13
Apoptosis May Be Either Suppressed or Enhanced with Strategic Combinations of Antineoplastic Drugs or Anti-IgM Ching-Kow E. Lin, Tam Thuan Nguyen, Thomas L. Morgan,* Rui-Lian Mei,* John S. Kaptein, Cosmas I. Kalunta, Cindy F. Yen, Eunhee Park, H. Yahong Zou, and P. M. Lad 1 Regional Research Laboratory, Kaiser Foundation Hospitals, 1515 N. Vermont Avenue, Los Angeles, California 90027; and *Department of Radiation Oncology, Southern California Permanente Medical Group, 4950 Sunset Boulevard, Los Angeles, California 90027 A variety of drugs have been used to treat B-lympho- cyte neoplasms, including both cell cycle-specific (CCS) and non-cell-cycle-specific drugs. Although the therapy for such cancers is complex and can include both types of drugs, the efficacy of these drugs in in- ducing cell death remains unclear. In this paper we have concentrated on specific CCS drugs and have examined their ability to induce programmed cell death (apoptosis) in Burkitt’s lymphoma cell lines de- rived from patients. The CCS drugs chosen were hy- droxyurea and aphidicolin (active in late G 1 , early S phase), the topoisomerase poisons camptothecin and etoposide (S, early G 2 phase) and vincristine and Taxol (late G 2 , M phase). These choices allow comparison of two drugs with differing modes of action for each of the various phases of the cell cycle. Our results indi- cate that the variation in apoptosis between drugs that act at the same phase of the cell cycle is negligi- ble. Both S/G 2 and G 2 /M blockers are very potent at inducing apoptosis whereas G 1 /S blockers are ineffec- tive in the induction of apoptosis. In addition, marked kinetic variations in the rate of apoptosis induction were observed, etoposide and camptothecin being more rapid in their action than the other agents. The order of effectiveness in inducing apoptosis on a ki- netic basis was S/G 2 agents @ G 2 /M agents @ G 1 /S agents. In this study we have also found that growth inhibition was induced by all the CCS agents chosen and by anti-IgM in various Burkitt’s lymphoma lines. Furthermore c-myc was down-regulated under similar conditions. Since apoptosis was only selectively in- duced by some of the CCS agents, it implies c-myc expression is associated with growth regulation and c-myc down-regulation is an insufficient condition for the induction of apoptosis. In addition, cotreatments using the CCS and other agents revealed the follow- ing: Cotreatment using two CCS drugs which act at the same stage in the cell cycle showed either no change or only additivity to the effects seen with either agent alone. However, cotreatment with CCS drugs showed that an inhibitory effect is found between G 1 /S and G 2 /M drugs or S/G 2 and G 2 /M drugs. No effect was found between G 1 /S and S/G 2 drugs. Anti-IgM, which by itself was capable of inducing apoptosis, was ob- served to augment apoptosis induced by very low con- centrations of G 2 /M-acting drugs but it has little effect on G 1 /S or the S/G 2 drugs. The inhibitory effect of anti-CD40 or TNF-a on anti-IgM-induced apoptosis did not carry over to an effect on apoptosis induction by the CCS agents. Thus specific combinations of agents may lead to either enhancement, inhibition, or no in- teractive effect on apoptosis. © 1998 Academic Press Key Words: apoptosis; antineoplastic agents; growth inhibition; cell cycle. INTRODUCTION Studies from several laboratories have been directed at understanding the regulation of growth and apopto- sis in B-lymphoma cells [1– 6]. Anti-IgM treatment of some Burkitt’s lymphoma cell lines (e.g., Ramos) re- sults in induction of apoptosis, while in others (e.g., Daudi) it results in retardation of growth without as- sociated apoptosis [7]. Although the precise mecha- nism(s) for apoptosis induction in these Burkitt’s lym- phoma cells remains unclear, c-myc levels seemed to be important to the overall process since (a) c-myc levels were rapidly decreased following anti-IgM treatment and (b) c-myc antisense oligodeoxynucleotides gave rise to apoptosis in Ramos cells while producing only growth inhibition in Daudi cells. Growth inhibition without associated apoptosis can also be achieved by other treatment conditions such as by the inhibition of ornithine decarboxylase using DFMO [8] or by starving the cells in the presence of DMSO [9]. These results demonstrated that growth inhibition can be uncoupled from the induction of apoptosis. Further support for these observations comes from studies which indicate that TNF-a and anti-CD40 selectively inhibit the apo- ptosis induced by anti-IgM [10, 11] but do so without altering the decline in c-myc levels. This suggests that some portion of the apoptosis pathway may lie distally 1 To whom correspondence and reprint requests should be ad- dressed. Fax: (323) 783-5275. 0014-4827/98 $25.00 1 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. EXPERIMENTAL CELL RESEARCH 244, 1–13 (1998) ARTICLE NO. EX984158

Apoptosis May Be Either Suppressed or Enhanced with Strategic Combinations of Antineoplastic Drugs or Anti-IgM

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Page 1: Apoptosis May Be Either Suppressed or Enhanced with Strategic Combinations of Antineoplastic Drugs or Anti-IgM

Apoptosis May Be Either Suppressed or Enhanced with StrategicCombinations of Antineoplastic Drugs or Anti-IgM

Ching-Kow E. Lin, Tam Thuan Nguyen, Thomas L. Morgan,* Rui-Lian Mei,* John S. Kaptein,Cosmas I. Kalunta, Cindy F. Yen, Eunhee Park, H. Yahong Zou, and P. M. Lad1

Regional Research Laboratory, Kaiser Foundation Hospitals, 1515 N. Vermont Avenue, Los Angeles, California 90027; and *Departmentof Radiation Oncology, Southern California Permanente Medical Group, 4950 Sunset Boulevard, Los Angeles, California 90027

