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

The effects of fluorodeoxyuridine were investigated during 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 inhibited, and there was limited action on other macromolecular syntheses. When DNA synthesis was slowed down, onset of the following increase of thymidine kinase synthesis occurred at approximately the same time as in the control, but mitosis was blocked in a very early prophase stage and metaphase was never observed. These effects were suppressed when the action of fluorodeoxyuridine was prevented by the addition of thymidine to the medium. In agreement with the action of aphidicolin and hydroxyurea, these observations show that: 1) perturbation of the S-phase does not prevent the nuclei from entering a very early prophase stage, but it does prevent them from proceeding through metaphase; 2) blockage of DNA synthesis does not perturb the normal timing of the triggering of thymidine kinase synthesis; and 3) the signal that triggers the arrest of thymidine kinase synthesis is postmitotic and does not require extensive DNA synthesis. In contrast with hydroxyurea and aphidicolin, in the presence of fluorodeoxyuridine metaphase was not observed. Thus, the triggering of thymidine kinase synthesis is unambiguously dissociated from metaphase and postmitotic events. Because synthesis of thymidine kinase remains under the control of temperature shifts from 22 to 32 degrees C, a simple model of the cell cycle involving two regulatory pathways could account for the triggering of thymidine kinase synthesis, early prophase stage, and metaphase.     

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.     

Heat sensitive factor necessary for mitosis onset in Physarum polycephalum (temperature shift/heat shock/cycloheximide/ts mutant)

Summary Physarum synchronous plasmodia were submitted to temperature shifts during the cell cycle and the onset of mitosis was followed at both temperatures. After 22 to 31 or 32° shifts, delays in mitosis onset, dependent upon protein synthesis, were observed at 32° and found to increase as the time separating the shift from the control mitosis decreases. The modification of a general metabolic process or the inactivation of a catalytic heat sensitive substance cannot account for such a result. The proposed model postulates a substance acting in a stoichiometric way, which can occur under three structural forms: two active forms synthesized at low and high temperatures respectively and an inactive one which comes from the transformation of the low temperature active form placed at high temperature. The constant delays observed after some shifts (29 to 32°) suggest that this substance is acting through a polymeric structure which would be necessary for the mitotic process and the initiation of the following DNA synthesis.     

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.     

Regulation of tubulin synthesis during the cell cycle in the synchronous plasmodia of Physarum polycephalum

Regulation of alpha- and beta-tubulin isotype synthesis during the cell cycle has been studied in the myxomycete Physarum polycephalum, by subjecting synchronous plasmodia to temperature shifts and pharmacological perturbations. Temperature shifts interfered with the regulation of tubulin synthesis. Inhibition of DNA synthesis prevents tubulin degradation after completion of the cell cycle (Ducommun and Wright, Eur. J. Cell Biol., 50:48-55, 1989) but did not perturb the initiation of tubulin synthesis. The constant increase of tubulin synthesis in the presence of tubulin-sequestering drugs and the decrease of tubulin synthesis during a treatment with aphidicolin in late G2 phase suggest the existence of an autoregulatory mechanism of tubulin synthesis. Moreover, the microtubule poison methyl benzimidazole carbamate dissociated synthesis of the alpha 1-tubulin isotype from the generally strictly coordinated synthesis of all tubulin isotypes during the transient interruption of mitosis. These observations show that a microtubular poison can perturb regulation of the synthesis of specific isotubulins.     

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.     

Cycloheximide resistance of Physarum polycephalum.

In the presence of cycloheximide, wild-type plasmodia of Physarum polycephalum exhibit an immediate decrease in deoxyribonucleic acid synthesis, a reduction in the incorporation of [3H]thymidine into thymidine triphosphate, and an increase in the level of thymidine triphosphate, as well as a decrease in protein synthesis. In this study, we have utilized a cycloheximide-resistant (Cycr) amoebic strain selected from a population of cells mutagenized with nitrosoguanidine. Segregation data indicate that the resistance is due to a single mutation. We have used this Cycr mutant to construct Cycr plasmodial strains. Ribosomes isolated from such Cycr plasmodia showed resistance to cycloheximide in vitro, in contrast to ribosomes isolated from wild-type plasmodia. The Cycr plasmodia showed none of the cycloheximide-induced biochemical effects. Plasmodia heterozygous for the resistance marker were sensitive to cycloheximide with regard to growth but showed an intermediate response in the biochemical parameters. Heterokaryons formed by fusion of various proportions of the sensitive and resistant plasmodia showed a resistance with regard to both growth and biochemical parameters which was directly related to the fraction of Cycr plasmodia present in the heterokaryons. The data are consistent with the hypothesis that the effects of cycloheximide on deoxyribonucleic acid synthesis and nucleoside metabolism are secondary to the effect of the drug on protein synthesis in this organism.     

