SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We previously reported that DNA topoisomerase II (topo II) is required for the G0-to-S phase transition in mammalian cells [Hossain et al. (2002) ICRF-193, a catalytic inhibitor of DNA topoisomerase II, inhibits re-entry into the cell division cycle from quiescent state in mammalian cells. Genes Cells 7, 285–294]. In this study, we examined whether the requirement for topo II is evolutionarily conserved in Drosophila and yeast. ICRF-193, a catalytic inhibitor of topo II, inhibited DNA synthesis in Drosophila Schneider cells released from the G0 (stationary) phase, whereas the drug did not inhibit DNA synthesis in Schneider cells released from the M phase. Depletion of topo II mRNA by RNA-interference (RNAi) in G0-phase Schneider cells resulted in significant inhibition of DNA synthesis after release from G0-arrest. In the yeast topo II temperature-sensitive (ts) mutant, the initial cycle of DNA synthesis occurred at a restrictive temperature after release from starvation-induced G0 phase and doubling of the DNA content in the cells was confirmed by both flow cytometry and fluorescence spectrophotometry. DNA synthesis in yeast cells after release from the G0 phase was also observed in the presence of ICRF-193. Doubling of the DNA content was observed during spore germination of topo II ts mutant yeast at a restrictive temperature as determined by fluorescence spectrophotometry. These results indicate that topo II is required for the G0-to-S phase transition in Drosophila Schneider cells, but not in yeast.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

DNA topoisomerases (topo) alter DNA topology and are necessary for releasing torsional stress generated in DNA during processes such as replication, transcription, recombination and chromosome segregation. There are two different types of topos: type I and II. Type I enzymes catalyse the cleavage and re-ligation reactions of DNA single strands, whereas type II enzymes catalyse these reactions with double-stranded DNA breaks (Wang 1996).

A typical eukaryotic cell division cycle consists of four phases: G1 (gap1), S (DNA synthesis), G2 (gap2) and M (mitosis) phases. Topo I acts as the major swivelase during progression through the S phase, whereas topo II is essential for the proper decatenation and segregation of newly replicated DNA during the G2/M phases (Holm et al. 1985; Uemura et al. 1987; Buchenau et al. 1993; Downes et al. 1994; Wang 1996; Akimitsu et al. 2003a). Yeast and Drosophila have one topo II whereas mammals have two isozymes—α and β. Topo IIα−/– mouse embryos terminate development at the 4- or 8-cell stage with catenated DNAs followed by apoptosis, indicating that topo IIα is essential for the cell division cycle (Akimitsu et al. 2003a,b). Topo IIβ−/– mouse embryos die shortly after birth as a result of abnormalities in the nervous system, indicating that this enzyme is not essential for the cell division cycle (Yang et al. 2000). Topo II is not essential for the G1-to-S phase transition in yeast and mammalian cells (DiNardo et al. 1984; Ishida et al. 1994). In exponentially proliferating Drosophila Schneider cells, depletion of topo II by RNA interference (RNAi) revealed that the enzyme is required for the proper segregation of sister chromatids during M phase, indicating that topo II is not essential at the other phases of cell cycle in Schneider cells (Chang et al. 2003).

The enzymatic action of topo II can be inhibited by two different classes of chemical inhibitors. The first type includes compounds that stabilize the ‘cleavable-complex’ between topo II and DNA resulting in DNA double-strand breaks (DSBs) (Wilstermann & Osheroff 2003). Examples of this type include etoposide, adriamycin and mitoxantrone. The second type of inhibitors are known as ‘catalytic inhibitors’ of topo II that inhibit the enzyme without causing DNA DSBs (Andoh & Ishida 1998). Examples of catalytic inhibitors include ICRF-193, merbarone and aclarubicin. ICRF-193 [meso-2,3-bis(3,5-dioxopiperazin-1-yl)butane], a derivative of bisdioxopiperazines, traps the topo II enzyme in the closed-clamp intermediate form in the presence of ATP resulting in the inhibition of ATPase activity (Roca et al. 1994). Both in vitro and in vivo, ICRF-193-treatment resulted in the formation of catenated dimers of simian virus 40 DNA (Ishimi et al. 1992, 1995). Genetic studies with yeast demonstrated that the target of ICRF-193 in vivo is topo II (Ishida et al. 1995). In vitro, ICRF-193 inhibits eukaryotic topo II enzymes, including that of Drosophila (Sato et al. 1997). In Drosophila syncytial embryos, ICRF-193 treatment resulted in the failure of chromosome segregation but, unlike DNA DSB-inducing topo II inhibitors, ICRF-193 did not trigger DNA damage-induced centrosome inactivation or mitotic delays (Takada et al. 2003). We previously reported that DNA DSB-inducing drugs, including ‘cleavable-complex’ stabilizing topo II inhibitors, induced myogenic differentiation of Drosophila Schneider cells, but no differentiation was observed with ICRF-193 (Hossain et al. 2003). Moreover, ICRF-193 strongly inhibited differentiation induced by etoposide, adriamycin and mitoxantrone, but not neocarzinostatin, a drug that causes DNA DSBs independent of topo II (M. S. Hossain, K. Kurokawa and K. Sekimizu; manuscript in preparation). These results indicate that ICRF-193 can be used to evaluate the biologic functions of topo II in Drosophila Schneider cells.

