Cultures Of Human Diploid Fibroblasts Research Articles

Glycopeptide hormone production by cultured human diploid fibroblasts.

Human diploid fibroblasts in culture were examined for production of glycopeptide hormones. Forty-one percent of the strains produced human chorionic gonadotropin (hCG) under normal growth conditions. Constitutive hCG synthesis was apparently unrelated to donor age, length of time in culture, or number of passages. Follicle stimulating hormone (FSH) was not found in any strain investigated. Only one cell strain produced free alpha-chains of glycopeptide hormones. Hydroxyurea (HU) at a concentration of 1 mM mediated a small, statistically significant increase in hCG production (p less than 0.01) in all constitutive strains, but had no effect on non-hCG-producing fibroblast strains. Sodium butyrate (Bu) was effective in increasing hCG synthesis in only one constitutive strain, derived from a newborn foreskin. HU treatment had no apparent effect on cell structure. All Bu-treated strains, both those producing hCG and the nonproducers, showed morphological alterations; cells were flattened and they contained ordered arrays of refractile granules. It is suggested that hCG synthesis in cultured human diploid fibroblasts may result from a localized chromosomal event in which the loci responsible for this hormone are activated. Human diploid fibroblasts in culture are shown to be amenable to the study of gene expression and its modulation.

Adaptive enhancement of cystine and glutamate uptake in human diploid fibroblasts in culture

The effect of cystine starvation on the transport system of cystine and glutamate was examined in cultures of human diploid fibroblasts. The 2-min uptake of cystine and glutamate increased progressively after a lag of 6 h of cystine starvation. There was approx. 2–3-fold increase, and the increased rate of uptake was accompanied by an increase in the V max and unchanged K m. The cystine starvation-induced enhancement appeared specific for the uptake of cystine and glutamate. Actinomycin D or cycloheximide completely blocked the time-related increase in the uptake. Depletion of glutamate did not lead to the enhanced uptake, whereas depletion of glycine and serine caused as much increase in the uptake as depletion of cystine did. The intracellular pool of glutathione was extremely reduced by depletion of cystine, or of glycine and serine, but to a far less extent by depletion of glutamate. The results indicate that the transport system for cystine and glutamate appears to undergo adaptive regulation. It is suggested that glutathione may function as a regulatory signal to this transport system.

Low density lipoprotein receptor binding in aging human diploid fibroblasts in culture.

High affinity cell surface receptors for low density lipoproteins (LDL) are inducible in cultured human lung fibroblasts by the removal of lipoproteins from the cell culture medium. The binding, uptake, and degradation of 125I-LDL by fibroblasts decrease with increasing number of population doublings. The affinity of LDL receptor binding, however, remained unchanged at different population doublings levels. Hence, the difference in LDL binding activity in the aging fibroblasts can be attributed to a reduction in the number of receptor sites on the cell membrane. Cellular uptake of [4-14C]cholesterol and 2-deoxy-D-[1-14C]glucose mediated through mechanisms independent of the LDL receptor pathway revealed no significant difference in early and late passage fibroblasts. This suggests that the alteration in the LDL receptor binding in serially passaged fibroblasts is an "age-related" phenomenon. The late population doubling fibroblasts require more LDL in the culture medium for feedback inhibition of LDL receptor synthesis. Thus, aging fibroblasts are both progressively less inducible and less suppressible in the regulation of their cell membrane LDL receptors. Similar results were also obtained with respect to the regulation of DL-3-hydroxy-3-methyl-glutaryl coenzyme A reductase in the aging fibroblasts in culture; the enzyme has become less inducible and less supressible as the fibroblasts approach the limit of their in vitro lifespan. These age-related alterations in the cellular metabolism of LDL and cholesterol might contribute to our understanding of the increased risk of athlerosclerosis in our aging population.

In vitro proliferation and lifespan of human diploid fibroblasts in serum-free BSA-containing medium.

