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PubMed1985

Lack of specific inhibition of DNA repair in WI-38 human diploid fibroblasts by sodium saccharin.

The ability of sodium saccharin (NaS) to inhibit the repair of DNA damaged by UV irradiation was examined in cultured WI-38 human diploid fibroblasts. Cesium chloride density gradient ultracentrifugation was used to measure DNA repair and DNA replication. NaS (10-10,000 micrograms/ml) did not specifically inhibit UV light-induced DNA repair. At doses of NaS (1785 and 10,000 micrograms/ml) that caused a 62-67% inhibition of semiconservative DNA replication, there was little or no inhibition of DNA repair synthesis. In cell cultures not exposed to UV irradiation, NaS failed to induce DNA repair. RNA synthesis and protein synthesis were unaffected by NaS at all doses tested. The inhibition of semiconservative DNA replication at higher doses of NaS may be a manifestation of cytotoxicity. In contrast to results with NaS, WI-38 cells were very sensitive to DNA repair inhibition by the well-studied inhibitor quinacrine-HCl. These results do not support mechanisms of saccharin-induced tumorigenesis involving either direct induction of DNA damage or inhibition of the repair of DNA damage caused by other agents.

Cells, Cultured↗

PubMed1979

Adenosine 3',5'-monophosphate-dependent protein kinase(s) in diploid and SV40 transformed human fibroblasts.

Cyclic AMP-dependent protein kinases (EC 2.7.1.37; ATP:protein phosphotransferase) in the human diploid fibroblast WI-38 and an SV40-transformant WI-38-VA13-2RA (VA13) have been compared on the basis of their concentrations in cells, isoenzyme composition and susceptibility to hormonal activation. In high population density cultures, total soluble cyclic AMP-dependent kinase activities measured with histone were essentially the same in WI-38 and VA13. Two soluble protein kinase forms separated by chromatography on DEAE-cellulose were present in both cell lines. The concentration of cyclic AMP required for half-maximal activation of both enzyme forms was 10-30 nM. Overall kinase stimulation was greater for the Peak I enzymes. Kinase activation induced in the presence of 0.5 M KCl was more rapid and complete for the Peak I enzymes. Under conditions which elevated the concentration of cyclic AMP in WI-38 and VA13 cells the activities of the soluble histone kinases were increased. Incubation of the cells with either of 5.7 micronM prostaglandin E1 or 1 micronM isopropylnorepinephrine induced complete activation of the cyclic AMP-dependent histone kinases within 5 min and maintained the effect for 20 min. When intracellular cyclic AMP levels were raised by prostaglandin E1, activation of glycogen phosphorylase (assayed-AMP) suggested that this enzyme cascade involving cyclic AMP-dependent protein kinase(s) was intact and responsive in both cell lines.

Cell Line↗

PubMed1981

Comparison of the oxidation of glutamine, glucose, ketone bodies and fatty acids by human diploid fibroblasts.

The contribution of glutamine, glucose, ketone bodies and fatty acids to the oxidative energy metabolism of human diploid fibroblasts ws studied. The rate of glutamine oxidation by fibroblasts was 98 nmol/h per mg cell protein compared to 2 nmol/h per mg cell protein or less for glucose, acetoacetate, D-3-hydroxybutyrate, octanoic acid and palmitic acid. Glucose inhibited glutamine oxidation by 85%, while the other substrates had no effect. Therefore, these cells meet their energy requirement almost solely by anaerobic glycolysis and glutamine oxidation.

Cell Line↗

PubMed1982

Abnormalities of lysosomes in human diploid fibroblasts from patients with Farber's disease.

