Comparative study of the enzymatic defense systems against oxygen-derived free radicals: the key role of glutathione peroxidase

Human WI-38 diploid fibroblasts have been cultivated under high toxic O2 pressure, and their survival curves are reported. Superoxide dismutase, catalase, or glutathione peroxidase provided some protection when injected in the cells exposed to O2. This protective effect, recorded after 3 or 4 days of incubation, was the most pronounced when cells were injected just before oxygen exposure. Quantitative injection assays have been performed for the three enzymes. Surprisingly, glutathione peroxidase was found to be much more effective than both catalase and superoxide dismutase, the latter being particularly inefficient.     

Overexpression of apolipoprotein J in human fibroblasts protects against cytotoxicity and premature senescence induced by ethanol and tert-butylhydroperoxide

Human diploid fibroblasts (HDFs) exposed to subcytotoxic stresses under H2O2, tert-butylhydroperoxide (t-BHP), and ethanol (EtOH) undergo stress-induced premature senescence (SIPS) characterized by many biomarkers of HDFs replicative senescence. Among these biomarkers are a growth arrest, an increase in the senescence-associated beta-galactosidase activity, a senescent morphology, an overexpression of p21waf-1 and the subsequent inability to phosphorylate pRb, the presence of the common 4977-bp mitochondrial deletion, and an increase in the steady-state level of several senescence-associated genes such as apolipoprotein J (apo J). Apo J has been described as a survival gene against cytotoxic stress. In order to study whether apo J would be protective against cytotoxicity SIPS and replicative senescence in human fibroblasts, a full-length complementary deoxyribonucleic acid of apo J was transfected into WI-38 HDFs and SV40-transformed WI-38 HDFs. The overexpression of apo J resulted in an increased cell survival after t-BHP and EtOH stresses at cytotoxic concentrations. In addition, when WI-38 HDFs were exposed to 5 subcytotoxic stresses with EtOH or t-BHP, in conditions that were previously shown to induce SIPS, a lower induction of 2 biomarkers of SIPS was observed in HDFs overexpressing apo J. No effect of apo J overexpression was observed on the proliferative life span of HDFs, even if apo J overexpression triggered osteonectin (SPARC) overexpression, which was shown to decrease the mitogenic potential of platelet-derived growth factor but not of other common growth-inducing conditions. Apo J senescence-related overexpression is proposed to have antiapoptotic rather than antiproliferative effects.     

Subcellular localization and modification with ageing of glutathione, glutathione peroxidase and glutathione reductase activities in human fibroblasts

Differential centrifugation and isopycnic equilibration in density gradients were used to localize glutathione (GSH), glutathione peroxidase and glutathione reductase in the subcellular organelles of WI-38 fibroblasts. GSH was present in all the subcellular fractions, whereas the glutathione peroxidase and reductase activities were restrained to the cytoplasm and the mitochondrial fractions. After equilibration in density gradients, the results showed the presence of GSH, glutathione peroxidase and glutathione reductase in both the cytoplasm and mitochondria. GSH was also located in plasma membranes and probably in peroxisomes, endoplasmic reticulum and lysosomal membranes. Evolution of GSH in ageing fibroblasts showed a sudden increase of its concentration just before cell death. The glutathione peroxidase activity already decreases in the early passages, while the decrease of the glutathione reductase activity was constant and reached a drastic low level at the end of the culture. In conclusion, GSH is probably involved in the cell degeneration associated with ageing but because of its multiple functions and its ubiquitous localization, it is difficult to assert to which extent this metabolite is implicated in the ageing process.     

Quantitative study of natural antioxidant systems for cellular nitrofurantoin toxicity

The toxicity of nitrofurantoin was studied on human WI-38 fibroblasts: this chemical was lethal when added at concentrations higher than 5.10(-5) M in the culture medium. The protection afforded by antioxidants was then tested: alpha-tocopherol gave at 10(-4) M a light protection in contrast to ascorbic acid which even became toxic at high concentrations. We also tested catalase, superoxide dismutase and glutathione peroxidase introduced intracellularly by the microinjection technique. On a molecular basis, glutathione peroxidase was 23-times more efficient than catalase and 3000-times more than superoxide dismutase. The results also showed that a similar range of enzyme concentrations was found for the protection against high oxygen pressure. This suggests that, in the case of both oxygen and nitrofurantoin toxicity, the peroxide derivatives are the most toxic intermediates of the free radical attacks.     