We have developed two serum-free chemically defined media (RITC 78-6 and RITC 80-7) that support the growth in culture of human diploid fibroblasts to the same extent as Eagle's basal medium (BME) supplemented with 10% fetal bovine serum (FBS). These two media contain modified Eagle's minimum essential medium (MEM) supplemented with nonessential amino acids, various trace metals, organic compounds and growth factors [insulin, mouse epidermal growth factor (m-EGF), transferrin and triiodothyronine (T3)]. RITC 80-7 medium differs from RITC 78-6 in that it contains thymidine, hypoxanthine, and vitamin B12 and supports the long-term serial cultivation of human diploid cultures. The addition of commercial bovine serum albumin (BSA, 5 g/liter) to the medium enhances cell growth. This effect is not observed if BSA is first delipidized, but reconstitution of BSA with certain lipids restores its ability to promote growth. BSA has an inhibitory effect on cellular attachment but this is overcome when fibronectin (FN, 10 mg/liter is added to the medium.

Exit of human diploid cells from low serum quiescence

We examined the time required for low serum-arrested cultures of human diploid fibroblasts (HDF) to exit G 0 and enter the DNA synthetic phase (S phase) of the cell cycle following serum stimulation. Regardless of whether cultures were recently confluent or had been quiescent for 7 or 21 days, DNA synthesis began at 10–12 hours and peaked at 21 hours after stimulation. Peak DNA synthesis levels were higher in arrested cultures as compared to confluent control cultures and could be at least partially accounted for by an increased fraction of arrested cells entering S phase as determined by autoradiography. We concluded that increasing the time of low serum arrest does not cause cells to enter “deeper phases” of G 0.

Interclonal variation in methylation patterns for expressed and non-expressed genes.

A mass culture of human diploid fibroblasts, and eight clones isolated from that mass culture, were examined for methylation patterns in several regions of DNA. Plasmid-inserted cDNA sequences were used as probes for alpha-hCG, beta-globin, A gamma- and G gamma-globin, and beta- and gamma-actin gene regions. Each probe revealed a different clone-specific pattern of DNA methylation, indicating a striking degree of inter-clonal heterogeneity, for those gene regions which are not normally expressed in diploid fibroblasts (alpha-hCG, gamma-globin and beta-globin). Intra-clonal variation was also evident in many instances, implying that heterogeneity could arise de novo in pure cell clones during serial passage. Thus methylation patterns, in particular for repressed genes, appear to be unstably inherited in these cells, and this instability may lead to random derepression in some cell lineages during mitotic growth.

Hydrocortisone effects on nucleic acid and nuclear protein content and protein metabolism in aging WI-38 cells

Confluent late passage WI-38 human diploid fibroblast cultures grown in the presence or absence of hydrocortisone for fifteen population doublings were measured cyto-chemically for DNA, nuclear RNA, basic nuclear protein and acidic nuclear protein content during a 12-hour interval following a medium-change stimulation. Radioisotope tracer studies were utilized to assess the effects of prolonged hydrocortisone treatment on protein synthesis and degradation in aging confluent and logarithmically growing cultures. Hydrocortisone-treated cultures exhibited an increased Feulgen—DNA stainability, no difference in basic nuclear protein content, a significantly larger initial decrease followed by an increase in acidic nuclear protein content, and elevated levels of nuclear RNA as compared to untreated controls. Hydrocortisone treatment also resulted in increased incorporation of leucine into protein and decreased breakdown of pre-labeled protein in both confluent and logarithmically growing cultures. These results indicate that many of the metabolic changes associated with proliferation are stimulated by prolonged hydrocortisone treatment of late passage WI-38 fibroblast cells.

DNA methylation in normal and SV40-transformed human fibroblasts

The 5-methylcytosine base content of DNA in both normal and SV40-transformed human diploid fibroblast cultures was measured by high performance liquid chromatography. In contrast to other reported studies comparing normal and oncogenically transformed cells, no apparent difference was observed in the 5-methylcytosine to cytosine base ratios in the two cell types. The advantages of this method are also discussed.

Role of proton dissociation in the transport of cystine and glutamate in human diploid fibroblasts in culture.

