Relationships between DNA synthesis and mitotic events in fertilized sea urchin eggs: aphidicolin inhibits DNA synthesis, nuclear breakdown and proliferation of microtubule organizing centers, but not cycles of microtubule assembly

The synthesis of DNA in fertilized eggs of the American Gulf Coast sea urchin Lytechinus variegatus is 90% inhibited in the presence of 5.0 micrograms/ml aphidicolin. This inhibition may be imposed immediately upon addition of aphidicolin to the external medium when embryos are in "S" phase. Observations of living embryos with Nomarski optics and time-lapse video microscopy reveal that when eggs are fertilized and cultured in the continuous presence of aphidicolin, nuclear envelope breakdown, chromosome condensation, and cytokinesis are inhibited. All other post-fertilization events observable with this technique, including the assembly and disassembly of a bipolar spindle, proceed in the presence of aphidicolin. Antitubulin immunofluorescence microscopy of aphidicolin-arrested embryos demonstrates that microtubules attempt to assemble a mitotic apparatus at the first cell cycle; the arrested intact zygote nucleus is embedded within this bipolar structure. Subsequent cycles of microtubule assembly and disassembly proceed roughly on schedule with later division cycles, but the microtubule organizing centers (MTOC's) are unable to duplicate properly and irregular monasters are observed. If aphidicolin is added to embryos after the first DNA synthetic period, nuclear envelope breakdown, chromosome condensation, and cytokinesis proceed for that cycle and the embryos arrest at the two-cell stage. These results suggest that the direct inhibitory effects of aphidicolin may well be limited to the synthesis of DNA, which itself regulates nuclear cycles independently from the subsequent generation of mitotic poles, and that cytoplasmic clocks regulate microtubule assembly cycles but not the configuration of microtubule arrays.     

Characteristics of the action of prolactin on [3H]-thymidine incorporation into DNA in mammary gland explants from virgin mice

The prolactin stimulation of the rate of [3H]-thymidine incorporation into DNA in mammary gland explants from virgin C3H mice was studied. The onset of this effect occurred between one and two days after adding prolactin to the culture medium. Prolactin effected an enhanced rate of [3H]-thymidine incorporation at all concentrations from 10 ng/ml to 10 micrograms/ml. The response is essentially an "all or none" phenomenon since the effect at 10 ng/ml was not different from that at 10 micrograms/ml. Hydrocortisone was not essential from the prolactin response, but it did significantly increase the basal rate of [3H]-thymidine incorporation. Both quinacrine (an inhibitor of phospholipase A2 activity) and indomethacin (an inhibitor of prostaglandin biosynthesis) abolished the action of prolactin on [3H]-thymidine incorporation into DNA.     

Relationship between nuclear DNA synthesis and centrosome reproduction in sea urchin eggs

The importance of nuclear DNA synthesis for the doubling, or reproduction, of centrosomes in cells that are not growth-limited, such as sea urchin eggs, has not been clearly defined. Studies of enucleated, fertilized eggs show that nuclear activities are not required at each cell cycle for the normal reproduction of the complete centrosome. However, other studies report that the inhibition of nuclear DNA synthesis in intact eggs by the drug aphidicolin prevents centrosome reproduction and entry into mitosis as seen by nuclear envelope breakdown. To resolve this paradox, we systematically characterized the effect of aphidicolin on cell division in eggs from three species of sea urchins. Eggs were continuously treated with 5 or 10 micrograms/ml aphidicolin starting 5 min after fertilization. This blocked total incorporation of 3H-thymidine into DNA by at least 90%, as previously reported. We found that the sperm aster always doubles prior to first mitosis. Over a period of several hours, the centrosomes reproduce in the normal 2-4-8-16 fashion, with a period that is longer and more variable than normal. In every culture, a variable percentage of the eggs undergoes nuclear envelope breakdown. Once broken down, the nuclear envelope never visibly reforms even though centrosomes continue to double. Fluorescent labeling of DNA revealed that the chromatin does not condense into discrete chromosomes. Whether or not the nuclear envelope breaks down, the chromatin appears as an amorphous mass of fibers stretched between first two and then four asters. Later, the nuclear envelope/chromatin loses its association with some or all centrosomes. Our results were the same for all eggs at both drug concentrations. Thus, nuclear DNA synthesis is not required for centrosome reproduction in sea urchin eggs.     

