Human Diploid Fibroblast Strains Research Articles

Induction of host DNA synthesis in senescent human diploid fibroblasts by infection with human cytomegalovirus

A human diploid fibroblast strain, TIG-1, ceased to proliferate at about 60–62 population doubling level. The percentage of nucleic incorporating [ 3H]thymidine during 24-h culture in fresh medium containing 10% fetal bovine serum was less than 2% in the senescent cells used in this study. Infection of these cells with human cytomegalovirus (HCMV), strain AD-169, increased the percentage of [ 3H]thymidine-labeled cells by about ten-fold. Equilibrium density gradient centrifugation analysis of purified DNA from infected cells showed that cellular DNA synthesis was stimulated preceded by the viral DNA synthesis. Ultraviolet irradiation of HCMV reduced the ability to induce DNA synthesis. Equilibrium density gradient centrifugation analysis of DNA which was labeled with 5-bromodeoxy-uridine indicated semiconservative replication rather than repair synthesis. These results suggested that in a considerable fraction of human senescent cells host DNA replication could be reinitiated by infection with HCMV, but not by the addition of fetal bovine serum.

Mitochondrial DNA in mortal and immortal human cells. Genome number, integrity, and methylation.

Mitochondrial DNA was quantitated in total DNA of various normal and mutant strains of human diploid fibroblasts (finite replicative lifespan) and permanent cell lines, using Southern-transfer hybridization to 32P-labeled pure mtDNA probe and saturation hybridization to 3H-labeled cRNA copied from mtDNA. In six normal fibroblast strains, mtDNA copy number increased during serial passage roughly in proportion to cell volume or protein content, whereas normalized mtDNA content per pg of protein depended upon in vivo donor age but not passage level ("in vitro" age). Copy numbers for mtDNA varied much more widely in individual fibroblast clones than in mass cultures, but were not well correlated with longevity or growth rate. Five mutant fibroblast strains associated with reduced replicative lifespan, and four permanent cell lines, were also examined; in each group, mtDNA values were observed both lower and higher than any obtained for normal fibroblasts. No evidence was found of petite-type deletions from human mtDNA, either at late passage or in individual clones of fibroblasts. Methylation of mtDNA genomes was strikingly non-random and apparently decreased with culture age.

A Comparison of the Lethal Effects of Intracellular Radionuclides in Human and Rodent Cells

The relative sensitivities of a human diploid fibroblast strain (AG1522) and an established line of contact-inhibited mouse embryo fibroblasts (10T1/2) were compared after incorporation of [3H]thymidine or [125I]iododeoxyuridine into the cellular DNA and growth at 37 degrees C. Whereas these cells show similar sensitivities to acute X irradiation, the D0 for clonal survival of the mouse fibroblasts was 5- to 10-fold higher than the D0 for the human fibroblasts for both 3H and 125I. A large difference in sensitivity between these two cell types was also seen after protracted irradiation from 3H2O at 37 degrees C, but this difference was markedly reduced when cells were exposed to 3H2O at 0 degree C to simulate an "acute" irradiation. An established human tumor cell line was similarly hypersensitive to killing by 3H2O at 37 degrees C as compared with early passage diploid hamster embryo cells. These results indicate that human cells are more radiosensitive than rodent cells when a radiation dose is protracted over several days rather than given as an "acute" exposure, and are consistent with the hypothesis that human cells are hypersensitive to low-dose-rate irradiation as compared with rodent cells.

Binding of human interferon alpha to cells of different sensitivities: studies with internally radiolabeled interferon retaining full biological activity.

The characteristics of interferon binding to various cells with different interferon sensitivity were studied by using [3H]leucine-labeled, pure human interferon alpha from Namalwa cells. Scatchard analysis of the binding data on cells sensitive to interferon alpha (human FL and fibroblasts and bovine MDBK) indicated the presence of two kinds of binding sites with high and low affinities. The binding constants of the high-affinity sites in these cells were similar (4 X 10(10) to 11 X 10(10) M-1). Cells insensitive to human interferon alpha (human HEC-1 and mouse L cells) were shown to have only low-affinity sites, suggesting that high-affinity binding sites are indispensable for interferon sensitivity and represent interferon receptors. However, the number of sites in three human diploid fibroblast strains and one strain trisomic for chromosome 21 were not proportionally correlated to the interferon sensitivity of the cells. The high-affinity binding to human cells was completely inhibited by both nonradioactive human interferons alpha and beta in a similar manner, but binding to bovine MDBK cells, on which human interferon beta is practically inactive, was inhibited effectively only by interferon alpha and not by beta. These results suggest that the receptor for human interferon alpha is common to human interferon beta in human cells, whereas the receptor on bovine cells binds only human interferon alpha.

