Effects of Cisplatin on the Induction of Apoptosis in Proliferating Hepatoma Cells and Nonproliferating Immature Thymocytes1

[CANCER RESEARCH S3. 2133-2139. May I. 1993] Effects of Cisplatin on the Induction of Apoptosis in Proliferating Hepatoma Cells and Nonproliferating Immature Thymocytes1

Dyfed L. Evans and Caroline Dive2

CRC Molecular and Cellular Pharmacolog\ Group. School of Biological Sciences. Manchester University; Manchester M LÃ¬9PT England

ABSTRACT merely the presence of a stimulus to engage the process. Other cell types, it has been argued, have to synthesize and appropriately locate A 2-h exposure of JB1 rat hepatoma cells in late log phase of growth to the "required machinery" to execute this active form of cell death 50 UMci.s-diamminedichloroplatinum (II) (cisplatin) resulted in the asyn (12). A hierarchy which reflects cell phenotype and predicts the ability chronous detachment of cells from the monolayer over 4 days. Detached but not monolayer cells exhibited condensed chromatin and DNA frag of cells to undergo apoptosis has been proposed ( 13). Where hepatoma mentation, which is indicative of endonuclease activation, the hallmarks of cells might be placed in such a hierarchy is unknown. apoptosis in epithelial cells. The number of cisplatin-treated cells identi Apoptosis is believed to be dependent on protein synthesis in many fied as apoptotic at any one time was never >1% of the total cell number but not all documented cases, an apparent paradox which may reflect present on addition of drug. Two days after drug addition there was a the "primed" status of each cell type (14â€”16).The inferred relation decrease from 85% to 29% cells in GÃŒphaseof the cell cycle, cells in S ship between apoptosis and protein synthesis stems from studies using phase increased from 9% to 18%, and cells in (Â¡,/Mphase increased from CX, although DNA and RNA synthesis are also inhibited by CX (17, 6% to 51% with respect to untreated cells. Previous studies by Eastman and colleagues demonstrated that cisplatin-induced apoptosis of Chinese 18). CX prevented CP-induced apoptosis in CHO cells (7). Unrepaired hamster ovary cells occurred in the G2 phase of the cell cycle [A. Eastman, CP-induced DNA lesions in the S phase of the cell cycle are thought Cancer Cells (Cold Spring Harbor), 2: 275-280, 1990]. Continuous expo to be the lethal events which predispose CHO cells to undergo apo sure of JB1 cells to cycloheximide (1 UM)during and after exposure to ptosis in the G2 phase of the cell cycle (19). The effect of CX could cisplatin prevented both drug-induced changes in cell cycle distribution therefore reflect a requirement to synthesize DNA repair enzymes, as and the engagement of apoptosis. Freshly isolated immature rat thy- yet unidentified "cell death-inducing" proteins, and/or the prevention mocytes are known to be exquisitely sensitive to the induction of apopto by CX of cell cycle-related events (cell cycle checkpoints) (20) which sis by multiple stimuli including dexamethasone, etoposide, and irradia may be important for the engagement of apoptosis by CP. tion. However, no significant increase in the amount of apoptosis above control levels was observed up to 36 h after a 2-h exposure to 50 UMcis The initial aim of this study was to determine whether hepatoma platin. JB1 cells have a doubling time of 24 h, whereas >90% of imma cells could undergo apoptosis in response to CP. Here we show that ture rat thymocytes are noncycling. The data presented here provide in cycling JB1 hepatoma cells undergo asynchronous apoptosis in re direct evidence that initiation of cisplatin-induced apoptosis may need to sponse to CP where apoptosis was detected only in cells which had be coupled to a cell cycle-mediated event. detached from the monolayer (that embryonic mouse fibroblasts which exhibited internucleosomal cleavage become nonadherent was INTRODUCTION previously shown by Kanter et al.) (21). In agreement with previous studies using CHO cells (7), both progression through the cell cycle Our laboratory is interested in the molecular regulation of apoptosis and the engagement of apoptosis induced by CP in hepatoma cells was in the liver and its putative role in the prevention of hepatocarcino- prevented by CX. We were unable to synchronize JB1 cells or halt genesis (1). Apoptosis is important during normal liver homeostasis them in their cycle long enough to determine whether CP induced (2) and during involution of hyperplasia following removal of liver apoptosis while they were in a nonproliferating state. We then tested mitogens such as phÃ©nobarbital(3, 4). Hepatocarcinomas are essen the hypothesis that CP cannot induce apoptosis in a quiescent cell tially refractory to chemotherapy (5), and we wanted to determine whether the pathway for the active process of apoptotic cell death is population by studying the effects of CP on immature rat thymocytes, retained by hepatoma cells. The withdrawal of serum, addition of a cell type known to rapidly undergo apoptosis in response to several ionomycin, and treatment with menadione, which previously were anticancer agents but which is predominantly nonproliferating (22, shown to induce apoptosis in normal rat hepatocytes (6), failed to 23). We report here that immature rat thymocytes, despite their ap induce apoptosis in our JB1 hepatoma cell line. CP3 induced apoptosis parent propensity to undergo apoptosis, did not exhibit levels of in epithelial cells (7), so we chose to determine whether this agent apoptosis greater than those seen in untreated thymocytes in response could induce apoptosis in JB1 hepatoma cells. to CP. We suggest that one reason for this lack of CP-induced apop Insight into the molecular mechanisms of apoptosis largely derives tosis could be due to the continued presence of thymocytes in the from studies using suspensions of hemopoietic cells, particularly of Go/G, phase of the cell cycle. the immature rat thymus (8). These thymocytes seem exquisitely sensitive to induction of apoptosis in response to a plethora of stimuli including glucocorticoids (9), etoposide (10), and irradiation (11). This cell type may be "primed" for apoptosis, perhaps requiring MATERIALS AND METHODS William's E medium, fetal bovine serum, antibiotics, glutamine, and tissue culture plastics were obtained from Gibco, Ltd. (Uxbridge, England). RPMI Received 2/19/92; accepted 2/19/93. 1640 was purchased from Imperial Laboratories (Andover, England). MP 1Supported by the Cancer Research Campaign (C. D.). D. L. E. holds a Medical (Solu-Medrone) was obtained from UpJohn (Crawley, England). PBS Research Council studentship. 2 To whom requests for reprints should be addressed, at CRC Molecular and Cellular (Dulbecco A) was obtained from Oxoid (Basingstoke, England). AO, propid- Pharmacology Group, School of Biological Sciences, Manchester University, Stopford ium iodide, and Hoechst 33342 were supplied by Molecular Probes, Inc. Building, Oxford Road, Manchester M13 9PT England. (Eugene, OR). [mE>r/iv/-'H]Thymidine and ["S)methionine were purchased ' The abbreviations used are: CP. cij-diamminedichloroplatinum (II) (cisplatin); AO, acridine orange; TB, trypan blue; PBS. phosphate-buffered saline; FCM. flow cytometry; from Amersham International (Aylesbury, England), and Optiphase Hisafe II CX. cycloheximide; CHO cells, Chinese hamster ovary cells; DMSO, dimethyl sulfoxide; scintillation fluid was supplied by FSA Laboratories (Loughborough, En MP, methylprednisolone sodium succinate. gland). All other materials were obtained from Sigma (Poole, England). 2133