The effect of heat shock on the cell cycle regulation of tubulin expression in Physarum polycephalum

In the myxomycete Physarum polycephalum, tubulin synthesis is subject to mitotic cycle control. Virtually all tubulin synthesis is limited to a 2-h period immediately preceding mitosis, and the peak of tubulin protein synthesis is accompanied by a parallel increase in the level of tubulin mRNA. The mechanism by which the accumulation of tubulin mRNA is turned on and off is not clear. To probe the relationship between tubulin regulation and cell cycle controls, we have used heat shocks to delay mitosis and have followed the pattern of tubulin synthesis during these delays. Two peaks of tubulin synthesis are observed after a heat shock. One occurs at a time when synthesis would have occurred without a heat shock, and a second peak immediately precedes the eventual delayed mitosis. These results are clearly due to altered cell cycle regulation. No mitotic activity is detected in delayed plasmodia at the time of the control mitosis, and tubulin behavior is shown to be clearly distinct from that of heat shock proteins. We believe that the tubulin family of proteins is subject to regulation by a thermolabile mitotic control mechanism but that once the cell has been committed to a round of tubulin synthesis the "tubulin clock" runs independently of the heat sensitive system. In delayed plasmodia, the second peak of synthesis may be turned on by a repeat of the commitment event.     

Regulation of thymidine kinase activity in the cell cycle by a labile protein

Previous studies have shown that the onset of DNA synthesis in Balb/c 3T3 cells appears to be regulated by a labile protein. We have found that induction of thymidine kinase (TK) activity, after quiescent cells are stimulated by the addition of serum, is similarly regulated by a labile protein. Eight hours after serum stimulation, a 6-h pulse of cycloheximide (CHM) caused an excess delay of 2 h in TK induction. A similar delay also was found in the induction of thymidylate synthase (TS). In contrast, the benzo(a)pyrene transformed 3T3 cell line, BP-A31, which had previously been shown to have no excess delay for the onset of DNA synthesis also had no excess delay for the induction of TK activity after a pulse of CHM. The induction of TK was inhibited by actinomycin D and dichlororibofuranosylbenzimidizole (DRB) suggesting a requirement for new RNA synthesis. It did not appear to depend on DNA synthesis as it was not blocked by aphidicolin. In conclusion, the induction of TK activity appears to be regulated by the same labile cellular signal as the onset of DNA synthesis, and to depend on an increase in the level of TK mRNA in late G1 or early S phase.     

Study of pp60v-src protein kinase activity in synchronized chicken embryo fibroblasts infected with Rous sarcoma virus

Chicken embryo fibroblast (CEF) cultures, synchronized by the addition of serum to stationary cells, were exposed to Schmidt-Ruppin strain of Rous Sarcoma Virus (SR-RSV) and the appearance of pp60v-src protein kinase activity was examined through the cell cycle. In cells infected either at the beginning or at the end of G1, the onset of pp60v-src protein kinase activity was coincidental, closely following mitosis, with a delay between the infection of cells with SR-RSV and the appearance of protein kinase activity of about 20 and 16 h, respectively. In cells infected during the S phase this delay was 16 h, as observed for late G1 cells. These experiments show that the activity of pp60v-src protein kinase, which cannot be detected before the first mitosis following infection does not depend on G1. The aphidicolin prevented protein kinase activity if added before or at the beginning of S phase, but not if added later, which is presumably related to the inhibition of S phase, required for provirus integration. The use of colcemid, which suppresses cell division, did not inhibit but delayed the appearance of protein kinase activity. These results show that the synthesis of an active oncogene product, such as pp60v-src protein kinase, depends on both S phase and mitosis.     

