2122.Full.Pdf

[CANCER RESEARCH 55, 2122-2128, May 15, 1995] Drug-induced Apoptosis Is Not Necessarily Dependent on Macromolecular Synthesis or Proliferation in the p53-negative Human Prostate Cancer Cell Line PC-31

Markus M. Borner,2 Charles E. Myers, Oliver Sartor, Yoshitatsu Sei, Toshiyuki Toko, Jane B. Trepel, and Erasmus Schneider Clinical Pharmacology Branch Â¡M.M. B.. C. E. M., O. S.. T. T.. J. B. TJ, and Medicine Branch [E. S.Â¡,National Cancer Institute, and Laboratory of Neuroscience IY. S.I, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland 20892

ABSTRACT as cycloheximide or actinomycin D had either no effect on cell death or even triggered apoptosis themselves (13, 22, 23). This apparent The propensity of a cell to undergo apoptosis has been proposed to be contradiction has been explained with the cell type-specific presence a determinant for chemotherapy sensitivity that is not directly dependent of different sets of predominant regulatory proteins, which either on specific drug-target interactions. Androgen-independent prostate can activate or inhibit apoptosis (2, 6, 8, 23). cer is typically refractory to cytotoxic drugs, and we tested whether this is due to a loss of the ability to undergo apoptosis. Exposure of the hormone- Prostate carcinoma is the second leading cause of cancer deaths in insensitive and p53-negative human prostate carcinoma cell line PC-3 to North American men (27). While more than 80% of the tumors 22 UMcisplatin, l UMcamptothecin, 10 UMtenoposide, 135 IIMvincristine, initially respond to hormonal interventions, most relapse and are then or 10 UM lovastatin for 72 h caused cell death, internucleosomal DNA refractory to further hormonal manipulations and cytotoxic drugs (28). fragmentation, and morphological changes typical for apoptosis. One UM It is well established that prostate regression after androgen with cycloheximide prevented anticancer drug-induced apoptosis, whereas drawal occurs by apoptosis (29-32), and high concentrations of high concentration (1 HIM) of cycloheximide alone induced apoptosis, fluoropyrimidines or infection with Sindbis virus have been shown to indicating that protein synthesis was not needed for these cells to undergo induce apoptosis in the androgen-independent rat prostatic carcinoma apoptosis. Since cycloheximide affected DNA synthesis and proliferation cell line AT-3 (15, 33). Results from experiments using cytotoxic of PC-3 cells, we tested whether the DNA polymerase inhibitor aphidicolin agents in human hormone-independent prostate cancer systems are could also suppress drug-induced apoptosis. In contrast to cycloheximide, aphidicolin inhibited only vincristine-induced apoptosis. Cycloheximide scarce (34), raising the question whether the relative drug insensitivity prevented drug-induced changes in cell cycle distribution except for vin of advanced prostate cancer is due to the loss of the potential to cristine, while aphidicolin led to an accumulation of cells at the G,-S undergo apoptosis. border independent of the drug used. These data indicate that macromo- Therefore, we tested whether anticancer drugs with different mech lecular synthesis, active cell cycling, and p53 expression are not required anisms of action can induce apoptosis in PC-3, a cell line with for apoptosis to proceed in this system. biological characteristics comparable to hormone-independent human prostate adenocarcinoma (35, 36). PC-3 cells are deficient in their p53 INTRODUCTION expression (37, 38), a tumor suppressor gene critically involved in The term "apoptosis" has been coined to distinguish active cell apoptosis induced by chemotherapeutic agents (39, 40). The following aspects of drug-induced cell death were examined: (a) induction of death, where cells participate in their own demise from passive Â¡nternucleosomal DNA fragmentation; (b) effect of the macromolec necrotic death, where severe environmental perturbations lead to membrane damage, loss of osmoregulation, and cell lysis (1-3). ular synthesis inhibitors cycloheximide and aphidicolin; (c) cell cycle phase specificity; and (d) morphology. The results of these studies Typically, apoptosis is characterized by distinct morphological show that all the anticancer drugs tested were able to induce apoptosis changes and internucleosomal DNA fragmentation (4, 5). Apoptosis in PC-3 cells. Furthermore, despite the fact that cycloheximide sup occurs in a variety of physiological situations, where an organism has pressed drug-induced cell death, neither macromolecular synthesis nor to remove unwanted cells (1-3, 6-8). However, apoptosis can also be active cell cycling were required for apoptosis to proceed in this induced by pathological stimuli, and most anticancer drugs have been shown to induce apoptotic features in normal and tumor cells (9-22). system. This suggests that the components of the apoptotic machinery were in place and ready to go upon stimulation. This suggests that a variety of biochemical events can trigger a conserved apoptotic machinery which is responsible for uniform morphological changes, DNA fragmentation, and cell death (2, 8, 23). MATERIALS AND METHODS The components of this putative apoptotic machinery have not been identified to date, and different biochemical pathways have been Materials. Agarose, RPMI 1640, fetal bovine serum, saturated phenol, 10X TAE buffer, and a 100-base pair DNA ladder marker were obtained from described to connect the inducing stimulus to the final apoptotic event GIBCO (Grand Island, NY). Glycogen and proteinase K were purchased (2, 6, 8). In view of potential pharmacological interventions, it is from Boehringer Mannheim (Indianapolis, IN). Chromomycin A, campto important to identify and distinguish between steps that are part of a thecin, cisplatin, cycloheximide, DMSO, pancreas RNase A, sulforhodamine specific signal transduction pathway and steps that are part of the B, Triton X-100, and vincristine were from Sigma Chemical Co. (St. Louis, conserved apoptotic machinery. Apoptosis has been shown to be MO). Tenoposide was a kind gift of Bristol-Myers Pharmaceutical Co. (Syr dependent on macromolecular synthesis, supporting the notion of an acuse, NY), and lovastatin was a gift of Merck, Sharp and Dohme Research active participation of a cell in its own demise (6, 9, 17, 19, 24-26). Pharmaceuticals (Rahway, NJ). [mci/iv/-'H]thymidine and [35S]methionine However, in other systems, macromolecular synthesis blockers such were from New England Nuclear (Wilmington, DE). Cell Culture and Drug Exposure. Human PC-3 cells (obtained from American Type Culture Collection, Rockville, MD) were grown at 37Â°Cin Received 12/5/94; accepted 3/15/95. RPMI 1640 containing 10% (v/v) heat-inactivated fetal bovine serum, 100 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with units/ml penicillin G, 100 fig/ml streptomycin, and 2 mM glutamine in an 18 U.S.C. Section 1734 solely to indicate this fact. atmosphere containing 5% (v/v) CO2. Under these conditions, the population 1This research was supported by grants from the Swiss Cancer League (Bern Section) doubling time was about 24 h. Cultures were regularly tested and found free of and the Swiss National Science Foundation (to M. M. B.). 2 To whom requests for reprints should be addressed, at Institute of Medical Oncology, Mycoplasma contamination. For drug exposure experiments, cells were grown University of Bern, Inselspital, CH-3010 Bern, Switzerland. to confluence in 175-cm2 culture flasks, passaged by trypsinization to twenty 2122

