Inhibition of P34cdc2kinase Activation, P34cdc2tyrosine Dephosphorylation, and Mitotic Progression in Chinese Hamster Ovary Cells Exposed to Etoposide1

[CANCER RESEARCH 52. 1817-1822. April 1. 1992] Inhibition of p34cdc2Kinase Activation, p34cdc2Tyrosine Dephosphorylation, and Mitotic Progression in Chinese Hamster Ovary Cells Exposed to Etoposide1

Richard B. Lock2

The J. Graham Brown Cancer Center, Departments of Medicine and Biochemistry, University of Louisville, Louisville, Kentucky 40292

ABSTRACT which is regulated by a complex series of phosphorylation/ II.M"''- kinase, an enzyme essential for mitosis in mammalian cells, dephosphorylation reactions (17, 18) and by its association with cyclin B (19). The high degree of conservation between human may play a role in etoposide-induced G2 phase arrest of Chinese hamster and Schizosaccharomyces pombe p34tdc2 proteins [63% amino ovary cells. In this study, etoposide is shown to cause inhibition of a specific p3-4"''-'kinase activation pathway, that of tyrosine dephosphoryl- acid homology (20)] and its identification as a component of ation. Exposure of asynchronous!}' dividing cells to etoposide caused a both maturation promoting factor and the growth-associated simultaneous rapid decline of both mitotic index and p.V4"''-'kinase histone HI kinase (21-24) suggest a central role for p34cdc2in activity, suggesting that the kinase was not activated and that the arrest mitotic progression. However, more direct evidence for its role point was in late Â»...phase. Using synchronized cells, p34cdc2kinase in mitosis of mammalian cells was obtained by the inhibition exhibited maximal activity at the Gz/M transition. Activation of the of cell division following microinjection of affinity-purified kinase and the onset of mitosis were accompanied by increased electro- p34Â«ic2antibodiesinto serum-stimulated rat fibroblasts (25) and phoretic mobility and tyrosine dephosphorylation of the p34"'' ' protein. the identification of a temperature-sensitive p34cdc2protein in A 1-h exposure to etoposide during early <â€¢..phaseinhibited p34cdc2 a mouse mammary carcinoma mutant cell line which arrests in kinase activation, its shift in electrophoretic mobility, and its tyrosine G2 at the restrictive temperature (26). dephosphorylation, all of which correlated with a delay in mitotic pro gression. The interaction between the p.M"'"' and cyclin B proteins p34cdc2kinase has been characterized in CHO cells (12) and found to share qualities common to other mammalian p34cdc2 appeared unaffected under etoposide exposure conditions which resulted in greater than 70% inhibition of p.M"''' kinase activity and almost kinases (16, 18, 27); it is a M, 34,000 histone HI kinase, complete cessation of transition into mitosis. These data suggest that maximally active at the G2/M transition, which exists in phos- mammalian cells express a DNA damage-responsive mechanism phorylated and unphosphorylated forms and associates with which controls mitotic progression at the level of p34rfc2 tyrosine higher-molecular-weight cyclin-like proteins. A brief exposure dephosphorylation. to etoposide caused significant inhibition of p34cdc2kinase and G2 arrest in CHO cells (12), leading to the proposal that INTRODUCTION etoposide-induced G2 arrest may result from the rapid inhibi tion of an enzyme required for mitosis. This paper describes The biochemical mechanisms responsible for drug- and ra further investigations regarding the role of p34cdc2kinase in the diation-induced G2 arrest of mammalian cells remain obscure. mitotic progression of CHO cells, its mechanism of activation, Contributing factors may include inhibition of synthesis of and the effects of etoposide thereupon. specific cellular (1) or nuclear (2) proteins, extensive chromo somal damage (3), failure of transcription of essential genes (4), MATERIALS AND METHODS or, Â¡nthe case of DNA topoisomerase II inhibitors, prevention of chromosome condensation by trapping a covalent DNA- Cell Culture, Synchronization, and Labeling. Wild-type CHO cells protein intermediate (5, 6). were maintained as a monolayer culture in Â»-minimalessential medium The antitumor epipodophyllotoxin etoposide' induces DNA supplemented with 5% fetal calf serum, penicillin (100 units/ml), and streptomycin (I00^g/ml) at 37Â°Cina humidified 5% CO2 atmosphere. single- and double-strand breaks in mammalian cells via inter action with topoisomerase II (reviewed in Ref. 7). Despite the Cells were exposed to etoposide (Sigma) or dimethyl sulfoxide control as described previously (12), except that the cells were washed twice observations in intact cells that DNA strand breaks rapidly with sterile 37Â°Ccalcium- and magnesium-free phosphate-buffered reseal following removal of etoposide (8, 9), probably due to saline following drug treatment and then incubated in drug-free medium the drug's swift rate of efflux (10), cells still progress into and at 37'C. are arrested in G2 phase (11, 12). This suggests, along with CHO cells were synchronized at the d/S boundary using a thymi- other data (13, 14), that a surveillance mechanism operates in dine/aphidicolin block. Exponentially dividing cells were incubated at mammalian cells, analogous to the Saccharomyces cerevisiae 37Â°Cfor 12 h in medium containing 2 mM thymidine (Sigma), followed