The effect of extracellular pH on the transport interaction of cystine and glutamate in cultured human diploid cells was examined over the pH range of 5.8-8.0. The initial rates of uptake of cystine increased with an increase in pH and glutamate potently inhibited the cystine uptake independently of pH. The uptake of glutamate was almost invariable within the pH range, but it was inhibited by cystine in a pH-dependent manner; the inhibition increased with an increase in pH. Regardless of pH, the uptake of cystine and glutamate was strongly inhibited by alpha-aminoadipate, alpha-aminopimelate, and homocysteate. From the pK values of cystine and other amino acids, it is suggested that cystine is transported in the same ionic form as is glutamate.

Laboratoriumspraxis bei Adenoviren

Six prototype strains of human adenoviruses (Ad1, 4, 7, 8, 12, 19) were passed and titrated comparatively in four strains of human diploid fibroblast cultures. The viruses were readily passed 5 times with cytopathic effect; no signs of adaptation or diminution of CPE were apparent. The virus yield was about equal in fibroblasts and in HeLa cells. High and low passage fibroblasts were of equal susceptibility. In endpoint titrations, fibroblast cultures showed an about 10-fold lower susceptibility towards Adl and Ad7; however, they compared favorably for Ad4, 8, 12, and 19 with HeLa or (for Ad 8) with human amnion cell cultures. In some instances differences in the susceptibility between fibroblast strains were observed. Fibroblast cultures and human amnion cells showed equal susceptibility for Ad8 from 9 original conjunctival specimens, while results with other types varied. For adenovirus isolation, fibroblast cultures are suitable as an adjunct to HeLa cell cultures, particularly for Ad8. However, the long duration till the appearance of CPE which sometimes exceeds 50 days is a disadvantage. Sechs Prototypstämme menschlicher Adenoviren (Ad1, 4, 7, 8, 12, 19) wurden vergleichend in 4 Stämmen von humanen diploiden Fibroblasten passagiert und titriert. Eine Passagierung mit cytopathischem Effekt über 5 Passagen war in allen Fällen möglich; Zeichen einer Adaptation oder Abschwächung des CPE fanden sich nicht. Der Virusertrag in Fibroblastenkulturen war etwa gleich wie in HeLa-Zellkulturen. Fibroblasten aus niedriger und hoher Passage zeigten gleiche Empfänglichkeit. In Endpunkttitrationen zeigten Fibroblastenkulturen bei Ad1 und Ad7 eine um etwa den Faktor 10 geringere Empfänglichkeit; sie waren dagegen bezüglich Ad4, Ad8, Ad 12 und Ad19 den Vergleichskulturen (HeLa- bzw. humane Amnionzellen für Ad 8) mindestens ebenbürtig. In einigen Fällen zeigten sich Unterschiede in der Empfänglichkeit zwischen den Fibroblastenstämmen. Fibroblastenkulturen erwiesen sich gegenüber Ad 8 in 9 Konjunktivalabstrichen als gleich empfänglich wie humane Amnionzellen, während Proben mit anderen Typen sich unterschiedlich verhielten. Fibroblastenkulturen sind als Ergänzungskultur für die Anzüchtung von Adenoviren (z.B. neben HeLa-Zellen) geeignet, insbesondere auch zum Nachweis von Ad8. Nachteilig ist die oft sehr lange Dauer bis zum Auftreten des CPE, die gelegentlich 50 Tage übersteigt.

The influence of labelling time and medium-condition on 14C-Prostaglandins (PG) release from human diploid fibroblasts in culture

The influence of labelling time and medium-condition on 14C-Prostaglandins (PG) release from human diploid fibroblasts in culture

Senescence in vitro and ionising radiations—the human diploid fibroblast model

The influence of ionising radiations on ageing is still controversial. Since Hayflick established the concept that diploid cells have finite lifespan in vitro, human diploid fibroblast (HDF) cultures have been recognised as a potent experimental model for cytogerontological investigations. In this study HDF cultures in phase II were exposed to acute irradiation with either X-rays on fast neutrons. The replicative potentials and labelling indices with [ 3H]thymidine were measured post irradiation until the cultures ceased growth in phase III. Cell mortality was measured by cloning. The apparent loss in replicative potential of irradiated mass cultures was wholly attributable to the loss of viable clonogenic cells. The current concept of precocious clonal senescence in vitro as a late effect of irradiation in clonogenic survivors is not supported by the present experiments. Instead, our results suggest that exposure to a single dose of ionising radiations either causes total replicative incapacitation (killing) of HDF cells and their progeny early after irradiation or leaves their replicative potentials unperturbed.