Fig. 1. Agarose gel electrophoresis of DNA from drug-treated PC-3 cells. Treatment with various drugs at the concentralions given in Table 1 for 24 h (Lane /), 48 h (Lane 2), 72 h (Lane 3), 96 h (Lane 4), 120 h (Lane 5), 144 h (Lane 6). A, lovastatin; B, vincristine; C, cisplatin. The pictures shown are representative of at least three independent experiments.

100 x 20 mm-style culture dishes, and grown for 48 h before adding the Table 1 Cytotoxicity of different anticancer drugs in PC-3 cells respective drug. At this point, each dish typically contained 1-2 X IO6 cells in DrugCisplatinCamptothecinTenoposideVincristincLovastatinModeactionAlkylatingof survival"32 logarithmic growth phase. Stock solutions were prepared in DMSO with AgentTopoisomerase UM1 Â±5*16Â±232 aphidicolin (11.8 HIM),tenoposide (10 mM), and camptothecin (1 mM) in PBS with inhibitorTopoisomerase1 UM10 cycloheximide (1 and 100 mM) and cisplatin (3.3 mM) in methanol with vincristin inhibitorAntimitoticII Â¿IM135 Â±342 (1.35 mM). Lovastatin (10 mM) was converted from the inactive lactone form into agentMevalonate nM10 Â±633*2 synthesis inhibitorConcentration22M"% its active dihydroxy open acid form as described (41). The solvents alone had no ' Seventy-two h exposure, measured with sulforhodamin B assay (42). cytotoxic effect at the concentrations used for the experiments. Mean Â±SDderived from six parallel experiments. Cytotoxicity Assays. To evaluate the number of live and dead cells, cells were stained with trypan blue and counted on a hemocytometer. Cells which showed trypan blue uptake were interpreted as nonviable, and the results were washed once in ice-cold PBS. The cells were fixed in cold 40% ethanol and expressed as percentage of the total cell number. To determine the relative growth stored at 4Â°C.At least 24 h before analysis, the fixed cells were washed once inhibitory activity of the drugs used, PC-3 cells were plated into 96-well microtiter with PBS and resuspended in 1 ml PBS containing 20 /ag/ml chromomycin A3 plates and treated with different concentrations of each drug for 72 h. Relative and 3 mg/ml MgCl2. The fluorescence of the samples was analyzed as toxicity was determined by the sulforhodamin B assay as described (42). single-parameter frequency histograms using a FACScan (Becton Dickinson, Analysis of DNA Integrity. To determine whether DNA fragmentation Mountain View, CA). occurred following treatment with various drugs, all cells were collected and Measurement of DNA Synthesis and Protein Synthesis. In order to the DNA extracted by a modification of the method originally described by measure DNA synthesis, 5000 cells/well were seeded into 96-well plates Arends el al. (43) and Wyllie (44). Attached cells were detached from the and grown 24 h before adding cycloheximide (1 JXMor 1 mM), aphidicolin culture dish with 5 mM EDTA, pooled with detached cells, spun down, and (11.8 /4.M), or complete medium alone. Cells were labeled with l /UM lysed in 5 mM Tris (pH 7.4), 5 mM EDTA, and 0.5% Triton X-100 for 2 h on [mer/iv/-1H]thymidine for 4 h. [35S]methionine was used instead of thymi- ice. The lysate was centrifuged at 27,000 x g for 20 min. The supernatant was incubated with 200 fig/ml proteinase K for l h at 50Â°Cand extracted with dine for measurement of protein synthesis. Cells were harvested on glass phenol/chloroform; then the DNA was precipitated overnight at â€”20Â°Cin 2 fiber filters using a Micromate 196 harvester and measured on a Matrix 9600 direct ÃŸcounter (Packard Instrument Company, Downers Grove, IL). volumes of ethanol and 0.13 M NaCl with 20 fig glycogen. After treatment with 1 mg/ml boiled bovine pancreatic RNase A for l h at 50Â°C,the DNA was loaded onto a 2% (w/v) horizontal agarose gel containing 0.3 fig/ml ethidium RESULTS bromide and run in IX TAE buffer at 2.5 V/cm. Gels were photographed under UV light with Polaroid 57 type film. DNA extracted from equal numbers of Induction of Internucleosomal DNA Fragmentation and Cell pretreated cells was loaded, which allowed us to semiquantitatively estimate Death in PC-3 Cells. Our preliminary experiments showed that a the relative amount of DNA fragmentation. variety of anticancer drugs induced internucleosomal DNA fragmen For quantification of DNA fragmentation, cells were labeled with \meth\l- tation in PC-3 cells. Lovastatin, a mevalonate synthesis inhibitor (41, 3H]thymidine to a final concentration of 1 /iCi/ml for 24 h prior to incubation 45, 46), was included in the drug panel because of its pronounced in with the respective anticancer agent. At the indicated times, attached and vitro activity against prostatic carcinoma cell lines.3 Compounds with detached cells were pooled and washed three times with ice-cold PBS, lysed, different cellular targets were selected, and the concentrations were and centrifuged as described above to separate high-molecular-weight and determined, at which the most pronounced DNA laddering could be low-molecular-weight (fragmented) DNA. The radioactivity of the supernatant (low-molecular-weight DNA) and the pellet (high-molecular-weight DNA) induced after 72 h drug exposure. These concentrations, which led to were quantified separately by liquid scintillation counting. The percentage of 58 to 84% growth inhibition, were used for all further experiments (Table 1). Continuous drug exposure for 48-72 h was necessary to fragmented DNA was determined by applying the formula: obtain these effects (Fig. 1) as shown by the dramatic increase in DNA Radioactivity of supernatant (cpm) % fragmented DNA = X 100 fragmentation at 72 h. At this time, quantitation of DNA fragmenta Radioactivity of supernatant and pellet tion revealed that less than 2.5% of total DNA from untreated cells Measurement of Cell Cycle Distribution. Attached cells were brought into suspension using 5 mM EDTA, pelleted together with detached cells, and 1J. Trepel, manuscript in preparation. 2123

