopodia in VA13A cultures.

Proliferation-dependent regulation of base excision repair in normal and SV-40 transformed human cells.

The effect of SV-40 viral transformation of human cells on the proliferation-dependent regulation of base excision repair was examined. Normal human diploid WI-38 fibroblasts and SV-40 transformed WI-38 cells were used to examine whether the process of transformation perturbed the regulation of base excision repair. The regulation of excision repair enzymes in each cell type was examined by quantitating: the specific activities of the base excision repair enzymes uracil DNA glycosylase and hypoxanthine DNA glycosylase as a function of cell growth; and immunological analysis of the uracil DNA glycosylase using monoclonal antibodies to human uracil DNA glycosylase. In each cell type, both DNA glycosylase activities were increased as a function of cell proliferation; the extent of enhancement being greater in the transformed cells. As defined by ELISA and immunoblot analysis, the induced uracil DNA glycosylases in both cell types share the same determinants. Although other repair processes are altered upon SV-40 transformation, these results suggest that the proliferation-dependent regulation of these base excision repair enzymes is retained in SV-40 transformed human cells.

Calcium effects on epidermal growth factor receptor‐mediated endocytosis in normal and SV40‐transformed human fibroblasts

Lowering of extracellular Ca2+ levels will reversibly arrest the growth of human fibroblasts (WI38). Simian virus40(SV40)-transformed WI38 cells do not exhibit this Ca2+-dependent arrest. One possibility for this difference in Ca2+ requirement is that extracellular or surface membrane-bound Ca2+ may be required for growth factor receptor-mediated endocytosis and this Ca2+ requirement may differ in normal versus transformed cells. In this study we have evaluated the role of Ca2+ in the binding, internalization, and degradation of epidermal growth factor (EGF) in the WI38 and SV40WI38 cell. The binding of [125I]EGF to the cell surface is not significantly altered by lowering of Ca2+ to 10(5)-M levels in either the normal or transformed cell. At this Ca2+ level, growth of the normal cell is inhibited. The subsequent internalization of EGF is reduced nearly threefold in the normal cell but not in the transformed cell following Ca2+ deprivation. Degradation of the EGF-receptor complex is also sensitive to Ca2+. A twofold reduction in the rate of release of acid-soluble 125I occurs in the normal but not the transformed cell under conditions of lowered medium Ca2+. In contrast, 2-chloro-10-3-aminopropyl phenothiazine (CP), an inhibitor of the Ca2+-dependent regulator protein calmodulin, causes an inhibition of [125I]EGF internalization and degradation in both the normal and transformed WI38 cell, and a marked inhibition of [125I]EGF binding to the cell surface receptor of the transformed cell but not the normal cell.

Inducers of DNA synthesis: levels higher in transformed cells than in normal cells.

The objective of this study was to determine whether transformed cells have greater DNA synthesis-inducing ability (DSIA) than normal cells when fused with G1 phase cells. HeLa cells synchronized in G1 phase, prelabeled with large latex beads, were fused separately with (a) quiescent human diploid fibroblasts (HDF), (b) HDF partially synchronized in late G1, and random populations of (c) HeLa, (d) WI-38, (e) SV-40 transformed WI-38, (f) CHO, (g) chemically transformed mouse cells (AKR-MCA), and (h) T98G human glioblastoma cells (all prelabeled with small latex beads) using UV-inactivated Sendai virus. The fusion mixture was incubated with [3H] thymidine, sampled at regular intervals, and processed for radioautography. Among the heterodikaryons, the frequency of those with a labeled and an unlabeled nuclei (L/U) were scored as a function of time after fusion. The faster the induction of DNA synthesis in HeLa G1, the steeper the drop in the L/U class and hence the higher DSIA in the S phase cells. The DSIA, which is indicative of the intracellular levels of the inducers of DNA synthesis, was the highest in HeLa and virally transformed WI-38 cells and the lowest in normal human diploid fibroblasts (HDF) while those of chemically and spontaneously transformed cells are intermediate between these two extremes. Higher level of DNA synthesis inducers appears to be one of the pleotropic effects of transformation by DNA tumor viruses. These studies also revealed that initiation of DNA synthesis per se is regulated by the presence of inducers and not by inhibitors.