A variety of drugs have been used to treat B-lympho-cyte neoplasms, including both cell cycle-specific(CCS) and non-cell-cycle-specific drugs. Although thetherapy for such cancers is complex and can includeboth types of drugs, the efficacy of these drugs in in-ducing cell death remains unclear. In this paper wehave concentrated on specific CCS drugs and haveexamined their ability to induce programmed celldeath (apoptosis) in Burkitt’s lymphoma cell lines de-rived from patients. The CCS drugs chosen were hy-droxyurea and aphidicolin (active in late G1, early Sphase), the topoisomerase poisons camptothecin andetoposide (S, early G2 phase) and vincristine and Taxol(late G2, M phase). These choices allow comparison oftwo drugs with differing modes of action for each ofthe various phases of the cell cycle. Our results indi-cate that the variation in apoptosis between drugsthat act at the same phase of the cell cycle is negligi-ble. Both S/G2 and G2/M blockers are very potent atinducing apoptosis whereas G1/S blockers are ineffec-tive in the induction of apoptosis. In addition, markedkinetic variations in the rate of apoptosis inductionwere observed, etoposide and camptothecin beingmore rapid in their action than the other agents. Theorder of effectiveness in inducing apoptosis on a ki-netic basis was S/G2 agents @ G2/M agents @ G1/Sagents. In this study we have also found that growthinhibition was induced by all the CCS agents chosenand by anti-IgM in various Burkitt’s lymphoma lines.Furthermore c-myc was down-regulated under similarconditions. Since apoptosis was only selectively in-duced by some of the CCS agents, it implies c-mycexpression is associated with growth regulation andc-myc down-regulation is an insufficient condition forthe induction of apoptosis. In addition, cotreatmentsusing the CCS and other agents revealed the follow-ing: Cotreatment using two CCS drugs which act at thesame stage in the cell cycle showed either no changeor only additivity to the effects seen with either agentalone. However, cotreatment with CCS drugs showed

that an inhibitory effect is found between G1/S andG2/M drugs or S/G2 and G2/M drugs. No effect wasfound between G1/S and S/G2 drugs. Anti-IgM, whichby itself was capable of inducing apoptosis, was ob-served to augment apoptosis induced by very low con-centrations of G2/M-acting drugs but it has little effecton G1/S or the S/G2 drugs. The inhibitory effect ofanti-CD40 or TNF-a on anti-IgM-induced apoptosis didnot carry over to an effect on apoptosis induction bythe CCS agents. Thus specific combinations of agentsmay lead to either enhancement, inhibition, or no in-teractive effect on apoptosis. © 1998 Academic Press

Key Words: apoptosis; antineoplastic agents; growthinhibition; cell cycle.

INTRODUCTION

Studies from several laboratories have been directedat understanding the regulation of growth and apopto-sis in B-lymphoma cells [1–6]. Anti-IgM treatment ofsome Burkitt’s lymphoma cell lines (e.g., Ramos) re-sults in induction of apoptosis, while in others (e.g.,Daudi) it results in retardation of growth without as-sociated apoptosis [7]. Although the precise mecha-nism(s) for apoptosis induction in these Burkitt’s lym-phoma cells remains unclear, c-myc levels seemed to beimportant to the overall process since (a) c-myc levelswere rapidly decreased following anti-IgM treatmentand (b) c-myc antisense oligodeoxynucleotides gave riseto apoptosis in Ramos cells while producing onlygrowth inhibition in Daudi cells. Growth inhibitionwithout associated apoptosis can also be achieved byother treatment conditions such as by the inhibition ofornithine decarboxylase using DFMO [8] or by starvingthe cells in the presence of DMSO [9]. These resultsdemonstrated that growth inhibition can be uncoupledfrom the induction of apoptosis. Further support forthese observations comes from studies which indicatethat TNF-a and anti-CD40 selectively inhibit the apo-ptosis induced by anti-IgM [10, 11] but do so withoutaltering the decline in c-myc levels. This suggests thatsome portion of the apoptosis pathway may lie distally

1 To whom correspondence and reprint requests should be ad-dressed. Fax: (323) 783-5275.

0014-4827/98 $25.001Copyright © 1998 by Academic Press

All rights of reproduction in any form reserved.

EXPERIMENTAL CELL RESEARCH 244, 1–13 (1998)ARTICLE NO. EX984158

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to c-myc, possibly at the level of free radical generation[12–14] or the caspase protease cascade [15–18].

B-cell lymphomas have been treated with many dif-ferent types of antineoplastic agents. Therapies thatuse both cell cycle-specific (CCS) and non-cell cycle-specific (NCCS) agents have been used [19]. However,induction of apoptosis by these agents alone or in com-bination has not been critically assessed and the path-ways associated with either growth inhibition or apo-ptosis remain unknown. In this paper we have chosenagents that are presumed to act on discrete phases ofthe cell cycle and are either already in common use orshow potential for future applications. Two agentswere chosen for each phase, each acting by a differentmechanism, so that the effect on the cell cycle phaserather than the individual mechanism of drug actioncould be assessed. The selected drugs were as follows:(i) hydroxyurea and aphidicolin as the G1/S phasedrugs. Hydroxyurea and aphidicolin have both beenreported to be chain elongation inhibitors which act toinhibit DNA synthesis and thus arrest cells at G1/Sboundary [20–24]. In addition, hydroxyurea was alsofound to be a ribonucleotide reductase inhibitor [25]while aphidicolin was shown to inhibit DNA poly-merases a, d, and e [26, 27]. (ii) Camptothecin andetoposide as the S/G2 phase drugs. Camptothecin [28–33] and etoposide [29, 30, 34, 35] are the topoisomeraseI and II poisons, respectively, which trap topoisomer-ase I or II activity involved in DNA replication, tran-scription, and recombination and therefore preventcells from entering G2 phase of the cell cycle. (iii) Vin-cristine and Taxol as the G2/M phase drugs. Vincris-tine and Taxol both interact with microtubule struc-tures, but function in different ways to inhibit celldivision and cause cells to be arrested at G2/M phase.Whereas Taxol binds to tubulin and thus stabilizes andprevents depolymerization of microtubules [36–38],vincristine acts to block polymerization of microtubulesthus preventing formation of mitotic spindle fiber ap-paratus [39, 40].

