Glucocorticoid receptors in WI-38 fibroblasts Characterization and changes with population doubling in culture

A high-affinity dexamethasone binding macromolecule was identified in WI-38 human fetal lung fibroblasts. High specificity of binding for glucocorticoidc was shown by competition studies in which binding of dexamethasone was inhibited by cortisol and corticosterone but not by testosterone of 17β-estradiol. WI-38 cells exposed to [³H]dexamethasone at 30°C were able to transfer the ³H-labeled steroid-receptor complex to the nuclear material. A reduction of 30–50% was observed in the number of [³H]dexamethasone-receptor binding sites per cell as well as in the nuclear fraction of the cells as a function of age (passage levels 27 and 54). However, in the same cells no significant changes in affinity of receptor for [³H]dexamethasone as a function of the two passage levels were detected.     

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

Summary Activities of three lysosomal enzymes—acid RNase,N-acetyl-β-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 five-fold; 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 cultured 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. This study was supported by Grant GM-21357 from the National Institutes of Health.     

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.     

Subcellular fractionation of actively growing protoplasts of Saccharomyces cerevisiae

Cell homogenates obtained from partially regenerated Saccharomyces cerevisiae protoplasts were fractionated by a procedure using a combination of continuous and discontinuous sucrose gradients, under experimental conditions that minimize possible artifacts due to centrifugation and resuspension. At least five different membranous organelle fractions (plasma membrane, mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum-like structures and small-sized particulated structures) were isolated. Subcellular fractions were characterized by assaying established marker enzymes. Radioactive labelled ([U-³H]uracil) ribosomes were also used as a further characterization criterion of the rough endoplasmic reticulum. Comparative SDS-polyacrylamide gel electrophoresis of the protein constituents of the isolated membrane-bound organelles suggest that the polypeptide pattern could also be used as an additional marker for some of these structures. Finally, subcellular distribution of chitin synthase was determined using this fractionation procedure, and two partially zymogenic enzyme pools (one inside the cell associated to particles which sediments at high speed, and the second one associated to the plasma membrane) were found.     

Critical adjustment of cysteine and glutamine concentrations for improved clonal growth of WI-38 cells

Summary Clonal growth of WI-38 cells with a plating efficiency of 45% has been achieved in a synthetic nutrient mixture (MCDB 102) supplemented with either whole or dialyzed fetal bovine serum. For optimum growth, the concentration of cysteine in the medium must be adjusted precisely. Deviation by a factor of three in either direction from the optimum concentration (9.0×10⁻⁵M) eliminates essentially all clonal growth. A high concentration of glutamine (2.5×10⁻³M) is also needed for, optimum clonal growth. Presented in preliminary form at the 26th Annual Meeting of the Tissue Culture Association, June 4, 1975. This work was supported by Grant No. HD-08181 from the National Institute of Child Health and Human Developement, Grant No. AG-00310 from the National Institute on Aging, and by Contract No. 223-74-1156 from the Bureau of Biologics, Food and Drug Administration.     

Tyrosine protein kinase expression in long-term quiescent WI-38 cells following growth factor simulation

We have used the WI-38 cell long-term quiescent model system to study the regulation of cell cycle progression at the molecular level. By modulating the length of time that WI-38 cells are density arrested, it is possible to proportionately alter the length of the prereplicative or G-1 phase which the cell traverses after growth factor stimulation in preparation for entry into DNA synthesis. Stimulation of long-and short-term density arrested WI-38 cells with different growth factors or higher concentrations of individual growth factors does not alter the time required by long-term cells to enter S after stimulation. However, the time during the prereplicative period for which these growth factors are needed is different. Long-term quiescent WI-38 cells require EGF to traverse the G-0/G-1 border but do not need and apparently cannot respond to IGF-1 during the first 10 h after EGF stimulation, the length of the prolongation of the prereplicative phase. This suggests that EGF stimulation of long-term quiescent WI-38 cells initiates a series of molecular events which make these cells “competent” to respond to the “progression” growth factor, IGF-1. In light of the well-established role of protein tyrosine kinases in signal transduction, we set out to identify, clone, and analyze the expression of receptor and non-receptor tyrosine kinases which potentially could play a role during the prolongation of the prereplicative phase in making the long-term quiescent WI-38 cells competent to respond to IGF-1. We obtained 49 clones representing 11 different receptor and non-receptor type protein tyrosine kinases. Analysis of expression of these clones revealed a variety of different patterns of expression. However, the most striking pattern was exhibited by IGF-1 receptor. Our results suggest that induction of IGF-1 receptor mRNA by EGF may be an important event in the establishment of competence by EGF in long-term density arrested WI-38 cells. © 1995 Wiley-Liss, Inc.     