An accumulation of ceramide associated with the deficiency of acid ceramidase has been demonstrated in cultured diploid skin fibroblasts from a patient with Farber's disease. We extend this observation to investigate the lysosomal localization of accumulated ceramide and the abnormalities of lysosomes caused by this ceramide accumulation in Farber's diseased fibroblasts. We have found that the lysosomal fraction isolated from Farber's diseased fibroblasts by a subcellular fractionation procedure is markedly low in density compared with that of normal fibroblasts and is separated from other subcellular organelles. Ultrastructural studies of the isolated lysosomal fraction from Farber's diseased fibroblasts showed a mixed population of intact and swollen vesicles with a lysosomal appearance. Examination under high magnification clearly demonstrated lysosomal inclusions which contain lamellar and curvilinear membranes and resembled those seen in the intact fibroblasts. Subcellular localization of Farber's fibroblasts showed that the accumulated [3H]ceramide from the culture medium was predominantly localized in the lysosomal fraction with a markedly low density and very little was found to be associated with other cellular membranes. Our finding that ceramide is accumulated in the lysosomal fraction of Farber's fibroblasts and that these cells also show membranous inclusions strongly suggests that the accumulation of ceramide is directly involved in the formation of lysosomal inclusions.

Acid Ceramidase↗

PubMed1983

Regulation of lipogenic enzymes in human diploid fibroblasts by hormones.

The hormonal regulation of two regulatory enzymes of fatty acid synthesis acetyl-CoA carboxylase (EC 6.4.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49), has been investigated in human diploid fibroblasts. There was a 35% increase in acetyl-CoA carboxylase activity, 72 h following addition of 10 microU/ml insulin to the culture medium. Addition of 1 microgram/ml of 3,3'5-triiodothyronine for 72 h resulted in an increase in acetyl-CoA carboxylase activity to 166% of the controls. The simultaneous addition of 1 microgram/ml triiodothyronine and 10 mU/ml insulin caused the enzyme activity to rise to 240% of the controls. A dose-dependent reduction in acetyl-CoA carboxylase activity was brought about by 1 X 10(-4) to 1 X 10(-3) M dibutyryl cyclic AMP. The earliest effect of dibutyryl cyclic AMP was observed within 24 h. Glucose-6-phosphate dehydrogenase followed qualitatively the same pattern of response, whereas the constitutive enzyme, lactate dehydrogenase (EC 1.1.1.27), did not show significant changes in these experiments. The data demonstrate common features of hormonal regulation of lipogenesis in human fibroblasts with liver and adipose tissue and substantiate the growing evidence that thyroid hormones are of major importance for the regulation of this process.

Acetyl-CoA Carboxylase↗

PubMed1981

Unscheduled DNA synthesis in human diploid cells of different donor ages.

Human diploid fibroblast-like cells from two donor of widely different ages were tested for their ability to perform unscheduled DNA synthesis (UDS) after exposure to ultraviolet irradiation. Cultures were assayed at various times throughout their in vitro lifespans as both confluent and mitotically arrested populations. Cells from both donors maintained their ability to perform UDS throughout their lifespans with arrested populations exhibiting increased levels. The appearance of elevated levels of UDS in arrested cells was directly related to the age of the donor. These results indicate that the loss of ability to repair DNA damage is not a primary cause of in vitro senescence. They do suggest, however, that the regulation of DNA repair synthesis is effected by increasing age.

Adult↗

PubMed1981

Pinocytosis of fluorescein-labelled growth-promoting alpha-globulin in human diploid cells.

Fluorescein isothiocyanate conjugated growth-promoting alpha-globulin (FITC-GPAG) binds to human diploid cells LEP 19 at 37 degrees C. After the initial binding step, labelled GPAG is progressively internalized by the cells. FITC-GPAG is present in pinocytic vesicles localized in the perinuclear region of the cells and its pinocytosis occurs without cell surface redistribution of GPAG.

Alpha-Globulins↗

PubMed1990

The relative biological effectiveness of photon radiation from encapsulated iodine-125, assessed in cells of human origin: I. Normal diploid fibroblasts.

