Cultures Of Human Diploid Fibroblasts Research Articles

Growth regulation in multilayered cultures of human diploid fibroblasts: the roles of contact, movement and matrix production.

Early subcultures of human embryonic lung fibroblasts are exceptional, as they grow far beyond confluence before growth ceases: the stationary dish may well contain 3-10 monolayer equivalents. Maximal growth rates, however, occur at about one-sixth confluence when doubling times are 15-20 hr; a density at which cell contacts begin to become frequent. The fact that a slowing down of growth is first apparent at such low densities argues against this regulation being due to diffusion effects. Confirmation of the role of short-range or contact interactions in growth regulation comes from an experiment using mixed cultures of fibroblasts: this shows that growth inhibition is not carried by medium-borne influences but depends on short-range (less than 1 mm) interactions. Evidence that cells can escape the effects of such contact interactions and so divide comes from time-lapse studies of dense cultures: there is a burst of motility soon after a fresh-medium change, which is followed by a burst of mitosis approximately 20 hr later. A medium change to conditioned medium supplemented with 10% foetal calf serum leads to neither the burst of motility nor the subsequent burst of mitosis, although this medium is better able to support the growth of sparse cells than is fresh medium. Data are also presented to show that the amount of collagen deposited in superconfluent cultures affects their growth: the stimulation of collagen production with ascorbic acid leads to an unexpectedly low stationary cell density and rather less movement in the culture. This result suggests that the collagen stabilizes cell contacts that are responsible for growth inhibition. The question of why these cells grow more slowly as density increases cannot be answered directly by these experiments; nevertheless, the results suggest that cell contact affects the permeability of the cell membrane to medium.

86] Measurement of antagonistic effects of growth factors and interferons

This chapter describes techniques for the measurement of interactions between interferons (IFNs) and growth factors (GFs) in vitro, in tissue culture of either human diploid fibroblasts or blasts of the cortisone resistant mouse thymocytes, and also in vivo, in mice infected with Moloney sarcoma virus. The proliferation of cells and tissues probably cannot occur without the participation of GFs or substances that either mimic or substitute for GFs. GFs have been defined as endogenous substances produced by various tissues that have multiplication stimulating activity and/or other trophic effects but are not nutrients, antibodies, enzymes, or attachment factors. The majority of GFs are polypeptides of various molecular weight and structure that have a hormone-like mode of action. These include the interleukins that stimulate the proliferation of cells of the immune system. IFNs, as well as GFs, fulfill the main biological and biochemical criteria of hormones. However, the response of various cells and tissues to IFNs is inhibition of the proliferation and other complex reactions not necessarily connected with the cell cycle but clearly antagonistic to GFs. Thus, it has been suggested that IFNs and GFs can be grouped together as nonclassical hormones with opposite actions. Human embryonic diploid fibroblasts (HEF) strains are used between fifth and twentieth passage levels in culture. The cells are found to be sensitive to both human IFN and several GFs.

Ribonucleotide reductase activity during the senescence of normal human diploid fibroblasts in culture

Changes in ribonucleotide reductase occur during the senescence of normal human diploid fibroblasts in culture. Enzyme activity is significantly lower in cells and extracts at high mean population doublings (MPD) as compared to fibroblasts at low MPD. Although many of the kinetic properties of the enzyme remain unaltered in cells at high MPD, changes in the extent and kinetic mechanism of inhibition of CDP and ADP reductase activity of dATP, the overall negative effector of ribonucleotide reductase, were observed. These results and a previous observation that the four deoxyribonucleotide pools are markedly altered during in vitro senescence of human diploid fibroblasts, provide evidence for a link between ribonucleotide reduction, doxyribonucleotide pools, and the establishment of the non-proliferative or senescent state.

SV40-transformed human fibroblasts: evidence for cellular aging in pre-crisis cells.

