Duration Of Cell Cycle Phases Research Articles

Effects of Environmental Stresses on the Cell Cycle of Two Marine Phytoplankton Species

Cell cycle phase durations of cultures of Hymenomonas carterae Braarud and Fagerl, a coccolithophore, and Thalassiosira weissflogii Grun., a centric diatom, in temperature-, light- or nitrogen-limited balanced growth were determined using flow cytometry. Suboptimal temperature caused increases in the duration of all phases of the cell cycle (though not equally) in both species, and the increased generation time of nitrogen-limited cells of both species was due almost wholly to expansion of G(1) phase. In H. carterae light limitation caused only G(1) phase to expand, but in T. weissflogii both G(2) + M and G(1) were affected. These results are discussed in relation to cell division phasing patterns of these two species and to models of phytoplankton growth. Simultaneous measurements of protein and DNA on individual cells indicated that under all conditions, the protein content of cells in G(1) was a constant proportion of that of G(2) + M cells. Simultaneous measurements of RNA and protein on each cell indicated that the amounts of these two cell constituents were always tightly correlated. Under conditions of nitrogen limitation both protein and RNA per cell decreased to less than one-third of the levels found in nonlimited cells. This indicates, at least for nitrogen-replete cells, that neither protein nor RNA levels are likely to act as the trigger for cell cycle progression. Strict control by cell size is also unlikely since mean cell volume decreased as growth rates were limited by light and nitrogen supply, but increased with decreasing temperature.

Autoradiographic analysis of the cell cycle: Howard and Pelc to the present day.

Although flow cytometry has become the popular way to examine the kinetics of cell proliferation, the technique of labelled mitoses remains an important and central technique. This is particularly the case when it is the durations of cell cycle phases that are required. Through simulation analysis it is possible to identify discrepancies that imply the existence either of artefacts or of unsuspected properties of the cell system under investigation: the implications of these discrepancies have often been inadequately explored. An important function of the modelling approach is to seek to identify aspects of the data that cannot be fitted, and to pursue the discrepancies either by eliminating artefacts or by seeking to describe a more comprehensive model.

Estimation of circadian variations in cell cycle phase durations in murine epidermal basal cells.

Circadian variations in the proliferative activity of squamous epithelia are well known. However, circadian variations in the duration of the various cell cycle phases (S, G2 and mitosis) have been disputed. The percent labelled mitoses method, which is traditionally used to obtain duration of cell cycle phases, is poorly suited for identification of circadian variations. Therefore methods combining changes in compartment size (cell cycle phase) and cellular flux through the compartments have been used. Three different methods using such data are presented. These incorporate various simplifying assumptions that cause methodological errors. Limits for use of the different methods are indicated. The use of all three methods gives comparable and pronounced circadian variations in the duration of S and G2 phase. These results are also compatible with circadian variations in the mitotic duration, but they may also represent artefacts due to sensitivity to model errors.

Can Nocodazole, an Inhibitor of Microtubule Formation, Be Used to Synchronize Mammalian Cells?

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 micrograms/l, cells could only be arrested in mitosis for about 7 hr and at 0.04 microgram/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 microgram/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.

The kinetics of human glioblastomas maintained in an organ culture system. An in vitro autoradiographic study.

Five human glioblastomas maintained in an organ culture system were studied by autoradiography to determine, after 8 days in vitro, the growth fraction (GF) of the explants, their total cell cycle time (TC) and cell cycle phase durations (TS,TG1,TG2 andTM), and their potential doubling time (Tpot) after pulse-labeling with [3H] TdR for 1 h. These parameters were derived from computer analysis of fraction of labeled mitoses (FLM) curves. The results fell into two groups.

Growth pattern of the human promyelocytic leukaemia cell line HL60.

In order to characterize the growth pattern of the human promyelocytic leukaemia cell line HL60, its kinetic parameters were studied. The doubling time was calculated from serial cell counts, the duration of the various cell cycle phases from the analysis of the labelled mitoses curve, and quiescent population from continuous labelling experiments. Proliferation in culture was exponential up to a saturation density of about 3.0 x 10(6) cells/ml, with a doubling time of 34.0 hr. The cell cycle duration was 24.3 +/- 4.1 hr (SD), and that of the cell cycle phases was: G1, 3.8 +/- 2.2 hr; S, 15.1 +/- 3 hr; and G2, 4 +/0 1.2 hr. The growth fraction was 0.85, and cell loss was restricted to the quiescent cells. The HL60 cell line, with fully characterized kinetics, provides a useful tool for the vitro study of substances which may affect human leukaemic myelopoietic proliferation.

