Effects of radical-scavenging enzymes and reduced oxygen exposure on growth and chromosome abnormalities of Werner syndrome cultured skin fibroblasts

Summary Two sister strains of skin fibroblast-like (FL) cells from a patient with Werner's syndrome (adult progeria) were grown in regular tissue culture medium or medium supplemented with the radical-scavenging enzymes superoxide dismutase and catalase. Differences in proliferative potential were shown to be due to strain variation rather than to treatment with the enzymes. Two independent strains were also grown in ambient (18%) or reduced (1%) concentrations of oxygen. All cultures (untreated, treated with enzymes, treated with reduced oxygen exposure) displayed the limited in vitro growth potential and cytogenetic abnormality characteristic of Werner's syndrome FL cells; thus the underlying defect in Werner's syndrome does not appear to be related to defective protection against or repair of damage by free radicals.     

Immortalization of Werner syndrome and progeria fibroblasts.

Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents.     

Galactosylceramide and galactosylsphingosine loading studies in cultured skin fibroblasts in human and murine globoid cell leukodystrophy

Cell level studies of 3H-galactosylceramide(GalCer) and 3H-galactosyl sphingosine (GalSph) have been carried out in cultured skin fibroblasts from human and murine globoid cell leukodystrophy (GLD). GalCer loading studies disclosed that the hydrolysis rates of GalCer in human control and GLD were 72% and 45%, respectively, and those from the murine control and GLD cells were 77% and 21%, respectively, on the 5th day of culture. On the other hand, GalSph loading studies showed that the hydrolysis rate of GalSph in the human control and GLD were 40% and 10%, respectively, and those from murine control and GLD cells were 38% and 10% on the 12th day of culture. These data suggest that both GalCer and GalSph degradations were impaired in cell level in human and murine GLD. Furthermore, when radioactive 3H-GalSph was loaded into cultured fibroblasts from murine and human GLD, 3H-GalCer band was formed via GalSph. These data strongly suggest that GalCer could be synthesized through the GalSph route as a minor pathway at least in cultured skin fibroblasts, although the major pathway to synthesize GalCer should be via ceramide.     

Proliferation dynamics in cultured skin fibroblasts from Down syndrome subjects

With a view to better understanding the role of oxidant/antioxidant variables in proliferation dynamics of somatic cells, we explored the relationships among superoxide dismutase (SOD) activity, glutathione peroxidase (Gpx) activity, reactive oxygen intermediates (ROI), and indices of cellular proliferation and senescence in cultured fibroblasts from Down syndrome and normal donors. We found that Down syndrome cells had a significantly slower proliferative rate, but attain replicative senescence at similar population doubling (PD) as control cells. Irrespective of donor origin, the number of PD until replicative senescence was positively correlated with Gpx activity (r = 0.784, P = 0.007). In addition, the presence of exogenous catalase in the growth medium significantly extended the number of PD until replicative senescence (P = 0.011). The loss of telomere repeats per PD was not different between Down syndrome cells and controls. However, SOD activity was inversely correlated with the loss of telomere repeats per PD. Collectively, these findings suggest that replicative senescence ultimately relates to mechanisms downstream to SOD (i.e., Gpx and catalase) and confirmed previous observations about inverse relationships between SOD activity and telomere repeat loss per cellular replication.     

Biochemical studies on the sulphated glycosaminoglycan fraction of skin fibroblasts cultured from a patient with the Hurler syndrome

