The effect of aphidicolin on adenovirus DNA synthesis.

Aphidicolin inhibits adenovirus DNA replication in HeLa cells and in a cell-free, infected, nuclear extract in which viral DNA is elongated. The compound inhibits alpha DNA polymerase, extensively purified from HeLa cells, but has little or no effect on the beta or gamma DNA polymerases similarly purified. Aphidicolin does not affect thymidine uptake by cells nor does synthesis as it also inhibits DNA replication in uninfected cells. The inhibition by aphidicolin is reversible if the drug is removed within 18 hrs after addition to HeLa or Chinese Hamster Ovary cells but the cells are irreversibly affected if the drug remains for 48 hours.     

Combined effects of aphidicolin and retinoic acid on proliferation and differentiation of human leukaemic (HL-60) cells

The relationships between replicative DNA synthesis and retinoic acid (RA)-induced differentiation of human promyelocytic leukaemic (HL-60) cells are evaluated with the use of Aphidicolin, a specific and reversible inhibitor of DNA polymerase alpha (alpha). Addition of a sublethal concentration of Aphidicolin (0.4 microM) in culture for 3 days suppresses DNA synthesis to a similar level of the resting stage (day 8) in control cultures. DNA synthesis is reactivated to the level observed in the growing stage of control cultures once Aphidicolin is removed after 3 days in culture. The level of DNA synthesis at the early stage of RA-induction (day 3) is suppressed by only 17% when compared to control cultures. The inhibitory effect of Aphidicolin on DNA synthesis in both control cultures and RA-induced cell cultures is similar. However, no reactivation of DNA synthesis is observed after removal of Aphidicolin on day 3 from RA-induced cell cultures. Flow cytometric analysis of DNA content on day 3 reveals that cells accumulate in G1 and early S phases of the cell cycle after exposure to Aphidicolin with or without RA. Of interest is the fact that, while Aphidicolin alone did not induce cells to differentiate, neither did it interfere with RA-induced cell differentiation (the rate of RA-induced cell differentiation in the presence of Aphidicolin is similar to that of RA-treated cultures in the absence of Aphidicolin). These results suggest that the combined use of Aphidicolin and RA may inhibit leukaemic cell proliferation more effectively without causing severe cytotoxicity and without interfering with RA-induced cell differentiation.     

Heteroduplex repair in extracts of human HeLa cells

A general repair process for DNA heteroduplexes has been detected in HeLa cell extracts. Using a variety of M13mp2 DNA substrates containing single-base mismatches and extra nucleotides, extensive repair is observed after incubation with HeLa cell cytoplasmic extracts and subsequent transfection of bacterial cells with the treated DNA. Most, but not all, mispairs as well as two frameshift heteroduplexes are repaired efficiently. Parallel measurements of repair in HeLa extracts and in Escherichia coli suggest that repair specificities are similar for the two systems. The presence of a nick in the molecule is required for efficient repair in HeLa cell extracts, and the strand containing the nick is the predominantly repaired strand. Mismatch-dependent DNA synthesis is observed when radiolabeled restriction fragments, produced by reaction of the extract with heteroduplex and homoduplex molecules, are compared. Specific labeling of fragments, representing a region of approximately 1,000 base pairs and containing the nick and the mismatch, is detected for the heteroduplex substrate but not the homoduplex. The repair reaction is complete after 20 min and requires added Mg2+, ATP, and an ATP-regenerating system, but not dNTPs, which are present at sufficient levels in the extract. An inhibitor of DNA polymerase beta, dideoxythimidine 5'-triphosphate, does not inhibit mismatch-specific DNA synthesis. Aphidicolin, an inhibitor of DNA polymerases alpha, delta, and epsilom, inhibits both semiconservative replication and repair synthesis in the extract. Butylphenyl-dGTP also inhibits both replicative and repair synthesis but at a concentration known to inhibit DNA polymerase alpha preferentially rather than delta or epsilon. This suggests that DNA polymerase alpha may function in mismatch repair.     

Involvement of DNA polymerase alpha in host cell reactivation of UV-irradiated herpes simplex virus.

