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From www.bloodjournal.org by guest on June 17, 2017. For personal use only. BCL-2 Expression and Mitochondrial Activity in Leukemic Cells With Different Sensitivity to Glucocorticoid-Induced Apoptosis By Lou A. Smets, Joop Van den Berg, Dennis Acton, Bert Top, Henny Van Rooij, and Manon Verwijs-Janssen The present study investigates the relationship between mitochondrial activity and the expression of the BCL-2 genein a panel of six human and murine leukemia/lymphoma cell lines. The cell lines all contained normal glucocorticoid receptors but differed widely in sensitivity to dexamethasone, ranging from very sensitive S49 lymphoma to completely resistant HL-60 acuteleukemiacells. In this panel,10- to 15-folddifferences in basaladenosinetriphosphate(ATP) content and adenosine diphosphate (ADP)/ATP ratio were correlated with up to fivefold differencesin bcl-2 protein (in human cells) and approximately 25-fold difference in bel-2 mRNA content (all cell lines). Moreover, ATP content and BCL-2 gene expression were inversely correlated with gluco- corticoid sensitivity and cell cycle length. In resistant cell lines, sensitivityto dexamethasonewas restored by the mitochondrial inhibitors rotenone andmeta-iodobenzylguanidine. This sensitization was not accompanied by detectable reductions in bcl-2 mRNA or protein content, suggesting that the inhibitors were capable of overriding BCL-2-mediated inhibition ofapoptosis.Increased mitochondrial activity and (overexpressed) BCL-2 appeared closely related properties of glucocorticoid-resistantcells, sharing common cellular targets in hormone-induced apoptosis. 0 1994 by The American Society of Hematology. T murine and human leukemic cell lines, selected for widely different susceptibility to the growth inhibitory and lytic effects of dexamethasone. In this report, we explain that the expression levels of the BCL-2 gene and cellular energy status are highly correlated phenomena and that both parameters are inversely related to sensitivity for GC-induced apoptosis and cell cycle length. HE TRANSCRIPTIONALLY deregulated BCL-2 gene increases the life-span of lymphoid cells'.' and confers resistance to various inducers of programmed cell death (apopt~sis).~.~ According to recent reports the bcl-2 protein complexes with death-accelerating homologs7 and functions in antioxidant pathways in preventing apoptosis.8The precise biochemical activities of bcl-2 protein and its homologs are not known, however. Because of its localization to mitochondrial membranes,' protection by BCL-2 has been previously associated with mitochondrial functions." An exclusive role of bcl-2 protein in mitochondria has been questioned, however, by the observation that in several cells the pattern of immunofluorescent staining is consistent with its localization to the endoplasmatic reticulum (ER) and the nuclear envelope as well.9"' In fact, neither mitochondrial localization nor an integral membrane position are absolute requirements for bcl-2 function according to Hockenberry et al.' Mitochondrial involvement has been specifically challenged by the observation that fibroblasts lacking mitochondrial DNA and thus deficient in oxidative phosphorylation, remain susceptible to protection by the transfected human BCL-2 gene." On the other hand, cells deficient in mitochondrial DNA still contain all nuclear encoded mitochondrial enzymes and retain important mitochondrial functions that include succinate dehydrogenase activity" and the generation of an electrochemical gradient.13There is circumstantial evidence for a critical role of mitochondrial activity in the susceptibility of leukemic cells to glucocorticoid (GC)-mediated lysis. Stru~tural'~ and functional" damage to mitochondria has been observed early in GC-induced lysis of lymphoid cells, although a causal sequence has not been established. However, several leukemic cell lines, including fully resistant variants, can be sensitized to dexamethasone by inhibitors of mitochondrial respiration.'"18 The observation that leukemic cells are protected from GC-induced apoptosis by a relative abundance of bcl-2 p r ~ t e i n but ~ . ~sensitized by respiration inhibitors adds to a notion of functional relationships between bcl-2 protein and mitochondrial activity in the lysis of leukemic cells. To investigate the possibility relationship between BCL2 and mitochondria, we have compared mitochondrial activity and BCL-2 expression in a panel of GC receptor-positive Blood, Vol 84, No 5 (September l), 1994: pp 1613-1619 MATERIALSANDMETHODS Cell lines and cytological assays. Cell lines HL-60, L1210 (subline 56.3), and S49 and culture conditions were as described in previous rep"t~.'