Abrogation of the effects of aphidicolin on NIH3T3 and V79 cells by caffeine

In this communication I show that caffeine (1,3,7-trimethylxanthine) stimulates [³H]thymidine incorporation in aphidicolin-treated V79 and NIH3T3 cells. Flow microfluorometric analysis showed that caffeine, partially or fully, abrogates the cell cycle progression block produced by aphidicolin. Increased cell growth is also observed in cultures treated with both aphidicolin and caffeine compared to cultures treated with aphidicolin only. Microscopic examination of V79 cultures treated with aphidicolin for 8 h showed a marked reduction in the freqeuncy of round mitotic cells, as is expected from a drug which inhibits progression through the cell cycle by inhibiting DNA replication; this effect of aphidicolin was also reduced by caffeine. Biochemical analysis showed that caffeine did not directly interfere with the inhibition of DNA polymerase-α by aphidicolin. Analysis of dNTP pools indicated that caffeine increased the level of dCTP in V79 cells. In aphidicolin-treated V79 cells, the increase in the dCTP level due to exogenous cytidine was almost completely blocked; caffeine also substantially overcame this effect of aphidicolin. These results indicate that caffeine produces its effects on aphidicolin-treated cells by altering the dCTP metabolism.     

Nucleic acid synthesis and development of human male pronucleus

Polyspermically penetrated human zona-free eggs prepared from oocytes that had failed to be fertilized in an in-vitro fertilization programme were used. The pronuclear synthetic activity was evaluated by high-resolution autoradiography and correlated with the development of pronuclear structure. Incorporation of [3H]-thymidine, signalling the occurrence of a DNA synthetic phase, was only detected in structurally fully developed pronuclei previously shown to appear no sooner than 12 h after gamete union. However, [3H]adenosine was incorporated into very early pronuclei which had not yet completed the development of their nuclear envelopes and which first appeared about 4 h after sperm-egg fusion. In the absence of DNA synthesis (shown by the lack of thymidine incorporation), this early adenosine incorporation apparently reflects an early pronuclear RNA synthesis. Taken together, these results indicate that nucleic acid synthesis in human male pronuclei is tightly bound to the development of a corresponding pronuclear structure and that DNA synthesis, beginning about 12 h after fertilization, is preceded by a slight but evident RNA synthesis taking place during an early stage of human male pronuclear formation.     

Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: evidence that DNA polymerases delta and beta are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

The involvement of DNA polymerases alpha, beta, and delta in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase alpha) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors on MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [3H]thymidine incorporated into repaired DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 micrograms of aphidicolin/mL, 6% by 10 microM BuPdGTP, 13% by anti-(DNA polymerase alpha) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 micrograms of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase alpha) antibodies into HF nuclei. These results indicate that both DNA polymerases delta and beta are involved in repairing DNA damage caused by MNNG.     

Specific inhibitors of eukaryotic DNA synthesis and DNA polymerase alpha, 3-deoxyaphidicolin and aphidicolin-17-monoacetate