When the conditions are not suitable for the continuation of proliferation, eukaryotic cells enter into an out-of-cell-cycle phase known as the G0 (stationary) phase (Pardee 1989). Cells arrested at the G0 phase synchronously re-enter the cell cycle when appropriate growth conditions are provided. The molecular mechanisms regulating this transition from the quiescent to the proliferating state are poorly understood.

We previously reported that ICRF-193 inhibited G0-to-S phase transition in ICRF-193-sensitive mammalian cells, but not in cell lines resistant to the drug because of point mutation in the topo IIα gene (Hossain et al. 2002). In the present study, we examined whether the requirement for topo II is evolutionarily conserved in lower eukaryotes. ICRF-193-treatment or topo II RNAi of G0 phase cells resulted in significant inhibition of DNA synthesis in Drosophila Schneider cells after release from the G0 phase. Using both topo II temperature-sensitive (ts) mutants and ICRF-193-sensitive yeast strains, we demonstrated that topo II is not required for the G0-to-S phase transition in yeast. We also showed that topo II is not required for spore germination in yeast. These results indicate that topo II is required for the G0-to-S phase transition in Drosophila, but not in yeast.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

ICRF-193, a catalytic inhibitor of DNA topoisomerase II, inhibits DNA synthesis in Drosophila Schneider cells after release from G0-arrest

Drosophila Schneider cells were arrested at the G0 (stationary) phase by keeping the cells at maximum density for 4 days in a spinner flask (Rizzino & Bluementhal 1978). When the G0 phase cells were diluted into fresh medium, DNA synthesis started after ∼18 h and maximum incorporation of [3H]thymidine was observed at 24 h, as determined by pulse-labelling (Fig. 1). Addition of 10 µm ICRF-193 at 0, 3, 6 or 9 h after release from G0-arrest resulted in significant inhibition of DNA synthesis (Fig. 1A–D), whereas addition of the drug after 12 or 15 h of release did not significantly inhibit the process (Fig. 1E,F). The results indicate that ICRF-193 inhibits G0-to-S phase transition in Drosophila Schneider cells, when added within 9 h of release from G0 arrest. Therefore, topo II might play a critical role in between 9 and 12 h to induce DNA replication after release from G0 arrest. This result might exclude the possibility that topo II plays any essential role in the expression of immediate–early genes.

image

Figure 1. ICRF-193, a catalytic inhibitor of DNA topoisomerase II (topo II), inhibits restoration of DNA synthesis in Drosophila Schneider cells after release from G0 arrest. Cells were arrested at the G0 phase by growing to maximum density and maintaining at full growth for four more days. Cells were then diluted in fresh medium and 10 µm ICRF-193 or equal volume of DMSO was added at (A) 0 h, (B) 3 h, (C) 6 h, (D) 9 h, (E) 12 h or (F) 15 h of release (as indicated by arrows). Cells were pulse-labelled with 5 µCi/mL [3H]thymidine for 1 h at the indicated times. The data shown are representative of two different experiments with similar results.

Download figure to PowerPoint

ICRF-193 does not inhibit G1-to-S phase transition in Schneider cells

Next, we examined whether ICRF-193 inhibits the G1-to-S phase transition in Schneider cells. Schneider cells were arrested at the M phase by nocodazole treatment followed by release in nocodazole-free medium for 3 h to allow the cells to enter the G1 phase. DNA synthesis was observed after 12 h of release from nocodazole-arrest (Fig. 2). Addition of ICRF-193 after 3 h of release from nocodazole-arrest did not significantly inhibit DNA synthesis (Fig. 2), indicating that ICRF-193 does not inhibit G1-to-S phase transition in Drosophila Schneider cells. This observation is similar to what we reported in mammalian cells (Hossain et al. 2002). We cannot exclude the possibility that topo II might be required within 3 h of release from M-phase arrest. We consider this is not the case because ICRF-193-treatment of Drosophila syncytial embryo (Takada et al. 2003) or topo II RNAi of exponentially proliferating Drosophila Schneider cells (Chang et al. 2003) resulted in M-phase arrest, indicating that topo II is required only at M phase in exponentially proliferating cells of Drosophila.

image

Figure 2. ICRF-193 does not inhibit G1-to-S phase transition in Drosophila Schneider cells. Cells were arrested at metaphase by nocodazole followed by release in nocodazole-free medium for 3 h. After 3 h, 10 µm ICRF-193 or an equal volume of DMSO was added. Cells were pulse-labelled with 5 µCi/mL [3H]thymidine for 1 h at the indicated times. The data shown are representative of two different experiments with similar results.

