EXPERIMENTAL CONTROL OF DNA SYNTHESIZING AND DIVIDING CELLS IN EXCISED ROOT TIPS OF PISUM

The number of dividing and DNA-synthesizing cells in excised pea roots can be regulated by eliminating the carbohydrate normally supplied in the culture medium. When the excised roots were allowed to remain for 24 hr in a medium lacking carbohydrate, the number of mitotic figures and tritiated thymidine (H³-T) labeled cells was reduced almost to zero. After an additional 24 hr in the incomplete culture medium, 15% of the interphase cells were H³-T labeled, the percentage of the cells that were dividing never exceeded 1.4, and 30% of these were H³-T labeled. When the roots remained in the deficient medium for 72 hr, neither cell division nor cells synthesizing DNA were observed. Upon addition of 2% sucrose, cell division and DNA synthesis were resumed in the roots that were maintained for 24 or 72 hr without an exogenous carbohydrate supply. It has been hypothesized that some proliferative systems consist of two cellular subpopulations which selectively stop or remain in either the pre-DNA synthetic (G₁) or post-DNA synthetic (G₂) periods of the mitotic cycle. The addition of sucrose, H³-T, and 5-aminouracil to the medium, after the roots had been maintained for 24 hr without a carbohydrate, indicated that most of the proliferative cells in the roots had accumulated in either G₁, a quasi-G₁ condition, i.e., DNA synthesis stopped sometime before completion, or G₂ periods of interphase; the majority, however, were in G₁ or quasi-G₁ conditions. The results suggested that DNA synthesis (S period) and mitosis or the onset of these processes have the highest metabolic requirements in the mitotic cycle and that G₁ and G₂ were the most probable states for proliferative cells in a meristem with a low metabolic level.     

Autoradiographic study of the effect of 5-aminouracil on the S phase and mitosis of barley root meristems

The effect of 5-aminouracil on the S phase and mitosis in root meristems of barley embryos cultivated in the liquid nutrient solution was followed. Embryos were cultivated in different concentrations of 5-aminouracil (200 ppm, 400 ppm and 750 ppm) for 48 h. The drug postponed the onset of mitosis. In the lowest concentration used, synchronization was observed even in the presence of 5-aminouracil. In higher concentrations, mitosis was suppressed irregularly with increasing concentration. 5-aminouracil slowed down the rate of DNA synthesis during S phase and prolonged the S phase, as measured by the utilization of [³H] thymidine. The drug does not influence considerably the entry of cells into the S phase. The transition from G₂ to mitosis is delayed in the presence of 5-aminouracil, especially in higher concentrations. After prolonged treatment with 5-aminouracil, all the effects of the drug on the mitotic cycle decrease continuously.     

High sensitivity of DNA replication to 5-aminouracil in the middle of the S period

Summary Cell distribution in different compartments of the cell cycle (G₁, early, middle and late S, G₂ and mitosis) has been studied during treatment with 0.5 mM 5-aminouracil and recovery inAllium cepa L. root meristems by cytophotometric and autoradiographic methods. At optimum conditions for obtaining mitotic synchronization, 5-aminouracil gives rise to cell accumulation in the S period, preferentially in its middle zone where the relative DNA content is 2.8 ± 0.1 C. After a 14-hour treatment 33% of the proliferative population is accumulated in this particular region.During recovery, a drastic reduction of the S phase and a clear increase of the mitotic frequency are the most important events observed. Apparently, the removal of the drug frees the blockage and the accumulated cells complete their interphase making up the mitotic wave.     

