A Mitotic Spindle Requirement for DNA Damage-Induced Apoptosis in Chinese Hamster Ovary Cells

[CANCER RESEARCH 59, 2696–2700, June 1, 1999] A Mitotic Spindle Requirement for DNA Damage-induced Apoptosis in Chinese Hamster Ovary Cells

Penny A. Johnson, Paula Clements, Kevin Hudson, and Keith W. Caldecott1 School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom [P. A. J., P. C., K. W. C.], and Zeneca Pharmaceuticals, Alderley Park, Alderley Edge, United Kingdom [K. H.]

ABSTRACT ascribed unequivocally to DNA damage. Furthermore, the use of mutant cells that are defective in specific repair pathways allows Promiscuously reactive electrophilic agents induce DNA and other cellular responses to be ascribed to specific DNA lesions (8–10). cellular damage. DNA repair-defective cells, when compared with genet- DNA single- and double-strand breaks can result in unwanted ically matched, repair-proficient parental cells, provide a means to distinguish cellular responses triggered by individual genetic lesions from recombination events, chromosomal abnormalities, and inhibition of other macromolecular damage. The Chinese hamster ovary (CHO) cell DNA replication and transcription and consequently pose a serious line EM9 is hypersensitive to the alkylating agent ethyl methanesulfonate threat to genetic stability. The mechanisms that couple this damage to (EMS) and is unable efficiently to repair DNA single strand breaks in loss of viability are thus likely to be important to maintaining genetic contrast to parental AA8 cells. EM9 was used to examine how CHO cells stability in vivo. In contrast to double strand breaks, little is known couple unrepaired DNA strand breaks to loss of viability. Flow cytometry about cellular responses to SSBs.2 This lack of understanding is revealed that EMS-treated EM9 cells underwent prolonged cell cycle despite the fact that SSBs arise more frequently than double strand arrest in G2, followed by entry into mitosis, micronucleation, and apop- breaks both spontaneously and in response to genotoxic stress. The tosis. EM9 cells synchronized in G prior to EMS treatment entered 1 most common source of spontaneous SSBs is as intermediates of the mitosis 24–36 h after release from synchrony, ϳ12 h after untreated DNA base excision repair pathways that remove DNA base damage control cells. Mitoses in EMS-treated cells were abnormal, involving Ͼ multipolar mitotic spindles and elongated and/or incompletely condensed that arises continuously in cells at a frequency of 1000 bases/cell/ chromosomes. The mitotic spindle poison nocodazole reduced DNA dam- day (11). A number of CHO cell lines have been isolated that exhibit age-induced apoptosis by >60%, whereas the frequency of micronucle- a defect specifically in rejoining SSBs. Most of these mutants harbor ation was similar in the presence or absence of nocodazole. Flow cytom- mutations within XRCC1, a protein required for binding and stability etry revealed that nocodazole-treated cells sustained a second round of of DNA ligase III-␣ polypeptide and for rejoining SSBs induced by DNA replication without intervening mitosis. These results demonstrate DNA base damage (12–17). Here, we have used the XRCC1 mutant that nuclear fragmentation and inappropriate DNA replication are insuf- cell line EM9 and its genetically matched, repair-proficient parent cell ficient to trigger apoptosis following DNA strand breakage and demon- line AA8 to examine how unrejoined SSBs are coupled to loss of strate a requirement for mitotic spindle assembly for this process in CHO viability in CHO cells. cells.

