WI-38 Cultures Research Articles

Sodium butyrate induction of apoptosis in human fibroblasts in culture

Tissue transglutaminase (tTg) is a multifunctional enzyme possessing two catalytic sites. One is a calcium‐dependent reaction that leads to formation of isodipeptide cross‐linkages. The cross‐linkages lead to the formation of a protein scaffold in cells undergoing apoptosis. GTP, a negative regulator for this cross‐linking activity, shifts the enzyme's catalytic activity to G‐protein activity.Human embryonic fibroblast cells (WI‐38) and their simian virus‐transformed counterparts (VA13A) grow adherent in vitro. Quantification of the amount of tTg using a fibronectin binding assay showed that VA13A cells contained 0.04493 ng tTg/uL sample in the supernatant and a non‐detectable amount in the pellet fraction. This amount increased to 8.2622ng tTg/uL sample in the supernatant fraction after the VA13A cells were exposed to sodium butyrate. These results were confirmed by indirect immunofluorescence microscopy, electrophoresis and Western blotting.Sodium butyrate induced apoptosis in VA13A cultures, but had no effect on apoptosis in WI‐38 cultures. Preliminary studies with sodium butyrate alone and in combination with calcium ionophore showed not only an increase in apoptosis but also an increased transglutaminase expression. Additionally, sodium butyrate treatment induced growth arrest in both cell lines and reverted the VA13A cells to a more “normal” morphology. These results taken collectively are indicative of a dual role of sodium butyrate; one involving growth arrest and the other apoptosis.

Green tea epigallocatechin gallate shows a pronounced growth inhibitory effect on cancerous cells but not on their normal counterparts

(−)-Epigallocatechin gallate (EGCG), a catechin polyphenol compound, represents the main ingredient of green tea extract. Although EGCG has been shown to be growth inhibitory in a number of tumor cell lines, it is not clear whether the effect is cancer-specific. In this study we compared the effect of EGCG on the growth of SV40 virally transformed WI38 human fibroblasts (WI38VA) with that of normal WI38 cells. The IC 50 value of EGCG was estimated to be 120 and 10 μM for WI38 and WI38VA cells, respectively. Thus, EGCG at 40 μM completely inhibited the growth of WI38VA cells, but had little or no inhibitory effect on the growth of WI38 cells. Similar differential growth inhibition was also observed between a human colorectal cancer cell line (Caco-2), a breast cancer cell line (Hs578T) and their respective normal counterparts. EGCG at a concentration range of 40–200 μM induced a significant amount of apoptosis in WI38VA cultures, but not in WI38 cultures, as determined by terminal deoxynucleotidyl transferase assay. After exposure to EGCG at 200 μM for 8 h, more than 50% of WI38VA cells in a confluent culture became apoptotic. In contrast, less than 1% of WI38 cells displayed apoptotic labeling under the same condition. EGCG did not affect the serum-induced expression of c-fos and c-myc genes in normal WI38 cells. However, it significantly enhanced their expression in transformed WI38VA cells. It is possible that differential modulation of certain genes, such as c-fos and c-myc, may cause differential effects of EGCG on the growth and death of cancer cells.

Differential gene expression between young and senescent, quiescent WI-38 cells

To investigate age-related changes in gene expression in WI-38 cells, we isolated RNA from young and senescent, quiescent cultures and made subtracted cDNA libraries. Density-arrested cells were incubated in serum-free MCDB-104 for 3 days. RNA was then isolated and subtracted cDNA libraries were made in the phagemid vector pCDM8. Both by picking clones at random from these subtracted libraries and by differential hybridization screening with subtracted cDNA probes from young and senescent cells, we have identified a total of 11 genes for which RNA is expressed differentially in these quiescent young and senescent WI-38 cultures. Two genes, EPC-1 and EPC-A2, with elevated RNA levels in young cells, have sequences which have not previously been identified. Two of the genes with elevated RNA expression in the senescent cells are the mitochodnria-coded genes for NADH dehydrogenase subunit 4 and for cytochrome b. We also identified seven other genes with elevated RNA levels in senescent cells. Three of these, LPC-1, LPC-14 and LPC-24, have been partially sequenced and have not previously been identified. These studies show that density-arrested, serum-deprived, quiescent young and senescent cells express a number of genes differentially. These differences are not growth-dependent, but are age-dependent. Our studies also show that the methods employed here, which include careful regulation of the cell cultures and subtraction of the libraries, result in libraries from which differentially expressed genes can be identified, either by random selection or by differential hybridization screening with subtracted probes.

