Human Fibroblast Strains Research Articles

Psoralen Plus Near-Ultraviolet Light: A Possible New Method for Measuring DNA Repair Synthesis

A new method is proposed to inhibit semiconservative DNA synthesis in cultured cells while DNA repair synthesis is being measured. The cells are treated with the DNA-crosslinking agent Trioxalen (4,5,8-trimethylpsoralen) plus near-ultraviolet light, and consequently 99.5% inhibition of replicative DNA synthesis is achieved. Additional DNA-damaging agents induce thymidine incorporation into the double-stranded regions of the DNA. The new method gave results very similar to those obtained with the benzoylated naphthoylated DEAE (BND) cellulose method using three human fibroblast strains, of which one had deficient capacity for DNA repair synthesis following treatment with gamma rays and methyl methanesulfonate. The advantages of the new method are simplicity and rapidity, as well as the high extent to which replicative DNA synthesis is inhibited.

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.

Action spectra for inactivation of normal and xeroderma pigmentosum human skin fibroblasts by ultraviolet radiations.

Abstract—Action spectra for UV‐induced lethality as measured by colony forming ability were determined both for a normal human skin fibroblast strain (lBR) and for an excision deficient xeroderma pigmentosum strain (XP4LO) assigned to complementation group A using 7 monochromatic wavelengths in the range 254‐365 nm. The relative sensitivity of the XP strain compared to the normal skin fibroblasts shows a marked decrease at wavelengths longer than 313 nm. changing from a ratio of about 20 at the shorter wavelengths to just greater than 1.0 at the longer wavelengths. The action spectra thus indicate that the influence on cell inactivation of the DNA repair defect associated with XP cells is decreased and almost reaches zero at longer UV wavelengths. This would occur, for example, if the importance of pyrimidine dimers as the lethal lesion decreased with increasing wavelength. In common with other studies both in bacterial and mammalian cells, our results are consistent with pyrimidine dimers induced in DNA being the major lethal lesion in both cell strains over the wavelength range 254‐313 nm. However, it is indicated that different mechanisms of inactivation operate at wavelengths longer than 313 nm.

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.

Similar repair of O 6-methylguanine in normal and ataxia-telangiectasea fibroblast strains : Deficient repair capacity of lymphobalstoid cell lines does not reflect a genetic polymorphism

The ability of human fibroblast strains to repair the mutagenic DNA adduct O 6-methylguanine (MNNG) was investigated. The repair reaction proceeded rapidly during the first hour after alkylation, followed by a slow, continuous phase of repair, and both processes were saturated by low doses of carcinogen. This was similar to what had previously been found in human lymphoblastoid lines. Three fibrobalst strains from healthy donors and six strains from patients with ataxia telangiectasia were all proficient in their capacity to repair O 6-MeG and had the same sensitivity to the cytotoxicity of MNNG and methyl methanesulphonate as normal cells. Three of these cell strains were derived from individuals whose lymphoblastoid lines were deficient in their ability to repair O 6-MeG. These lymphoblastoid lines were also extremely hypersensitive to killing by methylating carcinogens. Because non-transformed cells from the same donors behaved normally with regard to both parameters, we concluded that the repair deficiency accompanied by carcinogen hypersensitivity of the lymphoblastoid lines does not indicate a genetic deficiencu in the donor. These findings imply that lymphoblastoid lines may not always nbe the appropriate cell type for investigating genetic susceptibility to chemical mutagens.

Age dependent production of a competence factor by human fibroblasts

AbstractSeveral cell types such as Balb/c 3T3 have been shown to require platelet‐derived growth factor (PDGF); however, strains of human fibroblasts from fetal donors have been shown to divide in medium containing plasma free of PDGF. Since human fibroblasts have been demonstrated to secrete other peptide growth factors such as somatomedin‐C, we have undertaken a study to determine if fibroblasts derived from fetal donors are capable of producing a mitogen(s) which will substitute for PDGF and support growth in plasma alone. Quiescent human fibroblasts from donors ages 12‐wk embryo, newborn, and 3‐yr‐old were exposed to serum‐free minimum essential medium (MEM) for 24 hr. The conditioned media collected from embryonic and newborn fibroblast donors were demonstrated to stimulate growth in the 3‐yr‐old cells with the addition of plasma alone, whereas conditioned medium from the 3‐yr‐old donor cells was without effect. The increases in growth and DNA synthesis were dependent upon concentration of media used. Conditioned medium derived from newborn fibroblasts also supported 3‐yr‐old cell growth but embryonic conditioned medium was more potent. The embryonic conditioned medium factor was heat and acid stable but destroyed by trypsin and excluded by a 5,000 (MW) molecular weight filter. The factor(s) had full competence factor activity since transient exposure to fibroblasts (3‐yr‐old donor) stimulated 78% nuclear labeling vs. 81% with continuous exposure. These results support the concept that there is an age‐dependent production of a competence factor by human fibroblasts which may partially account for their capacity to grow in medium devoid of PDGF and supplemented with plasma alone.