Aphidicolin is a potent inhibitor of both host cell DNA polymerase alpha and herpes simplex virus (HSV)-induced DNA polymerase but has no effect on DNA polymerases beta and gamma of host cells. By using an aphidicolin-resistant mutant (Aphr) of HSV, a possible involvement of DNA polymerase alpha in host cell reactivation of UV-damaged HSV was studied. Plaque formation by UV-irradiated Aphr was markedly inhibited by 1 microgram of aphidicolin per ml, which did not affect the plating efficiency of nonirradiated Aphr. Aphidicolin added before 12 h postinfection inhibited plaque formation by irradiated Aphr, which became aphidicolin insensitive after 36 h postinfection. The results strongly suggest that host cell DNA polymerase alpha is involved in the repair of UV-irradiated HSV DNA.     

3-Aminobenzamide is lethal to MMS-damaged human fibroblasts primarily during S phase

3-Aminobenzamide (3-AB) interferes with DNA repair and enhances lethality in growing MMS (methyl methane sulfonate)-treated human fibroblasts. This sensitivity to 3-AB disappears slowly; MMS-treated cells are sensitive to 3-AB for up to 36 hours (Boorstein and Pardee, 1984). Evidence is now presented that 3-AB potentiates the effects of MMS primarily during S phase. When cells were synchronized at the G1/S boundary, released, and then treated with MMS, 3-AB caused very substantial lethality in only 4 hours, and a 12-hour treatment gave maximum lethality. These cells also lost sensitivity to 3-AB within 12 hours of growth minus 3-AB. In contrast, MMS-treated quiescent (G0) cells did not lose sensitivity to 3-AB nor did 3-AB cause lethality during G0. Enhanced lethality occurred when damaged G0-arrested cells were subsequently allowed to proceed through S phase in the presence of 3-AB; this 3-AB sensitivity was removed only during growth in the absence of 3-AB. The lethality of 3-AB to a population of asynchronously cycling cells treated with MMS is thus the summation of effects on the cells as they traverse S phase. Aphidicolin prevented lethality of 3-AB to cells released from G1/S and treated with MMS. It also inhibited the loss of sensitivity to added 3-AB later. Correlation with the inhibition of DNA synthesis by this drug suggests that DNA synthesis is essential for the lethality enhancement by 3-AB. Cells treated first with MMS and then with 3-AB accumulated in G2. This G2 arrest depended on S-phase events and correlated with cell lethality. Cells treated with a nonlethal dose of MMS at the G1/S boundary were delayed briefly (3 hours) in their passage through S and G2. These cells, when also exposed to 3-AB, were delayed 6-9 hours in S and they became arrested in G2. There was no G2 arrest when 3-AB was added only after these cells had reached G2. Treatment with 3-AB during S phase thus resulted in both enhanced lethality and G2 arrest. 3-AB inhibited repair of DNA single-strand damage, shown by alkaline elution analysis, in both S-phase and quiescent cells. Aphidicolin inhibited disappearance of breaks and eliminated the difference between 3-AB-treated and untreated cells. Lethality did not correlate well with the measured single-strand damage.(ABSTRACT TRUNCATED AT 400 WORDS)     

Combined Effects of Aphidicolin and Retinoic Acid On Proliferation and Differentiation of Human Leukaemic (HL-60) Cells

The relationships between replicative DNA synthesis and retinoic acid (RA)-induced differentiation of human promyelocytic leukaemic (HL-60) cells are evaluated with the use of Aphidicolin, a specific and reversible inhibitor of DNA polymerase alpha (α). Addition of a sublethal concentration of Aphidicolin (0.4 μM) in culture for 3 days suppresses DNA synthesis to a similar level of the resting stage (day 8) in control cultures. DNA synthesis is reactivated to the level observed in the growing stage of control cultures once Aphidicolin is removed after 3 days in culture. the level of DNA synthesis at the early stage of RA-induction (day 3) is suppressed by only 17% when compared to control cultures. the inhibitory effect of Aphidicolin on DNA synthesis in both control cultures and RA-induced cell cultures is similar. However, no reactivation of DNA synthesis is observed after removal of Aphidicolin on day 3 from RA-induced cell cultures. Flow cytometric analysis of DNA content on day 3 reveals that cells accumulate in G₁ and early S phases of the cell cycle after exposure to Aphidicolin with or without RA. of interest is the fact that, while Aphidicolin alone did not induce cells to differentiate, neither did it interfere with RA-induced cell differentiation (the rate of RA-induced cell differentiation in the presence of Aphidicolin is similar to that of RA-treated cultures in the absence of Aphidicolin). These results suggest that the combined use of Aphidicolin and RA may inhibit leukaemic cell proliferation more effectively without causing severe cytotoxicity and without interfering with RA-induced cell differentiation.     