INTRODUCTION MATERIALS AND METHODS

Cell death following exposure to a genotoxin can occur via several Cell Growth Conditions, Drug Treatment, and Cell Cycle Synchrony. routes, including apoptosis, extended or permanent cell cycle arrest, The CHO cell lines EM9 and AA8 (described previously by Thompson et al. necrosis, and mitotic catastrophe. Such variation can result from 18) were maintained either as monolayers or in suspension in ␣-MEM with differences between cell types, although different modes of cell death 10% FBS (Life Sciences, Paisley, United Kingdom). Clonogenicity assays can also occur within an individual cell type in response to a single were performed in duplicate with 200–300 cells plated per 60-mm dish genotoxin (1–3). In some cases, the mechanism by which cell death is (Nunc). After 4 h, cells were treated with EMS (1 mg/ml; Sigma Corp.) at 37°C 2ϩ triggered is dependent upon the dose of genotoxin used and/or dura- for 1 h. Treated cells were rinsed twice with PBS (Dulbecco’s minus Mg , 2ϩ tion of treatment. This may reflect promiscuity with respect to intra- Ca ; Life Technologies, Inc.) and incubated in drug-free medium for 10–14 days to allow formation of macroscopic colonies. cellular targets because most genotoxins can damage other macromol- Cells were synchronized at the G1-S transition essentially as described by ecules in addition to DNA, thereby eliciting multiple cell death Orren et al. (9). Briefly, cells were incubated for a minimum of 48 h in ␣-MEM pathways. For example, free radicals are induced by most genotoxins with 0.1% FBS. The low-serum medium was then replaced with ␣-MEM to some extent and can result in membrane lipid peroxidation and the containing 10% FBS and 300 ␮M mimosine (Sigma Corp.), and cells were hydrolytic formation of ceramide from sphingomyelin. Ceramide is a incubated overnight. Cells were then released into the cell cycle by replacing potent inducer of signal transduction cascades and of apoptosis (4). with normal medium. Synchronized cells were treated with EMS as described Indeed, the major UV-induced transcriptional stress response of mam- above before removal of mimosine-containing medium. Where indicated, the malian cells, and at least some X-ray-induced apoptosis, is triggered spindle microtubule poison, nocodazole (Sigma Corp.), was added to the fresh by membrane damage rather than DNA damage (5–7). To fully medium at a concentration of 20–40 ng/ml. understand the cell death responses of cells to DNA damage, it will be Flow Cytometry and Microscopy. To determine their DNA content, we fixed 1 ϫ 106 cells (70% methanol/30% PBS) at 4°C for 30 min, washed them necessary to distinguish effects induced by DNA damage from those twice, and resuspended the cells in PBS (1 ml). When monolayer cultures were induced by other macromolecular damage. One way to achieve this used, care was taken to harvest both adherent and floating cell populations. separation is through the use of DNA repair-defective cell lines, Fixed cells were incubated for 60 min at 37°C with 0.5 mg/ml RNase A (Sigma because the sensitivity of DNA repair mutants to genotoxins can be Corp.). Propidium iodide (Sigma Corp.) was added at a final concentration of 50 ␮g/ml, and 1 ϫ 104 cells were analyzed by flow cytometry (Becton- Received 11/4/98; accepted 3/29/99. Dickson). The costs of publication of this article were defrayed in part by the payment of page For microscopic investigation of nuclear morphology, cells (1 ϫ 104) were charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at School of Biological Sciences, 2 The abbreviations used are: SSB, single strand break; FBS, fetal bovine serum; EMS, G.38 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, ethylmethane sulfonate; CHO, Chinese hamster ovary; DAPI, 4Ј,6Ј-diamidino-2-phe- UK. nylindole; PARP, poly (ADP-ribose) polymerase. 2696