was low-molecular-weight DNA, whereas drug treatment increased 125 this fraction to 7-27% (Table 2). Fig. 2A shows that lovastatin induced the greatest amount of DNA laddering, although all drugs 100- caused a high degree of cell death. Interestingly, with our method of DNA preparation, in lanes of samples without significant DNA frag o mentation (i.e., controls), often no DNA was detectable, even in the o loading wells. However, appropriate loading could be verified by u>Â» incomplete RNA digestion (Fig. 1A, bottom). DNA fragmentation was detected only in detached cells as described (7, 19). Attached cells were >90% viable. s Effects of Macromolecular Synthesis Inhibition on Drug- K induced Apoptosis. The role of protein synthesis in this system was tested by exposing PC-3 cells for 72 h to the respective anticancer agent alone or in the presence of l /J.Mcycloheximide. Fig. 3 shows that the cytotoxic action of all drugs was reduced by cycloheximide, with tenoposide being the most affected and camptothecin being the least affected drug. Cotreatment with cycloheximide also resulted in a higher number of viable cells and less cells in A,, phase of the cell cycle at 72 h compared to treatment with the respective anticancer Fig. 3. Effect of cycloheximide and aphidicolin on drug-induced cytotoxicity. PC-3 cells were treated with the indicated drugs for 72 h at the concentrations given in Table drug alone (data shown below). Cycloheximide prevented cell death I. Drugs were either given alone (D) or together with continuous treatment of 11.8 JIM aphidicolin (D) or 1 /Â¿Mcycloheximide (â€¢)added 4 h before the respective drug. Viability was determined by trypan blue uptake. Effect of cotreatment with cycloheximide or Table 2 DNA fragmentation in PC-3 cells after treatment with various cytoloxic drugs aphidicolin on the effect of the indicated drug is shown as relative cytotoxicity. Cytotox icity of the respective drug alone is 100%. Relative cylotoxicity = (mean cytotoxicity of % DNA fragmentation anticanccr agent + macromolecular synthesis inhibitor/mean cytotoxicity of anticancer h"0.6* agent alone) X 100, determined from triplicates of a representative of at least three CytoloxicdrugsControlLovastatin'VincristineCisplatinCamptolhecin48 h2.4 experiments. All samples are from the same experiment. Â±0.15.7 Â±0.217.4 Â±1.81.2 Â±2.87.0 +0.62.0 Â±0.511.0 Â±0.58.2 Â±0.527.5 and DNA laddering, even when added 12 h after the anticancer drugs Â±1.272 Â±1.7 '' Duration of drug exposure. (Fig. 4), indicating that its principal action is not by interfering with * Percentage of DNA fragmentation (mean Â±SD) assessed as described in "MalcriÃ¡is drug uptake into the cells. Internucleosomal DNA fragmentation (Fig. and Methods" calculated from triplicates of a representative experiment. 2A) and cell death (Fig. 3) were affected by cycloheximide in a ' Concentrations of anticancer drugs as given in Table 1. parallel fashion. This suggests that cells mainly died by apoptosis in B

! i i i

Control V

AphidicolinCycloheximideM--+- -+- -+- -+- -+- -+- + -- + -- + -- + -M- + -- + -

Fig. 2. Agarose gel electrophoresis of DNA from drug-treated PC-3 cells. A. treatment with indicated drugs at the concentrations given in Table 1 for 72 h. Drugs were given alone or together with either continuous treatment of cycloheximide ( 1 ^IM)or aphidicolin ( 11.8 ;ÃŒM)added4 h before the respective drug. Marker. 100-base pair ladder marker. Control. untreated cells, same initial cell number as treated samples. B, cycloheximide, 1 mM for 72 h. The pictures shown are representative of at least three independent experiments. 2124

cycle) did not change the outcome of the experiment (data not shown). 1 A 2 Table 3 summarizes the effects of cycloheximide and aphidicoiin on protein and DNA synthesis. DNA synthesis inhibition was maximal within 4 h. Cell Cycle Distribution and Drug-induced Apoptosis. A variety of antitumor agents preferentially induce apoptosis in specific cell cycle phases (19, 48-50). Therefore, we tested whether cyclohexi mide and aphidicoiin differentially affect drug-induced changes in cell cycle distribution (Table 4). Exposure to aphidicoiin alone resulted in an increase from 48 to 70% cells at the G,-S border, while cyclohex-