Eugenol: liberation from dental materials and effect on human diploid fibroblast cells.

The amount of eugenol (4-allyl-2-methoxyphenol) released from four different dental materials immersed in phosphate buffer was measured by gas liquid chromatography. Maximal release of eugenol from zinc oxide-eugenol cement (ZOE) and IRM was attained within 5 h and corresponded to 5% and 4%, respectively, of the total amount of eugenol in each material. Both the rates and total amounts of eugenol released from Nobetec and Opotow were lower than for ZOE and IRM. Eugenol (0.67 mM) applied to growing cultures of human diploid fibroblasts reduced the number of cells recovered to a value which was 4% of that found for control cultures which grew to form monolayers. Confluent monolayers of 3H-uridine labeled cell cultures which were incubated for 1 h in the presence of 4 mM eugenol lost approximately 100% of this cytoplasmic label, indicating total cell death.

Sister-chromatid exchange induction by metabolically activated retinoids in human diploid fibroblast cultures

13- cis-Retinoic acid, retinyl-methyl-ether, retinyl-phenyl-ether, retinyl-thio-ether and axerophthene each induced dose-dependent sister-chromatid exchanges (SCE) in human diploid fibroblasts. The functional relationship between retinoid concentration and SCE rate was similar in each of the 5 retinoids tested. The relationship reached a plateau at concentrations exceeding 8 μg/ml. α-Naphthoflavone (ANF), an inhibitor of P448-dependent mono-oxygenase, prevented the retinoid-induced increase of the SCE rate, but had no inhibitory effect in the presence of 4-nitroquinoline-1-oxide, an ultimate carcinogen. ANF did not reduce the spontaneously increased SCE rate in fibroblasts of patients with Bloom's syndrome. Retinoids failed to induce SCE in V79 Chinese hamster cells, which lack mono-oxygenase. Thus, we conclude that the retinoid-induced SCE rate increases independently of structural changes in the molecular side-chain and that a metabolic activation of retinoids is required for SCE induction by cytochrome P448-dependent mono-oxygenase.

Age-related decline in histone H1 fraction in human diploid fibroblast cultures

The age-related increase in cell volume and nuclear size of cultured human diploid fibroblasts reflected the accumulation of proteins in cytoplasm and nuclei of growth-retarded fibroblasts. Determination of the amount of nuclear proteins, which were fractionated into 0.15 M NaCl-soluble proteins, 0.4 N H 2SO 4-extractable proteins and residual acidic proteins, indicated that age-related increase in nuclear proteins was due mainly to the accumulation of residual acidic proteins. However, electrophoretic fractionation of histones from various passages of fibroblast cultures on acid urea polyacrylamide gel revealed that the relative amount of H1 fraction decreased with in vitro aging. This was further confirmed by mixing experiments examining the distribution of radioactivity of the histones from cell mixtures of young and senescent cultures labeled with [ 3H]lysine or [ 14C]lysine. A pulse label and chase experiment indicated that the observed decrease in the amount of histone H1 was mainly due to decrease in synthesis of histone H1 in senescent human fibroblast cultures.

Suppression of cytidylate biosynthesis by protein synthesis antagonists.