Of several phytotoxins isolated from culture filtrates of Phoma betae Frank PS-13, an incitant of leaf spot disease of sugar beet, three have been identified as aphidicolin, 3-deoxyaphidicolin and aphidicolin-17-monoacetate. Aphidicolin is a selective inhibitor of eukaryotic DNA polymerase alpha (Ikegami et al. (1978) Nature 275, 458-460). Consequently, we studied the action mechanism of 3-deoxyaphidicolin and aphidicolin-17-monoacetate. These aphidicolin analogues markedly inhibited the in vivo DNA synthesis of sea urchin embryos and HeLa cells but not RNA and protein syntheses. Only DNA polymerase alpha, not DNA polymerase beta and gamma, was inhibited by these drugs. The mode of action of these analogues on DNA polymerase alpha from the sea urchin was competitive inhibition with respect to dCTP with Ki values of 0.44 micrograms/ml for deoxyaphidicolin and 0.89 micrograms/ml for aphidicolin monoacetate, respectively. None of the other three dNTPs competed with these drugs. A similar inhibitory mode was observed using the enzyme from HeLa cells and toad oocytes. These drugs at a concentration of 2 micrograms/ml caused a delay in the cleavage of fertilized eggs of the sea urchin and decomposition before blastulation, indicating the possibility of achromosomal cleavage because of the absence of DNA synthesis. Based on the above, it is concluded that these analogues can be used as other inhibitors of eukaryotic DNA synthesis and DNA polymerase alpha.     

Adenovirus DNA synthesized in the presence of aphidicolin

Adenovirus types 2 and 5 DNA synthesized in vivo and in vitro in the presence of aphidicolin were studied. Inhibition of adenoviral DNA synthesis by aphidicolin was only 70% even at a concentration of 30 micrograms/ml of aphidicolin, at which the cellular DNA synthesis was completely inhibited. When initiation of the viral DNA synthesis was synchronized with hydroxyurea and labeled with [3H]thymidine for 60 min, the viral DNA synthesized in the presence of 30 micrograms/ml of aphidicolin was not of full length (35 kb) but small (approximately 12 kb) by analysis of alkaline sucrose density gradient centrifugation. When initiation of the viral DNA synthesis was not synchronized, the viral DNAs ranging from full size to 12 kb were synthesized in the presence of aphidicolin, indicating that the nascent DNAs longer than about 12 kb can continue to elongate in the presence of aphidicolin. This 12 kb DNA was not derived from the degradation products of newly synthesized full size adenoviral DNA. The viral DNA synthesis was restored and the full size of adenoviral DNA was attained within 15 min following removal of aphidicolin. About 20% of the entire viral genome length from the 5'-end was not inhibited by aphidicolin, while the synthesis of interior fragments of the adenoviral DNA was markedly inhibited by aphidicolin, judging from the electrophoretic pattern on neutral agarose gel after digestion of DNA with Hind III. These results indicate that aphidicolin inhibits adenoviral DNA replication at the internal region located approximately 20-30% from both terminals.     

The effects of aphidicolin and α-amanitin on DNA synthesis in preimplantation mouse embryos

The effects of aphidicolin and α-amanitin on DNA synthesis by preimplantation mouse embryos were studied. It was found that both blastocyst and 8-cell embryos showed marked inhibition of ³H-thymidine incorporation into DNA by aphidicolin at concentrations of 20–50 μg/ml. However, aphidicolin did not inhibit the conversion of morula embryos to blastocyst embryos, although aphidicolin-treated blastocysts lost their blastocoel and collapsed into a compact form after prolonged exposure to the drug. Both 8-cell and blastocyst embryos were found to be susceptible to inhibition of DNA synthesis by α-amanitin.     

Dependence of mammalian DNA replication on DNA supercoiling. I. Effects of ethidium bromide on DNA synthesis in permeable Chinese hamster ovary cells.