Cell cycle analysis in a human cell line (EUE cells).

A new procedure to determine the durations of cell cycle phases is based on th autoradiography of labeled nuclei and a standard chemical determination of DNA content per cell. It has been used to study the cell cycle timing of a human established cell line, EUE cells, so far poorly characterized in its kinetic aspects. The cycle phase durations obtained with this method are compared with the DNA frequency profiles determined by flow cytometry and found to agree closely. The relevance of the growth conditions of the exponentially growing cultures for a correct estimation of the cycle phase duration is indicated. Cell size distribution has been shown to vary according to growth condition.

Cell kinetic characteristics in different parts of multicellular spheroids of human origin.

The growth fraction, the cell cycle time, and the duration of the individual cell cycle phases were determined as a function of distance from the surface of multicellular spheroids of the human cell line NHIK 3025. The techniques employed were percentage of labelled mitoses and labelling index measurements after autoradiography and flow cytometric measurements of DNA histograms. To separate cell populations from the different parts of the spheroid, fractionated trypsinization was employed. The results were compared with corresponding values in NHIK 3025 cell populations grown as monolayer cultures. While practically all cells in exponentially growing monolayer populations are proliferating, the growth fraction was between 0.6 and 0.7 in the outer parts of the spheroid. The inner region was mainly occupied by a necrotic mass. The proliferating fraction of the recognizable cells in the inner region was slightly below 0.5. The mean cell cycle time of NHIK 3025 cells in monolayer culture is 18 hr. The mean cell cycle time of proliferating cells in the periphery of the spheroid was 30 hr, compared to 41 hr in the inner region (150 micrometer from the spheroid surface). All phases of the cell cycle were prolonged compared to populations of exponentially growing monolayer cells. Within each part of the spheroid the distribution of cell cycle times was considerably broadened compared with monolayer populations.

Proliferation kinetics of Sezary cells

The proliferation kinetics of neoplastic T cells arising in Sezary syndrome (Sezary cells) are still poorly understood. Kinetic studies with 3H-thymidine as a DNA precursor revealed a low incorporation rate of the nucleotide into Sezary cells obtained from the peripheral blood versus Sezary cells from the skin. Using the double-label autoradiography we determined the duration of single cell cycle phases of blood and cutaneous Sezary cells. The results indicate the almost complete lack of proliferative activity in the blood but a considerable portion of proliferatively active Sezary cells in skin infiltrates. The removal of a large mass of quiescent blood cells by leukapheresis did not affect the proliferative state of the residual peripheral cell population implying that the procedure did not induce the migration of proliferating skin cells towards the blood. Terminally, the disease underwent transition into immunoblastic lymphoma. At this time, the kinetic behavior of the peripheral immunoblasts showed great similarity to that of cutaneous Sezary cells. The findings point towards a common extravascular production site of Sezary cells and immunoblasts probably located in the lymphatic tissue.

Proliferation Kinetics of Sézary Cells

AbstractThe proliferation kinetics of neoplastic T cells arising in Sezary syndrome (Sezary cells) are still poorly understood. Kinetic studies with 3H-thymidine as a DNA precursor revealed a low incorporation rate of the nucleotide into Sezary cells obtained from the peripheral blood versus Sezary cells from the skin. Using the double-label autoradiography we determined the duration of single cell cycle phases of blood and cutaneous Sezary cells. The results indicate the almost complete lack of proliferative activity in the blood but a considerable portion of proliferatively active Sezary cells in skin infiltrates. The removal of a large mass of quiescent blood cells by leukapheresis did not affect the proliferative state of the residual peripheral cell population implying that the procedure did not induce the migration of proliferating skin cells towards the blood. Terminally, the disease underwent transition into immunoblastic lymphoma. At this time, the kinetic behavior of the peripheral immunoblasts showed great similarity to that of cutaneous Sézary cells. The findings point towards a common extravascular production site of Sézary cells and immunoblasts probably located in the lymphatic tissue.

Estimating the proportion of proliferating cells in a population

In a population of cells that proportion which is actively engaged in the proliferative cycle is often estimated from the ratio of the observed labelling index to an expected labelling index, calculated, on the basis of all cells being in cycle, from the cell cycle phase durations and the age distribution. Ignoring the variability in cell cycle times may lead to large overestimates or underestimates in the expected labelling index. A method is given of obtaining a more accurate estimate of this variable, and hence of the proliferative proportion.

