Growth and nucleic acid synthesis in synchronously dividing populations of HeLa cells

Terasima, Toyozo; Tolmach, L. J.

doi:10.1016/0014-4827(63)90306-9

Cited by 734 publications

(184 citation statements)

References 23 publications

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“…Thymidine-arrest of RH TK+ -parasites occurs at the G1/S boundary and is consistent with the effect of high thymidine concentrations in animal cells, which causes dNTP depletion and growth arrest at a similar position in the animal cell cycle [18]. Thymidine blocked parasites possess a broad 1N to 1.2N DNA content, duplicated centrosomes, and they immediately progress through S phase when released into fresh media [3,12].…”

Section: Discussionsupporting

confidence: 71%

“…Growth synchrony has been achieved through the use of exogenous thymidine to reversibly block tachyzoites engineered to express the herpes simplex virus thymidine kinase (RH TK+ ), an enzyme these parasites normally lack. A short treatment of RH TK+ tachyzoites with exogenous thymidine, which is known to cause dNTP depletion [18], arrests asynchronous parasite populations in late G1/early S phase and is presumed to act via a checkpoint that governs commitment to chromosome replication in this parasite [3,12].…”

Section: Introductionmentioning

confidence: 99%

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Inhibition of Toxoplasma gondii growth by pyrrolidine dithiocarbamate is cell cycle specific and leads to population synchronization

Felipe

Lehmann

Jerome

et al. 2008

Molecular and Biochemical Parasitology

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Successful completion of the Toxoplasma cell cycle requires the coordination of a series of complex and ordered processes that results in the formation of two daughters by internal budding. Although we now understand the order and timing of intracellular events associated with the parasite cell cycle, the molecular details of the checkpoints that regulate each step in T. gondii division is still uncertain. In other eukaryotic cells, the use of cytostatic inhibitors that are able to arrest replication at natural checkpoints have been exploited to induce synchronization of population growth. Herein, we describe a novel method to synchronize T. gondii tachyzoites based on the reversible growth inhibition by the drug, pyrrolidine dithiocarbamate. This method is an improvement over other strategies developed for this parasite as no prior genetic manipulation of the parasite was required. RH tachyzoites blocked by pyrrolidine dithiocarbamate exhibited a near uniform haploid DNA content and single centrosome indicating that this compound arrests parasites in the G1 phase of the tachyzoite cell cycle with a minor block in late cytokinesis. Thus, these studies support the existence of a natural checkpoint that regulates passage through the G1 period of the cell cycle. Populations released from pyrrolidine dithiocarbamate inhibition completed progression through G1 and entered S phase ~2 hours postdrug release. The transit of drug-synchronized populations through S phase and mitosis followed a similar timeframe to previous studies of the tachyzoite cell cycle. Tachyzoites treated with pyrrolidine dithiocarbamate were fully viable and completed two identical division cycles post-drug release demonstrating that this is a robust method for synchronizing population growth in Toxoplasma.

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Section: Discussionsupporting

confidence: 71%

Section: Introductionmentioning

confidence: 99%

Inhibition of Toxoplasma gondii growth by pyrrolidine dithiocarbamate is cell cycle specific and leads to population synchronization

Felipe

Lehmann

Jerome

et al. 2008

Molecular and Biochemical Parasitology

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“…CHO cells were grown in the presence of 2 mM deoxythymidine (dThd) for 9 hr to synchronize them coarsely in S phase (5-7). The medium was replaced, and 5-7 hr later mitotic cells were collected by the shake-synchrony procedure (8,9). With this procedure generally 95-99% of the detached cells were in mitosis.…”

mentioning

confidence: 99%

Late S phase cells (Chinese hamster ovary) induce early S phase DNA labeling patterns in G1 phase nuclei.

Yanishevsky¹,

Prescott²

1978

Proc. Natl. Acad. Sci. U.S.A.