Identification of proteins undergoing expression level modifications in WI-38 SV40 fibroblasts overexpressing methionine sulfoxide reductase A

Methionine sulfoxide reductase A overexpressing WI-38 SV40 human fibroblasts have been previously shown to exhibit higher resistance to oxidative stress by decreasing intracellular reactive oxygen species content and oxidative damage to proteins [C.R. Picot, I. Petropoulos, M. Perichon, M. Moreau, C. Nizard, B. Friguet, Overexpression of MsrA protects WI-38 SV40 human fibroblasts against H(2)O(2)-mediated oxidative stress, Free Radic Biol Med 39 (2005) 1332-1341]. In order to get further insight into the molecular mechanisms underlying this resistance to oxidative stress, proteins that are differentially expressed in methionine sulfoxide reductase A overexpressing cells were identified by 2D gel and Western blot quantitative analyses. Five proteins were shown to be differentially expressed and were identified by mass spectrometry, some of them were related to either cellular protection against oxidative stress, apoptosis or premature ageing.     

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.     

The purification of plasma membranes from WI-38 fibroblasts Effects of ageing on their composition

A three-step method for the purification of plasma membranes from WI-38 fibroblasts was developed thus allowing the recovery of 36–44% of the plasma membrane. Except in the case of galactosyltransferase, the activity of the contaminating enzymes was very low. Morphological observations confirm the presence of a homogeneous population of vesicles.Preparations obtained from young and old cell cultures were compared for their enzymatic and protein contents. With ageing the activity of 5′-nucleotidase significantly increases whereas that of alkaline phosphodiesterase I decreases. Out of the 26 components detected after sodium dodecyl sulphate polyacrylamide gel electrophoresis, four decreased but only one increased. Cellular ageing seems to fulfil a specific and localized effect on the plasma membrane.     

Metabolic stabilization of MAP kinase phosphatase-2 in senescence of human fibroblasts

Cellular senescence is characterized by impaired cell proliferation. We have previously shown that, relative to the young counterpart, senescent WI-38 human fibroblasts display a decreased abundance of active phosphorylated ERK (p-ERK) in the nucleus. We have tested the hypothesis that this is due to elevated levels of nuclear MAP kinase phosphatase (MKP) activity in senescent cells. Our results indicate that the activity and abundance of MKP-2 is increased in senescent fibroblasts, compared to their young counterparts. Further analysis indicates that it is MKP-2 protein, but not MKP-2 mRNA level, that is increased in senescent cells. This increase is the result of the increased stability of MKP-2 protein against proteolytic degradation. The degradation of MKPs was impaired by proteasome inhibitors both in young and old WI-38 cells, indicating that proteasome activity is involved in the degradation of MKPs. Finally, our results indicate that proteasome activity, in general, is diminished in senescent fibroblasts. Taken together, these data indicate that the increased level and activity of MKP-2 in senescent WI-38 cells are the consequence of impaired proteosomal degradation, and this increase is likely to play a significant role in the decreased levels of p-ERK in the nucleus of senescent cells.     

WI-38 cell long-term quiescence model system: A valuable tool to study molecular events that regulate growth

A number of cell culture model systems have been used to study the regulation of cell cycle progression at the molecular level. In this paper we describe the WI-38 cell long-term quiescence model system. By modulating the length of time that WI-38 cells are density arrested, it is possible to proportionately alter the length of the prereplicative or G-1 phase which the cell traverses after growth factor stimulation in preparation for entry into DNA synthesis. Through studies aimed at understanding the cause and molecular nature of the prolongation of the prereplicative phase, we have determined that gene expression plays an important role in establishing growth factor “competence” and that once the cell becomes “competent” there is a defined order to the molecular events that follow during the remainder of G-1. More specifically, we have determined that the prolongation represents a delay in the ability of long term quiescent cells to become fully “competent” to respond to growth factors which regulate progression through G-1 into S. This prolongation appears to occur as a result of changes during long term quiescence in the ability of immediate early G-1 specific genes (such as c-myc) to activate the expression of early G-1 specific genes (such as ornithine decarboxylase). While ODC is the first and thus far only growth associated gene identified as a target of c-myc (and the Myc/Max protein complex), it is likely that further studies in this model system will reveal other early G-1 growth regulatory genes. We anticipate that future follow-up studies in this model system will provide additional valuable information abuot the function of growth-regulatory genes in controlling growth factor responsiveness and cell cycle progression.     