Dependence of timing of mitotic events on the rate of protein synthesis and DNA replication in sea urchin early cleavages

Abstract. To understand what processes affect the cell-cycle timing of mitotic events in early cleavage cycles of sea urchin embryos, a study was made on the effects of (a) reducing protein synthesis with emetine and (b) DNA replication with aphidi-colin, on the timing of nuclear envelope breakdown, anaphase onset and cytokinesis. When protein synthesis was slightly inhibited by administration of emetine, the delay in the mitotic events increased, with an increase in the delay in accumulation of proteins up to the levels to which cells must synthesize the proteins to execute the cleavage. This indicated that protein synthesis affects the timing of mitotic events. The delay in cleavage cycles caused by a slight inhibition of DNA replication with aphidicolin was in proportion to the concentration of aphidicolin administered, suggesting that DNA replication also affects the timing of mitotic events. Furthermore, it was confirmed that accumulation of the proteins to the levels required for execution of the first cleavage precedes completion of DNA replication as a requirement for execution of the first cleavage. These results imply the existence of process(es) affected by protein synthesis that are included in a feedback control system which prevents the initiation of mitosis until after the completion of DNA replication; it is the characteristic of a cell-cycle control system that has been predicted theoretically.     

Staurosporine overrides checkpoints for mitotic onset in BHK cells.

Under normal conditions, mammalian cells will not initiate mitosis in the presence of either unreplicated or damaged DNA. We report here that staurosporine, a tumor promoter and potent protein kinase inhibitor, can uncouple mitosis from the completion of DNA replication and override DNA damage-induced G2 delay. Syrian hamster (BHK) fibroblasts that were arrested in S phase underwent premature mitosis at concentrations as low as 1 ng/ml, with maximum activity seen at 50 ng/ml. Histone H1 kinase activity was increased to approximately one-half the level found in normal mitotic cells. Inhibition of protein synthesis during staurosporine treatment blocked premature mitosis and suppressed the increase in histone H1 kinase activity. In asynchronously growing cells, staurosporine transiently increased the mitotic index and histone H1 kinase activity but did not induce S phase cells to undergo premature mitosis, indicating a requirement for S phase arrest. Staurosporine also bypassed the cell cycle checkpoint that prevents the onset of mitosis in the presence of damaged DNA. The delay in mitotic onset resulting from gamma radiation was reduced when irradiation was followed immediately by exposure to 50 ng/ml of staurosporine. These findings indicate that inhibition of protein phosphorylation by staurosporine can override two important checkpoints for the initiation of mitosis in BHK cells.     

Effect of inhibition of the catalytic activity of cyclic AMP-dependent protein kinase on mitosis in PtK1 cells

Evidence has suggested that cyclic AMP, acting through activation of the type II cyclic AMP-dependent protein kinase, may play a role in the regulation of interphase and mitotic microtubules. In order to examine the potential role of the type II cAMP-dependent kinase during mitosis, dividing PtK1 cells were microinjected with two specific inhibitors of the catalytic activity of the type II kinase. These inhibitors were a specific protein inhibitor of cAMP-dependent protein kinase (PKI) and an affinity-purified polyclonal antiserum (anti-C) directed against the catalytic subunit of the kinase. Both have been shown previously to inhibit kinase activity in vitro. Microinjection of PKI during early- to mid-prophase significantly delayed the progression of the cells through mitosis, with the greatest delay occurring in metaphase. PKI injected during prometaphase also delayed progression through mitosis but to a lesser extent. Microinjection of anti-C during early- to mid-prophase also caused a significant delay in the completion of mitosis, with many cells becoming "hung up" in prometaphase. Anti-C injected during prometaphase had little effect on subsequent progression through mitosis. Microinjection of either anti-C or PKI during metaphase had no discernible effect. No effect on anaphase movement of chromosomes was observed with any treatment. These results provide further evidence that cAMP-dependent phosphorylation may be involved in the regulation of mitosis, although whether it acts directly through regulation of mitotic spindle microtubules is unclear.     