The 70 kDa subunit of replication protein A is required for the G1/S and intra-S DNA damage checkpoints in budding yeast.

The rfa1-M2 and rfa1-M4 Saccharomyces cerevisiae mutants, which are altered in the 70 kDa subunit of replication protein A (RPA) and sensitive to UV and methyl methane sulfonate (MMS), have been analyzed for possible checkpoint defects. The G1/S and intra-S DNA damage checkpoints are defective in the rfa1-M2 mutant, since rfa1-M2 cells fail to properly delay cell cycle progression in response to UV irradiation in G1 and MMS treatment during S phase. Conversely, the G2/M DNA damage checkpoint and the S/M checkpoint are proficient in rfa1-M2 cells and all the checkpoints tested are functional in the rfa1-M4 mutant. Preventing S phase entry by alpha-factor treatment after UV irradiation in G1 does not change rfa1-M4 cell lethality, while it allows partial recovery of rfa1-M2 cell viability. Therefore, the hypersensitivity to UV and MMS treatments observed in the rfa1-M4 mutant might only be due to impairment of RPA function in DNA repair, while the rfa1-M2 mutation seems to affect both the DNA repair and checkpoint functions of Rpa70.     

Comparison of synchronized Chinese hamster ovary cells obtained by mitotic shake-off, hydroxyurea, aphidicolin, or methotrexate

Synchronized cell populations are necessary to study many aspects of cell biology. We have developed a method to obtain highly synchronized Chinese hamster ovary cell populations in S phase or G2 phase by utilizing mitotic selection followed by incubation with either hydroxyurea, aphidicolin, or methotrexate for 12 h. Flow cytometry analysis shows that the coefficient of variation in the spread of the cell population in S phase is as low as 6%. Drug toxicity studies compare the effects of the various drugs on G1 and S phase cells. The use of aphidicolin or hydroxyurea results in the most highly synchronized cell populations, but methotrexate yields inadequate synchronization. These results demonstrate that both aphidicolin and hydroxyurea are useful drugs for obtaining highly synchronized cell populations after an initial synchrony in mitosis. Aphidicolin is perhaps the best choice because of less toxicity to S phase cells when used in low concentrations.     

Transforming growth factor-beta inhibits phosphorylation of the retinoblastoma susceptibility gene product in human monocytic leukemia cell line JOSK-I.

Proliferation of the human monocytic leukemia cell line JOSK-I is inhibited by transforming growth factor-beta (TGF-beta). Growth inhibition by TGF-beta was not due to either a toxic effect or to induction of differentiation. TGF-beta induced a cell cycle arrest at late G1 phase and was not found to be inhibitory to JOSK-I cells in S phase or G2/M. This G1 cell cycle arrest was associated with an accumulation of the unphosphorylated form of the retinoblastoma susceptibility gene product (Rb) in good correlation with inhibition of DNA synthesis. In contrast to the effects of TGF-beta, two other agents which induced a G1 arrest of JOSK-I cells had a different effect on Rb. Aphidicolin blocked cells at G1/S but could not reduce Rb phosphorylation as great as that seen with TGF-beta. 12-O-Tetradecanoylphorbol-13-acetate, an inducer of differentiation, did reduce Rb phosphorylation, but not until 72 h, when differentiation had already occurred. The identities of the Rb kinases are unknown, but recent evidence suggests that the cdc2 gene product could participate in Rb phosphorylation. Although cdc2 mRNA and total protein levels were not affected, TGF-beta inhibited the rate of translation and kinase activity of cdc2 in JOSK-I cells. These results suggest that growth inhibition of hematopoietic cells by TGF-beta is linked to suppression of Rb phosphorylation to retain Rb in an unphosphorylated, growth-inhibitory state. The suppression of Rb phosphorylation is suggested to be mediated through inhibition of cdc2 kinase activity by TGF-beta.     

Synchronisation of Giardia lamblia: identification of cell cycle stage-specific genes and a differentiation restriction point

The intestinal parasite Giardia lamblia undergoes cell differentiations that entail entry into and departure from the replicative cell cycle. The pathophysiology of giardiasis depends directly upon the ability of the trophozoite form to replicate in the host upper small intestine. Thus, cell proliferation is tightly linked to disease. However, studies of cell cycle regulation in Giardia have been hampered by the inability to synchronise cultures. Here we report that Giardia isolates of the major human genotypes A and B can be synchronised using aphidicolin, a mycotoxin that reversibly inhibits replicative DNA polymerases in eukaryotic cells. Aphidicolin arrests Giardia trophozoites in the early DNA synthesis (S) phase of the cell cycle. We identified a set of cell cycle orthologues in the Giardia genome using bioinformatic analyses and showed that synchronised parasites express these genes in a cell cycle stage-specific manner. The synchronisation method also showed that during encystation, exit from the ordinary cell cycle occurs preferentially in G(2) and defines a restriction point for differentiation. Synchronisation opens up possibilities for further molecular and cell biological studies of chromosome replication, mitosis and segregation of the complex cytoskeleton in Giardia.     