Effects of cell density and extracellular matrix on the lateral diffusion of major histocompatibility antigens in cultured fibroblasts.

We have studied the effect of cell density on the lateral diffusion of major histocompatibility (MHC) antigens in the plasma membranes of fibroblasts using fluorescence recovery after photobleaching. The percent recovery of fluorescence was decreased in fibroblasts grown in confluent cultures. While recovery of fluorescence was measurable in greater than 90% of the cells from sparse cultures, measurable recovery was detected in only 60-80% of the cells from dense cultures; no mobile antigens were detectable in 20-40% of cells examined. The diffusion coefficient on human skin fibroblast cells that did show recovery was the same for cells grown in sparse or dense conditions. In WI-38, VA-2, and c1 1d cultures the diffusion coefficients of mobile antigens were smaller in cells from dense cultures. Changes in lateral diffusion occurred with increased cell-cell contact and with age of cell culture but were not observed in growth-arrested cells or in sparse cells cultured in medium conditioned by confluent cells. Decreased mobile fractions of MHC antigens were observed when cells were plated on extracellular matrix materials derived from confluent cultures. Treatment of the extracellular matrix materials with a combination of proteolytic enzymes or by enzymes that degrade proteoglycans abolished this effect. Matrices produced by cells from other cell lines were less effective in inducing changes in mobile fractions and purified matrix components alone did not induce changes in lateral diffusion. Finally, there were no differences in the proportion of MHC antigens that were resistant to Triton X-100 extraction in sparse and dense cells. These results suggest that cell-cell interactions mediated through extracellular matrix materials can influence the lateral diffusion of at least part of the population of MHC antigens.

The effect of dexamethasone on epidermal growth factor binding and stimulation of proliferation in young and senescent WI38 cells

Addition of 0.14 μM dexamethasone (DEX) to young log-phase WI38 cultures seeded at various densities in serum-free medium containing 4.1 nM epidermal growth factor (EGF) resulted in a synergistic increase in proliferation and final cell density. The action of DEX plus EGF was stimulatory but not synergistic in young confluent cultures. DEX plus EGF had no synergistic effect on senescent cells either during log phase or at confluence. Analysis of the effect of DEX on [ 125I]EGF binding revealed no statistically significant changes in either the number of binding sites or the apparent dissociation constant of the EGF-receptor complex.

Enhanced proliferation of WI38 cells in the presence of glucocorticoid-conditioned medium

The addition of hydrocortisone (HC) or dexamethasone (DEX) to WI38 cells at subcultivation is known to result in increased saturation densities (20–40%). We report that maximal increases in saturation density are, however, only observed if HC is added to the culture shortly after subcultivation. We have found that the proliferative response of WI38 cells to glucocorticoids is mediated by a secondary stimulatory factor(s) present in medium-conditioned by cells in the presence of the hormone. Control cultures refed with medium conditioned in the presence of HC for the first 24 h after seeding (24 h HC-CM) achieve saturation densities 20–40% higher than cultures refed with either medium conditioned in the absence of the hormone (24 h CM) or 24 h CM supplemented with fresh HC. Furthermore, WI38 cultures remain responsive to the stimulatory activity present in 24 h HC-CM throughout the growth cycle. The stimulatory effects of 24 h HC-CM are enhanced by repeatedly refeeding cultures; WI38 cells refed every 2 days with 24 h HC-CM demonstrate an extended period of logarithmic growth and achieve densities 2–3 times higher than controls. A preliminary characterization of this activity shows it to be of low molecular weight (MW) (dialyzable using 12 000 MW cut-off tubing) and heat-stable (75°C).