1. The metabolism of the sulphated glycosaminoglycan fraction in cultured skin fibroblasts derived from a patient with the Hurler syndrome and from a normal subject was studied. Two labelled precursors, Na(2) (35)SO(4) and d-[2-(3)H]glucose, were used and their intracellular fates during uptake and ;chase' periods were assessed after separation of sulphated glycosaminoglycans from hyaluronic acid. After 4 or 8h of exposure to culture medium containing both labels, [(35)S]sulphate incorporation into the sulphated glycosaminoglycan fraction was twofold greater in Hurler-syndrome cells than in normal cells. At the same time, the rate of incorporation of [(3)H]glucose into the sulphated glycosaminoglycan fraction was approximately the same for both cell types. Consequently, an increased (35)S/(3)H ratio (nmol of [(35)S]sulphate incorporated/nmol of [(3)H]glucose incorporated) was observed for Hurler-syndrome cells compared with normal cells. 2. The results of ;chase' experiments revealed that although the expected loss and relative retention of labelled sulphate occurred in the sulphated glycosaminoglycan fraction of normal and Hurler-syndrome cells, both cell types retained all of their radioactivity derived from [(3)H]glucose. 3. After 34h exposure to a ;corrective-factor' preparation from urine, the sulphated glycosaminoglycan content (as hexosamine and [(35)S]sulphate) of the Hurler-syndrome cells approached normal values. At the same time, there was an increase in specific radioactivity of ;corrected' Hurler-syndrome cells.     

Effect of cholesterol and growth factors on the proliferation of cultured human skin fibroblasts

A cholesterol-deficient growth medium for human skin fibroblasts was prepared by adding to Eagle's Minimum Essential Medium a bovine serum treated with ultracentrifugation to remove bulk lipoproteins followed by silicic acid adsorption to remove residual lipoproteins and cholesterol. Cell growth was slow, but the daily cell doublings could be increased by 76% by including 7.5 micrograms purified cholesterol/ml in the medium. Cell growth in cholesterol-deficient culture medium could be increased to that seen with medium containing 15% untreated fetal bovine serum by the inclusion of the following growth factors: epidermal growth factor (EGF), cortisol, non-essential amino acids, insulin, transferrin and selenium. Cholesterol increased the proliferation of these rapidly-growing cultures by 19%. No effect of cholesterol was observed in transformed L-cell mouse fibroblasts.     

Comparative effects of glucose and fructose on growth and morphological aspects of cultured skin fibroblasts

The growth rate of human skin fibroblasts was evaluated when glucose was replaced by fructose in the culture medium. Four mediums containing respectively 5.5 mmol/l glucose (G1), 27.5 mmol/l glucose (G5), 5.5 mmol/l fructose (F1), and 27.5 mmol/fructose (F5) were used. Skin fibroblasts from fourteen subjects were continuously cultured for 20 days and the number of cells was counted at days 1, 3, 7, 10, 15 and 20 after plating. The morphological patterns were observed and compared, the pH values of the medium were calculated, as were hexose consumption and lactate production. The results established clear differences in cell growth, pH and morphology: up to day 7, the growth rate was lower in fructose than in glucose medium, and the pH values were higher. In addition, marked steatosis appeared, with increased pyruvate dehydrogenase (PDH) activity. After day 10, the mean values gave a significant increase in the number of cells grown in fructose mediums, even if variations occurred between different cell strains. This increase was accompanied by loss of density-dependent growth inhibition and a reduction in the quantity and size of the vacuoles caused by steatosis. These findings were also established for other cell types, like aponeurosis fibroblasts. In addition, the longevity of the strains increased. These observations indicate that intermediary metabolism is considerably influenced by the carbohydrate present in the cell culture medium and that there are also repercussions on the growth rate. Under our experimental conditions, metabolism pathways seemed to differ on day 7 and on day 20. The various metabolic events suggested by the differences in the pH values are now being studied in our laboratory.     

Insulin-like growth factor I-dependent regulation of prolidase activity in cultured human skin fibroblasts

The objectives of this article are to: (i) discuss the origins and the nature of ischemic injury, (ii) identify factors influencing the evolution of injury, (iii) consider various cellular targets for ischemic injury, (iv) assess the overall importance of reperfusion injury, (v) discuss conceptual approaches to cardioprotection and (vi) to identify new ideas and approaches in the realm of myocardial protection. In the human heart, myocardial ischemia may take many forms, it may exist for periods as short as a few seconds or minutes, it may last for hours or it may persist for years. In terms of discussing interventions to combat myocardial ischemia, this article will focus on: (i) regional ischemia as occurs during evolving myocardial infarction and (ii) whole heart or global ischemia as occurs during cardiac surgery and transplantation.     

