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

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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 »...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- p34«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 <•..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, ¡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 »-minimalessential medium The antitumor epipodophyllotoxin etoposide' induces DNA supplemented with 5% fetal calf serum, penicillin (100 units/ml), and streptomycin (I00^g/ml) at 37°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 37°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 37°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 ¿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 4°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 ±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),
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