Cell Culture Triton X-100 (pH 8)J before centrifugation at 27,000 X g for 20 min to collect high-molecular-weight DNA. The supernatant (containing low-molecular- JB1 Hepatoma Cells. Monolayer cultures of JBI rat hepatoma cells were weight DNA) was collected. Cell pellets were resuspended in 5 ml of buffer A maintained in William's E medium supplemented with 5% fetal bovine serum, (IO mM Tris, l mM EDTA, pH 8). DNA content was assessed in pellets and glutamine, and antibiotics at 37Â°C,in a humid atmosphere of 5% CO2/95% air supernatant using fluorochrome Hoechst 33342 according to the method of (24). Cells were routinely seeded on day 0 at 2 x IO5 cells/80-cm3 flask in 20 Laborea and Paigen (27). ml medium and passaged every 5 days by trypsinization. The doubling time of Measurement of Cell Cycle Distribution. Cell samples for FCM were JB1 cells cultured using this protocol was approximately 24 h. fixed in 0.1% paraformaldehyde and 0.1% Triton X-100 in PBS and stored at Freshly Isolated Immature Rat Thymocytes. Thymocyte suspensions 4Â°Cbefore analysis. Cell cycle distribution of trypsinized monolayer (but not were prepared as outlined by Wyllie et al. (9). Briefly, thymus glands from detached) JB 1 cells and thymocyte cell suspensions was performed at intervals 3-4-week-old Sprague-Dawley rats of either sex were minced in RPMI 1640 following CP treatment using a Coulter Epics V instrument with an argon laser supplemented with 1% fetal bovine serum, glutamine, and antibiotics. Cells set to excite at 488 nm. Propidium iodide (20 ul of a 2.5 mg/ml solution in were adjusted to a density of 1 x 107/ml appropriately treated and incubated PBS) was added to 5 X IO5 cells in 500 ul of fixative for 5 min before 20,000 at 37Â°Cin a humidified atmosphere of 5% CO2/air. cells were analyzed with respect to red fluorescence at a flow rate of approx imately 2500 cells/s. Red fluorescence due to propidium-bound DNA was Assessment of Cell Viability collected using a 630-nm-long bandpass filter. Data were analyzed as single- parameter frequency histograms. Uptake of Trypan Blue. Cell counts were performed on a hemocytometer Effect of CX on CP-induced JBI Cell Death. CX was dissolved in PBS using equal volumes of cell suspension and TB (0.3% in PBS). Cells with and added to cell cultures such that the final concentration was l UM.This membrane permeability allowing uptake of TB were interpreted as nonviable, concentration of CX was chosen because higher concentrations were cytotoxic although the mode of cell death could not be determined by this assay alone. over the time period of the experiment. CX was added simultaneously to CP Cells which permitted TB uptake were expressed as a percentage of the total and either maintained throughout the experiment or withdrawn 2-10 h after the cell number. 2-h CP exposure period. The effect of CX either alone or in combination with Staining with AO. One drop of cell suspension was added to one drop of CP on the cell cycle was also determined by FCM as described above. AO solution (10 ug/ml in PBS), mixed gently on a slide, and immediately Effects of CX and CP on JBI Cell DNA Synthesis Measured by [methyl- examined with an Olympus BH-2 microscope with fluorescence attachment. 'll| I hvniidine Incorporation. JBI cultures were incubated with 50 UMCP Green fluorescence was detected between 500 and 525 nm. Cells exhibiting with or without the continual presence of l UMCX for 8, 20, or 44 h as bright green fluorescent condensed nuclei (intact or fragmented) were inter described above. Cultures were subsequently incubated for 4 h in the presence preted as apoptotic cells and expressed as a percentage of the total cell number of 1 uC/ml of [mff/iv/-'H]thymidine. Cells were washed three times in both (25). Viable cells were interpreted as cells which exhibited a green, diffusely ice-cold PBS and then 10% (v/v) trichloroacetic acid and finally solubilized in stained intact nucleus. Occasionally, mitotic cells were recognized where the 100 ul of 0.1% sodium hydroxide/105 cells. Two hundred-ul aliquots were nucleus was in the process of division but remained diffusely stained and easily transferred into 3 ml of Optiphase Hisafe II scintillation fluid, and total [me- distinguished from apoptotic cells. Cell ghosts with an indeterminate mem r/iv/-1H]thymidine was measured using a Packard 1900 TR liquid scintillation brane and weak staining were not included in the cell count. analyzer. Clonogenic Assay. The effect of CP on the ability of