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Address correspondence to Conly L. Rieder, Lab of Cell Regulation, Division of Molecular Medicine, Wadsworth Center, P.O. Box 509, Albany, NY 12201-0509. Tel.: (518) 474-6774. Fax: (518) 486-4801. email: Rieder{at}Wadsworth.org
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Abstract |
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Key Words: mitosis; DNA; aclarubicin; merbarone; apicidin; ICRF-193
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Introduction |
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The arrest or reversal of chromosome condensation during prophase provides a unique visible cue that entry into mitosis has been delayed, and we have been using this feature to study how the G2/M transition is regulated. This assay has a numeric readout, the duration of prophase, and also a qualitative readout, the degree of chromatin condensation (a measure of CDK activity). In our initial studies we found that disassembling microtubules induces a 34-h delay in completing prophase (Rieder and Cole, 2000), a behavior that is likely mediated by a checkpoint involving the Chfr protein (Scolnick and Halazonetis, 2000; Chaturvedi et al., 2002; Matsusaka and Pines, 2004). Recently, we used this assay to explore how inhibiting topoisomerase II (topo II) and other enzymes involved in chromatin structure affect the G2/M transition. The results of these studies, which are described here, reveal that drugs which modify chromatin topology during late G2 delay entry into mitosis, independent of the ATM kinase, by activating the p38 MAPK checkpoint pathway.
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Results |
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Triggering p38 MAPK during antephase delays entry into mitosis
In addition to the ATM/DNA damage checkpoint, a caffeine-insensitive pathway appears to exist that delays cells in G2 in response to UV, IR, or -irradiation (Goldstone et al., 2001; Jha et al., 2002; Xu et al., 2002). In the case of
and UV irradiation this arrest is mediated by the p38 MAPK (Bulavin et al., 2001; Dmitrieva et al., 2002; for review see Bulavin et al., 2002). This prompted us to ask if activating p38 during antephase, with concentrations of anisomycin (57 ng/ml) that do not affect protein synthesis (Bunyard et al., 2003), delays entry into mitosis. We found that anisomycin rapidly induced early to mid prophase PtK1 cells to decondense their chromosomes and return to G2 for >3 h (Table I). Osmotic stress, which is also a potent activator of p38 (Han et al., 1994; Dmitrieva et al., 2002), similarly induced early to mid prophase cells to decondense their chromosomes and delay in antephase (unpublished data). By binding to the ATP site on p38, the small molecule SB203580 potently and selectively inhibits the downstream activity of p38 without preventing its activating phosphorylation (Gum et al., 1998; Lisnock et al., 1998). Not unexpectedly, if p38 activity was prevented in PtK1 with SB203580, before treating antephase cells with anisomycin (Table I) or hypertonic medium (not depicted), they entered prometaphase with near normal kinetics. Thus, activating p38 during antephase delays entry into mitosis, and this delay can be eliminated by inhibiting p38 with SB203580.
Topo II inhibitors activate the p38 pathway
To determine if topo II inhibitors activate p38 during G2/M we treated synchronized HeLa cells with adriamycin or aclarubicin. Western blots of whole cell extracts, immunostained for total and active p38 (T*GY*), confirm that p38 is not normally active in HeLa during S and G2/M (Fig. 3 B; for review see Deacon et al., 2003). However, it is clearly activated in a dose-dependent manner when G2/M cells are treated with adriamycin or aclarubicin (Fig. 3 B). Thus, inhibitors of topo II, including those that produce few if any DSBs, activate the p38 MAPK. P38 is highly conserved and antibodies against human p38 detected p38 in nonsynchronizable PtK1 cells (unpublished data). We also found that agents known to stimulate (anisomycin) or inhibit (SB203580) p38 in human cells also work on marsupial cells.
