Effect of procaine on cultivated human WI-38 fibroblasts

Procaine is a local anesthetic, also used in experimental gerontology and has been tested in cultivated human WI-38 fibroblasts. This molecule was found to enhance growth rate and cell densities in actively dividing cultures. As the cells aged, however, this stimulatory effect diminished and finally vanished. In a long term experiment the enhancement of growth of procaine treated cultures was finally replaced by a toxic effect even at low concentration. The amount of the thermolabile enzyme found in phase III cells did not change when procaine was added to the culture medium. In this cellular aging model, procaine behaved like a metabolic stimulator of actively dividing cells but not as an "antiaging" molecule as it is sometimes assumed.     

Release of somatomedin-like activity by cultured WI-38 human fibroblasts

Confluent cultures of normal diploid WI-38 human embryonic lung fibroblasts released somatomedin (SM)-like activity into their incubation medium during culture in serum-free medium. This postculture medium (conditioned medium) stimulated cell division in these same cultured WI-38 fibroblasts and 35SO4 uptake by hypophysectomized rat cartilage in vitro. The conditioned medium also contained immunoreactive SM (IRSM) activity which yielded parallel dose-response curves to human serum in a RIA for SM. The IRSM activity measured in conditioned medium was not the artifactual result of effects of possible SM-binding proteins or proteolytic enzymes in conditioned medium. These studies suggest that cultured WI-38 fibroblasts produce and release SM-like activity which has SM-like biological activity and is immunoreactive with a basic SM purified from human plasma Cohn fraction and having similarity with SM-C and insulin-like growth factor-I. Human GH appears to stimulate production and release of IRSM activity by these cells.     

Effect of cortisol on glycosaminoglycan synthesis and growth of diploid, human fibroblasts (WI-38) in relation to in vitro aging

Addition of cortisol (hydrocortisone; 1.4 X 10(-7) M) to the culture medium (BME with non-essential amino acids and 10% fetal calf serum) of human fibroblasts resulted in increased proliferative activity--in regard to cell number and incorporation rates of [3H]-thymidine into DNA and [14C]-leucine into protein--and in an increased saturation density. Glycosaminoglycan (GAG) synthesis rates (as measured by incorporation of [14C]-glucosamine or [35S]-sulfate) of the various GAG types secreted into the culture medium (hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate) were evenly inhibited by ca. 25% in the case of cortisol-treated Phase-II cultures. The dose-effect relationship revealed a median effective cortisol dose of ca. 0.01 microM for Phase-II cultures. Phase-III ("senescent") cultures revealed an elevated sensitivity as well in regard to cortisol concentration as to the extent of the inhibitory effect. Contrary to the medium GAGs, the pattern of the cell-bound GAGs was changed by cortisol (1.4 X 10(-7) M), with an increase of hyaluronic acid synthesis and a decrease of the sulfated GAGs. This cell-bound hyaluronic acid was predominantly removable by trypsin-treatment and therefore regarded to be localized at the cell surface or pericellularly. Also, following long-term cultivation (ca. 15 population-doublings) in the presence of cortisol, the synthesis of cell-bound hyaluronic acid was stimulated. Since cellular hyaluronic acid decreases during senescence of WI-38 cultures (Sluke et al., 1981), the cortisol effect in regard to GAG synthesis is in line with a "counter-aging" influence.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA synthesis in the human diploid cell strain WI-38 during in vitro aging: an autoradiography study.

The percent of cells in a WI-38 cell population which did not incorporate tritiated thymidine (3H-TdR) was determined by autoradiography from the measurement of percent nonlabeled nuclei and the ratio of total cell numbers at the initiation and at the termination of exposure to 3H-TdR. This percentage was minimally affected by factors influencing the proliferation of labeled cells, but was dependent on the population doubling level (PDL). The results suggest the presence, in a proliferating WI-38 population, of subpopulation(s) with an extremely slow rate of S phase entrance. The parameter was useful in estimating, empirically, the doubling potential of a cell population.     