INCORPORATION OF TRITIUM-LABELED THYMIDINE AND LYSINE INTO CHROMOSOMES OF CULTURED HUMAN LEUKOCYTES

The incorporation of thymidine-H³ and lysine-H³ into human leukocyte chromosomes was studied in order to determine the temporal relationships between the syntheses of chromosomal deoxyribonucleic acid and chromosomal protein. The labeled compounds were incorporated into nuclei of interphase cells. Label from both precursors became apparent over the chromosomes of dividing cells. Incorporation of thymidine-H³ occurred during a restricted period of midinterphase (S) which was preceded by a nonsynthetic period (G₁) and followed by a nonsynthetic period (G₂). Incorporation of lysine-H³ into chromosomal protein occurred throughout interphase. Grain counts made over chromosomes of dividing cells revealed that the rate of incorporation of lysine-H³ into chromosomal protein differed during various periods of interphase. The rate of incorporation was diminished during G₁. During early S period the rate of incorporation increased, reaching a peak in late S. The high rate continued into G₂. Thymidine-H³ incorporated into DNA was distributed to mitotic chromosomes of daughter cells in a manner which has been referred to as a "semi-conservative segregation." No such semi-conservative mechanism was found to affect the distribution of lysine-H³ to the mitotic chromosomes of daughter cells. Therefore, it is concluded that synthesis of chromosomal protein and its distribution to chromosomes of daughter cells are not directly influenced by synthesis and distribution of the chromosomal DNA with which the protein is associated.     

THE ONSET OF MITOSIS AND DNA SYNTHESIS IN ROOTS OF GERMINATING BEANS

The time of onset of mitosis and DNA synthesis has been determined in roots of germinating seeds of Vicia faba. Mitosis is not initiated in all roots simultaneously. Dividing cells are seen 36 hr from the beginning of germination, but they are present in low frequency (0.02%). Dividing cells do not become frequent, i.e., occurring as 5% or more of all cells, until 56 hr, and it is not until 66–68 hr that all roots in a sample of 10 are mitotically active. DNA synthesis shows a similar sporadic beginning. It occurs in a few cells by 28 hr, and by 40 hr all roots exposed to ³H–thymidine show active incorporation. For most cells in these germinating roots DNA synthesis precedes mitosis. In one root in 10, however, some cells are unlabeled when they enter mitosis, indicating that they had spent the dormant period in the G₂ phase of the mitotic cycle. The presence of these cells determines whether or not roots show chromatid and chromosome aberrations following irradiation during germination.     

Cell cycle duration and time of DNA synthesis in binucleate cells induced inV. faba meristems by caffeine or isobutyl-methylxanthine

Summary Lateral roots ofVicia faba were treated with a solution of 5-aminouracil (3.93×10^–3M) for 6 hours. After 15 hours roots were recovering from the temporary inhibition of mitosis induced by 5-AU and were approaching peak mitotic indices; they were then treated with 0.1% caffeine or 0.1% isobutylmethylxanthine (IBMX) for 1 hour. Treatment with methylxanthines when the mitotic index was high gave relatively high yields of binucleate cells, 3.8 to 7.5%. DNA synthesis, cell cycle duration and nuclear growth were determined for binucleate cells. Caffeine induced binucleate cells underwent a marked reduction in nuclear volume, from 1,074 m³ at 1+1 hours to 534 m³ at 1+14 hours. Only 15% of these binucleates entered S phase; those that did so were in mitosis or had divided by 1+14 hours. We conclude that 85% of the binucleate cells are so inhibited by caffeine that their G₁ is extended to>14 hours or that they are no longer proliferating cells. IBMX-induced binucleate cells, by contrast, did enter S phase and many of them also divided. Though in IBMX-induced binucleate cells there was also a decrease in nuclear volume up to 1+10 hours, subsequently mean nuclear volume increased e.g. at 1+16 and 1+18 hours. Both caffeine and IBMX treatments resulted in decreases in mean volume of prophase nuclei of mononucleate cells; this is further evidence that both methylxanthines inhibit the macromolecular synthesis required to sustain nuclear growth. It also suggests that nuclear division can be initiated at considerably lower nuclear volumes than those of untreated cells. We suggest that caffeine may act as a mimic of the normal mechanism that regulates the switch from a proliferating to a non-proliferative condition.     

EFFECTS OF NEAR ULTRAVIOLET AND VISIBLE RADIATIONS ON CELL CYCLE KINETICS IN EXCISED ROOT MERISTEMS OF PISUM SATIVUM

Near-ultraviolet and visible radiations increased the duration of the mitotic cycle in excised pea root meristems primarily by lengthening the duration of the pre-DNA synthetic period (G₁). All radiations tested shortened the duration of the post-DNA synthetic period (G₂). The most pronounced effects were exhibited by green radiation, which lengthened the duration of the cell cycle, G₁, DNA synthesis (S), and mitosis (M), and shortened the duration of G₂. Progression of cells arrested by starvation in G₁ and G₂ into DNA synthesis and mitosis was also affected by light treatments. Green radiation appeared to arrest a group of cells in DNA synthesis as well as in G₁ and G₂. Meristems receiving green and near-ultraviolet radiations exhibited the most rapid progression of G₁ cells through S and G₂.     