Comparison of mutagenicity of N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea in human diploid fibroblasts

We have studied the mutagenic efficiencies of N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) in four strains of human diploid fibroblasts by assaying induction of resistance to thioguanine (TG). MNU was observed to be a very weak mutagen whereas ENU was confirmed to be extremely potent in all strains. Resistance to the mutagenic effects of MNU was attributed to the efficient manner by which human fibroblasts can remove the hypothesized promutagenic base, O6-methylguanine, from their DNA following methylation.

Suppression of cytotoxic effect of mitomycin-C by superoxide dismutase in Fanconi's anemia and dyskeratosis congenita fibroblasts.

Survival curves were determined for several human diploid fibroblast strains treated with mitomycin-C (MMC) with or without concomitant incubation with superoxide dismutase (SOD). These included cells from patients with Fanconi's anemia (FA), dyskeratosis congenita (DC) a disorder related to FA, Gardner's syndrome (GS) and xeroderma pigmentosum grop C (XPC). Incubation with SOD had no effect on MMC-induced cytotoxicity in the two normal cell strains, XPC or GS. Although GM2053 (FA) and two DC strains showed intermediate sensitivity to killing by MMC similar to XPC and GS, the survival of these cell strains was increased approximately 2-4 times after 0.5 microgram/ml of MMC by concomitant treatment with SOD. Survival of the most MMC sensitive cell strain GM1309 (FA) was sharply increased by adding SOD; survival was enhanced about 15-fold after treatment with 0.1 microgram/ml of MMC. SOD induced no enhancement of survival in MMC treated normal cells at survival levels down to nearly 10(-3). We propose that the hypersensitivity to MMC cytoxicity in FA and DC cells may involve among other factors a deficiency in the inactivation of certain free radical intermediates.

Clonal selection in cultured human fibroblasts: role of protein synthetic errors.

Protein synthetic error frequency, determined in cell-free extracts as delta leu/delta phe incorporation following poly(U) stimulation, has been found to decrease progressively in several strains of human diploid fibroblasts during their limited replicative lifespan. To explore the basis of this phenomenon, we followed a mass (uncloned) culture of one normal strain at 13 stages of its replicative lifespan. We found a progressive tenfold decline in error frequency that was inversely correlated with passage level (r = -.93, p less than .001). This could not be ascribed to the slow rates of replication associated with fibroblast senescence because slowing of growth by serum deprivation did not change error frequency. Additionally, terminal mass cultures maintained for 16 wk at saturation density to minimize cell selection did not change error frequency over this time. Error frequencies in 12 individual clones purified from the parental culture did not decline on repeated passage, either remaining constant or, in two clones, rising abruptly three- to five-fold after initial assays. Error frequencies of clones showed a weak inverse correlation with growth vigor but not with the maximum doubling number. We conclude that selective pressures favor more vigorously dividing clones with low protein synthetic error frequencies leading to their predominance in mass cultures.

A model culture system with human gingival fibroblasts for evaluating the cytotoxicity of dental materials

