A Cytological Analysis of the Antimetabolite Activity of 5-Hydroxyuracil in Vicia faba Roots

The effects of 5-hydroxyuracil (5-HU) (isobarbituric acid) upon cell elongation, mitosis, and DNA synthesis were studied in Vicia faba roots. 5-HU had no consistent effect upon root elongation. It blocked DNA synthesis (analyzed by photometric measurements of Feulgen dye in nuclei) during the first 6 hours of treatment; the block spontaneously disappeared by the 12th hour of treatment. Uracil and thymine had no effect upon this block of synthesis. Both thymidine and uridine reversed the block in 6 and 9 hours respectively. In all cases blockage of DNA synthesis was followed by inhibition of mitosis (determined by changes in the percentage of cells in mitosis) and resumption of DNA synthesis was followed by resumption of mitosis. Inhibition indices calculated from the mitotic data indicated a competitive relationship between 5-HU and thymidine and 5-HU and uridine. 5-HU is considered to block DNA synthesis by competing with thymidine for sites on enzymes involved in the synthesis. It is suggested that uridine reverses the block in synthesis by undergoing a conversion to thymidine.     

A cytological analysis of the antimetabolite activity of 5-hydroxyuracil in Vicia faba roots

The effects of 5-hydroxyuracil (5-HU) (isobarbituric acid) upon cell elongation, mitosis, and DNA synthesis were studied in Vicia faba roots. 5-HU had no consistent effect upon root elongation. It blocked DNA synthesis (analyzed by photometric measurements of Feulgen dye in nuclei) during the first 6 hours of treatment; the block spontaneously disappeared by the 12th hour of treatment. Uracil and thymine had no effect upon this block of synthesis. Both thymidine and uridine reversed the block in 6 and 9 hours respectively. In all cases blockage of DNA synthesis was followed by inhibition of mitosis (determined by changes in the percentage of cells in mitosis) and resumption of DNA synthesis was followed by resumption of mitosis. Inhibition indices calculated from the mitotic data indicated a competitive relationship between 5-HU and thymidine and 5-HU and uridine. 5-HU is considered to block DNA synthesis by competing with thymidine for sites on enzymes involved in the synthesis. It is suggested that uridine reverses the block in synthesis by undergoing a conversion to thymidine.     

Regulation of mitosis onset and thymidine kinase activity during the cell cycle of Physarum polycephalum plasmodia: effect of hydroxyurea

The effects of hydroxyurea have been investigated on three events of the cell cycle, S-phase, mitosis, and the cyclic synthesis of thymidine kinase, in the synchronous plasmodium of the myxomycete Physarum. DNA synthesis was slowed down with limited action on other macromolecular syntheses and any increase of thymidine kinase that had already been triggered was indistinguishable from that of the control. When DNA synthesis was inhibited, the onset of the following cyclic increase of thymidine kinase synthesis occurred at the same time as in the control, but mitosis was delayed in a very early prophase stage. The arrest of thymidine kinase synthesis occurred after completion of the delayed mitosis. All these effects were suppressed when the action of hydroxyurea was prevented by the addition, to the medium, of the four deoxyribonucleosides. These observations show that (1). The blockage of S-phase does not prevent the nuclei from entering a very early prophase stage but does prevent them from proceeding through metaphase. (2) The transient blockage of DNA synthesis does not perturb the normal timing of the triggering of thymidine kinase synthesis. (3) The signal which triggers the arrest of thymidine kinase synthesis is postmitotic but does not require extensive DNA synthesis. The effect of hydroxyurea is not limited to an inhibition of S-phase. The blockage of DNA replication also led to the dissociation of the normal coordination between two other events of the cell cycle, mitosis and thymidine kinase synthesis. This observation could have strong implications in cell synchronization with chemical agents.     

Delays in prophase induced by adenosine 2-deoxyriboside and their relation with DNA synthesis

The rate of DNA synthesis in the course of the division cycle in root meristem ofAllium cepa growing under constant temperature and aeration conditions has been studied by means of treatment with AdR, as a specific inhibitor of the synthesis, as well as by the incorporation of tritiated thymidine. The one-hour treatment with AdR or tritiated thymidine was given at various hours in the course of the interphase of a synchronous population of binucleate cells induced by caffeine. In the case of AdR, sensitivity to the inhibition of DNA synthesis was studied by recording the delays produced by the treatment in the appearance of biprophases and bitelophases. The selection by the use of caffeine, of spontaneously synchronous populations of cells going through the telophase and becoming binucleate and the detection of the first biprophases in the subsequent mitosis provide a highly synchronized system with which to study the incorporation of tritiated thymidine during the interphase. The curves representing sensitivity to the inhibition of DNA synthesis by AdR and the rate of tritiated thymidine incorporation coincide, so that we can regard the delays, under our conditions, as proportional to the rate of DNA synthesis at the moment of the AdR treatment. This rate, in the S period, was found to be variable by both methods, being higher in the first and the last thirds of the S period (S₁ and S₃) and lower in the middle third (S₂).     