Subcellular fractionation of human liver reveals limits in global proteomic quantification from isolated fractions

The liver plays an important role in metabolism and elimination of xenobiotics, including drugs. Determination of concentrations of proteins involved in uptake, distribution, metabolism, and excretion of xenobiotics is required to understand and predict elimination mechanisms in this tissue. In this work, we have fractionated homogenates of snap-frozen human liver by differential centrifugation and performed quantitative mass spectrometry-based proteomic analysis of each fraction. Concentrations of proteins were calculated by the “total protein approach”. A total of 4586 proteins were identified by at least five peptides and were quantified in all fractions. We found that the xenobiotics transporters of the canalicular and basolateral membranes were differentially enriched in the subcellular fractions and that phase I and II metabolizing enzymes, the cytochrome P450s and the UDP–glucuronyl transferases, have complex subcellular distributions. These findings show that there is no simple way to scale the data from measurements in arbitrarily selected membrane fractions using a single scaling factor for all the proteins of interest. This study also provides the first absolute quantitative subcellular catalog of human liver proteins obtained from frozen tissue specimens. Our data provide quantitative insights into the subcellular distribution of proteins and can be used as a guide for development of fractionation procedures.     

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.     

Collagen prolyl hydroxylation in WI-38 fibroblast cultures: Action of hydralazine

Summary The action of hydralazine on collagen prolyl hydroxylation was studied in a cell culture system using WI-38 fibroblasts. The prolyl hydroxylation level was determined by a method involving the digestion of collagen by bacterial collagenase and the examination of specific peptides. The presence of low concentrations of hydralazine (0.2 mM) in both “young” and “old” fibroblast cultures strongly inhibited collagen prolyl hydroxylation. The degree of inhibition was greater in serum-deficient cultures. No significant improvement in the degree of hydroxylation was observed by increasing either ascorbate or iron levels in the hydralazine-containing cultures in which hydroxylation was inhibited. Some of the reported side effects of hydralazine seen in patients might be related to its inhibitory effects on mixed function oxidative (MFO) hydroxylation systems. While the ascorbate dependence of the prolyl hydroxylase system of WI-38 decreased with the “age” of the culture, hydralazine inhibition of hydroxylation was dramatic with cultures of all “ages”. This work was supported by NIH grants nos. AM15671, AM1675 and HD07376, and fellowship no. HD01998.     

Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: Characterization of subcellular fractions

Insulin stimulates glucose transport in rat adipose cells through the translocation of glucose transporters from an intracellular pool to the plasma membrane. A detailed characterization of the morphology, protein composition and marker enzyme content of subcellular fractions of these cells, prepared by differential ultracentrifugation, and of the distribution of glucose transporters among these fractions is now described. Glucose transporters were measured using specific d-glucose-inhibitable [³H]cytochalasin B binding. In the basal state, roughly 90% of the cells' glucose transporters are associated with a low-density microsomal, Golgi marker enzyme-enriched membrane fraction. However, the distributions of glucose transporters and Golgi marker enzyme activities over all fractions are clearly distinct. Incubation of intact cells with insulin increases the number of glucose transporters in the plasma membrane fraction 4–5-fold and correspondingly decreases the intracellular pool, without influencing any other characteristics of the subcellular fractions examined or the estimated total number of glucose transporters (3.7·10⁶/cell). Insulin does not influence the K_d of the glucose transporters in the plasma membrane fraction for cytochalasin B binding (98 nM), but lowers that in the intracellular pool (from 141 to 93 nM). The calculated turnover numbers of the glucose transporters in the plasma membrane vesicles from basal and insulin-stimulated cells are similar (15·10³ mol of glucose/min per mol of transporters at 37°C), whereas insulin appears to increase the turnover number in the plasma membrane of intact cells roughly 4-fold. These results suggest that (1) the intracellular pool of glucose transporters may comprise a specialized membrane species, (2) intracellular glucose transporters may undergo conformational changes during their cycling to the plasma membrane in response to insulin, and (3) the translocation of glucose transporters may represent only one component in the mechanism through which insulin regulates glucose transport in the intact cell.     