2000

-Oâ€” Control

-Oâ€” Cycloheximide Â» 1500 a "Oâ€” Aphidicoiin

1000

500

Fig. 4. Agarose gel electrophoresis of DNA from PC-3 cells treated with lovastatin (10 /AM)for 72 h alone (A) or together with continuous treatment of cycloheximide (1 /AM) added 4 h before lovastatin (Lane I) or 12 h after lovastatin (Lane 2). The picture shown 20 40 60 80 is representative of at least three independent experiments. Time (h)

Fig. 5. Effect of cycloheximide and aphidicoiin on growth of PC-3 cells for 72 h. Table 3 Effect of aphidicoiin and cycloheximide on DNA and protein synthesis Viable cells were counted at 24-h intervals with a hemocytometer using trypan blue. in PC-3 cells Cycloheximide (1 /AM),aphidicoiin (11.8 JAM),continuous exposure. Mean Â±SD (three DNA synthesis" synthesis" parallel samples) of a representative of at least three experiments. value)Exposure (% of control value)Aphidicoiin11.8(% of control

Table 4 Effect of cycloheximide and aphidicoiin on drug-induced changes in cell cycle phase distribution after 72-h treatment in PC-3 cells Duration4 (Â¿M3* /'M18 MIM3 JIM89 /AM41341 nr.l17 cyclephaseS13.1 h 24 hAphidicoiin11.81Cycloheximide1191 1Prolein 56Cycloheximide1 21 TreatmentControlCycloheximide +M39.2 " Determined by measuring radioactive precursor incorporation. Â±0.441.1 +1.014.6 h (Mean cpm of six parallel drug exposure experiments/mean of six parallel control Â±1.444.4 alone Â±0.7 Â±0.1 Â±0.8 experiments) X 100. AphidicoiinaloneLovastatin''Alone+Cycloheximidc'70.4 Â±0.670.2 9.1Â±0.34.5 20.5 Â±0.725.3

+1.445.8 this system. Interestingly, a 1000-fold higher concentration (1 ITIM)of Â±1.15.9 Â±0.248.3 Â±0.7 + 0.3 Â±0.5 cycloheximide alone for 72 h also induced internucleosomal DNA +Aphidicolin'VincristineAlone+Cycloheximide68.1 +0.512.2 8.3 Â±0.46.4 23.6 Â±0.581. laddering (Fig. 2B) and cell death. Thus, protein synthesis appeared not to be an absolute requirement for apoptosis in our system. +0.28.2 +0.55.9 4Â±0.585.9 Â±0.4 Â±0.4 + 0.1 Since cycloheximide also inhibited DNA synthesis (Table 3) and -t-AphidicolinTenoposide 60.0 +0.938.8 11.6 +0.618.1 28.4 Â±0.243.1 cell proliferation (Fig. 5), we tested whether this effect was respon sible for the prevention of drug-induced apoptosis. PC-3 cells were Aloneâ€¢fCycloheximide +1.042.6 +0.814.2 +0.443.2 treated with the various anticancer agents alone or in combination Â±0.3 Â±0.3 + 0.1 +AphidicolinCamptothecin 60.9 Â±0.758.1 11.1+0.312.6 28.1Â±0.929.2 with the DNA polymerase a inhibitor aphidicoiin (47), which was added 4 h before the anticancer agent. Aphidicoiin blocked DNA Alone+Cycloheximide Â±0.349.9 Â±0.110.7 +0.239.5 synthesis (Table 3) and proliferation (Fig. 5), even more efficiently Â±0.9 Â±0.4 Â±0.9 than cycloheximide. However, in contrast to the effect of cyclohexi +AphidicolinCisplatin 58.1Â±0.763.3 12.2Â±0.113.1 29.8 +0.623.6 mide, aphidicoiin prevented only the induction of apoptosis by vin- Alone+Cycloheximide Â±0.649.0 Â±0.111.0 Â±0.740.0 cristine and to a lesser degree by lovastatin (Figs. 2A and 3). The Â±0.6 Â±0.3 Â±0.4 cytotoxicity of the other agents was not significantly affected, and the tAphidicolinGÃŒ47.6" 63.2 Â±0.6Cell 11.6 Â±0.8G2 25.2 Â±0.8 slightly fainter DNA bands on agarose electrophoresis compared to " Percentage of cells (mean Â±SD) in respective cell cycle phase calculated from at least three experiments. drug treatment alone (Fig. 2/4) may reflect the lower cell number h Concentrations of anticancer drugs as given in Table 1. caused by the additive effect of aphidicoiin and the respective drug. ' Cycloheximide ( 1 /AM)or aphidicoiin ( 11.8 /IM) were added to the cells 4 h before the Pretreatment with aphidicoiin for 24 h (time to complete one cell respective anticancer drug. 2125

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1995 American Association for Cancer Research. DRUG-INDUCED APOPTOSIS IN HUMAN PROSTATE CANCER imide did not notably affect cell cycle distribution. Cisplatin, lovastatin, and camptothecin alone led to an accumulation of cells in G, vincristine led to a pronounced G2-M arrest, and tenoposide led to an accumulation of cells in S and G2-M phases. Low-dose cycloheximide prevented the cell cycle effect of all drugs except vincristine, despite the fact that it also suppressed apoptosis induced by this drug. Aphidi- colin led, in combination with all drugs, to an accumulation of cells in the G, phase, and it was not possible to distinguish the effect of aphidicolin from the effect of the G, arresting anticancer drugs alone. Drug treatment led to an accumulation of cells with a DNA content lower than that of G, cells (A,,), which have been described to represent apoptotic cells (51). The percentage of A,, cells did corre spond well to the cytotoxicity data shown in Fig. 3. However, the amount of A,, cells did not correlate with the loss of cells from another single cell cycle compartment, suggesting that, for a given drug, cells die in multiple phases of the cell cycle (Table 5). Analysis of Morphological Changes by Transmission Electron Microscopy. Apoptosis was originally defined by morphological characteristics, including condensation and margination of chromatin, contraction of the cytoplasm, and cell shrinkage, while mitochondria are still preserved (1). Therefore, we assessed by electron microscopy â€¢Ã•*- the morphological changes of PC-3 cells after a 72-h exposure to *â€¢- various anticancer agents. Aside from cells with the classical combi nation of condensed chromatin and intact mitochondria (Fig. 6B), many cells showed mixed features of apoptosis and necrosis (Figs. 6, C and D). It is known that cells maintain the typical apoptotic morphology for only a short period of time and then undergo "sec Fig. 6. Electron micrographs of detached PC-3 cells showing control, untreated PC-3 cells (A), typical apoptotic morphology (B), and secondary necrosis (C and D). ondary necrosis" if they escape phagocytosis (3, 7). A, X 3250; B, X 5000; C X 1750;D. X 2000.