JBI cells to survive Effects of CP and CX on JBI Cell Protein Synthesis Measured by and divide was assessed by clonogenic assay. The plating efficiency of un Incorporation of [35S]Methionine. [15S]Methionine uptake was determined treated JBI cells was 36%. Untreated cells were seeded at 500 cells/25-cm3 as described above for [mer/nV-'Hlthymidine incorporation with the exception culture flask in 10 ml medium. Cells treated with 50 UMCP for 2 h were seeded that after incubation with (!5S]methionine cells were washed 6 times in ice- at 1 x lOVflask. Cultures were incubated for 10 days, and the number of cold PBS only before solubilization. colonies containing more than 50 cells was determined and expressed as a percentage of the untreated control. Mode and Kinetics of Cisplatin-induced JBI and Thymocyte Cell RESULTS Death. CP was freshly prepared before each experiment as a stock solution at Mode of Cisplatin-induced Cell Death in JBI Hepatoma Cells. 10 Ã•TEMinDMSO. This was diluted in medium before addition to JBI cells and thymocyte such that the final concentration of cisplatin was 10-50 UMand that Following treatment with CP (10-50 UM)for 2 h, cells that remained of DMSO was maintained at 0.5% (v/v). In order to determine the mode of cell as an attached monolayer were assessed for viability over 48 h. Re death in CP-treated cultures, JBI cells were seeded at 2 x IO5 cells/80-cm1 gardless of CP concentration and time of analysis, monolayer cells flask on day 0. On day 4 the medium was replenished, and on day 5, when cells were 94-100% viable (x = 97%; n = 20; SE = 1.2) with respect to were in late log phase, they received 10-50 UMCP or the appropriate volume TB uptake and AO stain. In CP-treated cultures, cells detached from of DMSO alone. After 2 h the medium was discarded and replaced with the monolayer and were regularly collected in the medium at 2-h drug-free medium. At 2-h intervals up to 48 h. the medium was removed and intervals over the 48 h after drug exposure. In every case, detached centrifuged at 150 x g for 5 min. The cell pellet obtained (referred to as cells were 55-85% (x = 62%; n = 10; SE = 8.8) apoptotic as detached cells) was resuspended in 0.2-0.5 ml medium, counted on a hemocy measured by AO stain (Fig. 1), the remainder being cell ghosts and tometer, and examined for TB uptake. Detached cells were also stained with AO and examined by fluorescence microscopy. In parallel experiments, mono- debris (or occasional mitotic cells). That the cell ghosts had previously layer cells were trypsinized at each time point and similarly examined using undergone apoptosis could not be proved directly, but this seems TB and AO. Aliquots of monolayer and detached cells were stored as dry likely since at no time did we see evidence of primary necrosis. That pellets at -70Â°C and subsequently subjected to gel electrophoresis following apoptosis had occurred was confirmed by gel electrophoresis of ex DNA extraction using the method described by Smith et al. (26). tracted DNA, where a classical 180-200-base pair integer oligonu- In thymocyte preparations, cells were centrifuged at 700 X g for IO min cleosome "ladder" was observed for detached but not monolayer cells after a 2-h exposure to 1-50 UMCP and resuspended in drug-free medium. As (Fig. 2). At concentrations of CP up to 200 UM,the mode of JB 1 cell a positive control, thymocytes were treated continuously with 10~5 M MP. death was apoptosis where chromatin condensation was observed, Untreated, MP-treated, and CP-treated thymocytes were examined at inter rather than necrosis, where chromatin condensation was not seen (data vals between 0 and 36 h with respect to AO staining and TB uptake. In addition not shown). to gel electrophoresis of extracted DNA, which is only qualitative, the per Kinetics of Cisplatin-induced Apoptosis: Dependence on Drug centage of DNA fragmentation was also quantified as described below. Briefly, untreated thymocytes or those treated with MP ( 10~5 M) or CP (50 UMfor 2 h) Concentration and Time after Drug Exposure. Survival curves for were collected at 0, 6, 12, 24, and 36 h after treatment. Cells ( IO7) were spun JBI cells exposed for 2 h to 10-50 UMcisplatin are shown in Fig. 3. at 1500 x g for 10 min at 4Â°Cand washed twice with PBS. Pellets were Clonogenic assay showed that 10 days after a 2-h exposure to 50 UM resuspended in 5 ml lysis buffer [20 mM EDTA, 5 HIMTris-HCl, 0.5% (v/v) CP, only 0.28% cells were able to form colonies (average of 6 repeat 2134