Subcellular distribution of rat liver porin

The subcellular distribution of rat liver porin was investigated using the immunoblotting technique and monospecific antisera against the protein isolated from the outer membrane of rat liver mitochondria. Subfractionation of mitochondria into inner membranes, outer membranes and matrix fractions revealed the presence of porin only in the outer membranes. Porin was also not detected in highly purified subcellular fractions, including plasma membranes, nuclear membranes, Golgi I and Golgi II, microsomes and lysosomes. Thus, liver porin is located exclusively in the outer mitochondrial membrane.     

Analytical Subcellular Fractionation of Rat Cortex: Resolution of Serotonergic Nerve Endings and Receptors

Abstract: An analytical procedure for the subcellular fractionation of rat brain cortex is presented; it consists of a two-step procedure involving a differential centrifuga-tion using the five-fraction scheme and an isopycnic cen-trifugation in continuous sucrose gradients. All fractions obtained were analyzed for their content of various constituents, such as receptor binding, uptake, and several marker enzymes. Special attention was paid to the subcellular distribution of the serotonin S₂ receptors; they were mainly recovered in the microsomal P fraction, but a significant amount was also associated with the mito-chondrial (M and L) fractions. After equilibration in density gradients, serotonin S₂ receptors revealed two peaks, which were similarly affected after treatment with ami-triptyline and/or yohimbine. There is no evidence to suggest that serotonin S₂ receptors are associated with nerve endings containing the neurotransmitter serotonin. Although three main profiles, a microsomal, a mitochondrial, and a mixed one, clearly appear from the differential centrifugation, subgroups of these main profiles were also found. For instance, the microsomal distribution patterns of serotonin S₂ receptors and 5′-nucleoti-dase are very similar, but differ from that of UDP-galactosyltransferase. Similarly, the mitochondrial profiles of cytochrome oxidase and 5-HT (serotonin) uptake are different. An analytical approach for brain fractionation, when performed with appropriate measurements (cytochrome oxidase, amine uptake, 5′-nucleotidase, and receptor binding), is rapid and clearly differentiates pre-and postsynaptic constituents.     

Regional Localization and Subcellular Compartmentalization of Thyrotropin-Releasing Hormone in Adult Human Brain

In the current study, we sought to define the subcellular compartmentalization of thyrotropin-releasing hormone (TRH) in adult human brain tissues. Upon evaluating tissues (3-24 h post mortem) from 62 humans, ranging in age from 5 to 75 years, we found that TRH was widely distributed throughout the brain. The highest TRH concentration (ng/mg protein) was in the stalk-median eminence region of the hypothalamus (19.3 +/- 3.3, mean +/- SE); the TRH concentration in the hypothalamus, exclusive of the stalk-median eminence, was much lower (1.7 +/- 0.2). Substantial quantities of TRH also were detected in the medulla oblongata (0.26 +/- 0.08), mammillary bodies (0.33 +/- 0.25), and optic chiasm (0.14 +/- 0.07). Lower levels of TRH were found in the amygdala (0.060 +/- 0.015) and the corpus striatum (0.033 +/- 0.010). TRH was near or below the limits of detection in tissues of the cerebral and cerebellar cortices, the olfactory bulbs, the pons, and the hippocampus. When homogenates of medial basal hypothalamic tissue (prepared in 0.32 M sucrose-10 microM CaCl2) were fractionated by means of differential centrifugation, most of the TRH was recovered in subcellular particles which were pelleted at 10,000 X g and which contained the highest amounts of occluded LDH activity. When the nuclei-free supernatant fluid (900 X g S) was fractionated on discontinuous sucrose density gradients or continuous sucrose density gradients, most of the TRH was recovered in subcellular fractions containing synaptosomes. The subcellular distribution of TRH appeared to be stable for up to 24 h post mortem in rat and human brain tissue.(ABSTRACT TRUNCATED AT 250 WORDS)