DISCUSSION We were able to induce the typical hallmarks of apoptosis (4, 56) A wide variety of cytotoxic agents with different intracellular with alkylating agents and agents that interact with topoisomerases I targets can induce the uniform phenotype of apoptosis. This implies and II, microtubule apparatus assembly, and mevalonate synthesis that the cytotoxic activity of anticancer drugs is not solely dependent (45, 57). Although the drugs were used at approximately equitoxic on specific drug-target interactions but also on the activity of an concentrations, they induced various degrees of DNA fragmentation, apoptotic machinery (8, 18, 21, 52). The observation that the expres with lovastatin being the most potent inducer. A possible explanation sion of genes such as c-myc, p53, or bcl-2 is a critical determinant for for this finding is that the other drugs induced more nonapoptotic the threshold of a cell to undergo apoptosis (39, 40, 53-55) supports deaths than lovastatin. Alternatively, it is conceivable that some of the this view. This has led to the interesting concept that the intrinsic drugs increased membrane permeability, resulting in the loss of small propensity to undergo apoptosis is a general determinant for chemo DNA fragments (51). Electron microscopy did not help to definitely therapy sensitivity and could represent a target for pharmacological answer this question because the morphological features of the dying modulation (8, 18, 21, 39, 40, 52). For successful pharmacological cells transformed rapidly from apoptosis into secondary necrosis. modulation of chemotherapy sensitivity, however, it is important to be Thus, electron microscopy has to be interpreted with caution, since it able to differentiate between specific drug-target interactions leading might considerably underestimate the amount of apoptosis if only to the induction of apoptosis and biochemical events that are part of typical cells as shown in Fig. 6B are counted as apoptotic. the apoptotic machinery itself. This led us to study the effect of the Interestingly, the protein synthesis inhibitor cycloheximide exerted macromolecular synthesis inhibitors cycloheximide and aphidico an ambiguous effect on apoptosis in PC-3 cells. At low concentration lin on apoptosis induced by anticancer agents with different intracel (1 JU.M),drug-induced apoptosis was prevented, while at high concen lular targets in the human hormone-insensitive prostate carcinoma cell tration (1 min), cycloheximide itself induced apoptotic cell death. line PC-3. Apoptosis has been described before to be differentially affected by macromolecular synthesis inhibition, depending on the cell system under investigation, a fact which has been attributed to the presence of Table 5 Percentage <>fAHcells after treatment of PC-3 cells with various cytoloxic drugs Â±cycloheximide or aphidicolin different sets of regulatory proteins either stimulating or inhibiting apoptosis (2, 6, 8, 23). We show here inhibition and induction of CytotoxicDrugControlLovastatin'VincristineTenoposide apoptosis by cycloheximide in the same cell line. This observation Â±0.318.3 Â±0.511.6 Â±0.225.5 Â±0.310.7 Â±0.612.3 suggests that the phenotype of apoptosis, i.e., DNA fragmentation and Â±0.529.4 Â±0.39.8 Â±0.428.4 morphology, is not critically dependent on protein synthesis in PC-3 Â±0.7 Â±0.2 Â±0.5 cells and that the endonuclease involved in DNA fragmentation is Cisplatin 35.5 Â±0.3 19.6 Â±0.3 39.8 Â±0.9 CamptothecinAJone5.2*40.5 Â±0.4+Cycloheximide"3.710.15.023.4 Â±0.4+Aphidicolin"7.352.7 Â±0.5 already in place. This is supported by our recent finding that the "Cycloheximide (l /IM)or aphidicolin (11.8 U.M)wereadded to the cells 4 h before nonionic detergent Triton X-100 induces protein synthesis independ the respective anticancer drug. ent apoptosis in PC-3 cells within less than l h (58). Therefore, we b Percentageof cells (mean Â±SD)in A,, phase calculated from at least three experi tested alternative hypotheses to explain the interference of cyclohex ments. ' Concentrations of anticancer drugs as given in Table 1. imide at low concentration with drug-induced apoptosis. 2126