Arrest of Chinese hamster cells in G 2 following treatment with the anti-tumor drug bleomycin

Upon addition of bleomycin (BLM) to suspension cultures of Chinese hamster cells (line CHO), cells closer to prophase than 56 minutes continue dividing at the normal rate, whereas cells at earlier positions in the cell cycle either fail to reach mitosis altogether (at 200 μg/ml) or enter mitosis and divide at a reduced rate at lower drug concentrations. At 100 μg/ml of BLM (the rate of cell division slowed to a doubling time of 167 hours), initiation and termination of DNA synthesis occur at normal rates, resulting in an accumulation of cells with a G₂ DNA content in the first 130 minutes of G₂. Bleomycin effects are not readily reversible. The rates of incorporation of leucine, uridine, or thymidine into cells treated for six hours with 100 μg/ml of BLM were 90, 85, and 80%, respectively, of the values obtained in control cultures, suggesting that the effects of BLM on cell-cycle traverse cannot be correlated with gross inhibition of macromolecular synthesis.     

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.     

Spatial and temporal patterns of deoxyribonucleic acid synthesis and mitosis in the endometrial stroma during decidualization in the pseudopregnant rat

A quantitative study was made of the spatial patterns of stromal cell mitosis and DNA synthesis in the endometrium of the pseudopregnant rat before and during decidualization. A colchicine block was used for mitotic counts, and DNA synthesis was studied by [3H] thymidine autoradiography. Observations were also made on the subsequent fates of [3H] thymidine-labeled stromal cells. Before the onset of decidualization, on Days 3 and 4 (vaginal cornification = Day 0), mitosis was largely confined to the subepithelial stroma along the sides and around the antimesometrial pole of the lumen. [3H] thymidine labeling and stromal mitosis following a decidualizing stimulus at noon on Day 4 of pseudopregnancy were first seen close to the uterine lumen, with subsequent spread to deeper layers of the endometrium. At noon on Day 5, mitotic figures were numerous on all sides of the lumen and at all depths in the endometrium. At later stages, mitosis and the development of polyploidy continued in the decidual tissue, but little DNA synthesis or mitosis occurred in the basal zone of the stroma adjacent to the myometrium. In this zone, many cells in animals given [3H] thymidine 18 to 24 h after induction of decidualization remained heavily labeled throughout the growth and regression of deciduomata. Labeled cells derived from the basal zone and outer edge of the decidual capsule were present in the stroma of the regenerated endometrium following the regression of deciduomata. It was concluded that although cells at all depths in the endometrial stroma undergo DNA synthesis and mitosis in the early stages of response to a decidualizing stimulus, their subsequent behavior and fate depend upon their position in the endometrium.     

Stimulation of albumin synthesis in mouse hepatoma cells by Bt2cAMP: cell cycle specificity

Addition of 1 mm dibutyryl cyclic AMP (Bt₂cAMP) to cultures of mouse hepatoma cells, Hepa, specifically stimulates the synthesis of serum proteins including albumin. This stimulation is accompanied by an inhibition of cell proliferation. We have investigated these phenomena in synchronous cultures of Hepa. Proliferation of Hepa was arrested by isoleucine starvation. Synchronous growth was initiated by addition of complete growth medium or complete growth medium supplemented with 1 mm Bt₂cAMP. S phase and mitosis were estimated by determinations of [³H]thymidine incorporation and by cell numbers. The rate of albumin synthesis relative to total protein synthesis was measured by pulse labeling cultures for 30 min with [³H]leucine and comparing amounts of immunoprecipitable label with trichloroacetic acid-precipitable label. Treatment of synchronous cultures with Bt₂cAMP did not alter the duration of S phase or the onset of mitosis. The relative rate of albumin synthesis in Bt₂cAMP-treated culture began increasing after mitosis. The timing of the Bt₂cAMP stimulation of albumin synthesis was further investigated by adding Bt₂cAMP to cultures of Hepa at various times after the initiation of synchronous growth. The relative rate of albumin synthesis was then measured at a fixed postmitotic time. An increased relative rate of albumin synthesis was observed only in cultures exposed to Bt₂cAMP before or during S phase. Thus the postmitotic increase in the synthesis of albumin requires the presence of Bt₂cAMP during S phase.     