Thymidine triphosphate synthesis in senescent WI38 cells: Relationship to loss of replicative capacity

The effect of in vitro age on thymidine triphosphate (TTP) synthesis was assessed in WI38 cultures according to the following measurements: (1) thymidine kinase activity of broken cell preparations; (2) in situ incorporation of [ 3H]thymidine into acid-soluble material; and (3) total intracellular TTP content as determined by an enzymatic assay. All three parameters were maximal in exponentially proliferating populations and minimal in quiescent monolayers; no signifcant differences between young and old cultures were observed despite the reduced replicative capacity of the latter. The addition of serum to density-arrested cultures induced both TTP synthesis and DNA replication after a lag of approx. 12 h; although a greater percentage of young cells initiated replication as compared with old, pool sizes expanded to a similar extent in both populations. Pool expansion did not require entry into S phase; the pool sizes of control and cytosyl arabinoside-treated cultures were comparable. These findings suggest that senescent cells retain the ability to synthesize TTP, even though they are incapable of replicating DNA. Because TTP synthesis is a cell cycle-dependent event that normally begins in late G1, senescent cells might be blocked in the latter portion of the prereplicative phase and not in G0 as are quiescent cells.

Activity of 2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile (MDL-860) against picornaviruses in vitro.

The newly synthesized compound 2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile (MDL-860) has been found to inhibit picornavirus replication. In multiple growth cycle experiments, 1 microgram of MDL-860 per ml caused a reduction in virus yield of at least 1.0 log10 50% tissue culture infectious doses per 0.2 ml for 8 of 10 enteroviruses and 72 of 90 rhinovirus serotypes. This antiviral activity was dependent on both compound concentration and virus inoculum size. At concentrations that had no toxic effects on cell cultures, MDL-860 did not inhibit cytopathic effect or hemadsorption activity due to coronavirus 229-E, vesicular stomatitis virus, herpes simplex virus type 1, adenovirus, influenza virus A, or parainfluenza virus 1. Compound concentrations up to 25 micrograms/ml did not cause cytopathic effect in short-term cultures of rhesus monkey, WI-38, or HeLa cells; 10 micrograms/ml did not inhibit the replication of HeLa cells. The mechanism of action of MDL-860 has not been defined, although it was not directly virucidal and appeared to inhibit picornaviruses specifically at an early step in the virus-host cell interaction.

DNA synthesis in permeabilized WI38 and MRC5 cells

DNA synthesis was examined in cultures of growing WI38 and MRC5 cells made permeable to deoxyribonucleotides. Cells from late passage cultures showed a reduced rate of deoxythymidine triphosphate (dTTP) uptake as compared to cells from early- to mid-passage cultures. This reduction became evident earlier in WI38 cultures (passage 33) than in MRC5 cultures (passage 41). Although this reduced rate of incorporation appeared to be primarily due to a reduced percentage of replicating (S phase) cells in later passage cultures, some effect on the rate of DNA synthesis in replicating cells was also evident.

Inhibition of cyclic nucleotide phosphodiesterase during exposure to WI-38 cells to prostaglandin E1.

Short term incubation of WI-38 cultures with 5.7 micron prostaglandin E1 (PGE1) caused cyclic AMP phosphodiesterase activity in fibroblast homogenates to fall by 25 to 35% as compared to controls. The PGE1-induced decline in phosphodiesterase activity coincided with a rapid increase in intracellular cyclic AMP levels in response to the hormone and was rapidly reversed by washing the cultures free of the prostaglandin before homogenizing the cells. The effect of PGE1 on WI-38 phosphodiesterase activity was localized to the enzyme form(s) present in 27,000 times g supernatant fractions of cell homogenates. These data suggest that the pattern of cyclic AMP accumulation in WI-38 fibroblasts exposed to PGE1 may be related, at least in part, to decreased phosphodiesterase activity during hormone stimulation.