The relative biological effectiveness (RBE) of photon radiation from encapsulated Iodine-125 "seed" sources has not previously been investigated in human cells. The RBE of 125I photons relative to 137Cs gamma rays was examined in normal diploid human fibroblasts derived from lung and skin. The cells were irradiated in plateau phase using a specially designed incubator-irradiator which permitted simultaneous 125I and 137Cs irradiation. The cells were irradiated at various dose rates ranging from 7 to 70 cGy/hr. Dosimetry was performed using Monte Carlo computer calculations to simulate the 125I irradiations and the exposure-standardization measurements made by the U.S. National Bureau of Standards which are the basis for the specified strengths of 125I seeds. Simulation of the exposure standardization measurements revealed systematic errors due to the unrecognized presence of low-energy fluorescence X rays. The specified activity of the type of seeds used for this study (high-activity, no radiographic marker) was found to be too high by more than 10%. The RBE of 125I assessed with both lung fibroblast lines was found to be 1.2 and was 1.3 for the skin fibroblasts. The RBE did not change over the range of dose rates tested. In fact, for both 125I and 137Cs, the dose response curves did not change with dose rate over the range tested, implying full repair of sublethal damage at dose rates below 70 cGy/hr in these non-dividing cells.

Brachytherapy↗

PubMed1989

Amplification and direct nucleotide sequencing of cDNA from the lysate of low numbers of diploid human cells.

The polymerase chain reaction technique is widely employed to amplify short segments of genomic DNA to determine if a specific change has occurred. However, some investigators need to sequence the entire coding region of mammalian genes, e.g., cellular ras genes or the gene encoding hypoxanthine (guanine) phosphoribosyl transferase (HPRT), to determine what specific changes have occurred. To do so, they isolate RNA from large populations of cells, amplify cDNA from the gene of interest, subclone the product, and sequence two or more isolates to determine the common mutation. We have developed a method to simplify this procedure by copying mRNA of the hprt gene directly from the lysate of a clone of mutant diploid human fibroblasts (e.g., 100 cells). We amplified the first and second strand of the cDNA of the gene of interest 10(10)- to 10(11)-fold, obtained 5 to 10 micrograms of DNA in less than 10 h, and sequenced the coding region directly without the need for RNA extraction or DNA template purification. By our method cDNA can be amplified directly from the lysate of just one human cell, but to avoid detecting random changes introduced by the polymerase, we lysed approx. 200 cells from a clone, each containing the identical mutation, amplified the cDNA, and determined the consensus sequence by direct nucleotide sequencing.

Base Sequence↗

PubMed1982

Ganglioside changes during cell aging in human diploid fibroblast TIG-1.

Gangliosides of human diploid fibroblast, TIG-1, derived from fetal lung were analyzed during cell aging. The major gangliosides of TIG-1 cells were GM3 and GD3. During cell aging, GM3 and other gangliosides were decreased or below detection level. Lipid-bound sialic acid per mg protein was 2 micrograms at the early, middle and late passages, but between the middle and late passage it was twofold higher than the other passages.

Cell Count↗

PubMed1989

Decreased accuracy of protein synthesis in extracts from aging human diploid fibroblasts.

The accuracy of protein synthesis has been measured in extracts from human diploid fibroblasts of different ages. Extracts were supplied with purified mRNA for the coat protein of the cowpea variant of tobacco mosaic virus (CcTMV), which lacks codons for cysteine and methionine. The presence of 35S-cysteine in CcTMV coat protein synthesized during translation reactions therefore represents translational error. Translation reactions were performed with extracts from young fibroblasts (less than 50% of life span completed) and old fibroblasts (more than 90% of life span completed), and the translation products were purified by immunoprecipitation and analyzed by polyacrylamide gel electrophoresis. The error frequency increased from 4.2 x 10(-5) cysteines/amino acid in young cell extracts to 2.9 x 10(-4) cysteines/amino acid in old cell extracts. Cysteine incorporation was not due to nonspecific binding, and could be increased approximately sixfold by the addition of the misreading antibiotic, paromomycin. It is concluded that translational accuracy is not stable during aging in vitro, and it is proposed that this decrease in the fidelity of information transfer could be responsible for the variety of changes observed in aging cultured human cells.

