Transport ofl-histidine by human diploid fibroblasts in culture

Summary The transport ofl-histidine has been characterized in skin derived diploid human fibroblasts, cultured under strictly controlled conditions. The transport measurements were made on cells grown to subconfluency after 60 to 90 min timed preincubation. The data, at substrate concentrations ranging from 0.050 to 10 mmol/l, were analyzed by a computer program. A saturable transport system (K _m =0.25 mmol/l, V _max =17 nmol/mg protein per min) and a nonsaturable component of influx (K _d =1.6±0.4 nmol/mg protein/min per mmol) were found.l-Histidine displayed no Na⁺ requirement at either low or high concentrations. Inhibition analysis demonstrated thatl-histidine uptake at low concentration was poorly inhibited by amino acids known to be effective inhibitors of system A. The largest fraction ofl-histidine uptake was inhibited by 2-amino-bicyclo (2,2,1)-heptane-2-carboxylic acid (BCH), leucine, and tryptophan. These results indicated thatl-histidine is transported in human fibroblasts, mainly by the Na⁺ independent system L. The differences between this cell type and others studied previously are discussed. This work was supported in part by Grant 773 from UER de Médecine, Université Paris XI (France).     

The influence of culture medium volume on cell density and lifespan of human diploid fibroblasts

The volume of culture medium in which WI38 cells are grown affects the maximum cell number at stationary phase and in the vitro lifespan in terms of total population doublings. The saturation density at 0.53 ml/cm² (40 ml/T-75) is consistently about 2-fold higher than at 0.26 ml/cm² (20 ml/T-75).At a constant medium volume the cell yield at stationary phase is directly dependent on the amount of serum present. Thus the increased yields from greater medium volumes is probably due to a large extent on the increased amount of serum growth factor(s) present.For maximal cell yields in non-perfused WI38 cells, we suggest that routine subcultivation be carried out in medium containing10 % (v/v) serum and at 0.53 ml/cm² medium of surface area.     

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.     

Effect of antioxidants on cultured human diploid fibroblasts exposed to cystine-free medium

Logarithmically growing human embryonic diploid cells started to die in cystine-free medium within 18 hours. Glutathione accounted for almost all the acid-solube sulfhydryl compound of the cells and cellular glutathione level decreased rapidly after cystine depletion. By adding vitamin E the cells survived over 6 days in cystine-free medium, though glutathione content of the cells was reduced to less than 1% of the normal level. Synthetic antioxidants had similar effect, and mechanism by which cells die in cystine-free medium was suggested.     

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.     

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 α-chains of glycopeptide hormones. Hydroxyurea (HU) at a concentration of 1 mM mediated a small, statistically significant increase in hCG production (p < 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.     

Release of antigens into the culture medium by LS fibroblasts in culture]

LS fibroblasts cultivated for 1 or more days in unsupplemented Eagle's MEM release antigens into the medium, without showing any evidence of lysis, but on the contrary continuing to proliferate. These antigens give up to three precipitation lines when tested by means of immunodiffusion and immunoelectrophoresis against specific rabbit antisera; one or two of them give identity reactions with antigens obtained by repeatedly washing the fibroblasts with balanced salt solutions. Evidence has been obtained that they are surface components, easily stripped or spontaneously shed by the cells.     

Effects of some antibiotics on the growth of human diploid skin fibroblasts in cell culture

Summary During serial subcultures 50 μg per ml gentamicin and penicillin (100 U per ml)-streptomycin (100 μg per ml) depressed cell growth significantly 2 weeks after the addition of the antibiotics; gentamicin, but not penicillin-streptomycin, stimulated cell growth before it became inhibitory. Removal of the antibiotics resulted in the cell yield returning to normal. The results show that these antibiotics can be harmful to cells even at concentrations thought to be safe.     

Isolation of ouabain-resistant human diploid fibroblasts

Seventeen clones resistant to the cytotoxic action of ouabain were isolated in culture by direct selection from 5 independent strains of diploid human fibroblasts. Resistant clones were recovered at frequencies on the order of 10^?7 per wild type cell selected from populations treated with the mutagen EMS, but no resistant cells were detected among 10⁸ unmutagenized cells. Most selected clones remained ouabain-resistant following further propagation in the absence of drug. The growth of wild type cells was inhibited by 50% at ouabain concentrations of 2–5 × 10^?8 M, while resistant clones required 15–180 fold higher drug concentrations to cause equivalent inhibition. Ouabain-resistant clones showed increased resistance of K+ transport function to ouabain inhibition that paralleled their increased resistance to growth inhibition. Initial experiments suggest that under selective conditions the resistant diploid fibroblasts differ significantly from wild type in binding of ³H-ouabain per unit surface area. The ouabain-resistant cells were similar to wild type in transport properties unrelated to ouabain inhibition. Resistant cells had normal karyotypes and senesced with a lifespan similar to control clones. The ouabain-resistant phenotypes of these diploid human fibroblast isolates apparently reflect point mutations that specifically affect the Na+/K+ transport ATPase with respect to ouabain-binding and/or response to bound ouabain.     

