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.     

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.     

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.     

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.     

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)     

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).     

Selenium is an essential trace nutrient for growth of WI-38 diploid human fibroblasts.

The trace element selenium is essential for clonal growth of diploid fibroblasts from human fetal lung (WI-38) in media containing small amounts of serum protein. Maximum growth stimulation is obtained when 30 nM neutralized selenious acid is added to a synthetic medium containing 1.5 mg/ml of dialyzed fetal bovine serum protein (equivalent to a 3% serum concentration). Serum appears to be a source of selenium in most culture media, since higher concentrations of serum protein or whole serum mask the selenium requirement of WI-38 cells. Selenium is also required by a Chinese hamster cell line that can be grown in a protein-free synthetic culture medium.     

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.     

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。     

Oxidative damage to mitochondria and aging

Oxidative damage has been implicated to be a major factor in the decline in physiologic function that occurs during the aging process. Because mitochondria are a primary site of generation of reactive oxygen species, they have become a major focus of research in this area. Increased oxidative damage to mitochondrial proteins, lipid and DNA has been reported to occur with age in several tissues in a variety of organisms. Decreased activity of electron transport chain complexes and increased release of reactive oxygen species from the mitochondria with age suggest that alterations in mitochondrial function occur with age as a consequence of increased oxidative damage. In addition, age-related alterations in the mitochondrial pathway of apoptosis, which could have profound affects on the physiological function of a tissue, could arise from oxidative damage to mitochondria. Alterations in mitochondrial turnover with age could also contribute to an increase in the number of dysfunctional mitochondria with age.     

Proteasome inhibition increases HuR level, restores heat-inducible HSP72 expression and thermotolerance in WI-38 senescent human fibroblasts

At the end of their replicative potential in vitro, late passage WI-38 human diploid fibroblasts (HDF) have a low basal expression of heat shock protein 72 (HSP72) and an attenuated ability to induce it in response to heat shock. The transient exposure to the specific and reversible proteasome inhibitor MG132 during a mild heat shock induced late passage HDF to synthesize and accumulate high levels of HSP72. This HSP72 expression was long-lasting and appeared to result from both increased cytoplasmic levels and enhanced translation of HSP72 mRNA. The level of HuR, a stabilizing mRNA-binding protein, increased following the MG132 treatment. This result is consistent with the proposed role of HuR in assisting mRNA export to the cytoplasm and in antagonizing its degradation. Furthermore, the previous exposure of late passage HDF to a mild heat shock in the presence of MG132 protected these cells against the otherwise lethal effect of a subsequent severe heat shock. This acquisition of thermotolerance appeared to be correlated with the level of HSP72.     

CD38 as a prognostic marker in CLL

Cell-surface expression of CD38 in CLL has been recognised recently as a marker of progressive disease and poor outcome. In contrast to traditional staging systems, CD38 is able to identify progressive cases at an early stage. Measurement of CD38, in conjunction with other novel prognostic factors such as p53 and ZAP-70 helps to identify patients who might benefit from early and more intensive therapy. In addition, CD38 positivity can predict unmutated IgVH gene mutation status in most cases. These features, together with its easy applicability, render CD38 a valuable tool in the routine diagnostics of CLL. Questions remaining to be clarified about CD38 include the incidence and significance of its variations during the course of the disease, the optimal method to define CD38 positivity and the impact of different methodologies on results. Only after these issues are resolved can the definitive place of CD38 be defined in the diagnostics of CLL.     

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).     

CD38 as a prognostic marker in CLL

Cell-surface expression of CD38 in CLL has been recognised recently as a marker of progressive disease and poor outcome. In contrast to traditional staging systems, CD38 is able to identify progressive cases at an early stage. Measurement of CD38, in conjunction with other novel prognostic factors such as p53 and ZAP-70 helps to identify patients who might benefit from early and more intensive therapy. In addition, CD38 positivity can predict unmutated IgVH gene mutation status in most cases. These features, together with its easy applicability, render CD38 a valuable tool in the routine diagnostics of CLL. Questions remaining to be clarified about CD38 include the incidence and significance of its variations during the course of the disease, the optimal method to define CD38 positivity and the impact of different methodologies on results. Only after these issues are resolved can the definitive place of CD38 be defined in the diagnostics of CLL.     

May ultraviolet light-induced ornithine decarboxylase response in mitotic and postmitotic human skin fibroblasts serve as a marker of aging and differentiation?

Since Hayflick’s pioneering work in the early sixties, human diploid fibroblasts have become a widely accepted in vitro model system for gerontological research. Most recently, Bayreuther and co-workers extended this experimental approach showing that fibroblasts in culture resemble in their design the hemopoietic stem-cell differentiation system. Using this model, we have found in human skin fibroblasts following mitomycin-C (MMC) treatment characteristic morphological changes of the fibroblasts and specific shifts in the [³⁵S]methionine polypeptide pattern of the total cellular proteins which support the notion that MMC accelerates the differentiation pathway from mitotic (MF) to postmitotic fibroblasts (PMF). Ornithine decarboxylase (ODC) can be activated by ultraviolet light (UV) and is involved in the synthesis of polyamines which play a role in the regulation of DNA synthesis and cell proliferation. Therefore, ODC may participate in the modulation of gene expression. ODC may even serve as a biochemical marker of the mutagenic and carcinogenic effects of ultraviolet light; therefore, we tested this interesting enzyme in the fibroblast differentiation system. Indeed, we were able to show that UV-induced ODC response is significantly reduced in the MMC-induced postmitotic stage of fibroblasts originally derived from a 9-year-old female. We compared this finding with previous results from our laboratory, where we have demonstrated that ODC in human skin fibroblasts from younger donors can be significantly more stimulated by UV compared to the enzyme activities in fibroblasts from older donors. We conclude that UV-induced ODC response may serve as a marker of differentiation and aging. Our results also imply that the fibroblast differentiation system is a very useful tool to unravel the complex mechanisms of skin aging.     

Oxidative damage and mitochondrial decay in aging.

We argue for the critical role of oxidative damage in causing the mitochondrial dysfunction of aging. Oxidants generated by mitochondria appear to be the major source of the oxidative lesions that accumulate with age. Several mitochondrial functions decline with age. The contributing factors include the intrinsic rate of proton leakage across the inner mitochondrial membrane (a correlate of oxidant formation), decreased membrane fluidity, and decreased levels and function of cardiolipin, which supports the function of many of the proteins of the inner mitochondrial membrane. Acetyl-L-carnitine, a high-energy mitochondrial substrate, appears to reverse many age-associated deficits in cellular function, in part by increasing cellular ATP production. Such evidence supports the suggestion that age-associated accumulation of mitochondrial deficits due to oxidative damage is likely to be a major contributor to cellular, tissue, and organismal aging.     

The surface membrane proteins of the WI-38 fibroblast in relation to transformation and aging

The urea extractable cell surface proteins of WI-38 human embryonic fibroblasts and SV-40 transformed WI-38 fibroblasts were examined. Electrophoretic analysis on 5.6% SDS polyacrylamide gels revealed 10–15 discrete protein bands. Three of these bands, of molecular weight 50,000, 45,000 and 30,000 daltons, were found to be present in increased amounts in transformed fibroblasts. The expression of these proteins decreased with increasing culture age. Thus a direct relationship exists between the expression of these proteins and the proliferative state of the culture.