DNA damage-responsive RAD9 gene (15), which prevents a by a 6-h incubation in thymidine-free medium. Synchronization of cells mitotic catastrophe by inducing G2 arrest following detection at the Ci/S boundary' was then achieved by a further 12-h incubation of a variety of DNA lesions. The precise nature of this cell cycle in medium containing 5 /in ml aphidicolin (Sigma). Synchronous M control mechanism remains unknown. phase cells were obtained by treating exponentially dividing cells with Mammalian p34cdc2is a cell cycle-regulated serine/threonine 0.4 Â¿ig/mlnocodazole (Sigma) for 12 h. For labeling of proteins, cells were incubated for 2 h with 50 MCi/ml protein kinase maximally active at the G2/M transition (16), of Tran[35S]label (ICN) in methionine-free minimal essential medium

Received 10/2/91 ; accepted 1/23/92. (Sigma) containing 5% dialyzed fetal calf serum. The costs of publication of this article were defrayed in part by the payment Cell Cycle Analysis. The G,, S, and G2/M phases of the cell cycle of page charges. This article must therefore be hereby marked advertisement in were distinguished by flow cytometry, as described previously ( 12). Ten accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by USPHS Grant CA53184 from the NIH. thousand cells were analyzed for each data point. The percentage of 2To whom requests for reprints should be addressed, at The J. Graham Brown cells in mitosis was determined using standard procedures (28) as Cancer Center, 529 S. Jackson Street, University of Louisville, Louisville, KY follows. Approximately IO5 phosphate-buffered saline-washed cells 40292. were swollen in 75 m\i potassium chloride for 10 min at 4Â°C.Following JThe abbreviations used are: etoposide. 4'-demethyl-epipodophyllotoxin-9- (4,6-O-ethylidene-ii-D-glucopyranoside); CHO, Chinese hamster ovary; SDS, so centrifugation (200 x g for 5 min) cells were fixed by the dropwise dium dodecyl sulfate; TBS, 20 mM Tris (pH 7.5), 500 mM sodium chloride. addition of freshly prepared fixative (methanohacetic acid, 3:1), recen- 0.02% sodium azide; TTBS, TBS containing 0.05% Tween-20. trifuged, and resuspended in fixative. Cells were dropped onto a clean 1817 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1992 American Association for Cancer Research. INHIBITION OF P341*2 KINASE ACTIVATION BY ETOPOSIDE microscope slide and stained with a 1:25 dilution of Giemsa (Sigma) in RESULTS phosphate-buffered saline for 30 min. Mitotic indices were calculated The Effect of Etoposide on Mitotic Index and p34cdc2Kinase as the percentage of cells with condensed chromosomes, with at least Activity. Etoposide-induced G2 arrest of CHO cells may result 3000 cells being examined for each data point. The mitotic index was from the rapid inhibition of p34cdc2kinase (12), an enzyme then subtracted from the proportion of cells in G2/M (flow cytometry) to yield the percentage of cells in G2 phase. essential for mitosis in mammalian cells (25). To further define p34cdc2Kinase Assays. CHO cell extracts were prepared under non- the relationship between the effects of etoposide on the cell cycle and its effects on p34cdc2kinase activity, the mitotic indices denaturing conditions, and immunoprecipitation reactions were carried out using cdc2 peptide antisera (kindly provided by Drs. J. Bischoff of asynchronously dividing CHO cells exposed to 25 UM eto and D. Beach, Cold Spring Harbor Laboratory, Cold Spring Harbor, poside for up to 2 h were compared to immunoprecipitated p34cdc2histone HI kinase activity from cell lysates (Fig. 1). NY), as described previously (12), with the addition of 0.1 mM sodium orthovanadate to the immunoprecipitation lysis buffer [50 mM Tris Etoposide caused a simultaneous rapid decline in both mitotic (pH 7.4), 250 mivisodium chloride, 0.1% Nonidet P-40, 5 miviEDTA, index and p34cdc2kinase activity, compared to solvent-treated 50 mM sodium fluoride]. The histone HI kinase activities of immuno- control cells, which had a mitotic index of 2.3 Â±0.3. The precipitates from 100 ng of cellular protein (estimated by the Bradford decrease in mitotic index induced by etoposide is in agreement assay, Ref. 29) were assayed as described previously (30) in a final with data from other mammalian cell lines (33-35). Further reaction volume of 50 n\ containing 50 mM Tris (pH 8), 10 mM inhibition of histone HI kinase activity to less than approxi magnesium chloride, 1 mM dithiothreitol, and 50 //n/ml histone HI mately 24% of the control activity could not be achieved by (Boehringer Mannheim). Following a 5-min preincubation at 30Â°C, exposure times of longer than 60 min (Fig. 1), which did not reactions were started by the addition of 5 ^Ci of [7-32P]ATP to a final correlate with the observation that the mitotic index had concentration of 5 MM,incubated at 30Â°Cfor 10 min, and stopped by reached essentially zero by 90 min of etoposide exposure. This the addition of 50 M!of 2x SDS sample buffer [100 mM Tris (pH 6.8), suggests either a nonspecific background of histone HI kinase 4% SDS, 20 mM dithiothreitol, 20% (v/v) glycerol, 0.05% bromophenol activity present in reaction mixtures (unlikely, since immuno- blue]. Seventy-five n\ of this mixture were separated by SDS-polyacryl- precipitations carried out with preimmune sera or in the pres amide gel