DNA fragmentation detected was lower at 45%. In contrast, CP had no significant effect on thymocyte apoptosis by AO staining or percent age DNA fragmentation compared to untreated controls.

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Fig. 2. Agarose gel electrophoresis of DNA extracted from JBI hepatoma cells treated with 50 UMCP for 2 h. Lane I. DNA marker where the four bands seen indicate molecular weights of 1353. 1078. 872, and 603. respectively, and where 2 ug of marker DNA were loaded into the well. Lane 2. monolayer cells trypsinized 12 h after CP addition. Lanes 3 and 4. detached cells collected from above the drug-treated monolayer 6-12 h after drug addition. One million cells were loaded per well, and the gel is representative of at least 8 separate repeat experiments. Fig. I. AO staining of chromatin conformation in monolayer and detached JBI hepatoma cells following exposure to CP. A. monolayer cells exposed to 50 MMCP for 2 h. and 6-8 h later Irypsinized and stained with AO. Note the "mottled" fluorescence within intact nuclei typical of viable cells. B, AO-stained cells which have detached from the monolayer 4-14 h following exposure to CP. In contrast to monolayer cells, detached cells show condensed and fragmented nuclei typical of apoptotic cells. The results presented are typical of at least 8 separate experiments. experiments). The induction of apoptosis in JBI cells following ex 8 posure to CP was dependent on both the concentration of CP added 'S and the time after drug exposure (Fig. 4). After a 2-h exposure to 50 UMCP, the total number of apoptotic cells collected over each subse quent 2-h period increased to a maximum after 14-16 h. At this 2-h collection period, the number of apoptotic cells reflected only 0.6% of total cell number at drug addition. However, 48 h after drug exposure 57% (n = 6; SE = 4.2) of the cells present at the time of drug addition had detached from the monolayer. The percentage of detached cells with condensed or fragmented nuclei (with AO) was always greater than the percentage of detached cells permeable to TB, as shown in Table 1. Effects of CP on Immature Rat Thymocytes. The rate of spon taneous apoptosis in thymocytes and that induced by treatment with MP or CP are shown in Fig. 5. Fig. 5A shows the percentage of cells Time (days) recognized morphologically as being apoptotic using AO staining, and Fig. 5B shows the percentage of fragmented DNA present as evaluated Fig. 3. The percentage survival of JBI cells following a 2-h exposure to 10-50 UMCP calculated as the percentage of viable monolayer cells compared to untreated control by Hoechst 33342 and spectrofluorimetry. By 36 h 80% morpholog monolayer cell number. Viability was determined by trypan blue uptake. A. 10 MMCP; Q ical apoptosis was seen in response to MP, although the percentage of 25 UMCP; â€¢¿.50UMCP. Data shown are the mean Â±SE for 3 repeat experiments. 2135