~.'','~ Cell line JANEL was established by infection of normal human B cells with Epstein-Barr virus. The human nonHodgkin's follicular lymphoma cell line DoHH2;' carrying translocation t(14; 18), was kindly donated by H. Kluin-Nelemans (Leiden University, The Netherlands). Human T-cell leukemia CEM-C7 line was obtained by courtesy of T. Schmidt (University of Iowa). Dexamethasone (DEX) was added from 1,000-fold ethanol concentrates toafinal concentration of 10" m o w throughout and lysis was scored microscopically. DNA per cell recordings were made by flow cytometry on ethanol-fixed and ethidium bromide stained cells as described previously.2' Metabolic studies. DNAand protein synthesis was assessed from the incorporation of'H-thymidineandI4C-leucine, respectively, into acid-precipitable cell material and related to cellular protein according to routine procedures. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) content were determined in cold 0.5 N perchloric acid extracts" and the relative ADP content was expressed by the molar ratio ADP/ATP X 100%. 3H-dexamethasonebinding. Specific 3H-dexamethasonebinding From the Divisions of Experimental Therapy and Molecular Genetics, The Netherlands Cancer Institute, Amsterdam. Submitted January 20, 1994; accepted May 4, 1994. Supported in part by the Dutch Cancer Society, Grant No. NKI89-2. Address reprint requests to Lou A. Smets, PhD, The Division of Experimental Therapy, The Netherlands Cancer Institute, 121 Plesmanlaan, NL-1066 CX, Amsterdam, The Netherlands. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1994 by The American Society of Hematology. 0006-4971/94/8405-03$3.00/0 1613 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 1614 SMETS ET AL Table 1. Characterization of the Cell Lines With Different sites were measured in a whole-cell assay as described elsewhere." Briefly, cells were incubated with 2 pCi of 'H-dexamethasone (5 X Sensitivities t o Dexamethasone 10"' moliL) with or without a I ,000-fold excess of radioinert steroid DEX toassess nonspecific binding.After 20 minutespostincubationin Cell Cycle Binding Time Sites Sensitivity agonist-free growth medium the amount of cell-associated radioacCell Line Description (hours) (per cell) to DEX tivity was determined. Chemicals, antibodies, and buffers. Anti-receptor antibody HL-60 Human myeloid leukemia 48 18,000 GR49.1 was kindly donated by Dr H.M. Westphal (Marburg, GerDoHH2 Human N H lymphoma 36 15,000 + many): monoclonal MAI-510 (clone BuGR2) was purchased from Jane1 Human B-cell leukemia 36 ND 2 Affinity Bioreagents (Neshanic Station, NJ). Anti bcl-2 monoclonal CEM-C7 Human T-cell leukemia 18 20,000 + antibody (clone 124)'? was a kind gift of Drs Mason and Pezella L1210 Mouse leukemia 12 9,000 ++ (Oxford, U,K). Anti-actin antibody (clone C4) was purchased from S49 Mouse lymphoma 10-12 20,000 +++ Boehringer Mannheim Biochemica (Mannheim, Germany). HorseCell cycle time was estimated from the doubling time during exporadish peroxidase conjugated goat-anti-mouse (GAM-HRP) IgG was nential growth. Specific W D E X binding sites were determined as from Pierce (Rockford, IL), fluorescein isothiocyanate (FITC) coudescribed in Materials and Methods. Sensitivity to IO" mol/L of dexapled sheep-anti-mouse IgG was from Sigma (St Louis, MO), nitromethasone was rated as strong growth inhibition and massive cell cellulose filters werefromBio-Rad(Richmond,VA)andPVDF lysis after 24 hours (+++), growth inhibition with marginal cell lysis lmmobilonmembraneswerefromMillipore(Bedford,MA).Enhanced chemiluminescence (ECL) detection kit for Western blotting, after 48 hours (+) or no effect ( 6 ) . Abbreviation: ND. not done. 