Chinese hamster ovary cells labelled with [14C]thymidine were made permeable, incubated with various concentrations of the intercalating dye ethidium bromide, and centrifuged through neutral sucrose gradients. The gradient profiles of these cells were qualitatively similar to those obtained by centrifuging DNA from untreated, lysed permeable cells through gradients containing ethidium bromide. The sedimentation distance of DNA had a biphasic dependence on the concentration of ethidium bromide, suggesting that the dye altered the amount of DNA supercoiling in situ. The effect of ethidium bromide intercalation on incorporation of [3H]dTMP into acid-precipitable material in an in vitro DNA synthesis mixture was measured. The incorporation of [3H]dTMP was unaffected by less than 1 microgram/ml of ethidium bromide, enhanced up to two-fold by 1--10 microgram/ml, and inhibited by concentrations greater than 10 micrograms/ml. Alkaline sucrose gradient analysis revealed a higher percentage of small DNA fragments (6--20 S) in the cells treated with 2 micrograms/ml ethidium bromide than in control cells. These fragments attained parental size within the same time as the fragments in control cells. In cells treated with 2 micrograms/ml ethidium bromide, a significant fraction of newly synthesized DNA resulted from new starts, whereas in untreated cells practically none of the newly synthesized DNA resulted from new starts. These results suggest that relaxation of DNA supercoiled structures ahead of the replication fork generates spurious initiations of DNA synthesis and that in intact cells the rate of chain elongation is limited by supercoiled regions ahead of the growing point.     

Regulation of stage-specific gene expression during early mouse development: effect of cytochalasin B and aphidicolin on stage-specific protein synthesis in mouse eggs

Mechanisms regulating stage-specific translation in mouse embryos were studied by inhibitor experiments. When fertilized eggs were continuously treated with cytochalasin B, cleavage was prevented, whereas karyokinesis proceeded, resulting in protein synthesis patterns changing stage-specifically as in control embryos through preimplantation development. When fertilized eggs were continuously exposed to aphidicolin, cleavage and DNA synthesis were inhibited, thus keeping their protein synthesis at the level of fertilized eggs with few new polypeptides appearing after one day. The next day these eggs stopped translation almost completely. Stage-specific translation therefore might be controlled by nuclear replications rather than by cytoplasmic clock.     

Studies on fertilization of the teleost. II. Nuclear behavior and changes in histone H1 kinase

In order to understand the dynamic responses of gamete nuclei upon fertilization in the fish, Oryzias latipes, the relationship between changes in the activity of histone H1 kinase and nuclear behavior was examined during fertilization. Kinase activity rapidly decreased concomitant with the initiation of the propagative exocytosis of cortical alveoli following sperm attachment to the egg plasma membrane post-insemination (PI). Activity again increased 30 min PI. Similar changes in kinase activity, migration and syngamy of pronuclei, and subsequent cleavage were observed with aphidicolin or actinomycin D treatment, except that formation of abnormal metaphase chromosomes was retarded in aphidicolin-treated zygotes. Pretreatment of unfertilized eggs with cycloheximide or 6-dimethylaminopurine (6-DMAP) caused no nuclear changes. The activity of histone H1 kinase in these eggs rapidly declined following sperm penetration and exocytosis, but did not undergo subsequent increase in the presence of these inhibitors. In these eggs with low histone H1 kinase activity, the fertilization process from sperm penetration to syngamy occurred normally, but the pronuclear membrane did not break down and the chromosomes did not condense. The present data suggest that in fish eggs, DNA replication as well as the synthesis and phosphorylation of proteins, especially cyclin B, are required for normal formation of metaphase chromosomes at the first cleavage, but not for fertilization events from sperm penetration through to nuclear migration resulting in syngamy.     

Studies on differentiation without cleavage in Chaetopterus: effects of inhibition of DNA synthesis with aphidicolin

The effects of aphidicolin - a powerful inhibitor of DNA polymerase alpha and of DNA replication - on normal development and on differentiation without cleavage of Chaetopterus eggs have been studied with cytological, cytochemical, and biochemical methods. The experiments show that the initial period of pseudocleavage can take place in the absence of nuclear DNA synthesis, but further development (segregation, hatching, ciliation) requires DNA synthesis. However ciliated unicellular larvae can be obtained under conditions where the DNA content of the embryos in only 40% of the controls. In fertilized eggs, aphidicolin immediately stops cleavage. The significance of these results is discussed.     