electrophoresis (31) in a 12% gel. Following electrophoresis, ence of competing antigenic peptide contained essentially no phosphorylated histone HI was visualized by autoradiography and histone HI kinase activity; Ref. 12 and data not shown) or the quantified by Cerenkov counting of histone HI bands after staining presence of a p34cdc2kinase activity not associated with mitotic with Coomassie blue R-250 (BRL). Immunoprecipitation of [35S|Methionine-labeled Proteins and Western cells (see below). The etoposide-induced rapid decline of both mitotic index Blot Analysis. CHO cells were lysed under nondenaturing conditions and p34cdc2kinase activity not only confirms the close associa as described above. Alternatively, SDS-boiled lysates were prepared by tion between this histone HI kinase and mitosis in CHO cells, lysing the cells in 50 mM Tris (pH 6.8), 0.5% SDS, and 1 mM an association observed in a variety of other species (16, 18, 21, dithiothreitol, incubating for 5 min in a boiling water bath, and diluting 10-fold in the above immunoprecipitation lysis buffer. Analysis of [35S] 36), but also suggests that exposure to etoposide may result in a failure to activate the p34cdc2kinase in late G2, leading to G2 methionine-labeled proteins immunoprecipitated using either cdc2 pep- arrest. The mechanisms associated with activation of p34cdc2 tide antisera or human cyclin B antisera (kindly provided by Drs. J. Pines and T. Hunter, The Salk Institute, La Jolla, CA) was achieved as kinase and the effects of etoposide thereupon were studied using synchronous cell populations. described (12) using equal trichloroacetic acid-precipitable radioactivity Inhibition of pJM"1'' Kinase Activation by Etoposide. In order (IO7 cpm) per reaction. to further define the role of p34cdc2kinase in etoposide-induced For Western blot analysis, equivalent amounts of protein (100 Mg) from cells lysed under nondenaturing conditions (see above) were G2 arrest, CHO cells were synchronized at the Gi/S boundary electrophoresed in a 12% SDS-polyacrylamide gel and transferred to a using a thymidine/aphidicolin double block. This method of polyvinylidene difiuoride (PVDF) membrane (Immobilon-P supplied synchrony resulted in the peak of cells in S phase occurring at by Millipore; Ref. 32). The filter was then equilibrated in TBS for 10 2 h following release of the block (Fig. 2A). The highest min, and nonspecific binding sites were blocked by a 1-h incubation in proportion of G2/M-phase cells occurred between 5 and 6 h TBS containing 2% gelatin (Bio-Rad). Following two washes of 5 min following release, during which they were exposed to etoposide each in TTBS, the filter was incubated with a 1:1000 dilution in TTBS (25 MM)or solvent control. This 1-h etoposide treatment re of either cdc2 peptide antisera for 3 h or overnight with an anti- sulted in significant G2/M-phase delay compared to control phosphotyrosine monoclonal antibody (Upstate Biotechnology, Inc., cells (Fig. 2A), which began to progress out of G2/M and into Lake Placid, NY). Unbound antibody was removed by two washes of 5 min each in TTBS, and the filter was incubated for l h with a secondary antibody diluted 1:5000 in TTBS [anti-rabbit or anti-mouse Ig horse 100 -\ radish peroxidase-linked whole antibody (Amersham)]. Following four 80- washes of 5 min each in TTBS, bound antibody was detected using the enhanced chemiluminescence Western blotting detection system 60- (Amersham), according to the manufacturer's instructions. Autoradi- o

100 mitotic index (Fig. 3/1), and continued to rise as cells accumu lated in mitosis. p34cdc2 kinase was not activated following 80 etoposide treatment of G2 phase CHO cells (Fig. 3Ã„),consistent 60- with their failure to progress from G2 into mitosis (Fig. 3/1). 3I! 's 40- The Effect of Etoposide on Mechanisms of P34"1'2 Kinase Activation. The mammalian p34cdc2protein has been shown to 20 - exist in multiple forms of differing electrophoretic mobility (16, O 18, 19, 27). Similarly, it was possible to resolve three forms of 8 10 12 the p34cdc2protein from CHO cells by one-dimensional SDS- 20 polyacrylamide gel electrophoresis (Fig. 4A). Activation of M- phase-specific p34cdc2kinase from a variety of sources has been ., J 15 - I Â« shown to require both formation of a high-molecular-weight Â£Ã•-E complex between the p34cdc2protein and cyclin proteins (19, li &â€¢io-i .s Ã›.5 40, 41) and tyrosine dephosphorylation of the p34cdc2protein s S (18, 42, 43). Tyrosine dephosphorylation and activation of the p34cdc2kinase from Marthasterias and Xenopus oocytes or mam malian cells is accompanied by increased electrophoretic mo O 2 4 6 8 10 12 bility of the p34cdc2protein (17, 18, 41, 44). Asynchronously Hours Post Release dividing (mainly d phase) CHO cells contained predominantly Fig. 2. Cell cycle distribution and p34cdc2kinase activity in Ci-phase cells the faster-migrating form of the p34cdc2 protein (Fig. 4/4). exposed to etoposide. CHO cells were synchronized at the d/S boundary using a thymidine/aphidicolin double block and exposed to etoposide (25 Â»M)orsolvent However, synchronous G2-phase cells (>70% G2), which had control between 5 and 6 h following release of the block. At the appropriate time an immunoprecipitated p34cdc2histone HI kinase activity of cells were harvested and processed for phase