Pre-crisis SV40-transformed human diploid fibroblast (HDF) cultures have a finite proliferative lifespan, but they do not enter a viable senescent state at end of lifespan. Little is known about either the mechanism for this finite lifespan in SV40-transformed HDF or its relationship to finite lifespan in normal HDF. Recently we proposed that in normal HDF the phenomena of finite lifespan and arrest in a viable senescent state depend on two separate processes: 1) an age-related decrease in the ability of the cells to recognize or respond to serum and/or other mitogens such that the cells become functionally mitogen-deprived at the end of lifespan; and 2) the ability of the cells to enter a viable, G1-arrested state whenever they experience mitogen deprivation. In this paper, data are presented that suggest that pre-crisis SV40-transformed HDF retain the first process described above, but lack the second process. It is shown that SV40-transformed HDF have a progressively decreasing ability to respond to serum as they age, but they continue to traverse the cell cycle at the end of lifespan. Concomitantly, the rate of cell death increases steadily toward the end of lifespan, thereby causing the total population to cease growing and ultimately to decline. Previous studies have shown that when SV40-transformed HDF are environmentally serum deprived, they likewise exhibit continued cell cycle traverse coupled with increased cell death. Thus, these results support the hypothesis that pre-crisis SV40-transformed HDF still undergo the same aging process as do normal HDF, but they end their lifespan in crisis rather than in the normal G1-arrested senescent state because they have lost their ability to enter a viable, G1-arrested state in response to mitogen deprivation.

Platelet-derived growth factor stimulates rapid polyphosphoinositide breakdown in fetal human fibroblasts.

The addition of human platelet-derived growth factor (PDGF) to confluent, quiescent cultures of human diploid fibroblasts induced the rapid breakdown of cellular polyphosphoinositides. The levels of 32P-labeled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol (PI) decreased by 30 to 40% within 1 min after exposure of the cells to PDGF. The levels of PIP and PIP2 returned to their initial values within 3 and 10 min, respectively, after PDGF addition. The level of PI continued to increase after it had returned to control values and was up threefold within 30 min after PDGF addition. In cells prelabeled with myo-[3H]inositol PDGF caused an eightfold increase in the levels of inositol trisphosphate (IP3) within 2 min. Lesser increases, twofold and 1.3-fold, respectively, were seen in levels of inositol bisphosphate (IP2) and inositol monophosphate (IP). Within 10 min after PDGF addition the levels of all three inositol phosphates had decreased to control values. The levels of IP3 measured 2 min after PDGF addition depended on the PDGF concentration and were maximal at 5-10 ng/ml of PDGF. Similar concentrations of PDGF stimulate maximal cell growth and DNA synthesis in these cells.

Membrane glycoprotein changes during the senescence of normal human diploid fibroblasts in culture

The plasma membrane is intimately involved in a variety of cellular phenomena and may play an important role in the aging of human cells in culture. Significant differences in human diploid fibroblast surface glycoproteins were observed with in vitro aging. Senescent cells bound more concanavalin A (Con A) than young cells and exhibited two distinct classes of binding sites. Cell surfaces of senescent cells incorporated less labelled mannose and more labelled fucose and glucosamine than young cell surfaces. Membranes prepared from older cells were also less effective than membrane preparations from young cells in incorporating mannose from GDP-mannose into a group of oligolipid intermediates, required for the synthesis of glycoproteins with asparaginelinked oligosaccharides. These results demonstrate novel quantitative changes in membrane structure and lectin reactivity in aging human diploid fibroblasts, which must reflect the fundamental physiological modifications in the cell that occur during senescence.

Changes in protein synthesis and breakdown rates and responsiveness to growth factors with ageing in human lung fibroblasts

The effects of insulin, epidermal growth factor, insulin-like growth factor-2 and fetal bovine serum on protein synthesis and protein breakdown have been measured in mid-passage and senescent cultures of human diploid lung fibroblasts. Each of the individual growth factors was a potent inhibitor of protein breakdown with no difference in either the maximum response or sensitivity evidence between senescent cells and mid-passage cultures. Binding of 125I-labelled epidermal growth factor per mg of cell protein similarly showed no difference between senescent and confluent mid-passage fibroblasts, although sparse mid-passage cells bound more of the ligand. These results indicate normal binding and normal responsiveness to growth factors in senescent cultures. However, rates of protein synthesis are higher in confluent mid-passage cells and especially so in sparse mid-passage cells than in senescent cells of intermediate density. Furthermore, senescent cells differ from either growth state of mid-passage cells because protein synthesis in aged cells is much less responsive to any of the growth factors or to fetal bovine serum. It is suggested from these results that the reduced ability of serum or growth factors to initiate DNA synthesis or growth in ageing cells may be a consequence of an unresponsive protein synthesis pathway rather than a generalised defect in growth factor action.