A model experimental culture system and protocol are described to screen polymerized dental materials for diffusible toxic products. The system employs cultures of human gingival fibroblasts grown in plates containing immobilized samples of polymerized resins. Comparative cytoxicity is evaluated by counting viable cells with the aid of phase optics at several time periods up to 48 h. To achieve adequate statistical sampling, multiple counts are made in four different zones at 90 degrees angles from each sample and at three distances from the centers of samples. The most significant data were generated during a 24 to 48 h test period in climate. This cytotoxicity test measured cell death as a function of time of exposure and distance from the sample (24 h, 0 to 3 mm; 48 h, 3 to 6 mm) and permitted a calculation of the relative cytoxicity for each material, which is termed the viability index (VI). This can be expressed as a percentage related to the control, which is called the time-distance cytotoxicity index (TDCI). This method is simple to carry out because it used basic laboratory equipment, is rapid, and has a sound scientific basis. It focuses on times and distances when or where, or both, the greatest cellular changes are taking place. Some data illustrated are based on the screening of eight different restorative resins. The literature of cell culture testing of dental materials is reviewed. It is concluded that biotoxicity studies ideally should employ diploid human target cells from the oral cavity because the cells retain specialized features. Secondary cultures or strains of human diploid gingival fibroblasts, which are relatively easy to obtain and maintain, are recommended as cells of choice for screening dental restorative materials in vitro.

Sister chromatid exchange response of human diploid fibroblasts and chinese hamster ovary cells to dimethylnitrosamine and benzo(a)pyrene

In the search for relevant assays for mutagenicity testing, considerable attention has been given to the use of mammalian cells in vitro and the incorporation of metabolic activation in the protocol. Chinese hamster ovary (CHO) cells are commonly chosen as the target cells for cytogenetic tests because of their excellent growth characteristics and long lifespan in culture. However, there may be cellular factors affecting the uptake, metabolism, and repair of damage which are not the same in all cell lines. The response of CHO cells and three human diploid fibroblast strains (IMR-90, WI-38, S-3299) to benzo(a) pyrene (BP) and dimethylnitrosamine (DMN) were compared using sister chromatid exchange (SCE) analysis as a measure of genetic damage. For both BP and DMN the human cells and the CHO cells showed dose-response slopes that were significantly different from zero, except CHO cells treated with BP for 1 hr and S-3299 cells treated with DMN. Whereas human and CHO cells showed similar dose-responses to BP and the three human cell strains had similar dose-responses to BP and DMN, the dose-response of the human cells to DMN was statistically less significant than that of CHO cells. Reducing the duration of chemical treatment in CHO cells had no effect on the slope of the dose-response curves for BP or DMN. The observed differences between human and CHO cells may reflect differences in the fate of metabolic intermediates of DMN.

P-Isothiocyanatophenyl 6-phospho-alpha-D-mannopyranoside coupled to albumin. A model compound recognized by the fibroblast lysosomal enzyme uptake system. 2. Biological properties.

A conjugate of p-aminophenyl 6-phospho-alpha-D-mannopyranoside and bovine serum albumin was shown to interact with the uptake system for lysosomal enzymes in cultured human diploid fibroblasts. Radioiodinated conjugate containing 20 mol of mannose 6-phosphate/mol of albumin was taken up by the cells and degraded to trichloroacetic acid soluble fragments which were released into the medium. Unlabeled conjugate, mannose 6-phosphate, and a lysosomal enzyme, L-iduronidase, inhibited the uptake of the 125I-labeled conjugate (Ki = 2 X 10(-8), 5 X 10(-6), and 1.5 X 10(-9) M, respectively). Conversely, the uptake of L-iduronidase was competitively inhibited by the mannose 6-phosphate conjugate as well as by free mannose 6-phosphate; however, higher concentrations of these compounds were required (Ki = 10(-6) and 5 X 10(-5) M, respectively). These results suggest that although L-iduronidase and the conjugate are bound to the same receptor by mannose 6-phosphate residues, the uptake of the enzyme involves some additional structure that is not shared by the conjugate. Internalization of the radiolabeled mannose 6-phosphate albumin conjugate was observed only in human diploid fibroblast strains. An SV-40 transformed line of human fibroblasts as well as three permanent rodent fibroblast lines (CHO, NRK, and L cells) failed to take up the conjugate, presumably because they were deficient in receptors or in the ability to internalize receptor-conjugate complexes.

