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1.
Abstract. It has been predicted that whole-culture methods of synchronization cannot synchronize cells. We have tested whether thymidine block, one type of whole-culture synchronization, can synchronize L1210 cells. We demonstrate experimentally that the thymidine block method cannot produce a synchronized culture. Although thymidine-treated cells are arrested primarily with an S-phase amount of DNA, there is no narrowing of the cell size distribution and there is no synchronized division pattern following release from the thymidine block. In contrast to a whole-culture synchronization method, cells produced by a selective (i.e. non-whole-culture) method not only have a specific DNA content, but also have a narrow size distribution and divide synchronously. Generalizing the results to other cell lines, we suggest that these conclusions call into question experimental measurements of gene expression during the division cycle based on thymidine inhibition synchronization. 相似文献
2.
Accurate cell-size determinations support the prediction that serum starvation and related whole-culture methods cannot synchronize cells. Theoretical considerations predict that whole-culture methods of synchronization cannot synchronize cells. Upon serum starvation, the fraction of cells with a G1-phase amount of DNA increased, but the cell-size distribution is not narrowed. In true synchronization, the cell-size distribution should be narrower than the cell-size distribution of the original culture. In contrast, cells produced by a selective (i.e. non-whole-culture) method have a specific DNA content, a narrow size distribution, and divide synchronously. The general theory leading to the conclusion that whole-culture methods for synchronization do not work implies that one can generalize these serum-starvation results to other cell lines and other whole-culture methods, to conclude that these methods do not synchronize cells. 相似文献
3.
It has been predicted that nocodazole-inhibited cells are not synchronized because nocodazole-arrested cells with a G2-phase amount of DNA would not have a narrow cell-size range reflecting the cell size of some specific, presumably G2-phase, cell-cycle age. Size measurements of nocodazole-inhibited cells now fully confirm this prediction. Further, release from nocodazole inhibition does not produce cells that move through the cell cycle mimicking the passage of normal unperturbed cells through the cell cycle. Nocodazole, an archetypal whole-culture synchronization method, can inhibit growth to produce cells with a G2-phase amount of DNA, but such cells are not synchronized. Cells produced by a selective (i.e., non-whole-culture) method not only have a specific DNA content, but also have a narrow size distribution. The current view of cell-cycle control that is based on methods that are not suitable for cell-cycle analysis must therefore be reconsidered when results are based on whole-culture synchronization.This work was supported by the National Science Foundation (grant MCB–0323346) and (in part) by the National Institutes of Health (University of Michigan’s Cancer Center, support grant 5 P30 CA46592). G.I., M.T., and P. B. are associated with the Undergraduate Research Opportunity Program of the University of Michigan, which also supported this research. 相似文献
4.
Studies of gene expression during the eukaryotic cell cycle in whole-culture synchronized cultures have been published using many methodologies. These procedures alter the state of the cell cycle for a population of cells, rather than purifying a population of cells that are in the same state. Criticism of these methods (e.g. see Cooper, this issue, pp. 266-269, ) suggests that these studies are flawed, and posits that such methodologies cannot be used to study the cell cycle because they alter the size and age distributions of the cultures. We believe that whole-culture cell cycle studies work even though they alter the size and age distributions: these cells still progress through the cell cycle and although we do not suggest that the methods are perfect, we will explain how these microarray studies have successfully identified cell cycle regulated genes and why these results are biologically meaningful. 相似文献
5.
Cooper S 《Trends in biotechnology》2004,22(6):266-269
Whole-culture or batch synchronization cannot, in theory, produce a synchronized culture because it violates a fundamental law that proposes that no batch treatment can alter the cell-age order of a culture. In analogy with the history of perpetual-motion machines, it is suggested that the study of these whole-culture 'synchronization' methods might lead to an understanding of general biological principles even though these methods cannot be used to study the normal cell cycle. 相似文献
6.
Understanding the molecular and biochemical basis of cellular growth and division involves the investigation of regulatory events that most often occur in a cell-cycle phase-dependent fashion. Studies examining cell-cycle regulatory mechanisms and progression invariably require cell-cycle synchronization of cell populations. Thus, many methods have been established to synchronize cells at specific phases of the cell cycle. Several of the common methods involve pharmacological agents, which act at various points throughout the cell cycle. Because of adverse cellular perturbations resulting from many of the synchronizing drugs used, other synchrony methods that involve less perturbation of biological systems, such as serum deprivation, contact inhibition, and centrifugal elutriation have a significant advantage. The advantages and disadvantages of these cell synchronization methods are discussed in this review. 相似文献
7.