Aphidicolin: a specific inhibitor of DNA polymerases in the cytosol of rat liver.

Aphidicolin, a tetracyclic diterpenoid, is known to be antiviral and to inhibit the incorporation of thymidine into DNA of cultured human embryonic lung cells. We examined effects of the compound on the activity of several DNA polymerases obtained from subcellular fractions of rat liver. Aphidicolin at a concentration of 15 μg/ml caused a 85% reduction in level of the activity of crude and partially purified DNA polymerases from the cytosol. However, aphidicolin even at a concentration of 75 μg/ml failed to affect the activity of crude and partially purified DNA polymerases from nuclear and mitochondrial fractions.     

Characterization of mec1 kinase-deficient mutants and of new hypomorphic mec1 alleles impairing subsets of the DNA damage response pathway

DNA damage checkpoints lead to the inhibition of cell cycle progression following DNA damage. The Saccharomyces cerevisiae Mec1 checkpoint protein, a phosphatidylinositol kinase-related protein, is required for transient cell cycle arrest in response to DNA damage or DNA replication defects. We show that mec1 kinase-deficient (mec1kd) mutants are indistinguishable from mec1Delta cells, indicating that the Mec1 conserved kinase domain is required for all known Mec1 functions, including cell viability and proper DNA damage response. Mec1kd variants maintain the ability to physically interact with both Ddc2 and wild-type Mec1 and cause dominant checkpoint defects when overproduced in MEC1 cells, impairing the ability of cells to slow down S phase entry and progression after DNA damage in G(1) or during S phase. Conversely, an excess of Mec1kd in MEC1 cells does not abrogate the G(2)/M checkpoint, suggesting that Mec1 functions required for response to aberrant DNA structures during specific cell cycle stages can be separable. In agreement with this hypothesis, we describe two new hypomorphic mec1 mutants that are completely defective in the G(1)/S and intra-S DNA damage checkpoints but properly delay nuclear division after UV irradiation in G(2). The finding that these mutants, although indistinguishable from mec1Delta cells with respect to the ability to replicate a damaged DNA template, do not lose viability after UV light and methyl methanesulfonate treatment suggests that checkpoint impairments do not necessarily result in hypersensitivity to DNA-damaging agents.     

Cdc25B and Cdc25C differ markedly in their properties as initiators of mitosis.

We have used time-lapse fluorescence microscopy to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initiators of mitosis in human cells. To differentiate between the functions of the two proteins, we have microinjected expression constructs encoding Cdc25B or Cdc25C or their GFP-chimeras into synchronized tissue culture cells. This assay allows us to express the proteins at defined points in the cell cycle. We have followed the microinjected cells by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and assayed whether they enter mitosis prematurely or at the correct time. We find that overexpressing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mitosis, whether or not DNA replication is complete, whereas overexpressing Cdc25C does not cause premature mitosis. Overexpressing Cdc25C together with cyclin B1 does shorten the G2 phase and can override the unreplicated DNA checkpoint, but much less efficiently than overexpressing Cdc25B. These results suggest that Cdc25B and Cdc25C do not respond identically to the same cell cycle checkpoints. This difference may be related to the differential localization of the proteins; Cdc25C is nuclear throughout interphase, whereas Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. We have found that the change in subcellular localization of Cdc25B is due to nuclear export and that this is dependent on cyclin B1. Our data suggest that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in the controlling the initiation of mitosis.     

Auxin induction of cell cycle regulated activity of tobacco telomerase.