Long-term treatment with cortisol: influence on proliferation, aging and glycosaminoglycan synthesis of cultured human diploid fibroblasts (WI-38)

Long-term treatment (several weeks and months) of cultured human diploid fibroblasts (WI-38) with cortisol (1.4 x 10(-7) M) stimulated proliferative activity and cellular glycosaminoglycan synthesis, thus counteracting the normal in vitro aging process. Characterization of the individual glycosaminoglycan types revealed an increased portion of cellular hyaluronic acid in cells treated with cortisol. Elevated synthesis of total glycosaminoglycans and, especially, of hyaluronic acid was found in the percellular pool (as determined by the amount liberated from the cells by trypsin treatment).     

Oxidative DNA damage as a marker of aging in WI-38 human fibroblasts

Cause-effect relationships between oxidative stress, DNA damage and aging were investigated in WI-38 human diploid fibroblasts at 21, 41 or 58 population doublings (PDs), corresponding to young, middle age or old fibroblasts, respectively. Oxidative DNA damage was evaluated by immunohistochemical detection of 8-hydroxy-2'deoxyguanosine (8-OHdG) adducts or by single cell microgel electrophoresis (COMET assay). Aging was evaluated by growth rate, senescence-associated-beta-galactosidase (SA-beta galactosidase) activity, cell cycle distribution, and expression of p21. Our results demonstrate that (i) oxidative DNA damage is proportional to the age of cells (ii) DNA damage in old/58 PDs cells reflects both an increased susceptibility to oxidative stress, induced by acute exposure to sub-lethal concentrations of hydrogen peroxide (H(2)O(2)), and a reduced efficiency of repair mechanisms. We also show that mild chronic oxidative stress, induced by prolonged exposure to 5 microM H(2)O(2), accelerates aging in fibroblasts. In fact, this treatment increased 8-OHdG levels, SA-beta-galactosidase activity, and G0/G1 cell cycle arrest in middle age/41 PDs, making them similar to H(2)O(2)-untreated old/58 PDs cells. Although other mechanisms may concur in mediating the effects of H(2)O(2), these results lend support to the concept that oxidative stress may be a key determinant of aging. Measurements of oxidative DNA damage might therefore be exploited as reliable marker of cellular aging.     

Retrovirally mediated overexpression of peroxiredoxin VI increases the survival of WI-38 human diploid fibroblasts exposed to cytotoxic doses of tert-butylhydroperoxide and UVB

In this work, stable overexpression of peroxiredoxin VI was generated in WI-38 human diploid fibroblasts using a retrovirus-mediated transfection system. Estimation of cell survival showed that peroxiredoxin VI provides a significant protection against tert-butylhydroperoxide- or UVB-caused cytotoxicity. No protection was found against ethanol- or H₂O₂-caused cytotoxicity. These effects are correlated with the known functions of Prx VI. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ginsenoside Rg1 delays tert-butyl hydroperoxide-induced premature senescence in human WI-38 diploid fibroblast cells

Tert-butyl hydroperoxide (t-BHP), an analog of hydroperoxide, induced characteristic changes of senescence in human diploid fibroblasts WI-38 cells. It was reported that ginsenoside Rg1, an active ingredient of ginseng, ameliorated learning deficits in aged rats. The present study was aimed to investigate whether ginsenoside Rg1 can delay the premature senescence of WI-38 cells induced by t-BHP and to explore the underlying molecular mechanisms. First, Rg1 pretreatment markedly reversed senescent morphological changes in WI-38 cells induced by t-BHP. Second, t-BHP treatment alone resulted in an increase in the protein levels of P16 and P21, and a decline in intracellular adenosine 5'-triphosphate (ATP) level and mitochondrial complex IV activity. Ginsenoside Rg1 pretreatment had significant effects of attenuating these changes. These data indicate that ginsenoside Rg1 has an anti-aging effect on t-BHP-induced premature senescence in WI-38 cells. This effect may be mediated by regulating cell cycle proteins and enhancing mitochondrial functioning.     

Expression of cell cycle-dependent genes in young and senescent WI-38 fibroblasts.

We studied the expression of 11 cell cycle-dependent genes in senescent WI-38 fibroblasts and compared the results to those obtained in WI-38 cells from early passages (young cells). Every gene we examined is expressed in the senescent cells at levels similar to those in the young cells, including two genes maximally expressed at the G1/S phase boundary--genes for thymidine kinase and histone H3. The results clearly show that senescent, noncycling WI-38 cells are not similar to quiescent cells. Rather, such senescent WI-38 cells may be blocked just prior to the onset of DNA synthesis.     