Uwe Jandt Oscar Platas Barradas Ralf Pörtner An-Ping Zeng 《Applied microbiology and biotechnology》2014,98(10):4311-4319
The overall behavior of cell cultures is determined by the actions and regulations of all cells and their interaction in a mixed population. However, the dynamics caused by diversity and heterogeneity within cultures is often neglected in the study of cell culture processes. Usually, a bulk behavior is assumed, although heterogeneity prevails in most cases. It is, however, not valid to conclude from the bulk behavior to the single cell behavior. Instead, it is necessary to include the behavior and kinetics of subpopulations and their interactions into models in order to elucidate the dynamic effects occurring in typical cell cultures. Heterogeneity in cell cultures is largely caused by the progress of the cell cycle. Cell cycle-dependent dynamics resulting for example in variable transfection efficiencies or expression bistability have recently attracted attention. In order to elucidate cell cycle-dependent regulations in cell cultures, it is desirable to synchronize a culture with minimal perturbation, which is possible with different yield and quality using physical methods, but not possible for frequently used chemical, or whole-culture methods. Then, the culture is cultivated again under physiological conditions and subpopulation-resolved analysis and modeling approaches are applied. This should allow to account for the variable contributions of subpopulations to the whole behavior and also for obtaining hereto unaccessible dynamic information of cellular regulation. In this short review, we summarize techniques and key issues to be considered for successful synchronization, cultivation, and modeling in order to achieve the goal of better understanding cell culture at a population level. 相似文献
8.
Cooper S 《BioEssays : news and reviews in molecular, cellular and developmental biology》2002,24(6):499-501
A eukaryotic "baby machine" has been developed that produces synchronized cultures that display up to four synchronous cell cycles. That such cells can be produced implies that methods unable to produce successive synchronized cell cycles may not actually synchronize cells. But most important, the baby machine method now opens the way for the study of the cell cycle of minimally disturbed, artifact-free, well-synchronized, mammalian cells. 相似文献
9.
The purpose of the present study was to evaluate the effects of serum-starvation, contact-inhibition and roscovitine treatments on cell-cycle synchronization at the G0/G1 stage of ear skin fibroblasts isolated from transgenic cloned cattle. The developmental competence of re-cloned embryos was also examined. Our results showed that the proportion of G0/G1 cells from the serum-starved group at 3, 4 or 5 days was significantly higher compared with 1 or 2 days only (91.5, 91.7 and 93.5% versus 90.1 and 88.8%, respectively, p < 0.05); whilst there was no statistical difference among cells at 3, 4 or 5 days. For roscovitine-treated cells, the proportion of G0/G1 cells at 2, 3, 4 or 5 days was significantly higher than those treated for 1 day only (91.1, 90.1, 89.4 and 91.3% versus 86.51%, respectively, p < 0.05). The proportion of contact-inhibited G0/G1 cells rose significantly with treatment time, but was similar at 3, 4 and 5 days (89.4, 90.4, 91.4, 91.6 and 92.1%, respectively, p < 0.05). The efficiency of obtaining G0/G1 phase cells was lower when roscovitine treatment was employed to synchronize the cell cycle compared with the serum-starvation and contact-inhibition methods (89.7 versus 91.1% and 91.0%, p < 0.05). Moreover, obvious differences were observed in the rate of fused couplets and blastocysts (89.88 +/- 2.70 versus 87.40 +/- 5.13; 44.10 +/- 8.62 versus 58.38 +/- 13.28, respectively, p < 0.05), when nuclear transfer embryos were reconstructed using donors cells that had been serum starved or contact inhibited for 3 days. Our data indicate that 3 day treatment is feasible for harvesting sufficient G0/G1 cells to produce re-cloned transgenic bovine embryos, regardless of whether serum-starvation, contact-inhibition or roscovitine treatments are used as the synchronization methods. 相似文献
10.
Differing arresting agents and protocols can be used to synchronize cells in cultures to specific phases of the cell when studying cell-cycle gene expressions. Often, data derived from individual experiments are analyzed separately, since no appropriate statistical methodology is available at the moment to analyze the data from all such experiments simultaneously. The focus of this paper is to determine the association and coherence of the relative activation times of cell-cycling genes under different experimental conditions. Using a circular-circular regression model, we define two parameters, a rotation parameter for the angular difference between cells' arresting times (phases) in two cell-cycle experiments, and an association parameter to describe the correspondence between the cycle times of maximal expression (phase angles) for a set of genes studied in two experiments. Further, we propose a procedure to assess coherence across multiple experiments, i.e. to what extent the circular ordering of the phase angles of genes is maintained across multiple experiments. Coherence of genes across experiments suggests that functionally these genes tend to respond in a stereotypically sequenced way under different experimental conditions. Our proposed methodology is illustrated by applying it to a HeLa cell-cycle gene-expression data. 相似文献
11.