Metabolic stabilization of MAP kinase phosphatase-2 in senescence of human fibroblasts

Cellular senescence is characterized by impaired cell proliferation. We have previously shown that, relative to the young counterpart, senescent WI-38 human fibroblasts display a decreased abundance of active phosphorylated ERK (p-ERK) in the nucleus. We have tested the hypothesis that this is due to elevated levels of nuclear MAP kinase phosphatase (MKP) activity in senescent cells. Our results indicate that the activity and abundance of MKP-2 is increased in senescent fibroblasts, compared to their young counterparts. Further analysis indicates that it is MKP-2 protein, but not MKP-2 mRNA level, that is increased in senescent cells. This increase is the result of the increased stability of MKP-2 protein against proteolytic degradation. The degradation of MKPs was impaired by proteasome inhibitors both in young and old WI-38 cells, indicating that proteasome activity is involved in the degradation of MKPs. Finally, our results indicate that proteasome activity, in general, is diminished in senescent fibroblasts. Taken together, these data indicate that the increased level and activity of MKP-2 in senescent WI-38 cells are the consequence of impaired proteosomal degradation, and this increase is likely to play a significant role in the decreased levels of p-ERK in the nucleus of senescent cells.     

Changes in membrane transport function in G0 and G1 cells

Confluent quiescent monolayers of aneuploid and euploid cells in culture can be stimulated to proliferate by appropriate nutritional changes. In confluent monolayers of WI-38 human diploid fibroblasts the uptake of cycloleucine is increased three hours after these cells are stimulated to proliferate by a change of medium plus 10% serum. No changes in the uptake of cycloleucine are observed in logarithmically-growing WI-38 cells exposed to fresh medium plus 10% serum, or in WI-38 confluent monolayers in which the conditioned medium has been replaced by fresh medium with 0.3% serum (a change that does not cause stimulation of cellular proliferation in WI-38 cells). In 3T6 cells in the stationary phase stimulated to proliferate by nutritional changes, there is a prompt increase in the uptake of cycloleucine, within one hour after stimulation of cell proliferation. Similar results were obtained with stationary 2RA cells which are SV-40 transformed WI-38 fibroblasts. In addition, chromatin template activity which is known to increase in the early stages after stimulation of confluent WI-38 cells, was unchanged in confluent 3T6 or 2RA cells stimulated to proliferate. These results show that at least two of the very early biochemical events occurring in response to stimulation of cell proliferation are different in WI-38 diploid cells and in aneuploid 2RA or 3T6 cells. It is proposed that WI-38 cells in the stationary phase are arrested in the G₀ phase of the cell cycle, while 2RA and 3T6 cells are arrested in the G₁ phase.     

Quantitative study of natural antioxidant systems for cellular nitrofurantoin toxicity

The toxicity of nitrofurantoin was studied on human WI-38 fibroblasts: this chemical was lethal when added at concentrations higher than 5·10⁻⁵ M in the culture medium. The protection afforded by anitoxidants was then tested: α-tocopherol gave at 10⁻⁴ M a light protection in contrast to ascorbic acid which even became toxic at high concentrations. We also tested catalase, superoxide dismutase and glutathione peroxidase introduced intracellularly by the microinjection technique. On a molecular basis, glutathione peroxidase was 23-times more efficient than catalase and 3000-times more than superoxide dismutase. The results also showed that a similar range of enzyme concentrations was found for the protection against high oxygen pressure. This suggests that, in the case of both oxygen and nitrofurantoin toxicity, the peroxide derivatives are the most toxic intermediates of the free radical attacks.     

Effect of vitamin A on epithelial morphogenesis in vitro

Quiescent confluent monolayers of WI-38 human diploid fibroblasts and of 3T6 mouse fibroblasts were stimulated to proliferate by nutritional changes. WI-38 cells had a stringent requirement for serum factor(s) but 3T6 did not require serum in order to proliferate again. In both cell lines there was an early increase in the synthesis of non-histone chromosomal proteins shortly after stimulation of cellular proliferation and this increase was linearly correlated to the number of cells entering the S phase several hours later. Only WI-38 diploid fibroblasts, however, showed an early increase in chromatin template activity 1 h after stimulation of cellular proliferation, while chromatin template activity in 3T6 cells remained unchanged. It is suggested that the activation of gene function represents a critical step for the passage of WI-38 cells in the G0 resting phase to the G1 phase of the cell cycle. It is also suggested that 3T6 cells are unable to enter or stay in a G0 phase but can be arrested predominantly in the G1 phase by nutritional deficit, probably amino acid starvation.     