Changes in frequencies of chromosomal aberrations and sister chromatid exchanges during confluent holding recovery in X-irradiated normal human diploid fibroblasts.

The time course of the changes in survival and cytogenetic damage was studied in X-irradiated cultures of human diploid fibroblasts before release from density-inhibition of growth. The frequency of chromosomal aberrations declined rapidly during the initial 2 h recovery interval in parallel with an enhancement in survival. The frequency of sister chromatid exchanges (SCE) increased during this interval reaching a maximum of 2 h, then declined. These changes are discussed in relation to previous observations on X-ray induced cytogenetic damage and malignant transformation in rodent cells under similar conditions.

Ginseng extract inhibits protein degradation and stimulates protein synthesis in human fibroblasts

Aqueous extracts of Panax ginseng inhibit intracellular protein degradation in confluent cultures of IMR-90 human diploid fibroblasts. The magnitude of the inhibition is similar to that observed with insulin and polypeptide growth factors. Furthermore, the inhibition of proteolysis by ginseng, like that produced by insulin and growth factors, is selective in that it applies to long-lived proteins but not to short-lived proteins. Ginseng also stimulates protein synthesis in human fibroblasts indicating that components of ginseng extract are capable of acting directly on human cells to promote protein accumulation.

Methylation patterns in the gene for the alpha subunit of chorionic gonadotropin are inherited with variable fidelity in clonal lineages of human fibroblasts.

Cytosine methylation in DNA of an endogenous single-copy gene locus, the alpha-subunit of human chorionic gonadotropin (alpha-hCG), was assessed in a mass culture and individual clonal lineages of human diploid fibroblasts. Progressive hypomethylation at -CCGG- sites in this gene occurred during the replicative lifespan of the mass culture and was shown to arise randomly during clonal expansion. Thus, some clones and subclones lost -CCGG- methylation in the alpha-hCG gene region while others maintained essentially complete methylation. These data indicate significant interclonal variability in the fidelity with which DNA methylation is transmitted in an endogenous gene.

Selective inhibition of human diploid fibroblast collagen synthesis by interferons.

The effects of alpha- and gamma-interferons (IFNs) on collagen production by confluent human diploid fibroblasts in culture were examined. It was found that partially purified alpha-IFNs and affinity purified gamma-IFNs caused greater than 50% inhibition of collagen synthesis by these cells independently of their effect on cell proliferation. Recombinant alpha-IFNs showed a similar effect (38.8% inhibition), indicating that collagen synthesis inhibition was a constitutive property of IFNs. Collagen synthesis inhibition by IFNs was concentration dependent. Gel filtration chromatography of the newly synthesized proteins from the media of fibroblasts incubated with partially purified alpha-IFNs demonstrated a selective depression of molecules eluting in the region of procollagen. No detectable increase in collagen degradation products or underhydroxylation of procollagen was observed. Short-term kinetic studies further demonstrated that the major effect of IFNs was due to a net decrease in fibroblast collagen production rather than to impairment of secretion or increased extracellular degradation of the newly synthesized molecules. These results indicate that alpha- and gamma-IFNs are potent inhibitors of human fibroblast collagen production and suggest that they may play an important role in the regulation of normal and pathologic fibrogenesis.

Induction of replicative DNA synthesis in quiescent human fibroblasts by DNA damaging agents.