Inhibition by aphidicolin and dideoxythymidine triphosphate of a multienzyme complex of DNA synthesis from human cells

A multienzyme complex consisting of DNA polymerase and several DNA precursor-synthesizing enzymes was solubilized by gentle lysis of cultured human cells. This complex channelled the distal precursor [3H]dTMP into DNA. The patterns of inhibition of the complex by aphidicolin and dideoxythymidine triphosphate (ddTTP) suggested that the complex contained the replicative DNA polymerase, polymerase alpha. Inhibition by ddTTP was competitive with dTTP. This was exploited to estimate the effective concentration of [3H]dTTP at the site of DNA synthesis during channelling of [3H]dTMP into DNA. The estimated concentration (about 50 microM) was so high as to suggest that the solubilized complex was able to functionally compartmentalize DNA precursors.     

Early DNA synthesis during the germination of wheat embryos

Both [³H]thymidine and [³H]deoxyadenosine were found to be incorporated into the nuclear DNA of wheat embryos immediately after dry embryos were allowed to imbibe aqueous solutions of the radioactive precursors. The early labelled DNA sedimented in a manner suggesting that replicative intermediates were already formed within the first 90 min of germination. However, aphidicolin remained without any effect on this early DNA synthesis. Likewise, a cell-free system derived from early embryos incorporated [³H]dCTP into DNA independently of the presence of aphidicolin. On the contrary, dideoxyTTP inhibited the DNA synthesis considerably. It is concluded that a proportion of the resting wheat embryo cells is able to initiate a replicative DNA synthesis immediately upon imbibition. The synthesis seems, however, to proceed with the participation of a γ-like, rather than an α-like, DNA polymerase.     

Arrest of chain growth of replicon-sized intermediates by aphidicolin during rat fibroblast cell chromosome replication

The effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, on size maturation of nascent DNA intermediates was studied in cultured rat fibroblast cells. Results provided the first evidence of DNA synthesis associated with merging of intermediates of larger than replicon size. Aphidicolin at a concentration (1.4 micrograms/ml) causing 90-95% inhibition of [3H]thymidine incorporation, resulted in accumulation of intermediates of nearly the same size as the replicon (2-5 x 10(-7) Da); although the synthesis of short nascent fragments (referred to as Okazaki fragments) continued in the presence of aphidicolin, the rate of their elongation to the replicon size was greatly decreased. On removal of aphidicolin, these accumulated intermediates merged into high-molecular-weight DNA. This merging of the intermediates was associated with DNA synthesis in gaps between adjacent intermediates, as revealed by photolysis of bromodeoxyuridine-DNA leader with long-wave ultraviolet light; when the cells had been pulse-labeled for 5 min with bromodeoxyuridine immediately after removal of the drug, the large DNA arising from aphidicolin-arrested intermediates was cut into fragments of the original size by long-wave ultraviolet light irradiation. The arrest of chain elongation at the replicon-size by aphidicolin might be due to inhibition of this DNA synthesis in gaps, because aphidicolin did not cause degradation of nascent DNAs.     

Hypotonic buffer induces meiosis and formation of anucleate cytoplasmic islands in the egg of the two-spotted cricket Gryllus bimaculatus

In insects, egg activation is known to occur in vivo and independently of fertilization, but its mechanisms are poorly understood. To gain understanding of these mechanisms, an attempt was made to activate the egg of Gryllus bimaculatus in vitro. It was found that meiosis resumed and was completed in unfertilized eggs treated with hypotonic buffer. Early developmental processes in activated, unfertilized eggs were investigated and compared with those in fertilized eggs. Mitosis did not progress, resulting in formation of anucleate cytoplasmic islands (pseudoenergids). Development in the activated, unfertilized eggs stopped at this stage and both yolk subdivision and cellularization did not occur. To elucidate the role of the nucleus in the developmental process to the syncytial stage in fertilized eggs, eggs were treated with aphidicolin to inhibit DNA polymerization. It was found that pseudoenergids also formed in these aphidicolin-treated fertilized eggs. These results demonstrate that pseudoenergids can increase in number independently of nuclei, suggesting that the cytoplasm rather than the nucleus plays the primary role in development to the syncytial stage in G. bimaculatus.     