Both cell growth (cell mass increase) and progression through the cell division cycle are required for sustained cell proliferation. Proliferating cells in culture tend to double in mass before each division, but it is not known how growth and division rates are co-ordinated to ensure that cell size is maintained. The prevailing view is that coordination is achieved because cell growth is rate-limiting for cell-cycle progression. Here, we challenge this view. We have investigated the relationship between cell growth and cell-cycle progression in purified rat Schwann cells, using two extracellular signal proteins that are known to influence these cells. We find that glial growth factor (GGF) can stimulate cell-cycle progression without promoting cell growth. We have used this restricted action of GGF to show that, for cultured Schwann cells, cell growth rate alone does not determine the rate of cell-cycle progression and that cell size at division is variable and depends on the concentrations of extracellular signal proteins that stimulate cell-cycle progression, cell growth, or both. 相似文献
12.
In yeast, the Origin Recognition Complex (ORC) is bound to replication origins throughout the cell-cycle, but in animal cells, there are conflicting data as to whether and when ORC is removed from chromatin. We find ORC1, 2 and ORC4 to be metabolically stable proteins that co-fractionate with chromatin throughout the cell-cycle in Chinese hamster fibroblasts. Since cellular extraction methods cannot directly examine the chromatin binding properties of proteins in vivo, we examined ORC:chromatin interactions in living cells. Fluorescence loss in photobleaching (FLIP) studies revealed ORC1 and ORC4 to be highly dynamic proteins during the cell-cycle with exchange kinetics similar to other regulatory chromatin proteins. In vivo interaction with chromatin was not significantly altered throughout the cell-cycle, including S-phase. These data support a model in which ORC subunits dynamically interact with chromatin throughout the cell-cycle. 相似文献
13.
14.
Helmstetter and Cummings devised a technique of synchronization in which cells are implanted on a membrane filter and the membrane is subjected to reverse flow of liquid medium. The cells in the effluent stream have predominantly the characteristics of newborn cells. The advantage of this technique is that the population experiences a minimum of physiological stress; hence, the behavior of the synchronous culture should reflect the normal divisional cycle. The disadvantage is that strains other than Escherichia coli B/r cannot be synchronized. We have found that a modification of the method makes it possible to synchronize several strains of E. coli, including both male and female strains, as well as Salmonella typhimurium LT2. The principal difference in technique is a prolonged period (>400 doublings) of cultivation in glucose minimal medium at 30 C and at low density (<5 x 10(6) cells/ml) prior to implantation. This precaution was taken to insure that the bacterial growth population is in a steady state of balanced growth. From the resulting synchronous growth, the distribution of interdivision times has been computed; these distributions have coefficients of variation in the range 0.18 to 0.22 and are not appreciably skewed. 相似文献
15.
Recombinant bacterial cells express various levels of model product proteins if the genes of interest are regulated by controllable promoters. The level of gene expression influences the growth-rate differential between plasmid-bearing and plasmid-free cells, and thereby affects the culture dynamics of a plasmid-containing cell population. An expression system has been designed in which host Escherichia coli cells contain the pil operon controlled by a tac promoter; these cells are transformed with plasmids that contain the repressor gene, lacl, for the tac promoter, in combination with an expression system for a model protein, chloramphenicol acetyl tranferase (CAT). Experimental and theoretical results show that plasmid-bearing cells can be maintained as dominant in continuous cultures without selective pressure when 12% or less of the cells' total protein is the model product protein, CAT. This is because the segment cells produce pili greatly in excess of normal wild-type levels, and thus have more of a metabolic burden than do the plasmid-bearng cells that overproduce CAT. However, when the level of the plasmid-directed CAT expression is increased above 12% of the cells' total protein, the growth rate of the plasmid-bearing cells decreases to a value lower than that of the segregant cells. Therefore, plasmid-containing cells lose their selective advantage at this expression level, and cannot be maintained as the dominant cell type in a continuous culture unless antibiotic or other positive selection methods are used. By controlling the growth rate differential of this bacterial host/plasmid system, a variety of interesting competitive culture dynamics is investigated. All experimental measurements for continuous cultures are in very good agreement with theory using kinetic parameters determined from independent batch experiments. (c) 1992 John Wiley & Sons, Inc. 相似文献
16.