Changes of glycosaminoglycan synthesis during in vitro ageing of human fibroblasts (WI-38).

Synthesis rates of glycosaminoglycans by WI-38 cultures (diploid, human fibroblasts exhibiting a limited number of population doublings in vitro) were determined by incorporation of 35S-sulfate of 14C-glucosamine into cellular and extracellular glycosaminoglycans at different passage levels before phase out. A progressive decline in the synthesis of cellular and extracellular glycosaminoglycans occured during the last (about 4) population doublings. 35S-sulfate incorporation into extracellular glycosaminoglycans appeared to be somewhat more reduced than 14-C-glucosamine incorporation during the last passages. Analysis of the distribution pattern of incorporated label into various glycosaminoglycan types (hyaluronic acid, chondroitin sulfate, dermatan sulfate and possibly heparan sulfate) revealed an age-related relatively stonger decline of 14C-glucosamine incorporation into cellular and extracellular hyaluronic acid and of 35S-sulfate into extracellular chondroitin sulfate in comparison with the other glycosaminoglycan types. Addition of exogenous glycosaminoglycans (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, hyaluronic acid, heparan sulfate, heparin) at 100 microgram/ml to the culture media during the last 7 to 10 population doublings before phase out did not increase the total number of population-doublings. Heparin exhibited a significant growth inhibitory effect at 100 or 500 microgram/ml. The changes in glycosaminoglycan metabolism are interpreted as an expression of cellular ageing, and such an in vitro system offers a model for analyzing the factors involved in or causing the induction respectively prevention of this functional change.     

Zinc is an essential trace element for spermatogenesis

Zinc (Zn) plays important roles in various biological activities but there is little available information regarding its functions in spermatogenesis. In our current study, we further examined the role of Zn during spermatogenesis in the Japanese eel (Anguilla japonica). Human CG (hCG) was injected into the animals to induce spermatogenesis, after which the concentration of Zn in the testis increased in tandem with the progression of spermatogenesis. Staining of testicular cells with a Zn-specific fluorescent probe revealed that Zn accumulates in germ cells, particularly in the mitochondria of spermatogonia and spermatozoa. Using an in vitro testicular organ culture system for the Japanese eel, production of a Zn deficiency by chelation with N,N,N′,N′-tetrakis (2-pyridylemethyl)ethylenediamine (TPEN) caused apoptosis of the germ cells. However, this cell death was rescued by the addition of Zn to the cultures. Furthermore, an induced deficiency of Zn by TPEN chelation was found to inhibit the germ cell proliferation induced by 11-ketotestosterone (KT), a fish specific androgen, 17α,20β-dihydroxy-4-pregnen-3-one (DHP), the initiator of meiosis in fish, and estradiol-17β (E2), an inducer of spermatogonial stem-cell renewal. We also investigated the effects of Zn deficiency on sperm motility and observed that TPEN treatment of eel sperm suppressed the rate and duration of their motility but that co-treatment with Zn blocked the effects of TPEN. Our present results thus suggest that Zn is an essential trace element for the maintenance of germ cells, the progression spermatogenesis, and the regulation of sperm motility.     

Long-term growth of diploid human fibroblasts in low serum media

Hayflick and Moorhead demonstrated that diploid human fibroblasts have a limited life span when grown in media containing 10% bovine calf sera. Recent experiments have suggested that antigrowth factors in serum may be a potential contributor to the limited proliferative capacity of normal diploid cells. To reduce the concentration of inhibitory serum factors 10-fold, MRC-5 diploid fibroblasts were cultured in media with only 1% serum. Long-term culture in 1% serum requires the addition of purified growth factors to sustain proliferation. Although there are dramatic changes in cell morphology, we find that the long-term division potential of MRC-5 cells cultured in media containing 1% serum and growth factors differs little from that found with cells cultured in 10% serum. In contrast, MRC-5 cells cultured in 10% serum and added growth factors have a somewhat extended life span. These results suggest that negative growth factors are not responsible for the limited proliferative capacity of in vitro cultured human fibroblasts. Moreover, the evidence that human fibroblasts can undergo major changes in cell morphology and still retain a normal life span raises questions about the validity of using morphological changes as indicators of cellular senescence.     