resuspended in 4 ␮l of fixative solution containing 0.3 volume of 37% RESULTS formaldehyde (BDH, Laboratory Supplies, Poole, England), 0.6 volume of 80% glycerol, 0.1 volume of 10ϫ cell extract buffer (see below), and 1 ␮g/ml Unrejoined SSBs Trigger Apoptosis in EM9 CHO Cells. Apop- Hoechst 33342 or DAPI (Sigma Corp.). Both adherent and suspension frac- tosis is a genetically regulated mechanism leading to cell death in tions of cells for each sample were analyzed for nuclear morphology. Cells to response to a diversity of stimuli. Although a variety of genotoxins be used for ␣-tubulin staining were grown directly onto coverslips or, in the induce apoptosis, the contribution of individual lesions to this re- case of cells that had detached from coverslips, were centrifuged onto cover- sponse is poorly defined. To examine the relevance of DNA SSBs to slips. Cells were fixed in 70% methanol (Ϫ20°C) for at least 30 min. Fixed apoptosis, mutant EM9 cells and parental AA8 cells were compared cells were washed in PBS and then incubated at room temperature for 1 h with for biochemical and morphological hallmarks of apoptosis after treat- the anti-␣-tubulin monoclonal antibody, TAT-1 (19), diluted 1:5. After wash- ment with the alkylating agent, EMS. As observed previously (17), ing with PBS, the cells were stained with secondary antibody conjugated to treatment of asynchronous populations of AA8 and EM9 cells with 1 FITC (DAKO) for an additional h at room temperature. Coverslips were rinsed mg/ml EMS for 1 h reduced EM9 clonogenicity by Ͼ90% and ␮ with PBS, counterstained with DAPI (1 g/ml), and then mounted onto slides. repair-proficient parental AA8 cells by Ͻ10% (Fig. 1A). The presence Samples were analyzed by microscopy (Zeiss) and supporting softwear (Open- of condensed chromatin in EM9 cells 72 h after EMS treatment lab). Mitotic, apoptotic, and micronucleation indices (see “Results”) are the suggested that apoptosis contributed to cell death (Fig. 1B). This was result of assessing at least 10 individual microscopic fields at ϫ630. supported by the observation of nonrandom DNA degradation in EM9 Preparation of Cytosolic Extracts from CHO Cells and HeLa Cell Nuclei. Cytoplasmic extracts were prepared essentially as described (20). cells 24 h after EMS treatment (Fig. 1C) and development of sub-G1 Briefly, suspension cultures of AA8 or EM9 cells (1 ϫ 109 at a maximum DNA content within 72 h as measured by flow cytometry (Fig. 1D). density of 5 ϫ 105 cells/ml) were incubated with EMS (1 mg/ml) as The occurrence of apoptosis was further indicated by the observed described above or left untreated as controls. At various times after the induction of chromatin condensation (Fig. 2A) and nonrandom DNA removal of EMS, cells were collected by centrifugation and washed twice cleavage (Fig. 2B) in HeLa nuclei incubated with cytosol from EMS- in 10 volumes of ice-cold PBS and once in 10 volumes of ice-cold cell treated EM9 cells. Control cytosol prepared from untreated EM9 cells, extract buffer [CEB: 50 mM PIPES (pH 7.4), 50 mM KCl, 5 mM EGTA, 2 or EMS-treated AA8 cells, had little effect on HeLa nuclei. EMS- ␮ mM MgCl2,1mM DTT, 10 M cytochalasin B, 1 mM phenylmethylsulfonyl treated EM9 cytosol also contained more apoptotic caspase activity fluoride]. Excess CEB was removed from the cell pellet, which was then than did control extracts, as measured by cleavage of HeLa nuclear transferred to a prechilled Dounce homogenizer (2 ml) and left for 20 min on ice to swell. The cells were then quick frozen in a liquid nitrogen bath and lysed gently while thawing by 15 strokes in a Dounce homogenizer (2 ml) using a tight pestle. Cell lysis was monitored by microscopic exami- nation of an aliquot in the presence of trypan blue. Cell debris was removed by centrifugation at 10,000 ϫ g for 15 min at 4°C. The cleared lysate was carefully transferred to a fresh tube. The protein concentration of extracts prepared in this way was typically 40 mg/ml. The cell lysate was diluted (10–15 mg/ml) in extract dilution buffer [EDB: 10 mM HEPES (pH 7.0), 50