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.

Repair of potentially lethal and sublethal damage induced by neocarzinostatin in normal and ataxia-telangiectasia skin fibroblasts

Neocarzinostatin is a radiomimetic antibiotic with a potent cytotoxic effect which elicits a hypersensitive response in human cells homozygous or heterozygous for the gene for ataxia-telangiectasia. The extent and the time course of potentially lethal damage repair and sublethal damage repair following neocarzinostatin treatment were investigated in human skin fibroblast strains and were found to be remarkably similar to those obtained following X-irradiation. Ataxia-telangiectasia homozygous cells essentially lacked potentially lethal damage repair, but were able to perform some degree of sublethal damage repair following neocarzinostatin treatment. Ataxia-telangiectasia heterozygous cells which show an intermediate degree of neocarzinostatin sensitivity could perform both processes but with somewhat reduced efficiency as compared to normal cells. These observations provide further evidence for a DNA repair defect in ataxia-telangiectasia cells.

Hereditary orotic aciduria, Lesch-Nyhan syndrome, and xeroderma pigmentosum probed by herpes simplex virus: 125I-iododeoxycytidine incorporation as an assay for viral growth.

Abstract125I‐Iododeoxycytidine (125IdC) incorporation into acid‐insoluble material was a sensitive, rapid, and quantitative assay for the growth of herpes simplex virus type 1 (HSV‐1) in human fibroblasts. Cellular utilization of the isotope was 10 to 25% of the incorporation by infected cells and could be 80% inhibited by tetrahydrouridine (THU). Viral utilization was inhibited by acycloguanosine, thioguanine (TG), and cytosine arabinoside. Isotope was incorporated equally well by growing or quiescent infected cells. HSV‐1 was used to probe the metabolic capabilities of three mutant human fibroblast strains. 125IdC incorporation quantitatively measured the ability of the virus to grow in these cells. Viral 125IdC incorporation was sensitive to TG in normal fibroblasts but showed a 8‐ to 10‐fold greater resistance to TG in fibroblasts derived from patients with Lesch‐Nyhan syndrome (LN). Similarly, the growth of ultraviolet irradiated HSV‐1 in normal fibroblasts, as judged by 125IdC incorporation, was 5‐fold greater than in fibroblasts derived from patients with xeroderma pigmentosum. In fibroblasts derived from patients with hereditary orotic aciduria, viral 125IdC incorporation was sensitive to adenosine (AD) at concentrations which were slightly stimulatory in normal fibroblasts. This was a 2‐fold difference in AD sensitivity, which the radioassay reliably and quantitatively documented. Previous attempts to quantify and characterize this difference by immunofluorescence were unsuccessful. HSV‐1 infected cells could be individually identified by their incorporated 125IdC; such cells had blackened nuclei in autoradiograms prepared 12 hr after infection. Normal cells infected in the presence of TG had many fewer labeled nuclei than LN cells similarly infected in the presence of the drug. Thus, this viral assay can be a rapid and accurate probe of cellular function and has potential for the identification of mutant cells in amniocentesis samples or tumor speciments. Towards this end, we determined that the AD and TC dose responses of infected amniotic fluid cells closely paralleled that of the normal human fibroblasts.

Variation in response to mutagens amongst normal and repair-defective human cells.

Over the past five years we have been examining the mutability of a variety of human fibroblast strains established from both normal individuals and patients suffering from cancer-prone genetic diseases. The objective of these experiments was to determine if cancer proneness at the level of the individual is correlated with either hypersensitivity to the lethal effects or hypermutability to the mutagenic effects of DNA damaging agents. The existence of hypersensitivity and particularly hypermutability implies the presence of defects in repair of DNA damage and underlines the importance of effective repair processes for human health (1).

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.

Co-localization of 125I-epidermal growth factor and ferritin-low density lipoprotein in coated pits: a quantitative electron microscopic study in normal and mutant human fibroblasts.