'H-thymidine (25 Ci/mmol), I4C-leucine (300 mCi/mmol), 'H-dexamethasone(37Ciimmol),and '251-IgG sheep-anti-mouse ("'ISAM, 16 pCi/pg) were from Amersham International (Buckinghamshire, UK). rescenceafter fixation withglutaraldehyde (0.2% inPBSfor10 Buffers used included phosphate buffered saline (PBS); blocking minutes at 4 T ) , permeabilization with 0.2% Triton X-l00 (30 minbuffer no. I : Carnation (Los Angeles, CA) nonfat dry milk (5% (wt/ utes at room temperature) and reduction with NaBH4(3 X 5 minutes vol)) and 0.1% (vol/vol) Tween-20 in PBS; blocking buffer no. 2: at room temperature). The slides were then incubated in blocking 0.15% (wt/voi) bovine serum albumin (BSA), 0.5 mmol/L EDTA, buffer no. 2for 1 houratroomtemperatureandincubatedwith 0.5% (vol/vol) Tween-20 in PBS; SSC (20X): 88.2 g sodinmcitrate, undiluted anti bcl-2 antibody in the cold for 6 hours, followed by 175.3 g NaCI, pH 7; lysis buffer: 250 mmol/L sucrose, 20 mmol/L three washes with cold PBS and incubation in FITC-coupled RAM KH2P04 pH 7, 1 rnmol/L EDTA, CaCl, and MgCl,, both at 0.1 5 IgG for 10 hours in the cold (1:5000 in blocking buffer no. 2). DNA mmol/L. was stained by propidium iodide in a concentration of 0.02 pg/mL Western blotting. The intracellular distribution of glucocorticoid for 30 minutes at room temperature. A BioRad MRC 600 confocal receptors was determined by Western blotting. After disruption of laser microscope was used to visualize the protein (excitation at488 the cells in lysis buffer, cytosolic and nuclear fractions were preparednm) and nuclei (at 350 nm). by centrifugation at 14,000.g and cell equivalent amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel elecRESULTS trophoresis(SDS-PAGE)(7.5%)andtransferredtonitrocellulose Characterization of cell lines. The presence of GC-bindfilters. The filters were blocked with buffer no. I and probed with anti-receptor antibody GR49.1or BuGR2 (1:5,000 in blocking buffer ing sites, ranging from 9,000 in L1210 to 20,000 sitedcell in S49 and CEM-C7, wasconfirmed in conventional wholeno. 1) using GAM-HRP(1 :1000 in blocking buffer no.1) as secondcell binding assays. Nuclear translocation of DEX-liganded ary antibody. The immunoreactive proteins were visualized by the ECL technique according to instructions of the manufacturer. cytosolic receptors was also confirmed by immunoblotting Relative levels of bcl-2 protein were determined in samples of of the GC receptor protein in cytosolic and nuclear fractions IO" cells boiled in sample buffer, separated by SDS-PAGE (15%) before and after 2 hours of incubation with the steroid. In and transferred to Immobilon-P, PVDF membranes. The membranes spite of the presenceofnormal GC receptors and of the were incubated in blocking buffer no. 2 and probed with anti bcl-2 capability of nucleartranslocation, thecelllines differed antibody ( 1 :20 in blocking buffer no. 2). Detection was performed widelyinsusceptibility to the growthinhibitory and lytic by GAM-HRP (1:3,000 in blocking buffer no. 2) as a second antieffects of m o l L DEX. (Table 1). In S49 cells, 80% body and staining with 4-chloro-l-naphthol. Actin was detected on was lysed within 24 hours, whereas lysis of L1210 cells was the same membrane with anti-actin (1 :10,000 in blocking buffer no. observed only after 2 days.19 In CEM-C7 cells inhibition of 2) as primary antibody. For quantification, pre-stained bands were relabeled with "'l-SAM (2 pCiin blocking buffer no. 2), cut out, and growth started after 72 hours and was accompanied by the the associated radioactivity was measured by scintillation counting. appearance of 20% to 40%lysed cells after 92 hours. DoHH2 Northern blotting. TotalRNAwasisolatedfrom =50 X 10' and JANEL cellsresponded to DEXby a transient inhibition cells.24 For Northern blot analysis 20 pg of RNA was separated on of growth during the first 24 hours, accompanied by a mara 1 % agarose gel and transferred to nitrocellulose filters. Filters were ginal degree of cell death of 10% to 20%. Subsequently, hybridized with a mouse cDNA probe, derived from a mouse cDNA these cells resumed normal growth in the continuous preslibrary and comprising the bcl-2 codon region as described by Neence of DEX. HL-60 cells were completely insensitive to grini et al? and labeled by the random priming method. Filters were Overall, the cell DEX for 72 hours as reported previou~ly.'