Duration Of G1 Research Articles

THE INFLUENCE OF ZINC ON THE CELL CYCLE IN THE ROOT MERISTEM OF A ZINC‐TOLERANT AND A NON‐TOLERANT CULTIVAR OF FESTUCA RUBRA L.

SummarySeedlings of a zinc tolerant cultivar (Merlin) and a non‐tolerant cultivar (S59) of Festuca rubra L. were grown in aerated solution culture at various concentrations of zinc.The elongation of the longest root was much more inhibited by increasing Zn concentration in S59 than in Merlin. The mitotic index in the root meristem was reduced to a greater extent by increasing Zn concentration in S59 than in Merlin, whilst the cell doubling time (estimated by colchicine arrest) was increased by Zn much more in S59 than in Merlin.The kinetics of the cell cycle in the root meristem were determined by a pulse labelling experiment with low specific activity tritiated thymidine following a 4‐d exposure to three concentrations of Zn (0, 0.1 and 0.2 μg Zn cm−3). Treatment with 0.1 and 0.2 μg Zn cm−3increased the length of the cell cycle by 40 and 132 % respectively (compared with the zero Zn control treatment) in S59 but only by 6 and 16% respectively in Merlin. The increase in cell cycle length was due mainly to an increase in the duration of G1 in both cultivars; Zn had little effect on the duration of the other phases of the cell cycle.The growth fraction (the proportion of cycling cells in the meristem) was progressively reduced by increasing Zn concentration in S59 but was increased at 0.1 μg Zn cm−3in Merlin and then reduced to the control level at the higher Zn concentration.

Flow cytometric determination of cell cycle parameters of V79 cells by continuous labeling with bromodeoxyuridine.

A simple method with which to determine the cell cycle parameters, TG1, TS and TG2M (the durations of the G1, S and G2 + M phases) is described. V79 Chinese hamster lung cells were used to evaluate the method. After continuous labeling with bromodeoxyuridine (BrdU), V79 cells were stained with anti BrdU-monoclonal antibody with FITC (fluorescein isothiocyanate) and with PI (propidium iodide). The individual cells were checked by flow cytometry for green and red fluorescences whose signal intensities corresponded to the BrdU and cellular DNA contents. The durations of G1, S and G2 + M phases of V79 cells were determined by measuring the cell fractions containing the nonlabeled G1, labeled S and nonlabeled G2 + M phases. The reliability of this method is discussed.

Karyotype, growth, and cell cycle analysis of human embryonic palatal mesenchymal cells: relevance to the use of these cells in an in vitro teratogenicity screening assay.

Human embryonic palatal mesenchyme (HEPM) is an established cell line that is presently under investigation as an in vitro prescreening assay used to determine the teratogenic potential of chemicals. We describe here general growth characteristics, karyotype, and cell cycle analysis of these cells. HEPM cells had plating efficiencies of less than 95% and displayed notable contact growth inhibition following an exponential growth phase that lasted for approximately 6 days. These cells had the diploid karyotype of a female human embryo. The chromosomal complement showed no dramatic change between passage 5 and 14. Flow cytofluorometry analysis using bromodeoxyuridine (BrdU) pulse labeling and a direct immunofluorescence anti-BrdU FITC probe revealed that the total cell cycle transit time was approximately 22 hr: the duration of G1 was 12.2 hr, S was 6.1 hr, and G2-M lasted for 3.7 hr. The results indicate that HEPM cells met the criteria regarding karyotype stability that were assumed by the National Toxicology Program of the USA.

Cell cycle traverse and protein metabolism in human NHIK 3025 cells: the role of anchorage.