mM NaCl, 2 mM MgCl2,5mM EGTA, 1 mM DTT, 2 mM ATP, 10 mM phosphocreatine, and 50 ␮g/ml creatine kinase; Boehringer-Mannheim], quick-frozen, and stored in small aliquots under liquid nitrogen. HeLa cell nuclei were isolated as described (20). Cell-free Reactions and Analysis of Apoptosis in Vitro and in Vivo. Cell-free reactions were performed essentially as described by Martin et al. (20). Each 30-␮l reaction comprised 25 ␮l of cell extract (250–300 ␮g protein), 4 ␮l of EDB, and 1 ␮l of HeLa nuclei prewashed and resuspended in EDB (1 ϫ 105 nuclei/␮l). Reactions were incubated at 37°C for 2 h. At the indicated time points, an aliquot (10 ␮l) was removed for analysis of PARP protein cleavage or DNA degradation or 1 ␮l for analysis of chromatin condensation by microscopy (see above). To analyze DNA degradation, 1 ϫ 105 cells/nuclei were added to TE buffer [10 mM Tris-Cl (pH 8.0), 1 mM EDTA] containing 0.5% sodium lauryl sarkosyl and 0.5 mg/ml proteinase K (Sigma Corp.) and incubated for a minimum of3hat50°C. Lysates were then extracted once with an equal volume of aqueous-buffered phenol (Life Tech- nologies, Inc.); nucleic acids were precipitated and resuspended in TE. RNase A was added to 0.1 mg/ml, and incubation continued for 60 min. DNA was separated by agarose gel electrophoresis (1.5% agarose) in the presence of ethidium bromide (0.5 ␮g/ml). To detect PARP cleavage, nuclei/cells (3 ϫ 104) were lysed in 30 ␮lof SDS-PAGE sample buffer [50 mM Tris-Cl (pH 8.0), 2% SDS, 10% glycerol, 0.1% bromphenol blue, and 100 mM DTT] heated to 90°C for 5 min, and polypeptides were separated by SDS-PAGE (7.5%). After electroblotting,

nitrocellulose filters (Hybond C; Amersham Corp., Buckinghamshire, United Fig. 1. G2-M arrest and apoptosis in EM9 cells treated with EMS. A, 100–200 cells Kingdom) were immunoblotted and subjected to Enhanced Chemilumines- each of EM9 and AA8 were plated in duplicate and were left untreated (not shown) or were treated for 1 h with 1 mg/ml EMS. Macroscopic colonies of surviving cells were cence as described by the manufacturer (Amersham). Anti-PARP monoclonal fixed, stained, and counted 7–10 days later. B, AA8 and EM9 cells treated (as above) were antibody (kindly provided by Dr. Said Aoufouchi, Laboratory of Molecular fixed and stained with DAPI 24 h after EMS treatment, and their nuclear morphologies Biology, Hills Road, Cambridge CB2 2QLt, England, clone A6.4.7; now were assessed for chromatin condensation indicative of apoptosis. A representative available from Serotec, clone A6.4.12) was used as primary antibody at 1:100 nucleus of each type is shown. C, nonrandom DNA cleavage 24 h after EMS treatment of EM9 cells is not apparent in untreated controls or in treated AA8 cells. D, cell cycle dilution, followed by incubation with horseradish peroxidase-conjugated sec- analysis of EMS-treated AA8 and EM9 cells. The 2N (G1) and 4N (G2-M) DNA content ondary antibody at 1:5000 dilution (DAKO). peaks are indicated. Note that all axes are linear and to the same scale. 2697

treated for 1 h, and released into cell cycle. Under these conditions, the number of apoptotic EMS-treated cells again increased 24–72 h after release from synchrony (Fig. 3, A and B). An analysis of cellular DNA content by flow cytometry and cyclin B1 levels by immunoblotting confirmed that EMS-treated EM9 cells were again present in

G2-M during this period, concomitant with the onset of apoptosis (Fig. 3C). Cyclin B1 levels increased in untreated and EMS-treated cells

within 15 h, indicating rapid entry into G2-M after release from G1. However, whereas the level of cyclin B1 in untreated cells dropped after this period, consistent with exit from mitosis, the level in

EMS-treated cells remained high 15–48 h after release from G1, indicating their presence in G2-M during this period. This interpreta- tion was supported by flow cytometry, which confirmed that EMS-

treated cells were present in G2-M up to 48 h after EMS (data summarized in Fig. 3C). That EMS-treated EM9 cells attempted mitosis during this period was indicated by an increase in mitotic