Download figure to PowerPoint

Topo II RNAi in G0-phase Schneider cells inhibits DNA synthesis after release from G0 arrest

To further demonstrate that topo II is required for the G0-to-S phase transition, we performed topo II RNAi in G0-phase Schneider cells. Incubation of G0-phase Schneider cells with topo II dsRNA for 7 days resulted in significant depletion of topo II mRNA in comparison with control cells (Fig. 3A; 0 h) as determined by RT-PCR using PCR primers corresponding to a different region of topo II cDNA than that used for dsRNA preparation (see Experimental procedures). After release from G0-arrest, topo II expression was induced within 6 h with maximum expression at 24–30 h period in control cells, whereas no significant expression was detected until 12 h in topo II dsRNA-treated cells (Fig. 3A). Low induction of topo II expression, in comparison with control cells, was detected in topo II dsRNA-treated cells during an 18–30-h period (Fig. 3A), which could be because of the presence of cells that did not respond to dsRNA. When G0 phase cells were diluted and released in fresh medium, significant inhibition of DNA synthesis was observed in topo II dsRNA-treated cells in comparison with control cells as determined by [3H]thymidine incorporation (Fig. 3B). These results support the notion that topo II is required for the G0-to-S phase transition in Drosophila Schneider cells.

image

Figure 3. Topo II RNAi in G0-phase Schneider cells inhibits DNA synthesis after release from G0 arrest. (A) RT-PCR analysis of topo II expression during the G0-to-S phase transition in control and topo II dsRNA-treated cells. G0 phase cells were treated with dsRNA of either topo II or EGFP (control) for 7 days followed by dilution in fresh medium. For PCR amplification the primers used correspond to a different region of topo II cDNA than that used for dsRNA production (See Experimental procedures). The negative image of ethidium bromide-stained gel is shown. M indicates DNA marker. (B) Inhibition of DNA synthesis after release from G0 arrest in topo II dsRNA-treated cells. Cells were pulse-labelled with 5 µCi/mL [3H]thymidine for 1 h at the indicated times. The data shown are representative of two different experiments with similar results.

Download figure to PowerPoint

Restoration of DNA synthesis in yeast topo II ts mutant at a restrictive temperature

Exponentially proliferating cells of a haploid parent and its isogenic topo II ts mutant of Saccharomyces cerevisiae were starved from carbon sources (Granot & Snyder 1991). We used three criteria (Werner-Washburne et al. 1993) to judge whether yeast cells were in the G0 phase after starvation under our experimental conditions: (i) more than 90% of S. cerevisiae cells were unbudded as determined by microscopic analysis (data not shown); (ii) starved yeast cells were resistant to heat-shock at 50 °C in comparison with log-phase cells as determined by colony-forming ability (data not shown); and (iii) starved yeast cells were resistant to the cell-wall degrading enzyme, zymolyase-100T in comparison with log-phase cells (data not shown).

When carbon-starved cells were released at 25 °C in complete medium, DNA synthesis started to increase after 6 h and continued to increase until 12 h in both the parent and the topo II ts mutant as measured by alkali-resistant [3H]uracil incorporation (Fig. 4A,B). However, the increase of [3H] incorporation was terminated after 6 h at 37 °C in the topo II ts mutant (Fig. 4B). Rapamycin, a G1 phase-arresting drug (Zaragoza et al. 1998), strongly inhibited DNA synthesis in both the parent and the mutant (Fig. 4A,B). To measure the amount of [3H] incorporated only in the first round of DNA synthesis, we added benomyl, an M-phase-arresting drug, after 3 h of release from the G0 arrest. After 6 h, DNA synthesis was inhibited by benomyl in both the parent and mutant cells, suggesting that the first round of DNA synthesis was complete within 6 h (Fig. 4A,B). In the topo II ts mutant, no significant difference with respect to DNA synthesis was observed between those at 37 °C in the absence of benomyl and those at 25 °C in the presence of benomyl (Fig. 4B). The patterns of DNA synthesis between 37 °C and 25 °C were similar in the topo II ts mutant in the presence of benomyl (data not shown). These results indicate that the topo II ts mutant cells can initiate DNA synthesis after release from the G0 phase at a restrictive temperature (37 °C).

image

Figure 4. Topo II ts mutant of S. cerevisiae can restore DNA synthesis at a restrictive temperature (37 °C). (A) S. cerevisiae haploid strain, W303-1a, and (B) topo II ts mutant strain, RS191. Cells were carbon-starved for 14 days, followed by release in pre-warmed, complete medium. Rapamycin (10 µg/mL) and benomyl (40 µg/mL) were added at 0 h (rapamycin) and 3 h (benomyl) of release (indicated by arrows). Cells were continuously labelled with 15 µCi/mL [3H]uracil followed by alkaline lysis. The data shown are representative of four different experiments with similar results.

Download figure to PowerPoint

Doubling of the DNA content of yeast topo II ts mutant at 37 °C after release from G0 arrest

To examine whether the S. cerevisiae topo II ts mutant can double DNA content at 37 °C, we used flow cytometry. FACS profile revealed that both the parent and topo II ts mutant cells had 1C DNA content after 14 days’ starvation from carbon sources (Fig. 5; 0 h). After 3 h of release from G0 arrest, most cells showed 1C DNA content but a population of cells showed more than 1C DNA content in both the parent and the mutant (Fig. 5; 3 h). After 6 h of release, both the parent and mutant cells showed 2C DNA content at 37 °C in the presence of benomyl (Fig. 5C,G). Topo II ts mutant cells released at 37 °C showed a broad peak between 1C and 2C in the absence of benomyl (Fig. 5H). A possible interpretation of the broad peak is that, in the absence of benomyl, topo II ts mutant cells underwent enforced cytokinesis followed by breakage of thin connections between two cells (Uemura & Yanagida 1984; Holm et al. 1985; Uemura & Yanagida 1986; Holm et al. 1989). Benomyl might have prevented this enforced cytokinesis in the topo II ts mutant cells by arresting cells at metaphase and, therefore, a 2C peak was observed at 37 °C (Fig. 5G). Loss of viability of topo II ts mutant cells at 37 °C after 6 h of release from G0 arrest was prevented by addition of benomyl at 3 h of release as determined by its colony-forming ability on drug-free YPD agar plates (data not shown). The results of flow cytometry suggest that DNA content was doubled in the topo II ts mutant after release from G0 arrest at 37 °C in the presence of benomyl. Therefore, topo II is not required for the G0-to-S phase transition in yeast.