Immunofluorescent study of chromatin proteins in cultured fibroblasts

Antibodies against chromatin from 3T6 mouse fibroblasts and WI-38 human diploid fibroblasts were prepared by immunization of rabbits. Immunofluorescent studies showed species-specificity of these antichromatin antibodies. Furthermore, using anti-3T6 chromatin antibodies against 3T6 cells and anti-WI-38 chromatin antibodies against WI-38 cells, we observed, by immuno-fluorescent techniques, granular fluorescence in the diffusely stained nucleus and diffuse fluorescence in the cytoplasm. These results raise the possibility of the presence of a cytoplasmic pool of chromatin proteins.     

Low density lipoprotein receptor-related protein (LRP) is required for lactoferrin-enhanced collagen gel contractile activity of human fibroblasts

Fibroblasts plated on a type I collagen gel can reduce the size of the gel in a way that mimics the reorganization of the collagen matrix that accompanies the wound healing process. We demonstrated previously that lactoferrin (Lf) specifically binds to WI-38 human fibroblasts and enhances their collagen gel contractile activity. The effect of Lf correlated with the phosphorylation of myosin light chain (MLC), suggesting that Lf promotes fibroblast contractile activity by regulating MLC phosphorylation. We found here that the binding of Lf to WI-38 cells was inhibited by recombinant receptor-associated protein (RAP), a universal competitor for ligand binding to LRP (LDL receptor-related protein), and RAP can also promote the collagen gel contractile activity. These observations suggest that LRP is a receptor that mediates the Lf-induced enhancement of collagen gel contractile activity in WI-38 fibroblasts. To confirm the hypothesis, we utilized LRP antisense oligonucleotide, which was modified by morpholino linkage. Suppression of LRP expression abrogated the Lf-induced enhancement the contractile activity in fibroblasts. Treatment of fibroblasts with Lf enhanced the phosphorylation of ERK1/2 and the activation of MLC kinase (MLCK). These effects were attenuated by suppression of LRP expression. These findings suggest that LRP is involved in the Lf-enhanced collagen gel contractile activity of WI-38 fibroblasts by converting the Lf binding signal into the activation of ERK1/2 and MLCK.     

Dissimilar cyclic nucleotide phosphodiesterase activities in subcellular fractions from normal and SV40-transformed WI-38 fibroblasts.

Broken cell preparations of WI-38 and SV40-transformed WI-38 (VA13) fibroblasts were used to compare the cyclic nucleotide phosphodiesterase activities of the two cell strains. The bulk of the cAMP or cGMP phosphodiesterase activity of WI-38 and VA13 homogenates was found in the 100,000 x g fibroblast supernatant fractions. WI-38 and VA13 soluble phosphodiesterase activities showed anomalous kinetic behavior with either cAMP or cGMP as the substrate. At low substrate concentrations, e.g., 0.1 muM, WI-38 supernatant fractions hydrolyzed cGMP much more rapidly than cAMP. At high substrate concentrations, e.g., 100muM, the same enzyme preparations degraded cAMP more than twice as fast as cGMP. In contrast, VA13 soluble phosphodiesterase activity catalyzed the hydrolysis of a wide range of cAMP and cGMP concentrations at similar rates. Phosphodiesterase activity in WI-38 supernatant fractions was generally more sensitive than that of the comparable VA13 enzyme activity to inhibition by MIX and papaverine. The cAMP phosphodiesterase activity of both WI-38 and VA13 supernatant preparations was decreased by cGMP in a concentration-dependent manner. cAMP was an effective inhibitor of cGMP hydrolysis by VA13 soluble phosphodiesterase activity. Yet, the cGMP phosphodiesterase activity of WI-38 supernatant fractions was only slightly reduced in the presence of cAMP. DEAE-cellulose chromatography of WI-38 and VA13 supernatant preparations revealed two major peaks of phosphodiesterase activity for each cell type. WI-38 peak I showed much greater activity with 1muM cGMP than with 1muM cAMP and appeared to be composed of two different phosphodiesterase activities. WI-38 peak Ia included phosphodiesterase activity which could be stimulated by boiled, dialyzed fibroblast homogenates while WI-38 peak Ib coincided with column fractions which contained most of the cyclic GMP hydrolytic activity. VA13 peak I phosphodiesterase activity was eluted from DEAE cellulose columns at the same ionic strength as WI-38 peak Ia and hydrolyzed these two substrates at nearly identical rates. This enzyme activity was also increased in the presence of boiled, dialyzed fibroblast preparations. Peak II phosphodiesterase activities from both WI-38 and VA13 fibroblasts were relatively specific for cAMP as the substrate. Phosphodiesterase activity with the properties of WI-38 peak Ib was not isolated from VA13 supernatant fractions. These results suggested that the dissimilar patterns of cAMP accumulation in WI-38 and VA13 cultures may be at least partially related to different phosphodiesterase activities in the normal and the transformed fibroblasts.     