10 withdrawal of CX from CP-treated cultures, levels of apoptosis were seen that were closely similar to those observed in cells which had been treated with CP alone. The inhibitory effects of l UMCX alone or in combination with CP on both protein and DNA synthesis are shown in Table 4. CX also prevented CP-induced accumulation of JBI cells in S and G2/M phases of the cell cycle (Table 2).

DISCUSSION

Hepatocarcinomas are essentially refractory to cancer chemother apy (5). An objective of this study was to determine whether hepatoma 4 - cells retained the ability to undergo apoptosis in vitro. The engage ment of apoptosis in response to CP has been studied in proliferating

100 -t

10 20 30 40 SO 60

Time after exposure to CP (hours)

Fig. 4. The kinetics of apoptosis in JB1 hepatoma cells exposed for 2 h to varying !â€ž concentrations of CP. Apoptosis was detected in detached cells by acridine orange stain ing. The number of apoplolic cells detecied at single time points is shown. O, untreated il cells; A. 10 (IMCP; D, 25 MMCP; â€¢¿50UMCP. Results shown are the mean value of 3 -s. experiments Â±SE. Ã®!

Table 1 Analysis of cell death in JBI hepaloma cells which detach from the monolayer following a 2-h exposure lo 50 pM CP AO-positive cells indicates those cells with condensed and fragmented DNA. TB- positive cells indicates those cells which allow uptake of trypan blue.

TimeUMexposure after a 50 AO-positivedetached TB-positivedetached toCP(h)012243648% cellsmean cellsmean 4)46Â±SD (n = 4)20Â±SD (n = Â±11.063 Â±6.723 Â±7.666 Â±5.820 Â±6.161 Â±7.630 B Â±7.864 Â±6.227 Â±10.2^ Â±10.0 100-1