A marked induction of DNA replication was observed in confluent human diploid fibroblast cultures treated with low relatively nontoxic doses of UV radiation, N-methyl-N-nitrosourea (MNU), and N-acetoxy-2-acetylaminofluorene (AAAF). Isopycnic CsCl density gradient analysis of newly synthesized DNA labeled with BrdUrd indicated that most of the synthesis was semiconservative. The rate of semiconservative DNA synthesis was maximal 24 hr after damage. This induction of DNA replication was greatest at approximately equal to 3 J/m2 UV, 0.5 mM MNU, or 1.0 microM AAAF; was inhibited by hydroxyurea and aphidicolin; and also occurred in repair-deficient xeroderma pigmentosum fibroblasts. Autoradiographic examination of both confluent cultures and serum-arrested cultures showed a large increase in the fraction of densely labeled (S phase) cells after UV treatment. These densely labeled cells retain the capacity for cell division and subsequent proliferation. We conclude that low doses of at least three different DNA damaging agents are capable of recruiting quiescent cells into a state of DNA replication similar to that observed in the normal cell cycle.

Deoxyribonucleoside triphosphate pools in human diploid fibroblasts and their modulation by hydroxyurea and deoxynucleosides

Deoxyribonucleoside triphosphate (dNTP) pool levels were examined in synchronized and unsynchronized log phase cultures and in quiescent cultures of human diploid foreskin fibroblasts. dNTP levels were in good agreement with those previously published for human HeLa and lymphoblastic leukemia cells. dCTP and dGTP levels showed only a modest lowering in quiescent as compared to logphase cells, but dATP and dTTP levels were reduced dramatically in quiescent cultures. Cells synchronized by serum starvation and assayed at the peak DNA synthetic phase (18–21 hr post release) showed substantially higher pools of all four dNTPs. Hydroxyurea treatment reduced only purine dNTPs in both log phase and confluent cells while increasing dTTP and dCTP pools. The effects of deoxynucleosides on dNTP pools were also examined and are discussed in light of current models regarding regulation of purified ribonucleotide reductase formulated from in vitro studies.

Cytotoxicity, chromosome aberrations and unscheduled DNA synthesis in cultured human diploid fibroblasts induced by sodium fluoride

The effects of exposure of cultured human diploid fibroblasts (JHU-1 cells) to sodium fluoride have been studied with respect to cytotoxicity and induction of chromosome aberrations and unscheduled DNA synthesis (UDS) Cytotoxicity of NaF on JHU-1 cells, as determined by a decrease in colony-forming ability, linearly increased with increasing dose of NaF (50–150 μg/ml) or exposure time (1–24 h). Treatment of the cells with 50 μg/ml NaF for 24 h resulted in a lethality (∼70%) similar to that obtained with 100 μg/ml for 12 h. A linear increase in cytotoxicity was observed as a fraction of the product of NaF treatment time and dose. JHU-1 cells treated with 20–50 μg/ml NaF for 12 or 24 h were analyzed for chromosome aberrations. A significant increase in the frequency of chromosome aberrations at the chromatid level was observed in treated cells in a dose-dependent manner. For detection of UDS, confluent JHU-1 cells were cultured with medium containing low serum and then exposed to NaF in the presence of 10 mM hydroxyurea. Treatment with 100–400 μg NaF/ml for 4–24 h reproducibly elicited UDS in a dose-related fashion as determined by direct scintillation counting of [ 3H]thymidine incorporated into DNA during repair synthesis. These results suggest that NaF causes DNA damage in human diploid fibroblasts in culture.

Heterogeneity of normal human diploid fibroblasts: isolation and characterization of one phenotype.

Cultures of human diploid fibroblasts contain cells that respond to exposure to the first component of complement (C1) by initiating DNA synthesis and growth. The plasma membranes of these cells have specific binding sites for the C1q subcomponent of C1. A fluorescence-activated cell sorter was used to isolate a subset of cells with a high affinity for C1q, and the growth and synthesis activities of these high-affinity cells were studied after numerous replications in vitro. These cells synthesize DNA and grow faster than the parent cultures and low-affinity cells, and they produce two to three times as much protein. About 40 percent of their total protein synthesis activity is directed to collagen production, unusually high proportions of collagen types III and V being produced. These properties and the high affinity of the cells for C1q are retained for at least six cell transfers. This phenotype has the properties expected of fibroblasts in healing wounds and inflamed tissues.