Effects of some antibiotics on the growth of human diploid skin fibroblasts in cell culture.

During serial subcultures 50 micrograms per ml gentamicin and penicillin (100 U per ml)-streptomycin (100 micrograms per ml) depressed cell growth signficantly 2 weeks after the addition of the antibiotics; gentamicin, but not penicillin-streptomycin, stimulated cell growth before it became inhibitory. Removal of the antibiotics resulted in the cell yield returning to normal. The results show that these antibiotics can be harmful to cells even at concentrations thought to be safe.     

Chromosome replication in aging human diploid fibroblasts

The patterns of termination of DNA replication in human embryonic MRC-5 fibroblasts at four passage levels have been examined by autoradiography. Only chromosome 9 showed statistically significant differences in the time of replication among cultures of different ages. This chromosome terminated replication earlier at later passages than at earlier passages, primarily because of differences in the time of replication of the centromere region. Because very few differences were observed at different passage levels, we conclude that changes in the order of chromosome replication are unlikely to contribute to the phenomenon of in vitro senescence.     

Early changes in the synthesis of acidic nuclear proteins in human diploid fibroblasts stimulated to synthesize DNA by changing the medium

When resting WI-38 cells in a confluent monolayer were stimulated to proliferate by changing the medium, the incorporation of leucine-³H into nuclear acidic proteins was promptly stimulated, although its incorporation into total cellular proteins was unchanged or even decreased. Three fractions, all acidic by aminoacid analysis, were extracted from the nuclei: (1) ribonucleoproteins (RNP); (2) a fraction extractable with 0.15 M NaC1; and (3) a fraction tenaciously bound to the insoluble residue (residual fraction). A first increase occurred between one and three hours after stimulation in all three fractions. The synthesis of NaCl-soluble proteins then returned to control levels, while the synthesis of residual and RNP proteins remained high between 6 and 12 hours and increased even further at 18 hours, the peak of DNA synthesis. Pulse chase experiments indicated that the proteins synthesized in the first hour after stimulation have a turnover time of less than four hours, while the same fractions in non-proliferating cells were stable for at least 12 hours. 2-mercapto-1-(β-4-pyridethyl) benzimidazole, when added at the same time as the fresh medium, produced an inhibition of the increase in nuclear protein synthesis at one hour, but, if added at five hours after stimulation, it did not inhibit the increase in nuclear protein synthesis occurring at six hours. Actinomycin D (0.01 μg/ml) inhibited both the stimulation of DNA synthesis and the increases in nuclear acidic protein synthesis occurring at one and six hours after stimulation. These results seem to indicate that the serum factors responsible for the stimulation of WI-38 cells, after binding to cells, induce an early synthesis of acidic nuclear proteins which is sensitive to very low doses of actinomycin D. In turn, the newly synthesized proteins could in some way activate in the nuclei the genes that control DNA synthesis and cell division.     

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.     

Messenger RNA regulation in human diploid fibroblasts

In resting, non-growing human diploid fibroblasts the amount of rRNA is reduced 1.8-fold, cytoplasmic polysomes are disaggregated, and the level of poly-A RNA (mRNA) is reduced 1.8-fold in relation to growing cells. The distribution of poly-A RNA is altered in resting, non-growing cells so that an average of 64% of the total cytoplasmic poly-A RNA sediments along with particles lighter than 80S (prepolysomal) in sucrose density gradients. By comparison, in growing cells only 30% of the cytoplasmic poly-A RNA sediments in the prepolysomal region. In SDS sucrose gradients, the sedimentation profile of the prepolysomal poly-A RNA from resting cells resembles that of polysomal poly-A RNA from those cells. In contrast, the average size of prepolysomal poly-A RNA from growing cells is much smaller than that of the polysomal poly-A RNA from those cells. These data are compatible with the possibility that resting cell prepolysomal poly-A is untranslated mRNA. Also consistent with this interpretation are experiments which demonstrate that one-quarter to one-third of the prepolysomal poly-A RNA of resting cells is recruited into polysomes in the presence of cycloheximide.