A Mitotic Spindle Requirement for DNA Damage-Induced Apoptosis in Chinese Hamster Ovary Cells
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[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 dis- tinguish 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 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1999 American Association for Cancer Research. G2-M ARREST AND APOPTOSIS IN REPAIR-DEFECTIVE CELLS 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).