Unusual membrane bound intranuclear inclusions in human fibroblasts infected with Herpes simplex

Examination of brain tissue taken at autopsy from a 55-year old male showed the presence of herpes simplex viral antigen by immunofluorescence techniques. A suspension of this tissue was inoculated into cultures of human diploid fibroblasts. After 72 h, viral particles at different stages of development were seen inside the nuclei. Envelopment of the viral particles seemed to occur in association with the nuclear membranes, endoplasmic reticulum, cytoplasmic vacuoles and plasma membranes. Severely altered nuclear membranes, with as many as 4 to 8 layers of nuclear envelope, emphasized the common occurrence of membrane reduplication associated with herpes simplex infection. Unusual membrane-bound, dense, rounded inclusions, 100 to 500 nm in diameter, were present in the nucleus. These inclusions apparently were formed between the inner and outer lamellae of the nuclear membrane.

Influence of confluent holding time on UV light mutagenesis in human diploid fibroblasts

We have investigated the induction of mutants resistant to 6-thioguanine (6TG) following 254 nm ultraviolet light exposure of density-inhibited cultures of human diploid fibroblasts. Phenotypic expression of 6TG resistance was maximal within 9 days and remained stable through 19 days after irradiation. In reconstruction studies, complete recovery of 6TG-resistant mutants occurred at cell densities of up to 35 000 cells per 100-mm petri dish. The induced mutation frequency increased linearly with dose over the range of 3–9 J/m 2; the D 0 of the survival curve was 4.2 J/m 2. Delaying subculture to low density for 1.5–24 h after irradiation produced unexpected alterations in induced mutation frequencies. An increase in UV-induced mutations of approximately 3-fold was observed in cultures maintained in confluence for 3 h. This trend was reversed with longer holding times: the mutation frequency declined sharply in cultures held for 6 h compared to the 3-h value, and thereafter showed a steady and gradual diminution to background levels. These data suggest that the repair of potentíally mutagenic damage is a complex phenomenon which can lead to an increase or decrease in mutation frequency as a function of holding time. Although the decline in mutation frequency observed following longer holding intervals is consistent with the notion of an error-free process, we hypothesize that the increased mutation frequency produced by a short holding period reflects the existence of a cell-mediated process which enhances the mutagenic potential of at least some UV-induced DNA photoproducts.

Isolation by flow sorting of cytokinetic and morphological heterogeneity in late-passage cultures of human diploid fibroblasts (IMR-90).

Considerable structural, metabolic, and proliferative heterogeneity develops in populations of cultured diploid cells which have reached advanced levels of population doubling. Isolation of noncycling cells from late-passage cultures would permit more definitive investigation of the structure and behavior of individual senescent cells. In this paper, we report the viable sorting of late-passage cultures of human diploid fibroblasts (IMR-90) into two subpopulations of cells with different proliferative potentials. Sorting is based on cellular light-scattering properties and autofluorescence. Structural and behavioral features of the subpopulation exhibiting increased forward-angle light scatter are more characteristic of senescent cells than the subpopulation sorted by decreased forward-angle light scatter.

Transport of L-histidine by human diploid fibroblasts in culture.

The transport of L-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 (Km = 0.25 mmol/l, Vmax = 17 nmol/mg protein per min) and a nonsaturable component of influx (Kd = 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 that L-histidine uptake at low concentration was poorly inhibited by amino acids known to be effective inhibitors of system A. The largest fraction of L-histidine uptake was inhibited by 2-amino-bicyclo (2,2,1)-heptane-2-carboxylic acid (BCH), leucine, and tryptophan. These results indicated that L-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.

Effect of growth rate on phenotypic expression of 6-thioguanine resistance in human diploid fibroblasts

The influence of growth rate on the kinetics of phenotypic expression of 6-thioguanine resistance has been investigated by establishing 3 protocols for the maintenance of mutagenized stock cultures of human diploid fibroblasts. These protocols differed in the frequency with which subculture was performed during the phenotypic expression period. The resultant growth kinetics varied widely from continual exponential proliferation to density-inhibited growth for much of the expression period. With all of the substance protocols, a maximum mutation frequency was reached by 9 days after exposure of confluent cultures to 6 J/m 2 of 254 nm UV light. The maximum frequency was similar in all experimental groups and remained stable up to 17 days after irradiation, the last time point tested. These results indicate that continual proliferation is not required for optimal expression of 6-thioguanine resistance.