Down regulation and recovery of the epidermal growth factor receptor in serum supplemented versus defined medium.

The down regulation of surface membrane receptors for 125I) epidermal growth factor (EGF) has been evaluated in normal and SV40-transformed human fibroblasts (WI38) under conditions of serum-supplemented versus defined growth media. Both normal and transformed WI38 cells down regulate and recover the EGF receptor and these processes do not differ significantly in serum-supplemented versus defined media. These data are in contrast to a recent study that reported that the HeLa cell does not down regulate the EGF receptor in defined media, whereas it does in serum-supplemented media.

Assembly of secondary intermediates during deoxyribonucleic acid replication in transformed human fibroblasts

The elongation of replicative DNA was studied in transformed WI-38 cells (designated 2RA). Shear effects were avoided by use of an alkaline sucrose gradient sedimentation method whereby cells were lysed directly on top of gradients, at 4°C in the dark. The earliest detected intermediate is a short (2 S) piece of DNA which is converted first to a 25 S piece and then to a 100 S piece, within 10 min. The 100 S piece is next converted to a 212 S, and a 370 S, and finally to a chromosomal DNA of about 450 S. This pattern is quite different from that previously reported by us for normal WI-38 cells, where there was a 50 S intermediate which was not quickly converted into a much larger size, but which gradually elongated, by addition of smaller pieces, to a size of 100 S.; another difference was the time required for formation of the 100 S piece, i.e., 75 min (Rawles, J.W., Jr. and Collins, J.M. (1977) J. Biol. Chem. 252, 4762–4766).

Replicative potentials of various fusion products between WI-38 and SV40 transformed WI-38 cells and their components.

Hybrid cells derived from whole-cell fusions of replicating phase-II normal fibroblast cells (WI-38s) with SV40 transformed WI-38 fibroblast cells (CL-1s) demonstrated that the majority of the hybrid experimental cells still maintained a finite life-span. Approximately 2% demonstrated sustained and possibly indefinite replication. Experimental binucleate cells and subsequent hybrid synkaryons were also formed by fusing CL-1 karyoplasts into phase-II WI-38 replicating normal fibroblasts. In addition, viable cells were constructed from WI-38 fibroblast cytoplasts with CL-1 karyoplasts. Sustained replication was not observed in these crosses.

Alkaline phosphatase and an acid arylamidase as marker enzymes for normal and transformed WI-38 cells.

A survey of eleven enzyme activity levels in normal and SV40 transformed (VA-13) WI-38 cells revealed that the transformed cell enzymes differed by a quantitative and qualitative change of alkaline phosphatase and a quantitative loss of an arylamidase. Alkaline phosphatase activity was found to be elevated in the transformed cells at confluency but not in log phase cultures. This elevated activity was heat stable, L-homoarginine resistant and L-phenylalanine sensitive and is probably the term placental isoenzyme. In nontransformed WI-38 cells, the alkaline phosphatase was heat labile, L-homoarginine sensitive and L-phenylalanine resistant and so is probably the liver isoenzyme. While the arylamidase activity from both normal and transformed WI-38 cells had identical pH optima and Km values, the activity was approximately 20 times higher in confluent WI-38 cells than in confluent VA-13 cells. Cytochemical staining techniques for both activities are described that permit identification of fluorescent product within the cells, analysis of activity levels, and separation of cells with high and low activities. Mixtures of WI-38 cells and VA-13 cells separated by flow cytometry on the basis of arylamidase activity were subsequently evaluated for alkaline phosphatase isoenzyme and found to have been simultaneously separated into heat labile and heat stable samples.

Stimulation of clonal growth of normal fibroblasts with substrata coated with basic polymers.