Flow Cytometric Analysis of Cell Cycle Kinetics and Induction of Apoptosis. Cell cycle analysis by FCM revealed that CP-induced 80 changes in JBI cell cycle distribution were apparent 12 h after drug addition (Fig. 6/1). After 48 h, a 3-fold decrease in the percentage of cells in G, phase, a 2-fold increase in cells in S phase, and a 9-fold 60- increase in cells in G2/M phase were observed (Table 2). Continuous exposure to CX (I UM)completely abolished CP-induced changes in JBI cell cycle distribution (Table 2). 40- The effects of MP and CP on immature thymocytes are shown in Fig. 6B and Table 3. Regardless of treatment, the percentage of cells in S and G2/M phases was never >10%, and CP did not change cell cycle distribution over 24 h. The appearance of a peak to the left of the G, peak correlated with the onset of apoptosis as previously described by others (28-30). No such peak was seen for the JBI cell samples, which consisted of only trypsinized monolayer cells. Effect of Cycloheximide on Cisplatin-induced Apoptosis in JBI Cells. The continual presence of l UMCX prevented 84% (mean Time (hours) value; n = 3; SE = 1.5) of the apoptosis induced in JBI cells 48 h Fig. 5. The kinetics of apoptosis induced Â¡nimmature thymocytes by continuous after a 2-h exposure to 50 UMCP (Fig. 7). At this concentration CX exposure to MP( 10~s M)and by a 2-h exposure to CP(50 UM).In A, apoptosis was detected was not cytotoxic over 48 h. In order to suppress apoptosis CX was by acridine orange staining. In B, percentage DNA fragmented was assessed using Hoe chst 33342 fluorescence of purified high- and low-molecular-weight DNA. O, PBS required continually. Removal of CX at various time points after CP control; D, 0.5% (v/v) DMSO control; A, CP; â€¢¿.MP.Data are shown as the mean value exposure resulted in an increase in the rate of apoptosis. Six h after the of 3 repeat experiments Â±SE. 2136

Fig. 6. Row cytometric analysis of cell cycle distribution in JBI hepatoma cells treated with CP (2 h; 50 UM) (A} and immature rat thymocytes 121, treated with CP (2 h; 50 UM)or MP ( I0~5 M con tinuously) (fl). DNA histograms are of 20.000 fixed cells in 0.1* paraformaldehyde/0.1 % Triton X-100 in PBS and stained with propidium iodide at 100 ug/ml for 5 min. Results shown are represen tative of 3 separate repeat experiments each con taining duplicate samples. 24h100

100 100 100

Red Fluorescence (<630nm) Channel Number epithelial and hemopoietic cell types (7, 19, 31), where it occurred in with this morphological evidence, the characteristic DNA fragmenta G2 phase of the cell cycle. We wanted to investigate whether this was tion which confirms endonuclease activation typical of apoptosis was a general feature of CP-induced apoptosis and compared effects of CP seen in detached but not monolayer cells. Our data imply that chro- on the survival of cycling JBI hepatoma cells and noncycling imma matin condensation also occurs after or alongside the loss of cell ture thymocytes. The latter are the most extensively studied cell type adherence and cell-to-cell contacts in JBI cells. Comparison of the with respect to the induction of apoptosis. They readily undergo this indices of cell death by TB uptake and AO staining suggested that the active process in response to a plethora of stimuli (8-11 ). changes inside the nucleus preceded increased plasma membrane per The data presented here show that apoptosis was detectable in JBI meability (Table 1). This is consistent with reports of tissue trans- hepatoma cell cultures immediately after exposure to CP, but the glutaminase activation during hepatocyte apoptosis, which results in maximum levels observed at any one time point were not seen until the cross-linking of membrane proteins (32). 14-16 h after drug addition and reflected, at most, \% of the cells The rate of apoptosis in JB1 cells was dependent on the dose of CP present at drug addition. The small numbers of apoptotic cells present and the time elapsed after drug removal (Fig. 4). This presumably at any one point and their subsequent rapid disintegration made it reflected the time taken for cellular uptake of CP, drug-target inter necessary to collect and quantitate apoptotic cells every 2 h. JB1 action, the sensing of cellular damage, and triggering of the apoptotic hepatoma cells underwent apoptosis following CP treatment at con process. Although such a small percentage of cells underwent apop centrations similar to those required to induce this process in CHO tosis at each time point studied, the cells received the apoptotic stim cells (7). One to four % of cells remaining as an attached monolayer ulus during the 2-h drug exposure period, and by 72 h about 75% had were apoptotic compared to 62% for the detached cells. In accordance undergone apoptosis. In accordance with the studies of the effects of

Table 2 Flow cytometric analysis of cell cycle phase distribution in JBI hepatoma cells JBI cells were exposed to CP (50 UM,2-h exposure) with or without CX (1 UM,continuous exposure) or CX alone.