The effect of dexamethasone on the functioning of C3 receptor sites on the surfaces of human fibroblasts

The presence of C₃ receptor sites on the cell surfaces of WI-38 fibroblasts was reported in a previous paper. Here the effect of dexamethasone sodium sulfate of C₃ receptor site function was studied. The incubation of WI-38 fibroblasts with dexamethasone sodium sulfate produces the biphasic mode of action, i.e., the growth-stimulating phase with low doses (90–230 μg/ml) and the growth-inhibiting phase with high doses (450–900 μg/ml). The function of C₃ receptor sites on WI-38 fibroblasts seems to be very stable and cannot be suppressed by the pretreatment of WI-38 fibroblasts with dexamethasone in high concentrations, where the cell growth is inhibited.     

Involvement of glutathione and glutathione-related enzymes in the protection of normal and trisomic human fibroblasts against daunorubicin

We measured the glutathione content, and the activity of glutathione-related enzymes and DT-diaphorase in cultured normal (cell line: S-126) and trisomic (cell lines: S-158, S-240) human fibroblasts exposed to daunorubicin (DNR). Determination of reduced and total glutathione levels, and measurement of the activity of glutathione peroxidase, glutathione reductase, glutathione-S-transferase and DT-diaphorase were performed spectrophotometrically. Human fibroblasts were exposed to 4 microm DNR for 2 h, and the cells placed in drug-free medium for 6, 12, 24, 48, and 72 h. Cellular levels of GSH and total glutathione decreased following exposure to DNR. However, the ratio of GSH to total glutathione returned to control levels only in trisomic cells. These changes were concomitant with increasing glutathione-S-transferase and glutathione reductase activities. DNR also significantly increased the activity of Se-independent peroxidase and DT-diaphorase in trisomic fibroblasts. Marked increases in the activity of Se-dependent peroxidase and DT-diaphorase alone were seen in normal cells. The results provide the first evidence that DNR can induce alterations in the level of glutathione and glutathione-dependent enzymes in trisomic fibroblasts as compared to normal cells, which may provide additional protection against daunorubicin-induced oxidative stress in trisomic fibroblasts.     

Induction of replicative senescence biomarkers by sublethal oxidative stresses in normal human fibroblast

We tested the long-term effects of sublethal oxidative stresses on replicative senescence. WI-38 human diploid fibroblasts (HDFs) at early cumulative population doublings (CPDs) were exposed to five stresses with 30 microM tert-butylhydroperoxide (t-BHP). After at least 2 d of recovery, the cells developed biomarkers of replicative senescence: loss of replicative potential, increase in senescence-associated beta-galactosidase activity, overexpression of p21(Waf-1/SDI-1/Cip1), and inability to hyperphosphorylate pRb. The level of mRNAs overexpressed in senescent WI-38 or IMR-90 HDFs increased after five stresses with 30 microM t-BHP or a single stress under 450 microM H(2)O(2). These corresponding genes include fibronectin, osteonectin, alpha1(I)-procollagen, apolipoprotein J, SM22, SS9, and GTP-alpha binding protein. The common 4977 bp mitochondrial DNA deletion was detected in WI-38 HDFs at late CPDs and at early CPDs after t-BHP stresses. In conclusion, sublethal oxidative stresses lead HDFs to a state close to replicative senescence.     

