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Aleksandra Korcz Dianne Robert Soprano Kenneth J. Soprano 《Journal of cellular biochemistry》1995,59(1):42-52
We have used the WI-38 cell long-term quiescent model system to study the regulation of cell cycle progression at the molecular level. By modulating the length of time that WI-38 cells are density arrested, it is possible to proportionately alter the length of the prereplicative or G-1 phase which the cell traverses after growth factor stimulation in preparation for entry into DNA synthesis. Stimulation of long-and short-term density arrested WI-38 cells with different growth factors or higher concentrations of individual growth factors does not alter the time required by long-term cells to enter S after stimulation. However, the time during the prereplicative period for which these growth factors are needed is different. Long-term quiescent WI-38 cells require EGF to traverse the G-0/G-1 border but do not need and apparently cannot respond to IGF-1 during the first 10 h after EGF stimulation, the length of the prolongation of the prereplicative phase. This suggests that EGF stimulation of long-term quiescent WI-38 cells initiates a series of molecular events which make these cells “competent” to respond to the “progression” growth factor, IGF-1. In light of the well-established role of protein tyrosine kinases in signal transduction, we set out to identify, clone, and analyze the expression of receptor and non-receptor tyrosine kinases which potentially could play a role during the prolongation of the prereplicative phase in making the long-term quiescent WI-38 cells competent to respond to IGF-1. We obtained 49 clones representing 11 different receptor and non-receptor type protein tyrosine kinases. Analysis of expression of these clones revealed a variety of different patterns of expression. However, the most striking pattern was exhibited by IGF-1 receptor. Our results suggest that induction of IGF-1 receptor mRNA by EGF may be an important event in the establishment of competence by EGF in long-term density arrested WI-38 cells. © 1995 Wiley-Liss, Inc. 相似文献
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Critical adjustment of cysteine and glutamine concentrations for improved clonal growth of WI-38 cells 总被引:2,自引:0,他引:2
Dr. Richard G. Ham Susan L. Hammond Linda L. Miller 《In vitro cellular & developmental biology. Plant》1977,13(1):1-10
Summary Clonal growth of WI-38 cells with a plating efficiency of 45% has been achieved in a synthetic nutrient mixture (MCDB 102)
supplemented with either whole or dialyzed fetal bovine serum. For optimum growth, the concentration of cysteine in the medium
must be adjusted precisely. Deviation by a factor of three in either direction from the optimum concentration (9.0×10−5M) eliminates essentially all clonal growth. A high concentration of glutamine (2.5×10−3M) is also needed for, optimum clonal growth.
Presented in preliminary form at the 26th Annual Meeting of the Tissue Culture Association, June 4, 1975.
This work was supported by Grant No. HD-08181 from the National Institute of Child Health and Human Developement, Grant No.