distribution analysis or estimation of p34cdc2kinase activity. A, the proportion of cells in G, (A), S (â€¢),and G2/M 1.8 Â±0.2 relative to asynchronous cells, contained almost (O) phase. , phase distribution of solvent-treated cultures; , phase exclusively the slower-migrating form of p34cdc2.A marked shift distribution of etoposide-treated cultures. B, immunoprecipitated p34cdc2kinase in mobility of p34cdc2isoforms occurred between G2 and M activity of control (O) and etoposide-treated (â€¢)cultures. phase, with M-phase cells containing almost exclusively the faster-migrating form (Fig. 4/1). M-phase CHO cells (>55% GÃ¬at8 h following release of the aphidicolin block. Immuno mitotic) had an immunoprecipitated p34cdc2kinase activity of precipitated p34cdc2 kinase activity from control cultures 6.5 Â±0.4 relative to asynchronous cells. Overall levels of the reached a peak at 8 h following release (Fig. 2B), coincidental p34cdc2protein did not vary significantly between asynchronous, with the G2/M transition, and the kinase activity declined as G2- and M-phase CHO cells, consistent with published data cells traversed mitosis and progressed into GÃ¬(Fig. 2). This is (16, 36, 40). in agreement with the proposed central role for p34cdc2kinase The slower-migrating form of p34cdc2in G2-phase cell lysates in mitotic progression (16, 18, 37) and with previous studies of corresponded to a major M, 34,000 tyrosine phosphorylated p34cdc2histone HI kinase activity throughout the mammalian protein (Fig. 4Ã„),and the shift in mobility of the p34cdc2protein cell cycle (12, 16, 19). Activation of p34cdc2kinase did not occur observed as cells progressed from G2 into mitosis was accom in cells which had been exposed to etoposide for l h during panied by the disappearance of this tyrosine-phosphorylated early G2 (Fig. 2B), suggesting that a cellular response to eto- protein from M-phase lysates. Asynchronous cells contained a poside-induced DNA damage may prevent activation of p34cdc2 kinase, resulting in G2 arrest. To facilitate biochemical investigations into the mechanism of activation of p34cdc2kinase as CHO cells progressed from 62 into mitosis, and to study how etoposide may inhibit this process, cells were exposed to the microtubule-depolymerizing drug nocodazole immediately following solvent or etoposide treatment of G2 synchronized cells (Fig. 3). Thus, a distinction could be made between Gj- and M-phase cells using a combi nation of flow cytometry and mitotic index determinations (see "Materials and Methods"). Fig. 3/1 shows that control cells began to progress from G2 into mitosis at 8 h following release of the double block, where they accumulated in metaphase due to the presence of nocodazole (38). The mitotic index of non- drug-treated cells continued to increase until at least 12 h following release. In contrast, substantial inhibition of mitotic progression was observed in cells which had been exposed to 25 fiM etoposide for l h during early G2, consistent with the cell cycle perturbations induced by etoposide in a variety of other mammalian cell lines (11, 33, 35, 39). 4 6 8 10 A low level of immunoprecipitated p34cdc2histone HI kinase Ilours Post ReleasÂ« Fig. 3. Mitotic progression and p34cac2kinase activity of cells exposed to activity, not associated with mitotic cells, was detected between etoposide during GÂ¡phase. CHO cells were synchronized and drug treated as 3 and 6 h following release of the double block (Fig. 3). This described in Fig. 2. except that nocodazole (0.4 vg/ml) was included in the activity may be insufficient to initiate mitosis. However, the medium following exposure to solvent or etoposide. A, the proportion of cells in G2 (O) and M (â€¢)phase. , phase distribution following solvent exposure; kinase activity of control cultures increased significantly at 8 h , phase distribution following etoposide exposure. B, immunoprecipitated following release (Fig. 3Ã„),coincidental with the increase in p34cici |(Â¡naseactivity of solvent-treated (O) and etoposide-treated (â€¢)cells. 1819 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1992 American Association for Cancer Research. INHIBITION OF PÃŒ4Â«*1KINASEACTIVATION BY ETOPOSIDE A B phosphorylation, is inhibited following exposure of CHO cells to etoposide. As G2 M As Gz M To determine whether inhibition of activation of p34cdc2 kinase by etoposide may result from disruption of the p34cdc2/ â€”¿180â€”¿ cyclin B complex (16, 19, 41), in addition to inhibition of p34cdc2 tyrosine dephosphorylation, asynchronously dividing â€”¿97.4â€”¿ CHO cells were metabolically labeled with [35S]methionine followed by a 2-h exposure to 25 Â¿Â¿Metoposide.Immunoprecip- â€”¿66.2â€”¿ itations from SDS-boiled lysates using cyclin B or cdc2 peptide antisera revealed prominent doublets at M, ~57,000 and 34,000, respectively (Fig. 6). Anti-cyclin B immunoprecipita- â€”¿42.7â€”¿ tions carried out under nondenaturing conditions coprecipi- tated a protein corresponding to the slower-migrating form of the p34cdc2protein (Fig. 6), in agreement with published data (19, 27). The stability of this cyclin B/p34cdc2complex appeared unaffected following a 2-h exposure to etoposide compared to a solvent-treated control. Similarly, nondenaturing anti-cdc2 â€”¿31.0â€”¿ peptide precipitations identified a