Our experiments were directed at assessing whetherapoptosis, or inhibition of growth, or both occurredwith any of these CCS agents and whether the charac-teristics of cell death were similar to or different fromthose observed with antibody directed against B cellantigen receptor (i.e., anti-IgM). We evaluated the ef-fect of combinations of these drugs on the induction ofapoptosis and also whether c-myc was similarly down-regulated by these drugs.

MATERIALS AND METHODS

Materials

CA46 (CRL 1648), Daudi (CCL 213), Namalwa (CRL 1432), Raji(CCL 86), Ramos (CRL 1596), and ST486 (CRL 1647) cell lines andGAPDH plasmid DNA (57090) were obtained from American Type

Culture Collection (Rockville, MD). v-myc plasmid DNA was pre-pared from transfected Escherichia coli clones [41]. Goat anti-humanIgM (m chain specific) was purchased from Organon Technika(Durham, NC). Mouse monoclonal anti-human c-Myc antibody(9E10) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).Prestained low molecular weight protein markers, protein assayreagent, and horseradish peroxidase-conjugated anti-mouse IgGwere purchased from Bio-Rad (Hercules, CA). The enhanced chemi-luminescence detection kit (ECL) was obtained from AmershamCorp. (Arlington Heights, IL). [methyl-3H]Thymidine was obtainedfrom ICN Pharmaceuticals, Inc. Costa Mesa, CA). DNA size marker(HindIII digest of l DNA) was from United States Biochemical(Cleveland, OH). The RNA isolation kit (RNA STAT-60) was ob-tained from TEL-TEST “B” Inc. (Friendswood, TX). RPMI 1640 me-dium was from Irvine Scientific (Santa Ana, CA). Fetal calfserum was obtained from Gemini Bioproducts (Calabasas, CA). Vin-cristine was obtained from Janssen Biochemica (Spectrum ChemicalMfg., Gardena, CA), and Taxol (paclitaxel, NSC 125973-L) was ob-tained from the Drug Synthesis and Chemistry Branch, Develop-mental Therapeutics Program, Division of Cancer Treatment, Na-tional Cancer Institute (Bethesda, MD). Aphidicolin, hydroxyurea,camptothecin, etoposide, proteinase K, propidium iodide, and allother chemicals were obtained from Sigma Chemical Company (St.Louis, MO).

Methods

Cell culture and treatment. The Burkitt’s lymphoma cells werecultured in RPMI 1640 medium supplemented with penicillin G (100units/ml), streptomycin (100 mg/ml), fungizone (0.25 mg/ml), L-glu-tamine (2 mM), 10% heat-inactivated fetal calf serum, and Hepes (10mM, pH 7.4) at 37°C in a humidified 5% CO2 incubator [7]. Expo-nentially growing cells were then transferred to 12-well microtiterplates and treated with anti-IgM or various antineoplastic agents atthe concentrations and for the times indicated in the various exper-iments. The cells were collected into tubes by centrifugation at 70gfor 10 min, washed, and resuspended to approximately 106 cells/mlwith Hank’s balanced salt solution (HBSS) at room temperaturebefore analysis.

Apoptosis assessed by microscopic morphology. Cells werestained with ethidium bromide and acridine orange and examined byfluorescence microscopy. Cells were scored as either viable (brightgreen chromatin) or nonviable (bright orange chromatin) and aseither normal (chromatin in organized structure) or apoptotic (highlycondensed or fragmented nuclei). Viability and apoptotic index (per-centage of apoptotic cells) were both determined as previously pub-lished [7, 9, 42]

Apoptosis assessed by flow cytometry. The measurement of nu-clear DNA content for apoptosis and cell cycle distribution wascarried out as previously described using an ethanol fixation proce-dure [10, 43] and analyzed on a FACStarplus flow cytometer (BectonDickinson, Mountain View, CA). Briefly, washed cell pellets wereresuspended in 70% ethanol and incubated at 220°C for at least 20min. The fixed cells were then washed twice, resuspended in PBS,and incubated at 37°C for 20 min. The cells were stained withpropidium iodide (50 mg/ml) and analyzed by flow cytometry within6 h. Analyses of the resulting histogram profiles were performedusing PC LYSYS software. The histogram profile of untreated cellswas used to define the positions of the G1 and G2/M peaks. Theintervening region is defined as S region and the region below the G1

peak is defined as the A0 region. The delineation of regions fromuntreated cells was applied to profiles of treated cells.

Apoptosis assessed by DNA laddering. Apoptosis was also deter-mined by assessment of DNA laddering [42] in parallel to flowcytometry. About 5 3 106 cells were lysed with proteinase K and SDSbuffer and the DNAs were purified as previously reported [10]. TheDNA pellets were dissolved in 20 ml sterile water and quantitated by

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UV absorbance. Equivalent amounts of DNA samples (10 mg) weresubjected to electrophoresis on 1.2% agarose gels containingethidium bromide (0.5 mg/ml) at 5 V/cm in TBE buffer (45 mMTris–borate, 1 mM EDTA, pH 8.0). The gels were then visualized andphotographed under UV light. A HindIII digest of l DNA, ranging insize from 23 to 0.1 kb was used as size marker. DNA laddering wasdefined by the appearance of regularly-spaced nucleosomal bands atintervals of about 200 bp, in the region below 2.3 kb but above tRNA(0.1 kb).

Proliferation assays. Proliferation of cells was assessed by theincorporation of radiolabeled thymidine as described previously [7,44]. Briefly, cells (;2.5 3 105/ml) were cultured in a final volume of1 ml in 12-well microtiter plates (Costar, Cambridge, MA), treatedwith reagent for 24 h and then incubated with [3H]thymidine (0.5mCi/well) for an additional 4 h. Aliquots of 200 ml were then har-vested, lysed, and the trichloroacetic acid-precipitable materialswere collected onto glass filters using a semiautomated cell harvester(Skatron, Lier, Norway). Uptake of label was determined by countingthe disks using a liquid scintillation counter.