Changes of phospholipase A2 inhibitory activity in the K+-sensitive actin gelation factor during the differentiation of myeloid leukemia cells

The phospholipase A2 inhibitory activity of a 38 kDa K+-sensitive actin gelation factor in a murine leukemia cell line (M1) was examined. A specific antibody against 38 kDa protein was found to cross-react with 37 kDa protein (lipocortin) in rat peritoneal exudates. Although the native 38 kDa protein from M1 cells did not block phospholipase A2 activity, pretreatment with alkaline phosphatase produced a form that did inhibit this enzyme. However, a purified 38 kDa protein from differentiated M1 cells blocked phospholipase A2 activity without pretreatment with alkaline phosphatase. Phospholipase A2 inhibitory activity of the 38 kDa protein was not altered by addition of actin. These findings suggest that the phospholipase A2 inhibitory of our 38 kDa protein was induced during differentiation. We also proposed that our 38 kDa protein has the same epitope as lipocortin.     

Attenuation of NF-kappaB signaling response to UVB light during cellular senescence

The ability of cells to adapt to environmental stresses undergoes a progressive reduction during aging. NF-kappaB-mediated signaling is a major defensive system against various environmental challenges. The aim of this study was to find out whether replicative senescence affects the response of the NF-kappaB signaling pathway to UVB light in human WI-38 and IMR-90 fibroblasts. The exposure of early passage fibroblasts to UVB light inhibited the proliferation and induced a flat phenotype similar to that observed in replicatively senescent fibroblasts not exposed to UVB light. The UVB radiation dose used (153 mJ/cm2) did not induce apoptosis in either early or late passage WI-38 fibroblasts. UVB exposure induced a prominent activation of the NF-kappaB signaling pathway both in early and in late passage WI-38 and IMR-90 fibroblasts. Interestingly, the response to UVB light was significantly attenuated in late passage fibroblasts. This attenuation was most prominent in DNA binding activities of nuclear NF-kappaB complexes. Similar senescence-related attenuation was also observed in the DNA binding activities of nuclear AP-1 and Sp-1 factors after UVB treatment. Immunoblotting and -cytochemistry showed an increase in nuclear localization of p50 and p65 components of NF-kappaB complexes. Supershift experiments showed that the specific NF-kappaB complexes contain p50 and p65 protein components but not p52 and c-Rel proteins. Cytoplasmic IkappaBalpha showed a marked decrease at protein level but an increase in phosphorylation after UVB treatment. Transient transfection assays with TK5-CAT and TK10-CAT plasmids carrying NF-kappaB-responsive sites of the TNFalpha promoter were used to analyze the functional activity of the NF-kappaB complexes. Results showed that UVB exposure induced an increase in NF-kappaB-driven CAT expression both in early and in late passage fibroblasts though the response was significantly stronger in early passage fibroblasts. Our results show that the induction of NF-kappaB-mediated signaling by UVB light is highly attenuated in senescent fibroblasts. This attenuation may reduce the stress resistance during cellular senescence.     

Endothelial Matrix Assembly during Capillary Morphogenesis: Insights from Chimeric TagRFP-Fibronectin Matrix

Biologically relevant, three-dimensional extracellular matrix is an essential component of in vitro vasculogenesis models. WI-38 fibroblasts assemble a 3D matrix that induces endothelial tubulogenesis, but this model is challenged by fibroblast senescence and the inability to distinguish endothelial cell-derived matrix from matrix made by WI-38 fibroblasts. Matrices produced by hTERT-immortalized WI-38 recapitulated those produced by wild type fibroblasts. ECM fibrils were heavily populated by tenascin-C, fibronectin, and type VI collagen. Nearly half of the total type I collagen, but only a small fraction of the type IV collagen, were incorporated into ECM. Stable hTERT-WI-38 transfectants expressing TagRFP-fibronectin incorporated TagRFP into ~90% of the fibronectin in 3D matrices. TagRFP-fibronectin colocalized with tenascin-C and with type I collagen in a pattern that was similar to that seen in matrices from wild type WI-38. Human Umbilical Vein Endothelial Cells (HUVEC) formed 3D adhesions and tubes on WI38-hTERT-TagRFP-FN-derived matrices, and the TagRFP-fibronectin component of this new 3D human fibroblast matrix model facilitated the demonstration of concentrated membrane type 1 metalloprotease and new HUVEC FN and collagen type IV fibrils during EC tubulogenesis. These findings indicate that WI-38-hTERT- and WI-38-hTERT-TagRFP-FN-derived matrices provide platforms for the definition of new matrix assembly and remodeling events during vasculogenesis.