Fig. 2. Cytosolic extracts of EMS-treated EM9 cells induce apoptotic events in HeLa nuclei. HeLa nuclei were incubated in cytosolic extracts from EM9 and AA8 cells treated 24 h previously with EMS or in extracts from untreated controls. A, microscopic analysis of DAPI-stained nuclei incubated for2hintheindicated cytosolic extract. B, DNA extracted from HeLa nuclei incubated (2 h, 37°C) in buffer alone (Lane 1) or in cytosolic extract from untreated AA8 or EM9 cells (Lanes 2 and 4, respectively) or EMS-treated AA8 or EM9 cells (Lanes 3 and 5, respectively). C, top panel, HeLa nuclei were incubated for the time indicated in cytosolic extracts, immunoblotted, and analyzed with a specific monoclonal antibody that detects PARP. Lanes 1–3, nuclei incubated in cell extract buffer; Lanes 4–6, nuclei incubated in cytosol made from EMS-treated AA8 cells; Lanes 7–9, nuclei incubated in cytosol made from EMS-treated EM9 cells. The full-length (116 kDa) and cleavage (85 kDa) products of PARP are indicated: bottom panel, inhibition of caspase-3 activity by Ac-DEVD-CHO peptide inhibitor. HeLa nuclei were incubated for 2 h in cytosol from EMS-treated EM9 cells without addition of peptide inhibitors (Lane 2) or in the presence of either 0.5 ␮M Ac-DEVD-CHO (Lane 3), 0.5 ␮M Ac-YVAD-CHO (Lane 4), or 0.05 ␮M Ac-DEVD-CHO (Lane 5). Lane 1 shows the status of PARP cleavage in HeLa nuclei before incubation.

PARP (Fig. 2C, top panel). As observed in other experimental sys- tems (21–23), PARP cleavage was prevented by the peptide DEVD- CHO, but not by Ac-YVAD-CHO, suggesting the involvement of caspase-3 (Fig. 2C, bottom panel).

G2-M Arrest, Abnormal Mitosis, and Micronucleation Precede SSB-induced Apoptosis in EM9 CHO Cells. Apoptosis can be either cell cycle dependent or cell cycle independent. Because cell cycle dependency may be in part lesion specific, it was of interest to Fig. 3. Aberrant mitosis and micronucleation precede apoptosis in EMS-treated EM9 cells. A, EM9 cells were synchronized in G1 (see “Materials and Methods”), treated with determine which scenario applied to SSBs. Within asynchronous cell 1 mg/ml EMS (ϩEMS), or mock-treated as controls (ϪEMS), then released into cell cycle. populations, EMS-treated EM9 cells arrested with a G -M (4N) con- Apoptosis was measured at the times indicated after release from G1 by nonrandom DNA 2 degradation (A) and nuclear morphology (B). Cell cycle progression was measured at the tent of DNA within 18 h after EMS treatment (see Fig. 1D), before the indicated times after release from G1 by flow cytometric analysis of DNA content and appearance of cells with sub-G DNA content 24–72 h after EMS cyclin B1 content by immunoblotting (C) and by mitotic index (D). The flow cytometry 1 Ϫ treatment (Fig. 1, C and D). These results suggest that EMS-treated results are presented in summarized form only. E, untreated control ( EMS) and EMS- treated EM9 cells (ϩEMS) were harvested and fixed 48 h after treatment (see “Materials EM9 cells undergo apoptosis during G2 and/or mitosis, consistent and Methods”) and stained for DNA and mitotic spindles using DAPI and ␣-tubulin with the involvement of a cell cycle component in SSB-induced cell antibodies, respectively. F, micronucleation, defined as cells exhibiting decondensed death. To examine this relationship further, EM9 cells were synchro- chromatin present within multiple nuclei in a single cell, was scored at the indicated times after release from G1. Data shown in B, D, and F represent the mean of at least two nized in G1 by serum starvation/mimosine (9), mock-treated or EMS- independent experiments at each time indicated. 2698

index (Fig. 3D). However, the mitoses appeared abnormal, exhibiting incomplete or abnormal chromosome condensation and disorganized chromosome and spindle distribution (Fig. 3E). Furthermore, an increase in the number of micronucleated cells within the EMS-treated