Inhibiting p38 activity overrides the antephase delay caused by topo II inhibitors
We next incubated PtK1 cultures in SB203580 before treating them with topo II inhibitors. We found that inhibiting p38 with SB203580 completely abolished the antephase delay seen after treating cells with ICRF-193, merbarone or aclarubicin, and it significantly reduced the delay after adriamycin treatment (Table I; Fig. 5 A). During aclarubicin treatment the cells entered mitosis with little or no chromosome-bound topo II (Fig. 2 B). Pre-incubating PtK1 cells with SB202474, an inactive analogue of SB203580, did not prevent the antephase delay (unpublished data). We then repeated these experiments with another potent p38 inhibitor, 2-(4-chlorophenyl)-4-(4-fluorophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol-3-one (de Laszlo et al., 1998), and obtained the same results (unpublished data). Finally, the Jun-N-terminal (JNK) MAPK shares a high degree of structural and functional homology with p38. To determine if JNK is involved in the G2 delay induced by topo II inhibitors we inhibited this MAPK during prophase with 30 µM SP600125 (Bennett et al., 2001) and found that it did not prevent the antephase delay (unpublished data).
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Discussion |
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The synthesis of topo II starts in G1 and peaks in G2, whereas topo IIß expression is continuous throughout the cell cycle (Kellner et al., 2002). As a result, poisons like ICRF-193 and merbarone, that preferentially bind to and inhibit topo II
catalyzed decatenation (Perrin et al., 1998), delay cells selectively in G2 (Deming et al., 2002). In contrast, by intercalating directly into chromatin aclarubicin prevents decatenation by both topo II
and ß (Perrin et al., 1998). As a result, this drug delays progression through all stages of the cell cycle including G2 (Teillaud et al., 1998).
The mechanism(s) by which topo II inhibitors delay cell cycle progression are only vaguely understood. Work with the catalytic inhibitor ICRF-193 suggested that this delay is mediated by a caffeine-sensitive pathway that monitors chromatin decatenation (Downes et al., 1994; Clifford et al., 2003). Subsequent work suggested that this "decatenation checkpoint" delays entry into mitosis, independent of the p53 pathway (Kaufmann et al., 2002), by using the ATR kinase and BRCA1 to inhibit the polo-like kinase (Deming et al., 2001, 2002; Kaufmann et al., 2002). The idea that a G2 "decatenation" checkpoint exists, distinct from the DNA damage checkpoint, is based largely on indirect observations and biochemical data that ICRF-193 does not induce DSBs. However, recent work (Huang et al., 2001; Wang and Eastmond, 2002), as well as our own data (Fig. 2), reveals that many of these drugs, including ICRF-193 and merbarone, do damage DNA in vivo. Furthermore, it is now clear that the ATR kinase implicated in the so called decatenation checkpoint has considerable overlap with the ATM kinase which arrests cells in response to DSBs (Durocher and Jackson, 2001). Finally, because sister chromatids do not become fully decatenated until the metaphase/anaphase transition, it is difficult to envision how a checkpoint monitoring the state of DNA catenation can delay the G2/M transition when cells normally enter mitosis with catenated chromatids.
Topo II and histone deacetylase inhibitors delay the G2/M transition by activating the p38 pathway
Our data reveal that, when applied to antephase (late G2) cells, topo II inhibitors delay entry into mitosis via the p38 MAPK, and not the ATM, pathway. Because this delay is triggered in minutes, by a route that functions well into prophase, it does not require activation of transcription factors (like p53) or new protein synthesis. The p38 MAPK pathway fulfills the criteria for a checkpoint control, at least during late G2: at this time it is normally not active and, when activated, it delays cell cycle progression via a route that shows a relief of dependence. Importantly, under many conditions this delay is transient and it is ultimately bypassed by an adaptation process, even when the problem cannot be fixed. This control provides a mechanism during the G2/M transition, as it appears to also do during the G1/S transition (Kyriakis and Avruch, 2001), for quickly delaying cell cycle progression in response to diverse stresses. In the absence of such a rapid response system, such stresses may well lead to chromosome segregation problems during mitosis independent of those generated by DNA damage. The p38 checkpoint pathway thus gives the cell time to recover, just before important transitional events, in cases where the insult is transient. If need be, it also allows other checkpoints that require transcription time to work.
What does the "topo II checkpoint" monitor if not chromatin decatenation? Topo II inhibitors either bind to chromatin (aclarubicin) or lock the enzyme on chromatin in an inactive form (adriamycin, ICRF-193, merbarone). One possibility is that as these drugs bind, they induce a global change in chromatin topology that delays the G2/M transition by activating the p38 pathway. This idea is supported by our data, and those of others, that osmotic shock and histone deacetylase inhibitors, which similarly induce global changes in chromatin topology, also delay the G2/M transition via the p38 pathway. It is also consistent with our finding that selectively damaging chromatin in just a few regions of the antephase nucleus delays entry into mitosis via the ATM and not p38 kinase pathway.