c-myc mRNA determination. Total RNA was isolated from about3 3 107 cells using the RNA STAT kit according to the manufactur-er’s instructions. RNA pellets were resuspended in 100 ml TES (10mM Tris, 5 mM EDTA, 1% SDS, pH 7.4) and quantitated by UVabsorption. Northern blots of RNA from the cells were performed aspreviously reported [45, 46]. Twenty-five microliters of total RNA

from each sample were electrophoresed under denaturing conditionsand transferred to nylon membranes. These blots were probed with32P-labeled c-myc and GAPDH probes. Dried blots were exposed toKodak XAR-5 film over 48 h. Autoradiograms of the gels werescanned and analyzed using a densitometer (Biomed Instruments,Fullerton, CA).

c-Myc immunoblotting. Cells (1 3 107 ) were treated with variousreagents for 24 h and were collected by centrifugation at 70g for 10min. The cells were lysed in RIPA lysis buffer (50 mM Tris, pH 8.0,0.1% SDS, 0.5% deoxycholate, 1% NP-40, 150 mM NaCl , 10 mg/ml ofleupeptin, aprotinin, and PMSF) and the supernatants were col-lected by centrifugation at 16,000g for 10 min. Equal amounts ofprotein (10 mg), as determined by Bio-Rad protein assay, were boiledin Laemmli buffer for 10 min and then subjected to 10% SDS–polyacrylamide gel electrophoresis. Western blotting was performedas previously reported [7]. Proteins were transferred from the poly-acrylamide gel to a nitrocellulose membrane by electroblotting. Themembrane blots were blocked with 5% nonfat milk in 25 mM Trisbuffered saline (TBS), pH 7.4 with 0.1% Tween 20, incubated withmonoclonal antibody against c-Myc at 5 mg/ml in 1% nonfat milk inTBS overnight, followed by incubation with horseradish peroxidase-conjugated anti-mouse second antibody. Substrate for enhancedchemiluminescence was added and detection was by exposure toautoradiographic film.

FIG. 1. Dose responses and time courses of antineoplastic agents in Ramos cells. Cells were treated with antineoplastic agents at variousdoses and for varying times. Apoptotic cell morphology was evaluated by staining cells with ethidium bromide/acridine orange and examiningunder the fluorescence microscope, as described under Materials and Methods. (A) The percentage of apoptotic cells at 24 h after treatmentwith the G1/S-specific agents, aphidicolin and hydroxyurea. (B) the results with the S/G2-specific agents, etoposide and camptothecin. (C) theresults with the G2/M-specific agents, Taxol and vincristine. (D) The percentage of apoptotic cells at various times with aphidicolin treatmentat 10 mM or hydroxyurea at 1 mM. (E) The results with etoposide at 100 mM or camptothecin treatment at 1 mM. (F) The results with Taxolat 1 mM or vincristine treatment at 1 mM. Error bars, 6 1 standard deviation.

3ANTI-IgM ENHANCES AND G1/S OR S/G2 AGENTS INHIBIT G2/M AGENT-INDUCED APOPTOSIS

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RESULTS

Dose Responses and Time Courses of CCSAntineoplastic Agents on Ramos Cells

The effects of several antineoplastic agents on apo-ptosis in Ramos are shown in Fig. 1. The dose–responsecurves at the 24-h point are shown first (Fig. 1A–1C).

The 24-h time point was chosen because the doublingtime for Ramos is about 20 to 22 h and because there isminimal loss of viability at this time in control cellswhile still allowing enough time for appreciable apo-ptosis to occur in the treated cells. Apoptosis was mea-sured by fluorescence microscopy and morphologicevaluation as described in the Materials and Methods.Untreated cells (no drug added and/or cells at start oftime series) show minimal apoptosis and this basallevel does not vary over the time course studied (about3% in Ramos). Hydroxyurea and aphidicolin (Fig. 1A)at concentrations of up to 1 mM are without any effectand a progressive effect on apoptosis is observed atdoses ranging from 100 to 1000 mM. The results sug-gest that these agents, which act at the G1/S border,are minimal in their effect on apoptosis in Ramos cells.The effect of the G2/S agents, etoposide and camptoth-ecin, are shown in Fig. 1B. These agents induced sub-stantial apoptosis (up to 60–80%) at concentrations of100 and 1 mM, respectively. The G2/M agents, Taxoland vincristine, were both very effective, on a molarbasis, at inducing apoptosis (Fig. 1C) with significanteffects observable at 1 nM.

In the next experiment the cells were treated withantineoplastic agents at the concentrations indicatedin the figure legend (Figs. 1D–1F) for up to 48 h. Nearmaximal concentrations were chosen so that the timecourse of apoptosis development could be clearly exam-ined for each agent. Hydroxyurea and aphidicolin showminimal apoptosis at 24 h as shown in Fig. 1D. At 48 hsignificant apoptosis is observed. The kinetic curves forthe S/G2 agents (Fig. 1E) are remarkable. These agentsare, on a kinetic basis, the most rapidly acting of thethree groups tested. At time points as early as 4 hsignificant apoptosis is observed for both agents. Vin-cristine and Taxol (Fig. 1F), although being the mostpotent on a molar basis, are kinetically slower acting incomparison to etoposide and camptothecin. The rela-tive efficacy based on rate of apoptosis induction is thuscamptothecin . etoposide @ vincristine ; Taxol @hydroxyurea ; aphidicolin.

Validation of Apoptosis Measurements by FlowCytometry and DNA Laddering

In the previous section the results presented werebased on the microscopic and morphologic evaluationof the cells. However, flow cytometry and DNA ladder-ing were also carried out to validate these results. Thisis important in view of the gathering literature whichsuggests that certain forms of apoptosis may not al-ways be accompanied by identical changes in flow cy-tometric or DNA laddering profiles [47–50]. The resultsfor flow cytometry are shown in Fig. 2. In these exper-iments we examined whether these agents caused apo-ptosis as manifested by an A0 peak. The time point

FIG. 2. Flow cytometric representation of apoptosis caused byantineoplastic agents. Cells were treated with various antineoplasticagents for 24 h. Cell nuclei were stained with ethidium bromide forploidy and apoptosis evaluation by flow cytometry, as describedunder Materials and Methods. Panels show ploidy profiles for eitheruntreated Ramos cells or cells treated with various antineoplasticagents at the indicated concentrations. For each panel the x-axisdepicts the channel number of nuclear staining intensity (i.e., DNAcontent per cell). The y-axis depicts the number of cells observed foreach level (channel number) of staining intensity. Untreated cellsshow bimodal DNA content indicative of the diploid state (G1, aboutchannel 200) and the mitotic tetraploid state (G2/M, about channel400). S phase cells are intermediate between the G1 peak and theG2/M peak. Apoptosis is manifested by the presence of an A0 peakindicating cells with DNA content below that of the G1 peak. Thepercentage of the distribution in the apoptotic region is indicated.