Improved media have reduced the amount of serum protein required for clonal growth of normal human and chicken fibroblast-like cells. In the presence of limiting amounts of serum protein, attachment of colonies to tissue culture plastic surfaces is weak. Treatment of the culture surface with polylysine or other basic polymers causes the cells to adhere much more tightly. Growth is also improved on the surfaces treated with basic polymers, and further reductions in the concentration of serum as possible. At sufficiently low protein concentrations, growth of some types of cells is totally dependent on the use of a treated surface. Several different types of normal human and chicken fibroblast-like cells show improved growth on polylysine-coated surfaces, but no improvement was obtained in growth of a line of SV-40 transformed WI-38 cells. Acidic and neutral polymers are generally inactive. Collagen and gelatin improve growth slightly, but the effect is much less than that obtained with basic polymers. Both natural and synthetic polymers with an excess of basic groups are active, including histone, polyarginine, polyhistidine, polylysine, polyornithine, and protamine. The only critical requirement appears to be a polymer that carries a positive charge at a physiological pH.

Protein degradation in human fibroblasts (WI-38). Effects of aging, viral transformation, and amino acid analogs.

Protein degradation occurs more rapidly in senescent WI-38 cultures than in phase II cultures or in SV-40 transformed WI-38 cells (VA-13). The first differences are found in early phase III, when short lived but not long lived proteins are degraded more rapidly. At the end of phase III long lived proteins are also degraded more rapidly as shown by both intermittent perfusion and approach to equilibrium methods. By both methods the rates of protein degradation for the virally transformed derivative are the same as those for phase II WI-38, implying that transformation has not altered these characteristics of protein degradation. WI-38 cells incorporate canavanine, an analog of arginine, into protein. This analog, as well as p-fluorophenylalanine and azetidine carboxylic acid, accelerates the degradation of proteins labeled with [3H]leucine in their presence but does not alter the degradation rates of proteins prelabeled with [14C]leucine in the absence of the analogs. These results imply that the analogs increase the intracellular degradation rates of proteins because they render them more susceptible to the degradative system. Late phase III WI-38 cells may not selectively catabolize proteins containing canavanine as rapidly as do phase II and VA-13 cells. These results imply that the phase III protein degradative system becomes partially defective, thereby losing its ability to rapidly catabolize altered protein which leads to increased levels of abnormal proteins and decreased cell function.

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.

Properties of the genome in normal and SV40 transformed WI38 human diploid fibroblasts: II. Metabolism and binding of histones

Composition, metabolism and extractability of histone fractions from WI38 human diploid fibroblasts and SV40 transformed WI38 fibroblasts are compared. Two alternate procedures were used for isolation of nuclei which allow for either optimal recovery of arginine-rich histones F3 (III) and F2a1 (IV) or for optimal retention of lysine-rich F1 (I) and slightly lysine rich F2b (II b2). While the relative amount of each histone fraction was found to be similar in normal and SV40 transformed cells, substantial increases in the levels of F 3 acetylation and F1 and F2a2 phosphorylation are reported for the histones of SV40 transformed cells. Differences in extractability of arginine-rich histones with 0.25 M HCl are also reported. While F 3 is extracted more rapidly than F 2a1 from nuclei of normal WI38 fibroblasts, the reverse is true in SV40 transformed WI38 cells. These differences are discussed in relation to modification reactions, binding of histones to DNA and SV40-induced alterations in gene readout.

Modifications in the chromosomal proteins of SV-40 transformed WI-38 human diploid fibroblasts

Summary The composition and metabolism of chromosomal proteins associated with the genome of WI-38 human diploid fibroblasts and SV-40 transformed WI-38 fibroblasts were compared. While the five major histone fractions are present in normal and SV-40 transformed cells, variations in the relative contents and rates of synthesis of specific histone fractions are described. Differences in the protein contents and rates of synthesis of several molecular weight classes of nonhistone chromosomal proteins in normal and SV-40 transformed WI-38 cells are also reported.