In agreement with others (19, 59) we have found cycloheximide to but is rather involved in setting the threshold for apoptosis induction inhibit DNA synthesis, thus affecting cell cycle progression and directly or by affecting the transcription of other regulatory genes of proliferation. The relevance of this mechanism for the inhibition of apoptosis (39, 68). drug-induced apoptosis was tested by comparing the effect of cyclo heximide with aphidicolin, an even more potent inhibitor of DNA ACKNOWLEDGMENTS synthesis and proliferation in PC-3 cells. While cycloheximide re duced the cytotoxicity and the induction of internucleosomal DNA We thank Stefen Boehme and Catherine McLellan for their helpful fragmentation for all drugs tested, aphidicolin had this effect only on suggestions. vincristine and to a much lesser extent on lovastatin. Vincristine blocks mitosis by interfering with microtubule apparatus assembly REFERENCES (57), and it is conceivable that aphidicolin simply prevents the pro 1. Kerr, J. F. R.. Wyllie, A. H., and Curric, A. R. Apoptosis: a basic biological gression of the cells into the cell cycle phase, which is most sensitive phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer, 26: to the action of vincristine. Cisplatin, tenoposide, camptothecin, and 239-257, 1972. 2. Vaux, D. L. Towards an understanding of the molecular mechanism of physiological lovastatin alone, on the other hand, led to an accumulation of cells in cell death. Proc. Nati. Acad. Sci. USA, 90: 786-789, 1993. G!, suggesting drug action in this cell cycle compartment and this 3. Wyllie, A. H., Kerr J. F. R., and Currie, A. R. Cell death: the significance of process was favored rather than inhibited by aphidicolin. In addition, apoptosis. Int. Rev. Cytol., 68: 251-306, 1980. 4. Alles, A., Alley. K., Barrett, J. O. Buttyan, R.. Columbano, A., Cope, F. O., Copelan. it is possible that aphidicolin increased the cellular susceptibility to E. A., and Warner, H. R. Apoptosis: a general comment. FASEB J., 5: 2127-2128, some of these drugs by interfering with DNA repair (57). These 1991. 5. Wyllie, A. H., Arends, M. J.. Morris. R. G.. Walker. S. W., and Evan, G. The results support the notion that the position in the cell cycle is impor apoptosis endonuclease and its regulation. Semin. Immunol., 4: 389-397, 1992. tant for specific drug-target interactions to occur (57) and suggest that 6. Ellis. R. E., Yuan, J.. and Horvitz. H. R. Mechanisms and functions of cell death. apoptosis is not generally dependent on DNA synthesis or cell cycle Annu. Rev. Cell Biol., 7: 663-698, 1991. 7. Wyllie, A. H. Apoptosis and the regulation of cell numbers in normal and neoplastic progression per se. This last point could be of special importance for tissues: an overview. Cancer Metastasis Rev.. //: 95-103, 1992. the design of new therapy approaches for epithelial tumors which 8. Carson, D. A., and Ribeiro, J. M. Apoptosis and disease. Lancet, 341: 1251-1254, typically have a slow growth rate. 1993. 9. Barry, M. A., Behnke, C. A., and Eastman, A. Activation of programmed cell death Cycloheximide had a general suppressing effect on drug-induced by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem. Pharmacol.. apoptosis, despite the fact that it allowed drug-target interactions to 40: 2353-2362, 1990. occur as evidenced by the unaffected accumulation of cells in G3-M 10. Bertrand, R.. Sarang, M., Jenkin, J., Kerrigan, D., and Pommier, Y. Differential induction of secondary DNA fragmentation by topoisomerase II inhibitors in human in the case of vincristine treatment. This is in support of the concept tumor cell lines with amplified c-myc expression. Cancer Res., 51: 6280-6285, 1991. that drug-target interactions do not necessarily kill cells by themselves 11. Cotter, T. G., Lennon, S. V., Glynn, J. G., and Martin. S. J. Cell death via apoplosis and its relationship to growth: development and differentiation of both tumour and but rather trigger events, which finally lead to cell death (18, 21, 52, normal cells. Anticancer Res., 10: 1153-1160. 1990. 