AG-00310 from the National Institute on Aging, and by Contract No. 223-74-1156 from the Bureau of Biologics, Food and Drug
Administration. 相似文献
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Laroy N. Castor 《Journal of cellular physiology》1977,92(3):457-467
The overall rates of protein synthesis and degradation in perfusion-grown WI-38 cells were followed in the three days after a stepdown in the serum concentration of the culture medium, from 10% to 0.3%. Within three hours after the stepdown, the rate of protein synthesis had decreased and the rate of protein degradation had increased, the combined result being the cessation of protein accumulation. The degradation rate returned over the next three days to its original value, but a zero rate of accumulation was retained because the synthesis rate continued to decline. The rate of DNA synthesis remained constant for six hours after the stepdown. It then declined steadily until reaching a minimum about eight hours later. The results show that extracellular control of protein accumulation depends on adjustments in both protein synthesis and protein degradation, and that the adjustments take place rapidly. This behavior suggests that the cell cycle is arrested after a stepdown because post-mitotic cells are unable to accumulate additional protein. However, an alternative interpretation of the data is that at least part of the changed accumulation is the result, rather than the cause, of the cycle arrest, and that the arrest is caused by other, more specific, reactions than those of general protein metabolism. 相似文献
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Anne M. Donigan R. Christopher Cavalli Angel A. Pena C. Richard Savage Dianne Robert Soprano Kenneth J. Soprano 《Journal of cellular physiology》1993,155(1):164-170
WI-38 cells, density arrested for short periods of time, can be stimulated to re-enter the cell cycle by epidermal growth factor (EGF) alone. However, cells density arrested for longer periods have a prolonged prereplicative phase when serum stimulated and cannot be stimulated by EGF alone. Radio-ligand binding studies performed on WI-38 cells showed that actively growing cells bind [125I]EGF at relatively low levels that increase to a maximum as the cells become contact inhibited. As the cells enter a state of deeper quiescence, EGF binding falls to one-third to one-fifth the short-term growth arrested levels, remaining constant thereafter. The EGF-receptor complexes internalize more slowly in long-term growth arrested cells, and the rate of ligand association to the receptor is lower than short-term growth arrested cells. The amount of EGF receptor protein in lysates of equal numbers of both short- and long-term quiescent cells remains the same. These results suggest that the failure of long-term growth arrested cells to respond to EGF is not due to dramatic changes in the amount of receptor protein during prolonged quiescence but more likely to an alteration in the ability of these receptors to bind ligand and/or activate the EGF signal transduction pathway. © 1993 Wiley-Liss, Inc. 相似文献
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Background
Normal cells possess a limited proliferative life span after which they enter a state of irreversible growth arrest. This process, known as replicative senescence, is accompanied by changes in gene expression that give rise to a variety of senescence-associated phenotypes. It has been suggested that these gene expression changes result in part from alterations in the histone acetylation machinery. Here we examine the influence of HDAC inhibitors on the expression of senescent markers in pre- and post-senescent WI-38 cells. 相似文献8.
Stifanese R Averna M De Tullio R Salamino F Cantoni C Mingari MC Prato C Pontremoli S Melloni E 《Archives of biochemistry and biophysics》2008,479(2):145-152
In dividing cells calpastatin diffuses from aggregates into cytosol, indicating the requirement for a tight regulation of calpain. Accordingly, the involvement of the calpain-calpastatin system in cell proliferation and in the density-dependent growth arrest was studied in JA3 cells stably transfected with a calpastatin form permanently localized in cytosol.In calpastatin overexpressing cells, cell cycle rate is 50% reduced, and cells enter the ungrowing, still fully reversible, stage at a 3-fold higher cell density. Furthermore, in cell density growth arrest phase, down regulation of α- and θ-PKC isoforms, as well as FAK and talin occurs. In calpastatin overexpressing cells, degradation of these calpain substrate proteins is prevented and delayed. Thus, calpain activity plays a crucial role in inducing the cell entry into a functional quiescent phase. 相似文献
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Repair of x-ray damage in aging WI-38 cells 总被引:2,自引:0,他引:2