~ washed in 0.1 X standard sodium citrate (SSC), 1% SDS at 6VC, dried and exposedto x-ray film. The radioautograms were quantified lines with shorter cell cycle time were better responders to the steroid,but there was no relationwith the number of by densitometry. Total RNA and actin mRNA were used as loading specific GC binding sites. controls. Mitochondrial activity. The cell lines differed considerImmunocytochemistry of hcl-2. Intracellular distribution of bcl2 was investigated in cytospin preparationsby indirect immunofluoably in basal ATP content and energy status as reflected by From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 1615 bcl-2 AND GLUCOCORTICOIDSENSITIVITY 80 tein was not available, BCL-2 gene expression in all cell lines was assayed on Northern blots using a mouse cDNA probe. This probe detects a 7.5-and2.4-kb transcript as previously observed by Negrini et al. Because of the absence of cross-hybridization with the 28s rRNA, an additional 5kb message is also detected. The 7.5-kb bcl-2 mRNA levels in L12 IO and S49 cells were compared by densitometry, and normalized against the 2-kb actin mRNA signal. The results (Fig 3C) indicated at least three-fold higher levels in L1210 cells. Mouse L1210 cells werecomparedwithhuman HL-60DoHA2 Janel C E M L1210 S49 DoHH2 cells, using the mouse cDNA probe. At comparable total mRNA loading levels (Fig 3A), expression of the major Fig 1. ATPlevels (W) and relative ADP content (0)in celllines with (from left to right)increasing sensitivity to dexamethasone Id, 7.5-kb transcript in DoHH2 cells was about eight-fold higher Table 1). ADP content is expressed aspercentage of molar ATP conthan in L1 210 cells (Fig 3B). Considering the differences in tent and isdefined as the reciproque of cellular energy charge. Values the nucleotide sequences between mouse and human BCL2, are mean ? SEM of duplicates of six to eight independent experithis eight-fold higher level may be an underestimate. Howments. ever, no obvious differences in mRNA levels were observed between DoHH2and the other human cell lines HL-60. JANEL, and CEM-C7 (data not shown). ADP/ATP ratio (Fig l). ATP levels were expressed relative Reversal of GC-resistance by mitochondrial inhibitors. to protein content to compensate for differences among the To test if increased ATP levels wereinstrumental in GC various cell lines. The observed values were well above (HLresistance, the cells were assayed for dexamethasone-medi60) or below (S49; L1210) the average ATP content reported ated lysis in the presence of the inhibitors rotenone or metafor various leukemic and non-leukemic cell lines of -4 to iodobenzylguanidine (MIBG). Inhibition of mitochondrial 6 nmol/lO' cells,''~*' corresponding with -20 mg/g protein. respiration with these complex I inhibitors" or with doxycyLikewise, the relative ADP content ranged significantly becIineI8allows for a compensatory increase in glycolytic flux, low and above the average value of = 10% in actively growmaintaining ATP at lower but vital levels. MIBGcaused ing cells in vitro. Overall, sensitivity to dexamethasone was a dose-dependent cytolytic response to dexamethasone in inversely correlated with ATP content and proportional with refractory DoHH2 cells. This became already apparent as decreasing energy status, ie, increasing ADP/ATP ratio. early as 12 hours after combined treatment (Fig 4). Rotenone BCL-2 expression. The bcl-2/actin ratio was highest in in the non-toxic concentration range of 0.5 to 2.0 X IO-' HL-60 cells, exceeding by two-fold that of DoHH2 cells. molL also potentiated lysis (Fig 5). An excess of IO-' mol/ EBV-transformed JANEL cells contained lower levels of L of the GC-antagonist RU486 completely blocked rotenonebcl-2 protein but these were still higher thanin CEM-C7 induced sensitization to dexamethasone, indicating that lysis cells (Fig 2). The bcl-2/actin ratio of reference human leukowas initiated by the hormone and not caused by toxic side cytes was 0.05, indicating the relative abundance