Effects of oxygen,growth state,and senescence on the antioxidant responses of WI-38 fibroblasts

Mitotically active, growth-arrested cells and proliferatively senescent cultures of human fetal lung fibroblasts (WI-38) were exposed to six different oxygen tensions for various lengths of time and then analyzed to determine the responses of their antioxidant defense system. Glutathione (GSH) concentration increased as a function of ambient oxygen tension in early passage cultures; the effect was larger in exponentially growing cultures than in those in a state of contact-inhibited growth arrest, but was absent in senescent cells. Conversely, the activity of glutathione disulfide reductase was greater in growth-arrested cultures than in mitotically active cells irrespective of oxygen tension. Glucose-6-phosphate dehydrogenase was lowest in log-phase cells exposed to different oxygen tensions for 24 h and in senescent cells. Both hypoxia and hyperoxia depressed selenium-dependent glutathione peroxidase activity in early passage cultures, while the activity of the enzyme progressively declined with increasing oxygen in senescent cells. The GSH S-transferase activity was unresponsive to changes in ambient oxygen tension in either young or senescent cultures. Manganese-containing superoxide dismutase (MnSOD) activity was unaffected by oxygen tension, but was elevated in young confluent cultures as compared with cultures in log-phase growth. MnSOD activity was significantly higher in senescent cultures than in early passage cultures and was also responsive to increased oxygen tension in senescent cultures. Copper–zinc-containing superoxide dismutases activity was not affected by oxygen tension or the passage of time, but it declined in senescent cultures.     

人高亲和力钠依赖二羧酸转运蛋白3对人成纤维细胞衰老进程的影响

目的 探讨人高亲和力钠依赖二羧酸转运蛋白3(hNaDC3)在人胚肺二倍体成纤维细胞(WI38)衰老中的作用。方法 通过构建逆转录病毒载体．使用含有hNaDC3基因的逆转录病毒液将hNaDC3基因导入WI-38中．并获得表达．观察其对WI-38细胞衰老的影响。结果 与对照细胞相比，hNaDC3基因导入后．WI-38细胞传代数减少10～13代，生长速率降低40％．细胞周期阻滞于G期，细胞形态呈衰老细胞样变化．衰老相关-β-半乳糖苷酶(SA-β-gal)染色阳性率上升-线粒体膜电位下降。结论 hNaDC3可能促进人WI-38衰老。     

Alteration of the microtubule organization in aging WI-38 fibroblasts. A comparative study with embryonic hamster lung fibroblasts

The microtubule organization in human WI-38 fibroblasts subcultivated in vitro has been investigated using nocodazole, a reversible inhibitor of the microtubules. Two phenotypes were observed. The typical fibroblast cells, called Type 1 cells, showed, after nocodazole treatment, a centripetal depolymerization wave of the microtubules and the giant Type 2 cells which have a more heterogeneous behaviour. Some of the cells clearly showed a centrifugal depolymerization of the microtubules, others a mixed behavior and less than 1% displayed the same behavior as the Type 1 cells. Confirming previous data obtained with Hamster fibroblasts (Raes et al., 1983, 1984), these results suggest a modification in the microtubule organization which could account for the aberrant division of some WI-38 cells in aged cultures. The relevance of this observation for the emergence of the morphologically different Type 2 cells and for cell division impairment in serially in vitro cultivated cells is discussed.     

Energetic stress induces premature aging of diploid human fibroblasts (Wi-38) in vitro

Oxidative phosphorylation is the main endogenous source for the generation of reactive oxygen species (ROS). In order to investigate the influence of enhanced ROS production on the in vitro senescence of Wi-38 fibroblasts, cells were cultivated in medium with elevated (hypertonic) NaCl concentrations. The number of active Na(+)/K(+)-ATPase molecules per cell was found to be increased. A rise in both respiration and glycolysis as evidenced by the increases in oxygen and glucose consumption and lactate production was revealed. Cells stayed alive in medium with NaCl concentrations of up to 0.30 M and could be adapted to growth under these hypertonic conditions (high-NaCl tolerant cells). These cells exhibited an increased cell size and protein content. A growing number of cells showed stress fibers and granulation. The proliferation rate and the maximum number of cumulative population doublings of these high-NaCl tolerant cultures were reduced and saturation density was decreased. Thus, these cells under energetic stress due to increased energy requirements for active ion transport expressed features typical for aging in vitro. We conclude therefore that energetic stress induces premature aging in human diploid fibroblasts.     