Sensitivity to cell killing and the induction of cytogenetic damage following gamma irradiation in wild-type and thymidine kinase-deficient Friend mouse erythroleukaemia cells

Wild-type Friend erythroleukaemia (clone 707) cells and 2 thymidine kinase-deficient subclones, 707BUE and 707BUF, having thymidine kinase activities of 1.4% and 0.7% that of clone 707, were compared for sensitivity to killing and the induction of cytogenetic damage following irradiation. Three doses of gamma irradiation were used (150, 300 and 450 cGy), and cells were harvested for metaphase spreads after 4, 8, 12, 15, 29 and 43 h. G₂ delay was evident at 4 h following gamma irradiation in the 3 cell clones examined, and recovery of mitosis was observed to be dose-dependent. G₂ delay was found to be most prolonged in subclone 707BUE and most prompt in clone 707. Increased sensitivity to the induction of cytogenetic aberrations at all three doses was apparent in the 2 thymidine kinase-deficient subclones (as compared to wild-type cells) at 15, 29 and 43 h. Th thymidine kinase-deficient subclones also showed increased sensitivity to gamma radiation-induced cell killing. Furthermore, subclone 707BUE consistently exhibited greater to gamma irradiation than did the subclone with lower thymidine kinase activity, 707BUF. The importance of thymidine kinase levels and extended G₂ delay for DNA repair processes is discussed.     

Change of contractile behavior in plasmodia ofPhysarum polycephalum during mitosis

Summary Oscillations of ectoplasmic contraction in plasmodia of the myxomycetePhysarum polycephalum growing on agar containing semidefined medium were studied to determine if the contractile force is altered during the synchronous mitosis. In interphase the regular oscillations of contraction in the plasmodial sheet had an average period of 0.93 minutes in plasmodia growing at 24 °C. During mitosis the amplitude of these oscillations gradually decreased, ceasing for an average time of 2.7 minutes in 74% of the 23 plasmodia studied. Cessation of oscillating contractions in mitosis was accompanied by a decrease in the width of the channels embedded in the plasmodial sheet, and a decrease in the velocity of endoplasmic shuttle streaming usually to a complete standstill. Of 13 plasmodia in which the mitotic stage was very accurately determined, the stop in oscillating contractions occurred during metaphase in 10 plasmodia, and in prometaphase, anaphase, telophase in the 3 others. The cessation of contractile oscillations or of streaming did not occur absolutely simultaneously during mitosis in widely separated locations within one plasmodium, indicating mitotic asynchrony over a period of a few minutes within each plasmodium. We suggest that the halt of plasmodial migration during mitosis reported by others is caused by a decrease or cessation at slightly different times in the amplitude of ectoplasmic contractile oscillations in different areas of a plasmodium in mitosis resulting in an overall lack of coordination of endoplasmic flow throughout the plasmodium, thus temporarily halting migration. Possible physiological mechanisms linking a decrease in actomyosin contraction with the metaphase stage of mitosis are discussed.     

Stepwise effects of cytokinin activity and DNA synthesis upon mitotic cycle events in partially synchronized tobacco cells

Cytokinin addition to tobacco cell suspensions induced synchronous cell division after an 18 h lag period. Although continuous presence of the cytokinin in the culture medium during this lag period was essential to division, cytokinin was not required during mitosis itself. For each cell generation, cytokinin-dependent events are thus completed before mitosis occurs.Two experiments suggested that these cytokinin-dependent events are independent of DNA synthesis:

1. (i) With or without cytokinin, DNA synthesis proceeded normally in the presence of auxin, for at least the time required for one cell generation in complete medium.

2. (ii) In the presence of cytokinin, when DNA synthesis in the lag period was inhibited by FUdR, one normal cell division occurred when cytokinin was withdrawn and DNA synthesis restored by thymidine addition.

In cytokinin-starved cells, metaphase was greatly prolonged although prophase was unaffected.     