Genetic control of tumorigenicity in interspecific mammalian cell hybrids

The nature of genetic control of cellular malignancy was investigated by examining the tumorigenicity of a series of interspecific mouse-human cell hybrids in the athymic nude mouse. Two highly malignant but genetically distinct mouse cell lines, A9 and PG19, were hybridized with three normal human diploid fibroblast strains, and 19 independently arising hybrid clones were isolated. Each of these clones was capable of forming progressive lethal tumors in the nude mouse, and thus resembled the malignant parental mouse cells rather than the nonmalignant parental human cells. We failed to obtain any evidence for complete suppression of tumorigenicity in these cell hybrids. The absence of suppression was observed regardless of the extent and composition of the human chromosome complements retained in the hybrid clones; the results of detailed cytological and isoenzyme analyses would make it highly improbable that the observed lack of suppression was due to cellular selection in vivo for a more tumorigenic subpopulation in the injected hybrid cells. These data demonstrate that at least for the parental cell combinations used in this study, no human chromosome, when present singly in the mouse-human cell hybrids, can suppress the tumorigenic phenotype of the mouse cells. Our results are consistent with the view that the suppression of cellular malignancy previously demonstrated in intraspecific (mouse × mouse) somatic cell hybrids does not occur in interspecific (mouse-human) cell hybrids, or alternatively, genetic determinants located on two or more human chromosomes are required simultaneously to suppress the malignancy of the mouse cells in cell hybrids derived from malignant mouse cell and nonmalignant human cells.

Effects of cystine or glutamine restriction on human diploid fibroblasts in culture.

Amino-acid restriction was studied using a strain of human diploid fibroblasts (HDF) isolated from fetal skin. HDF populations were maintained at reduced proliferation rates by lowering cystine or glutamine concentrations of Eagle's MEM. Effects of this treatment on mean cell diameters, total cell protein and specific activity of acid phosphatase were assessed. Threshold levels of cystine required for maintenance were found to be markedly influenced by type and/or batch of serum used irrespective of their free cystine content. A 96-hr exposure of passage 25 cells to medium 0.01 or 0.005 mM in cystine resulted in an increased mean cell diameter and a greater heterogeneity of cell size. This was accompanied by an increase in the total cellular protein. A 48-hr exposure, followed by return to control serial cultivation, induced persistent change in mean cell diameter without affecting the total life span (49 passages over 165 days). The mean size of untreated cells increased with serial passage reaching a size similar to treated cells only at passage 44. Acid-phosphatase specific activities were higher in cells maintained under conditions restrictive in cystine. None of the above changes were observed when glutamine concentrations were reduced to 0.0025 mM, a level required to inhibit division rates. The possible relevance of these findings to the phenomenon of in vitro cell senescence is discussed.

Interferon affects both G1 and S+G2 in cells stimulated from quiescence to growth

THE interferons were originally described as proteins, produced by cells in response to virus infection, which could induce viral resistance in cells of the same species1. It is becoming clear that these proteins have other important biological activities2 and can inhibit cell division in both normal and malignant cells3–5. Although the phenomenon of growth inhibition by interferon is well established, data analysis of the effect in terms of the cell cycle is limited and not completely consistent. Killander et al.6 and Matarese and Rossi7, using asynchronously growing cultures of L1210 and Friend leukaemia cells respectively, have found that interferon-treated cells are delayed in both G1 and G2, and we have recently observed a similar effect of interferon on asynchronously growing MCF-7 cells8. Sokawa et al. have reported, however, that the only parameter affected in interferon-treated quiescent BALB/c 3T3 cells after serum stimulation, is their rate of entry into S (ref. 9). It is possible that the effect of interferon on the cell cycle of asynchronous transformed cells is different, in terms of cell cycle parameters, from its effect on quiescent untransformed cells. In an attempt to clarify this point, we have examined in detail the effect of interferon on the passage of serum-stimulated cells through the cell cycle, using BALB/c 3T3 and Swiss 3T3K cells, and a strain of human embryo lung diploid fibroblasts, HEL 27. In contrast to Sokawa et al., we have found that both G1 and S+G2 are extended by interferon treatment of all three cell types studied.