Rate of strand rejoining and the ability to perform repair replication were determined in young ad old X-irradiated WI-38 cells. No differences in either process were apparent and we conclude that reduced efficiency in one or both of them is not responsible for in vitro aging of human cells. 相似文献
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Replicative potentials of various fusion products between WI-38 and SV40 transformed WI-38 cells and their components 总被引:3,自引:0,他引:3
Hybrid cells derived from whole-cell fusions of replicating phase-II normal fibroblast cells (WI-38s) with SV40 transformed WI-38 fibroblast cells (CL-1s) demonstrated that the majority of the hybrid experimental cells still maintained a finite life-span. Approximately 2% demonstrated sustained and possibly indefinite replication. Experimental binucleate cells and subsequent hybrid synkaryons were also formed by fusing CL-1 karyoplasts into phase-II WI-38 replicating normal fibroblasts. In addition, viable cells were constructed from WI-38 fibroblast cytoplasts with CL-1 karyoplasts. Sustained replication was not observed in these crosses. 相似文献
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c-fos reduces growth factor requirements for mitogenic stimulation of L6 rat myoblasts 总被引:1,自引:0,他引:1
Addition of fetal calf serum (FCS) to serum-deprived L6J1 rat myoblasts increases fos-like immunoreactivity. The nuclear immunoreactivity reached a maximum 2 h after serum addition. Effects of the c-fos protein on myoblast proliferation were analyzed in L6J1 rat myoblasts transfected with the murine c-fos gene under control of a metallothionein promoter. L6J1 myoblasts with elevated expression of transfected c-fos reached higher cell densities than neo transfected control myoblasts when approaching a stationary phase in normal culture conditions (5% FCS). The differences in cell densities were even more pronounced at low serum concentrations (0.5% FCS). c-fos transfected cells also had a faster growth rate than did control cells in serum-free medium supplemented with calcium chloride, lithium chloride, sodium selenite, hydrocortisone, and insulin. The cell morphology of c-fos transfected L6J1 myoblasts was not affected compared to control myoblasts. These results suggest that c-fos protein expression in L6J1 myoblasts is activated by serum and that mitogenic stimulation of L6J1 myoblasts is facilitated by the presence of elevated amounts of c-fos protein. 相似文献
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Frank C. Praeger Vincent J. Cristofalo 《In vitro cellular & developmental biology. Plant》1986,22(6):355-359
Summary The growth of WI-38 cells in serum-free growth medium with and without hormone supplementation in the presence of elevated
Ca2+ concentrations was investigated. At 5 mM CaCl2, WI-38 cells seeded at low density without serum or hormone supplementation showed up to a 12-fold increased in cell number
at saturation density over that obtained at day 1. Saturation densities were comparable when either 5 mM CaCl2 or epidermal growth factor (1 mM CaCl2) was used in the presence of insulin, dexamethasone and transferrin. Combining suboptimal doses of epidermal growth factor
and CaCl2 resulted in an additive effect on saturation density. Thus, nornal human diploid cells are capable of substantial growth
in serum-free, hormone-free growth medium. In contrast, confluent cultures refed with the same medium are not responsive to
elevated Ca2+ concentrations. In fact, elevated Ca2+ concentrations inhibited the proliferative response of confluent cultures to epidermal growth factor, but enhanced their
response to the combined treatment of insulin, transferrin and dexamethasone.
This work was supported by the United States Public Health Society grants T-32, CA09171 and AG-00378.
Editor's Statement This paper rigorously dissects the interplay among external Ca2+ concentration, cell density and specific growth factors on fibroblast growth in defined medium. Wallace L. McKeehan 相似文献
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Synthesis of DNA-binding proteins was investigated in WI-38 human diploid fibroblast cultures after stimulation with serum containing medium. Density-inhibited confluent monolayers of young (phase II) and aging (phase III) WI-38 cells can be stimulated to synthesize DNA by replacing the medium with fresh medium containing 10% fetal calf serum. Of the phase II cells, 35–50% showed a partially synchronized burst of DNA-synthesizing activity between 15 and 24 h whereas only 4–6% of phase III cells showed DNA-synthesizing activity at 20 h, and that cell fraction was increasing even at 38 h. This suggests either an extremely prolonged G 1 in stimulated phase III cells, or a heterogeneity of the population (e.g., a mixed population of pre- and postmitotic cells) for phase III cells. At various times after the change of medium, DNA-binding protein synthesis was examined in these stimulated cultures. Protein of mol. wt 20 000–25 000 D accumulated rapidly during early G 1 and declined thereafter, whereas larger protein (40 000 and 68 000 D) accumulated during the late G 1 or G 1-S transition period indicating that accumulation of these proteins is associated with the onset of DNA synthesis in the serum-stimulated cells. In cultures where the DNA synthesis has been reduced or inhibited by an excess of thymidine, hydroxyurea or dibutyryl cAMP, the accumulation of the larger proteins (40 000 and 68 000 D) was neglible as compared with non-stimulated cultures. Hydrocortisone did not exert any effect on the DNA-binding protein synthesis in phase II cells. However, it seems to increase the cell fraction which can respond to the serum factor in phase III cells as evidenced from the pattern of DNA-binding proteins synthesis. 相似文献