Age-related decline in the synthesis of glycosaminoglycans by cultured human fibroblasts (WI-38)

A gradual decline in the synthesis of glycosaminoglycans, as evidenced by reduced rates of incorporation of [ 35S]-sulfate and [ 14C]-glucosamine into total cellular and extracellular glycosaminoglycans, occurred during the last 4 to 5 passages (1 : 2 splits) of WI-38 cultures before phase out. While labelling of cellular glycosaminoglycans by both radioactive precursors was reduced to about the same extent, a relatively greater decline in [ 35S]-sulfate than in [ 14C]-glucosamine incorporation into extracellular glycosaminoglycans was observed during the last passages. These changes in glycosaminoglycan metabolism are interpreted as an expression of cellular aging and a function of glycosaminoglycans in growth regulation (and possibly in the process of cellular senescence) is discussed.

Results of chromosome analysis of rabies virus infected human diploid cells (WI-38).

Fixed rabies virus strains did not induce mitotic or chromosome abnormalities in infected WI-38 cultures. Infected cells were shown to undergo mitosis thus indicating an endosymbiotic type of infection.

Prolyl and lysyl hydroxylase activation and cofactor specificity in young and senescent WI-38 fibroblast cultures

The activation of prolyl hydroxylase and lysyl hydroxylase by ascorbate was studied in young and senescent WI-38 fibroblast cultures using a tritium-release assay. Prolyl hydroxylase activity could be increased 3–4 fold in young cultures but remained unchanged in senescent cultures when these cultures underwent a two-hour preincubation in medium containing 0.2mM sodium ascorbate. Lysyl hydroxylase levels were unaffected both in young and senescent cultures. In another series of experiments, ascorbate was replaced with several other compounds in the tritium-release assay demonstrating that this reducing agent is not a specific cofactor of the partially purified enzymes from WI-38 cultures.

Comparison by autoradiography of macromolecular biosynthesis in “young” and “old” human diploid fibroblast cultures. A brief note

The biosynthetic abilities of WI-38 fibroblasts from early and late population-doubling-level cultures were compared by autoradiography of cells grown with labeled precursors of DNA, RNA, protein and lipids. Incorporation of radioactive thymidine, uridine, protein-hydrolysate, acetate, oleic acid and cholesterol, as measured by the number of grains per cell surface, decreased with the progressive aging of the culture. However, the decrease in the incorporation of acetate, oleic acid and cholesterol was much smaller than that of the other precursors, indicating that lipid synthesis is affected to a lesser degree that protein and nucleic acid synthesis on aging. This result is in accord with the higher lipid content and proliferation of intracellular membranes in cells of “old” WI-38 cultures reported by others.

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.

Protein alterations in aging WI38 cells as determined by proteolytic susceptibility

Summary In an effort to determine whether many different abnormal proteins are present in phase III W138 cells, we have determined the susceptibility to proteolysis of the total proteins isolated from phase II and phase III cultures. The results demonstrate that proteins with increased proteolytic susceptibility can be detected only at the last population doubling of W138 cultures, but not sooner. Such increased proteolytic susceptibility indicates that proteins from terminal phase III cells are modified in relation to proteins from phase II and early phase III cultures. The modification could be due to errors of transcription, translation, or to post-translational alterations that may be fundamental or incidental to the in vitro senescence phenomenon. These results suggest that biochemical studies of phase III WI38 cultures should not overlook the last population doubling.

Posttranslational protein modifications, with special attention to collagen and elastin.