protein, corresponding to the faster-migrating form of the cyclin B doublet, in complex with the p34cdc2protein (Fig. 6). This M, ~57,000 protein was shown previously to be phosphorylated in p34cdc2kinase assays carried out in the absence of exogenously added histone HI (12) and is recognized by cyclin B antisera in Western blots of anti-cdc2 â€”¿21.5â€”¿ peptide immunoprecipitations (data not shown). Again, the p34cdc2/cyclin g complex appeared unaffected by a 2-h exposure to etoposide (Fig. 6). Fig. 4. Western blots of cell extracts obtained from asynchronous (As), Gj- Therefore, despite the fact that a 2-h etoposide treatment phase (harvested at 5 h following release of a thymidine/aphidicolin block), and M-phase cultures probed with cdc2 peptide polyclonal antisera III and then resulted in greater than 70% inhibition of p34cdc2kinase activity stripped and reprobed with phosphotyrosine monoclonal antisera (B). The phase and almost complete inhibition of mitotic progression (Fig. 1), distributions were as follows: asynchronous cells, 56% G,, 23% S, 18% G2, 2.6% no disruption of the p34cdc2/cyclin B interaction was observed. M; G2-phase cells, 4% G,, 21% S, 74% G2, 0.4% M; M-phase cells, 3% G,, 11% S, 27% G2, 58% M. DISCUSSION proportion of the M, 34,000 tyrosine-phosphorylated protein This study has demonstrated the following in CHO cells: (a) that was intermediate between G2- and M-phase cells (Fig. 4Ã„), maximal activation of p34cdc2kinase occurs at the G2/M tran and the amount of this protein appeared to relate directly to sition; (b) p34cdc2is a major tyrosine phosphoprotein in G2 that of the slower-migrating p34cdc2 isoform (Fig. 4A). The phase cells; (c) activation of p34cdc2kinase is accompanied by identities of the tyrosine-phosphorylated proteins in asynchron ous, G2- and M-phase CHO cell lysates at M, 170,000, 56,000, and 53,000 were not determined. Control Etoposide These data imply that the major tyrosine-phosphorylated "9 4h 5h 6h 8h 10h 12h 6h 8h 10h 12h protein in G2-phase CHO cell lysates is the slower-migrating â€”¿180 form of the p34cdc2protein and that activation of the p34cdc2 -97.4 kinase in CHO cells is accompanied by a major shift in mobility and tyrosine dephosphorylation of the p34cdc2protein. â€”¿66.2 Confirmation that the prominent M, 34,000 tyrosine-phos phorylated protein in G2-phase CHO cell lysates was indeed the slower-migrating form of the p34cdc2protein was obtained -42.7 by its specific depletion using cdc2 peptide antisera (Fig. 5). The p34cdc2protein was fully tyrosine phosphorylated at 3 h cdc2 following release of the thymidine/aphidicolin double block ,<_p34 (data not shown), a point where over 70% of the cells were in â€”¿31.0 S phase (Fig. 2A). A gradual decrease in the amount of this tyrosine-phosphorylated protein was observed as p34cdc2kinase became activated and the cells progressed from G2 into mitosis -]_P34 cdc2 (Figs. 5A and 3). This was accompanied by a shift in mobility B of the p34cdc2protein from the slower- to the faster-migrating â€”¿31.0 form (Fig. 5B). Exposure of G2-phase cells to etoposide resulted Fig. 5. Western blots of cdc2 peptide antisera immunoprecipitations, p34coc2- in inhibition of both p34cdc2tyrosine dephosphorylation and its depleted lysates, or cell extracts from synchronous CHO cells probed with phosphotyrosine (A) or cdc2 peptide (B) antisera. Cells were synchronized and shift in mobility (Fig. 5), consistent with their inability to drug treated as described in Fig. 3. p34cdclwas precipitated from 500 fig of total activate p34cdc2kinase and progress into mitosis (Fig. 3). Overall protein from a G2-phase lysate (Pellet), harvested at 5 h following release of the cellular content of the p34cdc2protein appeared unaffected by block. One hundred ^g of protein from the resultant supernatant were electrophoresed (Super.), in addition to equal amounts of protein (100 HÃœ)fromcell etoposide treatment (Fig. 5B). These data suggest that a specific extracts prepared from control or etoposide-treated cells harvested at 4, 5, 6, 8, p34cdc2kinase activation pathway, that of p34cdc2tyrosine de 10. and 12 It following release of the block. 1820

anti-cyclin B anti-cdc2 but it appears to act posttranslationally (46). Such a mechanism in mammalian cells, which may function to control mitotic progression via activation of p34ccka kinase (25, 26), could also ff / * explain the inhibition of histone HI phosphorylation and chro .â€¢*//// -â€¢*,<5' ,? mosome condensation induced by teniposide in G2-phase baby hamster kidney cells (6). It is unlikely that these effects are due to a direct interaction between the epipodophyllotoxins and p34cdc2 kinase (6, 12). Regulators of mammalian p34cdc2remain undefined, although the human homologues of the p34ldc2 kinase activator cdc25 (47) and mitotic cyclin B (19) have been cloned. It appears that biochemical activation of mammalian p34cdc2 via its association â€”¿97.4 with cyclin B and its tyrosine dephosphorylation is similar to that of S. pombe (reviewed in Refs. 37 and 48). Therefore, it is â€”¿66.2 tempting to speculate that cdc25, which controls the tyrosine <â€”57 kDa dephosphorylation of p34cdc2 in S1.pombe and Xenopus (43, 49)