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ABST RAEC Cells (Chinese hamster ovary) in GI phase were fused with cells in late S phase to determine if a cell in late S phase can induce DNA synthesis in the nucleus of a GI cell and, if so, to determine if the DNA synthesis so induced in a GI phase nucleus has an autoradiographic pattern characteristic of early or of late S phase synthesis. The results indicate (i) that 89% of the GI nuclei in late-S/G1 binucleates synthesized DNA, while only 2% of the control unfused GI cells synthesized DNA, and (ii) that in all late-S/G1 binucleates the GI nucleus was induced to synthesize early S phase DNA. These results are compatible with the idea that a cytoplasmically transmissible factor initiates DNA synthesis but that an intranuclear mechanism defines the temporal order of replication.Two important problems in the study of cell reproduction are the mechanism governing the initiation of DNA replication and the mechanism responsible for the temporal ordering of DNA synthesis within the S phase. Evidence indicates that a cytoplasmically transmissible inducer of DNA synthesis is present in early S phase HeLa cells; when a Gi phase cell was fused with an early S phase cell 1 hr after its release from a double thymidine block, DNA synthesis was induced prematurely in the GI phase nucleus (1). In addition, fusion of erythrocytes (2) or macrophages (3) with S phase cells that had been selected at various times after mitosis has suggested that a cytoplasmically transmissible inducer is present throughout the S phase. However, the difficulty in positively identifying late S phase cells in these types of experiments leaves open the question of whether such an inducer of DNA synthesis is present in late S phase. If such an inducer is present in late S phase, does it induce the synthesis of early or late S phase DNA in a Gi nucleus; i.e., is DNA synthesis induced in the normal temporal order or are there specific late S phase initiators that can alter the normal replication sequence?To study these problems we fused Chinese hamster ovary (CHO) Cell Synchrony. CHO cells were grown in the presence of 2 mM deoxythymidine (dThd) for 9 hr to synchronize them coarsely in S phase (5-7). The medium was replaced, and 5-7 hr later mitotic cells were collected by the shake-synchrony procedure (8, 9). With this procedure generally 95-99% of the detached cells were in mitosis. The mitotic cells were grown in conditioned medium for 1 hr to obtain Gi phase cells. Alternatively, the mitotic cells were grown in conditioned medium containing 1 mM hydroxyurea for 9 hr to obtain early S phase cells (10, 11). The hydroxyurea was removed and the cells were allowed to proceed to late S phase.Labeling with Latex Beads. To identify fusion products between cells from S and GI phase populations we used latex beads (Dow Chemical Co., Indianapolis, IN) of two different sizes to prelabel the cytoplasms of the cells (approximately 1 Am diameter beads for the S phase cells and approximately 2 Am diameter beads for the GI phase cells; ref. 12). Cells were al...

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“…They have a doubling time of approximately 20 hr. The cells were synchronized by mitotic selection (22). The cells were held in mitosis at 0°C for a maximum of 1% hr during the selection procedure.…”

Section: Methodsmentioning

confidence: 99%

A model for the computer analysis of synchronous DNA distributions obtained by flow cytometry

Fox

1980

Cytometry

132

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Flow cytometry has become an important tool for research in cell biology (4, 15, 18, 21, 23). An important application of flow cytometry is to determine the distribution of DNA content per cell in a population of cells at a very rapid rate and with good statistics. However, the important parameters usually desired-the proportion of cells in each phase of the cell cycle-cannot be accurately determined without using a computer to model the distribution. Various mathematical models have been proposed to accomplish this, with varying degrees of success. These have been reviewed recently (12).These models may be categorized by two basic conceptual approaches. One approach is to develop a function that will accurately represent the GI, S and Gz + M components of the DNA histogram derived from flow cytometry, and thus obtain the relative proportion of cells in each phase (1,2, 5, 7, 8, 9). The other approach is more basic in the sense that it models the cell age distribution and then transforms that distribution into a DNA distribution (10, 11,17,24), usually by matrix manipulation. This approach requires knowledge of the rate of DNA synthesis during S-phase. It does allow the capability of determining additional information, such as the phase times, and in fact the percentage of cells in each phase is not usually the primary information desired from these models.' Supported by Grant CA2563601 awarded by the National Cancer Institute. DHEW. 71 The model presented here is based on the f i s t approach.Dean and Jett (5) presented the first serious attempt to model accurately the DNA distributions of asynchronous cell populations. With such populations, their model gives excellent results. The model fits normal (Gaussian) distributions to the GI and Gz + M peaks, and a broadened second-order polynomial to S-phase. The broadening accounts for dispersion from a variety of causes, including instrumental, staining and biologic factors. The limitation of the model is that it can only fit distributions where a second order polynomial can accurately represent the S-phase data. This leaves out nearly all synchronous populations. Fried (7-9) presented another model in which a series of normal curves is fitted to S-phase. The advantage of this model is that it can fit synchronous populations, with varying degrees of success. However, the model is very sensitive to the resolution of the data. Furthermore, it requires considerable operator monitoring to obtain proper parameters (12). The main difficulty is in choosing the spacing of the S-phase compartments. If this is not chosen carefully, the resulting fit can have large fluctuations in the estimated number of cells from one compartment to the next. The spacing is correlated with the coefficient of variation of the GI peak (8). This is a rather arbitrary factor because ideally there should be one compartment per channel since DNA synthesis is a continuous rather than discrete process.

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Growth and nucleic acid synthesis in synchronously dividing populations of HeLa cells

Cited by 734 publications

References 23 publications

Inhibition of Toxoplasma gondii growth by pyrrolidine dithiocarbamate is cell cycle specific and leads to population synchronization

Inhibition of Toxoplasma gondii growth by pyrrolidine dithiocarbamate is cell cycle specific and leads to population synchronization

Late S phase cells (Chinese hamster ovary) induce early S phase DNA labeling patterns in G1 phase nuclei.

A model for the computer analysis of synchronous DNA distributions obtained by flow cytometry

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