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201.
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Cell cycle perturbations in normal and transformed fibroblasts caused by detachment from the substratum 总被引:6,自引:0,他引:6
Exponentially growing, anchorage-dependent fibroblasts were impeded in their progress through the cell cycle as a result of brief trypsinization from the substratum followed by replating. Untransformed mouse (3T3, clone A31), hamster (CHEF/18-1) and human (FS2) fibroblasts were partially inhibited from entering the DNA synthetic (S) phase of the cell cycle for 8 or 12 hours after detachment, even though the cells reattached within an hour of replating and attained a spread morphology 5 or 8 hours later. The decline in the proportion of cells in S phase was accompanied accumulation of cells in G1 as measured by autoradiography and flow microfluorimetry. Cells removed from the substratum by EDTA alone showed identical disturbances of exponential growth. These cell cycle perturbations could be a result of the detachment per se, as opposed to the rounded morphology. Synchronized A31 cells, exposed to colcemid or cytochalasin B for two hours, were not delayed in their entry into S, whereas trypsinization delayed S phase entry by 4 to 5 hours. These drugs disrupt the cytoskeleton without causing detachment. Isotope incorporation experiments revealed no decreases in the rates of protein or RNA synthesis following replating. However, exponentially growing A31 cells, treated for 2 hours with an inhibitor of protein synthesis behaved similarly to those briefly detached from their substratum: 7 hours after treatment, there were fewer cells in S and more cells in G1 relative to untreated cells. Brief treatment with an inhibitor of hn-RNA synthesis did not alter the cell cycle distribution of these fibroblasts. Three tumorogenic A31 derivatives were less affected by brief detachment from the substratum than were the untransformed cells. The derivative exhibiting the least in vitro growth control (an SV-40 transformant) showed the least sensitivity to trypsinization, while that derivative having the most in vitro growth control (a Moloney sarcoma virus transformant) was most sensitive. A chemically [benzo(a)pyrene] transformed derivative gave intermediate results with respect to both growth control and sensitivity to detachment from the substratum. The results suggest that as yet unidentified protein(s) necessary for the normal transit through G1 may be quite sensitive to the presence of an anchoring substratum. 相似文献
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It is shown that the different onset of S phase as measured by autoradiography vs cumulative thymidine uptake is an artifact. We consequently propose that S phase-specific enzyme activities may accumulate a few hours prior to the actual initiation of DNA synthesis. A “pre-S” DNA synthesis that can be readily detected only by autoradiography has been proposed. Published data show that DNA synthesis in cultured animal cells is initiated approx. 2 h later when measured by cumulative incorporation of [3H]thymidine ([3H]TdR) as compared with autoradiography. We show here that the difference is in reality an artifact, owing to not taking into account both gradual, asynchronous entry of cells into S phase, as well as time-dependent accumulation of radioactivity into each cell after it has entered S phase. Combination of these two factors leads to the conclusion that [3H]TdR should be incorporated approximately as the square of time following entry of the first cell into S. Taking this into account, the two methods then are in agreement, as predicted. This argument also applies to the enzyme activities shown to increase with DNA synthesis in synchronized cultures. Such an enzyme accumulation really could begin some time earlier than indicated by conventional plots of cumulative enzyme activity vs time and may, in fact, precede the onset of S by a few hours. 相似文献
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The growth of herpes simplex virus type 1 (HSV-1), monitored by plaque assay, was inhibited by the cellular antimetabolites thioguanine (TG), cytosine arabinoside (AraC), methotrexate (MTX), and 5-fluorodeoxyuridine (5-FUdR). These results suggested a rapid means for assaying cellular drug sensitivity, based on the ability of infected cells to support viral replication. We have explored the feasibility of a virus-mediated assay for cellular metabolic function in two model systems. Using an immunofluorescence assay to assess viral growth, we found that all of the antimetabolites tested were effective in diminishing HSV-1 specific fluorescence in human fibroblasts. However, a DNA-damaging agent, bleomycin, was lethal to cells but was completely ineffective in reducing viral fluorescence. HSV-1 growth was markedly decreased by TG in a normal human fibroblast strain, FS-2. In contrast, a Lesch-Nyhan strain (LNF), resistant to TG owing to its genetic defect, showed no suppression of viral growth in the presence of TG. The drug's effect on viral fluorescence closely paralleled its effect on cellular colony forming ability and rate of cellular DNA synthesis. Thymidine kinase-deficient HSV-1 (TK?HSV-1) did not grow in a normal mouse fibroblast line (A31) in the presence of 5-FUdR. However, a TK? derivative of the A31 line allowed full production of the TK?HSV-1 antigens at low to moderate doses of 5-FUdR. Two potential applications for this assay are the prenatal diagnosis of some genetic disorders and the rapid detection of drug resistant populations in tumor specimens. Toward these ends, we demonstrated that human fibroblasts from patients with the hereditary disorder Xeroderma pigmentosum (group A) were easily distinguished from normal human fibroblasts by their inability to support the growth of UV-irradiated HSV-1. We also investigated the effects of TG upon HSV-1 fluorescence in two human tumor cell lines isolated from head and neck squamous cell carcinomas (SCC-15) and (SCC-25). Whereas TG was effective in reducing viral fluorescence in SCC-15 cells, it was only marginally so in SCC-25 cells. These latter cells showed the greater resistance to TG by growth and isotope incorporation experiments. 相似文献
207.
Martinez G. Carnazza M. L. Campisi A. Sorrenti V. Di Giacomo C. Perez-Polo J. R. Vanella A. 《Neurochemical research》1998,23(7):961-968
The present paper reports the effects of GSH depletion (diethylmaleate induced) on partial cerebral ischemia and reperfusion for 7 and 20 days. Our results confirm that there is a paradoxical protective effect of the GSH-depletor and suggest an improved neuronal trophism induced by diethylmaleate treatment. 相似文献
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Christopher D. Wiley Rishi Sharma Sonnet S. Davis Jose Alberto Lopez-Dominguez Kylie P. Mitchell Samantha Wiley Fatouma Alimirah Dong Eun Kim Therese Payne Andrew Rosko Eliezer Aimontche Sharvari M. Deshpande Francesco Neri Chisaka Kuehnemann Marco Demaria Arvind Ramanathan Judith Campisi 《Cell metabolism》2021,33(6):1124-1136.e5
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