of the proeffects of the inhibitor. The effects of rotenone and MIBG tein in the human cell lines. As expected, mouse L1210 cells in JANEL cells were similar to those observed in DoHH2 were negative for the human-specific antibody. Because an antiserum directed against murine bcl-2 procells. Sensitization of CEM-C7 and L1210 cells was as re- "1 T 1 .oo r I actin ] 0.80 Fig2.Levels of bcl-2 protein in human leukemic cell lines with increasing sensitivity to DEX es described in the legend to Fig 1. Bel-2 protein content is expressed relative to actin content in an arbitrary scale and values are mean ? SE of three to four independent experiments with mouse L1210 cells as a negative control (background levels). A representative immunoblot is shown in the insert and cell linesare indicatedby initials. The dashed line represents the bcl9lactin ratio in normal human peripheral lymphocytes. H J D C L 0.60 0.40 0.20 0.00 HL-60 DoHH2 Janel CEM L1210 S49 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. SMETS ET AL 1616 A B 1 2 3 1 2 3 C 1 2 S 100 . 80 kb kb - 7.6 -5 60 -7.6 40 -4.8 -2.1 20 - 2.4 .’ -.S - 0 0.1 1 1 10 rotenone (10-6M) Fig 5. Sensitization of DoHH2 cells by rotenone. Cells were incubated in graded concentrations of rotenonewith (VIor without (01 lo” mol/L DEX and lysis was scored after 24 hours of incubation. Mean values 2 SE of four independent experiments. Fig 3. Bcl-2 mRNA expression in leukemic cell lines. (A) TotalRNA loading control ofB. (B1 Northern blot of bcl-2 mRNA levels in mouse L1210 cells (lane l), control human DoHH2 cells (lane 2). and DoHH2 mol/L rotenone (lane 3). (Cl cells incubated for 21 hours with Northern blot of bcl-2 and actin mRNA levels in L1210 (lane 1) and S49 (lane 21 cells. ported previously” but observations in S49 cells were inconclusive because of toxicity of both inhibitors in the concentrations used, probably because of critically low basal ATP levels. However, HL-60 cells remained completely refractory to DEX in the presence of rotenone. With MIBG a weak and delayed (ie, after 4 days) response to the steroid was induced as reported earlier.“ ATP levels were reduced in 8o 6o t t 0 ’ 0 10 20 30 40 I 50 incubation time(hour) Fig 4. Sensitization of DoHH2 cells t o DEX (lo” mol/L) by MIBG; control: W 5 p g / m L (01, 10 p g / m L ( W , 20 p g / m L (01.Exponentially growing cells (4 x 1O5/mL1were incubatedwith DEX and MIBG and scored for thepercentage of apoptotic cells at regularintervals. Mean values f SE of four t o five independent experiments. cells grown with rotenone ( IOv6 mol/L) or MIBG ( I O pg/ mL). After 21 hours, the reductions variedbetween 70% (CEM-C7, L1210) and 40% (HL-60) and were all accompanied by a proportional decrease in energy charge. In HL-60 cells, refractory to sensitization by the inhibitors, the ATP levels (37 mg/g protein) and the relative ADP content (6%) of treated cells remained well above (ATP) or below (ADP/ ATP) the basal values in the sensitive L1210 and S49 cells (cf, Fig l ) . Efects on BCL-2 expression and cell proliferation. The effect of mitochondrial inhibitors was studied in more detail in DoHH2 cells. There was no reduction in the level of bcl2 protein during incubation in rotenone for 4 or 16 hours (Fig 6). In Northern blots, the major 7.5-kb messenger signal in DoHH2 wasnot detectably altered by incubation with MIBG or rotenone for 21 hours (Fig 3A,B). Immunocytochemistry with confocal laser scanning microscopy of bcl-2 protein distribution showed predominant perinuclear localization with the typical patchy staining as described for several other cells?” At this level of resolution, no gross effects of the inhibitors alone or in combination with dexamethasone on bcl-2 content nor its intracellular distribution could be observed (data not shown). Consistent with the finding on bcl-2 protein levels (Fig 6), the incorporation of I4C-leucine was not significantly affected during 21 hours of incubation with the inhibitors. The number of specific ‘H-DEX binding sites was only marginally reducedfrom 15,000 to 11,000 sites per cell, probably because of cell cycle Cell multiplication in 24 hours was reduced from 1.8 in controls to 1.2 in cells grown the