Autofluorescent structures in cultured WI-38 cells

Human fibroblasts (WI-38) were monitored for intracellular fluorescence at several passage levels. It was found that late-passage cells contained marked increments in particulate intracellular fluorescence when compared with early-passage cells. Intracellular hydrolysis of α-glycerophosphate at pH 5 was used to localize acid phosphatase activity. Most fluorescent structures contained acid phosphatase, and it was concluded that the fluorescence was associated with lysosomes. Autoradiographic analysis of labeled-thymidine uptake into nuclei was employed to distinguish dividing and nondividing cells in cultures of varying passage levels, and these results were correlated with intracellular fluorescence. It was found that cells containing high levels were unable to incorporate thymidine into nuclei. These results suggest a correlation between fluorescence accumulation in lysosomes during aging and inhibition of cell cycling.     

Glutamine synthetase activity in WI-38 cells

In the presence of complete growth media (Eagle's MEM), human diploid WI-38 cells have a low level of glutamine synthetase activity. The activity could be increased by depriving the cells of exogenous glutamine; addition of hydrocortisone to either glutamine-deficient or complete medium had no effect on the activity of the enzyme. Cell growth ceased under conditions that enhanced glutamine synthetase activity, and hydrocortisone could not reverse this inhibition.     

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.     

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.     

Clone size variation in the human diploid cell strain, WI-38

By mapping the location of isolated single cells; and then counting the number of cells at each location as a function of time. it was possible to accumulate data on the growth history for each of a large group of clones. The clone size distribution, its mean and standard deviation were computed for each day in culture. Variations in schedule of medium change and time of exposure to trypsin, did not measurably affect variation in clone size. Neither could clone size variation be accounted for on the basis of (1) occurrence of nondividing cells nor (2) presence of heritable growth rate variants in the population. It is probable that clone size variation under our conditions is primarily a consequence of a highly variable interdivision time among the constituent cells.     

HDACs and the senescent phenotype of WI-38 cells

Background  

Normal cells possess a limited proliferative life span after which they enter a state of irreversible growth arrest. This process, known as replicative senescence, is accompanied by changes in gene expression that give rise to a variety of senescence-associated phenotypes. It has been suggested that these gene expression changes result in part from alterations in the histone acetylation machinery. Here we examine the influence of HDAC inhibitors on the expression of senescent markers in pre- and post-senescent WI-38 cells.     

Repair of x-ray damage in aging WI-38 cells

Rate of strand rejoining and the ability to perform repair replication were determined in young ad old X-irradiated WI-38 cells. No differences in either process were apparent and we conclude that reduced efficiency in one or both of them is not responsible for _{in vitro} aging of human cells.     

Calcium, magnesium, and growth control in the WI-38 human fibroblast cell

WI-38 and SV40WI-38 cells have been synchronized using centrifugal elutriation. This technique allows for the rapid harvesting of early G1 phase cells from exponentially growing populations of both the normal and transformed cell. Using these cells, as well as WI-38 cells synchronized by serum deprivation, we have examined the effects of extracellular Ca and Mg levels on the progression of cells through G1 phase. A differential sensitivity to both Ca and Mg deprivation is observed between normal and transformed cells. The WI-38 cell requires higher levels of both ions for traversal of G1 phase and for continued proliferation as compared to the transformed cell. The temporal nature of the Ca and Mg requirements for the WI-38 cell has been examined during G1 phase. Ca is strictly required during early and late G1 phase, but not necessarily throughout mid-G1. An early as well as a late G1 Ca requirement is also found in serum-stimulated WI-38 cells. In contrast, the Mg requirement of WI-38 cells does not appear to be temporally well-defined. Mg appears to be a permissive factor, required throughout G1 phase rather than at certain prescribed intervals. On the basis of these data, it seems unlikely that these two cations exert their effects on cell growth entirely through a common competitive mechanism. Ca would appear to be involved in early serum or growth factor-mediated G1 events and later pre-S-phase events, as suggested in previous studies on other cell lines.     

Rhinovirus Plaque Formation in WI-38 Cells with Methylcellulose Overlay

A sensitive, reliable plaque assay system is described for five rhinoviruses using freshly prepared methylcellulose overlay and human embryonic diploid cells. Circular plaques with irregular edges, 2 mm in size, were formed by rhinoviruses 1A, 2, 6, and 13 after 6 or 7 days of incubation. A fifth rhinovirus, 17, formed a 1- to 2-mm feather plaque after 14 days of incubation. Plaque counts of rhinoviruses 1A and 13 were not affected by varying the pH of the overlay from 6.9 to 7.5. Plaque sizes and plaque-forming unit values of high passage rhinoviruses 1A and 13 were equivalent when tested at 26, 31, or 36 C. The rhinoviruses tested were sensitive to incubation at 40 C or heating at 50 C. Enhancement of plaques was observed when Mg(++) was incorporated into agar overlays, but enhancement did not occur when Mg(++) was added to methylcellulose overlays.     