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used in these studies was 24 h and the drug concen-trations used were near the maximal doses of the anti-neoplastic agents used in Figs. 1A–1C. Because of thehigh doses of etoposide and camptothecin used, the cellcycle stage specificity was not seen although lowerdoses of these agents do show S phase enrichment(Nguyen et al., unpublished data).

For hydroxyurea and aphidicolin, the A0 peak is dis-cernible because of the very high concentration of hy-droxyurea used. At concentrations equivalent to thoseused for the other agents, no apoptosis was observed.In short, the concentrations used were higher and yetthe apoptosis observed for these agents was markedlylower. The flow cytometry data also indicated that eto-poside and camptothecin were similar to vincristineand Taxol in inducing apoptosis, with all of these beingmore potent than hydroxyurea and aphidicolin. Thus,overall the relative efficacy observed by flow cytometryis similar for Ramos cells to that observed by micro-scopy, although small quantitative differences arenoted.

Another technique to assess apoptosis is DNA lad-dering. In these experiments the cells were treatedwith the antineoplastic agents and the DNA was di-rectly extracted and run on agarose gels. DNA hyper-fragmentation, if present, is seen as a stepwise ladderof DNA fragments at about 200-basepair incrementsbelow 2.3 kb. The data (Fig. 3) shows that in Ramoscells DNA laddering is pronounced for Taxol and vin-cristine. Minimal DNA laddering is observed with hy-droxyurea and aphidicolin. For etoposide and camp-tothecin large-sized DNA fragmentation is more

predominant than nucleosomal-sized fragments. Thishas been verified by field inversion gel electrophoresis(Yen et al., unpublished data).

Evaluation of Other Burkitt’s Lymphoma Cell Lines

In previous studies we have shown that some Bur-kitt’s lymphoma cell lines are far more susceptible toanti-IgM-induced apoptosis than are others [7]. To ex-amine whether similar differences may arise with anti-neoplastic agents, we evaluated additional Burkitt’slymphoma cell lines. The results are shown in Fig. 4.The cells were treated with the antineoplastic agentsfor 24 h at the indicated concentrations and the apo-ptotic index was assessed using flow cytometry andassessing the area under the A0 peak. In agreementwith our previous results using anti-IgM, induction ofapoptosis by the antineoplastic agents is highly cellline dependent. ST486 shows greatest sensitivity to theantineoplastic agents, similar to its sensitivity to anti-IgM. CA46, on the other hand, was most resistant tothe antineoplastic agents, similar to its resistance toanti-IgM. Other cell lines showed intermediate levelsof sensitivity to the antineoplastic agents which weredependent on both cell line and the specific agent beingexamined. Moreover, for Daudi cells only, the level ofapoptosis observed was dependent on the assay used.Morphologic changes characteristic of apoptosis werealways seen at a higher level than apoptosis seen byflow cytometry. Microscopy data indicates that Daudicells are sensitive to the antineoplastic agents whereasthe flow data may be underestimating the extent of

FIG. 3. Assessment of apoptosis by DNA laddering in Ramos cells. Cells were treated with G1/S, S/G2, and G2/M-specific antineoplasticagents at the indicated doses for 24 h. DNA extracts from treated cells were subjected to electrophoresis on 1.2% agarose gels to detectinternucleosomal cleavage, as described under Materials and Methods. Results are presented as indicated. Control, untreated cells.Standard, a set of size markers generated by HindIII digestion of l DNA.

5ANTI-IgM ENHANCES AND G1/S OR S/G2 AGENTS INHIBIT G2/M AGENT-INDUCED APOPTOSIS

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apoptosis, or apoptosis may be manifested in a differ-ent manner.

Inhibition of Apoptosis by Anti-CD40 or TNF-a

Anti-CD40 and TNF-a both cause a reduction inapoptosis induced by anti-IgM in Ramos cells [10, 11,18, 51–53]. We therefore tested whether these agentswould also inhibit apoptosis induced by the antineo-plastic drugs. In these studies the cells were treatedwith anti-CD40 or TNF-a simultaneously to exposureto the various antineoplastic drugs. Our results (Fig. 5)indicate that anti-CD40 or TNF-a did not inhibit apo-ptosis induced by any of the antineoplastic drugs tested

whereas they did inhibit apoptosis induced by anti-IgMas shown by the previous studies.

Growth Inhibition by Antineoplastic Agents

Effects of antineoplastic agents on apoptosis must beevaluated in relationship to effects on cell growth. Aquestion raised was whether inhibition of growth wasobligatorily associated with apoptosis. Ramos cellswere therefore treated with the various doses of anti-neoplastic agents used in the evaluation of apoptosis.Cell growth was examined by either direct cell countsover time or by determining thymidine incorporationinto DNA at a fixed time (24 h) as shown in Fig. 6.

FIG. 4. Comparison of apoptosis induced by antineoplastic agents in various Burkitt’s lymphoma cell lines. The Burkitt’s lymphoma celllines [Daudi (Da), Raji (Rj), Namalwa (Na), Ramos (Ra), ST486 (ST), and CA46 (CA)] were grown to exponential phase and treated withantineoplastic agents for 24 h at the doses indicated. The cells were then harvested and stained for ploidy analysis by flow cytometry asdescribed under Materials and Methods and the legend to Fig. 2. The fraction of the distribution under the A0 peak was determined. Thebasal level of the A0 peak was subtracted and plotted to show the net increase in apoptosis due to these agents on these six different Burkitt’slymphoma cell lines. Error bars, 61 standard deviation. For Daudi cells, apoptosis determined by microscopy [Da(M)] is compared toapoptosis determined by flow cytometry [Da(F)].