60-62). Furthermore, cell death, DNA fragmentation, and morpho Cotter, T. G. Induction of apoptosis in cells of the immune system by cytotoxic logical changes typical for apoptosis could be induced by 1 ITIM stimuli. Semin. Immunol., 4: 399-405, 1992. Cotter, T. G., Glynn, J. M., Echeverri, F., and Green, D. R. The induction of apoptosis cycloheximide alone, while 1 fÂ¿Mcycloheximide inhibited drug-in by chemotherapeutic agents occurs in all phases of the cell cycle. Anticancer Res., 12: duced apoptosis to an extent which varied for the different com 773-780, 1992. Lennon. S. V.. Martin, S. J.. and Cotter, T. G. Dose-dependent induction of apoptosis pounds. This suggests that protein synthesis is not needed for the in human tumour cell lines by widely diverging stimuli. Cell Prolif., 24: 203-214, apoptotic machinery itself. However, new protein synthesis appears to 1991. be required for the normal signal transduction induced by the various Kyprianou, N., and Isaacs, J. T. Thymineless death in androgen-independent prostatic cancer cells. Biochem. Biophys. Res. Commun., 165: 73-81, 1989. chemotherapeutic agents. Dyson, J. E. D., Simmons, D. M., Daniel, J., McLaughlin, J. M., Quirke, P., and Bird, Analogous to the present study, Schneider et al. (61) showed C. C. Kinetic and physical studies of cell death induced by chemotherapeutic agents cycloheximide but not aphidicolin to inhibit the cytotoxic action of the or hyperthermia. Cell Tissue Kinet., 19: 311-324, 1986. topoisomerase II drug amsacrine. However, d'Arpa et al. (49) dem Ling. Y-H., Priebe, W., and Perez-Soler, R. Apoptosis induced by anthracycline antibi otics in P388 parent and multidrug-resistant cells. Cancer Res., 53: 1845-1852, 1993. onstrated ongoing DNA synthesis to be important for the action of Dive, C., and Hickman, J. A. Drug-target interactions: only the first step in the commitment to a programmed cell death? Br. J. Cancer, 64: 192-196, 1991. topoisomerase I and II inhibitors in Chinese hamster lung fibroblasts. Evans, D. L., and Dive C. Effects of cisplatin on the induction of apoptosis in It is possible that the continuous drug treatment schedule used in our proliferating hepatoma cells and nonproliferating immature thymocytes. Cancer Res., study decreased the dependence of camptothecin and tenoposide on 53: 2133-2139, 1993. Searle, J., Lawson, T. A., Abbott, P. J., Harmon, B., and Kerr, J. F. R. An electron- DNA synthesis, as observed for the cell cycle dependence of T-cell microscope study of the mode of cell death induced by cancer-chemotherapeutic hybridoma cells to the action of actinomycin D, camptothecin, and agents in populations of proliferating normal and neoplastic cells, cell cycle, prolif aphidicolin (13). eration. J. Pathol., 116: 129-138, 1975. Eastman, A. Activation of programmed cell death by anticancer agents: cisplatin as The fact that continuous drug exposure was required to induce a model system. Cancer Cells, 2: 275-280, 1990. apoptosis in PC-3 cells might be an expression for a low propensity of Kaufmann, S. H. Induction of endonucleolytic DNA cleavage in human acute my- elogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer this cell line to undergo apoptosis. It can only be speculated at this drugs: a cautionary note. Cancer Res.. 49: 5870-5878. 1989. point that the absence of wild-type p53 in PC-3 cells was a contrib Martin. S. J. Apoptosis: suicide, execution or murder? Trends Cell Biol., 3: 141-144, uting factor, since this tumor suppressor gene has been shown to be an 1993. Martin, D. P., Schmidt, R. E., DiStefano. P. S., Lowry. O. H., Carter, J. G., and essential component of the apoptotic pathway induced by genotoxic Johnson, E. M. Inhibitors of protein synthesis and RNA synthesis prevent neuronal insult (63). Agents which are not known to induce DNA damage, such death caused by nerve growth factor deprivation. J. Cell Biol., 106: 829-844, 1988. Thakkar, N. S.. and Potten, C. S. Inhibition of doxorubicin-induced apoptosis in vivo as glucocorticoids, ionomycin, or phorbol esters, seem to be inde by 2-deoxy-D-glucose. Cancer Res., 53: 2057-2060, 1993. pendent from p53 since they readily induce apoptosis in lymphocytes Ayusawa, D., Shimizu K., Koyama H., Takeishi K., and Seno, T. Accumulation of of p53 deficient (p53~'~) mice (64-66). Castration-induced apopto DNA strand breaks during thymineless death in thymidylate synthase-negative mu sis of androgen-dependent prostatic glandular cells was also not tants of mouse FM3A cells. J. Biol. Chem., 25Â«:12448-12454, 1983. affected in p53~'~ mice (67). However, in the same system, trans Carter, H. B., and Coffey D. S. The prostate: an increasing medical problem. Prostate, 16: 39-48, 1990. formed or mitogen-activated lymphocytes undergo apoptosis if ex Isaacs, J., Schulze, H., and Coffey, D. Development of androgen resistance in prostatic cancer. In: G. Murphy, S. Khoury, I. Kuss. and C. ChÃ¢telain(eds.). Prostate posed to radiation or genotoxic anticancer drugs (64). Thus, wild-type Cancer, Part A: Research. Endocrine Treatment, and Histopathology, pp. 21-31. New p53 seems not to be an effector of DNA damage-induced apoptosis York: Alan R. Liss, Inc., 1987. 2127