STUDIES ON THE KINETICS OF INITIAL CYCLE PROGRESSION IN VITRO OF ASCITES TUMOUR CELLS SUBSEQUENT TO ISOLATION FROM ASCITES FLUID

A method is described for determining the duration of cell cycle phases traversed by cells responding to release from proliferation restraint. Experiments have been performed with arrested Yoshida ascites hepatoma cells allowed to re-enter the growing stage after transfer of cells from the late stage of ascites into an in vitro incubation system. Experimentally, this method requires information on the rate of incorporation of labelled thymidine and on the rate of increase in cell number. The rate of [14C]thymidine incorporation in vitro was shown to be directly proportional to the number of cells synthesizing DNA. This was shown by correlating data from measurements of the rate of thymidine incorporation with those from measurements of the labelling index of the cell population. Theoretically, the method is based on analysis of the region limited by two integral curves, one corresponding to the kinetics of cell entry into and the other to the kinetics of exit from the S-phase. From data on the actual rate of increase in the total number of cells and data on the S-phase duration it is possible to obtain information on the cytokinetics of growth resumption by the ascites cell population.

In vitro resistance testing of tumors in relation to cytostatics. 1. Animal experiments

By studying rat Yoshida sarcoma and Walker carcinoma, pertinent results have been obtained for chemotherapy drug (CD) resistance of human tumors. The incorporation rates of tritiated metabolic precusors of DNA and RNA synthesis could be shown to be reproducible in ascites tumor cells and in solid tumor cells, in the absence as well as in the presence of CDS. Fraction of proliferation cells, cell cycle phase duration and tumor generation time were equal before and after explanation. The in vitro CD concentration used did not promote cell death within the first 4 h post-explantation, as shown by supravital stain (with Lissamin green and Trypan blue) and by 51Cr-release studies.

Cell cycle distributions, growth characteristics, and variation in prolactin and growth hormone production in cultured rat pituitary cells

Clonal strains of rat pituitary tumour cells (GH3 cells) spontaneously produce and secrete prolactin and growth hormone. Chromosome analysis and DNA ploidy measurements revealed that the GH3 cells in the present study were triploid and had a decreased chromosome number compared to the parent strain. Monolayer cultures of these cells grow exponentially for 6-7 days with a mean doubling time of 54 h. Cell cycle distributions and phase durations were determined by micro-flow fluorometric measurements of cellular DNA content combined with computer calculations. During exponential growth the cell cycle distribution did not change (65.4% cells with a G1 phase DNA content, 24.9% with an S phase DNA content, and 9.7% with a (G2 + M) phase DNA content). Counting of mitoses gave 1.4% cells in M phase. The 3H-Tdr labeling indices were determined by autoradiography, and the results were in good agreement with the number of cells in S phase as calculated by micro-flow fluorometry. The phase durations were: Ts=15.9 h, TG2=6.2 h, TM=1.1 h, and TG1=30.9 h. TS and TM calculated from 3H-Tdr labeled and Colcemid treated cultures gave corresponding results. In plateau phase cultures the number of cells with a G1 DNA content increased to 80% and the number of cells with an S phase DNA content decreased to between 5% and 10%. The specific production of prolactin and growth hormone determined by radioimmunoassay showed two and four-fold increases respectively, during exponential growth. The hormone values decreased to initial or subinitial values (day 2 values) when approaching plateau phase. We conclude: that changes in the cell cycle distribution of the cell population cannot be responsible for the spontaneous alterations in hormone production during growth and that most of the hormone-producing cells must be in the G1 phase.

Alternative modes of population growth inhibition in a human lymphoid cell line growing in suspension

Using a human lymphoid cell line grown under continuous culture conditions, two distinct plateau states were induced, either by lack of sufficient medium-supplied nutrient, or by other unknown mechanisms dependent on cell density. Flow microfluorometric measurements show that growth arrest due to nutritional insufficiency results in an accumulation of cells with G1 DNA content. In contrast, growth arrest due to high cell density is not associated with an altered distribution of cells with respect to DNA content as the population progresses from exponential to plateau state growth. Cell size decreases with progression of the plateau state induced by either type of growth arrest. Cells in a plateau state induced by high cell density utilize glucose and incorporate exogenous amino acid into protein at approximately the same rate as exponential cells. Proliferating, high cell density, plateau state cells have cell cycle phase durations similar to exponential cells. The stable, plateau state cell density is maintained by cell loss. No stable, unbound growth inhibitory factor was found in the medium of density-inhibited plateau state cultures.