How could global changes in chromatin topology during antephase activate p38? One possibility is that regions of chromatin bind an unidentified factor that is released in response to abnormal chromatin topology. Once released this factor may interact with c-Abl and/or DNA-protein kinase (Kharbanda et al., 1997) to initiate a kinase cascade (Brancho et al., 2003) that activates p38. Active p38 can influence cell behavior by activating transcription factors or other kinases. Because the antephase response we describe is rapid, and occurs as chromosomes are condensing, it is not due to transcription factors like p53. Rather, the activation of p38 by abnormal chromatin topology likely initiates another kinase cascade, perhaps involving MNK1 (Fukunaga and Hunter, 1997), that produces the cell cycle delay. P38 can also directly interact with Cdc25B (Bulavin et al., 2001). The antephase checkpoint may work by ultimately blocking activation of cyclin A/CDK2 via Cdc25, which in response can occur independent of ATM/ATR (Goldstone et al., 2001; Mitra and Enders, 2004).
We find that the delay in entering mitosis induced in late G2 cells by topo II inhibitors is caffeine insensitive and does not involve the ATM kinase. Bakkenist and Kastan (2003) report that based primarily on immunofluorescence (IMF) data, osmotic stress, and histone deacetylase inhibitors induce a diffuse phosphorylation of ATM in the absence of DSBs. This suggested that ATM is activated globally by changes in chromatin structure, and then later accumulates at DSBs when present. Our results reveal that these same treatments delay the G2/M transition. However, we find that this delay is not overridden by inhibiting ATM with caffeine or wortmannin (or in / ATM cells), yet it is eliminated by preventing p38 kinase activity. We also find that topo II inhibitors which induce DSBs activate both ATM (as evidenced by H2AX foci formation) and p38, but that inhibitors that do not induce DSBs (aclarubicin) do not activate ATM. Regardless, with the exception of adriamycin, which induces massive numbers of DSBs, all of these inhibitors delay entry into mitosis via the p38 and not ATM pathway. These results imply that, by itself, the global activation of ATM by changes in chromatin topology does not produce a late G2 delay independent of the p38 pathway.
P38 activity is not required for entry into mitosis or the spindle assembly checkpoint
The spindle assembly checkpoint delays anaphase when kinetochores are not stably associated with the spindle. Work on 3T3 and HeLa cell populations suggests that p38 is activated in response to spindle poisons (Deacon et al., 2003), and that this activity is required for the spindle assembly checkpoint (Takenaka et al., 1998). However, in situ studies conclude that p38 is normally active during mitosis, and that this activity is required to overcome this checkpoint (Campos et al., 2002). Cell sorting studies even suggest that inhibiting p38 does not influence the mitotic arrest or slippage of HeLa cells treated with nocodazole (Tsuiki et al., 2001).
As reported by others (Deacon et al., 2003) we found that p38 is not activated as untreated HeLa cells transit from G2 into mitosis (Fig. 3 B). We also found that inhibiting p38 does not influence the rate at which CFPAC-1 or hTERT-RPE1 cells enter mitosis (Fig. 5, B and C), or the duration of the mitotic delay induced in live PtK1 or Indian muntjac cells by nocodazole or topo II inhibitors. This latter delay is, however, rapidly abrogated when cells are injected with a dominant negative construct of Mad2 (Mikhailov et al., 2002). From these observations we conclude that p38 activity is neither required for entry into mitosis, for normal mitotic progression, or for the spindle assembly checkpoint in PtK1 or Indian muntjac cells.
Many of the chemical or physical insults that delay the G2/M transition also delay the metaphase/anaphase transition. With few exceptions, most of these globally perturb chromatin topology. Good examples here include chromatin damage caused by radiation (Mikhailov et al., 2002), and inhibitors of topo II (Illidge et al., 2000; Mikhailov et al., 2002) or histone deacetylase (Cimini et al., 2003). We propose that topo II and histone deacetylase inhibitors delay entry into and exit from mitosis because they bind to and induce structural changes in chromatin. During antephase these changes are detected by the p38 pathway. During mitosis they impede satisfaction of the spindle assembly checkpoint by deleteriously affecting kinetochore structure and thus their stable attachment to the spindle.