Effects of cell density and cell growth alterations on cyclic nucleotide levels in cultured human diploid fibroblasts

cGMP and cAMP concentrations were studied in cultures of two strains of normal human diploid lung fibroblasts, WI38 and KL-2, under various conditions which alter growth rate. Higher levels of cAMP were found in fibroblasts grown in medium with low (0.1 – 1.0%) serum concentration and thus exhibiting a decreased rate of growth. A rise in cAMP also preceded the decreased growth rate when medium was not changed for 4 days or longer (starvation). The reinitiation of cell growth by addition of fresh medium containing the standard 10% serum to either starved or serum-restricted cells was preceded by a rapid drop in cAMP level. Cellular cAMP levels increased to a moderate extent as sparse cultures first increased in density, but did not continue to rise as the culture approached saturation density. cGMP levels were inversely related to cell density: much higher cellular cGMP levels were found at low density than at higher cell density, whether cells were rapidly proliferating under standard growth conditions or had their growth arrested by omission of medium change or restriction of serum. Thus, under these conditions the steady state levels of cGMP appear to be related to cell density rather than rate of cell proliferation. However, a transient but appreciable increase in cGMP did occur upon the addition of fresh medium containing 10% serum to starved or serum-restricted cells, a condition leading to reinitiation of cell proliferation. Smaller but significant increases in cGMP were also evident following routine addition of fresh medium with serum to growing cells fed every other day and following mild EDTA-trypsin treatment of confluent WI38 fibroblasts. Thus, at least dual control mechanisms appear to be involved in the regulation of cGMP levels. Comparison of mid- and late-passage WI38 cells revealed no significant differences either in the levels of cGMP at sparse densities or in the density-dependent change in levels. These results suggest that levels of both cAMP and cGMP are influenced by cell density and also by conditions which alter the rate of cell proliferation.

A study of the effect of ultraviolet light on the division potential of human diploid fibroblasts

Culture and UV (254 nm) irradiation conditions that are suggested as appropriate for a study of the effect of UV on the limited in vitro lifespan of a normal human diploid fibroblast (HDF) strain are first described. An inoculation density at each subcultivation of 1.8 × 10 4 viable cells/cm 2 permits the decline in proliferative capacity to occur with kinetics similar to that observed using a 1:2 split and prevents cell overlap at the time of irradiation. Doses of 5 and 10 J/m 2 have only a slight effect on initial growth rates and little or no effect on cell density achieved at confluence. With these conditions populations can be irradiated several times throughout the in vitro lifespan. No effect of UV on the limited division potential was observed. In the extreme, a population irradiated 14 times, once every second passage starting at P-18 with doses of 5 or 10 J/m 2 had the same lifespan as controls, as measured by lifespan determinations and thymidine labeling index. Transformed cells were not detected in the multi-irradiated populations. Evidently no accumulation in the populations of damage induced by UV that affected life span, thymidine labeling index, growth rates or confluent cell densities occurred. No selection of a population with altered sensitivity occurred. An argument that genome hits may not be a prime reason for the limited proliferative capacity of HDF populations is presented.

Transport of l-methionine in human diploid fibroblast strain WI38

The transport of L-methionine in human diploid fibroblast strain WI38 was investigated. The uptake of l-methionine was measured in sparse cell cultures in a simple balanced salt solution buffered with either Tris·HCl of N-2-hydroxyethylpiperazine- N′-2-ethanesulfonic acid (HEPES). Similar results were obtained with these two buffers. Cultures were allowed to equilibrate with the buffered saline before transport was measured. The presence of glucose in the buffered saline results in a slight reduction in the initial rate of transport for the first 2 h of equilibration in part buffered saline. l-Methionine is actively transported in WI38 by saturable, chemically specific mechanisms which are temperature, pH and, in part, Na + dependent, and are reactive with both l- and d-stereoisomers. Kinetic analysis of initial rates of transport at substrate concentrations from 0.0005 to 100 mM indicated the presence of two saturable transport systems. System 1 has an apparent K M of 21.7 μM and an apparent V of 3.57 nmol/mg per min. System 2 has an apparent K M of 547 μM and an apparent V of 22.6 nmol/mg per min. Kinetic analysis of initial rates of transport in Na +- free media or after treatment with ouabain suggested that system 1 is Na + independent and that system 2 is Na + dependent. Preloading of cells with unlabeled l-methionine greatly increases the initial rate of uptake. Efflux of transported methionine is temperature dependent, and is greatly increased in the presence of unlabeled l- or d-methionine or l-phenylalanine, but not in the presence of l-arginine. l-Methionine transport is strongly inhibited by other neutral amino acids, and is very weakly inhibited by dibasic amino acids, dicarboxylic amino acids, proline or glycine.