It is apparent that significant progress has been made in our understanding of the biosynthesis, modifications, and maturation of collagen and elastin. We now recognize and partially understand special reactions involved in hydroxylations within the cell and complex cross-linking processes occurring outside the cell. Recent experiments (191) have shown that in human diploid fibroblast cultures of limited doubling potential (191) the hydroxylation of collagen prolyl residues appears to be "age" or passage-level dependent. With increasing passage level of these cultures, both the ascorbate requirements and the extent of collagen hydroxylation decrease. "Young" cell cultures have a strong requirement for complete hydroxylation and without ascorbate there is only about 50% of the normal level. "Middle-aged" cultures show higher hydroxylation without and full hydroxylation with ascorbate, whereas "old" (or cultures close to "senescence") are incapable of full hydroxylation with or without ascorbic acid. Although the overall system may show some deterioration with increasing passage levels, it appears that with increasing passage levels other components in the cell replace the ascorbate dependence of the hydroxylase system to a greater exten. In some ways, aging WI-38 cultures begin to resemble some transformed cells in their biochemical reactions, although they continue to remain diploid and eventually lose the ability to replicate. It is not yet known whether old animals can produce collagen, which may now be underhydroxylated, perhaps contributing to certain senescent changes. Careful examination of the hydroxylation index of collagen produced in organoid cultures of tissue biopsies as a function of donor age might be informative, particularly if one looks at the quality of collagen by employing collagenase and other proteolytic digests with collagen (191). One could comare the levels of frequent and characteristic peptide triplet sequences such as Gly-Pro-Hyp to Gly-Pro-Pro, Gly-Ala-Hyp to Gly-Ala-Pro, or Gly-Pro-Hyl to Gly-Pro-Lys and others for evaluation of hydroxylation throughout the entire molecule or at selected sequences.

Genealogies of clones of diploid fibroblasts: Cinemicrophotographic observations of cell division patterns in relation to population age

Direct assessment of cell division patterns of human diploid fibroblasts (WI-38) has been achieved using techniques of time-lapse cinemicrophotography. Pedigrees of progeny cells in clones from middle and late passage WI-38 cultures are presented. The passage 28 clone exhibited a division pattern that was highly synchronous through four generations and had an average interdivision time that increased linearly from the second through sixth generations. Division was essentially logarithmic through the fourth generation. The passage 53 clone was much more variable in interdivision time, less synchronous, and less motile than the passage 28 clone. While there is considerable overlap in the interdivision times of cells of the passage 28 and 53 clones, the late passage cells generally exhibit more variation in interdivision times. The average population doubling time was 16.8 h in thepassage 28 clone and 32.0 h in the passage 53 clone.

Intermittent DNA synthesis and periodic expression of enzyme activity in the cell cycle of WI-38.

Synchronous cultures of WI-38 were obtained using an automated system for detachment and partitioning of mitotic cells which operates without the use of inhibitors, altered medium, or lowered temperatures. The generation time in synchronous WI-38 is 19.5 h and the duration of S phase when determined from the percentage of labeled metaphase cells or nuclei is 12 h. DNA replication in WI-38 occurs in three temporally distinct and rapid bursts separated by intervals of greatly reduced synthesis within what is nominally described as the DNA synthetic (S) period. Lactate dehydrogenase (LDH) displayed maxima in G(1) between 2 and 4 h and again at 10 and 16 h. Peaks in LDH activity were coordinated with DNA replication in a fashion similar to that reported for diploid Chinese hamster cells. Oscillations in LDH activity are more pronounced in normal diploid fibroblasts than in established and neoplastic lines.

Abortive infection of human diploid cells by murine cytomegalovirus.

Inoculation of human diploid cells (WI-38) with murine cytomegalovirus (MCMV) did not result in the synthesis of any infectious virions. However, morphological changes typical of the cytopathic effects (CPE) of MCMV were detectable within 12 hr of infection. The CPE included rounding, swelling, and detachment of cells. The nuclei of infected cells were enlarged, and intranuclear inclusions were visible by May Grunwald-Giemsa staining and by the indirect fluorescent-antibody test. All cells infected at a multiplicity of infection of 3 showed CPE, and these cells could not be passaged successfully. Cell lysates and exhausted media from infected WI-38 cultures did not produce any CPE in WI-38 cells. The virus absorbed to WI-38 cells with the same efficiency as to mouse embryo fibroblast cells (MEF). Samples of MCMV in which virus infectivity for MEF cells had been inactivated by ultraviolet irradiation or by exposure to 56 C failed to produce any of the above signs. MCMV-specific CPE did not occur in the presence of actinomycin D (1 mug/ml) or puromycin (20 mug/ml), but 5'-fluoro-2'-deoxyuridine at 1 x 10(-4)m did not prevent CPE or the development of intranuclear inclusions.