Nascent protein requirement for completion of meiotic maturation and pronuclear development: examination of fertilized and A-23187-activated surf clam (Spisula solidissima) eggs

The involvement of newly synthesized proteins and calcium in meiotic processes, sperm nuclear transformations, and pronuclear development was examined in emetine-treated, fertilized, and A-23187-activated Spisula eggs by observing changes in the morphogenesis of the maternal and paternal chromatin. Emetine treatment (50 micrograms/ml) initiated 30 min before fertilization or A-23187 activation inhibited incorporation of [3H]leucine into TCA-precipitable material and blocked second polar body formation. Sperm incorporation and the initial enlargement of the sperm nucleus were unaffected; however, the dramatic enlargement and transformation of the sperm nucleus into a male pronucleus, which normally follow polar body formation, were delayed 10 to 20 min. Unlike the situation in untreated, control eggs, male pronuclear development took place while the maternally derived chromosomes remained condensed. It was not until approximately 20 min after the normal period of pronuclear development that the maternal chromosomes dispersed and formed a female pronucleus in emetine-treated, fertilized eggs. Formation of pronuclei, however, was unaffected in both emetine-treated, A-23187-activated eggs and fertilized eggs incubated with A-23187. These observations indicate that germinal vesicle breakdown, first polar body formation, and initial transformations of the sperm nucleus are independent of newly synthesized proteins. Inhibition of second polar body formation and the delay in pronuclear development brought about by emetine, as well as the appearance of silver grains over pronuclei in autoradiographs of control eggs incubated with [3H]leucine demonstrate that nascent proteins are involved with the completion of meiotic maturation and the development of male and female pronuclei. The ability of A-23187 to override the inhibitory effects of emetine on pronuclear development suggests that both nascent protein and calcium signals are involved in regulating the status of the maternal and paternal chromatin during pronuclear development.     

Characterization of the effect of aphidicolin on adenovirus DNA replication: evidence in support of a protein primer model of initiation

Adenovirus DNA replication is inhibited by aphidicolin but the inhibition clearly has different parameters than the inhibition of purified DNA polymerase alpha. In adenovirus infected Hela cells, 10 micrograms/ml of aphidicolin reduced viral DNA synthesis by 80%. Cellular DNA synthesis was inhibited by 97% at 0.1 microgram/ml. 10 micrograms/ml of drug had no effect on virus yield or late protein synthesis though higher concentrations of drug (50 micrograms/ml) caused an abrupt cessation of late protein synthesis and 100 micrograms/ml reduced virus yield by 3 logs. Concentrations of the drug from 0.5 microgram/ml to 10 micrograms/ml were found to dramatically slow the rate of DNA chain elongation in vitro but not stop it completely, so that over a long period of time net incorporation was reduced only slightly compared to the control. 50 micrograms/ml or 100 micrograms/ml of drug completely inhibited incorporation in vitro. Initiation of viral DNA replication - covalent attachment of dCMP to the preterminal protein - occurs in vitro. This reaction was found to be insensitive to inhibition by aphidicolin. We thus conclude that aphidicolin exerts its effect on adenovirus DNA chain elongation, but not on the primary initiation event of protein priming.     

The effect of bleomycin on DNA synthesis in human cells: evidence for grouping of initiation of replicons in the S-period

The effect of bleomycin (Blm) on DNA synthesis has been studied in a synchronous culture of human embryonic lung cells. The data obtained suggest that in the Blm presence in a medium (20 micrograms/ml) DNA synthesis initiation in new replicons is suppressed. The Blm action at different S-phase intervals has been shown to inhibit DNA synthesis unequally. Four discrete time intervals have been singled out in the course of the 10-hr S-phase in which a grouped initiation of replicon portions can be supposed. Together with the data on DNA replication in large-size replicon units (50-500 microns), the obtained results account well for the uneven DNA synthesis in S-phase, manifested by 3 or 4 peaks of [3H]-thymidine incorporation in pulse-labelled cells.