â€”¿42.7 and provides the link between completion of DNA synthesis and initiation of mitosis in S. pombe (50), may constitute an element of the etoposide-induced G2 arrest response of CHO <â€”34 kDa cells. Certainly, tyrosine dephosphorylation of p34cdc2 accom â€”¿31.0 panies activation of the kinase and mitotic progression in these cells. The observation that all three processes are inhibited -21.5 following exposure to etoposide suggests that they share a functional relationship. In addition, it is necessary to consider the role of mammalian homologues of negative regulators of Fig. 6. Anti-cyclin B or anti-cdc2 peptide immunoprccipitations from [35S] p34cdc2 in S. pombe and Xenopus, such as weel, mikl, and sucl methionine-labeled cells. Asynchronously dividing cells were labeled for 2 h and (51-53). At least in 5. pombe, activation of p34cdc2 and the then harvested (SDS-Boiledand Control) or exposed to dimethyl sulfoxide solvent (control; 2 h DMSO) or 25 >iMetoposide (2 h Eloposide) for an additional 2 h. onset of mitosis are controlled by a balance between cdc25 phosphatase and weel kinase (reviewed in Ref. 37). By direct its tyrosine dephosphorylation; and (d) a brief exposure to analogy in mammalian cells, this balance may be affected by etoposide during G2 phase inhibits mitotic progression, p34cdc2 DNA damage, tilting it in the direction of p34cdc2 phosphoryl kinase activation, and its tyrosine dephosphorylation, appar ation and, consequently, inactivation. ently without affecting the p34cdc2/cyclin B interaction. In support of the hypothesis that a specific p34cdc2 kinase The rapidity with which etoposide inhibits both the transition activation pathway is inhibited in CHO cells following etopo into mitosis and the p34cdc2 kinase activity of CHO cells exposed side treatment, data were presented which showed that the p34cdc2/cyclin B interaction was unaffected by the drug. Okadaic continuously to the drug not only confirms the close association acid, a type 2A phosphatase inhibitor which induces p34cdc2 between these two processes, in agreement with data from other kinase activation and mitosis-specific phenomena in S-phase mammalian cell lines (16, 18), but also indicates that the arrest baby hamster kidney cells (27). caused activation of p34cdc2 point occurs late in G2 phase. This is consistent with the kinase and a rounded-up, mitosis-like state in G2 arrested CHO hypothesis that detection of topoisomerase II entrapped in a cleaved DNA intermediate results in the inhibition of a p34cdc2 cells (data not shown). A prerequisite for okadaic acid to induce activation of p34cdc2 kinase is formation of a complex between kinase activation pathway and G2 arrest and is supported by p34cdc2 and cyclin B (27). Therefore, it appears unlikely that the other investigations into the effects of epipodophyllotoxins on delay in p34cdc2 kinase activation induced by etoposide is due chromosome condensation and mitotic progression (5, 6, 45). to the breakdown of the p34"k'2/cyclin B complex, although However, it is important to differentiate between a biochemical cell cycle control mechanism which functions to prevent an studies are in progress to accurately quantify the proportion of the p34cdc2 protein retained in a high-molecular-weight complex aberrant mitosis following detection of DNA damage and a physical constraint on chromosome condensation placed by a before and after etoposide treatment. drug-induced DNA-toposiomerase II intermediate (5, 6, 45). Despite the finding that tyrosine dephosphorylation appears sufficient for the activation of p34cd<:2in S. pombe (42), threo- This study was designed to exclude the latter possibility by exposing cells briefly, rather than continuously (5, 6, 45), to nine dephosphorylation seems to be an additional requirement etoposide during G2 phase, thereby exploiting the fact that in mammalian cells (18). At this point it remains unknown DNA strand breaks rapidly reseal following removal of the drug whether the same phosphatase is responsible for both tyrosine and threonine dephosphorylation of p34cdc2 in mammalian cells, (8, 9). Indeed, only approximately 11% and 4% of initial etoposide-induced DNA single-strand breaks remain in G2- whether threonine dephosphorylation accompanies activation phase cells following a 3-h and 6-h repair period, respectively of the p34cdc:; kinase in CHO cells, and whether threonine (data not shown). The possibility remains, however, that a low dephosphorylation (in addition to tyrosine dephosphorylation) level of DNA lesions may prevent the inititation of mitotic is inhibited following exposure of CHO cells to etoposide. events. In summary, this paper has presented data regarding the role A more likely explanation, first documented in the X- or UV- of tyrosine dephosphorylation in p34cdc2 kinase activation in irradiation-responsive RAD9 gene of 5. cerevisiae (15), is that CHO cells, specific inhibition of which may be responsible for G2 arrest is an active cell cycle control mechanism which is etoposide-induced G2 arrest. Using etoposide as a reagent to switched on following detection of damage to the genome. The induce G2 arrest, it will be possible to study the positive and precise function of the RAD9 gene in 5. cerevisiae is unknown, negative regulators of p34cdc2 kinase activation which control 1821