Aging in vitro and large-scale interferon production by 15 new strains of human diploid fibroblasts.

To develop resources for large-scale production of human fibroblast interferon, we isolated, cryopreserved, and characterized 15 new strains of human diploid foreskin fibroblasts. Their life spans in vitro ranged from 52 to 72 population doublings. We based the selection of cell strains for mass interferon production on the number of population doublings during which consistently high yields of interferon were obtained after "superinduction" in roller bottles; our data show that aging in vitro leads to significant decline in amounts of interferon produced. In contrast, susceptibility to interferon remains largely unaffected by in vitro senescence. Karyotypic analysis indicated that the best interferon-producing strain, MLD (over 60,000 reference units/ml), has a translocation between chromosomes 5 and 15.

The replicative life spans of euploid hybrids derived from short-lived and long-lived human skin fibroblast cultures.

Two recent technical advances facilitate the derivation of proliferating hybrids from human diploid fibroblast strains without recourse to biochemical selection: (1) a new chemically-mediated method of somatic cell fusion (PEG-DMSO) yields hybrids at rates as high as 1 in 160 colonies after dilute plating of treated cell mixtures, and (2) a simple technology for assessment of DNA content (flow microfluorometry) permits rapid and highly sensitive monitoring of ploidy. Employing these techniques, we isolated 43 chromosomally stable hybrid clones from 12 crosses between seven different strains representing a wide range of longevities. Crosses between short-lived strains resulted in short-lived hybrid offspring, whereas hybrids derived from long-lived parents tended to be long-lived. Crosses between strains of contrasting longevities gave clones with intermediate growth potentials relative to the other types of hybrids. The failure to observe complementation (enhanced longevity) of hybrids argues against random recessive single-copy gene mutations as important determinants of clonal senescence.

Membrane function in cystic fibrosis. II. Methionine transport in normal and cystic fibrosis fibroblasts.

Initial rate kinetics of methionine transport, time course of accumulation of methionine, and efflux of accumulated methionine were studied in three normal and four CF human diploid fibroblast strains. The range of apparent Km's was 12.7-32.1 micrometer for the CF strains and 18.3-39.2 micrometer for the normal strains. The range of apparent Vmax's was 6.69-9.22 nmole mg-1 min-1 for the CF strains and 5.59-7.87 nmole mg-1 min-1 for the normal strains. The patterns of accumulation and efflux are quite similar in all the strains studied except for WI-38, which showed somewhat higher efflux and lower accumulation than for others. There was no significant difference in the kinetic parameters of methionine transport between CF and normal skin fibroblasts, and methionine transport will not serve as a marker for cystic fibrosis in cultured fibroblasts.

Mechanism of regulation of fibroblastic cell replication. IV. An analysis of the serum dependence of cell replication based on Michaelis-Menten kinetics.

AbstractThe quantitative dependence of the initiation of DNA synthesis in nonreplicating cultures of human diploid fibroblasts (HDFs) on the concentration of fresh serum present in the stimulating medium has been analyzed. A simple formal analysis of the system suggested that the theory of dissociation reactions should lead to a model analogous to the Michaelis‐Menten formulation. Accumulation of enough data for statistical analysis became possible by innovations in the culture techniques and isotope incorporation methods. The data were shown to conform to this model, and parameters Vm and K′m were calculated, together with appropriate statistical functions, from a weighted linear regression of the double reciprocal transformation of the data. Several variables in the culture system were assessed for their effects on the kinetic parameters. The results were consistent with the hypothesis that Vm is a measure of the maximum population of cells capable of responding to the stimulation, and K′m is an estimate of the interaction between the mitogenic factors in serum and the cell surface.Different strains of HDFs had different parameter values. Of several culture variables tested, the Vm was found to be influenced by the type of serum used, and by overall cell density as well as density variations within the monolayer. K′m showed significant changes only in response to cell density, and the evidence suggests that the affinity of cells for serum factors decreases as the cell density increases in confluence.