image

Figure 5. Topo II ts mutants of S. cerevisiae can double their DNA content at a restrictive temperature after release from G0 arrest as determined by flow cytometry. Carbon-starved S. cerevisiae cells were released in pre-warmed, complete medium. An aliquot was removed at the indicated times and the DNA content was analysed using flow cytometry. (A, E) Cells released at 25 °C with benomyl addition at 3 h of release; (B, F) cells released at 25 °C; (C, G) cells released at 37 °C with benomyl addition at 3 h of release; (D, H) cells released at 37 °C. The data shown are representative of three different experiments with similar results.

Download figure to PowerPoint

To further confirm that DNA content in the topo II ts mutant was doubled at 37 °C after release from G0 arrest, we determined the total DNA content using fluorescence spectrophotometry of 4′,6-diamidino-2-phenylindole (DAPI)-stained DNA isolated from cells. The amount of DNA was doubled after 6 h of release of carbon-starved topo II ts mutants of S. cerevisiae at 37 °C (Fig. 6), indicating that cells completed DNA replication after release from G0 arrest. Further increase of the DNA content was inhibited in the topo II ts mutant cells at 37 °C, but not at 25 °C. These results also support the notion that topo II is not required for the G0-to-S phase transition in yeast.

image

Figure 6. Topo II ts mutants of S. cerevisiae can double their DNA content after release from G0 arrest at a restrictive temperature as determined by fluorescence spectrophotometry. Carbon-starved cells were released in pre-warmed, complete medium. Total DNA was isolated from yeast cells followed by staining with DAPI (10 µg/mL). Fluorescence from DAPI-stained DNA was measured by fluorescence spectrophotometry. The data shown are representative of two different experiments with similar results.

Download figure to PowerPoint

ICRF-193 does not inhibit G0-to-S phase transition in yeast

Next, we tested whether ICRF-193 inhibits the G0-to-S phase transition in yeast. For this purpose, we used a topo II ts mutant whose growth is sensitive to 100 µm ICRF-193 at a semipermissive temperature (30 °C), whereas its isogenic parent remains resistant (Ishida et al. 1995). After release from the carbon-starved G0 phase, DNA synthesis started to increase at around 6 h in both the parent and the mutant (Fig. 7A,B). After 6 h, DNA synthesis in the presence of ICRF-193 was observed in the parent but not in the mutant (Fig. 7A,B). Moreover, the amounts of DNA synthesis in the presence of ICRF-193 with and without benomyl were almost the same in the mutant (Fig. 7). Given that benomyl arrests cells at the M phase, these results support the notion that topo II is not required for the G0-to-S phase transition in yeast. FACS analysis of the DNA content revealed a broad peak (similar to that observed in Fig. 5H) in the topo II ts mutant, JN394t2-1 in the presence of ICRF-193 at 30 °C after 6 h of release from G0 arrest (data not shown).

image

Figure 7. ICRF-193 does not inhibit the G0-to-S phase transition in yeast. (A) S. cerevisiae parent strain, JN394 (ICRF-193-resistant) and (B) ICRF-193-sensitive, topo II ts mutant strain, JN394t2-1. Carbon-starved yeast cells were re-suspended in complete medium pre-warmed at a semipermissive temperature (30 °C) and treated with either DMSO or 100 µm ICRF-193. Cells were continuously labelled with 15 µCi/mL [3H]uracil followed by alkaline lysis. Benomyl (40 µg/mL) was added after 3 h of release (indicated by the arrow). The data shown are representative of two different experiments with similar results.

Download figure to PowerPoint

Topo II is not required for spore germination in yeast

To test whether topo II is required for re-entry into the cell cycle during germination of spores of diploid yeast, we transformed the S. cerevisiae haploid yeast strain, W303–1a, and its isogenic topo II ts mutant strain, RS191 with the plasmid, YEp13 bearing the HO locus of S. cerevisiae (Jensen et al. 1983). HO locus promotes formation of diploid cells from haploid by stimulating mating-type switching (Jensen et al. 1983). Cells transformed with the empty vector, YEp13, or with YEp13-HO, were selected on SD(Leu) agar plates, grown up to log phase in liquid SD(Leu) medium followed by incubation in sporulation medium for seven more days at 25 °C. Microscopic observations revealed that YEp13-HO transformants formed asci in the sporulation medium at a freqeuncy of ∼80% in both the parent and topo II ts mutant, whereas no ascus was observed in cells transformed with the empty vector, YEp13 (data not shown).