Amino Acids↗

PubMed1989

Age and cell density dependent changes of gangliosides in human diploid fibroblasts.

The gangliosides in human diploid fibroblasts--TIG-1, TIG-7, and IMR-90--were analysed at different cell densities at early and late passages to clarify the relationship between age and cell density dependent changes of the gangliosides. In early passages, the ganglioside concentrations increased with increase in cell density. At late passages, however, the concentrations were lower than those at the early passages either in the growing or confluent phase, and slightly increased with increase in cell density. The pattern of ganglioside compositions were apparently different between early and late passage cells either in growing or confluent state. In the early passages, GM3 and GD3 were major constituents, and GM2, GD1a, or the other more complex gangliosides were detected as minor components. With increase in cell density, the content of GM3 decreased, whereas GD3 and the others increased. At the late passages, however, GM3 was the major component, and GD3, GM2, or GD1a were minor, but the others were hardly detectable. The ganglioside pattern did not change with increase in cell density. Thus, the age-dependent changes of gangliosides could be distinguished from the cell density dependent alterations.

Cell Count↗

PubMed1989

The relationship between cell size, the activity of DNA polymerase alpha and proliferative activity in human diploid fibroblast-like cell cultures.

In kinetic studies with human diploid fibroblast-like (HDFL) cells carried out in heterokaryons and in monokaryons, we have observed a first-order relationship between the level or concentration of DNA polymerase alpha and the rate of initiation of new rounds of DNA synthesis. Because cell size is inversely proportional to the concentration of DNA polymerase alpha and presumably other replication factors, it is inversely related to the initiation of new rounds of DNA synthesis. An inverse relationship between cell size and clonogenic activity was also observed in both unsorted HDFL cells and in HDFL cells sorted on the basis of size. Experimental enlargement of cells by serum deprivation at low density resulted in changes in colony-forming ability that would be predicted by these studies. A causal relationship between the observed increase in cell size with advancing passage level and the loss of proliferative activity is suggested by these studies; in addition, cell size may be a useful biophysical marker for cellular aging.

Cell Division↗

PubMed1989

Levels of DNA methylation in diploid and SV40 transformed human fibroblasts.

Total 5-methylcytosine (5mC) has been measured in the DNA of cultured human cells by HPLC chromatography. In agreement with other reports, the level of 5mC declines continuously during the serial passaging of diploid fibroblasts, strain MRC-5. On the contrary, fibroblasts infected with SV40, which have acquired most of the properties of transformed cells, maintain a constant level of 5mC, prior to dying out in the crisis period. MRC-5 and Werner's syndrome fibroblasts fully transformed and immortalised by SV40, also maintain a constant level of 5mC. These results indicate that the ability of cells to maintain DNA methylation is a necessary but not a sufficient step for immortalisation.

Cell Survival↗

PubMed1989

Polyamine metabolism and cell-cycle-dependent gene expression in IMR-90 human diploid fibroblasts during senescence in culture.

Aging of IMR-90 human diploid fibroblasts in culture is accompanied by specific changes of polyamine metabolism including: (a) a fivefold decrease of serum-induced activity of ornithine decarboxylase (ODC1 EC 4.1.1.17); (b) a six to tenfold increase of polyamine catabolism; and (c) a reduction of putrescine uptake. These changes apparently led to a significant reduction of putrescine accumulation in senescent cells following serum stimulation. Since the induction of ODC is a mid-G1 event, the change of polyamine metabolism may be related to changes of expression of other cell-cycle-dependent genes during cellular aging. In addition to ODC gene, we have examined the expression of two early G1 genes, c-erbB and c-myc, and one late G1/S gene thymidine kinase, at mRNA levels, in both young and old IMR-90 cells. We have also compared the enzyme activities of two late G1/S genes, thymidine kinase and thymidylate synthetase, in young and old cells following serum stimulation. We did not observe significant changes of c-erbB, c-myc, and ODC mRNA levels during cellular senescence. However, we found that serum-induced mRNA level of thymidine kinase gene in old IMR-90 cells was significantly reduced compared to that in the young cells. Results also demonstrate that aging of IMR-90 cells was accompanied by significant decrease of both thymidine kinase and thymidylate synthetase activities. In view of the recognized importance of polyamines in growth regulation, it is possible that alteration of polyamine metabolism may contribute to the impairment of expression of some key G1/S genes and such impairment may contribute to the ultimate loss of dividing potential in senescent cells.