We have studied the effect of cell anchorage on the human cell line NHIK 3025 in vitro, to see whether the growth regulating effect of cell anchorage primarily affected DNA division cycle or mass growth cycle. It was found that cell to cell anchorage had the same effect on cell cycle progression as anchorage to a solid surface, which indicates that it is anchorage per se and not cell shape that is important for growth control in NHIK 3025 cells. When NHIK 3025 cells were grown without attachment to a solid surface, both G1 and cell cycle duration was prolonged by 6 h, which means that the prolonged cell cycle was due to a prolonged G1. During the first part of the cell cycle the rate of protein synthesis and degradation was constant, and at the same level in cells grown with and without attachment. This means that the prolonged G1 was not due to a reduced protein accumulation or mass growth. Towards the end of the cell cycle protein accumulation was reduced. This effect was either due to a size control before cell division or a secondary effect of the prolonged G1. We therefore conclude that cell anchorage as a growth regulator primarily affects the DNA/cell division cycle.

Effects of light on the cell cycle in the shoot apex ofSilene coeli-rosa L. on the first day of floral induction

28-day-old plants ofSilene coeli-rosa were exposed, at 1,700 hours, to 5 minutes far-red light, 5 minutes red, 5 minutes far-red followed by 5 minutes red light, or maintained in darkness (short day controls). All plants were exposed to tritiated (methyl-3H-)thymidine for 2 hours (1645–1845) and subsequently sampled at 2-hour intervals for 24 hours. The length of the cell cycle (pulse-label mitoses (PLM) method) and changes in cell number were measured in the shoot apical meristems. The cell cycle in the short day controls was 16–17 hours compared with a mean cell generation time of 18 hours. Exposing plants to far-red light resulted in a shortening of the cell cycle to 11 hours, red light resulted in a shortened cycle of 12 hours whilst far-red, red gave a value of 9 hours. Mean cell generation times following each light treatment were approximetely 2–5 hours longer than the corresponding cell cycle times, suggesting that the shortened cell cycles reverted to longer cycles over the experimental period. Measurements of the proportions of cells with the 2C and 4C amounts of DNA in the apical meristems of unlabeled plants indicated that G1 shortened but G2 lengthened in response to far-red light. A measurement of the labeling index also indicated that S-phase shortened in response to far-red. These data also suggested that red light caused G1 to shorten and G2 to lengthen although the corresponding PLM curve was consistent with a dramatic shortening of G2. Far-red followed by red resulted in decreases in the durations of both G2 and G1.

Cell kinetic studies in the epidermis of mouse. III. The percent labelled mitosis (PLM) technique.

Full PLM curves have been obtained for four sites in the mouse. The first peaks have been analysed by computer and the duration of the G2 + M and S phases determined together with their standard deviations. The full curves showed a general similarity for all four sites with no clear second peak. The data are compared with the published data for mouse and human epidermis using the in vivo PLM technique. The timing and shape of the first peak can vary considerably even for one site in mice. Hence, both G2 + M and S can vary in their durations. Cells labelled at one time of day exhibit different kinetic properties to those labelled at another time of day. The duration of G2 + M is shortest in dorsum labelled at 03.00 hours (3 X 2 hr) and longest in tail (up to 7 X 5 hr). The S-phase is shortest in dorsum (6 X 3-7 X 2 hr) and longest in tail or ear (13 X 3-14 X 1 hr). There is also a very large standard deviation in tail and foot. There is little general variability when the psoriatic human data are considered, which is surprising. The general variability amongst the data from experimental mice might also be expected amongst humans which might make comparisons between the cell kinetics of normal and diseased skin difficult.

Autoradiographic studies of DNA replication in Werner's syndrome cells.

We have compared cultured fibroblasts of early passage derived from patients with the Werner syndrome and from normal subjects in several aspects of cell cycle time and DNA replication. The average cycle time was prolonged in Werner's syndrome cells compared with normal cells because of changes in the duration of S phase. The durations of G1 and G2 were unchanged. In addition, the labeling index was lower in Werner's syndrome cells, suggesting that there are two cell-cycle abnormalities in Werner's syndrome cells. The cause of the prolongation of S phase was investigated by DNA fiber autoradiography and alkaline sucrose density gradient sedimentation. The rate of DNA chain elongation was not different in Werner's syndrome cells from that in normal cells, but the frequency of replication initiation was decreased in Werner's syndrome cells.

The role of the G1 period in the life cycle of eukaryotic cells.