When the asci-containing cultures were diluted in complete medium at 25 °C, the amount of total DNA doubled after 6 h in both the parent and the topo II ts mutant, as measured by fluorescence spectrophotometry of DAPI-stained DNA isolated from cells, and the amount of DNA continued to increase until 12 h of release (Fig. 8A,B). Increase of the DNA amount was terminated in the topo II ts mutant after 6 h at 37 °C (Fig. 8B). Cycloheximide, a drug that inhibits spore germination (Herman & Rine 1997), inhibited any significant increase in the DNA amount in both the parent and the mutant (Fig. 8A,B). Microscopic observations revealed that asci in both the parent and the mutant started to disappear within 3 h of release at both 25 °C and 37 °C and the disappearance was almost complete within 4 h (Fig. 8C,D). The disappearance of asci was relatively rapid at 37 °C compared with that of 25 °C in both the parent and the mutant (Fig. 8C,D). In the presence of cycloheximide, asci remained in the culture of both the parent and the mutant until 6 h of release (Fig. 8C,D). The results indicate that topo II is not required for germination of spores in yeast.

image

Figure 8. Topo II is not required for the germination of spores of diploid yeast. Diploid yeast cells were obtained from haploid strains, W303-1a (parent) and its isogenic topo II ts mutant strain (RS191) by transformation with the plasmid, YEp13-HO. Transformants were grown in sporulation medium for 7 days. Asci containing cultures of (A) the parent, W303, and (B) the topo II ts mutant, RS191, were then diluted at A600−0.5 in pre-warmed, complete medium either at 25 °C or at 37 °C followed by isolation of total DNA. Fluorescence from DAPI-stained DNA was measured by fluorescence spectrophotometry. Cycloheximide (CHX), an inhibitor of germination, was added at 0 h of release to a final concentration of 40 µg/mL. Percentage of asci in (C) the parent and (D) the topo II ts mutant was determined microscopically. The data shown are representative of two different experiments with similar results.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In the present study, we showed that catalytic inhibition of topo II by ICRF-193 or depletion of topo II mRNA by RNAi inhibited re-entry into the cell division cycle from the quiescent (G0) phase in Drosophila Schneider cells. This finding is similar to our previous report on mammalian cells in which the α isoform of topo II is required for the G0-to-S phase transition (Hossain et al. 2002). We also found in the present study that topo II is not required for the G0-to-S phase transition in the budding yeast, S. cerevisiae.

The present findings, together with those in our previous report on mammalian cells (Hossain et al. 2002), suggest that mammals and flies have topo II-dependent step(s) during the G0-to-S phase transition that is not present in yeast. With respect to the requirement of topoisomerases, there are differences among mammals, flies and yeast. For example (i) the absence of topo I results in early embryonic lethality in multicellular mice and Drosophila, whereas topo I is not essential for the proliferation of unicellular yeast (Goto & Wang 1985; Lee et al. 1993; Morham et al. 1996); (ii) topo III, a type IA topo, is not essential for the proliferation of S. cerevisiae, but topo IIIα−/– mouse embryos die in utero and topo IIIβ−/– mice have a reduced mean lifespan (Wallis et al. 1989; Li & Wang 1998; Kwan & Wang 2001); (iii) mammalian topo IIα interacts with a number of transcription factors in vitro, resulting in its increased decatenation activity (Kroll et al. 1993) and topo IIβ is required for the expression of a set of genes involved in neuronal differentiation (Tsutsui et al. 2001). To our knowledge, however, there are no reports of yeast topo II interacting with transcription factors; (iv) there is a topo II-dependent decatenation checkpoint at the G2 phase in mammalian and plant cells, but such a checkpoint apparently does not exist in yeast (Downes et al. 1994; Gimenez-Abian et al. 2002). These findings illustrate that there are significant differences between multicellular and unicellular eukaryotes with respect to the roles of topoisomerases.

Yeast cells always proliferate under favourable conditions, whereas proliferation in mammal and fly is strictly regulated according to the demands of development. Most of the cells in fly and mammal remain quiescent (G0) and proliferate only under certain conditions. For re-entry into the cell cycle, G0 phase cells of mammal and fly require growth factors and nutrients, whereas G0-phase haploid yeast or spores of diploid yeast commit themselves to enter into the cell cycle in the presence of glucose only, even although the other essential nutrients are absent (Granot & Snyder 1991). Following the exit from the quiescent states in the presence of only glucose, yeast cells remain in the G1 phase of the cell cycle and have a decreased ability to survive without division relative to G0 phase cells (Granot & Snyder 1991). It is interesting to note that both the expression and the activity of topo IIα in mammalian cells is influenced by the mitogen-activated protein kinase, ERK (Chen et al. 1999; Shapiro et al. 1999). It is possible that topo II is required for the downstream events during mitogenic stimulation of G0-phase cells in fly and mammals, whereas such a topo II-dependent step during the G0-to-S phase transition is not present in yeast.