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.     

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: A reappraisal of translation initiation during mitosis

Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G₁/S boundary) or the Cdk1 inhibitor, RO3306 (G₂/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2α phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.     

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.     

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.

     

THE MECHANISM OF 5-AMINOURACIL-INDUCED SYNCHRONY OF CELL DIVISION IN VI CIA FABA ROOT MERISTEMS

Cessation of mitosis was brought about in Vicia faba roots incubated for 24 hours in the thymine analogue, 5-aminouracil. Recovery of mitotic activity began 8 hours after removal from 5-aminouracil and reached a peak at 15 hours. If colchicine was added 4 hours before the peak of mitoses, up to 80 per cent of all cells accumulated in mitotic division stages. By use of single and double labeling techniques, it was shown that synchrony of cell divisions resulted from depression in the rate of DNA synthesis by 5-aminouracil, which brought about an accumulation of cells in the S phase of the cell cycle. Treatment with 5-aminouracil may have also caused a delay in the rate of exit of cells from the G₂ period. It appeared to have no effect on the duration of the G₁ period. When roots were removed from 5-aminouracil, DNA synthesis resumed in all cells in the S phase. Although thymidine antagonized the effects of 5-aminouracil, an exogenous supply of it was not necessary for the resumption of DNA synthesis, as shown by incorporation studies with tritiated deoxycytidine.     

The duration of DNA synthetic (S) period in Zea mays: A genetic control

Summary The nuclear cycle among several diverse genetic stocks of Zea mays root meristem cells was compared and it was found that there were no significant differences among the nuclear cycle durations and its component phases. The durations of various periods of their mitotic cycles were studied by autoradiography of cells pulse-labelled with tritiated thymidine (3H-TdR). The total nuclear cycle was 10 to 11.5 hours and mitosis was 0.81 to 1.34 hours at 25°C. The S period is the longest interval (50% of the total time) of the nuclear cycle; of the rest of the cycle, G₂ is longer than G₁ or mitosis among all stocks. The constancy of the nuclear cycle among several stocks was adduced as evidence for strict genetic control of the cycle. Furthermore, it is demonstrated the DNA synthesis period is not dependent upon the amount of DNA present.This study is based on a portion of the dissertation presented by the senior author to the Graduate School, The University of Western Ontario, London, Canada, in partial fulfillment of the requirement for the Ph. D. degree     

Lethal effects of fluorodeoxyuridine on cultured mammalian cells at various stages of the cell cycle

The lethal damage induced by the exposure of synchronized Chinese hamster cells to various concentrations of 5-fluoro-2′deoxyuridine (FUdR) was not selectively restricted to cells exposed during the period of DNA synthesis S. The colony survival fraction observed after treatment for one hour with 5 × 10^?5 M FUdR was very low (0.0001–0.0003) whether the drug was administered during early G₁, late G₁, early S or in middle S. The survival of cells treated with the same concentration of FUdR during mitosis, however, was significantly higher (0.62) showing that mitotic cells were less sensitive to FUdR. Administration of 10^?7M thymidine or “conditioned” medium for one hour reversed the lethal effect of FUdR or improved the survival, depending on the time after removal of the FUdR at which these substances were given.     

Physarum thymidine kinase. A step or a peak enzyme depending upon temperature of growth.

The variations of thymidine kinase or ATP:thymidine 5'-phosphotransferase (EC 2.7.1.21) during the cell cycle of Physarum polycephalum plasmodia have been studied at two extreme physiological temperatures: 22 degrees C and 32 degrees C. At 22 degrees C the enzyme activity increases near mitosis and stays constant during late S and G2 phases, exhibiting the typical pattern of a 'step enzyme'. But at 32 degrees C thymidine kinase activity goes through a maximum 1 h 30 min after mitosis and decreases during the subsequent phases as expected for a 'peak enzyme'. The rate of enzyme degradation and/or inactivation, measured in the presence of metabolic poisons (cycloheximide or dinitrophenol), appears to follow a simple exponential function with a half-life of approximately 3 h and 1 h at 22 degrees C and 32 degrees C respectively. The effect of growth temperature on the decrease of thymidine kinase activity can account entirely for the differences in the pattern of enzyme activity at the two extreme temperatures. Tentative calculations indicate that the rate of enzyme synthesis is nearly constant during the cell cycle except near mitosis, where it is temporarily increased. The results suggest the existence of a regulatory mechanism able to modulate the rate of synthesis of thymidine kinase during the cell cycle.     