The objective of this study was to test the concept that the G1 period lacks any specific function in the life cycle of mammalian cells and hence could be drastically reduced without any effect on the generation time. HeLa cells were grown in medium containing an optimum dose (60 microM) of hydroxyurea at which the duration of S period was prolonged with little or no increase in generation time. At this concentration of hydroxyurea, we observed a maximum of 3 h (or 28.5%) reduction in the G1 period. We also studied the effects of synchronization in S phase by single and double thymidine blocks on cell size and its relationship to the duration of G1 in the subsequent cycle. By these treatments, we could reduce the G1 period by not more than 2 to 3 h. The reduction in G1 period was not directly proportional to the size (volume) of the G1 cells. These results suggest that G1 period has certain specific functions and cannot be eliminated by alterations in culture conditions.

Cell cycle parameters in radiation sensitive strains of Saccharomyces cerevisiae.

Cell cycle parameters in different radiation-sensitive strains of diploid yeast were determined by flow cytofluorometry. The cell generation time was increased in homozygous rad2 and rad51 mutants but was not significantly different from the wild type in rad9 and rad6 mutants. All mutants had a longer G1-phase than the wild type. A lengthened S-phase was found in rad2 cells. Rad51 mutants displayed a considerably longer duration of G2.

Triiodothyronine stimulates growth of cultured GC cells by action early in the G1 period.

T3 regulates the proliferation rate of GC cells, a rat GH-secreting pituitary tumor cell line, by modulating the duration of the G1 period. In GC cells which were synchronized at the beginning of G1 by mitotic selection, the G1 period was 11.7 +/- (SE)2.1 h, in agreement with the value we obtained in asynchronous cells by other methods (10.0 +/- 0.9 h). G1 was significantly prolonged in synchronized GC cells, previously maintained in the absence of T3 for more than 1 cycle time. G1 duration decreased when cells were exposed to T3 only during a discrete 4-6 h interval early in G1. Thus, T3 appeared to stimulate progression through the G1 period by action early in G1.

Kinetics of the nuclear division cycle of Aspergillus nidulans.

We have analyzed the cell cycle kinetics of Aspergillus nidulans by using the DNA synthesis inhibitor hydroxyurea (HU) and a temperature-sensitive cell cycle mutant nimT that blocks in G2. HU rapidly inhibits DNA synthesis (S), and as a consequence progression beyond S to mitosis (M) is blocked. Upon removal of HU the inhibition is rapidly reversible. Conidia (asexual spores) of nimT were germinated at restrictive temperature to synchronize germlings in G2 and then downshifted to permissive temperature in the presence of HU. This procedure synchronizes the germlings at the beginning of S in the second cell cycle after spore germination. We have measured the total duration of S, G2, and M as the time required for these cells to recover from the HU block and undergo the next nuclear division. The duration of S was defined by the time course of sensitivity to reintroduction of HU during recovery from the initial HU block. The cell cycle time was measured as the nuclear doubling time, and the duration of mitosis was determined from the mitotic index. The duration of G1 was calculated by subtracting the combined durations of S, G2, and M from the nuclear doubling time, and the length of G2 was calculated by subtracting S and M from the aggregate length of S, G2, and M. We have also determined the duration of the phases of the cell cycle during the first cycle after spore germination. In these experiments spores were germinated directly in HU without first being blocked in G2. Because the durations of G1, S, G2, and M for the first cell cycle after spore germination were identical with those previously determined for spores presynchronized at the beginning of S in the second cell cycle, we conclude that dormant conidia of A. nidulans are arrested at, or before, the start of S.