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Growth inhibition was observed with all of the CCSagents regardless of the assay used. In general, at thelowest concentrations of drugs, growth was minimallyaffected and no apoptosis was observed (see Fig. 1). Atincreasing dosages, growth inhibition was observedwhich was not necessarily accompanied by apoptosis.At the highest dosages or at longer times of incubation,cell growth was completely inhibited and apoptosis wasreadily evident. A notable exception is that hydroxy-urea and aphidicolin, agents which were relatively in-effective in causing apoptosis, caused growth inhibitionas readily as the other agents tested. Thus the inhibi-tion of growth can occur in the absence of, or possiblyprior to, the induction of apoptosis.

c-myc Expression

We have previously shown that, following anti-IgMtreatment, c-myc levels are decreased from an elevatedbasal level in the Burkitt’s lymphoma cell lines. Fur-thermore the decrease in c-myc leads to growth inhibi-tion which, in some cell lines (e.g., Ramos, ST486), isfollowed by apoptosis [7]. We therefore examined

whether the growth inhibition and apoptosis seen fol-lowing treatment with the antineoplastic agents wasalso associated with changes in c-myc expression. Theresults in Fig. 7 indicate that, compared to the highbasal level seen in untreated samples, there was adramatic dose-dependent reduction in c-myc mRNA forall CCS antineoplastic agents tested. Since hydroxy-urea and aphidicolin do not induce apoptosis, the de-cline in c-myc was primarily associated with growthinhibition. The western blots for c-Myc protein are alsoshown in Fig. 7. These results confirm that reductionsin mRNA levels are paralleled by reductions in c-Mycprotein which decrease after treatment with all of theagents tested, although the extent of the reductions areagent-specific to some degree.

Cotreatments with CCS Antineoplastic Agents and/orAnti-IgM

A point of interest is to examine whether combinedtreatment with specific CCS antineoplastic drugswould produce interactive effects which would be rele-vant in the clinical use of these drugs. We thereforetreated Ramos cells with different combinations of theselected CCS agents or anti-IgM and evaluated theinduction of apoptosis. The results summarized in Ta-ble 1 indicated the following: (i) Cotreatments withdifferent drugs acting on the same phase of the cellcycle showed that effects on apoptosis were at mostadditive, indicating that the contribution to apoptosisfrom each drug were equal and could be separated [e.g.,hydroxyurea and aphidicolin (both G1/S agents), camp-tothecin and etoposide (both S/G2 agents), or Taxol andvincristine (both G2/M agents)]. (ii) Cotreatments us-ing G1/S and S/G2 agents showed no significant changein apoptosis. (iii) Cotreatment using low doses of eitherG1/S or S/G2 agents together with G2/M agents showedthat G1/S or S/G2 agents significantly reduced the apo-ptotic effect of G2/M agents suggesting that interfer-ence with cell cycle progression prevented the action ofthe G2/M agents. This was most readily seen if signif-icant apoptosis was initiated by the G2/M agent. (iv)Cotreatments using anti-IgM with the CCS antineo-plastic agents resulted in three distinct patterns ofresponses—no interactive effect (anti-IgM with S/G2agents camptothecin or etoposide), slight augmenta-tion (anti-IgM with G1/S agents aphidicolin or hy-droxyurea), or enhanced effects (anti-IgM with lowdoses of G2/M agents Taxol or vincristine).

DISCUSSION

Cell-Cycle-Specific Reagents and the Induction ofApoptosis

The overall purpose of this study was to determinethe effects of CCS agents on apoptosis and growth

FIG. 5. Effects of TNF-a and anti-CD40 antibody on apoptosisinduced by anti-IgM and selected antineoplastic agents in Ramoscells. Ramos cells (;106/ml) were cultured in fresh RPMI 1640 me-dium in 12-well microtiter plates overnight. TNF-a (10 ng/ml) oranti-CD40 (1 mg/ml) was then added to the medium together with theagents that produce apoptosis—IgM, anti-IgM (10 mg/ml); Etop, eto-poside (100 mM); Camp, camptothecin (1 mM); Taxol (1 mM); Vin,vincristine (1 mM); or Cntl, no other additions. Incubation was con-tinued for an additional 24 h. The extent of apoptosis was thendetermined by fluorescence microscopy. Open bars, treated only withagent (or untreated for “Cntl”); diagonal hatched bars, treated withTNF-a plus agent; cross hatched bars, treated with anti-CD40 plusagent. Error bars, 61 standard deviation.

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inhibition in Burkitt’s lymphoma cells and to comparethese effects to those seen with anti-IgM. The cell cyclespecificity of the reagents involved was examined to alimited extent. Hydroxyurea and aphidicolin give en-richment of cells at G1/S and Taxol and vincristine giveenrichment of cells at G2/M (Fig. 2). The data withetoposide and camptothecin are dose dependent. Athigh doses, a considerable amount of apoptosis wasseen and cell cycle effects were difficult to evaluate(Fig. 2). At lower doses, S phase enrichment could bedemonstrated (unpublished data).

The picture that emerges for the induction of apopto-sis in Ramos cells is that the CCS agents differ in amanner dependent upon their cell cycle specificity. TheG2/M phase agents are active in the nano- to micromo-lar range; the S phase agents are active in the micro- tomillimolar range; and the G1/S phase agents inducelittle apoptosis even at millimolar concentrations. Thephase at which the agent acts was noted to be moreimportant than the individual mechanism by which itacts. For example vincristine acts to block polymeriza-tion of microtubules while Taxol acts to block depoly-merization [36–40], yet both seem effective in inducingapoptosis. The S phase specific reagents, etoposide andcamptothecin, act on different topoisomerases. Camp-tothecin is a poison of topoisomerase I while etoposideis a poison of topoisomerase II [28, 30, 33–35]. Here

again, regardless of the differences in individual mech-anisms, both agents induce marked apoptosis. Hy-droxyurea and aphidicolin, which are characterized asG1/S agents [20, 22, 24], are not potent inducers ofapoptosis in Ramos cells within 24 h. High concentra-tions and prolonged treatments (48 h or longer) areneeded for apoptosis to be observed.