Effect of ouabain on macromolecular synthesis during the cell cycle in mitogen-stimulated human lymphocytes

Ouabain inhibited in a concentration-dependent and completely reversible way, the synthesis of DNA, RNA and protein in phytohemagglutinin and concanavalin A-stimulated human lymphocytes without affecting the uptake of nucleosides and amino acids into the cells. On the other hand, ouabain even at very high concentrations was unable to interfere with the binding of [³H]concanavalin A. No correlation was found between the inhibition by ouabain of macromolecular synthesis and that of K⁺ transport. The inhibitor effect of ouabain on the stimulation of macromolecular synthesis could be partially reversed by higher concentrations of K⁺, due to the direct inhibition of ouabain binding. Ouabain added to the cultures at different stages of cell growth suppressed the incorporation of thymidine to various extents. Both ouabain sensitive stages fell in a period preceding the onset of mitosis and were characterized by very active thymidine incorporation. Lymphocytes were most sensitive to ouabain within the S phase. The results suggest that ouabain interferes with mitogen-triggered membrane-associated events, other than K⁺ transport, controlling mitosis at distinct phases of the cell cycle.     

Regulation of thymidine kinase synthesis during the cell cycle of Physarum polycephalum by the heat-sensitive system which triggers mitosis and S phase

Temperature shifts from 22 to 32 °C perturb one of the systems responsible for mitosis triggering in the plasmodia of Physarum (Myxomycetes). In order to determine if the same regulatory mechanism could also be involved in some other cell cycle events, the effects of temperature shifts on the peak of thymidine kinase (EC 2.7.1.21, ATP : thymidine 5′-phosphotransferase) synthesis have been studied. At 22 °C, the increase in thymidine kinase (tdk) activity begins shortly before mitosis and is thus always associated with the end of the G2 phase, the mitosis and the beginning of the S phase. The consequences of temperature shifts depend upon their position in the cell cycle. In all cases, a peak of tdk occurs concomitantly with the 32 °C mitosis. But, when the temperature shift is applied 90-15 min before the control metaphase at 22 °C, another peak of tdk is observed at 32 °C in absence of mitosis, but at the same time as the control mitosis at 22 °C. These results indicate that the increase in the synthesis of tdk is controlled by the heat-sensitive regulatory system which plays a role in the onset of mitosis and S phase. We further suggest that the increase in the synthesis of tdk and the triggering of mitosis are both controlled by the amount of a heat-sensitive effector. But the former takes place when the amount of the effector reaches a critical value lower than the value necessary to trigger mitosis.     

Effect of ouabain on macromolecular synthesis during the cell cycle in mitogen-stimulated human lymphocytes

Ouabain inhibited in a concentration-dependent and completely reversible way, the synthesis of DNA, RNa and protein in phytohemagglutinin and concanavalin A-stimulated human lymphocytes without affecting the uptake of nucleosides and amino acids into the cells. On the other hand, ouabain even at very high concentrations was unable to interfere with the binding of [3H]concanavalin A. No correlation was found between the inhibition by ouabain of macromolecular synthesis and that of K+ transport. The inhibitor effect of ouabain on the stimulation of macromolecular synthesis could be partially reversed by higher concentrations of K+, due to the direct inhibition of ouabain binding. Ouabain added to the cultures at different stages of cell growth suppressed the incorporation of thymidine to various extents. Both ouabain sensitive stages fell in a period preceding the onset of mitosis and were characterized by very active thymidine incorporation. Lymphocytes were most sensitive to ouabain within the S phase. The results suggest that ouabain interferes with mitogen-triggered membrane-associated events, other than K+ transport, controlling mitosis at distinct phases of the cell cycle.     

Life Cycle Analysis of Mammalian Cells: III. The Inhibition of Division in Chinese Hamster Cells by Puromycin and Actinomycin

Analysis of the effects of actinomycin and puromycin on the G₂ and mitotic parts of the life cycle in Chinese hamster ovary cells grown in suspension and synchronized by thymidine treatment has been carried out. Rates of division of partially synchronized cell populations were measured in the presence and absence of the drugs, and various controls were performed to test for absence of complex side effects. Actinomycin produces a block 1.9 hr before completion of division, while puromycin produces a block almost coinciding with the initiation of mitosis. Evidence is presented that the puromycin block may be a double one, inhibiting one kind of protein synthesis that virtually coincides with the beginning of mitosis and another that occurs about 8 min earlier. The data are interpreted in terms of the time interval between messenger formation and its associated protein synthesis in this region of the life cycle. The various events studied have been provisionally mapped in the G₂ and mitotic periods of the life cycle.