The G1 distribution of “G1-less” V79 Chinese hamster cells

The V79-8 line of Chinese hamster cells has been reported to lack a measurable G1 phase. However, using a combination of time-lapse cinemicroscopy and [ 3H]thymidine autoradiography, we have found these cells to have a median G1 duration ranging from 1.4 to 2.6 h in different experiments, accounting for more than 15% of the median cycle time. The youngest cell labelled (in seven experiments) was 0.73 h old at the time of fixation suggesting a minimum G1 of between 0.40 and 0.73 h (the duration of the [ 3H]thymidine pulse being 0.33 h). In those experiments where steady-state proliferation could be established unequivocally, variability in G1 times accounted for all of the variability in cycle times. In addition, the distribution of G1 times (and cycle times) was well described by the two-transition version of the transition probability model. Nevertheless, changes in the average duration of G1 (and hence changes in the transition probabilities) played a comparatively minor role in determining proliferation rate. Instead, the length of S + G2 was markedly influenced by the composition of the culture medium. For purposes of comparison with the ‘G1-less’ V79-8 line, we have also examined a revertant derived from it (G1 +5c) reported to have regained a substantial G1 phase. We are able to confirm that its G1 is indeed longer, the youngest labelled cell being 2.48 h old at the time of fixation. Unlike the parent line, there appeared to be more variability in G1 times than could be explained by two random transitions alone. The proliferation rate of the G1 +5c revertant was unusually sensitive to the composition of the culture medium, suggesting the possibility of a metabolic defect.

Microspectrophotometric and autoradiographic study of the timing and duration of pre-meiotic DNA synthesis in Paramecium caudatum.

Timing and duration of pre-meiotic G1, S and G2 phases in Paramecium caudatum have been clarified by microspectrophotometry of DNA content and autoradiography with [3H]thymidine ([3H]dThd) and [3H]deoxyuridinemonophosphate ([3H]dUMP). Microspectrophotometric measurement of DNA content showed that cells in stationary phase have a G1 micronucleus, and that pre-meiotic DNA synthesis begins with the micronuclear swelling and ends immediately before the nucleus enters meiotic prophase. The results indicate that the micronucleus is in G1 during the first 1.5 h after onset of mating reaction at 25 degrees C and in pre-meiotic S phase during the following 1 h, and that G2 phase is very short or lacking. Therefore, in P. caudatum, the transition point from pre-meiotic interphase to meiotic prophase is at or very close to the end of pre-meiotic DNA synthesis. Autoradiographic study showed that although [3H]dThd and [3H]dUMP were well utilized in pre-mitotic DNA synthesis, only [3H]dUMP was well utilized in pre-meiotic DNA synthesis. [3H]dThd was utilized very little in pre-meiotic DNA synthesis even if the precursor was taken into the cell, suggesting that the activity of thymidine kinase is low in these cells.

Growth and death of HeLa cells in the presence of caffeine.

Proliferation and death were measured in synchronously growing cultures of HeLa S3 cells during treatment with up to 30 mM caffeine. Changes in the number of colony-forming cells were determined by single-cell plating, while changes in the total number of cells were measured both by electronic counting and by monitoring cell division and physiological death cinemicrographically. At concentrations between 2 and 5 mM, cell killing occurs over several days during which the cells traverse the generation cycle once or a few times before losing colony-forming ability, with consequent proliferation of non-colony-forming cells. This indicates that lethal damage is accumulated with time. Death occurs more rapidly at higher concentrations, without proliferation, the kinetics of inactivation being strongly dependent on the phase of the cycle (cell age) at which treatment is initiated. G1 cells are killed more slowly in 10 mM caffeine than are S cells, but G1 cells respond rapidly to 20 mM caffeine, suggesting the inception of an additional mode of killing. The incidence of sister-cell fusion increases with increasing caffeine concentration above 1 mM. On addition of 10 mM caffeine to a culture prepared from collected mitotic cells, the cells undergo a transient rounding and then respread after several hours; with 20 mM, they never respread. The generation cycle is prolonged in a concentration-dependent fashion, as is the duration of G1; the generation time is doubled in 5-6 mM caffeine. G2 and M are also prolonged at concentrations above 3 mM, but S is not prolonged even by 10 mM caffeine.

BrdU-Hoechst-Giemsa analysis of DNA replication in synchronized lymphocyte cultures. Study of human X and Y chromosomes.