population was observed 36–72 h after release from G1 (Fig. 3F), consistent with exit from mitosis in the absence of normal chromosome segregation (1, 2). Micronucleation reflects the formation of discrete nuclear membranes around chromosomes that have not seg- regated properly and appears to be the result of aberrant mitosis (1). Assembly of a Mitotic Spindle Is Required for SSB-induced Apoptosis. Abnormal spindle formation and micronucleation was observed previously to precede apoptosis in cisplatin-treated UV41 CHO cells (3). However, the role of these phenomena in the induction of apoptosis was not addressed. To examine whether abnormal mitotic spindle formation activates SSB-induced apoptosis, EMS-treated EM9 cells were prevented from assembling mitotic spindles by the addition of nocodazole. Immunofluorescent analysis of EMS-treated ␣ EM9 cells, arrested in G2-M, with anti- -tubulin antibodies confirmed that nocodazole prevented formation of mitotic spindles (compare Fig. 3E and Fig. 4B, left panels). In addition, nocodazole prevented the defect in chromosome morphology observed previously (compare Fig. 3E and Fig. 4B, right panels). That nocodazole functioned as expected in these experiments was also indicated by the appearance, in asynchronous populations of EM9 cells, of cells with 8N DNA content after incubation for 48–72 h in the presence of this compound (Fig. 4A, right-hand panels). The appearance of octaploid cells results from the completion of a second round of DNA synthesis in cells in which an intervening cell division did not occur. A small subpopulation of 8N cells was also observed in EMS-treated cells in the presence of nocodazole, suggesting that nocodazole relieved EMS-induced apop-

tosis in these cells. A concomitant decrease in the number of sub-G1 cells was also noted in cells treated with both agents relative to those treated only with EMS (see especially Fig. 4A; 48 h), further suggesting that nocodazole relieved EMS-induced apoptosis. Finally, nocodazole reduced by ϳ60% the frequency of apoptotic cells present 48–72 h after EMS treatment, as measured by nuclear morphology (Fig. 4C). These results were observed in four independent experiments. The effect of nocodazole on SSB-induced apoptosis may have been greater than was apparent because nocodazole itself induces some apoptosis over this time, by a different mechanism (24) and may account for some of the residual apoptosis observed in cells treated with both agents. The results described above strongly suggest that a mitotic spindle was required both for the abnormalities in chromosome condensation/ organization visible during mitosis in EMS-treated EM9 cells and for the subsequent onset of apoptosis. In contrast, the overall frequency of micronucleated cells present 48–72 h after EMS treatment was not significantly altered by nocodazole, demonstrating that nuclear fragmentation was not sufficient to trigger apoptosis (Fig. 4D).

DISCUSSION DNA repair-defective cells provide a useful system by which the response to a genotoxin can be ascribed to a particular DNA lesion, rather than to other macromolecular damage capable of inducing apoptosis (e.g., membrane lipid peroxidation). Here, we have used the DNA repair mutant EM9 to examine how loss of viability is triggered by unrejoined DNA SSBs in CHO cells. Morphological and biochem-

Fig. 4. Nocodazole reduces apoptosis in EMS-treated EM9 cells. A, asynchronous as indicated. The characteristic nuclear morphology of EM9 cells, as measured by DAPI populations of EM9 cells incubated in the absence or presence of nocodazole (NCD) were staining and immunostaining with anti ␣-tubulin antibodies, is shown after EMS treatment examined for DNA content by flow cytometry at the indicated times after mock treatment and incubation in the presence of nocodazole (B). The percentage of apoptotic cells (C) or treatment with EMS, as indicated. The DNA content of 2N, 4N, and 8N peaks are and micronucleated cells (D) present at the indicated times after release from G1 are also indicated. B–D, EM9 cells were synchronized in G1, treated with 1 mg/ml EMS, or mock shown. Data shown in C and D represent the mean of at least two independent experiments treated as controls, then released into cell cycle in the presence or absence of nocodazole, at each time indicated. 2699

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1999 American Association for Cancer Research. A Mitotic Spindle Requirement for DNA Damage-induced Apoptosis in Chinese Hamster Ovary Cells

Penny A. Johnson, Paula Clements, Kevin Hudson, et al.

Cancer Res 1999;59:2696-2700.

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