Perhexiline maleate-induced lipidosis in cultured human fibroblasts: cell kinetics, ultrastructural and biochemical studies

Perhexiline maleate reduced the growth of human skin fibroblasts in cell culture at a concentration range of 0.3-3 micrograms/ml. At the highest concentration, the cells survived only four days. Pleomorphic inclusions characteristic of drug-induced phospholipidoses appeared in cultured cells. Analysis of the major lipid classes was performed on cells exposed to 3 micrograms/ml at four days. Gangliosides, phospholipids and cholesterol levels four to six times above controls were found. No major qualitative abnormalities were detected in phospholipids. On the contrary, an abnormal pattern of gangliosides was seen by densitometry of silica gel thin-layer plates with increases of GD3 and of an unknown ganglioside. Drug induced lipidosis may involve other lipids than phospholipids, particularly gangliosides.     

Hyaluronate synthesized by cultured skin fibroblasts derived from patients with Werner's syndrome.

Hyaluronate in cultured skin fibroblasts derived from patients with Werner's syndrome, who excrete large amounts of urinary hyaluronate, was investigated. The amount of hyaluronate secreted into the medium by Werner's fibroblasts was 2-3-times that of normal fibroblasts, whereas no difference in enzyme activities related to the degradation of hyaluronate was found. Werner's fibroblasts were then cultured in the presence of [3H]glucosamine, and the amount of [3H]hyaluronate and its chain lengths in the medium and matrix (trypsinate) fractions were compared with those of normal cells. No significant difference in the chain length of hyaluronate was observed between normal and Werner's fibroblasts. On the other hand, a significant increase of hyaluronate was found in the matrix fraction of Werner's fibroblasts when the cells reached confluency. In addition, a hyaluronate of small chain length was found in the matrix fraction of Werner's fibroblasts, although this was absent from that of normal cells. It was concluded that the constituents of the extracellular matrix of Werner's fibroblasts differed from those of normal cells, characterized by the presence of a large amount of hyaluronate and a relatively small hyaluronate chain.     

Rhizomelic chondrodysplasia punctata: biochemical studies of peroxisomes isolated from cultured skin fibroblasts.

Peroxisomes isolated from cultured skin fibroblasts of two patients with rhizomelic chondrodysplasia punctata (RCDP) and two controls were compared for biochemical studies. These experiments provided the following results: (1) peroxisomes isolated from RCDP-cultured skin fibroblasts had the same density (1.175 g/ml) as control peroxisomes; (2) dihydroxyacetone phosphate acyltransferase activity, the first enzyme in the synthesis of plasmalogens, was deficient (0.5% of control) in RCDP peroxisomes and this activity was not observed in any other region of the gradient; (3) the rate of activation (lignoceroyl-CoA ligase) and oxidation of lignoceric acid was normal in RCDP peroxisomes; and (4) peroxisomes from RCDP contained 3-ketoacyl-CoA thiolase in the unprocessed form (44-kDa protein), whereas control peroxisomes had both processed (41-kDa protein) and unprocessed forms of 3-ketoacyl-CoA thiolase. The presence of both processed and unprocessed 3-ketoacyl-CoA thiolase in control peroxisomes and the unprocessed form in RCDP peroxisomes suggests that processing of 3-ketoacyl-CoA thiolase takes place in peroxisomes. Although the specific activity and percentage of activity of 3-ketoacyl-CoA thiolase in RCDP peroxisomes was only 22-26% of control, the normal oxidation of lignoceric acid in RCDP peroxisomes indicates that unprocessed 3-ketoacyl-CoA thiolase is active. The remaining peroxisomal 3-ketoacyl-CoA thiolase activity in RCDP was observed in a protein fraction (peroxisome ghosts) lighter than peroxisomes. The normal oxidation of fatty acids in peroxisomes and the absence of such activity in peroxisome ghosts (d = 1.12 g/ml) containing peroxisomal proteins in RCDP suggest that RCDP has only one population of functional peroxisomes (d = 1.175 g/ml).     