When protein synthesis is effectively inhibited in Novikoff cell cultures by various drugs, there is a marked suppression of labeling of cytidiane nucleotides (mainly CTP) from exogenously supplied (/sup 3/H)uridine. The inhibition could be caused by a reduced activity of CTP synthetase, the enzyme catalyzing the conversion of UTP to CTP, since (/sup 3/H)UTP is readily produced. The variety of the drugs used appears to argue against a direct effect of the drugs on CTP synthetase, since three different glutarimide antibiotics (cycloheximide, streptimidone, and emetine) and puromycin, which has a quite different chemical structure, are equally effective. An increase of the cytidine nucleotide pool size is observed in most instances of drug treatment; however, feedback inhibition of CTP synthetase is considered unlikely because of a lack of correlation between the degree of inhibition of (/sup 3/H)CTP production and the extent of accumulation of the cytidine nucleotides. There is a marked inhibition of RNA synthesis when all protein synthesis inhibitors are present; but almost complete inhibition of RNA synthesis by actinomycin D produces only a slight inhibition in (/sup 3/H)CTP production, indicating that the block in the conversion of UTP to CTP results from inhibited protein synthesis rather than from inhibitedmore » RNA synthesis. Examination of the labeling pattern of both RNA and DNA nucleotides following drug treatment gave further evidence for inhibited CTP production. Inhibition of protein synthesis elicited a much greater reduction of the suppression ratio (experimental/control ratio) for RNA-CMP labeling than for RNA-UMP labeling and an even greater reduction of the suppression ratio for DNA-dCMP labeling compared to DNA-dTMP labeling. The effect was also obtained in HeLa cell cultures, but is not restricted to neoplastic cell lines since it was seen with normal human diploid fibroblast cultures.« less

Transport interaction of L-cystine and L-glutamate in human diploid fibroblasts in culture.

Transport interaction of L-cystine and L-glutamate in human diploid fibroblasts in culture.

Selective elimination of proliferating cells in human diploid cell cultures by treatment with BrdU, 33258 Hoechst and visible light

We have attempted to obtain pure populations of postmitotic cells from cultures of human diploid fibroblasts by the selective elimination of replicating cells. The selective system consists of treating cultures with 5-bromodeoxyuridine (5-BrdU) followed by exposure to the fluorescent dye 33258 Hoechst and irradiation with visible light. The percentage of survivors after the combined treatment was observed to be inversely proportional to the pretreatment [ 3H] thymidine labeling index of the culture. The posttreatment labeling indices, determined on the basis of the number of cells plated prior to the initiation of treatment, was higher in late passage cultures. Even after prolonged exposure to 5-BrdU (10 days), a small fraction of cells in early passage human fibroblastlike cultures was capable of incorporating [ 3H] thymidine. In contrast, actively replicating cells could be completely eliminated from HeLa cultures in this selective system. Studies with [ 3H] BrdU indicated that the cells incorporating this analog are preferentially killed. These observations suggest that human diploid cell cultures contain a subpopulation of very slowly cycling or transiently noncycling cells.

Rapid recovery in human diploid fibroblasts.

Rapid split dose and single dose recovery were examined in exponentially growing and in density inhibited, stationary phase culture of human diploid fibroblasts. The cultures were exposed to cobalt-60 gamma rays at a dose-rate of 50 rad/s:0-90 min were allowed between exposures or following a single exposure prior to subculture at low density. A rapid phase of recovery occurred which was of greater magnitude in the plateau phase cultures. Dose-dependent rapid recovery occurred within 2-10 min of the initial exposure, and was greater following single dose versus split dose irradiations. When the post-irradiation temperature was maintained at 37 degrees C, two phases of recovery were observed, one at early times (2-10 min) and one later (30-90 min). No recovery was observed with incubation at 4 degrees C, whereas incubation at room temperature (25 degrees C) allowed rapid recovery (2-10 min) to take place, but not the slower process (30-90 min). The hypothesis that this rapid recovery phenomenon is related to cell-to-cell contact and to repair of potentially lethal damage is discussed.

Retinoids inhibit 2,3,7,8-tetrachlorodibenzo-p-dioxine-induced activity of benzo[a]pyrene metabolizing enzymes in human diploid fibroblasts.

Retinoids are known to inhibit the substrate mediated enzyme induction of benzo[a]pyrene metabolizing enzymes. Consequently, the effect of two retinoids on the induction of benzo[a]pyrene metabolizing enzymes by the more potent inductor 2,3,7,8-tetrachlorodibenzo-p-dioxine (TCDD) was investigated. The studies were performed with human diploid fibroblasts in culture. Vitamin A palmitate and all-trans-retinoic-acid were found to prevent the TCDD induced increase of benzo[a]pyrene metabolism in a dose-dependent manner. The fact that this effect was immediately reversible makes it unlikely that it was due to non-specific toxic effects. The data suggest that retinoids cause a preferential inhibition of the de novo synthesis of benzo[a]pyrene metabolizing enzymes.