Finally, we found that cells arrested in G2 by drugs that prevent normal topo II function can be driven into a highly aberrant mitosis by simply overriding activation of the P38 MAPK. Many of these drugs are currently used as a primary or adjunct chemotherapy in cancer treatment (Froelich-Ammon and Osheroff, 1995; Kellner et al., 2002). One interesting avenue may therefore be to explore the clinical effects of combining topo II and p38 inhibitors.
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Materials and methods |
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Coverslip cultures of PtK1 and Indian muntjac cells were assembled into Rose chambers (Khodjakov and Rieder, 2004) at least 5 h before the start of each experiment, whereas those containing CFPAC, hTERT-RPE, and GM16666A cells at least 12 h before each experiment. Once assembled the Rose chambers were then incubated at 37°C.
Reagents
Adriamycin and caffeine were purchased from Sigma-Aldrich. Merbarone (5-(N-phenylcarboxamido)-2-thiobarbituric acid), aclarubicin (Aclacinomycin A), SB203580, SB 202474, JNK Inhibitor II (SP600125), apicidin, and 2-(4-chlorophenyl)-4-(4-fluorophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol-3-one were purchased from Calbiochem. ICRF-193 was a gift from A. Creighton (St. Bartholomew's Hospital College, London, UK). In all instances, reagents were added to conditioned media before use.
Western blotting
Cells were washed in ice-cold PBS and scraped from the culture into cold buffer containing 20 mM Hepes, pH 7.4, 2 mM EGTA, 50 mM ß-glycerophosphate, 2 mM EDTA, 137.5 mM NaCl, 10% glycerol, 1 mM DTT, 1 mM Na3VO4, 40 µM PMSF, 1% Triton X-100, and complete protease inhibitor cocktail (Roche Diagnostics Corp.). After 10 min on ice, the lysate was centrifuged at 14,000 rpm (4°C) for 10 min and the supernatant was used as whole cell extract. Equal amounts of protein were separated on reducing SDS-PAGE gels, immunoblotted and detected by ECL. To assay for general and active (T180GY-phosphorylated) p38, we used antibodies from Cell Signaling Technology, Inc. and Promega Biosciences, Inc. General and S1981-phosphorylated ATM was detected with antibodies from Cell Signaling ("5C2") and Rockland, Inc., correspondingly.
Immunochemical techniques
Rabbit antibody to human topo II was purchased from TopoGEN. Cells were fixed and stained for IMF, including H2AX, as detailed previously (Mikhailov et al., 2002).
Laser irradiation and live cell video microscopy
Laser irradiation of early prophase nuclei was conducted with pulses of 532 nm light (Rieder and Cole, 1998). Nuclei were irradiated with 50 pulses as they were translated in a linear fashion through the fixed laser beam. Each pulse contained 400 2 nJ of power as measured in the plane of the specimen.
All recordings were made on microscopes housed in a 37°C warm room, or inside custom built thermostatically regulated Plexiglas incubators.
For mitotic index studies coverslips of CFPAC-1 and hTERT-RPE1 cells were used at 70% confluence. For long-term recordings, Rose chambers were mounted on the stage of Nikon Diaphot or TMS microscopes housed in a 37°C warm room. Fields of cells were time lapsed with a 20x phase contrast objective, and one image was acquired every 10 min for 610 h using Image Pro Plus (Media Cybernetics) or Scion Image (Scion Corp.). Sequential images were then assembled into movie stacks which were then visually analyzed, during each hour of recording, for: (a) the total number of cells within the field of view (usually 200250); (b) the number of cells entering mitosis (i.e., undergoing NEB); and (c) the total number of mitotic cells.
Online supplemental material
One supplemental figure is included which illustrates that topo II inhibitors delay the G2/M transition in Indian muntjac cells via a p38-dependent mechanism. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200405167/DC1.
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Acknowledgments |
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This work was supported by National Institutes of Health/GMS grant 40198 to C.L. Rieder.
Submitted: 27 May 2004
Accepted: 9 July 2004
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