Heterogeneity in G2 duration during lateral root development

Developing lateral roots of V. faba were treated with 5-aminouracil for up to 6 hours using the 5-AU inhibition method discussed in this paper; the duration of G₂+mitosis/2 and the percentages of slow dividing cells were estimated from the fall in MI observed in just emerged meristems, very large primordia and large primordia. The results indicate that during the period of development studied here there are two subpopulations of dividing cells: 1) fast dividing population which makes up about 84 % of the dividing cells and which has a G₂+mitosis/2 duration of about 3.3 hours, and 2) a slow dividing population which constitutes about 16 % of the dividing cells and which has a G₂ duration in excess of 12 hours. This heterogeneity is discussed in relationship to the behaviour of different populations of proliferating cells during root morphogenesis.     

Cadmium cytotoxicity and variation in nuclear content of DNA in Euglena gracilis

Cultures of Euglena gracilis (strain Z from French CNRS collection) can be made cadmium resistant if grown in a medium with 5x10^-4M cadmium chloride. This resistance is reflected by the appearance of a second exponential growth phase. The development of this resistance was studied at the cellular level by determining the relative content of DNA at different stages of the cell cycle in an asynchronously grown culture. The culture was followed until the second, cadmium resistant, growth phase had reached its stationary state. During the first exponential growth phase, cells were mostly in the late period of DNA synthesis (stage S of the cell cycle), or in the gap preceding mitosis (stage G₂ of the cell cycle). In addition, some cells contained high multiples of the normal amount of DNA. In the beginning of the second exponential growth phase, a few cells were again in G₁ (the post mitotic stage of the cell cycle preceding DNA synthesis). These G₁ cells were predominant at the end of the second growth period. During the second stationary phase the DNA content of the cadmium treated cells was similar to the stationary phase of the control culture. Cells had stopped growing in G₁ with an unreplicated genome. The implications of these data are discussed.     

EFFECTS OF IRRADIATION ON THE GENERATIVE CYCLE OF THE ESTROGEN STIMULATED VAGINAL EPITHELIUM

The effects of irradiation (300, 500 and 1500 rads) on mitosis and DNA synthesis in the estrogen primed vaginal epithelium have been studied. Dose-effect relations and the time sequence of effects on the two processes were investigated. The technique of tritiated thymidine labeling of DNA with autoradiography was used, in conjunction with the mitotic count, to study alterations in the generative cycle. Prior to irradiation, ovariectomized female rats were treated daily with diethylstilbestrol for a period of 2 weeks to create a steady state in the vaginal cell population. It was observed that:

• 1 Within 1 hr following ionizing radiation, mitotic figures disappear from the population and reappear at a time that is dose dependent. Those cells that have begun mitosis at the time of irradiation were able to complete that phase but no cells which were in G₂ were able to begin mitosis. Therefore, a G₂ block occurs within 1 hr post-irradiation.
• 2 Radiation reduces the rate of DNA synthesis thus prolonging the S phase. There is no evidence of a radiation-induced G₁ to S block in this system.

Based on these observations, it was further hypothesized that:

• 1 Cells in G₁ at the time of irradiation are relatively insensitive and continue to progress through the generative cycle at a rate primarily determined by the level of estrogen stimulation.
• 2 Radiation may interfere with the estrogen priming mechanism in this hormonedependent system thereby reducing the effective level of estrogen stimulation. This is seen in the behavior of cells which were in G₁ at the time of irradiation. The extent of the blockage of estrogen increases with radiation dose and after 1500 rads, estrogen stimulation is essentially at castrate level.

     

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING ADHESIVE TAPE STRIPPING

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) (Laerum, 1969) and brought into a mono-disperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G^ S and (G₂+ M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. ³H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G₂ cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G₂ pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G₂ phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G₂ phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the Gj phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.     