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DLK, a serine/threonine kinase that functions as an upstream activator of the mitogen-activated protein kinase (MAPK) pathways, has been shown to play a role in development, cell differentiation, apoptosis and neuronal response to injury. Interestingly, recent studies have shown that DLK may also be required for cell proliferation, although little is known about its specific functions. To start addressing this issue, we studied how DLK expression is modulated during cell cycle progression and what effect DLK depletion has on cell proliferation in WI-38 fibroblasts. Our results indicate that DLK protein levels are low in serum-starved cells, but that serum addition markedly stimulated it. Moreover, RNA interference experiments demonstrate that DLK is required for ERK activity, expression of the cell cycle regulator cyclin D1 and proliferation of WI-38 cells. DLK-depleted cells also show a senescent phenotype as revealed by senescence-associated galactosidase activity and up-regulation of the senescence pathway proteins p53 and p21. Consistent with a role for p53 in this response, inhibition of p53 expression by RNA interference significantly alleviated senescence induced by DLK knockdown. Together, these findings indicate that DLK participates in cell proliferation and/or survival, at least in part, by modulating the expression of cell cycle regulatory proteins. 相似文献
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J A Golczewski 《Physiological chemistry and physics and medical NMR》1983,15(2):115-120
EGF receptors from young and old WI-38 cells were compared by immunoprecipitation of the receptor followed by electrophoresis. There was no difference in molecular weight between young and old receptors. Both were composed of two components with molecular weights of about 165,000 and 145,000. Young EGF receptors were phosphorylated when incubated with (gamma-32P)ATP. Old receptors had markedly reduced phosphorylation, indicating a loss of kinase activity with age. These results demonstrate a major metabolic difference between young and old cells which clarifies the nature of the decline in mitogenic response with age. 相似文献
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T Furukawa S Tanaka S A Plotkin 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1975,148(4):1249-1251
The growth of human CMV was inhibited by uv irradiation of cells prior to infection or during the 48-hr latent period of virus replication but not after virus synthesis began. The duration of uv exposure sufficient to inhibit CMV replication was insufficient to inhibit replication of Herpes simplex and did not prevent uninfected cells from dividing normally. The effect of uv irradiation on CMV replication may have been mediated through prevention of the virus on host cell RNA(s) synthesis. 相似文献
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Most nontransformed cell lines respond to confluence by arresting the cell cycle in a viable G(1) phase, whereas immortalized cell lines growing in monolayer do not stop cell cycle progression in response to high cell density and are subjected to density-dependent apoptosis. We have examined the effects, in terms of cell growth, apoptosis, and expression of adhesion molecules of culturing contact inhibition-deficient hamster cells in the presence of dimethyl sulfoxide (DMSO). Addition of 1.5% DMSO to the growth medium for 96 h arrested Chinese hamster ovary (CHO) cells in the G(1) phase as a confluent monolayer, associated with a remarkable increase in the expression of the cyclin-dependent kinase inhibitor p27. Cells cultured in DMSO-containing medium showed increased levels of cadherins and alpha5beta1 and beta1 integrin complexes. Cell exposure to DMSO also reduced both cell density-dependent apoptosis and necrosis and resulted in increased Bcl-2 expression. These results converge to indicate that DMSO restores contact inhibition-induced growth arrest and prevents high-density-dependent apoptosis and suggest that the effect of DMSO may be mediated by intracellular signaling triggered by cell-extracellular matrix and cell-cell interactions. Both p27 and bcl-2 appear to be involved in the resumption of growth control accompanying cell adhesion in DMSO-exposed CHO cells. 相似文献