Within the broad pattern of effective concentrationranges certain kinetic differences are observedamong the agents. For example etoposide and camp-tothecin produce apoptosis rapidly and produce hy-perfragmentation of the c-myc gene and other genesas well [54, 55]. By contrast Taxol and vincristineproduce similar levels of apoptosis to etoposide andcamptothecin but require more time (24 h). Thisdramatic kinetic difference between the topoisomer-ase poisons (S/G2) and the microtubule blockers(G2/M) may be due to the phenomenon of “early ap-optosis” seen with certain drugs [56]. On a kineticbasis, the time needed to reach significant apoptosisshows the following pattern: S/G2 phase agents @G2/M phase agents @ G1/S agents.

From these studies it was noted that agents thatact at the G1/S border (hydroxyurea and aphidicolin)were less effective on both a molar and kinetic basisthan the other agents tested. The cellular basis forthe lack of sensitivity to hydroxyurea and aphidico-

FIG. 6. Growth inhibition caused by antineoplastic agents. Growth of untreated and antineoplastic agent-treated cells was determinedby cell counts or by measurement of [3H]thymidine incorporation. (A–C) The 0- to 48-h growth curves of Ramos cells as determined by cellcounting. The doses used are as follows: for aphidicolin, 0 (F), 10 nM (E), 3 mM (‚), 10 mM (h); for hydroxyurea, 0 (F), 1 mM (E), 300 mM (‚),1 mM (h); for etoposide, 0 (F), 100 nM (E), 30 mM (‚), 100 mM (h); for camptothecin, Taxol, and vincristine, 0 (F), 1 nM (E), 300 nM (‚),1 mM (h). (D–F) The results of [3H]thymidine incorporation into Ramos cells treated with various antineoplastic agents at the indicateddoses.

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lin may be explained in part by the status of the p53gene product which exists in mutant form in theseBurkitt’s lymphoma cell lines [57, 58]. Because ofthis mutation, the checkpoint at G1/S may not befully operative [3, 59, 60]. A relative scarcity of cellsat this stage of the cell cycle may therefore contrib-ute to the diminished effect of these agents. A similarobservation was noted by Johnson et al. who haveshown that BM13674 cells treated with low doses ofhydroxyurea show growth inhibition without induc-ing apoptosis [61]. High concentration and prolongedtreatments were needed for hydroxyurea to be effec-tive in inducing apoptosis. Thus the potency andkinetic results indicate that in Burkitt’s lymphomacells, exit from the cell cycle and induction of apop-tosis most likely is a consequence of interferencewith normal cellular metabolism during S/G2 orG2/M phases. Exit to apoptosis prior to the G1/Scheckpoint probably occurs minimally due to the na-ture of the p53 mutation in these cells. This findingis very similar to that reported by Allday et al. [3].

Different Manifestations of Apoptosis

Apoptosis induction is both cell line and agentspecific. In addition, marked differences in overallsusceptibility are also observed (e.g., ST486 beingsusceptible and CA46 being resistant to apoptosis

induction by all of the agents tested). An observationmade during the course of these studies is that ap-parent resistance to apoptosis may in fact representan alternate manifestation of apoptosis not detect-able by certain techniques. For example, in Daudicells, treatment with the various antineoplasticagents showed morphologic changes (loss of nuclearintegrity) which were compatible with apoptosis butdid not generate comparable A0 peaks in flow cytom-etry or cause overt hyperfragmentation of DNA(DNA laddering). Also for Ramos cells, etoposide andcamptothecin induce apoptosis demonstrable by mi-croscopy and flow cytometry but very little DNAladdering is seen. In these instances one techniquemay underestimate the degree of apoptosis or fail todetect it at all. Other investigators have also shownthat morphologic changes and nuclear fragmentationmay occur without producing the classical profile ofDNA laddering [50, 61]. The presence of alternatemanifestations of apoptosis thus suggests a far morecomplex situation than previously supposed.

Inhibition of Growth and Apoptosis

The data presented here indicate that all of theantineoplastic agents tested induce an inhibition ofgrowth and a decline in c-myc (from an initial highbaseline), regardless of the cell cycle phase on which

FIG. 7. Effect of antineoplastic agents on c-myc. Ramos cells were treated with different antineoplastic agents for 24 h with various dosesas indicated. Control, untreated cells. (Top) Total RNA extracted from these cells was analyzed by Northern blotting for the expression ofc-myc mRNA and the housekeeping gene GAPDH, as described under Materials and Methods. (Bottom) Equal amounts of total cellularprotein extract were fractionated by 10% SDS–PAGE, transferred to nitrocellulose paper, and immunoblotted with mouse monoclonalantibody (9E10) against c-Myc protein, as described under Materials and Methods.

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they act and regardless of the method used to assessgrowth inhibition. However, they do not all induceapoptosis (e.g., the G1/S agents, hydroxyurea andaphidicolin). This implies that the decline in c-myc isprimarily associated with growth inhibition. Theseresults in turn support our previous observationsthat the inhibition of cell growth can be uncoupledfrom apoptosis [7]. Furthermore, DFMO, an orni-thine decarboxylase inhibitor, produces growth inhi-bition without apoptosis [8] as does DMSO [9]. Theconverse proposition of whether apoptosis can occurwithout growth inhibition remains controversial.Within 4 h after treatment with etoposide or camp-tothecin, apoptosis could be observed (Fig. 1E). Wewere unable to demonstrate either growth inhibi-tion or a lack thereof in this short time intervalbecause of the presence of apoptosis. Therefore, nodefinitive statement could be made as to whetherapoptosis is independent of growth inhibition or de-pendent upon it.

Inhibition of Apoptosis by Anti-CD40 or TNF-a

Neither the anti-IgM-mediated decline in c-myc norgrowth inhibition is inhibited by anti-CD40 or TNF-a [7,8]. However, these agents do inhibit anti-IgM-mediatedapoptosis [10, 11]. The step at which these agents act isunclear, although the inhibition of apoptosis probablyoccurs distally to the decline in c-myc mRNA. By contrastapoptosis produced by the antineoplastic agents is notsubject to this inhibition by anti-CD40 or TNF-a. Thusboth mechanisms (cell surface receptor-mediated agents,such as anti-IgM, and nonreceptor-initiated antineoplas-tic agents) cause a decline in c-myc and cause growthinhibition, but the steps leading to apoptosis are likelydifferent for the two classes of agents.