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1992 American Association for Cancer Research. INHIBITION OF P34"*2 KINASE ACTIVATION BY ETOPOSIDE mitotic progression in mammalian cells, in addition to a puta their modifications. CRC Crit. Rev. Biochem., 20: 201-263, 1986. 25. Riabowol, K., Draetta, G., Brizuela, L., Vandre, D., and Beach, D. The cdc2 tive DNA damage-sensing mechanism which induces negative kinase is a nuclear protein that is essential for mitosis in mammalian cells. growth control during GÃŒphase. Cell, 57:393-401, 1989. 26. Th'ng, J. P. H., Wright, P. S., Hamaguchi, J., Lee, M. G., Norbury, C. J., Nurse, P., and Bradbury, E. M. The FT210 cell line is a mouse G2 phase ACKNOWLEDGMENTS mutant with a temperature-sensitive CDC2 gene product. Cell, 63:313-324, 1990. Drs. Dan Sullivan and Terry Hadley and the personnel of the Cell 27. Yamashita, K., Yasuda, H., Pines, J., Yasumoto, K., Nishitani, H., Ohtsubo, M., Hunter, T., Sugimura, T., and Nishimoto, T. Okadaic acid, a potent Cycle and Drug Resistance groups of the Brown Cancer Center are inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/Hl thanked for their helpful discussions. Judy Hollkamp is appreciated for kinase and transiently induces a premature mitosis-like state in BHK21 cells. her excellent secretarial assistance. EMBO J., 9:4331-4338, 1990. 28. Worton. R. G., and Duff, C. Karyotyping. Methods Enzymol., 58: 322-344, 1979. 29. Bradford, M. M. A rapid and sensitive method for the quantitation of REFERENCES microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254, 1976. 1. Rao, P. N. The molecular basis of drug-induced G3 arrest in mammalian cells. Mol. Cell. Biochem., 29: 47-57, 1980. 30. Draetta, G., Brizuela, L., Potashkin, J., and Beach, D. Identification of p34 and pi 3, human homologsof the cell cycle regulators of fission yeast encoded 2. Holland, J. M., Wright, W. D., Higashikubo, R., and Roti Roti, J. L. Effects by cdc2+ and sud*. Cell, 50: 319-325, 1987. of irradiation on nuclear protein synthesis in G2 phase of the cell cycle. 31. Laemmli, U. K. Cleavage of structural proteins during the assembly of the RadiÃ¢t.Res., 122: 197-208, 1990. head of bacteriophage T4. Nature (Lond.), 227: 680-685, 1970. 3. Rao, A. P., and Rao, P. N. The cause of G2-arrest in Chinese hamster ovary cells treated with anticancer drugs. J. Nati. Cancer Inst., 57: 1139-1143, 32. Towbin, H., Staehelin, T., and Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some appli 1976. cations. Proc. Nati. Acad. Sci. USA, 76: 4350-4354, 1979. 4. Sorenson, C. M., and Eastman, A. Mechanism of c/i-diamminedichloropla- iHiimi(II) induced cytotoxicity: role of G2 arrest and DNA double-strand 33. Grieder, A., Maurer, R., and Stahelin, H. Effect of an epipodophyllotoxin derivative (VP 16-213) on macromolecular synthesis and mitosis in inasto breaks. Cancer Res., 48:4484-4488, 1988. cytoma cells in vitro. Cancer Res., 34: 1788-1793, 1974. 5. Charron, M., and Hancock, R. DNA topoisomerase II is required for for 34. Kalwinsky, D. A., Look, A. T., Ducore, J., and Fridland, A. Effects of the mation of mitotic chromosomes in Chinese hamster ovary cells: studies using the inhibitor 4'-demethylepipodophyllotoxin 9-(4,6-O-thenylidene-/3-D-glu- epipodophyllotoxin VP-16-213 on cell cycle traverse, DNA synthesis, and copyranoside). Biochemistry, 29: 9531-9537, 1990. DNA strand size in cultures of human leukemic lymphoblasts. Cancer Res., 6. Roberge, M., Th'ng, J., Hamaguchi, J.. and Bradbury, E. M. The topoisom 43: 1592-1597, 1983. 35. Misra, N. C., and Roberts, D. Inhibition by 4'-demethylepipodophyllotoxin erase II inhibitor VM-26 induces marked changes in histone HI kinase 9-(4,6-O-2-thenylidene-fl-D-glucopyranoside) of human lymphoblast cultures activity, histones HI and H3 phosphorylation, and chromosome condensa in G2 phase of the cell cycle. Cancer Res., 35: 99-105, 1975. tion in G2 phase and mitotic BHK cells. J. Cell Biol., ///: 1753-1762, 1990. 36. Moreno, S., Hayles, J., and Nurse, P. Regulation of p34cdc2protein kinase 7. Liu, L. F. DNA topoisomerase poisons as antitumor drugs. Annu. Rev. during mitosis. Cell, 5Â«:361-372, 1989. Biochem., SS: 351-375, 1989. 8. Lock, R. B., and Ross, W. E. Possible role for p34cdc2kinase in etoposide- 37. Nurse, P. Universal control mechanism regulating onset of M-phase. Nature (Lond.), 344: 503-508, 1990. induced cell death of Chinese hamster ovary cells. Cancer Res., JO: 3767- 38. De Brabander, M. J., Van de Veire, R. M. L., Aerts, F. E. M., Borgers, M., 3771, 1990. and Janssen, P. A. J. The effects of methyl-[5-(2-thienylcarbonyl)-l//-ben- 9. Long, B. H., Musial, S. T., and Brattain, M. G. Single- and double-strand zimidazol-2-yl]carbamate (R 17934; NSC 238159), a new synthetic antitu- DNA breakage and repair in human lung adenocarcinoma cells exposed to moral drug interfering with microtubules, on mammalian cells cultured in etoposide and teniposide. Cancer Res., 45: 3106-3112, 1985. vitro. Cancer Res., 36: 905-916, 1976. 10. Allen, L. M. Comparison of uptake and binding of two epipodophyllotoxin glucopyranosides, 4'-demethylepipodophyllotoxinthenylidene-/Ã®-D-glucoside 39. Krishan, A., and Frei, E. Cytofluorometric studies on the action of podo- and 4'-demethylepipodophyllotoxin ethylidene-,3-D-glucoside, in the L1210 phyllotoxin and epipodophyllotoxins (VM-26, VP-16-213) on the cell cycle traverse of human lymphoblasts. J. Cell Biol., 66: 521-530, 1975. leukemia cell. Cancer Res., 38: 2549-2554, 1978. Drewinko, B., and Barlogie, B. Survival and cycle-progression delay of human 40. Draetta, G., Luca, F., Westendorf. J., Brizuela, L., Ruderman, J., and Beach, 11 D. cdc2 protein kinase is complexed with both cyclin A and B: evidence for lymphoma cells in vitro exposed to VP-16-213. Cancer Treat. Rep., 60: proteolytic inactivation of MPF. Cell. 56: 829-838, 1989. 