INDEPENDENCE OF CENTRIOLE FORMATION AND DNA SYNTHESIS

The temporal relationship between cell cycle events and centriole duplication was investigated electron microscopically in L cells synchronized by mechanically selecting mitotic cells. The two mature centrioles which each cell received at telophase migrated together from the side of the telophase nucleus distal to the stem body around to a region of the cytoplasm near the stem body and then into a groovelike indention in the early G₁ nucleus, where they were found throughout interphase. Procentrioles appeared in association with each mature centriole at times varying from 4 to 12 h after mitosis. Since S phase was found to begin on the average about 9 h after mitotic selection, it appeared that cells generated procentrioles late in G₁ or early in S. During prophase, the two centriolar duplexes migrated to opposite sides of the nucleus and the daughter centrioles elongated to the mature length. To ascertain whether any aspect of centriolar duplication was contingent upon nuclear DNA synthesis, arabinosyl cytosine was added to mitotic cells at a concentration which inhibited cellular DNA synthesis by more than 99%. Though cells were thus prevented from entering S phase, the course of procentriole formation was not detectibly affected. However, cells were inhibited from proceeding to the next mitosis, and the centriolar elongation and migration normally associated with prophase did not occur.     

CELL-PRODUCTION RATES ESTIMATED BY THE USE OF VINCRISTINE SULPHATE AND FLOW CYTOMETRY I. AN IN VITRO STUDY USING MURINE TUMOUR CELL LINES

A method for the evaluation of cell-production rates is described which combines flow cytometry (FCM) and the stathmokinetic method. By means of FCM it is possible to estimate the distribution of cells with G₁, S and (G₂+ M) DNA content in a population. As this method gives the relative (G₂+ M) DNA content of cells within the cell cycle, it may be possible to evaluate cell-production rates by this technique. In the present study it was found that administration of a metaphase-arresting (stathmokinetic) agent, vincristine sulphate (VS), to asynchronous cell populations of three different murine tumour cell lines in vitro increased the peak representing cells with (G₂+ M) DNA content as the number of mitotic (M) cells increased during the period of treatment. The accumulation of mitotic cells was determined by cell counts on smears under the microscope and compared with the increase in the (G₂+ M) DNA peak measured by FCM as a function of time after the administration of VS. Good agreement was obtained between the cell-production rates as estimated by FCM and by mitotic counts in all three cell lines investigated.     

Culture conditions for arresting and stimulating the proliferation of a rainbow trout fibroblast cell line,RTG-2

Summary Conditions for arresting and stimulating the proliferation of the rainbow trout fibroblast cell line RTG-2 have been examined and the time course of events after stimulation determined. Quiescent populations were achieved in two ways. Cultures grown to confluency without a medium change for at least 7 d had fewer than 5% of the cells in S phase and few mitotic figures. Cultures deprived of serum, which could be done for up to 3 d without a loss in cell number, also achieved quiescence. After 3 d without serum, less than 1% of cells were in S phase and mitotic figures were infrequent. Addition to these cultures of fresh serum-containing medium brought about the synchronous entry of cells into S phase and mitosis. For cultures in which either the medium had been changed after 7 d without a change or serum-containing medium had been added after 3 d of serum deprivation, DNA synthesis increased after a lag period of 20 to 24 h, was pronounced between 30 and 45 h, and then declined. This was followed by a peak in the mitotic index. These protocols for arresting and subsequently stimulating RTG-2 proliferation should allow the G₁-S transition to be studied in a representative of teleosts. This research was supported by Natural Sciences and Engineering Research Council of Canada grant to N. C. B.     