Cotreatment with CCS Antineoplastic Agents andAnti-IgM

Our results using cotreatments with various CCSantineoplastic agents show that the therapeutic out-

TABLE 1

Apoptosis Affected by Cotreatment of Drugs

Treatment

Agentsa Apoptosis (%)b,c

A B A alone B alone A 1 B

Within same group Aphidicolin Hydroxyurea 5.3 6 2.6 4.0 6 2.8 9.5 6 0.7Camptothecin Etoposide 6.8 6 6.1 2.0 6 1.0 7.0 6 4.2Taxold Vincristined 25.8 6 11.8 23.8 6 11.1 67.0 6 14.0Taxole Vincristinee 90.8 6 4.6 .99 .99

G1/S vs S/G2 Aphidicolin Camptothecin 5.3 6 2.6 6.8 6 6.1 15.3 6 7.6Aphidicolin Etoposide 5.3 6 2.6 2.0 6 1.0 7.0 6 1.4Hydroxyurea Camptothecin 4.0 6 2.8 6.8 6 6.1 5.0 6 3.0Hydroxyurea Etoposide 4.0 6 2.8 2.0 6 1.0 4.0 6 1.0

G1/S, S/G2 vs G2/M Aphidicolin Taxole 5.3 6 2.6 90.8 6 4.6 34.3 6 34.8Hydroxyurea Taxole 4.0 6 2.8 90.8 6 4.6 30.3 6 27.0Aphidicolin Vincristinee 5.3 6 2.6 .99 25.7 6 8.2Hydroxyurea Vincristinee 4.0 6 2.8 .99 30.5 6 24.8Camptothecin Taxole 6.8 6 6.1 90.8 6 4.6 29.5 6 28.4Etoposide Taxole 2.0 6 1.0 90.8 6 4.6 15.0 6 17.0Camptothecin Vincristinee 6.8 6 6.1 .99 15.7 6 0.6Etoposide Vincristinee 2.0 6 1.0 .99 16.5 6 14.8

CCS agents vs anti-IgM Aphidicolin Anti-IgM 5.3 6 2.6 10.6 6 5.5 26.4 6 10.0Hydroxyurea Anti-IgM 4.0 6 2.8 10.6 6 5.5 17.0 6 4.2Camptothecin Anti-IgM 6.8 6 6.1 10.6 6 5.5 9.8 6 4.9Etoposide Anti-IgM 2.0 6 1.0 10.6 6 5.5 5.0 6 1.4Taxold Anti-IgM 25.8 6 11.8 10.6 6 5.5 77.3 6 25.8Vincristined Anti-IgM 23.8 6 11.1 10.6 6 5.5 70.0 6 18.0

a Doses for G1/S agents: 1 mM aphidicolin, 100 mM hydroxyurea. Doses for S/G2 agents: 50 nM camptothecin, 1 mM etoposide. Dose foranti-IgM: 2.5 mg/ml.

b Bold indicates enhanced interaction; italics indicate inhibitory interaction.c Results are compiled from four to six data sets.d Lower dose range for G2/M agents: 1 nM Taxol, 1 nM vincristine. Lower doses are used so that enhancement can be observed.e Higher dose range for G2/M agents: 10 nM Taxol, 10 nM vincristine. Higher doses are used so that inhibitory effects can be observed.

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comes are not easily predictable. Some agents antago-nize the action of other agents. For instance hydroxy-urea, aphidicolin, camptothecin, or etoposide canstrongly inhibit the action of Taxol or vincristine.Growth arrest caused by the G1/S and S/G2 agents mayprevent cells from entering into G2/M. This may in partexplain their inhibitory effect on G2/M agents.

Anti-IgM was found to induce apoptosis in Ramoscells and, in cotreatment with CCS antineoplasticagents, we found that it was synergistic selectivelywith G2/M agents. This suggests that antibodies di-rected against the B cell antigen receptor can be effec-tive drugs in augmenting the antineoplastic effects ofTaxol and vincristine. This enhancement suggests thatincreased potency can be achieved at tolerable doses ofthese highly toxic drugs. Maintaining efficacy whilealleviating the toxicity due to high dosages would be ofgreat clinical interest. Since G2/M agents are amongthe most widely used drugs, the development of syner-gistic conditions may be useful in multiple cancertreatment regimens. A recent report has indicated thatNCCS antineoplastic agents may function synergisti-cally with CCS agents [62]. The results reported hereindicate the concept and feasibility of using the syner-gistic action of antibodies together with G2/M agents.

These results are not a direct indication for clinicaltreatment modalities since further investigation shouldbe conducted to test whether the in vitro findings alsoapply to in vivo situations. In addition, the emphasis inthis study was to compare the ability of CCS antineoplas-tic agents to induce apoptosis during the initial 24 h (i.e.,during the first cell cycle after the onset of treatment).The effect of these agents on long-term survival and cellreproduction using assays such as clonogenicity as anend point were not investigated. The required drug dos-ages and drug effects which might affect the outcome inlong-term cell culture or in whole animal studies thuscannot be directly inferred from this study.

This paper introduces three novel concepts, namely,(a) agents that are acting at the same stage of cell cyclephase provide at most an additive effect. These condi-tions may not be advantageous since toxicity may behigher. (b) An agent which acts at one stage of the cellcycle may antagonize the action of another agent act-ing at another stage of the cell cycle resulting in aninhibitory effect. (c) Cell surface receptor-directed an-tibodies may be important cotreatment agents for en-hancing the apoptotic effect of widely used antineoplas-tic agents such as Taxol and vincristine, thus providingrelief from toxicity while at the same time enhancingcellular end effects.

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Received March 18, 1998Revised version received May 20, 1998

13ANTI-IgM ENHANCES AND G1/S OR S/G2 AGENTS INHIBIT G2/M AGENT-INDUCED APOPTOSIS