Low density lipoprotein (LDL) and epidermal growth factor (EGF) bind to receptors on the surface of human fibroblasts and are internalized in coated vesicles. Each of the ligands has been studied separately by electron microscopy in human fibroblasts using ferritin-LDL as one visual probe and 125I-EGF as a second visual probe. A mutant strain of human fibroblasts (J.D.) has been described in which LDL does not localize to coated pits and hence is not internalized. Because LDL and EGF do not compete with each other for binding, in the current studies we coincubated the two ligands with normal and mutant cells to visualize their cellular fates. In normal fibroblasts ferritin-LDL and 125I-EGF both bound preferentially to coated pits at 4 degrees C and both ligands were internalized into endocytotic vesicles and lysosomes. Quantitative studies in normal cells showed that 75% of the coated pits and vesicles that contained 125I-EGF also contained ferritin-LDL, indicating that both ligands enter the cell through the same endocytotic vesicles. In the LDL internalization-mutant J.D. cells, ferritin-LDL did not localize in coated pits and was not internalized, but 125I-EGF bound to coated pits and was internalized just as in normal fibroblasts.

Human fibroblast strains showing increased sensitivity or resistance to ethidium bromide

The sensitivity of a number of human fibroblast cell strains towards the DNA intercalating and mutagenic agent ethidium bromide has been examined. Among the cell strains investigated, 3 were of fetal origin, 6 from clinically normal adult persons, 2 from Lesch-Nyhan individuals and 1 each from persons with 3 genetic disorders, xeroderma pigmentosum, Fanconi anemia and Bloom syndrome, which are known to predispose to cancer. Results of our studies show that cells derived from 2 otherwise normal individuals exhibits a marked sensitivity towards ethidium bromide as compared to the rest of the group. At the same time all 3 cell strains of fetal origin were found to be highly resistant to killing by this agent. These results are discussed in relation to the toxic/mutagenic effects of ethidium bromide.

Colony-forming ability of ataxia-telangiectasia skin fibroblasts is an indicator of their early senescence and increased demand for growth factors

A gradual decrease in colony-forming ability of human fibroblast strains was found to be an early sign of cellular senescence. This decrease occurs in fibroblast strains from patients with ataxia-telangiectasia (A-T) at significantly earlier passage levels than with normal strains; accordingly, the lifespan of A-T cells in culture is shorter than that of normal strains. The colony-forming efficiency of A-T cells can serve as a quantitative measure for serum quality: A-T cells fail to form colonies, or have a markedly reduced colony-forming efficiency, in some batches of calf serum and newborn calf serum, whereas in other batches their clonal growth is maximal. Normal fibroblasts show the same colony-forming efficiency with all batches of serum. Fibroblast growth factor (FGF) and epidermal growth factor (EOF), but not insulin, were able to correct this deficiency in the growth-promoting ability of certain serum batches. In addition, conditioning medium containing these sera on confluent unirradiated or irradiated monolayers of any other human fibroblast strain had the same effect. A-T cells seem to have an increased demand for certain FGF- or EGF-like growth factors that are present in variable amounts in different serum batches and also in conditioned medium.

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.

Interferon action in human fibroblasts: induction of 2', 5'-oligoadenylate synthetase in the absence of detectable protein kinase activity.

In several strains of human fibroblasts, treatment with human alpha- or beta-interferon (IFN) resulted in an induction of 2', 5'-oligoadenylate synthetase but no protein kinase activity. The antiviral and anticellular effects of IFN are elicited in these cells in the absence of detectable IFN-induced protein kinase activity.

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.

Further Studies on the Survival of Non-proliferating Human Diploid Fibroblasts Irradiated with Ultraviolet Light

Labelling index data showed that in AG1518 cells, a diploid human fibroblast strain, there was a lag period of at least 14 hours between subculture from the density-inhibited plateau phase of growth and entry into DNA synthesis. Cells irradiated with 254 nm wavelength U.V. light 8 hours after subculture did not exhibit the same degree of resistance as cells irradiated in plateau phase and subcultured immediately. When cells were arrested from proliferation by maintenance in an arginine and glutamine deficient medium for 72 hours, they were nearly as resistant to U.V. light as plateau phase cells although maintenance in this medium for 24 hours after irradiation supported further recovery only after low U.V. doses. One strain of Cockayne syndrome fibroblasts was found to be resistant to U.V light in plateau phase while another strain was found to have the same survival response whether it was irradiated in the plateau or log phase of growth.