Heme content of normal and porphyric cultured skin fibroblasts

Partial deficiencies in enzyme activities of the heme biosynthetic pathway have been demonstrated in cultured skin fibroblasts and other tissues from patients with protoporphyria (PP) and acute intermittent porphyria (AIP). We have quantitatively and qualitatively assessed the heme and free porphyrin content in cultured PP, AIP, VP (variegate porphyria, in which an enzymatic deficiency has not been identified), and normal skin fibroblasts during routine culture conditions in order to assess the overall metabolism of heme in these cells. The total heme concentration was not significantly different between control and porphyric lines; 189 +/- 15 pmoles/mg protein (mean +/- SEM) in controls, 154 +/- 17 in PP, 175 +/- 20 in AIP, and 181 +/- 81 in VP. The hemoprotein difference spectra were similar in all lines. Free porphyrins were not detected in any of the disorders. Despite partial deficiencies in enzyme activities of the heme pathway, porphyric fibroblasts thus maintain normal heme content during routine culture conditions without detectable porphyrin accumulation.     

Functional role of the Werner syndrome RecQ helicase in human fibroblasts

Werner syndrome is an autosomal recessive human genetic instability and cancer predisposition syndrome that also has features of premature aging. We focused on two questions related to Werner syndrome protein (WRN) function in human fibroblasts: Do WRN‐deficient fibroblasts have a consistent cellular phenotype? What role does WRN play in the recovery from replication arrest? We identified consistent cell proliferation and DNA damage sensitivity defects in both primary and SV40‐transformed fibroblasts from different Werner syndrome patients, and showed that these defects could be revealed by acute depletion of WRN protein. Mechanistic analysis of the role of WRN in recovery from replication arrest indicated that WRN acts to repair damage resulting from replication arrest, rather than to prevent the disruption or breakage of stalled replication forks. These results identify readily quantified cell phenotypes that result from WRN loss in human fibroblasts; delineate the impact of cell transformation on the expression of these phenotypes; and define a mechanistic role for WRN in the recovery from replication arrest.     

Sericin enhances attachment of cultured human skin fibroblasts

Human skin fibroblasts were cultured on sericin prepared from cocoon shells. The living cell number after 72 h was enhanced to 250% of the no-sericin control. The increase was due to the acceleration of the initial attachment of the cells. It was found that sericin M, the main component of about 400 kDa, and its serine-rich repetitive domain were the active principles.     

Glucose transport and metabolism in cultured human skin fibroblasts

Human skin fibroblast cultures, seeded at $\text{10}{}^5\text{cells}\text{5}\text{cm}$ plate and allowed to grow to confluence at approx. $\text{10}{}^6\text{cells}\text{5}\text{cm}$ plate, utilized a glycolytic mode of metabolism where the ratio of glucose utilized to lactate produced wa 0.62±0.05 (Zielke, R.H., Ozand, P.T., Tyldon, J.I., Sevdalian, D.A. and Cornblath, M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4110–4114) (mean±S.E.). When the glucose in the medium was exhausted, the lactate produced during the highly glycolytic phase was then reutilized. In monolayer cultures that had been washed with phosphate-buffered saline, rates of glucose utilization were measured at 0.25 and 2 mM glucose by monitoring the appearance of ³H₂O from [5-³H]glucose. Rate of utilization for each concentration of glucose decreased markedly as the cultures became more confluent. This decrease also correlated with a reduced ability to transport glucose as measured by 2-deoxy-[³H]glucose uptake in washed monolayer cultures. In washed confluent culture of fibroblasts, glucose utilization was markedly decreased by the presence of pyruvate and lactate but not by glutamine. The respiratory inhibitors, rotenone and antimycin, did not increase the rate of glucose utilization except when added in combination with pyruvate. We conclude that cultured skin fibroblasts posses a highly glycolytic mode of metabolism but that this mode can become more oxidative in the presence of sufficient quantities of pyruvate and lactate.