The combination of a technique of reversible methotrexate (MTX) imposed G1/S block in cultures of human lymphocytes with the BrdU-Hoechst-Giemsa technique permitted the study of DNA replication patterns in individual chromosomes at different intervals of the S phase in a cell cohort with uniform S + G2 duration. The procedure did not increase either the frequency of chromosomal breakage or SCE frequency. The technique applied permitted visualization of the banding pattern in over 90% of mitoses. Examination of mitosis following different times of exposure to BrdU revealed a high degree of synchrony in the progression of the cell cohort examined through the S phase. The presence of two distinct late replication patterns of the allocyclic X chromosome was confirmed in studies on lymphocytes from normal human females by this technique. Interindividual and intercellular differences of the replication pattern have been demonstrated. The replicating patterns from one individual were relatively constant. The analysis of the Y chromosome has revealed marked differences of the termination of replication in individual cells. Euchromatic regions have been shown to complete DNA synthesis first, followed by the distal part of the long arm and, finally, by the region of Yq11/Yq12 junction. Lateral asymmetry was localised at this region.

RNA dependence in the cell cycle of V79 cells.

The cell cycle of V79 Chinese hamster lung cells synchronized by hydroxyurea was investigated by flow cytometry. The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA of V79 cells. Green and red fluorescence from individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. Periodic changes of cellular DNA and RNA contents were observed over nine cell cycles. The duration of G1, S, and G2 + M phases of synchronized V79 cells whose RNA content was close to that of the cells in balanced growth was 3, 4.5, and 1.5 hours, respectively. The duration of G1 and S phases of cells containing RNA above a certain threshold was inversely proportional to the RNA content. The RNA content of cells containing RNA above the normal level regressed to normal after a few generations. Coefficients of variation for RNA content were significantly larger than those for DNA. An explanation for the decay of synchrony in a synchronized cell population is proposed.

Growth Kinetics of Bp8 Mouse Ascites Sarcoma after Single Doses of Whole Body Irradiation: II. Analysis of the Progression of Cells Through the Cell Cycle

Bp8 ascites sarcoma cells growing in vivo were whole body irradiated with doses of 1.75 to 8 Gy. The inflow and outflow rates of cells in the various compartments of the cell cycle were estimated on the basis of sequential analysis of the total number of cells and from the proportion of cells in G1, S-phase, G2 and M. The flow through the compartments was calculated from these data and from the sizes of the cell pools. The durations of the mitotic delay, the G1-depletion and the early G2 blockage were linearly related to the dose. After release of the mitotic division delay the accumulation rate of cells in M and G1 decreased linearly with the dose; for G2, plateau values for the accumulation rate were found after 2.5 Gy. The outflow rates from G2 and M after release of the G2 blockage showed a shoulder type of dose-response with a D0 of 2.5 Gy and a n value of 1.5. In addition to an increase in the duration of G2, generally responsible for the increase in the total cell cycle time at about 24 hours after irradiation and an increase in the duration of the S-phase up to 12 hours after irradiation, an early increase in the duration of G1 and M was observed.

Cell Cycle Kinetics in the Primary Root Meristem ofVicia Faba: Influence of Different Ambient Water Levels

SUMMARYCell cycle kinetics were determined for roots grown either in tanks of water from 24 to 144 hr or in a moistened mixture of vermiculite and perlite (V/P) from 24 to 96 hr. For roots grown in tanks of water Tc was 23.4 hr (TG1 = 6.1 hr, TG2—5.5 hr, T2 = 9.6 hr, Tm = 2.3 hr) while roots grown in V/P had a shorter Tc of 17 hr (TG1 = 5.6 hr, TG2 = 4.0 hr, T, = 5.0 hr, Tm = 2.4 hr). Most of the difference in cycle times was due to a disproportionate increase in the duration of S for water grown roots while the duration of G1 and G2 showed relatively smaller changes. This difference in TS was coupled with an increase in L.I. in water grown roots over that seen in V/P grown roots, i.e. 31.0 ± 8.6 cf. 25.9 ± 6.5. Changes in the proportions of fast, slow and non-cycling cells also occurred; i.e. in V/P grown roots there were 83.2% fast, 11.4% slow and 5.4% non-cycling cells, while in tanks of water there were 52.9% fast, 22.7% slow and 24.4% non-cycling cells. The results are evidence of differences both in mean cycle duration and phase duration within the cell cycle for roots grown in different volumes of water. They also show that roots respond to different volumes of water by altering their GF and the proportions of fast, slow and non-cycling cells in their meristems. These changes may be part of a mechanism used by roots to regulate the overall rate of cell proliferation and thus to achieve different TDT values under different environmental conditions.