presence of rotenone or MIBG. Flow-cytometric analysis of DNA per cell content indicated that this inhibition of growth was accompanied byan increase in the fraction of cells in G, phase from 0.35 to 0.75. The biochemical and cell kinetic findings in DoHH2 cells were similar to the reported effects of doxycyclineIx and MIBGIh.17 ’In leukemic cells, showing that inhibitionof mitochondrial respiration with simultaneous glycolytic compen- From www.bloodjournal.org by guest on June 17, 2017. For personal use only. bcl-2 AND GLUCOCORTICOIDSENSITIVITY control 4 hrs 16 hrs 1 2 3 4 5 6 1617 kDa -55 - 26 -1 4 Fig 6. Effect of rotenone on bcl-2 protein levels in DoHH2 cells. lmmunoblot of bcl-2 protein in DoHH2 cells incubatedwithout (lanes l , 2) and with 10.' mol/L rotenone for 4 hours (lanes3,4) or 16 hours (lanes 5, 6). sation allows completion of the ongoing cell cycle but blocks cells in G, phase of the next division cycle. DISCUSSION The protective effect of the overexpressed RCL-2 gene against several apoptotic stimuli has been well documented:.' but the biochemical mechanisms involved are still a matter of intensive research. In view of its preferential association with mitochondrial membranes,' protection from apoptosis by bcl-2 has been previously associated with mitochondrial functions. According to recent reports, bcl-2 may act in the control of intracellular Ca" repartitioning','' and functions in antioxidant pathways.'.'' The present observations in a panel of leukemic cell lines revealed a correlation between BCL-2 expression (Figs 2 and 3) and mitochondrial activity as reflected by ATP levels and energy status (Fig l ) . Moreover, BCL-2 expression level and ATP content were inversely related to the susceptibility to dexamethasone and the rate of cell proliferation (Table l ) . Variation in sensitivity to dexamethasone was not associated with the amount of 'H-DEX binding sites nor with the nuclear translocation capability of the liganded GC-receptor. The notion thatincreased mitochondrial activity can interrupt the lytic signal, once initiated by binding of GC's to their cognate receptors, finds additional support in the present and previous observations'"'X that inhibitors of mitochondrial respiration can potentiate GC-action and even induce sensitivity to steroid-mediated lysis in resistant cell lines. A comparison of the basal ATP levels (Fig 1) and the sensitizing effect of inhibitors suggests that a level of about 20 mg ATP/ g protein (4 nmol/lO" cells) or the corresponding ADP/ATP ratio of about 0.20 are critical discriminators between sensitivity and insensitivity to GCs, irrespective of leukemic cell lineage. These observations agree with similar alterations in ATP levels and relative ADP content in Molt-4 leukemia cells sensitized to DEX by doxycycline.IxIn this view. failing sensitization of HL-60 cells by rotenone and MIBG can be ascribed to the inability of the drugs to lower high basal ATP levels below this critical level. Conversely, the sensitivity of morerapidly growing cell lines (CEM-C7, L1210. S49) is plausibly explained by an intrinsically lower energy status as a consequence of enhanced energy requirements for rapid protein and nucleic acid synthesis. Unlike in the human cell lines, the relative bcl-2 protein levels in themurine L1210 and S49 cells couldonly be estimated frommRNA levels. Althoughcomparison of mRNA levels can only show a trend in bcl-2 protein content, there was a marked, ie, 25-fold difference in BCL-2 gene expression between fully resistant HL-60 and moresensitive S49 cells detected with a mouse cDNA probe. Because of sequence differences betweenhumanandmurinemessengers, this difference is probably an underestimate. Despite this, the relationship between BCL-2 gene expression on the one hand and the correlated variations in ATP content, cell cycle time and DEX sensitivity (Fig 1: Table I ) on the other, is not an obvious one. However. abrogation of GC-resistance by the inhibitors in DoHH2 cells without detectable effects on bcl-2 protein (Fig 6) or mRNA (Fig 3) content may exclude a trivial explanation that a low energy status on its own reduces RCL-2 expression. Therefore, overexpression of RCL-2 