Studies on fertilization in the teleost IV. Effects of aphidicolin and camptothecin on chromosome formation in fertilized medaka eggs

To clarify the mechanisms of fish fertilization, the effects of inhibitors of DNA polymerase-alpha and DNA topoisomerases on nuclear behavior before and after fertilization were examined in eggs of the medaka, Oryzias latipes. Eggs underwent the fertilization process from sperm penetration to karyogamy of pronuclei, even when inseminated and incubated in the continuous presence of aphidicolin (DNA polymerase alpha inhibitor), camptothecin (DNA topoisomerase I inhibitor), etoposide, or beta-lapachone (DNA topoisomerase II inhibitor). However, continuous treatment with aphidicolin or camptothecin during fertilization inhibited the formation of sister chromosomes that were normally separated into blastomeres at the time of the subsequent cleavage. Sister chromosome formation appeared concomitantly with an increase in histone H1 kinase activity at the end of DNA synthesis, 30 min post insemination. However, non-activated eggs that were inseminated in saline containing anesthetic MS222 and aphidicolin had high levels of histone H1 kinase and MAP kinase activities, and transformation of the penetrated sperm nucleus to metaphase chromosomes occurred even in the presence of aphidicolin or camptothecin. The male chromosomes were normally separated into two anaphase chromosome masses upon egg activation. These results suggest that DNA polymerase alpha or DNA topoisomerase I, but not DNA topoisomerase II, may be required for the process by which the mitotic interphase nucleus transforms to separable metaphase chromosomes while the activity of MAP kinase is low, unlike the situation in meiotic division, during which MAP kinase activity is high and DNA replication is not required.     

DNA signals for G2 checkpoint response in diploid human fibroblasts

DNA (deoxyribonucleic acid) signals that induce the G2 checkpoint response were examined using proliferative secondary cultures of diploid human fibroblasts. Treatments that generated DNA double-strand breaks (DSBs) directly were effective inducers of checkpoint response, generally producing >80% inhibition of mitosis (G2 delay) and the kinase activity of M-phase-promoting factor within 2 h of treatment. Effective inducers of G2 checkpoint response included γ-irradiation and the cancer chemotherapeutic drugs, bleomycin and etoposide. Treatments that produced DNA single-strand breaks, directly or indirectly through nucleotide excision repair, were not effective inducers of G2 delay. Ineffective treatments included incubation with camptothecin, an inhibitor of topoisomerase I (topo I), and irradiation with sublethal fluences of UVC, followed by incubation with aphidicolin. Transient severe inhibition of DNA synthesis with aphidicolin did not affect mitosis substantially, suggesting that the replication arrest input to the G2 checkpoint required more than brief inhibition of DNA synthesis. In contrast, moderate camptothecin-induced inhibition of DNA synthesis was associated with a strong inhibition of mitosis that developed 4–12 h after drug treatment. This result suggested that G2 delay was not expressed until the cells that were in S-phase at the time of treatment with camptothecin proceeded into G2. DNA damage was not necessary for induction of mitotic delay. An inhibitor of topoisomerase II (topo II), ICRF-193, which inhibits chromatid decatenation in G2 cells without damaging DNA, induced a severe inhibition of mitosis and M-phase-promoting factor kinase activity. The results suggest that DNA double-strand breaks and insufficiency of chromatid decatenation effectively induce the G2 checkpoint response, but DNA single-strand breaks do not.     

The participation of elevated levels of cyclic GMP in the recovery from radiation-induced mitotic delay

The levels of cyclic AMP and cyclic GMP have been measured in Physarum plasmodia before and after treatment with gamma-radiation, 2 mM caffeine, or combinations of the two agents and compared to the length of the radiation-induced mitotic delay. Caffeine alone produces a rapid transient elevation of cyclic AMP and a slower delayed elevation of cyclic GMP. Irradiation elicits an immediate transient increase in cyclic AMP and a later cyclic GMP increase which accompanies or precedes the delayed mitosis. A composite pattern is produced by combinations of radiation and caffeine, a distinctive feature of which is an elevated level of cyclic GMP near the time of the radiation-delayed and caffeine-promoted mitosis. With pretreatment by caffeine, the least radiation-induced mitotic delay occurs when plasmodia are irradiated during the caffeine-elicited increase in cyclic GMP. The plasmodium becomes refractory to the reduction of mitotic delay by caffeine at approximately the time it becomes refractory to the further elevation of cyclic GMP by caffeine. The data support a role for cyclic AMP in the onset of and for cyclic GMP in the recovery from mitotic delay induced by ionizing radiation.