29. Kyprianou, N., English, H. F., and Isaacs, J. T. Programmed cell death during 50. Sorenson, C. M., Barry, M. A., and Eastman, A. Analysis of events associated with regression of PC-82 human prostate cancer following androgen ablation. Cancer Res., cell cycle arrest at G2 phase and cell death induced by cisplatin. J. Nati. Cancer Inst., 50: 3748-3753, 1990. 82: 749-755, 1990. 30. Kyprianou, N., and Isaacs, J. T. Activation of programmed cell death in the rat ventral 51. Darzynkiewicz, Z., Bruno, S., Bino, G. D., Gorczyca, W., Hotz, M. A., Lassota, P., prostate after castration. Endocrinology, 122: 552-562, 1988. and TrÃ¡ganos, F. Features of apoptotic cells measured by flow cytometry. Cytometry, 31. Kerr, J. F. R., and Searle, J. Deletion of cells by apoptosis during castration- 13: 795-808, 1992. induced involution of the rat prostate. Virchows Arch. Abt. B Zellpathol., 13: 52. Hickman, J. A. Apoptosis induced by anticancer drugs. Cancer Metastasis Rev., 11: 87-102, 1973. 121-139, 1992. 32. Sandford, N. L., Searle, J. W., and Kerr, J. F. R. Successive waves of apoptosis in the 53. Lotem, J., and Sachs, L. Regulation by bcl-2, c-myc, and p53 of susceptibility to rat prostate after repeated withdrawal of testosterone stimulation. Pathology, 16: induction of apoptosis by heat shock and cancer chemotherapy compounds in differ 406-410, 1984. entiation-competent and -defective myeloid leukemic cells. Cell Growth & Differ., 4: 33. Levine, B., Huang, 0., Isaacs, J. T., Reed, J. C, Griffin, D. E., and Hardwick, J. M. 41-47, 1993. Conversion of lytic to persistent alphavirus infection by the bcl-2 cellular oncogene. 54. Evan, G. I., Wyllie, A. H., Gilbert, C. S., Littlewood, T. D., Land, H., Brooks, M., Nature (Lond.), 361: 739-743, 1993. Waters, C. M., Penn, L. Z., and Hancock, D. C. Induction of apoptosis in fibroblasts 34. Kyprianou, N., Bains, A., and Jacobs, S. Induction of apoptosis in androgen-inde- by c-myc protein. Cell, 69: 119-128, 1992. pendent human prostate cancer cells undergoing thymineless death. Prostate, 25: 55. Kamesaki, S., Kamesaki, H., Jorgensen, T. J., Tanizawa, A., Pommier, Y., and 66-75, 1994. Cossman, J. Bcl-2 inhibits etoposide-induced apoptosis through its effects on events 35. Kozlowski, J. M., Fidler, I. J., Campbell, D., Xu, Z-L., Kaighn, M. E., and Hart, I. R. subsequent to topoisomerase II-induced DNA strand breaks and their repair. Cancer Metastatic behavior of human tumor cell lines grown in the nude mouse. Cancer Res., Res., 53: 4251-4256, 1993. 44: 3522-3529, 1984. 56. Compton, M. M. A biochemical hallmark of apoptosis: internucleosomal degradation 36. Kaighn, M. E., Narayan, K. S., Ohnuki, Y., Lechner, J. F., and Jones, L. W. of the genome. Cancer Metastasis Rev., ;/: 105-119, 1992. Establishment and characterization of a human prostatic carcinoma cell line (PC-3). 57. Chabner, B. A. Anticancer Drugs. In: V. T. De Vita, S. Hellman, and S. A. Rosenberg Invest. Urol., 17: 16-23, 1979. (eds.), Cancer, Principles and Practice of Oncology, pp. 325-417. Philadelphia: J. B. 37. Rubin, S. J., Hallahan, D. E., Ashman, C. R., Brachman, D. G., and Weichselbaum, Lippincott Co., 1993. R. R. Two prostate carcinoma cell lines demonstrate abnormalities in tumor suppres 58. Borner, M., Schneider, E., Pirnia, F., Sartor, O., Trepel, J., and Myers, C. The sor genes. J. Surg. Oncol., 46: 31-36, 1991. detergent Triton X-100 induces a death pattern in human carcinoma cell lines that 38. Isaacs, W. B., Carter, B. S., and Ewing, C. M. Wild-type p53 suppresses growth resembles cytotoxic lymphocyte-induced apoptosis. FEES Lett., 353: 129-132,1994. of human prostate cancer cells containing mutant p53 alÃeles. Cancer Res., 51: 59. Stimac, E., Housman, D., and Huberman, J. A. Effects of inhibition of protein 4716-4720, 1991. synthesis on DNA replication in cultured mammalian cells. J. Mol. Biol., 115: 39. Fisher, D. Apoptosis in cancer therapy: crossing the threshold. Cell, 78: 539-542, 485-511, 1977. 1994. 60. Kung, A. L., Zetterberg, A., Sherwood, S. W., and Schimke, R. T. Cytotoxic effects 40. Lowe, S. W., Ruley, H. E., Jacks, T., and Housman, D. E. p53-dependent apoptosis of cell cycle phase specific agents: result of cell cycle perturbation. Cancer Res., 50: modulates the cytotoxicity of anticancer agents. Cell, 74: 957-967, 1993. 7307-7317, 1990. 41. Keyomarsi, K., Sandoval, L., Band, V., and Pardee, A. B. Synchronization of tumor 61. Schneider, E., Lawson, P. A., and Ralph, R. K. Inhibition of protein synthesis reduces and normal cells from G| to multiple cell cycles by lovastatin. Cancer Res., 51: the cytotoxicity of 4'-(9-acridinylamino)methane-sulfon-m-anisidide without affect 3602-3609, 1991. ing DNA breakage and DNA topoisomerase II in a murine mastocytoma cell line. 42. Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, Biochem. Pharmacol., 38: 263-269, 1989. J. T., Bokesch, H., Kenney, S., and Boyd, M. R. New colorimetrie cytotoxicity assay 62. Fisher, T. C., Milner, A. E., Gregory, C. D., Jackman, A. L., Aherne, G. W., Hartley, for anticancer-drug screening. J. Nati. Cancer Inst., 82: 1107-1112, 1990. J. A., Dive, C., and Hickman, J. A. bcl-2 modulation of apoptosis induced by 43. Arends, M. J., Morris, R. G., and Wyllie, A. H. Apoptosis: the role of the endonu- anticancer drugs: resistance to thymidylate stress is independent of classical resistance clease. Am. J. Pathol., 136: 593-608, 1990. pathways. Cancer Res., 53: 3321-3326, 1993. 44. Wyllie, A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endog 63. Lane, D. P. A death in the life of p53. Nature (Lond.), 362: 786-787, 1993. enous endonuclease activation. Nature (Lond.), 284: 555-556, 1980. 64. Strasser, A., Harris, A., Jacks, T., and Cory, S. DNA damage can induce apoptosis in 45. Jakobisiak, M., Bruno, S., Skierski, J. S., and Darzynkiewicz, Z. Cell cycle-specific proliferating lymphoid cells via p53-independent mechanisms inhibitable by bcl-2. effects of lovastatin. Proc. Nati. Acad. Sci. USA, 88: 3628-3632, 1991. Cell, 79: 329-339, 1994. 46. Fenton, R. G., Kung, H., Longo, D. L., and Smith, M. R. Regulation of intracellular 65. Clarke, A. R., Purdie, C. A., Harrison, D. J., Morris, R. G., Bird, C. C., Hooper, M. L., actin polymerization by prenylated cellular proteins. J. Cell Biol., 117: 347-356, and Wyllie, A. H. Thymocyte apoptosis induced by p53-dependent and independent 1992. pathways. Nature (Lond.), 362: 849-852, 1993. 47. Lee, M. Y., Tan, C. K., Downey, K. M., and So, A. Structure and function of 66. Lowe, S. W., Schmitt, E. M., Smith, S. W., Osborne, B. A., and Jacks, T. p53 is calf thymus DNA polymerase 8. Prog. Nucleic Acid Res. Mol. Biol., 26: 83-96, required for radiation-induced apoptosis in mouse thymocytes. Nature (Lond.), 362: 1981. 847-849, 1993. 48. Gorczyca, W., Gong, J., Ardelt, B., TrÃ¡ganos, F., and Darzynkiewicz, Z. The cell 67. Berges, R. R., Furuya, Y., Remington, L., English, H. F., Jacks, T., and Isaacs, J. T. cycle related differences in susceptibility of HL-60 cells to apoptosis by various Cell proliferation, DNA repair, and p53 function are not required for programmed antitumor agents. Cancer Res., 53: 3186-3192, 1993. death of prostatic glandular cells induced by androgen ablation. Proc. Nati. Acad. Sci. 49. D'Arpa, P., Beardmore, C., and Liu, L. F. Involvement of nucleic acid synthesis in USA, 90: 8910-8914, 1993. cell killing mechanisms of topoisomerase poisons. Cancer Res., JO: 6919-6924, 68. Miyashita, T., and Reed, J. Tumor suppressor p53 is a direct transcriptional activator 1990. of the human box gene. Cell, 80: 293-299, 1995.

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Markus M. Borner, Charles E. Myers, Oliver Sartor, et al.

Cancer Res 1995;55:2122-2128.

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