Effect of n-butyrate on cell cycle progression and in situ chromatin structure of L1210 cells

In the presence of 1–5 mM n-butyrate, murine leukemic L1210 cells cease proliferation and become arrested in the G1A compartment of the G1 phase. Cells in this compartment, in comparison with the remaining cells of the G1 phase (G1B), are characterized by low RNA content and more condensed chromatin. During unperturbed growth the cell residence times in G1A are of indeterminate duration (exponentially distributed); the half-time of L1210 cell residence in G1A is about 1.4 h. The effect of n-butyrate in arresting cells in G1A was concentration-dependent. However, the sensitivity of L1210 cells to this drug was markedly enhanced when cells were treated for longer than one generation (12 h). Cells arrested in G1A remained viable and when n-butyrate was removed, after a lag period, they resumed progression through the cycle. The effect of n-butyrate on cell progression through various parts of the cycle was studied in a stathmokinetic experiment. The rate of cell entrance into mitosis was decreased by 30, 60 and 110%, in the presence of 1, 2.5 and 5 mM n-butyrate respectively, thus indicating a slowdown in cell progression through G2 and S. The duration of G2 was prolonged by 20, 70 and 140% at 1, 2.5 and 5 mM n-butyrate respectively. The half-time of cell residence in G1A was increased by as much as 1.5-, 6.3- and 15.6-fold by 1, 2.5 and 5 mM n-butyrate. Progression through late G1 (G1B) was not affected at 1 mM, and could not be estimated at higher drug concentrations. The effects on cell cycle progression were evident 1 h after addition of n-butyrate. DNA in situ in nuclei of n-butyrate-treated cells had lowered (by 2–8 °C) stability to thermal denaturation and increased (by 15%) accessibility to DNase I. The decrease in DNA stability to heat was more pronounced when permealized cells were heated in the presence of 1 mM MgCl 2 rather than EDTA. DNA in situ in the nuclei of n-butyrate-treated cells also showed decreased sensitivity to acid-induced denaturation. Changes in chromatin were seen in all cells, regardless of cell cycle phase, within the first hours after addition of n-butyrate. Mitotic cells, however, reacted to n-butyrate more rapidly than interphase cells. The observed changes in L1210 cells are most likely a consequence of histone modifications (acetylation of inner histones, dephosphorylation of histone H1) induced by n-butyrate.

The role of protein accumulation in the cell cycle control of human NHIK 3025 cells.

The cell cycle kinetics of NHIK 3025 cells, synchronized by mitotic selection, was studied in the presence of cycloheximide at concentrations (0.125-1.25 microM) which inhibited protein synthesis partially and slowed down the rate of cell cycle traverse. The median cell cycle duration was equal to the protein doubling time in both the control cells and in the cycloheximide-treated cultures at all drug concentrations. This conclusion was valid whether protein synthesis was continuously depressed by cycloheximide throughout the entire cell cycle, or temporarily inhibited during shorter periods at various stages of the cell cycle. These results may indicate that cell division does not take place before the cell has reached a critical size, or has completed a protein accumulation-dependent sequence of events. When present throughout the cell cycle, cycloheximide increased the median G1 duration proportionally to the total cell cycle prolongation. However, the entry of cells into S, once initiated, proceeded at an almost unaffected rate even at cycloheximide concentrations which reduced the rate of protein synthesis 50%. The onset of DNA synthesis seemed to take place in the cycloheximide-treated cells at a time when the protein content was lower than in the control cells. This might suggest that DNA synthesis in NHIK 3025 cells is not initiated at a critical cell mass.