+CX% Time(h)06122448%G,84.6 S8.3 S8.3 G2/M6.3 S7.3 S7.6 G2/M6.0 Â±2.385.6 Â±0.78.7 Â±1.65.6 Â±1.878.3 Â±1.311.5 Â±1.07.2 Â±1.587.1 Â±1.46.3 Â±I.I4.6 Â±1.485.3 Â±0.78.4 Â±0.76.4 Â±1.484.8 Â±1.28.7 Â±1.66.5 Â±4.881.1 Â±2.510.1 Â±3.07.5 Â±1.286.5 Â±2.07.3 Â±0.65.2 Â±0.685.4 Â±1.57.3 Â±1.85.4 Â±0.685.2 Â±1.68.5 Â±0.45.9 Â±5.741.5 Â±1.420.3 Â±3.936.2 Â±1.784.2 Â±1.86.8 Â±0.57.0 Â±2.184.6 Â±1.08.2 Â±1.46.9 Â±2.485.2 Â±1.89.0 Â±1.55.6 Â±1.929.2 Â±2.317.4 Â±2.751.1 Â±4.188.4 Â±2.35.3 Â±1.46.2 Â±1.385.1 Â±1.47.9 Â±1.26.1 Â±1.9Untreated%Â±0.7%G2/M7.0Â±1.3%G,85.2 Â±7.0CP% Â±2.4% Â±5.4%G,84.6 Â±1.0CP Â±0.4%G2/M6.7Â±1.1%G,84.0 Â±1.6CX% Â±1.4% Â±1.2

Table 3 Flow cytometric analysis of cell cycle phase distribution in immature rat thymocyles Immature thymocytes were exposed to MP (H)~5 M. continuous exposure) or CP (50 UJM,2-h exposure). The percentage of apoptotic cells was detected as a hypodiploid peak below that of G0/i coinciding with the appearance of AO-positive cells in culture.

apoptoticcellsMP0 Time (h)0 S3.5 G,91.6 S3.5 G2fM5.0 G,9 6 Â±0.6 Â±0.5 Â±0.7 Â±0.6 Â±0.5 Â±0.7 1.6 Â±0.6 Â±0.5 Â±0.7 1224%G,91.81.3 Â±3.2 4.5 Â±1.4 5.2 Â±0.6 62.8 Â±2.3 4.0 Â±0.3 4.3 Â±0.3 83.6 Â±3.2 3.2 Â±0.7 3.58 Â±0.42 9.7Â±1.224.0 28.9 Â±1.8 9.6 Â±1.7 67.7 Â±1.3Untreated%3.8 Â±0.2%G2/M5.04.4 Â±0.3% 29.2 Â±2.9MP%3.9 Â±0.9<7r 5.9 Â±0.9<7r 64.5 Â±1.7CP%s3.53.4 Â±0.4%G2/M5.05.1 Â±0.7No.Control0Â±0.8of 6 1.0 Â±4.2CP0 27.0 Â±3.0

2137

Table 4 Effect of continuous exposure lo CX (I UM)alone or in combination with a 2-h exposure to 50 fiu CP on JBÃŒhepatoma cell DNA synthesis and protein synthesis using radioactive precursor incorporation Results shown are the mean value Â±SD from 3 separate repeat experiments each containing triplicate samples. [mÂ«'iAv/-1H]Thymidineincorporation [15S]Methionine incorporation (expressed as % of control value) (expressed as % of control value) Time after initial exposure(h)1224 +CP41.8 +CP56.8 Â±3.2 Â±5.7 Â±3.8 Â±7.3 Â±9.0 Â±3.1 50.2 Â±1.4 59.7 Â±11.4 36.5 Â±3.3 65.4 Â±10.0 109.6 Â±9.5 44.3 Â±1.3 48CX54.4 40.2 Â±5.8CP81.7 61.7 Â±10.0CX 15.5 Â±7.3CX73.1 58.2 Â±12.8CP106.7 102.8 Â±5.2CX 40.7 Â±5.5