4-Hydroxy-3-(3'-methyl-2'-butenyl)-benzoic acid (HMBA) was previously isolated from Curvularia sp. KF119 as a cell-cycle inhibitor. However, the present study used a novel and practical synthetic method to prepare a large quantity of HMBA. The synthetic HMBA was found to inhibit the cell-cycle progression of HeLa cells with a comparable potency to the natural fungal metabolite. The inhibition of the cell-cycle progression by the synthetic HMBA involved both the activation of p21(WAFI) and the inhibition of cyclin Dl expression in the cells. Consequently, this new synthetic procedure provides an easy and convenient way to produce or manipulate the original fungal metabolite. 相似文献
17.
George P. Studzinski John F. Gierthy Jolanta J. Cholon 《In vitro cellular & developmental biology. Plant》1973,8(6):466-472
Summary Autoradiographic detection of incorporation of tritiated thymidine into the cytoplasm of cultured mammalian cells has been
evaluated as a test of contamination of the cultures by cell-associated microorganisms, which usually are mycoplasmas. Criteria
which indicate the presence of cell-associated mycoplasmas have been established, and the reliability of the standardized
autoradiographic method has been assessed by testing the same cultures by two colony formation methods of mycoplasmal detection.
The autoradiographic method demonstrated cell-associated microorganisms in all cultures from which characteristic colonies
were grown on mycoplasma agar. The autoradiographic method did not produce false positive results, and the outcome of this
test was evident in 3 days as opposed to 7 to 14 days by agar culture methods. Some applications of the autoradiographic method
are shown, and it is suggested that this method be employed for routine surveillance for mycoplasmal contamination in laboratories
where facilities for frequent agar culture tests are not easily available.
This research was supported by U.S. Public Health Service Grants CA 12351-02 and CA 12334-01 from the National Cancer Institute. 相似文献
18.
Synchronization of 30–80 Hz oscillatory activity of the principle neurons in the olfactory bulb (mitral cells) is believed to be important for odor discrimination. Previous theoretical studies of these fast rhythms in other brain areas have proposed that principle neuron synchrony can be mediated by short-latency, rapidly decaying inhibition. This phasic inhibition provides a narrow time window for the principle neurons to fire, thus promoting synchrony. However, in the olfactory bulb, the inhibitory granule cells produce long lasting, small amplitude, asynchronous and aperiodic inhibitory input and thus the narrow time window that is required to synchronize spiking does not exist. Instead, it has been suggested that correlated output of the granule cells could serve to synchronize uncoupled mitral cells through a mechanism called “stochastic synchronization”, wherein the synchronization arises through correlation of inputs to two neural oscillators. Almost all work on synchrony due to correlations presumes that the correlation is imposed and fixed. Building on theory and experiments that we and others have developed, we show that increased synchrony in the mitral cells could produce an increase in granule cell activity for those granule cells that share a synchronous group of mitral cells. Common granule cell input increases the input correlation to the mitral cells and hence their synchrony by providing a positive feedback loop in correlation. Thus we demonstrate the emergence and temporal evolution of input correlation in recurrent networks with feedback. We explore several theoretical models of this idea, ranging from spiking models to an analytically tractable model. 相似文献
19.
20.
Can Nocodazole,an Inhibitor of Microtubule Formation,Be Used to Synchronize Mammalian Cells? 总被引:6,自引:0,他引:6
Abstract Nocodazole, a temporary inhibitor of microtubule formation, has been used to partly synchronize Ehrlich ascites tumour cells growing in suspension. the gradual entry of cells into mitosis and into the next cell cycle without division during drug treatment has been studied by flow cytometric determination of mitotic cells, analysing red and green fluorescence after low pH treatment and acridine orange staining. Determination of the mitotic index (MI) by this method has been combined with DNA distribution analysis to measure cell-cycle phase durations in asynchronous populations growing in the presence of the drug. With synchronized cells, it was shown that in the concentration range 0.4–4.0 μg/l, cells could only be arrested in mitosis for about 7 hr and at 0.04 μg/ml, for about 5 hr. After these time intervals, the DNA content in nocodazole-blocked cells was found to be increased, and, in parallel, the ratio of red and green fluorescence was found to have changed, showing entry of cells into a next cell cycle without division (polyploidization). It was therefore only possible to partially synchronize an asynchronous population by nocodazole. However, a presynchronized population, e.g. selected G1 cells or metabolically blocked G1/S cells, were readily and without harmful effect resynchronized in M phase by a short treatment (0.4 μg/ml, 3–4 hr) with nocodazole; after removal of the drug, cells divided and progressed in a highly synchronized fashion through the next cell cycle. 相似文献