The role of topo II that is required for the G0-to-S phase transition in Drosophila Schneider cells remains unknown. As reported earlier with mammalian cells (Hossain et al. 2002), there are several possibilities. First, it is possible that negatively supercoiled DNA formed by topo II is essential for the initiation of DNA replication after release from G0-arrest. Negatively supercoiled DNA is implicated in the initiation of DNA replication in vitro (Baker et al. 1986). It is possible that proliferating cells retain negatively supercoiled DNA, but upon entering into the G0 phase, the DNA of Drosophila Schneider cells relaxes. Although eukaryotic topo II cannot introduce negative supercoiling in DNA under standard assay conditions, it can introduce negative supercoils in the presence of a supercoiling factor in Drosophila melanogaster (Kobayashi et al. 1998). Second, topo II might be required for the transcription of specific genes whose products are essential for re-entry into the cell division cycle from the G0 phase. In mouse, topo IIβ is required for the expression of a set of genes essential for neural development (Tsutsui et al. 2001). The decatenation activity of topo IIαin vitro is stimulated by interacting with transcription factors CREB, ATF-2 and c-jun (Kroll et al. 1993). Topo IIα is required for the transcription by RNA polymerase II on chromatin templates in vitro (Mondal & Parvin 2001).

Topo IIα is present in the centrosomes of G0-phase mammalian cells with unknown biological significance (Berthelmes et al. 2000). It would be worthwhile determining whether Drosophila Schneider cells also have topo II in the centrosomes. It is possible that upon appropriate stimulation of G0 phase cells, centrosomal topo II is translocated to the nucleus where it either performs an essential function in the initiation of DNA replication or participates in the transcription of genes whose products, in turn, are essential for re-entry into the cell division cycle.

In summary, we have shown that topo II is required for the G0-to-S phase transition in Drosophila Schneider cells, but not in yeast. We previously reported that mammalian cells require topo IIα for the G0-to-S phase transition (Hossain et al. 2002). It would be worthwhile not only investigating the precise functions of topo II that are required for the G0-to-S phase transition in fly and mammal, but also determining why such a topo II-dependent step is absent in yeast.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Materials

ICRF-193 (Zenyaku Kogyo Co. Ltd, Tokyo, Japan), nocodazole (Wako, Osaka, Japan), benomyl (Sigma-Aldrich Chemical Co., St Louis, MO) and rapamycin (Calbiochem, Darmstadt, Germany) were dissolved in dimethylsulfoxide (DMSO; Wako). Cycloheximide (Sigma-Aldrich) was dissolved in distilled water. Phosphate-buffered saline without MgCl2 and CaCl2[PBS(–)] was obtained from Invitrogen (Grand Island, NY, USA). [methyl-3H]Thymidine (1 mCi/mL) and [5, 6–3H]uracil (1 mCi/mL) were obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). All other chemicals were reagent grade.

Cell culture and G0-phase synchronization of Drosophila Schneider line 2 cells

Drosophila Schneider line 2 cells (Schneider 1972; Aozasa et al. 2002) were cultured at 27 °C in Schneider's Drosophila medium (Invitrogen) supplemented with 0.5% (w/v) polypeptone (Nihon Seiyaku, Tokyo, Japan), 10% (v/v) heat-inactivated foetal bovine serum (FBS; Cell Culture Laboratories, Cleveland, OH), 1% penicillin-streptomycin (Invitrogen). Cells were routinely cultured in tightly capped tissue culture flasks (Iwaki) up to ∼70% confluence and the attached cells were passaged every 3–4 days at 1 : 5 dilution in fresh media after gentle scraping with a cell scraper (Falcon). For synchronization at the G0 phase (Rizzino & Bluementhal 1978), exponentially proliferating Schneider cells were collected and re-suspended into 60 mL of fresh medium at a density of 8 × 105 cells/mL. Cells were allowed to grow up to maximum density in spinner flasks. Cell density was maximum (∼3 × 106 cells/mL) after 4 days and cell viability was ∼99% as determined by Trypan Blue (Sigma-Aldrich Chemical Co.) staining. Cells were maintained at full growth for four more days (cell viability after 4 days was ∼90%). Cells were then collected, washed once with PBS(–) and re-suspended at a density of 8 × 105 cells/mL in fresh medium and 100 µL was plated per well of 96-well suspension culture plates (Iwaki). Cells were treated with either 10 µm ICRF-193 or an equal volume of DMSO at 0, 3, 6, 9, 12 or 15 h of release from G0 arrest. To measure the rate of DNA synthesis, cells were pulse-labelled with 5 µCi/mL [3H]thymidine for 1 h. Cells were then lysed by adding 100 µL Tris-EDTA containing 2% sodium dodecyl sulphate (SDS) in each well followed by precipitation with 10% trichloroacetic acid (TCA). Acid-insoluble materials were collected on a glass filter (Whatman GF/C) and the radioactivity was counted using a liquid scintillation counter (Beckman). ICRF-193 inhibited DNA synthesis of exponentially proliferating Schneider cells in a dose-dependent manner (data not shown). ICRF-193 (10 µm) strongly inhibited DNA synthesis but did not significantly decrease the viability of log-phase cells, as determined by MTT assay, after 24 h incubation (data not shown).

Metaphase synchronization of Schneider cells

Exponentially proliferating Schneider cells were treated with 20 µm nocodazole, a microtubule-depolymerizing drug, for 24 h to arrest cells at M phase. Cells were then washed three times with PBS(–), re-suspended in fresh medium at a density of 8 × 105 cells/mL in 100 µL and plated on 96-well suspension culture plates followed by incubation at 27 °C for 3 h. The cells were then treated with either 10 µm ICRF-193 or an equal volume of DMSO. [3H]Thymidine pulse-labelling was performed as described above.