Biological Transport↗

PubMed1991

Isozyme polymorphisms in human diploid cell strains for research on cellular aging.

Six human lung diploid cell strains established for the study of in vitro cellular aging (TIG-1, TIG-7, WI-38, IMR-90, MRC-5, MRC-9, and HeLa cells as a control) were studied by cellulose acetate membrane electrophoresis for allozymic differences at 18 enzyme loci. Eight enzyme loci (G6PD, PGM1, PGM3, PepA, PGD, ADA, GLO1, and ME), proved to be informative in establishing unique allozyme genetic signatures for all of the cell strains established from the same species and from the same organ. Changes in the allozyme genetic signatures were not observed throughout the life span of TIG-1 and MRC-9 cells. The allozyme genetic signatures can be used as a quick monitor of cell identification and intraspecific cell contamination.

Cell Line↗

PubMed1991

A new human male diploid cell strain, TIG-7: its age-related changes and comparison with a matched female TIG-1 cell strain.

A new human diploid cell strain, TIG-7, which has the male karyotype, was established and characterized. Isozyme and histocompatibility typing of the cell strain was performed. The average in vitro life span of the cells is 73 population doublings. Changes in cell volume, doubling time, saturation density, the efficiency of cell attachment, plating efficiency, and relative DNA content were examined during in vitro cellular aging. Hydrocortisone slightly prolongs the life span of the cell strain when the hormone is administered to the cultures during middle passages. The age-related changes in the parameters of TIG-7 are not appreciably different from those of the previously established TIG-1 cell strain. These results show that this cell strain is useful for research on cellular aging; further profit is anticipated from research using a combination of these two sexually different cell strains.

Cell Line↗

PubMedDate not supplied

Metallothionein expression and stress responses in aging human diploid fibroblasts.

Metallothioneins (MTs) are low molecular weight proteins with a high cysteine content that are inducible by heavy metals and by other conditions of environmental stress. This laboratory was investigated in human diploid fibroblasts the induction of MTs by cadmium and by dexamethasone, and the induction of heat shock proteins, as models for age-related changes in gene expression that reflect the ability of old cells to respond to environmental stress. Old cells were more sensitive to the toxic effects of CdCl2 in the concentration range 100-175 microM. Analysis of 35S-cysteine-labelled cell extracts by polyacrylamide gel electrophoresis and fluorography showed that in the absence of any inducer, old cells have a 3.7-fold increase over young cells in the basal level of MT. The rate and extent of induction of MT by CdCl2 was reduced in old cells: Exposure of old cells to 100 microM CdCl2 for 18 h resulted in MT levels about 33% of the amount in young cells. Northern blot analysis showed that the changes in MT protein levels occurred in parallel with changes in mRNA levels, which implicates transcriptional control as the origin of these aging changes. These young/old differences in MT synthesis were maintained in density-arrested cultures, indicating that the aging changes were not due to differences in the cell cycle status of these cell populations. The rate and extent of induction of a 68-kDa heat shock protein were also reduced in old cells, which showed an increase in basal, uninduced level of this protein similar to MT. In contrast, old cells retained the ability to synthesize MTs in response to dexamethasone at a rate similar to that in young cells.

Blotting, Northern↗