抑制细胞间通讯功能可加速人成纤维细胞的衰老

目的研究间隙连接蛋白connexin43在人成纤维细胞衰老过程中的调节作用。方法设计并合成针对人connexin43的双链RNA(siRNA),抑制人二倍体成纤维细胞WI-38细胞的connexin43表达和细胞间通讯功能,观察对其衰老相关β-半乳糖苷酶(SA-β-gal)染色阳性率、细胞增殖能力、细胞周期调节蛋白P27、P21表达水平等衰老表型的影响。结果我们合成的siRNA-cx43可高效、特异的抑制connexin43 mRNA和蛋白质表达及细胞间通讯功能。转染WI-38细胞后,细胞增殖能力降低、细胞SA-β-gal染色阳性率(75.32±5.17)较对照组(32.48±3.94)和转染siRNA-con组(37.81±4.1 2)细胞升高(P<0.05),P27、P21表达增加,转染siRNA-cx43的WI-38细胞增殖能力显著降低,细胞变扁平、肥大,细胞SA-β-gal染色阳性率达100%,细胞提前出现衰老。结论间隙连接蛋白connexin43对WI-38细胞的衰老进程有重要的调节作用,其表达减少和细胞间通讯功能降低,可以促进WI-38细胞衰老进程。     

Corrugated attachment membrane in WI-38 fibroblasts: alternating fibronectin fibers and actin-containing focal contacts.

The distributions of both fibronectin (LETS, CSP) fibers and focal contacts to the substratum, as viewed by fluorescence and reflection contrast microscopy, respectively, have been compared in freshly plated WI-38 human fibroblasts. Most frequently, the actual focal attachment plaques did not contain fibronectin fluorescence and, furthermore, fibronectin spots and fibers often alternated with focal contacts. Overlap, however, was observed between focal contacts and the endings of actin-containing stress fibers [see also Wehland, J., Osborn, M. & Weber, K. (1979) J. Cell Sci. 37, 257-273]. Thus, the fibroblast attachment membrane might best be described as a corrugated sheet that undulates between alternating microfilaments and fibronectin fibers, at the points of closest and farthest distance to the substratum, respectively.     

Increase of cellular hyaluronic acid synthesis following continuous exposure of cultured human fibroblasts (WI-38) to hydrocortisone

Continuous (long-term) exposure of cultured normal (diploid) human fibroblasts (WI-38) to hydrocortisone (1.4 X 10(-7) M) resulted, as originally described by Macieira-Coelho (1966) and Cristofalo (1970), in a stimulation of proliferative activity and an increase of population doublings. Stimulation of DNA-synthesis by hydrocortisone, as measured by 3H-thymidine incorporation, required the presence of serum in the culture medium. Analysis of the cellular glycosaminoglycan (GAG) pattern, as measured by 14C-glucosamine incorporation into the various GAG types (hyaluronic acid, heparan sulfate, chondroitin sulfate, dermatan sulfate) revealed a significant increase of cell-bound hyaluronic acid (it appears to be predominantly located at the cell surface or pericellular, since it is removable to a large extent by trypsin treatment), while the distribution pattern of sulfated GAGs did not exhibit a significant change. This increase of cellular hyaluronic acid synthesis was regarded largely as an adaptive response to hydrocortisone, since its removal from the culture medium of hydrocortisone pretreated cultures resulted in a significant decrease of cellular hyaluronic acid. Possible functions of cell-bound hyaluronic acid were suggested in regard to cell surface properties (cell-cell and cell-substratum adhesion; migratory activity). Thus, decreased adhesiveness (by elevated cellular hyaluronic acid) might be a decisive factor for the well-known increase in cell saturation density caused by hydrocortisone. Generally, the present findings support a concept (Sluke et al., 1981; Schachtschabel and Sluke, 1984) that an increase of cellular hyaluronic acid synthesis is "growth-favorable", which is in line with previous findings of a decrease of hyaluronic acid synthesis by growth restriction in the course of in vitro ageing (Sluke et al., 1981).