Topo II RNAi in G0-phase Schneider cells

Schneider cells were synchronized at G0 phase as described above. Cells were kept at full-growth condition for 1 day in a spinner flask and 1 mL was plated in a 35-mm suspension culture dish followed by addition of 15 µg/mL dsRNA for topo II or EGFP (control). Cells were incubated at 27 °C for seven more days. Cell viability was ∼80% after 7-day treatment with either topo II or EGFP dsRNA as determined by trypan-blue staining. After 7 days, cells were diluted at a density of 8 × 105 cells/mL in fresh medium and 100 µL was plated per well of 96-well suspension culture plates. [3H]thymidine pulse-labelling was performed as described above. Drosophila melanogaster full-length cDNA of topo II (kindly provided by Dr T-s. Hsieh, Duke University Medical Center, Durham, NC, USA) was used as PCR reaction template to amplify a 772-bp cDNA fragment corresponding to 932–1703 nt for the preparation of dsRNA using the following set of primers: forward, 5′-CCAAGTTCAAGATGGACCGT-3′; and reverse, 5′-TGACTTGGCTGAGTCTCCCT-3′ with T7 RNA polymerase binding site (5′-GAATTAATACGACTCACTATAGGGAGA-3′) attached to the 5′ end of each primer. Full-length EGFP dsRNA was prepared from the plasmid, pKE515 (Li et al. 2004). Topo II dsRNA was prepared as previously described (Clemens et al. 2000), except that a T7 Ribomax Express kit from Promega was used. Total RNA was isolated from Schneider cells by using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. For RT-PCR analysis, cDNAs were synthesized from 1 µg of total RNA using the reverse transcriptase kit of Stratagene (La Jolla, CA) with oligo-dT primer according to the manufacturer's instructions. The PCR reaction mixture consisted of 125 ng cDNA, 200 µm each of dNTPs, 1.25 mm MgCl2, 50 pmol of each of the primers and 1.25 U AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA) in a reaction volume of 50 µL. The PCR amplification cycles consist of 35 cycles of denaturing at 94 °C for 1 min, annealing at 44 °C for 1 min and extension at 72 °C for 2 min, followed by a final extension at 72 °C for 7 min. A 782-bp fragment of topo II cDNA (corresponding to 2096–2877 nt) was amplified by PCR. The sequences of primers used for PCR amplification are as follows: topo II, forward: 5′-GATCGCCATCGCATCTTATT-3′, reverse: 5′-TACCACGGATGCATCACACT-3′; rp49, forward: 5′-TACAGGCCCAAGATCGTGAA-3′, reverse: 5′-ACCGTTGGGGTTGGTGAG-3′. No significant difference in rp49 expression was observed with 25, 30 or 35 PCR amplification cycles among G0-phase cells and those after release from G0 arrest (data not shown). The PCR products were resolved by 1.5% agarose gel electrophoresis and stained with ethidium bromide. The identities of PCR products were confirmed by restriction digestion analyses (data not shown).

G0-phase synchronization of yeast

The budding yeast, S. cerevisiae strain, W303-1a (ade2-1 ura3-1 his3-11 trp1-1 leu2-3112 can1-100) and its isogenic topo II ts strain, RS191 (ade2-1 ura3-1 his3-11 trp1-1 leu2-3112 can1-100 top2-1) were provided by Dr R. Sternglanz (Stony Brook University, Stony Brook, NY, USA) and the temperature-sensitivity of the mutant at 37 °C was confirmed (data not shown). Cells were grown at 25 °C in a shaking water bath up to A600 = 0.4 in 100 mL of SD medium [0.67% yeast nitrogen base without amino acids (Difco), 2% D-(+)-glucose (Kanto Chemical, Tokyo, Japan)] supplemented with 100 µg/mL each of adenine, uracil, histidine, tryptophan and leucine in 500-mL long-neck, flat-bottom flasks. Cells were then collected, washed with distilled water and re-suspended in SD (–C) (SD medium without glucose) medium at A600−0.3 followed by incubation at 25 °C for 14 days.

Measurement of DNA synthesis in yeast

G0-phase yeast cells were collected, washed once with distilled water and re-suspended in 12 mL of YM5 medium (Hartwell 1967) pre-warmed either at 25 °C or 37 °C at A600−0.3 in a 225-mL conical graduated polypropylene tube (Falcon). [3H]Uracil was added to a final concentration of 15 µCi/mL at 0 h and 2 mL aliquots were taken at 0, 3, 6, 9 and 12 h. Cells were then treated with 1 N NaOH for 16 h at room temperature followed by precipitation of DNA with 10% TCA, glass fibre filtration and liquid scintillation counting. Rapamycin, a drug that causes G1-arrest (Zaragoza et al. 1998), was added to a final concentration of 10 µg/mL at 0 h of release, whereas benomyl (40 µg/mL), a microtubule depolymerizing drug (Hoyt et al. 1991), was added after 3 h, the time after which cells started budding, as determined by microscopic observation.