Telomerase activity was measured at each phase of the cell cycle in synchronized tobacco (Nicotiana tabacum) BY-2 cells in suspension culture with the use of the telomeric repeat amplification protocol assay. The activity was low or undetectable at most phases of the cell cycle but showed a marked increase at early S phase. The induction of telomerase activity was not affected by the S phase blockers aphidicolin (which inhibits DNA polymerase alpha) or hydroxyurea (which inhibits ribonucleotide reductase), but it was prevented by olomoucine, an inhibitor of Cdc2/Cdk2 kinases that blocks G(1)-S cell cycle transition. These results suggest that the induction of telomerase activity is not directly coupled to DNA replication by conventional DNA polymerases, but rather is triggered by the entry of cells into S phase. Various analogs of the plant hormone auxin, including indole-3-acetic acid, alpha-naphthaleneacetic acid, and 2,4-dichlorophenoxyacetic acid, potentiated the increase in telomerase activity at early S phase; the growth-inactive analog 2,3-dichlorophenoxyacetic acid, however, had no such effect. Potentiation by indole-3-acetic acid of the induction of telomerase activity was dose dependent. Together, these data indicate that telomerase activity in tobacco cells is regulated in a cell cycle-dependent manner, and that the increase in activity at S phase is specifically inducible by auxin.     

Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools

The relationship between dNTP levels and DNA synthesis was investigated using alpha factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU). Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G1/S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G1 levels. When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37 degrees C before being released to 25 degrees C in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G1 levels. Accumulation of dNTPs at G1/S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25 degrees C were able to replicate when subsequently switched to 37 degrees C in the absence of HU. The replication arrest mechanism was not dependent on the Mec1/Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU. The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold.     

Apoptosis in erythroid progenitors deprived of erythropoietin occurs during the G1 and S phases of the cell cycle without growth arrest or stabilization of wild-type p53.

Erythropoietin (Epo) inhibits apoptosis in murine proerythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells). We have shown that the apoptotic process in FVA cell populations deprived of Epo is asynchronous as a result of a heterogeneity in Epo dependence among individual cells. Here we investigated whether apoptosis in FVA cells correlated with cell cycle phase or stabilization of p53 tumor suppressor protein. DNA analysis in nonapoptotic FVA cell subpopulations cultured without Epo demonstrated little change in the percentages of cells in G1,S, and G2/M phases over time. Analysis of the apoptotic subpopulation revealed high percentages of cells in G1 and S, with few cells in G2/M at any time. When cells were sorted from G1 and S phases prior to culture without Epo, apoptotic cells appeared at the same rate in both populations, indicating that no prior commitment step had occurred in either G1 or S phase. Steady-state wild-type p53 protein levels were very low in FVA cells compared with control cell lines and did not accumulate in Epo-deprived cultures; however, p53 protein did accumulate when FVA cells were treated with the DNA-damaging agent actinomycin D. These data indicate that erythroblast apoptosis caused by Epo deprivation (i) occurs throughout G1 and S phases and does not require cell cycle arrest, (ii) does not have a commitment event related to cell cycle phase, and (iii) is not associated with conformational changes or stabilization of wild-type p53 protein.     

Laser flow cytofluorometric analysis of HTC cells synchronized with hydroxyurea, nocodazole and aphidicolin

The technique of laser flow cytofluorometry has been used to monitor the arrival in G1 and the subsequent progression through the cell cycle of HTC cells accumulated in metaphase with colcemid alone or after treatment with hydroxyurea and Nocodazole. Under the experimental conditions used in this study, the latter procedure gives much better results, avoiding in particular the extensive formation of micronucleated cells. Aphidicolin, an inhibitor of DNA polymerase, in combination with Nocodazole, provides a useful method to tightly synchronize these cells at the G1/S border.     

Cell synchronization effect on mammalian cell permeabilization and gene delivery by electric field

Electropermeabilization is a promising nonviral method for gene therapy. However, despite the fact that it is widely used to transfer genes into living cells, the steps that limit DNA transfer remain to be determined. Here, we report the effect of cell synchronization on membrane permeabilization and gene delivery by electric fields.Chinese hamster ovary (CHO) cells were synchronized by aphidicolin or butyrate treatment. Electro-mediated transfection of these cells was evaluated under electric field conditions leading to the same level of membrane permeabilization.Aphidicolin cell synchronization in G2/M phase leads to a slight increase in plasma membrane permeabilization but to a three-fold increase in percentage of transfected cells and to an eight-fold increase in gene expression. This increase in cell transfection is specifically due to the G2/M synchronization process. Indeed, cell synchronization in G1 phase by sodium butyrate has no effect on cell permeabilization and transfection.Our results suggest that the enhanced transfection level in G2/M phase is not simply due to enhanced permeabilization, but reinforce the statement that the melting of the nuclear membrane facilitates direct access of plasmid DNA to the nucleus.