andelevated energy status may each afford protection by totally different mechanisms and the observed correlations could be coincidental. ie, the result of co-selection for two unrelated mechanisms of GC-resistance. On the other hand, there are some grounds for the hypothesis that mitochondrial activity and bcl-2 protein may cooperate in modulating GC sensitivity. In cultured hematopoietic cells and in the B-cell compartment of transgenic mice, overexpression of the bcl-2 proteindoes not stimulate cell proliferation per se, but promotes survival by preventing irreversible cell cycle exit leading to cell death."' In fact. the inverse correlation in our cell panel between RCL-2 expression and cell cycle length suggests that the proto-oncogene can reduce the rate of cell proliferation, allowing the recovery of energy charge required for the acquisition of resistance toDEX. The notion that the BCL-2 gene can suppress proliferation finds support in the observations that elevated levels of bcl2 protein are associated with slowly growing, indolent tumors3" and can afford protectionagainst the lethal effects of excessive or inappropriate mitogenic signals.." Finally, the observation that mitochondrial inhibitors were capable of overriding RCL-2-mediated protection in DoHH2 cells could indicate a more direct relationship between bcl2 proteinandmitochondrial activity. Current explanations on bcl-2action" concentrate on twoequally attractive hypotheses. namely the regulation of intracellular Ca'' repartioning and scavenging of reactive oxygen species. It is conceivable that any role of bcl-2 in antioxidant pathways will be dependent on the cellular redox state, and thus, sensitive to complex I inhibitors in cells with a functional respiratory chain. Likewise, mitochondria are directly and indirectly implicated in Ca" homeostasis. It is of note that incubation of hepatocytes with MIBG results in an increased size of the mitochondrial Ca2+pool,33a process that is typically blocked From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 1618 SMETS ET AL by bcl-2 overproduction in a hematopoietic cell line resistant to apoptosis induced by growth factor ~ithdrawal.~’ In spite of suggestive correlations, the present experiments cannot provide for direct evidence that bcl-2 protein level is a metabolic checkpoint of GC-sensitivity, acting through mitochondrial functions. However, it is obvious that the physiologic role of bcl-2 in the response of leukemic cells to GC treatment can be fully appreciated only in the context of cell cycle control and cellular energy status. Irrespective of the precise mechanisms involved, the apparent possibility to override resistance to GC hormones in bcl-2 expressing human leukemicflymphoma cells may be of clinical relevance. Increased bcl-2 protein levels are found inan expanding spectrum of hematologic malignancies, often without involvement of translocation t( 14;18).23,34In tissue culture models, the proto-oncogene appears a potential arbiter of response to GC hormones and various antileukemic drug^^.^ and BCL-2 expression has been recently associated with poor outcome of chemotherapy in acute myeloid leukemia.35Accordingly, antagonism of bcl-2 action by pharmacologic interventions of the type described in this report would appear a feasible strategy in the chemotherapy of leukemid lymphoma with high expression of bcl-2 protein. ACKNOWLEDGMENT The authors gratefully acknowledge Dr H. Westphal for providing the anti-GR antibody, Drs F. Pezella andD. Mason for the antibcl-2 Moab, Dr H. Kluin-Nelemans for the DoHH2 cells, Nienke Oldenburg for assistance in the ATP assays, and Dr C. van den Bogert for valuable suggestions. REFERENCES 1 . 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For personal use only. 1994 84: 1613-1619 BCL-2 expression and mitochondrial activity in leukemic cells with different sensitivity to glucocorticoid-induced apoptosis LA Smets, J Van den Berg, D Acton, B Top, H Van Rooij and M Verwijs-Janssen Updated information and services can be found at: http://www.bloodjournal.org/content/84/5/1613.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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