Flow cytometric analysis of the DNA content of yeast

After starvation, G0-phase cells were collected and re-suspended in YM5 medium pre-warmed either to 25 °C or 37 °C followed by incubation in a shaking water bath at either 25 °C or 37 °C. Benomyl (40 µg/mL) was added after 3 h of release from G0 arrest. An aliquot was removed at 0, 6 and 9 h after release into fresh medium. Cells were washed with distilled water, fixed with ice-cold 70% ethanol and incubated at 4 °C for 12 h. Cells were then washed and suspended in 1.0 mL of filter-sterilized 50 mm Na-citrate containing 0.5 mg/mL Rnase A (Sigma-Aldrich Chemical Co.) and incubated at 50 °C for 1 h. Propidium iodide (Molecular Probes, Eugene, OR) was added to a final concentration of 50 µg/mL and 10 000 cells for each sample were analysed by FACScalibur (Becton-Dickinson).

Fluorescence spectrophotometric measurement of the total DNA content of yeast

Total yeast DNA was isolated as previously described (Ausubel et al. 1998). Briefly, yeast cells were lysed with breaking buffer [2% (v/v) Triton X-100, 1% (w/v) SDS, 100 mm NaCl, 10 mm Tris.HCl, 1 mm EDTA, pH 8.0] in the presence of glass beads (106 µ and finer) followed by treatment with phenol : chloroform : isoamyl alcohol, ethanol precipitation, Rnase A treatment and ethanol precipitation again. Total DNA was dissolved in Tris-EDTA buffer and stained with 10 µg/mL of DAPI (Sigma-Aldrich Chemical Co.). Fluorescence was measured at λexc = 356 nm and λemm = 452 nm in a Hitachi F-4500 fluorescence spectrophotometer (Mizushima et al. 1997).

ICRF-193 treatment of yeast

The drug-permeable S. cerevisiae strain, JN394 (MATa ura3-52 leu2 trp1 his7 ade1-2 ISE2 rad52::LEU2) and its isogenic topo II ts mutant strain, JN394t2-1 (MATa ura3-52 leu2 trp1 his7 ade1-2 ISE2 rad52::LEU2 top2-1) were provided by Dr T. Andoh (Soka University, Tokyo, Japan). We confirmed the resistance and sensitivity of log phase cells of JN394 and JN394t2-1, respectively, to 100 µm ICRF-193 at a semipermissive temperature (data not shown). Cells were arrested at the G0 phase by carbon-starvation in SD (–C) medium for 7 days and released at A600 = 0.5 into pre-warmed YM5 medium at 30 °C with either 100 µm ICRF-193 or an equal volume of the solvent, DMSO. Benomyl (40 µg/mL) was added after 3 h of release from G0 arrest. Aliquots (2 mL) were removed at the indicated times and the incorporation of alkali-resistant [3H]uracil was determined as described above.

Spore germination of yeast

S. cerevisiae haploid strains, W303–1a (parent) and RS191 (topo II ts mutant) were transformed with plasmid, YEp13 or YEp13-HO (kindly provided by Dr T. Hisatomi, Fukuyama University, Hiroshima, Japan) by the modified lithium acetate method as previously described (Kaiser et al. 1994). Transformants were selected on SD(Leu) agar plates. Diploidization of the transformants was confirmed by FACS analysis of the DNA content (data not shown). Temperature-sensitivity at 37 °C of single colonies obtained from the topo II ts mutant strain was confirmed (data not shown). Single colonies were grown in liquid SD(Leu) medium up to A600 = 0.5 at 25 °C in a shaking water bath followed by washing with distilled water and incubation in a 500-mL long neck, flat-bottom flask in 25 mL sporulation medium [1% potassium acetate, 0.1% yeast extract, 0.05% D-(+)-glucose, pH 7.0] supplemented with auxotrophic requirements except leucine (Ausubel et al. 1998). Cells were incubated in sporulation medium for seven more days at 25 °C. The efficiency of ascus formation in cells transformed with YEp13-HO was ∼80% in both the parent and the mutant as determined by microscopic observation, whereas no ascus was observed in cells transformed with the empty vector, YEp13 (data not shown). For germination, asci-containing cultures were centrifuged, washed once with distilled water and re-suspended in 30 mL of YM5 medium (A600 = 0.5) pre-warmed at either 25 °C or 37 °C in a 225-mL conical graduated polypropylene tube (Falcon). Total DNA was isolated as described above. Percentage of asci in culture at a given time was determined by microscopic observation. Cycloheximide, a protein synthesis inhibitor that blocks germination, was added to a final concentration of 40 µg/mL at 0 h of release.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Dr T. Kubo (The University of Tokyo) for kindly providing Drosophila Schneider line 2 cells. We are grateful to Dr T. Andoh (Soka University, Tokyo, Japan) and Dr R. Sternglanz (Stony Brook University, NY, USA) for kindly providing us with the yeast strains used in this study. We are indebted to Dr T-s. Hsieh (Duke University Medical Center, Durham, NC, USA) for generously providing us with the full-length cDNA of Drosophila melanogaster topo II. We are also grateful to Dr T. Hisatomi (Fukuyama University, Hiroshima, Japan) for kindly providing us with the plasmids, YEp13 and YEp13-HO.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References