Transition probability and the division pattern of WI-38 Cells

Analysis of the pedigrees of WI-38 clones reveals a division pattern in accord with the transition probability model of the cell cycle. This is in distinct contrast to a recent report by METS and VERDONK (1978).     

Interplay between Selenium Levels,Selenoprotein Expression,and Replicative Senescence in WI-38 Human Fibroblasts

Selenium is an essential trace element, which is incorporated as selenocysteine into at least 25 selenoproteins using a unique translational UGA-recoding mechanism. Selenoproteins are important enzymes involved in antioxidant defense, redox homeostasis, and redox signaling pathways. Selenium levels decline during aging, and its deficiency is associated with a marked increase in mortality for people over 60 years of age. Here, we investigate the relationship between selenium levels in the culture medium, selenoprotein expression, and replicative life span of human embryonic lung fibroblast WI-38 cells. Selenium levels regulate the entry into replicative senescence and modify the cellular markers characteristic for senescent cells. Whereas selenium supplementation extends the number of population doublings, its deficiency impairs the proliferative capacity of WI-38 cells. We observe that the expression of several selenoproteins involved in antioxidant defense is specifically affected in response to cellular senescence. Their expression is selectively controlled by the modulation of mRNA levels and translational recoding efficiencies. Our data provide novel mechanistic insights into how selenium impacts the replicative life span of mammalian cells by identifying several selenoproteins as new targets of senescence.     

Immunochemical quantitation of enzymes in human diploid cell line WI-38

A quantitative immunochemical study was carried out of four enzymes, cathodal esterase, acid phosphatase, glucosaminidase and β-glucuronidase. In homogenates of the human diploid cell line WI-38, the relative amounts of the enzymes increased with the passage number of the culture, although great variation was found in later passages just before death of the culture.     

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.     

Propagation of Rhinovirus on WI-38 Cell Monolayers in Rolling Bottles

Techniques are described for the propagation of rhinoviruses on WI-38 monolayers in rolling bottles. High yields of viruses were obtained, as indicated by infectivity titers and electron microscopy. When crude harvests were subjected to low-speed centrifugation and then filtered through a 0.45-μ membrane filter, little or no loss in infectivity titer was observed. However, electron microscopic examination indicated that the concentration of viral physical particles was reduced below detectable levels after filtration. The guinea pig potency test on the lot of unfiltered rhinovirus 14 vaccine prepared in rolling bottles indicated that this vaccine stimulated higher reciprocal serum-neutralizing titers than a rhinovirus 14 vaccine prepared in stationary monolayers.     

Comparison of EGF receptors from young and old WI-38 cells

EGF receptors from young and old WI-38 cells were compared by immunoprecipitation of the receptor followed by electrophoresis. There was no difference in molecular weight between young and old receptors. Both were composed of two components with molecular weights of about 165,000 and 145,000. Young EGF receptors were phosphorylated when incubated with (gamma-32P)ATP. Old receptors had markedly reduced phosphorylation, indicating a loss of kinase activity with age. These results demonstrate a major metabolic difference between young and old cells which clarifies the nature of the decline in mitogenic response with age.     

Respiration and glycolysis in the human diploid cell strain WI-38

Assessment of the respiratory and glycolytic capacity of non-growing WI-38 cells shows that, in the absence and presence of added glucose, the mean rates of oxygen consumption were 247 (QO₂ = 5.61) and 208 (QO₂ = 4.73) mμmoles/mg dry wt/hr., respectively. Mean glucose consumption was 225 mμmoles/mg dry wt/ hr. With uniformly labeled ¹⁴C glucose as substrate, 36 mμg atoms of carbon dioxide were produced, corresponding to 15–20% of the total cellular respiration. Mean values for lactate production in the presence and absence of glucose were 345 (Q_L^O2 = 7.85) and 196 (Q_L^O2 = 4.45) mμmoles/mg dry wt/hr., respectively. Human diploid cells in culture age, in the sense that their ability to proliferate decreases with time during serial subcultivation. Studies of their respiratory and glycolytic capacity as a function of the aging process showed that total respiration, glucose respiration and glycolytic capacity were relatively constant for cells in the middle and late passages and indicate that aging in this sense is not directly related to the respiratory and glycolytic capacity of the cell.