Nucleic acids methylation of synchronized BHK 21 HS 5 fibroblasts during the mitotic phase

The methylation of nucleic acids has been investigated during the cell cycle of an asparagine dependent strain of transformed fibroblasts (BHK 21 HS 5). The synchrony was carried out by a partial asparagine starvation of cells for 24 hours. The amino acid supply induced all cells to enter synchronously the G₁ phase. Methylation and DNA synthesis were respectively measured by pulsed [methyl-¹⁴C] methionine and [methyl-³H] thymidine incorporation. DNA methylation followed a biphasic pattern with maximal methyl incorporations during both S phase and mitosis. A partial desynchronisation induced the S phase of the second cycle to proceed before all the cells have achieved their division. Hydroxyurea was used in order to inhibit the DNA synthesis of cells entering the second cell cycle, which might interfer with the mitosis of the first one. The inhibitor was added either at the first beginning of cell division or during all the G₁ phase. In both conditions it suppressed ³H thymidine incorporation of the second cycle. However, mitosis took place and methylations occurred as in previous experiments. The DNA methylation of the mitotic phase in the first cell cycle could thus be dissociated from the classical post-synthetic DNA maturation and did not correspond to any DNA methylation appearing in the course of the second cell cycle.     

5-AMINOURACIL TREATMENT : A Method for Estimating G2

Treatment of Vicia faba lateral roots with a range of concentrations of 5-aminouracil (5-AU) indicate that cells are stopped at a particular point in interphase. The timing of the fall in mitotic index suggests that cells are held at the S - G₂ transition. When cells are held at this point, treatments with 5-AU can be used to estimate the duration of G₂ + mitosis/2 of proliferating cells. Treatment with 5-AU can also be used to demonstrate the presence of subpopulations of dividing cells that differ in their G₂ duration. Using this method, 5-AU-induced inhibition, we have confirmed that in V. faba lateral roots there are two populations of dividing cells: (a) a fast-dividing population, which makes up ~85% of the proliferating cell population and has a G₂ + mitosis/2 duration of 3.3 hr, and (b) a slow-dividing population, which makes up ~15% of dividing cells and has a G₂ duration in excess of 12 hr. These estimates are similar to those obtained from percentage labeled mitosis (PLM) curves after incorporation of thymidine-³H.     

STUDIES ON THE REGULATION OF LYMPHOCYTE PRODUCTION IN THE MURINE THYMUS AND SOME EFFECTS OF A CRUDE THYMUS EXTRACT

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0–8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering Go/Gi cells into DNA synthesis, the inhibition of G₂ efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.     

The effect of lead on the cell cycle in the root meristem ofAllium cepa L.

Summary Allium cepa (L.) adventitious roots were treated with lead (2.5 mg of Pb²⁺ [from Pb(NO₃)₂] per dm³) for 30–72 h. The cell cycle was studied by pulse labeling with [³H]thymidine. Mitotic activity kinetics, occurrence of disturbed mitoses (c-mitoses), and level of DNA synthesis were examined. It was found that lead prolonged the cell cycle and that cells in two phases of the cycle, G₂ and S, differed in their sensitivity to lead. Cells in G₂ were more sensitive; lead lengthened their cycle by 216% and disturbed the course of cell division by causing c-mitoses. Cells in S phase were less sensitive. Their cell cycle was longer by 55%. They went through their G₂ phase without major disturbances, mitosis in these cells was normal. During treatment ofA. cepa with lead, its destructive effects on cells were exerted only during the first few hours (around 6 h) of incubation. That is when the inhibition of mitotic activity, numerous disturbances of cell division, a decline in the number of cells synthesizing DNA, and a lower level of DNA synthesis were observed. As the incubation continued, the above processes were found to return to normal. In the discussion, data are presented supporting the hypothesis that during the initial period of exposure ofA. cepa to lead, this metal enters both the root apoplast and symplast, exerting a destructive effect on cells, while later, lead penetrates only into the root apoplast, and in this way remains harmless to cells.     

STATIONARY PHASE OF CULTURED MAMMALIAN CELLS (L5178Y)

The stationary phase of the mammalian cells L5178Y in culture can be divided into two stages: (a) an early phase characterized by the decline of mitotic index, followed by a stabilization of the cell number, and (b) a late stage, occurring several hours after the flattening of the growth curve, during which dead or dying cells appear in the cultures. The estimates of rates of cell progress showed that the rates from G₁ to S and from G₂ to M were affected in the early stationary phase. The main cause of cessation of increase in cell number in the early stationary phase is resulted from the decline in mitotic index, which is caused by prolongation of the G₂ period. The importance of the G₂ stage in regulating the cell growth is discussed in relation to other known situations in the literature.