The duration of the cell cycle of Daucus carota L. in vivo and in vitro

The duration of the cell cycle in Daucus carota seedling root-tips was measured by scoring the fraction of labelled metaphases after pulse labelling with ( 3H-TdR). This gave a total cycle duration of 7.5 h almost equally divided between the component stages, G 1, S, G 2 and M. The duration of the cell cycle in established diploid and tetraploid suspension cultures grown in the presence of 2,4-D was measured by continuous labelling with 3H-TdR. This gave mean cell cycle times almost identical with the mean cell number doubling times (specific growth rates) obtained from cell number growth curves. Specific growth rates were then used as estimates of the mean cell cycle duration for diploid and tetraploid lines grown in the presence and absence of 2,4-D. As the continuous labelling experiments showed that 93% of cells could incorporate 3H-TdR, cell cycle phase durations were calculated from measurements of the proportions of cells in G 1, S, G 2 and M. These proportions were determined by a combination of autoradiography, microdensitometry and mitotic index determination on culture samples incubated for short periods with 3H-TdR. In the presence of 2,4-D, both diploid and tetraploid cultures had cell cycle times of 45–58 h. In the absence of 2,4-D the diploid culture actively produced embryo plantlets with a mean cell cycle time of 33 h, whereas the tetraploid culture showed a cell cycle time of 82 h and remained undifferentiated. The extension and variation of the cell cycle in culture relative to the root-tip meristem was due almost entirely to changes in the duration of G 1. However, except in the root-tip and the differentiating diploid culture, G 1 and G 2 occupied constant proportions of the cell cycle. S phase duration under all cultural conditions was similar to that found in the root-tips but mitotic duration was variably increased. Despite this variation in mitotic duration, mitotic phase proportions in culture were mostly similar to those of the root-tip.

Selective chromosomal elimination during haploid formation in barley following interspecific hybridization

Cytological observations were made on embryo and endosperm tissues with different genome combinations that were produced by crossing the diploid and tetraploid cytotypes of Hordeum vulgare and H. bulbosum. The high frequency of barley haploids results from hybridization followed by the selective elimination of bulbosum chromosomes during the early development of embryos which initially contained a ratio of 1 vulgare to 1 bulbosum genomes. Elimination is gradual as indicated by the increase in the percentage of cells with the gametic chromosome number. However, the balance between genetic factors of the two parents appears to regulate the stability or elimination of chromosomes. Triploid embryos containing 1 vulgare to 2 bulbosum genomes are relatively stable. The most stable endosperm tissues examined had a ratio of 1 vulgare to 4 bulbosum genomes. Evidence of genetic control in both the vulgare and bulbosum chromosomes and their interaction is discussed. As has been suggested by Lange (1971) and also found in mammalian somatic cell hybrids, the most probable basis for selective chromosome elimination relates to mitotic rhythm and the duration of cell cycle phases.

Perturbation of Generation Cycle of Human Leukemic Myeloblasts In Vivo by Methotrexate

Abstract The cytokinetic effects of Methotrexate (MTX) on leukemic myeloblasts in vivo in man have been studied, utilizing in vitro and in vivo labeling with 3H-TDR, mitotic index, stathmokinetic index after VCR, measurements of single-cell DNA content, and in vitro labeling with 3H-UDR. It is demonstrated that MTX arrests cells in S for a period (about 20 hr) that corresponds to their DNA synthesis time. This S-phase arresting effect of MTX seems restricted to those cells that are in S at the time of MTX exposure. Cells in G1, G2, and M are not directly influenced by MTX. Some implications of these observations for combination drug therapy and for exploiting differences in the duration of cell cycle phases between leukemic blast cells and differentiating hemopoietic cells to obtain preferential killing of leukemic cells are briefly discussed. In one case, erythroblasts hardly incorporated exogenous TDR and UDR, although their mitotic activity was high. After MTX there was a transient but very pronounced increase in TDR and UDR ultilization. It is suggested that these cells may have had a large intracellular pool of thymidine triphosphate that was temporarily reduced by the action of MTX. Whether these erythroblasts were of leukemic origin remains undecided.

Depression of DNA synthesis and mitotic index by colchicine in cultured human lymphocytes

Colchicine, at concentrations of 5 μg/ml to 15 μg/ml administered for 1 h to 6 h caused significant depressions of DNA synthesis and of the mitotic index in PHA-stimulated human lymphocytes. Colchicine appeared both to prevent cells from beginning DNA synthesis and to reduce the rate of synthesis of cells already in S. The reduced mitotic index probably resulted to a large extent from depressed DNA synthesis, but there was evidence that colchicine also slowed the progress of cells throught G2. The use of colchicine at these concentrations can be a source of serious error in determinations of the duration of cell cycle phases and in kinetic studies based on metaphase counts after a mitotic block.