an intrinsic "default" program. Alternatively, since CX halts passage 12 -i through the cell cycle, cells with platinated DNA may not approach a critical cycle check point which prevents further proliferation and instead couples DNA damage to the engagement of apoptosis. How ever, the effects of CX upon the modulation of apoptosis are clearly complex, and in some cell types CX induces apoptosis (14). Recently Chang et al. (35) demonstrated that diphtheria toxin but not CX (both potent inhibitors of protein synthesis) induced internucleosomal cleavage and cell death. Moreover, Sandvig and van Deurs (36) showed that CX protected against apoptosis induced by ricin (also a protein synthesis inhibitor) in Madin-Darby canine kidney cells (36). We have not been able to halt JB 1 cells in cycle long enough to test whether CP is ineffective at inducing apoptosis in these cells during quiescence. However, we have shown that CP did not induce levels of apoptosis above those seen for untreated controls in immature rat thymocytes which do undergo apoptosis in response to glucocorticoids (9), toxins such as tetrachlorodibenzo-p-dioxin (37), inhibitors of topoisomerases I (23) and II (10), and irradiation (11). That thy mocytes readily undergo apoptosis is clear from rapid induction of the process by MP and the relatively high levels of spontaneous ap optosis seen by 36 h in untreated controls seen in Fig. 5. Previous re ports (22, 23) and our FCM data show that >90% of these cells re Time after exposure to CP (hours) side in the Go/G, phase of the cell cycle. One of several possible explanations of these data is that CP does not induce apoptosis in Fig. 7. Inhibition of CP-induced apoptosis in JBI hepatoma cells by CX (l UM).The number of apoptotic cells collected above the CP-treated monolayer was determined by nonproliferating cells, although more detailed experiments are obvi acridine orange staining. CX was either maintained throughout the experiment or removed ously required to thoroughly test this hypothesis. Experiments are at various lime points after CP exposure. â€¢¿untreated cells; â€¢¿50UMCP for 2 h with no now under way to quantitate cellular levels of CP, the type and CX; O, 50 UMCP for 2 h with continuous CX; D, 50 UMCP for 2 h with CX removal at 4 h; A, 50 UMCP for 2 h with CX removal at 12 h. amount of DNA adducts formed, and the resultant number of DNA strand breaks in cycling compared to quiescent thymocytes. Such studies will shed light on whether the drug-target interaction itself CP on CHO cells described by Eastman (19), CP was shown to cause predicts the degree of apoptosis or whether events downstream of cell accumulation in the S and G2/M phases of the cell cycle in JBI DNA platination are important determinants of the cellular response. cultures. Concurrent exposure of JB1 hepatoma CP-treated cells with Interestingly, a recent report by Colombe! et al. (38) concluded that CX prevented accumulation in the S and G2/M phases of the cell cycle quiescent epithelial prostate cells do undergo apoptosis following and completely suppressed the engagement of apoptosis. That CX testosterone depletion but that apoptosis occurred after exit from the prevented uptake of CP into JBI cells seems unlikely since rapid GO state into a defective cell cycle. If platinated DNA must be apoptosis followed removal of the protein synthesis inhibitor several "sensed" at a cell cycle check point, presumably at the G2/M bound hours after the removal of CP (Fig. 7). The prevention of cell cycle ary, and this "sensing" is required to trigger apoptotic cell death, there events and induction of apoptosis are in agreement with the findings are important ramifications for CP treatment of only slowly prolifer of Kung et al. (33), who showed that the cytotoxicity of vincristine ating or quiescent tumour cells in vivo. and aphidocolin, although not identified as resulting in apoptosis, was similarly abolished by CX. They concluded that cell death was evoked, not by the biochemical lesion per se, but by the dissociation ACKNOWLEDGMENTS of normally integrated cell cycle events. We have previously sug We thank John A. Hickman for his constructive criticism of the manuscript, gested that the drug-target interaction is only the first step in the Robert Legg for the supply of JBI hepatoma cells, and Jaleel Myan for his commitment of a cell to apoptotic cell death (34). Here we suggest that assistance with fluorescence microscopy and photography. platination of DNA is not per se the event which precipitates apoptosis but that this lesion must be present at a time when the cell makes the REFERENCES decision regarding whether it will undergo mitosis. 1. Bursch, W., Oberhammer. F.. and Schulte-Hermann. R. Cell death by apoptosis and We have shown that CX protects against CP-induced apoptosis in its protective role against disease. Trends Pharmacol. Sci., 13: 245-251, 1992. cycling JBI cells. There are several hypotheses which might explain 2. Bursch, W.. Paffe, S., Putz, B., Barthel, G., and Schulle-Hermann, R. Determination this effect. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1993 American Association for Cancer Research. Effects of Cisplatin on the Induction of Apoptosis in Proliferating Hepatoma Cells and Nonproliferating Immature Thymocytes

Dyfed L. Evans and Caroline Dive

Cancer Res 1993;53:2133-2139.

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