1295-1306, 1976. 41. Pondaven, P., Meijer, L., and Beach, D. Activation of M-phase-specific 12 Lock, R. B., and Ross, W. E. Inhibition of p34coc2kinase activity by etoposide histone HI kinase by modification of the phosphorylation of its p34clic2and or irradiation as a mechanism of G2 arrest in Chinese hamster ovary cells. cyclin components. Genes Dev., 4: 9-17, 1990. Cancer Res., 50: 3761-3766, 1990. 42. Gould, K. L., and Nurse, P. Tyrosine phosphorylation of the fission yeast 13. Lau, C. C., and Pardee, A. B. Mechanism by which caffeine potentiates cdc2+ protein kinase regulates entry into mitosis. Nature (Lond.), 342: 39- lethality of nitrogen mustard. Proc. Nati. Acad. Sci. USA, 79: 2942-2946, 45, 1989. 1982. 43. Gould, K. L., Moreno, S., Tonks, N. K., and Nurse, P. Complementation of 14. Tobey, R. A. Different drugs arrest cells at a number of distinct stages in G2. the mitotic activator, pSO00025,bya human protein-tyrosine phosphatase. Nature (Lond.), 254: 245-247, 1975. Science (Washington DC), 250: 1573-1576, 1990. 15. Weinert, T. A., and Hartwell, L. H. The RAD9 gene controls the cell cycle 44. Jessus, C, Ducommun, B., and Beach, D. Direct activation of cdc2 with response to DNA damage in Saccharomyces cerevisiae. Science (Washington phosphatase: identification of pl3sll<:'-sensitive and insensitive steps. FEBS DC), 24/: 317-322, 1988. Lett., 266: 4-8, 1990. 16. Draetta, G., and Beach, D. Activation of cdc2 protein kinase during mitosis 45. Wright, S. J., and Schatten, G. Teniposide, a topoisomerase II inhibitor, in human cells: cell cycle-dependent phosphorylation and subunit re prevents chromosome condensation and separation but not decondensation arrangement. Cell, 54:17-26, 1988. in fertilized surf clam (Spisula solidissima) oocytes. Dev. Biol., 142: 224- 17. Draetta, G., Piwnica-Worms, H., Morrison, D., Druker, B., Roberts, T., and 232, 1990. Beach, D. Human cdc2 protein kinase is a major cell-cycle regulated tyrosine 46. Weinert, T. A., and Hartwell, L. H. Characterization of RAD9 of Saccharo kinase substrate. Nature (Lond.), 336: 738-744, 1988. myces cerevisiae and evidence that its function acts posttranslationally in cell 18. Moria, A. O., Draetta, G., Beach, D., and Wang, J. Reversible tyrosine cycle arrest after DNA damage. Mol. Cell. Biol., 10:6554-6564, 1990. phosphorylation of cdc2: dephosphorylation accompanies activation during 47. Sadhu, K., Reed, S. I., Richardson, H., and Russell, P. Human homolog of entry into mitosis. Cell, 5Â«:193-203, 1989. fission yeast cdc25 mitotic inducer is predominantly expressed in G2. Proc. 19. Pines, J., and Hunter, T. Isolation of a human cyclin cDNA: evidence for Nati. Acad. Sci. USA, 87: 5139-5143, 1990. cyclin mRNA and protein regulation in the cell cycle and for interaction with 48. Draetta, G., and Beach, D. The mammalian cdc2 protein kinase: mechanisms 034Â«".Cell, 58: 833-846, 1989. of regulation during the cell cycle. J. Cell Sci. Suppl., 12: 21-27, 1989. 20. Lee, M., and Nurse, P. Complementation used to clone a human homologue 49. Kumagai, A., and Dunphy, W. G. The cdc25 protein controls tyrosine of the fission yeast cell cycle control gene cdc2+. Nature (Lond.), 327: 31- dephosphorylation of the cdc2 protein in a cell-free system. Cell, 64: 903- 35, 1987. 914, 1991. 21. Arion, D., Meijer, L., Brizuela, L., and Beach, D. cdc2 is a component of the 50. Enoch, T., and Nurse, P. Mutation of fission yeast cell cycle control genes M phase-specific histone HI kinase: evidence for identity with MPF. Cell, abolishes dependence of mitosis on DNA replication. Cell, 60: 665-673, 55:371-378, 1988. 1990. 22. Labbe, J. C., Picard, A., Peaucellier, G., Cavadore, J. C., Nurse, P., and 51. Dunphy, W. G., and Newport, J. W. Fission yeast pi3 blocks mitotic Doree, M. Purification of MPF from starfish: identification as the H l histone activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase p34cdc2and a possible mechanism for its periodic activation. Cell, 57: kinase. Cell, 58:181-191, 1989. 253-263, 1989. 52. Lundgren, K., Walworth, N., Booher, R., Dembski, M., Kirschner, M., and 23 Masui, Y., and Marker!, C. L. Cytoplasmic control of nuclear behaviour Beach, D. mikl and weel cooperate in the inhibitory tyrosine phosphoryla during meiotic maturation of frog oocytes. J. Exp. Zool., 777: 129-146, tion of cdc2. Cell, 64: 1111-1122, 1991. Russell, P., and Nurse, P. Negative regulation of mitosis by weel*, a gene 1971. 53. 24. Wu, R. S., Panusz, H. T., Hatch, C. L., and Bonner, W. H. Histones and encoding a protein kinase homolog. Cell, 49: 559-567, 1987. 1822

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1992 American Association for Cancer Research. Inhibition of p34cdc2 Kinase Activation, p34cdc2 Tyrosine Dephosphorylation, and Mitotic Progression in Chinese Hamster Ovary Cells Exposed to Etoposide

Richard B. Lock

Cancer Res 1992;52:1817-1822.

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